ObjectiveTo investigate the influence of Ataxia-telangiectasia mutated (ATM) activation on cellular oxidative stress induced by high glucose in bovine retinal capillary endothelial cells(BRECs). Methods The BRECs were treated by different culture medium with various glucose concentrations (5 mmol/L glucose, 30 mmol/L glucose, 30 mmol/L glucose+10 μmol/L KU55933) as normal glucose group, high glucose group and treatment group respectively.After the cells incubated for 48 hours, the protein expression of ATM, P-ATM, Mitogen-Activated Protein Kinase P38(P38), P-P38, Extracellular signal-regulated kinases(ERKs), P-ERKs was detected by Western blot; cellular ROS level was detected by Reactive Oxygen Species Assay Kit; propidium iodide/Hoechst staining was used for analysis of apoptosis; the expression of vascular endothelial growth factor (VEGF) in the supernatant was determined by Enzyme-Linked Immunosorbent Assay (ELISA); the paracellular permeability between endothelium cells was detected by FITC-dextran. ResultsCompared with the protein level of P-ATM, P-P38 and P-ERKs in high glucose group increased. Especially, P-P38, P-ERKs expressed much more than in high glucose group. The secretion of VEGF in high glucose group was higher than that in the normal glucose group but less than that in treatment group. The same tendency existed in ROS assay, apoptosis assay and paracellular permeability measuring. ConclusionsHigh glucose induced altered activation of ATM which might play a protective role in cellular oxidative stress. Deficiency of ATM might lead to ROS explosion, cell apoptosis and dysfunction of endothelial barrier. The mechanism might be associated with P38, ERKs and VEGF.
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.
Abstract: Objective To investigate the messenger ribonucleic acid (mRNA) expression level of tissue-type plasminogen activator (t-PA) in endothelial cells derived from adult mesenchymal stem cells (MSCs) after fluid shear stress loading which is within the physiological range. Methods After culturing in vitro, bone marrow MSCs of SD rats were seeded on slides.When it come to 80% confluence,26 slides were exposed to 5dyn/cm2 fluid shear stress for 3h in a flow chamber, and then induced to endothelial cells. Among them,13 slides constituted group Ⅰ, and the rest 13 slides set up group Ⅱ, which would be cultured for 3-4d further and passaged in 1∶3. At the same time, control group was set up, which including the cells never exposed to fluid shear stress before the endothelial differentiation. Fluid shear stress were exerting to cells in a specially made flow chamber. The expression level of t-PA mRNA of all groups were measured by real-time fluorescent quantitation reverse transcriptionpolymerase chain reaction (RTPCR). Results After endothelial differentiation for 7 days, the SD rats bone marrow MSCs acquired typical endothelial cell appearance. The t-PA mRNA expression level of group Ⅰ and group Ⅱ have an obviously enhance compared with control group(P＜0.05). The t-PA mRNA expression level of group Ⅱ step down a little (P＞0.05), but it is still significantly higher than that of control group (P＜0.05). Conclusion Fluid shear stress could provide a protective action on the endothelial cells induced from MSCs in vitro, and the effect maintains with the cells passages. This formulates a theoretical foundation to the therapeutics of atherosclerosis and selection of seed cells in vascular tissue engineering.
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 To discuss the endothelial cell which was modified by exogenous anticoagulant genes contribute to the increase of antithrombosis activity of lined vascular prosthesis and the influence to other physiological functions of endothelial cells. Methods This summarized paper was made on literature review of recent years. Results The transfection of genes, including plasminogen activator (tPA, uPA, Urokinase), thrombomoduline (TM) and hirudin, etc, to endothelial cells resulted in not only the increase of antithrombosis activity of local vascular, but also the decrease of endothelial cell function in adherence and proliferation. Conclusion The increase of antithrombosis activity of lined vascular prosthesis has been done by exogenous genes. However, this technique ought to be studied, intensively.
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 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.
ObjectiveTo observe RNA-Seq analysis of gene expression profiling in human retinal vascular endothelial cells after anti-vascular endothecial growth factor (VEGF) treatment.MethodsCultured the retinal vascular endothelial cells in vitro and logarithmic growth phase cells were used for experiments. The cells were divided into VEGF group and VEGF combined with anti-VEGF drugs group. The VEGF group cells were treated with 50 ng/ml VEGF for 72 h to simulate the high VEGF survival conditions of vascular endothelial cells in diabetic retinopathy. VEGF combined with anti-VEGF drug group cells was treated with 50 ng/ml VEGF and 2.5 μg/ml anti-VEGF drugs for 72 h to imitate the microenvironment of cells following the anti-VEGF drugs treatment, and whole transcriptome sequencing approach was applied to the above two groups of cells through RNA-Seq. Now with biological big data obtained as a basis, to analyze the differentially expressed genes (DEGs). And through enrichment analysis to explain the differential functions of DEGs and their signal pathways.ResultsThe gene expression profiles of the two groups of cells were obtained. Through analysis, 328 DEGs were found, including 194 upregulated and 133 downregulated ones. The functions of DEGs were influenced by regulations over molecular biological process, cellular energy metabolism and protein synthesis, etc. Among these genes, SI,PRX and HPGD were related to protein synthesis, BIRCT to cellular apoptosis, and ABLIM1 and CRB2 to retinal development, and ABCG1, ABCA9 and ABCA12 were associated with the cholesterol of macrophage and the transfer of phospholipid. GO enrichment analysis showed that DEGs mainly act in three ways: regulating biological behavior, organizing cellular component and performing molecular function. Pathway enrichment analysis showed that gene expressions of the two cell groups were differentiated in ECM receptor pathway, and Notch, mitogen-activated protein kinase, transforming growth factor (TGF)-β and Wnt signal pathways. Among them, the gene expression in TGF-β signal pathway attracts most attention, where the DEGs, such as CAMK2B, COL3A1, CYGB, PTGER2 and HS6ST2, among others, were closely related to fibrosis process.ConclusionThe anti-VEGF drugs may enhance the expression of CAMK2B, COL3A1, CYGB, PTGER2 and others genes related to TGF-β signal pathway and aggravate retinal fibrosis disease.
ObjectiveTo construct the connective tissue growth factor (CTGF) recombinant interference vector (shRNA) and observe its inhibitory effect on the expression of endogenous CTGF in retinal vascular endothelial cells. Methods The human CTGF shRNA was constructed and the high-titer CTGF shRNA lentivirus particles was acquired via three-plasmid lentivirus packaging system to infect retinal vascular endothelial cells. The optimal multiplicity and onset time of lentivirus infection were identified by tracing down the red florescent protein in interference vector. The cells were classified into three groups: blank control group, infection control group and CTGF knockdown group. The differences in cells migrating ability was observed through Transwell allay. The mRNA and protein expression of CTGF, fibronectin, α-smooth muscle actin (α-SMA) and collagen Ⅰ (Col Ⅰ) were quantified through real-time PCR testing and Western blot system. Data between the three groups were examined via one-way analysis of variance. ResultsThe result showed that an optimal multiplicity of 20 and onset time of 72 hours were the requirements to optimize lentivirus infection. Transwell allay result showed a contrast in the number of migrated cells in the CTGF knockdown group and that in the blank control group and infection control group (F=20.64, P=0.002). Real-time PCR testing showed a contrast in related gene expression (CTGF, fibronectin, α-SMA and Col Ⅰ) in the CTGF knocked-down group and that in the blank control group and infection control group (F=128.83, 124.44, 144.76, 1 374.44; P=0.000, 0.000, 0.000, 0.000). Western blot system showed the statistical significance of the contrasted number of related protein expression (CTGF, fibronectin, α-SMA and Col Ⅰ) in the knockdown group and that in the blank control group (F=22.55, 41.60, 25.73, 161.68; P=0.002, 0.000, 0.001, 0.000). ConclusionThe success in producing CTGF shRNA lentivirus particle suggests that CTGF shRNA lentivirus can effectively knock down CTGF 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.