| IntroductionC-reactive protein (CRP) has received substantial attention in current years as a promising risk factor of cardiovascular disease which has been demonstrated by numerous prospective epidemiologic studies. And many studies demonstrate that CRP is not merely a nonspecific marker of inflammation but also plays a direct role in the pathogenesis of cardiovascular disease. For example, it is reported lately that CRP inhibits angiogenesis which is key to the outcome of coronary heart disease. Therefore, it is of significance to explore the mechanism of the effects of CRP on angiogenesis.Hypoxia inducible factor (HIF) is the critic material for angiogenesis. It is a heterodimer composed of HIF-lα and HIF-lβ, and the former regulates the effect of HIF by being degraded under normoxia. Although a lot of factors have been founded to affect the level of HIF-lα in cells, the association between CRP and the expression of HIF-lα has not been documented yet. So it is necessary to investigate the effect of CRP on the expression of HIF-lα and to explore the prohibitive mechanism of CRP in angiogenesis. Endothelial proliferation is also critical to angiogenesis, the effect of CRP on it is observed to get a further understanding of the-6-mechanism.Objectives(1) To simulate hypoxia condition for the culture by using CoC12 and identify HIF-la level under various extent of hypoxia condition.(2) To evaluate quantitatively the effect of CRP on the expression of HIF-la under the CoC12 mimic hypoxia condition.(3) To detect whether there is any time depended association between CPR and HIF-la.(4) To observe the effect of CRP on the proliferation of the human umbilical vein endothelial cells (HUVECs).Methods(1) HUVECs were cultured under various concentrations of CoCh (0 50, 100, 200, 400M) for 24 hours. Then, HUVECs were lysed and the concentration of protein was measured using Bradford method. HIF-la protein expressions were determined by Western Blotting.(2) HUVECs were stimulated by different level of CRP (0,5 10 50,1 00uM) for 24 hours in the CoCl2 (l00uM) induced hypoxic condition. Then the cells were treated as described in (1).(3) CRP was used to stimulate HUVECs 12 hours after the CoCl2 (l00uM) preincubation. The cells were harvested at different time point (0, 6, 12, 24, 48 hours ) and then were treated as (1).(4) HUVECs were passed on 96 well assay plate and were stimulated by different level of CRP (0, 5 10 50 l00ug/ml) for 24 hours. MTS-7-technique was used to determine HUVECs proliferation.ResultsHIF-la was not detected in HUVECs cultured without CoCl2; CoCl2 stimulated the expression of HIF-la with a significant positive dose-response association at 50-200uM levels.CRP inhibited HUVECs to produce HIF-la under hypoxia-mimic condition. The levels of HIF-la stimulated by CRP were significant lower than the control one without CRP (P<0.05). l00ug/ml CRP caused the strongest inhibitive effect with about a 50% reduction for HIF-la. Despite no significant difference between Sug/ml and l0ug/ml group, the remains of experimental groups had significant difference with a significant dose-response association.CRP inhibited the expression of HIF-la after 6 hours under the preincubation of CoCh (p<0.05 vs control). The inhibition got its utmost after 48 hours. There was a time dependent effect within 48 hours incubation.There was not any effect of CRP on the proliferation of HUVECs at the level of 5ug/ml. CRP showed the inhibitive role on proliferation with a positive dose-response manner above the level of lOug/ml.ConclusionCoCU effectively induced the expression of HIF-la under normoxic condition, therefore it could be used as a hypoxia-mimic material. Moderate CRP elevation decreased the expression of HIF-la in HUVECs and inhibited the cell proliferation, and thereafter, it produced negative effect on-8-angiogenesis. The most obvious effects of CRP were found at the level similar to that in the condition of acute myocardium infarction, which demonst...
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