Abstract-Unidirectional laminar flow is atheroprotective, in part by inhibiting cytokine-mediated endothelial cell (EC) inflammation and apoptosis. Previously, we showed that flow inhibited TNF-␣ signaling by preventing activation of JNK. Recently, PKC was identified as the PKC isoform most strongly regulated by flow pattern, with increased PKC activity in regions of disturbed flow versus unidirectional flow. Interestingly, PKC is cleaved by caspases after TNF-␣ stimulation to generate a 50-kDa truncated form (CAT, catalytic domain of PKC) with a higher kinase activity than the full-length protein. We hypothesized that flow would inhibit TNF-␣-mediated PKC cleavage and thereby CAT formation. We found that PKC activity was required for TNF-␣-mediated JNK and caspase-3 activation in ECs. PKC was rapidly cleaved to generate CAT in cultured bovine and human aortic ECs and in intact rabbit vessels stimulated with TNF-␣. This truncated form of PKC enhanced JNK and caspase-3 activation. Interestingly, PKC cleavage was prevented by inhibitors of PKC, JNK, and caspase activities, suggesting that these enzymes, via regulating CAT formation, modulate caspase-3 activity in ECs. Finally, we found that flow reduced caspase-dependent processing of PKC and caspase-3 activation. These results define a novel role for PKC as a shared signaling mediator for flow and TNF-␣, and important for flow-mediated inhibition of proinflammatory and apoptotic events in ECs.

Abstract-Big MAP kinase 1 (BMK1 or ERK5) is a key mediator of endothelial cell (EC) function as shown by impaired embryonic angiogenesis and vascular collapse in BMK1 knockout mice. Hypoxia inducible factor 1␣ (HIF1␣), a potent mediator of angiogenesis, is positively regulated by the MAP kinases, ERK1/2. Because BMK1 deficiency is associated with impaired angiogenesis we hypothesized that BMK1 might regulate HIF1␣. To test this hypothesis, bovine lung microvascular ECs (BLMECs) were transfected with HIF1␣ and BMK1 cDNAs, and stimulated by hypoxia. HIF1␣ activity was measured by a reporter gene assay in which luciferase expression was driven by HIF1␣ activation. Understanding the specific mechanisms that contribute to each step of the angiogenic response may offer the best approach to developing therapies that will augment angiogenesis in diseases such as stroke and myocardial infarction or inhibit angiogenesis in cancer. An important master regulator of angiogenesis (especially induced by hypoxia) is hypoxia inducible factor-1␣ (HIF1␣), which controls critical proangiogenic genes such as vascular endothelial growth factor (VEGF). [3][4][5][6] Targeted inactivation of HIF1␣ in mice results in abnormal vascular development and embryonic lethality, demonstrating its key role in the angiogenic processes. 7 Multiple mechanisms regulate HIF1␣ including both transcriptional and posttranslational processes. In EC, posttranslational mechanisms include both protein degradation (mediated by proline hydroxylases and ubiquitination) and protein phosphorylation. 8 -11 Previous studies suggest that HIF1␣ function is regulated by mitogen activated protein (MAP) kinases. 6,12,13 Specifically it was shown that ERK1/2 was activated by hypoxia, phosphorylated HIF1␣ directly, and enhanced HIF1␣ transcriptional activity as measured by VEGF expression. 6,12 The mechanisms by which ERK1/2 stimulates HIF1␣ transcriptional activity remain unknown because HIF1␣ phosphorylation by ERK1/2 did not change HIF1␣ protein stability. However, the ERK1 knockout mouse did not exhibit any defects in angiogenesis. 14 In contrast, several recent articles 15-17 suggest a critical role for the closely related MAP kinase, BMK1, in angiogenesis. Sohn et al showed in the BMK1 knockout mouse that initial vascularization occurred normally but subsequent remodeling and maintenance of the vasculature was adversely affected. 15 A similar result was reported by Hayashi et al who showed in a conditional BMK1 knockout that vascular integrity was compromised and EC apoptosis increased. 16 We hypothesized that the abnormalities in BMK1 knockout vasculature may reflect BMK1-mediated regulation of HIF-1␣. This hypothesis derived from an article showing that VEGF expression was upregulated in BMK1 knockout embryonic fibroblasts and overexpression of BMK1 cDNA inhibited hypoxia induced VEGF promoter activity. 15 The authors suggested that the phenotype of BMK1 null mice was possibly because of disregulation of HIF activity, which disrupted normal vasculogenesis and an...

The focal development of atherosclerosis in the vascular tree may be explained in part by the local nature of blood flow. Bifurcations and branching points, prone to early atherogenesis, experience disturbed and oscillatory flow, whereas straight vascular regions, resistant to atherosclerosis, are exposed to steady laminar flow. A large number of studies suggest that the antiatherosclerotic effects of laminar flow are in part due to the ability of flow to modulate endothelial cell phenotype. Under steady laminar flow, endothelial cells generate molecules that promote a vasoactive, anticoagulant, anti-inflammatory, and growth-inhibitory surface. In contrast, disturbed flow induces a proliferative, prothrombotic, and adhesive phenotype. Endothelial cells are able to sense the variations of flow via mechanosensitive cell surface proteins and to transduce these signals via intracellular pathways to transcription factors in the nucleus leading to phenotypic changes. This review summarizes the "outside-in" signaling events initiated by flow that modulate endothelial cell phenotype.