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LACK OF ANGIOPOIETIN LIKE-2 PROTECTS AGAINST ANGIOTENSIN II-INDUCED CEREBRAL ENDOTHELIAL DYSFUNCTION IN MICE
S162
Canadian Journal of Cardiology Volume 30 2014
Canadian Society for Atherosclerosis, Thrombosis and Vascular Biology (CSATVB) Oral ENDOTHELIAL FUNCTION AND DYSFUNCTION Sunday, October 26, 2014
Trainee Research Award Finalist e Basic Science 212 SIRTUIN 1 PLAYS A CRUCIAL ROLE IN MODULATING THE PULMONARY AND SYSTEMIC HYPOXIC RESPONSE IN MICE: NOVEL ROLE FOR HYPOXIA INDUCIBLE FACTOR-3ALPHA? M Taha, Y Deng, MW McBurney, DJ Stewart Ottawa, Ontario BACKGROUND:
Pulmonary Hypertension (PH) is caused by occlusive remodelling of pulmonary arterioles leading to increased pulmonary vascular resistance, right ventricle hypertrophy and eventually failure. Sirtuin (Sirt)-1 is an NAD+-dependent deacetylase that has been strongly implicated in maintaining endothelium homeostasis in systemic vessels, but little is known about its role in hypoxia sensing and the lung vasculature. Thus, the goal of this study was to understand the mechanisms by which Sirt1 regulates the pulmonary vascular and systemic response to chronic hypoxia (CH)-induced PH. METHODS AND RESULTS: Mice lacking Sirt1 catalytic activity (sirt1Y/Y, H355Y) and their wild type (WT) littermates were exposed to chronic hypoxia (10% O2) for 1, 7, 14 or 21 days. Sirt1Y/Y exhibited an exaggerated increase in right ventricle systolic pressure (RVSP), apparent within the first week of hypoxic exposure (Day 7: 332 sirt1Y/Y vs. 272 WT; n¼8-10, p< 0.05) which progressively increased over the 3 week CH exposure period (41.51.8 sirt1Y/Y vs. 29.70.8 WT; n¼27 both, p< 0.001). Right ventricular (RV) hypertrophy, assessed by the RV/LV+S weight ratio was also increased (Day 7: 0.520.01 sirt1Y/Y vs. 0.400.02 WT, n¼8-10; p< 0.0001). Hemtaocrit levels were similar in Sirt1Y/Y and WT mice at baseline; however, there was a delayed increase after three weeks of CH in Sirt1Y/Y mice relative to WT mice (Day 7: 552% sirt1Y/Y vs. 522% WT, n¼8-9) (Day 21: 712% sirt1Y/Y vs. 631% WT, n¼17 both; p<0.001). Plasma levels of erythropoietin (EPO), a hypoxic responsive protein responsible for hematopoiesis, were assessed by ELISA at each time point. EPO was markedly increased at Day 7 in Sirt1Y/Y vs. WT mice (492240 vs. 8116pg/mL, respectively; n¼7-9, p< 0.05) but normalized at later time points (Day 21: 759 sirt1Y/Y vs. 9612pg/mL WT; n¼4 both). Lung expression of the hypoxia inducible factors (HIF) 1a and 2a subunits was not increased by western blot analysis. Moreover, HIF1a and HIF2a responsive target genes showed no increase in transcript levels. However, there was a dramatic increase in HIF3a expression in Sirt1 mutant vs. WT lungs, which has been
implicated both in repression of other HIFs transcription, activity as well as a direct mediator hypoxic signaling. CONCLUSION: Our data supports a role for Sirt1 in modulating the response to hypoxia, with loss of deacetylation activity leading to an exaggerated pulmonary and systemic induction of the hypoxic response genes. This appears to be mediated by HIF3a as a potential novel downstream target for Sirt1 activity in hypoxia. CIHR
213 LACK OF ANGIOPOIETIN LIKE-2 PROTECTS AGAINST ANGIOTENSIN II-INDUCED CEREBRAL ENDOTHELIAL DYSFUNCTION IN MICE C Yu, N Thorin-Trescases, E Thorin Montréal, Québec BACKGROUND:
Angiopoietin like-2 (angptl2) is a recently identified pro-inflammatory and pro-oxidative protein that is elevated in patients with chronic inflammatory and cardiovascular diseases. Its role in regulating endothelial function is largely unknown, and there is no report of the impact of angptl2 in the cerebrovasculature. We hypothesized that the lack of angptl2 improves endothelial cell stress resistance and prevents cerebral endothelial dysfunction induced by a subpressor dose of angiotensin II (angII). METHOD AND RESULTS: Subcutaneous infusion of angII (200 ng/kg/min, n¼15; osmotic mini-pump), or saline as control (n¼15), was performed in 20- to 22-week-old angptl2 knockdown (KD) mice and wild-type (WT) littermates for 14 days. In isolated cerebral arteries of saline-treated KD and WT mice, global acetylcholine (ACh)-induced vasodilations were similar. However, the contribution of endothelium-derived relaxing factors differed: while eNOS-derived O2-/H2O2 contributed to dilation in WT, eNOS-derived NO (P<0.001) was involved in KD mice. Furthermore, angII induced endothelial dysfunction only in WT mice (P<0.01). This was reversed by acute addition of either N-acetylcysteine (10 mM), an anti-oxidant, apocynin (10 mM), a non-selective NADPH oxidase (Nox) inhibitor, or indomethacin (10 mM), a nonselective cyclooxygenase (COX) inhibitor, suggesting that angII-induced endothelial dysfunction in WT mice was related to oxidative stress, Nox activity and/or COX-derived constrictor-derivatives. In KD mice treated with angII, on the other hand, apocynin worsened endothelium-dependent dilation, while N-acetylcysteine and indomethacin were ineffective, suggesting remodeling of the Nox systems, low levels of oxidative stress and COX-derived constrictor derivatives. CONCLUSION: The demonstration that the lack of angptl2 expression is associated with NO-dependent dilation, prevented angII-induced endothelial dysfunction, and led to a deleterious effect of apocynin, suggests that lowering angptl2 protects cerebral endothelial function and leads to a potential compensatory Nox dilatory pathway, ultimately reinforcing cerebral endothelial cell stress resistance. NSERC
Canadian Journal of Cardiology Volume 30 2014
Canadian Society for Atherosclerosis, Thrombosis and Vascular Biology (CSATVB) Oral ENDOTHELIAL FUNCTION AND DYSFUNCTION Sunday, October 26, 2014
Trainee Research Award Finalist e Basic Science 212 SIRTUIN 1 PLAYS A CRUCIAL ROLE IN MODULATING THE PULMONARY AND SYSTEMIC HYPOXIC RESPONSE IN MICE: NOVEL ROLE FOR HYPOXIA INDUCIBLE FACTOR-3ALPHA? M Taha, Y Deng, MW McBurney, DJ Stewart Ottawa, Ontario BACKGROUND:
Pulmonary Hypertension (PH) is caused by occlusive remodelling of pulmonary arterioles leading to increased pulmonary vascular resistance, right ventricle hypertrophy and eventually failure. Sirtuin (Sirt)-1 is an NAD+-dependent deacetylase that has been strongly implicated in maintaining endothelium homeostasis in systemic vessels, but little is known about its role in hypoxia sensing and the lung vasculature. Thus, the goal of this study was to understand the mechanisms by which Sirt1 regulates the pulmonary vascular and systemic response to chronic hypoxia (CH)-induced PH. METHODS AND RESULTS: Mice lacking Sirt1 catalytic activity (sirt1Y/Y, H355Y) and their wild type (WT) littermates were exposed to chronic hypoxia (10% O2) for 1, 7, 14 or 21 days. Sirt1Y/Y exhibited an exaggerated increase in right ventricle systolic pressure (RVSP), apparent within the first week of hypoxic exposure (Day 7: 332 sirt1Y/Y vs. 272 WT; n¼8-10, p< 0.05) which progressively increased over the 3 week CH exposure period (41.51.8 sirt1Y/Y vs. 29.70.8 WT; n¼27 both, p< 0.001). Right ventricular (RV) hypertrophy, assessed by the RV/LV+S weight ratio was also increased (Day 7: 0.520.01 sirt1Y/Y vs. 0.400.02 WT, n¼8-10; p< 0.0001). Hemtaocrit levels were similar in Sirt1Y/Y and WT mice at baseline; however, there was a delayed increase after three weeks of CH in Sirt1Y/Y mice relative to WT mice (Day 7: 552% sirt1Y/Y vs. 522% WT, n¼8-9) (Day 21: 712% sirt1Y/Y vs. 631% WT, n¼17 both; p<0.001). Plasma levels of erythropoietin (EPO), a hypoxic responsive protein responsible for hematopoiesis, were assessed by ELISA at each time point. EPO was markedly increased at Day 7 in Sirt1Y/Y vs. WT mice (492240 vs. 8116pg/mL, respectively; n¼7-9, p< 0.05) but normalized at later time points (Day 21: 759 sirt1Y/Y vs. 9612pg/mL WT; n¼4 both). Lung expression of the hypoxia inducible factors (HIF) 1a and 2a subunits was not increased by western blot analysis. Moreover, HIF1a and HIF2a responsive target genes showed no increase in transcript levels. However, there was a dramatic increase in HIF3a expression in Sirt1 mutant vs. WT lungs, which has been
implicated both in repression of other HIFs transcription, activity as well as a direct mediator hypoxic signaling. CONCLUSION: Our data supports a role for Sirt1 in modulating the response to hypoxia, with loss of deacetylation activity leading to an exaggerated pulmonary and systemic induction of the hypoxic response genes. This appears to be mediated by HIF3a as a potential novel downstream target for Sirt1 activity in hypoxia. CIHR
213 LACK OF ANGIOPOIETIN LIKE-2 PROTECTS AGAINST ANGIOTENSIN II-INDUCED CEREBRAL ENDOTHELIAL DYSFUNCTION IN MICE C Yu, N Thorin-Trescases, E Thorin Montréal, Québec BACKGROUND:
Angiopoietin like-2 (angptl2) is a recently identified pro-inflammatory and pro-oxidative protein that is elevated in patients with chronic inflammatory and cardiovascular diseases. Its role in regulating endothelial function is largely unknown, and there is no report of the impact of angptl2 in the cerebrovasculature. We hypothesized that the lack of angptl2 improves endothelial cell stress resistance and prevents cerebral endothelial dysfunction induced by a subpressor dose of angiotensin II (angII). METHOD AND RESULTS: Subcutaneous infusion of angII (200 ng/kg/min, n¼15; osmotic mini-pump), or saline as control (n¼15), was performed in 20- to 22-week-old angptl2 knockdown (KD) mice and wild-type (WT) littermates for 14 days. In isolated cerebral arteries of saline-treated KD and WT mice, global acetylcholine (ACh)-induced vasodilations were similar. However, the contribution of endothelium-derived relaxing factors differed: while eNOS-derived O2-/H2O2 contributed to dilation in WT, eNOS-derived NO (P<0.001) was involved in KD mice. Furthermore, angII induced endothelial dysfunction only in WT mice (P<0.01). This was reversed by acute addition of either N-acetylcysteine (10 mM), an anti-oxidant, apocynin (10 mM), a non-selective NADPH oxidase (Nox) inhibitor, or indomethacin (10 mM), a nonselective cyclooxygenase (COX) inhibitor, suggesting that angII-induced endothelial dysfunction in WT mice was related to oxidative stress, Nox activity and/or COX-derived constrictor-derivatives. In KD mice treated with angII, on the other hand, apocynin worsened endothelium-dependent dilation, while N-acetylcysteine and indomethacin were ineffective, suggesting remodeling of the Nox systems, low levels of oxidative stress and COX-derived constrictor derivatives. CONCLUSION: The demonstration that the lack of angptl2 expression is associated with NO-dependent dilation, prevented angII-induced endothelial dysfunction, and led to a deleterious effect of apocynin, suggests that lowering angptl2 protects cerebral endothelial function and leads to a potential compensatory Nox dilatory pathway, ultimately reinforcing cerebral endothelial cell stress resistance. NSERC