Polydatin enhances blood vessel relaxation and reduces NLRP3-mediated inflammation in hyperglycemia by lowering vascular cell adhesion molecule expression

Endothelial dysfunction leading to cardiovascular disease risk involves a decrease in nitric oxide (NO) production. In physiological conditions shear stress is a potent stimulation of endothelium-derived NO production and flow mediated NO production is regulated by the activation of endothelial NO synthase (eNOS). In endothelial cells, eNOS, aldose reductase (AR), a rate limiting enzyme of polyol pathway, and glutathione reductase (GR) share a NADPH as an obligate cofactor. In diabetec condition intracellular polyol pathway is activated and this may decrease shear stress-induced endothelial NO production and increase intracellular oxidative stress via inhibition of eNOS and GR by NADPH consumption. Therefore we investigated whethter AR inhibitor epalrestat improved endothelial NO production under high glucose condition to elucidate the mechanism of endothelial dysfunction in diabetes. We incubated human umbilical vein endothelial cells (HUVECs) in normal (5mM) and high (30mM) glucose condition for 72 hours, with or without epralrestat, or 100U/ml superoxide dismutase (SOD), respectively. After exchange of medium for Krebs’ buffer, HUVECs were exposed to 12dyne/cm2 steady laminar fluid shear stress for 5 minutes. NO release from HUVECs was measured as NO2 using a NOx analyzing HPLC system by Griess reaction. Next we harvested the cells in lysis buffer and analyzed phosphorylation of Akt (shear induced intracellular signal transduction) and eNOS by western blotting, and measured intracellular 8-OHdG and ratio of NADPH/NADP. In high glucose condition NO2 was decreased and 8-OHdG increased compared to low glucose. NO2 was restored and 8-OHdG was reduced by epalrestat significantly (p<0.01, p<0.05, respectively, vs. high glucose condition). In SOD-treated HUVECs, NO2 was not restored (n.s. vs. high glucose condition) despite of complete reduction of 8-OHdG (p<0.01). Both Akt and eNOS phosphorylation by shear stress was affected neither by high glucose, epalrestat nor SOD. Intracellular NADPH/NADP ratio was decreased in high glucose condition, but this reduction was restored by epalrestat. These results showed that polyol pathway activation plays a key role in endothelial NO production under high glucose condition via a cofactor NADPH.

The ability of different phosphatidylcholine (PC) species to inhibit cytokine-induced expression of vascular cell adhesion molecule 1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) was investigated. PC species containing palmitoyl- in the sn-1 position and palmitoyl- (DPPC), arachidonyl- (PAPC), linoleoyl- (PLPC) or oleoyl- (POPC) in the sn-2 position were compared. These PC species were studied as components of reconstituted high density lipoproteins (rHDL) (containing apolipoprotein A-I [apoA-I] as the sole protein) or as small unilamellar vesicles (SUVs). The rHDL containing PLPC and PAPC inhibited VCAM-1 expression in activated HUVECs by 95 and 70%, respectively, at an apoA-I concentration of 16 micrometer. At this concentration of apoA-I, POPC rHDL inhibited by only 16% and DPPC rHDL did not inhibit at all. These differences could not be explained by differential binding of the rHDL to HUVECs. The same hierarchy of inhibitory activity was observed when these PC species were presented to the cells as SUVs but only when the SUVs also contained an antioxidant. It was concluded that rHDL PC is responsible for their inhibitory activity and that this varies widely with different PC species.

Objective To investigate the effects of interaction between human umbilical vein endothelial cells (HUVECs) which were infected with dengue virus type 2 (DENV-2) and CD4+ T cells on the expression of ICAM-1 (intercellular adhesion molecule 1), VCAM-1 (vascular cell adhesion molecule 1), IL-10 and TGF-β1 at mRNA level for further understanding the immunological mechanism of DENV infection. Methods HUVECs were treated with CYM-5442, a selective agonist for sphingosine-1-phosphate receptor 1 (S1P1), for 24 hours and then infected with 103 TCID50 (50% tissue culture infective dose) of DENV-2 before co-culturing with CD4+ T cells. Changes in the expression of NS1 (DENV-2 nonstructural protein), SPHK1 (sphingosine kinase 1, phosphorylating sphingosine to S1P), ICAM-1, VCAM-1, IL-10 and TGF-β1 at mRNA level were detected by real-time PCR after 4, 8, 12, 24, 48 and 72 hours of co-culturing. Results There was a certain timeliness in the expression of NS1 at mRNA level after infecting HUVECs with DENV-2 and the expression reached a peak at 24 h. Treating HUVECs with or without CYM-5442 had no significant influence on the expression of DENV-2 NS1 at mRNA level. The expression of SPHK1 at mRNA level was significantly increased after treating HUVECs with CYM-5442 and DENV-2 (P<0.05). Compared with DENV-2-infected or untreated HUVECs, Co-culturing DENV-2-infected HUVECs with CD4+ T cells increased the expression of ICAM-1 and VCAM-1 in HUVECs at mRNA level (P<0.01) as well as the expression of IL-10 in CD4+ T cells at mRNA level (P<0.05), but had no significant influence on the expression of TGF-β1 in CD4+ T cells at mRNA level. Conclusion This study shows that DENV-2 can replicate and proliferate in HUVECs, but CD4+ T cells inhibit the replication and proliferation. CD4+ T cells play an important role in promoting the expression of VCAM-1 and ICAM-1 in DENV-2-infected HUVECs at mRNA level, activating HUVECs and increasing inflammation, which may be associated with increased vascular permeability induced by DENV-2 infection. Co-culturing CD4+ T cells with DENV-2-infected HUVECs promotes the expression of IL-10 in CD4+ T cells at mRNA level, but has no significant effect on TGF-β1. Key words: HUVEC; CD4+ T cell; DENV-2; Cytokine; Immunoregulation

This study aimed at verifying whether thiamine, a co-enzyme which decreases intracellular glycolysis metabolites by allowing pyruvate and glyceraldheyde 3-phosphate to enter the Krebs cycle and the pentose-phosphate shunt, respectively, corrects delayed replication caused by high glucose concentrations in cultured human umbilical vein (HUVEC) and bovine retinal endothelial cells (BREC). After incubation in physiological (5.6 mmol/l) or high (28.0 mmol/l) glucose with or without 150 mumol/l thiamine, cells were counted and proliferation assessed by mitochondrial dehydrogenase activity. Lactate was measured in both cell types as an index of glycolytic activity and fluorescent advanced glycosylation end-products (AGE) concentration was determined in the HUVEC lysate. Both cell counts and proliferation assays in either of the cell types confirmed the impairment to cell replication induced by high glucose. When thiamine was added to cells kept under high glucose conditions, the number of surviving cells was significantly increased and the reduced cell proliferation appeared to be corrected. Lactate assays confirmed the increased production of this metabolite by BREC and HUVEC in high glucose, which was reduced by thiamine. Fluorescent AGE determination showed that thiamine may prevent non-enzymatic glycation in HUVEC. Thiamine restores cell replication, decreases the glycolytic flux and prevents fluorescent AGE formation in endothelial cells cultured in high glucose, suggesting that abnormal levels of glycolytic metabolite(s) may damage cells.

The protective effect of rosuvastatin in human umbilical endothelial cells exposed to constant or intermittent high glucose

Objective To investigate effect of radix hedysari polysaccharide (HPS) on secretion of nitric oxide (NO) and endothelin-1 (ET-1) on human umbilical vein endothelial cells(HUVECs) induced by high glucose and its mechanism. Methods HUVECs were isolated from umbilical vein, and cultured cells were divided into normal control group, high glucose group, HPS group, MTT assay were used to analyze the effect of HPS on HUVECs viability under high glucose. The levels of NO, nitric oxide synthase (NOS) and inducible nitric oxide synthase (iNOS) were measured by colorimetric analysis. The content of ET-1 in clear supernatant were detected by enzyme linked immunosorbent assay (ELISA). The expression of endothelial nitric oxide synthase (eNOS), ET-1 mRNA, c-Jun kinase-1(JNK1) mRNA were examined by real-time quantitative RT-PCR. Results The cells viability were markedly decreased when HUVECs were treated with 30 mmol/L-glucose for 6 h((82.4±3.5)%), 24 h((68.2±1.4)%), 48 h((63.0±2.9)%), HPS could efficiently prevented cells viability lost(P<0.05), especially in 50 mg/L((85.3±4.6)%), 100 mg/L ((89.6±1.1)%), 200 mg/L ((88.8±3.6)%), P<0.05. In high glucose group, the levels of NO ((24.84±1.34)μmol/L)and NOS((0.54±0.06) U/ml) were increased at early stage and decreased at advanced stage compared with normal control group (P<0.05). The contents of iNOS((0.08±0.020) U/ml) and ET-1 ((0.710±0.030) ng/ml) were upgraded under high glucose at any time, nevertheless, HPS could balanced the levels of NO((23.20±0.55)μmol/L), NOS((0.46±0.10) U/ml), iNOS((0.08±0.020) U/ml) and ET-1((0.710±0.030) μg/L), P<0.05. At equal pace, the expressions of eNOS mRNA was up-regulated and ET-1 mRNA was down-regulated in HPS group compared with high glucose group(0.33±0.02 vs 0.23±0.04, 2.28±0.31 vs 2.79±0.29). The contents of JNK1 mRNA in HUVECs were shown to increased after exposure to high glucose(2.95±0.05), P<0.05, which were markedly prevented by HPS(1.45±0.05), P<0.05. Conclusion HPS can protect the injury of HUVECs induced by high glucose in vitro. The mechanism of protection may be associated with blocked JNK signaling pathway. Key words: Radix hedysari polysaccharide; Human umbilical vein endothelial cells; High glucose; Nitric oxide; Endothelin-1

The present study is aimed to investigate the regulatory effect of microRNA (miRNA or miR)-503 on endothelial functions, as well as the mechanism by which high glucose leads to injury of endothelial cells. When reaching 80% confluency, human umbilical vein endothelial cells (HUVECs) were subjected to non-serum synchronization for 12 h, and medium of cells in high-glucose (HG) group was replaced by normal medium supplemented with 25 mmol/L D-glucose. HUVECs cultured in normal glucose (NG) medium were used as control. To overexpress miR-503, HUVECs were transfected with miR-503 mimics. To silence insulin-like growth factor-1 receptor (IGF-1R) mRNA, HUVECs were transfected with small interfering RNA (siRNA). To predict whether miR-503 targets IGF-1R, bioinformatics was performed. Quantitative Real-time polymerase chain reaction was used to determine miR-503 and IGF-1R mRNA expression, and Western blotting was employed to measure IGF-1R protein expression. Cell-Counting Kit 8 assay was used to determine HUVECs proliferation, while wound-healing assay was used to evaluate HUVECs migration. HUVECs apoptosis was investigated by measuring caspase 3 activity. Expression of IGF-1R in HUVECs in high glucose was decreased compared to that in normal glucose. miR-503 was predicted to target IGF-1R mRNA, and miR-503 expression in HUVECs in high glucose was higher than that in normal glucose. Overexpression of miR-503 inhibited the transcription and the translation of IGF-1R gene reducing migration, suppressed proliferation and promoted apoptosis. Transfection with IGF-1R siRNA decreased IGF-1R protein expression in HUVECs. Down-regulated IGF-1R expression reduced migration and proliferation, but promoted apoptosis of HUVECs. The present study demonstrates that miR-503 expression in HUVECs is elevated in high glucose condition. Also, miR-503 reduces migration and proliferation, but promotes apoptosis of HUVECs by inhibiting IGF-1R expression.

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In this study, we aimed to investigate whether an aqueous extract of Buddleja officinalis (ABO) suppresses high-glucose-induced vascular inflammatory processes in the primary cultured human umbilical vein endothelial cells (HUVEC). The high-glucose-induced increase in expression of cell adhesion molecules (CAMs) such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial-selectin (E-selectin) was significantly attenuated by pretreatment with ABO in a dose-dependent manner. Enhanced cell adhesion caused by high glucose in co-cultured U937 and HUVEC was also blocked by pretreatment with ABO. Pretreatment with ABO also blocked formation of high-glucose-induced reactive oxygen species (ROS). In addition, ABO suppressed the transcriptional activity of NF-kappaB and IkappaB phosphorylation under high-glucose conditions. Pretreatment with N(G)-nitro-l-arginine methyl ester (L-NAME), an endothelial nitric oxide (NO) synthase inhibitor, attenuated the protective action of ABO on high-glucose-induced CAM expression, suggesting a potential role of NO signaling. The present data suggest that ABO could suppress high-glucose-induced vascular inflammatory processes, and ABO may be closely related with the inhibition of ROS and NF-kappaB activation in HUVEC.

Background: Smoking cigarettes decreases expression of eNOS in the endothelium, resulting in lower nitric oxide (NO) secretion and decreased flow-mediated dilation in the conducting arteries. In contrast, the effects of e-cigarettes (e-cigs) on endothelial function are just beginning to be studied and the mechanisms of action are unclear. Aim: To test the hypothesis that circulating factors from e-cigarette users decrease endothelial eNOS protein levels and NO secretion in primary endothelial cell cultures, relative to nonsmokers and cigarette smokers. Methods: 36 healthy individuals were recruited and grouped as nonsmokers (n=12), cigarette smokers (n=7), and e-cig users (n=17). Human umbilical vein endothelial cells (HUVECs) were cultured at 20,000 cells per well in 24-well culture plate. Serum samples from individual subjects and endothelial growth media were added at a 1:1 ratio to confluent cells and incubated for 12 hours (basal condition), followed by fresh medium containing VEGF at 50 ng/ml for 30 min (stimulated condition). The amount of NO liberated from cells was measured in culture supernatants by the chemilumin- escence method using a NO analyzer. Endothelial NO synthase (eNOS) protein levels in HUVECs was measured in cell lysates by ELISA. NO and eNOS results were normalized to cell number. Results: The cells treated with serum from e-cig users vs. nonsmokers produced less NO upon stimulation (P&lt;.03) and contained less eNOS protein (P&lt;.03). eNOS protein level was lower in the e-cig user serum group than in the cigarette smoker serum group (P&lt;.05), although stimulated NO production was decreased less by e-cig user serum than by smoker serum. Lower eNOS levels in the cigarette group vs. non-smoker group was not significant, but stimulated NO was significantly lower in the cigarette group vs. nonsmoker (P&lt;.006; see figure). Conclusion: Exposure of cultured endothelial cells to circulating factors from e-cig users, relative to non-users, leads to lower eNOS protein levels and decreased NO production.

Objective To investigate the effects of sitagliptin and glucagon like peptide-1 (GLP-1) on the iron metabolism in human umbilical vein endothelial cells (HUVECs) under normal and high glucose conditions. Methods HUVECs were treated with sitagliptin (1 μmol/L) and/or GLP-1 (100 nmol/L) under normal (5.5 mmol/L) and high glucose (30.0 mmol/L) conditions, and the corresponding control groups were also included. Cell viability was evaluated. After treatment for 24 h in normal glucose and 48 h in high glucose, mRNA and protein expressions of frataxin (FXN) and aconitase1 (ACO1) were measured by real-time PCR and Western blotting, respectively. Multiple-group comparisons were performed using one-way variance analysis.The two groups were compared using the Bonferroni test (when the variance homogeneity test was performed) or the Dunnett T3 test (when the variance homogeneity test was not satisfied). Results (1) Comparing with the control group (assuming its expression level was 1) under normal glucose condition, cell viability had no significance among those groups treated with GLP-1, sitagliptin and the combinations of sitagliptin and GLP-1 (0.99±0.04, 1.07±0.08, 1.06±0.05, F=4.059, all P>0.05); mRNA expressions of FXN were significantly decreased by combinations of sitagliptin and GLP-1 administration (0.68±0.18, t=3.59, P 0.05). (2) Comparing with the control group (assuming its expression level was 1) under high glucose condition, mRNA expressions of FXN were significantly increased by combinations of sitagliptin and GLP-1 administration (1.75±0.26, t=-5.71, P<0.05), while mRNA expressions of ACO1 showed no much significance after treatment with sitagliptin and/or GLP-1. Conclusions Without affecting cell proliferation in normal state of endothelial conditions, sitagliptin and GLP-1 may affect the intracellular iron metabolism by regulating the expression of FXN and ACO1 in HUVECs in normal or hyperglycemic conditions. Key words: Sitagliptin; Glucagon like peptide-1; Frataxin; Aconitase1; Human umbilical vein endothelial cells

Anagliptin ameliorates high glucose- induced endothelial dysfunction via suppression of NLRP3 inflammasome activation mediated by SIRT1

Effects of endomorphins on human umbilical vein endothelial cells under high glucose

Motorcycle exhaust particles up-regulate expression of vascular adhesion molecule-1 and intercellular adhesion molecule-1 in human umbilical vein endothelial cells

Although experimental animal and clinical trials have suggested that additional mechanisms other than protein kinase C (PKC)-β activation are involved in the vascular pathology of diabetic complications, current knowledge on the role of PKC-delta is incomplete and inconclusive. Human umbilical vein endothelial cells (HUVECs) were cultured in both high and normal glucose conditions and infected with recombinant adenovirus to overexpress PKC-delta. PKC-delta expression was also down-regulated using the PKC-delta inhibitor, rottlerin. Using flow cytometric analysis, we showed that PKC-delta is activated and translocates to the nucleus under high glucose conditions. Augmented cell apoptosis and cell cycle arrest were observed in a PKC-delta-dependent manner in the HUVECs. Furthermore, proteomic analyses identified 51 high glucose-induced and PKC-delta-associated proteins, and subsequent matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis uncovered a total of 37 unique proteins. The majority of identified proteins were previously unknown targets of PKC-delta signaling and were involved in the regulation of the cell cycle and apoptosis, tumor suppression, transcription, stress and signal transduction within the nucleus. Our data show that PKC-delta is an important mediator of cell apoptosis and cell cycle arrest in HUVECs under high glucose stress.