Rho signaling and mechanical control of vascular development

PURPOSE OF REVIEW: To discuss how mechanical cues and Rho signaling contribute to control of vascular development and hematopoiesis. RECENT FINDINGS: Rho guanine trinucleotide phosphatases are ubiquitious regulators of cytoskeletal structure and tension generation. Recent work shows that Rho-dependent mechanical interactions between cells and extracellular matrix regulate cell fate switching in capillary endothelial cells and megakaryocytes in vitro, as well as angiogenesis, vascular permeability, leukocyte migration and platelet formation in vivo. Signaling pathways that link integrins and tension-dependent changes in cytoskeletal structure to Rho have also begun to be delineated. SUMMARY: Mechanical force generation by cells and simultaneous sensing of these physical forces play critical roles in vascular development by estimating whether individual cells will grow, differentiate, move or undergo apoptosis in the local tissue microenvironment. Future work in the vascular field therefore needs to incorporate physical control mechanisms into existing biochemical concepts of cell and tissue regulation.     

Rho and Rho kinase mediate thrombin-stimulated vascular smooth muscle cell DNA synthesis and migration.

Aberrant regulation of smooth muscle cell proliferation and migration is associated with the pathophysiology of vascular disorders such as hypertension, atherosclerosis, restenosis, and graft rejection. To elucidate molecular mechanisms that regulate proliferation and migration of vascular smooth muscle cells, we determined whether signaling through the small G protein Rho is involved in thrombin- and phenylephrine-stimulated proliferation and migration of rat aortic smooth muscle cells (RASMCs). Thrombin and the thrombin peptide SFLLRNP stimulated DNA synthesis of RASMCs as measured by [3H]thymidine incorporation. Both ligands also increased cell migration as measured by the Boyden chamber method. L-Phenylephrine failed to induce either of these responses but increased inositol phosphate accumulation and mitogen-activated protein kinase activation in these cells, which indicated that the cells were responsive to alpha1-adrenergic stimulation. The C3 exoenzyme, which ADP-ribosylates and inactivates Rho, fully inhibited both thrombin-stimulated proliferation and migration but had no effect on inositol phosphate accumulation. In addition, Y-27632, an inhibitor of the Rho effector p160ROCK/Rho kinase, decreased thrombin-stimulated DNA synthesis and migration. To directly examine Rho activation, Rho-[35S]GTPgammaS binding was measured. The addition of the thrombin peptide SFLLRNP, but not phenylephrine, to RASMC lysates resulted in a significant increase in Rho-[35S]GTPgammaS binding. Thrombin and SFLLRNP, but not phenylephrine, also increased membrane-associated Rho in intact RASMCs, consistent with selective activation of Rho by thrombin. These results indicate that thrombin activates Rho in RASMCs and establish Rho as a critical mediator of thrombin receptor effects on DNA synthesis and cell migration in these cells.     

Rho激酶与缺血性脑血管病

缺血性脑血管病可导致严重的神经功能缺损.甚至死亡.Rho激酶是Rho蛋白的重要下游底物,参与血管平滑肌收缩、细胞迁移、炎症细胞浸润以及血管内皮功能障碍等多种生物学效应,在缺血性脑血管病的发生、发展和梗死体积扩大等过程中起着重要作用.Rho激酶抑制剂可有效舒张痉李血管,减轻局部炎症反应,保护内皮功能,增加缺血区域血流,缩小梗死体积,改善神经功能.     

Rho激酶与缺血性脑血管病

缺血性脑血管病可导致严重的神经功能缺损.甚至死亡.Rho激酶是Rho蛋白的重要下游底物,参与血管平滑肌收缩、细胞迁移、炎症细胞浸润以及血管内皮功能障碍等多种生物学效应,在缺血性脑血管病的发生、发展和梗死体积扩大等过程中起着重要作用.Rho激酶抑制剂可有效舒张痉李血管,减轻局部炎症反应,保护内皮功能,增加缺血区域血流,缩小梗死体积,改善神经功能.     

Rho/Rho kinase signal transduction pathway in cardiovascular disease and cardiovascular remodeling

The small guanosine triphosphatase Rho and its target, Rho kinase, play important roles in both blood pressure regulation and vascular smooth muscle contraction. Rho is activated by agonists of receptors coupled to cell membrane G protein, such as angiotensin II and phenylephrine. Once Rho is activated, it translocates to the cell membrane where it, in turn, activates Rho kinase. Activated Rho kinase phosphorylates myosin light chain phosphatase, which is then inhibited. This sequence stimulates vascular smooth muscle contraction, stress fiber formation,and cell migration. In this way, Rho and Rho kinase activation have important effects on several cardiovascular diseases. Currently available substances that specifically inhibit this signaling pathway could offer clinical benefits in several cardiovascular, as well as noncardiovascular diseases, such as arterial hypertension, pulmonary hypertension, cerebral or coronary spasm, post-angioplasty restenosis, and erectile dysfunction.     

Contribution of Rho A and Rho kinase to platelet-derived growth factor-BB-induced proliferation of vascular smooth muscle cells

In order to identify small G protein (s) which contributes to the proliferation of vascular smooth muscle cells (VSMCs), we examined the effect of an HMG-CoA reductase inhibitor (cerivastatin), a farnesyltransferase inhibitor (FTI-277), a geranyl geranyl transferase inhibitor (GGTI-286) and a Rho kinase inhibitor (Y-27632) on the proliferation of cultured rat VSMCs stimulated with 20ng/ml platelet-derived growth factor (PDGF)-BB. Cerivastatin and GGTI-286, but not FTI-277, suppressed the PDGF-BB-induced activation of extracellular signal related kinase (ERK1/2). The inhibitory effect of cerivastatin on the PDGF-BB-induced activation of ERK1/2 was fully recovered by the addition of geranylgeranyl pyrophosphate (GGPP), but not farnesyl pyrophosphate (FPP). Cerivastatin and GGTI-286, but not FTI-277, suppressed the PDGF-BB-induced [3H] thymidine incorporation and activation of ornitine decarboxylase (ODC), both of which were fully recovered by the addition of GGPP, but not FPP. These data indicate that the PDGF-BB-induced activation of ERK1/2 and proliferation of VSMCs depend upon geranylgeranylated small G protein. Immunoblotting analysis revealed the upregulation of Rho A protein in the membrane fractions of VSMCs stimulated by PDGF-BB. Furthermore, Y-27632 suppressed the PDGF-BB-induced activation of ERK1/2 and proliferation of VSMCs. On the basis of these data, we conclude that PDGF-BB stimulates the proliferation of VSMCs via the activation of Rho A. Rho kinase plays an important role in this process as an effector of Rho A.     

Platelet Rho GTPase regulation in physiology and disease

Rho GTPases are master orchestrators of cytoskeletal dynamics and serve critical roles in platelet physiology to promote hemostasis or pathology in thrombotic, inflammatory and other disease states. Over the past 25 years, specific platelet cell biological outputs have been linked to the activities of Rho GTPases, including RhoA, Rac1, Cdc42, and RhoG in shape change and secretion as well as cytoskeletal assembly events underlying platelet aggregation and thrombus stability. Rho GTPases have also more recently been noted to serve more specialized roles in platelet function and to cooperate with one another in mediating essential platelet responses. The evolving molecular mechanisms regulating platelet Rho GTPase functions are increasingly complex, involving an interdependent array of signal transduction molecules, including several protein kinases as well as numerous Rho GEFs, GAPs, and GDI proteins such as LARG, ARHGEF6 (Cool-2, α-Pix), ARHGEF10, GIT1, ARHGAP17 (Nadrin, Rich1), OPHN1, and Ly-GDI. In this review, we provide an update of recent work and developing hypotheses further establishing more specialized as well as cooperative roles for Rho GTPases in platelet physiology and emerging regulatory and downstream effector mechanisms whereby Rho GTPases participate in platelet activation programs in physiology and an expanding set of platelet-associated disease states.     

Ca2+-dependent activation of Rho and Rho kinase in membrane depolarization-induced and receptor stimulation-induced vascular smooth muscle contraction

Ca2+ sensitization of vascular smooth muscle (VSM) contraction involves Rho-dependent and Rho-kinase-dependent suppression of myosin phosphatase activity. We previously demonstrated that excitatory agonists in fact induce activation of RhoA in VSM. In this study, we demonstrate a novel Ca2+-dependent mechanism for activating RhoA in rabbit aortic VSM. High KCl-induced membrane depolarization as well as noradrenalin stimulation induced similar extents of sustained contraction in rabbit VSM. Both stimuli also induced similar extents of time-dependent, sustained increases in the amount of an active GTP-bound form of RhoA. Consistent with this, the Rho kinase inhibitors HA1077 and Y27632 inhibited both contraction and the 20-kDa myosin light chain phosphorylation induced by KCl as well as noradrenalin, with similar dose-response relations. Either removal of extracellular Ca2+ or the addition of a dihydropyridine Ca2+ channel antagonist totally abolished KCl-induced Rho stimulation and contraction. The calmodulin inhibitor W7 suppressed KCl-induced Rho activation and contraction. Ionomycin mimicked W7-sensitive Rho activation. The expression of dominant-negative N19RhoA suppressed Ca2+-induced Thr695 phosphorylation of the 110-kDa regulatory subunit of myosin phosphatase and phosphorylation of myosin light chain in VSM cells. Finally, either the combination of extracellular Ca2+ removal and depletion of the intracellular Ca2+ store or the addition of W7 greatly reduced noradrenalin-induced and the thromboxane A2 analogue-induced Rho stimulation and contraction. Taken together, these results indicate the existence of the thus-far unrecognized Ca2+-dependent Rho stimulation mechanism in VSM. Excitatory receptor agonists are suggested to use this pathway for simulating Rho.     

Elastin and vascular disease

Supravalvular aortic stenosis (SVAS) is a vascular disease that primarily affects large arteries, like the aorta and pulmonary arteries. SVAS can be inherited as an isolated, autosomal dominant trait or as part of a complex developmental disorder, Williams syndrome. Molecular genetic studies indicate that mutations affecting part of an elastin allele cause autosomal dominant SVAS while submicroscopic deletions that disrupt the entire elastin gene (and presumably adjacent loci) are responsible for Williams syndrome. These studies suggest that loss of vascular elasticity from any cause may contribute to vascular obstruction.     

Phytosterols and vascular disease

Phytosterols or plant sterols have long been known to lower serum cholesterol concentrations by competing with dietary and biliary cholesterol for intestinal absorption. Food products containing phytosterols and phytostanols are now available to assist in lowering blood cholesterol levels. In contrast to these possibly beneficial effects of plant sterols, a rare genetic condition called sitosterolemia, an autosomal recessive disorder also known as phytosterolemia, is characterized by over absorption of phytosterols and premature coronary artery and aortic valve disease. Phytosterols have also recently been identified in atheromatous plaque obtained from individuals with apparently normal absorption of plant sterols raising the possibility that phytosterols are a novel atherosclerotic risk factor. This article reviews phytosterols and their relationship to vascular disease.     

Oestrogen and vascular disease

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in postmenopausal women. Epidemiological studies consistently suggest that oestrogen administered to postmenopausal women confers an estimated 30–50% reduction in risk of development and progression of CVD. The long term effect on the cardiovascular system of the addition of a progestogen to the replacement regimen is currently unknown. In addition, it may be argued that it remains to be proven whether the magnitude of the oestrogen-induced cardioprotective effect demonstrated in these observational studies is a real biological phenonumon. No prospective, randomised, controlled studies examining the effect of oestrogen on primary and secondary prevention of CVD have been completed. However, a large number of biologically plausible mechanisms have been identified which provide evidence to support the proposed oestrogen-induced cardioprotection. These include oestrogen mediated favourable changes in metabolic profile, in particular changes in lipid metabolism, insulin resistance and the fibrinolytic system. In addition, recent data have shown that oestrogen may affect vascular structure and function by a variety of mechanisms. It has been shown that oestrogen may induce acute and chronic coronary and cerebral vasodilation through both direct (vascular smooth muscle) and indirect (endothelium dependent) mechanisms. Oestrogen also has recently been shown to have complex anti-atherogenic and antioxidant properties. Much less is known of the vascular effects of progestogens. Progestogens currently in clinical use have androgenic properties and may attenuate the beneficial effects of oestrogen by neutralising or opposing the lipid lowering, vasodilatory and anti-atherogenic actions of oestrogen. Thus further studies are required to elucidate the effects on arterial physiology and CVD outcome of the oestrogens and progestogens of different types, doses and routes of administration which are collectively referred to as postmenopausal ‘hormone replacement therapy’.     

Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction

Thrombin-induced barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contraction, and gap formation. Phosphorylation of regulatory myosin light chains (MLC) is a key mechanism of endothelial cell (EC) contraction and barrier dysfunction, which is triggered by Ca(2+)/calmodulin-dependent MLC kinase (MLCK) and Rho-associated kinase (Rho-kinase). The role of MLCK in EC barrier regulation has been previously described; however, Rho-mediated pathway in thrombin-induced pulmonary EC dysfunction is not yet precisely characterized. Here, we demonstrate that thrombin-induced decreases in transendothelial electrical resistance (TER) indicating EC barrier dysfunction are universal for human and bovine pulmonary endothelium, and involve membrane translocation and direct activation of small GTPase Rho and its downstream target Rho-kinase. Transient Rho membrane translocation coincided with translocation of upstream Rho activator, guanosine nucleotide exchange factor p115-RhoGEF. Rho mediated activation of downstream target, Rho-kinase induced phosphorylation of the EC MLC phosphatase (MYPT1) at Thr(686) and Thr(850), resulting in MYPT1 inactivation, accumulation of diphospho-MLC, actin remodeling, and cell contraction. The specific Rho-kinase inhibitor, Y27632, abolished MYPT1 phosphorylation, MLC phosphorylation, significantly attenuated stress fiber formation and thrombin-induced TER decrease. Furthermore, expression of dominant-negative Rho and Rho-kinase abolished thrombin-induced stress fiber formation and MLC phosphorylation. Our data, which provide comprehensive analysis of Rho-mediated signal transduction in pulmonary EC, demonstrate involvement of guanosine nucleotide exchange factor, p115-RhoGEF, in thrombin-mediated Rho regulation, and suggest Rho, Rho-kinase, and MYPT1 as potential pharmacological and gene therapy targets critical for prevention of thrombin-induced EC barrier disruption and pulmonary edema associated with acute lung injury.     

Rho kinase as a therapeutic target in cardiovascular disease

Rho kinase (ROCK) belongs to the AGC (PKA/PKG/PKC) family of serine/threonine kinases and is a major downstream effector of the small GTPase RhoA. ROCK plays central roles in the organization of the actin cytoskeleton and is involved in a wide range of fundamental cellular functions such as contraction, adhesion, migration, proliferation and gene expression. Two ROCK isoforms, ROCK1 and ROCK2, are assumed to be functionally redundant, based largely on the major common activators, the high degree of homology within the kinase domain and studies from overexpression with kinase constructs and chemical inhibitors (e.g., Y27632 and fasudil), which inhibit both ROCK1 and ROCK2. Extensive experimental and clinical studies support a critical role for the RhoA/ROCK pathway in the vascular bed in the pathogenesis of cardiovascular diseases, in which increased ROCK activity mediates vascular smooth muscle cell hypercontraction, endothelial dysfunction, inflammatory cell recruitment and vascular remodeling. Recent experimental studies, using ROCK inhibitors or genetic mouse models, indicate that the RhoA/ROCK pathway in myocardium contributes to cardiac remodeling induced by ischemic injury or persistent hypertrophic stress, thereby leading to cardiac decompensation and heart failure. This article, based on recent molecular, cellular and animal studies, focuses on the current understanding of ROCK signaling in cardiovascular diseases and in the pathogenesis of heart failure.