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.
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.
Intravitreal injection of anti-VEGF drugs has gradually become the first-line treatment for diabetic retinopathy (DR). However, diabetic macular edema (DME) caused by DR blood-retinal barrier damage is less sensitive to anti-VEGF drugs.Therefore, it is necessary to find supplementary drugs or alternative drugs that can effectively protect the structure of the blood vessel wall. Melatonin is a hormone mainly secreted by the pineal gland, which can play a number of functions in the human body such as regulating biological rhythms, scavenging free radicals, and anti-inflammatory. In recent years, studies have shown that melatonin can improve neuronal degeneration and protect blood vessel structure through multiple mechanisms in retinopathy. In terms of its protective effect on the retinal capillary structure, melatonin can improve the damage of early DR endothelial cells and pericytes through anti-oxidative stress, anti-inflammatory, and inhibiting cell apoptosis so as to protect the integrity of the blood-retinal barrier structure. It suggests that melatonin may provide new ideas for the prevention and treatment of DR, especially with DME.
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.
Objective To observe the expression of vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 in hypoxic chorioretinal endothelial cells of monkeys (RF/6A), and to evaluate the effect of minocycline. Methods RF/6A was cultured and divided into four groups: control group, hypoxia group, hypoxia and low dose of minocycline group (0.5 mu;mol/L), hypoxia and medium dose of minocycline group (5 mu;mol/L), and hypoxia and high dose of minocycline group (50 mu;mol/L). Real-time reverse transcriptionpolymerase chain reaction (RT-PCR) and immunohistopathological staining were used to measure the mRNA and protein expression of VEGFR-1 and VEGFR-2, respectively. Results RT-PCR showed that the expression of VEGFR-1 mRNA did not vary significantly between groups (F 24 h=0.17,F 48 h=1.53,F72 h=2.04;P＞0.05). Compared with hypoxia group, the expression of VEGFR-2 mRNA in all minocycline treated groups were significantly downregulated (low minocycline, medium minocycline, high minocycline: t=4.69, 20.16, 17.12; P＜0.001). The immunohistopathological study showed the cells with positive staining of VEGFR-1 can be observed in all groups, and the staining was relatively weak and mainly located in cell membrane and cytoplasm. The optical density value analysis showed that the protein expression of VEGFR-1 did not vary significantly between groups at all time points（F 24 h=0.251,F 48 h=0.340,F72 h=0.589;P＞0.05）. The VEGFR-2 positive staining cells were also observed in all groups, and the staining was relatively high. Brown staining particles of VEGFR-2 were observed in the cell membrane with minor staining particles in cytoplasm. The staining density of VEGFR-2 was significantly higher in hypoxia group than control group. Compared with the hypoxia group, the protein expression of VEGFR-2 in minocycline treated groups was significantly lower（F 24 h=19.147,F 48 h=14.893,F72 h==11.984; P＜0.05). Conclusion The expression of VEGFR-2 is upregulated in RF/6A, and minocycline somewhat shows an inhibition effect.
ObjectiveTo observe the effects of Curcumin on the cellular apoptosis of rat retinal vascular endothelial cells (RRVEC) induced by high glucose.MethodsGeneration 4 cultured RRVEC were used in this experiment, and identified with anti-vWF factor antibody by immunochemistry and immunofluorescence. The RRVEC were divided into control group (5.5 mmol/L glucose), high glucose group (30 mmol/L glucose), and treatment group (30 mmol/L glucose+30 μmol/L Curcumin), respectively. Flow cytometry was used to measure the cellular reactive oxygen species (ROS) level and apoptosis. The expression intensity and location of nuclear factor (NF)-κB p65 in the cells of the three groups were detected by immunochemistory. The expression of Bcl-2 and Bax protein was detected by Western blot test.ResultsImmunostaining showed that RRVEC were positive for vWF factor. The flow cytometry showed that the cellular ROS level in treatment group was higher than that in the control group (t=8.677, P=0.000), but less than that in the high glucose group (t=40.957, P=0.000). Compared with the high glucose group, the cellular ROS level in the treatment group was decreased significantly (t=6.568, P=0.000). The cellular apoptosis were significantly different among the three groups (F=325.137, P=0.000). Compared with the high glucose group, the cellular apoptosis in the treatment group was decreased significantly (t=12.818, P=0.000). Immunochemistry showed that NF-κB p65 was expressed strongly in the cellular nuclei and cytoplasm in the high glucose group than that in the control group and the treatment group with the significant differences (t=8.322, P=0.000). Western blot results demonstrated that compared with the control group, the expression of Bcl-2 of RRVEC and Bcl-2/Bax ratio decreased (t=4.362, 6.449; P=0.005, 0.001) and Bax increased (t=3.813, P=0.009）in the high glucose group, with statistically significant differences. Compared with the high glucose group, the expression of NF-κB and Bax decreased (t=2.577, 3.059; P=0.042, 0.022) and Bcl-2/Bax ratio increased significantly (t=3.831, P=0.009) in the treatment group.ConclusionCurcumin could suppress the cellular apoptosis of RRVEC induced by high glucose. The mechanism of Curcumin protecting RRVEC may be via regulating NF-κB signal pathway.
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.
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.
ObjectiveTo investigate the effects of thrombospondin-1 active fragment (TSP-1) synthetical peptide VR-10 on proliferation and migration of rhesus choroidal-retinal endothelial (RF/6A) cell and the expressions of apoptosis relative genes in RF/6A cell. MethodsThe survival rate of RF/6A cell were detected by methyl thiazolyl tetrazolium, and migration ability was measured by transwell chamber after exposure to 1.0 μg/ml TSP-1 and synthetic peptide VR-10 (0.1, 1.0, 10.0 μg/ml) for different times (6, 12, 24, 48 hours). Caspase-3 and factor associated suicide (FAS) protein levels were measured by Western blot. The mRNA level of bcl-2 and FAS ligand (FASL) were measured by reverse transcription-polymerase chain reaction (RT-PCR). ResultsThe survival rate of RF/6A cells was determined by the treatment time and concentration of TSP-1(1.0 μg/ml) and the synthetic peptide VR-10 (0.1, 1.0, 10.0 μg/ml). The lowest survival ratio of RF/6A was 78% (P < 0.001) when cells were treated by 10 μg/ml synthetic peptide VR-10 after 48 hours. TSP-1 and synthetic peptide VR-10 could inhibit migration of RF/6A cells in transwell chamber (P < 0.001). 10.0 μg/ml synthetic peptide VR-10 had the strongest effect, 1.0 μg/ml TSP-1 was the next. Migration inhibition rate was increase with the increase of the concentration of VR-10 (P < 0.001). There was no significant differences between 0.1 μg/ml and 1.0 μg/ml VR-10 (P=0.114). Western bolt showed that RF/6A cell in control group mainly expressed the 32×103 procaspase-3 forms. To 10.0 μg/ml VR-10 treated group, it showed decreased expression of procaspase-3 (32×103) and concomitant increased expression of its shorter proapoptotic forms (20×103). Compared with control group, expression of FAS peptides were significantly increased in 10.0 μg/ml VR-10 treated group. Compared with control group, expression of FasL mRNA was significantly increased in 10.0 μg/ml VR-10 treated group(t=39.365, P=0.001), but the expression of bcl-2 mRNA was decreased(t=-67.419, P=0.000). ConclusionTSP-1 and synthetic peptide VR-10 had the ability to inhibit proliferation and migration of endothelial cell, and also induce apoptosis by increasing FAS/FASL expression and repressing bcl-2 expression.