Regulatory Effects Of HIF-1α On Vasodilation Reactivity And The Relationship To MEGJ Following Hemorrhagic Shock In Rats

After sever trauma or shock, vascular reactivity to vasoconstrictors and vasodilators is greatly reduced, which results in hypotension, disorder of perfusion and damage of cell and tissue. Previous studies showed that it may be related to the increased level of nitric oxide (NO), the functional disorder of the K+ and Ca+ channels on the vascular smooth muscle cell (VSMC) membrane and the hyperpolarization of cell membrane. Furthermore, our previous study showed that calcium desensitization (the decrease of force/Ca2+ ratio) existed in the myocardium cell following severe trauma or shock.Hypoxia-inducible factor 1α(HIF-1α) is the oxygen-regulated subunit of the transcriptional activator HIF-1, which mediates the changes of gene expression in response to the cellular oxygen concentrations. Our early study revealed that HIF-1αplayed an important role in hemorrhagic shock-induced vasoconstrictive hyporeactivity. And the possible mechanism of HIF-1αregulating vascular reactivity includes the pathway of eNOS/iNOS—NO, HO-1—CO, COX-2—PGI. But it's not clear whether HIF-1αtakes part in the regulation of vasodilative reactivity following hemorrhagic shock and its mechanism. However, we had found that MEGJ played very important role in the regulation of vasodilative reactivity following hemorrhagic shock before.Thus, using oligomycin as the tool drug and rat hemorrhagic shock model, we obversed the regulatory effect of HIF-1αon vasodilative reactivity and the relationship to MEGJ following hemorrhagic shock in the present study.Methods:The experiments were conducted in three parts. In the first part, we observed the role of HIF-1αin the development of vasodilative hyporeactivity following hemorrhagic shock in rats. Superior mesenteric artery (SMA) from rats at different time after shock (shock 0h, 0.5h, 1h, 2h, 3h and 4h) was adopted to assay the vasodilative reactivity via observing the dilatation initiated by sodium nitroprusside (SNP) and acetylcholine (Ach) with isolated organ perfusion system with or without the HIF-1αinhibitor oligomycin. Meanwhile, the changes of HIF-1αmRNA were measured by RT-PCR.In the second part, we observed the relationship between the role of HIF-1αin the development of vasodilative hyporeactivity and myoendothelial gap junction (MEGJ).We imitated the ischemic SMA of hemorrhagic shock in vitro by using the treatment of oxygen-glucose deprivation. And SMA at different time after oxygen-glucose deprivation (0h, 0.5h, 1h, 2h, 3h and 4h) was adopted to assay the vasodilative reactivity by Ach with isolated organ perfusion system with or without oligomycin.Then we treated the SMA rings with connexin 40 and connexin 43 antisense oligodeoxyuncleotide (Cxs AODN) and measured the vasodilative reactivity by Ach.After using extract of ginkgo biloba leaf EGb761 and oligomycin to induce and inhibit HIF-1αexpression, we semi-quantitated Cx40 and Cx43 mRNA expression of SMA by RT-PCR and observed their relationship to HIF-1αvia co-immunoprecipitaion and immunoblot analysis.In the third part, we observed the role of HIF-1αin the development of calcium desensitization following hemorrhagic shock in rats. Superior mesenteric artery (SMA) from rats at different time after shock (shock 0h, 0.5h, 1h, 2h, 3h and 4h) was adopted to assay the calcium sensitivity via observing the contraction initiated by Ca2+ with isolated organ perfusion system with or without the HIF-1αinhibitor oligomycin. Meanwhile, the phosphorylation level of MLC20 was measured by western blot.Results:1. The HIF-1αwas responsible for the regulation of the vasodilative reactivity following hemorrhagic shock: The vascular reactivity of SMA to SNP in each shock group was significantly decreased at lower concentration (10-9, 10-8, 10-7mol/L) of SNP (P<0.05 or P<0.01); oligomycin further depressed the response of SMA to SNP. HIF-1αplayed a positive role to the non-endothelium-dependent vasodilation reactivity in the earlier stage of shock.The response of SMA to Ach in shock groups was transiently decreased after shock, and then began to increase during the compensatory period of shock, which reached the peak level at 2h after shock, and then decreased again. Oligomycin decreased the increase trend of Ach induced relaxing response in the earlier period of hemorrhagic shock, and increased the decrease trend in the late period of shock. HIF-1αmRNA exhibited a time-dependent increase following shock, and peaked at 4h, which was positively correlated with the endothelium-dependent vascular relaxation reactivity in the earlier period of hemorrhagic shock, and negatively correlated with in the late period of shock. HIF-1αplayed a positive role to the endothelium-dependent vasodilation reactivity in the earlier stage of sock, but show negative effect in the late period.2. The relationship between MEGJ and HIF-1αas regulators of the vasodilative reactivity:(1) The vascular reactivity of SMA to Ach in oxygen-glucose deprivation has similar changes to that after shock. The response of SMA to Ach reached the peak level at 1h after hypoxia, and then decreased. Oligomycin decreased the increase trend of Ach induced relaxing response in the short time of hypoxia, and increased the decrease trend in long time of hypoxia. The vasodilative reactivity of oligomycin group was lower than those of hypoxia-control group immediately after hypoxia (P<0.01), and higher in 3h and 4h after hypoxia (P<0.05). HIF-1αplayed a positive role to the endothelium-dependent vasodilatation reactivity in the earlier stage of hypoxia, but showed negative effect in the late period.(2) Cx40 AODN improved the vascular response of SMA to Ach in 4h hypoxia groups (P<0.01), and Cx43 AODN decreased the reactivity in non-hypoxia groups (P<0.05).These results showed that Cx40 and Cx43 took part in the regulation of vascular reactivity after hemorrhagic shock.Cx40 had a suppressing effect on vasodilator reactivity, while Cx43 had an enhancing effect on vasodilator reactivity.(3) EGb761 incresad the expression of HIF-1αmRNA at non-hypoxia groups and 4h hypoxia groups (P<0.05 or P<0.01). And expression of Cx40 mRNA increased by the effect of EGb761, while expression of Cx43 mRNA decreased. As compared with the control groups, oligomycin made the expression of Cx40 mRNA in non-hypoxia groups and 4h hypoxia groups decrease significantly (P<0.01), and made the expression of Cx43 mRNA in 4h hypoxia groups increase significantly(P<0.01). HIF-1αwas present in the immunoprecipitates of Cx43 in all groups,and HIF-1αwas present weakly in the immunoprecipitates of Cx40 in control groups and 1h hypoxia groups. HIF-1αcan up-regulate the expression of Cx40 mRNA, and down-regulate the expression of Cx43 mRNA. HIF-1αmight directly act on Cx43 and might act on Cx40 weakly following shock.3. The HIF-1αwas responsible for the regulation of the calcium sensitivity following hemorrhagic shock.As compared with the control group, the cumulative dose-response curves of SMA to Ca2+at 0.5h after shock shifted to the left, the maximal contractions (Emax) of Ca2+ were increased significantly (P<0.05 or P<0.01). But the cumulative dose-response curves of SMA to Ca2+ at late shock(2h to 4h after shock) shifted to the right, Emax of Ca2+ were significantly decreased (P<0.05 or P<0.01). Oligomycin made the cumulative dose-response curves of SMA to Ca2+ at 1h and 2h after shock shift to the right, and the calcium sensitivity was decreased in the period of early shock (0.5h and 1h after shock) (P<0.05),and increased in the period of late shock. The phosphorylation of MLC20 was increased during the compensatory period of shock, and then decreased. As compared with the shock group, oligomycin inhibited the high phosphorylation level of MLC20 at early shock (immediate after shock) significantly (P<0.05), and the phosphorylation of MLC20 was increased at late shock (3h and 4h after shock) significantly (P<0.01) by the treatment of oligomycin. HIF-1αup-regulates the calcium sensitivity in the earlier period of hemorrhagic shock, and down-regulates it in the late stage.Conclusions:1. The endothelium-dependent vascular relaxation reactivity in the earlier period of hemorrhagic shock is increased, and decreased in the late stage. Endothelium-independent vascular relaxation reactivity is markedly decreased following hemorrhagic shock. HIF-1αplays a positive role to the non-endothelium-dependent vasodilatation reactivity in the earlier stage of sock, and plays a positive role to the endothelium-dependent vasodilatation reactivity in the earlier stage of sock, but show negative effect in the late period.2. Cx40 can depress endothelium-dependent vasodilator in late hemorrhagic shock, Cx43 can enhance it in earlier stage. HIF-1αhas an obivious effect on Cx40 and Cx43, which can up-regulate the expression of Cx40 mRNA, and down-regulate the expression of Cx43 mRNA. HIF-1αmight directly act on Cx43 and might act on Cx40 weakly following shock.3. HIF-1αup-regulates the calcium sensitivity in the earlier period of hemorrhagic shock, and down-regulates it in the late stage through regulating the phosphorylation level of MLC20.