ObjectiveTo observe the immunological regulation effects of human umbilical cord mesenchymal stem cells (hUCMSC) on glucose-damaged rhesus retinal vascular endothelial cells (RF/6A). MethodshUCMSC and RF/6A were co-culture according to 1:1 ratio in the co-culture system (Transwell plates), hUCMSC cells were added to upper chamber, while the lower chamber containing 25mmol/L glucose and RF/6A. There were three groups including RF/6A blank control group, high glucose treated RF/6A group, and high glucose treated RF/6A with hUCMSC co-culture group. MTT was used to measure the RF/6A cell viability. Western blot was used to to detect protein level of Foxp3. Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration of interleukin (IL)-17. ResultsMTT assay revealed that at the first day, the survival rate of the three groups had no significant difference (F=0.030, P > 0.05). On day 3 and day 7, the cell viability of the high glucose group was significantly lower than that of the control group (t=36.072, 27.890; P < 0.05), the cell viability of the high glucose treated RF/6A with hUCMSC co-culture group was higher than that of high glucose group (t=36.072, 19.650; P < 0.05).Western blot analysis showed that Foxp3 in high glucose RF/6A group was significantly lower than that in the control group at day 7 after culture (t=7.826, P < 0.05) and high glucose RF/6A with hUCMSC group (t=19.936, P < 0.05). ELISA showed that IL-17 in the high glucose group, high glucose with hUCMSC co-culture group was significantly higher than that of the control group (F=1 267.503, P < 0.05), while IL-17 in the hUCMSC co-culture group was significantly lower than that in high glucose group (t=17.386, P < 0.05). ConclusionhUCMSC can regulate the expression of Foxp3 and IL-17 to increase the proliferative ability of RF/6A, which was suppressed by high glucose.
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.
OBJECTIVE: To explore the possibility of detergent acellularized porcine heart valve serving as a scaffold for tissue engineering valve. METHODS: The porcine aortic valves were acellularized by use of trypsin-EDTA. Triton X-100, RNase and DNase treatment. Biomechanical characteristics of fresh valves and acellularized valve were tested; also fresh valves, acellularized valve and valves treated with method of bioprothetic treatment were implanted subcutaneously in rats; frequently seeded with bovine aortic endothelial cells(BAECs), and then cultured for 7 days. RESULTS: The acellularization procedure resulted in complete removal of the cellular components while the construction of matrix was maintained. The matrix could be successfully seeded with in vitro expanded BAECs, which formed a continuous monolayer on the surface. There is no significant difference of PGI2 secretion of BAECs between cells seeded onto the acellular leaflets and that onto the wells of 24-wells plate (P gt; 0.05). CONCLUSION: Acellularied porcine aortic valve can be applied as a scaffold to develop tissue engineering heart valve.
Objective To investigate the effect of small interfering RNA(siRNA) targeting hypoxia inducible factor1alpha; (HIF1alpha;) and vascular endothelial growth factor (VEGF) on expression of VEGF in human vascular endothelial cells. Methods HIF-1alpha; siRNA recombinant plasmid was constructed. Human vascular ndothelial cells were cultured in vitro and divided into normoxia group (20% O2) and hypoxia group (1% O2). Hypoxia group was then divided into control group, vector group, HIF-1alpha; group (HIF-1alpha; siRNA), VEGF group ( VEGF165 siRNA) and cotransfection group (HIF-1alpha; siRNA+VEGF165 siRNA). LipofectamineTM 2000 (LF2000) mediated vector plasmid was transfected to cells in each group except the control group. The expression of HIF-1alpha; siRNA and VEGF165 siRNA recombinant plasmid were identified by reverse transcriptasepolymerase chain reaction (RT-PCR). The expression of VEGF mRNA and protein were detected by RTPCR and immunocytochemical method. Results The expression of HIF-1alpha; siRNA and VEGF165 si RNA recombinant plasmid were detected 24 hours after transfected. The expression of VEGF mRNA and protein was faint in the normoxia group, but increased obviously in hypoxia group. The expression of VEGF mRNA and protein in the HIF1alpha;, VEGF and cotransfection groups were lower than which in the control group. Cotransfection group showed the highest inhibitory effect. Conclusion HIF-1alpha; and VEGF165 siRNA can effectively inhibit the expression of VEGF in human vascular endothelial cells.
Objective To observe the effect of ginsenoside Rg3 on the proliferation, migration, and tube formation of human retinal capillary endothelial cell (HRCEC) cultured in normal and hypoxia condition. Methods HRCEC was cultured in normal condition and treated with 0.0 mmol/L (group A), 0.1 mmol/L (group B) and 0.5 mmol/L (group C) ginsenoside Rg3. HRCEC was also cultured in hypoxia condition and treated with 0.0 mmol/L (group D), 0.1 mmol/L (group E) and 0.5 mmol/L (group F) ginsenoside Rg3. The effects of ginsenoside Rg3 on HRCEC proliferation were measured by methylthiazoletrazolium assay in 24, 48 and 72 hours after culture. In 24 hours after culture, the effect of cell migration was evaluated by transwell chamber; the effect of tube formation was evaluated by Matrigel; the expression of vascular endothelial growth factor (VEGF) protein and mRNA were detected by Western blot and real-time quantitative reverse transcription-polymerase chain reaction. Results Ginsenoside Rg3 could inhibit proliferation of HRCEC, depending on the concentration (F=30.331 and 33.402 in normal and hypoxia condition, respectively; P＜0.05) and time (F=85.462 and 136.045 in normal and hypoxia condition, respectively; P＜0.05). The number of cell migration was 103.33plusmn;3.54, 92..25plusmn;3.68, 78.64plusmn;4.66 in group A, B and C, the difference among three groups was statistically significant (F=28.801, P＜0.05). The number of cell migration was 125.76plusmn;3.11, 90.27plusmn;3.55, 77.81plusmn;5.01 in group D, E and F, the difference among three groups was statistically significant (F=117.594, P＜0.05). The number of tube formed in Matrigel was 24.3plusmn;2.2, 15.7plusmn;1.7, 10.1plusmn;2.3 in group A, B and C, the difference among three groups was statistically significant (F=35.364, P＜0.05). The number of tube formed in Matrigel was 26.2plusmn;1.9, 15.1plusmn;2.6, 8.6plusmn;1.9 in group D, E and F, the difference among three groups was statistically significant (F=50.989, P＜0.05). The expression of VEGF mRNA was 1.00plusmn;0.06, 0.79plusmn;0.06, 0.68plusmn;0.02 in group A, B and C, the difference among three groups was statistically significant (F=31.303, P＜0.05). The expression of VEGF mRNA was 3.88plusmn;0.12, 2.83plusmn;0.09, 1.15plusmn;0.05 in group D, E and F, the difference among three groups was statistically significant (F=682.668, P＜0.05). The expression of VEGF protein was 0.62plusmn;0.03, 0.41plusmn;0.02, 0.32plusmn;0.02 in group A, B and C, the difference among three groups was statistically significant (F=125.471, P＜0.05). The expression of VEGF protein was 0.91plusmn;0.03, 0.82plusmn;0.03, 0.71plusmn;0.02 in group D, E and F, the difference among three groups was statistically significant (F=41.045, P＜0.05). Conclusion Ginsenoside Rg3 can inhibit the proliferation, migration, and tube formation of HRCEC through the inhibition of VEGF expression.
ObjectiveTo explore repressive effects of transthyretitin (TTR) on the growth of human retinal endothelial cells (hREC) under high glucose and hypoxia environment.MethodshRECs were divided into 8 groups, including normal glucose group (5.5 mmol/L glucose), hypoxia group, high glucose group (25.0 mmol/L glucose), high glucose and hypoxia group, normal glucose group+TTR, normal glucose and hypoxia group+TTR, high glucose group+TTR, high glucose and hypoxia group+TTR. Flow cytometry was used to analyze cellular apoptosis. The expression level of Akt, p-Akt, eNOS, Bcl-2 and Bax protein were measured by Western blot.ResultsHypoxia could induce apoptosis as the apoptosis rate of normal and hypoxia group was higher than normal group (χ2=25.360, P＜0.05), high glucose and hypoxia group was higher that high glucose group (χ2=17.400, P＜0.05). The cell apoptosis rate of high glucose and hypoxia group+TTR were increased significantly as compared with high glucose and hypoxia group (χ2=9.900, P＜0.05). There was no statistically significant difference on the cell apoptosis rate between normal group and high glucose group, normal group+TTR and normal group, high glucose group+TTR and high glucose group, normal and hypoxia group+TTR and normal and hypoxia group (P＞0.05). Western blot showed that the expression of Akt did not change significantly in all eight groups（F=2.450, P＞0.05). Compared to normal group, the expression of p-Akt, eNOS, Bcl-2 in normal and hypoxia group were decreased (t=9.406, 5.306, 4.819), and the expression of Bax (t=−4.503) was increased (P＜0.05). Compared to high glucose group, same trend was found in high glucose and hypoxia group (t=8.877, 7.723, 6.500, −14.646; P＜0.05). The expression of p-Akt in normal and hypoxia group+TTR was higher than normal and hypoxia group (t=−5.024, P＜0.05) , but there was no difference on the expression of eNOS, Bcl-2, Bax between these two groups (t=−2.235, −2.656, −0.272; P＞0.05). Compared to high glucose and hypoxia group, the expression of p-Akt and Bcl-2 in high glucose and hypoxia group+TTR were decreased (t=4.355, 4.308; P＜0.05), the expression of Bax was increased (t=−4.311, P＜0.05), and there was no difference on the expression of eNOS between these two groups (t=−1.590, P＞0.05). There was no statistically significant difference in the expression of p-Akt, eNOS, Bcl-2, Bax between high glucose group and normal group (t=−3.407, −4.228, −4.302, −2.076; P＞0.05), normal group+TTR and normal group (t=−4.245, −4.298, −2.816, −1.326; P＞0.05), high glucose group+TTR and high glucose group (t=4.016, −0.784, 0.707, −0.328; P＞0.05).ConclusionUnder high glucose and hypoxia, transthyretitin suppress the growth of hREC through Akt/Bcl-2/Bax, but not Akt/eNOS signaling pathway.
ObjectiveTo observe the stoichiometry of vascular endothelial growth factor receptor 2 (VEGFR2) on the retinal vascular endothelial cell membrane by single-molecule fluorescence imaging.MethodsRhesus monkey retinal vascular endothelial cells (RF/6A) were divided into blank control group (normal culture) and plasmid transfection group [transfected with VEGFR2-green fluorescent protein (GFP) recombinant plasmid]. The expression of GFP in the plasmid transfected group was observed by confocal microscope, and the expression of VEGFR2 in the cells was detected by real-time fluorescent quantitative polymerase chain reaction (qPCR) and Western blot. The fluorescence intensity distribution and bleaching steps of single VEGFR2-GFP molecule on the cell membrane were recorded by single-molecule imaging. The distribution of fluorescence intensity and the number of fluorescence bleaching steps of GFP were recorded.ResultsGFP green fluorescence was observed in the transfected cells 12 hours after transfection. qPCR results showed that the expression of VEGFR2 and GFP mRNA in the plasmid transfected group was significantly higher than that in the blank control group (t=11.240, 12.330; P＜0.001, 0.001). Western blot results showed that the expression of VEGFR2 protein in the plasmid transfected group was significantly higher than that in the blank control group (t=8.346, P＜0.01). The results of single-molecule imaging showed that the fluorescence intensity distribution of VEGFR2-GFP on the surface of RF/6A cell membrane without ligand stimulation was bimodal, in which monomer and dimer were 86.0% and 14.0% respectively. By counting the steps of GFP fluorescence bleaching, the proportions of receptor monomer, dimer, trimer, and tetramer were 81.4%, 12.9%, 5.5%, and 0.3% respectively.ConclusionIn the absence of ligands, VEGFR2 coexists in the form of monomers and dimers on the surface of RF/6A cell membrane, and monomers are dominant.
Objective To explore the effect and mechanism of ultrashort wave (USW) for prevention and treatment of vascular crisis after rat tail replantation. Methods Eighty 3-month old female Sprague Dawley rats (weighing 232.8-289.6 g) were randomly divided into 5 groups. In each group, based on the caudal vein and the coccyx was retained, the tail was cut off. The tail artery was ligated in group A; the tail artery was anastomosed in groups B, C, D, and E to establish the tail replantation model. After surgery, the rats of group B were given normal management; the rats of group C were immediately given intraperitoneal injection (3.125 mL/kg) of diluted papaverine hydrochloride injection (1 mg/mL); the rats of groups D and E were immediately given the local USW treatment (once a day) at anastomotic site for 5 days at the dosage of 3 files and 50 mA for 20 minutes (group D) and 2 files and 28 mA for 20 minutes (group E). The survival rate of the rat tails was observed for 10 days after the tail replantation. The tail skin temperature difference between proximal and distal anastomosis was measured at pre- and post-operation; the change between postoperative and preoperative temperature difference was calculated. The blood plasma specimens were collected from the inner canthus before operation and from the tip of the tail at 8 hours after operation to measure the content of nitric oxide (NO). Results The survival rates of the rat tails were 0 (0/14), 36.4% (8/22), 57.1% (8/14), 22.2% (4/18), and 75.0% (9/12) in groups A, B, C, D, and E, respectively, showing significant overall differences among 5 groups (χ2=19.935, P=0.001); the survival rate of group E was significantly higher than that of group B at 7 days (P lt; 0.05), but no significant difference was found between the other groups by pairwise comparison (P gt; 0.05). At preoperation, there was no significant difference in tail skin temperature difference among 5 groups (P gt; 0.05); at 8 hours, 5 days, 6 days, and 7 days after operation, significant overall difference was found in the change of the skin temperature difference among groups (P lt; 0.05); pairwise comparison showed significant differences after operation (P lt; 0.05): group B gt; group D at 8 hours, group C gt; group D at 5 days, groups A, B, and C gt; group D at 6 days, groups B and C gt; groups A and E, and group B gt; group D at 7 days; but no significant difference was found between the other groups at the other time points (P gt; 0.05). Preoperative plasma NO content between each group had no significant difference (P gt; 0.05). The overall differences had significance in the NO content at postopoerative 8 hours and in the change of the NO content at pre- and post-operation among groups (P lt; 0.05). Significant differences were found by pairwise comparison (P lt; 0.05): group D gt; groups A, B, and C in the plasma NO content, group D gt; groups A and B in the change of the NO content at pre- and post-operation; but no significant difference was found between the other groups by pairwise comparison (P gt; 0.05). Conclusion Rat tail replantation model in this experiment is feasible. USW therapy can increase the survival rate of replanted rat tails, reduce skin temperature at 7 days, improve blood supply, increase the content of nitric oxide at the early period and prevent vascular crisis.
Objective To study the differentially expressed genes (DEG) during the differentiation of human induced pluripotent stem cells (hiPSC) and human embryonic stem cells (hESC) into pericytes and endothelial cells, and to identify key molecules and signaling pathways that may regulate this differentiation process. MethodshiPSC and hESC were selected and expanded using mTeSR medium. A "two-step method" was used to induce the differentiation of hiPSC and hESC into pericytes and endothelial cells. Pericytes were identified using immunofluorescence staining, while endothelial cells were isolated and identified using flow cytometry. Total RNA samples were extracted on days 0, 4, 7, and 10 of differentiation and consistently significant DEGs were screened. Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis were performed on the screened DEGs. ResultsBoth hiPSCs and hESCs successfully differentiated into pericytes and endothelial cells under induction conditions. Transcriptome sequencing results showed that with the extension of differentiation time, the DEGs in hiPSCs and hESCs were significantly upregulated or downregulated, following a generally consistent trend. During the differentiation process, marker genes for pericytes and endothelial cells were significantly upregulated. A total of 491 persistent DEGs were detected in both hiPSC and hESC, with 164 unique to hiPSCs and 335 to hESCs, while 8 DEGs were co-expressed in both cell lines. Among these, SLC30A3, LCK, TNFRSF8, PRDM14, and GLB1L3 showed sustained downregulation, whereas CLEC18C, CLEC18B, and F2RL2 exhibited sustained upregulation. GO enrichment analysis revealed that DEGs with sustained upregulation were primarily enriched in terms related to neurogenesis, differentiation, and developmental proteins, while DEGs with sustained downregulation were enriched in terms related to membrane structure and phospholipid metabolic processes. KEGG pathway analysis showed that upregulated genes were primarily enriched in cancer-related pathways, pluripotency regulatory pathways, the Wnt signaling pathway, and the Hippo signaling pathway, whereas downregulated genes were predominantly enriched in metabolism-related pathways. ConclusionsDuring the differentiation of hiPSC and hESC into pericytes and endothelial cells, 8 DEGs exhibit sustained specific expression changes. These changes may promote pericyte and endothelial cell differentiation by activating the Wnt and Hippo pathways, inhibiting metabolic pathways, releasing the maintenance of stem cell pluripotency, affecting the cell cycle, and inhibiting cell proliferation.
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.