Overdistention of lung tissue during mechanical ventilation may be one of the factors that initiates ventilator-induced lung injury (VILI). We hypothesized that cyclic mechanical stretch (CMS) of the lung epithelium is involved in the early events of VILI through the production of reactive oxygen species (ROS). Cultures of an immortalized human airway epithelial cell line (16HBE), a human alveolar type II cell line (A549), and primary cultures of rat alveolar type II cells were cyclically stretched, and the production of superoxide (O2-) was measured by dihydroethidium fluorescence. CMS stimulated increased production of O2- after 2 h in each type of cell. 16HBE cells exhibited no significant stimulation of ROS before 2 h of CMS (20% strain, 30 cycles/min), and ROS production returned to control levels after 24 h. Oxidation of glutathione (GSH), a cellular antioxidant, increased with CMS as measured by a decrease in the ratio of the reduced GSH level to the oxidized GSH level. Strain levels of 10% did not increase O2- production in 16HBE cells, whereas 15, 20, and 30% significantly increased generation of O2-. Rotenone, a mitochondrial complex I inhibitor, partially abrogated the stretch-induced generation of O2- after 2 h CMS in 16HBE cells. NADPH oxidase activity was increased after 2 h of CMS, contributing to the production of O2-. Increased ROS production in lung epithelial cells in response to elevated stretch may contribute to the onset of VILI.

Objectives-We have previously reported that vascular injury or treatment of cultured vascular smooth muscle cells with platelet-derived growth factor-BB (PDGF-BB) or fibroblast growth factor-2 (FGF2) increases the levels of protein tyrosine phosphatase (PTP)1B. The current study was designed to test the hypothesis that PTP1B attenuates PDGF-or FGF-induced motility and proliferation of cultured cells, as well as neointima formation in injured rat carotid arteries. Methods and Results-Treatment of cultured cells with adenovirus expressing PTP1B decreased PDGF-BB-or FGF2-induced cell motility and blocked PDGF-BB-or FGF2-induced proliferation, whereas expression of dominant negative PTP1B (C215S-PTP1B) uncovered the motogenic effect of subthreshold levels of PDGF-BB or FGF2, increased neointimal and medial cell proliferation, and induced neointimal enlargement after balloon injury. The inhibitory effect of PTP1B directed against PDGF in cultured cells was associated with dephosphorylation of the PDGF␤ receptor. Conclusions-PTP1B suppresses cell proliferation and motility in cultured smooth muscle cells treated with PDGF-BB or FGF2, and the phosphatase plays a counter-regulatory role in vascular injury-induced cell proliferation and neointima formation. Taken together with previous studies indicating increased PTP1B levels in cells treated with growth factors, the current findings are the first to report the existence of an inhibitory feedback loop involving PDGF or FGF, and PTP1B in blood vessels. Key Words: PTP1B Ⅲ growth factors Ⅲ neointima formation Ⅲ cell motility Ⅲ cell proliferation M igration and proliferation of smooth muscle cells are of critical importance in neointima formation and remodeling occurring in response to vascular injury. 1 Increased release of platelet-derived growth factor (PDGF) and/or fibroblast growth factor-2 (FGF2), followed by activation of PDGF and/or FGF2 receptor tyrosine kinase activities, are thought to be major events contributing to vascular remodeling. 2 Several reports indicate that injury-induced movement of smooth muscle cells from media to intima and the proliferation of smooth muscle cells in intima are significantly reduced by pharmacological antagonists of the function or availability of PDGF or FGF2. [3][4][5][6][7] Conversely, administration of PDGF-BB or FGF2 has been reported to enhance smooth muscle cell movement from media to intima, followed by cell proliferation in vessels with minimal endothelial damage. 7,8 These studies indicate that PDGF and FGF are important mediators of neointima formation in models of vascular injury. Tyrosyl phosphorylation of growth factor receptors via their intrinsic tyrosine kinase activities is a pivotal event for activation of downstream signaling that mediates increased motility and proliferation in cultured cells. Furthermore, balloon injury or treatment in vivo with PDGF also induces PDGF receptor tyrosyl phosphorylation in vascular smooth muscle, 6,9 a finding consistent with experiments in vitro.Protein tyrosine phosphatases ...

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Contrary to the antimotogenic effect of NO in dedifferentiated vascular smooth muscle cells (VSMCs), we have reported that NO stimulates the motility of differentiated cultured VSMC isolated from adult rats. This process involves upregulation of protein tyrosine phosphatase SHP2, followed by downregulation of RhoA activity. In the present study, we tested the hypothesis that insulin alters the motogenic phenotype of cultured rat aortic smooth muscle cells exposed to NO from inhibition to stimulation of cell motility. We demonstrate for the first time that NO stimulates the motility of VSMCs cultured for several days in the presence but not the absence of insulin. Moreover, we show that NO blocks PDGF-induced cell motility in insulin-naive but not in insulin-treated cells. We also demonstrate that the scaffold adapter protein Gab1, considered a physiological activator of protein tyrosine phosphatase SHP2, increases cell motility in the presence but not the absence of insulin. In cells cultured in the presence of insulin, overexpression of Gab1 mimics, whereas a dominant-negative allele of Gab1 (Gab1YF) blocks, the motility-stimulatory effect of NO. Cotransfection experiments with dominant-negative Gab1 and wild-type SHP2 or wild-type Gab1 and dominant-negative SHP2 indicate that the two proteins work together as a functional unit to induce motility. Because chronic insulin can increase the levels of phosphatidylinositol 3 (PI3) kinase in several models of hyperinsulinemia, we also tested the potential involvement of this enzyme in mechanisms leading to increased cell motility. We found that the motogenic effect of NO, Gab1, and SHP2 was blocked by the selective PI3 kinase inhibitor LY294002, suggesting a requirement of PI3 kinase in mediating motogenesis. These observations may be relevant to molecular mechanisms related to the pathogenesis of vascular disease in hyperinsulinemic diabetes. The full text of this article is available online at http://www.circresaha.org.

Hyperinsulinemia plays a major role in the pathogenesis of vascular disease. Restenosis occurs at an accelerated rate in hyperinsulinemia and is dependent on increased vascular smooth muscle cell movement from media to neointima. PDGF plays a critical role in mediating neointima formation in models of vascular injury. We have reported that PDGF increases the levels of protein tyrosine phosphatase PTP1B and that PTP1B suppresses PDGF-induced motility in cultured cells and that it attenuates neointima formation in injured carotid arteries. Others have reported that insulin enhances the mitogenic and motogenic effects of PDGF in cultured smooth muscle cells and that hyperinsulinemia promotes vascular remodeling. In the present study, we tested the hypothesis that insulin amplifies PDGF-induced cell motility by suppressing the expression and function of PTP1B. We found that chronic but not acute treatment of cells with insulin enhances PDGF-induced motility in differentiated cultured primary rat aortic smooth muscle cells and that it suppresses PDGF-induced upregulation of PTP1B protein. Moreover, insulin suppresses PDGF-induced upregulation of PTP1B mRNA levels, PTP1B enzyme activity, and binding of PTP1B to the PDGF receptor-beta, and it enhances PDGF-induced PDGF receptor phosphotyrosylation. Treatment with insulin induces time-dependent upregulation of phosphatidylinositol 3-kinase (PI3-kinase)-delta and activation of Akt, an enzyme downstream of PI3-kinase. Finally, inhibition of PI3-kinase activity, or its function, by pharmacological or genetic means rescues PTP1B activity in insulin-treated cells. These observations uncover novel mechanisms that explain how insulin amplifies the motogenic capacity of the pivotal growth factor PDGF.