Vascular endothelial-cadherin downregulation as a feature of endothelial transdifferentiation in monocrotaline-induced pulmonary hypertension

Increased pulmonary vascular resistance in pulmonary hypertension (PH) is caused by vasoconstriction and obstruction of small pulmonary arteries by proliferating vascular cells. In analogy to cancer, subsets of proliferating cells may be derived from endothelial cells transitioning into a mesenchymal phenotype. To understand phenotypic shifts transpiring within endothelial cells in PH, we injected rats with alkaloid monocrotaline to induce PH and measured lung tissue levels of endothelial-specific protein and critical differentiation marker vascular endothelial (VE)-cadherin. VE-cadherin expression by immonoblotting declined significantly 24 h and 15 days postinjection to rebound to baseline at 30 days. There was a concomitant increase in transcriptional repressors Snail and Slug, along with a reduction in VE-cadherin mRNA. Mesenchymal markers alpha-smooth muscle actin and vimentin were upregulated by immunohistochemistry and immunoblotting, and alpha-smooth muscle actin was colocalized with endothelial marker platelet endothelial cell adhesion molecule-1 by confocal microscopy. Apoptosis was limited in this model, especially in the 24-h time point. In addition, monocrotaline resulted in activation of protein kinase B/Akt, endothelial nitric oxide synthase (eNOS), nuclear factor (NF)-kappa B, and increased lung tissue nitrotyrosine staining. To understand the etiological relationship between nitrosative stress and VE-cadherin suppression, we incubated cultured rat lung endothelial cells with endothelin-1, a vasoconstrictor and pro-proliferative agent in pulmonary arterial hypertension. This resulted in activation of eNOS, NF-kappa B, and Akt, in addition to induction of Snail, downregulation of VE-cadherin, and synthesis of vimentin. These effects were blocked by eNOS inhibitor N-omega-nitro-Larginine methyl ester. We propose that transcriptional repression of VE-cadherin by nitrosative stress is involved in endothelial-mesenchymal transdifferentiation in experimental PH

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

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