ObjectiveTo review the research progress on the role and mechanism of matrix stiffness in regulating endothelial cell sprouting. MethodsThe related literature at home and abroad in recent years was extensively reviewed, and the behaviors of matrix stiffness related endothelial cell sprouting in different cell cultivation conditions were analyzed, and the specific molecular mechanism of matrix stiffness regulating related signal pathways in endothelial cell sprouting was elaborated. Results In two-dimensional cell cultivation condition, increase of matrix stiffness stimulates endothelial cell sprouting within a certain range. However, in three-dimensional cell cultivation condition, the detailed function of matrix stiffness in regulating endothelial cell sprouting and angiogenesis are still unclear. At present, the research of the related molecular mechanism mainly focuses on YAP/TAZ, and roles of its upstream and downstream signal molecules. Matrix stiffness can regulate endothelial cell sprouting by activating or inhibiting signal pathways to participate in vascularization. ConclusionMatrix stiffness plays a vital role in regulating endothelial cell sprouting, but its specific role and molecular mechanism in different environments remain ambiguous and need further study.
ObjectiveTo evaluate the effects of icariin on autophagy induced by low-concentration of glucocorticoid and exosome production in bone microvascular endothelial cells (BMECs).MethodsBMECs were isolated from femoral heads resected in total hip arthroplasty and then intervened with hydrocortisone of low concentration (0, 0.03, 0.06, 0.10 mg/mL), which were set as groups A, B, C, and D, respectively. On the basis of hydrocortisone intervention, 5×10?5 mol/L of icariin was added to each group (set as groups A1, B1, C1 and D1, respectively). Western blot was used to detect the expressions of microtubule-associated protein 1 light chain 3B (LC3B) and dead bone slice 1 (p62) after 24 hours. Exosomes were extracted from BMECs treated with icariin (intervention group) and without icariin (non-intervention group), and the diameter and concentration of exosomes were evaluated by nanoparticle tracking analysis technique. The total protein content of exosomes was detected by BCA method, and the expressions of proteins carried by exosomes including CD9, CD81, transforming growth factor β1 (TGF-β1), and vascular endothelial growth factor A (VEGFA) were assessed by Western blot. The BMECs were further divided into three groups: BMECs in the experimental group and the control group were co-cultured with exosomes secreted by BMECs treated with or without icariin, respectively; the blank control group was BMECs without exosome intervention. The three groups were treated with hydrocortisone and Western blot was used to detect the expressions of LC3B and p62. The scratching assay was used to detect cell migration ability; angiogenic ability of BMECs was also assessed.ResultsWith the increase of hydrocortisone concentration, the protein expression of LC3B-Ⅱ increased gradually, and the protein expression of p62 decreased gradually (P<0.01). Compared with group with same concentration of hydrocortisone, the protein expression of LC3B-Ⅱ decreased and the protein expression of p62 increased after the administration of icariin (P<0.01). The concentration of exosomes in the intervention group was significantly higher than that in the non-intervention group (t=?10.191, P=0.001); and there was no significant difference in exosome diameter and total protein content between the two groups (P>0.05). CD9 and CD81 proteins were highly expressed in the non-intervention group and the intervention group, and the relative expression ratios of VEGFA/CD9 and TGF-β1/CD9 proteins in the intervention group were significantly higher than those in the non-intervention group (P<0.01). After co-culture of exosomes, the protein expression of p62 increased in blank control group, control group, and experimental group, while the protein expression of LC3B-Ⅱ decreased. There were significant differences among groups (P<0.05). When treated with hydrocortisone for 12 and 24 hours, the scratch closure rate of the control group and experimental group was significantly higher than that of the blank control group (P<0.05), and the scratch closure rate of the experimental group was significantly higher than that of the control group (P<0.05). When treated with hydrocortisone for 4 and 8 hours, the number of lumens, number of sprouting vessels, and length of tubule branches in the experimental group and the control group were significantly greater than those in the blank control group (P<0.05); the length of tubule branches and the number of lumens in the experimental group were significantly greater than those in the control group (P<0.05).ConclusionIcariin and BMECs-derived exosomes can improve the autophagy of BMECs induced by low concentration of glucocorticoid.
Objective To investigate the role and relative mechanism of stromal cell derived factorl (SDF-1) secreted by nucleus pulposus cells (NPCs) on the proliferation of vascular endothelial cells (VECs). Methods The NPCs were isolated from the degenerated disc specimens after discectomy. NPCs at passage 1 were transfected with lentivirus-mediated SDF-1 over-expression; transfected and untransfected NPCs at passage 2 were cultured in the three-dimensional alvetex? scaffold, then they were co-cultured with HMEC-1 cells. The morphology of NPCs was observed by scanning electron microscope (SEM), and the apoptosis of HMEC-1 cells was detected by Annexin V/propidiumiodide staining after 72 hours co-culutre. The proliferation of HMEC-1 cells was detected by cell counting kit 8 at 12, 24, 48, and 72 hours in transfected group and untransfected group, respectively. ELISA was used to measure the vascular endothelial growth factor (VEGF) expression level. The virus transfection efficiency and relative Akt pathway were determined by Western blot. Results The NPCs maintained cell phenotype and secreted much extracellular matrix in three-dimensional-culture by SEM observation. In the co-culutre system, after NPCs were transfected with SDF-1 over-expression lentivirus, the proliferation of HMEC-1 cells was significantly increased, while the apoptosis was decreased obviously. The ELISA results demonstrated that the amount of VEGF was remarkably increased in the culture medium. Furthermore, SDF-1 promoted the up-regulation of phosphorylate Akt expression; after inhibition of Akt expression by GSK690693, the proliferation rate of VECs decreased significantly. Conclusion Over-expression of SDF-1 by NPCs is beneficial for VECs proliferation, which is involved in SDF-1-Akt signalling pathway.
Objective To investigate the effects of vascular endothelial growth factor C (VEGF-C) gene modified lymph nodes on promoting proliferation of lymphatic endothelial cells in the surrounding tissues. Methods Thirty-six Sprague Dawley rats, weighing 200.1-271.5 g, were randomly divided into 2 groups (n=18). After the in situ axillary lymph nodes transplantation models were established in both groups, 1.5 × 108 PFU Ad-VEGF-C-Flag and Ad-Flag were injected into the transplanted lymph nodes in experimental group and control group, respectively. At 3 days after injection, the axillary lymph nodes were harvested to observe the expression of Flag; at 1, 2, and 4 weeks after injection, the axillary lymph nodes and the surrounding tissues were harvested to observe the expression of Prxo-1 protein and to calculate the fluorescence density; at 2 and 4 weeks after injection, the absorbance (A) value of treated blood at 620 nm was calculated to observe lymphatic back-flow function improvement; the rats without treatment served as normal control group, and the rats with in situ axillary lymph nodes transplantation model served as blank control group. Results At 3 days after injection, the expression of Flag could be detected in experimental group and control group. The fluorescence density of Prox-1 protein in experimental group increased at 1, 2, and 4 weeks, and it was significantly higher than that in control group (P lt; 0.05). The A values of normal control group and blank control group were 0.539 ± 0.020 and 0.151 ± 0.007, respectively. The A values of experimental group and control group were 0.170 ± 0.011 and 0.168 ± 0.010 at 2 weeks, and 0.212 ± 0.016 and 0.197 ± 0.006 at 4 weeks, which were significantly lower than those of normal control group (P lt; 0.05), but no significant difference was found when compared with blank control group, and between the experimental group and control group (P gt; 0.05). Conclusion The VEGF-C gene modified lymph nodes can stimulate the proliferation of lymphatic endothelial cells in the surrounding tissues. However, it has no improved effect on lymphatic back-flow function in the affected limb.
ObjectiveTo explore a new strategy for constructing three-dimensional dermoid tissue in vitro by using cell sheets technology.MethodsRabbit bone marrow mesenchymal stem cells (rBMSCs) were isolated from bone marrow of New Zealand white rabbits and cultured by whole bone marrow adherent method. Human dermal fibroblasts (HDFs) were cultured and passaged in vitro. The 2nd generation rBMSCs and the 3rd generation HDFs were cultured in a culture dish for 2 weeks with cell sheets conditioned medium respectively to obtain a monolayer cell sheets. Human umbilical vein endothelial cells (HUVECs) were inoculated on rBMSCs sheet to construct pre-vascularized cell sheet. During the culture period, the morphological changes of the cell sheet were observed under an inverted phase contrast microscope. At 1, 3, 7, and 14 days, HE staining and CD31 immunofluorescence staining were performed to observe the cell distribution and microvascular network formation. The rBMSCs sheet was used as control. The pre-vascularized cell sheet (experimental group) and rBMSCs sheet (control group) cultured for 7 days were placed in the middle of two HDFs sheets, respectively, to prepare three-dimensional dermoid tissues. After 24 hours of culture, CD31 immunofluorescence staining and collagen type Ⅰ and collagen type Ⅲ immunohistochemical stainings were performed to evaluate cell distribution and collagen expression.ResultsHDFs and rBMSCs sheets were successfully prepared after 2 weeks of cell culture. After inoculation of HUVECs on rBMSCs sheet for 3 days, HUVECs could be seen to rearrange on rBMSCs sheet and forming vacuoles. The reticular structure was visible at 7 days and more obvious at 14 days. The formation of vacuoles between the cell sheets was observed by HE staining, and the vacuoles became more and more obvious, the thickness of the membranes increased significantly with time. CD31 immunofluorescence staining showed the microvascular lumen formation. However, only the thickness of rBMSCs sheet increasing was observed, with no changes in cell morphology or cavitation structure. The three-dimensional dermoid tissue observation showed that the endothelial cells in the experimental group were positive expressions, and the rBMSCs, HDFs, and HUVECs cells were arranged neatly. The endothelial cells were negative expressions and randomly arranged in the control group. The collagen type Ⅰ and collagen type Ⅲ were positive expression in the experimental group and the control group. But compared with control group, experimental group presented a " honeycomb” network connection, where the matrix was distributed regularly, and cells were arranged tightly. The difference in the expression of collagen type Ⅰ and collagen type Ⅲ between the experimental group and the control group was not significant (P>0.05).ConclusionThree-dimensional dermoid tissue is successfully constructed by using cell sheet technology. The cell matrix distribution of the pre-vascularized cell sheet constructed by HUVECs and rBMSCs sheet is relatively regular, which has the potential to form tissue engineered dermis.
Objective To investigate the effect of Nodal protein on retinal neovascularization under hypoxia. MethodsIn vivo animal experiment: 48 healthy C57BL/6J mice were randomly divided into normal group, oxygen-induced retinopathy (OIR) group, OIR+dimethyl sulfoxide (DMSO) group and OIR+SB431542 group, with 12 mice in each group. Retinal neovascularization was observed in mice at 17 days of age by retina flat mount. Counts exceeded the number of vascular endothelial nuclei in the retinal inner boundary membrane (ILM) by hematoxylin eosin staining. In vivo cell experiment: human retinal microvascular endothelial cells (hRMEC) were divided into normal group, hypoxia group, hypoxia+DMSO group and hypoxia +SB431542 group. The cell proliferation was detected by thiazolyl blue colorimetry (MTT). The effect of SB431542 on hRMEC lumen formation was detected by Matrigel three-dimensional in vitro molding method. Cell migration in hRMEC was detected by cell scratch assay. The Seahorse XFe96 Cell Energy Metabolism analyzer measured extracellular acidification rate (ECAR) of intracellular glycolysis, glycolysis reserve, and glycolysis capacity. One-way analysis of variance was used to compare groups. ResultsIn vivo animal experiment: compared with normal group, the neovascularization increased in OIR group (t=41.621, P＜0.001). Compared with OIR group, the number of vascular endothelial nuclei breaking through ILM in OIR+SB431542 group was significantly reduced, and the difference was statistically significant (F=36.183, P＜0.001). MTT test results showed that compared with normal group and hypoxia+SB431542 group, the cell proliferation of hypoxia group and hypoxia+DMSO group was significantly increased, and the difference was statistically significant (F=39.316, P＜0.01). The cell proliferation of hypoxia+SB431542 group was significantly lower than that of hypoxia+DMSO group, and the difference was statistically significant (t=26.182, P＜0.001). The number of intact lumen formation and migration cells in normal group, hypoxia group, hypoxia+DMSO group and hypoxia+SB431542 group were statistically significant (F=34.513, 41.862; P＜0.001, ＜0.01). Compared with the hypoxia+DMSO group, the number of intact lumen formation and migrating cells in the hypoxia+SB431542 group decreased significantly, and the differences were statistically significant (t=44.723, 31.178; P＜0.001, ＜0.01). The results of cell energy metabolism showed that compared with the hypoxia +DMSO group, the ECAR of intracellular glycolysis and glycolysis reserve in the hypoxia +SB431542 group was decreased, and the ECAR of glycolysis capacity was increased, with statistical significance (t=26.175, 33.623, 37.276; P＜0.05). ConclusionSB431542 can inhibit the proliferation, migration and the ability to form lumens, reduce the level of glycolysis of hRMECs cells induced by hypoxia.
ObjectiveTo observe the effect of interleukin-8 (IL-8) on the adhesion and migration of retinal vascular endothelial cells (RCEC). MethodsA cell experiment. Human RCEC (hRCEC) was divided into normal control group (N group), advanced glycation end product (AGE) treatment group (AGE group), and AGE-induced combined IL-8 antagonist SB225002 treatment group (AGE+SB group). The effect of AGE on IL-8 expression in hRCEC was observed by Western blot. The effect of SB225002 on hRCEC migration was observed by cell scratch assay. The effects of SB225002 on leukocyte adhesion and reactive oxygen species (ROS) on hRCEC were detected by flow cytometry. Student-t test was performed between the two groups. One-way analysis of variance was performed among the three groups. ResultsCompared with group N, the expression level of IL-8 in cells of AGE group was significantly increased, with statistical significance (t=25.661, P＜0.001). Compared with N group and AGE+SB group, cell mobility in AGE group was significantly increased (F=29.776), leukocyte adhesion number was significantly increased (F=38.159, 38.556), ROS expression level was significantly increased (F=22.336), and the differences were statistically significant (P＜0.05). ConclusionIL-8 antagonist SB225002 may down-regulate hRCEC adhesion and migration by inhibiting ROS expression.
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.
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.