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.
ObjectiveTo investigate the heterotopic osteogenesis of tissue engineered bone using the co-culture system of vascular endothelial cells (VECs) and adipose-derived stem cells (ADSCs) as seed cells.MethodsThe partially deproteinized biological bone (PDPBB) was prepared by fibronectin combined with partially deproteinized bone (PDPB). The ADSCs of 18-week-old Sprague Dawley (SD) rats and VECs of cord blood of full-term pregnant SD rats were isolated and cultured. Three kinds of tissue engineered bone were constructed in vitro: PDPBB+VECs (group A), PDPBB+ADSCs (group B), PDPBB+co-cultured cells (VECs∶ADSCs was 1∶1, group C), and PDPBB was used as control group (group D). Scanning electron microscopy was performed at 10 days after cell transplantation to observe cell adhesion on scaffolds. Forty-eight 18-week-old SD rats were randomly divided into groups A, B, C, and D, with 12 rats in each group. Four kinds of scaffolds, A, B, C, and D, were implanted into the femoral muscle bags of rats in corresponding groups. The animals were killed at 2, 4, 8, and 12 weeks after operation for gross observation, HE staining and Masson staining histological observation, and the amount of bone collagen was measured quantitatively by Masson staining section.ResultsScanning electron microscopy showed that the pores were interconnected in PDPB materials, and a large number of lamellar protein crystals on the surface of PDPBB modified by fibronection were loosely attached to the surface of the scaffold. After 10 days of co-culture PDPBB and cells, a large number of cells attached to PDPBB and piled up with each other to form cell clusters in group C. Polygonal cells and spindle cells were mixed and distributed, and some cells grew along bone trabeculae to form cell layers. Gross observation showed that the granulation tissue began to grow into the material pore at 2 weeks after operation. In group C, a large number of white cartilage-like substances were gradually produced on the surface of the material after 4 weeks, and the surface of the material was uneven. At 12 weeks, the amount of blood vessels on the surface of group A increased, and the material showed consolidation; there was a little white cartilage-like material on the surface of group B, but the pore size of the material did not decrease significantly; in group D, the pore size of the material did not decrease significantly. Histological observation showed that there was no significant difference in the amount of bone collagen between groups at 2 weeks after operation (F=2.551, P=0.088); at 4, 8, and 12 weeks after operation, the amount of bone collagen in group C was significantly higher than that in other 3 groups, and that in group B was higher than that in group D (P<0.05); there was no significant difference between group A and groups B, D (P>0.05).ConclusionThe ability of heterotopic osteogenesis of tissue engineered bone constructed by co-culture VECs and ADSCs was the strongest.
To study the potential molecular mechanism of tumor angiogenesis in its microenvironment, we investigated the effects of HepG2 conditioned medium on the proliferation of vascular endothelial cell and vascular angiogenesis in our laboratory. Human umbilical vein endothelial EA.hy926 cells were co-cultured with HepG2 conditioned medium in vitro. The proliferation and the tubulogenesis of EA.hy926 cells were detected by teramethylazo salt azole (MTT) and tube formation assay, respectively. The results showed that the survival rate of the EA.hy926 cells was significantly increased under the co-culture condition. HepG2 conditioned medium also enhanced the angiogenesis ability of EA.hy926 cells. In addition, the expressions of intracellular VEGF and extracellular VEGFR (Flk-1) were regulated upward in a time-dependent manner. In conclusion, the proliferation of vascular endothelial cells and Vascula angiogenesis were improved under the condition of indirect co-culture.
Objective To investigate the effect of microRNA-22-3p (miR-22-3p) on the inflammation of human pulmonary microvascular endothelial cells (HPMEC) induced by lipopolysaccharide (LPS) by regulating the HMGB1/NLRP3 pathway. Methods miRNA microarray was taken from peripheral blood of patients with acute respiratory distress syndrome (ARDS) caused by abdominal infection and healthy controls for analysis, and the target miRNA was selected. miRNA mimics, inhibitor and their negative controls were transfected in HPMECs which were stimulated with LPS. Real time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to detect the mRNA and protein levels of high mobility group box-1 protein (HMGB1) and nucleotide binding oligomerization segment like receptor family 3 (NLRP3). RT-qPCR and enzyme linked immunosorbent assay were used to detect the levels of inflammatory factors in the cells and supernatant. Results miRNA microarray showed that miR-22-3p was down-regulated in the plasma of patients with ARDS. Compared with the negative control group, after miR-22-3p over-expression, the protein and mRNA levels of HMGB1 and NLRP3 decreased significantly. Similarly, the level of cleaved-caspase-1 decreased significantly. At the same time, interleukin (IL)-6, IL-8 and IL-1β mRNA level in cytoplasm and supernatant were down-regulated by miR-22-3p mimics. After transfected with miR-22-3p inhibitor, the expression levels of HMGB1, NLRP3, caspase-1 protein and inflammatory factors were significantly up-regulated. Conclusion miR-22-3p is significantly downregulated in peripheral blood of ARDS patients caused by abdominal infection, which can inhibit the expression of HMGB1 and NLRP3 and its downstream inflammatory response in HPMECs.
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.
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.
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.
Coronary heart disease is a kind of heart disease that is caused by atherosclerosis.The lipid deposition in the vessel wall results in occlusion of coronary artery and stenosis, which could induce myocardial ischemia and oxygen deficiency. Intervention therapies like percutaneous coronary intervention (PCI) and coronary stent improve myocardial perfusion using catheter angioplasty to reduce stenosis and occlusion of coronary artery lumen. Accordingly, intervention therapies are widely applied in clinic to treat ischemic cardiovascular disease, arterial intima hyperplasia and other heart diseases, which could save the patients′ life rapidly and effectively. However, these interventions also damage the original endothelium, promote acute and subacute thrombosis and intimal hyperplasia, and thus induce in stent restenosis (ISR) eventually. Studies indicated that the rapid reendothelialization of damaged section determined postoperative effects. In this review, reendothelialization of implants after intervention therapy is discussed, including the resource of cells contributed on injured artery, the influences of implanted stents on hemodynamic, and the effects of damaged degree on reendothelialization.
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.
ObjectiveTo investigate the effect of ADAM33 gene silencing in VSMCs on the proliferation and lumen formation of airway vascular endothelial cells (VECs) in a co-culture system and the possible regulatory mechanism. MethodsThe Human aortic smooth muscle cells (HASMCs) and human pulmonary microvascular endothelial cells (HPMECs) were used to construct a cell co-culture system. ADAM33 gene expression was silenced by lentivirus transfection technique, and the subjects were divided into endothelial cell blank group, co-culture group, co-culture +shRNA negative control group, and co-culture + ADAM33-SHRNA group. The expressions of sADAM33, VEGFA,VEGER2, ang-1 and ang-2 in co-culture system were detected by ELISA. The proliferation and lumen formation of HPMECs were observed by CCK-8 and Transwell experiments. The protein expression of Tie2, PI3K, Akt, and mTOR key molecules in Tie2/PI3K/Akt/mTOR signaling pathway and the phosphorylation levels of AKT and mTOR were detected by Western-blotting method. Results① Compared with the co-culture group (0.851±0.036) and the co-culture + shRNA negative control group (0.828±0.047), the OD value of the co-culture + ADAM33shRNA group (0.699±0.038) was significantly decreased (P<0.05). ② Compared with the co-culture group (159.169±15.740) and the co-culture +shRNA negative control group (157.357±21.612), the tube length of the co-culture +ADAM33shRNA group (120.812±2.791) was also significantly decreased (P<0.05). ③ After ADAM33 gene expression of HASMCs was silted in co-culture system, the expression levels of VEGFA, VEGFR2, ang-1 and ang-2 were significantly decreased (P<0.05), while the expression levels of Tie2, PI3K, P-Akt and P-mtor were decreased (P<0.05). ConclusionsSilencing the expression of the ADAM33 gene could reduce the release of sADAM33 from the membrane of the airway VSMCs, regulate the proliferation and lumen formation of airway VECs by reducing the expression of VEGF/VEGFR and inhibiting the activities of the Tie2/PI3K/Akt/mTOR signaling pathways,and then participate in airway vascular remodeling in asthma.
