Investigation On The Antagonist Role Against Vascular Calcification By C-type Natriuretic And Its Mechanism

BACKGROUND Vascular calcification is a common phenomenon in hypertension, atherosclerosis,diabetes mellitus,and is believed to be an important risk factor of cardiovascular diseases.Recent studies point out that vascular calcification is an active,regulated process,similar to osteogenesis.It was reported that some vasoactive peptides,which indirectly influenced calcium and phosphorus metabolism, are involved in regulation of calcification pathogenesis.C-type natriuretic peptide (CNP) has recently been identified as an important anabolic regulator of endochondral bone growth.CNP is produced by the endothelium and the vascular smooth muscle cells(SMC) in injured and atherosclerotic arteries,where it may exert paracrine/autocrine control over SMC by binding to its principal receptors,NPR-B and NPR-C,but few studies have examined the effect of CNP on vascular calcification.The present study carries forward by assessing the effects of CNP on vascular calcification.It's reported that supplement of CNP obviously attenuated cardiovascular calcification in vivo and in vitro,suggesting that endogenous CNP may be a target of treating vascular calcification as an endogenous prevention and cure system.However,the alterations and their significance of CNP/CNP receptor in cardiovascular calcification are unclear,and the mechanism of CNP inhibiting vascular calcification was poorly elucidated.OBJECTIVE:we developed an in vivo typical vascular calcification model treated with Vitamin D3 and nicotine to induce cardiovascular calcification of rats and in isolated culture VSMC in vitro.In these models,we attempted to demonstrate the changes of CNP/CNP receptors(NPRA,NPRB and NPRC) in vascular tissues and isolated culture VSMC and contribution of exogenous CNP to vascular calcification, and advance to approach the mechanism of CNP to inhibit vascular calcification in cultured vascular smooth muscle cell(VSMC) derived from rat thoracic aorta.METHODS:Rats received Vitamin D3(300 000 U/kg,i.m.) and nicotine(25 mg/kg,5 mL /kg in peanut oil,p.o.)(VDN group),to place Mini-osmotic pump(500ng/kg.h) bump subcutaneously on the back of VDN group rats for 4 weeks. Rats in control group received an injection of normal saline(i.m.) and two gavages of medium oil.All of the ratswere then allowed to recover for 4 weeks and given standard rodent chow.After the measurement of cardiac function the ratswere sacrificed and the calcium content in myocardium and aorta were measured.Von Kossa staining was used to detect the deposit of calcium in myocardium and aorta. Cultured smooth muscle cell(SMC) derived from rat thoracic aorta was treated withβ-glycerophosphate for 7 days,then the calcium content and deposit were measured.The CNP content and cGMP production was measured by radioimmunoassay(RIA).The CNP/NPRs mRNA levels were determined by RT-PCR,and OPN,MGP,BMP-2,cbfα-1 mRNA expression by Real Time PCR. OPN protein levels were assayed by Western blotting.Male Sprague-Dawley rats were randomly divided into three groups,(1) Control group(CON):rats in control group received an injection of normal saline(i.m.) and two gavages of peanut oil;(2) VDN group:Rats received Vitamin D3(300 000 U/kg, i.m.) and nicotine(25 mg/kg,5 mL/kg in peanut oil,p.o.) at 8 a.m.on day 1.The nicotine administration was repeated at 6 p.m.;(3) VDN+CNP:to place Mini-osmotic pump(500ng/kg.h) subcutaneously on the back of rats from VDN group for 4 weeks.Rat culture VSMCs were cultured for 7 days and replace cell culture medium every 2 days,and treated as follows:(1) Control group(CON):VSMCs were incubated cultivation in common cell culture medium;(2) Calcified VSMCs group(Cal):VSMCs culture medium were supplemented with 10mmol/Lβ-Glycerophosphate calcified medium;(3) Cal+H7 group:VSMCs were incubated cultivation with PKG inhibitor H7(1×10^-5mol/L);(4) Cal+CNP1 group:VSMCs were incubated cultivation in calcified medium with CNP(10^-9mol/L);(5) Ca7l+CNP2 group: VSMCs were incubated cultivation in calcified medium with CNP(10^-7 mol/L);(6) Cal+CNP+H7 group:VSMCs were incubated cultivation in calcified medium with CNP(10^-7 mol/L) and H7(1×10^-5mol/L);(7) CON+CNP group:VSMCs were incubated in common cell culture medium with CNP(10^-7mol/L);(8) CON+CNP+H7 group:VSMCs were incubated in common cell culture medium with CNP(10^-7 mol/L) and H7(1×10^-5 mol/L).RESULTS:1.Von Kossa staining for calcification revealed positive staining shown as black/brown spots within the main,large,nodular structures in extracellular matrix and cytoplasma.The aortic calcium content and vascular ALP activity in rats of VDN group were obviously increased by 73%and 126%(all P＜0.01),respectively, compared with the control rats.CNP concentrations in plasma and vessels of rats in VDN group were increased.Furthermore,it was found that the levels of CNP and NPR-B mRNA in calcified aorta were decreased by 16%and 95.7%,respectively, while that of NPR-B mRNA was 21-fold higher(all P＜0.05),and the expression of MGP and BMP-2 mRNA were elevated by 55.2%and 150%(all P＜0.05), respectively.Western blot indicated the OPN expression in the aorta of rats with vascular calcification was 1.4-fold higher(P＜0.01).Four weeks of CNP treatment attenuated the increase of calcium content and alkaline phosphatase activity in the aorta of rats with vascular calcification by 39.2%and 51.9%(all P＜0.01), respectively.Compared with VDN group,the levels of MGP and BMP-2 mRNA in VDN group were decreased by 35.7%and 64.8%(all P＜0.05),respectively.The OPN expression,compared with VDN group,were decreased by 44.6%(P＜0.01). CNP upregulated cyclic guanosine monophosphate(cGMP) content by 46.8%and 57.1%(aU P＜0.01) respectively,compared with the control and VDN group,which suggested CNP prevented vascular calcification in a cGMP/PKG pathway.2.The content of calcium and ALP activity was increased by 111.3%and 80.9%(all P＜0. 01),respectively,in calcified VSMCs induced byβ-glycerophosphate for 7 days. Content of CNP in cell culture fluid were increased obviously.Von Kossa staining for calcification revealed positive staining shown as black/brown areas within the main, large,nodular structures in extracellular matrix and cytoplasma.Western blot indicated VSMCs developed phenotype transformation and calcification,andα-actin protein expression obviously decreased.The mRNA levels of CNP and its receptor NPR-B were decreased by 35.3%and 64.6%(all P＜0.05),respectively,while that of NPR-C was increased by 32.7%(P＜0.05) in calcified VSMCs.OPN and BMP-2 mRNA were elevated by 92.4%and 112%(all P＜0.01),respectively,but cbfα-1 mRNA were lowered by 31.8%(P＜0.05) in calcified VSMCs.The OPN protein expression in calcified VSMCs was 1.03-fold higher(P＜0.01).Exgenous CNP(10^-9 and 10^-7M) decreased the calcium content by 40.4%and 53.4%(all P＜0.01), respectively,and ALP activity by 36.3%and 58.3%(all P＜0.01).CNP downregulated OPN mRNA and protein level in a dose-dependent manner,compared with calcified VSMCs group,with a decrease by 36.4%(P＜0.05) and 48.3%(P＜0.01),respectively,which suggested CNP significantly prevented VSMCs from developing phenotype transformation and calcification.CNP decreased the mRNA level of BMP-2 by 33.2%(P＜0.05) in calcified VSMCs,while cbfα-1 mRNA expression showed no remarkable change,suggesting CNP downregulated BMP-2 to inhibit VSMCs phenotype transformation and calcification.Administration of PKG inhibitor H7 did not show contribution to the calcium content and OPN protein expression in calcified VSMCs,but H7 obviously blocked the effect of CNP on decreasing the calcium content and OPN protein expression in calcified VSCMs, while H7 and CNP did not to affect the OPN protein expression in normal VSMCs. Real Time-PCR disclosed that OPN and BMP-2 mRNA level were increased by 51.3%and 53.8%(P＜0.05) respectively,compared with simply CNP stimulation in calcified VSCMs,and cbfα-1mRNA expression did not show significant changes, which suggested that CNP regulated OPN and BMP-2mRNA expression in a cGMP/PKG pathway.CONCLUSION:1.Exogenous CNP can inhibit vascular calcification and VSMCs phenotype transformation obviously.2.Vascular calcification may result in an alternation of vascular CNP/CNP receptor/cGMP pathway.Treatment with exogenous CNP can inhibit vascular calcification by improving vascular CNP-CNP receptor-cGMP pathway.3.Exogenous CNP may inhibit calcification and phenotype transformation by down-regulating BMP-2,the high-performance osteoinductive factor in cGMP/PKG pathway,and improve vascular structure and function, suggesting that CNP/CNP system is an important defensive and protective factor for blood vessel.Therefore endogenous vascular paracrine and/or autocrine factor CNP/ CNP receptor system may be a new target to elucidate the mechanism of vascular calcification and retrieve new strategy for preventing and curing diseases related to vascular calcification.