Associations Between Changes In Hyoxia-inducible Factor 1α And Micro Vessel Density In The Hair Cycle Of C57BL/6 Mice

ObjectiveThe aim of present study is to observe the relationships between the fluctuations of HIF-1α, various angiogenic factors and changes in micro vessel densities in the entire synchronized hair cycle of C57BL/6 mice. The possible effects of PHD inhibition on changes in HIF-la expression and micro vessel densities in the hair cycle are also investigated.Materials and MethodsSynchronized hair cycles were induced on the dorsal skins of 6 to 8 weeks old female C57BL/6 mice by depilation using a 1:1 rosin-wax mixture. The back skins were harvested on specific days after depilation in accordance to different stages of hair growth cycle. RT-PCR, Western blot and immunohistochemistry were performed to observe the fluctuations in mRNA and protein levels of HIF-la, VEGF, Ang-1 and Ang-2, as well as changes in micro vessel densities during the entire hair cycle. By employing the same animal model and similar methods, we then gave daily injections of PHD inhibitor EDHB at the dosage of 100mg/kg to determine the effects of PHD inhibitions on protein expressions of HIF-1αand micro vessel densities.ResultsLevels of HIF-1αmRNA did not show marked changes post-depilation. The expression of VEGF mRNA was up-regulated after depilation but differences between each day point were not statistically significant (P>0.05). Levels of Ang-1 mRNA showed pattern of gradual decrease, comparing to day O, expressions were significantly lower on day 5,8 and 25(P<0.05). Levels of Ang-2 mRNA showed pattern of gradual increase post depilation, comparing to day 0,3,5 and 25, the rise at day 19 was statistically significant(P<0.05). Proteins levels of HIF-1αpeaked at day 8, and significantly decreased at day 17 and 19(P<0.05).VEGF proteins showed pattern of gradual decrease followed by gradual increase, the lowest point occurred at day 12 (P<0.05). Ang-1 proteins showed significant increase at day 12(P<0.05), whereas the peaks for Ang-2 proteins occurred at day 5 and 8(P<0.05). Anti-CD31 stains showed increase in micro vessel density post depilation; after reaching the peak at day 12, density of vessels then gradually decreased to about pre-depilation levels. Under the intervention of EDHB injections, HIF-1αproteins showed gradual increase post-depilation and reached the peak at day 5. Comparing to day 5 and 8, the expression levels of HIF-la proteins at day 12 and 17 were significantly lowered (P<0.05). The observed changes in micro vessel density of the EDHB group were similar to that of the observational group.ConclusionPhysiological angiogenesis was observed in the hair cycle of C57BL/6 mice. Pattern of protein expression of HIF-1αwas similar to that of changes in micro vessel density with the exception of an earlier peak. This suggested that HIF-1αmight function as a pro-angiogenic factor during the hair cycle. During the cycle, VEGF mRNA was up-regulated post depilation and that its protein level was at the lowest when vessel density reached the peak. Judging from the pattern of expression and its relations with HIF-la, multiple factors are likely involved in the regulation of VEGF in the hair cycle. Peak of Ang-1 proteins coincided with the peak of vessel density at anagen, and Ang-2 proteins showed greater expressions during the periods of vessel density changes during early anagen and telogen. This observation suggested that Ang-1 and Ang-2 might exert opposite effects in the control of angiogenesis during the hair cycle. Lastly, we have found that systemic administration of EDHB at the dosage of 100mg/kg/day had no significant effect on protein expression of HIF-1αand micro vessel density of the skin tissues in the synchronized hair cycle.