The aim of the study is to identify the effects and underlying mechanisms of visfatin on inflammation and necroptosis in vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with visfatin or pretreated with Polyinosinic acid (LOX-1 inhibitor). By using the Western blot, RT-PCR, immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), MTT and flow cytometry technique, the occurrence of inflammation and necroptosis in HUVECs were evaluated. Our results showed that 100 ng/mL visfatin significantly increased the mRNA and protein expression of monocyte chemotactic protein 1 (MCP-1) and LOX-1 after 24 hours’ treatment in HUVECs. However, pretreatment with Polyinosinic acid could significantly reduce the expression of MCP-1 compared with visfatin group. Additionally, 100 ng/mL visfatin could induce the production of necrotic features and increase the mRNA expression of BMF (one of the markers of necroptosis), while pretreating with Polyinosinic acid markedly downregulated the mRNA expression of BMF gene and promoted the cell proliferation. These results indicate that visfatin might induce inflammation and necroptosis via LOX-1 in HUVECs, suggesting that visfatin plays a central role in the development of atherosclerosis.
Objective To study the differenation of adult marrow mesenchymal stem cells(MSCs) into vascular endothelial cells in vitro and to explore inducing conditions. Methods MSCs were isolated from adult marrow mononuclear cells by attaching growth. MSCs were divided into 4 groups to induce: the cells seeded at a density of 5×103/cm2 in 2% and 15% FCS LDMEM respectively (group1 and group 2)， at a density of 5×104/cm2 in 2% and 15% FCS LDMEM respectively (group 3 and group 4); vascular endothelial growth factor(VEGF) supplemented with Bovine pituitary extract was used to induce the cell differentiation. The differentiated cells were identified by measuring surfacemarks (CD34, VEGFR2, CD31 and vWF ) on the 14th day and 21st day and performed angiogenesis in vitroon the 21st day.The cell proliferation index（PI）of different inducing conditions were measured. Results After induced in VEGF supplemented with Bovine pituitary extract, the cells of group 3 expressed the surface marks CD34, VEGFR-2, CD31 and vWF on the 14th day, the positive rates were 8.5%, 12.0%, 40.0% and 30.0% respectively, and on the 21st day the positive ratesof CD34 and VEGFR2 increased to 15.5％ and 20.0％, while the other groups did not express these marks; the induced cells of group 3 showed low proliferating state(PI was 10.4%) and formed capillary-like structure in semisolid medium. Conclusion Adult MSCs can differentiate into vascular endothelial cellsafter induced by VEGF and Bovine pituitary extract at high cell densities and low proliferatingconditions，suggesting that adult MSCs will be ideal seed cells forthe therapeutic neovascularization and tissue engineering.
Objective To study the biological behavior of osteoblast and vascular endothelial cell culture. Methods The osteoblasts and vascular endothelial cells were obtained from calvarial bone and renal cortox of 2-week rabbits respectively. The experiment were divided into group A (osteoblasts), group B (vascular endothelial cells) and group C(co-cultured osteoblasts and vascular endothelial cells). The cells were identified with cytoimmunochemical staining. The cellular biological behavior and compatibilitywere observed under inverted phase contrast microscope and with histological staining. The cells viability and alkaline phosphatase(ALP) activity were measured. Results The cytoimmunochemical staining showed that the cultured cells were osteoblasts and vascular endothelial cells .The cellular compatibility of osteoblasts and vascular endothelial cells was good. The ALP activity was higher in group C than in group A and group B(P＜0.01), and it was higher in group A than in group B(P＜0.05). In group C, the cellproliferation were increased slowly early, but fast later. Conclusion Thecellular compatibility of osteoblasts and vascular endothelial cells were good. The vascular endothelial cells can significantly increased the osteoblast viability and ALP activity,and the combined cultured cells have greater proliferation ability.
ObjectiveTo investigate the mechanism of G protein coupled receptor kinase interacting protein 1 (GIT1) affecting angiogenesis by comparing the differentiation of bone marrow mesenchymal stem cells (BMSCs) differentiated into endothelial cells between GIT1 wild type mice and GIT1 gene knockout mice.MethodsMale and female GIT1 heterozygous mice were paired breeding, and the genotypic identification of newborn mice were detected by PCR. The 2nd generation BMSCs isolated from GIT1 wild type mice or GIT1 gene knockout mice were divided into 4 groups, including wild type control group (group A), wild type experimental group (group A1), GIT1 knockout control group (group B), and GIT1 knockout experimental group (group B1). The cells of groups A1 and B1 were cultured with the endothelial induction medium and the cells of groups A and B with normal cluture medium. The expressions of vascular endothelial growth factor receptor 2 (VEGFR-2), VEGFR-3, and phospho-VEGFR-2 (pVEGFR-2), and pVEGFR-3 proteins were detected by Western blot. The endothelial cell markers [von Willebrand factor (vWF), platelet-endothelial cell adhesion molecule 1 (PECAM-1), and vascular endothelial cadherin (VE-Cadherin)] were detected by flow cytometry. The 2nd generation BMSCs of GIT1 wild type mice were divided into 4 groups according to the different culture media: group Ⅰ, primary cell culture medium; group Ⅱ, cell culture medium containing SAR131675 (VEGFR-3 blocker); group Ⅲ, endothelial induction medium; group Ⅳ, endothelial induction medium containing SAR131675. The endothelial cell markers (vWF, PECAM-1, and VE-Cadherin) in 4 groups were also detected by flow cytometry.ResultsWestern blot results showed that there was no obviously difference in protein expressions of VEGFR-2 and pVEGFR-2 between groups; and the expressions of VEGFR-3 and pVEGFR-3 proteins in group A1 were obviously higher than those in groups A, B, and B1. The flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group A1 than in groups A, B, and B1 (P<0.05), and in group B1 than in groups A and B (P<0.05); but no significant difference was found between groups A and B (P>0.05). In the VEGFR-3 blocked experiment, the flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group Ⅲ than in groupsⅠ, Ⅱ, and Ⅳ, and in group Ⅳ than in groups Ⅰ and Ⅱ (P<0.05); but no significant difference was found between groups Ⅰ and Ⅱ (P>0.05).ConclusionGIT1 mediates BMSCs of mice differentiation into endothelial cells via VEGFR-3, thereby affecting the angiogenesis.
Objective To study whether the porcine endothelial cells (PECs) lines transfected by HLA-G1 can alter the lysis mediated by human peripheral blood mononuclear cell (PBMC) and natural killer cell 92(NK-92). Methods By use of liposomes pack, the pcDNA3.0 eukaryotic expression vector carrying HLA-G1 was transfected into PECs. Using indirect immunofluorescence and RT-PCR assays, the HLA-G1 expression in PECs was detected. The alteration of the lysis mediated by PBMC and NK-92 was detected by51Cr-release assays. Results HLA-G1 expression could be detected in PECs after transfection of HLA-G1 at the levels of protein andRNA. It also could be found that the survival rate of transfected PECs was muchhigher than that of non-transfected PECs, when both of them faced the lysismediated by human PBMC and NK-92.After transfecting the expression of HLA-G1 could be found in the transfected PECs and the lysis mediated by PBMC and NK-92 to PECs decreased obviously (Plt;0.05). Conclusion The PECs- transfected by HLAG1 can decrease the NK lysis, so that it may provide us a new thought to inhibit the xeno-cell-rejection.
Objective To investigate the protocols of combined culture of human placenta-derived mesenchymal stem cells (HPMSCs) and human umbilical vein endothelial cells (HUVECs) from the same and different individuals on collagen material, to provide the. Methods Under voluntary contributions, HPMSCs were isolated and purified from human full-term placenta using collagenase IV digestion and lymphocyte separation medium, and confirmed by morphology methods and flow cytometry, and then passage 2 cells were cultured under condition of osteogenic induction. HUVECs were isolated from fresh human umbilical vein by collagenase I digestion and subcultured to purification, and cells were confirmed by immunocytochemical staining of von Willebrand factor (vWF). There were 2 groups for experiment. Passage 3 osteoblastic induced HPMSCs were co-cultured with HUVECs (1 ∶ 1) from different individuals in group A and with HUVECs from the same individual in group B on collagen hydrogel. Confocal laser scanning microscope was used to observe the cellular behavior of the cell-collagen composites at 1, 3, 5, and 7 days after culturing. Results Flow cytometry showed that HPMSCs were bly positive for CD90 and CD29, but negative for CD31, CD45, and CD34. After induction, alizarin red, alkaline phosphatase, and collagenase I staining were positive. HUVECs displayed cobble-stone morphology and stained positively for endothelial cell marker vWF. The immunofluorescent staining of CD31 showed that HUVECs in the cell-collagen composite of group B had richer layers, adhered and extended faster and better in three-dimension space than that of group A. At 7 days, the class-like microvessel lengths and the network point numbers were (6.68 ± 0.35) mm/mm2 and (17.10 ± 1.10)/mm2 in group A, and were (8.11 ± 0.62) mm/mm2 and (21.30 ± 1.41)/mm2 in group B, showing significant differences between the 2 groups (t=0.894, P=0.000; t=0.732, P=0.000). Conclusion Composite implant HPMSCs and HUVECs from the same individual on collagen hydrogel is better than HPMSCs and HUVECs from different individuals in integrity and continuity of the network and angiogenesis.
ObjectiveTo observe the changes of follistatin-like protein 1 (FSTL1) in serum of patients with proliferative diabetic retinopathy (PDR).MethodsTwenty PDR patients confirmed by clinical examination and 20 normal people were included in the study. Human retinal vascular endothelial cells (HRCEC) were divided into HRCEC blank control group, 3 h hypoxia group, 6 h hypoxia group. Human umbilical vein endothelial cell (HUVEC) were divided into HUVEC blank control group, 3h hypoxia group, 6h hypoxia group. Real-time quantitative PCR (RT-PCR) and ELISA were used to determine the expression of FSTL1, TGF-β, VEGF, connective tissue growth factor (CTGF) mRNA and protein in peripheral blood and cells of all groups from all subjects.ResultsThe expressions of FSTL1, TGF-β1, CTGF, VEGF mRNA in blood samples of patients with PDR were 1.79±0.58, 0.97±0.21, 1.85±0.69 and 1.38±0.44. The expressions of FSTL1, TGF-β1 protein were 1.19±0.50, 0.71±0.24 ng/ml and 734.03±116.45, 649.36±44.23 ng/L. Compared with normal people, the differences were statistically significant (tmRNA=0.90, 0.21, 2.85, 1.77; P=0.00, 0.00, 0.04, 0.02. tprotein=1.88, 7.68; P=0.00, 0.02). The cell viability of HRCEC cells in the 3 h hypoxia group and the 6 h hypoxia group were 0.66±0.05 and 0.64±0.04, respectively. Compared with the blank control group, the difference was statistically significant (F=13.02, P=0.00). The cell viability of HUVEC cells in the 3 h hypoxia group and the 6 h hypoxia group were 0.63±0.06 and 0.68±0.06, respectively. Compared with the blank control group, the difference was statistically significant (F=26.52, P=0.00). Comparison of FSTL1, TGF-β1, CTGF, and VEGF mRNA expression in HRCEC blank control group and 3 h hypoxia group, the differences were statistically significant (F=14.75, 44.93, 85.54, 6.23; P=0.01, 0.00, 0.00, 0.03). Compared with the HRCEC blank control and 3 h hypoxia group, the expressions of FSTL1 and TGF-β1 protein were statistically significant (P＜0.05). There was a statistically significant difference in TGF-β1 protein expression in the hypoxic 6 h group (P=0.03) and no significant difference in FSTL1 protein expression (P=0.68). Comparison of FSTL1, TGF-β1, CTGF, and VEGF mRNA expression in HUVEC blank control group and 3h hypoxia group, the differences were statistically significant (F=19.08, 25.12, 22.89, 13.07; P=0.00, 0.00, 0.00, 0.01). Immunofluorescence staining results showed that FSTL1, TGF-β1, CTGF, and VEGF proteins were positively expressed in cells in the 3h hypoxia and 6h hypoxia groups.ConclusionThe expression of FSTL1 gene and protein in serum of PDR patients was significantly higher than that of normal people.
ObjectiveTo explore the effect of Kaempferol on bone microvascular endothelial cells (BMECs) in glucocorticoid induced osteonecrosis of the femoral head (GIONFH) in vitro. MethodsBMECs were isolated from cancellous bone of femoral head or femoral neck donated voluntarily by patients with femoral neck fracture. BMECs were identified by von Willebrand factor and CD31 immunofluorescence staining and tube formation assay. The cell counting kit 8 (CCK-8) assay was used to screen the optimal concentration and the time point of dexamethasone (Dex) to inhibit the cell activity and the optimal concentration of Kaempferol to improve the inhibition of Dex. Then the BMECs were divided into 4 groups, namely, the cell group (group A), the cells treated with optimal concentration of Dex group (group B), the cells treated with optimal concentration of Dex+1 μmol/L Kaempferol group (group C), and the cells treated with optimal concentration of Dex+5 μmol/L Kaempferol group (group D). EdU assay, in vitro tube formation assay, TUNEL staining assay, Annexin Ⅴ/propidium iodide (PI) staining assay, Transwell migration assay, scratch healing assay, and Western blot assay were used to detect the effect of Kaempferol on the proliferation, tube formation, apoptosis, migration, and protein expression of BMECs treated with Dex. ResultsThe cultured cells were identified as BMECs. CCK-8 assay showed that the optimal concentration and the time point of Dex to inhibit cell activity was 300 μmol/L for 24 hours, and the optimal concentration of Kaempferol to improve the inhibitory activity of Dex was 1 μmol/L. EdU and tube formation assays showed that the cell proliferation rate, tube length, and number of branch points were significantly lower in groups B-D than in group A, and in groups B and D than in group C (P<0.05). TUNEL and Annexin V/PI staining assays showed that the rates of TUNEL positive cells and apoptotic cells were significantly higher in groups B-D than in group A, and in groups B and D than in group C (P<0.05). Scratch healing assay and Transwell migration assay showed that the scratch healing rate and the number of migration cells were significantly lower in groups B-D than in group A, and in groups B and D than in group C (P<0.05). Western blot assay demonstrated that the relative expressions of Cleaved Caspase-3 and Bax proteins were significantly higher in groups B-D than in group A, and in groups B and D than in group C (P<0.05); the relative expressions of matrix metalloproteinase 2, Cyclin D1, Cyclin E1, VEGFA, and Bcl2 proteins were significantly lower in groups B-D than in group A, and in groups B and D than in group C (P<0.05). Conclusion Kaempferol can alleviate the damage and dysfunction of BMECs in GIONFH.
ObjectiveTo prepare polyurethane (PU) microspheres and evaluate its physicochemical properties and biocompatibility for biomedical applications in vitro. MethodsThe PU microspheres were prepared by self-emulsification procedure at the emulsification rates of 1 000, 2 000, 3 000, and 4 000 r/min. The molecular structure was tested by Fourier transform infrared spectrometer and the surface and interior morphology of PU microspheres were observed by scanning electron microscopy (SEM). PU microspheres prepared at best emulsification rate were selected for the subsequent experiment. The human umbilical vein endothelial cells (HUVECs) were cultured and seeded on the materials, then cell morphology and adhesion status were observed by calcein-acetoxymethylester/pyridine iodide (Calcein-AM/PI) staining. The cells were cultured in the H-DMEM containing 10%FBS with additional 1% phenol (group A), in the extracts of PU prepared according to GB/T 16886.12 standard (group B), and in H-DMEM containing 10%FBS (group C), respectively. Cell counting kit 8 (CCK-8) assay was used to detect the cell viability. The blood compatibility experiments were used to evaluate the blood compatibility, the PU extracts as experimental group, stroke-physiological saline solution as negative control group, and distilled water as positive control group. The hemolytic rate was calculated. ResultsThe SEM results of PU microspheres at the emulsification rate of 2 000 r/min showed better morphology and size. The microstructure of the PU was rough on the surface and porous inside. The Calcein-AM/PI staining showed that the HUVECs attached to the PU tightly and nearly all cells were stained by green. CCK-8 assays demonstrated that group B and group C presented a significantly higher cell proliferative activity than group A (P<0.05), indicating low cytotoxicity of the PU. The absorbance value was 0.864±0.002 in positive control group and was 0.015±0.001 in negative control group. The hemolysis rate of the PU extracts was 0.39%±0.07% (<5%), indicating no hemolysis. ConclusionThe PU microspheres are successfully prepared by self-emulsification. The scaffold can obviously promote cell attachments and proliferation and shows low cytotoxicity and favorable blood compatibility, so it might be an ideal filler for soft tissue.
Objective To observe the effect of Nodal on the biological behavior of retinal vascular endothelial cells (RF/6A cells) in monkeys with high glucose. MethodsRF/6A cells were divided into normal group, mannitol group, high glucose group, high glucose combined with non-specific small interfering RNA treatment group (HG+NC group), high glucose combined with small interfering Nodal treatment group (HG+siNodal group). The transfection efficiency of siNodal was observed by real-time fluorescence quantitative PCR and western blot protein immunoblotting. The effect of Nodal on the proliferation of RF/6A cells was detected by thiazole blue colorimetry. The effect of Nodal on migration ability of RF/6A cells was detected by cell scratch assay. The effect of Nodal on the formation of RF/6A cell lumen was measured by Matrigel three-dimensional in vitro. The expression of extracellular signal phosphorylated regulated kinase 1/2 (pERK1/2) in RF/6A cells was detected by western blot protein immunoblotting. One-way analysis of variance was used to compare groups. ResultsCompared with HG+NC group, Nodal protein (F=33.469) and mRNA relative expression levels (F=38.191) in HG+siNodal group were significantly decreased, cell proliferation was significantly decreased (F=28.548), and cell migration ability was significantly decreased (F=24.182). The number of cell lumen formation was significantly decreased (F=52.643), and the differences were statistically significant (P＜0.05). Compared with HG+NC group, the relative expression of pERK1/2 protein in HG+siNodal group was significantly decreased, and the difference was statistically significant (F=44.462, P＜0.01). ConclusionsSilencing Nodal expression can inhibit proliferation, migration and tube formation of RF/6A cells induced by high glucose. It may act by inhibiting pERK1/2 expression.