Increased RhoA Activity Research Articles

Cigarette smoke extract induces p38-mediated expression and ROS/rho-mediated translocation of alpha 2C adrenoceptor in human microvascular smooth muscle cells.

Raynaud's phenomenon (RP) is a vascular disease characterized by exaggerated vasoconstriction in response to stressors, mainly cold and emotional stress. This vasoconstriction is mediated solely by alpha 2C-adrenoceptors (α2C-AR) expressed in vascular smooth muscle cells of dermal arterioles. Several factors, among which is cigarette smoking, are associated with aggravated symptoms of and increased risk for RP. Evidence shows that cigarette smoking induces the production of reactive oxygen species (ROS), which is a major driver of RP pathogenesis. However, the exact mechanism by which smoking contributes to RP or α2C-AR remains unclear. Here, we show that cigarette smoke extract (CSE) upregulates the expression of α2C-AR in a concentration- and time-dependent manner in VSMCs extracted from human dermal arterioles. This increase is associated with the activation of p38 MAPK, as pretreatment with SB-202190, a p38 specific inhibitor, attenuated CSE-induced α2C-AR expression. Furthermore, our results show that CSE induces ROS production followed by increased RhoA activation. We also show that CSE induces translocation of vascular α2C-AR to the plasma membrane, and that this mobilization is attenuated by inhibiting ROS via N-acetylcysteine or apocynin. Similarly, inhibition of Rho kinase via H- 11522 abolished CSE-induced α2C-AR translocation. Collectively, these results indicate that CSE activates two different signaling pathways to induce the expression and the translocation of α2C-AR. While CSE activates a p38-dependent mechanism to increase α2C-AR expression, it initiates the receptor's spatial and functional rescue via a ROS/RhoA signaling pathway. These results provide mechanistic insight into the effect of cigarette smoking on RP, and further reinforce that smoking avoidance/cessation is critical to manage this disease, especially in the absence of a definitive drug for RP.

Silk Sericin is a Potent, Multifunctional Biomaterial for Endothelialization of Tissue‐Engineered Heart Valves

AbstractEndothelialization is important for offering an anticoagulant surface, which is crucial for the construction of tissue‐engineered heart valves. Silk sericin is extensively investigated in the biomedical field due to its remarkable biological activities and diverse functional groups, favoring chemical modifications for the formation of versatile constructs. Herein, a sericin covalently modified valve (SCMV) is successfully fabricated by coupling maleylated silk sericin (MSS) with thiolated decellularized heart valve (DHV) through a Michael addition reaction. The results demonstrate that SCMV exhibits a smooth surface, higher hydrophilicity, and excellent resistance to platelets adhesion compared to DHV. Additionally, SCMV promotes endothelial cells (ECs) adhesion under both static and flow conditions and enhances their survival in a mouse subcutaneous implant model. The study also discovered that MSS‐stimulated ECs exhibit increased RhoA activation, expression of rho‐associated protein kinase 2 (ROCK2) and phosphorylated myosin light chain 2 (MLC2), actin polymerization, and cell adhesion; whereas actin organization and cell adhesion are significantly reduced by treatment with Y‐27632, a ROCK inhibitor. Furthermore, several integrin subunits, including α1, α2, α3, α5, α6, β1, β3, and β5, are involved in this regulatory process. Collectively, the findings highlight the potential application of silk sericin in promoting endothelialization of DHV while uncovering novel mechanisms underlying its regulatory effect on ECs adhesion.

Abstract 6225: Tumor evolution through selection by ECM stiffness

Abstract In this study, we used experimental evolution to investigate the extent to which the mechanical stiffness of the tumor extracellular matrix (ECM) exerts selection pressure on a genetically diverse population of breast cancer cells. Polyacrylamide hydrogels were fabricated and conjugated with collagen type I to create the ECM with different stiffness. MDA-MB-231 cells were continuously cultured on the hydrogels of the same corresponding stiffness. Growth rate was determined at certain time points by harvesting and counting the cells. Abundance of clonal MDA-MB-231 cells labeled with heritable DNA barcodes underwent the same selection was identified with sequencing of DNA barcode tags. Cellular phenotypes were characterized through immunofluorescence staining and confocal microscopy. 3D migration of the cancer cells was examined using Boyden chamber assay. Cellular contractility was measured through traction force microscopy. Whole genome transcriptome sequencing (RNA-seq), assay for transposase-accessible chromatin with sequencing, and reduced representation bisulfite sequencing were performed for transcriptomic and epigenetic analysis. Sustained culture of MDA-MB-231 breast cancer cells overall three months on soft ECM resulted in an increased proliferation rate and beta1 integrin-dependent spreading area of the cells. Such changes did not occur in clonal cells with low genetic variation and did not occur in ancestral cells on stiff substrates. Barcode analysis revealed two distinct surviving populations post-genetic selection, with one demonstrating robust proliferation on both soft and stiff ECM, and the other exhibiting preferential adaptation to the soft matrix. The soft-selected populations exhibited nuclear localization of the transcriptional regulator yes-associated protein and less wrinkled nuclei compared with ancestral populations when both populations were grown on soft ECM. Aggressive migration was observed in the soft-selected populations, indicating a more malignant selected phenotype. The soft-selected cells showed increased RhoA activity and contractile tension on a soft substrate compared to the ancestral cells. RNA-seq analysis revealed systematic differences between ancestral, stiff-selected, and soft-selected populations corresponding to enriched biological processes related to cell adhesion and ECM organization. Significant differences in DNA methylation and chromatin accessibility were observed in soft selected populations which were correlated with changes in RNA expression. ECM stiffness exerts selection pressure on genetically variable cancer cell populations, selecting specific genetic variants in a cell population optimally adapted for that stiffness. Selected variants on soft ECM tend to be highly migratory, proliferative and generate high traction even on soft ECM. Our results highlight the importance of ECM stiffness-driven evolution in cancer development. Citation Format: Ting-Ching Wang, Suchitaa Sawhney, Daylin Morgan, Richa Rashmi, Marcos R. Estecio, Amy Brock, Irtisha Singh, Richard L. Bennett, Charles F. Baer, Jonathan D. Licht, Tanmay P. Lele. Tumor evolution through selection by ECM stiffness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6225.

The CPLANE protein Fuzzy regulates ciliogenesis by suppressing actin polymerization at the base of the primary cilium via p190A RhoGAP.

The primary cilium decorates most eukaryotic cells and regulates tissue morphogenesis and maintenance. Structural or functional defects of primary cilium result in ciliopathies, congenital human disorders affecting multiple organs. Pathogenic variants in the ciliogenesis and planar cell polarity effectors (CPLANE) genes FUZZY, INTU and WDPCP disturb ciliogenesis, causing severe ciliopathies in humans and mice. Here, we show that the loss of Fuzzy in mice results in defects of primary cilia, accompanied by increased RhoA activity and excessive actin polymerization at the basal body. We discovered that, mechanistically, Fuzzy interacts with and recruits the negative actin regulator ARHGAP35 (also known as p190A RhoGAP) to the basal body. We identified genetic interactions between the two genes and found that a mutant ArhGAP35 allele increases the severity of phenotypic defects observed in Fuzzy-/- mice. Based on our findings, we propose that Fuzzy regulates ciliogenesis by recruiting ARHGAP35 to the basal body, where the latter likely restricts actin polymerization and modifies the actin network. Our study identifies a mechanism whereby CPLANE proteins control both actin polymerization and primary cilium formation.

Essential role of obscurin kinase-1 in cardiomyocyte coupling via N-cadherin phosphorylation.

Obscurins are giant cytoskeletal proteins with structural and regulatory roles. Obscurin-B (~870 kDa), the largest known isoform, contains 2 enzymatically active Ser/Thr kinase (kin) domains, kin1 and kin2, which belong to the myosin light chain kinase family. Kin1 binds to and phosphorylates N-cadherin, a major component of the intercalated disc, the unique sarcolemmal microdomain that mediates the mechanochemical coupling of adjacent cardiomyocytes. Obscurin-B containing kin1 and N-cadherin colocalize at cell junctions in embryonic rat ventricular myocytes (ERVMs), and their codistribution is regulated by Ca2+. Phosphoproteomics analysis revealed that obscurin-kin1 phosphorylates N-cadherin at Ser-788 located within the juxtamembrane region of its cytoplasmic domain, with an apparent Kcat of approximately 5.05 min-1. Overexpression of obscurin-kin1 or phosphomimic-Ser-788-Glu N-cadherin in ERVMs markedly increases cell adhesion and chemical coupling. Importantly, phosphomimic Ser-788-Glu N-cadherin exhibits significantly reduced binding to p120-catenin, while overexpression of phosphoablated Ser-788-Ala N-cadherin increases RhoA activity. Consistent with an essential role of the obscurin-kin1/N-cadherin axis in cardiomyocyte coupling, it is deregulated in end-stage human heart failure. Given the nearly ubiquitous expression of obscurin and N-cadherin, our findings may have broad applicability in deciphering the obscurin-kin1/N-cadherin axis that likely mediates cell coupling in diverse tissues and organs.

DNAR-06. RHO GTPASES AS A POTENTIAL CHEMOSENSITIZER OF TMZ AND CISPLATIN-RESISTANT GBM TUMOR CELLS THROUGH A P53-DEPENDENT MECHANISM

Abstract Rho GTPases regulate cellular processes related to tumor progression and their expression/activity determines the invasiveness and aggressiveness of GBM and, despite current aggressive therapies, effective treatments remain desired. Here we proposed that Rho GTPase acts on the genomic stability of GBM and its resistance to TMZ and cisplatin drugs in a p53-dependent mechanism. We selected GBM cell lines with different p53 status – U87-MG and A172 (wt-p53), T98G and U138-MG (mut-p53) – and subjected them to Rho inhibition by C3 toxin and subsequent DNA damage by TMZ treatments or cisplatin. Bioinformatic analysis showed that prolonged exposure to cisplatin induces the transcription of regulatory genes of Rho GTPases. Pulldown assays corroborated this analysis showing an increase in Rho activity after exposure to cisplatin or TMZ. IC50 for TMZ and cisplatin, as well as survival and proliferation assays, were all decreased by Rho inhibition in wt-p53 cells, while HCR assays showed that DNA repair capacity for both genotoxic drugs is decreased by Rho inhibition only in wt-p53 cells. To verify the role of p53 in this mechanism, we knocked down p53 in U87-MG cells, thus avoiding Rho inhibition-induced chemosensitivity and impaired DNA repair. Then, U87-MG cells were subjected to various treatment regimens with low doses of TMZ or cisplatin to obtain a subpopulation of cells resistant to these drugs. Further exposure to DNA damage led to increased p53(Ser15) phosphorylation, p53 nuclear translocation, and F-actin-induced polymerization. Two TMZ-resistant cell clones were selected, both showing a significant increase in TMZ IC50 for survival, while Rho inhibition reduced IC50 in both clones, significantly reversing the acquired resistance of these cells. These results suggest that Rho GTPase plays a role in DNA repair and in the acquired GBM wt-p53 resistance to TMZ and show that this pathway may be a fragile-point against current therapies.

The Muscarinic Acetylcholine M2 Receptor-Induced Nitration of p190A by eNOS Increases RhoA Activity in Cardiac Myocytes.

p190RhoGAP, which exists in two paralogs, p190RhoGAP-A (p190A) and p190RhoGAP-B (p190B), is a GTPase activating protein (GAP) contributing to the regulation of the cellular activity of RhoGTPases. Recent data showed that M2 muscarinic acetylcholine receptor (M2R) stimulation in neonatal rat cardiac myocytes (NRCM) induces the binding of p190RhoGAP to the long isoform of the regulator of G protein signaling 3 (RGS3L). This complex formation alters the substrate preference of p190RhoGAP from RhoA to Rac1. By analyzing carbachol-stimulated GAP activity, we show herein that p190A, but not p190B, alters its substrate preference in NRCM. Based on data that the RhoGAP activity of p190A in endothelial cells is diminished upon nitration by endothelial nitric oxide synthase (eNOS)-derived peroxynitrite, we studied whether carbachol-induced NO/peroxynitrite formation contributes to the carbachol-induced RhoA activation in NRCM. Interestingly, the carbachol-induced RhoA activation in NRCM was suppressed by the eNOS-preferring inhibitor L-NIO as well as the non-selective NOS inhibitor L-NAME. Using L-NIO, we firstly verified the carbachol-induced NO production concurrent with eNOS activation and, secondly, the carbachol-induced nitration of p190A in NRCM. By co-immunoprecipitation, the carbachol-induced complex formation of eNOS, p190A, RGS3L and caveolin-3 was detected. We thus conclude that the NO production by M2R-induced eNOS activation in caveolae in NRCM is required for the nitration of p190A, leading to the binding to RGS3L and the change in substrate preference from RhoA to Rac1. In line with this interpretation, the disruption of caveolae in NRCM by methyl-β-cyclodextrin suppressed carbachol-induced RhoA activation in NRCM to a similar extent as the inhibition of NO production.

Genome-Wide CRISPR Screens Identify Multiple Synthetic Lethal Targets That Enhance KRASG12C Inhibitor Efficacy.

Identification of synthetic lethal genes with KRASG12C using genome-wide CRISPR/Cas9 screening and credentialing of the ability of TEAD inhibition to enhance KRASG12C efficacy provides a roadmap for combination strategies. See related commentary by Johnson and Haigis, p. 4005.

#3119 DEEP LEARNING IDENTIFIES SUBPHENOTYPES OF DIABETIC KIDNEY DISEASE DRIVEN BY GENETIC VARIATIONS IN THE RHO PATHWAY

Abstract Background and Aims Diabetic kidney disease (DKD) is a leading cause of end-stage kidney disease (ESKD), however therapies targeting causal pathways have been limited by disease heterogeneity. Integrating electronic health record (EHR) data and genomics may uncover hidden subphenotypes in DKD. In this study, we use deep learning to identify a novel genetic variant of ARHGEF18 associated with significantly higher risk of DKD and ESKD (Figure 1A). We further employed quantitative microscopy techniques and biochemical assays to elucidate the mechanistic role of ARHGEF18 and its variant in podocytes. Method DKD patients from the Mount Sinai BioMe Biobank were used in this study. Unsupervised clustering and accounting for population structure in a deep learning framework identified two clusters: Cluster M (mild) and S (severe). We then performed a genome wide association study (GWAS) of patients within each cluster compared with healthy controls. For mechanistic studies of the novel variant, cytoplasmic, focal adhesion, cytoskeletal morphometrics as well as live-cell motility, Rho GTPase activity, and protein degradation experiments were performed using confocal and total internal reflection fluorescence (TIRF) microscopy as well as cell-free biochemical assays using immortalized human podocytes expressing ARHGEF18 wild-type (WT) and mutant transcripts. Results We employed autoencoders and unsupervised clustering of EHR data on 1,372 DKD patients to establish two clusters with differential prevalence of ESKD. There was a greater prevalence of proteinuria in Cluster S compared to Cluster M (Figure 1B). Further exome sequencing study in these patients identified a novel variant in ARHGEF18, a Rho guanine exchange factor highly enriched in podocytes (Figure 1A). Nephroseq database showed an increased ARHGEF18 expression in chronic kidney disease (CKD) kidney biopsy samples compared to healthy controls (Figure 1C). Overexpression of ARHGEF18 mutant transcripts in human immortalized podocytes led to impairments in cell adhesion, focal adhesion architecture, and cell motility. Live TIRF microscopy experiments showed preferential subcellular localization of GEF18 mutant to the periphery of migrating podocytes whereas GEF18 WT localized at the perinuclear/cytoplasmic region (Figure 2A, B). GEF18 mutant cells also displayed an increased RhoA activation (Figure 2C). Upon inhibition of protein synthesis using cycloheximide (CHX), we observed a significantly slower degradation of GEF18 mutant protein over a 12h period indicating increased protein stability (Figure 2D). GEF18 mutant also showed resistance to ubiquitin mediated degradation leading to pathologically increased protein levels. Conclusions We report a novel gain of function variant of ARHGEF18 that drives podocyte dysfunction through impaired protein localization and degradation. Targeting this pathway could help regulate RhoA activation and cytoskeletal rearrangements preventing podocyte effacement in DKD.

Tumoricidal Activity of Simvastatin in Synergy with RhoA Inactivation in Antimigration of Clear Cell Renal Cell Carcinoma Cells.

Among kidney cancers, clear cell renal cell carcinoma (ccRCC) has the highest incidence rate in adults. The survival rate of patients diagnosed as having metastatic ccRCC drastically declines even with intensive treatment. We examined the efficacy of simvastatin, a lipid-lowering drug with reduced mevalonate synthesis, in ccRCC treatment. Simvastatin was found to reduce cell viability and increase autophagy induction and apoptosis. In addition, it reduced cell metastasis and lipid accumulation, the target proteins of which can be reversed through mevalonate supplementation. Moreover, simvastatin suppressed cholesterol synthesis and protein prenylation that is essential for RhoA activation. Simvastatin might also reduce cancer metastasis by suppressing the RhoA pathway. A gene set enrichment analysis (GSEA) of the human ccRCC GSE53757 data set revealed that the RhoA and lipogenesis pathways are activated. In simvastatin-treated ccRCC cells, although RhoA was upregulated, it was mainly restrained in the cytosolic fraction and concomitantly reduced Rho-associated protein kinase activity. RhoA upregulation might be a negative feedback effect owing to the loss of RhoA activity caused by simvastatin, which can be restored by mevalonate. RhoA inactivation by simvastatin was correlated with decreased cell metastasis in the transwell assay, which was mimicked in dominantly negative RhoA-overexpressing cells. Thus, owing to the increased RhoA activation and cell metastasis in the human ccRCC dataset analysis, simvastatin-mediated Rho inactivation might serve as a therapeutic target for ccRCC patients. Altogether, simvastatin suppressed the cell viability and metastasis of ccRCC cells; thus, it is a potentially effective ccRCC adjunct therapy after clinical validation for ccRCC treatment.

Abstract 2461: The cell migration inhibitor dihydromotuporamine C regulates actin-myosin contractility and actin polymerization

Abstract The motuporamines are a promising class of anti-metastatic compounds. Dihydromotuporamine C (Motu33) has been shown to activate the small GTPase RhoA, however, little is known about subsequent downstream events leading to cell migration inhibition. In the present study, we investigated the mechanism of action of Motu33 and a synthetic derivative, Motu-(CH2)-33, in Drosophila by manipulating the gene dose of positive and negative regulators of actin dynamics. Consistent with previous findings, reduced gene dose of Rho1 (the Drosophila RhoA ortholog) attenuates motuporamine activity, confirming that RhoA/Rho1 is targeted by these compounds. Actin-myosin contraction is controlled by the RhoA-ROCK-myosin regulatory light chain (MRLC) pathway. Reduced gene dose of the myosin binding subunit of myosin phosphatase, a negative regulator of the RhoA-ROCK-MRLC pathway, enhances motuporamine activity indicating that the motuporamines stimulate actin-myosin contraction through activation of the myosin regulatory light chain. RhoA also activates diaphanous (dia) to control actin polymerization. Surprisingly, reduced gene dose of dia enhances motuporamine activity, suggesting that the motuporamines act on dia in a RhoA-independent manner. Reduction in gene dose of the Drosophila Rac orthologs also enhances motuporamine activity. In contrast, motuporamine activity is unaltered by reduced gene dose of slingshot (ssh) which acts to trigger actin severing and depolymerization. Since ssh is directly regulated by Rac, the enhanced activity of motuporamines observed when Rac gene dose is reduced may reflect an indirect mode of action on the Rac GTPases leading to increased RhoA activity. In summary, these findings demonstrate that motuporamines act through RhoA and diaphanous to regulate actin-myosin contractility and actin polymerization. Citation Format: Laurence von Kalm, Corey Seavey. The cell migration inhibitor dihydromotuporamine C regulates actin-myosin contractility and actin polymerization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2461.

CAR links hypoxia signaling to improved survival after myocardial infarction

The coxsackievirus and adenovirus receptor (CAR) mediates homo- and heterotopic interactions between neighboring cardiomyocytes at the intercalated disc. CAR is upregulated in the hypoxic areas surrounding myocardial infarction (MI). To elucidate whether CAR contributes to hypoxia signaling and MI pathology, we used a gain- and loss-of-function approach in transfected HEK293 cells, H9c2 cardiomyocytes and CAR knockout mice. CAR overexpression increased RhoA activity, HIF-1α expression and cell death in response to chemical and physical hypoxia. In vivo, we subjected cardiomyocyte-specific CAR knockout (KO) and wild-type mice (WT) to coronary artery ligation. Survival was drastically improved in KO mice with largely preserved cardiac function as determined by echocardiography. Histological analysis revealed a less fibrotic, more compact lesion. Thirty days after MI, there was no compensatory hypertrophy or reduced cardiac output in hearts from CAR KO mice, in contrast to control mice with increased heart weight and reduced ejection fraction as signs of the underlying pathology. Based on these findings, we suggest CAR as a therapeutic target for the improved future treatment or prevention of myocardial infarction.

Induced Remodelling of Astrocytes In Vitro and In Vivo by Manipulation of Astrocytic RhoA Activity.

Structural changes of astrocytes and their perisynaptic processes occur in response to various physiological and pathophysiological stimuli. They are thought to profoundly affect synaptic signalling and neuron-astrocyte communication. Understanding the causal relationship between astrocyte morphology changes and their functional consequences requires experimental tools to selectively manipulate astrocyte morphology. Previous studies indicate that RhoA-related signalling can play a major role in controlling astrocyte morphology, but the direct effect of increased RhoA activity has not been documented in vitro and in vivo. Therefore, we established a viral approach to manipulate astrocytic RhoA activity. We tested if and how overexpression of wild-type RhoA, of a constitutively active RhoA mutant (RhoA-CA), and of a dominant-negative RhoA variant changes the morphology of cultured astrocytes. We found that astrocytic expression of RhoA-CA induced robust cytoskeletal changes and a withdrawal of processes in cultured astrocytes. In contrast, overexpression of other RhoA variants led to more variable changes of astrocyte morphology. These induced morphology changes were reproduced in astrocytes of the hippocampus in vivo. Importantly, astrocytic overexpression of RhoA-CA did not alter the branching pattern of larger GFAP-positive processes of astrocytes. This indicates that a prolonged increase of astrocytic RhoA activity leads to a distinct morphological phenotype in vitro and in vivo, which is characterized by an isolated reduction of fine peripheral astrocyte processes in vivo. At the same time, we identified a promising experimental approach for investigating the functional consequences of astrocyte morphology changes.

SHOCK INDUCES ENDOTHELIAL PERMEABILITY AFTER TRAUMA THROUGH INCREASED ACTIVATION OF RHOA GTPASE.

Introduction: Severely injured patients develop a dysregulated inflammatory state characterized by vascular endothelial permeability, which contributes to multiple organ failure. To date, however, the mediators of and mechanisms for this permeability are not well established. Endothelial permeability in other inflammatory states such as sepsis is driven primarily by overactivation of the RhoA GTPase. We hypothesized that tissue injury and shock drive endothelial permeability after trauma by increased RhoA activation leading to break down of endothelial tight and adherens junctions. Methods: Human umbilical vein endothelial cells (HUVECs) were grown to confluence, whereas continuous resistance was measured using electrical cell-substrate impedance sensing (ECIS) Z-Theta technology, 10% ex vivo plasma from severely injured trauma patients was added, and resistance measurements continued for 2 hours. Areas under the curve (AUCs) were calculated from resistance curves. For GTPase activity analysis, HUVECs were grown to confluence and incubated with 10% trauma plasma for 5 minutes before harvesting of cell lysates. Rho and Rac activity were determined using a G-LISA assay. Significance was determined using Mann-Whitney tests or Kruskal-Wallis test, and Spearman ρ was calculated for correlations. Results: Plasma from severely injured patients induces endothelial permeability with plasma from patients with both severe injury and shock contributing most to this increased permeability. Surprisingly, Injury Severity Score (ISS) does not correlate with in vitro trauma-induced permeability (-0.05, P > 0.05), whereas base excess (BE) does correlate with permeability (-0.47, P = 0.0001). The combined impact of shock and injury resulted in a significantly smaller AUC in the injury + shock group (ISS > 15, BE < -9) compared with the injury only (ISS > 15, BE > -9; P = 0.04) or minimally injured (ISS < 15, BE > -9; P = 0.005) groups. In addition, incubation with injury + shock plasma resulted in higher RhoA activation ( P = 0.002) and a trend toward decreased Rac1 activation ( P = 0.07) compared with minimally injured control. Conclusions: Over the past decade, improved early survival in patients with severe trauma and hemorrhagic shock has led to a renewed focus on the endotheliopathy of trauma. This study presents the largest study to date measuring endothelial permeability in vitro using plasma collected from patients after traumatic injury. Here, we demonstrate that plasma from patients who develop shock after severe traumatic injury induces endothelial permeability and increased RhoA activation in vitro . Our ECIS model of trauma-induced permeability using ex vivo plasma has potential as a high throughput screening tool to phenotype endothelial dysfunction, study mediators of trauma-induced permeability, and screen potential interventions.

Abstract P3012: Syndecan 1 Regulates Lung Endothelial Barrier Function In Homeostatic And Injury Conditions

Introduction: Acute lung injury (ALI) is an inflammatory syndrome that may occur secondarily to bacterial infection. Pulmonary edema is a severe complication of ALI and it may result from endothelial barrier disruption. The mechanisms that initiate ALI during bacterial infection remains unresolved. Syndecan 1 (Sdc1) is a heparan sulfate proteoglycan that contributes to endothelial barrier stability and is cleaved during inflammatory processes. Whether Sdc1 contributes to ALI progression is unresolved. Herein we investigated if Sdc1 mediates LPS-induced ALI in mice. Methods: Male and Female Sdc1 knockout (Sdc1KO) and wild type (WT) mice, with 5-8 weeks of age, were injected with lipopolysaccharide&amp;nbsp;(LPS, 10mg/Kg, IP) or PBS and euthanized after 6hr. Bronchoalveolar lavage fluid (BAL) and lung tissue were collected and assessed for myeloperoxidase (MPO) activity, albumin content, RhoA activity using commercially available spectrometric and ELISA kits. Lung edema was assessed by lung wet/dry ratio (W/D). Results and Conclusions: LPS increased lung W/D by 1.5-fold (p=0.0042, n≥5/group) in WT but not Sdc1KO mice. Sdc1KO showed higher basal lung W/D vs WT (Sdc1KO=3.55 vs WT=2.21, p=0.044, n≥5/group). Similarly, a trend increase in basal lung MPO activity was seen in Sdc1KO when compared to WT (p=0.33, n≥5/group). A significant increase in MPO activity was seen in LPS-treated WT (LPS=0.47±0.03 vs. PBS=0.26±0.03, p=0.003) but not LPS-treated Sdc1KO mice (p=0.11; n≥5/group) when compared to matched PBS-treated strain controls. Albumin content was increased by LPS only in WT BAL (LPS=106.9±8.9 vs PBS=63.65±6.15, p=0.03, n≥5/group). LPS increased RhoA activity exclusively in WT lungs. (LPS=0.04±0.002 vs PBS= 0.02±0.003, p=0.04, n≥5/group). A trend increase in RhoA activity was seen in PBS-treated Sdc1KO when compared to PBS-treated WT (p=0.06, n≥4/group). Collectively, our data suggest that Sdc1KO show basal lung endothelial barrier failure. This phenotype is associated with decreased response to LPS. The mechanisms whereby Sdc1 regulate barrier function may involve RhoA-dependent pathway. Sdc1 may participate in lung edema development during ALI.

RhoA as a Signaling Hub Controlling Glucagon Secretion From Pancreatic α-Cells.

Glucagon hypersecretion from pancreatic islet α-cells exacerbates hyperglycemia in type 1 diabetes (T1D) and type 2 diabetes. Still, the underlying mechanistic pathways that regulate glucagon secretion remain controversial. Among the three complementary main mechanisms (intrinsic, paracrine, and juxtacrine) proposed to regulate glucagon release from α-cells, juxtacrine interactions are the least studied. It is known that tonic stimulation of α-cell EphA receptors by ephrin-A ligands (EphA forward signaling) inhibits glucagon secretion in mouse and human islets and restores glucose inhibition of glucagon secretion in sorted mouse α-cells, and these effects correlate with increased F-actin density. Here, we elucidate the downstream target of EphA signaling in α-cells. We demonstrate that RhoA, a Rho family GTPase, plays a key role in this pathway. Pharmacological inhibition of RhoA disrupts glucose inhibition of glucagon secretion in islets and decreases cortical F-actin density in dispersed α-cells and α-cells in intact islets. Quantitative FRET biosensor imaging shows that increased RhoA activity follows directly from EphA stimulation. We show that in addition to modulating F-actin density, EphA forward signaling and RhoA activity affect α-cell Ca2+ activity in a novel mechanistic pathway. Finally, we show that stimulating EphA forward signaling restores glucose inhibition of glucagon secretion from human T1D donor islets.

Active RhoA Exerts an Inhibitory Effect on the Homeostasis and Angiogenic Capacity of Human Endothelial Cells.

BackgroundThe small GTPase RhoA (Ras homolog gene family, member A) regulates a variety of cellular processes, including cell motility, proliferation, survival, and permeability. In addition, there are reports indicating that RhoA‐ROCK (rho associated coiled‐coil containing protein kinase) activation is essential for VEGF (vascular endothelial growth factor)‐mediated angiogenesis, whereas other work suggests VEGF‐antagonistic effects of the RhoA‐ROCK axis.Methods and ResultsTo elucidate this issue, we examined human umbilical vein endothelial cells and human coronary artery endothelial cells after stable overexpression (lentiviral transduction) of constitutively active (G14V/Q63L), dominant‐negative (T19N), or wild‐type RhoA using a series of in vitro angiogenesis assays (proliferation, migration, tube formation, angiogenic sprouting, endothelial cell viability) and a human umbilical vein endothelial cells xenograft assay in immune‐incompetent NOD scid gamma mice in vivo. Here, we report that expression of active and wild‐type RhoA but not dominant‐negative RhoA significantly inhibited endothelial cell proliferation, migration, tube formation, and angiogenic sprouting in vitro. Moreover, active RhoA increased endothelial cell death in vitro and decreased human umbilical vein endothelial cell‐related angiogenesis in vivo. Inhibition of RhoA by C3 transferase antagonized the inhibitory effects of RhoA and strongly enhanced VEGF‐induced angiogenic sprouting in control‐treated cells. In contrast, inhibition of RhoA effectors ROCK1/2 and LIMK1/2 (LIM domain kinase 1/2) did not significantly affect RhoA‐related effects, but increased angiogenic sprouting and migration of control‐treated cells. In agreement with these data, VEGF did not activate RhoA in human umbilical vein endothelial cells as measured by a Förster resonance energy transfer–based biosensor. Furthermore, global transcriptome and subsequent bioinformatic gene ontology enrichment analyses revealed that constitutively active RhoA induced a differentially expressed gene pattern that was enriched for gene ontology biological process terms associated with mitotic nuclear division, cell proliferation, cell motility, and cell adhesion, which included a significant decrease in VEGFR‐2 (vascular endothelial growth factor receptor 2) and NOS3 (nitric oxide synthase 3) expression.ConclusionsOur data demonstrate that increased RhoA activity has the potential to trigger endothelial dysfunction and antiangiogenic effects independently of its well‐characterized downstream effectors ROCK and LIMK.

Soluble VE‐cadherin disrupts endothelial barrier function via VE‐PTP/RhoA signalling

Increased levels of soluble VE‐cadherin fragments (sVE‐cadherin) have previously been linked to inflammation‐induced loss of endothelial barrier function. We hypothesized that sVE‐cadherin independent of pro‐inflammatory stimuli may be critically involved in the context of endothelial barrier dysfunction.Recombinant human sVE‐cadherin (extracellular domains EC1‐5) was generated and verified by sequencing and immunoblotting. Application of sVE‐cadherin on human dermal microvascular endothelial cells (HDMECs) dose‐dependently induced loss of endothelial barrier function as revealed by measurements of transendothelial electrical resistance (TER) and flux of 70 kDa FITC‐dextran across HDMEC monolayers. In an in vivo rat model i.v. application of sVE‐cadherin resulted in loss of endothelial barrier function and reduced microcirculatory flow, which was comparable to the conditions when bacterial Lipopolysaccharide (LPS) was applied in vivo. Immunofluorescence staining and western blot analysis revealed that the functional effects of sVE‐cadherin were paralleled by decreased expression of VE‐cadherin and reduction of α‐/ β‐/ γ‐/ δ‐catenin and tight‐junction associated protein ZO‐1 at the cell borders. VE‐protein tyrosine phosphatase (VE‐PTP) which is associated with VE‐cadherin under resting conditions was removed from the cell borders following application of sVE‐cadherin to HDMEC. These changes were accompanied by intercellular gap formation and increased stress fibers in the actin cytoskeleton. On a mechanistic level, sVE‐cadherin did not induce cell death but increased RhoA activity and reduced extracellular VE‐cadherin interaction as revealed by atomic force measurements. Rho‐kinase inhibitor Y27632 blocked all sVE‐cadherin‐induced effects on endothelial barrier function. Furthermore, VE‐PTP inhibitor AKB9778 attenuated both RhoA activation and all sVE‐cadherin‐induced effects.In summary, sVE‐cadherin disrupts endothelial barrier function by dismantling the VE‐cadherin complex at the cell border via VE‐PTP‐dependent RhoA activation. This points to a novel pathophysiological role of sVE‐cadherin in the context of endothelial barrier dysfunction in inflammation.

Exosomes derived from human umbilical cord mesenchymal stem cells reduce tendon injuries via the miR-27b-3p/ARHGAP5/RhoA signaling pathway.

Tendon injuries are common clinical issues resulted from tissue overuse and age-related degeneration. Previous sutdies have suggested that exosomes secreted by mesenchymal stem cells (MSCs) contribute to tissue injury repair. Here, we provide evidence for a critical role of human umbilical cord mesenchymal stem cell (hucMSC)-derived exosomes in reducing tendon injury by activating the RhoA signaling. Treatment of primary injured tenocytes with hucMSC exosomes increases cell proliferation and invasion, which correlates with increased RhoA activity. RhoA mediates the effects of hucMSC exosomes, as treatment of primary injured tenocytes with the RhoA inhibitor, CCG-1423, abolishes the effects of hucMSC exosomes on cell proliferation and invasion. Mechanistically, we observe that hucMSC exosomes induce the expression of a microRNA, miR-27b-3p, which targets and suppresses ARHGAP5, a negative regulator of RhoA. Consistent with this observation, ARHGAP5 overexpression suppresses the effects of hucMSC exosomes on cell proliferation and invasion, while knockdown of ARHGAP5 rescues these effects. Finally, we demonstrate the functional significance of our findings using an Achilles tendon injury model and show that treatment with exosomes reduces tendon injury in rats, which correlates with increased RhoA activity and reduced ARHGAP5 expression. Taken together, our findings highlight a critical role of hucMSC exosomes in reducing tendon injury via miR-27b-3p-mediated suppression of ARHGAP5, resulting in RhoA activation, and leading to increased cell proliferation and invasion of primary injured tenocytes.

Abstract 13551: RhoA Mediates Pressure-Induced Endothelial Hyperpermeability

Pulmonary edema is a severe complication of heart failure. It is often caused by the abrupt increase in lung pulmonary capillary pressure that leads to endothelial barrier instability and disruption. The mechanisms whereby high pressure causes pulmonary edema is unresolved. However, eNOS uncoupling and downstream protein nitration may play a role. We have previously shown that eNOS uncoupling, leading to RhoA activation, is key to the development of pulmonary edema in a model of hypertensive heart failure. Herein, we sought to determine if RhoA activation plays a role in pressure-induced pulmonary edema and the molecular mechanisms that govern this process. Human lung microvascular endothelial cells (HLMEC) were exposed to 0cm (control) or 30cm (high pressure) H 2 O pressure for 5 to 60 minutes. Phosphorylation of eNOS at T495 and PKCα activation were assessed by Western blot, RhoA activation and nitration was assessed by ELISA and dot blot assays and endothelial barrier stability was assessed by VE-cadherin staining. The effects of pressure on barrier function were assessed by transendothelial electrical resistance (TEER). High hydrostatic pressure induced PKCα activation (1.42±0.2 vs 0.8±0.05 control, p=0.029; N=5) and the phosphorylation of eNOS at T495 (1.6±0.1 vs 0.62±0.08 control; p=0.0319; N=3), which was used as a correlation to eNOS uncoupling. This was associated with increased RhoA activity (0.009±0.001 vs. 0.005±0.001 control; p=0.0247; N=5), RhoA nitration (2.1±0.4 vs 1.0±0.02 control, p=0.0511; N=4) and VE-cadherin internalization. HLMEC exposed to control and high pressure conditions were transferred to gold electrodes (ECIS assay) to monitor TEER as indices of barrier recovery. High pressure caused a delay in barrier recovery and a decrease in the maximum TEER after 24 hours (0.952±0.16 vs 1.55±.019 control; p=0.0412; N=4). Fasudil, a Rho inhibitor, mitigated the effects of pressure on barrier recovery (1.996±0.79 vs 0.968±0.44 pressure; p&lt;0.0001; N=4). Altogether, these findings suggest that pressure induces endothelial barrier disruption via PKCα-mediated eNOS uncoupling and subsequent activation of RhoA. This may be important in conditions in which pulmonary pressure can increase abruptly, including heart failure.