The Effects Of Adiponectin On Calcification In Cultured Rat Vascular Smooth Muscle Cell

Objective: Vascular calcification, an process similar to osteogenesis,often lead to the phenotypic transformation of vascular smooth musclecells (VSMCs) in the medial vascular wall. The phenotype of calcifiedVSMC dramatically upregulates osteoblastic specific molecules. Thus,the present study was carried out to establish calcification system in thepresence of 13-glycerophosphate and oberserved the expression ofosteogenic proteins such as alkaline phosphatase (ALP), typeⅠcollagenand osteocalcin (OC) in calcified VSMCs.Methods: The VSMCs were identified by immunocytochemistry.ALP activity was determined by using p-nitrophenyl phosphate assubstrate. TypeⅠcollagen was analyzed by ELISA. OC was measured byradioimmunoassay.Results: (1) The immunocytochemical stain for smooth muscleα-actin confirmed VSMC phenotype. (2) The calcification system invitro was induced by incubating VSMCs withβ-glycerophosphate, andin calcified VSMCs, the ALP activity and the secretion of typeⅠcollagenand OC were enhanced. Conclusion: Some phenotypic markers of osteoblasts, such as ALPactivity, typeⅠcollagen and OC production, were enhanced in theprocess ofβ-glycerophosphate-induced VSMC calcification. Chapter Two Adiponectin Inhibits Calcification of RatVascular Smooth Muscle Cells in CulturedObjective: Adiponectin, the biological effects of which aremediated by adiponectin receptor (AdipoR), has anti-atherogenic andanti-inflammatory effects and plays a protective role in vascular disease.However, whether adiponectin has beneficial effect on vascularcalcification is not yet known. Therefore, the present study investigatedthe expression of AdipoR in rat VSMCs and the effect of adiponectin onβ-glycerophosphate-induced VSMCs calcification.Methods: The expression of AdipoR mRNA and protein in VSMCswere detected by RT-PCR and Western blot. ALP activity was determinedby using p-nitrophenyl phosphate as substrate. TypeⅠcollagen wasanalyzed by ELISA. OC was measured by radioimmunoassay. Thecalcified nodules were stained by Alizarin Red S.Results: (1) Both AdipoR1 mRNA and protein were expressed in ratVSMCs. (2) Adiponectin concentration- and time-dependently suppressedALP activity, typeⅠcollagen and OC secretion in calcified VSMCs. (3)Adiponectin also inhibited the formation of calcified nodules.Conclusion: AdipoR1 can be transcripted and translated in ratVSMCs, and adiponectin significantly inhibitedβ-glycerophosphate-induced VSMCs calcification in vitro. Chapter Three Adiponectin Inhibits Rat Vascular SmoothMuscle Cells Calcification by ERK signal pathwayObjective: Our previous study showed that adiponectin can inhibitcalcification of cultured VSMCs, but the mechanism of the effect ofadiponectin on VSMCs was unclear. In the present study, we investigatedthe signal pathway by which adiponectin inhibited VSMCs calcificationin vitro.Methods: P-ERK, ERK, p-p38, p38, p-JNK, JNK were analysed byWestern blot. ALP activity was determined by using p-nitrophenylphosphate as substrate.Results: Adiponectin induced activation of ERK, but not activationof p38 and JNK. The effect that adiponectin inhibited ALP activity wasblocked by pretreatment of VSMCs with ERK inhibitor. These datesuggested that ERK played an important role in inhibiting thecalcification of VSMCs.Conclusion: Adiponectin inhibited calcification of VSMCs inculture through ERK signal pathway.