SIRT1 Inhibits Blood Flow Recovery Following Hindlimb Ischemia In Mice

Blood vessels allow blood to flow through every tissue to supply oxygen and nutrients and to dispose of waste.Ischemia-induced angiogenesis and arteriogenesis are critical for blood flow recovery,and are strictly controlled by pro-angiogenic and antiangiogenic factors.SIRT1 is a nicotinamide adenine dinucleotide(NAD)-dependent deacetylase,which is widely involved in physiological and pathological processes,such as embryonic development,damage healing,angiogenesis,neuranagenesis.However,the mechanism underlying SIRT1 regulating angiogenesis and arteriogenesis remains unclear.1 Activation of SIRT1 impairs effective blood flow recovery after hindlimb ischemia in mice.1.1 Overexpression of SIRT1 significant inhibits the blood flow recovery following femoral artery ligation.To investigate the role of SIRT1 in neovascularization of adult tissues,the hindlimb ischemia was surgically induced by ligation of femoral artery in mice.Blood flow was measured by Laser Speckle Contrast Analysis.We found a delayed blood flow recovery in SIRT1 transgenic(Tg)mice,compared with wild type(WT)mice.However,the limb perfusion was moderately improved in SIRT1-Tg mice treated with SIRT1 inhibitor EX527.1.2 Activation of SIRT1 inhibits blood flow recovery following hindlimb ischemia.SIRT1 agonist resveratrol(RSV)was intraperitoneal injected into WT mice at different time points following femoral artery ligation,and attenuated the blood flow recovery in ischemic hindlimb.Conversely,EX527 accelerated the blood flow recovery.These findings suggest that activation of SIRT1 impairs the blood flow recovery in the hindlimb ischemia model.1.3 Overexpression of SIRT1 inhibits arteriogenesis.The arteriogenesis is a prerequisite for the blood flow recovery after ischemia.To identify the function of SIRT1 in this process,the expression of CD31(endothelial marker)and smooth muscle ?-actin(smooth muscle marker)in the ischemic gastrocnemius was detected using immunohistochemistry assay.The density of capillaries(positive for CD31)and arterioles(positive for smooth muscle ?-actin)in the ischemic gastrocnemius of SIRT1-Tg mice were significantly decreased compared with WT mice.Meanwhile,the mural cell coverage(NG2 positive)was reduced in SIRT1-Tg mice.These data indicate that SIRT1 activation is closely associated with reduced arteriogenesis.2 Activation of SIRT1 impairs angiogenesis.2.1 Activation of SIRT1 inhibits the proliferation and tube formation of endothelial cells.To investigate the relationship between SIRT1 and the functions of endothelial cells,the effect of SIRT1 activation on the response of endothelial cells was tested.Human umbilical vein endothelial cells(HUVECs)were preincubated with RSV or EX527 to activation or inhibition of SIRT1.The results showed that RSV treatment resulted in a reduced Brd U incorporation which was increased by EX527.Similarly,overexpression of SIRT1 was accompanied by decreased Brd U incorporation in primary endothelial cells of SIRT1-Tg mice.To confirm the role of SIRT1 in angiogenesis,the Matrigel assay was used to detect in vitro tube formation.The tube formation was inhibited in RSV-treated HUVECs,and accelerated tube formation in HUVECs treated with EX527.The in vitro functional angiogenesis assays support that the activation of SIRT1 inhibits the proliferation,migration and tube formation of endothelial cells.2.2 Overexpression of SIRT1 inhibits angiogenesis in vivo.To address the effect of SIRT1 activity on angiogenesis,the Matrigel plugs containing VEGFA was subcutaneously injected into opposite iliac regions of mice,and the effects of SIRT1 on endothelial cell migration,network formation and mural cell coverage were examined.Compared with WT mice,the CD31-positive(endothelial cells)and NG2 positive(mural cells)area was markedly reduced in SIRT1-Tg mice.The wound closure rate was decreased in SIRT1-Tg mice compared with WT mice.To determine the critical regulatory role of SIRT1 in angiogenesis,the retinas were isolated from postnatal 5-day mice.The retinas of SIRT1-Tg mice displayed decreased number of vascular branching points and shortened length of vascular tubules,compared with WT mice.Similar changes were observed in the retinas from RSV-treated WT mice.These results demonstrate that the activation of SIRT1 inhibits angiogenesis.2.3 SIRT1 inhibits VEGFA expression in endothelial cells through Wnt/?-catenin signaling pathway.VEGFA is one of the target genes of the Wnt/?-catenin signaling pathway,and plays an important role in angiogenesis.Western blot analysis showed that the expression of ?-catenin and its upstream DVL3 was decreased in the ischemic gastrocnemius tissues of SIRT1-Tg mice compared with WT mice,accompanied by a significant reduced VEGFA expression.In the in vitro study,the activation of SIRT1 significantly repressed hypoxia-induced VEGFA,?-catenin and DVL3 expression in HUVECs.These results suggest that SIRT1 downregulates the expression of VEGFA by inhibiting of the Wnt/?-catenin signaling pathway.3 SIRT1 retards arteriogenesis through inhibiting hypoxia-induced PDGFB expression in vascular smooth muscle cells.3.1 Activation of SIRT1 inhibits PDGFB expression in ischemic hindlimb.Western blot analysis showed that the expression of PDGFB was decreased in the ischemic gastrocnemius of SIRT1-Tg mice compared with WT mice,accompanied by a significant reduced ERK activation.Quantitative PCR analysis showed that overexpression of SIRT1 distinctly reduced PDGFB m RNA levels.Conversely,EX527 upregulated the expression of PDGFB at the protein and m RNA levels,and increased the phosphorylation of ERK1/2 in the ischemic gastrocnemius of SIRT1-Tg mice.These results show that activation of SIRT1 inhibits PDGFB expression,which is responsible for the decline of blood flow following hindlimb ischemia. 3.2 SIRT1 inhibits hypoxia-inducible factor 1?(HIF-1?)-mediated PDGFB transcription in VSMCs.HIF-1? triggers a coordinated response of arteriogenesis by inducing expression of PDGFB.To address the effect of SIRT1 on PDGFB expression,VSMCs were exposured to hypoxia.The results showed that HIF-1? expression increased,which was positively correlated with the expression of PDGFB.Ch IP assay was performed,and showed that overexpression of SIRT1 significantly inhibited hypoxia-induced HIF-1? binding to HBS1 site of PDGFB gene.Moreover,the reporter gene assay showed a significantly decreased luciferase activity in RSV-treated HUVECs under the same conditions.Conversely,it was increased in HUVECs treated with EX527.These results suggest that SIRT1 inhibits PDGFB expression through decreasing the binding activity of HIF-1? to the regulatory element in PDGFB gene.3.3 SIRT1 abolishes HIF-1? activity by its deacetylase activity in VSMCs.To investigate the effect of SIRT1 on HIF-1?-mediated PDGFB expression,Lysyl-acetylated HIF-1? was detected by immunoprecipitation.The results showed that overexpression or activation of SIRT1 in VSMCs inhibited HIF-1? acetylation,and inhibition of SIRT1 activity increased the acetylated HIF-1?.To further verify that HIF-1? is deacetylated by SIRT1,the interaction between HIF-1? and SIRT1 was examined by coimmunoprecipitation.The interaction of SIRT1 with HIF-1? was decreased under hypoxia,overexpression or activation of SIRT1 increased the binding of HIF-1? to SIRT1.Conversely,inhibition of SIRT1 activity abolished this interaction.These results indicate that HIF-1? is inactivated by SIRT1-dependent deacetylation,subsequently,inhibits PDGFB expression.Conclusions: 1 Activation of SIRT1 delays the blood flow recovery through impairing arteriogenesis after hindlimb ischemia in mice.2 SIRT1 suppresses angiogenesis via inhibiting VEGFA expression through Wnt/?-catenin signaling pathway.3 HIF-1? is inactivated by SIRT1-dependent deacetylation,which inhibits PDGFB expression and arteriogenesis.