Jeremy D. Ollerenshaw

The signaling pathways involved in the long-term metabolic effects of angiotensin II (Ang II) in vascular smooth muscle cells are incompletely understood but include the generation of molecules likely to affect oxidase activity. We examined the ability of Ang II to stimulate superoxide anion formation and investigated the identity of the oxidases responsible for its production. Treatment of vascular smooth muscle cells with Ang II for 4 to 6 hours caused a 2.7 +/- 0.4-fold increase in intracellular superoxide anion formation as detected by lucigenin assay. This superoxide appeared to result from activation of both the NADPH and NADH oxidases. NADPH oxidase activity increased from 3.23 +/- 0.61 to 11.80 +/- 1.72 nmol O2-/min per milligram protein after 4 hours of Ang II, whereas NADH oxidase activity increased from 16.76 +/- 2.13 to 45.00 +/- 4.57 nmol O2-/min per milligram protein. The NADPH oxidase activity was stimulated by exogenous phosphatidic and arachidonic acids and was partially inhibited by the specific inhibitor diphenylene iodinium. NADH oxidase activity was increased by arachidonic and linoleic acids, was insensitive to exogenous phosphatidic acid, and was inhibited by high concentrations of quinacrine. Both of these oxidases appear to reside in the plasma membrane, on the basis of migration of the activity after cellular fractionation and their apparent insensitivity to the mitochondrial poison KCN. These observations suggest that Ang II specifically activates enzyme systems that promote superoxide generation and raise the possibility that these pathways function as second messengers for long-term responses, such as hypertrophy or hyperplasia.

Blood vessels respond to injury by initiating cell proliferation and migration that result in vascular lesion formation. To determine the roles of thrombin and the thrombin receptor in this process, we characterized thrombin receptor expression in normal and injured arteries, thrombin receptor-mediated smooth muscle cell mitogenesis, and the regulation of thrombin receptor mRNA expression in vitro. Thrombin receptor mRNA was not detected in normal rat or baboon arteries by in situ hybridization. Immunohistochemistry using an antithrombin receptor antibody (TR-R9), directed against the thrombin cleavage site of the rat aortic smooth muscle cell thrombin receptor, revealed low-level staining for thrombin receptor protein in endothelial cells and smooth muscle cells of normal arteries. In contrast, balloon catheter injury increased thrombin mRNA expression in medial smooth muscle cells within 6 hours. This increased thrombin receptor expression continued within the media and in neointimal cells throughout vascular lesion formation, predominantly in areas of active cell proliferation. In vitro, alpha-thrombin stimulates rat aortic smooth muscle cell proliferation in a concentration-dependent manner. That thrombin receptor activation is required for the mitogenic response was confirmed by demonstrating that the polyclonal antibody TR-R9 inhibits thrombin-induced cell proliferation. Thrombin receptor mRNA synthesis was induced by both basic fibroblast growth factor (maximal stimulation of 1.8-fold at 1 hour) and platelet-derived growth factor (maximal stimulation of 2.4-fold at 8 and 24 hours) in quiesced cultured rat aortic smooth muscle cells. In summary, upregulation of smooth muscle cell thrombin receptor expression occurs very early after vascular injury and continues throughout neointimal development.(ABSTRACT TRUNCATED AT 250 WORDS)

The objective of this study was to investigate if function and durability of connective tissue grafts stems from in vivo revascularization and recellularization. Viability is important for durable valve performance, demonstrated by pulmonary autografts. A pattern of in vivo recellularization occurs in xenogeneic or allogeneic heart valves decellularized prior to implantation, dictated by the tissue matrix and functional biomechanics. Porcine or sheep heart valves were decellularized with the SynerGraft antigen reduction process (a common treatment process to remove all histologically demonstrable leaflet cells), and implanted as pulmonary (n = 11) or aortic valve (n = 9) replacements in sheep. Sheep allograft pulmonary valves (n = 4) were implanted as pulmonary valve replacements. Recellularization was evaluated histologically after 3, 4, 5, 6, and 11 months, with cell phenotypes identified using specific antibodies. SynerGraft heart valves were progressively recellularized beginning with an initial cellular infiltrate, and subsequent repopulation with mature interstitial cells. This process occurs in the conduit and then in the leaflet, and is associated with revascularization of the graft. Functional, fully developed fibrocytes, actively synthesizing type I procollagen (antibody probe) were present within 3 months. As the process matured cell density and distribution became similar to native valve leaflets with localization of smooth muscle actin positive cells at the ventricularis/spongiosa interface. After 11 months, leaflet explants had no detectable inflammatory cells, were as much as 80% repopulated, and had a distribution of smooth muscle actin positive cells similar to that of the natural leaflet. SynerGraft- treated heart valve implants are repopulated by a process typical of adaptive remodeling following implantation. This antigen reduction treatment is the first successful tissue engineering effort obtaining an implant with mature recipient cells capable of matrix protein synthesis. Normal early valve function and durability is maintained.

Agonist-induced receptor phosphorylation plays a role in transmembrane signal transduction systems. Although the cDNA for the rat vascular type 1 angiotensin II receptor (AT1AR) encodes a G protein-coupled receptor with several potential phosphorylation sites for serine/threonine and tyrosine kinases, little is known about the phosphorylation of this receptor. The aim of this study was to determine the effects of angiotensin II (Ang II) on phosphorylation of the AT1AR in rat aortic vascular smooth muscle cells. Using [32P]orthophosphate-labeled cells, immunoprecipitates with anti-AT1AR antibody revealed a labeled band of molecular weight 52 kD, corresponding to the Ang II receptor. Ang II induced a rapid and significant increase in phosphorylation of the Ang II receptor, with a peak at 20 minutes. Phosphoamino acid analysis showed that the major phosphoamino acid is serine, in both the basal and Ang II-stimulated states. Constitutive and agonist-stimulated tyrosine phosphorylation is also observed to a lesser extent. Immunoblotting of anti-phosphotyrosine immunoprecipitates with anti-AT1AR antibody showed that Ang II caused a delayed tyrosine phosphorylation of the receptor with a peak at 20 minutes in a dose-dependent manner. Forskolin increased total phosphorylation of AT1AR but had no effect on tyrosine phosphorylation. Neither phorbol 12-myristate-13-acetate nor ionomycin altered receptor phosphorylation. These findings suggest that Ang II induces the phosphorylation of its own G protein-coupled receptor through both serine and tyrosine kinases and raise the possibility that phosphorylation of the AT1AR is an important regulator of receptor function.