ObjectiveTo observe the effects of overexpression of S100A4 protein on retinal capillary cells and retinal ganglion cells (RGC) after retinal ischemia-reperfusion injury (RIRI). MethodsOne hundred healthy adult male C57BL/6 mice were randomly divided into normal control group (group C), RIRI group, adeno-associated virus (AAV2)-S100A4 green fluorescent protein (GFP) intravitreal injection group (group S), RIRI+AAV2-GFP intravitreal injection group (group GIR), and RIRI+AAV2-S100A4-GFP intravitreal injection group (group SIR), with 20 mice in each group. The RIRI model was established using the high intraocular pressure anterior chamber method in the RIRI, GIR and SIR groups of mice. Eyes were enucleated 3 days after modelling by over anaesthesia. The number of retinal capillary endothelial cells and pericytes in the retinal capillaries of mice in each group was observed by retinal trypsinised sections and hematoxylin-eosin and periodic acid-Schiff staining; immunofluorescence staining was used to observe endothelial cell, pericyte coverage and RGC survival; The relative expression of Toll-like receptor 4 (TLR4), p38 MAPK and nuclear factor erythroid 2-related factor 2 (NRF2) in retinal tissues was measured by Western blot. One-way analysis of variance was used to compare data between groups. ResultsThree days after modeling, the endothelial cell to pericyte ratio in group C was compared with group S and SIR, and the difference was not statistically significant (F=106.30, P＞0.05); the SIR group was compared with group RIRI and GIR, and the difference was statistically significant (F=106.30, P＜0.000 1). Comparison of endothelial cell coverage in each group, the difference was not statistically significant (F=3.44, P＞0.05); compared with the pericyte coverage in group C, the RIRI group and the GIR group were significantly lower, and the difference was statistically significant (F=62.69, P＜0.001). Compared with the RGC survival rate in group C, it was significantly lower in RIRI and GIR groups, and the difference was statistically significant (F=171.60, P＜0.000 1); compared with RIRI and GIR groups, the RGC survival rate in SIR group was significantly higher, and the difference was statistically significant (F=171.60, P＜0.000 1). The relative expression levels of TLR4, p38 and NRF2 proteins were statistically significant among all groups (F=42.65, 20.78, 11.55; P＜0.05). ConclusionsPericytes are more sensitive to ischemia than endothelial cells after retinal RIRI in mice, and early vascular cell loss is dominated by pericytes rather than endothelial cells. The overexpression of S100A4 protein protects against loss of pericytes and RGC after RIRI by inhibiting the TLR4/p38/NRF2 signaling pathway.
Objective To investigate the effect of surface propertyof different polyether-ester block copolymers［poly(ethylene glycol-terephthalate)/poly(butylene terephthalate), PEGT/PBT］ on the growth of smooth muscle cells (SMCs) and endothelial cells(ECs). Methods Three kinds of copolymers were synthesized, which were 1000-T20 (group A), 1000PEGT70/PBT30 (group B) and 600PEGT70/PBT30 (group C). The water-uptake and contact angle of three polyether-ester membranes were determined. The canine aorta smooth muscle cells and external jugular vein endothelial cells were primarily harvested, subcultured, and then identified. The proliferation of SMCs and ECs on the different polyether-ester membranes were investigated. Results The water-uptake of three copolymers arranged as the sequence of group C＜group A＜group B, and contact angle as the sequence of group C＞group A＞group B, indicating group B being more hydrophilic. However, smooth musclecells andendothelial cells grew poorly on the membrane of group B after low density seeding, but proliferated well on the membranes of group A and group C. Conclusion In contrast with more hydrophilic 1000PEGT70/PBT30, moderately hydrophilic 1000-T20 and 600PEGT70/PBT30 has better compatibility with vascular cells. The above results indicate that the vascular cells can grow well on moderately hydrophilic PEGT/PBT and that PEGT/PBT can be used in vascular tissue engineering.
ObjectiveTo observe the ultrastructural changes of vasa vasorum endothelial cells in the walls of the great saphenous vein and splenic vein, and to evaluate the effect of high hydrostatic pressure and hypoxia upon vasa vasorum endothelial cells.
MethodsThirty-four varicose great saphenous vein samples and splenic vein samples with portal hypertension were obtained, and the same number of normal great saphenous vein and splenic vein were used as the control groups. Semi-thin sections stained with HE staining vasa vasorum of the adventitia in great saphenous vein and splenic vein were observed for light microscopy. Samples were made into ultrathin-slices again. The ultrastructural changes of endothelial cells were observed under transmission electron microscopy.
ResultsIn varicose great saphenous veins and diseased splenic veins, the nuclear architecture of endothelial cells in vasa vasorum were integrity and the distribution of chromatin were normal. In some mitochondria, the trachychromatic groundplasm, undefined and ruptured cristae were found.
ConclusionUnder high hydrostatic pressure and hypoxia conditions, the ultrastructure of vasa vasorum endothelial cells between the great saphenous vein and the splenic vein may appear remodeling phenomenon, and both changes are similar.
ObjectiveTo observe the effect of pyrimidine bundle-binding protein-associated splicing factors (PSF) on the function of hypoxia-induced human retinal microvascular endothelial cells (hRMECs).MethodsA three-plasmid system was used to construct lentivirus (LV)-PSF. After LV-PSF infected hRMECs in vitro, the infection efficiency was measured by flow cytometry. Real-time quantitative PCR (RT-PCR) was used to detect the expression of PSF mRNA in hRMECs infected with LV-PSF. The experiment was divided into two parts, in vivo and in vitro. In vivo experiments: 20 healthy C57B/L6 mice at the age of postnatal 7 were randomly divided into normal group, oxygen-induced retinopathy (OIR) group, OIR+LV-Vec group, and OIR+LV-PSF group, each group has five mice. Mice in 3 groups were constructed with OIR models except the normal group and the mice in OIR group were not treated. The mice in the OIR + LV-Vec group and the OIR+LV-PSF group were injected with an empty vector (LV-Vec) or LV-PSF in the vitreous cavity, respectively. The effect of LV-PSF on the formation of retinal neovascularization (RNV) was observed then. In vitro experiments: hRMECs were divided into normal group, hypoxia group, vector group, and PSF high expression group. HRMECs in the normal group were cultured in vitro; hRMECs in the hypoxic group were restored to normal culture conditions for 3 h after 3 h of hypoxia stimulation; hRMECs in the vector group and PSF high expression group were infected with LV-Vec and LV-PSF for 48 h, and hRMECs were returned to normal culture conditions for 24 h with hypoxia stimulation for 3 h. The effect of PSF on cell proliferation was observed by MTT colorimetry. Cell scratch test and Transwell migration experiment were used to observe the effect of PSF on cell migration ability under hypoxia stimulation. RT-PCR was used to observe the mRNA expression of HIF-1α, VEGF and PSF in each group of cells.ResultsThe LV-PSF of stably expressing PSF was successfully constructed. The infection efficiency was 97% determined by flow cytometry. The level of PSF mRNA in hRMECs infected with LV-PSF was significantly increased and detected by RT-PCR. In vivo experiments: The RNV area of the mice in the OIR group and the OIR + LV-Vec group was significantly increased compared to the normal group (t=18.31, 43.71), and the RNV area of the mice in the OIR + LV-PSF group was smaller than that in the OIR group (t=11.30) and OIR + The LV-Vec group (t=15.47), and the differences were statistically significant (P＜0.05). In vitro experiments: MTT colorimetry results showed that the proliferative capacity of hRMECs in the hypoxic group was significantly enhanced compared with the normal group (t=2.57), and the proliferative capacity of hRMECs in the PSF high expression group was significantly lower than that of the normal, hypoxic, and vector groups (t=5.26, 5.46, 3.73), the differences were statistically significant (P＜0.05). The results of cell scratch test showed that the hRMECs could be stimulated by the hypoxia stimulation for 3 hours to restore the normal condition for 24 hours or 48 hours (t=8.35, 13.84; P＜0.05). Compared with the vector group, cell migration rate in the PSF-high expression group was not significant (t=10.99, 18.27, 9.75, 8.93, 26.94, 7.01; P＜0.05). Transwell experiments showed that the number of cells stained on the microporous membrane was higher in the normal group and the vector groups, while the number of cells stained in the PSF high expression group was significantly reduced (t=9.33, 6.15; P＜0.05). The results of RT-PCR showed that the mRNA expression of HIF-1α and VEGF in hRMECs in the hypoxic and vector groups increased significantly compared with the normal group (t=15.23, 21.09; P＜0.05), but no change in the mRNA expression of PSF (t=0.12, 2.15; P>0.05); compared with the hypoxia group and the vector group, the HIF-1α and VEGF mRNA expression in hRMECs in the PSF high expression group were significantly decreased (t=10.18, 13.10; P＜0.05), but the PSF mRNA expression increased (t=65.00, 85.79; P<0.05).ConclusionPSF can reduce the RNV area in OIR model mice. PSF may inhibit hypoxia-induced proliferation and migration of hRMECs through the HIF-1α/VEGF signaling pathway.
Objective To observe the effect of tetramethypyrazine (TMP) on the expression of hypoxia-related factors in human umbilical vein endothelial cells (HUVECs). Methods The second to fifth passage cultured HUVECs were divided into five groups: control group, CoCl2induced hypoxic group and 50, 100, 200 mu;mol/L TMP treatment groups. HUVECs in control group were not treated. HUVECs inCoCl2induced hypoxic group were treated with 150 mu;mol/LCoCl2for four hours. HUVECs in 50, 100, 200 mu;mol/L TMP treated groups were pretreated with 150 mu;mol/LCoCl2 for four hours, followed by treatment with 50, 100, 200 mu;mol/L TMP for eight hours. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of prolyl hydroxylase 2 (PHD2), hypoxia-induced factor-1alpha;(HIF-1alpha;) and vascular endothelial growth factor (VEGF). Protein levels of PHD2, HIF-1alpha;, and VEGF were detected using Western blot. Results Compared with the control group, theCoCl2 induced hypoxic group showed decreased mRNA and protein levels of PHD2 (t=3.734, 3.122;P＜0.05), while those of HIF-1alpha; and VEGF increased (HIF-1alpha; mRNA:t=4.589,P＜0.05; HIF-1alpha; protein:t=3.778,P＜0.05. VEGF mRNA:t=3.926,P＜0.05; VEGF protein:t=3.257,P＜0.05). Compared with theCoCl2 induced hypoxic group, 50, 100, 200 mu;mol/L TMP treated groups showed increased mRNA and protein levels of PHD2 (PHD2 mRNA: t=3.286, 3.617, 3.886;P＜0.05. PHD2 protein: t=2.813, 3.026, 3.078; P＜0.05); while those of VEGF decreased (VEGF mRNA: 50 mu;mol/L TMP: t=1.696,P＞0.05; 100 mu;mol/L TMP:t=2.974,P＜0.05; 200 mu;mol/L TMP: t=3.492,P＜0.05; VEGF protein: 50 mu;mol/L TMP: t=1.986,P＞0.05; 100 mu;mol/L TMP: t=2.976,P＜0.05; 200 mu;mol/L TMP:t=3.136,P＜0.05); although changes in HIF-1alpha;mRNA levels were not statistically significant (t=1.025, 0.726, -1.386;P＞0.05), showed a decrease in HIF-1alpha;protein levels (50 mu;mol/L TMP: t=2.056,P＞0.05; 100 mu;mol/L TMP:t=3.058,P＜0.05; 200 mu;mol/L TMP:t=3.828,P＜0.05). ConclusionIn HUVECs, TMP can upregulate the mRNA and protein expression of PHD2, while down regulating HIF-1alpha; protein expression and VEGF mRNA and protein expression under acute hypoxic conditions.
The autograft and non-autograft cannot meet the needs of clinical vascular surgery. Since there are possibilities of thrombus formation in artificial vascular grafts, the methods for deposing the graft using physical and chemical ways or simply seeding with endothelial cells cannot produce satisfactory grafts for vascular operations until now. In order to increase the anticoagulative capacity of artificial vascular graft, it is rational to use genetic engineering methods modifying the endothelial cells to make it express anticoagulative factors stably. Although seeding artificial graft with the genetically engineered endothelial cells can possibly produce a satisfactory graft for vascular surgery, some problems still need to be solved.
OBJECTIVE: To explore an ideal way of small vessel anastomosis for microsurgery. METHODS: Anastomosis of both carotid arteries were performed in 20 rabbits. One side of the arteries were anastomosed with anastomotic clips, the other side of the arteries, as comparison, were anastomosed with suture. The vessels were harvested at first and 14th day after operation and were evaluated using operating microscope, light microscope and electronic microscope. RESULTS: The average anastomotic time for suture was about 15 minutes, while for the clips was 2 to 5 minutes. There were no difference in patency between the two techniques. Endothelialization at the anastomotic sites were both completed 14 days postoperatively. However, for the anastomotic clips, there were no endothelia damage and foreign bodies formation inside the vessels. CONCLUSION: This experiment has confirmed that the anastomotic clip’s procedure provides a very safe and easy way to perform anastomosis and reduce the incidence of thromboses.
Objective To clarify that the vascular endothelial cell injury caused by obstructive sleep apnoea hypopnea syndrome (OSAHS) is partly mediated by miRNA-92a. Methods Serum miRNA-92a level was measured in patients who underwent polysomnography between January 2018 and December 2018. The correlation between miRNA-92a and OSAHS was analyzed. Meanwhile, endothelial cells were cultured in vitro, and morphological changes and JC-1 staining results of endothelial cells were observed after OSAHS serum stimulation, so as to further clarify the injury of endothelial cells. The changes of miRNA-92a target gene were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot to further clarify the mechanism of endothelial cell injury. Results Seventy-two patients received polysomnography, including 22 cases in the non-OSAHS group, 18 in the mild OSAHS group, 10 in the moderate OSAHS group, and 22 in the severe OSAHS group. Serum miRNA-92a level was significantly increased in the OSAHS patients, and it also increased with the aggravation of OSAHS severity. OSAHS serum significantly damaged endothelial cells. Endothelial cells were swollen, disordered arrangement, and unclear boundaries. JC-1 staining showed that green fluorescence was significantly enhanced compared with the control group. RT-PCR and Western blot showed that the expressions of Krüppel-like factor-2 (KLF-2), Krüppel-like factor-4 (KLF-4) and endothelial nitric oxide synthase (eNOS) were significantly decreased under OSAHS serum stimulation. Conclusion Serum miRNA-92a of OSAHS patients is significantly increased, and reduces the expression of target genes KLF-2, KLF-4 and eNOS, affects the mitochondrial function of endothelial cells, and injures endothelial cells.
Objective The observe the effects of interferon-inducible protein-10 (IP-10) on proliferation, migration and capillary tube formation of human retinal vascular endothelial cells (HREC) and human umbilical vein endothelial cells (HUVEC). Methods The chemokine receptor (CXCR3) mRNA of HREC and HUVEC were quantified by reverse transcriptase polymerase chain reaction (RT-PCR). In the presence of the different concentrations of IP-10, the difference in proliferation capacity of HREC and HUVEC were analyzed by cell counting kit-8 (CCK-8) methods. Wound scratch assay and threedimensional in vitro matrigel assay were used for measuring migration and capillary tube formation of HREC and HUVEC, respectively. Results RT-PCR revealed both HREC and HUVEC expressed CXCR3. The proliferation of HREC in the presence of IP-10 was inhibited in a dosagedependent manner (F=6.202,P＜0.05), while IP-10 showed no effect on the inhibitory rate of proliferation of HUVEC (F=1.183,P＞0.05). Wound scratch assay showed a significant reduction in the migrated distance of HREC and HUVEC under 10 ng/ml or 100 ng/ml IP-10 stimulation (F=25.373, 23.858; P＜0.05). There was no effect on the number of intact tubules formed by HREC in the presence of 10 ng/ml or 100 ng/ml IP-10. The number of intact tubules formed by HREC in the presence of 1000 ng/ml IP-10 was remarkably smaller. The difference of number of intact tubules formed by HREC among 10, 100, 1000 ng/ml IP-10 and nonintervention group was statistically significant (F=5.359,P＜0.05). Conclusion IP-10 can inhibit the proliferation, migration and capillary tube formation ability of HREC and the migration of HUVEC.
ObjectiveTo observe the MiSeq sequencing analysis results of fulvic acid (FA) intervention in hypoxia-induced human retinal microvascular endothelial cell (hRMEC) gene expression profile.MethodshRMEC were cultured in vitro and divided into the hypoxia group (hypoxia treatment) and the FA intervention group (FA intervention after hypoxia). The MTT colorimetric method was used to detect the influence of different concentrations and different modes of FA on hRMEC activity. The optimal concentration of FA was chosen. RT-PCR was used to investigated the effect of FA on hypoxia-induced intercellular adhesion molecule-1 (ICAM-1), IL-1β, IL-4, IL-6, IL-6, IL-8, IL-10, MMP-2, TNF-α, TNF-β, other inflammatory factors in hRMEC, and inflammation-related factors mRNA expression. Cells in the hypoxia group and FA intervention group in the logarithmic growth phase were collected. MiSeq sequencing technology was applyed to complete the whole transcriptome sequencing of the two groups of cells, biological data were obtained, and the differentially expressed miRNA were analyzed on this basis. Gene annotation (GO) functionally significant enrichment analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway significant enrichment analysis were used to analyze the functions and signal pathways of differential miRNAs. The expression of inflammatory factors and inflammation-related factors were compared between groups. The expression level of the corresponding miRNA in the cell was regulated by miRNA mimic, and its effect on cell function was observed, so as to judge the effect of the miRNA.ResultsDifferent concentrations and different modes of action of FA had no effect on the cell viability of hRMEC. The mRNA expression of ICAM-1, IL-1β, IL-6 and TNF-β in the hypoxia group hRMEC were significantly up-regulated compared with the normal group, and the difference was statistically significant (t=3.426, 6.011, 5.282, 6.500; P=0.027, 0.004, 0.006, 0.003); the mRNA expression of ICAM-1, IL-6, TNF-α and TNF-β in the FA intervention group hRMEC was significantly lower than that of the hypoxia group, and the difference was statistically significant (t=9.961, 3.676, 3.613, 3.387; P=0.001, 0.021, 0.023, 0.028). There were 14 differentially expressed miRNAs between the hypoxia group and the FA intervention group, of which 9 were up-regulated genes and 5 were down-regulated genes. The predicted target genes of 4 differential miRNAs (hsa-miR-1285-3p, hsa-miR-30d-3p, hsa-miR-3170, hsa-miR-7976) were all ICAM-1. The results of significant enrichment analysis of GO function showed that the functions of differential genes were mainly enriched in the process of cell development, cell differentiation and single organism development. Significant enrichment analysis of the KEGG pathway showed that the differential miRNA expression was highly enriched in the proteoglycan pathway and the cytokine-cytokine receptor interaction pathway in cancer, and the arachidonic acid metabolism pathway and the amphetamine pathway were the more obvious differential expressions.ConclusionFA may affect the expression level of downstream ICAM-1 mRNA by regulating the expression of multiple miRNAs, thereby affecting the inflammatory state of cells after hypoxia-stimulated hRMEC.
