p190RhoGAP mediates protective effects of oxidized phospholipids in the models of ventilator-induced lung injury

Products resulting from oxidation of cell membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) exhibit potent protective effects against lung endothelial cell (EC) barrier dysfunction caused by pathologically relevant mechanical forces and inflammatory agents. These effects were linked to enhancement of peripheral cytoskeleton and cell adhesion interactions mediated by small GTPase Rac and inhibition of Rho-mediated barrier-disruptive signaling. However, the mechanism of OxPAPC-induced, Rac-dependent Rho downregulation critical for vascular barrier protection remains unclear. This study tested the hypothesis that Rho negative regulator p190RhoGAP is essential for OxPAPC-induced lung barrier protection against ventilator-induced lung injury (VILI), and investigated potential mechanism of p190RhoGAP targeting to adherens junctions (AJ) via p120-catenin. OxPAPC induced peripheral translocation of p190RhoGAP, which was abolished by knockdown of Rac-specific guanine nucleotide exchange factors Tiam1 and Vav2. OxPAPC also induced Rac-dependent tyrosine phosphorylation and association of p190RhoGAP with AJ protein p120-catenin. siRNA-induced knockdown of p190RhoGAP attenuated protective effects of OxPAPC against EC barrier compromise induced by thrombin and pathologically relevant cyclic stretch (18% CS). In vivo, p190RhoGAP knockdown significantly attenuated protective effects of OxPAPC against ventilator-induced lung vascular leak, as detected by increased cell count and protein content in the bronchoalveolar lavage fluid, and tissue neutrophil accumulation in the lung. These results demonstrate for the first time a key role of p190RhoGAP for the vascular endothelial barrier protection in VILI.     

Rac1 activity is required for cardiac myocyte alignment in response to mechanical stress

Mechanical stretch is essential for the cardiac growth. The exposure of cardiac myocytes to the mechanical stretch leads to the cell alignment in parallel to the stretch direction, determining the cell polarity, though it remains to be addressed how mechanical stretch regulates cell orientation. In the present study, we investigated the signal transduction pathways responsible for the cell orientation response to mechanical stretch, focusing on Rho family proteins. Neonatal rat cardiomyocytes were cultured on silicon chambers and exposed to artificial uniaxial cyclic stretch. The pull-down assays revealed that Rac1 was rapidly activated by stretch, but not RhoA. To analyze the roles of Rho family proteins in cardiomyocyte orientation, adenoviral vectors expressing dominant-negative (dn) RhoA and Rac1 were generated. The transfection with adenovirus vector expressing dnRac1, but not dnRhoA, inhibited stretch-induced cell alignment. In conclusion, Rac1 activity is necessary for cardiomyocyte alignment in response to directional stretch.     

An efficient full space-time discretization method for subject-specific hemodynamic simulations of cerebral arterial blood flow with distensible wall mechanics

A computationally inexpensive mathematical solution approach using orthogonal collocations for space discretization with temporal Fourier series is proposed to compute subject-specific blood flow in distensible vessels of large cerebral arterial networks. Several models of wall biomechanics were considered to assess their impact on hemodynamic predictions. Simulations were validated against in vivo blood flow measurements in six human subjects. The average root-mean-square relative differences were found to be less than 4.3% for all subjects with a linear elastic wall model. This discrepancy decreased further in a viscoelastic Kelvin-Voigt biomechanical wall. The results provide support for the use of collocation-Fourier series approach to predict clinically relevant blood flow distribution and collateral blood supply in large portions of the cerebral circulation at reasonable computational costs. It thus opens the possibility of performing computationally inexpensive subject-specific simulations that are robust and fast enough to predict clinical results in real time on the same day.     

Regional distribution of layer-specific circumferential residual deformations and opening angles in the porcine aorta

Information on the layer-specific residual deformations of aortic tissue and how these vary throughout the vessel is important for understanding the regionally-varying aortic functions and pathophysiology, but not so much can be found in the literature. Toward this end, porcine aortas were sectioned into eighteen rings, with one ring from each anatomical position radially cut to obtain the zero-stress state for the intact wall and the other ring dissected into intimal-medial and adventitial layers; these rings were then radially cut to reach the zero-stress state for the intima-media and adventitia. Peripheral variations in internal/external circumferences, thickness, and opening angle of the intact wall and its layers were measured through image analysis at the no-load and zero-stress states. Intact wall and layer circumferences at both states significantly declined along the aorta, as did intact wall and intimal-medial but not adventitial thickness. Adventitia exhibited the greatest opening angles, approaching 180 deg all over the aorta. The opening angles of the intima-media and intact wall were quite similar, with the highest values in the ascending aorta, the lowest at the diaphragm, and increasing subsequently. Bending-related residual stretches were released by radial cutting that were compressive internally and tensile externally, displaying distinct axial variation for the intima-media and intact wall, and non-significant variation for the adventitia. Evidence is provided for the release upon layer separation of compressive stretches in the intima-media and of tensile stretches in the adventitia, whose values were smallest in the descending thoracic aorta and highest near the iliac artery bifurcation.     

高糖环境下持续性牵张力对子宫平滑肌细胞白介素-1和白介素-6表达的影响

目的:探讨高糖环境下持续性牵张力对大鼠子宫平滑肌细胞白介素-1(IL-1)、白介素-6(IL-6)表达的影响。方法:体外培养大鼠子宫平滑肌细胞,高糖作用不同时间后,观察高糖状态下大鼠子宫平滑肌细胞IL-1、IL-6表达变化情况。对高糖状态下的肌细胞施加持续性牵张力,明确牵张力对肌细胞IL-1、IL-6表达的影响以及高糖和牵张力之间的协同作用,同时采用晚期糖基化终末产物(AGEs)抑制剂拮抗高糖作用作为参照,并对结果进行分析。结果:随着高糖作用时间增加,肌细胞IL-1、IL-6表达也随之升高。牵张力也可促进肌细胞IL-1、IL-6表达增加,并可与高糖状态产生协同作用,这一过程可被高糖抑制剂部分阻断,但不能完全阻断。结论:高糖状态及牵张力均可促进肌细胞IL-1、IL-6表达增加,并有一定协同作用,AGEs参与了这一过程,但并不是唯一途径。     

Membrane Reserves and Hypotonic Cell Swelling

To accommodate expanding volume (V) during hyposmotic swelling, animal cells change their shape and increase surface area (SA) by drawing extra membrane from surface and intracellular reserves. The relative contributions of these processes, sources and extent of membrane reserves are not well defined. In this study, the SA and V of single substrate-attached A549, 16HBE14o⁻, CHO and NIH 3T3 cells were evaluated by reconstructing cell three-dimensional topology based on conventional light microscopic images acquired simultaneously from two perpendicular directions. The size of SA reserves was determined by swelling cells in extreme 98% hypotonic (∼6 mOsm) solution until membrane rupture; all cell types examined demonstrated surprisingly large membrane reserves and could increase their SA 3.6 ± 0.2-fold and V 10.7 ± 1.5-fold. Blocking exocytosis (by N-ethylmaleimide or 10°C) reduced SA and V increases of A549 cells to 1.7 ± 0.3-fold and 4.4 ± 0.9-fold, respectively. Interestingly, blocking exocytosis did not affect SA and V changes during moderate swelling in 50% hypotonicity. Thus, mammalian cells accommodate moderate (<2-fold) V increases mainly by shape changes and by drawing membrane from preexisting surface reserves, while significant endomembrane insertion is observed only during extreme swelling. Large membrane reserves may provide a simple mechanism to maintain membrane tension below the lytic level during various cellular processes or acute mechanical perturbations and may explain the difficulty in activating mechanogated channels in mammalian cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Stretch-activated currents in cardiomyocytes isolated from rabbit pulmonary veins

Evidence is growing of a relationship between atrial dilation and atrial fibrillation (AF), the most prevalent type of arrhythmia. Pulmonary veins, which are important ectopic foci for provoking AF, are of increasing interest in relation to the early development of AF. Here, using single cardiomyocytes isolated from rabbit pulmonary veins, we characterised the stretch-activated currents induced by swelling and axial mechanical stretching. Swelling induced both a stretch-activated nonselective cationic current (NSC) and a Cl(-) current. The swelling-induced Cl(-) current (I Cl,swell) was inhibited by DIDS, whereas the swelling-induced NSC (I NSC,swell) was inhibited by Gd3+. The cationic selectivity of the I NSC,swell was K+ >Cs+ >Na+ >Li+, whilst the PK/PNa, PCs/PNa, and PLi/PNa permeability ratios were 2.84, 1.86, and 0.85, respectively. Activation of the I NSC,swell was faster than that of the I Cl,swell. Given a high K+ concentration in the bath solution, the I NSC,swell showed limited amplitude (<-70 mV). Mechanical stretching induced an immediate Gd3+- and streptomycin-sensitive NSC (I NSC,stretch) that was permeable to Na+, K+, Cs+ and NMDG. Persistent stretching activated a DIDS-sensitive current (I Cl,stretch). The I NSC,stretch, but not the I NSC,swell, was completely blocked by 400 microM streptomycin; therefore, the two currents may not be associated with the same channel. In addition, the type of current induced may depend on the type of stretching. Thus, stretch-induced anionic and cationic currents are functionally present in the cardiomyocytes of the main pulmonary veins of rabbits, and they may have pathophysiological roles in the development of AF under stretched conditions.     

Role of muscle progenitor cells in maintaining morphological characteristics of rat soleus muscle during gravitational unloading by means of passive stretch

Working hypertrophy of skeletal muscle is usually coupled with activation of satellite cells with subsequent incorporation of their nuclei into muscle fibers. Earlier, it has been repeatedly shown that muscle stretching prevents the development of atrophic alterations and is accompanied by an intensification of protein synthesis. We suggested that the elimination of the proliferative abilities of progenitor cells by γ-irradiation would lead to a partial loss of the ability of muscle fibers to maintain their size. To evaluate the role of progenitor cells in the development of the preventive effect of passive stretching, an experiment was carried out with the 2500 rad local irradiation of a rat shin and subsequent hind-limb suspension or hind-limb suspension with stretch. Passive stretching during hind-limb suspension completely prevented atrophy, the transformation of fibers, and a decrease in the myonuclear number observed in the hind-limb-suspension group. Irradiation produced no action of the preventive effect of passive stretch. The conclusion is made that passive stretch preventive action is also realized in the absence of proliferating satellite cells.     

Infrared Methods for Monitoring the Protonation State of Carboxylic Amino Acids in the Photocycle of Bacteriorhodopsin

This review deals with the role of carboxylic amino acids in the proton-transport activity of bacteriorhodopsin. The main focus is on the infrared data, which allow direct monitoring of the protonation/deprotonation of specific residues during the proton movement in the course of the photocycle. Additional attention is paid to the potential use of carboxylic acids in proteins as internal sensors, based on the sensitivity of their IR frequencies to the immediate environment.     

Cyclic stretch-induced stress fiber dynamics - Dependence on strain rate, Rho-kinase and MLCK

Stress fiber realignment is an important adaptive response to cyclic stretch for nonmuscle cells, but the mechanism by which such reorganization occurs is not known. By analyzing stress fiber dynamics using live cell microscopy, we revealed that stress fiber reorientation perpendicular to the direction of cyclic uniaxial stretching at 1 Hz did not involve disassembly of the stress fiber distal ends located at focal adhesion sites. Instead, these distal ends were often used to assemble new stress fibers oriented progressively further away from the direction of stretch. Stress fiber disassembly and reorientation were not induced when the frequency of stretch was decreased to 0.01 Hz, however. Treatment with the Rho-kinase inhibitor Y27632 reduced stress fibers to thin fibers located in the cell periphery which bundled together to form thick fibers oriented parallel to the direction of stretching at 1 Hz. In contrast, these thin fibers remained diffuse in cells subjected to stretch at 0.01 Hz. Cyclic stretch at 1 Hz also induced actin fiber formation parallel to the direction of stretch in cells treated with the myosin light chain kinase (MLCK) inhibitor ML-7, but these fibers were located centrally rather than peripherally. These results shed new light on the mechanism by which stress fibers reorient in response to cyclic stretch in different regions of the actin cytoskeleton.