| Objectives: As the pathological foundation of varied diseases like atherosclerosis, old people isolated systolic hypertension, arteriosclerotic kidney and diabetes , the vascular calcification occurs frequently in old people retrogression cardiac disease, coronary artery disease , diabetes and chronic renal insufficiency patients.It is an important Risk factor to lead to clinic heart blood vessel events like myocardial infarction, postangioplasty endomembrane tearing and so forth.The aortal rigidity has been increased and the vascular compliance depressed by aorta vascular calcification,It can induce those heart blood vessel events like heart muscle ischemia, left ventricular hypertrophy ,congestive heart failure and stroke. Nowadays, the process of vascular calcification was confirmed to be active by a lot of in vitro and in vivo experiments.It is suggested that vascular calcification is result from vascular smooth muscle cells experienced transconformation ,differentiation under gene regulation and influenced by many kinds of factors.It might have substaintial clinical significance to lucubrate the pathogenesis of the vascular smooth muscle cells calcification,and to prevent, postpone and reverse the vascular smooth muscle cells calcification is equally important.It is reported that GATA-6 and Calcineurin (CaN) play a certain regulatory role in some changes for example apoptosis, Bone metabolism and cardiomyocyte hypertrophy.In different vascular smooth muscle cells, dephosphorylated CaN stimulates the NFATs, which is the substrate of CaN has been found recently to interact with intranuclear GATA-6.They stimulate the expression of smooth muscle specific Sm-MHC together.And the expression and transcriptional activity of the smooth muscle myosin heavy chain can be degraded accordingly the down regulation of GATA-6 conjunction with DNA by CaN signal in the regulation process. Thus it prompts that GATA-6 and CaN were involved in VSMCs specific transcription process of transformation, cell differentiation and hyperplasy, and play an important role in maintainning different VSMCs phenotypic differentiation. As calcification of vascular smooth muscle cells is relation with VSMCs proliferation, CaN and GATA-6 play a role in the proliferation of VSMCs. GATA-6 and CaN are likely involved in the process of the vascular smooth muscle cells calcification.This hypothesis has not been confirmed at present.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. activity of the CaN,mRNA. protein of the GATA-6, 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,mRNA.protein of the GATA-6 and vascular smooth muscle cells calcification. The purpose of this study was to approach the regulatory role of CaN and GATA-6 on rat aortic smooth muscle cell calcification and to provide a new way of thinking for the prevention and treatment of vascular smooth muscle cells calcification.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). Group A:GMDM medium culture cells Were used. Group B: GMDM+High phosphorus (2mmol/l) medium culture cells Were used. GroupC: GMDM+High phosphorus(2mmol/l)+CsA (5ug/ml) medium culture cells were used. They were cultivated for 6 days respectively, medium was changed once every 3 days. After six days the cells were collected.Using alizarin bordeaux staining, the calcification situation of rat thoracic aortic vascular smooth muscle cells from each group were observed by light microscope. Using colorimetry to detect the alkaline phosphatase (ALP) activity. Using ELISA, calcium activity and calcineurin phosphatase (CaN) activity of the cells from rat thoracic aortic vascular smooth muscle cells were evaluated. The expression of CaNB mRNA and GATA-6 mRNA were detected by RT-PCR. And The expression of CaNB protein and GATA- 6 protein were detected by Western blot method. 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) Cells fusiform, orderly, closely, evenly arranged and presented no calcification 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, presented 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) Using ELISA method, significantly increased Ca2+ quantitation of the rat aortic VSMCs in calcification group(9.01±0.32pg/ml) and CsA group (10.39±0.70pg/ml) were observed when compared respectively to the control group(6.03±0.33pg/m)(p<0.01); and significantly increased Ca2+ quantitation of the rat aortic VSMCs in CsA group was observed when compared to the calcification group(p<0.05). (3) Using Colorimetric, significantly increased ALP activity of the rat aortic VSMCs in calcification group(146.04±9.02 U·g-1protein) and CsA group (216.69±14.0U·g-1protein) were observed when compared respectively to the control group(38.74±2.23 U·g-1 protein)(p<0.01); and significantly increased ALP activity of the rat aortic VSMCs in CsA group was observed when compared to the calcification group(p<0.01). (4) Using ELISA method, significantly increased CaN activity of the rat aortic VSMCs in calcification group(21.47±1.86 IU/L) were observed 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 of the rat aortic VSMCs in CsA group was observed when compared to the control group (p>0.05). (5) Using RT-PCR method, significantly increased CaNB mRNA of the rat aortic VSMCs in calcification group(6.27±1.45) and CsA group(3.38±0.77) were observed when compared respectively to the control group(1.10±0.28) (p<0.01); and significantly increased CaNB mRNA of the rat aortic VSMCs in calcification group was observed when compared to the CsA group (p<0.01). Significantly decreased GATA-6 mRNA of the rat aortic VSMCs in calcification group(0.30±0.06)and CsA group(0.69±0.12)were observed when compared respectively to the control group(1.09±0.27) (p<0.01); and significantly increased GATA-6 mRNA of the rat aortic VSMCs in the CsA group was observed when compared to the calcification group (p<0.01). (6) Using Western blot, significantly increased CaN B protein of the rat aortic VSMCs in calcification group(69.11±2.51) and CsA group(48.22±2.47)were observed when compared respectively to the control group(29.9±1.39) (p<0.01), and significantly increased CaN B protein of the rat aortic VSMCs in calcification group was observed when compared to the CsA group (p<0.01). Significantly decreased GATA-6 protein of the rat aortic VSMCs in calcification group(0.40±0.05) and CsA group(0.85±0.21)were observed when compared respectively to the control group(1.67±0.20) (p<0.01); and significantly increased GATA-6 protein of the rat aortic VSMCs in the CsA group was observed 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 , structural changes of the calcification cells were observed by optical microscopy. (2) In rat aortic calcific VSMCs , the test was firstly found that CaN mRNA and protein expression and activity were significantly increased, while GATA-6 mRNA and protein expression were significantly reduced,calciumion and alkaline phosphatase activity were decreased relative to the quantitative cyclosporine A group, indicating that CaN and GATA-6 would participate in the regulation of vascular smooth muscle cell calcification, GATA-6 might be inhibited by CaN. (3)Immunosuppressive drugs CsA might promote vascular smooth muscle cell calcification.
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