| Objectives: Bone, teeth outside the non-osseous tissue calcification, with or without tissue necrosis, are referred to as ectopic calcification.Clinical ectopic calcification involving heart valve calcification, vascular calcification, soft tissue calcification, etc. In recent years, studies of vascular biology and cardiovascular imaging (such as electron beam computed tomography, etc) found that vascular calcification is common associated with the pathologic of atherosclerosis, hypertension, diabetes and other diseases, but also one of the important factors to the formation of lumen narrow caused by arterial plaque. The survey data of epidemiology show that vascular calcification is one of the important risk factor to cardiovascular disease.Although vascular calcification is the important characteristics of a lot of clinical diseases, the mechanism of vascular calcification are poorly understood. Vascular calcification once was thought heterotopic ossification during the aging period, passive deposition of calcium salts in the cells and extracellular matrix . However, in recent study, we found that vascular calcification is not a simple and passive deposition of calcium phosphate crystal in the blood vessel wall, but an actively regulated process for the formation of similar bone and cartilage, the normal contraction phenotype of vascular cells (especially the vascular smooth muscle cells) are transformed into osteoblast-like phenotype, atherosclerotic plaques and calcified lesion containing a variety of bone matrix protein and osteoblast markers.Calcineurin(CaN)is a serine/threonine protein phosphatase and known as the only phosphatase that regulated by Ca2+ and calmodulin. CaN is expressed mainly in cytoplasm and distributed widely in eukaryotic tissues. CaN was mainly expressed in cytoplasm,the tissue are widely distributed,and they activate the downstream gene transcription and participate in many regulation of cell function through its most important substrate NFATs(nuclear factor of activated T cells, NFATs) dephosphorylation and nuclear translocation. Research has shown that CaN/NFATc pathyway play important roles in myocardial hypertrophy and bone metabolism, but also widely in the immune system, regulate lymphocyte growth, differentiation and development. CaN/NFATc pathyway not only promote osteoblast differentiation and bone formation , but also it is required for the genesis of osteoclast. Whether CaN/NFATc pathyway play a role in vascular calcification is not yet known, and no report has been seen at present. Therefore, it is envisaged that we can regulate the vessel of calcification and improve the clinical prognosis of patients through this pathway,which demonstrate applications prospects for clinical treatment .This study is to construct a rat vacular calcification model by subcutaneously injection of warfarin and Vitamin D3, further to detect CaN,NFATc mRNA and protein expression as well as enzyme activity of CaN and ALP. Furthermore to know whether cyclosporinA (CsA), inhibitor of CaN activity, influence CaN/NFATc pathway expression in rat aorta and the extent of vascular calcification, rat in group C were treated with CsA. Through this test, we aim to investigate the role of CaN/NFATc pathway in vascular calcification, so that a new thought for clinical treatment of vascular calcification will be presented.Methods: 36 four weeks male Sprague-Dawlay rats, 80～90g in weight, were randomly divided into 3 groups: group A (normal control group), group B (calcification group) and group C (CsA group). Each group contain 12 rats. From day 1 to day 8 during the test, rats in each group were subcutaneously injected of vatamin k1 at dose of 15mg·kg-1·d-1. CsA, 10mg·kg-1·d-1, were also administered to group C by intraperitoneal injection, while group A and B were injected of isodose normal saline. From day 3 on, group B and C were subcutaneously injected of vatamin D3 at dose of 3×105U·kg-1·d-1 till day 5, as well as warfarin (15mg·100g-1 body weight) every 12 hours till day 8, and group A were injected of isodose normal saline. At day 9, all rats were mercyly killed after anaesthesia. Thoracic aorta and abdominal aorta of each rat were isolated. Samples from 6 rats of each group were used for immunohistochemistry to examine expression of CaNB1,NFATc protein and for in situ hybridization to detect expression of CaNAα,NFATcmRNA,as well as morphological observation by , the others were used to detect activities of CaN and alkaline phosphatase(ALP)in rat aorta. Results of immunohistochemistry and in situ hybridization were analyzed by Image-Pro Plus image analysis software. Integrated optic density (IOD) of positive cells in same area of aorta from each group were calculated respectively. All numerical data were presented with mean±standard deviation( x±s). SPSS13.0 statistics analysis software was used. Test of normality and homogeneity test for variance were performed firstly, then one-way analysis of variance was carried out, LSD-t test was used to compare differences among groups. P<0.05 was thought of statistical significance.Results: (1) Hematoxylin Eosin staining shows the structure of rat aorta in group A is integrity, the arrangement of VSMCs are in order; however in group B and C, the structure of rat aorta is not very integrity, the arrangement of VSMCs are disorder, elastic fibers are crooked and breakage. Black calcium deposition among the elastic fibers is seen in the medial layer of rat aorta in group B and C as shown by Von Kossa staining, while not in group A.(2)As shown by Immumohistochemistry,a low level expression Of CaNB1,NFATc protein is seen in normal control rat aorta, mainly located in cell cytoplasm. Expression Of CaNB1,NFATc protein in rat aorta from calcification group is elevated significantly compared with control group; Expression Of CaNB1,NFATc protein in rat aorta from CsA group is elevated compared with control group, but depressed compared with calcification group. (3) In situ hybridization shows a low level expression Of CaNAα,NFATc mRNA in normal control rat aorta, mainly located in cell cytoplasm. Expression Of CaNAα,NFATc mRNA in rat aorta from calcification group is elevated compared with control group; Expression Of CaNAα,NFATcmRNA in rat aorta from CsA group also increased compared with control group, but decreased compared with calcification group. (4) Compared with control group, rat aorta CaN activity from calcification group increased; rat aorta CaN activity from CsA group also increased compared with control group, however compared with calcification group, the difference is not significant. (5) ALP activity of rat aorta from calcification group also increased compared with control group; compared with control group, rat aorta ALP activity from CsA group is elevated, however compared with calcification group, ALP activity of CsA group has a tendency to increase, but the difference is not significant.Conclusions: (1)This experiment successfully established a rat vacular calcification model by warfarin and Vitamin D3 subcutaneously injection as evidenced by vonKossa staining. (2)It is discovered for the first time in this study that expression of CaN,NFATc in both protein level and mRNA level is elevated, as well as the activity of CaN increased in calcified rat aorta, which suggest that CaN/NFATc pathway plays a role in the regulation of vascular calcificatin, although its exact mechanisms is not yet known. (3)Immunosupressive agent CsA may facilitate vascular calcification.
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