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Regulation of Rho GTPases by N-Cadherin Cell-Cell Contact Formation in Vascular Smooth Muscle Cells
S172
Canadian Journal of Cardiology Volume 29 2013
212 REGULATION OF RHO GTPASES BY N-CADHERIN CELL-CELL CONTACT FORMATION IN VASCULAR SMOOTH MUSCLE CELLS
213 DYSFUNCTIONAL CARDIAC LIPID METABOLISM IN CYSTATHIONINE-BETA-SYNTHASE DEFICIENT MICE WITH DIETINDUCED WEIGHT GAIN
S Xu, M Bendeck
MB Glier, JD Olson, SL Gerrard, RE Aleliunas, DC Sulistyoningrum, TJ Green, AM Devlin, AM Devlin
Toronto, Ontario BACKGROUND:
The directional migration of medial smooth muscle cells (SMC) into the vessel intima in vascular diseases such as atherosclerosis leads to stenosis and occlusion of the arterial lumen. An important initiating factor is the loss of direct SMC contacts with the overlying endothelial cells and with adjacent SMCs. We have shown that N-cadherin, a calcium-dependent cell-cell adhesion molecule, becomes asymmetrically distributed around the cell periphery when SMCs lose contact with endothelial cells, and that restoration of symmetric N-cadherin distribution may arrest migration. In addition, we detected activation of Cdc42 and Rac1, Rho GTPases that regulate actin-mediated processes such as lamellipodia formation and membrane ruffling, after wounding an SMC monolayer. We hypothesize that N-cadherin regulates Rho GTPases to establish and stabilize cell-cell contacts in SMCs. METHODS: Mouse aortic vascular smooth muscle (MOVAS) cells were grown to near confluence in DMEM supplemented with 10% fetal bovine serum. Cells were either treated with 4mM EGTA or serum starved for 24 hours and then incubated in calcium-free DMEM to remove calcium and disrupt cadherin contacts. After 1 hour of calcium removal, fresh media was added to restore calcium for 0, 1/4, 1 /2, 1, 2, 3, 6, 16, 20, and 24 hours to allow re-establishment of cadherin contacts. The cells were immunofluorescently stained for N-cadherin and b-catenin. Cdc42 activity was measured using pull-down assays with PAK-PBD beads followed by Western blotting. Confluent MOVAS monolayers were linearly wounded down the centre and immunofluorescently stained for N-cadherin, b-catenin and Cdc42 at 0, 1, 3, 6, 16, 20, 24, 32, and 48 hours postwounding. RESULTS: Before calcium removal, both N-cadherin and bcatenin are co-localized at cell-cell junctions. N-cadherin and b-catenin staining disappears from the cell periphery after 1 hour of calcium removal, indicating contact disruption, and both are completely restored after 30 minutes of calcium add-back, indicating contact re-establishment. Increased levels of active Cdc42 are detected after contact disruption, which decrease after 3 hours of contact re-establishment. In wounded MOVAS cultures, posteriorlateral staining of N-cadherin and b-catenin is observed in wound edge cells, accompanied by staining of Cdc42 at the leading edge lamellipodia that disappears after 16 hours post-wounding. CONCLUSION: The results suggest that the absence or removal of N-cadherin cell-cell contacts in SMCs may increase the activity of Cdc42, while N-cadherin cell-cell contact formation may suppress Cdc42 activation.
Vancouver, British Columbia
Obesity is characterized by ectopic cardiac lipid deposition. Excess cardiac lipid accumulation is associated with lipotoxicity and eventually cardiac dysfunction. The molecular mechanisms linking obesity and the development of cardiac lipotoxicity are unknown. Cysteine is a sulfur-containing amino acid required for synthesis of the antioxidant glutathione. Cysteine is supplied by the diet or the transsulfuration of homocysteine by cystathioninebeta-synthase (Cbs). We previously reported mice heterozygous for targeted disruption of Cbs (Cbs +/-) with diet-induced weight gain and glucose intolerance have enhanced cardiac lipotoxicity. We hypothesize that Cbs +/- mice diet-induced weight gain have altered myocardial fatty acid uptake, activation, and oxidation. To address our hypothesis, Cbs +/+ and Cbs +/- mice were fed a high-fat diet (HFD) from weaning for 20 weeks to induce weight gain and glucose intolerance. Cbs +/- mice fed HFD had higher fasting blood glucose concentrations (P<0.01) and greater glucose intolerance (P<0.01) than Cbs +/+ mice fed the HFD. Cbs +/+ and Cbs +/- mice fed the HFD had higher fasting insulin concentrations (P<0.01) compared to mice fed chow diet. Cbs +/- mice had lower total adiponectin concentrations (P<0.06) in plasma compared to Cbs +/+ mice. An interaction was observed between diet and genotype for high molecular weight (HMW) adiponectin concentrations in plasma, with lower HMW adiponectin concentrations (P<0.01) in Cbs +/- mice than Cbs +/+ mice fed chow diet. When assessing the heart, Cbs +/- mice had higher heart weights (P<0.01) compared to Cbs +/+ mice. Interventricular septum thickness in Cbs +/+ and Cbs +/- fed HFD was greater during both end systole (P<0.01) and diastole (P<0.01). Left ventricle performance was also higher for measurements such as ejection fraction (P<0.01) and fractional shortening (P<0.01) than in chow fed mice. This was accompanied by increased expression of markers of fatty acid uptake (CD36, P<0.01); activation (long-chain acyl CoA synthetase 1, P<0.01); and oxidation (carnitine palmitoyltransferase 1 and acyl CoA oxidase, P<0.01) in heart. These findings demonstrate a novel role for Cbs in the pathology of cardiac lipotoxicity. NSERC 214 INTERLEUKIN 15 PLAYS AN IMPORTANT ROLE IN THE DEVELOPMENT OF ATHEROSCLEROSIS IN MICE O Dadoo, A Ashkar, C Richards, B Trigatti Hamilton, Ontario ABSTRACT:
IL-15 is an essential cytokine for the development of NK and CD8+T cells and also involved in activation of inflammation in macrophages. Therefore, we hypothesized that IL-15 may affect the development of atherosclerosis via
Canadian Journal of Cardiology Volume 29 2013
212 REGULATION OF RHO GTPASES BY N-CADHERIN CELL-CELL CONTACT FORMATION IN VASCULAR SMOOTH MUSCLE CELLS
213 DYSFUNCTIONAL CARDIAC LIPID METABOLISM IN CYSTATHIONINE-BETA-SYNTHASE DEFICIENT MICE WITH DIETINDUCED WEIGHT GAIN
S Xu, M Bendeck
MB Glier, JD Olson, SL Gerrard, RE Aleliunas, DC Sulistyoningrum, TJ Green, AM Devlin, AM Devlin
Toronto, Ontario BACKGROUND:
The directional migration of medial smooth muscle cells (SMC) into the vessel intima in vascular diseases such as atherosclerosis leads to stenosis and occlusion of the arterial lumen. An important initiating factor is the loss of direct SMC contacts with the overlying endothelial cells and with adjacent SMCs. We have shown that N-cadherin, a calcium-dependent cell-cell adhesion molecule, becomes asymmetrically distributed around the cell periphery when SMCs lose contact with endothelial cells, and that restoration of symmetric N-cadherin distribution may arrest migration. In addition, we detected activation of Cdc42 and Rac1, Rho GTPases that regulate actin-mediated processes such as lamellipodia formation and membrane ruffling, after wounding an SMC monolayer. We hypothesize that N-cadherin regulates Rho GTPases to establish and stabilize cell-cell contacts in SMCs. METHODS: Mouse aortic vascular smooth muscle (MOVAS) cells were grown to near confluence in DMEM supplemented with 10% fetal bovine serum. Cells were either treated with 4mM EGTA or serum starved for 24 hours and then incubated in calcium-free DMEM to remove calcium and disrupt cadherin contacts. After 1 hour of calcium removal, fresh media was added to restore calcium for 0, 1/4, 1 /2, 1, 2, 3, 6, 16, 20, and 24 hours to allow re-establishment of cadherin contacts. The cells were immunofluorescently stained for N-cadherin and b-catenin. Cdc42 activity was measured using pull-down assays with PAK-PBD beads followed by Western blotting. Confluent MOVAS monolayers were linearly wounded down the centre and immunofluorescently stained for N-cadherin, b-catenin and Cdc42 at 0, 1, 3, 6, 16, 20, 24, 32, and 48 hours postwounding. RESULTS: Before calcium removal, both N-cadherin and bcatenin are co-localized at cell-cell junctions. N-cadherin and b-catenin staining disappears from the cell periphery after 1 hour of calcium removal, indicating contact disruption, and both are completely restored after 30 minutes of calcium add-back, indicating contact re-establishment. Increased levels of active Cdc42 are detected after contact disruption, which decrease after 3 hours of contact re-establishment. In wounded MOVAS cultures, posteriorlateral staining of N-cadherin and b-catenin is observed in wound edge cells, accompanied by staining of Cdc42 at the leading edge lamellipodia that disappears after 16 hours post-wounding. CONCLUSION: The results suggest that the absence or removal of N-cadherin cell-cell contacts in SMCs may increase the activity of Cdc42, while N-cadherin cell-cell contact formation may suppress Cdc42 activation.
Vancouver, British Columbia
Obesity is characterized by ectopic cardiac lipid deposition. Excess cardiac lipid accumulation is associated with lipotoxicity and eventually cardiac dysfunction. The molecular mechanisms linking obesity and the development of cardiac lipotoxicity are unknown. Cysteine is a sulfur-containing amino acid required for synthesis of the antioxidant glutathione. Cysteine is supplied by the diet or the transsulfuration of homocysteine by cystathioninebeta-synthase (Cbs). We previously reported mice heterozygous for targeted disruption of Cbs (Cbs +/-) with diet-induced weight gain and glucose intolerance have enhanced cardiac lipotoxicity. We hypothesize that Cbs +/- mice diet-induced weight gain have altered myocardial fatty acid uptake, activation, and oxidation. To address our hypothesis, Cbs +/+ and Cbs +/- mice were fed a high-fat diet (HFD) from weaning for 20 weeks to induce weight gain and glucose intolerance. Cbs +/- mice fed HFD had higher fasting blood glucose concentrations (P<0.01) and greater glucose intolerance (P<0.01) than Cbs +/+ mice fed the HFD. Cbs +/+ and Cbs +/- mice fed the HFD had higher fasting insulin concentrations (P<0.01) compared to mice fed chow diet. Cbs +/- mice had lower total adiponectin concentrations (P<0.06) in plasma compared to Cbs +/+ mice. An interaction was observed between diet and genotype for high molecular weight (HMW) adiponectin concentrations in plasma, with lower HMW adiponectin concentrations (P<0.01) in Cbs +/- mice than Cbs +/+ mice fed chow diet. When assessing the heart, Cbs +/- mice had higher heart weights (P<0.01) compared to Cbs +/+ mice. Interventricular septum thickness in Cbs +/+ and Cbs +/- fed HFD was greater during both end systole (P<0.01) and diastole (P<0.01). Left ventricle performance was also higher for measurements such as ejection fraction (P<0.01) and fractional shortening (P<0.01) than in chow fed mice. This was accompanied by increased expression of markers of fatty acid uptake (CD36, P<0.01); activation (long-chain acyl CoA synthetase 1, P<0.01); and oxidation (carnitine palmitoyltransferase 1 and acyl CoA oxidase, P<0.01) in heart. These findings demonstrate a novel role for Cbs in the pathology of cardiac lipotoxicity. NSERC 214 INTERLEUKIN 15 PLAYS AN IMPORTANT ROLE IN THE DEVELOPMENT OF ATHEROSCLEROSIS IN MICE O Dadoo, A Ashkar, C Richards, B Trigatti Hamilton, Ontario ABSTRACT:
IL-15 is an essential cytokine for the development of NK and CD8+T cells and also involved in activation of inflammation in macrophages. Therefore, we hypothesized that IL-15 may affect the development of atherosclerosis via