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...
Our purpose was to determine whether L-arginine was involved in vascular communication between downstream and upstream locations within a defined microvascular region. Arteriolar diameter was measured for the branches along a transverse arteriole in the superfused cremaster of anesthetized (pentobarbital sodium, 70 mg/kg i.p.) hamsters (N = 53). The upstream branch arterioles dilated significantly to locally applied L-arginine (100 mumol/L pipette concentration) only if the downstream branches (approximately 1400 microns away) were preexposed. With exposure order downstream to upstream, diameter change was last branch, -3.8 +/- 1.5% (of baseline); third, +58.1 +/- 27%; first, +92 +/- 26% (n = 5); with exposure order upstream to downstream: first branch, -0.4 +/- 3%; third, +5 +/- 11%; last, -5.6 +/- 7.5% (n = 4). Thus, downstream preexposure to L-arginine altered the responsivity upstream to locally applied L-arginine. Downstream-applied L-arginine also induced a conducted vasodilation (+17.8 +/- 2.8%; n = 14) 1327 +/- 166 microns upstream. This response was completely blocked by simultaneous sucrose (600 mOsm), halothane (0.0345%), or N omega-nitro-L-arginine (L-NNA, 100 mumol/L) exposure to the feed vessel (second micropipette) midway between the downstream site of L-arginine exposure and the upstream observation site. An acetylcholine-induced conducted vasodilation (+18.1 +/- 2.6%, n = 8) was also completely blocked by sucrose, halothane, or L-NNA.(ABSTRACT TRUNCATED AT 250 WORDS)
The identification of the physical mechanism(s) by which cells can sense vibrations requires the determination of the cellular mechanical environment. Here, we quantified vibration-induced fluid shear stresses in vitro and tested whether this system allows for the separation of two mechanical parameters previously proposed to drive the cellular response to vibration – fluid shear and peak accelerations. When peak accelerations of the oscillatory horizontal motions were set at 1g and 60Hz, peak fluid shear stresses acting on the cell layer reached 0.5Pa. A 3.5-fold increase in fluid viscosity increased peak fluid shear stresses 2.6-fold while doubling fluid volume in the well caused a 2-fold decrease in fluid shear. Fluid shear was positively related to peak acceleration magnitude and inversely related to vibration frequency. These data demonstrated that peak shear stress can be effectively separated from peak acceleration by controlling specific levels of vibration frequency, acceleration, and/or fluid viscosity. As an example for exploiting these relations, we tested the relevance of shear stress in promoting COX-2 expression in osteoblast like cells. Across different vibration frequencies and fluid viscosities, neither the level of generated fluid shear nor the frequency of the signal were able to consistently account for differences in the relative increase in COX-2 expression between groups, emphasizing that the eventual identification of the physical mechanism(s) requires a detailed quantification of the cellular mechanical environment.
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