Regulatory Effects Of Hypoxia-inducible Factor 1α On Vascular Reactivity And Its Mechanisms Following Hemorrhagic Shock In Rats

Patients of severe trauma,shock and sepsis appear to suffer from irreversible organ damage in the decompensatory period after the"multiple attacks"of ischemia ,ischemia-reperfusion,translocation of intestinal bacteria and release of endotoxin,followed by systemic inflammatory response syndrome(SIRS),acute respiratory distress syndrome(ARDS),and even multiple organ failure(MOF).Besides uncontrolled systemic inflammation, vascular hyporeactivity after hemorrhagic shock is another important cause,resulting in inefficient promotion of blood pressure and progressing damage of organ function. Hence, vascular hyporeactivity impacts greatly on the incidence,development and the outcome of shock and interferes with the trerapy of shock. Current research suggested that vascular hyporeactivity is closely related to the desensitization of adrenergic receptors,functional disorder of potassium and calcium ions of vascular smooth muscle cell and hyperpolarization of cell membrane. Our previous study demonstrated that Rho kinase regulated the vascular reactivity following hemorrhagic shock via regulating the activity of MLCP ,the phosphorylation level of MLC20 and calcium sensitivity.As one of the most crucial molecules involved in regulating the expression of hypoxia-related genes, HIF-1αcan modulate the adaptive reponse of cells to hypoxia,and so far it is the unique regulatory molecule perfoming specific activity under hypoxic condition,coordinated by the influence of activated protein 1, nuclear factorκB and p53. And it is involved in the energy metabolism, cell proliferation, hemogenesis and vessel formation and remodeling.Ischemia and hypoxia are the basic pathophysiological change of shock,so HIF-1αshould be one of the most conspicuous molecules in expression and activity changes after shock.Lots of research demonstrated that hemorrhagic shock caused obvious uncontrolled systemic inflammation and ischemia-reperfusion,and HIF-1αtook part in the above-mentioned courses and served for their formation and development.However,there is little research and report about the relation between HIF-1αand vascular tone and reactivity.Hence, using Oligomycin as the tool drug, we obversed the regulatory effect of HIF-1αon vascular reactivity and its mechanism following hemorrhagic shock in the present study.Methods:The experiments were conducted in two parts. In the first part, we observed the changes of HIF-1αmRNA expression of mesenteric arteries (SMA) in rats and its role in vascular reactivity following hemorrhagic shock. SMA from rats at different time after shock (shock 0.5h, 1h,2h,3h,4h and 6h were adopted to analyze the vascular reactivity via observing the contraction initiated by norepinephrine(NE) in isolated organ perfusion system. HIF-1αmRNA expression of SMA were semi-quantitated by RT-PCR.Meanwhile, HIF-1αmRNA expression and vascular reactivity of SMA were tested after oligomycin administration,which is a specific antagonist of HIF-1α.In the second part, we observed the possible mechanism of HIF-1αin regulating vascular reactivity both in vivo and in vitro. In vivo experiment, phasic changes of eNOS,iNOS,HO-1,COX-2 mRNA expression of SMA from hemorrhagic shock rats were analysed by RT-PCR. Meanwhile blood NO,CO and PGI were measured by means of Nitric Acid deoxidase,Na2S2O4 deoxidation and radioimmunoassay (RIA)respectively. In vitro experiment, VSMC (vascular smooth muscle cell) and VEC (vascular endothelial cell) were co-cultrued in Transwell models under hypoxia. The contractile response of VSMC to NE were determined by measuring the fluorescent infiltraton rate in the lower chamber. The phasic changes of mRNA expression of HIF-1α,eNOS,iNOS,HO-1 and COX-2 were determined by RT-PCR.Results:1. HIF-1αmRNA exhibited a time-dependent increase following HS, and peaked at 4h(P <0.01), compared with the low level-expressed HIF-1αmRNA in normoxia. Vascular reactivity exihibited a trend of biphasic change, which was increased compensatorily at the early stage of HS, while decreased gradually at the decompensatory period after 4.0h of HS.Oligomycin treatment partly inhibited the vascular reactivity at early stage (0.0h-1.0h), while improved at decompensated period at 4.0h to 6.0h(P <0.01). 2. eNOS,iNOS,HO-1,COX-2 mRNA also exhibited a time-dependent increase following HS, and peaked at 1.0h,2.0h,3.0h and 4.0h respectively(P <0.01). And eNOS,HO-1,COX-2 mRNA of oligomycin-treated group basically fluctuated between the normal range, while enhanced iNOS mRNA expression during 1.0h-3.0h of HS was suppressed but expression of late period(4.0h - 6.0h) was improved significantly compared to corresponding shock point of HS(P <0.01).3. With the progress of HS course,plasma NO,PGI and whole blood CO were significantly increased(P <0.05 or P <0.01),while oligomycin administration sustained CO in the normal level,and NO and PGI content were partly inhibited, exihibiting a trend of less increase compared to the control group(P <0.01).4. VSMC contraction following hypoxia was increased at the early stage(0.0-1.0h) with a 1.53-fold increase at 0.5h as compared to the control group(P <0.01),and decreased gradually at the prolonged period of hypoxia (4.0-6.0h) with the drop of 30% at 6h as compared to the normal group(P <0.05). Oligomycin treatment totally inhibited the increase of VSMC contraction at early stage(P <0.05 or P <0.01), while improved it at late hypoxic period with the 6h increase of 12.8%( P <0.05).5. HIF-1α,iNOS,COX-2 and HO-1 mRNA of co-cultured VSMC and VEC exhibited a time-dependent increase following hypoxia, and peaked at 6h,2h,3h and 4h respectively, they were increased 1.62,3.23,2.26 and 2.86-folds as compared with normal group(P <0.01 or P <0.05). Comparatively,iNOS,COX-2 and HO-1 mRNA expression were suppressed to fluctuate in the normal range following oligomycin administration,while eNOS mRNA expression of both hypoixa and oligomycin-treated group were influenced little or mildly by oligomycin treatment.Conclusion:1. HIF-1αmRNA expression was increased as the progress of HS,and decreased at the late stage of HS.And the vascular reactivity presented a biphasic change after HS. Oigomycin treatment can weaken the amplitude of vasoreactivity.This result suggest that HIF-1αplays an important role in the formation of biphasic change of vascular reactivity in rats following HS.its mild increase at the early stage is positively related to vasoreactivity,while they are negatively related at the final stage.2. The possible mechanism of HIF-1αregulating vascular reactivity includes the pathways of eNOS/iNOS—NO,HO-1—CO,COX-2—PGI. HIF-1αcan modulate the gene expression of vasotone,and impact on the production and release of vasoactive substances.In the early period of HS,the compensatory increase of HIF-1αand its related molecules are conducive to the adaptive protection of vasoreactivity.While at the late stage,the overactivation,expression and accumulation of HIF-1αbrings about the paralysis of the production and release of NO,CO and PGI, finally resulting multiple organ damage.3. Hypoxic stimulation to VSMC in vitro resulted in a similar change of contratile response in vivo experiment,and oligomycin incubation weakened the amplitude of contratile reactivity.This result shows that HIF-1αregulates VSMC contration also via iNOS—NO,HO-1—CO,COX-2—PGI pathways.