HIF-1α activation results in actin cytoskeleton reorganization and modulation of Rac-1 signaling in endothelial cells

Abstract

BackgroundHypoxia is a major driving force in vascularization and vascular remodeling. Pharmacological inhibition of prolyl hydroxylases (PHDs) leads to an oxygen-independent and long-lasting activation of hypoxia-inducible factors (HIFs). Whereas effects of HIF-stabilization on transcriptional responses have been thoroughly investigated in endothelial cells, the molecular details of cytoskeletal changes elicited by PHD-inhibition remain largely unknown. To investigate this important aspect of PHD-inhibition, we used a spheroid-on-matrix cell culture model.ResultsMicrovascular endothelial cells (glEND.2) were organized into spheroids. Migration of cells from the spheroids was quantified and analyzed by immunocytochemistry. The PHD inhibitor dimethyloxalyl glycine (DMOG) induced F-actin stress fiber formation in migrating cells, but only weakly affected microvascular endothelial cells firmly attached in a monolayer. Compared to control spheroids, the residual spheroids were larger upon PHD inhibition and contained more cells with tight VE-cadherin positive cell-cell contacts. Morphological alterations were dependent on stabilization of HIF-1α and not HIF-2α as shown in cells with stable knockdown of HIF-α isoforms. DMOG-treated endothelial cells exhibited a reduction of immunoreactive Rac-1 at the migrating front, concomitant with a diminished Rac-1 activity, whereas total Rac-1 protein remained unchanged. Two chemically distinct Rac-1 inhibitors mimicked the effects of DMOG in terms of F-actin fiber formation and orientation, as well as stabilization of residual spheroids. Furthermore, phosphorylation of p21-activated kinase PAK downstream of Rac-1 was reduced by DMOG in a HIF-1α-dependent manner. Stabilization of cell-cell contacts associated with decreased Rac-1 activity was also confirmed in human umbilical vein endothelial cells.ConclusionsOur data demonstrates that PHD inhibition induces HIF-1α-dependent cytoskeletal remodeling in endothelial cells, which is mediated essentially by a reduction in Rac-1 signaling.

Kidney injury is independent of endothelial HIF-1α

“…Cells were cultured at 37 °C and 7.5 % CO 2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal calf serum (FCS) and routinely split in a 1:5 ratio. The stable knockdown of HIF-1α was induced by lentiviral transduction with shRNA producing constructs as described previously [21]. Cells with stable knockdown of HIF-1α (shHIF-1α) were compared to cells with stable transfection of an irrelevant shRNA (shGFP).…”

“…Sections were counterstained with DAPI (1:5000; Life Technologies). Immunoblots were performed as described [21]. …”

Современные Возможности Идентификации Новых Молекул-Мишеней Для Фармакологической Коррекции Проницаемости Гематоэнцефалического Барьера

“…Ïðè ýòîì îòìå÷àåòñÿ î÷åíü áûñòðàÿ äåñòàáèëèçàöèÿ áåëêîâ ïëîòíûõ êîíòàêòîâ, ïîâðåaeäå-íèå ýíäîòåëèîöèòîâ è àêòèâèçàöèÿ ìèãðàöèè ëåéêîöè-òîâ â òêàíü ãîëîâíîãî ìîçãà.  ðàáîòå [77] äîêàçàíî, ÷òî èñïîëüçîâàíèå èíãèáèòîðîâ Rac1 ïðè ñîõðàíåíèè àêòèâíîñòè HIF-1 ïðèâîäèò ê ñòàáèëèçàöèè ìåaeêëå-òî÷íûõ êîíòàêòîâ ýíäîòåëèîöèòîâ è óìåíüøåíèþ ìè-ãðàöèè êëåòîê â òêàíü ãîëîâíîãî ìîçãà.  ñâîþ î÷å-ðåäü, èìåþòñÿ äîêàçàòåëüñòâà òîãî, ÷òî Rac1 ÿâëÿåòñÿ îäíèì èç èíäóöèðóþùèõ è àêòèâèðóþùèõ ôàêòîðîâ äëÿ HIF-1.…”

Dephosphorylation of Y685-VE-Cadherin Involved in Pulmonary Microvascular Endothelial Barrier Injury Induced by Angiotensin II

“…Wessel et al showed that Tyr685 and Tyr731 of VE-cadherin distinctly and selectively regulate the induction of vascular permeability or leukocyte extravasation [12]. Meanwhile, several factors have been reported to induce phosphorylation of tyrosine of the VE-cadherin including VEGF, TNF- α , and HIF-1 α , which may subsequently result in elevation of vascular permeability [13–15]. Our previous study revealed that the serum Angiotensin (AngII) was remarkably elevated in AAD patients, together with elevation of pulmonary vascular permeability.…”