Study On Development And Mechanism Of Arterial Calcification In Adiponectin-deficient Mice Under High-fat/High-sucrose Diet

Part one Observation on pathological character of arterial calcification in adiponectin knockout miceObjective to observe on histopathologic and histochemical characters in adiponectin knockout (Adipo-/-) mice.MethodsForty of 6 weeks old mice were divided at random into 5 groups (n=8 per group). Group 1:wild type (WT) mice fed high-fat/high-sucrose (HF/HS)diet. Group 2:Adipo-/-mice fed HF/HS diet. group3:WT mice fed normal chow diet. Group 4:Adipo-/-mice fed normal diet. Goup 5: WT mice intraperitoneal injected streptozotocin (30mg/kg body weight) 1 time and then fed HF/HS diet developing a model of type 2 diabetes mellitus. After 10 weeks, the mice were euthanized.Blood was collected from retro-orbital venous plexus, and plasma concentrations of glucose were determined enzymatically.Insulin 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. 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.Runx2 protein expression was analyzed by Western-blot.ResultsAdipo-/-mice and the model mice of type 2 diabetes mellitus from the HF/HS diet and displayed high fasting glucose levels, and high fasting plasma insulin levels, these were significantly higher than those in other mice. Severe aortic calcification could be seen in Adipo-/-mice, and aortic calcium contents in Adipo-/-mice with HF/HS diet increased more obviously than WT mice and streptozotocin injected mice compared with the normal diet.WT mice with HF/HS showed significantly elevated ALP activity, but Adipo-/-mice with HF/HS diet increased more obviously. The expression of Runx2 protein in aorta of Adipo-/-mice with HF/HS diet was higher than that of WT diabetic mice (group 5).ConclusionOur work demonstrated that Adipo-/-mice developed severe diabetic arterial calcification, and the mechanism was associated with the elevated ALP activity and Runx2 protein expression in the arota. Part two The effects of adiponectin on cultured calcifying vascular smooth muscle cellsObjectiveTo investigate the expression of adiponectin receptor 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 high-fat/high-sucrose (HF/HS) diet diet for 10 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 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, the alkaline phosphatase (ALP) activity was measured by spectrophotometric measurement of P-nitorphenol release, osteocalcin(OC) was detected by radioimm-unoassay, 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 muscle a-actin confirmed vascular smooth muscle cells (VSMCs) pheotype and the multicellular nodules spontaneously appeared in VSMCs culture. Adiponectin receptor R1 (AdipoRl) and Adiponectin receptor R2 (AdipoR2) mRNA were expressed in CVSMCs, but only AdipoRl Protein was detected. Adiponectin suppressed ALP activity, OC secretion and Runx2 protein expression in a dose-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. 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, then AdipoRl, p-p38, p38 were analysed. 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. These effects were blocked by suppression of AdipoRl with SiRNA. Furthermore, pretreatment of CVSMCs with p38 inhibitor (SB203580) or SiRNAs-AdipoRl abolished adiponectin induced ALP activity. These date suggested that p38 signaling pathway played an important role in inhibiting the calcification of CVSMCs. ConclusionOur date indicated adiponectin inhibited osteoblastic calcification of CVSMCs via AdipoR1/p38 signaling pathway.