Rho Kinase Activity Research Articles

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity.

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

426-P: ROCK2 Regulates Monocytic Migration and Cell-to-Cell Adhesion in Vascular Endothelial Cells

A pro-inflammatory lipid mediator, lysophosphatidic acid (LPA), is a potent activator of the RhoA/Rho-kinase signaling pathway and has been shown to induce the expression of chemokines and cell adhesion molecules (CAMs). LPA levels are elevated in the circulation of patients with acute coronary syndrome and have also been detected inside human atherosclerotic specimens. However, the involvement of Rho-kinase isoforms (i.e., ROCK1, ROCK2) in LPA-mediated expression of chemokines and CAMs in endothelial cells is not fully understood. In this study, we investigated isoform-specific roles of Rho-kinase in LPA-mediated MCP-1 and E-selectin expression using human aortic endothelial cells (HAEC). Exposure of HAEC to LPA resulted in an increase of MCP-1 and E-selectin expression at both mRNA and protein levels. Rho-kinase inhibitor abolished MCP-1 and E-selectin protein expression, indicating that LPA induces MCP-1 and E-selectin expression via Rho-kinase activation. From a mechanistic standpoint, phosphorylation of IκBα, a well-characterized initial step in NF-κB activation, was decreased by Rho-kinase inhibition in the LPA-stimulated cells. Furthermore, using isoform specific siRNAs, we found that Rho-kinase isoform ROCK2 is the key mediator of LPA-induced MCP-1 and E-selectin expression. Consistently, ROCK2 but not ROCK1 controlled LPA-induced monocytic migration and adhesion in functional assays using HAECs and human monocytic cells (THP-1). In the present study, we demonstrate that endothelial ROCK2 mediates monocyte migration and cell adhesion induced by lipid mediator. In order to further explore endothelial-specific role of ROCK2 in vivo, we generated endothelial ROCK2 knockout mouse (EC-ROCK2KO) by mating of ROCK2 flox mice with VE-cadherin-cre mice. Further investigation using HFD (high-fat diet) mouse model is ongoing. ROCK2 could be an important therapeutic target against atherosclerosis. Disclosure Y. Takeda: None. K. Matoba: Research Support; Self; Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Sanofi, Shionogi & Co., Ltd., Takeda Pharmaceutical Company Limited. D. Kawanami: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. Y. Nagai: None. K. Utsunomiya: None. Funding Japan Society for the Promotion of Science; Uehara Memorial Foundation; MSD Life Science Foundation

Protein Tyrosine Phosphatase Inhibitor, Orthovanadate, Induces Contraction <i>via</i> Rho Kinase Activation in Mouse Thoracic Aortas

Orthovanadate (OVA), a protein tyrosine phosphatase inhibitor, induces contraction in endothelium-denuded mouse thoracic aortas. OVA-induced contraction was significantly (vs. control rings) suppressed by Rho kinase (Y-27632, 10 µM), extracellular signal-regulated kinase 1 and 2 (Erk1/2, FR180204, 10 µM), Erk1/2 kinase (MEK, PD98059, 10 µM), epidermal growth factor receptor (EGFR, AG1478, 10 µM), and Src inhibitors, and was partially suppressed by c-Jun N-terminal kinase (JNK, AS601245, 10 µM) and p38 (SB203580, 10 µM) inhibitors. However, a myosin light chain kinase inhibitor (ML-7, 10 µM) and a metalloproteinase inhibitor (TAPI-0, 10 µM) had no effect on OVA-induced contraction in mouse thoracic aortas. Phosphorylation of myosin phosphatase target subunit 1 (MYPT1) was abolished by inhibitors of Src, EGFR, MEK, Erk1/2, and Rho kinase, but not by inhibitors of JNK and p38. Erk1/2 phosphorylation by OVA was blocked by inhibitors of EGFR, Src, MEK, and Erk1/2, but not by Rho kinase inhibition. Src phosphorylation at Tyr-416 was abrogated by only Src inhibitor. EGFR phosphorylation at Tyr-1173 was suppressed by a Src inhibitor. These findings suggest that OVA induces contraction via activation of Src, EGFR, MEK, Erk1/2, and Rho kinase, leading to inactivation of myosin light chain phosphatase via MYPT1 phosphorylation.

Urotensin II receptor antagonist reduces hepatic resistance and portal pressure through enhanced eNOS-dependent HSC vasodilatation in CCl4-induced cirrhotic rats.

Increased serum urotensin II (UII) levels in human cirrhotic populations have been recently shown, but the long-term effects of UII receptor antagonist on the cirrhosis have not been investigated. To investigate the therapeutic effects of urotensin II receptor (UT) antagonist palosuran on rats with carbon tetrachloride (CCl4)-induced cirrhosis, the hepatic and systemic hemodynamics, liver fibrosis, the metalloproteinase-13 (MMP-13)/tissue inhibitor of metalloproteinase-1 (TIMP-1) ratio, hepatic Rho-kinase activity, and the endothelial nitric oxide synthase (eNOS) activity are measured in CCl4-cirrhotic rats treated with palosuran or vehicle for 4 weeks. Primary hepatic stellate cells (HSCs) are used to investigate the changes in UII/UT expression and the in vitro effect of palosuran. Compared with the vehicle-treated cirrhotic rats, treatment with palosuran can reduce the portal pressure (PP), decrease the risk of liver fibrosis and the level of α smooth muscle actin, collagen-I (COL-I), and transforming growth factor β expression. However, treatment with palosuran can increase MMP-13/TIMP-1, pvasodilator-stimulated phosphoprotein (p-VASP), and p-eNOS expression. Moreover, in vitro UII/UT mRNA expression increases during HSC activation. MMP-13/TIMP-1, COL-I, and p-VASP are inhibited after palosuran treatment. Our data indicate that long-term administration of palosuran can decrease PP in cirrhosis, which results from decreased hepatic fibrosis and enhanced eNOS-dependent HSC vasodilatation.

MASP-1 Increases Endothelial Permeability.

Pathologically increased vascular permeability is an important dysfunction in the pathomechanism of life-threatening conditions, such as sepsis, ischemia/reperfusion, or hereditary angioedema (HAE), diseases accompanied by uncontrolled activation of the complement system. HAE for example is caused by the deficiency of C1-inhibitor (the main regulator of early complement activation), which leads to edematous attacks threatening with circulatory collapse. We have previously reported that endothelial cells become activated during HAE attacks. A natural target of C1-inhibitor is mannan-binding lectin-associated serine protease-1 (MASP-1), a multifunctional serine protease, which plays a key role in the activation of complement lectin pathway. We have previously shown that MASP-1 induces the pro-inflammatory activation of endothelial cells and in this study we investigated whether MASP-1 can directly affect endothelial permeability. All experiments were performed on human umbilical vein endothelial cells (HUVECs). Real-time micro electric sensing revealed that MASP-1 decreases the impedance of HUVEC monolayers and in a recently developed permeability test (XperT), MASP-1 dose-dependently increased endothelial paracellular transport. We show that protease activated receptor-1 mediated intracellular Ca2+-mobilization, Rho-kinase activation dependent myosin light chain (MLC) phosphorylation, cytoskeletal actin rearrangement, and disruption of interendothelial junctions are underlying this phenomenon. Furthermore, in a whole-transcriptome microarray analysis MASP-1 significantly changed the expression of 25 permeability-related genes in HUVECs—for example it up-regulated bradykinin B2 receptor expression. According to our results, MASP-1 has potent permeability increasing effects. During infections or injuries MASP-1 may help eliminate the microbes and/or tissue debris by enhancing the extravasation of soluble and cellular components of the immune system, however, it may also play a role in the pathomechanism of diseases, where edema formation and complement lectin pathway activation are simultaneously present. Our findings also raise the possibility that MASP-1 may be a promising target of anti-edema drug development.

MON-114 Effect of Selective Mineralocorticoid Receptor Antagonist on liver Fat and Metabolism in Patients with Type 2 Diabetes: A Randomized Controlled Trail

OBJECTIVE Non-alcoholic fatty liver disease (NAFLD) is highly prevalent in patients with type 2 diabetes. A growing body of evidence points at a beneficial effect of MR antagonists (MRAs) on adipocyte function via the inhibition of adipocyte differentiation, downregulation of rho-kinase activity, pro-inflammatory proteins and increase of adiponectin expression in visceral fat. Therefore, intervention with MRAs may improve dysfunctional adipose tissue metabolism and reverse NAFLD. The primary hypothesis of the MIRAD trial was that treatment with high dose of the selective MRA eplerenone as an add-on to standard therapy, would improve liver fat content, lipid and glucose metabolism in patients with type 2 diabetes. RESEARCH DESIGN AND METHODS In this investigator-initiated, double-blinded clinical trial, 140 patients with type 2 diabetes and high cardiovascular disease risk were randomized 1:1 to eplerenone 100 - 200 mg or placebo for 26 weeks. The primary outcome was change in liver fat assessed by proton magnetic resonance spectroscopy. Secondary outcomes were changes in fat distribution assessed by DXA scan, measures of lipid and glucose metabolism by 4 point OGTT with measurements of glucose and insulin, and safety as evaluated by incident hyperkalaemia and increase in serum creatinine. RESULTS The mean ± SD age was 63.4 ± 9 years, BMI 30.7 ± 4.2 kg/m2, HbA1c 7.6 ± 3.5% (59.6 ± 14.5 mmol/mol) and duration of diabetes was 11.7 ± 7.2 years. At baseline, more than 80% of the patients were on ACE-inhibitor or ARB. The prevalence of NAFLD, defined as liver fat content ≥5.6%, was 48%. Liver fat content did not change after 26 weeks of treatment with eplerenone (1.1% [0.68; 1.66], P = 0.776) or with a placebo (-0.8% [-1.4; 0.5], P = 0.500), while a minor difference between groups was observed (P=0.026). Furthermore, there was no beneficial impact on supporting secondary outcome measures of lipid and glucose metabolism as abdominal fat distribution, lipids, circulating free fatty acids, plasma adiponectin or IR. Despite a high dosage of eplerenone (164 vs 175 mg) in patients treated with placebo (P = 0.228), the incidence of hyperkalaemia during the 26 weeks was low (p-potassium ≥5.5 mmol/L; 10 vs 2) for eplerenone vs placebo (P = 0.038). Only four patients had an increase ≥ 30% in s-creatinine during titration, with no difference between groups. At baseline, no differences between groups in liver fibrosis score NFS was observed, while at week 26, the NFS score decreased in the eplerenone group compared to placebo (P = 0.021). CONCLUSIONS We report no metabolic beneficial effect from eplerenone on liver fat or parameters of glucose and lipid metabolism in patients with type 2 diabetes, although a potential beneficial effect on liver fibrosis was found.

Elevated Platelet Galectin-3 and Rho-Associated Protein Kinase Activity Are Associated with Hemodialysis Arteriovenous Shunt Dysfunction among Subjects with Diabetes Mellitus.

Introduction Hyperglycemia is a major factor in influencing the patency rate of arteriovenous shunts, potentially associated with the RhoA/Rho-associated protein kinase (ROCK) pathway. Besides, galectin-3 mediates thrombotic mechanisms in venous thrombosis and peripheral artery disease. We hypothesized that high ROCK activity and galectin-3 levels are associated with arteriovenous shunt dysfunction. Methods We prospectively enrolled 38 patients diagnosed with arteriovenous shunt dysfunction. 29 patients received a complete follow-up and each provided two blood samples, which were collected at the first visit for occluded status of arteriovenous shunts and 1 month later for patent status. A Western blot assay for a myosin phosphatase target subunit (MYPT) was performed to examine Rho-kinase activity. A Western blot assay for platelet galectin-3 and enzyme-linked immunosorbent assay (ELISA) for circulating galectin-3 were completed. Results Higher platelet MYPT ratios and galectin-3 levels were identified at occluded arteriovenous shunts (MYPT ratio: 0.5 [0.3–1.4] vs. 0.4 [0.3–0.6], p = 0.01; galectin-3: 1.2 [0.4–1.6] vs. 0.7 [0.1–1.2], p = 0.0004). The plasma galectin-3 binding protein ELISA was also higher at occluded arteriovenous shunts (8.4 [6.0–9.7] μg/mL vs. 7.1 [4.5–9.1] μg/mL, p = 0.009). Biomarker ratios (occluded/patent status) trended high in patients with poorly controlled diabetes (MYPT ratio: 1.7 [1.0–3.0] vs. 1.1 [0.7–1.3], p = 0.06; galectin-3: 1.6 [1.3–3.4] vs. 1.1 [0.8–1.9], p = 0.05). Conclusion High platelet ROCK activity and galectin-3 levels are associated with increased risk in arteriovenous shunt dysfunction, especially in patients with poorly controlled diabetes.

Cardiac Lymphatic Dysfunction Causes Drug-Eluting Stent-Induced Coronary Hyperconstricting Responses in Pigs In Vivo.

Objective- We have previously demonstrated that coronary adventitial inflammation plays important roles in the pathogenesis of coronary vasomotion abnormalities, including drug-eluting stent (DES)-induced coronary hyperconstricting responses. Importantly, the adventitia also harbors lymphatic vessels, which may prevent inflammation by transporting extravasated fluid and inflammatory cells. We thus aimed to examine the roles of coronary adventitial lymphatic vessels in the pathogenesis of DES-induced coronary hyperconstricting responses in a porcine model in vivo. Approach and Results- We performed 2 experimental studies. In protocol 1, 15 pigs were divided into 3 groups with or without DES and with bare metal stent. Nonstented sites 20 mm apart from stent implantation also were examined. In the protocol 2, 12 pigs were divided into 2 groups with or without lymphatic vessels ligation followed by DES implantation at 2 weeks later (n=6 each). We performed coronary angiography 4 weeks after DES implantation, followed by immunohistological analysis. In protocol 1, the number and the caliber of lymphatic vessels were greater at only the DES edges after 4 more weeks. In protocol 2, coronary hyperconstricting responses were further enhanced in the lymphatic vessels ligation group associated with adventitial inflammation, Rho-kinase activation, and less adventitial lymphatic vessels formation. Importantly, there were significant correlations among these inflammation-related changes and enhanced coronary vasoconstricting responses. Conclusions- These results provide evidence that cardiac lymphatic vessel dysfunction plays important roles in the pathogenesis of coronary vasoconstrictive responses in pigs in vivo.

Role of Protein Kinase‐C and Rho Kinase in the Cytotoxic Effects of Bitter Melon Extract on Metastatic Breast Cancer Cells

IntroductionBitter melon is a common vegetable in the kitchens of most of Asian countries. Mitter melon Extract (BME) is known to inhibit proliferation of cancer cells including MCF‐7 cells which are metastatic breast cancer cells. The PKC and ROK play critical roles in cell division, migration, and survival. However, the roles of protein kinase‐c (PKC) and rho kinase (ROK) inhibition on MCF‐7 cells have not been established. Moreover, whether potential effects of BME's effects on MCF‐7 cells are mediated by PKC and ROK are unknown.ObjectiveWe aimed to investigate if BME exerts cytotoxic effects on breast cancer cells (MCF‐7 cells) and if PKC and ROK mediate BME's effects.HypothesisBME inhibits proliferation of MCF‐7 cells by decreasing PKC activity and increasing ROK activity.MethodsFresh bitter melons were purchased from an Asian grocery store and the extract (BME) was extracted, centrifuged, and filter sterilized. Equal number of MCF‐7 cells was plated in culture flasks containing DMEM medium and increasing amount of BME [0%, 0.5%, 1%, 2%, 5%, and 10% of BME (v/v)] was added to the culture flasks. To study whether PKC or ROK play any roles in mediating BME's effects, MCF‐7 cells were cultured in the following conditions; 0% BME, 1% BME, PKC inhibitor (GF109203x; 0.5μM)/ROK inhibitor (H‐1152; 1 μM), and inhibitor combined with 1% BME. After culturing cells for 4 days (for the dose‐response study) and 2 days (for the inhibitor studies), pictures of cultures were taken, and cell viability was determined using Trypan blue dye and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. After culturing cells for the required number of days, culture was centrifuged and cells were collected, protein was extracted using RIPA buffer and proteins candidates of the ROK pathways were detected by western blotting using HRP‐conjugated chemiluminescence system. Multiple comparison ANOVA was used to determine statistical significance.Results and ConclusionsBME dose‐dependently inhibited viability of MCF‐cells (N=3) starting form 1% (v/v), which was measured by two independent viability assay methods. Also, BME dose‐dependently decreased expression of MYPT1 protein, which is a target of ROK (N=1; duplicate) but the beta‐actin expression (N=1, duplicate) was not altered. Neither PKC inhibitor (N=5) nor ROK inhibitor (N=6) alone had significant effects on cell viability. However, when these inhibitors were combined with 1% BME, the effect on cell viability was of the same magnitude that the 1% BME alone had. In the future, we plan to measure phosphorylation of ROK's and PKC's target proteins by western blotting and also test if the BME exerts any cytotoxic effects on non‐cancerous breast epithelial cells.Support or Funding InformationBiomedical science master's thesis funding, Marian University College of Osteopathic Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Thrombin stimulates gene expression and secretion of IL-11 via protease-activated receptor-1 and regulates extravillous trophoblast cell migration

Extravillous trophoblast (EVT) migration and invasion is the crucial step for normal placental development. IL-11 is a cytokine regulating cell migration and invasion in cells and is a critical factor for successful implantation of an embryo. Higher expression of thrombin receptor PAR-1 was reported in early pregnancy. The precise role of thrombin in trophoblast functions is not well understood. In this study, we asked whether thrombin can induce IL-11 secretion in trophoblasts if yes, which physiological cell functions are possibly affected? In this study, HTR-8/SVneo cells, which were originally derived from first-trimester villous explants of early pregnancy were used as the extravillous trophoblast (EVT) model. BeWo cells were used as the cytotrophoblast model. For gene silencing, qPCR and ELISA, each experiment was performed in triplicates for minimum three times.Here, we found that thrombin stimulates IL-11 gene expression and protein secretion in HTR-8/SVneo cells but not in BeWo cells. PAR-1 was the only receptor which was highly expressed in HTR-8/SVneo cells. Thrombin-mediated expression and secretion of IL-11 were mainly activated via PAR-1 receptor. Rac1, but not Rho-kinase activation is required for thrombin-induced IL-11 secretion. We also found that thrombin stimulation significantly enhanced cell migration that was inhibited after silencing the IL-11 gene. In conclusion, this study demonstrates the role of thrombin in regulating human EVT migration via IL-11 secretion. We propose that thrombin might regulate EVT migration through the decidua and spiral artery remodeling. Failure of thrombin-dependent EVT migration results in pregnancy disorder, such as preeclampsia.

Paying the Toll for Inflammation

Paying the Toll for Inflammation

Sleep disorders, nocturnal blood pressure, and cardiovascular risk: A translational perspective.

Cardiovascular disease (CVD) represents the first cause of death globally. The nighttime is generally a period of relative protection from CVD events such as myocardial infarction, sudden cardiac death, and stroke, at least compared to the early morning period. The nighttime also generally entails lower values of arterial blood pressure (ABP) and heart rate (HR) and higher cardiac parasympathetic modulation. These day-night cardiovascular rhythms are ultimately driven by circadian molecular oscillators in the hypothalamic suprachiasmatic nucleus and in peripheral cells, including those in the heart, blood vessels, and kidneys. The wake-sleep states are intermediate mechanisms of circadian cardiovascular regulation, with non-REM sleep decreasing ABP and HR and increasing cardiac parasympathetic modulation at the beginning of the night. Obstructive sleep apnea, insomnia, and the restless legs syndrome have high prevalence in the general population and may increase nighttime cardiovascular activity and CVD risk. CVD risk is better predicted by ABP values during nighttime sleep than during daytime wakefulness. Higher nighttime values of ABP and HR increase cardiac work and vessel wall stress. During the night, circadian rhythms may enhance cardiac responses to hypertrophic stimuli, increase vascular smooth muscle Rho kinase activity and contractility, decrease endothelial nitric oxide production and vascular responses to vasodilators, and increase circulating monocytes with the potential to infiltrate atherosclerotic plaques. Together, these factors configure a "perfect storm" scenario that may make increased cardiovascular activity during the night a final common mechanism linking sleep disorders to CVD risk.

Rho-kinase inhibitor coupled to peptide-modified albumin carrier reduces portal pressure and increases renal perfusion in cirrhotic rats

Rho-kinase (ROCK) activation in hepatic stellate cells (HSC) is a key mechanism promoting liver fibrosis and portal hypertension (PTH). Specific delivery of ROCK-inhibitor Y-27632 (Y27) to HSC targeting mannose-6-phosphate-receptors reduces portal pressure and fibrogenesis. In decompensated cirrhosis, presence of ascites is associated with reduced renal perfusion. Since in cirrhosis, platelet-derived growth factor receptor beta (PDGFRβ) is upregulated in the liver as well as the kidney, this study coupled Y27 to human serum albumin (HSA) substituted with PDGFRβ-recognizing peptides (pPB), and investigated its effect on PTH in cirrhotic rats. In vitro collagen contraction assays tested biological activity on LX2 cells. Hemodynamics were analyzed in BDL and CCl4 cirrhotic rats 3 h, 6 h and 24 h after i.v. administration of Y27pPBHSA (0.5/1 mg/kg b.w). Phosphorylation of moesin and myosin light chain (MLC) assessed ROCK activity in liver, femoral muscle, mesenteric artery, kidney and heart. Three Y27 molecules were coupled to pPBHSA as confirmed by HPLC/MS, which was sufficient to relax LX2 cells. In vivo, Y27pPBHSA-treated rats exhibited lower portal pressure, hepatic vascular resistance without effect on systemic vascular resistance, but a tendency towards lower cardiac output compared to non-treated cirrhotic rats. Y27pPBHSA reduced intrahepatic resistance by reduction of phosphorylation of moesin and MLC in Y27pPBHSA-treated cirrhotic rats. Y27pPBHSA was found in the liver of rats up to 6 hours after its injection, in the HSC demonstrated by double-immunostainings. Interestingly, Y27pPBHSA increased renal arterial flow over time combined with an antifibrotic effect as shown by decreased renal acta2 and col1a1 mRNA expression. Therefore, targeting the ROCK inhibitor Y27 to PDGFRβ decreases portal pressure with potential beneficial effects in the kidney. This unique approach should be tested in human cirrhosis.

Involvement of RhoA/Rho-kinase in l-cysteine/H2S pathway-induced inhibition of agonist-mediated corpus cavernosal smooth muscle contraction

Rho-kinase activity is a key regulator in the maintenance of corporal vasoconstriction and penile detumescense. Also, importance of l-cysteine/H2S pathway in erectile tissue has been shown; however it is currently unknown the role RhoA/Rho-kinase pathway in H2S-induced inhibition in cavernosal tissue. We investigated the role of RhoA/Rho-kinase pathway in the inhibitory effect of l-cysteine and NaHS, as endogenous and exogenous H2S, respectively, on phenylephrine-induced contractions of mouse cavernosal strips. Phenylephrine, α1 receptor agonist, (10 nM-100 μM) induced a concentration-dependent contraction in CC. l-cysteine (endogenous H2S substrate; 10 mM) and exogenous H2S (NaHS; 1 mM) significantly inhibited the contractile response to phenylephrine (P < 0.05). Inhibition of CSE and CBS enzymes by PAG (10 mM) and AOAA (1 mM), respectively, significantly reversed the inhibitory effects of l-cysteine on phenylephrine-induced contraction (P < 0.05). Y-27632 (1 μM), a specific Rho-kinase inhibitor, significantly augmented the inhibitory effect of l-cysteine and NaHS on phenylephrine-induced contraction, and this inhibition was reversed by PAG and AOAA (P < 0.05). In addition, the formation of H2S was increased by approximately 1.8 fold over basal values after incubation of tissue homogenates with l-cysteine. Y-27632 significantly increased both basal and l-cysteine-induced H2S formation and this augmentation diminished by PAG and AOAA (P < 0.05). Furthermore, the pMYPT-1 expression was significantly decreased by l-cysteine, NaHS or Y-27632 alone. Also, pMYPT-1 expression was completely abolished by the l-cysteine/NaHS plus Y-27632 combination, and this inhibition was reversed by PAG and AOAA (P < 0.05). These results suggest that there is an interaction between Rho-kinase and H2S pathways. Rho-kinase may be, at least in part, inhibits CSE/CBS enzymes in mouse corpus cavernosal tissue; however, it is not excluded the other kinases such as PKC and Zip-kinase.

Patterning of human epidermal stem cells on undulating elastomer substrates reflects differences in cell stiffness

In human skin the junction between epidermis and dermis undulates, the width and depth of the undulations varying with age and disease. When primary human epidermal keratinocytes are seeded on collagen-coated polydimethylsiloxane (PDMS) elastomer substrates that mimic the epidermal-dermal interface, the stem cells become patterned by 24 h, resembling their organisation in living skin. We found that cell density and nuclear height were higher at the base than the tips of the PDMS features. Cells on the tips not only expressed higher levels of the stem cell marker β1 integrin but also had elevated E-cadherin, Desmoglein 3 and F-actin than cells at the base. In contrast, levels of the transcriptional cofactor MAL were higher at the base. AFM measurements established that the Young’s modulus of cells on the tips was lower than on the base or cells on flat substrates. The differences in cell stiffness were dependent on Rho kinase activity and intercellular adhesion. On flat substrates the Young’s modulus of calcium-dependent intercellular junctions was higher than that of the cell body, again dependent on Rho kinase. Cell patterning was influenced by the angle of the slope on undulating substrates. Our observations are consistent with the concept that epidermal stem cell patterning is dependent on mechanical forces exerted at intercellular junctions in response to undulations in the epidermal-dermal interface. Statement of significanceIn human skin the epidermal-dermal junction undulates and epidermal stem cells are patterned according to their position. We previously created collagen-coated polydimethylsiloxane (PDMS) elastomer substrates that mimic the undulations and provide sufficient topographical information for stem cells to cluster on the tips. Here we show that the stiffness of cells on the tips is lower than cells on the base. The differences in cell stiffness depend on Rho kinase activity and intercellular adhesion. We propose that epidermal stem cell patterning is determined by mechanical forces exerted at intercellular junctions in response to the slope of the undulations.

The antifibrotic potential of a sustained release formulation of a PDGFβ-receptor targeted rho kinase inhibitor

Rho kinase activity in hepatic stellate cells (HSCs) is associated with activation, transformation and contraction of these cells, leading to extracellular matrix production and portal hypertension in liver cirrhosis. Inhibition of rho kinase activity can reduce these activities, but may also lead to side effects, for instance systemic hypotension. This can be circumvented by liver-specific delivery of a rho kinase inhibitor to effector cells. Therefore, we targeted the rho kinase inhibitor Y27632 to the key pathogenic cells in liver fibrosis, i.e. myofibroblasts including activated HSCs that highly express the PDGFβ-receptor, using the drug carrier pPB-MSA. This carrier consists of mouse serum albumin (MSA) covalently coupled to several PDGFβR-recognizing moieties (pPB). We aimed to create a prolonged release system of such a targeted construct, by encapsulating pPB-MSA-Y27632 in biodegradable polymeric microspheres, thereby reducing short-lasting peak concentrations and the need for frequent administrations. Firstly, we confirmed the vasodilating potency of PDGFβ-receptor targeted Y27632 in vitro in a contraction assay using HSCs seeded on a collagen gel. We subsequently demonstrated the in vivo antifibrotic efficacy of pPB-MSA-Y27632-loaded microspheres in the Mdr2−/− mouse model of progressive biliary liver fibrosis. A single subcutaneous microsphere administration followed by organ harvest one week later clearly attenuated liver fibrosis progression and significantly suppressed the expression of fibrosis related genes, such as several collagens, profibrotic cytokines and matrix metalloproteinases. In conclusion, we demonstrate that polymeric microspheres are suitable as drug delivery system for the sustained systemic delivery of targeted protein constructs with antifibrotic potential, such as pPB-MSA-Y27632. This formulation appears suitable for the sustained treatment of liver fibrosis and possibly other chronic diseases.

M108 - A NOVEL RARE VARIANT R292H IN RTN4R AFFECTS GROWTH CONE FORMATION AND POSSIBLY CONTRIBUTES TO SCHIZOPHRENIA SUSCEPTIBILITY

Background RTN4R (Reticulon 4 receptor) plays an essential role in regulating axonal regeneration and plasticity in the central nervous system through the activation of rho kinase, and is located within chromosome 22q11.2, a region that is known to be a hot-spot for Schizophrenia (SCZ) and Autism Spectrum Disorder (ASD). Recently, rare variants such as Copy Number Variants (CNV) and Single Nucleotide Variants (SNV) have been a focus of research because of their large effect size associated with increased susceptibility to SCZ and ASD and the possibility of elucidating the pathophysiology of mental disorder through functional analysis of the discovered rare variants. Methods In order to discover rare variants with large effect size and to explore their role in the etiopathophysiology of SCZ and ASD, we performed mutation screening of RTN4R coding exons using a sample comprised of 370 SCZ and 192 ASD patients, and association analysis using a large number of unrelated individuals (1716 SCZ, 382 ASD and 4009 controls). Results Through this mutation screening, we discovered four rare (minor allele frequency Discussion This study strengthens the evidence for association between rare variants within RTN4R and SCZ, and may shed light on the molecular mechanisms underlying the neurodevelopmental disorder.

Angiotensin II-induced constriction via Rho kinase activation in pressure-overloaded rat thoracic aortas

Angiotensin II-induced constriction via Rho kinase activation in pressure-overloaded rat thoracic aortas

MR (Mineralocorticoid Receptor) Induces Adipose Tissue Senescence and Mitochondrial Dysfunction Leading to Vascular Dysfunction in Obesity.

Adipose tissue (AT) senescence and mitochondrial dysfunction are associated with obesity. Studies in obese patients and animals demonstrate that the MR (mineralocorticoid receptor) contributes to obesity-associated cardiovascular complications through its specific role in AT. However, underlying mechanisms remain unclear. This study aims to elucidate whether MR regulates mitochondrial function in obesity, resulting in AT premature aging and vascular dysfunction. Obese (db/db) and lean (db/+) mice were treated with an MR antagonist or a specific mitochondria-targeted antioxidant. Mitochondrial and vascular functions were determined by respirometry and myography, respectively. Molecular mechanisms were probed by Western immunoblotting and real-time polymerase chain reaction in visceral AT and arteries and focused on senescence markers and redox-sensitive pathways. db/db mice displayed AT senescence with activation of the p53-p21 pathway and decreased SIRT (sirtuin) levels, as well as mitochondrial dysfunction. Furthermore, the beneficial anticontractile effects of perivascular AT were lost in db/db via ROCK (Rho kinase) activation. MR blockade prevented these effects. Thus, MR activation in obesity induces mitochondrial dysfunction and AT senescence and dysfunction, which consequently increases vascular contractility. In conclusion, our study identifies novel mechanistic insights involving MR, adipose mitochondria, and vascular function that may be of importance to develop new therapeutic strategies to limit obesity-associated cardiovascular complications.