Thymosin beta4 regulation, expression and function in aortic valve interstitial cells.

BACKGROUND AND AIMS OF THE STUDY: Previous research has demonstrated that tenascin-C, an extracellular matrix protein involved in bone development and mineralization, was specifically present in calcified aortic valves, always in association with matrix metalloproteinase (MMP)-2, and was not detectable in non-calcified human aortic valves. The aim of the present study was to identify downstream targets of tenascin-C in aortic valve interstitial cells. METHODS: Subtractive hybridization was performed using sheep aortic valve interstitial cells (SAVIC) grown on a substrate of collagen plus tenascin-C, versus cells grown on type I collagen alone. RESULTS: Subtractive hybridizations revealed that nearly 70% of the clones isolated contained the sequence for thymosin beta-4, an actin-binding protein, also associated with mineralization, regulation of MMPs and inflammation. In cell culture studies, it was shown that both thymosin beta4 and thymosin beta4 sulfoxide are produced by SAVIC. When aortic valve interstitial cells were grown on a substrate of tenascin-C, thymosin beta4 expression was up-regulated. The addition of thymosin beta4 to aortic valve interstitial cell cultures resulted in a re-orientation of cytoskeletal F-actin, and induction of MMP-2. However, thymosin beta4 antisense oligonucleotide transfection did not suppress MMP-2 expression. CONCLUSION: Thymosin beta4 is up-regulated by tenascin-C, and may be involved in the primary initiation of valvular calcification.

Novel Rho kinase inhibitors with anti-inflammatory and vasodilatory activities.

Increased Rho kinase (ROCK) activity contributes to smooth muscle contraction and regulates blood pressure homeostasis. We hypothesized that potent and selective ROCK inhibitors with novel structural motifs would help elucidate the functional role of ROCK and further explore the therapeutic potential of ROCK inhibition for hypertension. In this article, we characterized two aminofurazan-based inhibitors, GSK269962A [N-(3-{[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4, 5-c]pyridin-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]-oxy}benzamide] and SB-7720770-B [4-(7-{[(3S)-3-amino-1-pyrrolidinyl]carbonyl}-1-ethyl-1H-imidazo[4,5-c]pyridin-2-yl)-1,2,5-oxadiazol-3-amine], as members of a novel class of compounds that potently inhibit ROCK enzymatic activity. GSK269962A and SB-772077-B have IC50 values of 1.6 and 5.6 nM toward recombinant human ROCK1, respectively. GSK269962A also exhibited more than 30-fold selectivity against a panel of serine/threonine kinases. In lipopolysaccharide-stimulated monocytes, these inhibitors blocked the generation of inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-alpha. Furthermore, both SB-772077-B and GSK269962A induced vasorelaxation in preconstricted rat aorta with an IC50 of 39 and 35 nM, respectively. Oral administration of either GSK269962A or SB-772077-B produced a profound dose-dependent reduction of systemic blood pressure in spontaneously hypertensive rats. At doses of 1, 3, and 30 mg/kg, both compounds induced a reduction in blood pressure of approximately 10, 20, and 50 mm Hg. In addition, administration of SB-772077-B also dramatically lowered blood pressure in DOCA salt-induced hypertensive rats. SB-772077-B and GSK269962A represent a novel class of ROCK inhibitors that have profound effects in the vasculature and may enable us to further evaluate the potential beneficial effects of ROCK inhibition in animal models of cardiovascular as well as other chronic diseases.