CLIC4 Regulates Endothelial Barrier Control by Mediating PAR1 Signaling via RhoA.

BACKGROUND: Endothelial CLICs (chloride intracellular channel proteins) CLIC1 and CLIC4 are required for the GPCRs (G-protein-coupled receptors) S1PR1 (sphingosine-1-phosphate receptor 1) and S1PR3 to activate the small GTPases Rac1 (Ras-related C3 botulinum toxin substrate 1) and RhoA (Ras homolog family member A). To determine whether CLIC1 and CLIC4 function in additional endothelial GPCR pathways, we evaluated CLIC function in thrombin signaling via the thrombin-regulated PAR1 (protease-activated receptor 1) and downstream effector RhoA. METHODS: We assessed the ability of CLIC1 and CLIC4 to relocalize to cell membranes in response to thrombin in human umbilical vein endothelial cells (HUVEC). We examined CLIC1 and CLIC4 function in HUVEC by knocking down expression of each CLIC protein and compared thrombin-mediated RhoA or Rac1 activation, ERM (ezrin/radixin/moesin) phosphorylation, and endothelial barrier modulation in control and CLIC knockdown HUVEC. We generated a conditional murine allele of Clic4 and examined PAR1-mediated lung microvascular permeability and retinal angiogenesis in mice with endothelial-specific loss of Clic4. RESULTS: Thrombin promoted relocalization of CLIC4, but not CLIC1, to HUVEC membranes. Knockdown of CLIC4 in HUVEC reduced thrombin-mediated RhoA activation, ERM phosphorylation, and endothelial barrier disruption. Knockdown of CLIC1 did not reduce thrombin-mediated RhoA activity but prolonged the RhoA and endothelial barrier response to thrombin. Endothelial-specific deletion of Clic4 in mice reduced lung edema and microvascular permeability induced by PAR1 activating peptide. CONCLUSIONS: CLIC4 is a critical effector of endothelial PAR1 signaling and is required to regulate RhoA-mediated endothelial barrier disruption in cultured endothelial cells and murine lung endothelium. CLIC1 was not critical for thrombin-mediated barrier disruption but contributed to the barrier recovery phase after thrombin treatment.

CDK5RAP3 Inhibition by Hypoxia Activates P38MAPK to Facilitate Angiogenesis.

CDK5RAP3 is a recognized tumor suppressor that inhibits Chk1 and Chk2 and activates p53, all of which are involved with mediating toxin-induced apoptosis of cancer cells. CDK5RAP3 also inhibits p38MAPK phosphorylation and activity via mediating a p38 interaction with wild-type p53-induced phosphatase 1. This study aimed to investigate the antiangiogenic activity of CDK5RAP3 and its molecular mechanisms in human umbilical vein endothelial cells (HUVECs) under conditions of hypoxic conditions. Angiogenesis was induced in HUVECs mainly by vascular endothelial growth factor (VEGF). The CDK5RAP3 levels of HUVECs were reduced in a time-dependent manner in response to hypoxic treatment at 2% O2. The reduction of CDK5RAP3 was accompanied with increased p38MAPK phosphorylation and activation. Moderate hypoxia was found to significantly increase secreted VEGF concentrations, and the hypoxic conditioned medium (HCM) markedly enhanced proliferation, migration, and tube formation. Our findings indicate that moderate hypoxia facilitates angiogenesis by inhibiting CDK5RAP3. CDK5RAP3 exhibits a clear regulatory role in vascular regeneration, as downregulating its expression in endothelial cells enhances VEGF synthesis and subsequently improves cell migration and lumen formation capability. This study presents evidence indicating that moderate hypoxia facilitates angiogenesis by inhibiting CDK5RAP3, demonstrating the potential for CKD5RAP3 to be a potent antiangiogenic agent in angiogenesis regulation of cancer, ischemic diseases, and wound healing.

[Effects of propranolol on the biological behavior of human umbilical vein endothelial cells and the expression of SOX18, MMP-7, and VEGFA]. / æ™®è˜æ´›å°”å¯¹äººè„é™è„‰å† çš®ç»†èƒžç”Ÿç‰©å­¦è¡Œä¸ºåŠSOX18、MMP-7、VEGFA表达的影响.

OBJECTIVES: To investigate the effects of propranolol on the proliferation, apoptosis, migration, and tube formation ability of human umbilical vein endothelial cells (HUVEC), as well as its impact on the expression of sex-determining region Y-box 18 (SOX18), matrix metalloproteinase-7 (MMP-7), and vascular endothelial growth factor A (VEGFA). METHODS: HUVEC were treated with different concentrations of propranolol, and cell viability was assessed using the CCK-8 method to determine the optimal concentration and treatment duration. The experiment consisted of a control group and groups treated with different concentrations of propranolol (50, 100, 150 µmol/L). Apoptosis, migration, and tube formation of HUVEC were observed using flow cytometry, wound healing assays, and tube formation assays. Western blot and real-time quantitative PCR were used to detect the expression levels of SOX18, MMP-7, and VEGFA proteins and mRNA. RESULTS: Compared to the control group, the apoptosis rate in the propranolol treatment groups increased significantly (P<0.05), and it rose significantly with increasing drug concentration (P<0.05). The wound healing rate decreased in the propranolol treatment groups, and both the number of tube formation nodes and total tube length were reduced (P<0.05). The expression levels of SOX18, MMP-7, and VEGFA proteins and mRNA were downregulated in the propranolol treatment groups (P<0.05). CONCLUSIONS: Propranolol can inhibit the proliferation, migration, and tube formation ability of HUVEC and promote cell apoptosis, resulting in decreased expression levels of SOX18, MMP-7, and VEGFA.

System to screen and purify active ingredients from herbal medicines using hydrogel-modified human umbilical vein endothelial cell membrane chromatography coupled with semi-preparative high-performance liquid chromatography-offline-high-performance liquid chromatography-mass spectrometry.

Analytical screening and validation systems based on a combination of cell membrane chromatography and two-dimensional chromatography-tandem mass spectrometry are incapable of providing prepared samples containing the active ingredients found in traditional Chinese medicine; therefore, these samples cannot be directly used in subsequent studies. In this study, a semi-preparative cell membrane chromatography column was developed using a hydrogel-modified carrier and human umbilical vein endothelial cells to optimize prepared conditions, such as hydrogel polymerization, cell fragmentation, and cell membrane volume. This increased the binding ratio of membrane protein and carrier to 15.79 mg/g. The column was systematically evaluated using multitarget tyrosine kinase inhibitors that displayed good specificity and reproducibility. Subsequently, using the column coupled with a semi-preparative high-performance liquid chromatography-offline-high-performance liquid chromatography-mass spectrometry system, 15 active ingredients were screened and purified from Indigo naturalis, and five main components were identified: l-lysine, oxyresveratrol, tryptanthrin, isorhamnetin, and indirubin. Furthermore, the pharmacological effects of the ingredients were confirmed using cell proliferation and apoptosis assays. Results revealed potent proliferation-inhibiting and apoptosis-promoting abilities on human chronic myelogenous leukemic cells and human promyelocytic leukemic cells (p < 0.001). Overall, the system presented screening and purification functions that could be used to prepare I. naturalis samples acting on the epidermal growth factor receptor and vascular endothelial cell growth factor.

Fucoidan promotes angiogenesis and accelerates wound healing through AKT/Nrf2/HIF-1α signalling pathway.

After skin injury, wound repair involves a complex process in which angiogenesis plays a crucial role. Previous research has indicated that fucoidan may aid in wound healing; we therefore hypothesised that fucoidan may speed up the process by promoting angiogenesis. In this study, we investigated the potential molecular mechanism underlying fucoidan's ability to accelerate wound healing by promoting angiogenesis. Using a full-cut wound model, we observed that fucoidan significantly intensified wound closure and promoted granulation formation and collagen deposition. Immunofluorescence staining revealed that fucoidan also promoted wound angiogenesis, specifically by accelerating the migration of new blood vessels to the middle area of the wound. Furthermore, fucoidan demonstrated the ability to enhance the proliferation of human umbilical vein endothelial cells (HUVECs) damaged by hydrogen peroxide (H2 O2 ) and to improve the formation of endothelial tubes. Mechanistic studies revealed that fucoidan upregulated the protein levels of the AKT/Nrf2/HIF-1α signalling pathway, which plays a crucial role in angiogenesis. This was further confirmed using the inhibitor LY294002, which reversed the promotion of endothelial tube formation by fucoidan. Overall, our findings suggest that fucoidan can promote angiogenesis via the AKT/Nrf2/HIF-1α signalling pathway and accelerate wound healing.

[Effect of procalcitonin on lipopolysaccharide-induced expression of nucleotide-binding oligomerization domain-like receptor protein 3 and caspase-1 in human umbilical vein endothelial cells]. / é™é’™ç´ åŽŸå¯¹è„‚å¤šç³–è¯±å¯¼çš„äººè„é™è„‰å† çš®ç»†èƒžNLRP3和caspase-1表达的影响.

OBJECTIVES: To study the effect of procalcitonin (PCT) on lipopolysaccharide (LPS)-induced expression of the pyroptosis-related proteins nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and caspase-1 in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were induced by LPS to establish a model of sepsis-induced inflammatory endothelial cell injury. The experiment was divided into two parts. In the first part, HUVECs were randomly divided into four groups: normal control, LPS (1 µg/mL), PCT (10 ng/mL), and LPS+PCT (n=3 each). In the second part, HUVECs were randomly grouped: normal control, LPS, and LPS+PCT of different concentrations (0.1, 1, 10, and 100 ng/mL) (n=3 each). Quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression levels of NLRP3 and caspase-1 in each group. RESULTS: In the first experiment: compared with the normal control group, the PCT, LPS, and LPS+PCT groups had significantly upregulated mRNA and protein expression levels of NLRP3 and caspase-1 (P<0.05); compared with the LPS group, the LPS+PCT group had significantly downregulated mRNA and protein expression levels of NLRP3 and caspase-1 (P<0.05). In the second experiment: compared with those in the LPS group, the mRNA and protein expression levels of NLRP3 and caspase-1 in the LPS+PCT of different concentrations groups were significantly downregulated in a concentration-dependent manner (P<0.05). CONCLUSIONS: LPS can promote the expression of the pyroptosis-related proteins NLRP3 and caspase-1 in HUVECs, while PCT can inhibit the LPS-induced expression of the pyroptosis-related proteins NLRP3 and caspase-1 in HUVECs in a concentration-dependent manner.

Long noncoding TUG1 promotes angiogenesis of HUVECs in PE via regulating the miR-29a-3p/VEGFA and Ang2/Tie2 pathways.

BACKGROUND: Preeclampsia (PE) is a pregnancy-specific disease that is associated with oxidative stress-induced endothelial dysfunction. Long noncoding RNAs (lncRNAs) are related to PE progression. The purpose is to study whether lncRNA taurine-upregulated gene 1 (TUG1) takes part in endothelial dysfunction in PE. METHODS: The placenta tissues were collected from PE patients and normal subjects. Human umbilical vein endothelial cells (HUVECs) were suffered from hypoxia-reoxygenation (H/R). TUG1, miR-29a-3p and vascular endothelial growth factor A (VEGFA) were detected via qRT-PCR. soluble fms-related tyrosine kinase-1 (sFLT1) and soluble endoglin (sENG) levels were detected by ELISA. Cell proliferation, migration, invasion and angiogenesis were examined via MTT, wound healing analysis, transwell and tube formation analysis. The proteins in VEGFA and angiopoietin 2 (Ang2)/tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Tie2) signaling were measured by western blot. The binding relationship was analyzed via Starbase, Jefferson and dual-luciferase reporter analysis. RESULTS: TUG1 and VEGFA levels were downregulated, and levels of miR-29a-3p, sFLT1 and sENG were increased in PE patients. TUG1 abundance was reduced in H/R-stimulated HUVECs, and TUG1 overexpression increased proliferation, migration, invasion and angiogenesis, and activated the VEGFA and Ang2/Tie2 signaling in H/R-stimulated HUVECs. TUG1 sponged miR-29a-3p, and miR-29a-3p overexpression reversed the function of TUG1 on H/R-induced HUVECs dysfunction. MiR-29a-3p knockdown attenuated H/R-induced inhibition of proliferation, migration, invasion, angiogenesis and activation of the VEGFA and Ang2/Tie2 signaling in HUVECs. VEGFA and Ang2 were targeted by miR-29a-3p, and VEGFA or Ang2 silence weakened the role of miR-29a-3p knockdown in H/R-caused HUVECs dysfunction. CONCLUSION: TUG1 facilitates proliferation, migration, invasion and angiogenesis in H/R-stimulated HUVECs via activating the VEGFA and Ang2/Tie2 signaling by regulating miR-29a-3p.

Bisdemethoxycurcumin Inhibits VEGF-Induced HUVECs Proliferation, Migration and Invasion through AMPK/mTOR Pathway-Dependent Autophagy Activation and Cell Cycle Arrest.

Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis, which plays a key role in the proliferation, migration and invasion of endothelial cell. Bisdemethoxycurcumin (BDMC) is a natural demethoxy curcumin derivative. In this study, we explored the mechanisms whereby BDMC is able to influence the proliferative, migratory and invasive activity of human umbilical vein endothelial cells (HUVECs) in response to VEGF treatment. These experiments revealed that BDMC at 10 and 20 µM suppressed HUVECs proliferation in response to VEGF (10 ng/mL) without impacting the proliferation in absence of VEGF. BDMC treatment also signifantly suppressed VEGF-induced migratory and invasive activity in HUVECs. However, the selective AMP-activated protein kinase (AMPK) inhibitor compound C (3 µM) treatment signifantly reversed all of these effects. Flow cytometric assay showed BDMC treatment was found to induce G0/G1 phase cell cycle arrest. Western blotting further indicated that BDMC treatment increased the ratios of p-AMPK/AMPK and LC3B/LC3A, up-regulated the expression of Beclin-1, decreased the ratio of p-mammalian target of rapamycin (mTOR)/mTOR, down-regulated the expression of cyclin D1 and CDK4. Overall, these data suggested that BDMC may exert benefical effect on HUVECs activation by activating autophagy and inducing cell cycle arrest through regulation of the AMPK/mTOR pathway, which could provide a potential compound candidate for the treatment of diseases related to VEGF overproduction.

Salvia miltiorrhiza Prevents Methylglyoxal-Induced Glucotoxicity via the Regulation of Apoptosis-Related Pathways and the Glyoxalase System in Human Umbilical Vein Endothelial Cells.

Methylglyoxal (MGO), which is produced as a byproduct of glucose metabolism, is the leading to diabetic cardiovascular complications. Salvia miltiorrhiza Bunge (Lamiaceae) has been reported as a potential plant to control diabetes and cardiovascular disease. However, no report exists on the effect of Salvia miltiorrhiza Bunge extract (SME) on MGO-induced glucotoxicity in human umbilical vein endothelial cells (HUVECs). We demonstrated the protective effects of SME (1, 5, and 10 µg/mL) and its components against MGO-induced endothelial dysfunction in HUVECs. Cytotoxicity was evaluated using the several in vitro experiments. Additionally, the protein expression of receptor of advanced glycation end-products (RAGE), mitogen-activated protein kinase (MAPK) pathway and glyoxalase system were measured. Then, the inhibitory effects of SME and its main components on MGO-induced oxidative stress, radical scavenging, formation of MGO-derived advanced glycation end products (AGEs), and MGO-AGEs crosslinking were evaluated. SME (10 µg/mL) strongly prevented expressed levels of RAGE, MGO-induced apoptosis and reduced reactive oxygen species (ROS) generation in HUVECs, comparing with 1 mM aminoguanidine. Additionally, SME (5 and 10 µg/mL) reduced the expression of proteins (e.g., p-extracellular signal-regulated kinase (ERK) and p-p38) in the MAPKs pathway and upregulated the glyoxalase system in HUVECs. SME (0.5-10 mg/mL), dihydrotanshinone (0.4 mM), and rosmarinic acid (0.4 mM) prevented MGO-AGEs formation and broke the MGO-AGE crosslinking. These results show that S. miltiorrhiza has protective effects against MGO-induced glucotoxicity by regulating the proteins involved in apoptosis, glyoxalase system and antioxidant activity. We expect that S. miltiorrhiza is a potential natural resource for the treatment of MGO-induced vascular endothelial dysfunction.

Titanium implant alters the effect of zoledronic acid on the behaviour of endothelial cells.

OBJECTIVES: To evaluate the effect of zoledronic acid (ZA) on human umbilical vein endothelial cells (HUVECs) attached to different surfaces. MATERIALS AND METHODS: A total of three groups were evaluated in this study: sandblasting and acid etching (SLA) + HUVECs; mechanically polished (MP) + HUVECs; and plastic cell culture plates + HUVECs. Scanning electron microscopy, energy-dispersive X-ray spectroscopy, surface roughness and water contact angle were tested for titanium surface characterisation. ZA was added at different concentrations (0, 1, 10, 50 and 100 µM). Cell adhesion, proliferation, viability, apoptosis and gene expression were evaluated. RESULTS: Mechanically polished and SLA surfaces showed negative effects on cell adhesion and proliferation and promoted cell apoptosis with 100 µM ZA (p < .05). The highest expression of intercellular adhesion molecule-1 (ICAM-1) and angiopoietin-1 was found on SLA surfaces (p < .01). The lowest expression of platelet-endothelial cell adhesion molecule-1 and ICAM-1 was found on MP surfaces (p < .05). A significant decrease in von Willebrand factor was detected on MP and SLA surfaces (p < .001). CONCLUSIONS: Zoledronic acid has an anti-angiogenic effect on HUVECs attached to titanium implants, while the SLA surface might stimulate HUVECs to express angiogenic and adhesive factor genes despite ZA treatment.

Phenolic Profile and Fingerprint Analysis of Akebia quinata Leaves Extract with Endothelial Protective Activity.

In contrast to the stem and fruit of Akebia quinata, A. quinata leaves as a source rich in phenolic compounds with potentially beneficial pharmacological activities have been largely overlooked. To develop and use A. quinata leaves as a resource, we evaluated its potential as a cardiovascular-protective agent. Herein, we investigated the effects and potential mechanisms of A. quinata leaves extract on lipopolysaccharide (LPS)-induced inflammatory responses in human umbilical vein endothelial cells. We found that A. quinata leaves extract pretreatment of 10 µg/mL significantly attenuated LPS-induced protein expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1. Furthermore, this extract also suppressed LPS-induced phosphorylation of nuclear factor-κB p65. In order to elucidate the chemical profiles of the samples, the HPLC fingerprint was established, and prominent peaks were identified via HPLC-electrospray ionization-mass spectrometry. Multivariate statistical analyses, including hierarchical cluster analysis, principal component analysis, and partial least-squares discriminant analysis, were performed to evaluate the clustering of the samples. It was found that isochlorogenic acid C was a key marker for the classification of A. quinata leaves from the Gongju and Muju city in Korea. Collectively, this study not only suggested the potential of A. quinata leaves as a novel therapeutic candidate for inflammatory cardiovascular disease but also developed a quality control method for A. quinata leaves, which could help to expand the application of A. quinata.

Extracellular vesicle-encapsulated microRNA-424 exerts inhibitory function in ovarian cancer by targeting MYB.

BACKGROUND: Recent studies have suggested a crucial role of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) in ovarian cancer treatment. We, therefore, set out to explore the mechanism through which MSC-derived EVs delivered microRNA-424 (miR-424) to influence the development of ovarian cancer. METHODS: Bioinformatics analyses were first performed to screen ovarian cancer-related differentially expressed genes and to predict regulatory miRNAs. Then, dual-luciferase reporter gene assay was carried out to verify the relationship between miR-424 and MYB. Subsequently, the characterized MSCs and isolated EVs were co-cultured with ovarian cancer cells, followed by determination of the expression patterns of miR-424, MYB, vascular endothelial growth factor (VEGF), and VEGF receptor (VEGFR), respectively. In addition, the effects of EVs-delivered miR-424 on the proliferation, migration, invasion and tube formation of ovarian cancer cells were assessed using gain- and loss-of-function approaches. Lastly, tumor xenograft was induced in nude mice to illustrate the influence of EVs-loaded miR-424 on ovarian cancer in vivo. RESULTS: Our data exhibited that MYB was highly-expressed and miR-424 was poorly-expressed in ovarian cancer. More importantly, MYB was identified as a target gene of miR-424. Additionally, the transfer of miR-424 by MSC-derived EVs was found to repress the proliferation, migration, and invasion of ovarian cancer cells, with a reduction in the expressions of VEGF and VEGFR. Furthermore, MSC-derived EVs over-expressing miR-424 could inhibit the proliferation, migration, and tube formation of human umbilical vein endothelial cells, and also suppressed tumorigenesis and angiogenesis of ovarian tumors in vivo. CONCLUSION: Collectively, our findings indicate that MSC-derived EVs transfer miR-424 to down-regulate MYB, which ultimately led to the inhibition of the tumorigenesis and angiogenesis of ovarian cancer. Hence, this study offers a potential prognostic marker and a therapeutic target for ovarian cancer.

Psoriatic mesenchymal stem cells stimulate the angiogenesis of human umbilical vein endothelial cells in vitro.

OBJECTIVE: To investigate the regulation of psoriatic dermal mesenchymal stem cells (p-DMSCs) in the expression of vascular growth factor (VEGF), and migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: A co-culture model of HUVECs and dermal mesenchymal stem cells (DMSCs)was used in this study. After 7-day co-culture, changes in expression levels of VEGF mRNA and protein in HUVECs were assessed using RT-PCR and Western Blotting, respectively. Migration and tubular formation of HUVECs were also assessed following co-culture of DMSCs and HUVECs. RESULTS: In comparison to either HUVECs alone or co-culture of n-DMSCs and HUVECs, co-culture of HUVECs and p-DMSCs significantly increased expression levels of both VEGF mRNA (p < 0.01 vs. HUVECs alone) and protein in HUVECs (p < 0.001 vs. both HUVECs alone and HUVECs co-cultured with n-DMSCs). Moreover, p-DMSCs stimulated HUVEC migration and vascular formation (p < 0.05 vs. both HUVECs alone and co-culture of n-DMSCs and HUVECs). CONCLUSION: Psoriatic DMSCs can upregulate VEGF expression, and stimulate migration and angiogenesis of HUVECs, suggesting a pathogenic role of p-DMSCs in psoriasis.

Tetrahydroxystilbene glucoside alleviates angiotensin II induced HUVEC senescence via SIRT1.

Tetrahydroxystilbene glucoside (TSG), an active ingredient of Polygonum multiflorum, has been known for certain anti-aging effects. In this study, the possible protective mechanism of TSG on human umbilical vein endothelial cell (HUVEC) senescence induced by angiotensin II (Ang II) was investigated. The results revealed that TSG pretreatment could reduce the percentage of senescence-associated ß-galactosidase (SA-ß-gal) positive cells and decrease the expression levels of the cellular senescence biomarker proteins p53 and PAI-1. At the same time, the expression of SIRT1 in senescent cells showed an upward trend due to TSG treatment. When inhibiting the expression of SIRT1 by EX527, our results showed that TSG reversed the effect of EX527 by promoting the expression level of SIRT1, reducing the expression of SA-ß-gal positive cells and the expression level of p53 and PAI-1 proteins. The present study demonstrated that TSG could protect against HUVEC senescence induced by Ang II, potentially through modulation of SIRT1 activity.

Febuxostat Attenuates the Induction of Vascular Cell Adhesion Protein 1 by TNF-α in Human Umbilical Vein Endothelial Cells.

In a randomized trial, higher all-cause and cardiovascular mortality was observed in treatment with febuxostat than with allopurinol in patients with coexisting gout and serious cardiovascular conditions. In this study, we focus on an intervention of febuxostat or allopurinol as an anti-inflammatory treatment to reduce the transcription of nuclear factor-kappa B (NF-κB) and production of relevant inflammatory factors. We evaluated the effect of febuxostat on vascular cell adhesion protein 1 (VCAM-1) induction in cultured human umbilical vein endothelial cells (HUVECs). Cells were exposed to tumor necrosis factor (TNF)-α (10 ng/mL) treatment for 24 h. Febuxostat or allopurinol (0.1-100 µM) was added to the bath medium 15 min before TNF-α treatment. VCAM-1 levels in HUVECs increased after 24-h TNF-α treatment (n = 4). Febuxostat and allopurinol significantly suppressed VCAM-1 induced by treatment with TNF-α in a dose-dependent manner (p < 0.05, n = 4). Furthermore, these drugs suppressed the NF-κB protein levels in the nucleus 4 h after TNF-α treatment (n = 3 or 4). Our results suggest that TNF-α induces VCAM-1 production via NF-κB, which can be blocked by febuxostat or allopurinol. The effect of febuxostat treatment on cardiovascular events may be associated with protection against the infiltration of lymphocytes or monocytes through VCAM-1 induction in inflamed endothelial cells such as arterial sclerosis.

The effect of gestational diabetes on identification of key genes and pathways in human umbilical vein endothelial cell by integrated bioinformatics analysis.

Maternal diabetes may lead to long-term risks for the offspring. The study aims at identifying the potential crucial genes and pathways associated with foetal metabolism and malformation of gestational diabetes mellitus (GDM). Gene Expression Series 49524 and 87295 were downloaded from Gene Expression Omnibus database, including eight from GDM and eight from non-GDM. A total of 35 differentially expressed genes were identified. Gene ontology functional annotation and signalling pathway analyses were performed. Four hub genes were identified by protein-protein interaction network: SHH, E2F1, STAT1, and HOXA9. The four hub genes were assessed by western blot and real-time quantitative PCR in clinical samples. The results of this data mining and integration help to reveal the pathophysiologic and molecular mechanism imprinted in primary umbilical cord-derived cells from GDM offspring. These genes and pathways identified are potential stratification biomarkers and provide further insight for developing therapeutic intervention for the offspring of diabetic mothers.Impact statementWhat is already known on this subject? Maternal diabetes may lead to long-term risks for the offspring. A high glucose environment might change the umbilical cord expression of genes implicated in foetal metabolism and development. However, underlying molecular mechanisms have not been investigated thoroughly.What do the results of this study add? GO functional annotation showed that the biological functions of differentially expressed genes mainly involved in metanephros development, salivary gland morphogenesis, fat cell differentiation, vasculogenesis, muscle cell proliferation, heart morphogenesis and Wnt signalling pathway. Signalling pathway analyses found that these differentially expressed genes mainly implicated in the apoptosis, cell cycle, Hedgehog, P53, and NOTCH signalling pathway. Four hub genes were identified by protein-protein interaction network: SHH, E2F1, STAT1 and HOXA9.What are the implications of these findings for clinical practice and/or further research? The genes and pathways identified in the present study are potential stratification biomarkers and provide further insight for developing therapeutic intervention for the offspring of diabetic mothers.

Sitagliptin attenuates endothelial dysfunction independent of its blood glucose controlling effect.

Although the contributions of sitagliptin to endothelial dysfunction in diabetes mellitus were previously reported, the mechanisms still undefined. Autophagy plays an important role in the development of diabetes mellitus, but its role in diabetic macrovascular complications is unclear. This study aims to observe the effect of sitagliptin on macrovascular endothelium in diabetes and explore the role of autophagy in this process. Diabetic rats were induced through administration of high-fat diet and intraperitoneal injection of streptozotocin. Then diabetic rats were treated with or without sitagliptin for 12 weeks. Endothelial damage and autophagy were measured. Human umbilical vein endothelial cells were cultured either in normal glucose or in high glucose medium and intervened with different concentrations of sitagliptin. Rapamycin was used to induce autophagy. Cell viability, apoptosis and autophagy were detected. The expressions of proteins in c-Jun N-terminal kinase (JNK)-Bcl-2-Beclin-1 pathway were measured. Sitagliptin attenuated injuries of endothelium in vivo and in vitro. The expression of microtubuleassociated protein 1 light chain 3 II (LC3II) and beclin-1 were increased in aortas of diabetic rats and cells cultured with high-glucose, while sitagliptin inhibited the over-expression of LC3II and beclin-1. In vitro pre-treatment with sitagliptin decreased rapamycin-induced autophagy. However, after pretreatment with rapamycin, the protective effect of sitagliptin on endothelial cells was abolished. Further studies revealed sitagliptin increased the expression of Bcl-2, while inhibited the expression of JNK in vivo. Sitagliptin attenuates injuries of vascular endothelial cells caused by high glucose through inhibiting over-activated autophagy. JNK-Bcl-2-Beclin-1 pathway may be involved in this process.

Elevated Expression of Vascular Adhesion Molecule-1, Plasminogen Activator Inhibitor-1, Cyclooxygenase-2, and Thrombomodulin in Human Umbilical Vein Endothelial Cells from Hospitalized Gestational Diabetes Mellitus Patients.

Protein expression in human umbilical vein endothelial cells (HUVECs) is a useful indicator of maternal condition and the intrauterine environment during pregnancy. Therefore, we investigated protein expression in HUVECs obtained from patients with gestational diabetes mellitus (GDM). HUVECs were prepared from the umbilical cords of GDM patients and controls who underwent planned cesarean section between 2013 and 2014 at Teikyo University Hospital (Tokyo, Japan). There were no differences in blood glucose levels between the GDM patients and controls at admission. However, pre-pregnancy body mass index (BMI) was higher in GDM patients, although the changes in gestational BMI were smaller during hospitalization. To evaluate the state of the endothelium, we examined the protein expression levels of vascular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1, thrombomodulin (TM), endothelial nitric oxide synthase, plasminogen activator inhibitor-1 (PAI-1), cyclooxygenase-2 (COX-2), and VE-cadherin, which are altered by various factors in endothelial tissue. VCAM-1, PAI-1, and COX-2 expression was higher in HUVECs from patients with GDM than the controls. Because the pre-pregnancy BMI was higher in GDM patients, we examined the relationship between BMI and protein expression. However, the expression levels of these proteins were not correlated with pre-pregnancy BMI and were higher in HUVECs from BMI-matched GDM patients than from BMI-matched controls. Intriguingly, TM expression was also higher in HUVECs from BMI-matched GDM patients. Thus, expression of VCAM-1, PAI-1, COX-2, and TM may reflect certain factors in the intrauterine environment that are altered in hospitalized GDM patients with controlled body weight.

Ultravist Induces the Expression of MCP-1 and VCAM-1 in IL-4-Stimulated HUVECs.

The goal of the present study focused on the adverse reaction of contrast medium (CM) via the induction of inflammatory molecules in human umbilical vein endothelial cells (HUVECs). Ultravist-induced monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) gene expression was markedly increased in interleukin-4 (IL-4)-pretreated HUVECs in a time- and dose-dependent manner and was paralleled by concomitant production of MCP-1 and VCAM-1 proteins. MCP-1 and VCAM-1 gene expression by Ultravist in combination with IL-4 was mediated by the c-Jun N-terminal kinases (JNK1/2) signaling pathway. IL-4-pretreated Ultravist-stimulated HUVECs showed greatly increased migration and adhesion of THP-1 cells. Cell migration was decreased by treatment of CCR2 antagonist, and cell adhesion was also decreased by VCAM-1 blocking antibody. Furthermore, when tested in vivo under similar conditions, MCP-1 protein was significantly increased in Ultravist combined with IL-4-injected mice. Taken together, our findings suggest that MCP-1 blocking may be crucial in preventing the endothelial dysfunction induced by contrast medium in patients with inflammatory disease and atherosclerosis.

Epimedin C Protects H2O2-Induced Peroxidation Injury by Enhancing the Function of Endothelial Progenitor HUVEC Populations.

Endothelial cell injury and apoptosis induced by oxidative stress serve important roles in many vascular diseases. The repair of endothelial cell vascular injury relies on the function of local endothelial progenitor cells (EPCs). Our previous study indicated that epimedin C, a major flavonoid derived from Herba epimedii (yin yang huo), could promote vascularization by inducing endothelial-like differentiation of mesenchymal stem cells C3H/10T1/2 both in vivo and in vitro. In view of the significant cardiovascular protective effects of Herba epimedii, we detected a protective effect of epimedin C on hydrogen peroxide (H2O2)-induced peroxidation injury in human umbilical vein endothelial cells (HUVECs) and the role of EPC in this process. The results show that epimedin C increased the expression of the stem cell marker, CD34 and PROM1, and subsequently enhanced the expression and function of vascular endothelial growth factor and matrix metalloproteinase (MMP)-2 in local vascular endothelial cells. In conclusion, epimedin C protects H2O2-induced peroxidation injury by enhancing the function of endothelial progenitor HUVEC populations.