ABSTRACT
Objective To investigate the mechanism that receptor activator of NF-κB ligand (RANKL) promotes arterial calcification.Methods Firstly,RANKL was added into the culture media,in which the monocyte precursor cells alone were cultured.Morphological observation and tartrate resistant acid phosphatase(TRAP)stain were used to assess whether RANKL could induce the monocyte precursor cells to differentiate into osteoclast-like cells.During arterial calcification,both in vivo and in vitro expressions of RANKL and osteoprotegerin (OPG,as RANKL inhibitor)were measured via real-time PCR.The extent of osteoclast-like cell differentiation was also assessed.Results It was found that RANKL could induce osteoclast-like cell differentiation.There were no both in vivo and in vitro expressions of osteoclast-like cells in the early stage of calcification.At that time,the ratio of RANKL to OPG was very low.In the late stage of calcification,a small amount of osteoclast-like cell expression coincided with a relatively high ratio of RANKL to OPG.According to the results,the ratio of RANKL to OPG was very low during most of the arterial calcification period.This made it possible for OPG to completely inhibit RANKL-induced osteoclast-like cell differentiation.The ratio of RANKL to OPG was (0.36 ± 0.08) (F =36) and (1.68 ± 0.08) (F =36) respectively in the early and late subgroup of calcification group in the animal model,but was zero in the control group(both P＜0.05).The ratio of RANKL to OPG was(0.42±0.09) (F=16)and(1.50 ± 0.10)(F=16)respectively in the early and late subgroup of calcification group in the cell model,but was zero in the control group(both P＜0.05).Conclusions Our result likely explains why RANKL has the ability to induce osteoclast-like cell differentiation,but acts as a promoter of calcification.
ABSTRACT
Objective To study the differentiation of bone marrow mesenchymal stem cells in artery tissue under different calcification environments.Methods We made a vascular calcification model using warfarin,vitamin K1 and vitamin D3.After the model was successfully made,we took artery tissue of normal SD rat arteries and calcified arteries co-cultured with MSC,which were divided into three groups.The normal group included normal artery tissue with MSC; calcified inducers group included calcified inducers (dexamethasone with β-glycerophosphate and ascorbic acid),normal arterial tissue and MSC; calcification group included calcified artery tissue and MSC.Each group was cultured for 3 weeks.On the 10th day of the experiment,osteoprotegerin (OPG) protein secretion was detected by ELISA.After three weeks,changes of cell morphology were observed using inverted microscope,and total protein content and alkaline phosphatase (ALP) activity were detected by ELISA.In additional,the Ror2 (receptor tyrosine kinase-like orphan receptor 2) mRNA expression was detected by using RT-PCR method.Results MSC in calcification group spontaneously proliferated and differentiated to osteoblast-like cells.Compared with normal group,calcification group showed that the total protein content,ALP activity of bone metabolism markers,OPG were significantly elevated,while Ror2 mRNA expression was significantly decreased.MSCs in calcified inducers group did not differentiate to osteoblast-like cells,and the total protein content and OPG were increased,while ALP activity had no significant difference as compared with the normal group.However,Ror2 mRNA expression was lower in calcified inducers group than in normal group,while was higher than that in calcification group.Conclusions MSCs proliferate into bone-like differentiation in vascular calcification environment and aggravate the vascular calcification.And in normal vascular with calcified inducers environment,MSCs proliferate into smooth muscle cell differentiation and rehabilitate the vascular calcification.These phenomenons may be related to the Ror2 expression in artery.
ABSTRACT
Recent studies showed that activation of Wnt/β-catenin pathway promoted the differentiation of osteoblast-like cells in the arterial calcification, but its mechanism remains unknown. In this study, the hypothesis that Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by upregulating the expression of receptor activator of NF-κB ligand (RANKL) was examined. LiCl was used to activate the Wnt/β-catenin pathway. The differentiation of osteoblast-like cells was observed by Von Kossa staining, calcium content assay, alkaline phosphatase (ALP) activity assay, and detection of osteocalcin expression. Real-time PCR was performed to detect the expression of RANKL and osteoprotegerin (OPG, the decoy receptor of RANKL) during the osteoblast-like cell differentiation. Different concentrations of OPG were added to the culture media respectively to inhibit the function of RANKL, and the change in the differentiation of osteoblast-like cells was evaluated. The results showed that when the Wnt/β-catenin pathway was activated by LiCl, the expression of RANKL was significantly increased, which coincided with the differentiation of osteoblast-like cells (P<0.05), and the OPG treatment could partly attenuate the promoting effect of Wnt/β-catenin pathway on the differentiation of osteoblast-like cells (P<0.05), but it failed to completely abolish such effect. It was concluded that activation of Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by both RANKL-dependent and RANKL-independent mechanisms.
Subject(s)
Animals , Rats , Cell Differentiation , Cells, Cultured , Osteoblasts , Metabolism , Osteogenesis , RANK Ligand , Metabolism , Signal Transduction , Wnt Signaling Pathway , beta Catenin , MetabolismABSTRACT
Objective To study the change and significance of osteoprotegerin(OPG)/receptor activator of NF κB ligand(RANKL)during the process of rat arterial smooth muscle cells (SMC) to differentiate subgroups:7 d (early) subgroup and 14 d (late) subgroup.into osteoblast-like cells.Methods The rat arterial SMCs were divided randomly into the SMC group,atorvastatin group,and osteoblast-like group.Each group was also divided into 2.The osteoblast-like group was given β-glycerophosphate and vitamin C in culture medium,the atorvastatin group was given both β-glycerophosphate,vitamin C and atorvastatin.Von Kossa staining,Ca2+ content assay,ALP activity assay and osteocalcin assayed with Western blot were used to check the level of calcification.Real-time PCR was used to check the mRNA expressions of OPG and RANKL.Results There was high expression of OPG (early 2.71 ±0.08,late 2.69 ±0.02) but no RANKL in the SMC group all the time.The expressions of OPG were increased in the early subgroup and decreased in late subgroup in the atorvastatin group (early 3.52±0.05,late 2.50±0.03) and osteoblast-like group (early 4.18±0.10,late 2.30 ± 0.11).And the expressions of RANKL were both increased in the atorvastatin group and osteoblast-like group dnring the calcification process (from 1.01 ± 0.19 to 2.40 ± 0.10,and from 1.70±0.07 to 3.22±0.11,respectively).Among the three groups,the ratio of OPG/RANKL was decreased with the increasing calcification level (F =52.93,2.33,both P＜0.05).And compared with the early subgroups,the ratio of OPG/RANKL in the late subgroups was also decreased along with the increase of calcification level in each group(F=38.71,1.74,both P＜0.05).Conclusions The ratio of OPG/RANKL has a negative correlation with the level of the osteoblast-like cells differentiation,and atorvastatin could inhibit the calcification.
ABSTRACT
Recent studies showed that activation of Wnt/β-catenin pathway promoted the differentiation of osteoblast-like cells in the arterial calcification, but its mechanism remains unknown. In this study, the hypothesis that Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by upregulating the expression of receptor activator of NF-κB ligand (RANKL) was examined. LiCl was used to activate the Wnt/β-catenin pathway. The differentiation of osteoblast-like cells was observed by Von Kossa staining, calcium content assay, alkaline phosphatase (ALP) activity assay, and detection of osteocalcin expression. Real-time PCR was performed to detect the expression of RANKL and osteoprotegerin (OPG, the decoy receptor of RANKL) during the osteoblast-like cell differentiation. Different concentrations of OPG were added to the culture media respectively to inhibit the function of RANKL, and the change in the differentiation of osteoblast-like cells was evaluated. The results showed that when the Wnt/β-catenin pathway was activated by LiCl, the expression of RANKL was significantly increased, which coincided with the differentiation of osteoblast-like cells (P<0.05), and the OPG treatment could partly attenuate the promoting effect of Wnt/β-catenin pathway on the differentiation of osteoblast-like cells (P<0.05), but it failed to completely abolish such effect. It was concluded that activation of Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by both RANKL-dependent and RANKL-independent mechanisms.
ABSTRACT
AIM:To investigate the effects of Compound Rhizoma Coptidis ( Rhizoma eoptidis,Radix suctellariae,Cortex phellodendri Chinensis,Fructus gardeniae and Radix et Rhizoma glycyrrhizae) on vascular calcification in rats treated with warfarin and vitamin D_3.METHODS: Thirty-two male SD rats were assigned randomly into control group,calcified group,Compound Rhizoma Coptidis prevented group and treated group.The later three groups were treated with warfarin,and subcutaneously injected with vitamin K_1 and vitamin D_3 for one week to induce extensive calcification of the aorta.Compound Rhizoma Coptidis was given before the first warfarin dose in prevented group and the drug was given after the modeling in the treated group.The control group was treated with normal saline.The calcification in the aorta was analyzed and osteoprotegerin (OPG) mRNA and protein were determined using histomorphometry,RT-PCR and immunohistoehemistry after 4 weeks of drug intervention.RESULTS : The results of 32 rats was analyzed compared to the control group,the area of darkly stained regions by Von Kossa staining and the level of calcium content in aorta wall increased significantly[(608.32±42.29) μg/g vs (1 139.47±230.03) μg/g,P ＜0.05].The OPGmRNA and protein decreased in aortic sections.No artery calcification could be detected in Compound Rhizoma Coptidis prevented group and a little artery calcification could be detected in Compound Rhizoma Coptidis treated group.The level of calcium content in aorta wall significantly lower[(854.77±12.99) μg/g,(875.78±27.23 ) μg/g].The expresion of OPGmRNA and the protein significanfly increased in Compound Rhizoma Coptidis prevented and treated groups.CONCLUSION:Compound Rhizo-ma Coptidis may prevent or regress the vegcular calcifiation,that seems dependent on the upregression of aortic OPG levels.
ABSTRACT
In order to investigate the origin of neointimal smooth muscle cells in transplant arteriosclerosis in rat aortic allograft, sex-mismatched bone marrow transplantation was performed from male Wistar rats to female Wistar rats. Four weeks after transplantation, the aortic transplant model was established by means of micro-surgery in rats. The recipients were divided into 4 groups: female Wistar-female Wistar aortic isografts, female SD female Wistar aortic allografts, male SD-male Wistar aortic allografts, female SD-chimera Wistar aortic allografts. Eight weeks after transplantation, aortic grafts were removed at autopsy and processed for histological evaluation and immunohistochemistry. The results indicated that excessive accumulation of alpha-SMA-positive smooth muscle cells resulted in significant neointima formation and vascular lumen stricture in rat aortic allografts. Neointima assay revealed that the neointimal area and NIA/MA ratio of transplanted artery were significantly increased in all of aortic allograft groups as compared with those in aortic isograft group (P<0.01). Neointimal smooth muscle cells were harvested from cryostat sections of aortic allograft by microdissection method. The Sry gene-specific PCR was performed, and the result showed that a distinct DNA band of 225 bp emerged in the male-male aortic allograft group and chimera aortic allograft group respectively, but not in the female-female aortic allograft group. It was suggested that recipient bone-marrow cells, as the origin of neointimal smooth muscle cells, contributed to the pathological neointimal hyperplasia of aortic allograft and transplant arteriosclerosis.
ABSTRACT
In order to investigate the origin of neointimal smooth muscle cells in transplant arteriosclerosis in rat aortic allograft, sex-mismatched bone marrow transplantation was performed from male Wistar rats to female Wistar rats. Four weeks after transplantation, the aortic transplant model was established by means of micro-surgery in rats. The recipients were divided into 4 groups: female Wistar-female Wistar aortic isografts, female SD-female Wistar aortic allografts, male SD-male Wistar aortic allografts, female SD-chimera Wistar aortic allografts. Eight weeks after transplantation, aortic grafts were removed at autopsy and processed for histological evaluation and immunohistochemistry. The results indicated that excessive accumulation of α-SMA-positive smooth muscle cells resulted in significant neointima formation and vascular lumen stricture in rat aortic allografts.Neointima assay revealed that the neointimal area and NIA/MA ratio of transplanted artery were significantly increased in all of aortic allograft groups as compared with those in aortic isograft group (P＜0.01). Neointimal smooth muscle cells were harvested from cryostat sections of aortic allograft by microdissection method. The Sry gene-specific PCR was performed, and the result showed that a distinct DNA band of 225 bp emerged in the male-male aortic allograft group and chimera aortic allograft group respectively, but not in the female-female aortic allograft group. It was suggested that recipient bone-marrow cells, as the origin of neointimal smooth muscle cells, contributed to the pathological neointimal hyperplasia of aortic allograft and transplant arteriosclerosis.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of fosinopril and valsartan on the expression of intercellular adhesion molecule-1 (ICAM-1) and nitric oxide (NO) induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells.</p><p><b>METHODS</b>The levels of NO, ICAM-1, and nitric oxide synthase (NOS) were determined using the nitrate reductase method, ELISA, immunohistochemical and image analyses.</p><p><b>RESULTS</b>The ox-LDL can significantly increase the expression of ICAM-1 and inhibit the expression of NO and NOS in a dose-dependent manner. Fosinopril and valsartan can significantly inhibit these roles of ox-LDL. The roles of fosinopril and valsartan were not significantly different.</p><p><b>CONCLUSION</b>Fosinopril and valsartan inhibit oxidized LDL-induced expression of ICAM-1 and increase the expression of NO in human umbilical vein endothelial cells, which is one of the mechanisms of antiatherosclerosis.</p>
Subject(s)
Humans , Arteriosclerosis , Cells, Cultured , Endothelium, Vascular , Chemistry , Cell Biology , Fosinopril , Pharmacology , Intercellular Adhesion Molecule-1 , Lipoproteins, LDL , Pharmacology , Nitric Oxide , Nitric Oxide Synthase , Tetrazoles , Pharmacology , Umbilical Veins , Cell Biology , Valine , Pharmacology , ValsartanABSTRACT
Objective: To construct the eurokaryotic expression vector of KCNE1 gene and express recombinant KCNE1 in HEK293 cells.Methods:Human KCNE1 gene fragment was amplified from human placenta total RNA by RT-PCR and cloned into the vector of pCR2.1 TOPO by means of T-A cloning.KCNE1 cDNA was obtained from pCR2.1-KCNE1 by restriction enzyme digestion and inserted into the same restriction site of pEGFP-N1.Thus pEGFP-N1-KCNE1 was constructed and transfected into HEK293 cells with Effectene transfection reagent.Results:The eukaryotic expression vector pEGFP-N1-KCNE1 was successfully constructed by gene cloning and recombinant method and expressed in HEK293 cells.Conclusion:The cloning of KCNE1 gene and the construction and expression of its eukaryotic expression vector may shed some light on further functional study of KCNE1 gene.
ABSTRACT
AIM: To study the effects of low density lipoproteins (LDLs) on insulin-like growth factor-1 receptor (IGF-1R), phosphorylated extracellular signal-regulated kinase (p-ERK), Bcl-2 and Bax protein expression in mouse peritoneal macrophages (MPMs). METHODS: Using the methods of immunocytochemistry and Western blotting, the expression of IGF-1R, p-ERK, Bcl-2 and Bax protein in MPMs treated with LDLs, anti-IGF-1R antibody (?-IR-3) or ERK inhibitor (PD98059) were detected. RESULTS: oxLDL significantly increased the IGF-1R protein expression in a dose and time-dependent manner. P-ERK protein expression induced by oxLDL peaked at 5 min. Moreover, oxLDL could induced ERK translocation from cytoplasm to nuclear. When given ?-IR-3, p-ERK protein expression was significantly inhibited, and ERK translocation disappeared. oxLDL increased Bcl-2 protein expression and reduced Bax expression in a dose and time-dependent manner. When given PD98059, Bcl-2 and Bax protein expression induced by oxLDL altered significantly. Native LDL had no significant effect on the expression of these four proteins. CONCLUSIONS: oxLDL may promote cultured MPMs survival at least by enhancing IGF-1R expression and ERK phosphorylation, and there may be many pathways involved in MPMs survival induced by oxLDL, Whereas native LDL had no effects on culture MPMs.