Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells

Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB.     

Involvement of Rho/ROCK signalling in small cell lung cancer migration through human brain microvascular endothelial cells

Small cell lung cancer (SCLC) cells migration across human brain microvascular endothelial cells (HBMECs) is an essential step of brain metastases. Here we investigated signalling pathways in HBMECs contributing to the process. Inhibition of endothelial Rho kinase (ROCK) with Y27632 and overexpression of ROCK dominant-negative mutant prevented SCLC cells, NCI-H209, transendothelial migration and the concomitant changes of tight junction. Conversely, inhibition of phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) had no effects. Furthermore, endothelial RhoA protein was activated during NCI-H209 cells transendothelial migration. Rho/ROCK participated in NCI-H209 cells transendothelial migration through regulating actin cytoskeleton reorganization. These results suggested that Rho/ROCK was required for SCLC cells transendothelial migration.     

Roles of Rho/ROCK and MLCK in TNF-alpha-induced changes in endothelial morphology and permeability

Tumor necrosis factor-alpha (TNF-alpha) is known to induce changes in endothelial cell morphology and permeability, but the mechanisms have not been extensively characterized. TNF-alpha rapidly induced RhoA activation and myosin light chain phosphorylation, but caused only small changes to cortical F-actin, without significantly increasing paracellular permeability up to 30 min after stimulation. TNF-alpha subsequently caused a progressive increase in permeability and in stress fiber reorganization, cell elongation, and intercellular gap formation over 8-24 h. Consistent with the increased permeability, Occludin and JAM-A were removed from tight junctions and ZO-1 was partially redistributed. Rho/ROCK but not MLCK inhibition prevented the long-term TNF-alpha-induced changes in F-actin and cell morphology, but ROCK inhibition did not affect permeability. These results suggest that the gradual increase in permeability induced by TNF-alpha does not reflect contractile mechanisms mediated by Rho, ROCK, and MLCK, but involves long-term reorganization of tight junction proteins.     

Rac and Rho play opposing roles in the regulation of hypoxia/reoxygenation-induced permeability changes in pulmonary artery endothelial cells

Hypoxia/reoxygenation-induced changes in endothelial permeability are accompanied by endothelial actin cytoskeletal and adherens junction remodeling, but the mechanisms involved are uncertain. We therefore measured the activities of the Rho GTPases Rac1, RhoA, and Cdc42 during hypoxia/reoxygenation and correlated them with changes in endothelial permeability, remodeling of the actin cytoskeleton and adherens junctions, and production of ROS. Dominant negative forms of Rho GTPases were introduced into cells by adenoviral gene transfer and transfection, and inhibitors of NADPH oxidase, PI3 kinase, and Rho kinase were used to characterize the signaling pathways involved. In some experiments constitutively activated forms of RhoA and Rac1 were also used. We show for the first time that hypoxia/reoxygenation-induced changes in endothelial permeability result from coordinated actions of the Rho GTPases Rac1 and RhoA. Rac1 and RhoA rapidly respond to changes in oxygen tension, and their activity depends on NADPH oxidase- and PI3 kinase-dependent production of ROS. Rac1 acts upstream of RhoA, and its transient inhibition by acute hypoxia leads to activation of RhoA followed by stress fiber formation, dispersion of adherens junctions, and increased endothelial permeability. Reoxygenation strongly activates Rac1 and restores cortical localization of F-actin and VE-cadherin. This effect is a result of Rac1-mediated inhibition of RhoA and can be prevented by activators of RhoA, L63RhoA, and lysophosphatidic acid. Cdc42 activation follows the RhoA pattern of activation but has no effect on actin remodeling, junctional integrity, or endothelial permeability. Our results show that Rho GTPases act as mediators coupling cellular redox state to endothelial function.     

Indoxyl sulfate potentiates endothelial dysfunction via reciprocal role for reactive oxygen species and RhoA/ROCK signaling in 5/6 nephrectomized rats

Accumulative indoxyl sulfate (IS) retained in chronic kidney disease (CKD) can potentiate vascular endothelial dysfunction, and herein, we aim at elucidating the underlying mechanisms from the perspective of possible association between reactive oxygen species (ROS) and RhoA/ROCK pathway. IS-treated nephrectomized rats are administered with antioxidants including NADPH oxidase inhibitor apocynin, SOD analog tempol, and mitochondrion-targeted SOD mimetic mito-TEMPO to scavenge ROS, or ROCK inhibitor fasudil to obstruct RhoA/ROCK pathway. First, we find in response to IS stimulation, antioxidants treatments suppress increased aortic ROCK activity and expression levels. Additionally, ROCK blockade prevent IS-induced increased NADPH oxidase expression (mainly p22phox and p47phox), mitochondrial and intracellular ROS (superoxide and hydrogen peroxide) generation, and decreased Cu/Zn-SOD expression in thoracic aortas. Apocynin, mito-TEMPO, and tempol also reverse these markers of oxidative stress. These results suggest that IS induces excessive ROS production and ROCK activation involving a circuitous relationship in which ROS activate ROCK and ROCK promotes ROS overproduction. Finally, ROS and ROCK depletion attenuate IS-induced decrease in nitric oxide (NO) production and eNOS expression levels, and alleviate impaired vasomotor responses including increased vasocontraction to phenylephrine and decreased vasorelaxation to acetylcholine, thereby preventing cardiovascular complications accompanied by CKD. Taken together, excessive ROS derived from NADPH oxidase and mitochondria coordinate with RhoA/ROCK activation in a form of positive reciprocal relationship to induce endothelial dysfunction through disturbing endothelium-dependent NO signaling upon IS stimulation in CKD status.     

RhoA GTPase regulates radiation-induced alterations in endothelial cell adhesion and migration

Endothelial cells of the microvasculature are major target of ionizing radiation, responsible of the radiation-induced vascular early dysfunctions. Molecular signaling pathways involved in endothelial responses to ionizing radiation, despite being increasingly investigated, still need precise characterization. Small GTPase RhoA and its effector ROCK are crucial signaling molecules involved in many endothelial cellular functions. Recent studies identified implication of RhoA/ROCK in radiation-induced increase in endothelial permeability but other endothelial functions altered by radiation might also require RhoA proteins. Human microvascular endothelial cells HMEC-1, either treated with Y-27632 (inhibitor of ROCK) or invalidated for RhoA by RNA interference were exposed to 15 Gy. We showed a rapid radiation-induced activation of RhoA, leading to a deep reorganisation of actin cytoskeleton with rapid formation of stress fibers. Endothelial early apoptosis induced by ionizing radiation was not affected by Y-27632 pre-treatment or RhoA depletion. Endothelial adhesion to fibronectin and formation of focal adhesions increased in response to radiation in a RhoA/ROCK-dependent manner. Consistent with its pro-adhesive role, ionizing radiation also decreased endothelial cells migration and RhoA was required for this inhibition. These results highlight the role of RhoA GTPase in ionizing radiation-induced deregulation of essential endothelial functions linked to actin cytoskeleton.     

Rho/ROCK-dependent pseudopodial protrusion and cellular blebbing are regulated by p38 MAPK in tumour cells exhibiting autocrine c-Met activation

BACKGROUND INFORMATION: The c-Met-dependent, beta-actin-rich, blebbed pseudopodia of MSV-MDCK-INV (invasive Moloney-sarcoma-virus-transformed Madin-Darby canine kidney) cells are induced by Rho/ROCK (Rho kinase) activation, and are morphologically distinct from flat extended lamellipodia. RESULTS: Microtubules were shown to extend to these actin-rich pseudopodial domains, and microtubule depolymerization by nocodazole treatment resulted in progressive cellular blebbing, initiating in the pseudopodial domains and resulting in transient cellular rounding and blebbing after 30 min. The blebbing response was dependent on autocrine HGF (hepatocyte growth factor) activation of c-Met and prevented by inhibition of RhoA, ROCK and p38 MAPK (p38 mitogen-activated protein kinase), but not ERK (extracellular-signal-regulated kinase) or PI3K (phosphoinositide 3-kinase). Phospho-p38 MAPK was present in pseudopodia, localizing activation of this signalling pathway to this protrusive membrane structure. In serum-starved cells, LPA (lysophosphatidic acid) activation of RhoA induced p38 MAPK-dependent pseudopodial protrusions, and inhibition of p38 MAPK prevented pseudopodial protrusion and displacement of MSV-MDCK-INV cells. MSV-MDCK-INV cells exhibited intermittent blebbing and rounding, which may represent an integral part of their motile behaviour. CONCLUSIONS: The localized activation of an autocrine HGF/c-Met loop regulates Rho/ROCK activation of p38 MAPK signalling to stimulate both membrane blebbing and pseudopod formation.     

Role of the Rho pathway in regulating valvular interstitial cell phenotype and nodule formation

The differentiation of valvular interstitial cells (VICs) to a myofibroblastic or osteoblast-like phenotype is commonly found in calcific valvular stenosis, although the molecular-level mechanisms of this process remain poorly understood. Due to the role of the Rho pathway in various vascular diseases and in the expression of a myofibroblast phenotype, the present study was inspired by the hypothesis that Rho activation is involved in regulating cellular processes related to valve calcification. It was found that increased RhoA and Rho kinase (ROCK) activity was associated with increased nodule formation in VIC cultures in vitro, and intentional induction of RhoA activity led to a further increase in nodules and expression of α-smooth muscle actin. VICs treated with ROCK inhibitors were also examined for nodule formation, proliferation, apoptosis, and expression of myofibroblastic or osteoblastic markers. ROCK inhibition dramatically reduced myofibroblast-regulated nodule formation in VIC cultures, as evidenced by a decrease in nodule number, total nodule area, α-smooth muscle actin-positive stress fibers, apoptosis, and gene expression of myofibroblast-related phenotypic markers. Meanwhile, ROCK inhibition was less effective at reducing nodule formation associated with osteogenic activity. In fact, ROCK inhibition increased the expression of alkaline phosphatase and effected only a modest decrease in nodule number when applied to VIC cultures with higher osteogenic activity. Thus, the Rho pathway possesses a complex role in regulating the VIC phenotype and nodule formation, and it is hoped that further elucidation of these molecular-level events will lead to an improved understanding of valvular disease and identification of potential treatments.     

RhoA-Rho kinase pathway mediates thrombin- and U-46619-induced phosphorylation of a myosin phosphatase inhibitor, CPI-17, in vascular smooth muscle cells

Protein kinase C-potentiated phosphatase inhibitor of 17 kDa (CPI-17) mediates some agonist-induced smooth muscle contraction by suppressing the myosin phosphatase in a phosphorylation-dependent manner. The physiologically relevant kinases that phosphorylate CPI-17 remain to be identified. Several previous studies have shown that some agonist-induced CPI-17 phosphorylation in smooth muscle tissues was attenuated by the Rho kinase (ROCK) inhibitor Y-27632, suggesting that ROCK is involved in agonist-induced CPI-17 phosphorylation. However, Y-27632 has recently been found to inhibit protein kinase C (PKC)-, a well-recognized CPI-17 kinase. Thus the role of ROCK in agonist-induced CPI-17 phosphorylation remains uncertain. The present study was designed to address this important issue. We selectively activated the RhoA pathway using inducible adenovirus-mediated expression of a constitutively active mutant RhoA (V14RhoA) in primary cultured rabbit aortic vascular smooth muscle cells (VSMCs). V14RhoA caused expression level-dependent CPI-17 phosphorylation at Thr38 as well as myosin phosphatase phosphorylation at Thr853. Importantly, we have shown that V14RhoA-induced CPI-17 phosphorylation was not affected by the PKC inhibitor GF109203X but was abolished by Y-27632, suggesting that ROCK but not PKC was involved. Furthermore, we have shown that the contractile agonists thrombin and U-46619 induced CPI-17 phosphorylation in VSMCs. Similarly to V14RhoA-induced CPI-17 phosphorylation, thrombin-induced CPI-17 phosphorylation was not affected by inhibition of PKC with GF109203X, but it was blocked by inhibition of RhoA with adenovirus-mediated expression of exoenzyme C3 as well as by Y-27632. Taken together, our present data provide the first clear evidence indicating that ROCK is responsible for thrombin- and U-46619-induced CPI-17 phosphorylation in primary cultured VSMCs. protein kinase C; signal transduction; adenovirus     

Protein kinase C-alpha signals rho-guanine nucleotide dissociation inhibitor phosphorylation and rho activation and regulates the endothelial cell barrier function

The Rho-GDP guanine nucleotide dissociation inhibitor (GDI) complexes with the GDP-bound form of Rho and inhibits its activation. We investigated the role of protein kinase C (PKC) isozymes in the mechanism of Rho activation and in signaling the loss of endothelial barrier function. Thrombin and phorbol 12-myristate 13-acetate induced rapid phosphorylation of GDI and the activation of Rho-A in human umbilical venular endothelial cells. Inhibition of PKC by chelerythrine chloride abrogated the thrombin-induced GDI phosphorylation and Rho activation. Depletion of PKC prevented the thrombin-induced GDI phosphorylation and Rho activation, thereby indicating that these events occurred downstream of phorbol ester-sensitive PKC isozyme activation. The depletion of PKC or inhibition of Rho by C3 toxin also prevented the thrombin-induced decrease in transendothelial electrical resistance (a measure of increased transendothelial permeability), thus indicating that PKC-induced barrier dysfunction was mediated through Rho-dependent pathway. Using inhibitors and dominant-negative mutants, we found that Rho activation was regulated by PKC-alpha. Moreover, the stimulation of human umbilical venular endothelial cells with thrombin induced rapid association of PKC-alpha with Rho. Activated PKC-alpha but not PKC-epsilon induced marked phosphorylation of GDI in vitro. Taken together, these results indicate that PKC-alpha is critical in regulating GDI phosphorylation, Rho activation, and in signaling Rho-dependent endothelial barrier dysfunction.     

Rho protein inactivation induced apoptosis of cultured human endothelial cells

Small GTP-binding Rho GTPases regulate important signaling pathways in endothelial cells, but little is known about their role in endothelial cell apoptosis. Clostridial cytotoxins specifically inactivate GTPases by glucosylation [Clostridium difficile toxin B-10463 (TcdB-10463), C. difficile toxin B-1470 (TcdB-1470)] or ADP ribosylation (C. botulinum C3 toxin). Exposure of human umbilical cord vein endothelial cells (HUVEC) to TcdB-10463, which inhibits RhoA/Rac1/Cdc42, or to C3 toxin, which inhibits RhoA, -B, -C, resulted in apoptosis, whereas inactivation of Rac1/Cdc42 with TcdB-1470 was without effect, suggesting that Rho inhibition was responsible for endothelial apoptosis. Disruption of endothelial microfilaments as well as inhibition of p160ROCK did not induce endothelial apoptosis. Exposure to TcdB-10463 resulted in activation of caspase-9 and -3 but not caspase-8 in HUVEC. Moreover, Rho inhibition reduced expression of antiapoptotic Bcl-2 and Mcl-1 and increased proapoptotic Bid but had no effect on Bax or FLIP protein levels. Caspase-3 activity and apoptosis induced by TcdB-10463 were abolished by cAMP elevation. In summary, inhibition of Rho in endothelial cells activates caspase-9- and -3-dependent apoptosis, which can be antagonized by cAMP elevation.     

GEF-H1/RhoA signalling pathway mediates lipopolysaccharide-induced intercellular adhesion molecular-1 expression in endothelial cells via activation of p38 and NF-κB

The purpose of study is to investigate the effects of GEF-H1/RhoA pathway in regulating intercellular adhesion molecule-1 (ICAM-1) expression in lipopolysaccharide (LPS)-activated endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) to LPS induced GEF-H1 and ICAM-1 expression in dose- and time-dependent up-regulating manners. Pretreatment with Clostridium difficile toxin B-10463 (TcdB-10463), an inhibitor of Rho activity, reduced LPS-related phosphorylation of p65 at Ser 536 in a dose-dependent manner. Inhibition of TLR4 expression significantly blocked LPS-induced RhoA activity, NF-κB transactivation, GEF-H1 and ICAM-1 expression. Coimmunoprecipitation assay indicated that LPS-activated TLR4 and GEF-H1 formed a signalling complex, suggesting that LPS, acting through TLR4, stimulates GEF-H1 expression and RhoA activity, and thereby induces NF-κB transactivation and ICAM-1 gene expression. However, GEF-H1/RhoA regulates LPS-induced NF-κB transactivation and ICAM-1 expression in a MyD88-independent pathway because inhibition of MyD88 expression could not block LPS-induced RhoA activity. Furthermore, pretreatment with Y-27632, an inhibitor of ROCK, significantly reduced LPS-induced p38, ERK1/2 and p65 phosphorylation, indicating that ROCK acts as an upstream effector of p38 and ERK1/2 to promote LPS-induced NF-κB transactivation and ICAM-1 expression. What is more, the p38 inhibitor (SB203580) but not ERK1/2 inhibitor (PD98059) blocked LPS-induce NF-κB transactivation and ICAM-1 expression, which demonstrates that RhoA mediates LPS-induced NF-κB transactivation and ICAM-1 expression dominantly through p38 but not ERK1/2 activation. In summary, our data suggest that LPS-induced ICAM-1 synthesis in HUVECs is regulated by GEF-H1/RhoA-dependent signaling pathway via activation of p38 and NF-κB.     

PKC and RhoA signals cross-talk in Escherichia coli endotoxin induced alterations in brain endothelial permeability

Escherichia coli endotoxin LPS regulates blood-brain barrier permeability by disrupting the tight junction (TJ) complex between brain endothelial cells. This study used Bend.3 cells to examine the signaling networks involved in the hyperpermeability of the brain endothelial barrier caused by LPS. The LPS-induced alterations in the brain endothelial barrier were associated with PKC (a, β, ζ) and RhoA, but were independent of PI3K and the tyrosine kinase pathway. Inhibition of PKC (a, β, ζ) and RhoA activity using shRNA and dominant negative mutants diminished the effects of LPS on the brain's endothelial TJs. The interactions between the PKC and Rho pathways were therefore examined. PKC-a and PKC-ζ, but not PKC-β interacted with RhoA in Bend.3 cells stimulated by LPS. PKC-a acted as the upstream molecule for Rho and PKC-ζ acted as the downstream target for Rho. Comparing the effect of double inhibition of "Rho and PKC" and single inhibition of "Rho" or "PKC" confirmed that this interaction is critical for LPS-induced brain endothelial cell hyperpermeability. Collectively these data are the first to suggest that LPS affects the brain's endothelial TJ barrier via PKC (a, β, ζ)- and RhoA, independent of the PI3K and tyrosine kinase pathways. In addition, PKC-a and PKC-ζ, respectively, act as the upstream and downstream regulator for RhoA in the process.     

RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.     

RhoA/rho kinase signaling reduces connexin43 expression in high glucose-treated glomerular mesangial cells with zonula occludens-1 involvement

RhoA/Rho kinase (ROCK) signaling has been suggested to be involved in diabetic nephropathy (DN) pathogenesis. Altered expression of connexin43 (Cx43) has been found in kidneys of diabetic animals. Both of them have been found to regulate nuclear factor kappa-B (NF-κB) activation in high glucose-treated glomerular mesangial cells (GMCs). The aim of this study was to investigate the relationship between RhoA/ROCK signaling and Cx43 in the DN pathogenesis. We found that upregulation of Cx43 expression inhibited NF-κB p65 nuclear translocation induced by RhoA/ROCK signaling in GMCs. Inhibition of RhoA/ROCK signaling attenuated the high glucose-induced decrease in Cx43. F-actin accumulation and an enhanced interaction between zonula occludens-1 (ZO-1) and Cx43 were observed in high glucose-treated GMCs. ZO-1 depletion or disruption of F-actin formation also inhibited the reduction in Cx43 protein levels induced by high glucose. In conclusion, activated RhoA/ROCK signaling induces Cx43 degradation in GMCs cultured in high glucose, depending on F-actin regulation. Increased F-actin induced by RhoA/ROCK signaling promotes the association between ZO-1 and Cx43, which possibly triggered Cx43 endocytosis, a mechanism of NF-κB activation in high glucose-treated GMCs.     

Cdc42 and RhoA have opposing roles in regulating membrane type 1-matrix metalloproteinase localization and matrix metalloproteinase-2 activation

Proteolysis of the basement membrane and interstitial matrix occurs early in the angiogenic process and requires matrix metalloproteinase (MMP) activity. Skeletal muscle microvascular endothelial cells exhibit robust actin stress fibers, low levels of membrane type 1 (MT1)-MMP expression, and minimal MMP-2 activation. Depolymerization of the actin cytoskeleton increases MT1-MMP expression and MMP-2 activation. Rho family GTPases are regulators of actin cytoskeleton dynamics, and their activity can be modulated in response to angiogenic stimuli such as vascular endothelial growth factor (VEGF). Therefore, we investigated their roles in MMP-2 and MT1-MMP production. Endothelial cells treated with H1152 [an inhibitor of Rho kinase (ROCK)] induced stress fiber depolymerization and an increase in cortical actin. Both MMP-2 and MT1-MMP mRNA increased, which translated into greater MMP-2 protein production and activation. ROCK inhibition rapidly increased cell surface localization of MT1-MMP and increased PI3K activity, which was required for MMP-2 activation. Constitutively active Cdc42 increased cortical actin polymerization, phosphatidylinositol 3-kinase activity, MT1-MMP cell surface localization, and MMP-2 activation similarly to inhibition of ROCK. Activation of Cdc42 was sufficient to decrease RhoA activity. Capillary sprout formation in a three-dimensional collagen matrix was increased in cultures treated with RhoAN19 or Cdc42QL and, conversely, decreased in cultures treated with dominant negative Cdc42N17. VEGF stimulation also induced activation of Cdc42 while inhibiting RhoA activity. Furthermore, VEGF-dependent activation of MMP-2 was reduced by inhibition of Cdc42. These results suggest that Cdc42 and RhoA have opposing roles in regulating cell surface localization of MT1-MMP and MMP-2 activation.     

Tetramethylpyrazine Protects Against Oxygen-Glucose Deprivation-Induced Brain Microvascular Endothelial Cells Injury via Rho/Rho-kinase Signaling Pathway

Tetramethylpyrazine (TMP, also known as Ligustrazine), which is isolated from Chinese Herb Medicine Ligustium wollichii Franchat (Chuan Xiong), has been widely used in China for the treatment of ischemic stroke by Chinese herbalists. Brain microvascular endothelial cells (BMECs) are the integral parts of the blood–brain barrier (BBB), protecting BMECs against oxygen-glucose deprivation (OGD) which is important for the treatment of ischemic stroke. Here, we investigated the protective mechanisms of TMP, focusing on OGD-injured BMECs and the Rho/Rho-kinase (Rho-associated kinases, ROCK) signaling pathway. The model of OGD-injured BMECs was established in this study. BMECs were identified by von Willebrand factor III staining and exposed to fasudil, or TMP at different concentrations (14.3, 28.6, 57.3 µM) for 2 h before 24 h of OGD injury. The effect of each treatment was examined by cell viability assays, measurement of intracellular reactive oxygen species (ROS), and transendothelial electric resistance and western blot analysis (caspase-3, endothelial nitric oxide synthase (eNOS), RhoA, Rac1). Our results show that TMP significantly attenuated apoptosis and the permeability of BMECs induced by OGD. In addition, TMP could notably down-regulate the characteristic proteins in Rho/ROCK signaling pathway such as RhoA and Rac1, which triggered abnormal changes of eNOS and ROS, respectively. Altogether, our results show that TMP has a strong protective effect against OGD-induced BMECs injury and suggest that the mechanism might be related to the inhibition of the Rho/ROCK signaling pathway.     

Chronic intrauterine pulmonary hypertension increases endothelial cell Rho kinase activity and impairs angiogenesis in vitro

Persistent pulmonary hypertension of the newborn (PPHN) is characterized by endothelial dysfunction and decreased vascular growth. The role of Rho kinase activity in modulating endothelial function and regulating angiogenesis during normal lung development and in PPHN is unknown. We hypothesized that PPHN increases Rho kinase activity in fetal pulmonary artery endothelial cells (PAECs) and impairs angiogenesis in vitro. Proximal PAECs were harvested from fetal sheep with partial ligation of the ductus arteriosus in utero (PPHN) and age-matched controls. Rho kinase activity was measured by RhoA, Rho GTP, and phosphorylated MYPT-1 protein content. The effects of Rho kinase activity on angiogenesis, endothelial nitric oxide (NO) synthase (eNOS) protein expression, and NO production were determined in normal and PPHN PAECs. Angiogenesis was assessed by tube formation in vitro with/without Y-27632 (a Rho kinase inhibitor) and calpeptin (a Rho kinase activator) in the presence/absence of N-nitro-l-arginine (l-NA, an NOS inhibitor). RhoA, Rho GTP, and phosphorylated MYPT-1 protein were increased in PPHN PAECs. Tube formation was reduced 29% in PPHN PAECs (P < 0.001) and increased with Y-27632 treatment in normal and PPHN PAECs, with PPHN PAECs achieving levels similar to those of normal PAECs. l-NA inhibited the Y-27632-induced increase in tube formation in normal, but not PPHN, PAECs. Calpeptin reduced tube formation in normal and PPHN PAECs. eNOS expression was reduced 42% in PPHN PAECs (P < 0.01). Y-27632 increased eNOS protein and NO production in normal and PPHN PAECs. Calpeptin decreased eNOS protein only in normal PAECs but reduced NO production in normal and PPHN PAECs. We conclude that Rho kinase activity is increased in PPHN PAECs and impairs angiogenesis and downregulates eNOS protein and NO production in vitro.