Signaling Involved With Cbfα1in VSMC Calcification Induced By High Phosphate

【Objective】To observe the possible molecular signaling that regulates the Cbfα1on high phosphate induced vascular smooth muscle cells(VSMC)calcification. Our objective is to clarify the mechanism of AKt in VSMCcalcification by examining the phosphorylation of AKt and therelationship with Cbfα1in VSMC calcification induced by high phosphate【Methods】1. The explants derived from thoracic aorta were used for primary cultureof VSMC and identification of cells was carried on by morphologicalidentification and direct immunohistochemical staining of α-SMA,Passage3to5of VSMC were used for experiments.2. VSMC were divided into two groups:(1) normal phosphate group(Pi1.3mmol/L),(2) high phosphate group (Pi2.6mmol/L). Calcium depositionwas visualized by Alizarin stain method at day7. Cbfα1and OPN mRNAlevels were determined by Real-Time PCR. p-AKt(ser473), Cbfα1and OPNprotein expressions were semi-quantified by Western Blot.3. The p-AKt(ser473) expression of VSMC enhanced by high Phosphate wasexamined. VSMC were treated with AKt inhibitor, Wortmannin. VSMC weredivided into six groups:(1)high phosphate group (Pi2.6mmol/L),(2)high phosphate+Wortmannin（5nmol/L)，（3）high phosphate+Wortmannin（10nmol/L)，（4）high phosphate+Wortmannin（30nmol/L)，（5）highphosphate+Wortmannin（50nmol/L)，（6）high phosphate+Wortmannin（100nmol/L)，after24h, Cbfα1and OPN mRNA levels were determinedby Real-Time PCR. p-AKt(ser473), Cbfα1and OPN protein expressionswere quantified by Western Blot. All of the experiments were repeatedfor3times at least. 【Results】1. We found the VSMC was characterized by the ribbon spindle shape andshowed“hill and valley”morphological appearance under inverted phasecontrast microscope. direct immunohistochemical staining of α-SMAshowed positive staining in the VSMC.2. After7days, compared with the normal phosphate group,alizarin stainresults showed that the calcium deposition was obvious in highphosphate group； Cbfα1and OPN mRNA expression was significantlyincreased in high phosphate group by Real-Time PCR (P<0.05)；theexpression of p-AKt, Cbf α1and OPN protein was significantlyincreased in high phosphate group by Western Blot(P<0.05).3. After treated with Wortmannin24h, compared with the high phosphategroup, Alizarin stain showed that the calcium deposition was notdecreased in high phosphate+Wortmannin（5nmol/L) and high phosphate+Wortmannin(10nmol/L) group. But in high phosphate+Wortmannin(30nmol/L)group, calcium deposition was decreased slightly. Andcalcium deposition was significantly decreased in high phosphate+Wortmannin(50nmol/L) group and high phosphate+Wortmannin(100nmol/L)group. Compared with the high phosphate group, Real-Time PCR showedthat Cbf α1and OPN mRNA expression was not decreased in highphosphate+Wortmannin(5nmol/L) group and high phosphate+Wortmannin(10nmol/L) group (P>0.05)，but the Cbfα1and OPN mRNA expression wasslightly decreased in high phosphate+Wortmannin(30nmol/L) group(P<0.05), while the Cbfα1and OPN mRNA expression was significantlydecreased in high phosphate+Wortmannin(50nmol/L) group and highphosphate+Wortmannin(100nmol/L) group(P<0.05). Compared with thehigh phosphate group, Western blot showed that p-AKt(ser473), Cbfα1and OPN protein expression was not decreased in high phosphate+Wortmannin(5nmol/L) group and high phosphate+Wortmannin(10nmol/L) group (P>0.05)，but p-AKt(ser473), Cbfα1and OPN protein expressionwas slightly decreased in high phosphate+Wortmannin(30nmol/L) group(P<0.05), while the expression of this proteins was significantlydecreased in high phosphate+Wortmannin(50nmol/L) group and highphosphate+Wortmannin(100nmol/L) group(P<0.05)【Conclusion】1. High phosphate induces VSMC calcification in vitro. Cbfα1and OPNmRNA expression is increased and p-AKt(ser473),Cbfα1and OPNprotein expression is elevated in calcificated VSMC.2. Wortmannin may inhibit VSMC calcification. Meanwhile, Cbfα1and OPNmRNA expression is decreased. The protein expression ofp-AKt(ser473)、Cbfα1and OPN is also decreased.3. AKt is involved in VSMC calcification induced by high phosphate, andAKt may work by activating Cbfα1.