We examined the effect of hypoxia on proliferation and osteopontin (OPN) expression in cultured rat aortic vascular smooth muscle (VSM) cells. In addition, we determined whether hypoxia-induced increases in OPN and cell proliferation are altered under hyperglycemic conditions. Quiescent cultures of VSM cells were exposed to hypoxia (3% O 2 ) or normoxia (18% O 2 ) in a serum-free medium, and cell proliferation as well as the expression of OPN was assessed. Cells exposed to hypoxia for 24 h exhibited a significant increase in [ 3 H]thymidine incorporation followed by a significant increase in cell number at 48 h in comparison with respective normoxic controls. Exposure to hypoxia produced significant increases in OPN protein and mRNA expression at 2 h followed by a gradual decline at 6 and 12 h, with subsequent significant increases at 24 h. Neutralizing antibodies to either OPN or its receptor ␤3 integrin but not neutralizing antibodies to ␤5 integrin prevented the hypoxia-induced increase in T he first descriptions of a link between systemic hypoxia and pulmonary artery smooth muscle cell proliferation came from both in vivo and in vitro models of pulmonary hypertension (1-3). Increased proliferation of aortic vascular smooth muscle (VSM) cells is also a key feature in the progression of atherosclerosis (4). Both systemic and local hypoxia contributes to the development of atherosclerotic lesions (5-11). Recent in vivo studies found a direct correlation of local arterial wall hypoxia, VSM cell proliferation, and atherosclerosis (12,13). However, the underlying signaling mechanisms whereby hypoxia induces VSM cell proliferation and subsequent atherosclerotic lesions remain poorly defined. Part of the problem has been in the demonstration of a mitogenic effect of hypoxia in cultured VSM cells in vitro. Hypoxia has only been shown to induce the proliferation of bovine pulmonary artery smooth muscle cells in culture when they are costimulated either with an activator of protein kinase C (PKC) or serum (3,14). To examine the effect of local hypoxia on cell proliferation, our laboratory has developed an appropriate cell culture model system in which cultured cells exhibited differentiated morphology and function by improved oxygenation (15). Using this culture model, we reported that hypoxia induces proliferation, dedifferentiation, and/or extracellular matrix synthesis in cultured renal tubular epithelial and glomerular mesangial cells (16 -18). In the present study, we examined the effect of hypoxia on the proliferation of cultured rat aortic VSM cells to determine whether hypoxia directly alters their proliferative behavior.Enhanced proliferation of VSM cells has also been demonstrated in both human and experimental models of diabetes (19,20). In addition, cultured VSM cells grown in high media glucose concentration (to mimic hyperglycemia of diabetes) have exhibited increased cell proliferation (21). The pathophysiological mechanisms responsible for accelerated VSM cell proliferation and progression into diabetic...

The effect of hypoxia on the proliferation and collagen synthesis of cultured rat mesangial cells was examined under normal-glucose (NG, 5 mM) and high-glucose (HG, 25 mM)-media conditions. In addition, a role for osteopontin (OPN) in mediating these processes was assessed. Quiescent cultures were exposed to hypoxia (3% O(2)) and normoxia (18% O(2)) in a serum-free medium with NG or HG, and cell proliferation, collagen synthesis, and OPN expression were assessed. Cells exposed to hypoxia in NG medium resulted in significant increases in [(3)H]thymidine incorporation, cell number, and [(3)H]proline incorporation, respectively. HG incubations also produced significant stimulation of these parameters under normoxic conditions, which were markedly enhanced in cells exposed to hypoxia in HG medium. In addition, hypoxia and HG stimulated the mRNA levels of type IV collagen, and the combination of hypoxia and HG resulted in additive increases in type IV collagen expression. Hypoxia and HG also stimulated OPN mRNA and protein levels in an additive fashion. A neutralizing antibody to OPN or its beta(3)-integrin receptor significantly blocked the effect of hypoxia and HG on proliferation and collagen synthesis. In conclusion, these results demonstrate for the first time that hypoxia in HG medium produces exaggerated mesangial cell growth and type IV collagen synthesis. In addition, OPN appears to play a role in mediating the accelerated mesangial cell growth and collagen synthesis found in a hyperglycemic and hypoxic environment.