Abstract-Fluid shear stress generated by blood flowing over the endothelium is a major determinant of arterial tone, vascular remodeling, and atherogenesis. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an essential role in regulation of vascular function and structure by blood flow, but the molecular mechanisms that transduce mechanical force to eNOS activation are not well understood. In this study, we found that laminar flow (shear stressϭ12 dyne/cm 2 ) rapidly activates vascular endothelial growth factor receptor 2 (VEGFR2) in a ligand-independent manner and leads to eNOS activation in cultured endothelial cells. Flow-stimulated VEGFR2 recruits phosphoinositide 3-kinase and mediates activation of Akt and eNOS. Inhibiting VEGFR2 kinase with selective inhibitors blocks flow-induced activation of Akt and eNOS and production of NO. Decreasing VEGFR2 expression with antisense VEGFR2 oligonucleotides significantly attenuates activation of Akt and eNOS. Furthermore, Src kinases are involved in flow-stimulated VEGFR2 because inhibiting Src kinases by PP2, a selective inhibitor for Src kinases, abolishes flow-induced VEGFR2 tyrosine phosphorylation and downstream signaling. Finally, we show that inhibiting VEGFR2 kinase significantly reduces flow-mediated NO-dependent arteriolar dilation in vivo. These data identify VEGFR2 as a key mechanotransducer that activates eNOS in response to blood flow. Key Words: vascular endothelial growth factor receptor Ⅲ shear stress Ⅲ mechanotransduction Ⅲ endothelial nitric oxide synthase Ⅲ vasodilation V ascular endothelial cells (ECs), which form the inner lining of the blood vessel wall, are exposed to fluid shear stress, the dragging force generated by the flowing blood. Fluid shear stress modulates endothelial structure and function and is a major determinant of vascular remodeling, arterial tone, and atherogenesis. 1,2 It has been shown that atherosclerotic lesions preferentially develop in regions of low shear stress, whereas laminar flow generating high shear stress is atheroprotective. 1,2 Although the exact mechanisms by which flow prevents atherosclerosis are not known, nitric oxide (NO) plays an essential role in mediating many effects of flow, including vessel relaxation, 3 inhibition of apoptosis, 4 inhibition of platelet coagulation, 5 and antiinflammation. 6,7 Physiologically, fluid shear stress is the most important stimulus for the continuous formation of NO in vessels. 8,9 Endothelial-derived NO has a critical role in the local regulation of vascular homeostasis. A decrease in the bioavailability of NO is a characteristic feature in patients with coronary artery disease 10 and promotes the development of atherosclerotic lesions. 11 In addition, blood flow and NO appear to play important roles in angiogenesis. [12][13][14] Flow stimulates production of NO via endothelial nitricoxide synthase (eNOS) both in cultured ECs and in intact vessels. 8,9,[15][16][17][18] We and others have previously reported flowstimulated phosphorylation of eNOS regulat...

Endothelial cells release nitric oxide (NO) acutely in response to increased laminar fluid shear stress, and the increase is correlated with enhanced phosphorylation of endothelial nitric-oxide synthase (eNOS). Phosphoamino acid analysis of eNOS from bovine aortic endothelial cells labeled with [ 32 P]orthophosphate demonstrated that only phosphoserine was present in eNOS under both static and flow conditions. Fluid shear stress induced phosphate incorporation into two specific eNOS tryptic peptides as early as 30 s after initiation of flow. The flow-induced tryptic phosphopeptides were enriched, separated by capillary electrophoresis with intermittent voltage drops, also known as "peak parking," and analyzed by collision-induced dissociation in a tandem mass spectrometer. Two phosphopeptide sequences determined by tandem mass spectrometry, TQpSFSLQER and KLQTRPpSPGPPPAEQLLSQAR, were confirmed as the two flow-dependent phosphopeptides by co-migration with synthetic phosphopeptides. Because the sequence (RIR)TQpSFSLQER contains a consensus substrate site for protein kinase B (PKB or Akt), we demonstrated that LY294002, an inhibitor of the upstream activator of PKB, phosphatidylinositol 3-kinase, inhibited flow-induced eNOS phosphorylation by 97% and NO production by 68%. Finally, PKB phosphorylated eNOS in vitro at the same site phosphorylated in the cell and increased eNOS enzymatic activity by 15-20-fold.Endothelial nitric-oxide synthase (eNOS 1 or type III NOS) is one of three isoenzymes that converts L-arginine to L-citrulline and nitric oxide (NO). Endothelial cells synthesize NO tonically and increase NO production in response to agonists and increased fluid shear stress (FSS). Endothelial NO contributes to blood vessel homeostasis by regulating vessel tone (1), cell growth (2), platelet aggregation (3), and leukocyte binding to endothelium (4). In vivo eNOS is both myristoylated and palmitoylated. These modifications increase eNOS compartmentalization to plasmalemmal caveolae and facilitate release of NO from cells (5-7). In caveolae, which are small plasmalemmal invaginations that sequester signaling proteins (8), eNOS specifically interacts with the scaffolding protein caveolin-1 through a caveolin (9, 10) binding motif (11), located near the domain that binds Ca 2ϩ /calmodulin. Recent studies suggest that the activity of eNOS is regulated in a reciprocal manner through caveolin-1 inhibition and Ca 2ϩ /calmodulin stimulation (12-14).Increased FSS stimulates an increase in free intracellular calcium [Ca 2ϩ ] i from intracellular stores (15, 16) leading to a Ca 2ϩ /calmodulin-dependent increase in eNOS activity. However, recent investigations show that increases in [Ca 2ϩ ] i do not fully explain the rapid rise in NO production in response to FSS (17). Exposure of bovine aortic endothelial cells (BAEC) to 25 dynes/cm 2 FSS for 30 s caused a 7-fold rise in NO production and a corresponding 2-fold increase in eNOS phosphorylation, whereas the calcium ionophore A23187 neither caused rapid NO production...

The present study investigates whether lower-limb dominant exercise training in patients with chronic heart failure (CHF) improves endothelial function primarily in the trained lower extremities or equally in the upper and lower extremities. Twenty-eight patients with CHF were randomized to the exercise or control group. The exercise group underwent cycle ergometer training for 3 months while controls continued an inactive sedentary lifestyle. Exercise capacity (6-min walk test) and flow-mediated vasodilation in the brachial and posterior tibial arteries were evaluated. After 3 months, walking performance increased only in the exercise group (488+/-16 to 501+/-14 m [control]; 497+/-23 to 567+/-39 m [exercise, p<0.05]). The flow-mediated vasodilation in the brachial arteries did not change in either group (4.2+/-0.5 to 4.5+/-0.4% [control]; 4.3+/-0.5 to 4.6+/-0.4% [exercise]), but that in the posterior tibial arteries increased only in the exercise group (4.1+/-0.5 to 4.1+/-0.3% [control]; 3.6+/-0.3 to 6.4+/-0.6% [exercise, p<0.01]). Cycle ergometer training improved flow-mediated vasodilation in the trained lower limbs, but not in the untrained upper limbs. Exercise training appears to correct endothelial dysfunction predominantly by a local effect in the trained extremities.