SummaryVascular endothelial growth factor (VEGF) plays a crucial role in developmental and pathological angiogenesis. Expression of VEGF in quiescent adult tissue suggests a potential role in the maintenance of mature blood vessels. We demonstrate, using a Vegf-lacZ reporter mouse model, that VEGF is expressed by arterial but not by venous or capillary endothelial cells (ECs) in vivo. Using an in vitro model, we show that arterial shear stress of human umbilical vein ECs (HUVECs) decreases apoptosis and increases VEGF expression, which is mediated by the induction of Krüppel-like factor 2 (KLF2). Additionally, shear stress stimulates the expression of VEGF receptor 2 (VEGFR2) and is associated with its activation. Knockdown of VEGF in shear stressed HUVECs blocks the protective effect of shear stress, resulting in EC apoptosis equivalent to that in control ECs cultured under static conditions. Similarly, treatment of ECs subjected to arterial shear stress with the VEGF receptor tyrosine kinase inhibitor SU1498, or VEGFR2 neutralizing antiserum, led to increased apoptosis, demonstrating that the mechanoprotection from increased shear is mediated by VEGFR2. Taken together, these studies suggest that arterial flow induces VEGF-VEGFR2 autocrine-juxtacrine signaling, which is a previously unidentified mechanism for vascular EC survival in adult arterial blood vessels.

Vascular endothelial cells (ECs) form the inner lining of all blood vessels from the largest artery and veins, viz., the aorta and venae cavae, respectively, to the capillaries that connect the arterial and venous systems. Because these two major conducting systems of the cardiovasculature differ functionally, it is not surprising that the physical makeup of arteries and veins, including the ECs that line their lumina, are also distinct. Although few would argue that the local environment contributes to the differences between arteries and veins, recent evidence has shown that the specification of arterial and venous identity is largely genetically determined.

Vascular endothelial cells play an important role in the regulation of vascular function in response to mechanical stimuli in both healthy and diseased states. Prostaglandin I (PGI) is an important antiatherogenic prostanoid and vasodilator produced in endothelial cells through the action of the cyclooxygenase (COX) isoenzymes COX-1 and COX-2. However, the mechanisms involved in sustained, shear-induced production of COX-2 and PGI have not been elucidated but are determined in the present study. We used cultured endothelial cells exposed to steady fluid shear stress (FSS) of 10 dyn/cm for 5 h to examine shear stress-induced induction of COX-2/PGI Our results demonstrate the relationship between the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1) and the intracellular mechanoresponsive molecules phosphatidylinositol 3-kinase (PI3K), focal adhesion kinase (FAK), and mitogen-activated protein kinase p38 in the FSS induction of COX-2 expression and PGI release. Knockdown of PECAM-1 (small interference RNA) expression inhibited FSS-induced activation of αβ-integrin, upregulation of COX-2, and release of PGI in both bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs). Furthermore, inhibition of the PI3K pathway (LY294002) substantially inhibited FSS activation of αβ-integrin, upregulation of COX-2 gene and protein expression, and release of PGI in BAECs. Inhibition of integrin-associated FAK (PF573228) and MAPK p38 (SB203580) also inhibited the shear-induced upregulation of COX-2. Finally, a PECAM-1 mouse model was characterized by reduced COX-2 immunostaining in the aorta and reduced plasma PGI levels compared with wild-type mice, as well as complete inhibition of acute flow-induced PGI release compared with wild-type animals. In this study we determined the major mechanotransduction pathway by which blood flow-driven shear stress activates cyclooxygenase-2 (COX-2) and prostaglandin I (PGI) release in endothelial cells. Our work has demonstrated for the first time that COX-2/PGI mechanotransduction is mediated by the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1).

Objective Vascular endothelial cells (ECs) are continuously exposed to blood flow that contributes to the maintenance of vessel structure and function; however, the effect of hemodynamic forces on transforming growth factor-β (TGF-β) signaling in the endothelium is poorly described. We examined the potential role of TGF-β signaling in mediating the protective effects of shear stress on ECs. Approach and Results Human umbilical vein endothelial cells (HUVECs) exposed to shear stress were compared to cells grown under static conditions. Signaling through the TGF-β receptor ALK5 was inhibited with SB525334. Cells were examined for morphological changes and harvested for real-time PCR, western blot analysis, apoptosis, proliferation and immunocytochemistry. Shear stress resulted in ALK5-dependent alignment of HUVECs as well as attenuation of apoptosis and proliferation compared to static controls. Shear stress lead to an ALK5-dependent increase in TGF-β3 and Krüppel-like factor 2 (KLF2), phosphorylation of endothelial nitric oxide synthase (eNOS) and NO release. Addition of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) rescued the cells from apoptosis due to ALK5 inhibition under shear stress. Knockdown of TGF-β3, but not TGF-β1, disrupted the HUVEC monolayer and prevented the induction of KLF2 by shear. Conclusions Shear stress of HUVECs induces TGF-β3 signaling and subsequent activation of KLF2 and NO, and represents a novel role for TGF-β3 in the maintenance of HUVEC homeostasis in a hemodynamic environment.

Endomucin is a membrane-bound glycoprotein expressed luminally by endothelial cells that line postcapillary venules, a primary site of leukocyte recruitment during inflammation. Here we show that endomucin abrogation on quiescent endothelial cells enables neutrophils to adhere firmly, via LFA-1-mediated binding to ICAM-1 constitutively expressed by endothelial cells. Moreover, TNF-α stimulation downregulates cell surface expression of endomucin concurrent with increased expression of adhesion molecules. Adenovirus-mediated expression of endomucin under inflammatory conditions prevents neutrophil adhesion in vitro and reduces the infiltration of CD45+ and NIMP-R14+ cells in vivo. These results indicate that endomucin prevents leukocyte contact with adhesion molecules in non-inflamed tissues and that downregulation of endomucin is critical to facilitate adhesion of leukocytes into inflamed tissues.

Background:The mechanisms by which the PECAM-1⅐G␣ q/11 mechanosensitive complex mediates endothelial flow responses remain unclear. Results: The PECAM-1⅐G␣ q/11 complex contains heparan sulfate proteoglycans (HSPGs) and is disrupted by inhibition of HS. Conclusion:The interaction between PECAM-1 and G␣ q/11 may be mediated by the HS of the HSPG syndecan-1. Significance: Targeting specific HSPGs may be an effective strategy for the therapeutic treatment of vascular diseases.

Piezo1 is a mechanosensitive cation channel that is activated by shear stress in endothelial cells (ECs). It has been shown to mediate shear-induced EC responses, including increased calcium influx, and vascular functions, such as vascular tone and blood pressure. Yoda1, a selective Piezo1 activator, has been shown to mimic shear-induced responses in ECs. Since shear-induced calcium influx causes Akt and ERK1/2 activation in ECs, we examined the effects of Yoda1 and the role of Piezo1 on their activation. Here, we show that Yoda1 robustly activates Akt and ERK1/2 in ECs. Additionally, the Piezo1 antagonists, gadolinium and ruthenium red, but not GsMTx4, effectively blocks Yoda1-induced Akt activation. Our results suggest that Yoda1-induced Akt and ERK1/2 activation is not dependent on Piezo1.

Maintenance of the cells of the vessel wall in a quiescent