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Hirano M, Hirano K. Critical role of Rho proteins in myosin light chain di-phosphorylation during early phase of endothelial barrier disruption. J Physiol Sci 2022; 72:32. [PMID: 36476233 PMCID: PMC10717653 DOI: 10.1186/s12576-022-00857-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2022] [Accepted: 11/22/2022] [Indexed: 12/12/2022]
Abstract
We previously reported the Rho-associated coiled-coil containing protein kinase (ROCK)-mediated di-phosphorylation of myosin light chain (MLC) and actin bundle formation at the cell periphery as early events of the endothelial barrier disruption. We herein examined the role of RhoA during early events of barrier disruption. Treatment of cultured porcine aortic endothelial cells with simvastatin prevented the decrease in trans-endothelial electrical resistance, MLC di-phosphorylation and peripheral actin bundle formation seen 3 min after thrombin stimulation. Co-treatment with geranylgeranyl pyrophosphate rescued the thrombin-induced events. Thrombin increased a GTP-bound form of RhoA and phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at the ROCK site. The intracellular introduction of the inhibitory protein of RhoA inhibited the thrombin-induced di-phosphorylation of MLC. However, knockdown of either one of RhoA, RhoB or RhoC failed to inhibit thrombin-induced MLC di-phosphorylation. The findings suggest that Rho proteins play a critical role during early events of thrombin-induced barrier disruption.
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Affiliation(s)
- Mayumi Hirano
- Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, Miki-cho, Kita-Gun, Kagawa, Japan
| | - Katsuya Hirano
- Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, Miki-cho, Kita-Gun, Kagawa, Japan.
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2
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Kamiya T, Omae T, Nakabayashi S, Takahashi K, Tanner A, Yoshida A. Effect of Rho Kinase Inhibitor Ripasudil (K-115) on Isolated Porcine Retinal Arterioles. J Ocul Pharmacol Ther 2020; 37:104-111. [PMID: 33351704 DOI: 10.1089/jop.2020.0082] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Purpose: To investigate the vasorelaxation effect of ripasudil (K-115), a novel Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, on isolated retinal arterioles. We determined whether the actions of ripasudil on the retinal microvascular diameter were dependent on the endothelium and/or potassium channels in the smooth muscle, with the goals of uncovering the signaling mechanisms required for this vasomotor activity and inhibiting the action of endothelin-1 (ET-1). Methods: In this in vitro study, we isolated porcine retinal arterioles, which were cannulated and pressurized without flow. We recorded diametric changes using videomicroscopic techniques. Results: In a dose-dependent (10 nM-30 μM) manner, retinal arterioles were relaxed in response to ripasudil [maximum % resting diameter, 160.3% ± 7.7% (mean ± standard error of the mean)]. The ripasudil-induced vasorelaxation was unaffected by endothelium removal, using nonselective potassium channel blocker tetraethylammonium, Ca2+-activated large-conductance potassium channel blocker iberiotoxin, voltage-gated potassium channel blocker 4-AP, ATP-sensitive potassium channel blocker glibenclamide, and inward rectifier potassium channel blocker BaCl2. Ripasudil prevented ET-1-caused vasoconstriction of the retinal arterioles regardless of the presence of endothelium to a similar extent. Conclusion: The ROCK inhibitor ripasudil elicits endothelium-independent relaxation and inhibits the action of ET-1 on the retinal arterioles. Determining the relaxation properties of ripasudil on the retinal microvasculature will likely support the development of potential therapies for glaucoma.
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Affiliation(s)
- Takayuki Kamiya
- Department of Ophthalmology, Asahikawa Medical University, Asahikawa, Japan
| | - Tsuneaki Omae
- Department of Ophthalmology, Asahikawa Medical University, Asahikawa, Japan
| | - Seigo Nakabayashi
- Department of Ophthalmology, Asahikawa Medical University, Asahikawa, Japan
| | - Kengo Takahashi
- Department of Ophthalmology, Asahikawa Medical University, Asahikawa, Japan
| | - Akira Tanner
- Department of Ophthalmology, Asahikawa Medical University, Asahikawa, Japan
| | - Akitoshi Yoshida
- Department of Ophthalmology, Asahikawa Medical University, Asahikawa, Japan
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3
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NADPH oxidases and oxidase crosstalk in cardiovascular diseases: novel therapeutic targets. Nat Rev Cardiol 2019; 17:170-194. [PMID: 31591535 DOI: 10.1038/s41569-019-0260-8] [Citation(s) in RCA: 370] [Impact Index Per Article: 61.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 08/19/2019] [Indexed: 02/07/2023]
Abstract
Reactive oxygen species (ROS)-dependent production of ROS underlies sustained oxidative stress, which has been implicated in the pathogenesis of cardiovascular diseases such as hypertension, aortic aneurysm, hypercholesterolaemia, atherosclerosis, diabetic vascular complications, cardiac ischaemia-reperfusion injury, myocardial infarction, heart failure and cardiac arrhythmias. Interactions between different oxidases or oxidase systems have been intensively investigated for their roles in inducing sustained oxidative stress. In this Review, we discuss the latest data on the pathobiology of each oxidase component, the complex crosstalk between different oxidase components and the consequences of this crosstalk in mediating cardiovascular disease processes, focusing on the central role of particular NADPH oxidase (NOX) isoforms that are activated in specific cardiovascular diseases. An improved understanding of these mechanisms might facilitate the development of novel therapeutic agents targeting these oxidase systems and their interactions, which could be effective in the prevention and treatment of cardiovascular disorders.
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Saccà SC, Corazza P, Gandolfi S, Ferrari D, Sukkar S, Iorio EL, Traverso CE. Substances of Interest That Support Glaucoma Therapy. Nutrients 2019; 11:E239. [PMID: 30678262 PMCID: PMC6412416 DOI: 10.3390/nu11020239] [Citation(s) in RCA: 31] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2018] [Revised: 01/11/2019] [Accepted: 01/15/2019] [Indexed: 12/14/2022] Open
Abstract
Glaucoma is a multifactorial disease in which pro-apoptotic signals are directed to retinal ganglion cells. During this disease the conventional outflow pathway becomes malfunctioning. Aqueous humour builds up in the anterior chamber, leading to increased intraocular pressure. Both of these events are related to functional impairment. The knowledge of molecular mechanisms allows us to better understand the usefulness of substances that can support anti-glaucoma therapy. The goal of glaucoma therapy is not simply to lower intraocular pressure; it should also be to facilitate the survival of retinal ganglion cells, as these constitute the real target tissue in this disease, in which the visual pathway is progressively compromised. Indeed, an endothelial dysfunction syndrome affecting the endothelial cells of the trabecular meshwork occurs in both normal-tension glaucoma and high-tension glaucoma. Some substances, such as polyunsaturated fatty acids, can counteract the damage due to the molecular mechanisms - whether ischemic, oxidative, inflammatory or other - that underlie the pathogenesis of glaucoma. In this review, we consider some molecules, such as polyphenols, that can contribute, not only theoretically, to neuroprotection but which are also able to counteract the metabolic pathways that lead to glaucomatous damage. Ginkgo biloba extract, for instance, improves the blood supply to peripheral districts, including the optic nerve and retina and exerts a neuro-protective action by inhibiting apoptosis. Polyunsaturated fatty acids can protect the endothelium and polyphenols exert an anti-inflammatory action through the down-regulation of cytokines such as TNF-α and IL-6. All these substances can aid anti-glaucoma therapy by providing metabolic support for the cells involved in glaucomatous injury. Indeed, it is known that the food we eat is able to change our gene expression.
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Affiliation(s)
- Sergio Claudio Saccà
- Ophthalmology Unit, Department of Head/Neck Pathologies, Policlinico San Martino Hospital, IRCCS Hospital-University San Martino, Viale Benedetto XV, 16132 Genoa, Italy.
| | - Paolo Corazza
- Eye Clinic, Department of Neuroscience and Sensory Organs, University of Genoa, Policlinico San Martino Hospital IRCCS Hospital-University San Martino, Viale Benedetto XV, 16132 Genoa, Italy.
| | - Stefano Gandolfi
- Ophthalmology Unit, Department of Biological, Biotechnological and Translational Sciences, University of Parma, 43121 Parma, Italy.
| | - Daniele Ferrari
- Ophthalmology Unit, Department of Head/Neck Pathologies, Policlinico San Martino Hospital, IRCCS Hospital-University San Martino, Viale Benedetto XV, 16132 Genoa, Italy.
| | - Samir Sukkar
- U.O. di Dietetica e Nutrizione Clinica, Policlinico San Martino Hospital IRCCS Hospital-University San Martino, 35122 Genoa, Italy.
| | - Eugenio Luigi Iorio
- International Observatory of Oxidative Stress, Via Paolo Grisignano 21, 84127 Salerno, Italy.
| | - Carlo Enrico Traverso
- Eye Clinic, Department of Neuroscience and Sensory Organs, University of Genoa, Policlinico San Martino Hospital IRCCS Hospital-University San Martino, Viale Benedetto XV, 16132 Genoa, Italy.
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Abstract
Many diseases are related to age, among these neurodegeneration is particularly important. Alzheimer's disease Parkinson's and Glaucoma have many common pathogenic events including oxidative damage, Mitochondrial dysfunction, endothelial alterations and changes in the visual field. These are well known in the case of glaucoma, less in the case of neurodegeneration of the brain. Many other molecular aspects are common, such as the role of endoplasmic reticulum autophagy and neuronal apoptosis while others have been neglected due to lack of space such as inflammatory cytokine or miRNA. Moreover, the loss of specific neuronal populations, the induction of similar mechanisms of cell injury and the deposition of protein aggregates in specific anatomical areas are very similar events between these diseases. Intracellular and/or extracellular accumulation of protein aggregates is a key feature of many neurodegenerative disorders. The existence of abnormal protein aggregates has been documented in the RGCs of glaucomatous patients such as the anomalous Tau protein or the β-amyloid accumulations. Intra-cell catabolic processes also appear to be common in both glaucoma and neurodegeneration. They also help us to understand how the basis between these diseases is common and how the visual aspects can be a serious problem for those who are affected.
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Affiliation(s)
- Sergio Claudio Saccà
- Department of Head/Neck Pathologies, St Martino Hospital, Ophthalmology Unit, Genoa, Italy.
| | - Carlo Alberto Cutolo
- Department of Neuroscience, Rehabilitation, Ophthalmology, Genetics and Maternal and Child Science, University of Genoa, Policlinico San Martino Hospital, Eye Clinic Genoa, Genoa, Italy
| | - Tommaso Rossi
- Department of Head/Neck Pathologies, St Martino Hospital, Ophthalmology Unit, Genoa, Italy
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Zhang B, Naik JS, Jernigan NL, Walker BR, Resta TC. Reduced membrane cholesterol after chronic hypoxia limits Orai1-mediated pulmonary endothelial Ca 2+ entry. Am J Physiol Heart Circ Physiol 2017; 314:H359-H369. [PMID: 29101179 DOI: 10.1152/ajpheart.00540.2017] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Endothelial dysfunction in chronic hypoxia (CH)-induced pulmonary hypertension is characterized by reduced store-operated Ca2+ entry (SOCE) and diminished Ca2+-dependent production of endothelium-derived vasodilators. We recently reported that SOCE in pulmonary arterial endothelial cells (PAECs) is tightly regulated by membrane cholesterol and that decreased membrane cholesterol is responsible for impaired SOCE after CH. However, the ion channels involved in cholesterol-sensitive SOCE are unknown. We hypothesized that cholesterol facilitates SOCE in PAECs through the interaction of Orai1 and stromal interaction molecule 1 (STIM1). The role of cholesterol in Orai1-mediated SOCE was initially assessed using CH exposure in rats (4 wk, 380 mmHg) as a physiological stimulus to decrease PAEC cholesterol. The effects of Orai1 inhibition with AnCoA4 on SOCE were examined in isolated PAEC sheets from control and CH rats after cholesterol supplementation, substitution of endogenous cholesterol with epicholesterol (Epichol), or vehicle treatment. Whereas cholesterol restored endothelial SOCE in CH rats, both Epichol and AnCoA4 attenuated SOCE only in normoxic controls. The Orai1 inhibitor had no further effect in cells pretreated with Epichol. Using cultured pulmonary endothelial cells to allow better mechanistic analysis of the molecular components of cholesterol-regulated SOCE, we found that Epichol, AnCoA4, and Orai1 siRNA each inhibited SOCE compared with their respective controls. Epichol had no additional effect after knockdown of Orai1. Furthermore, Epichol substitution significantly reduced STIM1-Orai1 interactions as assessed by a proximity ligation assay. We conclude that membrane cholesterol is required for the STIM1-Orai1 interaction necessary to elicit endothelial SOCE. Furthermore, reduced PAEC membrane cholesterol after CH limits Orai1-mediated SOCE. NEW & NOTEWORTHY This research demonstrates a novel contribution of cholesterol to regulate the interaction of Orai1 and stromal interaction molecule 1 required for pulmonary endothelial store-operated Ca2+ entry. The results provide a mechanistic basis for impaired pulmonary endothelial Ca2+ influx after chronic hypoxia that may contribute to pulmonary hypertension.
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Affiliation(s)
- Bojun Zhang
- Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center , Albuquerque, New Mexico
| | - Jay S Naik
- Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center , Albuquerque, New Mexico
| | - Nikki L Jernigan
- Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center , Albuquerque, New Mexico
| | - Benjimen R Walker
- Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center , Albuquerque, New Mexico
| | - Thomas C Resta
- Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center , Albuquerque, New Mexico
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Saccà SC, Gandolfi S, Bagnis A, Manni G, Damonte G, Traverso CE, Izzotti A. From DNA damage to functional changes of the trabecular meshwork in aging and glaucoma. Ageing Res Rev 2016; 29:26-41. [PMID: 27242026 DOI: 10.1016/j.arr.2016.05.012] [Citation(s) in RCA: 98] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2015] [Revised: 05/25/2016] [Accepted: 05/26/2016] [Indexed: 12/24/2022]
Abstract
Glaucoma is a degenerative disease of the eye. Both the anterior and posterior segments of the eye are affected, extensive damage being detectable in the trabecular meshwork and the inner retina-central visual pathway complex. Oxidative stress is claimed to be mainly responsible for molecular damage in the anterior chamber. Indeed, oxidation harms the trabecular meshwork, leading eventually to endothelial cell decay, tissue malfunction, subclinical inflammation, changes in the extracellular matrix and cytoskeleton, altered motility, reduced outflow facility and (ultimately) increased IOP. Moreover, free radicals are involved in aging and can be produced in the brain (as well as in the eye) as a result of ischemia, leading to oxidation of the surrounding neurons. Glaucoma-related cell death occurs by means of apoptosis, and apoptosis is triggered by oxidative stress via (a) mitochondrial damage, (b) inflammation, (c) endothelial dysregulation and dysfunction, and (d) hypoxia. The proteomics of the aqueous humor is significantly altered in glaucoma as a result of oxidation-induced trabecular damage. Those proteins whose aqueous humor levels are increased in glaucoma are biomarkers of trabecular meshwork impairment. Their diffusion from the anterior to the posterior segment of the eye may be relevant in the cascade of events triggering apoptosis in the inner retinal layers, including the ganglion cells.
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Affiliation(s)
- Sergio Claudio Saccà
- IRCCS San Martino University Hospital, Department of Neuroscience and Sense Organs, San Martino Hospital, Ophthalmology Unit, Viale Benedetto XV, 16132 Genoa, Italy.
| | - Stefano Gandolfi
- Ophthalmology Unit, Department of Biological, Biotechnological and Translational Sciences, University of Parma, Parma, Italy
| | - Alessandro Bagnis
- University of Genoa, Eye Clinic, Department of Neuroscience and Sense Organs, Viale Benedetto XV, 5, 16148 Genoa, Italy
| | - Gianluca Manni
- Dept. of Clinical Science and Translational Medicine, University Tor Vergata, Rome, Italy
| | - Gianluca Damonte
- Dept. of Experimental Medicine, Section of Biochemistry and Center of Excellence for Biomedical Research, University of Genoa, Viale Benedetto XV 1, 16132 Genoa, Italy
| | - Carlo Enrico Traverso
- University of Genoa, Eye Clinic, Department of Neuroscience and Sense Organs, Viale Benedetto XV, 5, 16148 Genoa, Italy
| | - Alberto Izzotti
- Mutagenesis Unit, IRCCS San Martino University Hospital, IST National Institute for Cancer Research, Department of Health Sciences, University of Genoa, Via A. Pastore 1, Genoa I-16132, Italy
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8
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Abstract
In past decades, growing evidence from basic and clinical researches reveal that small guanosine triphosphate binding protein ras homolog gene family, member A (RhoA) and its main effector Rho-associated kinase (ROCK) play central and complex roles in cardiovascular systems, and increasing RhoA and ROCK activity is associated with a broad range of cardiovascular diseases such as congestive heart failure, atherosclerosis, and hypertension. Favorable outcomes have been observed with ROCK inhibitors treatment. In this review, we briefly summarize the pathophysiological roles of RhoA/ROCK signaling pathway on cardiovascular system, displaying the potential benefits in the cardiovascular system with controlling RhoA/ROCK signaling pathway.
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9
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Abstract
Vascular endothelial growth factor (VEGF) plays a fundamental role in angiogenesis and endothelial cell biology, and has been the subject of intense study as a result. VEGF acts via a diverse and complex range of signaling pathways, with new targets constantly being discovered. This review attempts to summarize the current state of knowledge regarding VEGF cell signaling in endothelial and cardiovascular biology, with a particular emphasis on its role in angiogenesis.
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Affiliation(s)
- Ian Evans
- Centre for Cardiovascular Biology and Medicine, Division of Medicine, University College London, Rayne Building, 5 University Street, London, WC1E 6JF, UK,
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10
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Mu ZH, Jiang Z, Lin XJ, Wang LP, Xi Y, Zhang ZJ, Wang YT, Yang GY. Vessel Dilation Attenuates Endothelial Dysfunction Following Middle Cerebral Artery Occlusion in Hyperglycemic Rats. CNS Neurosci Ther 2016; 22:316-24. [PMID: 26842484 DOI: 10.1111/cns.12500] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2015] [Revised: 11/26/2015] [Accepted: 11/27/2015] [Indexed: 12/26/2022] Open
Abstract
OBJECTIVES Dynamically observe cerebral vascular changes in hyperglycemic rats in vivo and explore the effect of diabetes on endothelial function after ischemic stroke. BACKGROUND Diabetes affects both large and small vessels in the brain, but the dynamic process and mechanism are unclear. METHODS We investigated the structural and functional changes of brain vasculature in living hyperglycemic rats and their impact on stroke outcomes via a novel technique: synchrotron radiation angiography. We also examined the effect of prolonged fasudil treatment on arterial reactivity and hemorrhagic transformation. Adult Sprague Dawley rats were treated by streptozotocin to induce type 1 diabetes. These hyperglycemic rats received fasudil pretreatment and then underwent transient middle cerebral artery occlusion. RESULTS We found that diabetes caused arteries narrowing in the circus Willis as early as 2 weeks after streptozotocin injection (P < 0.05). These vessels were further constricted after middle cerebral artery occlusion. L-NAME could induce regional constrictions and impaired relaxation in hyperglycemic animals. Furthermore, hemorrhagic transformation was also increased in the hyperglycemic rats compared to the control (P < 0.05). In fasudil-treated rats, the internal carotid artery narrowing was ameliorated and L-NAME-induced regional constriction was abolished. Importantly, stroke prognosis was improved in fasudil-treated rats compared to the control (P < 0.05). CONCLUSIONS Our dynamic angiographic data demonstrated that diabetes could impair the cerebral arterial reactivity. Prolonged fasudil treatment could attenuate arterial dysfunction and improve the prognosis of ischemic stroke by affecting both the large and small vasculature.
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Affiliation(s)
- Zhi-Hao Mu
- Department of Neurology, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Zhen Jiang
- Department of Neurology, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Xiao-Jie Lin
- Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China
| | - Li-Ping Wang
- Department of Neurology, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Yan Xi
- Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China.,Center for Biotechnology and Interdisciplinary Studies, Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA
| | - Zhi-Jun Zhang
- Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China
| | - Yong-Ting Wang
- Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China
| | - Guo-Yuan Yang
- Department of Neurology, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.,Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China
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Tillery LC, Epperson TA, Eguchi S, Motley ED. Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells. Exp Biol Med (Maywood) 2016; 241:569-80. [PMID: 26729042 DOI: 10.1177/1535370215622584] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2015] [Accepted: 11/06/2015] [Indexed: 11/17/2022] Open
Abstract
Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632, suggesting protease-activated receptor coupling to Gq and G12/13, respectively. These data suggest a vascular bed specific differential coupling of protease-activated receptors to the signaling pathways that regulate endothelial nitric oxide synthase and nitric oxide production that may be responsible for endothelial dysfunction associated with cardiovascular disease.
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Affiliation(s)
- Lakeisha C Tillery
- Department of Microbiology and Immunology, Meharry Medical College, Nashville, TN 37208, USA
| | - Tenille A Epperson
- Department of Physiology, School of Medicine, Meharry Medical College, Nashville, TN 37208, USA
| | - Satoru Eguchi
- Department of Physiology, Temple University School of Medicine, Philadelphia, PA 19140, USA
| | - Evangeline D Motley
- Department of Physiology, School of Medicine, Meharry Medical College, Nashville, TN 37208, USA
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12
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Saccà SC, Pulliero A, Izzotti A. The Dysfunction of the Trabecular Meshwork During Glaucoma Course. J Cell Physiol 2014; 230:510-25. [DOI: 10.1002/jcp.24826] [Citation(s) in RCA: 69] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2014] [Accepted: 09/05/2014] [Indexed: 12/22/2022]
Affiliation(s)
- Sergio Claudio Saccà
- Department of Head/Neck Pathologies; St Martino Hospital; Ophthalmology Unit; Genoa Italy
| | - Alessandra Pulliero
- Department of Health Sciences; Section of Hygiene and Preventive Medicine; University of Genoa; Genoa Italy
| | - Alberto Izzotti
- Department of Health Sciences; Section of Hygiene and Preventive Medicine; University of Genoa; Genoa Italy
- Mutagenesis Unit; IST National Institute for Cancer Research; IRCCS Hospital-University San Martino Company; Genoa Italy
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13
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WEI LAI, YANG JUAN, WANG MIN, XU SHENGNAN, LIANG HUAMIN, ZHOU QI. Sodium ferulate lowers portal pressure in rats with secondary biliary cirrhosis through the RhoA/Rho-kinase signaling pathway: a preliminary study. Int J Mol Med 2014; 34:1257-67. [PMID: 25174394 PMCID: PMC4199412 DOI: 10.3892/ijmm.2014.1905] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2014] [Accepted: 07/25/2014] [Indexed: 12/12/2022] Open
Abstract
Cirrhotic rats show higher expression levels of hepatic RhoA and Rho-kinase than normal healthy rats, and the activation of this signaling pathway leads to portal hypertension. Sodium ferulate (SF) has been shown to decrease the production of geranylgeranyl pyrophosphate (GGPP), a substance essential for RhoA activation. In the present study, to investigate the effects of SF on fibrosis, portal hypertension and the RhoA/Rho-kinase pathway, hepatic cirrhosis was induced in rats by bile duct ligation. Liver function and fibrogenesis-related biochemical parameters, the hepatic hydroxyproline content, the pathological characteristics of the liver sections and the levels of hepatic α-smooth muscle actin (α-SMA; by immunohistochemistry) were analyzed to assess effects of SF on hepatic fibrosis. In addition, hepatic RhoA, Rho-kinase and endothelial nitric oxide synthase (eNOS) expression was examined by immunohistochemistry. Apoptosis in the SF-treated and SF + GGPP-treated rat primary hepatic stellate cells (HSCs) and a human stellate cell line (LX-2) was examined by flow cytometry. Intrahepatic resistance and responsiveness to the α1-adrenoceptor agonist, methoxamine, were investigated by in situ liver perfusion. Treatment with SF did not affect fibrosis-related biochemical parameters or the hydroxyproline content; however, SF reduced the histological evidence of fibrosis and hepatocyte damage. The SF-treated rats had a significantly lower expression of α-SMA and Rho-kinase, as well as an increased hepatic eNOS content; however, SF did not affect RhoA expression. The SF-treated HSCs had a significantly increased apoptotic rate compared to the untreated rats. Following the addition of GGPP, the rate apoptotic rate decreased. SF reduced basal intrahepatic resistance and the responsiveness of hepatic vascular smooth muscle to methoxamine. Therefore, our data demonstrate that SF reduces fibrogenesis, decreases portal pressure in cirrhotic rats and inhibits the activation of the RhoA/Rho-kinase signaling pathway.
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Affiliation(s)
- LAI WEI
- Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, P.R. China
| | - JUAN YANG
- Department of Digestive Diseases, Chengdu First People’s Hospital, Chengdu, Sichuan, P.R. China
| | - MIN WANG
- Department of Emergency Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, P.R. China
| | - SHENG-NAN XU
- Department of Digestive Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, P.R. China
| | - HUA-MIN LIANG
- Department of Physiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, P.R. China
| | - QI ZHOU
- Department of Digestive Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, P.R. China
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14
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Liu Y, Huang C, Ceng C, Zhan H, Zheng D, Han W. Metformin enhances nitric oxide production and diminishes Rho kinase activity in rats with hyperlipidemia. Lipids Health Dis 2014; 13:115. [PMID: 25028180 PMCID: PMC4109376 DOI: 10.1186/1476-511x-13-115] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2014] [Accepted: 06/18/2014] [Indexed: 12/30/2022] Open
Abstract
BACKGROUND Rho kinase over-activation is associated with nitric oxide (NO) reduction and atherosclerosis. Metformin is favorable for endothelial function improvement and cardiovascular outcomes. Whether cardio-protective effect of metformin is associated with Rho kinase activity is unknown. METHODS Hyperlipidemia model of rats were established accordingly. Thereafter, medical interventions in terms of atorvastatin, metformin or combined therapy were administered for 4 weeks. Laboratory parameters were compared among each groups at initial, 6 weeks of high-fat and high-cholesterol diet administration, and 4 weeks of medical intervention. Lineal regression analyses were performed. RESULTS No significant difference of laboratory parameters was observed initially. Six weeks of high-fat and high-cholesterol diet administration, serum levels of cholesterol, C-reactive protein (CRP) level, and Rho kinase activity were significantly increased while NO production was concomitantly reduced in comparison to the sham group. After 4 weeks of medical intervention, CRP level and Rho kinase activity were profoundly diminished while NO production was significantly enhanced in the atorvastatin and metformin groups, and these benefits were further enhanced with combined therapy. Lineal regression analyses showed that Rho kinase activity was negatively correlated with NO production but positively correlated with CRP level. CONCLUSION In rats with hyperlipidemia, metformin and atorvastatin therapy is favorable for NO production and CRP reduction, which might be associated with Rho kinase activity decrease.
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Affiliation(s)
- Yan Liu
- The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui Province, China
- The Third people's hospital of Huizhou, Huizhou, Guangdong Province, China
| | - Congwu Huang
- The Department of Internal Medicine, the Second Affiliated Hospital of Shantou University, Shantou, Guangdong Province, China
| | - Chuan Ceng
- The Department of Internal Medicine, the Second Affiliated Hospital of Shantou University, Shantou, Guangdong Province, China
| | - Haiyong Zhan
- The Department of Internal Medicine, the Second Affiliated Hospital of Shantou University, Shantou, Guangdong Province, China
| | - Dongdan Zheng
- The Department of Cardiology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China
| | - Weixing Han
- The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui Province, China
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15
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Sawada N, Liao JK. Rho/Rho-associated coiled-coil forming kinase pathway as therapeutic targets for statins in atherosclerosis. Antioxid Redox Signal 2014; 20:1251-67. [PMID: 23919640 PMCID: PMC3934442 DOI: 10.1089/ars.2013.5524] [Citation(s) in RCA: 66] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
SIGNIFICANCE The 3-hydroxy-methylglutaryl coenzyme A reductase inhibitors or statins are important therapeutic agents for lowering serum cholesterol levels. However, recent studies suggest that statins may exert atheroprotective effects beyond cholesterol lowering. These so-called "pleiotropic effects" include effects of statins on vascular and inflammatory cells. Thus, it is important to understand whether other signaling pathways that are involved in atherosclerosis could be targets of statins, and if so, whether individuals with "overactivity" of these pathways could benefit from statin therapy, regardless of serum cholesterol level. RECENT ADVANCES Statins inhibit the synthesis of isoprenoids, which are important for the function of the Rho/Rho-associated coiled-coil containing kinase (ROCK) pathway. Indeed, recent studies suggest that inhibition of the Rho/ROCK pathway by statins could lead to improved endothelial function and decreased vascular inflammation and atherosclerosis. Thus, the Rho/ROCK pathway has emerged as an important target of statin therapy for reducing atherosclerosis and possibly cardiovascular disease. CRITICAL ISSUES Because atherosclerosis is both a lipid and an inflammatory disease, it is important to understand how inhibition of Rho/ROCK pathway could contribute to statins' antiatherosclerotic effects. FUTURE DIRECTIONS The role of ROCKs (ROCK1 and ROCK2) in endothelial, smooth muscle, and inflammatory cells needs to be determined in the context of atherogenesis. This could lead to the development of specific ROCK1 or ROCK2 inhibitors, which could have greater therapeutic benefits with less toxicity. Also, clinical trials will need to be performed to determine whether inhibition of ROCKs, with and without statins, could lead to further reduction in atherosclerosis and cardiovascular disease.
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Affiliation(s)
- Naoki Sawada
- 1 GCOE Program and Department of Molecular Endocrinology and Metabolism, Tokyo Medical and Dental University , Tokyo, Japan
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16
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Abstract
Renal afferent arterioles (AFF) regulate glomerular capillary pressure through two main mechanisms: the myogenic response (MYO) and tubuloglomerular feedback (TGF). Because Rho-kinase and nitric oxide synthase (NOS) are established factors that modulate vascular tone, we examined the role of these factors in pressure-induced AFF tone in Wistar-Kyoto rats and in spontaneously hypertensive rats (SHR) using an intravital CCD camera. Elevated renal perfusion pressure elicited marked AFF constriction that was partially inhibited by gadolinium, furosemide and fasudil, which inhibit MYO, TGF and Rho-kinase, respectively; however, this AFF constriction was completely blocked by combined treatment with fasudil+gadolinium or fasudil+furosemide. S-methyl-L-thiocitrulline (SMTC) partially reversed the fasudil-induced inhibition of TGF-mediated, but not that of MYO-mediated, AFF constriction. In SHR, the pressure-induced AFF response was enhanced, and MYO- and TGF-induced constriction were exaggerated. In the presence of gadolinium, SMTC partially mitigated the fasudil-induced inhibition of TGF-mediated AFF constriction. Immunoblot analyses demonstrated that both Rho-kinase activity and neuronal NOS were augmented in SHR kidneys. In conclusion, Rho-kinase contributes to MYO- and TGF-mediated AFF responses, and these responses are enhanced in SHR. Furthermore, neuronal NOS-induced nitric oxide modulates the TGF mechanism. This mechanism constitutes a target for Rho-kinase in TGF-mediated AFF constriction.
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17
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Targeting of Rho kinase ameliorates impairment of diabetic endothelial function in intrarenal artery. Int J Mol Sci 2013; 14:20282-98. [PMID: 24129169 PMCID: PMC3821615 DOI: 10.3390/ijms141020282] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2013] [Revised: 07/11/2013] [Accepted: 09/09/2013] [Indexed: 02/04/2023] Open
Abstract
Endothelial dysfunction in kidney vasculature is the initial and key element for nephropathy in diabetes mellitus. Accumulating evidence suggests the protective role of Rho kinase inhibitors in endothelial dysfunction via modulating eNOS activity and NO production. However, the role of Rho kinase in diabetes-related endothelial dysfunction in kidney vasculature and the relevant mechanisms remain unknown. We assessed whether pharmacological inhibition of Rho kinase attenuates endothelial dysfunction in intrarenal arteries from type 1 diabetic rats. Fasudil, a Rho kinase inhibitor effectively decreased the phosphorylated level of MYPT1 without affecting the expression of ROCKs in the kidney. Fasudil treatment showed no improvement in diabetes-related abnormality in metabolic indices, but it significantly ameliorated endothelial dysfunction in intrarenal arteries and lessened the mesangial matrix expansion in the kidney cortex. Mechanistically, superoxide production in the intrarenal artery and NOX4 member of NADPH oxidase in the renal cortex that contribute to diabetic nephropathy were also prevented by the Rho kinase inhibitor. In conclusion, the present results indicate that Rho kinase is involved in endothelial dysfunction in type 1 diabetes via enhancement of oxidative stress and provides new evidence for Rho kinase inhibitors as potential therapeutic agents for the treatment of diabetic nephropathy.
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18
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The vasorelaxant mechanisms of a Rho kinase inhibitor DL0805 in rat thoracic aorta. Molecules 2012; 17:5935-44. [PMID: 22609784 PMCID: PMC6268074 DOI: 10.3390/molecules17055935] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2012] [Revised: 05/11/2012] [Accepted: 05/11/2012] [Indexed: 11/21/2022] Open
Abstract
Rho-kinase has been suggested as a potential therapeutic target in the treatment of cardiovascular diseases. The Rho-kinase signaling pathway is substantially involved in vascular contraction. The aim of the present study was to evaluate the vasorelaxant effects of Rho kinase inhibitor DL0805 in isolated rat aortic rings and to investigate its possible mechanism(s). It was found that DL0805 exerted vasorelaxation in a dose-dependent manner in NE or KCl-induced sustained contraction and partial loss of the vasorelaxation under endothelium-denuded rings. The DL0805-induced vasorelaxation was significantly reduced by the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester, the guanylate cyclase inhibitor methylene blue and the cyclooxygenase inhibitor indomethacin. The voltage-dependent K+ channel blocker 4-aminopyridine remarkably attenuated DL0805-induced relaxations. However, the ATP-sensitive K+ channel blocker glibenclamide and Ca2+-activated K+ channel blocker tetraethylammonium did not affect the DL0805-induced relaxation. In the endothelium-denuded rings, DL0805 also reduced NE-induced transient contraction and inhibited contraction induced by increasing external calcium. These findings suggested that DL0805 is a novel vasorelaxant compound associated with inhibition of Rho/ROCK signaling pathway. The NO-cGMP pathway may be involved in the relaxation of DL0805 in endothelium-intact aorta. The vasorelaxant effect of DL0805 is partially mediated by the opening of the voltage-dependent K+ channels.
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Lamers ML, Almeida MES, Vicente-Manzanares M, Horwitz AF, Santos MF. High glucose-mediated oxidative stress impairs cell migration. PLoS One 2011; 6:e22865. [PMID: 21826213 PMCID: PMC3149607 DOI: 10.1371/journal.pone.0022865] [Citation(s) in RCA: 108] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2011] [Accepted: 06/30/2011] [Indexed: 11/18/2022] Open
Abstract
Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we evaluate the hypothesis that high glucose concentrations inhibit cell migration. Using CHO.K1 cells, NIH-3T3 fibroblasts, mouse embryonic fibroblasts and primary skin fibroblasts from control and diabetic rats cultured in 5 mM D-glucose (low glucose, LG), 25 mM D-glucose (high glucose, HG) or 25 mM L-glucose medium (osmotic control - OC), we analyzed the migration speed, protrusion stability, cell polarity, adhesion maturation and the activity of the small Rho GTPase Rac1. We also analyzed the effects of reactive oxygen species by incubating cells with the antioxidant N-Acetyl-Cysteine (NAC). We observed that HG conditions inhibited cell migration when compared to LG or OC. This inhibition resulted from impaired cell polarity, protrusion destabilization and inhibition of adhesion maturation. Conversely, Rac1 activity, which promotes protrusion and blocks adhesion maturation, was increased in HG conditions, thus providing a mechanistic basis for the HG phenotype. Most of the HG effects were partially or completely rescued by treatment with NAC. These findings demonstrate that HG impairs cell migration due to an increase in oxidative stress that causes polarity loss, deficient adhesion and protrusion. These alterations arise, in large part, from increased Rac1 activity and may contribute to the poor wound healing observed in diabetic patients.
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Affiliation(s)
- Marcelo L. Lamers
- Department of Morphological Sciences, Institute of Basic Health Science, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
- Department of Cell and Developmental Biology, Biomedical Sciences Institute, University of São Paulo, São Paulo, São Paulo, Brazil
| | - Maíra E. S. Almeida
- Department of Cell and Developmental Biology, Biomedical Sciences Institute, University of São Paulo, São Paulo, São Paulo, Brazil
| | - Miguel Vicente-Manzanares
- Department of Cell Biology, University of Virginia, Charlottesville, Virginia, United States of America
| | - Alan F. Horwitz
- Department of Cell Biology, University of Virginia, Charlottesville, Virginia, United States of America
| | - Marinilce F. Santos
- Department of Cell and Developmental Biology, Biomedical Sciences Institute, University of São Paulo, São Paulo, São Paulo, Brazil
- * E-mail:
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20
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Yang G, Lucas R, Caldwell R, Yao L, Romero MJ, Caldwell RW. Novel mechanisms of endothelial dysfunction in diabetes. J Cardiovasc Dis Res 2011; 1:59-63. [PMID: 20877687 PMCID: PMC2945199 DOI: 10.4103/0975-3583.64432] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Diabetes mellitus is a major risk factor for cardiovascular morbidity and mortality. This condition increases the risk of developing coronary, cerebrovascular, and peripheral arterial disease fourfold. Endothelial dysfunction is a major contributor to the pathogenesis of vascular disease in diabetes mellitus patients and has recently received increased attention. In this review article, some recent developments that could improve the knowledge of diabetes-induced endothelial dysfunction are discussed.
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Affiliation(s)
- Guang Yang
- Department of Vascular Biology Center, Medical College of Georgia, Augusta, GA, USA
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21
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Shatanawi A, Romero MJ, Iddings JA, Chandra S, Umapathy NS, Verin AD, Caldwell RB, Caldwell RW. Angiotensin II-induced vascular endothelial dysfunction through RhoA/Rho kinase/p38 mitogen-activated protein kinase/arginase pathway. Am J Physiol Cell Physiol 2011; 300:C1181-92. [PMID: 21289285 DOI: 10.1152/ajpcell.00328.2010] [Citation(s) in RCA: 111] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Enhanced vascular arginase activity impairs endothelium-dependent vasorelaxation by decreasing l-arginine availability to endothelial nitric oxide (NO) synthase, thereby reducing NO production. Elevated angiotensin II (ANG II) is a key component of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. We determined signaling mechanisms by which ANG II increases endothelial arginase function. Results show that ANG II (0.1 μM, 24 h) elevates arginase activity and arginase I expression in bovine aortic endothelial cells (BAECs) and decreases NO production. These effects are prevented by the arginase inhibitor BEC (100 μM). Blockade of ANG II AT(1) receptors or transfection with small interfering RNA (siRNA) for Gα12 and Gα13 also prevents ANG II-induced elevation of arginase activity, but siRNA for Gαq does not. ANG II also elevates active RhoA levels and induces phosphorylation of p38 MAPK. Inhibitors of RhoA activation (simvastatin, 0.1 μM) or Rho kinase (ROCK) (Y-27632, 10 μM; H1152, 0.5 μM) block both ANG II-induced elevation of arginase activity and phosphorylation of p38 MAPK. Furthermore, pretreatment of BAECs with p38 inhibitor SB-202190 (2 μM) or transfection with p38 MAPK siRNA prevents ANG II-induced increased arginase activity/expression and maintains NO production. Additionally, inhibitors of p38 MAPK (SB-203580, 5 μg·kg(-1)·day(-1)) or arginase (ABH, 8 mg·kg(-1)·day(-1)) or arginase gene knockout in mice prevents ANG II-induced vascular endothelial dysfunction and associated enhancement of arginase. These results indicate that ANG II increases endothelial arginase activity/expression through Gα12/13 G proteins coupled to AT(1) receptors and subsequent activation of RhoA/ROCK/p38 MAPK pathways leading to endothelial dysfunction.
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Affiliation(s)
- Alia Shatanawi
- Dept. of Pharmacology and Toxicology, Georgia Health Sciences University, Augusta, GA 30912, USA
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22
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Chen GP, Li L, Yang Y, Fu M, Yao L, Wu T, Zhang XQ, Hu SJ. Chronic inhibition of farnesyl pyrophosphate synthase improves endothelial function in spontaneously hypertensive rats. Biochem Pharmacol 2010; 80:1684-9. [DOI: 10.1016/j.bcp.2010.08.015] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2010] [Revised: 08/18/2010] [Accepted: 08/19/2010] [Indexed: 10/19/2022]
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23
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Atorvastatin Protects Against Angiotensin II–induced Injury and Dysfunction in Human Umbilical Vein Endothelial Cells Through Bradykinin 2 Receptors. J Cardiovasc Pharmacol 2010; 56:171-6. [DOI: 10.1097/fjc.0b013e3181e5f2e2] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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24
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Wirth A. Rho kinase and hypertension. Biochim Biophys Acta Mol Basis Dis 2010; 1802:1276-84. [PMID: 20460153 DOI: 10.1016/j.bbadis.2010.05.002] [Citation(s) in RCA: 98] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2010] [Revised: 04/16/2010] [Accepted: 05/01/2010] [Indexed: 11/26/2022]
Abstract
Arterial hypertension is a multifactorial disease that is characterised by increased peripheral vascular resistance often accompanied by smooth muscle cell hypertrophy and proliferation. Rho kinases (ROCKs) are the most extensively studied effectors of the small G-protein RhoA and abnormalities in RhoA/ROCK signalling have been observed in various cardiovascular disease including hypertension. The RhoA/ROCK-pathway is a key player in different smooth muscle cell functions including contractility, proliferation and migration. Furthermore, there is extensive crosstalk between RhoA/ROCK- and NO-signalling. Therefore, not only ROCK inhibitors but also NO-donators or pleiotropic agents like statins exert their beneficial effects on the cardiovascular system at least in part via Rho/Rho-kinase.
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Affiliation(s)
- Angela Wirth
- Max-Planck-Institute for Heart and Lung Research, Dept. of Pharmacology, Ludwigstraße 43, 61231 Bad Nauheim, Germany.
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25
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Vascular dysfunction in cerebrovascular disease: mechanisms and therapeutic intervention. Clin Sci (Lond) 2010; 119:1-17. [PMID: 20370718 DOI: 10.1042/cs20090649] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The endothelium plays a crucial role in the control of vascular homoeostasis through maintaining the synthesis of the vasoprotective molecule NO* (nitric oxide). Endothelial dysfunction of cerebral blood vessels, manifested as diminished NO* bioavailability, is a common feature of several vascular-related diseases, including hypertension, hypercholesterolaemia, stroke, subarachnoid haemorrhage and Alzheimer's disease. Over the past several years an enormous amount of research has been devoted to understanding the mechanisms underlying endothelial dysfunction. As such, it has become apparent that, although the diseases associated with impaired NO* function are diverse, the underlying causes are similar. For example, compelling evidence indicates that oxidative stress might be an important mechanism of diminished NO* signalling in diverse models of cardiovascular 'high-risk' states and cerebrovascular disease. Although there are several sources of vascular ROS (reactive oxygen species), the enzyme NADPH oxidase is emerging as a strong candidate for the excessive ROS production that is thought to lead to vascular oxidative stress. The purpose of the present review is to outline some of the mechanisms thought to contribute to endothelial dysfunction in the cerebral vasculature during disease. More specifically, we will highlight current evidence for the involvement of ROS, inflammation, the RhoA/Rho-kinase pathway and amyloid beta-peptides. In addition, we will discuss currently available therapies for improving endothelial function and highlight future therapeutic strategies.
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26
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Chiba Y, Sato S, Misawa M. GGTI-2133, an inhibitor of geranylgeranyltransferase, inhibits infiltration of inflammatory cells into airways in mouse experimental asthma. Int J Immunopathol Pharmacol 2010; 22:929-35. [PMID: 20074456 DOI: 10.1177/039463200902200408] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Statins have been proposed as a novel treatment of respiratory diseases including asthma. Although the mechanism of anti-inflammatory effect of statins is still unclear, an inhibition of protein prenylation by depleting the downstream metabolites of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase might be involved. To test the hypothesis, the effects of GGTI-2133, a direct inhibitor of geran ylgeranyltransferase (GGTase), on antigen-induced airway inflammation were investigated in a murine model of allergic bronchial asthma. Mice were sensitized and repeatedly challenged with ovalbumin antigen (OA). Animals were also treated with GGTI-2133 (5 mg/kg/day, i.p.) once a day before and during the antigen inhalation period. Repeated antigen inhalation caused an infiltration of inflammatory cells, especially eosinophils, into airways. Significant increases in interleukin (IL)-4, IL-13, eotaxin, thymus and activation-regulated chemokine (TARC) and leukotriene B4 (LTB4) in bronchoalveolar lavage fluids and total and OA-specific IgE in sera were also found in the antigen-exposed animals. The systemic treatments with GGTI-2133 inhibited the antigen-induced eosinophil infiltration into airways almost completely. However, interestingly, the GGTI-2133 treatment did not affect the levels of these chemotactic factors and IgE. These findings suggest that selective inhibition of GGTase is effective for eosinophilic airway inflammation such as asthma.
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Affiliation(s)
- Y Chiba
- Department of Pharmacology, School of Pharmacy, Hoshi University, Tokyo, Japan.
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27
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Liu CQ, Wong SL, Leung FP, Tian XY, Lau CW, Lu L, Yao X, Chen ZY, Yao T, Huang Y. Prostanoid TP receptor-mediated impairment of cyclic AMP-dependent vasorelaxation is reversed by phosphodiesterase inhibitors. Eur J Pharmacol 2010; 632:45-51. [PMID: 20096281 DOI: 10.1016/j.ejphar.2010.01.008] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2009] [Revised: 12/18/2009] [Accepted: 01/12/2010] [Indexed: 11/18/2022]
Abstract
Activation of the thromboxane prostanoid (TP) receptor produces potent vasoconstriction, which contributes to the increased vascular tone and blood pressure. The present study was designed to examine the hypothesis that stimulation of prostanoid TP receptors impairs endothelium-independent relaxations to cyclic AMP-elevating agents via increasing the activity of phosphodiesterases (PDEs). Rat carotid arteries without endothelium were isolated and suspended in myograph for the measurement of changes in isometric tension; the tissue content of cyclic AMP was assayed by enzyme immunoassay kit; and prostanoid TP receptor was detected in vascular wall by immunohistochemistry and Western blot. In phenylephrine-contracted rings without endothelium, relaxations induced by isoprenaline (receptor-mediated) and forskolin (receptor-independent) were markedly reduced by the presence of a prostanoid TP receptor agonist, U46619; the attenuated relaxations were prevented by acute treatment with S18886, the selective prostanoid TP receptor antagonist, but not by protein kinase C inhibitors. The reduced relaxations were partially restored by IBMX (non-selective PDE inhibitor), cilostazol (PDE3 inhibitor), rolipram (PDE4 inhibitor) or by Y27632 (Rho kinase inhibitor), but not by T0156 (PDE5 inhibitor). U46619 diminished isoprenaline- or forskolin-stimulated rise in cyclic AMP and this effect was inhibited by cilostazol, rolipram or Y27632. The present results suggest that activation of prostanoid TP receptors impairs cyclic AMP-dependent vasorelaxations partly via PDE- and RhoA/Rho kinase-dependent mechanisms. Inhibitors of PDEs and Rho kinase may be useful in the treatment of cardiovascular complications.
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Affiliation(s)
- Cui Qing Liu
- Department of Physiology, Hangzhou Normal University, China
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28
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Chiba Y, Sato S, Hanazaki M, Sakai H, Misawa M. Inhibition of geranylgeranyltransferase inhibits bronchial smooth muscle hyperresponsiveness in mice. Am J Physiol Lung Cell Mol Physiol 2009; 297:L984-91. [PMID: 19717551 DOI: 10.1152/ajplung.00178.2009] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Recent studies revealed an involvement of RhoA/Rho-kinase in the contraction of bronchial smooth muscle (BSM), and this pathway has now been proposed as a new target for asthma therapy. A posttranslational geranylgeranylation of RhoA is required for its activation. Thus selective inhibition of geranylgeranyltransferase may be a novel strategy for treatment of the BSM hyperresponsiveness in asthmatics. To test this hypothesis, we investigated the effect of a geranylgeranyltransferase inhibitor, GGTI-2133, on antigen-induced BSM hyperresponsiveness by using mice with experimental asthma. Mice were sensitized and repeatedly challenged with ovalbumin antigen. Animals also were treated with GGTI-2133 (5 mg/kg ip) once a day before and during the antigen inhalation period. Repeated antigen inhalation caused a BSM hyperresponsiveness to acetylcholine with the increased expressions of RhoA and the anti-farnesyl-positive 21-kDa proteins, probably geranylgeranylated RhoA. The in vivo GGTI-2133 treatments significantly inhibited BSM hyperresponsiveness induced by antigen exposure. In another series of experiments, BSM tissues isolated from the repeatedly antigen-challenged mice were cultured for 48 h in the absence or presence of GGTI-2133. Under these conditions, the putative geranylgeranylated RhoA was decreased in a GGTI-2133 concentration-dependent manner. The in vitro incubation with GGTI-2133 also inhibited BSM hyperresponsiveness induced by antigen exposure. These findings suggest that GGTI-2133 inhibits antigen-induced BSM hyperresponsiveness, probably by reducing downstream signal transduction of RhoA. Selective geranylgeranyltransferase inhibitors may be beneficial for the treatment of airway hyperresponsiveness, one of the characteristic features of allergic bronchial asthma.
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Affiliation(s)
- Yoshihiko Chiba
- Department of Pharmacology, School of Pharmacy, Hoshi University, Shinagawa-ku, Tokyo 142-8501, Japan.
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Liu CQ, Leung FP, Wong SL, Wong WT, Lau CW, Lu L, Yao X, Yao T, Huang Y. Thromboxane prostanoid receptor activation impairs endothelial nitric oxide-dependent vasorelaxations: the role of Rho kinase. Biochem Pharmacol 2009; 78:374-81. [PMID: 19409373 DOI: 10.1016/j.bcp.2009.04.022] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2009] [Revised: 04/15/2009] [Accepted: 04/17/2009] [Indexed: 12/27/2022]
Abstract
Activation of thromboxane prostanoid (TP) receptors causes potent vasoconstriction, which contributes to increased vascular tone and blood pressure. The present study examined the hypothesis that stimulation of TP receptor impaired endothelial nitric oxide-mediated vasorelaxation via a Rho kinase-dependent mechanism. The common carotid arteries of Sprague-Dawley rats were isolated and suspended in myograph for measurement of changes in isometric tension. The production of nitric oxide in primary cultured aortic endothelial cells was assayed with an imaging technique and phosphorylated levels of endothelial NOS were determined by Western blot analysis. 9,11-dideoxy-11alpha,9alpha-epoxy-methanoprostaglandin F(2alpha) (U46619) inhibited isoprenaline-induced relaxations in rings with or without endothelium. Treatment with Rho kinase inhibitors, Y27632 (2 microM) or HA 1077 (10 microM) prevented the effect of U46619 only in rings with endothelium while protein kinase C inhibitors were without effect. Rho kinase inhibitors did not affect isoprenaline-induced relaxations in endothelium-intact rings treated with L-NAME or 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one (ODQ). Isoprenaline stimulated rises in nitric oxide (NO) production in cultured rat endothelial cells. The increased NO production was inhibited by U46619 (100 nM) and this effect was prevented by treatment with Y27632 but unaffected by the absence of extracellular calcium ions. U46619 attenuated isoprenaline-stimulated phosphorylation of eNOS, which was sensitive to inhibition by Y27632 and HA 1077. U46619-mediated effects were abolished by TP receptor antagonist, S18886 and the TP receptor was present in endothelial cells. The present results demonstrate that Rho kinase activation is likely to be the primary mechanism that underlies the U46619-stimulated TP-receptor-mediated inhibition of endothelial NO production and subsequent endothelium-dependent relaxations to isoprenaline.
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Affiliation(s)
- Cui Qing Liu
- Department of Physiology, Hangzhou Normal University, China.
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30
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Rodríguez C, Alcudia JF, Martínez-González J, Guadall A, Raposo B, Sánchez-Gómez S, Badimon L. Statins normalize vascular lysyl oxidase down-regulation induced by proatherogenic risk factors. Cardiovasc Res 2009; 83:595-603. [PMID: 19406911 DOI: 10.1093/cvr/cvp136] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
AIMS Statins are lipid-lowering drugs widely used in the management of vascular diseases. Clinical and experimental evidence suggest that statins improve endothelial function by both cholesterol-lowering-dependent and -independent mechanisms. We have previously shown that endothelial dysfunction induced by risk factors and proinflammatory cytokines is associated with down-regulation of lysyl oxidase (LOX), a key enzyme modulating extracellular matrix maturation and vascular integrity. Our aim was to analyse whether statins could normalize LOX expression impaired by proatherogenic risk factors. METHODS AND RESULTS We observed that pharmacological concentrations of statins (atorvastatin and simvastatin) modulated LOX transcriptional activity, counteracting the down-regulation of LOX (at the mRNA, protein, and activity level) caused by tumour necrosis factor-alpha (TNFalpha) in porcine, bovine, and human aortic endothelial cells. Geranylgeraniol but not farnesol reversed this effect, suggesting the involvement of geranylgeranylated proteins. In accordance, inhibitors of RhoA/Rho kinase also counteracted LOX down-regulation caused by TNFalpha, and over-expression of a RhoA dominant-negative mutant mimicked statin effects. Statins were also able to counteract the decrease in LOX expression produced by atherogenic concentrations of LDL by a similar mechanism and to partially prevent the increase in endothelial permeability elicited by these lipoproteins. Finally, in the in vivo porcine model of hypercholesterolaemia, we observed that statins abrogated the reduction of vascular LOX expression triggered by high plasma levels of LDL. CONCLUSION These data indicate that statins normalize vascular LOX expression altered by atherogenic risk factors through a RhoA/Rho kinase-dependent mechanism. Thus, modulation of LOX by statins could contribute to vascular protection and to the cardiovascular risk reduction achieved by this therapy.
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Affiliation(s)
- Cristina Rodríguez
- Centro de Investigación Cardiovascular, Antoni M Claret 167, Barcelona 08025, Spain.
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Jarai R, Kaun C, Weiss TW, Speidl WS, Rychli K, Maurer G, Huber K, Wojta J. Human cardiac fibroblasts express B-type natriuretic peptide: fluvastatin ameliorates its up-regulation by interleukin-1alpha, tumour necrosis factor-alpha and transforming growth factor-beta. J Cell Mol Med 2009; 13:4415-21. [PMID: 19228263 PMCID: PMC4515057 DOI: 10.1111/j.1582-4934.2009.00704.x] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
B-type natriuretic peptide (BNP) is a cardiac hormone, which plays a major role in body fluid and cardiovascular homeostasis. Produced by cardiac ventricles, its expression is highly regulated by various mediators. Canine cardiac fibroblasts have been identified as a source of BNP. Cardiac fibroblasts are key regulators of myocardial structure and function. We treated cultured human adult cardiac fibroblasts (HACF) with 2000 U/ml tumour necrosis factor-α (TNF-α), 200 U/ml interleukin-1α (IL-1α) or 50 ng/ml transforming growth factor-β (TGF-β) in the presence or absence of 500 nM fluvastatin. N-terminal pro-BNP (Nt-proBNP) concentration was determined by a competitive enzyme immunoassay. RealTime polymerase chain reaction (real-time PCR) was performed to investigate changes in BNP mRNA expression. Nt-proBNP peptide was present in the conditioned media of HACF and incubation with fluvastatin significantly reduced Nt-proBNP peptide levels. Treatment of HACF with TNF-α, IL-1α or TGF-β significantly increased Nt-proBNP levels compared with untreated cells. This effect was completely abolished in the presence of fluvastatin. Real-time PCR analysis confirmed these changes at the level of mRNA expression. Our data suggest that cardiac fibroblasts are a potential source of BNP in the human heart. Pro-inflammatory cytokines, associated with ventricular dysfunction and cardiac fibrosis, seem to be major inducers of BNP production in cardiac fibroblasts. This effect can be reverted by a statin. Based on our data, we speculate that elevated plasma BNP levels might not only reflect increased myocardial stretch but also inflammatory and remodelling processes. A possible benefit of statin-induced reduction in BNP production requires further studies.
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Affiliation(s)
- Rudolf Jarai
- Department of Cardiology and Emergency Medicine, Wilhelminenhospital, Vienna, Austria
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Suzuki H, Motley ED, Eguchi K, Hinoki A, Shirai H, Watts V, Stemmle LN, Fields TA, Eguchi S. Distinct roles of protease-activated receptors in signal transduction regulation of endothelial nitric oxide synthase. Hypertension 2009; 53:182-8. [PMID: 19064814 PMCID: PMC2679177 DOI: 10.1161/hypertensionaha.108.125229] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2008] [Accepted: 11/13/2008] [Indexed: 01/29/2023]
Abstract
Protease-activated receptors (PARs), such as PAR1 and PAR2, have been implicated in the regulation of endothelial NO production. We hypothesized that PAR1 and PAR2 distinctly regulate the activity of endothelial NO synthase through the selective phosphorylation of a positive regulatory site, Ser(1179), and a negative regulatory site, Thr(497), in bovine aortic endothelial cells. A selective PAR1 ligand, TFLLR, stimulated the phosphorylation of endothelial NO synthase at Thr(497). It had a minimal effect on Ser(1179) phosphorylation. In contrast, a selective PAR2 ligand, SLIGRL, stimulated the phosphorylation of Ser(1179) with no noticeable effect on Thr(497). Thrombin has been shown to transactivate PAR2 through PAR1. Thus, thrombin, as well as a peptide mimicking the PAR1 tethered ligand, TRAP, stimulated phosphorylation of both sites. Also, thrombin and SLIGRL, but not TFLLR, stimulated cGMP production. A G(q) inhibitor blocked thrombin- and SLIGRL-induced Ser(1179) phosphorylation, whereas it enhanced thrombin-induced Thr(497) phosphorylation. In contrast, a G(12/13) inhibitor blocked thrombin- and TFLLR-induced Thr(497) phosphorylation, whereas it enhanced the Ser(1179) phosphorylation. Although a Rho-kinase inhibitor, Y27632, blocked the Thr(497) phosphorylation, other inhibitors that targeted Rho-kinase failed to block TFLLR-induced Thr(497) phosphorylation. These data suggest that PAR1 and PAR2 distinctly regulate endothelial NO synthase phosphorylation and activity through G(12/13) and G(q), respectively, delineating the novel signaling pathways by which the proteases act on protease-activated receptors to potentially modulate endothelial functions.
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Affiliation(s)
- Hiroyuki Suzuki
- Cardiovascular Research Center and Department of Physiology, Temple University School of Medicine, 3420 N Broad St, Philadelphia, PA 19140, USA
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Hu YF, Chen YC, Cheng CC, Higa S, Chen YJ, Chen SA. Fluvastatin reduces pulmonary vein spontaneous activity through nitric oxide pathway. J Cardiovasc Electrophysiol 2008; 20:200-6. [PMID: 18775044 DOI: 10.1111/j.1540-8167.2008.01281.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
INTRODUCTION Pulmonary veins (PVs) are the most important focus for the generation of atrial fibrillation. The HMG-CoA reductase inhibitors (statins) can reduce the occurrence of atrial fibrillation. The purposes of this study were to evaluate whether statins may inhibit the PV arrhythmogenic activity to prevent atrial arrhythmias from PVs and to investigate the link between fluvastatin, nitric oxide synthase (NOS) activity, mechanical activity, and electrical activity. METHODS Conventional microelectrodes and Western blot were used to record the electrical activity, diastolic tension, contractility and expression of Akt, endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and phosphorylated Akt and eNOS before and after the administration of fluvastatin in rabbit PVs or atria. RESULTS Fluvastatin decreased the PV spontaneous activity, diastolic tension, and contractility, but did not change the action potential duration or resting membrane potential. The effects of fluvastatin on the PV firing rate and diastolic tension were attenuated in the presence of L-NAME (100 microM), wortmannin (100 nM), and ODQ (3 microM). Fluvastatin (1 muM) increased the phosphorylated Akt and eNOS, but did not change the total Akt or eNOS in the PVs and atria. In contrast, fluvastatin (1 microM) decreased the total nNOS in the PVs and atria. CONCLUSIONS AND IMPLICATIONS Fluvastatin produced nitric oxide through the PI3kinase/Akt pathway, thus reducing the PV vascular diastolic tension and PV spontaneous activity. These results may contribute to the beneficial effects of statins.
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Affiliation(s)
- Yu-Feng Hu
- Division of Cardiology and Cardiovascular Research Center, Veterans General Hospital-Taipei, Taiwan
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Tourneux P, Chester M, Grover T, Abman SH. Fasudil inhibits the myogenic response in the fetal pulmonary circulation. Am J Physiol Heart Circ Physiol 2008; 295:H1505-13. [PMID: 18676688 DOI: 10.1152/ajpheart.00490.2008] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
In addition to high pulmonary vascular resistance (PVR) and low pulmonary blood flow, the fetal pulmonary circulation is characterized by mechanisms that oppose vasodilation. Past work suggests that high myogenic tone contributes to high PVR and may contribute to autoregulation of blood flow in the fetal lung. Rho-kinase (ROCK) can mediate the myogenic response in the adult systemic circulation, but whether high ROCK activity contributes to the myogenic response and modulates time-dependent vasodilation in the developing lung circulation are unknown. We studied the effects of fasudil, a ROCK inhibitor, on the hemodynamic response during acute compression of the ductus arteriosus (DA) in chronically prepared, late-gestation fetal sheep. Acute DA compression simultaneously induces two opposing responses: 1) blood flow-induced vasodilation through increased shear stress that is mediated by NO release and 2) stretch-induced vasoconstriction (i.e., the myogenic response). The myogenic response was assessed during acute DA compression after treatment with N(omega)-nitro-L-arginine, an inhibitor of nitric oxide synthase, to block flow-induced vasodilation and unmask the myogenic response. Intrapulmonary fasudil infusion (100 microg over 10 min) did not enhance flow-induced vasodilation during brief DA compression but reduced the myogenic response by 90% (P<0.05). During prolonged DA compression, fasudil prevented the time-dependent decline in left pulmonary artery blood flow at 2 h (183+/-29 vs. 110+/-11 ml/min with and without fasudil, respectively; P<0.001). We conclude that high ROCK activity opposes pulmonary vasodilation in utero and that the myogenic response maintains high PVR in the normal fetal lung through ROCK activation.
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Affiliation(s)
- Pierre Tourneux
- The Pediatric Heart Lung Center, Sections of Neonatology and Pulmonary Medicine, Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado, USA.
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Aoki T, Kataoka H, Ishibashi R, Nozaki K, Hashimoto N. Simvastatin suppresses the progression of experimentally induced cerebral aneurysms in rats. Stroke 2008; 39:1276-85. [PMID: 18309148 DOI: 10.1161/strokeaha.107.503086] [Citation(s) in RCA: 100] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
BACKGROUND AND PURPOSE The pathophysiology of cerebral aneurysms (CAs) is linked to chronic inflammation and degradation of extracellular matrix in vascular walls. Because statins have protective effects on various vascular diseases independent of their lipid-lowering effects, we investigated the effect of simvastatin on CA progression. METHODS CAs were induced in Sprague-Dawley rats with or without oral administration of simvastatin. The size and media thickness of CAs was evaluated 3 months after aneurysm induction. Expression of macrophage chemoattractant protein-1, vascular cell adhesion molecule-1, endothelial nitric oxide synthase, interleukin-1beta, inducible nitric oxide synthase, matrix metalloproteinase-2, and matrix metalloproteinase-9 in aneurysmal walls was examined by reverse transcriptase-polymerase chain reaction and immunohistochemistry. To examine whether simvastatin has a suppressive effect on preexisting CAs, simvastatin administration started at 1 month after aneurysm induction. RESULTS Rats treated with simvastatin exhibited a significant increase in media thickness and a significant reduction in aneurysmal size compared with control rats. Treatment with simvastatin resulted in reduced expression of macrophage chemoattractant protein-1 and vascular cell adhesion molecule-1, increased expression of endothelial nitric oxide synthase, and reduced the number of macrophage infiltration. In quantitative polymerase chain reaction and immunohistochemistry, simvastatin significantly inhibited upregulated expression of interleukin-1beta, inducible nitric oxide synthase, matrix metalloproteinase-2, and matrix metalloproteinase-9 associated with CA progression. Gelatin zymography revealed decreased activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 in aneurysmal walls by simvastatin treatment. Simvastatin also effectively inhibited aneurysm enlargement and thinning of the media of preexisting CAs. CONCLUSIONS Treatment with simvastatin suppresses the development of CAs by inhibiting inflammatory reactions in aneurysmal walls. Simvastatin also has a preventive effect on the progression of preexisting CAs. Simvastatin is a promising candidate of a novel medical treatment for the prevention of CA progression.
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Affiliation(s)
- Tomohiro Aoki
- Department of Neurosurgery, Kyoto University Graduate School of Medicine, Kyoto, Japan
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Haydont V, Bourgier C, Pocard M, Lusinchi A, Aigueperse J, Mathé D, Bourhis J, Vozenin-Brotons MC. Pravastatin Inhibits the Rho/CCN2/extracellular matrix cascade in human fibrosis explants and improves radiation-induced intestinal fibrosis in rats. Clin Cancer Res 2007; 13:5331-40. [PMID: 17875761 DOI: 10.1158/1078-0432.ccr-07-0625] [Citation(s) in RCA: 102] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
PURPOSES Intestinal complications after radiotherapy are caused by transmural fibrosis and impair the quality of life of cancer survivors. Radiation fibrosis was considered permanent and irreversible, but recently, its dynamic nature was shown, providing new opportunities for the development of antifibrotic therapies. Among these new targets, we identified the Rho/ROCK pathway and thought to investigate whether pravastatin treatment inhibits Rho pathway activation and elicits an antifibrotic action. EXPERIMENTAL DESIGN Rho and ROCK activities were monitored in human explants presenting radiation fibrosis remodeling after incubation with pravastatin. Subsequent modulation of CCN2, type I collagen, and fibronectin expression were assessed ex vivo and in intestinal smooth muscle cells derived from radiation enteropathy. Then, the therapeutic relevance of the antifibrotic action of pravastatin was explored in vivo in a rat model of chronic radiation fibrosis (19 Gy X-rays) treated with 30 mg/kg/d pravastatin in the drinking water. RESULTS The results obtained with human explants show that pravastatin specifically inhibits Rho activity in submucosal mesenchymal cells. Pravastatin also elicits ROCK inhibition, and subsequent CCN2 production in human explants and smooth muscle cells isolated from radiation enteropathy. Inhibition of type I collagen and fibronectin does occur, showing that pravastatin modulates the secretory phenotype of mesenchymal cells. Lastly, curative pravastatin administration improves radiation enteropathy in rats. This structural improvement is associated with decreased deposition of CCN2 and subsequent decreased extracellular matrix deposition. CONCLUSION Targeting established fibrosis with pravastatin is an efficient and safe antifibrotic strategy in radiation-induced enteropathy, and is easily transferable into the clinic.
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Affiliation(s)
- Valérie Haydont
- UPRES EA 27-10 Radiosensibilité des Tumeurs et Tissus Sains, Institut de Radioprotection et de Sûreté Nucléaire/Institut Gustave Roussy, Villejuif, France
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Sun X, Ku DD. Rosuvastatin provides pleiotropic protection against pulmonary hypertension, right ventricular hypertrophy, and coronary endothelial dysfunction in rats. Am J Physiol Heart Circ Physiol 2007; 294:H801-9. [PMID: 18055512 DOI: 10.1152/ajpheart.01112.2007] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
We recently reported that increased vascular endothelial nitric oxide production could protect against the development of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) and right ventricular hypertrophy (RVH) in rats (32). The present study investigated whether the pleiotropic action of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors in upregulating endothelial function could also protect against the MCT-induced end-organ damages. Rosuvastatin (2 mg kg(-1) day(-1) via oral gavage) or placebo was initiated 1 wk before or 1 wk after MCT (60 mg/kg ip) administration. One month after MCT, significant PAH developed in the placebo rats, which were accompanied by histological evidence of pulmonary vascular thickening and right ventricular hypertrophy. The coronary endothelial vasodilatory function, assessed with endothelial/nitric oxide-dependent responses to acetylcholine and N(G)-nitro-L-arginine methyl ester (L-NAME), was depressed, while the constrictory responses to known coronary constrictors was enhanced. In rats that received rosuvastatin treatment 1 wk before MCT administration, a significantly reduced PAH and RVH was observed, as well as reduced pulmonary vascular and right ventricular remodelings. Rosuvastatin 1-wk posttreatment had no effect on PAH, but inhibited RVH. Right coronary endothelial dysfunction, which was shown in placebo rats, was effectively prevented by both pre- and postrosuvastatin treatment, while this effect was more dramatic in the pretreated group. Left coronary endothelial function, which was not affected by MCT, also showed an upregulation by rosuvastatin. Taken together, our results demonstrated the pleiotropic protection of rosuvastatin against the development of PAH and RVH and confirmed our previous finding that the targeted preservation of coronary endothelial function and vasoactivity may provide a novel approach to protect against cardiac remodeling.
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Affiliation(s)
- Xiaowei Sun
- Department of Pharmacology and Toxicology, University of Alabama at Birmingham 35294-0019, USA
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Liesmaa I, Kokkonen JO, Kovanen PT, Lindstedt KA. Lovastatin induces the expression of bradykinin type 2 receptors in cultured human coronary artery endothelial cells. J Mol Cell Cardiol 2007; 43:593-600. [PMID: 17900611 DOI: 10.1016/j.yjmcc.2007.08.007] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/29/2007] [Revised: 08/06/2007] [Accepted: 08/10/2007] [Indexed: 11/17/2022]
Abstract
Cardioprotective bradykinin type-2 receptors (BK-2Rs) are downregulated in the myocardial endothelium of both human and rat failing hearts. Statins are cardioprotective drugs that reduce the level of plasma cholesterol but also exert cholesterol-independent pleiotropic effects. Here we examined the effect of lovastatin on BK-2R expression in cultured human coronary artery endothelial cells. The effect of lovastatin on the expression of BK receptors in human coronary artery endothelial cells (HCAECs) was examined by real-time PCR, Western blot analysis and immunocytochemistry. Lovastatin induced a time- and concentration-dependent increase in both BK-2R and BK-1R mRNA expression in the cultured HCAECs. Also, the number of functional BK-2Rs capable of inducing BK-mediated NO production and cGMP signaling was increased in the lovastatin-treated HCAECs. Mevalonate, the direct metabolite of HMG-CoA reductase, reversed the effect of lovastatin. Furthermore, lovastatin inhibited Rho activation and a selective inhibitor of Rho-associated kinases, Y-27632, induced a similar increase in BK-2R expression as lovastatin. In contrast, a specific inhibitor of COX-2, NS398, significantly inhibited the lovastatin-induced expression of BK-2Rs. Here we show for the first time that lovastatin induces the expression of BK-2Rs in cultured human coronary artery endothelial cells through a novel cholesterol-independent pleiotropic mechanism that involves RhoA kinase inhibition and COX-2 activation. Thus, reported beneficial effects of statins in cardiovascular diseases may be partly mediated by an increased expression of cardioprotective BK-2Rs in the endothelial cells of the coronary tree. Moreover, the use of COX-2 inhibitors may affect the level of endothelial BK-2Rs in a negative fashion.
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Affiliation(s)
- Inka Liesmaa
- Wihuri Research Institute, Kalliolinnantie 4, FI-00140 Helsinki, Finland
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Hirano M, Kanaide H, Hirano K. Rac1-dependent transcriptional up-regulation of p27Kip1 by homophilic cell-cell contact in vascular endothelial cells. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2007; 1773:1500-10. [PMID: 17868934 DOI: 10.1016/j.bbamcr.2007.08.001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/10/2007] [Revised: 07/18/2007] [Accepted: 08/01/2007] [Indexed: 01/24/2023]
Abstract
The mechanism for the transcriptional up-regulation of p27Kip1 due to the formation of the cell-cell contact was investigated in vascular endothelial cells. The induction of the cell-cell contact by adding an extra number of endothelial cells activated Rac1, up-regulated p27Kip1 mRNA and protein, and also facilitated the cell cycle arrest. Transduction of the Rac1 inhibitor protein using the cell-penetrating peptide or treatment with a Rac1 inhibitor NSC23766 inhibited the p27Kip1 up-regulation and delayed the cell cycle arrest. Rac1 was therefore suggested to mediate the contact-induced transcriptional up-regulation of p27Kip1. The role of Rac1 in the regulation of the p27Kip1 promoter activity was next examined with a luciferase reporter assay. The promoter activity was increased by inducing the cell-cell contact, which was significantly inhibited by the Rac1 inhibitory protein and NSC23766. The evaluation of various truncated promoter regions determined region -620 to -573 nucleotides from the initiation codon to be responsible for the contact-induced, Rac1-dependent activation of the p27Kip1 promoter. The present study thus demonstrated for the first time that the activation of Rac1 due to the cell-cell contact plays a critical role in the transcriptional up-regulation of p27Kip1 in vascular endothelial cells.
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Affiliation(s)
- Mayumi Hirano
- Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
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Hirano K, Nomoto N, Hirano M, Momota F, Hanada A, Kanaide H. Distinct Ca2+ requirement for NO production between proteinase-activated receptor 1 and 4 (PAR1 and PAR4) in vascular endothelial cells. J Pharmacol Exp Ther 2007; 322:668-77. [PMID: 17494865 DOI: 10.1124/jpet.107.121038] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Proteinase-activated receptors 1 and 4 (PAR(1) and PAR(4)) are the major receptors mediating thrombin-induced NO production in endothelial cells. The intracellular signaling following their activation still remains to be elucidated. The present study provides the first evidence for the distinct Ca(2+) requirement for the NO production between PAR(1) and PAR(4). The activation of PAR(1) by the activating peptide (PAR(1)-AP) elevated cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and activated NO production in porcine aortic and human umbilical vein endothelial cells, whereas it had little effect on bovine aortic endothelial cells. PAR(4) activation by PAR(4)-AP consistently induced NO production without an appreciable [Ca(2+)](i) elevation in three types of endothelial cells. The PAR(1)-mediated NO production was significantly inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), whereas the PAR(4)-mediated NO production was resistant. NO production following the PAR(1) and PAR(4) activation was significantly inhibited by pertussis toxin, but it was resistant to a Galpha(q/11) inhibitor, YM254890 [(1R)-1-[(3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-[(1R)-1-methoxyethyl]-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl]-2-methylpropyl rel-(2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate]. However, YM254890 abrogated the PAR(1)-mediated Ca(2+) signal. PAR(4)-mediated NO production was substantially inhibited by the inhibitors of phosphotidylinositol-3 kinase (PI3K) and Akt, as well as by the dominant negative mutant of Akt. The PAR(1)-mediated NO production was relatively resistant to inhibitors of PI3K. An immunoblot analysis revealed a transient increase in the phosphorylation of Akt and endothelial NO synthase following the PAR(4) stimulation. In conclusion, PAR(1) and PAR(4) engage distinct signal transduction mechanisms to activate NO production in vascular endothelial cells. PAR(4) preferably activates Galpha(i/o) and induced NO production in a manner mostly independent of Ca(2+) but dependent on the PI3K/Akt pathway, whereas PAR(1) activates both the Ca(2+)-dependent and -independent mechanisms.
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Affiliation(s)
- Katsuya Hirano
- Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
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Abstract
Rho kinase (ROCK1 and ROCK2) is a serine/threonine kinase that serves as an important downstream effector of Rho GTPase, and plays a critical role in regulating the contractile tone of smooth muscle tissues in a calcium-independent manner. Several lines of experimental evidence indicate that modulating ROCK activity within the aqueous humor outflow pathway using selective inhibitors could achieve very significant benefits for the treatment of increased intraocular pressure in patients with glaucoma. The rationale for such an approach stems from experimental data suggesting that both ROCK and Rho GTPase inhibitors can increase aqueous humor drainage through the trabecular meshwork, leading to a decrease in intraocular pressure. In addition to their ocular hypotensive properties, inhibitors of both ROCK and Rho GTPase have been shown to enhance ocular blood flow, retinal ganglion cell survival and axon regeneration. These properties of the ROCK and Rho GTPase inhibitors indicate that targeting the Rho GTPase/ROCK pathway with selective inhibitors represents a novel therapeutic approach aimed at lowering increased intraocular pressure in glaucoma patients.
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Affiliation(s)
- Vasantha P Rao
- Department of Ophthalmology, Duke University School of Medicine, Durham, North Carolina, USA.
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Haydont V, Vozenin-Brotons MC. Maintenance of radiation-induced intestinal fibrosis: cellular and molecular features. World J Gastroenterol 2007; 13:2675-83. [PMID: 17569135 PMCID: PMC4147115 DOI: 10.3748/wjg.v13.i19.2675] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/28/2006] [Revised: 01/11/2007] [Accepted: 02/25/2007] [Indexed: 02/06/2023] Open
Abstract
Recent advances in cell and molecular radiobiology clearly showed that tissue response to radiation injury cannot be restricted to a simple cell-killing process, but depends upon continuous and integrated pathogenic processes, involving cell differentiation and crosstalk between the various cellular components of the tissue within the extracellular matrix. Thus, the prior concept of primary cell target in which a single-cell type (whatever it's epithelial or endothelial cells) dictates the whole tissue response to radiation injury has to be replaced by the occurrence of coordinated multicellular response that may either lead to tissue recovery or to sequel development. In this context, the present review will focus on the maintenance of the radiation-induced wound healing and fibrogenic signals triggered by and through the microenvironment toward the mesenchymal cell compartment, and will highlight how sequential and sustained modifications in cell phenotypes will in cascade modify cell-to-cell interactions and tissue composition.
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Affiliation(s)
- Valérie Haydont
- Laboratoire UPRES EA 27-10, Radiosensibilite des tumeurs et tissus sains, Institut de Radioprotection et de Sûreté Nucléaire/Institut Gustave Roussy, Villejuif, France
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Patel CA, Rattan S. RhoA prenylation inhibitor produces relaxation of tonic smooth muscle of internal anal sphincter. J Pharmacol Exp Ther 2007; 321:501-8. [PMID: 17322025 DOI: 10.1124/jpet.107.119339] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
RhoA prenylation is a critical step for the translocation of RhoA to the membrane and its activation in response to agonist-induced sustained contraction of the smooth muscle. However, the effect and role of RhoA prenylation in the spontaneously tonic smooth muscle, such as internal anal sphincter (IAS), is not known. Present studies determined RhoA prenylation and its association with the basal tone in the IAS before and after the RhoA prenylation inhibitor, geranylgeranyl transferase inhibitor GGTI-297 [N-4-[2(R)-amino-3-mercaptopropyl]amino-2-naphthylbenzoyl-(L)-leucine,TFA]. Western blot analyses of cytosolic and membrane fractions determined the effects of RhoA prenylation inhibition on the cellular distribution of the RhoA. Additional studies were performed to determine the relationship between RhoA prenylation and Rho kinase (ROCK) activity. GGTI-297 decreased prenylation of RhoA, decreased ROCK activity, and caused a corresponding fall in the IAS tone. These inhibitory effects following RhoA prenylation blockade were demonstrated to be directly on the spontaneously contracted IAS smooth muscle cells. Western blot analysis revealed high levels of RhoA in the IAS smooth muscle cellular membrane in the basal state, and GGTI-297 shifted the RhoA localization to the cytosol. RhoA prenylation may play an important role in the translocation of RhoA to the smooth muscle cell membrane leading to its activation and for the maintenance of basal tone in the IAS.
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Affiliation(s)
- Chirag A Patel
- Department of Medicine, Division of Gastroenterology and Hepatology, Jefferson Medical College, Thomas Jefferson University, 1025 Walnut Street, Philadelphia, PA 19107, USA
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Ozacmak VH, Sayan H, Igdem AA, Cetin A, Ozacmak ID. Attenuation of contractile dysfunction by atorvastatin after intestinal ischemia reperfusion injury in rats. Eur J Pharmacol 2007; 562:138-47. [PMID: 17335801 DOI: 10.1016/j.ejphar.2007.01.061] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2006] [Revised: 01/16/2007] [Accepted: 01/17/2007] [Indexed: 10/23/2022]
Abstract
Growing number of studies implicate that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, have beneficial effects on ischemia/reperfusion injury that are unrelated to their cholesterol-lowering action. In the present study, we aimed to evaluate possible effects of atorvastatin on oxidative stress, neutrophil accumulation, and contractile response of terminal ileum segments in rats subjected to intestinal ischemia/reperfusion. Intestinal ischemia/reperfusion model was generated by clamping the superior mesenteric artery for 30 min followed by reperfusion for 3 h. Oral administration of atorvastatin at a dose of 10 mg/kg/day lasted 3 days just before induction of intestinal ischemia. At the end of reperfusion period, terminal ileum samples were removed to determine the concentrations of malondialdehyde, reduced glutathione, and myeloperoxidase. Samples were collected also to assess histopathological alterations and contractile response to agonists. Ischemia/reperfusion significantly decreased contractile responses, and this decrease was attenuated by atorvastatin. Pretreatment with atorvastatin caused remarkable decrease in both oxidative stress and neutrophil accumulation. Atorvastatin appeared to be restoring amount of reduced glutathione back to about control level. Furthermore, the pretreatment lowered mucosal damage at histopathological level. Our results suggested that pretreatment with atorvastatin attenuated intestinal muscle dysfunction associated with ischemia/reperfusion. This remarkable effect of atorvastatin is accomplished at least by decreasing oxidative stress and neutrophil accumulation as well as preventing the depletion of reduced glutathione.
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Affiliation(s)
- V Haktan Ozacmak
- Zonguldak Karaelmas University, School of Medicine, Department of Physiology, 67600 Kozlu, Zonguldak, Turkey.
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Girgis RE, Mozammel S, Champion HC, Li D, Peng X, Shimoda L, Tuder RM, Johns RA, Hassoun PM. Regression of chronic hypoxic pulmonary hypertension by simvastatin. Am J Physiol Lung Cell Mol Physiol 2007; 292:L1105-10. [PMID: 17277047 DOI: 10.1152/ajplung.00411.2006] [Citation(s) in RCA: 81] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
The 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitor, simvastatin, has been shown to attenuate chronic hypoxic pulmonary hypertension (CHPH). Here, we assess whether simvastatin is capable of inducing regression of established CHPH and explore potential mechanisms of statin effect. Rats ( n = 8 in each group) were exposed to chronic hypoxia (10% FiO2) for 2 or 4 wk. Simvastatin treatment (20 mg·kg−1·day−1) commenced after 2 wk of hypoxia, at which time CHPH was fully established, reduced mean pulmonary artery pressure (19 ± 0.5 vs. 27 ± 0.9 mmHg; P < 0.001), the ratio of right ventricular free wall to left ventricular plus septal weight (0.41 ± 0.03 vs. 0.54 ± 0.03; P < 0.001), and medial thickening of small pulmonary arteries (13 ± 0.4 vs. 16 ± 0.4%; P < 0.01) compared with 4-wk hypoxic controls. Supplementation with mevalonate (50 mg·kg−1·day−1) prevented the attenuation of CHPH induced by simvastatin during 2 wk of hypoxia. Because statins are known to inhibit Rho-kinase (ROCK), we determined expression of ROCK-1 and -2 in whole lung by Western blot and ROCK activity by phosphorylation of the myosin-binding subunit of myosin phosphatase. Expression of both ROCK-1 and -2 were markedly diminished in simvastatin-treated animals during normoxia and hypoxia (2- and 4-wk) exposure ( P < 0.01). ROCK activity was increased threefold under hypoxic conditions and normalized with simvastatin treatment ( P < 0.001). We conclude that simvastatin attenuates and induces regression of established CHPH through inhibition of HMG-CoA reductase. Inhibition of ROCK expression and activity may be an important mechanism of statin effect.
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Affiliation(s)
- Reda E Girgis
- Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
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Wojciak-Stothard B, Torondel B, Tsang LYF, Fleming I, Fisslthaler B, Leiper JM, Vallance P. The ADMA/DDAH pathway is a critical regulator of endothelial cell motility. J Cell Sci 2007; 120:929-42. [PMID: 17327280 DOI: 10.1242/jcs.002212] [Citation(s) in RCA: 75] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Asymmetric dimethylarginine (ADMA) is an inhibitor of nitric oxide production associated with abnormal blood vessel growth and repair, however, the mechanism of action of ADMA is not well understood. We studied the role of exogenous and endogenous ADMA in the regulation of cell motility and actin cytoskeleton in porcine pulmonary endothelial cells (PAECs) and pulmonary microvascular endothelial cells (PMECs) from knockout mice that lack one of the enzyme metabolising ADMA, dimethylarginine dimethylaminohydrolase I (DDAHI) as well as endothelial cells overexpressing DDAH in vitro. We show that ADMA induced stress fibre and focal adhesion formation and inhibited cell motility in primary pulmonary endothelial cells. The effects of ADMA depended on the activity of RhoA and Rho kinase and were reversed by overexpression of DDAH, nitric oxide donors and protein kinase G activator, 8-bromo-cGMP. ADMA also inhibited the activities of Rac1 and Cdc42 in cells but these changes had a minor effect on cell motility. Endogenous ADMA increased RhoA activity and inhibited cell motility in PMECs from DDAHI knockout mice and inhibited angiogenesis in vitro. These results are the first demonstration that metabolism of cardiovascular risk factor ADMA regulates endothelial cell motility, an important factor in angiogenesis and vascular repair.
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Affiliation(s)
- Beata Wojciak-Stothard
- BHF Laboratories, Department of Medicine, University College London, 5 University Street, London, WC1 E6JJ, UK.
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Calò LA, Pessina AC. RhoA/Rho-kinase pathway: much more than just a modulation of vascular tone. Evidence from studies in humans. J Hypertens 2007; 25:259-64. [PMID: 17211228 DOI: 10.1097/hjh.0b013e328010d4d2] [Citation(s) in RCA: 86] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
RhoA/Rho-kinase signaling and its relationship/balance with the nitric oxide level, angiotensin II and vasopressors for cardiovascular pathophysiology is of increasing importance, and its involvement goes far beyond blood pressure regulation. The deep involvement of this pathway in cardiovascular biology is now known to include a wide spectrum of conditions relating to the long-term complications of hypertension, and in general of cardiovascular pathophysiology, such as changes in cardiovascular structure (remodeling) and the induction of atherosclerosis, involvement in the pathophysiological relationships between inflammation and hypertension, and in those between hypertension, glucose metabolism and insulin resistance. Studies from our laboratory have made an important contribution to the understanding of the cellular and molecular mechanisms mediated by the RhoA/Rho-kinase pathway, which include all the aspects of cardiovascular pathophysiology in which this pathway plays a role. In addition, if it is considered that our contribution to the clarification of these mechanisms only comes from studies in humans, their impact on the scenario of the RhoA/Rho-kinase pathway's biology, essentially supported by studies 'in vitro' or in animal models, is immediate. This review examines all the aspects of RhoA/Rho-kinase signaling in the light of the available data, and gives an updated and useful overall picture of its involvement in cardiovascular pathophysiology.
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Affiliation(s)
- Lorenzo A Calò
- Department of Clinical and Experimental Medicine, Clinica Medica 4, University of Padova, Padua, Italy.
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Xing XQ, Gan Y, Wu SJ, Chen P, Zhou R, Xiang XD. Statins may ameliorate pulmonary hypertension via RhoA/Rho-kinase signaling pathway. Med Hypotheses 2006; 68:1108-1113. [PMID: 17097823 DOI: 10.1016/j.mehy.2006.09.034] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2006] [Accepted: 09/06/2006] [Indexed: 02/08/2023]
Abstract
Statins are the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors that function as potent inhibitors of cholesterol biosynthesis and have been used for many years for the treatment of hypercholesterolemia. However, accumulating experimental and clinical studies have revealed that the health benefits associated with statins treatment, particularly those conferred on the cardiovascular system, were the cholesterol-independent. Because statins inhibit an early step in the cholesterol biosynthetic pathway, they also inhibit the synthesis of isoprenoids such as farnesylpyrophosphate and geranylgeranylpyrophosphate, which are important postranslational lipid attachments for intracellular signaling molecules such as the Rho GTPases. The isoprenylation of Rho is a prerequisite for Rho activation, facilitating its interaction with the plasma membrane, undergoing GDP-GTP exchange and be activated. Inhibition of RhoA geranylgeranylation by statins decreases membrane GTP-bound active RhoA and subsequent Rho-kinase activity. Activated RhoA via its downstream effector Rho-kinase is involved in a wide range of cellular functions, such as cell migration, proliferation and apoptosis. Recently, rising evidences suggested that RhoA/Rho-kinase pathway was essentially involved in various models of pulmonary hypertension and statins effectively ameliorated pulmonary hypertension. Based on this findings, we hypothesis that statins attenuate pulmonary hypertension via RhoA/Rho-kinase signaling pathway in vivo.
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Affiliation(s)
- Xi-Qian Xing
- Department of Respiratory Medicine, The Second Xiangya Hospital, Central South University, Middle Renmin Road, No. 86, Changsha, Hunan 410011, PR China
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Abstract
BACKGROUND AND PURPOSE The small G protein rhoA and its downstream effector rho-kinase are both expressed in vascular cells and are involved in several cellular processes. One of these processes is the regulation of the phosphorylation state of myosin light chain in vascular muscle and thus, the development of force. Recently, considerable evidence for increased activity of this pathway in cerebral and noncerebral vessels has been reported in several cardiovascular diseases associated with increased vascular tone. SUMMARY OF REVIEW The main aim of this brief review is to summarize current evidence for the involvement of rhoA/rho-kinase signaling in dysfunction of the cerebral circulation in disease states, such as cerebral vasospasm, hypertension, diabetes, and ischemic brain injury. We will also briefly consider the novel hypothesis that augmented activity of endothelial rho-kinase decreases nitric oxide production and contributes to increased vascular tone in disease and the possibility of this action being a key therapeutic target of statins (inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase) in cerebral and noncerebral arteries. CONCLUSIONS Considerable evidence indicates that rhoA/rho-kinase activity is commonly increased in cerebral vascular disease, not only in vascular muscle, but also in the endothelium and possibly in inflammatory cells and neurons.
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Ramasubbu K, Mann DL. The Emerging Role of Statins in the Treatment of Heart Failure⁎⁎Editorials published in the Journal of the American College of Cardiologyreflect the views of the authors and do not necessarily represent the views of JACCor the American College of Cardiology. J Am Coll Cardiol 2006; 47:342-4. [PMID: 16412858 DOI: 10.1016/j.jacc.2005.10.031] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
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