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Afshar Y, Ma F, Quach A, Jeong A, Sunshine HL, Freitas V, Jami-Alahmadi Y, Helaers R, Li X, Pellegrini M, Wohlschlegel JA, Romanoski CE, Vikkula M, Iruela-Arispe ML. Transcriptional drifts associated with environmental changes in endothelial cells. eLife 2023; 12:e81370. [PMID: 36971339 PMCID: PMC10168696 DOI: 10.7554/elife.81370] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2022] [Accepted: 03/26/2023] [Indexed: 03/29/2023] Open
Abstract
Environmental cues, such as physical forces and heterotypic cell interactions play a critical role in cell function, yet their collective contributions to transcriptional changes are unclear. Focusing on human endothelial cells, we performed broad individual sample analysis to identify transcriptional drifts associated with environmental changes that were independent of genetic background. Global gene expression profiling by RNA sequencing and protein expression by liquid chromatography-mass spectrometry directed proteomics distinguished endothelial cells in vivo from genetically matched culture (in vitro) samples. Over 43% of the transcriptome was significantly changed by the in vitro environment. Subjecting cultured cells to long-term shear stress significantly rescued the expression of approximately 17% of genes. Inclusion of heterotypic interactions by co-culture of endothelial cells with smooth muscle cells normalized approximately 9% of the original in vivo signature. We also identified novel flow dependent genes, as well as genes that necessitate heterotypic cell interactions to mimic the in vivo transcriptome. Our findings highlight specific genes and pathways that rely on contextual information for adequate expression from those that are agnostic of such environmental cues.
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Affiliation(s)
- Yalda Afshar
- Department of Obstetrics and Gynecology, University of California, Los AngelesLos AngelesUnited States
- Molecular Biology Institute, University of California, Los AngelesLos AngelesUnited States
| | - Feyiang Ma
- Molecular Biology Institute, University of California, Los AngelesLos AngelesUnited States
- Department of Molecular, Cell, and Developmental Biology, University of California, Los AngelesLos AngelesUnited States
| | - Austin Quach
- Department of Molecular, Cell, and Developmental Biology, University of California, Los AngelesLos AngelesUnited States
| | - Anhyo Jeong
- Department of Obstetrics and Gynecology, University of California, Los AngelesLos AngelesUnited States
| | - Hannah L Sunshine
- Department of Molecular, Cellular and Integrative Physiology, University of California, Los AngelesLos AngelesUnited States
- Department of Cell and Developmental Biology, Northwestern University Feinberg School of MedicineChicagoUnited States
| | - Vanessa Freitas
- Departament of Cell and Developmental Biology, Institute of Biomedical Science, University of Sao PauloLos AngelesUnited States
| | - Yasaman Jami-Alahmadi
- Department of Biological Chemistry, University of CaliforniaLos AngelesUnited States
| | - Raphael Helaers
- Human Molecular Genetics, de Duve Institute, University of LouvainBrusselsBelgium
| | - Xinmin Li
- Department of Pathology and Laboratory Medicine, University of CaliforniaLos AngelesUnited States
| | - Matteo Pellegrini
- Molecular Biology Institute, University of California, Los AngelesLos AngelesUnited States
- Department of Molecular, Cell, and Developmental Biology, University of California, Los AngelesLos AngelesUnited States
| | - James A Wohlschlegel
- Department of Biological Chemistry, University of CaliforniaLos AngelesUnited States
| | - Casey E Romanoski
- Department of Cellular and Molecular Medicine, University of ArizonaTucsonUnited States
| | - Miikka Vikkula
- Human Molecular Genetics, de Duve Institute, University of LouvainBrusselsBelgium
- WELBIO department, WEL Research InstituteWavreBelgium
| | - M Luisa Iruela-Arispe
- Department of Molecular, Cell, and Developmental Biology, University of California, Los AngelesLos AngelesUnited States
- Department of Cell and Developmental Biology, Northwestern University Feinberg School of MedicineChicagoUnited States
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Ceccherini E, Cecchettini A, Gisone I, Persiani E, Morales MA, Vozzi F. Vascular Calcification: In Vitro Models under the Magnifying Glass. Biomedicines 2022; 10:biomedicines10102491. [PMID: 36289753 DOI: 10.3390/biomedicines10102491] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2022] [Revised: 10/01/2022] [Accepted: 10/04/2022] [Indexed: 11/16/2022] Open
Abstract
Vascular calcification is a systemic disease contributing to cardiovascular morbidity and mortality. The pathophysiology of vascular calcification involves calcium salt deposition by vascular smooth muscle cells that exhibit an osteoblast-like phenotype. Multiple conditions drive the phenotypic switch and calcium deposition in the vascular wall; however, the exact molecular mechanisms and the connection between vascular smooth muscle cells and other cell types are not fully elucidated. In this hazy landscape, effective treatment options are lacking. Due to the pathophysiological complexity, several research models are available to evaluate different aspects of the calcification process. This review gives an overview of the in vitro cell models used so far to study the molecular processes underlying vascular calcification. In addition, relevant natural and synthetic compounds that exerted anticalcifying properties in in vitro systems are discussed.
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Affiliation(s)
- Elisa Ceccherini
- Institute of Clinical Physiology, National Research Council (CNR), 56124 Pisa, Italy
| | - Antonella Cecchettini
- Institute of Clinical Physiology, National Research Council (CNR), 56124 Pisa, Italy
- Department of Clinical and Experimental Medicine, University of Pisa, 56126 Pisa, Italy
| | - Ilaria Gisone
- Institute of Clinical Physiology, National Research Council (CNR), 56124 Pisa, Italy
| | - Elisa Persiani
- Institute of Clinical Physiology, National Research Council (CNR), 56124 Pisa, Italy
| | - Maria Aurora Morales
- Institute of Clinical Physiology, National Research Council (CNR), 56124 Pisa, Italy
| | - Federico Vozzi
- Institute of Clinical Physiology, National Research Council (CNR), 56124 Pisa, Italy
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Oncel S, Basson MD. Gut homeostasis, injury, and healing: New therapeutic targets. World J Gastroenterol 2022; 28:1725-1750. [PMID: 35633906 PMCID: PMC9099196 DOI: 10.3748/wjg.v28.i17.1725] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/04/2021] [Revised: 12/12/2021] [Accepted: 03/27/2022] [Indexed: 02/06/2023] Open
Abstract
The integrity of the gastrointestinal mucosa plays a crucial role in gut homeostasis, which depends upon the balance between mucosal injury by destructive factors and healing via protective factors. The persistence of noxious agents such as acid, pepsin, nonsteroidal anti-inflammatory drugs, or Helicobacter pylori breaks down the mucosal barrier and injury occurs. Depending upon the size and site of the wound, it is healed by complex and overlapping processes involving membrane resealing, cell spreading, purse-string contraction, restitution, differentiation, angiogenesis, and vasculogenesis, each modulated by extracellular regulators. Unfortunately, the gut does not always heal, leading to such pathology as peptic ulcers or inflammatory bowel disease. Currently available therapeutics such as proton pump inhibitors, histamine-2 receptor antagonists, sucralfate, 5-aminosalicylate, antibiotics, corticosteroids, and immunosuppressants all attempt to minimize or reduce injury to the gastrointestinal tract. More recent studies have focused on improving mucosal defense or directly promoting mucosal repair. Many investigations have sought to enhance mucosal defense by stimulating mucus secretion, mucosal blood flow, or tight junction function. Conversely, new attempts to directly promote mucosal repair target proteins that modulate cytoskeleton dynamics such as tubulin, talin, Ehm2, filamin-a, gelsolin, and flightless I or that proteins regulate focal adhesions dynamics such as focal adhesion kinase. This article summarizes the pathobiology of gastrointestinal mucosal healing and reviews potential new therapeutic targets.
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Affiliation(s)
- Sema Oncel
- Department of Biomedical Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, United States
| | - Marc D Basson
- Department of Biomedical Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, United States
- Department of Surgery, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, United States
- Department of Pathology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, United States
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Lust ST, Shanahan CM, Shipley RJ, Lamata P, Gentleman E. Design considerations for engineering 3D models to study vascular pathologies in vitro. Acta Biomater 2021; 132:114-128. [PMID: 33652164 PMCID: PMC7611653 DOI: 10.1016/j.actbio.2021.02.031] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2020] [Revised: 01/28/2021] [Accepted: 02/18/2021] [Indexed: 12/15/2022]
Abstract
Many cardiovascular diseases (CVD) are driven by pathological remodelling of blood vessels, which can lead to aneurysms, myocardial infarction, ischaemia and strokes. Aberrant remodelling is driven by changes in vascular cell behaviours combined with degradation, modification, or abnormal deposition of extracellular matrix (ECM) proteins. The underlying mechanisms that drive the pathological remodelling of blood vessels are multifaceted and disease specific; however, unravelling them may be key to developing therapies. Reductionist models of blood vessels created in vitro that combine cells with biomaterial scaffolds may serve as useful analogues to study vascular disease progression in a controlled environment. This review presents the main considerations for developing such in vitro models. We discuss how the design of blood vessel models impacts experimental readouts, with a particular focus on the maintenance of normal cellular phenotypes, strategies that mimic normal cell-ECM interactions, and approaches that foster intercellular communication between vascular cell types. We also highlight how choice of biomaterials, cellular arrangements and the inclusion of mechanical stimulation using fluidic devices together impact the ability of blood vessel models to mimic in vivo conditions. In the future, by combining advances in materials science, cell biology, fluidics and modelling, it may be possible to create blood vessel models that are patient-specific and can be used to develop and test therapies. STATEMENT OF SIGNIFICANCE: Simplified models of blood vessels created in vitro are powerful tools for studying cardiovascular diseases and understanding the mechanisms driving their progression. Here, we highlight the key structural and cellular components of effective models and discuss how including mechanical stimuli allows researchers to mimic native vessel behaviour in health and disease. We discuss the primary methods used to form blood vessel models and their limitations and conclude with an outlook on how blood vessel models that incorporate patient-specific cells and flows can be used in the future for personalised disease modelling.
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Affiliation(s)
- Suzette T Lust
- Centre for Craniofacial and Regenerative Biology, King's College London, London SE1 9RT, United Kingdom; School of Biomedical Engineering and Imaging Sciences, King's College London, London SE1 7EH, United Kingdom
| | - Catherine M Shanahan
- School of Cardiovascular Medicine and Sciences, King's College London, London SE5 9NU, United Kingdom
| | - Rebecca J Shipley
- Institute of Healthcare Engineering and Department of Mechanical Engineering, University College London, London WC1E 7JE, United Kingdom
| | - Pablo Lamata
- School of Biomedical Engineering and Imaging Sciences, King's College London, London SE1 7EH, United Kingdom
| | - Eileen Gentleman
- Centre for Craniofacial and Regenerative Biology, King's College London, London SE1 9RT, United Kingdom.
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Small molecule FAK activator promotes human intestinal epithelial monolayer wound closure and mouse ulcer healing. Sci Rep 2019; 9:14669. [PMID: 31604999 PMCID: PMC6789032 DOI: 10.1038/s41598-019-51183-z] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2019] [Accepted: 09/24/2019] [Indexed: 01/23/2023] Open
Abstract
GI mucosal healing requires epithelial sheet migration. The non-receptor tyrosine kinase focal adhesion kinase (FAK) stimulates epithelial motility. A virtual screen identified the small drug-like FAK mimic ZINC40099027, which activates FAK. We assessed whether ZINC40099027 promotes FAK-Tyr-397 phosphorylation and wound healing in Caco-2 monolayers and two mouse intestinal injury models. Murine small bowel ulcers were generated by topical serosal acetic acid or subcutaneous indomethacin in C57BL/6J mice. One day later, we began treatment with ZINC40099027 or DMSO, staining the mucosa for phosphorylated FAK and Ki-67 and measuring mucosal ulcer area, serum creatinine, ALT, and body weight at day 4. ZINC40099027 (10–1000 nM) dose-dependently activated FAK phosphorylation, without activating Pyk2-Tyr-402 or Src-Tyr-419. ZINC40099027 did not stimulate proliferation, and stimulated wound closure independently of proliferation. The FAK inhibitor PF-573228 prevented ZINC40099027-stimulated wound closure. In both mouse ulcer models, ZINC40099027accelerated mucosal wound healing. FAK phosphorylation was increased in jejunal epithelium at the ulcer edge, and Ki-67 staining was unchanged in jejunal mucosa. ZINC40099027 serum concentration at sacrifice resembled the effective concentration in vitro. Weight, creatinine and ALT did not differ between groups. Small molecule FAK activators can specifically promote epithelial restitution and mucosal healing and may be useful to treat gut mucosal injury.
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Han X, Sakamoto N, Tomita N, Meng H, Sato M, Ohta M. Influence of TGF-β1 expression in endothelial cells on smooth muscle cell phenotypes and MMP production under shear stress in a co-culture model. Cytotechnology 2019; 71:489-496. [PMID: 30707337 PMCID: PMC6465372 DOI: 10.1007/s10616-018-0268-7] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2018] [Accepted: 10/13/2018] [Indexed: 12/28/2022] Open
Abstract
Recently, our group has contrasted an endothelial cell-smooth muscle cell (EC-SMC) co-culture model with 3D-cultured SMCs and found that SMCs could respond to high shear stress (SS), which has not been explored before. SMCs were not directly exposed to the flow but were under an EC monolayer; therefore, it is necessary to explore the influence of EC on SMC behaviors under high SS for understanding the mechanism of SMC response to various magnitudes of SS. In the present study, TGF-β1 expression in ECs in an EC-SMC co-culture model was suppressed by an siRNA transfection method. Next, phenotypic changes were observed and MMP-2 and -9 productions were measured in SMCs in the co-culture model after 72-h flow exposure to different SS levels. We confirmed that TGF-β1 expression in ECs could influence SMC phenotypic change under SS conditions and that TGF-β1 expression in ECs could also change MMP-2 production but not MMP-9 production in SMCs under SS conditions in the co-culture model. These results could be useful for understanding the mechanisms of SMC response to SS, particularly for understanding signal transduction emanating from ECs.
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Affiliation(s)
- Xiaobo Han
- Graduate School of Biomedical Engineering, Tohoku University, 2-1-1, Katahira, Aoba-ku, Sendai, Japan
| | - Naoya Sakamoto
- Department of Intelligent Mechanical Systems, Tokyo Metropolitan University, Hachioji, Japan
| | - Noriko Tomita
- Institute of Fluid Science, Tohoku University, 2-1-1, Katahira, Aoba-ku, Sendai, Miyagi, 980-8577, Japan
| | - Hui Meng
- Toshiba Stroke and Vascular Research Center, Department of Mechanical and Aerospace Engineering, University at Buffalo, State University of New York, New York, USA
| | - Masaaki Sato
- Graduate School of Biomedical Engineering, Tohoku University, 2-1-1, Katahira, Aoba-ku, Sendai, Japan
| | - Makoto Ohta
- Institute of Fluid Science, Tohoku University, 2-1-1, Katahira, Aoba-ku, Sendai, Miyagi, 980-8577, Japan.
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7
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Han X, Sakamoto N, Tomita N, Meng H, Sato M, Ohta M. Influence of shear stress on phenotype and MMP production of smooth muscle cells in a co-culture model. ACTA ACUST UNITED AC 2017. [DOI: 10.17106/jbr.31.50] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Affiliation(s)
- Xiaobo Han
- Graduate School of Biomedical Engineering, Tohoku University
| | - Naoya Sakamoto
- Department of Medical Engineering, Kawasaki University of Medical Welfare
- Department of Intelligent Mechanical Systems, Tokyo Metropolitan University
| | | | - Hui Meng
- Toshiba Stroke and Vascular Research Center, Department of Mechanical and Aerospace Engineering, University at Buffalo, State University of New York
| | - Masaaki Sato
- Graduate School of Biomedical Engineering, Tohoku University
| | - Makoto Ohta
- Institute of Fluid Science, Tohoku University
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8
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Li Y, Guo Y, Chen Y, Wang Y, You Y, Yang Q, Weng X, Li Q, Zhu X, Zhou B, Liu X, Gong Z, Zhang R. Establishment of an interleukin-1β-induced inflammation-activated endothelial cell-smooth muscle cell-mononuclear cell co-culture model and evaluation of the anti-inflammatory effects of tanshinone IIA on atherosclerosis. Mol Med Rep 2015; 12:1665-76. [PMID: 25936371 PMCID: PMC4464412 DOI: 10.3892/mmr.2015.3668] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2014] [Accepted: 02/19/2015] [Indexed: 02/02/2023] Open
Abstract
Increasing evidence supports the hypothesis that inflammatory reactions serves an important function in the formation, progression and plaque rupture of atherosclerosis. Interleukin (IL)-1 primarily induces inflammation and is closely associated with the inflammatory environment and the formation of atherosclerosis. The present study aimed to establish an in vitro model for the evaluation of drug efficacy in the intervention of atherosclerosis from the inflammatory perspective, and to observe the anti-inflammatory effects of tanshinone IIA and andrographolide on atherosclerosis. The IL-1β-induced inflammation-activated endothelial cell (EC)-smooth muscle cell (SMC)-mononuclear cell (MC) co-culture model was established, based on the changes in a series of atherosclerosis-associated inflammatory markers secreted by ECs and SMCs. The expression of connexin in ECs, adhesion of MCs and changes in inflammatory signalling molecules were selected as evaluation indices for the inflammatory microenvironment of atherosclerosis. The use of this model revealed that tanshinone IIA exhibited significant efficacy against atherosclerosis and its inflammatory reactions. Inflammatory reactions were regarded as the primary mechanism underlying atherosclerosis. The established model simulated a series of relevant changes in the arterial wall under the inflammatory cytokines with oxidized low-density lipoprotein during the atherosclerotic process. The present study presented a reliable method for the identification of drugs with potential anti-inflammatory activity in atherosclerosis, for investigating the mechanisms of action, considering the improvement of the inflammatory state and the increase in plaque stability observed.
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Affiliation(s)
- Yujie Li
- Department of Pharmacokinetics of Chinese Materia Medica, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, P.R. China
| | - Yan Guo
- Department of Pharmacokinetics of Chinese Materia Medica, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, P.R. China
| | - Ying Chen
- Department of Pharmacokinetics of Chinese Materia Medica, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, P.R. China
| | - Yajie Wang
- Department of Pharmacokinetics of Chinese Materia Medica, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, P.R. China
| | - Yun You
- Department of Pharmacokinetics of Chinese Materia Medica, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, P.R. China
| | - Qing Yang
- Department of Pharmacokinetics of Chinese Materia Medica, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, P.R. China
| | - Xiaogang Weng
- Department of Pharmacokinetics of Chinese Materia Medica, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, P.R. China
| | - Qi Li
- Department of Pharmacokinetics of Chinese Materia Medica, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, P.R. China
| | - Xiaoxin Zhu
- Department of Pharmacokinetics of Chinese Materia Medica, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, P.R. China
| | - Bingbing Zhou
- Department of Pharmacokinetics of Chinese Materia Medica, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, P.R. China
| | - Xucen Liu
- Department of Pharmacokinetics of Chinese Materia Medica, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, P.R. China
| | - Zaipeng Gong
- Department of Pharmacokinetics of Chinese Materia Medica, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, P.R. China
| | - Ruijie Zhang
- Department of Pharmacokinetics of Chinese Materia Medica, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, P.R. China
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Chaturvedi L, Sun K, Walsh MF, Kuhn LA, Basson MD. The P-loop region of Schlafen 3 acts within the cytosol to induce differentiation of human Caco-2 intestinal epithelial cells. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2014; 1843:3029-37. [PMID: 25261706 DOI: 10.1016/j.bbamcr.2014.09.017] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/05/2014] [Revised: 09/05/2014] [Accepted: 09/17/2014] [Indexed: 01/26/2023]
Abstract
Schlafen 3 (Slfn3) mediates rodent enterocyte differentiation in vitro and in vivo, required for intestinal function. Little is known about Schlafen protein structure-function relationships. To define the Slfn3 domain that promotes differentiation, we studied villin and sucrase isomaltase (SI) promoter activity in Slfn3-null human Caco-2BBE cells transfected with full-length rat Slfn3 DNA or truncated constructs. Confocal microscopy and Western blots showed that Slfn3 is predominantly cytosolic. Villin promoter activity, increased by wild type Slfn3, was further enhanced by adding a nuclear exclusion sequence, suggesting that Slfn3 does not affect transcription by direct nuclear action. We therefore sought to dissect the region in Slfn3 stimulating promoter activity. Since examination of the Slfn3 N-terminal region revealed sequences similar to both an aminopeptidase (App) and a divergent P-loop resembling those in NTPases, we initially divided Slfn3 into an N-terminal domain containing the App and P-loop regions, and a C-terminal region. Only the N-terminal construct stimulated promoter activity. Further truncation indicated that both the App and the smaller P-loop constructs enhanced promoter activity similarly to the N-terminal sequence. Point mutations within the N-terminal region (R128L, altering a critical active site residue in the App domain, and L212D, conserved in Schlafens but variable in P-loop proteins) did not affect activity. These results show that Slfn3 acts in the cytosol to trigger a secondary signal cascade that elicits differentiation marker expression and narrows the active domain to the third of the Slfn3 sequence homologous to P-loop NTPases, a first step in understanding its mechanism of action.
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Affiliation(s)
| | - Kelian Sun
- Department of Surgery, Michigan State University, East Lansing, MI, USA.
| | - Mary F Walsh
- Department of Surgery, Michigan State University, East Lansing, MI, USA.
| | - Leslie A Kuhn
- Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA; Computer Science & Engineering, Michigan State University, East Lansing, MI, USA.
| | - Marc D Basson
- Department of Surgery, Michigan State University, East Lansing, MI, USA.
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Bhattacharyya A, Lin S, Sandig M, Mequanint K. Regulation of vascular smooth muscle cell phenotype in three-dimensional coculture system by Jagged1-selective Notch3 signaling. Tissue Eng Part A 2014; 20:1175-87. [PMID: 24138322 PMCID: PMC3993058 DOI: 10.1089/ten.tea.2013.0268] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2013] [Accepted: 10/16/2013] [Indexed: 12/21/2022] Open
Abstract
The modulation of vascular smooth muscle cell (VSMC) phenotype is an essential element to fabricate engineered conduits of clinical relevance. In vivo, owing to their close proximity, endothelial cells (ECs) play a role in VSMC phenotype switching. Although considerable progress has been made in vascular tissue engineering, significant knowledge gaps exist on how the contractile VSMC phenotype is induced at the conclusion of the tissue fabrication process. The objectives of this study were as follows: (1) to establish ligand presentation modes on transcriptional activation of VSMC-specific genes, (2) to develop a three-dimensional (3D) coculture model using human coronary artery smooth muscle cells (HCASMCs) and human coronary artery endothelial cells (HCAECs) on porous synthetic scaffolds and, (3) to investigate EC-mediated Notch signaling in 3D cultures and the induction of the HCASMC contractile phenotype. Whereas transcriptional activation of VSMC-specific genes was not induced by presenting soluble Jagged1 and Jagged1 bound to protein G beads, a direct link between HCAEC-bound Jagged1 and HCASMC differentiation genes was observed. Our 3D culture results showed that HCASMCs seeded to scaffolds and cultured for up to 16 days readily attached, infiltrated the scaffold, proliferated, and formed dense confluent layers. HCAECs, seeded on top of an HCASMC layer, formed a distinct, separate monolayer with cell-type partitioning, suggesting that HCAEC growth was contact inhibited. While we observed EC monolayer formation with 200,000 HCAECs/scaffold, seeding 400,000 HCAECs/scaffold revealed the formation of cord-like structures akin to angiogenesis. Western blot analyses showed that 3D coculture induced an upregulation of Notch3 receptor in HCASMCs and its ligand Jagged1 in HCAECs. This was accompanied by a corresponding induction of the contractile HCASMC phenotype as demonstrated by increased expression of smooth muscle-α-actin (SM-α-actin) and calponin. Knockdown of Jagged1 with siRNA showed a reduction in SM-α-actin and calponin in cocultures, identifying a link between Jagged1 and the expression of contractile proteins in 3D cocultures. We therefore conclude that the Notch3 signaling pathway is an important regulator of VSMC phenotype and could be targeted when fabricating engineered vascular tissues.
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Affiliation(s)
- Aparna Bhattacharyya
- Graduate Program of Biomedical Engineering, The University of Western Ontario, London, Canada
| | - Shigang Lin
- Department of Chemical and Biochemical Engineering, The University of Western Ontario, London, Canada
| | - Martin Sandig
- Graduate Program of Biomedical Engineering, The University of Western Ontario, London, Canada
- Department of Anatomy and Cell Biology, The University of Western Ontario, London, Canada
| | - Kibret Mequanint
- Graduate Program of Biomedical Engineering, The University of Western Ontario, London, Canada
- Department of Chemical and Biochemical Engineering, The University of Western Ontario, London, Canada
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11
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Development of vascular smooth muscle contractility by endothelium-derived transforming growth factor β proteins. Pflugers Arch 2013; 466:369-80. [PMID: 23887380 DOI: 10.1007/s00424-013-1329-6] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2013] [Revised: 07/12/2013] [Accepted: 07/13/2013] [Indexed: 10/26/2022]
Abstract
It is well established that the release of vasodilators and vasoconstrictors from vascular endothelium regulates vascular smooth muscle contraction. In this report, we investigate the role of the endothelium in the development and maintenance of constitutive vascular contractility. For that purpose, contractile activity of cultured bovine aortic smooth muscle cells (BASMCs) embedded in collagen gels was monitored by changes in gel diameter. After culturing for 5 days, ATP- and high KCl solution-induced contractions were significantly enhanced in the gels that were overlaid with bovine aortic endothelial cells (BAECs) or were cultured with conditioned medium of cultured BAECs. ATP-induced Ca(2+) transients, recorded in BASMCs cultured with conditioned medium of BAECs, were markedly augmented, but high KCl-induced Ca(2+) transients were not affected. BASMCs in control gels were spindle shaped, and those in endothelium-treated gels were more elongated and interconnected. The endothelial conditioned medium also strongly affected the intracellular distribution of actin fibers. Conditioned medium of BAECs contained TGFβ1 and TGFβ2. The TGFβ receptor antagonist SB431542 as well as simultaneous treatment with TGFβ1 and TGFβ2 neutralizing antibodies completely reversed the above effects of endothelial conditioned medium on BASMCs. BAECs medium induced phosphorylation of Smad2 and increased ATP-induced phosphorylation of myosin light chain in BASMCs. The present results indicate that the release of TGFβ1 and TGFβ2 from vascular endothelium affects the contractility of vascular smooth muscle cells by altering their morphology and agonist-induced Ca(2+) mobilization.
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12
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Truskey GA. Endothelial Cell Vascular Smooth Muscle Cell Co-Culture Assay For High Throughput Screening Assays For Discovery of Anti-Angiogenesis Agents and Other Therapeutic Molecules. ACTA ACUST UNITED AC 2010; 2010:171-181. [PMID: 21278926 DOI: 10.2147/ijhts.s13459] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Drug development for many diseases would be aided greatly by accurate in vitro model systems that replicate key elements of in vivo physiology. The recent development of co-culture systems of endothelial cells and vascular smooth muscle cells can be extended to high throughput systems for the identification of compounds for angiogenesis, vascular repair and hypertension. In this review, the various co-culture systems are reviewed and biological interactions between endothelial cells and vascular smooth muscle cells are discussed. Key considerations in the design of high throughput systems are presented and selected examples are discussed.
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Affiliation(s)
- George A Truskey
- Department of Biomedical Engineering Duke University Durham, NC 27708-0281 USA
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13
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Chaterji S, Park K, Panitch A. Scaffold-free in vitro arterial mimetics: the importance of smooth muscle-endothelium contact. Tissue Eng Part A 2010; 16:1901-12. [PMID: 20088699 PMCID: PMC2949266 DOI: 10.1089/ten.tea.2009.0271] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2009] [Accepted: 01/20/2010] [Indexed: 12/19/2022] Open
Abstract
We have developed an in vitro endothelial cell (EC)-smooth muscle cell (SMC) coculture platform that can mimic either the healthy or diseased state of blood vessels. Transforming growth factor-beta1 (TGF-beta1) and heparin were introduced to the SMC cultures to upregulate the SMC differentiation markers, alpha-smooth muscle actin (alpha-SMA) and calponin (homotypic model). Interestingly, seeding of near-confluent concentrations of ECs on the SMCs (heterotypic model) induced higher levels of alpha-SMA and calponin expression in the SMC cultures than did the addition of heparin and TGF-beta1 alone. The expression levels increased further on pretreating the SMCs with TGF-beta1 and heparin before adding a near-confluent monolayer of ECs. In contrast, seeding of sparse concentrations of ECs forced the SMCs into a more hyperplastic state as determined by alpha-SMA and calponin expression. This study highlights the importance of both soluble factors and EC seeding densities when considering culture conditions; in vivo SMCs are in close proximity with and interact with a monolayer of ECs. Our study suggests that this architecture is important for healthy vascular tissue function. In addition, it shows that disruption of this architecture can be used to mimic diseased states. As the EC-SMC coculture model can mimic either a diseased or a healthy blood vessel it may be useful as a test bed for evaluating cardiovascular therapeutics.
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MESH Headings
- Actins/metabolism
- Arteries/cytology
- Arteries/metabolism
- Calcium-Binding Proteins/metabolism
- Cells, Cultured
- Coculture Techniques/methods
- Endothelial Cells/cytology
- Endothelial Cells/drug effects
- Endothelial Cells/metabolism
- Endothelium, Vascular/cytology
- Endothelium, Vascular/metabolism
- Heparin/pharmacology
- Humans
- Microfilament Proteins/metabolism
- Muscle, Smooth, Vascular/cytology
- Muscle, Smooth, Vascular/metabolism
- Myocytes, Smooth Muscle/cytology
- Myocytes, Smooth Muscle/drug effects
- Myocytes, Smooth Muscle/metabolism
- Tissue Engineering/methods
- Transforming Growth Factor beta1/pharmacology
- Calponins
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Affiliation(s)
- Somali Chaterji
- Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
| | - Kinam Park
- Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
- Department of Pharmaceutics, Purdue University, West Lafayette, Indiana
- Oncological Sciences Center, Purdue University, West Lafayette, Indiana
| | - Alyssa Panitch
- Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
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14
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Ruiz M, Singh P, Thomson SC, Munger K, Blantz RC, Gabbai FB. L-arginine-induced glomerular hyperfiltration response: the roles of insulin and ANG II. Am J Physiol Regul Integr Comp Physiol 2008; 294:R1744-51. [PMID: 18353876 DOI: 10.1152/ajpregu.00871.2007] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Infusion of L-arginine produces an increase in glomerular filtration via kidney vasodilation, correlating with increased kidney excretion of nitric oxide (NO) metabolites, but the specific underlying mechanisms are unknown. We utilized clearance and micropuncture techniques to examine the whole kidney glomerular filtration rate (GFR) and single nephron GFR (SNGFR) responses to 1) L-arginine (ARG), 2) ARG+octreotide (OCT) to block insulin release, 3) ARG+OCT+insulin (INS) infusion to duplicate ARG-induced insulin levels, and 4) losartan (LOS), an angiotensin AT-1 receptor blocker, +ARG+OCT. ARG infusion increased GFR, while increasing insulin levels. OCT coinfusion prevented this increase in GFR, but with insulin infusion to duplicate ARG induced rise in insulin, the GFR response was restored. Identical insulin levels in the absence of ARG had no effect on GFR. In contrast to ARG infusion alone, coinfusion of OCT with ARG reduced proximal tubular fractional and absolute reabsorption potentially activating tubuloglomerular feedback. Losartan infusion, in addition to ARG and OCT (LOS+ARG+OCT), restored the increase in both SNGFR and proximal tubular reabsorption, without increasing insulin levels. In conclusion, 1) hyperfiltration responses to ARG require the concurrent, modest, permissive increase in insulin; 2) inhibition of insulin release after ARG reduces proximal reabsorption and prevents the hyperfiltration response; and 3) inhibition of ANG II activity restores the hyperfiltration response, maintains parallel increases in proximal reabsorption, and overrides the arginine/octreotide actions.
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Affiliation(s)
- Mario Ruiz
- Division of Nephrology-Hypertension, VA San Diego Healthcare System and the University of California, San Diego School of Medicine, La Jolla, CA 92161, USA
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15
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Abstract
The overall production of nitric oxide (NO) is decreased in chronic kidney disease (CKD) which contributes to cardiovascular events and further progression of kidney damage. There are many likely causes of NO deficiency in CKD and the areas surveyed in this review are: 1. Limitations on substrate (l-Arginine) availability, probably due to impaired renal l-Arginine biosynthesis, decreased transport of l-Arginine into endothelial cells and possible competition between NOS and competing metabolic pathways, such as arginase. 2. Increased circulating levels of endogenous NO synthase (NOS) inhibitors, in particular asymmetric dimethylarginine (ADMA). Increased methylation of proteins and their subsequent breakdown to release free ADMA may contribute but the major culprit is probably reduced ADMA catabolism by the enzymes dimethylarginine dimethylaminohydrolases. 3. Reduced renal cortex abundance of the neuronal NOS (nNOS)α protein correlates with injury while increasing nNOSβ abundance may provide a compensatory, protective response. Interventions that can restore NO production by targeting these various pathways are likely to reduce the cardiovascular complications of CKD as well as slowing the rate of progression.
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16
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Piotrkowski B, Fraga CG, de Cavanagh EMV. Mitochondrial function and nitric oxide metabolism are modified by enalapril treatment in rat kidney. Am J Physiol Regul Integr Comp Physiol 2006; 292:R1494-501. [PMID: 17185409 DOI: 10.1152/ajpregu.00540.2006] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The renal and cardiac benefits of renin-angiotensin system (RAS) inhibition in hypertension exceed those attributable to blood pressure reduction, and seem to involve mitochondrial function changes. To investigate whether mitochondrial changes associated with RAS inhibition are related to changes in nitric oxide (NO) metabolism, four groups of male Wistar rats were treated during 2 wk with a RAS inhibitor, enalapril (10 mg x kg(-1) x day(-1); Enal), or a NO synthase (NOS) inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME) (1 mg x kg(-1) x day(-1)), or both (Enal+L-NAME), or were untreated (control). Blood pressure and body weight were lower in Enal than in control. Electron transfer through complexes I to III and cytochrome oxidase activity were significantly lower, and uncoupling protein-2 content was significantly higher in kidney mitochondria isolated from Enal than in those from control. All of these changes were prevented by L-NAME cotreatment and were accompanied by a higher production/bioavailability of kidney NO. L-NAME abolished mitochondrial NOS activity but failed to inhibit extra-mitochondrial kidney NOS, underscoring the relevance of mitochondrial NO in those effects of enalapril that were suppressed by L-NAME cotreatment. In Enal, kidney mitochondria H(2)O(2) production rate and MnSOD activity were significantly lower than in control, and these effects were not prevented by L-NAME cotreatment. These findings may clarify the role of NO in the interactions between RAS and mitochondrial metabolism and can help to unravel the mechanisms involved in renal protection by RAS inhibitors.
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Affiliation(s)
- Barbara Piotrkowski
- Physical Chemistry-PRALIB, Univ. of Buenos Aires, Junín 956, 1113-Buenos Aires, Argentina
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17
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Bai Y, Ye S, Mortazavi R, Campese V, Vaziri ND. Effect of renal injury-induced neurogenic hypertension on NO synthase, caveolin-1, AKt, calmodulin and soluble guanylate cyclase expressions in the kidney. Am J Physiol Renal Physiol 2006; 292:F974-80. [PMID: 17122386 DOI: 10.1152/ajprenal.00157.2006] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Single injection of a small quantity of phenol into the cortex of one kidney in rats results in development of persistent hypertension (HTN) which is thought to be mediated by activation of renal afferent and efferent sympathetic pathways and sodium retention. Nitric oxide (NO) plays a major role in regulation of renal vascular resistance, tubular Na(+) reabsorption, pressure natriuresis, and thereby systemic arterial pressure. The present study was performed to test the hypothesis that chronic renal injury-induced HTN may be associated with dysregulation of NO system in the kidney. Accordingly, urinary NO metabolite (NO(x)) and cGMP excretions as well as renal cortical tissue (right kidney) expressions of NO synthase (NOS) isoforms [endothelial, neuronal, and inducible NOS, respectively (eNOS, nNOS, and iNOS)], NOS-regulatory factors (Caveolin-1, phospho-AKt, and calmodulin), and second-messenger system (soluble guanylate cyclase [sGC] and phosphodiesterase-5 [PDE-5]) were determined in male Sprague-Dawley rats 4 wk after injection of phenol (50 mul of 10% phenol) or saline into the lower pole of left kidney. The phenol-injected group exhibited a significant elevation of arterial pressure, marked reductions of urinary NO(x) and cGMP excretions, downregulations of renal tissue nNOS, eNOS, Phospho-eNOS, iNOS, and alpha chain of sGC. However, renal tissue AKt, phospho-AKT, Calmodulin, and PDE-5 proteins were unchanged in the phenol-injected animals. In conclusion, renal injury in this model results in significant downregulations of NOS isoforms and sGC and consequent reductions of NO production and cGMP generation by the kidney, events that may contribute to maintenance of HTN in this model.
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Affiliation(s)
- Y Bai
- Division of Nephrology and Hypertension, University of California, Irvine, CA 92868, USA
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18
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Jie KE, Verhaar MC, Cramer MJM, van der Putten K, Gaillard CAJM, Doevendans PA, Koomans HA, Joles JA, Braam B. Erythropoietin and the cardiorenal syndrome: cellular mechanisms on the cardiorenal connectors. Am J Physiol Renal Physiol 2006; 291:F932-44. [PMID: 16885153 DOI: 10.1152/ajprenal.00200.2006] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
We have recently proposed severe cardiorenal syndrome (SCRS), in which cardiac and renal failure mutually amplify progressive failure of both organs. This frequent pathophysiological condition has an extremely poor prognosis. Interactions between inflammation, the renin-angiotensin system, the balance between the nitric oxide and reactive oxygen species and the sympathetic nervous system form the cardiorenal connectors and are cornerstones in the pathophysiology of SCRS. An absolute deficit of erythropoietin (Epo) and decreased sensitivity to Epo in this syndrome both contribute to the development of anemia, which is more pronounced than renal anemia in the absence of heart failure. Besides expression on erythroid progenitor cells, Epo receptors are present in the heart, kidney, and vascular system, in which activation results in antiapoptosis, proliferation, and possibly antioxidation and anti-inflammation. Interestingly, Epo can improve cardiac and renal function. We have therefore reviewed the literature with respect to Epo and the cardiorenal connectors. Indeed, there are indications that Epo can diminish inflammation, reduce renin-angiotensin system activity, and shift the nitric oxide and reactive oxygen species balance toward nitric oxide. Information about Epo and the sympathetic nervous system is scarce. This analysis underscores the relevance of a further understanding of clinical and cellular mechanisms underlying protective effects of Epo, because this will support better treatment of SCRS.
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Affiliation(s)
- Kim E Jie
- Dept. of Nephrology and Hypertension, F03.223, Univ. Medical Ctr. Utrecht, Utrecht, The Netherlands
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19
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Wang HQ, Huang LX, Qu MJ, Yan ZQ, Liu B, Shen BR, Jiang ZL. Shear stress protects against endothelial regulation of vascular smooth muscle cell migration in a coculture system. ACTA ACUST UNITED AC 2006; 13:171-80. [PMID: 16840173 DOI: 10.1080/10623320600760282] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
Abstract
Vascular endothelial cells (ECs) are constantly exposed to blood flow-induced shear stress; these forces strongly influence the behaviors of neighboring vascular smooth muscle cells (VSMCs). VSMC migration is a key event in vascular wall remodeling. In this study, the authors assessed the difference between VSMC migration in VSMC/EC coculture under static and shear stress conditions. Utilizing a parallel-plate coculture flow chamber system and Transwell migration assays, they demonstrated that human ECs cocultured with VSMCs under static conditions induced VSMC migration, whereas laminar shear stress (1.5 Pa, 15 dynes/cm2) applied to the EC side for 12 h significantly inhibited this process. The changes in VSMC migration is mainly dependent on the close interactions between ECs and VSMCs. Western blotting showed that there was a consistent correlation between the level of Akt phosphorylation and the efficacy of shear stress-mediated EC regulation of VSMC migration. Wortmannin and Akti significantly inhibited the EC-induced effect on VSMC Akt phosphorylation and migration. These results indicate that shear stress protects against endothelial regulation of VSMC migration, which may be an atheroprotective function on the vessel wall.
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Affiliation(s)
- Han Qin Wang
- School of Life Sciences and Biotechnology, Institute of Mechanobiology and Medical Engineering, Shanghai Jiao Tong University, Shanghai, China
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20
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Erdely A, Freshour G, Tain YL, Engels K, Baylis C. DOCA/NaCl-induced chronic kidney disease: a comparison of renal nitric oxide production in resistant and susceptible rat strains. Am J Physiol Renal Physiol 2006; 292:F192-6. [PMID: 16896184 DOI: 10.1152/ajprenal.00146.2006] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Recent studies show nitric oxide (NO) deficiency is both a cause and consequence of chronic kidney disease (CKD). Reduced renal neuronal NO synthase (nNOS) abundance and activity parallel development of CKD with different models in the Sprague-Dawley (SD) rats, whereas Wistar Furth (WF) rats are protected against CKD and show preserved renal NO production. In this study, we compared renal NO in response to DOCA/salt-induced injury between the WF and SD. Studies were conducted on sham WF (n = 6) and SD (n = 6) and uninephrectized (UNX)+75 mg DOCA+1% NaCl (WF n = 9; SD n = 10) rats followed for 5 wk. Kidneys were harvested for Western blot, NOS activity, and histology. Other measurements included creatinine clearance and 24-h total NO production and urinary protein excretion. Absolute values of kidney weight were lower in WF than SD rats that showed similar percent increases with UNX+DOCA/NaCl. Proteinuria and decreased creatinine clearance were present in the SD but not the WF rats following UNX+DOCA/NaCl. Glomerular injury was mild in the WF compared with SD rats that showed many globally damaged glomeruli. Although renal nNOS abundance was decreased in both strains (higher baseline in WF), soluble NOS activity was maintained in the WF but significantly reduced in the SD rats. Renal endothelial NOS abundance and membrane NOS activity were unaffected by treatment. In summary, WF rats showed resistance to UNX+DOCA/NaCl-induced CKD with maintained renal NO production despite mild reduction in nNOS abundance. Further studies are needed to evaluate how WF rats maintain renal NO production despite similar changes in abundance as the vulnerable SD strain.
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Affiliation(s)
- Aaron Erdely
- Department of Physiology and Pharmacology, West Virginia University, USA
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21
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Vaziri ND. Dyslipidemia of chronic renal failure: the nature, mechanisms, and potential consequences. Am J Physiol Renal Physiol 2006; 290:F262-72. [PMID: 16403839 DOI: 10.1152/ajprenal.00099.2005] [Citation(s) in RCA: 307] [Impact Index Per Article: 16.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Chronic renal failure (CRF) results in profound lipid disorders, which stem largely from dysregulation of high-density lipoprotein (HDL) and triglyceride-rich lipoprotein metabolism. Specifically, maturation of HDL is impaired and its composition is altered in CRF. In addition, clearance of triglyceride-rich lipoproteins and their atherogenic remnants is impaired, their composition is altered, and their plasma concentrations are elevated in CRF. Impaired maturation of HDL in CRF is primarily due to downregulation of lecithin-cholesterol acyltransferase (LCAT) and, to a lesser extent, increased plasma cholesteryl ester transfer protein (CETP). Triglyceride enrichment of HDL in CRF is primarily due to hepatic lipase deficiency and elevated CETP activity. The CRF-induced hypertriglyceridemia, abnormal composition, and impaired clearance of triglyceride-rich lipoproteins and their remnants are primarily due to downregulation of lipoprotein lipase, hepatic lipase, and the very-low-density lipoprotein receptor, as well as, upregulation of hepatic acyl-CoA cholesterol acyltransferase (ACAT). In addition, impaired HDL metabolism contributes to the disturbances of triglyceride-rich lipoprotein metabolism. These abnormalities are compounded by downregulation of apolipoproteins apoA-I, apoA-II, and apoC-II in CRF. Together, these abnormalities may contribute to the risk of arteriosclerotic cardiovascular disease and may adversely affect progression of renal disease and energy metabolism in CRF.
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Affiliation(s)
- N D Vaziri
- Division of Nephrology and Hypertension, UCI Medical Center, Orange, CA 92868, USA.
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22
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Erdely A, Freshour G, Baylis C. Resistance to renal damage by chronic nitric oxide synthase inhibition in the Wistar-Furth rat. Am J Physiol Regul Integr Comp Physiol 2006; 290:R66-72. [PMID: 16352862 PMCID: PMC2756821 DOI: 10.1152/ajpregu.00444.2005] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Chronic nitric oxide synthase inhibition (NOSI) causes chronic kidney disease (CKD) in the Sprague Dawley (SD) rat. We previously showed that the Wistar-Furth (WF) rats are resistant to several models of CKD and maintain renal nitric oxide (NO) production compared with SD rats, whereas low-dose NOSI caused progression of CKD in WF rats. Here, we evaluate the impact of high-dose chronic NOSI in WF and SD rats, as well as intrarenal responses to an acute pressor dose of NOSI in the normal WF. Rats were given N(G)-nitro-l-arginine methyl ester (l-NAME) (150 and 300 mg/l for 6-10 wk) in the drinking water after an initial bolus tail vein injection. Both strains showed significant reductions in total NO production with chronic l-NAME. SD given 150 mg/l l-NAME for 6 wk developed proteinuria and renal injury, whereas WF rats receiving 150 mg/l l-NAME for 6-10 wk or 300 mg/l for 6 wk developed no proteinuria and minimal renal injury. Blood pressure was significantly elevated with chronic NOSI in both strains but was higher in the SD rat. There was little impact on renal nitric oxide synthase expression with l-NAME, except that cortical endothelial nitric oxide synthase abundance increased in WF after 6 wk (150 mg/l). Micropuncture experiments with acute pressor NOSI resulted in similar increases in systemic blood pressure in SD and WF rats, whereas WF rats showed a much smaller increment in glomerular blood pressure compared with SD rats. In conclusion, WF rats do not develop renal injury after chronic NOSI at, or above, a dose that causes significant injury in the SD rat. This protection may be associated with protection from glomerular hypertension.
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Affiliation(s)
- Aaron Erdely
- Department of Physiology and Functional Genomics, 1600 SW Archer Rd., P. O. Box 100274, University of Florida, Gainesville, FL 32610-0274, USA
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23
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Liang K, Kim CH, Vaziri ND. HMG-CoA reductase inhibition reverses LCAT and LDL receptor deficiencies and improves HDL in rats with chronic renal failure. Am J Physiol Renal Physiol 2005; 288:F539-44. [PMID: 15507547 DOI: 10.1152/ajprenal.00074.2004] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Dyslipidemia is a prominent feature of chronic renal failure (CRF) and a major risk factor for atherosclerosis and the progression of renal disease. CRF-induced dyslipidemia is marked by hypertriglyceridemia and a shift in plasma cholesterol from HDL to the ApoB-containing lipoproteins. Several studies have demonstrated a favorable response to administration of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors (statins) in CRF. This study was intended to explore the effect of statin therapy on key enzymes and receptors involved in cholesterol metabolism. Accordingly, CRF ( nephrectomized) and sham-operated rats were randomized to untreated and statin-treated (rosuvastatin 20 mg·kg−1·day−1) groups and observed for 6 wk. The untreated CRF rats exhibited increased total cholesterol-to-HDL cholesterol ratio, diminished plasma lecithin:cholesterol acyltransferase (LCAT) and the hepatic LDL receptor, elevated hepatic acyl-CoA:cholesterol acyltransferase (ACAT), and no change in hepatic HMG-CoA reductase, cholesterol 7α-hydroxylase, or HDL receptor (SRB-1). Statin administration lowered HMG-CoA reductase activity, normalized plasma LCAT, total cholesterol-to-HDL cholesterol ratio, and hepatic LDL receptor but did not significantly change either plasma total cholesterol, hepatic cholesterol 7α-hydroxylase, total ACAT activity, or SRB-1 in the CRF animals. Statin administration to the normal control rats led to significant increases in plasma LCAT and hepatic LDL receptor, significant reductions of total cholesterol-to-HDL cholesterol ratio, hepatic HMG-CoA reductase activity, and cholesterol 7α-hydroxylase abundance with virtually no change in plasma cholesterol concentration. Thus administration of rosuvastatin reversed LCAT and LDL receptor deficiencies and promoted a shift in plasma cholesterol from ApoB-containing lipoproteins to HDL in CRF rats.
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Affiliation(s)
- K Liang
- Div. of Nephrology and Hypertension, Univ. of California, Irvine Medical Ctr., Bldg. 53, Rm. 125, 101 The City Dr., Rt. 81, Orange, CA 92868, USA
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24
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Vaziri ND, Liang K. ACAT inhibition reverses LCAT deficiency and improves plasma HDL in chronic renal failure. Am J Physiol Renal Physiol 2004; 287:F1038-43. [PMID: 15280162 DOI: 10.1152/ajprenal.00150.2004] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
Chronic renal failure (CRF) is associated with increased risk of arteriosclerotic cardiovascular disease and profound alteration of plasma lipid profile. Uremic dyslipidemia is marked by increased plasma concentration of ApoB-containing lipoproteins and impaired high-density lipoprotein (HDL)-mediated reverse cholesterol transport. These abnormalities are, in part, due to acquired LCAT deficiency and upregulation of hepatic acyl-CoA:cholesterol acyltransferase (ACAT). ACAT catalyzes intracellular esterification of cholesterol, thereby promoting hepatic production of ApoB-containing lipoproteins and constraining HDL-mediated cholesterol uptake in the peripheral tissues. In view of the above considerations, we tested the hypothesis that pharmacological inhibition of ACAT may ameliorate CRF-induced dyslipidemia. 5/6 Nephrectomized rats were treated with either ACAT inhibitor IC-976 (30 mg.kg(-1).day(-1)) or placebo for 6 wk. Sham-operated rats served as controls. Key cholesterol-regulating enzymes, plasma lipids, and creatinine clearance were measured. The untreated CRF rats exhibited increased plasma low-density lipoprotein (LDL) and very LDL (VLDL) cholesterol, unchanged plasma HDL cholesterol, elevated total cholesterol-to-HDL cholesterol ratio, reduced liver microsomal free cholesterol, and diminished creatinine clearance. This was accompanied by reduced plasma LCAT, increased hepatic ACAT-2 mRNA, ACAT-2 protein and ACAT activity, and unchanged hepatic HMG-CoA reductase and cholesterol 7alpha-hydroxylase. ACAT inhibitor raised plasma HDL cholesterol, lowered LDL and VLDL cholesterol, and normalized total cholesterol-to-HDL cholesterol ratio without changing total cholesterol concentration (hence, a shift from ApoB-containing lipoproteins to HDL). This was accompanied by normalizations of hepatic ACAT activity and plasma LCAT. In conclusion, inhibition of ACAT reversed LCAT deficiency and improved plasma HDL level in CRF rats. Future studies are needed to explore the efficacy of ACAT inhibition in humans with CRF.
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Affiliation(s)
- N D Vaziri
- Irvine Medical Center, Division of Nephrology and Hypertension, University of California, 101 The City Drive, Bldg. 53, Rm. 125, Rt. 81, Orange, CA 92868, USA.
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25
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Erdely A, Freshour G, Smith C, Engels K, Olson JL, Baylis C. Protection against puromycin aminonucleoside-induced chronic renal disease in the Wistar-Furth rat. Am J Physiol Renal Physiol 2004; 287:F81-9. [PMID: 15039144 PMCID: PMC2756808 DOI: 10.1152/ajprenal.00349.2003] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
The Wistar-Furth (WF) rat is protected against chronic renal disease (CRD) following 5/6th ablation/infarction vs. the Sprague-Dawley (SD) rat, and protection was associated with preserved renal nitric oxide (NO) production. This study examined CRD induced with repeated administration of puromycin aminonucleoside (PAN). SD PAN developed nephrotic range proteinuria (>1 g/24 h), and at 15 wk severe renal injury developed and the glomerular filtration rate (GFR) was reduced to approximately 10% of sham. Total NO production, renal NO synthase (NOS) activity, and renal neuronal (n) and medullary endothelial (e)NOS abundance were reduced in the SD PAN. WF PAN exhibited less severe initial proteinuria (>400 mg/24 h), which abated within weeks, whereas GFR was normal and injury was minimal at 15 wk. Total NO production and renal NOS activity and abundance were significantly elevated compared with SD PAN. NOS mRNA (nNOS, eNOS, and inducible NOS) was not altered in WF, whereas SD showed significant increases in NOS gene expression with PAN. In conclusion, WF showed resistance to a second model of CRD with maintained renal NOS activity compared with SD.
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Affiliation(s)
- Aaron Erdely
- Department of Physiology, West Virginia University, Morgantown, West Virginia 26506, USA.
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26
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Bakker AD, Joldersma M, Klein-Nulend J, Burger EH. Interactive effects of PTH and mechanical stress on nitric oxide and PGE2 production by primary mouse osteoblastic cells. Am J Physiol Endocrinol Metab 2003; 285:E608-13. [PMID: 12746215 DOI: 10.1152/ajpendo.00501.2002] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Parathyroid hormone (PTH) and mechanical stress both stimulate bone formation but have opposite effects on bone resorption. PTH increased loading-induced bone formation in a rat model, suggesting that there is an interaction of these stimuli, possibly at the cellular level. To investigate whether PTH can modulate mechanotransduction by bone cells, we examined the effect of 10-9 M human PTH-(1-34) on fluid flow-induced prostaglandin E2 (PGE2) and nitric oxide (NO) production by primary mouse osteoblastic cells in vitro. Mechanical stress applied by means of a pulsating fluid flow (PFF; 0.6 +/- 0.3 Pa at 5 Hz) stimulated both NO and PGE2 production twofold. In the absence of stress, PTH also caused a twofold increase in PGE2 production, but NO release was not affected and remained low. Simultaneous application of PFF and PTH nullified the stimulating effect of PFF on NO production, whereas PGE2 production was again stimulated only twofold. Treatment with PTH alone reduced NO synthase (NOS) enzyme activity to undetectable levels. We speculate that PTH prevents stress-induced NO production via the inhibition of NOS, which will also inhibit the NO-mediated upregulation of PGE2 by stress, leaving only the NO-independent PGE2 upregulation by PTH. These results suggest that mechanical loading and PTH interact at the level of mechanotransduction.
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Affiliation(s)
- Astrid D Bakker
- Department of Oral Cell Biology, Academic Center for Dentistry Amsterdam, Vrije Universiteit, The Netherlands
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27
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Vouyouka AG, Salib SS, Cala S, Marsh JD, Basson MD. Chronic high pressure potentiates the antiproliferative effect and abolishes contractile phenotypic changes caused by endothelial cells in cocultured smooth muscle cells. J Surg Res 2003; 110:344-51. [PMID: 12788664 DOI: 10.1016/s0022-4804(03)00025-8] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
UNLABELLED High in vitro pressures have been reported to alter smooth muscle cell (SMC) and endothelial cell (EC) phenotype, while endothelial cells (ECs) can influence the proliferation, phenotype, and contractile features of smooth muscle cells (SMC) in coculture systems. However, little is known about the in vitro effects of pressure on EC/SMC cocultures. We therefore sought to compare SMC proliferation in independent and EC coculture under ambient and high pressure, and identify changes in the contractile phenotype of SMCs by measuring levels of the L-type Ca(2+) channel a(1) subunit (dihydropyridine-DHP receptor) which is critical for Ca(2+) transients, differentiation and contractility in SMC. METHODS Rat aortic SMCs in independent culture (SMC/0) and coculture with ECs (SMC/EC) were maintained in 5% CO(2) under either atmospheric or high pressure (130 mmHg). SMC were counted at 0, 1, 3, and 5 days and compared to initial cell counts of day 0 before the exposure to experimental conditions. DHP receptor levels were quantitated by Western blotting (three similar studies). RESULTS ECs suppressed SMC proliferation on day 1 of coculture in both atmospheric and high pressure (20% inhibition vs independent culture, P < or = 0.05). By day 3, cocultured SMC under atmospheric pressure displayed no EC-mediated inhibition, and at day 5, atmospheric cocultured SMCs revealed statistically significant enhanced proliferation as compared with SMCs in independent cultures. However, cocultured SMCs exposed to 130 mmHg pressure displayed sustained sensitivity to EC growth inhibition at both days 3 and 5 of the experiment. Coculture decreased SMC DHP-receptor levels under atmospheric pressure. However, this effect was abolished in cocultures exposed to high pressure. CONCLUSIONS High pressure substantially alters the regulatory influence of EC on SMC proliferation and contractile potential. This pressure/coculture model should increase our understanding of cellular interaction in hypertensive vasculopathy.
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Affiliation(s)
- Angela G Vouyouka
- Departments of Surgery and Cardiology, John D. Dingell VA Medical Center, Detroit, MI 48201-1932, USA.
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Korff T, Kimmina S, Martiny-Baron G, Augustin HG. Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness. FASEB J 2001; 15:447-57. [PMID: 11156960 DOI: 10.1096/fj.00-0139com] [Citation(s) in RCA: 268] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Paracrine interactions between endothelial cells (EC) and mural cells act as critical regulators of vessel wall assembly, vessel maturation and define a plasticity window for vascular remodeling. The present study was aimed at studying blood vessel maturation processes in a novel 3-dimensional spheroidal coculture system of EC and smooth muscle cells (SMC). Coculture spheroids differentiate spontaneously in a calcium-dependent manner to organize into a core of SMC and a surface layer of EC, thus mimicking the physiological assembly of blood vessels with surface lining EC and underlying mural cells. Coculture of EC with SMC induces a mature, quiescent EC phenotype as evidenced by 1) a significant increase in the number of junctional complexes of the EC surface layer, 2) a down-regulation of PDGF-B expression by cocultured EC, and 3) an increased resistance of EC to undergo apoptosis. Furthermore, EC cocultured with SMC become refractory to stimulation with VEGF (lack of CD34 expression on VEGF stimulation; inability to form capillary-like sprouts in a VEGF-dependent manner in a 3-dimensional in gel angiogenesis assay). In contrast, costimulation with VEGF and Ang-2 induced sprouting angiogenesis originating from coculture spheroids consistent with a model of Ang-2-mediated vessel destabilization resulting in VEGF responsiveness. Ang-2 on its own was able to stimulate endothelial cells in the absence of Ang-1 producing SMC, inducing lateral sheet migration as well as in gel sprouting angiogenesis. Taken together, the data establish the spheroidal EC/SMC system as a powerful cell culture model to study paracrine interactions in the vessel wall and provide functional evidence for smooth muscle cell-mediated quiescence effects on endothelial cells.
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MESH Headings
- Antigens, CD34/analysis
- Apoptosis/drug effects
- Calcium/pharmacology
- Calcium/physiology
- Cell Differentiation
- Cells, Cultured
- Coculture Techniques
- Culture Media, Serum-Free
- Culture Techniques/methods
- DNA Fragmentation
- Endothelial Growth Factors/pharmacology
- Endothelium, Vascular/cytology
- Endothelium, Vascular/drug effects
- Endothelium, Vascular/physiology
- Fibroblast Growth Factor 2/pharmacology
- Humans
- Intercellular Junctions/drug effects
- Intercellular Junctions/physiology
- Intercellular Junctions/ultrastructure
- Kinetics
- Lymphokines/pharmacology
- Models, Cardiovascular
- Muscle, Smooth, Vascular/cytology
- Muscle, Smooth, Vascular/physiology
- Neovascularization, Physiologic/drug effects
- Neovascularization, Physiologic/physiology
- Recombinant Proteins/pharmacology
- Time Factors
- Umbilical Veins
- Vascular Endothelial Growth Factor A
- Vascular Endothelial Growth Factors
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Affiliation(s)
- T Korff
- Cell Biology Laboratory, Department of Gynecology and Obstetrics, University of Göttingen Medical School, 37075 Göttingen, Germany
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