Long-term cyclic stretch controls pulmonary endothelial permeability at translational and post-translational levels

Abstract

We have previously described differential effects of physiologic (5%) and pathologic (18%) cyclic stretch (CS) on agonist-induced pulmonary endothelial permeability. This study examined acute and chronic effects of CS on agonist-induced intracellular signaling and cell morphology in the human lung macro- and microvascular endothelial cell (EC) monolayers. Endothelial permeability was assessed by analysis of morphological changes, parameters of cell contraction and measurements of transendothelial electrical resistance. Exposure of both microvascular and macrovascular EC to 18% CS for 2–96 h increased thrombin-induced permeability and monolayer disruption. Interestingly, the ability to promote thrombin responses was present in EC cultures exposed to 48–96 h of CS even after replating onto non-elastic substrates. In turn, physiologic CS preconditioning (72 h) attenuated thrombin-induced paracellular gap formation and MLC phosphorylation in replated EC cultures. Long-term preconditioning at 18% CS (72 h) increased the content of signaling and contractile proteins including Rho GTPase, MLC, MLC kinase, ZIP kinase, PAR1, caldesmon and HSP27 in the pulmonary microvascular and macrovascular cells. We conclude that short term CS regulates EC permeability via modulation of agonist-induced signaling, whereas long-term CS controls endothelial barrier at both post-translational level and via magnitude-dependent regulation of pulmonary EC phenotype, signaling and contractile protein expression.

Introduction

Pathologic lung over-distention caused by mechanical ventilation at high tidal volumes compromises the blood-gas barrier, increases lung permeability, and may culminate in ventilator-induced lung injury (VILI) and pulmonary edema [1], [2]. Vascular leak observed in VILI patients is also associated with increased levels of edemagenic agents and inflammatory cytokines such as thrombin, histamine, TNF-alpha, IL-8, and IL-1 in the lung [1], [3], [4], [5]. The significance of the interactions between the edemagenic agents and pathological mechanical distension of the lung tissue in progression of VILI-associated vascular leak and pulmonary edema becomes increasingly recognized. Therefore, two-hit animal models, which combine lung inflammation induced by pro-inflammatory agents and mechanical ventilation at high tidal volumes reflect more appropriately common co-morbidities and risk factors present in patients with acute lung injury [6]. Importantly, in vitro models of pulmonary cells exposed to pathologic or physiologic mechanical stimulation and edemagenic agonists may provide vital information about molecular mechanisms regulating lung endothelial or epithelial permeability in VILI patients.

Biomechanical forces acting on vascular endothelium stimulate a variety of signaling pathways including MAP kinase cascades (Erk-1,2, JNK, p38), non-receptor tyrosine kinases (p60^Src, FAK), integrin-mediated signaling, and ion channels [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. Small GTPases Rac and Rho directly regulate cytoskeletal reorganization and endothelial permeability and can be activated by mechanical stimuli of different origin and magnitude [17], [18]. We have recently shown the differential effects of physiologic and pathologic magnitudes of cyclic stretch (CS) applied to pulmonary endothelial cell (EC) monolayers on the agonist-induced EC barrier disruption [19], [20]. Consistent with differential effects on monolayer integrity, 18% CS enhanced thrombin-induced Rho activation, whereas 5% CS promoted Rac activation critical for EC recovery phase. These studies revealed critical roles of the amplitude of applied cyclic stretch on the Rac/Rho GTPase balance and mechano-chemical regulation of the lung EC barrier.

However, the role of long-term exposure to physiologic or pathologic levels of cyclic stretch on pulmonary EC permeability responses, phenotypic expression and cell signaling relevant to more prolonged periods of mechanical ventilation in vivo remains to be investigated. Microarray analysis of mRNA expression profiles in endothelial cells exposed to various levels of cyclic stretch and shear stress reported by our group and others indicates significant effects of different types of mechanical stimulation on gene expression patterns [21], [22], [23], [24].

This study directly tested effects of chronic CS preconditioning at physiologic and pathologic amplitudes on sustained changes in the pulmonary EC signaling, thrombin-induced permeability responses, and expression of contractile and signaling proteins.