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Zhao L, Zhang Y, Tian Y, Ding X, Lin R, Xiao L, Peng F, Zhang K, Yang Z. Role of ENPP1 in cancer pathogenesis: Mechanisms and clinical implications (Review). Oncol Lett 2024; 28:590. [PMID: 39411204 PMCID: PMC11474142 DOI: 10.3892/ol.2024.14722] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2024] [Accepted: 09/17/2024] [Indexed: 10/19/2024] Open
Abstract
Cancer is a significant societal, public health and economic challenge in the 21st century, and is the primary cause of death from disease globally. Ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) serves a crucial role in several biochemical processes, including adenosine triphosphate hydrolysis, purine metabolism and regulation of signaling pathways. Specifically, ENPP1, a type II transmembrane glycoprotein and key member of the ENPP family, may be upregulated in tumor cells and implicated in the pathogenesis of multiple human cancers. The present review provides an overview of the structural, pathological and physiological roles of ENPP1 and discusses the potential mechanisms of ENPP1 in the development of cancers such as breast, colon, gallbladder, liver and lung cancers, and also summarizes the four major signaling pathways in tumors. Furthermore, the present review demonstrates that ENPP1 serves a crucial role in cell migration, proliferation and invasion, and that corresponding inhibitors have been developed and associated with clinical characterization.
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Affiliation(s)
- Lujie Zhao
- School of Basic Medical Sciences, Shandong Second Medical University, Weifang, Shandong 261021, P.R. China
| | - Yu Zhang
- School of Basic Medical Sciences, Shandong Second Medical University, Weifang, Shandong 261021, P.R. China
| | - Yahui Tian
- School of Basic Medical Sciences, Shandong Second Medical University, Weifang, Shandong 261021, P.R. China
| | - Xin Ding
- School of Clinical Medical Sciences, Shandong Second Medical University, Weifang, Shandong 261021, P.R. China
| | - Runling Lin
- School of Basic Medical Sciences, Shandong Second Medical University, Weifang, Shandong 261021, P.R. China
| | - Lin Xiao
- School of Basic Medical Sciences, Shandong Second Medical University, Weifang, Shandong 261021, P.R. China
| | - Fujun Peng
- School of Basic Medical Sciences, Shandong Second Medical University, Weifang, Shandong 261021, P.R. China
- Weifang Key L2aboratory of Collaborative Innovation of Intelligent Diagnosis and Treatment and Molecular Diseases, School of Basic Medical Sciences, Shandong Second Medical University, Weifang, Shandong 261053, P.R. China
| | - Kai Zhang
- Genetic Testing Centre, Qingdao University Women's and Children's Hospital, Qingdao, Shandong 266000, P.R. China
| | - Zhongfa Yang
- School of Basic Medical Sciences, Shandong Second Medical University, Weifang, Shandong 261021, P.R. China
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Ryder S. Integrated Applied Clinical Pharmacology in the Advancement of Rare and Ultra-Rare Disease Therapeutics. Clin Pharmacol Ther 2024; 116:1485-1495. [PMID: 39034754 DOI: 10.1002/cpt.3382] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2024] [Accepted: 06/29/2024] [Indexed: 07/23/2024]
Abstract
The introduction of safe and effective rare/ultra-rare disease treatments is a focus of many biotherapeutic enterprises. Despite this increased activity, a significant unmet need remains, and the responsibility to meet this need is augmented by enhanced genomic, biologic, medical, analytical, and informatic tools. It is recognized that the development of an effective and safe rare/ultra-rare disease therapeutic faces a number of challenges with an important role noted for clinical pharmacology. Clinical pharmacology is foundationally an integrative discipline which must be embedded in and is interdependent upon understanding the pathogenic biology, clinical presentation, disease progression, and end-point assessment of the disease under study. This manuscript presents an overview and two case examples of this integrative approach, the development of C5-targeted therapeutics for the treatment of generalized myasthenia gravis and asfotase alpha for the treatment of hypophosphatasia. The two presented case examples show the usefulness of understanding the biological drivers and clinical course of a rare disease, having relevant animal models, procuring informative natural history data, importing assessment tools from relevant alternative areas, and using integrated applied clinical pharmacology to inform target engagement, dose, and the cascade of pharmacodynamic and clinical effects that follow. Learnings from these programs include the importance of assuring cross-validation of assays throughout a development program and continued commitment to understanding the relationship among the array of Pd end points and clinical outcomes. Using an integrative approach, substantive work remains to be done to meet the unmet needs of patients with rare/ultra-rare disease.
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Noor Ul Ayan H, Nitschke Y, Mughal AR, Thiele H, Malik NA, Hussain I, Haider SMI, Rutsch F, Erdmann J, Tariq M, Aherrahrou Z, Ahmad I. Homozygous splice-site variant in ENPP1 underlies generalized arterial calcification of infancy. BMC Pediatr 2024; 24:733. [PMID: 39538190 PMCID: PMC11558987 DOI: 10.1186/s12887-024-05123-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/23/2024] [Accepted: 09/30/2024] [Indexed: 11/16/2024] Open
Abstract
ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) plays a critical role by converting extracellular ATP to AMP, generating extracellular PPi, a potential inhibitor of calcification. Pathogenic variants in the ENPP1 cause generalized arterial calcification of infancy (GACI [OMIM 208000]). GACI, is an ultra-rare disease characterized by early-onset calcification of large and medium-sized arteries, leading to severe cardiovascular complications such as heart failure, pulmonary stenosis (PS), hypertension, and more. In this study, we report a novel homozygous splice-site pathogenic variant in ENPP1 (NM_006208, c.2230 + 5G > A; p.Asp701Asnfs*2) residing in C-terminal nuclease-like domain (NLD) of ENPP1 protein in a Pakistani family diagnosed with severe valvular PS and mild right ventricular hypertrophy (RVH). cDNA assays confirmed the skipping of exon 21, and the splice product underwent nonsense-mediated decay. Functional studies on fibroblasts from the patient demonstrated increased calcification and decreased enzymatic activity of ENPP1, recapitulating the hallmarks of GACI. By combining genetic analysis with the in vitro study, we substantiate that ENPP1:c.2230 + 5G > A variant is pathogenic, underscoring its role in the development of GACI.
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Affiliation(s)
- Hafiza Noor Ul Ayan
- Institute for Cardiogenetics, University of Lübeck, Lübeck, 23562, Germany
- National Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS), Faisalabad, 38000, Pakistan
- DZHK (German Research Center for Cardiovascular Research), Partner Site Hamburg/Lübeck/Kiel, Lübeck, 23562, Germany
| | - Yvonne Nitschke
- Department of General Pediatrics, Muenster University Children's Hospital, Muenster, 48149, Germany
| | | | - Holger Thiele
- Cologne Center for Genomics (CCG), University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, 50931, Germany
| | - Naveed Altaf Malik
- National Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS), Faisalabad, 38000, Pakistan
| | - Ijaz Hussain
- Peshawar Institute of Cardiology, Peshawar, 25000, Pakistan
| | - Syed Muhammad Ijlal Haider
- Institute for Cardiogenetics, University of Lübeck, Lübeck, 23562, Germany
- DZHK (German Research Center for Cardiovascular Research), Partner Site Hamburg/Lübeck/Kiel, Lübeck, 23562, Germany
| | - Frank Rutsch
- Department of General Pediatrics, Muenster University Children's Hospital, Muenster, 48149, Germany
| | - Jeanette Erdmann
- Institute for Cardiogenetics, University of Lübeck, Lübeck, 23562, Germany
- DZHK (German Research Center for Cardiovascular Research), Partner Site Hamburg/Lübeck/Kiel, Lübeck, 23562, Germany
| | - Muhammad Tariq
- National Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS), Faisalabad, 38000, Pakistan
| | - Zouhair Aherrahrou
- Institute for Cardiogenetics, University of Lübeck, Lübeck, 23562, Germany
- DZHK (German Research Center for Cardiovascular Research), Partner Site Hamburg/Lübeck/Kiel, Lübeck, 23562, Germany
| | - Ilyas Ahmad
- Institute for Cardiogenetics, University of Lübeck, Lübeck, 23562, Germany.
- DZHK (German Research Center for Cardiovascular Research), Partner Site Hamburg/Lübeck/Kiel, Lübeck, 23562, Germany.
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Rutsch F, Salusky IB, Ferreira CR. Commentary on: The use of burosumab to treat autosomal-recessive hypophosphatemic rickets type 2: rationale and a first clinical experience. J Nephrol 2024; 37:2059-2060. [PMID: 39162952 PMCID: PMC11649749 DOI: 10.1007/s40620-024-02057-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2024] [Accepted: 07/10/2024] [Indexed: 08/21/2024]
Affiliation(s)
- Frank Rutsch
- Department of General Pediatrics and Center for Rare Diseases, Muenster University Children's Hospital, Muenster, Germany.
| | - Isidro B Salusky
- Department of Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
| | - Carlos R Ferreira
- Metabolic Medicine Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA
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Stabile J, Fürstenau CR. Platelets isolation and ectonucleotidase assay: Revealing functional aspects of the communication between the vasculature and the immune system. J Immunol Methods 2024; 533:113746. [PMID: 39181235 DOI: 10.1016/j.jim.2024.113746] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2024] [Revised: 08/17/2024] [Accepted: 08/19/2024] [Indexed: 08/27/2024]
Abstract
Platelets are enucleated fragments of cells with a diversity of internal granules. They are responsible for functions related to hemostasis, coagulation, and inflammation. The activation of these processes depends on a cascade coordinated by cytokines, chemokines, and components of purinergic signaling, such as ATP, ADP, and adenosine. Platelets express distinct components of the purinergic system: P2X1, P2Y1, PY12, and P2Y14 receptors; and the ectonucleotidases NTPDase, NPP, and 5NTE (ecto-5'-nucleotidase). Except for P2Y14, which has not yet exhibited a known function, all other components relate to the biological processes mentioned before. Platelets are known to display specific responses to microorganisms, being capable of recognizing pathogen-associated molecular patterns (PAMPs), engulfing certain classes of viruses, and participating in NETosis. Platelet function dysregulation implicates various pathophysiological processes, including cardiovascular diseases (CVDs) and infections. In COVID-19 patients, platelets exhibit altered purinergic signaling and increased activation, contributing to inflammation. Excessive platelet activation can lead to complications from thrombosis, which can affect the circulation of vital organs. Therefore, controlling the activation is necessary to end the inflammatory process and restore homeostasis. Ectonucleotidases, capable of hydrolyzing ATP, ADP, and AMP, are of fundamental importance in activating platelets, promising pharmacological targets for clinical use as cardiovascular protective drugs. In this review, we revisit platelet biology, the purinergic receptors and ectonucleotidases on their surface, and their importance in platelet activity. Additionally, we describe methods for isolating platelets in humans and murine, as well as the main techniques for detecting the activity of ectonucleotidases in platelets. Considering the multitude of functions revealed by platelets and their potential use as potent bioreactors able to secrete and present molecules involved in the communication of the vasculature with the immune system, it is crucial to deeply understand platelet biology and purinergic signaling participation to contribute to the developing of therapeutic strategies in diseases of the cardiovascular, inflammatory, and immune systems.
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Affiliation(s)
- Jeferson Stabile
- Laboratory of Vascular Biochemistry, Center for Natural and Human Sciences, Federal University of ABC, Santo André, SP, Brazil
| | - Cristina Ribas Fürstenau
- Laboratory of Vascular Biochemistry, Center for Natural and Human Sciences, Federal University of ABC, Santo André, SP, Brazil.
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Ju O, Ko SY, Jang YJ. Regulation of TGF-β1-induced fibroblast differentiation of human periodontal ligament stem cells through the mutually antagonistic action of ectonucleotide pyrophosphatase/phosphodiesterase 1 and 2. Front Cell Dev Biol 2024; 12:1426762. [PMID: 39291269 PMCID: PMC11405333 DOI: 10.3389/fcell.2024.1426762] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2024] [Accepted: 06/28/2024] [Indexed: 09/19/2024] Open
Abstract
Human periodontal ligament stem cells (hPDLSCs) differentiate into periodontal ligament (PDL) fibroblasts, osteoblasts, and cementoblasts. To identify inducers of PDL fibroblastic differentiation, monoclonal antibody series were developed a series of against membrane/extracellular matrix (ECM) molecules through decoy immunization. The anti-PDL13 antibody targets ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), renowned for regulating skeletal and soft tissue mineralization. ENPP1 accumulates in the periodontal ligament region of tooth roots, and specifically localizes to the cell boundaries and elongated processes of the fibroblastic cells. As ENPP1 expression increases during fibroblastic differentiation, mineralization induced by tissue-nonspecific alkaline phosphatase (TNAP), a pyrophosphate-degrading enzyme, is completely inhibited. This is consistent with ENPP1 and TNAP acting in opposition, and TGF-β1-induced ENPP1 expression creates an essential environment for PDL fibroblast differentiation. Representative fibroblastic differentiation markers decrease with endogenous ENPP1 inhibition by siRNA and antibody blocking. ENPP2 generates lipid signaling molecules. In contrast to ENPP1, ENPP2 disappears in TGF-β1-induced PDL fibroblasts. Ectopic expression of ENPP2 hinders TGF-β1-induced PDL fibroblastic differentiation. Suppression of ENPP1 and ENPP2 leads to severe defects in undifferentiated and differentiated cells, demonstrating that these two factors play opposing roles in soft and hard tissue differentiation but can complement each other for cell survival. In conclusion, increased ENPP1 is crucial for TGF-β1-induced PDL differentiation, while ENPP2 and TNAP can inhibit ENPP1. ENPP1 and ENPP2 exhibit complementary functions in the cell survival.
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Affiliation(s)
- Onyou Ju
- Department of Nanobiomedical Science and BK21 FOUR NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan, Republic of Korea
| | - Seon-Yle Ko
- Department of Oral Biochemistry, School of Dentistry, Dankook University, Cheonan, Republic of Korea
| | - Young-Joo Jang
- Department of Nanobiomedical Science and BK21 FOUR NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan, Republic of Korea
- Department of Oral Biochemistry, School of Dentistry, Dankook University, Cheonan, Republic of Korea
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Ansh AJ, Stabach PR, Ciccone C, Cao W, De La Cruz EM, Sabbagh Y, Carpenter TO, Ferreira CR, Braddock DT. Quantitative correlation of ENPP1 pathogenic variants with disease phenotype. Bone 2024; 186:117136. [PMID: 38806089 PMCID: PMC11227391 DOI: 10.1016/j.bone.2024.117136] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/05/2024] [Revised: 04/07/2024] [Accepted: 05/24/2024] [Indexed: 05/30/2024]
Abstract
Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein which hydrolyzes extracellular phosphoanhydrides into bio-active molecules that regulate, inter alia, ectopic mineralization, bone formation, vascular endothelial proliferation, and the innate immune response. The clinical phenotypes produced by ENPP1 deficiency are disparate, ranging from life-threatening arterial calcifications to cutaneous hypopigmentation. To investigate associations between disease phenotype and enzyme activity we quantified the enzyme velocities of 29 unique ENPP1 pathogenic variants in 41 patients enrolled in an NIH study along with 33 other variants reported in literature. We correlated the relative enzyme velocities with the presenting clinical diagnoses, performing the catalytic velocity measurements simultaneously in triplicate using a high-throughput assay to reduce experimental variation. We found that ENPP1 variants associated with autosomal dominant phenotypes reduced enzyme velocities by 50 % or more, whereas variants associated with insulin resistance had non-significant effects on enzyme velocity. In Cole disease the catalytic velocities of ENPP1 variants associated with AD forms trended to lower values than those associated with autosomal recessive forms - 8-32 % vs. 33 % of WT, respectively. Additionally, ENPP1 variants leading to life-threatening vascular calcifications in GACI patients had widely variable enzyme activities, ranging from no significant differences compared to WT to the complete abolishment of enzyme velocity. Finally, disease severity in GACI did not correlate with the mean enzyme velocity of the variants present in affected compound heterozygotes but did correlate with the more severely damaging variant. In summary, correlation of ENPP1 enzyme velocity with disease phenotypes demonstrate that enzyme velocities below 50 % of WT levels are likely to occur in the context of autosomal dominant disease (due to a monoallelic variant), and that disease severity in GACI infants correlates with the more severely damaging ENPP1 variant in compound heterozygotes, not the mean velocity of the pathogenic variants present.
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Affiliation(s)
- Anenya Jai Ansh
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - Paul R Stabach
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - Carla Ciccone
- Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA
| | - Wenxiang Cao
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA
| | - Enrique M De La Cruz
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA
| | - Yves Sabbagh
- Inozyme Pharma, 321 Summer St., Suite 400, Boston, MA 02201, USA
| | - Thomas O Carpenter
- Department of Pediatrics (Endocrinology), Yale University School of Medicine, New Haven, CT, USA
| | - Carlos R Ferreira
- Metabolic Medicine Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA
| | - Demetrios T Braddock
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
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Ferreira CR, Carpenter TO, Braddock DT. ENPP1 in Blood and Bone: Skeletal and Soft Tissue Diseases Induced by ENPP1 Deficiency. ANNUAL REVIEW OF PATHOLOGY 2024; 19:507-540. [PMID: 37871131 PMCID: PMC11062289 DOI: 10.1146/annurev-pathmechdis-051222-121126] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/25/2023]
Abstract
The enzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein that hydrolyzes extracellular ATP to generate pyrophosphate (PPi) and adenosine monophosphate, thereby contributing to downstream purinergic signaling pathways. The clinical phenotypes induced by ENPP1 deficiency are seemingly contradictory and include early-onset osteoporosis in middle-aged adults and life-threatening vascular calcifications in the large arteries of infants with generalized arterial calcification of infancy. The progressive overmineralization of soft tissue and concurrent undermineralization of skeleton also occur in the general medical population, where it is referred to as paradoxical mineralization to highlight the confusing pathophysiology. This review summarizes the clinical presentation and pathophysiology of paradoxical mineralization unveiled by ENPP1 deficiency and the bench-to-bedside development of a novel ENPP1 biologics designed to treat mineralization disorders in the rare disease and general medical population.
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Affiliation(s)
- Carlos R Ferreira
- Metabolic Medicine Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA
| | - Thomas O Carpenter
- Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Demetrios T Braddock
- Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA;
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Arima T, Sugimoto K, Taniwaki T, Maeda K, Shibata Y, Tateyama M, Karasugi T, Tokunaga T, Sueyoshi T, Hisanaga S, Masuda T, Uehara Y, Yugami M, Matsushita K, Yonemitsu R, Kawakami J, Yoshimura N, Tanimura S, Kato H, Ito N, Inoue K, Bando K, Nakamura T, Miyamoto T. Cartilage tissues regulate systemic aging via ectonucleotide pyrophosphatase/phosphodiesterase 1 in mice. J Biol Chem 2024; 300:105512. [PMID: 38042486 PMCID: PMC10777000 DOI: 10.1016/j.jbc.2023.105512] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2023] [Revised: 11/09/2023] [Accepted: 11/21/2023] [Indexed: 12/04/2023] Open
Abstract
Aging presents fundamental health concerns worldwide; however, mechanisms underlying how aging is regulated are not fully understood. Here, we show that cartilage regulates aging by controlling phosphate metabolism via ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1). We newly established an Enpp1 reporter mouse, in which an EGFP-luciferase sequence was knocked-in at the Enpp1 gene start codon (Enpp1/EGFP-luciferase), enabling detection of Enpp1 expression in cartilage tissues of resultant mice. We then established a cartilage-specific Enpp1 conditional knockout mouse (Enpp1 cKO) by generating Enpp1 flox mice and crossing them with cartilage-specific type 2 collagen Cre mice. Relative to WT controls, Enpp1 cKO mice exhibited phenotypes resembling human aging, such as short life span, ectopic calcifications, and osteoporosis, as well as significantly lower serum pyrophosphate levels. We also observed significant weight loss and worsening of osteoporosis in Enpp1 cKO mice under phosphate overload conditions, similar to global Enpp1-deficient mice. Aging phenotypes seen in Enpp1 cKO mice under phosphate overload conditions were rescued by a low vitamin D diet, even under high phosphate conditions. These findings suggest overall that cartilage tissue plays an important role in regulating systemic aging via Enpp1.
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Affiliation(s)
- Takahiro Arima
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Kazuki Sugimoto
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Takuya Taniwaki
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Kazuya Maeda
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Yuto Shibata
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Makoto Tateyama
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Tatsuki Karasugi
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Takuya Tokunaga
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Takanao Sueyoshi
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Satoshi Hisanaga
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Tetsuro Masuda
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Yusuke Uehara
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Masaki Yugami
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Kozo Matsushita
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Ryuji Yonemitsu
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Junki Kawakami
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Naoto Yoshimura
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Shuntaro Tanimura
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Hajime Kato
- Division of Nephrology and Endocrinology, The University of Tokyo Hospital, Tokyo, Japan
| | - Nobuaki Ito
- Division of Nephrology and Endocrinology, The University of Tokyo Hospital, Tokyo, Japan
| | - Kenichi Inoue
- Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, Kobe, Hyogo, Japan
| | - Kana Bando
- Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, Kobe, Hyogo, Japan
| | - Takayuki Nakamura
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
| | - Takeshi Miyamoto
- Department of Orthopedic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
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Nakamichi Y, Liu Z, Mori T, He Z, Yasuda H, Takahashi N, Udagawa N. The vitamin D receptor in osteoblastic cells but not secreted parathyroid hormone is crucial for soft tissue calcification induced by the proresorptive activity of 1,25(OH) 2D 3. J Steroid Biochem Mol Biol 2023; 232:106351. [PMID: 37352941 DOI: 10.1016/j.jsbmb.2023.106351] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/30/2023] [Revised: 05/16/2023] [Accepted: 06/14/2023] [Indexed: 06/25/2023]
Abstract
The vitamin D receptor (VDR) is expressed most abundantly in osteoblasts and osteocytes (osteoblastic cells) in bone tissues and regulates bone resorption and calcium (Ca) and phosphate (P) homeostasis in association with parathyroid hormone (PTH). We previously reported that near-physiological doses of vitamin D compounds suppressed bone resorption through VDR in osteoblastic cells. We also found that supra-physiological doses of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced bone resorption and hypercalcemia via VDR in osteoblastic cells. Here, we report that the latter, a proresorptive dose of 1,25(OH)2D3, causes soft tissue calcification through VDR in osteoblastic cells. High concentrations of vitamin D affect multiple organs and cause soft tissue calcification, with increases in bone resorption and serum Ca levels. Such a variety of symptoms is known as hypervitaminosis D, which is caused by not only high doses of vitamin D but also impaired vitamin D metabolism and diseases that produce 1,25(OH)2D3 ectopically. To clarify the biological process hierarchy in hypervitaminosis D, a proresorptive dose of 1,25(OH)2D3 was administered to wild-type mice in which bone resorption had been suppressed by neutralizing anti-receptor activator of NF-κB ligand (RANKL) antibody. 1,25(OH)2D3 upregulated the serum Ca x P product, concomitantly induced calcification of the aorta, lungs, and kidneys, and downregulated serum PTH levels in control IgG-pretreated wild-type mice. Pretreatment of wild-type mice with anti-RANKL antibody did not affect the down-regulation of PTH levels by 1,25(OH)2D3, but inhibited the increase of the serum Ca x P product and soft tissue calcification induced by 1,25(OH)2D3. Consistent with the effects of anti-RANKL antibody, VDR ablation in osteoblastic cells also did not affect the down-regulation of PTH levels by 1,25(OH)2D3, but suppressed the 1,25(OH)2D3-induced increase of the serum Ca x P product and calcification of soft tissues. Taken together with our previous results, these findings suggest that bone resorption induced by VDR signaling in osteoblastic cells is critical for the pathogenesis of hypervitaminosis D, but PTH is not involved in hypervitaminosis D.
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Affiliation(s)
- Yuko Nakamichi
- Institute for Oral Science, Matsumoto Dental University, Shiojiri, Nagano 399-0781, Japan.
| | - Ziyang Liu
- Graduate School of Oral Medicine, Matsumoto Dental University, Shiojiri, Nagano, Japan
| | - Tomoki Mori
- Graduate School of Oral Medicine, Matsumoto Dental University, Shiojiri, Nagano, Japan
| | - Zhifeng He
- Graduate School of Oral Medicine, Matsumoto Dental University, Shiojiri, Nagano, Japan
| | | | - Naoyuki Takahashi
- Institute for Oral Science, Matsumoto Dental University, Shiojiri, Nagano 399-0781, Japan
| | - Nobuyuki Udagawa
- Department of Biochemistry, Matsumoto Dental University, Shiojiri, Nagano, Japan
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Witz A, Effertz D, Goebel N, Schwab M, Franke UFW, Torzewski M. Pro-Calcifying Role of Enzymatically Modified LDL (eLDL) in Aortic Valve Sclerosis via Induction of IL-6 and IL-33. Biomolecules 2023; 13:1091. [PMID: 37509127 PMCID: PMC10377083 DOI: 10.3390/biom13071091] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2023] [Revised: 06/25/2023] [Accepted: 07/03/2023] [Indexed: 07/30/2023] Open
Abstract
One of the contributors to atherogenesis is enzymatically modified LDL (eLDL). eLDL was detected in all stages of aortic valve sclerosis and was demonstrated to trigger the activation of p38 mitogen-activated protein kinase (p38 MAPK), which has been identified as a pro-inflammatory protein in atherosclerosis. In this study, we investigated the influence of eLDL on IL-6 and IL-33 induction, and also the impact of eLDL on calcification in aortic valve stenosis (AS). eLDL upregulated phosphate-induced calcification in valvular interstitial cells (VICs)/myofibroblasts isolated from diseased aortic valves, as demonstrated by alizarin red staining. Functional studies demonstrated activation of p38 MAPK as well as an altered gene expression of osteogenic genes known to be involved in vascular calcification. In parallel with the activation of p38 MAPK, eLDL also induced upregulation of the cytokines IL-6 and IL-33. The results suggest a pro-calcifying role of eLDL in AS via induction of IL-6 and IL-33.
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Affiliation(s)
- Annemarie Witz
- Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, 70376 Stuttgart, Germany
| | - Denise Effertz
- Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, 70376 Stuttgart, Germany
| | - Nora Goebel
- Department of Cardiovascular Surgery, Robert-Bosch-Hospital, 70376 Stuttgart, Germany
| | - Matthias Schwab
- Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, 70376 Stuttgart, Germany
- Department of Clinical Pharmacology, University of Tuebingen, 72076 Tuebingen, Germany
- Department of Biochemistry and Pharmacy, University of Tuebingen, 72076 Tuebingen, Germany
| | - Ulrich F W Franke
- Department of Cardiovascular Surgery, Robert-Bosch-Hospital, 70376 Stuttgart, Germany
| | - Michael Torzewski
- Department of Laboratory Medicine and Hospital Hygiene, Robert-Bosch-Hospital, 70376 Stuttgart, Germany
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12
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Mae T, Hasegawa T, Hongo H, Yamamoto T, Zhao S, Li M, Yamazaki Y, Amizuka N. Immunolocalization of Enzymes/Membrane Transporters Related to Bone Mineralization in the Metaphyses of the Long Bones of Parathyroid-Hormone-Administered Mice. MEDICINA (KAUNAS, LITHUANIA) 2023; 59:1179. [PMID: 37374382 DOI: 10.3390/medicina59061179] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/11/2023] [Revised: 06/15/2023] [Accepted: 06/16/2023] [Indexed: 06/29/2023]
Abstract
The present study aimed to demonstrate the immunolocalization and/or gene expressions of the enzymes and membrane transporters involved in bone mineralization after the intermittent administration of parathyroid hormone (PTH). The study especially focused on TNALP, ENPP1, and PHOSPHO1, which are involved in matrix vesicle-mediated mineralization, as well as PHEX and the SIBLING family, which regulate mineralization deep inside bone. Six-week-old male mice were subcutaneously injected with 20 μg/kg/day of human PTH (1-34) two times per day (n = 6) or four times per day (n = 6) for two weeks. Additionally, control mice (n = 6) received a vehicle. Consistently with an increase in the volume of the femoral trabeculae, the mineral appositional rate increased after PTH administration. The areas positive for PHOSPHO1, TNALP, and ENPP1 in the femoral metaphyses expanded, and the gene expressions assessed by real-time PCR were elevated in PTH-administered specimens when compared with the findings in control specimens. The immunoreactivity and/or gene expressions of PHEX and the SIBLING family (MEPE, osteopontin, and DMP1) significantly increased after PTH administration. For example, MEPE immunoreactivity was evident in some osteocytes in PTH-administered specimens but was hardly observed in control specimens. In contrast, mRNA encoding cathepsin B was significantly reduced. Therefore, the bone matrix deep inside might be further mineralized by PHEX/SIBLING family after PTH administration. In summary, it is likely that PTH accelerates mineralization to maintain a balance with elevated matrix synthesis, presumably by mediating TNALP/ENPP1 cooperation and stimulating PHEX/SIBLING family expression.
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Affiliation(s)
- Takahito Mae
- Department of Developmental Biology of Hard Tissue, Graduate School of Dental Medicine, Faculty of Dental Medicine, Hokkaido University, Sapporo 060-8586, Japan
- Department of Gerontology, Graduate School of Dental Medicine, Faculty of Dental Medicine, Hokkaido University, Sapporo 060-8586, Japan
| | - Tomoka Hasegawa
- Department of Developmental Biology of Hard Tissue, Graduate School of Dental Medicine, Faculty of Dental Medicine, Hokkaido University, Sapporo 060-8586, Japan
| | - Hiromi Hongo
- Department of Developmental Biology of Hard Tissue, Graduate School of Dental Medicine, Faculty of Dental Medicine, Hokkaido University, Sapporo 060-8586, Japan
| | - Tomomaya Yamamoto
- Department of Developmental Biology of Hard Tissue, Graduate School of Dental Medicine, Faculty of Dental Medicine, Hokkaido University, Sapporo 060-8586, Japan
- Northern Army Medical Unit, Camp Makomanai, Japan Ground Self-Defense Forces, Sapporo 005-8543, Japan
| | - Shen Zhao
- Department of Endodontics and Operative Dentistry, Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Minqi Li
- Center of Osteoporosis and Bone Mineral Research, Department of Bone Metabolism, School of Stomatology, Shandong University, Jinan 250012, China
| | - Yutaka Yamazaki
- Department of Gerontology, Graduate School of Dental Medicine, Faculty of Dental Medicine, Hokkaido University, Sapporo 060-8586, Japan
| | - Norio Amizuka
- Department of Developmental Biology of Hard Tissue, Graduate School of Dental Medicine, Faculty of Dental Medicine, Hokkaido University, Sapporo 060-8586, Japan
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13
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Histological Assessment of Endochondral Ossification and Bone Mineralization. ENDOCRINES 2023. [DOI: 10.3390/endocrines4010006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Finely tuned cartilage mineralization, endochondral ossification, and normal bone formation are necessary for normal bone growth. Hypertrophic chondrocytes in the epiphyseal cartilage secrete matrix vesicles, which are small extracellular vesicles initiating mineralization, into the intercolumnar septa but not the transverse partitions of the cartilage columns. Bone-specific blood vessels invade the unmineralized transverse septum, exposing the mineralized cartilage cores. Many osteoblast precursors migrate to the cartilage cores, where they synthesize abundant bone matrices, and mineralize them in a process of matrix vesicle-mediated bone mineralization. Matrix vesicle-mediated mineralization concentrates calcium (Ca) and inorganic phosphates (Pi), which are converted into hydroxyapatite crystals. These crystals grow radially and are eventually get out of the vesicles to form spherical mineralized nodules, leading to collagen mineralization. The influx of Ca and Pi into the matrix vesicle is regulated by several enzymes and transporters such as TNAP, ENPP1, PiT1, PHOSPHO1, annexins, and others. Such matrix vesicle-mediated mineralization is regulated by osteoblastic activities, synchronizing the synthesis of organic bone material. However, osteocytes reportedly regulate peripheral mineralization, e.g., osteocytic osteolysis. The interplay between cartilage mineralization and vascular invasion during endochondral ossification, as well as that of osteoblasts and osteocytes for normal mineralization, appears to be crucial for normal bone growth.
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14
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Kato H, Ansh AJ, Lester ER, Kinoshita Y, Hidaka N, Hoshino Y, Koga M, Taniguchi Y, Uchida T, Yamaguchi H, Niida Y, Nakazato M, Nangaku M, Makita N, Takamura T, Saito T, Braddock DT, Ito N. Identification of ENPP1 Haploinsufficiency in Patients With Diffuse Idiopathic Skeletal Hyperostosis and Early-Onset Osteoporosis. J Bone Miner Res 2022; 37:1125-1135. [PMID: 35340077 PMCID: PMC9177665 DOI: 10.1002/jbmr.4550] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/22/2021] [Revised: 03/14/2022] [Accepted: 03/21/2022] [Indexed: 11/10/2022]
Abstract
Homozygous ENPP1 mutations are associated with autosomal recessive hypophosphatemic rickets type 2 (ARHR2), severe ossification of the spinal ligaments, and generalized arterial calcification of infancy type 1. There are a limited number of reports on phenotypes associated with heterozygous ENPP1 mutations. Here, we report a series of three probands and their families with heterozygous and compound heterozygous ENPP1 mutations. The first case (case 1) was a 47-year-old male, diagnosed with early-onset osteoporosis and low-normal serum phosphate levels, which invoked suspicion for hypophosphatemic rickets. The second and third cases were 77- and 54-year-old females who both presented with severe spinal ligament ossification and the presumptive diagnosis of diffuse idiopathic skeletal hyperostosis (DISH). Upon workup, fibroblast growth factor 23 (FGF23) was noted to be relatively high in case 2 and serum phosphorous was low-normal in case 3, and the diagnoses of X-linked hypophosphatemic rickets (XLH) and ARHR2 were considered. Genetic testing for genes related to congenital hypophosphatemic rickets was therefore performed, revealing heterozygous ENPP1 variants in cases 1 and 2 (case 1, c.536A>G, p.Asn179Ser; case 2, c.1352A>G, p.Tyr451Cys) and compound heterozygous ENPP1 variants in case 3 constituting the same variants present in cases 1 and 2 (c.536A>G, p.Asn179Ser and c.1352A>G, p.Tyr451Cys). Several in silico tools predicted the two variants to be pathogeneic, a finding confirmed by in vitro biochemical analysis demonstrating that the p.Asn179Ser and p.Tyr451Cys ENPP1 variants possessed a catalytic velocity of 45% and 30% compared with that of wild-type ENPP1, respectively. Both variants were therefore categorized as pathogenic loss-of-function mutations. Our findings suggest that ENPP1 mutational status should be evaluated in patients presenting with the diagnosis of idiopathic DISH, ossification of the posterior longitudinal ligament (OPLL), and early-onset osteoporosis. © 2022 American Society for Bone and Mineral Research (ASBMR).
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Affiliation(s)
- Hajime Kato
- Division of Nephrology and Endocrinology, The University of Tokyo Hospital, Tokyo, Japan
- Osteoporosis center, The University of Tokyo Hospital, Tokyo, Japan
| | - Anenya J. Ansh
- Department of Pathology, Yale University, New Haven, CT, USA
| | - Ethan R. Lester
- Department of Pathology, Yale University, New Haven, CT, USA
| | - Yuka Kinoshita
- Division of Nephrology and Endocrinology, The University of Tokyo Hospital, Tokyo, Japan
- Osteoporosis center, The University of Tokyo Hospital, Tokyo, Japan
| | - Naoko Hidaka
- Division of Nephrology and Endocrinology, The University of Tokyo Hospital, Tokyo, Japan
- Osteoporosis center, The University of Tokyo Hospital, Tokyo, Japan
| | - Yoshitomo Hoshino
- Division of Nephrology and Endocrinology, The University of Tokyo Hospital, Tokyo, Japan
- Osteoporosis center, The University of Tokyo Hospital, Tokyo, Japan
| | - Minae Koga
- Division of Nephrology and Endocrinology, The University of Tokyo Hospital, Tokyo, Japan
- Osteoporosis center, The University of Tokyo Hospital, Tokyo, Japan
| | - Yuki Taniguchi
- Department of Orthopedic Surgery, The University of Tokyo Hospital, Tokyo, Japan
| | - Taisuke Uchida
- Division of Neurology, Respirology, Endocrinology and Metabolism, Department of Internal Medicine, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
| | - Hideki Yamaguchi
- Division of Neurology, Respirology, Endocrinology and Metabolism, Department of Internal Medicine, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
| | - Yo Niida
- Division of Genomic Medicine, Department of Advanced Medicine, Medical Research Institute, Kanazawa Medical University, Ishikawa, Japan
| | - Masamitsu Nakazato
- Division of Neurology, Respirology, Endocrinology and Metabolism, Department of Internal Medicine, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
| | - Masaomi Nangaku
- Division of Nephrology and Endocrinology, The University of Tokyo Hospital, Tokyo, Japan
| | - Noriko Makita
- Division of Nephrology and Endocrinology, The University of Tokyo Hospital, Tokyo, Japan
- Osteoporosis center, The University of Tokyo Hospital, Tokyo, Japan
| | - Toshinari Takamura
- Department of Endocrinology and Metabolism, Kanazawa University Graduate School of Medical Sciences, Ishikawa, Japan
| | - Taku Saito
- Osteoporosis center, The University of Tokyo Hospital, Tokyo, Japan
- Department of Orthopedic Surgery, The University of Tokyo Hospital, Tokyo, Japan
| | | | - Nobuaki Ito
- Division of Nephrology and Endocrinology, The University of Tokyo Hospital, Tokyo, Japan
- Osteoporosis center, The University of Tokyo Hospital, Tokyo, Japan
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15
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Carozza JA, Cordova AF, Brown JA, AlSaif Y, Böhnert V, Cao X, Mardjuki RE, Skariah G, Fernandez D, Li L. ENPP1's regulation of extracellular cGAMP is a ubiquitous mechanism of attenuating STING signaling. Proc Natl Acad Sci U S A 2022; 119:e2119189119. [PMID: 35588451 PMCID: PMC9173814 DOI: 10.1073/pnas.2119189119] [Citation(s) in RCA: 50] [Impact Index Per Article: 16.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2021] [Accepted: 04/01/2022] [Indexed: 11/18/2022] Open
Abstract
The metazoan innate immune second messenger 2′3′-cGAMP is present both inside and outside cells. However, only extracellular cGAMP can be negatively regulated by the extracellular hydrolase ENPP1. Here, we determine whether ENPP1’s regulation of extracellular cGAMP is a ubiquitous mechanism of attenuating stimulator of interferon genes (STING) signaling. We identified ENPP1H362A, a point mutation that cannot degrade the 2′-5′ linkage in cGAMP while maintaining otherwise normal function. The selectivity of this histidine is conserved down to bacterial nucleotide pyrophosphatase/phosphodiesterase (NPP), allowing structural analysis and suggesting an unexplored ancient history of 2′-5′ cyclic dinucleotides. Enpp1H362A mice demonstrated that extracellular cGAMP is not responsible for the devastating phenotype in ENPP1-null humans and mice but is responsible for antiviral immunity and systemic inflammation. Our data define extracellular cGAMP as a pivotal STING activator, identify an evolutionarily critical role for ENPP1 in regulating inflammation, and suggest a therapeutic strategy for viral and inflammatory conditions by manipulating ENPP1 activity.
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Affiliation(s)
- Jacqueline A. Carozza
- ChEM-H Institute, Stanford University, Stanford, CA 94301
- Department of Chemistry, Stanford University, Stanford, CA 94301
| | - Anthony F. Cordova
- ChEM-H Institute, Stanford University, Stanford, CA 94301
- Cancer Biology Program, Stanford University, Stanford, CA 94301
| | - Jenifer A. Brown
- ChEM-H Institute, Stanford University, Stanford, CA 94301
- Department of Biophysics, Stanford University, Stanford, CA 94301
| | - Yasmeen AlSaif
- ChEM-H Institute, Stanford University, Stanford, CA 94301
- Department of Biology, Stanford University, Stanford, CA 94301
| | - Volker Böhnert
- ChEM-H Institute, Stanford University, Stanford, CA 94301
- Department of Biochemistry, Stanford University, Stanford, CA 94301
| | - Xujun Cao
- ChEM-H Institute, Stanford University, Stanford, CA 94301
- Department of Chemistry, Stanford University, Stanford, CA 94301
| | - Rachel E. Mardjuki
- ChEM-H Institute, Stanford University, Stanford, CA 94301
- Department of Chemistry, Stanford University, Stanford, CA 94301
| | - Gemini Skariah
- ChEM-H Institute, Stanford University, Stanford, CA 94301
- Department of Biochemistry, Stanford University, Stanford, CA 94301
| | - Daniel Fernandez
- ChEM-H Institute, Stanford University, Stanford, CA 94301
- Department of Biochemistry, Stanford University, Stanford, CA 94301
- Macromolecular Structural Knowledge Center, Stanford University, Stanford, CA 94301
| | - Lingyin Li
- ChEM-H Institute, Stanford University, Stanford, CA 94301
- Department of Biochemistry, Stanford University, Stanford, CA 94301
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16
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Nam HK, Emmanouil E, Hatch NE. Deletion of the Pyrophosphate Generating Enzyme ENPP1 Rescues Craniofacial Abnormalities in the TNAP−/− Mouse Model of Hypophosphatasia and Reveals FGF23 as a Marker of Phenotype Severity. FRONTIERS IN DENTAL MEDICINE 2022; 3. [PMID: 35909501 PMCID: PMC9336114 DOI: 10.3389/fdmed.2022.846962] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Hypophosphatasia is a rare heritable metabolic disorder caused by deficient Tissue Non-specific Alkaline Phosphatase (TNAP) enzyme activity. A principal function of TNAP is to hydrolyze the tissue mineralization inhibitor pyrophosphate. ENPP1 (Ectonucleotide Pyrophosphatase/Phosphodiesterase 1) is a primary enzymatic generator of pyrophosphate and prior results showed that elimination of ENPP1 rescued bone hypomineralization of skull, vertebral and long bones to different extents in TNAP null mice. Current TNAP enzyme replacement therapy alleviates skeletal, motor and cognitive defects but does not eliminate craniosynostosis in pediatric hypophosphatasia patients. To further understand mechanisms underlying craniosynostosis development in hypophosphatasia, here we sought to determine if craniofacial abnormalities including craniosynostosis and skull shape defects would be alleviated in TNAP null mice by genetic ablation of ENPP1. Results show that homozygous deletion of ENPP1 significantly diminishes the incidence of craniosynostosis and that skull shape abnormalities are rescued by hemi- or homozygous deletion of ENPP1 in TNAP null mice. Skull and long bone hypomineralization were also alleviated in TNAP−/−/ENPP1−/− compared to TNAP−/−/ENPP1+/+ mice, though loss of ENPP1 in combination with TNAP had different effects than loss of only TNAP on long bone trabeculae. Investigation of a relatively large cohort of mice revealed that the skeletal phenotypes of TNAP null mice were markedly variable. Because FGF23 circulating levels are known to be increased in ENPP1 null mice and because FGF23 influences bone, we measured serum intact FGF23 levels in the TNAP null mice and found that a subset of TNAP−/−/ENPP1+/+ mice exhibited markedly high serum FGF23. Serum FGF23 levels also correlated to mouse body measurements, the incidence of craniosynostosis, skull shape abnormalities and skull bone density and volume fraction. Together, our results demonstrate that balanced expression of TNAP and ENPP1 enzymes are essential for microstructure and mineralization of both skull and long bones, and for preventing craniosynostosis. The results also show that FGF23 rises in the TNAP−/− model of murine lethal hypophosphatasia. Future studies are required to determine if the rise in FGF23 is a cause, consequence, or marker of disease phenotype severity.
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17
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Hao1 Is Not a Pathogenic Factor for Ectopic Ossifications but Functions to Regulate the TCA Cycle In Vivo. Metabolites 2022; 12:metabo12010082. [PMID: 35050204 PMCID: PMC8780519 DOI: 10.3390/metabo12010082] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2021] [Revised: 01/10/2022] [Accepted: 01/11/2022] [Indexed: 02/01/2023] Open
Abstract
Ossification of the posterior longitudinal ligament (OPLL), a disease characterized by the ectopic ossification of a spinal ligament, promotes neurological disorders associated with spinal canal stenosis. While blocking ectopic ossification is mandatory to prevent OPLL development and progression, the mechanisms underlying the condition remain unknown. Here we show that expression of hydroxyacid oxidase 1 (Hao1), a gene identified in a previous genome-wide association study (GWAS) as an OPLL-associated candidate gene, specifically and significantly decreased in fibroblasts during osteoblast differentiation. We then newly established Hao1-deficient mice by generating Hao1-flox mice and crossing them with CAG-Cre mice to yield global Hao1-knockout (CAG-Cre/Hao1flox/flox; Hao1 KO) animals. Hao1 KO mice were born normally and exhibited no obvious phenotypes, including growth retardation. Moreover, Hao1 KO mice did not exhibit ectopic ossification or calcification. However, urinary levels of some metabolites of the tricarboxylic acid (TCA) cycle were significantly lower in Hao1 KO compared to control mice based on comprehensive metabolomic analysis. Our data indicate that Hao1 loss does not promote ectopic ossification, but rather that Hao1 functions to regulate the TCA cycle in vivo.
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18
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Vilmundarson RO, Duong A, Soheili F, Chen HH, Stewart AFR. IRF2BP2 3'UTR Polymorphism Increases Coronary Artery Calcification in Men. Front Cardiovasc Med 2021; 8:687645. [PMID: 34760935 PMCID: PMC8573268 DOI: 10.3389/fcvm.2021.687645] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2021] [Accepted: 09/28/2021] [Indexed: 12/15/2022] Open
Abstract
Interferon regulatory factor 2 binding protein 2 (IRF2BP2) suppresses the innate inflammatory response of macrophages. A 9-nucleotide deletion (rs3045215) in the 3' untranslated region (3'-UTR) of human IRF2BP2 mRNA confers risk of coronary artery disease (CAD) in the Ottawa Heart Genomics Study (OHGS). Here, we sought to identify regulatory mechanisms that may contribute to this risk. We tested how lipopolysaccharides (LPS) affects IRF2BP2 expression in human THP-1 macrophages and primary aortic smooth muscle cells (HAoSMC) genotyped for the deletion allele. Both cell types are implicated in coronary atherosclerosis. We also examined how the deletion affects interaction with RNA binding proteins (RBPs) to regulate IRF2BP2 expression. LPS altered allele-specific binding of RBPs in RNA gel shift assays with the THP-1 macrophage protein extracts. The RBP ELAVL1 suppressed the expression of a luciferase reporter carrying the 3'UTR of IRF2BP2 with the deletion allele. Other RBPs AUF1 or KHSRP did not confer such allele specific regulation. Since it is co-inherited with a risk variant for osteoporosis, a condition tied to arterial calcification, we examined the association of the deletion allele with coronary artery calcification in individuals who had undergone computed tomography angiography in the OHGS. In 323 individuals with a minimal burden of atherosclerosis (<30% coronary stenosis) and 138 CAD cases (>50% stenosis), Mendelian randomization revealed that the rs3045215 deletion allele significantly increased coronary artery calcification in men with minimal coronary stenosis. Thus, not only does the rs3045215 deletion allele predict atherosclerosis, but it also predisposes to early-onset calcification in men.
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Affiliation(s)
- Ragnar O Vilmundarson
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada.,Laboratory of Translational Genomics, John and Jennifer Ruddy Canadian Cardiovascular Genetics Centre, University of Ottawa Heart Institute, Ottawa, ON, Canada
| | - An Duong
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada.,Laboratory of Translational Genomics, John and Jennifer Ruddy Canadian Cardiovascular Genetics Centre, University of Ottawa Heart Institute, Ottawa, ON, Canada
| | - Fariborz Soheili
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada.,Laboratory of Translational Genomics, John and Jennifer Ruddy Canadian Cardiovascular Genetics Centre, University of Ottawa Heart Institute, Ottawa, ON, Canada
| | - Hsiao-Huei Chen
- Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada.,The Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Alexandre F R Stewart
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada.,Laboratory of Translational Genomics, John and Jennifer Ruddy Canadian Cardiovascular Genetics Centre, University of Ottawa Heart Institute, Ottawa, ON, Canada
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19
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Tintut Y, Honda HM, Demer LL. Biomolecules Orchestrating Cardiovascular Calcification. Biomolecules 2021; 11:biom11101482. [PMID: 34680115 PMCID: PMC8533507 DOI: 10.3390/biom11101482] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2021] [Revised: 09/28/2021] [Accepted: 10/03/2021] [Indexed: 01/12/2023] Open
Abstract
Vascular calcification, once considered a degenerative, end-stage, and inevitable condition, is now recognized as a complex process regulated in a manner similar to skeletal bone at the molecular and cellular levels. Since the initial discovery of bone morphogenetic protein in calcified human atherosclerotic lesions, decades of research have now led to the recognition that the regulatory mechanisms and the biomolecules that control cardiovascular calcification overlap with those controlling skeletal mineralization. In this review, we focus on key biomolecules driving the ectopic calcification in the circulation and their regulation by metabolic, hormonal, and inflammatory stimuli. Although calcium deposits in the vessel wall introduce rupture stress at their edges facing applied tensile stress, they simultaneously reduce rupture stress at the orthogonal edges, leaving the net risk of plaque rupture and consequent cardiac events depending on local material strength. A clinically important consequence of the shared mechanisms between the vascular and bone tissues is that therapeutic agents designed to inhibit vascular calcification may adversely affect skeletal mineralization and vice versa. Thus, it is essential to consider both systems when developing therapeutic strategies.
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Affiliation(s)
- Yin Tintut
- Department of Medicine, University of California-Los Angeles, Los Angeles, CA 90095, USA; (Y.T.); (H.M.H.)
- Department of Physiology, University of California-Los Angeles, Los Angeles, CA 90095, USA
- Department of Orthopaedic Surgery, University of California-Los Angeles, Los Angeles, CA 90095, USA
- Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USA
| | - Henry M. Honda
- Department of Medicine, University of California-Los Angeles, Los Angeles, CA 90095, USA; (Y.T.); (H.M.H.)
| | - Linda L. Demer
- Department of Medicine, University of California-Los Angeles, Los Angeles, CA 90095, USA; (Y.T.); (H.M.H.)
- Department of Physiology, University of California-Los Angeles, Los Angeles, CA 90095, USA
- Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USA
- Department of Bioengineering, University of California-Los Angeles, Los Angeles, CA 90095, USA
- The David Geffen School of Medicine, University of California-Los Angeles, 10833 Le Conte Ave, Los Angeles, CA 90095, USA
- Correspondence: ; Tel.: +1-(310)-206-2677
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20
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Understanding the Stony Bridge between Osteoporosis and Vascular Calcification: Impact of the FGF23/Klotho axis. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2021; 2021:7536614. [PMID: 34539972 PMCID: PMC8448600 DOI: 10.1155/2021/7536614] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/23/2021] [Accepted: 08/07/2021] [Indexed: 12/11/2022]
Abstract
A relationship between osteoporosis (OP) and vascular calcification (VC) is now proposed. There are common mechanisms underlying the regulation of them. Fibroblast growth factor- (FGF-) 23 and Klotho are hormones associated with the metabolic axis of osteovascular metabolism. Most recently, it was suggested that the FGF23-klotho axis is associated with increasing incidence of fractures and is potentially involved in the progression of the aortic-brachial stiffness ratio. Herein, we discussed the potential role of the FGF23/Klotho axis in the pathophysiology of OP and VC. We want to provide an update review in order to allow a better understanding of the potential role of the FGF23/Klotho axis in comorbidity of OP and VC. We believe that a better understanding of the relationship between both entities can help in proposing new therapeutic targets for reducing the increasing prevalence of OP and VC in the aging population.
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21
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Lu Y, Yuan T, Min X, Yuan Z, Cai Z. AMPK: Potential Therapeutic Target for Vascular Calcification. Front Cardiovasc Med 2021; 8:670222. [PMID: 34046440 PMCID: PMC8144331 DOI: 10.3389/fcvm.2021.670222] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2021] [Accepted: 04/08/2021] [Indexed: 12/18/2022] Open
Abstract
Vascular calcification (VC) is an urgent worldwide health issue with no available medical treatment. It is an active cell-driven process by osteogenic differentiation of vascular cells with complex mechanisms. The AMP-activated protein kinase (AMPK) serves as the master sensor of cellular energy status. Accumulating evidence reveals the vital role of AMPK in VC progression. AMPK is involved in VC in various ways, including inhibiting runt-related transcription factor 2 signaling pathways, triggering autophagy, attenuating endoplasmic reticulum stress and dynamic-related protein 1-mediated mitochondrial fission, and activating endothelial nitric oxide synthase. AMPK activators, like metformin, are associated with reduced calcification deposits in certain groups of patients, indicating that AMPK is a potential therapeutic target for VC.
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Affiliation(s)
- Yi Lu
- Department of Cardiology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Tan Yuan
- Department of Cardiology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Xinjia Min
- Department of Cardiology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Zhen Yuan
- Department of Cardiology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Zhejun Cai
- Department of Cardiology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.,Jiaxing Key Laboratory of Cardiac Rehabilitation, Jiaxing, China
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22
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Ferreira CR, Kavanagh D, Oheim R, Zimmerman K, Stürznickel J, Li X, Stabach P, Rettig RL, Calderone L, MacKichan C, Wang A, Hutchinson HA, Nelson T, Tommasini SM, von Kroge S, Fiedler IA, Lester ER, Moeckel GW, Busse B, Schinke T, Carpenter TO, Levine MA, Horowitz MC, Braddock DT. Response of the ENPP1-Deficient Skeletal Phenotype to Oral Phosphate Supplementation and/or Enzyme Replacement Therapy: Comparative Studies in Humans and Mice. J Bone Miner Res 2021; 36:942-955. [PMID: 33465815 PMCID: PMC8739051 DOI: 10.1002/jbmr.4254] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/08/2020] [Revised: 01/11/2021] [Accepted: 01/15/2021] [Indexed: 12/14/2022]
Abstract
Inactivating mutations in human ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) may result in early-onset osteoporosis (EOOP) in haploinsufficiency and autosomal recessive hypophosphatemic rickets (ARHR2) in homozygous deficiency. ARHR2 patients are frequently treated with phosphate supplementation to ameliorate the rachitic phenotype, but elevating plasma phosphorus concentrations in ARHR2 patients may increase the risk of ectopic calcification without increasing bone mass. To assess the risks and efficacy of conventional ARHR2 therapy, we performed comprehensive evaluations of ARHR2 patients at two academic medical centers and compared their skeletal and renal phenotypes with ENPP1-deficient Enpp1asj/asj mice on an acceleration diet containing high phosphate treated with recombinant murine Enpp1-Fc. ARHR2 patients treated with conventional therapy demonstrated improvements in rickets, but all adults and one adolescent analyzed continued to exhibit low bone mineral density (BMD). In addition, conventional therapy was associated with the development of medullary nephrocalcinosis in half of the treated patients. Similar to Enpp1asj/asj mice on normal chow and to patients with mono- and biallelic ENPP1 mutations, 5-week-old Enpp1asj/asj mice on the high-phosphate diet exhibited lower trabecular bone mass, reduced cortical bone mass, and greater bone fragility. Treating the Enpp1asj/asj mice with recombinant Enpp1-Fc protein between weeks 2 and 5 normalized trabecular bone mass, normalized or improved bone biomechanical properties, and prevented the development of nephrocalcinosis and renal failure. The data suggest that conventional ARHR2 therapy does not address low BMD inherent in ENPP1 deficiency, and that ENPP1 enzyme replacement may be effective for correcting low bone mass in ARHR2 patients without increasing the risk of nephrocalcinosis. © 2021 American Society for Bone and Mineral Research (ASBMR).
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Affiliation(s)
- Carlos R Ferreira
- Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA
| | - Dillon Kavanagh
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - Ralf Oheim
- Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Kristin Zimmerman
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - Julian Stürznickel
- Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Xiaofeng Li
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - Paul Stabach
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - R Luke Rettig
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - Logan Calderone
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - Colin MacKichan
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - Aaron Wang
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - Hunter A Hutchinson
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - Tracy Nelson
- Department of Orthopaedics and Rehabilitation, Yale University School of Medicine, New Haven, CT, USA
| | - Steven M Tommasini
- Department of Orthopaedics and Rehabilitation, Yale University School of Medicine, New Haven, CT, USA
| | - Simon von Kroge
- Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Imke Ak Fiedler
- Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Ethan R Lester
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - Gilbert W Moeckel
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - Björn Busse
- Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Thorsten Schinke
- Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Thomas O Carpenter
- Department of Pediatrics, Yale University School of Medicine, New Haven, CT, USA
| | - Michael A Levine
- Department of Pediatrics, University of Pennsylvania Perlman School of Medicine, Philadelphia, PA, USA.,Division of Endocrinology and Diabetes and Center for Bone Health, The Children's Hospital of Philadelphia, Philadelphia, PA, USA
| | - Mark C Horowitz
- Department of Orthopaedics and Rehabilitation, Yale University School of Medicine, New Haven, CT, USA
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23
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Shimada BK, Pomozi V, Zoll J, Kuo S, Martin L, Le Saux O. ABCC6, Pyrophosphate and Ectopic Calcification: Therapeutic Solutions. Int J Mol Sci 2021; 22:ijms22094555. [PMID: 33925341 PMCID: PMC8123679 DOI: 10.3390/ijms22094555] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2021] [Revised: 04/20/2021] [Accepted: 04/21/2021] [Indexed: 12/11/2022] Open
Abstract
Pathological (ectopic) mineralization of soft tissues occurs during aging, in several common conditions such as diabetes, hypercholesterolemia, and renal failure and in certain genetic disorders. Pseudoxanthoma elasticum (PXE), a multi-organ disease affecting dermal, ocular, and cardiovascular tissues, is a model for ectopic mineralization disorders. ABCC6 dysfunction is the primary cause of PXE, but also some cases of generalized arterial calcification of infancy (GACI). ABCC6 deficiency in mice underlies an inducible dystrophic cardiac calcification phenotype (DCC). These calcification diseases are part of a spectrum of mineralization disorders that also includes Calcification of Joints and Arteries (CALJA). Since the identification of ABCC6 as the “PXE gene” and the development of several animal models (mice, rat, and zebrafish), there has been significant progress in our understanding of the molecular genetics, the clinical phenotypes, and pathogenesis of these diseases, which share similarities with more common conditions with abnormal calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into inorganic pyrophosphate (PPi) and adenosine by the ectonucleotidases NPP1 and CD73 (NT5E). PPi is a potent endogenous inhibitor of calcification, whereas adenosine indirectly contributes to calcification inhibition by suppressing the synthesis of tissue non-specific alkaline phosphatase (TNAP). At present, therapies only exist to alleviate symptoms for both PXE and GACI; however, extensive studies have resulted in several novel approaches to treating PXE and GACI. This review seeks to summarize the role of ABCC6 in ectopic calcification in PXE and other calcification disorders, and discuss therapeutic strategies targeting various proteins in the pathway (ABCC6, NPP1, and TNAP) and direct inhibition of calcification via supplementation by various compounds.
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Affiliation(s)
- Briana K Shimada
- Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96817, USA
| | - Viola Pomozi
- Institute of Enzymology, RCNS, Hungarian Academy of Sciences, 1117 Budapest, Hungary
| | - Janna Zoll
- Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96817, USA
| | - Sheree Kuo
- Department of Pediatrics, Kapi'olani Medical Center for Women and Children, University of Hawaii, Honolulu, HI 96826, USA
| | - Ludovic Martin
- PXE Consultation Center, MAGEC Reference Center for Rare Skin Diseases, Angers University Hospital, 49100 Angers, France
- BNMI, CNRS 6214/INSERM 1083, University Bretagne-Loire, 49100 Angers, France
| | - Olivier Le Saux
- Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96817, USA
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24
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Ullah S, El-Gamal MI, El-Gamal R, Pelletier J, Sévigny J, Shehata MK, Anbar HS, Iqbal J. Synthesis, biological evaluation, and docking studies of novel pyrrolo[2,3-b]pyridine derivatives as both ectonucleotide pyrophosphatase/phosphodiesterase inhibitors and antiproliferative agents. Eur J Med Chem 2021; 217:113339. [PMID: 33744686 DOI: 10.1016/j.ejmech.2021.113339] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2021] [Revised: 02/25/2021] [Accepted: 02/26/2021] [Indexed: 11/17/2022]
Abstract
Ecto-nucleotide pyrophosphatases/phosphodiesterases (NPPs) together with nucleoside triphosphate diphosphohydrolases (NTPDases) and alkaline phosphatases (APs) are nucleotidases located at the surface of the cells. NPP1 and NPP3 are important members of NPP family that are known as druggable targets for a number of disorders such as impaired calcification, type 2 diabetes, and cancer. Sulfonylurea derivatives have been reported as antidiabetic and anticancer agents, therefore, we synthesized and investigated series of sulfonylurea derivatives 1a-m possessing pyrrolo[2,3-b]pyridine core as inhibitors of NPP1 and NPP3 isozymes that are over-expressed in cancer and diabetes. The enzymatic evaluation highlighted compound 1a as selective NPP1 inhibitor, however, 1c was observed as the most potent inhibitor of NPP1 with an IC50 value of 0.80 ± 0.04 μM. Compound 1l was found to be the most potent and moderately selective inhibitor of NPP3 (IC50 = 0.55 ± 0.01 μM). Furthermore, in vitro cytotoxicity assays of compounds 1a-m against MCF-7 and HT-29 cancer cell lines exhibited compound 1c (IC50 = 4.70 ± 0.67 μM), and 1h (IC50 = 1.58 ± 0.20 μM) as the most cytotoxic compounds against MCF-7 and HT-29 cancer cell lines, respectively. Both of the investigated compounds showed high degree of selectivity towards cancer cells than normal cells (WI-38). Molecular docking studies of selective and potent enzyme inhibitors revealed promising mode of interactions with important binding sites residues of both isozymes i.e., Thr256, His380, Lys255, Asn277 residues of NPP1 and His329, Thr205, and Leu239 residues of NPP3. In addition, the most potent antiproliferative agent, compound 1h, doesn't produce hypoglycemia as a side effect when injected to mice. This is an additional merit of the promising compound 1h.
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Affiliation(s)
- Saif Ullah
- Centre for Advanced Drug Research, COMSATS University Islamabad, Abbottabad Campus, Abbottabad, 22060, Pakistan
| | - Mohammed I El-Gamal
- Department of Medicinal Chemistry, College of Pharmacy, University of Sharjah, Sharjah, 27272, United Arab Emirates; Sharjah Institute for Medical Research, University of Sharjah, Sharjah, 27272, United Arab Emirates; Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura, 35516, Egypt
| | - Randa El-Gamal
- Department of Medical Biochemistry, Faculty of Medicine, University of Mansoura, Mansoura, 35516, Egypt
| | - Julie Pelletier
- Centre de Recherche du CHU de Québec - Université Laval, Québec, QC, G1V 4G2, Canada
| | - Jean Sévigny
- Centre de Recherche du CHU de Québec - Université Laval, Québec, QC, G1V 4G2, Canada; Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université Laval, Québec, QC, G1V 0A6, Canada
| | - Mahmoud K Shehata
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah, 27272, United Arab Emirates
| | - Hanan S Anbar
- Department of Clinical Pharmacy and Pharmacotherapeutics, Dubai Pharmacy College for Girls, Dubai, 19099, United Arab Emirates.
| | - Jamshed Iqbal
- Centre for Advanced Drug Research, COMSATS University Islamabad, Abbottabad Campus, Abbottabad, 22060, Pakistan.
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25
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Maulding ND, Kavanagh D, Zimmerman K, Coppola G, Carpenter TO, Jue NK, Braddock DT. Genetic pathways disrupted by ENPP1 deficiency provide insight into mechanisms of osteoporosis, osteomalacia, and paradoxical mineralization. Bone 2021; 142:115656. [PMID: 32980560 PMCID: PMC7744330 DOI: 10.1016/j.bone.2020.115656] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/31/2020] [Revised: 09/17/2020] [Accepted: 09/18/2020] [Indexed: 12/16/2022]
Abstract
Ectonucleotide phosphatase/phosphodiesterase 1 (ENPP1) deficiency results in either lethal arterial calcifications ('Generalized Arterial Calcification of Infancy' - GACI), phosphate wasting rickets ('Autosomal Recessive Hypophosphatemic Rickets type 2' - ARHR2), early onset osteoporosis, or progressive spinal rigidity ('Ossification of the Posterior Longitudinal Ligament' - OPLL). As ENPP1 generates a strong endogenous mineralization inhibitor - extracellular pyrophosphate (PPi) - ENPP1 deficiency should not result in reduced bone volume, and therefore the mechanism ENPP1 associated osteoporosis is not apparent given current understanding of the enzyme's function. To investigate genetic pathways driving the skeletal phenotype of ENPP1 deficiency we compared gene expression in Enpp1asj/asj mice and WT sibling pairs by RNAseq and qPCR in whole bones, and in the liver and kidney by qPCR, directly correlating gene expression with measures of bone microarchitectural and biomechanical phenotypes. Unbiased analysis of the differentially expressed genes compared to relevant human disease phenotypes revealed that Enpp1asj/asj mice exhibit strong signatures of osteoporosis, ARHR2 and OPLL. We found that ENPP1 deficient mice exhibited reduced gene transcription of Wnt ligands in whole bone and increased transcription of soluble Wnt inhibitors in the liver and kidney, suggestive of multiorgan inhibition of Wnt activity. Consistent with Wnt suppression in bone, Collagen gene pathways in bone were significantly decreased and Fgf23 was significantly increased, all of which directly correlated with bone microarchitectural defects and fracture risk in Enpp1asj/asj mice. Moreover, the bone findings in 10-week old mice correlated with Enpp1 transcript counts but not plasma [PPi], suggesting that the skeletal phenotype at 10 weeks is driven by catalytically independent ENPP1 function. In contrast, the bone findings in 23-week Enpp1asj/asj mice strongly correlated with plasma PPi, suggesting that chronically low PPi drives the skeletal phenotype in older mice. Finally, correlation between Enpp1 and Fgf23 transcription suggested ENPP1 regulation of Fgf23, which we confirmed by dosing Enpp1asj/asj mice with soluble ENPP1-Fc and observing suppression of intact plasma FGF23 and ALP. In summary, our findings suggest that osteoporosis associated with ENPP1 deficiency involves the suppression of Wnt via catalytically independent Enpp1 pathways, and validates Enpp1asj/asj mice as tools to better understand OPLL and Paradoxical Mineralization Disorders.
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Affiliation(s)
- Nathan D Maulding
- Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA
| | - Dillon Kavanagh
- Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA
| | - Kristin Zimmerman
- Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA
| | - Gianfilippo Coppola
- Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA
| | - Thomas O Carpenter
- Department of Pediatrics at Yale University School of Medicine, New Haven, CT 06510, USA
| | - Nathaniel K Jue
- Department of Biology and Chemistry, California State University, Monterey Bay, CA, USA.
| | - Demetrios T Braddock
- Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA.
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26
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Schlesinger PH, Braddock DT, Larrouture QC, Ray EC, Riazanski V, Nelson DJ, Tourkova IL, Blair HC. Phylogeny and chemistry of biological mineral transport. Bone 2020; 141:115621. [PMID: 32858255 PMCID: PMC7771281 DOI: 10.1016/j.bone.2020.115621] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/26/2020] [Revised: 08/24/2020] [Accepted: 08/24/2020] [Indexed: 02/08/2023]
Abstract
Three physiologically mineralizing tissues - teeth, cartilage and bone - have critical common elements and important evolutionary relationships. Phylogenetically the most ancient densely mineralized tissue is teeth. In jawless fishes without skeletons, tooth formation included epithelial transport of phosphates, a process echoed later in bone physiology. Cartilage and mineralized cartilage are skeletal elements separate from bone, but with metabolic features common to bone. Cartilage mineralization is coordinated with high expression of tissue nonspecific alkaline phosphatase and PHOSPHO1 to harvest available phosphate esters and support mineralization of collagen secreted locally. Mineralization in true bone results from stochastic nucleation of hydroxyapatite crystals within the cross-linked collagen fibrils. Mineral accumulation in dense collagen is, at least in major part, mediated by amorphous aggregates - often called Posner clusters - of calcium and phosphate that are small enough to diffuse into collagen fibrils. Mineral accumulation in membrane vesicles is widely suggested, but does not correlate with a definitive stage of mineralization. Conversely mineral deposition at non-physiologic sites where calcium and phosphate are adequate has been shown to be regulated in large part by pyrophosphate. All of these elements are present in vertebrate bone metabolism. A key biological element of bone formation is an epithelial-like cellular organization which allows control of phosphate, calcium and pH during mineralization.
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Affiliation(s)
- Paul H Schlesinger
- Dept of Cell Biology, Washington University, Saint Louis, MO, United States of America
| | - Demetrios T Braddock
- Dept. of Pathology, Yale New Haven Hospital, 310 Cedar Street, New Haven, CT, United States of America
| | - Quitterie C Larrouture
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, Windmill Road, Oxford OX3 7LD, UK
| | - Evan C Ray
- Renal Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, United States of America
| | - Vladimir Riazanski
- Dept of Neurobiology, Pharmacology & Physiology, University of Chicago, Chicago, IL, United States of America
| | - Deborah J Nelson
- Dept of Neurobiology, Pharmacology & Physiology, University of Chicago, Chicago, IL, United States of America
| | - Irina L Tourkova
- Veteran's Affairs Medical Center, Pittsburgh PA and Department of Pathology, University of Pittsburgh, Pittsburgh, PA, United States of America
| | - Harry C Blair
- Veteran's Affairs Medical Center, Pittsburgh PA and Department of Pathology, University of Pittsburgh, Pittsburgh, PA, United States of America.
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27
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Carozza JA, Brown JA, Böhnert V, Fernandez D, AlSaif Y, Mardjuki RE, Smith M, Li L. Structure-Aided Development of Small-Molecule Inhibitors of ENPP1, the Extracellular Phosphodiesterase of the Immunotransmitter cGAMP. Cell Chem Biol 2020; 27:1347-1358.e5. [PMID: 32726585 PMCID: PMC7680421 DOI: 10.1016/j.chembiol.2020.07.007] [Citation(s) in RCA: 72] [Impact Index Per Article: 14.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2020] [Revised: 06/28/2020] [Accepted: 07/08/2020] [Indexed: 11/23/2022]
Abstract
Cancer cells initiate an innate immune response by synthesizing and exporting the small-molecule immunotransmitter cGAMP, which activates the anti-cancer Stimulator of Interferon Genes (STING) pathway in the host. An extracellular enzyme, ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), hydrolyzes cGAMP and negatively regulates this anti-cancer immune response. Small-molecule ENPP1 inhibitors are much needed as tools to study the basic biology of extracellular cGAMP and as investigational cancer immunotherapy drugs. Here, we surveyed structure-activity relationships around a series of cell-impermeable and thus extracellular-targeting phosphonate inhibitors of ENPP1. In addition, we solved the crystal structure of an exemplary phosphonate inhibitor to elucidate the interactions that drive potency. This study yielded several best-in-class inhibitors with Ki < 2 nM and excellent physicochemical and pharmacokinetic properties. Finally, we demonstrate that an ENPP1 inhibitor delays tumor growth in a breast cancer mouse model. Together, we have developed ENPP1 inhibitors that are excellent tool compounds and potential therapeutics.
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MESH Headings
- Animals
- Antineoplastic Agents/chemical synthesis
- Antineoplastic Agents/chemistry
- Antineoplastic Agents/pharmacology
- Cell Survival/drug effects
- Cells, Cultured
- Dose-Response Relationship, Drug
- Drug Screening Assays, Antitumor
- Enzyme Inhibitors/chemical synthesis
- Enzyme Inhibitors/chemistry
- Enzyme Inhibitors/pharmacology
- Female
- Humans
- Mice
- Mice, Inbred C57BL
- Molecular Structure
- Neoplasms, Experimental/drug therapy
- Neoplasms, Experimental/metabolism
- Neoplasms, Experimental/pathology
- Neurotransmitter Agents/chemistry
- Neurotransmitter Agents/isolation & purification
- Neurotransmitter Agents/metabolism
- Nucleotides, Cyclic/chemistry
- Nucleotides, Cyclic/isolation & purification
- Nucleotides, Cyclic/metabolism
- Phosphoric Diester Hydrolases/metabolism
- Pyrophosphatases/antagonists & inhibitors
- Pyrophosphatases/metabolism
- Small Molecule Libraries/chemical synthesis
- Small Molecule Libraries/chemistry
- Small Molecule Libraries/pharmacology
- Structure-Activity Relationship
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Affiliation(s)
- Jacqueline A Carozza
- Department of Chemistry, Stanford University, Stanford, CA 93405, USA; Stanford ChEM-H, Stanford University, Stanford, CA 93405, USA
| | - Jenifer A Brown
- Stanford ChEM-H, Stanford University, Stanford, CA 93405, USA; Biophysics Program, Stanford University, Stanford, CA 93405, USA
| | - Volker Böhnert
- Stanford ChEM-H, Stanford University, Stanford, CA 93405, USA; Department of Biochemistry, Stanford University, Stanford, CA 93405, USA
| | - Daniel Fernandez
- Stanford ChEM-H Macromolecular Structure Knowledge Center, Stanford University, Stanford, CA 93405, USA
| | - Yasmeen AlSaif
- Stanford ChEM-H, Stanford University, Stanford, CA 93405, USA; Department of Biology, Stanford University, Stanford, CA 93405, USA
| | - Rachel E Mardjuki
- Department of Chemistry, Stanford University, Stanford, CA 93405, USA; Stanford ChEM-H, Stanford University, Stanford, CA 93405, USA
| | - Mark Smith
- Stanford ChEM-H Medicinal Chemistry Knowledge Center, Stanford, CA 93405, USA
| | - Lingyin Li
- Stanford ChEM-H, Stanford University, Stanford, CA 93405, USA; Department of Biochemistry, Stanford University, Stanford, CA 93405, USA.
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28
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Rutsch F, Buers I, Nitschke Y. Hereditary Disorders of Cardiovascular Calcification. Arterioscler Thromb Vasc Biol 2020; 41:35-47. [PMID: 33176451 DOI: 10.1161/atvbaha.120.315577] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Arterial calcification is a common phenomenon in the elderly, in patients with atherosclerosis or renal failure and in diabetes. However, when present in very young individuals, it is likely to be associated with an underlying hereditary disorder of arterial calcification. Here, we present an overview of the few monogenic disorders presenting with early-onset cardiovascular calcification. These disorders can be classified according to the function of the respective disease gene into (1) disorders caused by an altered purine and phosphate/pyrophosphate metabolism, (2) interferonopathies, and (3) Gaucher disease. The finding of arterial calcification in early life should alert the clinician and prompt further genetic work-up to define the underlying genetic defect, to establish the correct diagnosis, and to enable appropriate therapy.
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Affiliation(s)
- Frank Rutsch
- Department of General Pediatrics, Muenster University Children's Hospital, Germany
| | - Insa Buers
- Department of General Pediatrics, Muenster University Children's Hospital, Germany
| | - Yvonne Nitschke
- Department of General Pediatrics, Muenster University Children's Hospital, Germany
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29
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Cui F, Yin G, Yang R, Guo X. A colorimetric chemosensor for pyrophosphate based on mono-pyrenylurea in aqueous media. SPECTROCHIMICA ACTA. PART A, MOLECULAR AND BIOMOLECULAR SPECTROSCOPY 2020; 241:118658. [PMID: 32650244 DOI: 10.1016/j.saa.2020.118658] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Revised: 06/15/2020] [Accepted: 06/28/2020] [Indexed: 06/11/2023]
Abstract
Research on pyrophosphate ions detection remains important because it plays crucial roles in various fields. A simple and new colorimetric sensor for pyrophosphate (PPi) based on mono-pyrenylurea ligand (L) has been designed and synthesized by a simple reaction of 1-pyrenemethylamine hydrochloride with p-nitrophenylisocyanate. In DMSO-15% H2O solution and DMSO-15% HEPES (10 mM, pH = 7.2) buffer solution, L displayed a selective colorimetric response for pyrophosphate (PPi) against other anions by changing color from colorless to yellow. This recognition process was confirmed by UV-vis spectroscopy. Also, the colorimetric properties of L are attributed to the anion-induced deprotonation of the urea subunit as demonstrated by 1H NMR titration method. Moreover, convenient test strips coated with L could be utilized to detect PPi in aqueous solution by naked-eye.
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Affiliation(s)
- Fengjuan Cui
- College of Chemistry and Chemical Engineering, Qiqihar University, Qiqihar, Heilongjiang 161006, PR China.
| | - Guangming Yin
- College of Chemistry and Chemical Engineering, Qiqihar University, Qiqihar, Heilongjiang 161006, PR China
| | - Rui Yang
- College of Chemistry and Chemical Engineering, Qiqihar University, Qiqihar, Heilongjiang 161006, PR China
| | - Xiangfeng Guo
- College of Chemistry and Chemical Engineering, Qiqihar University, Qiqihar, Heilongjiang 161006, PR China.
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Stabach PR, Zimmerman K, Adame A, Kavanagh D, Saeui CT, Agatemor C, Gray S, Cao W, De La Cruz EM, Yarema KJ, Braddock DT. Improving the Pharmacodynamics and In Vivo Activity of ENPP1-Fc Through Protein and Glycosylation Engineering. Clin Transl Sci 2020; 14:362-372. [PMID: 33064927 PMCID: PMC7877847 DOI: 10.1111/cts.12887] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2020] [Accepted: 08/24/2020] [Indexed: 02/06/2023] Open
Abstract
Enzyme replacement with ectonucleotide pyrophosphatase phospodiesterase‐1 (ENPP1) eliminates mortality in a murine model of the lethal calcification disorder generalized arterial calcification of infancy. We used protein engineering, glycan optimization, and a novel biomanufacturing platform to enhance potency by using a three‐prong strategy. First, we added new N‐glycans to ENPP1; second, we optimized pH‐dependent cellular recycling by protein engineering of the Fc neonatal receptor; finally, we used a two‐step process to improve sialylation by first producing ENPP1‐Fc in cells stably transfected with human α‐2,6‐sialyltransferase (ST6) and further enhanced terminal sialylation by supplementing production with 1,3,4‐O‐Bu3ManNAc. These steps sequentially increased the half‐life of the parent compound in rodents from 37 hours to ~ 67 hours with an added N‐glycan, to ~ 96 hours with optimized pH‐dependent Fc recycling, to ~ 204 hours when the therapeutic was produced in ST6‐overexpressing cells with 1,3,4‐O‐Bu3ManNAc supplementation. The alterations were demonstrated to increase drug potency by maintaining efficacious levels of plasma phosphoanhydride pyrophosphate in ENPP1‐deficient mice when the optimized biologic was administered at a 10‐fold lower mass dose less frequently than the parent compound—once every 10 days vs. 3 times a week. We believe these improvements represent a general strategy to rationally optimize protein therapeutics.
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Affiliation(s)
- Paul R Stabach
- Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Kristin Zimmerman
- Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Aaron Adame
- Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Dillon Kavanagh
- Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Christopher T Saeui
- Department of Biomedical Engineering, Translational Tissue Engineering Center, The Johns Hopkins University, Baltimore, Maryland, USA
| | - Christian Agatemor
- Department of Biomedical Engineering, Translational Tissue Engineering Center, The Johns Hopkins University, Baltimore, Maryland, USA
| | - Shawn Gray
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA
| | - Wenxiang Cao
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA
| | - Enrique M De La Cruz
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA
| | - Kevin J Yarema
- Department of Biomedical Engineering, Translational Tissue Engineering Center, The Johns Hopkins University, Baltimore, Maryland, USA
| | - Demetrios T Braddock
- Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA
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Calcinosis in Systemic Sclerosis: Updates in Pathophysiology, Evaluation, and Treatment. Curr Rheumatol Rep 2020; 22:73. [DOI: 10.1007/s11926-020-00951-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
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Boyce AM, Gafni RI, Ferreira CR. Generalized Arterial Calcification of Infancy: New Insights, Controversies, and Approach to Management. Curr Osteoporos Rep 2020; 18:232-241. [PMID: 32172442 PMCID: PMC9506683 DOI: 10.1007/s11914-020-00577-4] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
PURPOSE OF REVIEW This review summarizes current understanding of generalized arterial calcification of infancy (GACI), emphasizing pathophysiology, clinical presentation, and approaches and controversies in management. RECENT FINDINGS Identification of causative ENPP1 mutations revealed that GACI arises from deficiencies in inorganic pyrophosphate (leading to calcifications) and adenosine monophosphate (leading to intimal proliferation). Identification of genotypic and phenotypic overlap with pseudoxanthoma elasticum and autosomal recessive hypophosphatemic rickets further advanced understanding of GACI as a complex, multisystemic disease. Clinical data is limited to small, retrospective samples; it is therefore unknown whether commonly used medications, such as bisphosphonates and hypophosphatemia treatment, are therapeutic or potentially harmful. ENPP1-Fc replacement represents a promising approach warranting further study. Knowledge gaps in natural history place clinicians at high risk of assigning causality to interventions that are correlated with changes in clinical status. There is thus a critical need for improved natural history studies to develop and test targeted therapies.
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Affiliation(s)
- Alison M Boyce
- Skeletal Diseases and Mineral Homeostasis Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive Room 218 MSC 4320, Bethesda, MD, 20892, USA.
| | - Rachel I Gafni
- Skeletal Diseases and Mineral Homeostasis Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive Room 218 MSC 4320, Bethesda, MD, 20892, USA
| | - Carlos R Ferreira
- Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA
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Agarwal N, Agarwal U, Alfirevic Z, Lim J, Kaleem M, Landes C, Mughal MZ, Ramakrishnan R. Skeletal abnormalities secondary to antenatal etidronate treatment for suspected generalised arterial calcification of infancy. Bone Rep 2020; 12:100280. [PMID: 32490054 PMCID: PMC7256299 DOI: 10.1016/j.bonr.2020.100280] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/04/2019] [Revised: 04/27/2020] [Accepted: 05/11/2020] [Indexed: 12/01/2022] Open
Abstract
Background Generalised arterial calcification of infancy (GACI) is a rare disorder characterised by the deposition of hydroxyapatite crystals within the vessel walls. It is associated with a high mortality rate. Bisphosphonates have been used with some success in the treatment of GACI. However, there is a paucity of data on the antenatal use of bisphosphonates for GACI. In this paper, we report development of the skeletal changes suggestive of hypophosphatasia (HPP) in an infant with GACI, whose mother was treated with etidronate during pregnancy. Case report A Caucasian infant boy had a suspected antenatal diagnosis of GACI based on the findings suggestive of calcification of the annulus of the tricuspid valve and wall of the right ventricular (RV) outflow tract and main pulmonary artery on foetal echocardiography and the genetic analysis which showed a pathogenic heterozygous mutation in ABCC6. Based on these findings, mother was started on etidronate treatment from 26 weeks of gestation. A healthy male baby was delivered at 38 weeks of gestation. Initial postnatal echocardiogram on day 1 of life was normal with good biventricular function; subtle changes suggestive of microcalcifications were detected on the CT angiography. Serum calcium, phosphate, alkaline phosphatase and renal profile were normal. Further, the serum inorganic pyrophosphate (PPi) level was significantly low. Skeletal changes suggestive of HPP were seen on the radiographs. The baby developed cardiac dysfunction on day 4 of life with evidence of ischaemic changes on electrocardiogram (ECG).Treatment with etidronate was started in view of probable evolving coronary calcifications. Despite treatment with cardiac supportive measures and bisphosphonate, he succumbed to death in the third week of life. Discussion We believe, this is the first report of skeletal changes suggestive of HPP, arising secondary to antenatal etidronate (first generation bisphosphonate) used for the treatment of suspected GACI due to a heterozygous ABCC6 mutation.
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Affiliation(s)
- Neha Agarwal
- Department of Paediatric Endocrinology, Alder Hey Children's NHS Foundation Trust, Liverpool, UK
| | - Umber Agarwal
- Department of Obstetrics and Maternal-Fetal Medicine, Liverpool Women's NHS Foundation Trust, Liverpool, UK
| | - Zarko Alfirevic
- Department of Women's and Children's Health, Obstetrics, Maternal & Fetal Medicine, University of Liverpool and Liverpool Women's Hospital NHS Foundation Trust, Liverpool, UK
| | - Joyce Lim
- Department of Paediatric Cardiology, Alder Hey Children's NHS Foundation Trust, Liverpool, UK
| | - Musa Kaleem
- Department of Paediatric Radiology, Alder Hey Children's NHS Foundation Trust, Liverpool, UK
| | - Caren Landes
- Department of Paediatric Radiology, Alder Hey Children's NHS Foundation Trust, Liverpool, UK
| | - M Zulf Mughal
- Department of Paediatric Endocrinology, Royal Manchester Children's Hospital, Manchester University Hospital's NHS Trust, Oxford Road, Manchester, UK
| | - R Ramakrishnan
- Department of Paediatric Endocrinology, Alder Hey Children's NHS Foundation Trust, Liverpool, UK
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Orriss IR. Extracellular pyrophosphate: The body's "water softener". Bone 2020; 134:115243. [PMID: 31954851 DOI: 10.1016/j.bone.2020.115243] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/10/2019] [Revised: 01/08/2020] [Accepted: 01/16/2020] [Indexed: 12/18/2022]
Abstract
Extracellular pyrophosphate (ePPi) was first identified as a key endogenous inhibitor of mineralisation in the 1960's by Fleisch and colleagues. The main source of ePPi seems to be extracellular ATP which is continually released from cells in a controlled way. ATP is rapidly broken down by enzymes including ecto-nucleotide pyrophosphatase/phosphodiesterases to produce ePPi. The major function of ePPi is to directly inhibit hydroxyapatite formation and growth meaning that this simple molecule acts as the body's own "water softener". However, studies have also shown that ePPi can influence gene expression and regulate its own production and breakdown. This review will summarise our current knowledge of ePPi metabolism and how it acts to prevent pathological soft tissue calcification and regulate physiological bone mineralisation.
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Affiliation(s)
- Isabel R Orriss
- Department of Comparative Biomedical Sciences, Royal Veterinary College, London NW1 0TU, UK.
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35
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Abstract
Over the last decades, the association between vascular calcification (VC) and all-cause/cardiovascular mortality, especially in patients with high atherogenic status, such as those with diabetes and/or chronic kidney disease, has been repeatedly highlighted. For over a century, VC has been noted as a passive, degenerative, aging process without any treatment options. However, during the past decades, studies confirmed that mineralization of the arteries is an active, complex process, similar to bone genesis and formation. The main purpose of this review is to provide an update of the existing biomarkers of VC in serum and develop the various pathogenetic mechanisms underlying the calcification process, including the pivotal roles of matrix Gla protein, osteoprotegerin, bone morphogenetic proteins, fetuin-a, fibroblast growth-factor-23, osteocalcin, osteopontin, osteonectin, sclerostin, pyrophosphate, Smads, fibrillin-1 and carbonic anhydrase II.
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Oheim R, Zimmerman K, Maulding ND, Stürznickel J, von Kroge S, Kavanagh D, Stabach PR, Kornak U, Tommasini SM, Horowitz MC, Amling M, Thompson D, Schinke T, Busse B, Carpenter TO, Braddock DT. Human Heterozygous ENPP1 Deficiency Is Associated With Early Onset Osteoporosis, a Phenotype Recapitulated in a Mouse Model of Enpp1 Deficiency. J Bone Miner Res 2020; 35:528-539. [PMID: 31805212 PMCID: PMC7184798 DOI: 10.1002/jbmr.3911] [Citation(s) in RCA: 41] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/07/2019] [Revised: 09/27/2019] [Accepted: 10/14/2019] [Indexed: 12/15/2022]
Abstract
Biallelic ENPP1 deficiency in humans induces generalized arterial calcification of infancy (GACI) and/or autosomal recessive hypophosphatemic rickets type 2 (ARHR2). The latter is characterized by markedly increased circulating FGF23 levels and renal phosphate wasting, but aberrant skeletal manifestations associated with heterozygous ENPP1 deficiency are unknown. Here, we report three adult men with early onset osteoporosis who presented with fractures in the thoracic spine and/or left radius, mildly elevated circulating FGF23, and hypophosphatemia. Total hip bone mineral density scans demonstrated osteoporosis (Z-score < -2.5) and HRpQCT demonstrated microarchitectural defects in trabecular and cortical bone. Next-generation sequencing revealed heterozygous loss-of-function mutations in ENPP1 previously observed as biallelic mutations in infants with GACI. In addition, we present bone mass and structure data as well as plasma pyrophosphate (PPi) data of two siblings suffering from ARHR2 in comparison to their heterozygous and wild-type family members indicative of an ENPP1 gene dose effect. The skeletal phenotype in murine Enpp1 deficiency yielded nearly identical findings. Ten-week-old male Enpp1 asj/asj mice exhibited mild elevations in plasma FGF23 and hypophosphatemia, and micro-CT analysis revealed microarchitectural defects in trabecular and cortical bone of similar magnitude to HRpQCT defects observed in humans. Histomorphometry revealed mild osteomalacia and osteopenia at both 10 and 23 weeks. The biomechanical relevance of these findings was demonstrated by increased bone fragility and ductility in Enpp1 asj/asj mice. In summary, ENPP1 exerts a gene dose effect such that humans with heterozygous ENPP1 deficiency exhibit intermediate levels of plasma analytes associated with bone mineralization disturbance resulting in early onset osteoporosis. © 2019 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.
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Affiliation(s)
- Ralf Oheim
- Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Kristin Zimmerman
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - Nathan D Maulding
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - Julian Stürznickel
- Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Simon von Kroge
- Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Dillon Kavanagh
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - Paul R Stabach
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - Uwe Kornak
- Institute of Medical Genetics and Human Genetics, Charité Universitätsmedizin Berlin, Berlin, Germany
| | - Steven M Tommasini
- Department of Orthoaedics and Rehabilitation, Yale University School of Medicine, New Haven, CT, USA
| | - Mark C Horowitz
- Department of Orthoaedics and Rehabilitation, Yale University School of Medicine, New Haven, CT, USA
| | - Michael Amling
- Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | | | - Thorsten Schinke
- Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Björn Busse
- Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Thomas O Carpenter
- Department of Orthoaedics and Rehabilitation, Yale University School of Medicine, New Haven, CT, USA.,Department of Pediatrics, Yale University School of Medicine, New Haven, CT, USA
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37
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Carozza JA, Böhnert V, Nguyen KC, Skariah G, Shaw KE, Brown JA, Rafat M, von Eyben R, Graves EE, Glenn JS, Smith M, Li L. Extracellular cGAMP is a cancer cell-produced immunotransmitter involved in radiation-induced anti-cancer immunity. NATURE CANCER 2020; 1:184-196. [PMID: 33768207 PMCID: PMC7990037 DOI: 10.1038/s43018-020-0028-4] [Citation(s) in RCA: 212] [Impact Index Per Article: 42.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/27/2019] [Accepted: 01/15/2020] [Indexed: 12/18/2022]
Abstract
2'3'-cyclic GMP-AMP (cGAMP) is an intracellular second messenger that is synthesized in response to cytosolic double-stranded DNA and activates the innate immune STING pathway. Our previous discovery of its extracellular hydrolase ENPP1 hinted at the existence of extracellular cGAMP. Here, we detected that cGAMP is continuously exported but then efficiently cleared by ENPP1, explaining why it has previously escaped detection. By developing potent, specific, and cell impermeable ENPP1 inhibitors, we found that cancer cells continuously export cGAMP in culture at steady state and at higher levels when treated with ionizing radiation (IR). In mouse tumors, depletion of extracellular cGAMP decreased tumor-associated immune cell infiltration and abolished the curative effect of IR. Boosting extracellular cGAMP with ENPP1 inhibitors synergized with IR to delay tumor growth. In conclusion, extracellular cGAMP is an anti-cancer immunotransmitter that could be harnessed to treat cancers with low immunogenicity.
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Affiliation(s)
- Jacqueline A Carozza
- Department of Chemistry, Stanford University, Stanford, CA, USA
- Stanford ChEM-H, Stanford University, Stanford, CA, USA
| | - Volker Böhnert
- Stanford ChEM-H, Stanford University, Stanford, CA, USA
- Department of Biochemistry, Stanford University, School of Medicine, Stanford, CA, USA
| | - Khanh C Nguyen
- Departments of Medicine and Microbiology & Immunology, Stanford University, School of Medicine, Stanford, CA, USA
| | - Gemini Skariah
- Stanford ChEM-H, Stanford University, Stanford, CA, USA
- Department of Biochemistry, Stanford University, School of Medicine, Stanford, CA, USA
| | - Kelsey E Shaw
- Stanford ChEM-H, Stanford University, Stanford, CA, USA
- Department of Biochemistry, Stanford University, School of Medicine, Stanford, CA, USA
| | - Jenifer A Brown
- Stanford ChEM-H, Stanford University, Stanford, CA, USA
- Biophysics Program, Stanford University, Stanford, CA, USA
| | - Marjan Rafat
- Department of Radiation Oncology, Stanford University, School of Medicine, Stanford, CA, USA
| | - Rie von Eyben
- Department of Radiation Oncology, Stanford University, School of Medicine, Stanford, CA, USA
| | - Edward E Graves
- Department of Radiation Oncology, Stanford University, School of Medicine, Stanford, CA, USA
| | - Jeffrey S Glenn
- Departments of Medicine and Microbiology & Immunology, Stanford University, School of Medicine, Stanford, CA, USA
| | - Mark Smith
- Stanford ChEM-H, Stanford University, Stanford, CA, USA
| | - Lingyin Li
- Stanford ChEM-H, Stanford University, Stanford, CA, USA.
- Department of Biochemistry, Stanford University, School of Medicine, Stanford, CA, USA.
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38
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Gourgas O, Cole GB, Muiznieks LD, Sharpe S, Cerruti M. Effect of the Ionic Concentration of Simulated Body Fluid on the Minerals Formed on Cross-Linked Elastin-Like Polypeptide Membranes. LANGMUIR : THE ACS JOURNAL OF SURFACES AND COLLOIDS 2019; 35:15364-15375. [PMID: 31729882 DOI: 10.1021/acs.langmuir.9b02542] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/10/2023]
Abstract
Deposition of calcium phosphate minerals on the elastin-rich medial layers of arteries can cause severe cardiovascular complications. There are no available treatments for medial calcification, and the mechanism of mineral formation on elastin layers is still unknown. We recently developed an in vitro model of medial calcification using cross-linked elastin-like polypeptide (ELP) membranes immersed in simulated body fluid (SBF). While mineral phase evolution matched that observed in a mouse model of medial calcification, the long incubation required was a practical limitation of this model. Using higher SBF ion concentrations could be a solution to speed up mineral deposition, but its effect on the mineralization process is still not well understood. Here we analyze mineral formation and phase transformation on ELP membranes immersed in high concentration SBF. We show that while mineral deposition is significantly accelerated in these conditions, the chemistry and morphology of the minerals deposited on the ELP membranes and the overall mineralization process are strongly affected. Overall, this work suggests that while the use of low concentration SBF in this in vitro model is more appropriate to study medial calcification associated with the loss of calcification inhibitors, higher SBF ion concentration may be more relevant to study medial calcification in patients with life-threatening diseases such as chronic kidney disease.
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Affiliation(s)
- Ophélie Gourgas
- Department of Mining and Materials Engineering , McGill University , Montreal , Quebec H3A 0C5 , Canada
| | - Gregory B Cole
- Molecular Medicine , Hospital for Sick Children , Toronto , Ontario M5G 0A4 , Canada
- Department of Biochemistry , University of Toronto , Toronto , Ontario M5S 1A8 , Canada
| | - Lisa D Muiznieks
- Molecular Medicine , Hospital for Sick Children , Toronto , Ontario M5G 0A4 , Canada
| | - Simon Sharpe
- Molecular Medicine , Hospital for Sick Children , Toronto , Ontario M5G 0A4 , Canada
- Department of Biochemistry , University of Toronto , Toronto , Ontario M5S 1A8 , Canada
| | - Marta Cerruti
- Department of Mining and Materials Engineering , McGill University , Montreal , Quebec H3A 0C5 , Canada
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Disthabanchong S, Srisuwarn P. Mechanisms of Vascular Calcification in Kidney Disease. Adv Chronic Kidney Dis 2019; 26:417-426. [PMID: 31831120 DOI: 10.1053/j.ackd.2019.08.014] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2019] [Revised: 08/18/2019] [Accepted: 08/19/2019] [Indexed: 02/07/2023]
Abstract
The increase in prevalence and severity of vascular calcification in chronic kidney disease is a result of complex interactions between changes in the vascular bed, mineral metabolites, and other uremic factors. Vascular calcification can occur in the intima and the media of arterial wall. Under permissive conditions, vascular smooth muscle cells (VSMCs) can transform to osteoblast-like phenotype. The membrane-bound vesicles released from transformed VSMCs and the apoptotic bodies derived from dying VSMCs serve as nucleating structures for calcium crystal formation. Alterations in the quality and the quantity of endogenous calcification inhibitors also give rise to an environment that potentiates calcification.
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Affiliation(s)
- Sinee Disthabanchong
- Division of Nephrology, Department of Medicine, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
| | - Praopilad Srisuwarn
- Division of Nephrology, Department of Medicine, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
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Dursun F, Atasoy Öztürk T, Güven S, Kırmızıbekmez H, Seymen Karabulut G, Kalın S, Sözeri B. Magnesium and Anti-phosphate Treatment with Bisphosphonates for Generalised Arterial Calcification of Infancy: A Case Report. J Clin Res Pediatr Endocrinol 2019; 11:311-318. [PMID: 30525344 PMCID: PMC6745454 DOI: 10.4274/jcrpe.galenos.2018.2018.0204] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Generalized arterial calcification of infancy (GACI) is a rare autosomal-recessive disorder, characterized by calcification of the internal elastic lamina, fibrotic myointimal proliferation of muscular arteries and resultant arterial stenosis. Treatment with bisphosphonates has been proposed as a means of reducing arterial calcifications in GACI patients, although there is no formalized treatment approach. The case reported here was a patient with severe GACI diagnosed at three months of age who had no response to bisphosphonate treatment, but clinically improved after the initiation of magnesium and anti-phosphate (using calcium carbonate) treatments. In patients unresponsive to bisphosphonate, magnesium and anti-phosphate treatment may be attempted.
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Affiliation(s)
- Fatma Dursun
- Ümraniye Training and Research Hospital, Clinic of Pediatric Endocrinology, İstanbul, Turkey,* Address for Correspondence: Ümraniye Training and Research Hospital, Department of Pediatric Endocrinology, İstanbul, Turkey Phone: +90 505 267 14 03 E-mail:
| | - Tülay Atasoy Öztürk
- Ümraniye Training and Research Hospital, Clinic of Radiology, İstanbul, Turkey
| | - Serçin Güven
- Ümraniye Training and Research Hospital, Clinic of Pediatric Nephrology, İstanbul, Turkey
| | - Heves Kırmızıbekmez
- Ümraniye Training and Research Hospital, Clinic of Pediatric Endocrinology, İstanbul, Turkey
| | - Gülcan Seymen Karabulut
- Ümraniye Training and Research Hospital, Clinic of Pediatric Endocrinology, İstanbul, Turkey
| | - Sevinç Kalın
- Ümraniye Training and Research Hospital, Clinic of Radiology, İstanbul, Turkey
| | - Betül Sözeri
- Ümraniye Training and Research Hospital, Clinic of Pediatric Rheumatology, İstanbul, Turkey
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42
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Villa-Bellosta R. Synthesis of Extracellular Pyrophosphate Increases in Vascular Smooth Muscle Cells During Phosphate-Induced Calcification. Arterioscler Thromb Vasc Biol 2019; 38:2137-2147. [PMID: 30002059 DOI: 10.1161/atvbaha.118.311444] [Citation(s) in RCA: 39] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Objective- Hydroxyapatite deposition on the medial layer of the aortic walls is the hallmark of vascular calcification and the most common complication in aging individuals and in patients with diabetes mellitus and those undergoing hemodialysis. Extracellular pyrophosphate is a potent physicochemical inhibitor of hydroxyapatite crystal formation. This study analyzed changes in extracellular pyrophosphate metabolism during the phosphate-induced calcification process. Approach and Results- Phosphate-induced calcification of ex vivo-cultured aortic rings resulted in calcium accumulation after 7 days. This accumulation was enhanced when aortic walls were devitalized. BMP2 (bone morphogenic protein 2) expression was associated with calcium accumulation in cultured aortic rings, as well as in cultured vascular smooth muscle cells (VSMCs) and in calcitriol-induced calcification in rats. Hydroxyapatite dose dependently induced BMP2 overexpression in VSMCs. Moreover, TNAP (tissue nonspecific alkaline phosphatase) mRNA levels and activity were found to be downregulated in early phases and upregulated in later phases of calcification in all 3 models studied. eNPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) increased from early to later phases of calcification, whereas eNTPD1 (ectonucleoside triphosphate diphosphohydrolase 1) was downregulated during later phases. Synthesis of pyrophosphate in VSMCs increased significantly over time, in all 3 models studied. Because the rate of pyrophosphate hydrolysis was 10× slower than the rate of pyrophosphate synthesis, pyrophosphate synthesis is determined mainly by the ratio of eNPP1 to eNTPD1 activity. Hydroxyapatite also induces increments both in TNAP and eNPP1/eNTPD1 ratio in VSMCs. Conclusions- Pyrophosphate synthesis increases in VSMCs during phosphate-induced calcification because of compensatory regulation of extracellular pyrophosphate metabolism.
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Affiliation(s)
- Ricardo Villa-Bellosta
- From the Fundación Instituto de Investigación Sanitaria, Fundación Jiménez Díaz, Madrid, Spain
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43
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Azpiazu D, Gonzalo S, Villa-Bellosta R. Tissue Non-Specific Alkaline Phosphatase and Vascular Calcification: A Potential Therapeutic Target. Curr Cardiol Rev 2019; 15:91-95. [PMID: 30381085 PMCID: PMC6520574 DOI: 10.2174/1573403x14666181031141226] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/06/2018] [Revised: 10/16/2018] [Accepted: 10/23/2018] [Indexed: 12/17/2022] Open
Abstract
Vascular calcification is a pathologic phenomenon consisting of calcium phosphate crystal deposition in the vascular walls. Vascular calcification has been found to be a risk factor for cardiovascular diseases, due to its correlation with cardiovascular events and mortality, and it has been associated with aging, diabetes, and chronic kidney disease. Studies of vascular calcification have focused on phosphate homeostasis, primarily on the important role of hyperphosphatemia. Moreover, vascular calcification has been associated with loss of plasma pyrophosphate, one of the main inhibitors of calcification, thus indicating the importance of the phosphate/pyrophosphate ratio. Extracellular pyrophosphate can be synthesized from extracellular ATP by ecto-nucleotide pyrophosphatase/ phosphodiesterase, whereas pyrophosphate is hydrolyzed to phosphate by tissuenonspecific alkaline phosphatase, contributing to the formation of hydroxyapatite crystals. Over the last decade, vascular calcification has been the subject of numerous reviews and studies, which have revealed new agents and activities that may aid in explaining the complex physiology of this condition. This review summarizes current knowledge about alkaline phosphatase and its role in the process of vascular calcification as a key regulator of the phosphate/pyrophosphate ratio.
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Affiliation(s)
- Daniel Azpiazu
- Fundacion Instituto de Investigacion Sanitaria, Fundacion Jimenez Diaz, Avenida Reyes Catolicos 2, Madrid, Spain
| | - Sergio Gonzalo
- Fundacion Instituto de Investigacion Sanitaria, Fundacion Jimenez Diaz, Avenida Reyes Catolicos 2, Madrid, Spain
| | - Ricardo Villa-Bellosta
- Fundacion Instituto de Investigacion Sanitaria, Fundacion Jimenez Diaz, Avenida Reyes Catolicos 2, Madrid, Spain
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44
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Patel JJ, Bourne LE, Davies BK, Arnett TR, MacRae VE, Wheeler-Jones CP, Orriss IR. Differing calcification processes in cultured vascular smooth muscle cells and osteoblasts. Exp Cell Res 2019; 380:100-113. [PMID: 31004580 PMCID: PMC6520648 DOI: 10.1016/j.yexcr.2019.04.020] [Citation(s) in RCA: 40] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2018] [Revised: 04/11/2019] [Accepted: 04/15/2019] [Indexed: 11/15/2022]
Abstract
Arterial medial calcification (AMC) is the deposition of calcium phosphate mineral, often as hydroxyapatite, in the medial layer of the arteries. AMC shares some similarities to skeletal mineralisation and has been associated with the transdifferentiation of vascular smooth muscle cells (VSMCs) towards an osteoblast-like phenotype. This study used primary mouse VSMCs and calvarial osteoblasts to directly compare the established and widely used in vitro models of AMC and bone formation. Significant differences were identified between osteoblasts and calcifying VSMCs. First, osteoblasts formed large mineralised bone nodules that were associated with widespread deposition of an extracellular collagenous matrix. In contrast, VSMCs formed small discrete regions of calcification that were not associated with collagen deposition and did not resemble bone. Second, calcifying VSMCs displayed a progressive reduction in cell viability over time (≤7-fold), with a 50% increase in apoptosis, whereas osteoblast and control VSMCs viability remained unchanged. Third, osteoblasts expressed high levels of alkaline phosphatase (TNAP) activity and TNAP inhibition reduced bone formation by to 90%. TNAP activity in calcifying VSMCs was ∼100-fold lower than that of bone-forming osteoblasts and cultures treated with β-glycerophosphate, a TNAP substrate, did not calcify. Furthermore, TNAP inhibition had no effect on VSMC calcification. Although, VSMC calcification was associated with increased mRNA expression of osteoblast-related genes (e.g. Runx2, osterix, osteocalcin, osteopontin), the relative expression of these genes was up to 40-fold lower in calcifying VSMCs versus bone-forming osteoblasts. In summary, calcifying VSMCs in vitro display some limited osteoblast-like characteristics but also differ in several key respects: 1) their inability to form collagen-containing bone; 2) their lack of reliance on TNAP to promote mineral deposition; and, 3) the deleterious effect of calcification on their viability.
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Affiliation(s)
- Jessal J Patel
- Department of Comparative Biomedical Sciences, Royal Veterinary College, London, UK; School of Life & Medical Sciences, University of Hertfordshire, Hatfield, UK
| | - Lucie E Bourne
- Department of Comparative Biomedical Sciences, Royal Veterinary College, London, UK
| | - Bethan K Davies
- Department of Comparative Biomedical Sciences, Royal Veterinary College, London, UK
| | - Timothy R Arnett
- Department of Cell and Developmental Biology, University College London, London, UK
| | - Vicky E MacRae
- The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, UK
| | | | - Isabel R Orriss
- Department of Comparative Biomedical Sciences, Royal Veterinary College, London, UK.
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45
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ENPP1 in the Regulation of Mineralization and Beyond. Trends Biochem Sci 2019; 44:616-628. [PMID: 30799235 DOI: 10.1016/j.tibs.2019.01.010] [Citation(s) in RCA: 61] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2018] [Revised: 01/26/2019] [Accepted: 01/29/2019] [Indexed: 12/24/2022]
Abstract
ENPP1 is well known for its role in regulating skeletal and soft tissue mineralization. It primarily exerts its function through the generation of pyrophosphate, a key inhibitor of hydroxyapatite formation. Several previous studies have suggested that ENPP1 also contributes to a range of human diseases including diabetes, cancer, cardiovascular disease, and osteoarthritis. In this review, we summarize the pathological roles of ENPP1 in mineralization and these soft tissue disorders. We also discuss the underlying mechanisms through which ENPP1 exerts its pathological effects. A fuller understanding of the pathways through which ENPP1 acts may help to develop novel therapeutic strategies for these commonly diagnosed morbidities.
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46
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Tian X, Hussain S, de Pace C, Ruiz-Pérez L, Battaglia G. Zn II Complexes for Bioimaging and Correlated Applications. Chem Asian J 2019; 14:509-526. [PMID: 30716209 DOI: 10.1002/asia.201801437] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2018] [Revised: 12/31/2018] [Indexed: 11/09/2022]
Abstract
Zinc is a biocompatible element that exists as the second most abundant transition metal ion and an indispensable trace element in the human body. Compared to traditional metal-organic complexes systems, d10 metal ZnII complexes not only exhibit a large Stokes shift and good photon stability but also possess strong emission and low cytotoxicity with a relatively small molecular weight. The use of ZnII complexes has emerged in the last decade as a versatile and convenient tool for numerous biological applications, including bioimaging, molecular and protein recognition, as well as photodynamic therapy. Herein, we review recent developments involving ZnII metal complexes applied as specific subcellular compartment imaging probes and their correlated utilizations.
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Affiliation(s)
- Xiaohe Tian
- School of life science, Anhui University, Hefei, 230039, P.R. China
| | - Sajid Hussain
- School of life science, Anhui University, Hefei, 230039, P.R. China.,School of Applied Sciences and Humanities (NUSASH), National University of Technology, Sector I-12, Islamabad, Pakistan
| | - Cesare de Pace
- Department of Chemistry, University College London, London, WC1H 0AJ, UK
| | - Lorena Ruiz-Pérez
- Department of Chemistry, University College London, London, WC1H 0AJ, UK
| | - Giuseppe Battaglia
- School of life science, Anhui University, Hefei, 230039, P.R. China.,Department of Chemistry, University College London, London, WC1H 0AJ, UK
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47
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Bowden SA, Foster BL. Alkaline Phosphatase Replacement Therapy for Hypophosphatasia in Development and Practice. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1148:279-322. [PMID: 31482504 DOI: 10.1007/978-981-13-7709-9_13] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Hypophosphatasia (HPP) is an inherited disorder that affects bone and tooth mineralization characterized by low serum alkaline phosphatase. HPP is caused by loss-of-function mutations in the ALPL gene encoding the protein, tissue-nonspecific alkaline phosphatase (TNSALP). TNSALP is expressed by mineralizing cells of the skeleton and dentition and is associated with the mineralization process. Generalized reduction of activity of the TNSALP leads to accumulation of its substrates, including inorganic pyrophosphate (PPi) that inhibits physiological mineralization. This leads to defective skeletal mineralization, with manifestations including rickets, osteomalacia, fractures, and bone pain, all of which can result in multi-systemic complications with significant morbidity, as well as mortality in severe cases. Dental manifestations are nearly universal among affected individuals and feature most prominently premature loss of deciduous teeth. Management of HPP has been limited to supportive care until the introduction of a TNSALP enzyme replacement therapy (ERT), asfotase alfa (AA). AA ERT has proven to be transformative, improving survival in severely affected infants and increasing overall quality of life in children and adults with HPP. This chapter provides an overview of TNSALP expression and functions, summarizes HPP clinical types and pathologies, discusses early attempts at therapies for HPP, summarizes development of HPP mouse models, reviews design and validation of AA ERT, and provides up-to-date accounts of AA ERT efficacy in clinical trials and case reports, including therapeutic response, adverse effects, limitations, and potential future directions in therapy.
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Affiliation(s)
- S A Bowden
- Division of Endocrinology, Department of Pediatrics, Nationwide Children's Hospital/The Ohio State University College of Medicine, Columbus, OH, USA.
| | - B L Foster
- Division of Biosciences, College of Dentistry, The Ohio State University, Columbus, OH, USA
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48
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Borst P, Váradi A, van de Wetering K. PXE, a Mysterious Inborn Error Clarified. Trends Biochem Sci 2018; 44:125-140. [PMID: 30446375 DOI: 10.1016/j.tibs.2018.10.005] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2018] [Revised: 10/07/2018] [Accepted: 10/15/2018] [Indexed: 12/15/2022]
Abstract
Ever since Garrod deduced the existence of inborn errors in 1901, a vast array of metabolic diseases has been identified and characterized in molecular terms. In 2018 it is difficult to imagine that there is any uncharted backyard left in the metabolic disease landscape. Nevertheless, it took until 2013 to identify the cause of a relatively frequent inborn error, pseudoxanthoma elasticum (PXE), a disorder resulting in aberrant calcification. The mechanism found was not only biochemically interesting but also points to possible new treatments for PXE, a disease that has remained untreatable. In this review we sketch the tortuous road that led to the biochemical understanding of PXE and to new ideas for treatment. We also discuss some of the controversies still haunting the field.
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Affiliation(s)
- Piet Borst
- Division of Oncogenetics, The Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands.
| | - András Váradi
- Institute of Enzymology, Research Center for Natural Sciences (RCNS), Hungarian Academy of Sciences, 1117 Budapest, Hungary
| | - Koen van de Wetering
- Department of Dermatology and Cutaneous Biology and PXE International Center of Excellence in Research and Clinical Care, Thomas Jefferson University, Philadelphia, PA 19107, USA
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49
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Nitschke Y, Yan Y, Buers I, Kintziger K, Askew K, Rutsch F. ENPP1-Fc prevents neointima formation in generalized arterial calcification of infancy through the generation of AMP. Exp Mol Med 2018; 50:1-12. [PMID: 30369595 PMCID: PMC6204430 DOI: 10.1038/s12276-018-0163-5] [Citation(s) in RCA: 38] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2018] [Revised: 05/08/2018] [Accepted: 07/12/2018] [Indexed: 12/12/2022] Open
Abstract
Generalized arterial calcification of infancy (GACI) is associated with widespread arterial calcification and stenoses and is caused by mutations in ENPP1. ENPP1 encodes for ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which cleaves ATP to generate inorganic pyrophosphate (PPi) and adenosine monophosphate (AMP) extracellularly. The current study was designed to define the prevalence of arterial stenoses in GACI individuals and to identify the mechanism through which ENPP1 deficiency causes intimal proliferation. Furthermore, we aimed to effectively prevent and treat neointima formation in an animal model of GACI through the systemic administration of recombinant human (rh)ENPP1-Fc protein. Based on a literature review, we report that arterial stenoses are present in at least 72.4% of GACI cases. We evaluated the effect of rhENPP1-Fc on ENPP1-silenced human vascular smooth muscle cells (VSMCs) and on induced intimal proliferation in Enpp1-deficient ttw/ttw mice treated with carotid ligation. We demonstrate that silencing ENPP1 in VSMCs resulted in a tenfold increase in proliferation relative to that of cells transfected with negative control siRNA. The addition of rhENPP1-Fc, AMP or adenosine restored the silenced ENPP1-associated proliferation. In contrast, neither PPi nor etidronate, a current off-label treatment for GACI, had an effect on VSMC proliferation. Furthermore, subcutaneous rhENPP1-Fc protein replacement was effective in preventing and treating intimal hyperplasia induced by carotid ligation in an animal model of GACI. We conclude that ENPP1 inhibits neointima formation by generating AMP. RhENPP1-Fc may serve as an approach for the effective prevention and treatment of arterial stenoses in GACI. A protein replacement therapy may prove useful in tackling calcification and narrowing of the arteries in babies with a severe genetic disorder. Generalized Arterial Calcification of Infancy (GACI) is a rare condition in which infants’ arteries become calcified and their blood vessels internally scarred. It often leads to congestive heart failure. The ENPP1 gene encodes a protein that is crucial to preventing excess calcium build-up in the body. Mutations in the ENPP1 gene lead to GACI, but no therapies for the condition exist. Now, Frank Rutsch at Muenster University Children’s Hospital in Germany and co-workers have shown that administering a protein replacement can inhibit blood vessel scarring and arterial clogging in GACI mice models and in human stem cell cultures. The protein replacement boosts production of a key metabolic molecule called adenosine monophosphate.
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Affiliation(s)
- Yvonne Nitschke
- Department of General Pediatrics, Münster University Children's Hospital, Albert-Schweitzer-Campus 1, D-48149, Münster, Germany.,Cells in Motion Cluster of Excellence, Münster University, Münster, Germany
| | - Yan Yan
- Alexion Pharmaceuticals, 100 College St, New Haven, CT, USA
| | - Insa Buers
- Department of General Pediatrics, Münster University Children's Hospital, Albert-Schweitzer-Campus 1, D-48149, Münster, Germany.,Cells in Motion Cluster of Excellence, Münster University, Münster, Germany
| | - Kristina Kintziger
- Department of General Pediatrics, Münster University Children's Hospital, Albert-Schweitzer-Campus 1, D-48149, Münster, Germany
| | - Kim Askew
- Alexion Pharmaceuticals, 100 College St, New Haven, CT, USA
| | - Frank Rutsch
- Department of General Pediatrics, Münster University Children's Hospital, Albert-Schweitzer-Campus 1, D-48149, Münster, Germany. .,Cells in Motion Cluster of Excellence, Münster University, Münster, Germany.
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50
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Bowden SA, Foster BL. Profile of asfotase alfa in the treatment of hypophosphatasia: design, development, and place in therapy. Drug Des Devel Ther 2018; 12:3147-3161. [PMID: 30288020 PMCID: PMC6161731 DOI: 10.2147/dddt.s154922] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Hypophosphatasia (HPP) is a multi-systemic metabolic disorder caused by loss-of-function mutations in the ALPL gene that encodes the mineralization-associated enzyme, tissue-nonspecific alkaline phosphatase (TNSALP). HPP is characterized by defective bone and dental mineralization, leading to skeletal abnormalities with complications resulting in significant morbidity and mortality. Management of HPP has been limited to supportive care until the introduction of a recently approved enzyme replacement therapy employing bone-targeted recombinant human TNSALP, asfotase alfa (AA). This new therapy has been transformative as it improves survival in severely affected infants, and overall quality of life in children and adults with HPP. This review provides an overview of HPP, focusing on important steps in the development of AA enzyme replacement therapy, including the drug design, preclinical studies in the HPP mouse model, and outcomes from clinical trials and case report publications to date, with special attention given to response to therapy of skeletal manifestations, biochemical features, and other clinical manifestations. The limitations, adverse effects, and outcomes of AA are outlined and the place in therapy for individuals with HPP is discussed.
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Affiliation(s)
- Sasigarn A Bowden
- Division of Endocrinology, Department of Pediatrics, Nationwide Children's Hospital/The Ohio State University College of Medicine, Columbus, OH 43205, USA,
| | - Brian L Foster
- Division of Biosciences, College of Dentistry, The Ohio State University, Columbus, OH 43205, USA
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