RhoA guanine exchange factor expression profile in arteries: evidence for a Rho kinase-dependent negative feedback in angiotensin II-dependent hypertension.

Sustained overactivation of RhoA is a common component for the pathogenesis of several cardiovascular disorders, including hypertension. Although activity of Rho proteins depends on Rho exchange factors (Rho-GEFs), the identity of Rho-GEFs expressed in vascular smooth muscle cells (VSMC) and participating in the control of Rho protein activity and Rho-dependent functions remains unknown. To address this question, we analyzed by quantitative RT-PCR the expression profile of 28 RhoA-GEFs in arteries of normotensive (saline-treated) and hypertensive (ANG II-treated) rats. Sixteen RhoA-GEFs were downregulated in mesenteric arteries of hypertensive rats, among which nine are also downregulated in cultured VSMC stimulated by ANG II (100 nM, 48 h), suggesting a direct effect of ANG II. Inhibition of type 1 ANG II receptors (losartan, 1 µM) or Rho kinase (fasudil, 10 µM) prevented ANG II-induced RhoA-GEF downregulation. Functionally, ANG II-induced downregulation of RhoA-GEFs is associated with decreased Rho kinase activation in response to endothelin-1, norepinephrine, and U-46619. This work thus identifies a group of RhoA-GEFs that controls RhoA and RhoA-dependent functions in VSMC, and a negative feedback of RhoA/Rho kinase activity on the expression of these RhoA-GEFs that may play an adaptative role to limit RhoA/Rho kinase activation.

Identification of differentially expressed genes in human varicose veins: involvement of matrix gla protein in extracellular matrix remodeling.

This study was designed to identify the global pattern of differentially expressed genes in human varicose veins. Using suppressive subtractive hybridization, we identified overexpression of genes known to be associated with extracellular matrix remodeling, including collagen III, tissue inhibitor of metalloproteinases I, dermatopontin, matrix Gla protein (MGP) and tenascin C. Real-time polymerase chain reaction analysis confirmed the differential expression of these genes. The overexpression of MGP transcript was associated with increased MGP level in varicose veins, in particular the undercarboxylated form of the protein. Smooth muscle cells from varicose veins showed increased proliferation rate and enhanced matrix mineralization. This observation correlated with the presence of ectopic mineralization areas in the varicose vein walls. The use of warfarin, to inhibit MGP activity, or siRNA targeting MGP transcript induced a reduction in the exacerbated proliferation of varicose vein smooth muscle cells. Our results suggest that high expression of MGP in varicose veins may contribute to venous wall remodeling by affecting proliferation and mineralization processes probably through impaired carboxylation of MGP. In addition, suppressive subtractive hybridization results also produce a profile of differentially expressed genes in varicose veins, in particular extracellular matrix components. Further study of these genes will provide insights into their specific roles in the etiology of venous disease.

Role of Rho kinase signalling in healthy and varicose human saphenous veins.

1. The present study was performed to determine the role of Rho-Rho kinase signalling pathway in smooth muscle cells from both healthy and varicose human saphenous vein. 2. The Rho kinase inhibitor Y-27632 inhibited the noradrenaline (NA)-induced contraction in human saphenous veins with IC(50) corresponding to 0.5 microM and 10.9 microM in control and varicose veins, respectively. The maximal amplitude of the NA-induced contraction was smaller in varicose vein compared to control (1263+/-172 mg versus 1974+/-245 mg, P<0.05). 3. In beta-escin permeabilized strips, GTPgammaS induced a rise in tension that was inhibited by Y-27632. The amplitude of the GTPgammaS-induced contraction was smaller in varicose compared to control veins (23.1+/-2.4% versus 41.3+/-2.2%, P<0.002). 4. In smooth muscle cells, Y-27632 induced disassembly of both actin cytoskeleton and extracellular fibronectin matrix. In comparison to control cells, varicose vein smooth muscle cells show decreased actin cytoskeleton organization and reduction of fibronectin matrix deposition. 5. The Rho proteins Rnd1 and RhoA, and Rho kinase 1 are expressed in human saphenous veins. A 2.6 fold reduction of Rho kinase expression was found in varicose veins. 6. These results indicate that RhoA-Rho kinase mediated Ca(2+) sensitization of the contraction and regulated actin cytoskeleton and extracellular fibronectin matrix assembly in human saphenous smooth muscle. The decrease of Rho kinase expression and Rho kinase-dependent functions detected in smooth muscle from varicose veins supports a role of this signalling pathway in the functional alterations of the vein wall occurring in the course of the disease.

Fetal muscle contains different CD34+ cell subsets that distinctly differentiate into adipogenic, angiogenic and myogenic lineages.

We have previously demonstrated that CD34(+) cells isolated from fetal mouse muscles are an interesting source of myogenic progenitors. In the present work, we pinpoint the tissue location of these CD34(+) cells using cell surface and phenotype markers. In order to identify the myogenic population, we next purified different CD34(+) subsets, determined their expression of relevant lineage-related genes, and analyzed their differentiation capacities in vitro and in vivo. The CD34(+) population comprised a CD31(+)/CD45(-) cell subset exhibiting endothelial characteristics and only capable of forming microvessels in vivo. The CD34(+)/CD31(-)/CD45(-)/Sca1(+) subpopulation, which is restricted to the muscle epimysium, displayed adipogenic differentiation both in vitro and in vivo. CD34(+)/CD31(-)/CD45(-)/Sca1(-) cells, localized in the muscle interstitium, transcribed myogenic genes, but did not display the characteristics of adult satellite cells. These cells were distinct from pericytes and fibroblasts. They were myogenic in vitro, and efficiently contributed to skeletal muscle regeneration in vivo, although their myogenic potential was lower than that of the unfractionated CD34(+) cell population. Our results indicate that angiogenic and adipogenic cells grafted with myogenic cells enhance their contribution to myogenic regeneration, highlighting the fundamental role of the microenvironment on the fate of transplanted cells.

The Rho protein exchange factor Vav3 regulates vascular smooth muscle cell proliferation and migration.

AIMS: Rho guanine nucleotide exchange factors (Rho GEFs) are responsible for Rho protein activation by catalyzing the exchange of GDP for GTP. Although overactivation of Rho proteins is a common component of the pathogenesis of vascular disorders, the molecular mechanisms and the Rho GEFs regulating Rho protein activity and Rho-dependent functions in vascular smooth muscle cells (VSMC) are still unknown. The aim of this study was thus to identify Rho GEFs involved in the regulation of VSMC functions. METHODS AND RESULTS: By a functional screening based on small interfering RNA (siRNA)-mediated silencing of 27 Rho GEFs, we found that only silencing of the Rho GEF Vav3 inhibited rat VSMC proliferation. Conversely, overexpression of Vav3 potentiated VSMC proliferation, whereas the catalytically inactive Vav3 mutant had no effect. The stimulatory effect of Vav3 on VSMC proliferation was inhibited by the Src tyrosine kinase inhibitor SU6656 and by co-expression of the dominant-negative Rac1-N17 mutant. In agreement with this observation, expression of Vav3 induced enrichment of Rac1 to the membrane, activation of its effector PAK, and stimulated VSMC migration. Increased levels of Vav3 transcripts were found in stented arteries and arteries from hypertensive rats. Furthermore, siRNA targeting Vav3 decreased arterial wall cell proliferation. CONCLUSION: The Rho GEF Vav3 controls VSMC proliferation and migration through activation of Rac1/PAK signalling. Vav3 is a convergent point mediating Rac1 activation in response to different upstream mediators that promote VSMC proliferation and migration and thus appears to be a new potential therapeutic target that could be used to limit vascular proliferative diseases.

Rho kinase blockade prevents inflammation via nuclear factor kappa B inhibition: evidence in Crohn's disease and experimental colitis.

BACKGROUND & AIMS: Rho proteins are involved in the regulation of several cellular functions. Data from in vitro studies suggest that RhoA could be involved in the inflammatory response. We investigated the role of RhoA and its downstream effector Rho kinase in intestinal inflammation. METHODS: Activation of RhoA was assessed by pull-down assays. A specific inhibitor of Rho kinase, Y-27632, was used to examine the role of Rho kinase in inflammatory response in vivo and in vitro by molecular biology and by immunological and biochemical approaches. RESULTS: Increased activation of RhoA was found in inflamed intestinal mucosa of patients with Crohn's disease and of rats with 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Oral administration of Y-27632 in rats significantly reduced the colonic inflammation. In vitro, activation of RhoA alone was sufficient to induce tumor necrosis factor production. Y-27632 inhibited production of tumor necrosis factor-alpha and interleukin-1 beta by lamina propria and peripheral blood mononuclear cells. Rho kinase inhibition prevented nuclear factor kappa B activation and I-kappa B phosphorylation and degradation. We showed that Rho kinase associates with and activates I-kappa B kinase alpha and that Y-27632 prevents I-kappa B kinase activation. CONCLUSIONS: Our study provides the first evidence that Rho kinase activates I-kappa B kinase and, thus, nuclear factor kappa B, suggesting a key role of Rho kinase in inflammatory responses and intestinal inflammation. Specific inhibition of Rho kinase may be a promising approach for the treatment of patients with Crohn's disease.