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The intracellular signaling events by which NADPH oxidase-generated reactive oxygen species (ROS) modulate vascular smooth muscle cell (VSMC) function and atherogenesis are yet to be entirely elucidated. We previously demonstrated that NADPH oxidase deficiency decreased atherosclerosis in apoE(-/-) mice and identified adhesion protein CD44 as an important ROS-sensitive gene expressed in VSMC and atherosclerotic lesions. Here, we examined the molecular mechanisms by which NADPH oxidase-generated ROS regulate the expression of CD44 and its principal ligand, hyaluronan (HA), and how CD44-HA interaction affects VSMC proliferation and migration and inflammatory gene expression in apoE(-/-) mice aortas. Thrombin-induced CD44 expression is mediated by transcription factor AP-1 in a NADPH oxidase-dependent manner. NADPH oxidase-mediated ROS generation enhanced thrombin-induced HA synthesis, and hyaluronan synthase 2 expression in VSMC. Hyaluronidase, which generates low molecular weight HA (LMW-HA), is induced in VSMC in a NADPH oxidase-dependent manner and LMW-HA stimulated ROS generation and cell proliferation in wild-type but not p47(phox-/-) VSMC, effects that were enhanced by thrombin pretreatment. Haptotactic VSMC migration toward HA was increased by thrombin in a CD44-dependent manner. HA expression in atherosclerotic lesions and plasma-soluble CD44 and HA levels were higher in apoE(-/-) compared with apoE(-/-)/p47(phox-/-) mice. HA-regulated pro-inflammatory gene expression was higher in apoE(-/-) than apoE(-/-)/p47(phox-/-) mouse aortas. GKT136901, a specific inhibitor of Nox1- and Nox4-containing NADPH oxidase activity, attenuated ROS generation and atherosclerosis and decreased CD44 and HA expression in atherosclerotic lesions. Together, these data suggest that increased CD44 and HA expression and CD44-HA-dependent gene regulation may play a role in atherosclerosis stimulated by NADPH oxidase activation.

Reactive oxygen species (ROS) have been implicated in vascular smooth muscle cell (VSMC) apoptosis, a hallmark of advanced atherosclerotic lesions. Transient oxidation and inactivation of protein-tyrosine phosphatases play a critical role in cellular response to ROS production. However, the function of leukocyte antigen-related (LAR) protein-tyrosine phosphatase in ROS signaling is not known. To determine the expression of LAR in ROS-induced apoptosis, we investigated hydrogen peroxide-induced cell death and signaling in aortic VSMCs from wild-type and LAR(-/-) mice. Histone-associated DNA fragmentation and caspase-3/7 activity were significantly enhanced, mitochondrial membrane integrity was compromised, and cell viability was significantly decreased following H(2)O(2) treatment in LAR(-/-) VSMCs compared with wild-type cells. Stronger and sustained increase in autophosphorylation and activity of Fyn, an Src family tyrosine kinase, was observed in LAR(-/-) cells compared with wild-type cells following H(2)O(2) treatment. LAR binds to activated Fyn in H(2)O(2)-treated VSMCs, and recombinant LAR dephosphorylates phosphorylated-Fyn in vitro. In addition, LAR deficiency enhanced H(2)O(2)-induced phosphorylation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), and p38 mitogen-activated protein kinase (MAPK). PP2, a Fyn-specific inhibitor, blocked JAK2, STAT3, and p38 MAPK activation and significantly attenuated apoptosis induced by H(2)O(2). AG490, a JAK2-specific inhibitor, significantly attenuated H(2)O(2)-induced apoptosis, and blocked H(2)O(2)-induced activation of STAT3, but not p38 MAPK in both wild-type and LAR(-/-) VSMCs. Attenuation of Fyn expression by short hairpin RNA significantly decreased H(2)O(2)-induced downstream signaling and apoptosis in VSMCs. Together, these data indicate that LAR regulates Fyn/JAK2/STAT3 and Fyn/p38 MAPK pathways involved in ROS-induced apoptosis.

The development of therapeutic strategies to inhibit reactive oxygen species (ROS)-mediated damage in blood vessels has been limited by a lack of specific targets for intervention. Targeting ROS-mediated events in the vessel wall is of interest, because ROS play important roles throughout atherogenesis. In early atherosclerosis, ROS stimulate vascular smooth muscle cell (VSMC) growth, whereas in late stages of lesion development, ROS induce VSMC apoptosis, causing atherosclerotic plaque instability. To identify putative protective genes against oxidative stress, mouse aortic VSMC were infected with a retroviral human heart cDNA expression library, and apoptosis was induced in virus-infected cells by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) treatment. A total of 17 different, complete cDNAs were identified from the DMNQ-resistant VSMC clones by PCR amplification and sequencing. The cDNA encoding PP1cgamma1 (catalytic subunit of protein phosphatase 1) was present in several independent DMNQ-resistant VSMC clones. DMNQ increased mitochondrial ROS production, caspase-3/7 activity, DNA fragmentation, and decreased mitochondrial transmembrane potential in VSMC while decreasing PP1cgamma1 activity and expression. Depletion of PP1cgamma1 expression by short hairpin RNA significantly enhanced basal as well as DMNQ-induced VSMC apoptosis. PP1cgamma1 overexpression abrogated DMNQ-induced JNK1 activity, p53 Ser(15) phosphorylation, and Bax expression and protected VSMC against DMNQ-induced apoptosis. In addition, PP1cgamma1 overexpression attenuated DMNQ-induced caspase-3/7 activation and DNA fragmentation. Inhibition of p53 protein expression using small interfering RNA abrogated DMNQ-induced Bax expression and significantly attenuated VSMC apoptosis. Together, these data indicate that PP1cgamma1 overexpression promotes VSMC survival by interfering with JNK1 and p53 phosphorylation cascades involved in apoptosis.

Increase in the expression of leukocyte antigen-related (LAR) protein causes insulin resistance, an important contributor to atherosclerosis. However, the function of LAR in atherosclerosis is not known. To address whether LAR is important in the response of vascular cells to atherogenic stimuli, we investigated cell proliferation, migration, and insulin-like growth factor-1 receptor (IGF-1R) signaling in wild-type and LAR(-/-) mouse vascular smooth muscle cells (VSMC) treated with IGF-1. Absence of LAR significantly enhanced proliferation and migration of VSMC compared with wild-type cells after IGF-1 treatment. U0126 and LY249002, specific inhibitors of MAPK/ERK kinase (MEK) and phosphoinositide 3-kinase, respectively, inhibited IGF-1-induced DNA synthesis and migration in both wild-type and LAR(-/-) VSMC. IGF-1 markedly enhanced IGF-1R phosphorylation in both wild-type and LAR(-/-) VSMC, but the phosphorylation was 90% higher in knock-out cells compared with wild-type cells. Absence of LAR enhanced phosphorylation of insulin receptor substrate-1 and insulin receptor substrate-1-associated phosphoinositide 3-kinase activity in VSMC treated with IGF-1. IGF-1-induced phosphorylation of ERK1/2 also increased significantly in LAR(-/-) VSMC compared with wild-type cells. Furthermore, LAR directly binds to IGF-1R in glutathione S-transferase-LAR pull-down and IGF-1R immunoprecipitation experiments and recombinant LAR dephosphorylates IGF-1R in vitro. Neointima formation in response to arterial injury and IGF-1R phosphorylation in neointima increased significantly in LAR(-/-) mice compared with wild-type mice. A significant decrease in body weight, fasting insulin, and IGF-1 levels were observed in LAR(-/-) mice compared with wild-type mice. Together, these data indicate that LAR regulates IGF-1R signaling in VSMC and dysregulation of this phosphatase may lead to VSMC hyperplasia.

Tie2 is an endothelial receptor tyrosine kinase that is required for both embryonic vascular development and tumor angiogenesis. There is considerable interest in understanding the mechanisms of Tie2 activation for therapeutic purposes. The recent solution of the Tie2 crystal structure suggests that Tie2 activity is autoinhibited by its carboxyl terminus. Here we investigated the role of the C tail in Tie2 activation, signaling, and function both in vitro and in vivo by deleting the C terminus of Tie2 (Delta CT). Compared to wild type Tie2, in vitro autophosphorylation and kinase activity were significantly enhanced by the Delta CT mutation. In NIH 3T3 cells expressing chimeric Tie2 receptors, both basal and ligand-induced tyrosine phosphorylation were markedly enhanced compared to wild type in several independent clones of Tie2-Delta CT. Moreover, the Delta CT mutation enhanced basal and ligand-dependent activation of Akt and extracellular signal-regulated kinase. Enhanced Akt activation correlated with significant inhibition of staurosporine-induced apoptosis. These findings demonstrate that the Tie2 C tail performs a novel negative regulatory role in Tie2 signaling and function, and they provide important insights into the mechanisms by which the Tie2 kinase is activated.