| Objective:A well-functioning vascular access is very important for hemodialysis patients,and Autogenous arteriovenous fistula（AVF）is the preferred choice for hemodialysis patients,but its failure ratio is high.AVF stenosis is the main cause of its failure,It can cause the decrease of blood flow,which eventually leads to thrombosis and the occlusion of AVF.There are many causes of AVF stenosis,including vascular calcification.It can increase the stiffness of AVF and cause blood pressure instability during dialysis,which lead to AVF stenosis.Calcification of radial arteries has been studied in the past,and the calcification of cephalic vein at AVF was also an important cause of AVF expansion limitation and power loss.Its exact pathophysiological mechanism is not yet very clear,but several previous experiments and clinical studies have found,vascular calcification is associated with calcium and phosphorus metabolic disorders,inflammatory reactions,apoptosis,oxidative stress and genetic disorders.Bone morphogenetic proteins bind to hydroxyapatite and collagen in bone matrix and is a highly effective calcification and bone formation inducer.It plays an important role in calcification and bone regeneration through several intracellular signaling pathways.There are several species of BMPs,of which BMP-2 is the strongest bone inducer.Wnt gene is a large family that encodes a large class of secretory glycoprotein factors,which including 19 human proteins.Wnt3a is one of them,which plays an important role in regulating cellular functions such as self-renewal,proliferation,differentiation and activity.According to its functional characteristics,the Wnt family can be divided into two categories:Wnt1 and Wnt5a.Among them,Wnt3a belongs to the class of Wnt1 and plays an important role in regulating cell function through Wnt/β-catenin signaling pathway.In this experiment,BMP-2,Wnt3a,β-catenin was detected by immunohistochemistry.At the same time,the fasting serum calcium,serum phosphorus,parathyroid hormone,alkaline phosphatase and C-reactive protein were detected.In order to investigate the relationship between AVF cephalic vein calcification and BMP-2,Wnt3a andβ-catenin.Methods:From June 2016 to June 2017,we recruit ten patients who were referred for the reconstruction of failing arteriovenous fistula and another ten patients who were referred for their first operation of AVF,they were set up experimental group and the control group respectively.When the patients in the experimental group underwent AVF reconstruction,the abandoned cephalic vein and its proximal tissue were removed about 0.5 cm each as experimental group 1 and experimental group 2.The patients in the control group were treated with AVF end-to-side anastomosis for the first time.After ligation and transection of the cephalic vein,about 0.5 cm of complete vein was removed from the distal end of the heart as the control group.we use von kossa to detect vascular calcification,Immunohistochemical to detect of BMP-2,Wnt3a andβ-catenin.At the same time,we test fasting serum calcium,serum phosphorus,parathyroid hormone i PTHN,alkaline phosphatase（ALP）and C-reactive protein（CRP）.All selected patients signed informed consent and approved by the ethics committee.All data was analyzed by statistical analysis software SPSS 21.0,and the measurement data in accordance with normal distribution are expressed as mean±standard deviation,adopt single factor variance analysis and T test for comparison of mean numbers of two samples.The data of non-normal distribution are expressed as median,and application of nonparametric test.Spearman analysis for correlation analysis,Probability values less than0.05 were required for statistical significance.Results:1 There was no significant difference in age between the experimental group and the control group（t=-0.015,p=0.988）;And no significant difference in sex between the two groups（χ2=0.22,p=0.639）.The serum Ca in the experimental group was lower than that in the control group（1.90±0.13VS2.22±0.06）,and the difference is statistically significant（t=-6.933,p=0.000）.The serum levels of P,iPTH,CRP and ALP in the experimental group were higher than those in the control group（2.41±0.36VS1.87±0.18,418.01±264.95VS213.01±105.68,4.06±4.27VS2.28±1.03,106.30±46.57VS72.10±11.45）,and the difference was statistically significant(tP=4.139,pP=0.001;tiPTH=2.273,piPTH=0.043;tCRP=1.279,pCRP=0.023;tALP=2.255,pALP=0.048).2 Von kossa staining Calcium deposit is black and its background is red.According to calcium score,Calcium deposition was observed in tunicae media vasorum of experimental group 1 and experimental group 2,and there was no significant difference between the two groups（z=-0.847,p=0.436）.The control group was weaker than the experimental group（z1=-2.202,p1=0.029;z2=-1.595,p2=0.023）.3 Immunohistochemical staining3.1 BMP-2 immunohistochemical staining The expression of BMP-2 is mainly in the cytoplasm of smooth muscle cell.The strong positive expression was found mainly in the endometrium and the media in the experimental group 1and 2,and there was no significant difference between the two groups（z=-0.746,p=0.481）.The expression of BMP-2 was weakly positive in the cephalic vein of the control group,which was located in the medial membrane and mainly in the intima.The expression level of BMP-2 in the experimental group was significantly higher than that in the control group,and the difference was statistically significant（z1=-2.387,p1=0.019;z2=-2.009,p2=0.042）.3.2 Wnt3a immunohistochemical staining The expression of Wnt3a was mainly in cytoplasm of smooth muscle cell.The strong positive expression was found mainly in the endometrium and the media in the experimental group 1and 2,and there was no significant difference between the two groups（z=-0.990,p=0.353）.The expression of Wnt3a in the cephalic vein of the control group was weakly positive,located in the inner membrane,and mainly located in the intima.The expression level of Wnt3a in the experimental group was significantly higher than that in the control group,the difference was statistically significant（z1=-3.228,p1=0.001;z2=-3.116,p2=0.002）.3.3β-catenin immunohistochemical staining The expression ofβ-catenin is mainly located in the cytoplasm of smooth muscle cell.The strong positive expression was found mainly in the endometrium and the media in the experimental group 1 and 2,and there was no significant difference between the two groups（z=-1.513,p=0.165）.The expression ofβ-catenin in the cephalic vein of the control group was weakly positive,mainly in the intima,and the expression ofβ-catenin in the experimental group was significantly higher than that in the control group（z1=-3.474,p1=0.000;z2=-3.232,p2=0.001）.3.4 Negative control with immunohistochemical staining In the negative control group,there were no positive staining in intima and middle membrane,but no positive cells in the adventitia,which were considered as nonspecific staining.4 The BMP-2,Wnt3a andβ-catenin staining scores of blood vessels gradually increased with the increase of the degree of calcification,the difference was statistically significant(zBMP-2=-2.405,p1=0.000;zWnt3a=-3.89,p2=0.001;zβ-catenin=-2.405,pβ-catenin=0.016),and the serum Ca and P level gradually increased with the increase of calcification degree（2.12±0.14VS1.98±0.22;1.91±0.22VS2.41±0.39）,and the difference was statistically significant(tCa=1.794,pCa=0.040;tP=-3.443,pP=0.005),but there was no significant difference in Ca,iPTH,CRP,ALP with the severity of calcification（p>0.05）.5 correlation analysis5.1 There was a positive correlation between vascular calcification score with serum Ca and P(rca=-0.455,pca=0.004;rP=0.821,p P=0.000);There was no correlation with iPTH and ALP and CRP and age(riPTH=0.200,pca=0.399;rALP=0.102,pALP=0.669;rCRP=0.281,pCRP=0.230).5.2 There was a positive correlation between BMP-2,Wnt3a,β-catenin staining scores and serum Ca,serum P(rBMP-2=-0.478,pBMP-2=0.033;rWnt3a=-0.652,pWnt3a=0.002;rβ-catenin=-0.660,pβ-catenin=0.002;rBMP-2=0.744,pBMP-2=0.000;rWnt3a=0.785,pWnt3a=0.000;rβ-catenin=0.730,pβ-catenin=0.000).There was no correlation with iPTH and ALP and CRP and age（p>0.05）.5.3 There was a positive correlation between Wnt3a/β-catenin staining integral and BMP-2 staining integral(rWnt3a=0.909,pWnt3a=0.00;rβ-catenin=0.05,pβ-catenin=0.00).Conclusions:1 Vascular calcification of AVF’s cephalic vein in hemodialysis patients is a process similar to bone formation.BMP-2 and Wnt3a/β-catenin signaling pathway may be involved in the occurrence of vascular calcification of the cephalic vein.2 Wnt3a/β-catenin signaling pathway can mediate BMP-2 expression and regulate the occurrence of vascular calcification together.3 The high expression of BMP-2,Wnt3a andβ-catenin is closely related to venous calcification and may be a target for future intervention. |
