The Role Of Advanced Oxidation Protein Products In Vascular Calcification And Its Possible Mechanisms

PARTⅠThe relationship between Advanced Oxidation Protein Products (AOPP) andvascular calcification in uremic patientsBackgroundOxidative stress is commonly observed in patients with chronic kidney diseases(CKD) and has been shown to predict overall and cardiovascular mortality. Vascularcalcification is a non-traditional risk factor which plays an important role incardiovascular diseases and it is now considered to be an actively regulated processsimilar to bone formation. Our research first to study the relationship between plasmaadvanced oxidation protein products (AOPP) and vascular calcification in uremicpatients.MethodsFifty patients with CKD stage V were included in our study and pieces of radialarteries were taken during the arteriovenous fistula operation. Ten patients withthymoma and normal renal function were chosen as control. Pieces of internalthoracic artery were taken in the operation. The vessels were examined forcalcification by alizarin red staining and for the presence of bone matrix protein(osteopontin, OPN) by immunohistochemistry. Measurements of intima-mediathickness (IMT) and wall-to-lumen ratio of radial arteries, as assessed in H.E. staining,were obtained in all these patients. Plasma AOPP and blood chemistry, includingserum calcium, phosphate, iPTH were all analyzed.ResultsPlasma AOPP levels in patients with CKD stage V was significantly higher thanthose in controls (90.22±55.88 vs. 35.79±4.31umol/L, P＜0.01). Vascularcalcification was found in the media of the vessels of 24 uremic patients (48%), whilenot in controls. OPN was expressed in the media of the vessels of 44 uremic patients(88%), but there was little staining in control vessels. OPN was observed in all the 24 vessels that were calcified, and in 20 of the 26 non-calcified vessels (76.9%). Patientswith calcified vessels showed remarkably higher levels of AOPP as compared withpatients without calcified vessels (122.52±66.9 vs. 61.29±31.23umol/L, P＜0.01).Pearson correlation analysis showed that plasma levels of AOPP were stronglyassociated with the extent of calcification (r=0.791, P＜0.001), IMT (r=0.691, P＜0.001) and wall-to-lumen ratio (r=0.354, P＜0.05) of radial arteries. The extent ofcalcification was associated with AOPP (r=0.791, P＜0.001), serum phosphate (r=0.602, P＜0.01) and serum iPTH (r=0.549, P＜0.05).ConclusionIncreased plasma AOPP and vascular calcification were found in patients withCKD stage V. Plasma levels of AOPP were closely related to vascular calcification. PARTⅡAOPP induced in vitro vascular calcification by promoting osteoblasticdifferentiation of vascular smooth musclesBackgroundIn the first part, we have demonstrated that plasma levels of AOPP were closelyrelated to vascular calcification in uremic patients. We wonder whether AOPP is onlythe marker of oxidative stress, or it promotes initiation and/or progression ofvascular calcification? Now more and more findings confirm that vascularcalcification is an actively regulated process similar to bone formation. The facts thatoxidative stress can amplify the smooth muscle cell differentiation process have beenconfirmed by several studies. The present in vitro study we are to investigate whetherAOPP promotes the osteoblastic differentiation and calcification of Human aorticsmooth muscle cells (HASMC).MethodsAOPP-HSA was prepared in vitro with human serum albumin (HSA) andhypochloric acid. We examined the effect of AOPP-HSA on the expression of OPNand core binding factorα1 (Cbfα1 ) by Real-time PCR after 72 hours in culturedHASMC in vitro, and the expression of OPN and smooth muscleαactin (SM-α-actin) by immunostaining after 14 days. The quantification of calcium deposition wasalso performed, and the calcium content of the cell layer was normalized to proteincontent.ResultsCompared with control, AOPP significantly induced the expression of cbf—α(16.03±0.45 vs. 1.06±0.43, P＜0.001) and OPN (4.62±0.34 vs. 1.06±0.43, P＜0.01) after 72 hours by Real-time PCR, while there was no differences between HSAgroup and control group. Data from Western blot assay revealed an increase in thelevel of OPN and a decrease in SM-α-actin in cells cultured in AOPP after 14 days,and HSA had no such effects. The amount of calcium deposition in AOPP group wassignificantly higher than that in control group (42±2.65 vs. 4.23±0.21 ug/mg protein,P＜0.001), and that in HSA group and control group were similar (4.3±0.35 vs. 4.23±0.21 ug/mg protein, P＞0.05).Conclusion AOPP directly increased the expression of osteoblastic differentiation markersincluding OPN and cbfα, a key transcription factor in osteoblastic differentiation,and decreased the expression of smooth muscle cells contractile phenotype markerssuch as SM-α-actin. These suggested that AOPP enhanced vascular calcification bypromoting the osteoblastic differentiation of vascular smooth muscle cells. PARTⅢAOPP enhances in vitro osteoblastic differentiation and calcification of vascularsmooth muscle cells through oxidative stress and ERK pathwayBackgroundIt is known that oxidative stress in uremia can increase AOPP formation, and AOPPaccumulation may constitute a new molecular basis for enhanced oxidative stress. Soin this part we are to investigate whether AOPP enhance osteoblastic differentiationand calcification of vascular smooth muscle cells through promoting oxidative stress.In addition, the mitogen-activated protein kinase (MAPK) pathway, especiallyERK1/2, is an important cellular signal transduction pathway in regulatingproliferation and differentiation. It was shown that adult human mesenchymal stemcell differentiation to the osteogenic or adipogenic lineage is regulated by MAPK. Sowe are further to examine whether AOPP enhances osteoblastic differentiation ofvascular smooth muscle cells via MAPK pathway.MethodsHASMC was cultured in media containing AOPP for 2h, 4h, 6h, 8h, 12h, 16h, 24h,48h, 72h respectively. 2,7 Dichlorofluorescein diacetate (2,7-DCFH-DA) is a cellpermeable dye which becomes fluorescent upon reaction with some oxidants.Intracellular oxidative stress was measured fluorometrically by monitoring theoxidation of intracellular DCFH using flow cytometer. To determine whether theAOPP effect is due to oxidative stress, we examined the effects of antioxidant VitaminE. HASMC were pretreated with Vitamin E for 24h, followed by treatment withAOPP for 6h for examination of intracellular oxidative stress, and for 14 days fordetermination of OPN expression by Western blot and calcium deposition.Immunoblotting was used to determine the expression of p44/42 MAPK,phospho-p44/42 MAPK, SAPK/JNK, phospho—SAPK/JNK, p38MAPK, phospho—p38MAPK in cultured HASMC treated with AOPP for 10min, 20min, 30min,40min, 50min, 60min respectively. In addition, HASMC were pretreated with aspecific inhibitor of ERK, PD98059, followed by treatment with AOPP for 14 daysfor determination of OPN expression by Western blot and calcium deposition.ResultsIn response to AOPP, DCF fluorescence increased in HASMC compared to control at 2h, and it reached the peak at 6h. HSA itself had no effects on the increase of DCFfluorescence. Pretreatment of Vitamin E could reduce intracellular oxidative stress at6h. Vitamin E inhibited AOPP-induced OPN expression (P＜0.01) and calciumdeposition (20±1.00 vs. 42±2.65 ug/mg protein, P＜0.01) after 14 days.Immunoblotting assays showed that AOPP activated phosphor-ERK, and the inhibitorof ERK (PD98059) inhibited both osteoblastic differentiation markers OPN (P＜0.01)and HASMC calcification (17±1.00 vs. 42±2.65 ug/mg protein, P＜0.01).ConclusionAOPP enhances in vitro osteoblastic differentiation and calcification of vascularsmooth muscle cells through oxidative stress and ERK pathway.