ABSTRACT
AIM:To investigate the RELMα/FIZZ1 signaling pathway on the regulation of the Ca 2+/CaM sig-naling pathway in the mouse aortic smooth muscle cells MOVAS and on the formation of blood vessels in the mouse aortic endothelial cells(MAEC).METHODS:The MAEC cultured in vitro were divided into pGSadeno-HK group,pGSadeno-RELMα/FIZZ1 group,pGSadeno-shRNA control group and pGSadeno-shRNA RELMα/FIZZ1 group.MTT assay was used to detect the viability of the MAEC.The formation of stroma tubes was observed for determining angiogenesis.The MOVAS cells cultured in vitro were also divided into pGSadeno-HK group,pGSadeno-RELMα/FIZZ1 group,pGSadeno-shRNA con-trol group and pGSadeno-shRNA RELMα/FIZZ1 group.The viability of MOVAS cells was measured by MTT assay.Fluo-3 AM fluorescence probe was used to detect intracellular Ca 2+concentration.The expression of calmodulin(CaM)and my-osin light chain kinase(MLCK)at mRNA and protein levels was determined by real-time PCR and Western blot.RE-SULTS:Over-expression of RELMα/FIZZ1 significantly promoted the viability of the MAEC.The number of lumen forma-tion was increased significantly(P<0.05).Knockdown of the RELM α/FIZZ1 expression inhibited the viability and lumen formation of MAEC(P <0.05).Over-expression of RELMα/FIZZ1 significantly promoted the viability of the MOVAS cells,enhanced the mean fluorescence intensity of intracellular Ca 2+,and the expression of CaM and MLCK at mRNA and protein levels was significantly increased.Knockdown of the RELMα/FIZZ1 expression significantly inhibited the viability of the MOVAS cells,the mean fluorescence intensity of Ca 2+was decreased, the expression of CaM and MLCK at mRNA and protein levels was decreased significantly(P<0.05).CONCLUSION:RELMα/FIZZ1 signaling pathway is involved in the angiogenesis,and the mechanism may be related to the changes of intracellular Ca 2+concentration and then to regu-late the intracellular CaM and MLCK expression.
ABSTRACT
Objective To explore the expression of resistin-like molecules-a (RELMa) in atherosclerotic plaque of ApoE-/- mouse, and to study the effects of RELMa on the proliferation and migration of vascular smooth musclee cells (VSMCs) . Method Nine ApoE-/- mice and nine C57BL/6J mice were fed with high fat diet. All mice were sacrificed 24 weeks after force feeding. Vessels were dissected from to abdominal aorta. Sections of aortic tissue were stained with HE dyeing and RELMa in aortic tissue was assayed by immunohistochemistry. The expression of RELMa mRNA in vessels was detected by RT-PCR. The effects of different concentration RELMa in different concentrations on the proliferation and migration of VSMCs were detected. Data was expressed as mean ± standard deviation. ANOVA were used for comparison in SPSS 11.0, and changes were considered as statistically significant if P value was less than 0.05. Results Atherosclerosis plaque formed in aortic root of ApoE-/- mice after they were fed with high fat diet for 24 weeks. RELMa protein and RELMa mRNA were found by immunohistochemistry and RT-PCR in atherosclerotic plaque of ApoE-/- mouse. RELMa protein didn't be found in vessels of control mouse. RELMa promoted the proliferation of VSMCs (RELMa groups: 2811. 21 ± 216. 89,4056. 87 ±220.65,5061.45 ± 335.86, vs. control 1609.58 ± 203.53, P < 0.01). RELMa promoted the migration of VSMCs (RELMa groups: 130.54±12.98,158.39±11.58,203.50± 17.37 vs. control:70.54± 11.92, P<0.01).Conclusions RELMa expresses in atherosclerotic plaque of ApoE-/- mouse. RELMa enhances the proliferation and migration of VSMCs of aorta.