Ca²⁺ signalling and PKCα activate increased endothelial permeability by disassembly of VE—cadherin junctions

Abstract

The role of intracellular Ca2+ mobilization in the mechanism of increased endothelial permeability was studied. Human umbilical vein endothelial cells (HUVECs) were exposed to thapsigargin or thrombin at concentrations that resulted in similar increases in intracellular Ca2+ concentration ([Ca2+]i). The rise in [Ca2+]i in both cases was due to release of Ca2+ from intracellular stores and influx of extracellular Ca2+. Both agents decreased endothelial cell monolayer electrical resistance (a measure of endothelial cell shape change) and increased transendothelial 125I‐albumin permeability. Thapsigargin induced activation of PKCα and discontinuities in VE‐cadherin junctions without formation of actin stress fibres. Thrombin also induced PKCα activation and similar alterations in VE‐cadherin junctions, but in association with actin stress fibre formation. Thapsigargin failed to promote phosphorylation of the 20 kDa myosin light chain (MLC20), whereas thrombin induced MLC20 phosphorylation consistent with formation of actin stress fibres. Calphostin C pretreatment prevented the disruption of VE‐cadherin junctions and the decrease in transendothelial electrical resistance caused by both agents. Thus, the increased [Ca2+]i elicited by thapsigargin and thrombin may activate a calphostin C‐sensitive PKC pathway that signals VE‐cadherin junctional disassembly and increased endothelial permeability. Results suggest a critical role for Ca2+ signalling and activation of PKCα in mediating the disruption of VE‐cadherin junctions, and thereby in the mechanism of increased endothelial permeability.

Soluble CD146 is generated by ectodomain shedding of membrane CD146 in a calcium-induced, matrix metalloprotease-dependent process

“…Inflammatory mediators such as thrombin and histamine induce vascular leakage defined as increased permeability to plasma proteins and other solutes. Many of these inflammatory mediators trigger increased vascular permeability by increasing intracellular calcium concentrations which then mediate cytoskeletal reorganization and disassembly of VE cadherin at the adherens junctions (Sandoval et al, 2001;Tiruppathi et al, 2002;Vanden broucke et al, 2008;Schulz et al, 2008). In accordance with this, we observed increased permeability for albumin when confluent HMVEC-L were cultured on collagen-coated cell culture inserts for 5 days.…”

“…Elevated intracellular calcium concentrations in endothelial cells are known to cause increased vascular permeability (Sandoval et al, 2001;Tiruppathi et al, 2002;Vandenbroucke et al, 2008). We could show that calcium influx also induced shedding of CD146.…”

Agonist of growth hormone-releasing hormone reduces pneumolysin-induced pulmonary permeability edema

“…Cytoplasmic hemolytic PLY is released during autolysis, such as occurs because of antibiotic treatment (7,8), and subsequently binds to cholesterol in cell membranes, upon which oligomerization and pore formation take place. These pores facilitate increased intracellular Ca 2+ levels (9), which in turn activate mechanisms leading to contraction of endothelial cells (10,11). These pathways include both myosin light chain (MLC)-dependent mechanisms and microtubule depolymerization, the latter of which can cause disassembly of adherens junction proteins, such as vascular endothelial (VE)-cadherin (12).…”

Cellular Dielectric Spectroscopy: A Powerful New Approach to Label-Free Cellular Analysis

“…At each frequency, the difference in magnitude of the reflected signal, as compared to the incident wave, is measured. In cell biology, this technology is better known as bioimpedance and has been successfully applied to monitoring cell membrane conductivity, [11][12][13][14][15] monolayer permeability, [16][17][18][19] morphology, [20][21][22] migration, 23,24 and cellular micromotion. 25 Our goal was to extend the use of bioimpedance measurements in an embodiment of this technology to the label-free, noninvasive monitoring of the cellular consequences of ligandreceptor interaction.…”

“…In concurrence with this work, we are investigating the cellular physiological events that manifest themselves in the biophysical parameters that we measure. The literature has revealed that changes in the cell membrane, 11,14,15 in cell-cell junctions, 17,18 in the degree of cellular adhesion, [16][17][18][19]21,23,24,25 and in the state of the actin cytoskeleton 16,19 can all contribute to bioimpedance measurements. Work is progressing to define our biophysical measurements in terms of cellular physiological changes including, but not restricted to, those mentioned above.…”