AB, Chatterjee S. PECAM-1 and caveolae form the mechanosensing complex necessary for NOX2 activation and angiogenic signaling with stopped flow in pulmonary endothelium. Am J Physiol Lung Cell Mol Physiol 305: L805-L818, 2013. First published September 27, 2013 doi:10.1152/ajplung.00123.2013.-We showed that stop of flow triggers a mechanosignaling cascade that leads to the generation of reactive oxygen species (ROS); however, a mechanosensor coupled to the cytoskeleton that could potentially transduce flow stimulus has not been identified. We showed a role for KATP channel, caveolae (caveolin-1), and NADPH oxidase 2 (NOX2) in ROS production with stop of flow. Based on reports of a mechanosensory complex that includes platelet endothelial cell adhesion molecule-1 (PECAM-1) and initiates signaling with mechanical force, we hypothesized that PECAM-1 could serve as a mechanosensor in sensing disruption of flow. Using lungs in situ, we observed that ROS production with stop of flow was significantly reduced in PECAM-1 Ϫ/Ϫ lungs compared with lungs from wild-type (WT) mice. Lack of PECAM-1 did not affect NOX2 activation machinery or the caveolin-1 expression or caveolae number in the pulmonary endothelium. Stop of flow in vitro triggered an increase in angiogenic potential of WT pulmonary microvascular endothelial cells (PMVEC) but not of PECAM-1 Ϫ/Ϫ PMVEC. Obstruction of flow in lungs in vivo showed that the neutrophil infiltration as observed in WT mice was significantly lowered in PECAM-1 Ϫ/Ϫ mice. With stop of flow, WT lungs showed higher expression of the angiogenic marker VEGF compared with untreated (sham) and PECAM-1 Ϫ/Ϫ lungs. Thus PECAM-1 (and caveolae) are parts of the mechanosensing machinery that generates superoxide with loss of shear; the resultant ROS potentially drives neutrophil influx and acts as an angiogenic signal. mechanotransduction; stop of flow; pulmonary endothelium; PECAM; K atp (Kir6.2) channel; NOX2; angiogenic potential CELLS SENSE THE PHYSICAL STIMULUS in their environment and translate these physical forces into biochemical signals (20, 37). Sensing and responding to a physical force require specialized structures and machinery that can engage in signal transduction (12,23,42).In the vascular system, with a highly distributed network of blood vessels, mechanical forces arising from blood flow initiate signaling that helps maintain vascular structure and function. Indeed, shear associated with blood flow is sensed by the endothelium and the resultant signaling regulates normal vascular physiology (such as embryonic morphogenesis and organization of the vascular tree) while irregular or abnormal shear can lead to vascular dysfunction and disease (19,27). Thus the mechanosignaling that accompanies various shear profiles and patterns, regular or aberrant, governs susceptibility to atherosclerosis, by inducing athero-protective or atheroprone phenotypes in endothelial cells (10,22). It thus becomes important to understand the link among the mechanical force, the shear sensing machinery and...
Vascular responses to shear stress arising from blood flow entail the involvement of mechanosensors on endothelial cells. We showed earlier that the KATP channel is an important component of the flow sensing machinery in the pulmonary endothelium. Based on previous studies (E. Tzima, Nature, 2005), we posited that the platelet endothelial cell adhesion molecule 1 (PECAM‐1), by virtue of its junctional location and cytoskeletal linkage, could serve as a mechanosensor that senses loss of blood flow. Using an isolated perfused mouse lung and confocal imaging of ROS probes in the pulmonary microvasculature, we detected ROS generation upon loss of shear (ischemia) in wild type lungs that was significantly attenuated both in KATP and PECAM‐1 knockout (KO) mouse lung. By image analysis, KATP KO and PECAM‐1 KO were capable of generating only 62% and 51% of the ROS level detected in wild type (WT), respectively. High‐performance liquid chromatography (HPLC) of dihydroethidium extracted from the ischemic lung was used to detect superoxide (SO). In agreement with the imaging data, HPLC analysis showed a partial attenuation of SO in the PECAM‐1 KO, indicating SO as the parent oxidant species and reinforcing the confocal imaging method. These results indicate that KATP channel and PECAM‐1 may be a part of a mechanosensing machinery that contributes to the generation of SO in a shear‐dependent manner. NHLBI‐T32‐S54932
Feline calicivirus (FCV) is a nonenveloped, spherical animal virus of the Calicivirus family. It contains a positive sense, single-stranded RNA genome and one major capsid protein. The virion has a diameter of 405 A and exhibits T=3 icosahedral symmetry. The FCV crystals belong to an or~horhombic srystal system with unit-cell dimensions a=889.0 A, b=998.3 A, c=437.6A. Based on the Vm value ( 3.4 A3/Da), it was estimated that there is one FCV particle in one crystallographic asymmetric unit, which implies the presence of 60-fold noncrystallographic redundm1cy.The orientations of the FCV particles in the unit cell were determined with a self-rotation as well as the locked rotation function using the GLRF program. Data between l 5-7 A and 7-4 A, respectively, were used to search for 5-, 3-and 2-fold axes. Two sets of 5-, 3-, and 2-fold noncrystallographic symmetry axes were obtained. The orientations of the 5-, 3-, and 2-fold noncrystallographic symmetry axes of each set were those expected for an icosahedron. This indicated that two different particle orientations exist in the crystallographic asymmetric unit. Combining this with the packing consideration and the Vm value, it was concluded that there is one unique particle content from two differently 01iented FCV particles in one crystallographic asymmetric unit. The precise particle orientations were refined by optimizing the fit between the directions of the observed selfrotation function peaks from one of the virus particle and the corTesponding axes of a standard icosahedron.In order to precisely define the particle positions in the unit cell, heavy atom derivatives of FCV crystals are in pursuit.
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