| Objectives: Cardiovascular calcification is often seen in the aging as well as patients with hypertension, atherosclerosis, diabetes mellitus or chronic renal desease, including artery intima calcification (atherosclerotic calcification), medial calcification (Mo¨nckeberg's sclerosis) and cardiac valvular calcification. Regardless in which anatomic compartment it occurs, vascular calcification leads to altered hemodynamics and mechanical properties, causing increased wall stiffness, heightened systolic blood pressure, depressed diastolic blood pressure, augmentation of pulse pressure, decreased coronary perfusion, ultimately left ventricular hypertrophy, myocardial ischemia and cardiac arrhythmia. Moreover, vascular calcification also increases risk for stroke and sudden death. Prediction value of coronary artery calcification caused great attention recent years. Compared with those without calcification, total relative risk reaches 4.3 in patients with coronary artery calcification no matter its extension, and incidents of coronary artery disease including myocardial infarction and sudden cardiac death four times increase within three to five years. Therefore investigating regulation mechanisms of vascular calcification to prevent, to delay or even to reverse its development will have great clinical significance.Vascular calcification was once thought passive deposition of supersaturated calcium and phosphate in the vessel wall, it was proved recent decades to be an active regulated process involving multiple cells and molecules, resembling osteogenesis. 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 involves in cell function regulation of multiple cell types by dephosphorylating its substrates such as member of NFAT (nuclear factor of activated T cells) family and transposition from cytoplasm to nucleus. Evidences show that CaN play important roles in myocardial hypertrophy and bone metabolism recent years. CaN not only promote osteoblast differentiation and bone formation , but also it is required for the genesis of osteoclast. Whether CaN plays an role in vascular calcification is not yet known, and no report has been seen at present.This study is to construct a rat vascular calcification model by subcutaneously injection of warfarin and Vitamin D3, further to detect CaN mRNA and protein expression as well as enzyme activity of CaN. Furthermore to know whether cyclosporin A (CsA), inhibitor of CaN activity, influence CaN 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 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 vitamin 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 vitamin 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 protein and for in situ hybridization to detect expression of CaNAαmRNA, as well as morphological observation and changes of ultramicrostructure of calcified rat aorta under transmission electron microscope, 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 vascular smooth muscle cells (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. (2) 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. (3) Changes in ultramicrostructure of calcified rat aorta are observed by transmission electron microscope as shown in Fig.3 Vacuolar degeneration of endothelial cells occurred, along with the mitochondria hydropsia, mitochondrial cristae decreased and part of its membrane coalesced with cytomembrane or disappeared. At the same time, slight dilation of the rough endoplasm reticulum with the phenomenon of degranulation is seen as well as part of its membrane coalesced or disappeared. Pinocytosis vesicles of endothelial cells reduced. Meantime, cell nucleus became irregular and slightly hydropsia with part of its double-layer nuclear membrane coalesced or disappeard. As regard smooth muscle cells, large part of the mitochondrial cristae and part of the nuclear membrane coalesced or disappeared. While slight dilation of the rough endoplasm reticulum with the phenomenon of degranulation is also seen in smooth muscle cells. Calcium deposion which located in the extra cellular matrix of smooth muscle cell is seen with increased electron density under transmission electron microscope and surrounded by a membrane-like strucure. (4) As shown by Immunohistochemistry, a low level expression Of CaNB1 protein is seen in normal control rat aorta, mainly located in cell cytoplasm. Expression Of CaNB1 protein in rat aorta from calcification group (8731.93±469.92) is elevated significantly compared with control group (5500.95±982.57) (P<0.01). Expression Of CaNB1 protein in rat aorta from CsA group (7712.79±539.20) is elevated compared with control group (5500.95±982.57) (P<0.01), but depressed compared with calcification group (8731.93±469.92) (P<0.05). (5) In situ hybridization shows a low level expression Of CaNAαmRNA in normal control rat aorta, mainly located in cell cytoplasm. Expression Of CaNAαmRNA in rat aorta from calcification group (26420.97±1697.97) is elevated compared with control group (10302.17±2636.06) (P<0.01). Expression Of CaNAαmRNA in rat aorta from CsA group (16775.25±4029.59) also increased compared with control group (10302.17±2636.06) (P<0.01), but decreased compared with calcification group (26420.97±1697.97) (P<0.01). (6) Compared with control group (0.90±0.39μmolPi·mgprot-1·h-1), rat aorta CaN activity from calcification group (1.53±0.29μmolPi·mgprot-1··h-1) increased(P<0.01). Rat aorta CaN activity from CsA group (1.54±0.40μmolPi·mgprot-1·h-1) also increased compared with control group (0.90±0.39μmolPi·mgprot-1·h-1) (P<0.01), however compared with calcification group(1.53±0.29μmolPi·mgprot-1··h-1), the difference is not significant (P>0.05). (7) ALP activity of rat aorta from calcification group (463.38±65.09U·gprot-1) also increased compared with control group (360.04±49.01U·gprot-1) (P<0.05); compared with control group (360.04±49.01U·gprot-1), rat aorta ALP activity from CsA group (479.78±74.67U-1gprot) is elevated (P<0.01), however compared with calcification group (463.38±65.09U·gprot-1), ALP activity of CsA group (479.78±74.67U-1gprot) has a tendency to increase, but the difference is not significant (P>0.05).Conclusions: (1) This experiment successfully established a rat vascular calcification model by warfarin and Vitamin D3 subcutaneously injection as evidenced by vonKossa staining. Ultramicrostructure changes of calcified rat aorta are observed under transmission electron microscope. (2) It is discovered for the first time in this study that expression of CaN 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 plays a role in the regulation of vascular calcification, although its exact mechanisms is not yet known. (3) Immunosupressive agent CsA may facilitate vascular calcification.
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