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.
Objective To investigate the effect and potential mechanism of bone marrow mesenchymal stem cells (BMSCs) - derived extracellular vesicles (EVs) on lung tissue injury in mice with severe acute pancreatitis (SAP). Methods A total of 24 specific pathogen free grade male C57BL/6 mice and primary mouse lung microvascular endothelial cells (PMVECs) were selected. The mice were divided into sham group, SAP group, and BMSC group, with 8 mice in each group. The mouse primary PMVECs were divided into model group [sodium taurocholate (NaTC) group], BMSC-EV group, and control group. Extraction and characterization of healthy mouse BMSCs and their derived extracellular vesicles (BMSC-EVs) were conducted. A mouse model of SAP was established, and BMSC-EVs were injected into SAP mice by tail vein or intervened in PMVECs in vitro, to observe the pathological damage of pancreatic and lung tissues, the changes of serum amylase, lipase, and inflammatory factors [tumor necrosis factor α (TNF-α), interleukin-6 (IL-6)], the expression of inflammatory factors of lung tissues and PMVECs, and the endothelial cell barrier related proteins [E-cadherin, ZO-1, intercellular cell adhesion molecule-1 (ICAM-1)], and tight junctions between PMVECs to explore the effects of BMSC-EVs on pancreatic and lung tissues in SAP mice and PMVECs in vitro. Results BMSCs had the potential for osteogenic, chondrogenic, and lipogenic differentiation, and the EVs derived from them had a typical cup-shaped structure with a diameter of 60-100 nm. BMSC-EVs expressed the extracellular vesicle-positive proteins TSG101 and CD63 and did not express the negative protein Calnexin. Compared with the mice in the sham group, the SAP mice underwent significant pathological damage to the pancreas (P<0.05), and their serum amylase, lipase, inflammatory factor IL-6, and TNF-α levels were significantly up-regulated (P<0.05); whereas, BMSC-EVs markedly ameliorated the pancreatic tissue damage in the SAP mice (P<0.05), down-regulated the levels of peripheral serum amylase, lipase, IL-6 and TNF-α (P<0.05), and up-regulated the level of anti-inflammatory factor IL-10 (P<0.05). In addition to this, the SAP mice showed significant lung histopathological damage (P<0.05), higher neutrophils and macrophages infiltration (P<0.05), higher levels of the inflammatory factors TGF-β and IL-6 (P<0.05), as well as reduced barrier protein E-cadherin, ZO-1 expression and elevated expression of ICAM-1 (P<0.05). BMSC-EVs significantly ameliorated lung histopathological injury, inflammatory cells infiltration, inflammatory factor levels, and expression of barrier proteins, and suppressed ICAM-1 expression (P<0.05). In the in vitro PMVECs experiments, it was found that intercellular tight junctions were broken in the NaTC group, and the levels of inflammatory factors TNF-α and IL-6 were significantly up-regulated (P<0.05), the protein expression of E-cadherin and ZO-1 was significantly down-regulated (P<0.05), and the expression of ICAM-1 was significantly up-regulated (P<0.05). BMSC-EVs significantly improved intercellular tight junctions in the NaTC group and inhibited the secretion of TNF-α and IL-6 (P<0.05), up-regulated the expression of the barrier proteins E-cadherin and ZO-1, and down-regulated the expression of ICAM-1 (P<0.05). Conclusion BMSC-derived EVs ameliorate lung tissue injury in SAP mice by restoring the lung endothelial cell barrier and inhibiting inflammatory cell infiltration.
Coronary atherosclerotic heart disease is a serious threat to human life and health. In recent years, the main treatment for it is to implant the intravascular stent into the lesion to support blood vessels and reconstruct blood supply. However, a large number of experimental results showed that mechanical injury and anti-proliferative drugs caused great damage after stent implantation, and increased in-stent restenosis and late thrombosis risk. Thus, maintaining the integrity and normal function of the endothelium can significantly reduce the rate of thrombosis and restenosis. Stem cell mobilization, homing, differentiation and proliferation are the main mechanisms of endothelial repair after vascular stent implantation. Vascular factor and mechanical microenvironmental changes in implanted sites have a certain effect on re-endothelialization. In this paper, the process of injury caused by stent implantation, the repair mechanism after injury and its influencing factors are expounded in detail. And repairing strategies are analyzed and summarized. This review provides a reference for overcoming the in-stent restenosis, endothelialization delay and late thrombosis during the interventional treatment, as well as for designing drug-eluting and biodegradation stents.
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 relationship of cluster of differentiation 40L (CD40L) between inflammatory response mediated by vascular endothelial injury and Stanford A type aortic dissection (STAAD). Methods In this study from August 2016 to February 2017, a total of 215 blood samples from 95 STAAD patients (67 males and 28 females aged 48.33±12.19 years) and 120 healthy volunteers (94 males and 26 females aged 48.64±10.13 years) were collected. The patients with aortic dissection were taken blood 1 hour before the operation and the healthy volunteers were taken blood from the elbow vein. All STAAD patients were diagnozed by computed tomography angiography (CTA) and patients with Marfan syndrome were excluded. Blood samples were tested by enzyme-linked immunosorbent assay (ELISA) for CD40L, vascular cell adhesion molecule (VCAM-1), E-selectin, interleukin-1 (IL-1) beta, IL-6, tumor necrosis factor-alpha (TNF-α) and so on. ResultsCompared with the healthy population, the level of SCD40L(26.87±5.50 ng/ml vs. 13.39±4.03 ng/ml, P<0.001) in the STAAD patients was significantly higher. E-Selectin (116.62±25.24 ng/ml vs. 77.05±14.30 ng/ml, P<0.001), VCAM-1 (P<0.001), TNF-α (55.35±9.12 ng/ml vs. 37.33±5.61 pg/ml, P<0.001), IL-1β (62.12±13.37 ng/ml vs. 48.68±9.86 pg/ml, P<0.001), IL-6 (499.54±90.45 ng/ml vs. 422.44±34.00 pg/ml, P<0.001) significantly increased. Conclusion The increased expression of SCD40L in STAAD patients and the inflammatory reaction induced by endothelial injury in aortic dissection patients are obvious.
Objective To observe the effect of metformin (Met) on inflammatory bodies and focal death in human retinal microvascular endothelial cells (hRMEC) in diabetes mellitus (DM) microenvironment. MethodsExperimental research was divided into in vivo animal experiment and in vitro cell experiment. In vivo animal experiments: 9 healthy C57BL/6J male mice were randomly divided into DM group, normal control group, and DM+Met group, with 3 mice in each group. DM group and DM+Met group mice were induced by streptozotocin to establish DM model, and DM+Met group was given Met 400 mg/ (kg · d) intervention. Eight weeks after modeling, the expression of NLRP3, cleaved-membrane perforating protein D (GSDMD) and cleaved-Caspase-1 in the retina of mice in the normal control group, DM group and DM+Met group were observed by immunohistochemical staining. In vitro cell experiments: hRMEC was divided into conventional culture cell group (N group), advanced glycation end products (AGE) group, and AGE+Met group. Joining the AGE, AGE+Met groups cells were induced by 150 μg/ml of glycation end products, and 2.0 mmol/L Met was added to the AGE+Met group. Pyroptosis was detected by flow cytometry; 2',7'-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe was used to detect the expression of reactive oxygen species (ROS) in cells of each group. Real-time fluorescence quantitative polymerase chain reaction and Western blot were used to detect the relative mRNA and protein expression levels of NLRP3, cleaved-GSDMD, cleaved-Caspase-1 in each group of cells. Single factor analysis of variance was used for comparison among the three groups. ResultsIn vivo animal experiments: compared with the DM group, the expression of NLRP3, cleaved-GSDMD, and cleaved-Caspase-1 in the retina of normal control group and DM+Met group mice was significantly reduced, with significant difference among the 3 groups (F=43.478, 36.643, 24.464; P＜0.01). In vitro cell experiment and flow cytometry showed that the pyroptosis rate of AGE group was significantly higher than that of N group and AGE+Met group (F=32.598, P＜0.01). The DCFH-DA detection results showed that the intracellular ROS levels in the N group and AGE+Met group were significantly lower than those in the AGE group, with the significant difference (F=47.267, P＜0.01). The mRNA (F=51.563, 32.192, 44.473; P＜0.01) and protein levels (F=63.372, 54.463, 48.412; P＜0.01) of NLRP3, cleaved-GSDMD, and cleaved-Caspase-1 in hRMEC of the AGE+Met group were significantly reduced compared to the N group. ConclusionMet can down regulate the expression of NLRP3 inflammatory body related factors in hRMEC and inhibit pyroptosis.
Objective To observe the effects of overexpression of polypyrimidine tract binding protein-associated splicing factor (PSF) on the endoplasmic reticulum (ER) oxidative stress damage of human retinal microvascular endothelial cells (hRMEC) under high concentration of 4-hydroxynonenal (4-HNE). MethodsThe logarithmic growth phase hRMEC cultured in vitro was divided into normal group, simple 4-HNE treatment group (simple 4-HNE group), empty plasmid combined with 4-HNE treatment group (Vec+4-HNE group), and PSF high expression combined with 4-HNE treatment group (PSF+4-HNE group). In 4-HNE group, Vec+4-HNE group, and PSF+4-HNE group cell culture medium, 10 μmol/L 4-HNE was added and stimulated for 12 hours. Subsequently, the Vec+4-HNE group and PSF+4-HNE group were transfected with transfection reagent liposome 2000 into pcDNA empty bodies and pcDNA-PSF eukaryotic expression plasmids, respectively, for 24 hours. Flow cytometry was used to detect the effects of 4-HNE and PSF on cell apoptosis. The effect of PSF overexpression on the expression of reactive oxygen species (ROS) in hRMEC was detected by 2', 7'-dichlorodihydrofluorescein double Acetate probe. Western blot was used to detect ER oxide protein 1 (Ero-1), protein disulfide isomerase (PDI), C/EBP homologous transcription factor (CHOP), glucose regulatory protein (GRP) 78, protein kinase R-like ER kinase (PERK)/phosphorylated PERK (p-PERK), and Eukaryotic initiation factor (eIF) 2α/the relative expression levels of phosphorylated eIF (peIF) and activated transcription factor 4 (ATF4) proteins in hRMEC of normal group, 4-HNE group, Vec+4-HNE group, and PSF+4-HNE group. Single factor analysis of variance was performed for inter group comparison. ResultsThe apoptosis rates of the simple 4-HNE group, Vec+4-HNE group, and PSF+4-HNE group were (22.50±0.58)%, (26.93±0.55)%, and (11.70±0.17)%, respectively. The intracellular ROS expression levels were 0.23±0.03, 1.60±0.06, and 0.50±0.06, respectively. The difference in cell apoptosis rate among the three groups was statistically significant (F=24.531, P＜0.05). The expression level of ROS in the Vec+4-HNE group was significantly higher than that in the simple 4-HNE group and the PSF+4-HNE group, with a statistically significant difference (F=37.274, P＜0.05). The relative expression levels of ER Ero-1 and PDI proteins in the normal group, simple 4-HNE group, Vec+4-HNE group, and PSF+4-HNE group were 1.25±0.03, 0.45±0.03, 0.63±0.03, 1.13±0.09, and 1.00±0.10, 0.27±0.10, 0.31±0.05, and 0.80±0.06, respectively. The relative expression levels of CHOP and GRP78 proteins were 0.55±0.06, 1.13±0.09, 0.90±0.06, 0.48±0.04 and 0.48±0.04, 1.25±0.03, 1.03±0.09, 0.50±0.06, respectively. The relative expression levels of Ero-1 (F=43.164), PDI (F=36.643), CHOP (F=42.855), and GRP78 (F=45.275) proteins in four groups were compared, and the differences were statistically significant (P＜0.05). Four groups of cells ER p-pERK/pERK (F=35.755), peIF2 α/ The relative expression levels of eIF (F=38.643) and ATF4 (F=31.275) proteins were compared, and the differences were statistically significant (P＜0.05). ConclusionPSF can inhibit cell apoptosis and ROS production induced by high concentration of 4-HNE, and its mechanism is closely related to restoring the homeostasis of ER and down-regulating the activation level of PERK/eIF2α/ATF4 pathway.
Objective To explore the effect of natural hirudin on proliferation of human microvascular endothelial cells (HMVECs) and its preliminary mechanism of promoting angiogenesis. Methods Three-dimensional culture models of HMVECs were established in vitro and observed by inverted phase contrast microscopy after 24 hours of culturing. Then, the three-dimensional culture models of HMVECs were treated with different concentrations (1, 4, and 7 ATU/mL) of the natural hirudin, respectively, and Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum as control. The cell proliferations of 4 groups were detected by cell counting kit 8 (CCK-8) method at 24, 48, and 72 hours; the angiogenesis of 4 groups were observed by tube formation assay at 24 hours; the expressions of vascular endothelial growth factor (VEGF) and Notch1 of HMVECs in 4 groups were observed by immunofluorescence staining at 24 hours. Results The observation of cells in three-dimensional culture models showed that HMVECs attached to Matrigel well, and the cells formed tube structure completely after 24 hours. The results of CCK-8 test showed that the absorbance (A) value of 1 and 4 ATU/mL groups were higher than that of control group at each time point (P<0.05), andA value of 4 ATU/mL group was the highest. The A value of 7 ATU/mL group was significantly lower than those of 1 and 4 ATU/mL groups and control group (P<0.05). The tube formation assay showed that the tube structure was more in 1 and 4 ATU/mL groups than in 7 ATU/mL group and control group, and in 4 ATU/mL group than in 1 ATU/mL group, showing significant differences (P<0.05). There was no significant difference between 7 ATU/mL group and control group (P>0.05). The results of immunofluorescence staining showed that compared with control group, the Notch1 expression was higher in 1 and 4 ATU/mL groups and lower in 7 ATU/mL group; and there was significant difference between 4 and 7 ATU/mL groups and control group (P<0.05). The VEGF expression was higher in 1, 4, and 7 ATU/mL groups than in control group, in 4 ATU/mL group than in 1 and 7 ATU/mL groups, showing significant differences (P<0.05). Conclusion Natural hirudin can promote angiogenesis at low and medium concentrations, but suppress angiogenesis at high concentrations. Its mechanism may be related to the VEGF-Notch signal pathway.
Objective To observe the effect of high expression of polypyrimidine tract-binding protein-associated splicing factor (PSF) on low concentration of 4-hydroxynonenal (4-HNE) induced human retinal microvascular endothelial cells (HRMECs), and explore the possible mechanism. MethodsThe HRMECs cultured in vitro were divided into 4-HNE treated group, PSF overexpression group combined with 4-HNE group (PSF+4-HNE group), PSF overexpression+ML385 treatment combined with 4-HNE group (PSF+ML385+4-HNE group), and 4-HNE induced PSF overexpression group with LY294002 pretreatment (LY294002+4-HNE+PSF group). Cell culture medium containing 10 μmmol/L 4-HNE was added into 4-HNE treatment group, PSF+4-HNE group, PSF+ML385+4-HNE group for 12 hours to stimulate oxidative stress. 1.0 μg of pcDNA-PSF eukaryotic expression plasmid were transfected into PSF+4-HNE group and PSF+ML385+4-HNE group to achieve the overexpression of PSF. Also cells were pretreated with ML385 (5 μmol/L) for 48 hours in the PSF+ML385+4-HNE group, meanwhile within the LY294002+4-HNE+PSF group, after pretreatment with LY294002, cells were treated with plasmid transfection and 4-HNE induction. Transwell detects the migration ability of PSF to HRMECs. The effect of PSF on the lumen formation of HRMECs was detected by using Matrigel in vitro three-dimensional molding method. Flow cytometer was used to detect the effect of PSF overexpression on reactive oxygen (ROS) level in HRMECs. Protein immunoblotting was used to detect the relative expression of PSF, nuclear factor E2 related factor 2 (Nrf2), heme oxygenase-1 (HO-1) protein, and phosphoserine threonine protein kinase (pAkt) protein. The comparison between the two groups was performed using a t-test. ResultsThe number of live cells, migrating cells, and intact lumen formation in the 4-HNE treatment group and the PSF+4-HNE group were 1.70±0.06, 0.80±0.13, 24.00±0.58, 10.00±0.67, and 725.00±5.77, 318.7±12.13, respectively. There were significant differences in the number of live cells, migrating cells, and intact lumen formation between the two groups (t=12.311, 15.643, 17.346; P＜0.001). The results of flow cytometry showed that the ROS levels in the 4-HNE treatment group, PSF+4-HNE group, and PSF+ML385+4-HNE group were 816.70±16.67, 416.70±15.44, and 783.30±17.41, respectively. There were statistically significant differences between the two groups (t=16.311, 14.833, 18.442; P＜0.001). Western blot analysis showed that the relative expression levels of pAkt, Nrf2, and HO-1 proteins in HRMECs in the 4-HNE treatment group, PSF+4-HNE group and LY294002+4-HNE+PSF group were 0.08±0.01, 0.57±0.04, 0.35±0.09, 0.17±0.03, 1.10±0.06, 0.08±0.11 and 0.80±0.14, 2.50±0.07, 0.50±0.05, respectively. Compared with the PSF+4-HNE group, the relative expression of pAkt, Nrf2, and HO-1proteins in the LY294002+4-HNE+PSF group decreased significantly, with significant differences (t=17.342, 16.813, 18.794; P＜0.001). ConclusionPSF upregulates the expression of HO-1 by activating the phosphatidylinositol 3 kinase/Akt pathway and inhibits cell proliferation, migration, and lumen formation induced by low concentrations of 4-HNE.
OBJECTIVE: To determine an optimal co-culture ratio of the rabbit periosteal osteoblasts (RPOB) and rabbit renal vascular endothelial cells(RRVEC) without direct contact for future study of bone tissue engineering. METHODS: RPOB and RRVEC in the ratios of 1:0(control group), 2:1(group 1), 1:1(group 2) and 1:2(group 3) were co-cultured by six well plates and cell inserts. Four days later, the proliferation of RPOB and RRVEC were examined through cell count. Differentiated cell function was assessed by alkaline phosphatase (ALP) activity assay and 3H proline incorporation assay. RESULTS: When RPOB and RRVEC were indirectly co-cultured, the proliferation of RPOB and 3H proline incorporation was higher in group 1 than in the other experimental groups and control group (P lt; 0.05). ALP activity of RPOB was higher in group 1 than in control group and group 3 (P lt; 0.05), but there was no significant difference between group 1 and group 2 (P gt; 0.05). CONCLUSION: These results suggest that RPOB and RRVEC co-cultured in a ratio of 2:1 is optimal for future study of bone tissue engineering.