HIF-1α regulates Cx40-dependent vasodilatation following hemorrhagic shock in rats

Abstract

HIF-1α plays an essential role in hemorrhagic shock-induced vasoconstriction. However, the underlying mechanisms remain poorly understood. Here, we studied both the role of HIF-1α in regulating vasodilatation, and the involvement of Cx40 in this process. We found that endothelium-dependent vasodilatation exhibited an overall decline after hemorrhagic shock: at the beginning of shock vasodilatation reactivity significantly decreased, followed by a slight increase from 0.5 h to 2 h after shock. After 2 h vasodilatation dropped again. Throughout this process, protein levels of HIF-1α gradually increased. In the late period of shock, vasodilatation reactivity was enhanced by oligomycin, an HIF-1α inhibitor, suggesting that HIF-1α may promote vasoconstriction. Moreover, in the late period of shock Cx40 levels gradually increased and exhibited a negative correlation with endothelium-dependent vasoconstriction reactivity. Furthermore, Cx40 AODN significantly improved vasoconstriction reactivity and could be regulated by either an HIF-1α inhibitor or an agonist. Together, these data suggest that HIF-1α may inhibit endothelium-dependent vasodilatation reactivity following hemorrhagic shock by up-regulating Cx40, especially in the late period of shock.

Keywords: Cx40; HIF-1α; endothelium-dependent vasodilatation reactivity; hemorrhagic shock.

Figure 1

Changes in vasodilatation reactivity in hemorrhagic shock. A: The cumulative dose-response curves to Ach after shock. B: The cumulative dose-response curves to SNP after shock. C: The maximal vasodilatation reactivity (Emax) of SMA to Ach and SNP after shock. D: The cumulative dose-response curves to Ach after anoxia. E: The cumulative dose-response curves to SNP after anoxia. F: The maximal vasodilatation reactivity (Emax) of SMA after anoxia. *P<0.05, **P<0.01 as compared with control group.

Figure 2

The expression of HIF-1α after hemorrhagic shock. A: Electrophoresis of HIF-1α mRNA and β-actin mRNA. B: Statistical graph of graph A. C: Western blot of HIF-1α after shock. D: Statistical graph of graph C. M, molecular marker; Sham, sham-operated group; S, shocked groups. *P<0.05, **P<0.01 as compared with the sham-operated group.

Figure 3

The effect of HIF-1α on vasodilatation reactivity in hemorrhagic shock. A: The cumulative dose-response curves to Ach after shock treated with oligomycin. B: The maximal vasodilatation reactivity (Emax) of SMA to Ach in shock group (data from section 3.1) and shock + oligomycin group. C: The cumulative dose-response curves to Ach after anoxia treated with oligomycin. D: The maximal vasodilatation reactivity (Emax) of SMA to Ach in anxoia group (data from section 3.1) and anoxia + oligomycin group. E: The cumulative dose-response curves to SNP after shock treated with oligomycin. F: The maximal vasodilatation reactivity (Emax) of SMA to SNP in shock group (data from section 3.1) and shock + oligomycin group. *P<0.05, **P<0.01 as compared with normal group. #P<0.05, ##P<0.01 as compared with the same shock time group.

Figure 4

The expression of Cx40 in hemorrhagic shock and the effect on vasodilatation reactivity after Cx40 AODN. A: Protein expression of Cx40 after shock. B: Statistical graph of graph A. C: The expression of Cx40 mRNA in control group and Cx40 AODN group. D: Statistical graph of graph C. E: The cumulative dose-response curves to Ach in normal conditions after Cx40 AODN. F: The cumulative dose-response curves to Ach in 1 h anoxia after Cx40 AODN. G: The cumulative dose-response curves to Ach in 4 h anoxia after Cx40 AODN. H: The maximal vasodilatation reactivity (Emax) of SMA to Ach after anoxia treated with Cx40 AODN. *P<0.05, **P<0.01 as compared with sham-operated group. #P<0.05, ##P<0.01 as compared with the same anoxia time group.

Figure 5

Relationship between HIF-1α and Cx40 in the regulation of vasodilatation reactivity after anoxia. A: RT-PCR electrophoregrams of HIF-1α, Cx40 and β-actin mRNA. M: DNA marker; 1: non-anoxia control; 2: 1 h anoxia control; 3: 4 h anoxia control; 4: non-anoxia + EGb761; 5: 1 h anoxia + EGb761; 6: 4 h anoxia + EGb761; 7: non-anoxia + EGb761 + oligomycin; 8: 1 h anoxia + EGb761 + oligomycin; 9: 4 h anoxia + EGb761 + oligomycin. B: Bar graph of HIF-1α RT-PCR. C: Bar graph of Cx40 RT-PCR. *P<0.05, **P<0.01 as compared with the same anoxia time control group.