Role Of Calcineurin And Nfat In Vascular Smooth Muscle Cells Calcification From Rat Aorta

Objectives: It now appears that vascular calcification is a consequence of pathophysiology processes that precipitation of hydroxyapatite crystals in the vascular wall arises from high calcium/phosphate products and dysregulated locally or systemically of the normal balance between promotion and inhibition of calcification .Calcification may occur at several sites in the cardiovascular system, including the intima and media of vessels and cardiac valves. It occurs frequently in old people and hypertension, coronary artery disease and patients with diabetes and chronic renal insufficiency.The degree of calcification was positively correlated with the severity of disease and showed a progressive deterioration with aging. Nowadays ,studys found that vascular calcification is an active process involving multiple cells , similar to that in bone with active endochondral and intramembranous ossification.In the development process of this disease, after the blood vessel wall mineralization defense mechanisms are weakened or even depletion, vascular smooth muscle cells (VSMCs) and endothelial cells (EC) shift into osteoblasts phenotype, and release lipid vesicles. Lipid vesicles in the arterial wall exists at least in two forms:matrix vesicles and apoptotic bodies, it provides a suitable nucleation and micro-environment to calcium crystal, the structure of hydroxyapatite deposition is supported by the flexibility protein of the vessel wall. Therefore,after the balance between calcium deposition (osteoblast-like cell-mediated) and calcium absorption (osteoclast-like cell-mediated) disruption ,the blood vessel wall or arterial valve may form ectopic calcification.Calcineurin(CaN),called protein phosphatase 2B(PP2B), is one member of serine-threonine protein phosphatase groups. It is so far only one serine-threonine protein phosphatase which is mediated by Ca²⁺/ Calmodulin and directly regulated by Ca²⁺ in the process of aignal transduction as a function of dephosphorylation. It is well known that CaN is one kind of multifunctional signal peptidase,which is widespreadly distributed in the cells of brain tissue, myocardium, vascular smooth muscle, blood vessel endothelium, skeletal muscle and T- lymphocyte and participates in the regulation of function in mult-cytes and mult-system. The role of CaN is educed by the dephosphorylation and intranuclear transposition of its important substrate, nuclear factor of activated T cells(NFAT). Many studies has indicated that CaN/NFAT signaling pathway did not only paticipate in the development of many kinds of immune system disease, regulate the genetic transcription and expression in the course of tumorigenesis and mediate the pathophysiology of the cardiac musclar hypertrophy, but also participate in the differentiation of the osteoclast and osteoblastic. It is rarely reported about the relationship between the CaN/NFAT signaling pathway and vascular calification.Using an experimental vascular smooth muscle cells(VSMCs) calcification model in rat aortic was established successfully induced by high phosphorus medium, the expressions of mRNA. protein of the NFAT, activity of the CaN, calcium ion activity in vascular smooth muscle cells and alkaline phosphatase(ALP) activity were observed.And the influences of CaN inhibitor cyclosporin A (CsA) on the expressions of mRNA .protein of the CaN and vascular smooth muscle cells calcification. The purpose of this study was to approach the regulatory role of CaN/NFAT signaling pathway on rat aortic smooth muscle cell calcification and to provide a new way of thinking for the prevention and treatment of Vascular calcification-related clinical disease.Methods: Rat aortic VSMCs(A7r5) were randomly divided into 3 groups after subcultivation: group A (normal control group), group B (calcification group) and group C (CsA group). After cells covered the bottom, GMDM medium culture cells were used in Group A; High phosphorus(2mmol/l) was added into medium to induce calcification of the culture cells in Group B; High phosphorus(2mmol/l) and CsA (5ug/ml) were added into medium in Group C. They were cultivated for 6 days respectively, medium was changed once every 3 days. After six days the cells were collected.Using western blotting, the protein level of NFAT was assayed; Using RT-PCR , the mRNA level of NFAT was assayed; Using ELISA, calcium activity and calcineurin phosphatase (CaN) activity of the cells from rat thoracic aortic vascular smooth muscle cells were detected. Using colorimetry, the alkaline phosphatase (ALP) activity was detected. Using alizarin red staining, the calcification of rat thoracic aortic vascular smooth muscle cells from each group were observed by light microscope. All data are expressed as mean±standard deviation ( (x|-)±s),statistical analysis was used SPSS13.0 software. Group comparison was determined by one-way ANOVA. pairwise comparison was determined by LSD-t test. Differences with P < 0.05 values were considered statistically significant.Results: (1)Fusiform, orderly, closely, evenly arranged and presented no calcification of the cells were observed in aorta smooth muscle cells from control group by Alizarin bordeaux staining, however, cells are in spindle or polygon, normal cells form disorderly and breakage, brown calcium deposition were observed in aorta smooth muscle cells from calcification group and CsA group by Alizarin bordeaux staining, while were not observed in control group. (2) Significantly increased the activity of CaN in the rat aortic VSMCs from calcification group(21.47±1.86 IU/L) were detected by ELISA when compared respectively to the control group(14.31±0.24 IU/L) and CsA group (15.34±0.14IU/L) (p<0.01), and no significantly change of CaN activity in the rat aortic VSMCs from CsA group was observed when compared to the control group (p>0.05). (3) Significantly increased of the Ca²⁺ quantitation in the rat aortic VSMCs from calcification group(9.01±0.32pg/ml) and CsA group (10.39±0.70pg/ml) were detected by ELISA when compared respectively to the control group(6.03±0.33pg/m)(p<0.01), and significantly increased of the Ca²⁺ quantitation in the rat aortic VSMCs from CsA group was detected by ELISA when compared to the calcification group(p<0.05). (4) Significantly increased of the mRNA level of NFAT in the rat aortic VSMCs from calcification group(6.39±0.45) and CsA group(2.16±0.46)were assayed by RT-PCR when compared respectively to the control group(1.10±0.45)(p<0.01), and significantly increased of the mRNA level of NFAT in the rat aortic VSMCs from calcification group was assayed by RT-PCR when compared to the CsA group (p<0.01). (5) Significantly increased of the protein level of NFAT in the rat aortic VSMCs from calcification group(40.90±2.07)and CsA group(27.45±2.59)were assayed by western blotting when compared respectively to the control group(17.27±1.36) (p<0.01), and significantly increased of the protein level of NFAT in the rat aortic VSMCs from calcification group was assayed by western blotting when compared to the CsA group (p<0.01). (6) Significantly increased of the ALP activity in the rat aortic VSMCs from calcification group(146.04±9.02 U·g^-1protein) and CsA group (216.69±14.0U·g^-1protein) were detected by colorimetric when compared respectively to the control group(38.74±2.23 U·g^-1 protein)(p<0.01), and significantly increased of the ALP activity in the rat aortic VSMCs from CsA group was detected by colorimetric when compared to the calcification group(p<0.01).Conclusion: (1) Using an experimental aortic smooth muscle cell calcification model in rats was established successfully induced by high phosphorus medium , changes of the structural in calcification cells were observed by optical microscopy.(2) In rat aortic calcific VSMCs , we found that the activity of CaN, the mRNA and protein level of CaN and NFAT were significantly increased compared to the control group, while the calciumion and the ALP activity were significantly decreased compared to the CsA group. It indicates that the CaN/NFAT may participate in the regulation of vascular smooth muscle cell calcification, immunosuppressive drugs CsA may promote calcification of the vascular smooth muscle cell.