Role Of HIF-1α-mediated ROCK Pathway Activation In Vascular Endothelial Hyperpermeability After Hypoxia

Background & Aims: Vascular endothelium damage caused by severe burn injury leads to the changes of actin and intercellular junctions, and the increased vascular permeability, resulting in the leakage of fluids across blood vessels, decreased blood volume and shock. Thus, the increase of vascular endothelial permeability is the main cause of burn shock. However, the machanisms involved in the pathogenesis of vascular endothelial permeability following severe burn injury have not yet been elucidated.The phosphorylation of myosin light chain (MLC) induced by both the activated myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) is the key event of vascular endothelial hyperpermeability. The activation of MLCP is negatively regulated by Rho-associated kinase (ROCK). In other words, the activation of ROCK leads to the increased MLC phosphorylation. Thus, ROCK signaling cascade plays very important role in the regulation of vascular endothelial hyperpermeability.Hypoxia-inducible factor-1 (HIF-1) is a heterodimer complex consisting of HIF-1α, whose expression is tightly regulated by oxygen concentration, and HIF-1β, which is a constitutively expressed aryl hydrocarbon receptor nuclear translocator. HIF-1αis believed to be the master regulator of oxygen homeostasis, and induces a network of genes involved in angiogenesis, erythropoiesis, and glucose metabolism after hypoxia. Prevous studies have shown that HIF-1αis involved in the regulation of both intestinal and vascular permeability. However, the mechanisms by which HIF-1αregulates permeability are not well understood. Thus, in this study, the mechanism by which HIF-1αregulates vascular endothelial permeability after hypoxia was studied, with the focus on ROCK signaling cascade, so as to further elucidate the mechanisms of vascular endothelial hyperpermeability at the early stage of severe burn injury.Materials & Methods1. The permeability of cultured vascular endothelial cell monolayers was measured after hypoxia. Meanwhile, the expression of HIF-1α, tight junction protein occludin, RhoA, ROCKΙ, ROCKⅡ, phosphorylated MYPT1 and phosphorylated MLC was detected in endothelial cells exposed to hypoxia.2. The effects of ROCK inhibition by ROCKOUT, a specific ROCK inhibitor, on MLC phosphorylation and permeability were studied in hypoxic endothelial cells.3. The effects of DMOG, a specific inducer of HIF-1α, on the expression of HIF-1α, occludin, ROCKII, phosphorylated MYPT1 and phosphorylated MLC were investigated in endothelial cells in normoxia.4. The effects of YC-1 and oligomyci, two kinds of specific HIF-1αinhibitor, on permeability and expression of HIF-1α, occludin, RhoA, ROCK1, ROCKII, phosphorylated MYPT1 and phosphorylated MLC were studied in hypoxic endothelial cells.5. pcDNA6.2-GW/EmGFP-miR-siHIF-1α, a expressing vector of huaman HIF-1αRNA interference was constructed and stably trasfected into endothelial cells. Then, the effects of HIF-1αRNA interference on permeability, ROCK activity and the expression of HIF-1α, RhoA, ROCK1, ROCKII, phosphorylated MYPT1 and phosphorylated MLC were determined in endothelial cells exposed to hypoxia.Main Results1. Hypoxia treatment induced a significant increase of permeability in endothelial cell monolayers, which was accompanied by the increased expression of RhoA, ROCKΙ, ROCKⅡ, phosphorylated MYPT1 and phosphorylated MLC.2. HIF-1αprotein expression in endothelial cells was significantly increased after hypoxia exposure.3. Hypoxia exposure had no significant effect on the expression of occludin, a tight junction protein.4. The hypoxi-induced increase of MLC phosphorylation and permeability was significantly inhibited by the treatment of ROCKOUT, a specific ROCK inhibitor.5. In normoxic endothelial cells, DMOG, a specific inducer of HIF-1α, not only significantly induced HIF-1αexpression, but also caused a significant increase of ROCKII, phosphorylated MYPT1 and phosphorylated MLC. However, DMOG treatment had no significant effect on occludin proein expression.6. In endothelial cells exposed to hypoxia, YC-1 and oligomyci, two kinds of specific HIF-1αinhibitor, significantly inhibited the hypoxia-induced increase of HIF-1α, RhoA, ROCK1, ROCKII, phosphorylated MYPT1 and hosphorylated MLC. Both YC-1 and oligomyci also partially inhibited permeability increase induced by hypoxia, but had no significant effect on occludin expression.7. The expressing vector of human HIF-1αRNA interference was successfully constructed and stably transfected into endothelial cells. In endothelial cells stably transfected with expressing vector of HIF-1αRNA interference, the hypoxia-induced increases of HIF-1α, RhoA, ROCK1, ROCKII, phosphorylated MYPT1 and hosphorylated MLC expression, ROCK activity and permeability were significantly.Conclusions1. The hypoxia-induced increase of permeability in endothelial cell monolayers was accompanied by the activation of ROCK-MLC phosphorylation pathway. ROCK inhibition is capable of inhibiting the hypoxia-induced increase of MLC phosphorylation and permeability. MLC phosphorylation induced by the activated ROCK pathway is involved in the hypoxia-induced increase of vascular endothelium.2. The increased endothelial permeability induced by the activated ROCK-MLC phosphorylaion is accompanied by the increased HIF-1αexpression. In normoxia, HIF-1αinduction leads to the activation of ROCK-MLC phosphorylation. It is suggested that HIF-1αmight be important to hypoxia-induced activation of ROCK-MLC phosphorylation pathway.3. HIF-1αinhibition with chemical inhibitor results in the inhibition of hypoxi-induced activation of ROCK-MLC phosphorylation. The hypoxia-induced endothelial permeability increase is partially inhibited by HIF-1αinhibition. It is suggested that HIF-1αmay be involved in the hypoxi-induced increase of endothelial permeability associated with ROCK-MLC phosphorylation pathway.4. HIF-1αRNA interference not only inhibits the hypoxia-induced activation of ROCK-MLC phosphorylation pathway, but also inhibits endothelial permeability increase induced by hypoxia. It is concluded that HIF-1α-induced activation of ROCK-MLC phosphorylation pathway is involved in the endothelial permeability increase induced by hypoxia.