Study On Development And Mechanism Of Arterial Calcification In Adiponectin-deficient Mice

Part one Observation on pathological character of arterial calcification in adiponectin knockout miceObjectiveto observe on histopathologic and histochemical characters in adiponectin knockout (Adipo-/-)mice.MethodsThirty-six of 6 weeks old mice were divided at random into 6 groups (n=6 per group).Group 1:Adiponectin-/-mice fed chow diet for 10 weeks.Group 2:Adiponectin-/-mice fed chow diet for 20 weeks.Group 3:Adiponectin-/- mice fed chow diet for 30 weeks. Group 4: Adiponectin-/-mice treated with P-galactosidase adenovirus for 30 weeks. Group 5:Adiponectin-/-mice treated with adiponectin adenovirus for 30 weeks.Group 6:Wild-type mice fed chow diet for 30 weeks.The mice were euthanized, blood was collected from retro-orbital venous plexus, and plasma concentrations of glucose were determined enzymatically. Insulin and adiponectin concentrations were determined by radioimmunoassay. The thoracic aortas were dissected, and then aortas were fixed in 4% paraformaldebyde.Alizarin Red S staining was used to detect calcification. Immunohistochemistry was used to detect the expression of OPG and RANKL protein.Aortic segments from aorta arch to the iliac bifurcation were removed, then calcium was extracted with 10% formic acid and the colorimetric quantification of calcium was achieved. The thoracic aorta was homogenized by ultrasounds, the ALP activity of supernatant was measured by spectrophotometric measurement of P-nitrophenol release.Total protein was determined using Bradford protein assay.ResultsThere was no significant difference in body weight, serum fasting glucose and insulin levels between Adiponectin-/- mice and wild type mice.Adiponectin-/-mice developed slight arterial calcification after fed with normal chow diet for 30 weeks,the aortic calcium contents and ALP activity increased, and the expression of OPG decreased. There were no visible arterial calcification observed in Adiponectin-/- mice with chow diet for 10 or 20 weeks.After adenovirus-mediated supplement of adiponectin for Adiponectin-/- mice,no arterial calcification were shown. The aortic calcium and ALP activity contents also decreased, and the expression of OPG increased. It showed that adenovirus-mediated supplement of adiponectin attenuated arterial calcification. RANKL protein wasn't detected in these mice.ConclusionOur findings demonstrated that Adiponectin-/-mice developed arterial calcification, and this could be attributed to the elevated ALP activity and the decreased expression of OPG, and adenovirus-mediated supplement of adiponectin attenuated arterial calcification.It suggested that adiponectin play a protective role against arterial calcification. Part two The effects of adiponectin on cultured calcifying vascular smooth muscle cellsObjectiveTo investigate the expression of adiponectin receptor and OPG/RANKL/RANK system in culured calcifying vascular smooth muscle cells (CVSMCs) in vitro and the effects of adiponectin on CVSMCs.Methods6 weeks old male adiponectin knockout mice (Adipo-/-) were fed cow diet for 30 weeks, then these mice were sacrificed, the aorta removed, and the CVSMCs were cultured. The culured CVSMCs were identifed by their positive staining with monoclonal antibody a-actin.The expression of adiponectin receptor and OPG/RANKL/RANK mRNA and protein in CVSMCs were detected using RT-PCR and western-blot.When the cells were treated with adiponectin at 3μg/mL,10μg/mL,30μg/mL for 48h, or treated with 30μg/mL adiponectin for 12-48h, to investigate the dose effect and time effect of adiponectin, the OPG mRNA was measured by Real-time quantitative PCR and OPG protein was measured by ELISA; alkaline phosphatase (ALP) activity was measured by spectrophotometric measurement of P-nitorphenol release, osteocalcin(OC) was detected by radioimmunoassay, and Runx2 expression was analyzed by Western-blot. The calcified nodules were stained by 1% Alizarin Red S in the presence of 30μg/mL for 20 days.ResultsThe immunocytochemical stain for smooth muscleα-actin confirmed vascular smooth muscle cells (VSMCs) pheotype and the multicellular nodules spontaneously appeared in VSMCs culture. Adiponectin receptor R1 (AdipoR1)and Adiponectin receptor R2 (AdipoR2) mRNA were expressed in CVSMCs, but only AdipoRl Protein was detected; OPG,RANK mRNA and protein was detected,but RANKL mRNA and protein was not detected;and Adiponectin elevated the level of OPG mRNA and protein, but suppressed ALP activity, OC secretion and Runx2 protein expression in a dose-dependent and time-dependent manner. Adiponectin also decreased calcified nodules formation in at 30μg/mL concentration for 20 days.ConclusionThese data indicated that adiponectin significantly inhibited cultured CVSMCs calcification in vitro may mediate by AdipoR pathway, thereby promoted the expression of vessel protecting factor OPG and blocked the transform from VSMCs to osteoblasts phenotype. Part three Adiponectin inhibited osteoblastic calcification of CVSMCs via AdipoRl/p38 signaling pathway in vitroObjectiveTo investigate mechanisms of adiponectin inhibited osteoblastic calcification of cultured calcifying vascular smooth muscle cells (CVSMCs) in vitro.MethodsAfter in the presence of 30μg/mL adiponectin for 0,5,30,60min, the total protein were extracted. The protein of P-JNK, JNK, P-p38,p38, P-ERK, ERK were detected using Western-blot method. Small interfering RNA (SiRNAs) was used to down-regulate the expression of AdipoRl in CVSMCs.The OPG mRNA was measured by Real-time quantitative PCR and OPG protein was measured by ELISA, and the ALP activity was measured by spectrophotometric measurement of p-nitrophenol release when SB 203580 blocked p38 or SiRNAs blocked AdipoRl.ResultsAdiponectin induced activation of p38,but not JNK and ERK in CVSMCs.Furthermore,pretreatment of CVSMCs with p38 inhibitor (SB203580) or SiRNAs-AdipoRl abolished adiponectin induced the expression of OPG and ALP activity. These date suggested that p38 signaling pathway played an important role in inhibiting the calcification of CVSMCs. ConclusionOur date indicated adiponectin induced the expression of OPG and inhibited osteoblastic calcification of CVSMCs via AdipoR1/p38 signaling pathway.