CaN In Vascular Smooth Muscle Cell Calcification In The Regulation Of PKG I

Objectives: Vascular calcification is a pathological basis of many diseases, and it is most closely related to atherosclerosis, it makes the mechanical properties of arteries changed, can cause thrombosis, plaque rupture, peripheral vascular plug ischemia, aortic stenosis , myocardial ischemia and infarction and other serious consequences, it increases the risk of vascular rupture in balloon angioplasty and cardiovascular failure of biological repairment. Lead to heart valve calcification is mainly due to mechanical damage and inflammation, involving the main part of the aortic valve and mitral valve, calcification of the results can be caused by stenosis and / or regurgitation, is an important reason of heart valve replacement surgery. Above the blood vessels and heart valve calcification is a common site of cardiovascular calcification occurred.Vascular cell calcification in the past that the calcium is the passive deposition of inside the cell and extracellular matrix, is a sign of aging of vascular cells, but recent studies have found calcification of vascular cells is a complex, active, can regulate the biological process is vascular structure of the active ossification, similar to bone and cartilage formation during ossification.Calcineurin phosphatase (Calcineurin, CaN) is a serine - threonine protein phosphatase. CaN is widely distributed in many tissues, is a multifunctional signaling enzyme, mainly through the NFAT and other substrates to phosphorylation and nuclear translocation, activation of its downstream gene transcription, involved in regulating a variety of cell functions. Recent studies have confirmed that, CaN signal pathway involved in VSMC proliferation and function are maintained. Different stimuli (such as PDGF2BB, thrombin and Angâ…¡, etc.) can induce NF-AT transmission of extracellular signals, activated VSMC nuclear gene transcription, and ultimately stimulate VSMC proliferation, a process that can be blocked by the inhibitor CsA of CaN.CGMP-dependent protein kinase (cGMP-dependent protein kinase, PKG) is widely present in eukaryotic cells within a serine / threonine protein kinase. NO (nitric oxide, NO) can regulate a variety of signaling pathways, can promote the synthesis of intracellular cGMP, and cGMP activation of PKG can modulate the local and systemic signals. Recently there are many studies in the PKG CaN and the relationship between vascular calcification experiments, and CaN in the calcification of vascular smooth muscle cells (VSMCs) within the cytoplasm of the relationship with the PKG I have not been reported at home and abroad.This experiment, sodium dihydrogen phosphate in rat aortic VSMCs calcification model, we determined cell PKG I and CaN mRNA, protein expression and activity, and to observe the expression of CaN effect on PKG I, discuss PKG I and CaN to calcification of vascular cells regulatory role for the prevention and treatment of calcification of vascular cells to provide a new way of thinking.Methods: Rats aortic VSMCs are randomly divided into 3 groups: group A (normal control group), group B (calcification group) and group C (CsA group). Group A:We use GMDM medium culture cells. Group B: We use GMDM+ NaH2PO4 medium culture cells; GroupC: We use GMDM+ NaH2PO4+CsA medium culture cells;It is respectively trained for 6 days, and we collect cells after six days. After that samples used for Western blot to examine expression of CaNB protein and PKGâ… protein as well as for in PCR to detect expression of CaNB mRNA and PKGâ… mRNA, The others are used to detect activities of CaN and Ca²⁺. All numerical data are presented with mean±standard deviation( x±s). SPSS13.0 statistics analysis software is used. Test of normality and homogeneity test for variance are performed firstly, then one-way analysis of variance is carried out, LSD-t test is used to compare differences among groups, P<0.05 is thought of statistical significance.Results: (1)Alizarin bordeaux staining shows the structure of rat aorta smooth muscle cells in control group, there are fusiform,orderly, closely, evenly arranged and presented no calcification; however in calcification group and treatment group, the structure of rat aorta smooth muscle cells are in spindle or polygon, normal cells form disorderly and breakage, presented brown calcium deposition, while not in control group. (2)In PCR shows the expression of CaNB and PKGâ… mRNA in three groups of rat aortic VSMCs.Expression Of CaNB mRNA from calcification group(6.27±1.45)is elevated compared with control group(1.10±0.28)(P<0.01), and expression of PKGâ… mRNA (0.26±0.07)is reduced compared with control group(0.89±0.17)(P<0.01); Expression Of CaNB mRNA from CsA group (3.38±0.77) increased compared with control group(1.10±0.28)(P<0.01), and expression of PKGâ… mRNA(0.60±0.12) also reduced compared with control group(0.89±0.17) (P<0.01), but CaNB mRNA decreased compared with calcification group (6.27±1.45)(P<0.01), while PKGâ… mRNA increased significantly compared with calcification group(0.26±0.07)(P<0.01). (3)As shown by Western blot, the expression of CaNB and PKGâ… protein is seen in three groups of rat aortic VSMCs. Expression of CaNB protein from calcification group (69.11±2.51) is elevated significantly compared with control group (29.9±1.39) (P<0.01), and expression of PKGâ… protein(0.32±0.01)is reduced significantly compared with control group(1.54±0.02) (P<0.01). Expression of CaNB protein from CsA group(48.22±2.47)is elevated compared with control group (29.9±1.39) (P<0.01), and expression of PKGâ… protein(0.77±0.03)is reduced compared with control group (1.54±0.02)(P<0.01), but CaNB protein depressed compared with calcification group (69.11±2.51)(P<0.01), while PKGâ… protein increased significantly compared with calcification group(0.32±0.01)(P<0.01). (4)Compared with control group(14.31±0.24 IU/L), rat aortic VSMCs CaN activity from calcification group (21.47±1.86 IU/L) increased (P<0.01). Rat aortic VSMCs CaN activity from CsA group(15.34±0.14 IU/L)decreased compared with calcification group(21.47±1.86 IU/L)(P<0.01). However compared with control group(14.31±0.24 IU/L), CaN activity of CsA group (15.34±0.14 IU/L)has a tendency to increase, but the difference is not significant(P>0.05). (5)Ca²⁺ concentration of rat aortic VSMCs from calcification group(9.01±0.32pg/ml) also increased compared with control group (6.03±0.33 pg/m) (P<0.01). Compared with control group (6.03±0.33 pg/ml), rat aortic VSMCs Ca²⁺ concentration from CsA group (10.39±0.70pg/ml)is elevated (P<0.01), CsA group(10.39±0.70 pg/ml)increased compared with calcification group(9.01±0.32pg/ml)(P<0.05).(6)ALP activity of rat aortic VSMCs from calcification group (146.04±9.02 U·g-1protein)also increased compared with control group (38.74±2.23 U·g-1protein)(P<0.01). Compared with control group(38.74±2.23U·g-1protein), rat aortic VSMCs ALP activity from CsA group (216.69±14.0U·g-1protein) iselevated (P<0.01), CsA group (216.69±14.0U·g-1protein) increased compared with calcification group (146.04±9.02U·g-1protein) (P<0.01).onclusion: (1)The present study, sodium dihydrogen phosphate successfully established a rat aortic VSMCs calcification model. (2)This study found that in rat aortic VSMCs calcification CaN mRNA, protein expression and activity was significantly increased when, PKG mRNA, protein expression was significant reduced, indicating that CaN inhibited the PKG of the calcification in VSMCs. (3) CsA may promote cell calcification.