Human Umbilical Cells Research Articles

Differences in anti-endothelial and anti-retinal antibody titers: implications for the pathohysiology of acute and chronic central serous chorioretinopathy.

The aim of the study was to evaluate the prevalence of serum anti-retinal (ARAs) and anti-endothelial cell antibodies (ACEAs) in patients with acute and chronic central serous chorioretinopathy (CSC). We enrolled 28 patients with acute CSC, 42 patients with chronic CSC, and 40 healthy controls. The presence of ARAs was determined by indirect immunofluorescence using monkey retina as an antigen substrate, while the presence of AECAs was determined using cultivated human umbilical vein endothelial cells (HUVECs) and primate skeletal muscle according to the manufacturer's instructions (Euroimmun AG). There were no differences in the prevalence of antibodies against rods, cones, cytoplasmic components of retinal nuclear layer cells, and retinal vessels between the acute and chronic CSC groups and the control group (P = 0.27, P = 0.16, P = 0.71, and P = 0.06, respectively). However, AECAs reactive with HUVECs were observed in 46% of patients with acute CSC, 45% of those with chronic CSC, and 22% of controls, whereas AECAs reactive with the skeletal muscle were present in 46%, 45%, and 15%, respectively (difference between groups: P = 0.045 for HUVECs and P = 0.005 for the skeletal muscle). Furthermore, AECA titers were higher in CSC patients than in controls (P = 0.004). This study provides evidence for the possible involvement of an autoimmune process directed against vessel antigens in the pathogenesis of CSC. AECAs may be more important than ARAs in this disease and may be involved in endothelial damage in the choroidal vessels and choriocapillaris, leading to hyperpermeability, which is central to the pathophysiology of CSC.

Circ-100338 induces angiogenesis after myocardial ischemia-reperfusion injury by sponging miR-200a-3p.

To investigate the effect of circular RNA circ-100338 on angiogenesis of human umbilical vein endothelial cells (HUVEC) cells after hypoxia/reoxygenation (H/R) and its molecular mechanism. We evaluated the role of circ-100338 in coronary artery endothelial cells using human coronary endothelial cells (HCAEC). Then, we verified the function of circ-100338 in HUVEC cells through cell counting kit-8 (CCK8), scratch test, Tube forming experiment, 5-Ethynyl-2'-deoxyuridine (EdU) staining. Dual-Luciferase reporter gene experiment and RNA Pull-Down experiments were used to detect the binding effect of circ-100338 and miR-200a-3p, miR-200a-3p and FUS. QRT-PCR results showed that the expression of circ-100338 decreased in HCAEC after H/R treatment. Overexpression of circ-100338 promotes angiogenesis. The Dual-Luciferase reporter gene assay and RNA pull-down assay consistently indicated the specific binding effect between circ-100338 and miR-200a-3p, miR-200a-3p and FUS, and circ-100338 promoted the angiogenesis phenotype in HUVEC cells. CircRNA-100338 may inhibit the function of miRNA-200a-3p by combining with miRNA-200a-3p, and then miRNA-200a-3p plays a role in regulating FUS, thereby regulating the state of angiogenesis.

Preliminary discussion on the potential mechanism of follistatin-like protein 1 in the process of proliferative diabetic retinopathy

Objective To observe the changes of follistatin-like protein 1 (FSTL1) in serum of patients with proliferative diabetic retinopathy (PDR). Methods Twenty PDR patients confirmed by clinical examination and 20 normal people were included in the study. Human retinal vascular endothelial cells (HRCEC) were divided into HRCEC blank control group, 3 h hypoxia group, 6 h hypoxia group. Human umbilical vein endothelial cell (HUVEC) were divided into HUVEC blank control group, 3h hypoxia group, 6h hypoxia group. Real-time quantitative PCR (RT-PCR) and ELISA were used to determine the expression of FSTL1, TGF-β, VEGF, connective tissue growth factor (CTGF) mRNA and protein in peripheral blood and cells of all groups from all subjects. Results The expressions of FSTL1, TGF-β1, CTGF, VEGF mRNA in blood samples of patients with PDR were 1.79±0.58, 0.97±0.21, 1.85±0.69 and 1.38±0.44. The expressions of FSTL1, TGF-β1 protein were 1.19±0.50, 0.71±0.24 ng/ml and 734.03±116.45, 649.36±44.23 ng/L. Compared with normal people, the differences were statistically significant (tmRNA=0.90, 0.21, 2.85, 1.77; P=0.00, 0.00, 0.04, 0.02. tprotein=1.88, 7.68; P=0.00, 0.02). The cell viability of HRCEC cells in the 3 h hypoxia group and the 6 h hypoxia group were 0.66±0.05 and 0.64±0.04, respectively. Compared with the blank control group, the difference was statistically significant (F=13.02, P=0.00). The cell viability of HUVEC cells in the 3 h hypoxia group and the 6 h hypoxia group were 0.63±0.06 and 0.68±0.06, respectively. Compared with the blank control group, the difference was statistically significant (F=26.52, P=0.00). Comparison of FSTL1, TGF-β1, CTGF, and VEGF mRNA expression in HRCEC blank control group and 3 h hypoxia group, the differences were statistically significant (F=14.75, 44.93, 85.54, 6.23; P=0.01, 0.00, 0.00, 0.03). Compared with the HRCEC blank control and 3 h hypoxia group, the expressions of FSTL1 and TGF-β1 protein were statistically significant (P<0.05). There was a statistically significant difference in TGF-β1 protein expression in the hypoxic 6 h group (P=0.03) and no significant difference in FSTL1 protein expression (P=0.68). Comparison of FSTL1, TGF-β1, CTGF, and VEGF mRNA expression in HUVEC blank control group and 3h hypoxia group, the differences were statistically significant (F=19.08, 25.12, 22.89, 13.07; P=0.00, 0.00, 0.00, 0.01). Immunofluorescence staining results showed that FSTL1, TGF-β1, CTGF, and VEGF proteins were positively expressed in cells in the 3h hypoxia and 6h hypoxia groups. Conclusion The expression of FSTL1 gene and protein in serum of PDR patients was significantly higher than that of normal people. Key words: Follistatin-related proteins; Human umbilical vein endothelial cells; Diabetic retinopathy

Co-culture of mesenchymal stem cells and human umbilical vein endothelial cells on heparinized polycaprolactone/gelatin co-spun nanofibers for improved endothelium remodeling

Endothelization of a tissue-engineered substrate is important for its application as an artificial vascular graft. Despite recent advancements in artificial graft fabrication, a graft of <5 mm is difficult to fabricate owing to insufficient endothelization that results in thrombosis after transplantation. We aimed to perform a co-culture of adipose-derived mesenchymal stem cells (MSCs) with human umbilical vein endothelial cells (HUVECs) on antithrombogenic polycaprolactone (PCL)/heparin-gelatin co-spun nanofibers to evaluate the role of co-culturing in promoting quick endothelization of vascular substrates without surface modification by growth factors or other ECM proteins that trigger the endothelization process. Using a co-axial electrospinning technique, we attempted to fabricate our scaffold balancing between mechanical properties and biocompatibility. Antithrombogenic characteristics were then imparted to the fabricated nanofiber substrate by grafting of heparin. Finally, we performed a co-culture of MSCs and HUVECs on the fabricated co-spun nanofiber substrate to obtain proper endothelization of our material under the in-vitro culture. Staining for CD-31 at seven days of culture revealed enhanced CD-31 expression under the co-culture condition; actin staining revealed healthy cobblestone HUVEC morphology, suggesting that MSCs can aid in proper endothelization. Hence, we conclude that co-culture is effective for quick endothelization of vascular substrates.

Effects of exosomal circular RNA-100338 derived from hepatocellular carcinoma on angiogenesis of human umbilical vein endothelial cells

Objective To observe the effects of exosomal circular RNA (circRNA)-100338 derived from hepatocellular carcinoma (HCC) on angiogenesis of human umbilical vein endothelial cells (HUVEC). Methods The exosomes from the supernatant of MHCC97H cells were extracted, purified and identified, and co-incubated with HUVECs (37 ℃, 5% CO2). The exosome tracer experiments traced whether the exosomes could be transformed into HUVECs. MHCC97H cells were transfected with Lipofectamine™ 2000 transfection reagent to extract exosomes from the supernatant of HCC cells after interfering with circRNA-100338 (1 g/L). Proliferation assay was used to detect the changes of proliferation ability of HUVECs [The absorbance (A) values were measured at a wavelength of 450 nm], and angiogenesis assay was applied to evaluate the tubular formation of HUVECs (×100). Results The results of transmission electron microscopy showed that most exosomes from MHCC97H cells were round or oval, and the peak size distribution was 120 nm, which accorded with the characteristics of exosome size. Western blotting was used to detect exosome marker proteins. MHCC97H cells-derived exosomes were internalized by HUVECs. The A values of HUVECs co-cultured with circRNA-100338 interfering group and control group were 0.358±0.005 and 0.436±0.011 (t=-16.227, P<0.01), 0.661±0.052 and 0.888±0.031 (t =-9.146, P<0.01), 1.191±0.084 and 1.542±0.038 (t=-9.296, P<0.01) at 48, 72 and 96 h, respectively. The proliferation ability of HUVECs in interfering group was significantly lower than that in control group after 48-h treatment. In the interferening group, the ability of HUVECs to form tubules was decreased, and the tubular nodules were thin and small. Conclusion HCC-derived exosomal circRNA-100338 enhanced the proliferation ability of HUVECs and promoted endothelial angiogenesis. Key words: Carcinoma, hepatocellular; Exosome; Circular RNA-100338; Proliferation; Angiogenesis

LncRNA ANRIL regulates cell proliferation and migration via sponging miR-339-5p and regulating FRS2 expression in atherosclerosis.

Atherosclerosis (AS), with high risk of stroke or cerebrovascular disease, is one of the most common causes of death worldwide. Increasing evidence shows that long non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) is related to atherothrombotic stroke susceptibility and contributes to AS progression. However, the underlying mechanism was not explained yet. Human aorta vascular smooth muscle cells (HA-VSMCs) and human umbilical vein endothelial cells (HUVECs) were treated with oxidized Low Density Lipoprotein (ox-LDL) and considered as AS cell models. Quantitative reverse transcriptase PCR (qRT-PCR) and Western blotting were employed to investigate the mRNA and protein expression level, respectively. Microscopic examination through fluorescent in situ hybridization (FISH) was used to determine the location of ANRIL. Cell proliferation and migration assays were demonstrated to evaluate the functional role of ANRIL in AS. Potential target of ANRIL was determined using Luciferase reporter assay and RNA immunoprecipitation (RIP). ANRIL was upregulated and miR-399-5p was down-regulated in both human atherosclerotic plaques and ox-LDL-induced cells. ANRIL was located in cytoplasm and promoted cell proliferation and migration by sponging miR-399-5p. Further analysis identified fibroblast growth factor receptor substrate 2 (FRS2) as a direct target of miR-399-5p. Finally, RAS/RAF/ERK signal pathway was proved to be involved in the regulation of ANRIL on the progression of AS. These results revealed the underlying mechanism that ANRIL promoted AS progression by sponging miR-399-5p and regulating RAS/RAF/ERK signal pathway, suggesting that ANRIL might be a potential target for the therapeutic strategy of AS.

Saponins from Tribulus terrestris L. Extract Down-regulate the Expression of ICAM-1, VCAM-1 and E-selectin in Human Endothelial Cell Lines.

Atherosclerosis is an inflammatory disease in which intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin (SELE) are consistently expressed in the vascular endothelium. Several evidence support the crucial role of adhesion molecules in the development of atherosclerosis and plaque instability. Due to the anti-inflammatory activity of Tribulus terrestris (TT), the present study investigated the effect of aqueous extract and saponin fraction of TT on the expression of ICAM-1, VCAM-1, and SELE genes in endothelial cells during normal and lipopolysaccharide (LPS) induced conditions. Human umbilical vein endothelial cells (HUVEC) and human bone marrow endothelial cells (HBMEC) were cultured, stimulated by LPS, and treated with aqueous extract and saponin fraction of TT. Finally, the expression of ICAM-1, VCAM-1, and SELE genes were measured using quantitative real-time polymerase chain reaction. LPS-induced HUVECs and HBMECs significantly increased the expression of ICAM-1, VCAM-1, and SELE in comparison with control groups (P<0.001). Treatment of LPS-induced HUVECs and HBMECs by aqueous extract and saponin fraction of TT decreased the expression of all three mentioned genes significantly (P<0.001) in comparison with LPS-induced cells. Taken together, our data suggest that TT has an anti-inflammatory effect. In vivo study about anti-inflammatory effect of this herb may provide new insights into the development of a herbal drug for the prevention/therapy of atherosclerosis.

MiR-126 on mice with coronary artery disease by targeting S1PR2.

To screen the differentially expressed micro ribonucleic acids (miRNAs) in the serum of coronary atherosclerosis patients, and to investigate their possible mechanisms of action. The differentially expressed serum miRNAs were screened from 3 coronary artery disease (CAD) patients and 3 healthy controls using miRNA expression profiles, which were verified using low-throughput quantitative Reverse Transcription-Polymerase Chain Reaction (RT-qPCR) assay. 60 apolipoprotein E (ApoE)-/- mice were divided into model group, agomir-126 group, agomir-control (con) group, and antagomir-126 group using a random number table. They were fed with high-fat diets (21% fat and 0.15% cholesterol) ad libitum for 15 weeks to establish the mouse model of CAD. Then, hematoxylin and eosin (HE) staining was applied to detect the impact of miR-126 expression level on the tissue morphology in the thoracic aortic region. The influences of miR-126 expression level on the secretion levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-10 were determined via enzyme-linked immunosorbent assay (ELISA). Western blotting assay was performed to examine the effects of miR-126 expression level on the expression levels of nuclear factor-kappa B (NF-κB) and vascular cell adhesion molecule-1 (VACM-1) in the tissues of the thoracic aortic region of the mice. The correlation between miR-126 expression level and sphingosine-1-phosphate receptor 2 (S1PR2) in the serum of CAD patients and animal models was analyzed by the Pearson correlation coefficient method. The targets of miR-126 were predicted using the bioinformatics method, and the direct targets were verified through investigations. Western blotting assay and ELISA were adopted to detect the impacts of miR-126 expression level on the expression and secretion levels of TNF-α, IL-1β, and IL-10 in S1P + oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs). Lentivirus-small hairpin RNA (shRNA) was utilized to knock down the expression level of S1RP2 to determine whether miR-126 affected the increase in the inflammation level in S1P + ox-LDL-induced HUVECs by targeting S1RP2. Compared with those in control group, 4 miRNAs (miR-126, miR-206, miR-4297, and miR-3646) in the serum of CAD patients exhibited the most significant expression differences, which increased by 6.72, 7.11, 13.57, and 21.22 times, respectively. The verification results of low-throughput RT-qPCR assay indicated that there were remarkable changes in the expression levels of the 4 selected miRNAs with differential expressions in comparison with those in control group, displaying statistically significant differences (p<0.01). The results of HE staining manifested that the coronary atherosclerotic plaques were reduced markedly in agomir-126 group, while notably more coronary atherosclerotic plaques were formed in the thoracic aortic region in antagomir-126 group. Meanwhile, the elevated expression level of miR-126 evidently lowered the expressions of serum TNF-α and IL-1β, but significantly increased the expression of IL-10 in the mouse model of CAD. According to the analysis results of the Pearson correlation coefficient method, the miR-126 expression level was negatively correlated with S1PR2 expression level in the serum of both CAD patients and animal models (r=-0.6123, r=-5.37). It was shown in bioinformatics prediction and luciferase reporter gene assay that miR-126 negatively regulated the S1PR2 expression by targeting the 3' untranslated region (UTR) of S1PR2 messenger RNA (mRNA). In the in vitro inflammation model, the increased expression level of miR-126 could relieve the inflammation in cells induced by S1P + ox-LDL. Based on the results of both Western blotting assay and ELISA, the differences in the expression and secretion levels of TNF-α, IL-1β, and IL-10, as well as the expression levels of signaling molecules of the NF-κB signaling pathway, in the cells were not statistically significant among miR-126 mimic treatment group, sh-S1PR2 group, and miR-126 mimic + sh-S1PR2 group, indicating that miR-126 affects the inflammation level in HUVECs by targeting S1PR2. MiR-126 represses the progression of coronary atherosclerosis in the mice by binding to S1PR2. The results of this research may propose a new mechanism of miR-126 in exerting its therapeutic effects and possess potential value for the treatment of CAD in the future.

Inhibitory effect of exosomes secreted by human umbilical cord mesenchymal stem cells on apoptosis of oxygen-glucose deprived reoxygenation model of venous endothelial cells

Objective To explore the inhibitory effect of exosomes secreted by human umbilical cord mesenchymal stem cells(HUCMSC) on apoptosis of human umbilical vein endothelial cells(HUVEC) after model group(oxygen-glucose deprivation reoxygenation), and to clarify its possible mechanism. Methods Human umbilical cord mesenchymal stem cells were cultured. The collected cell supernatant was stored in a centrifugal tube. The exosomes secreted by human umbilical cord mesenchymal stem cells were extracted by ultracentrifugation and identified. Human umbilical vein endothelial cells were randomly divided into control group, model group and different concentrations of HUCMSC-EXO(20 μg/ml, 40 μg/ml, 60 μg/ml) treatment groups(adding HUCMSC-EXO into the model group) . The morphological changes of HUVEC cells in each group were observed by inverted phase contrast microscope, and the proliferation inhibition rate of HUVEC in each group was measured by CCK-8 reagent. Western blot was used to detect the expression of apoptosis-related proteins Caspase-3, Bax, Bcl-2 and hypoxia-associated protein hypoxia inducible factor 1α(HIF-1α). Inhibitor(HIF-1α inhibitor) + model group and HUCMSC-EXO + inhibitor + model group were added on the basis of the above experiments. Western blot analysis was performed to observe the effects of HUCMSC-EXO, inhibitor and both of them on HIF-1α and Bax expressions in HUVEC. Results HUCMSC-EXO was successfully extracted and identified. Compared with the control group, the volume of HUVEC in the model group and the HUCMSC-EXO group with different concentrations decreased, became round, connected and evacuated, and the growth state was poor under the inverted phase contrast microscope.CCK-8 detection showed that the cell viability in the HUCMSC-EXO group was significantly higher than that in the model group, the difference was statistically significant (t=9.23, P 0.05)and Bax protein ((0.363±0.069), (0.370±0.064); t=0.18, P>0.05). But both of them were down-regulated compared with the model group (HIF-1α protein (0.919±0.064), Bax protein (0.902±0.071)), the differences were significant( t=13.56, t=13.03, both P<0.05). Conclusion HUCMSC-EXO has a protective effect on OGD/R model of HUVEC, and its mechanism may be related to the down-regulation of HIF-1α expression. Key words: Human umbilical mesenchymal stem cells; Exosomes; Oxygen-glucose deprivation reoxygenation; Human umbilical venous endothelial cells; Apoptosis

Effects of TET2-targeting miR-27b on oxidized low-density lipoprotein-induced inflammatory responses and apoptosis in endothelial cells

Objective To investigate the effects of miR-27b targeting ten-eleven translocation methylcytosine dioxygenase 2 (TET2) on oxidized low-density lipoprotein (ox-LDL) induced inflammatory responses and apoptosis in endothelial cells. Methods Double luciferase reporter gene analysis verified the targeting effect of miR-27b on TET2. Human umbilical vein endothelial cells (HUVECs) were induced by ox-LDL in vitro. Eight groups were set up including control group, ox-LDL group, ox-LDL+ anti-miR-con group, ox-LDL+ anti-miR-27b group, ox-LDL+ pcDNA group, ox-LDL+ pcDNA-TET2 group, anti-miR-27b+ si-con group and anti-miR-27b+ si-TET2 group. qRT-PCR was used to detect the expression of miR-27b and TET2 at mRNA level. Cell viability was detected by MTT assay. Cell apoptosis rate was detected by flow cytometry. Western blot was used to detect the expression of TET2, Cyclin D1 and caspase-3 at protein level. Results TET2 was the target gene of miR-27b. TET2 expression could be negatively regulated by miR-27b. ox-LDL increased the expression of miR-27b and reduced the expression of TET2 in HUVECs. The secretion of inflammatory factors and apoptosis rates of HUVECs in the control, ox-LDL+ anti-miR-27b, ox-LDL+ pcDNA-TET2 and anti-miR-27b+ si-con groups were significantly lower than those in the ox-LDL, ox-LDL+ anti-miR-con, ox-LDL+ pcDNA and anti-miR-27b+ si-TET2 groups, respectively (P<0.05). Conclusions miR-27b promoted ox-LDL-induced inflammatory responses and apoptosis in endothelial cells through down-regulating the expression of TET2. Key words: miR-27b; ox-LDL; TET2; HUVEC; Inflammatory response; Apoptosis

Rhizoma Pleionis inhibits of breast cancer epithelial-mesenchymal transition by VEGF-A related angiogenesis

Objective To investigate the Rhizoma Pleionis mediating vascular endothelial growth factor A (VEGF-A)-related angiogenesis in breast cancer cells to inhibit epithelial-mesenchymal transition (EMT) in breast cancer. Methods Firstly, the proliferation activity of MDA-MB-231 and 4T1 of breast cancer cells was detected by methyl thiazolyl tetrazolium (MTT) method at different concentrations (0.2 g/ml, 0.4 g/ml, 0.6 g/ml, 0.8 g/ml, 1.0 g/ml); secondly, the specific concentration of Rhizoma Pleionis was selected and tested by Transwell test for its effect on breast cancer cell metastasis and invasion. The formation of human umbilical vein endothelial cells (HUVEC) capillary wall was measured to detect the effect of Rhizoma Pleionis on cell angiogenesis. The EMT-related protein and angiogenesis-related molecules were detected by Western blot. Results MTT results showed that Rhizoma Pleionis of 0.8 g/ml and 1.0 g/ml can inhibit the MDA-MB-231 and 4T1 cells proliferation in a dose-dependent manner, with statistically significant difference (P<0.05). Rhizoma Pleionis can inhibit the invasion of breast cancer cells and inhibit the formation of blood vessel walls in HUVECs. Western blot confirmed that the expression level of EMT-related proteins: E-cadherin was significantly increased and the expression level of N-cadherin, Vimentin and Snail was significantly decreased after treatment with Rhizoma Pleionis (P<0.05); and the level of the related molecule hypoxia inducible factor-1α (HIF-1α), VEGF-A and vascular endothelial growth factor receptor-2 (VEGFR-2) was significantly reduced (P<0.05). Conclusions Rhizoma Pleionis inhibits the epithelial-mesenchymal transition of breast cancer by influencing the angiogenesis of VEGF-A, and inhibits the invasion and metastasis of breast cancer. Key words: CREMASTRA VARIABILIS; Vascular endothelial growth factor A; Breast neoplasms; Tumor cells, cultured; Epithelial-mesenchymal transition

Effects of usnic acid on the proliferation and migration activity of human umbilical vein endothelial cells and hypertrophic scar fibroblasts

Objective To Explore the effect of usnic acid on the proliferation and migration of human umbilical vein endothelial cells (HUVECs) and human hypertrophic scar fibroblasts (HSFBs) . Methods HSFBs were isolated and cultured in vitro, and their morphological features were analyzed. Different concentrations of usnic acid (0, 5, 10, 20, 50 μmol/L) were added to the HUVECs and HSFBs, control group was treated with Dimethyl sulfoxide (DMSO), after culture for 48 h, the proliferation activity of cells was detected with the MTS kit. The migration ability of two cells was analyzed by wound healing assay. The experimental data were analyzed by SPSS19.0 software, with P<0.05 as the statistical significance. Results Compared with the control group (100%), 5, 10, 20, 50 μmol/L usnic acid could significantly inhibit the proliferation of HUVECs, and the cell viability was reduced to (91.67±10.99)%, t=0.068, P>0.05; (40.68±8.87)%, t=5.280, P<0.01; (33.97±2.12)%, t=1.140, P<0.01; (20.39±0.84)%, t=8.280, P<0.01. respectively. Under the same experimental conditions, 20 μmol/L and 50 μmol/L usnic acid could inhibit the proliferation of HSFBs, cell viability was (76.16±11.03)% and (51.59±6.82)% respectively. 1, 5, 10 μmol/L usnic acid had no inhibitory effect on HSFBs cell proliferation. The migration of HUVECs was inhibited by 1, 10, 20 μmol/L of usnic acid (81.26±10.82, t=0.000, P<0.01; 71.81±3.96, t=3.780, P<0.01; 54.91±8.47, t=5.280, P<0.01). Meanwhile, usnic acid has no inhibition effect on HSFBs migration. Conclusion Usnic acid had the ability to inhibit HUVECs and HSFBs proliferation, and the ability to inhibit HUVECs migration. Key words: Usnic acid; Human umbilical vein endothelial cells; Human hypertrophic scar fibroblasts; Cell proliferation; Cell migration

Crocetin promotes angiogenesis in human endothelial cells through PI3K-Akt-eNOS signaling pathway.

Previous studies proved the pro-angiogenic effect of Crocetin, a natural carotenoid dicarboxylic acid, in both in vivo and in vitro models. However, the exact mechanism of Crocetin action has not completely been elucidated yet. The current experiment was designed to find the activity of PI3K-Akt-eNOS axis after the treatment of endothelial cells with Crocetin in vitro. Human Umbilical Vein Endothelial Cells (HUVECs) were incubated with various concentrations of Crocetin (1, 5, 25, 50, and 100 µM) over a period of 72 h. Crocetin significantly increased HUVECs viability after 72 h as compared with the control group. We also found that Crocetin promoted the formation of the capillary-like structure compared to the control (p<0.05). Moreover, an improved migration rate and increased MMP-9 activity were observed in HUVECs that received 50 µM Crocetin (p<0.05). Crocetin enhanced the uptake of Ac-LDL which is correlated with increased lipid metabolism. Based on the data from the current experiment, protein level of VEGFR-1, -2 and p-Akt/Akt, p-eNOS/eNOS ratios were increased 72 h after the treatment of HUVECs with Crocetin (p<0.05). In contrast, the transcription level of VEGF was reduced in Crocetin-treated cells. These data demonstrated that Crocetin promotes HUVECs angiogenesis potential by the modulation of VEGF signaling pathway and increased cell viability. The PI3K/Akt/eNOS axis is required for a Crocetin-associated activity in endothelial cells.

Ephrin type-A receptor 2 on tumor-derived exosomes enhances angiogenesis through the activation of MAPK signaling.

Exosomes are potent players in the development of metastases and they play an important role in cancer angiogenesis and exacerbation. However, it is unclear how proteins on exosomes affect development of blood vessel networks. In this study, we focused on relationships between membrane proteins on exosomes and angiogenesis using human umbilical vein endothelial cells (HUVEC). Lung tumor cell-derived exosomes induced tube formation and growth of endothelial cells in vitro in a dose-dependent manner involving MAPK activation, but this was not seen in normal lung epithelial cells. Ephrin type-A receptor 2 (EphA2) was identified by proteomic analysis and an inhibition assays showed it is a major MAPK activator on exosomes. Thus EphA2 on exosomes participates in angiogenesis as a ligand of the ephrin signaling pathway. These results support the development of novel therapeutic strategies such as blockade of remote cancer communications through exosomes.

Protective effect of cycloartenyl ferulate on lipopolysaccharide induced endothelial cell apoptosis and its mechanism

Objective To investigate the effect of cycloartenyl ferulate (CF) on lipopolysaccharide (LPS)-induced apoptosis in human umbilical vein endothelial cells (HUVEC), and to explore its relative mechanism. Methods Human umbilical vein endothelial cells were cultured in vitro, and the experiment was divided into the normal group, CF group, LPS group, and LPS+CF groups at different concentrations. After the corresponding treatment of cells in each group, the cell viability and apoptosis of each group were tested by the cell counter Kit-8 (CCK-8) assay and TdT-mediated dUTP nick end labeling (TUNEL) assay. The protein expression of Bax, Bcl-2, caspase-3 and the effect of CF on the protein expression of Nrf2/HO-1 pathway were determined by Western blot. One-way ANOVA were performed in multigroup mean comparison, LSD-t test was used to compare the mean values of two samples, and P<0.05 was considered statistically significant. Results Compared with the LPS group, the survival rate of HUVECs cells in the CF group was significantly increased with different doses, and the survival rate increased significantly in the 320 μmol/L CF group [(69.85±1.2)% vs ( 100.2±1.824)%, P< 0.01]. TUNEL staining showed that compared with the LPS group, the number of apoptotic positive cells in the 320 μmol/L CF group was significantly reduced [(27.33 ± 3.40) vs (11.67 ± 2.04), P<0.01]. Compared with the LPS group, Bcl-2 protein expression level was significantly increased in the CF group at different doses (P<0.01), caspase-3 and Bax protein expression were significantly decreased (P<0.01). Nrf2 protein expression level increased significantly (P<0.01), and HO-1 protein level increased significantly (P<0.01). Conclusion CF can alleviate LPS-induced apoptosis of HUVEC, which may be related to the increase of bcl-2 expression, the decrease of caspase-3 and Bax protein expression and the activation of Nrf2/ HO-1 signaling pathway. Key words: Cycloartenyl ferulate; Lipopolysaccharide; Vascular endothelial cells; Apoptosis; Nrf2/HO-1 signaling pathway

Senescent HUVECs-secreted exosomes trigger endothelial barrier dysfunction in young endothelial cells.

Accumulation of senescent endothelial cells can cause endothelium dysfunction which eventually leads to age-related vascular disorders. The senescent-associated secretory phenotype (SASP) cells secrete a plethora of soluble factors that negatively influence the surrounding tissue microenvironment. The present study sought to investigate the effects of exosomes, which are nano-sized extracellular vesicles known for intercellular communications secreted by SASP cells on young endothelial cells. Exosomes were isolated from the condition media of senescent human umbilical vein endothelial cells (HUVECs) and then confirmed by the detection of exosome specific CD63 and CD9 expressions, electron microscopy and acetylcholinesterase assay. The purified exosomes were used to treat young HUVECs. Exposure to exosomes repressed the expression of adherens junction proteins including vascular endothelial (VE)-cadherin and beta-catenin, decreased cell growth kinetics and impaired endothelial migration potential of young endothelial cells. These findings suggest that senescent HUVECs-secreted exosomes could disrupt barrier integrity that underpins endothelial barrier dysfunction in healthy young endothelial cells.

The impact of inflammatory factors and related cell growth factors of human umbilical cord mesenchymal stem cells for model rats with diabetic foot ulcer

Objective To study effect of inflammatory factors and related cell growth factors of human umbilical cord mesenchymal stem cells (HUCMSCs) for model rats with diabetic foot ulcer (DFU), and analyzed the possible action mechanism of HUCMSCs therapy DFU. Methods 50 specific pathogen free (SPF)-level male rats were fed with high-fat beverage for 4 weeks. The DFU models were established by means of intraperitoneal injection of 1% streptozotocin (40 mg/kg) and steam fumigation. 30 successful model rats were divided into model group, high-dose HUCMSCs group (high-dose group: femoral artery injection with HUCMSCs 2.0×106/0.2 ml) and low-dose HUCMSCs group (low-dose group: femoral artery injection with HUCMSCs 1.0×106/0.2 ml), 10 rats/each group. Another 10 SPF-level male rats were selected and set as blank control group (blank group). Then wound healing rate, serum inflammatory factors, related cell growth factors, granulation tissue fibroblasts and newly born capillaries were compared between the groups. Results (1) Wound healing rate: The model group, low-dose group, high-dose group respectively were (26.31±4.32)%, (66.92±7.24)%, (74.74±8.32)%. low-dose group and high-dose group wound healing rate were significantly higher than model group (t=8.424, 11.201, P<0.01), high-dose group wound healing rate were significantly higher than low-dose group (t=3.148, P<0.05). (2) Serum inflammatory factors: model group, low-dose group and high-dose group interleukin (IL)-1, tumor necrosis factor-α (TNF-α), hs-CRP respectively were (6.12±0.25), (4.54±0.36), (4.20±0.33) ng/L; (29.45±3.12), (21.13±3.24), (18.20±2.65) ng/L; (0.61±0.08), (0.45±0.07), (0.37±0.06) ng/L. low-dose group and high-dose group IL-1, TNF-α, hs-CRP were significantly lower than model group (t=11.400, 5.849, 5.111, 14.666, 8.691, 8.222, P<0.05, P<0.01), high-dose group were significantly lower low-dose group (t=2.202, 2.214, 2.744, P<0.05); (3) Related cell growth factors: model group, low-dose group and high-dose group basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) respectively were (46.42±6.36), (61.35±7.20), (68.24±8.12) ng/L; (546.32±30.24), (722.36±32.17), (772.45±34.26) μg/L; (2.24±0.32), (3.65±0.41), (4.26±0.55) μg/L. Low-dose group and high-dose group bFGF, EGF, VEGF were significantly higher than model group (t=4.915, 12.609, 8.573, 6.690, 15.648, 10.039, P<0.05, P<0.01). high-dose group bFGF, EGF and VEGF were significantly higher than low-dose group (t=2.031, 3.740, 2.812, P<0.05); (4) Granulation tissue fibroblasts and newly born capillaries: model group, low-dose group and high-dose group granulation tissue fibroblasts and newly born capillaries were respectively (155.24±8.45), (278.65±9.12), (305.12±10.25) cells/visual field; (4.36±0.45), (6.45±0.62), (7.24±0.60)×10/μm2. The low-dose group and high-dose group were significantly higher than model group (t=31.389, 8.627, 35.679, 12.143, P<0.01), high-dose group were significantly higher than low-dose group (t=6.101, 2.896, P<0.05). Conclusion HUMSCs help to promote the ulcerative healing of DFU rats. Key words: Diabetic foot ulcer; Human umbilical cord mesenchymal stem cells; Inflammatory factor; Growth factor

Vitamin D attenuates lipopolysaccharide-induced inflammatory response in endothelial cells through inhibition of PI3K/Akt/NF-κB signaling pathway.

Inflammation and endothelial cells contribute to the pathogenesis of various cardiovascular diseases (CVDs). Vitamin D is associated with a decreased risk of inflammation related diseases including CVDs. However, the links between vitamin D, endothelial cells and vascular inflammation remain to be elucidated. In this study, we investigated the anti-inflammatory effect of vitamin D in lipopolysaccharide (LPS)-activated endothelial cells and the related signaling pathways. 1,25(OH)₂D₃ treatment significantly suppressed LPS-induced upregulation of COX-2, TNF-α, IL-6 and PGE2 in Human umbilical vein endothelial cells (HUVECs). 1,25(OH)₂D₃ inhibited LPS-stimulated phosphorylation of Akt at S473 and T308, IκBα phosphorylation, and nuclear accumulation of NF-κB. Blocking PI3K signaling abolished the inhibitory effect of 1,25(OH)₂D₃ on LPS-induced increase of Akt S473 phosphorylation and COX-2 expression. Blocking NF-κB signaling blunted the suppression effect of 1,25(OH)₂D₃ on IκBα phosphorylation, NF-κB accumulation in the nucleus and COX-2 expression. These results indicate that 1,25(OH)₂D₃ suppresses inflammatory response in LPS-induced HUVECs through PI3K/Akt/NF-κB signaling pathway.

Influences of interaction between dengue virus type 2-infected human umbilical vein endothelial cells and macrophages on major inflammatory cytokines

Objective To study the influences on the production of major inflammatory cytokines after co-culturing macrophages with human umbilical vein endothelial cells (HUVECs) that were infected with dengue virus type 2 (DENV-2). Methods Density gradient centrifugation was used to isolate peripheral blood mononuclear cells (PBMC) from concentrated human leukocytes. Adherent monocytes in culture flasks were obtained and stimulated with macrophage colony-stimulating factor (M-CSF) to prepare macrophages. The purity of CD14+ CD11b+ cells was measured by flow cytometry. Changes in the expression of NS1 at mRNA level in HUVECs were detected by real-time PCR following DENV-2 infection. DENV-2-infected HUVECs were co-culture with macrophages in Transwell chambers. A control group was set up by pretreating HUVECs with sphingosine-1-phosphate (S1P) type 1 (S1P1)-specific receptor agonist CYM-5442 for 24 h to remove the drug before infection and then co-culturing the infected cells with macrophages. Real-time PCR was used to detect the expression at mRNA level of IL-6 and IL-8 in HUVECs and IL-6, IL-8, TNF-α and IL-1β in macrophages. A double-antibody sandwich ELISA was used to detect the expression of above cytokines in culture supernatants. Results After HUVECs were infected with DENV-2, expression of NS1 gene at mRNA level gradually increased to the peak at 24 h (2.66±0.53, P<0.05) and then decreased. The purity of macrophages detected by flow cytometry was (89.16±2.07) %. Expression of IL-6 and IL-8 at mRNA level in DENV-2-infected HUVECs was up-regulated. The peak values reached at 24 h of IL-6 and IL-8 expression were 16.10±0.17 and 29.76±0.58, while the expression levels at 24 h in the uninfected group were 1.46±0.67 and 1.60±0.54, respectively. Expression of IL-6, IL-8, TNF-α and IL-1β at mRNA level in DENV-2-infected macrophages was increased significantly. The levels of IL-6, IL-8, TNF-α and IL-1β expression at 24 h were 45.82±3.72, 52.34±1.69 (12 h), 8.94±1.75 and 30.96±1.44 in the infected macrophages, and 1.16±0.22, 1.15±0.21, 1.11±0.09 and 1.47±0.31 in the uninfected group. Expression of these cytokines was decreased at every time points after co-culturing of DENV-2-infected HUVECs with macrophages, but still significantly higher than that in the uninfected group. In the co-culture group with DENV-2 infection, CYM-5442 pretreatment significantly decreased the expression at mRNA level of IL-6 and IL-8 in HUVECs (P<0.01) and that of IL-6, IL-8, TNF-α and IL-1β in macrophages (P<0.01). Conclusions DENV-2 could infect primary HUVECs, and then activate macrophages to promote the secretion of large amounts of IL-6, IL-8, TNF-α and IL-1β. Moreover, the activated macrophages could reduce the production of inflammatory cytokines in HUVECs to a certain extent. Key words: Dengue virus type 2 (DENV-2); Human umbilical vein endothelial cell (HUVEC); Macrophage; Cytokine; Immunoregulation

1α, 25-dihydroxyvitamin D3 inhibits tumor necrosis factor-α induced activation of human umbilical vein endothelial cells via NF-κB signaling pathway

Objective To investigate the effect of 1α, 25-dihydroxyvitamin D3[1α, 25-(OH)2D3] on tumor necrosis factor-α (TNF-α) induced activation of human umbilical vein endothelial cells (HUVECs). The mechanism involved in this process was also studied. Methods HUVECs were cultured and treated with TNF-α (40 ng/ml), 1α, 25-(OH)2D3(10-8 mol/L), and SN50 as indicated. Vascular cell adhesion molecule (VCAM) and E-selectin were used as markers of endothelial activation, which were detected by Western blotting and realtime PCR (RT-PCR). NF-κB signaling pathway was investigated to study the mechanism. Western blotting, RT-PCR, immunofluorescence assay, and chromatin immunoprecipitation (ChIP) method were used to evaluate the effects of 1α, 25-(OH)2D3 on its early activation, nuclear transport, and binding to VCAM and E-selectin promoters. Results (1)Western blotting and RT-PCR showed that TNF-α could significantly up-regulate the expression of VCAM and E-selectin in HUVECs, which can be inhibited by specific NF-κB blocker SN50. 1α, 25-(OH)2D3 down-regulated the expression of VCAM and E-selectin induced by TNF-α. (2) Western blotting showed that TNF-α induces I-κBα phosphorylation, thereby activating NF-κB p65 subunit. Immunofluorescence showed that 1α, 25-(OH)2D3 significantly inhibited the nuclear translocation of NF-κB p65 subunit. ChIP analysis revealed that 1α, 25-(OH)2D3 inhibited the binding of NF-κB p65 to VCAM and E-selectin promoters and thus affected gene expression. Conclusions TNF-α enhanced the expression of E-selectin and VCAM in HUVECs via NF-κB signaling pathway. 1α, 25-(OH)2D3 may inhibit NF-κB early activation, nuclear transport and the binding of NF-κB p65 to VCAM and E-selectin promoters, thereby inhibiting TNF-α-induced endothelial cell activation. Key words: 1α, 25-dihydroxyvitamin D3; Endothelial activation; NF-κB signaling pathway; Adhesion molecule