Rap1 signaling in endothelial barrier control

The small G-protein Rap1 plays an important role in the regulation of endothelial barrier function, a process controlled largely by cell–cell adhesions and their connection to the actin cytoskeleton. During the various stages of barrier dynamics, different guanine nucleotide exchange factors (GEFs) control Rap1 activity, indicating that Rap1 integrates multiple input signals. Once activated, Rap1 induces numerous signaling cascades, together responsible for the increased endothelial barrier function. Most notably, Rap1 activation results in the inhibition of Rho to decrease radial stress fibers and the activation of Cdc42 to increase junctional actin. This implies that Rap regulates endothelial barrier function by dual control of cytoskeletal tension. The molecular details of the signaling pathways are becoming to be elucidated.     

Asef controls vascular endothelial permeability and barrier recovery in the lung

Increased levels of hepatocyte growth factor (HGF) in injured lungs may reflect a compensatory response to diminish acute lung injury (ALI). HGF-induced activation of Rac1 GTPase stimulates endothelial barrier protective mechanisms. This study tested the involvement of Rac-specific guanine nucleotide exchange factor Asef in HGF-induced endothelial cell (EC) cytoskeletal dynamics and barrier protection in vitro and in a two-hit model of ALI. HGF induced membrane translocation of Asef and stimulated Asef Rac1-specific nucleotide exchange activity. Expression of constitutively activated Asef mutant mimicked HGF-induced peripheral actin cytoskeleton enhancement. In contrast, siRNA-induced Asef knockdown or expression of dominant-negative Asef attenuated HGF-induced Rac1 activation evaluated by Rac-GTP pull down and FRET assay with Rac1 biosensor. Molecular inhibition of Asef attenuated HGF-induced peripheral accumulation of cortactin, formation of lamellipodia-like structures, and enhancement of VE-cadherin adherens junctions and compromised HGF-protective effect against thrombin-induced RhoA GTPase activation, Rho-dependent cytoskeleton remodeling, and EC permeability. Intravenous HGF injection attenuated lung inflammation and vascular leak in the two-hit model of ALI induced by excessive mechanical ventilation and thrombin signaling peptide TRAP6. This effect was lost in Asef^−/− mice. This study shows for the first time the role of Asef in HGF-mediated protection against endothelial hyperpermeability and lung injury.     

Cyclic AMP blocks bacterial lipopolysaccharide-induced myosin light chain phosphorylation in endothelial cells through inhibition of Rho/Rho kinase signaling

During Gram-negative sepsis bacterial LPS induces endothelial cell contraction, actin reorganization, and loss of endothelial integrity by an unknown signal mechanism. In this study, we provide evidence that LPS-stimulation of endothelial cells (HUVEC) decreases myosin light chain (MLC) phosphatase, resulting in an increase in MLC phosphorylation followed by cell contraction. All of these LPS effects could be blocked by the Rho-GTPase inhibitor C3 transferase from Clostridium botulinum or the Rho kinase inhibitor Y-27632. These data suggest that LPS induces MLC phosphorylation via Rho/Rho kinase-mediated inhibition of MLC phosphatase in HUVEC. Furthermore, we observed that cAMP-elevating drugs, known to exert a vasoprotective function, mimicked the effects of C3 transferase and Y-27632, i.e., inhibited LPS-induced MLC phosphatase inactivation and MLC phosphorylation. cAMP elevation did not inhibit myosin phosphorylation induced by constitutively active V14Rho or the MLC phosphatase inhibitor calyculin and did not induce phosphorylation of RhoA in HUVEC, indicating inhibition of an upstream regulator of Rho/Rho kinase. Taken together, Rho/Rho kinase appears to be a central target for inflammatory mediators causing endothelial cell contraction such as bacterial toxins, but also for vasoprotective molecules elevating intracellular cAMP.     

Spatial control of Rho (Rac-Rop) signaling in tip-growing plant cells

Spatially restricted signaling by Rho GTPases is essential for the polarization of eukaryotic cells, which is required for the morphogenesis, mobility and division of single cells, and for the development of multicellular organisms. Rac-Rop GTPases, which constitute a plant-specific Rho GTPase subfamily, accumulate at the apical plasma membrane of pollen tubes and root hairs, where they control rapid polar cell expansion by a process known as tip growth. Here, recent insights into the spatial control of Rac-Rop-dependent signaling in tip-growing plant cells by regulatory proteins (i.e. Rho GTPase-activating proteins, Rho guanine nucleotide dissociation inhibitors, Rho guanine nucleotide-exchange factors and phosphoinositide-specific phospholipase C) and lipids [phosphatidylinositol (4,5)-bisphosphate and diacyl glycerol] are summarized. A model is presented, which integrates the current knowledge concerning the molecular mechanisms that maintain the polarization of Rho signaling in plant cells.     

Rho/Rho激酶信号通路与脉管收缩

Rho/Rho激酶信号通路在正常血管、淋巴管等脉管收缩过程中发挥重要的调节作用,并参与休克后血管反应性和钙敏感性的双相调节以及休克淋巴管低反应性的发生.以Rho/Rho激酶为靶点,对于干预休克脉管系统低反应性的发生具有重要意义.本文综述Rho/Rho激酶信号通路在脉管收缩中的调节机制.     

IL-8 activates endothelial cell CXCR1 and CXCR2 through Rho and Rac signaling pathways

Stimulation of microvascular endothelial cells with interleukin (IL)-8 leads to cytoskeletal reorganization, which is mediated by combined activation of the CXCR1 and the CXCR2. In the early phase actin stress fibers appear, followed by cortical actin accumulation and cell retraction leading to gap formation between cells. The early response (between 1 and 5 min) is inhibited by an antibody that blocks the CXCR1. The later phase (from about 5 to 60 min), which is associated with cell retraction, is prevented by anti-CXCR2 antibody. Furthermore, anti-CXCR2, but not anti-CXCR1, antibody blocked IL-8-mediated haptotaxis of endothelial cells on collagen. The later phase of the IL-8-mediated actin response is inhibited by pertussis toxin, indicating that the CXCR2 couples to G(i). In contrast, the early phase is blocked by C3 botulinum toxin, which inactivates Rho, and by Y-27632, which inhibits Rho kinase, but not by pertussis toxin. Furthermore, the early CXCR1-mediated formation of stress fibers was prevented by dominant negative Rho. Dominant negative Rac on the other hand initially translocated to actin-rich filopodia after stimulation with IL-8 and later prevented cell retraction by blocking the CXCR2-mediated cytoskeletal response. These results indicate that IL-8 activates both the CXCR1 and the CXCR2 on microvascular endothelial cells, using different signal transduction cascades. The retraction of endothelial cells due to activation of the CXCR2 may contribute to the increased vascular permeability observed in acute inflammation and during the angiogenic response.     

Advanced glycation end products increase endothelial permeability through the RAGE/Rho signaling pathway

Although increased vascular permeability is known to be a major characteristic of diabetic vasculopathy, the precise mechanisms and relevance of advanced glycation end products (AGE) to hyperpermeability of vessels remains unclear. Here, we studied changes in cytoskeletal configuration and the signaling mechanism induced by AGE in human endothelial cells. AGE-BSA stimulation induced Rho activation, intercellular gap formation, prominent actin stress fiber and cell contraction without changing VE-cadherin, and subsequently transendothelial diffusion of FITC-labeled dextran. These processes induced by AGE-BSA were inhibited by either Rho-kinase inhibitor Y27632 or anti-RAGE antibody. We also showed that RhoA and RAGE spontaneously formed a complex. These findings suggest that activation of RAGE/Rho is involved in AGE-BSA-induced hyperpermeability through gap formation and actin reorganization in diabetes.

Structured summary

MINT-7301170: Rhotekin (uniprotkb:Q9BST9) physically interacts (MI:0915) with RhoA (uniprotkb:P61586) by pull down (MI:0096)MINT-7301204, MINT-7301186: RhoA (uniprotkb:P61586) physically interacts (MI:0915) with RAGE (uniprotkb:Q15109) by anti bait coimmunoprecipitation (MI:0006)     

Vascular endothelial growth factor stimulates neurite outgrowth from cerebral cortical neurons via Rho kinase signaling

Vascular endothelial growth factor (VEGF),which is prominently involved in angiogenesis, also exerts direct effects on neurons, leading to neurite extension, neuroprotection, and neurogenesis. However, the signal transduction pathways employed by VEGF in neurons are incompletely understood. We investigated the molecular mechanisms through which VEGF stimulates neurogenesis in primary cultures of rat cerebral cortical neurons. VEGF increased neurite outgrowth, measured using a colorimetric assay for cresyl violet staining of neuronal processes, with half-maximal enhancement at 10 ng/mL and maximal, approximately 60% enhancement at 30-100 ng/mL. The effect of VEGF was not reproduced by VEGF-B or placental growth factor, but was blocked by SU1498, consistent with a VEGFR2 receptor-mediated process. VEGF-induced neurite outgrowth was also blocked by the ROK inhibitor Y27632 and the Rho inhibitors sulindac and Clostridium botulium exoenzyme C3, and was accompanied by Y27632-sensitive phosphorylation of cofilin, a downstream mediator of Rho/ROK signaling. We conclude that VEGF promotes neurite outgrowth from cerebral cortical neurons by interacting with VEGFR2 and activating Rho/ROK signaling pathways.     

Rho GTPases in the regulation of pulmonary vascular barrier function

Pulmonary endothelial permeability is an important determinant of vascular adaptation to changes in oxygen tension, blood pressure, levels of growth factors or inflammatory cytokines. The Ras homologous (Rho) family of guanosine triphosphate phosphatases (Rho GTPases), key regulators of the actin cytoskeleton, regulate endothelial barrier function in response to a variety of environmental factors and signalling agents via the reorganization of the actin cytoskeleton, changes in receptor trafficking or the phosphorylation of junctional proteins. This review provides a brief summary of recent knowledge on Rho-GTPase-mediated effects on pulmonary endothelial barrier function and focuses in particular on their role in pulmonary vascular disorders, including pulmonary hypertension, chronic obstructive pulmonary disease, acute lung injury and acute respiratory distress syndrome.     

Structural basis of the Rho GTPase signaling

Small GTPases of the Rho family serve as conformational switches in a wide variety of signal transduction pathways that regulate diverse cellular functions. The GTP-bound forms of Rho GTPases are capable of interacting with downstream effectors that control cytoskeletal rearrangements. Regulators that stimulate nucleotide exchange, the hydrolytic cycle and distribution between the membrane and cytosol control the switch. Detailed pictures of Rho GTPase switching, effector recognition and regulation by regulators have emerged from recent structural investigations. These include the most extensively studied Rho GTPases, RhoA, Rac1, 2 and Cdc42, and their complexes with effectors and regulators. These studies have revealed the general diversity of effector and regulator structures, and in particular the structural features concerning the specific interactions involved in Rho effector recognition and regulator interactions with Rho GTPase. These findings provide a critical insight into the nature of Rho GTPase activity and consequently allow for a detailed manipulation of signaling pathways mediated by these proteins.     

Rho/ROCK信号通路与神经可塑性

在神经网络中,神经可塑性是大脑响应内在和外在刺激的重要特征。越来越多的研究已经阐明了神经可塑性与神经损伤性疾病之间的相关性。Rho/Rho相关卷曲螺旋形成蛋白激酶(Rho/Rho associ-ated coiledcoil forming protein kinase, Rho/ROCK)通路是生物体广泛存在的经典信号通路,参与细胞迁移、树突发育和轴突延伸,并且与帕金森、精神发育迟滞和阿尔茨海默症等多种神经退行性或损伤性疾病有关。本文对Rho/ROCK信号通路与神经可塑性的研究进展予以综述,讨论了ROCK抑制剂对各种神经疾病的潜在治疗前景。     

Wnt5A/Ryk signaling critically affects barrier function in human vascular endothelial cells

Satisfactory therapeutic strategies for septic shock are still missing. Previously we found elevated levels of Wnt5A in patients with severe sepsis and septic shock. Wnt5A is released by activated macrophages but knowledge of its effects in the vascular system remains scant. Here we investigate the response of human coronary artery endothelial cells (HCAEC) to Wnt5A. We used a genome-wide differential expression approach to define novel targets regulated by Wnt5A. Gene ontology analysis of expression profiles revealed clusters of genes involved in actin cytoskeleton remodeling as the predominant targets of Wnt5A. Wnt5A targeted Rho-associated protein serine/threonine kinase (ROCK), leading to phosphorylation of LIM kinase-2 (LIMK2) and inactivation of the actin depolymerization factor cofilin-1 (CFL1). Functional experiments recording cytoskeletal rearrangements in living cells showed that Wnt5A enhanced stress fiber formation as a consequence of reduced actin depolymerization. The antagonist Wnt inhibitory factor 1 (WIF1) that specifically interferes with the WIF domain of Ryk receptors prevented actin polymerization. Wnt5A disrupted β-catenin and VE-cadherin adherens junctions forming inter-endothelial gaps. Functional experiments targeting the endothelial monolayer integrity and live recording of trans-endothelial resistance revealed enhanced permeability of Wnt5A-treated HCAEC. Ryk silencing completely prevented Wnt5A-induced endothelial hyperpermeability. Wnt5A decreased wound healing capacity of HCAEC monolayers; this was restored by the ROCK inhibitor Y-27632. Here we show that Wnt5A acts on the vascular endothelium causing enhanced permeability through Ryk interaction and downstream ROCK/LIMK2/CFL1 signaling. Wnt5A/Ryk signaling might provide novel therapeutic strategies to prevent capillary leakage in systemic inflammation and septic shock.     

Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta

Evidence indicates that both the Rho/Rho kinase signaling pathway and reactive oxygen species (ROS) such as superoxide and H(2)O(2) are involved in the pathogenesis of hypertension. This study aimed to determine whether ROS-induced vascular contraction is mediated through activation of Rho/Rho kinase. Rat aortic rings (endothelium denuded) were isolated and placed in organ chambers for measurement of isometric force development. ROS were generated by a xanthine (X)-xanthine oxidase (XO) mixture. The antioxidants tempol (3 mM) and catalase (1,200 U/ml) or the XO inhibitor allopurinol (400 microM) significantly reduced X/XO-induced contraction. A Rho kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl-N-4-pyridil)cyclohexanecarboxamide dihydrochloride (Y-27632), decreased the contraction in a concentration-dependent manner; however, the Ca(2+)-independent protein kinase C inhibitor rottlerin did not have an effect on X/XO-induced contraction. Phosphorylation of the myosin light chain phosphatase target subunit (MYPT1) was increased by ROS, and preincubation with Y-27632 blocked this increased phosphorylation. Western blotting for cytosolic and membrane-bound fractions of Rho showed that Rho was increased in the membrane fraction by ROS, suggesting activation of Rho. These observations demonstrate that ROS-induced Ca(2+) sensitization is through activation of Rho and a subsequent increase in Rho kinase activity but not Ca(2+)-independent PKC.     

Cyclic AMP potentiates vascular endothelial cadherin-mediated cell-cell contact to enhance endothelial barrier function through an Epac-Rap1 signaling pathway

Cyclic AMP (cAMP) is a well-known intracellular signaling molecule improving barrier function in vascular endothelial cells. Here, we delineate a novel cAMP-triggered signal that regulates the barrier function. We found that cAMP-elevating reagents, prostacyclin and forskolin, decreased cell permeability and enhanced vascular endothelial (VE) cadherin-dependent cell adhesion. Although the decreased permeability and the increased VE-cadherin-mediated adhesion by prostacyclin and forskolin were insensitive to a specific inhibitor for cAMP-dependent protein kinase, these effects were mimicked by 8-(4-chlorophenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate, a specific activator for Epac, which is a novel cAMP-dependent guanine nucleotide exchange factor for Rap1. Thus, we investigated the effect of Rap1 on permeability and the VE-cadherin-mediated cell adhesion by expressing either constitutive active Rap1 or Rap1GAPII. Activation of Rap1 resulted in a decrease in permeability and enhancement of VE-cadherin-dependent cell adhesion, whereas inactivation of Rap1 had the counter effect. Furthermore, prostacyclin and forskolin induced cortical actin rearrangement in a Rap1-dependent manner. In conclusion, cAMP-Epac-Rap1 signaling promotes decreased cell permeability by enhancing VE-cadherin-mediated adhesion lined by the rearranged cortical actin.     

Rho/Rho kinase signaling mediates increased basal pulmonary vascular tone in chronically hypoxic rats

Recent evidence suggests that Rho/Rho kinase signaling plays an important role in the sustained vasoconstriction induced by many agonists and is involved in the pathogenesis of systemic vascular diseases. However, little is known about its role in increased vascular tone in hypoxic pulmonary hypertension (PH). The purpose of this study was to examine whether Rho/Rho kinase-mediated Ca2+ sensitization contributed to sustained vasoconstriction and increased vasoreactivity in hypoxic PH in rats. Acute intravenous administration of Y-27632, a Rho kinase inhibitor, nearly normalized the high pulmonary arterial blood pressure and total pulmonary resistance in chronically hypoxic rats. In contrast to nifedipine, Y-27632 also markedly decreased elevated basal vascular tone in hypertensive blood-perfused lungs and isolated pulmonary arteries. Y-27632 and another Rho kinase inhibitor, HA-1077, completely reversed nitro-L-arginine-induced vasoconstriction in physiological salt solution-perfused hypertensive lungs, whereas inhibitors of myosin light chain kinase (ML-9), protein kinase C (GF-109203X), phosphatidylinositol 3-kinase (LY-294002), and tyrosine kinase (tyrphostin A23) caused only partial or no reversal of the vasoconstriction. Vasoconstrictor responses to KCl were augmented in hypertensive physiological salt solution-perfused lungs and pulmonary arteries, and the augmentation was eliminated by Y-27632. These results suggest that Rho/Rho kinase-mediated Ca2+ sensitization plays a central role in mediating sustained vasoconstriction and increased vasoreactivity in hypoxic PH.     

Glucocorticoids and endothelial cell barrier function

Glucocorticoids (GCs) are steroid hormones that have inflammatory and immunosuppressive effects on a wide variety of cells. They are used as therapy for inflammatory disease and as a common agent against edema. The blood brain barrier (BBB), comprising microvascular endothelial cells, serves as a permeability screen between the blood and the brain. As such, it maintains homeostasis of the central nervous system (CNS). In many CNS disorders, BBB integrity is compromised. GC treatment has been demonstrated to improve the tightness of the BBB. The responses and effects of GCs are mediated by the ubiquitous GC receptor (GR). Ligand-bound GR recognizes and binds to the GC response element located within the promoter region of target genes. Transactivation of certain target genes leads to improved barrier properties of endothelial cells. In this review, we deal with the role of GCs in endothelial cell barrier function. First, we describe the mechanisms of GC action at the molecular level. Next, we discuss the regulation of the BBB by GCs, with emphasis on genes targeted by GCs such as occludin, claudins and VE-cadherin. Finally, we present currently available GC therapeutic strategies and their limitations.