To determine the role of phosphoinositide 3-OH kinase (PI3K) pathways in the acute vascular permeability increase associated with ventilator-induced lung injury, we ventilated isolated perfused lungs and intact C57BL/6 mice with low and high peak inflation pressures (PIP). In isolated lungs, filtration coefficients ( Kf) increased significantly after ventilation at 30 cmH2O (high PIP) for successive periods of 15, 30 (4.1-fold), and 50 (5.4-fold) min. Pretreatment with 50 μM of the PI3K inhibitor, LY-294002, or 20 μM PP2, a Src kinase inhibitor, significantly attenuated the increase in Kf, whereas 10 μM Akt inhibitor IV significantly augmented the increased Kf. There were no significant differences in Kf or lung wet-to-dry weight (W/D) ratios between groups ventilated with 9 cmH2O PIP (low PIP), with or without inhibitor treatment. Total lung β-catenin was unchanged in any low PIP isolated lung group, but Akt inhibition during high PIP ventilation significantly decreased total β-catenin by 86%. Ventilation of intact mice with 55 cmH2O PIP for up to 60 min also increased lung vascular permeability, indicated by increases in lung lavage albumin concentration and lung W/D ratios. In these lungs, tyrosine phosphorylation of β-catenin and serine/threonine phosphorylation of Akt, glycogen synthase kinase 3β (GSK3β), and ERK1/2 increased significantly with peak effects at 60 min. Thus mechanical stress activation of PI3K and Src may increase lung vascular permeability through tyrosine phosphorylation, but simultaneous activation of the PI3K-Akt-GSK3β pathway tends to limit this permeability response, possibly by preserving cellular β-catenin.

Objective We hypothesized that cofilin activation by members of the slingshot (SSH) phosphatase family is a key mechanism regulating VSMC migration and neoinitima formation following vascular injury. Methods and Results Scratch wound and modified Boyden chamber assays were used to assess VSMC migration following downregulation of the expression of cofilin and each slingshot phosphatase isoforms (SSH1,-2,-3) by siRNA, respectively. Cofilin siRNA greatly attenuated the ability of VSMC migration into the “wound” and PDGF-induced migration was virtually eliminated versus a 3.5-fold increase in non-treated VSMCs, establishing a critical role for cofilin in VSMC migration. Cofilin activation (dephosphorylation) was increased in PDGF-stimulated VSMCs. Thus, we assessed the role of the SSH family of phosphatases on cofilin activation and VSMC migration. Treatment with either SSH1 or SSH2 siRNA attenuated cofilin activation, while SSH3 siRNA had no effect. Only SSH1 siRNA significantly reduced wound healing and PDGF-induced VSMC migration. Both SSH1 (4.7 fold) and cofilin (3.9 fold) expression were increased in balloon injured versus non-injured carotid arteries and expression was prevalent in the neointima. Conclusions These studies demonstrate that the regulation of VSMC migration by cofilin is SSH1 dependent, and that this mechanism potentially contributes to neointima formation following vascular injury in vivo.

We hypothesized that transgenic mice overexpressing the p22phox subunit of the NADPH oxidase selectively in smooth muscle (Tgp22smc) would exhibit an exacerbated response to transluminal carotid injury compared to wild-type mice. To examine the role of reactive oxygen species (ROS) as a mediator of vascular injury, the injury response was quantified by measuring wall thickness (WT) and cross-sectional wall area (CSWA) of the injured and noninjured arteries in both Tgp22smc and wild-type animals at days 3, 7, and 14 after injury. Akt, p38 MAPK, and Src activation were evaluated at the same time points using Western blotting. WT and CSWA following injury were significantly greater in Tgp22smc mice at both 7 and 14 days after injury while noninjured contralateral carotids were similar between groups. Apocynin treatment attenuated the injury response in both groups and rendered the response similar between Tgp22smc mice and wild-type mice. Following injury, carotid arteries from Tgp22smc mice demonstrated elevated activation of Akt at day 3, while p38 MAPK and Src activation was elevated at day 7 compared to wild-type mice. Both increased activation and temporal regulation of these signaling pathways may contribute to enhanced vascular growth in response to injury in this transgenic model of elevated vascular ROS.

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These studies tested the hypothesis that transgenic mice overexpressing the p22phox subunit of the NAD(P)H oxidase selectively in smooth muscle (Tgp22smc) would exhibit exacerbated neointimal formation following mechanically induced transluminal vascular injury compared to wild‐type control mice. Tgp22smc mice have a two‐fold increase in p22phox expression and H2O2 production; thus providing a model to examine the effects of elevated vascular ROS production on VSMC function in vivo. To induce vascular injury, a 0.36 mm steel catheter was guided 4–5 mm into the left common carotid via the external carotid. Following one minute the catheter was removed and reperfusion verified. The degree of neointimal formation was assessed by morphological analysis of wall thickness (WT) and cross‐sectional wall area (CSWA) in male Tgp22smc mice and littermate controls 14 days following injury. WT following injury indicated significantly greater neoinitimal formation in Tgp22smc mice (54.3 ± 7.3 vs. 43.7 ± 4.1 μm), while non‐injured contralateral carotids were similar between groups (28.5 ± 3.3 vs. 33.8 ± 2.8 μm). Moreover, the increase in carotid artery CSWA following injury was approximately four‐fold greater in Tgp22smc mice compared to control animals. Findings from these in vivo studies strongly support in vitro data which has implicated ROS as a crucial mediator of both VSMC proliferation and migration. AHA 03300119N