Human Umbilical Vein Endothelial Cells Lines Research Articles

Role of UDP-glucose ceramide glucosyltransferase in venous malformation.

Venous malformation (VM) results from the abnormal growth of the vasculature; however, the detailed molecular mechanism remains unclear. As a glycosyltransferase, UDP-glucose ceramide glucosyltransferase (UGCG) is localized to the Golgi body and is a key enzyme in the first step of glycosphingolipid synthesis. Here, we aimed to explore the relationship between UGCG and the development of VM. First, investigations using RT-qPCR and Western blotting on the diseased vasculature of VM patients and normal vascular tissues revealed that UGCG expression was markedly elevated in the diseased vessels. Subsequently, immunofluorescence assay showed that UGCG was co-localized with CD31, an endothelial cell marker, in tissues from patients with VM and healthy subjects. Then, we established TIE2-L914F-mutant human umbilical vein endothelial cells (HUVECs) by lentivirus transfection. Next, Western blotting revealed that UGCG expression was considerably higher in HUVECsTIE2-L914F. In addition, we established a UGCG-overexpressing HUVECs line by plasmid transfection. With the CCK8 cell proliferation experiment, wound healing assay, and tube formation assay, we found that UGCG could promote the proliferation, migration, and tube formation activity of HUVECs, whereas the inhibition of UGCG could inhibit the proliferation, migration, and tube formation activity of HUVECsTIE2-L914F. Finally, Western blotting revealed that UGCG regulates the AKT/mTOR pathway in HUVECs. These data demonstrated that UGCG can affect the activity of vascular endothelial cells and regulate the AKT/mTOR signaling pathway; this is a potential mechanism underlying VM pathogenesis.

Ultrasound-Stimulated Microbubbles Enhance Radiation-Induced Cell Killing.

Recent in vivo studies using ultrasound-stimulated microbubbles as a localized radiosensitizer have had impressive results. While in vitro studies have also obtained similar results using human umbilical vein endothelial cells (HUVEC), studies using other cell lines have had varying results. This study was aimed at investigating any increases in radiation-induced cell killing in vitro using two carcinoma lines not previously investigated before (metastatic follicular thyroid carcinoma cells [FTC-238] and non-small cell lung carcinoma cells [NCI-H727]), in addition to HUVEC. Cells were treated using a combination of 1.6% (v/v) microbubbles, ∼90 s of 2-MHz ultrasound (mechanical index=0.8) and 0-6 Gy of kilovolt or MV X-rays. Cell viability assays obtained 72 h post-treatment were normalized to untreated controls, and analysis of variance was used to determine statistical significance. All cells treated with combined ultrasound-stimulated microbubbles and radiation exhibited decreased normalized survival, with statistically significant effects observed for the NCI-H727 cells. No statistically significant differences in effects were observed using kV compared with MV radiation. Further studies using increased microbubble concentrations may be required to achieve statistically significant results for the FTC-238 and HUVEC lines.

Nociceptin/Orphanin FQ receptor expression in primary human umbilical vein endothelial cells is not regulated by exposure to breast cancer cell media or angiogenic stimuli.

Opioid receptors are naloxone-sensitive (MOP [mu: μ], DOP [delta: δ], and KOP [kappa: κ]) and naloxone-insensitive Nociceptin/Orphanin FQ (N/OFQ) peptide receptor (NOP). Clinically, most opioid analgesics target MOP. Angiogenesis is the formation of new blood vessels and involves endothelial cell activation, proliferation, and migration. The effect of opioids on this process is controversial with no data for NOP receptor ligands. We used patient-derived human umbilical vein endothelial cells (HUVECs) treated with media from the Michigan Cancer Foundation-7 (MCF-7) breast cancer cells or vascular endothelial growth factor (VEGF; 10 ng ml-1) and fibroblast growth factor (FGF; 10 ng ml-1) as angiogenic stimuli. We have measured (i) NOP/MOP messenger RNA, (ii) receptor protein using N/OFQATTO594 and DermorphinATTO488 as fluorescent probes for NOP and MOP, and (iii) NOP/MOP function in a wound healing assay (crude measure of migration that occurs during angiogenesis). HUVEC lines from 32 patients were used. Using all 32 lines, mRNA for NOP but not MOP was detected. This was unaffected by media from MCF-7 cells or VEGF/FGF. There was no binding of either N/OFQATTO594(NOP) or DermorphinATTO488(MOP) in the absence or presence of angiogenic stimuli (six lines tested). In the absence of MOP mRNA, this was expected. Whilst MCF-7 conditioned medium (not VEGF/FGF) reduced wound healing per se (14 lines tested), there was no effect of N/OFQ (NOP ligand) or morphine (MOP ligand). Media from MCF-7 breast cancer cells or VEGF/FGF as angiogenic stimuli did not influence NOP translation into receptor protein. MOP was absent. In the absence of constitutive or inducible MOP/NOP, there was no effect on wound healing as a measure of angiogenesis.

In vitro sepsis induces Nociceptin/Orphanin FQ receptor (NOP) expression in primary human vascular endothelial but not smooth muscle cells.

Sepsis is a dysregulated host response to infection that can cause widespread effects on other organs including cardiovascular depression, hypotension and organ failure. The receptor for Nociceptin/Orphanin FQ (N/OFQ), NOP is expressed on immune cells and these cells can release the peptide. Exogenous N/OFQ can dilate blood vessels and this peptide is increased in animal and human sepsis. We hypothesise that NOP receptors are present on vascular endothelial cells and therefore provide the target for released N/OFQ to cause vasodilation and hence hypotension. Using human umbilical vein endothelial cells (HUVEC) and human vascular smooth muscle cells (HVSMC) freshly prepared from umbilical cords and up to passage 4, we assessed NOP mRNA expression by Polymerase Chain Reaction (PCR), NOP surface receptor expression using a fluorescent NOP selective probe (N/OFQATTO594) and NOP receptor function with N/OFQ stimulated ERK1/2 phosphorylation. As an in vitro sepsis mimic we variably incubated cells with 100ng/ml Lipopolysaccharide and Peptidoglycan G (LPS/PepG). HUVECs express NOP mRNA and this was reduced by ~80% (n = 49) after 24–48 hours treatment with LPS/PepG. Untreated cells do not express surface NOP receptors but when treated with LPS/PepG the reduced mRNA was translated into protein visualised by N/OFQATTO594 binding (n = 49). These NOP receptors in treated cells produced an N/OFQ (1μM) driven increase in ERK1/2 phosphorylation (n = 20). One (of 50) HUVEC lines expressed NOP mRNA and receptor protein in the absence of LPS/PepG treatment. In contrast, HVSMC expressed NOP mRNA and surface receptor protein (n = 10) independently of LPS/PepG treatment. These receptors were also coupled to ERK1/2 where N/OFQ (1μM) increased phosphorylation. Collectively these data show that an in vitro sepsis mimic (LPS/PepG) upregulates functional NOP expression in the vascular endothelium. Activation of these endothelial receptors as suggested from in vivo whole animal work may contribute to the hypotensive response seen in sepsis. Moreover, blockade of these receptors might be a useful adjunct in the treatment of sepsis.

Spatially guided endothelial tubulogenesis by laser-induced side transfer (LIST) bioprinting of HUVECs

The ability to bioprint microvasculature networks is central for drug screening and for tissue engineering applications. Here we used a newly developed bioprinting technology, termed laser-induced side transfer (LIST), to print human umbilical vein endothelial cells (HUVECs) and to spatially guide endothelial tubulogenesis. We investigated the effect of three bioprinting matrices (fibrin, Matrigel and Matrigel/thrombin) on HUVECs self-assembly. Furthermore, we studied the effect of pro- and anti-angiogenic compounds on sprouting angiogenesis and tubulogenesis. We found that HUVECs self-assembly is optimal on Matrigel/thrombin due to the formation of fibrin stripes that enhance HUVECs confinement and adhesion. Importantly, we showed that treatment of printed HUVEC lines with the anti-angiogenic factor bone morphogenetic protein 9 (BMP9) significantly improves the percentage of lumen coverage. Our results showcase LIST as a powerful bioprinting technology to study tubulogenesis and to screen compounds targeting microvasculature pathologies.

Absorption characteristics of ilexgenin A and ilexsaponin B1 in human umbilical vein endothelial cells after administration of the total triterpenoid saponins from Ilex pubescens.

Ilex pubescens is a famous Chinese herbal medicine, frequently used to treat cardiovascular disease in South China. In this study, we aim to explore the absorption properties of ilexgenin A (C1) and ilexsaponin B1 (C3) in vascular endothelial cells after administration of the total triterpenoid saponins from Ilex pubescens (IPTS) and clarify the possible transport mechanisms. A UPLC-qTOF-MS/MS system was used to identify the components in IPTS that could be intracellularly transported by human umbilical vein endothelial cells (HUVECs). Afterwards, a rapid, highly selective and sensitive method was established to simultaneously quantify the concentration of C1 and C3 in HUVECs after administration of IPTS. The results demonstrate that pretreatment with IPTS could promote the survival of HUVECs and reduce the damage caused by TNF-α to HUVECs. Among the main 11 components in IPTS, eight components could be absorbed by HUVECs, including seven triterpenoids and one phenolic acid. The uptake of C1 and C3 by HUVECs occurred in a time-, temperature- and concentration-dependent manner, indicating the participation of passive diffusion and active transportation mechanisms. The two triterpenoid saponins all exhibited rapid absorption and a bimodal phenomenon in their concentration-time profiles, and equilibrium could be achieved after 6h. Furthermore, C1 and C3 intracellular transportation was regulated by serum proteins, sodium-dependent glucose transporter 1 and P-glycoprotein. The current research for the first time demonstrates the in vitro pharmacokinetics characteristics of C1 and C3 in HUVECs lines, which could supply a new way of understanding the treatment of cardiovascular diseases.

Abstract 11232: Role of Line-1 Retrotransposition in Atherosclerosis

Introduction: LINE-1 (L1) retrotransposons are a class of transposable elements comprising ~21% of the human genome. L1-retrostransposed elements move within the genome, where they profoundly alter gene function. Somatic L1 insertions, or retrotranspositions (RTNs), were implicated in multiple diseases including cancer. However, this literature has been mainly confined to epithelial tissue, and the role of L1 in cardiovascular diseases is unknown. Specifically, whether L1 presence and activity in vascular cells has a functional role in atherosclerosis, a disease characterized by extensive vascular remodeling, inflammation and plaque formation, remains unexplored. Hypothesis: L1 expression and RTN in vascular endothelial cells (ECs) leads to endothelial activation in atherosclerosis. Methods: L1 mRNA expression was analyzed by qRT-PCR in postmortem coronary artery specimens from patients with atherosclerosis and non-diseased controls. Immunofluorescence (IF) in coronary tissue was performed for ORF1p, a L1 encoded polypeptide required for active RTN. In vitro, qRT-PCR was used to assess L1 expression in primary human aortic ECs (HAEC) treated with proatherogenic inflammatory stimuli TNFα, IL6, oxidized LDL (oxLDL) and IL1β. Active RTN was assessed by FACS and IF in a purpose-built human umbilical vein EC line (CRL2922) L1 RTN detection system. Results: L1 mRNA was increased in atherosclerotic human coronary arteries and positively correlated with age and histologically-assessed disease severity. ORF1p expression increased in EC of atherosclerotic plaques indicative of increased L1 activity. L1 expression and retrotransposition was induced in ECs in vitro by TNFα, IL-6 and oxLDL. Finally, de novo L1 insertion alone resulted in upregulation of genes involved in EC activation and atherosclerosis (ICAM1, MCP-1, IL6, NOR), and this effect was further enhanced by co-treatment with TNFα, IL-6 and oxLDL. Conclusions: Our results identify a disease-related increase in L1 in the vascular endothelium under atherosclerotic conditions that contributes to EC activation. These data support a permissive environment during atherosclerosis for L1 activity and unveil a previously unrecognized role for L1 in this disease’s pathophysiology.

Potency and selectivity indices of Myristica fragrans Houtt. mace chloroform extract against non-clinical and clinical human pathogens

Abstract This study assessed the antimicrobial, toxicity, and phytochemical profiles of Myristica fragrans extracts. Different solvent extracts were tested for antimicrobial activity against clinical and reference microbial strains, using disc and well diffusion assays and microdilution techniques. Antioxidant potential was investigated using 2,2-diphenyl-1-picryhydrazyl (DPPH) assays. Cytotoxicity assay was conducted against human umbilical vein endothelial cells (HUVECs) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Acute toxicity was assessed in laboratory Swiss albino mice at a single dose of 2,000 mg/kg body weight for 14 days. To assess the phytochemical constituents, spectrophotometric and gas chromatography-mass spectrometry (GC-MS) methods were used. The chloroform extract revealed antimicrobial potencies against the Gram-positive bacteria and C. albicans with minimum inhibitory concentrations. In the DPPH assay, the IC50 value of the chloroform extract was determined to be 1.49 mg/mL. The phenolic and flavonoid contents were 26.64 ± 0.1 mg of gallic acid equivalents/g and 8.28 ± 0.1 mg quercetin equivalents/g, respectively. The IC50 value was determined to be 49 µg/mL against the HUVEC line. No mortality or morbidity was observed. GC-MS analysis indicated the presence of 2-cyclopenten-1-one (44.72%) as a major compound. The current results provide scientific support for the use of M. fragrans in folk medicine.

The Effect of a Unique Region of Parvovirus B19 Capsid Protein VP1 on Endothelial Cells.

Parvovirus B19 (B19V) is a widespread human pathogen possessing a high tropism for erythroid precursor cells. However, the persistence or active replication of B19V in endothelial cells (EC) has been detected in diverse human pathologies. The VP1 unique region (VP1u) of the viral capsid has been reported to act as a major determinant of viral tropism for erythroid precursor cells. Nevertheless, the interaction of VP1u with EC has not been studied. We demonstrate that recombinant VP1u is efficiently internalized by rats’ pulmonary trunk blood vessel-derived EC in vitro compared to the human umbilical vein EC line. The exposure to VP1u was not acutely cytotoxic to either human- or rat-derived ECs, but led to the upregulation of cellular stress signaling-related pathways. Our data suggest that high levels of circulating B19V during acute infection can cause endothelial damage, even without active replication or direct internalization into the cells.

Investigation of biological activities of plant extract and green synthesis silver nanoparticles obtained from Gilaburu (Viburnum opulus L.) fruits.

In this study, biological activities of silver nanoparticles (AgNP) synthesized by green synthesis method using the fruit extract of Gilaburu (Viburnum opulus L.) plant were investigated. The characterization of the synthesized AgNPs was performed by ultraviolet visible region spectroscopy (UV-Vis), Fourier transform - infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and X-ray diffraction (XRD) techniques. The crystal size was found to be 52.32 nm when the XRD data were calculated with the Debye-Scherrer's equation. The antimicrobial activities of extract and AgNPs were investigated by microdilution and disk diffusion methods. Antibiofilm activities were examined by the crystal violet technique. The cytotoxic effects of the extract and AgNPs against MCF-7(human breast cancer) and HUVEC (human umbilical vein endothelial cells) cell lines were evaluated by MTT assay. The IC50 valuesfor the HUVEC line were found to be 0.97 mg/mL (AgNP) and 85.24 mg/mL (extract), while the IC50 values for the MCF-7 line were determined as 0.022 μg/mL (AgNP) and 0.021 μg/mL (extract). To our knowledge, this is the first report to comprehensively examine the biological activities of V. opulus L. extract and biosynthetic AgNPs.

MicroRNA-142-5p is Up-regulated on Allogeneic Immune Responses and Up-regulates MHC Class II Expression in Human Umbilical Vein Endothelial Cells

PurposeMicroRNA could be biomarker and therapeutic target for rejection. The aim of this study was to investigate the role of miR-142-5p in allogeneic immune responses using in vitro and in vivo models. Materials and MethodsPrimary and immortalized human umbilical vein endothelial cells (HUVECs) were cultured with unrelated blood mononuclear cells to induce allogeneic immune responses. Syngeneic and allogeneic skin graft was performed in mice. Flow cytometry, quantitative reverse transcription–polymerase chain reaction, and Western blotting was performed to understand the underlying mechanisms. ResultsmiR-142-5p was up-regulated in primary HUVEC and a HUVEC line when allogeneic immune responses were elicited. miR-142-5p was also up-regulated in the murine allogeneic skin graft. Overexpression of miR-142-5p in HUVEC increased the expression of HLA-ABC and HLA-DR additively to allogeneic immune responses, suggesting a possible increase in alloantigen presentation. Inhibition of miR-142-5p reduced the expression of HLA-DR. ZEB1, a putative target gene of miR-142-5p, was down-regulated in HUVEC on allogeneic immune response as well as in murine allogeneic skin graft. ConclusionThese results suggest that the up-regulation of miR-142-5p on allogeneic immune response might facilitate endothelial activation to exacerbate rejection.

Role of Junction-Mediating and Regulatory Protein in the Pathogenesis of Glucocorticoid-Induced Endothelial Cell Lesions.

ObjectiveNontraumatic osteonecrosis of the femoral head (ONFH) is one of the most common diseases in orthopaedics. The damage to vascular endothelial (VE) cells caused by glucocorticoids (GC) has been reported as a possible mechanism of pathogenesis for ONFH. Junction‐mediating and regulatory protein (JMY), originally identified as a p53 coactivator, plays prominent roles in the DNA damage response and in cell motility. This study aimed to discover the role of JMY in the pathogenesis of GC‐induced endothelial cell lesions.MethodsHigh‐throughput RNA sequencing was performed to identify the differentially expressed genes between GC‐treated human umbilical vein endothelial cells (HUVEC) and control cells. JMY knockdown and overexpressing HUVEC lines were treated with GC. Cell proliferation was examined with a survival cell count assay (Cell Counting Kit‐8, CCK‐8); cell apoptosis was measured by flow cytometry; a scarification assay was used to detect the capability of cell migration; a Transwell chamber assay was done to detect the cell motility . Differential expression of cell protein was detected by western blot.ResultsA total of 1561 differential genes were obtained through transcription sequencing, of which 789 mRNA were upregulated and 772 mRNA were downregulated in the GC‐treated HUVEC compared with the control cells. CCK‐8 assay results showed that: without GC treatment, overexpression or knockdown of JMY did not affect the proliferation activity of HUVEC. In the presence of GC treatment, the proliferation activity of HUVEC in the JMY knockdown group was significantly higher than that in the control group (P < 0.01). The proliferation activity of HUVEC in the overexpression JMY group was significantly lower than that in the control group (P < 0.01). The results of flow cytometry showed that without GC treatment, overexpression or knockdown of JMY did not affect the apoptosis proportion of HUVEC. With GC treatment, the apoptosis proportion of HUVEC in the JMY knockdown group was significantly lower than that in the control group (P < 0.01), and the apoptosis proportion of HUVEC in the overexpression JMY group was significantly higher than that in the control group (P < 0.01). Western blot results showed that with GC treatment, the JMY expression level of HUVEC increased with the reaction time. Moreover, the distribution of JMY was mainly concentrated in the nucleus. The expression level of Bax also increased with the reaction time. With GC treatment, overexpression of JMY could significantly increase the expression of Bax in HUVEC. JMY knockdown could reduce the expression of Bax in HUVEC. In the absence of GC treatment, HUVEC overexpression or knockdown of JMY did not affect the expression of Bax. The results of scarification and Transwell chamber assays showed that: without GC treatment, JMY knockdown could significantly decrease the cell motility and increase the expression level of VE‐cadherin in HUVEC; with GC treatment, JMY knockdown in HUVEC had lower cell motility compared with the control group (P < 0.01).ConclusionGlucocorticoids can induce the HUVEC apoptosis, and reduce its proliferation, cell motility. Our results mainly confirmed the role of JMY in the pathogenesis of GC‐inducing lesions in HUVEC. GC act on HUVEC, inducing cell damage. Following the event of cell damage, JMY levels upregulate in the nucleus to induce transcription of Bax, triggering apoptosis. JMY can also regulate HUVEC motility via its regulation of VE‐cadherin levels.

Teucrium polium Extract Enhances the Anti-Angiogenesis Effect of Tranilast on Human Umbilical Vein Endothelial Cells.

Purpose: Angiogenesis plays an important role in numerous pathophysiological events like cancer. As a result of this, tranilast as an anti-fibrotic drug induces the promising antitumor activities through the inhibition of angiogenesis. Further, Teucrium polium (TP) is a herbal medicine (family Lamaceae) with antitumor properties. This study was conducted to investigate the combination effects of tranilast and T. polium on human umbilical vein endothelial cells (HUVECs) viability and apoptotic genes expression.Methods: The HUVECs line was treated using different doses of tranilast and T. polium alone or their combination. The cell cytotoxicity was evaluated using MTT and LDH assays; apoptosis was examined using acridine orange/ethidium bromide staining, nitric oxide (NO) production was evaluated using Griess reaction and the expression of BAX and BCL-2 genes were detected using real-time RT-PCR. One-way analysis of variance (ANOVA) test was used to compare the data in different groups.Results: The survival rate of HUVECs was significantly reduced (p<0.05) in a dose dependent manner by tranilast and T. polium. However, T. polium and tranilast combination significantly (p<0.001) reduced cell viability and increased apoptotic cells as compared to each drug alone. Also, HUVECs treated with Tranilast / T. polium combination showed a reduced level of NO as regards to cells exposed only to Tranilast or T. polium (p<0.05). Furthermore, a significant increase in BAX and a decrease in BCL-2 mRNA expression were observed in combination group (p<0.001).Conclusion: T. polium synergistically increased the antiangiogenic effect of tranilast on in vitro angiogenic model of HUVECs.

Isolation of Rickettsia amblyommatis in HUVEC line.

Rickettsia amblyommatis, formerly named Rickettsia amblyommii and ‘Candidatus Rickettsia amblyommii’ is an intracellular bacterium belonging to the spotted fever group Rickettsia. It is highly prevalent in Amblyomma americanum and in other Amblyomma spp. throughout the Western Hemisphere. R. amblyommatis has been cultivated in chicken fibroblast, primary embryonated chicken eggs, Vero cells and arthropod-derived cells. Because of the affinity of rickettsiae to invade vascular endothelial cells, we tried to isolate R. amblyommatis from a nymph of Amblyomma cajennense s.l. collected in Saltillo (Coahulia, Mexico) using human umbilical vein endothelial cells (HUVEC). One tick half was analysed by ompA PCR and was found to be positive for R. amblyommatis. The other half was selected for in vitro culture of Rickettsia spp. It was triturated in 1 mL of endothelial cell growth medium with 1% antibiotic–antimycotic solution, and the homogenate was inoculated into a HUVEC line. Culture was maintained at 33°C in endothelial cell growth medium plus 2 mM l-glutamine and 2% fetal calf serum, with 5% CO2. The medium was changed weekly. Culture was checked by Gimenez stain for Rickettsia-like intracellular organisms. After 48 days of incubation, Rickettsia-like organisms were observed in HUVEC. PCR assays and sequencing of ompA gene in the culture suspension showed 100% identity with R. amblyommatis. This isolate was successfully established in HUVEC, and it has been deposited in the collection of the Center of Rickettsioses and Arthropod-Borne Diseases, Infectious Diseases Department, Hospital San Pedro–Center of Biomedical Research from La Rioja, Logroño, Spain. The HUVEC line is a useful tool for the isolation of R. amblyommatis.

Nitric oxide synthesis-promoting effects of valsartan in human umbilical vein endothelial cells via the Akt/adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway.

Valsartan (VAL), an antagonist of angiotensin II receptor type 1, has antihypertensive and multiple cardiovascular protective effects. The pleiotropic functions of VAL are related to the increased synthesis and biological activity of intravascular nitric oxide (NO). In this study, the role and mechanisms of VAL in the synthesis of NO were examined in human umbilical vein endothelial cells (HUVECs). Ten µmol/L of VAL was used to treat EA.hy926 cells for 30 minutes, 1, 3, 6, 12, and 24 hours, and three concentrations of VAL (i.e., 10, 1, and 0.1 µmol/L) were used to treat EA.hy926 cells for 24 hours. The cells were divided into five groups: control, VAL, VAL + Compound C (adenosine monophosphate-activated protein kinase [AMPK] inhibitor, 1 µmol/L), VAL + LY294002 (Akt [protein kinase B] inhibitor, 10 µmol/L), and VAL + L-nitro-arginine methyl ester (L-NAME, endothelial NO synthase [eNOS] inhibitor, 500 µmol/L) groups. The NO content in the VAL-treated HUVEC line (EA.hy926) was detected using the nitrate reductase method, and western blot was used to detect the phosphorylation of Akt, AMPK, and eNOS, as well as the changes in total protein levels. VAL increased NO synthesis in EA.hy926 cells in time- and dose-dependent manners (p < 0.05) and the intracellular phosphorylation levels of Akt, AMPK, and eNOS at the corresponding time points. LY294002, Compound C, and L-NAME could inhibit the VAL-promoted NO synthesis. VAL activated Akt, AMPK, and eNOS, thus promoting NO synthesis and playing a protective role in endothelial cells. These results partially explained the mechanisms underlying the cardiovascular protective effects of VAL.

EGCG Attenuates Uric Acid-Induced Inflammatory and Oxidative Stress Responses by Medicating the NOTCH Pathway.

Background. The aim of this study is to investigate whether (-)-epigallocatechin-3-gallate (EGCG) can prevent the UA-induced inflammatory effect of human umbilical vein endothelial cells (HUVEC) and the involved mechanisms in vitro. Methods. HUVEC were subjected to uric acid (UA) with or without EGCG treatment. RT-PCR and western blots were performed to determine the level of inflammation marker. The antioxidant activity was evaluated by measuring scavenged reactive oxygen species (ROS). Functional studies of the role of Notch-1 in HUVEC lines were performed using RNA interference analyses. Results. UA significantly increased the expressions of IL-6, ICAM-1, TNF-α, and MCP-1 and the production of ROS in HUVEC. Meanwhile, the expression of Notch-1 and its downstream effects significantly increased. Using siRNA, inhibition of Notch-1 signaling significantly impeded the expressions of inflammatory cytokines under UA treatment. Interestingly, EGCG suppressed the expressions of inflammatory cytokines and the generation of ROS. Western blot analysis of Notch-1 showed that EGCG significantly decreased the expressions of inflammatory cytokines through Notch-1 signaling pathways. Conclusions. In summary, our findings indicated that Notch-1 plays an important role in the UA-induced inflammatory response, and the downregulation of Notch-1 by EGCG could be an effective approach to decrease inflammation and oxidative stress induced by UA.

Pseudopterosin A ‐ an Atypical Antagonist of Adenosine A2B Receptors that Accelerates Angiogenesis

SummaryPseudopterosins are known anti‐inflammatory and anti‐microbial agents, isolated from the soft coral, Pseudopterogorgia elisabethae. We report pseudopterosins as activators of cell proliferation in human umbilical vein endothelial cells (HUVEC), a cellular model of angiogenesis. A hydroxypropyl‐β‐cyclodextrin (HPβCD) formulation of pseudopterosin A (PsA) elicited a 25% increase in cell proliferation (EC50 = 1.34 ×10−8 M) in growth factor depleted HUVEC lines. This constitutes a &gt;200 fold increase in potency over DMSO formulations. We demonstrate that the effect of PsA is mediated through inhibition of adenosine A2B receptors which leads to decreased synthesis of cAMP and that this effect can be reversed with a selective A2 agonist, CV‐1808. In addition we show that PsA can induce protein kinase B (AKT) phosphorylation which has been shown to activate proliferation via the phosphatidylinositol 3‐kinase (PI3K) pathway. Collectively, this indicates that pseudopterosins are promising compounds for adenosine receptor mediated pathologies.

In Vitro Screening for Angiostatic Potential of Herbal Chemicals

Herbal medicine has long been used in traditional medicinal systems. The authors carried out a first-line screening of four herbal chemicals with reported antioxidative properties and capabilities to suppress endothelial cell growth and migration. These herbal chemicals were isoliquiritigenin (ISL) from licorice, epigallocatechin gallate (EGCG) from green tea, resveratrol (Rst) from grapes, and gambogic acid (GA) from the resin of Garcinia hanburyi. Cytotoxicity was studied by MTT cell viability/proliferation assay on human retinal pigment epithelial cells (ARPE19). Effects on vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation and migration were investigated by a scratch-wound model using human umbilical vein endothelial cells (HUVECs). The effects on VEGF signaling in HUVECs were analyzed by Western blotting. At sub-cytotoxic levels, ISL (10 μM), EGCG (50 μM), and Rst (10 μM) suppressed HUVEC proliferation and migration under VEGF (20 ng/mL) stimulation in our scratch-wound model. HUVEC migration was reduced more by ISL and EGCG than bevacizumab, a humanized monoclonal antibody against VEGF. The efficiency of Rst was similar to that of bevacizumab. GA, however, was toxic to cells even at nanomolar concentrations. Western blot analysis showed that these chemicals affected focal adhesion kinase activation and expression of pigment epithelial growth factor. ISL, EGCG, and Rst are highly effective and efficient in suppressing endothelial cell proliferation and migration, with low cytotoxicity on ARPE19 and HUVEC lines. They are potentially useful for further investigation to develop antiangiogenic therapies by virtue of their small molecular sizes for easy penetration through tissue cells and their low effective dosages.

Overexpression of Allograft Inflammatory Factor-1 Promotes the Proliferation and Migration of Human Endothelial Cells (HUV-EC-C) Probably by Up-Regulation of Basic Fibroblast Growth Factor

Our previous study demonstrated that allograft inflammatory factor-1 (AIF-1) is present in the vessels of infantile hemangiomas but neither in the vessels of vascular malformations, pyogenic granulomas, normal skin, placental tissues nor in the neovessels of squamous cell carcinomas of the tongue. The purpose of this study was to explore the impact of AIF-1 alterations on endothelial cells (EC). Stable introduction of AIF-1 to the human umbilical vein EC line (HUV-EC-C) in vitro revealed that AIF-1 enhances the proliferation and migration of the EC and promotes G0/G1-to-S-phase transition, accompanied by up-regulation of basic fibroblast growth factor (p < 0.05). In contrast, AIF-1 did not affect the expression of granulocyte colony-stimulating factor, VEGF-a, monocyte chemoattractant protein-1, or tissue inhibitor of metalloproteinase-1. AIF-1 expression was not induced by hypoxia, VEGF-a, basic fibroblast growth factor, or insulin-like growth factor-2 in EC. Taken together, these findings suggest that the impact of AIF-1 on EC would stimulate angiogenesis and consequently affect the progression of infantile hemangiomas.

Role of Protein Kinase C in the Expression of Endothelin Converting Enzyme-1

Increased expression of endothelin converting enzyme-1 (ECE-1) is associated with diabetic nephropathy. The molecular mechanisms underlying this association, as yet unknown, possibly involve protein kinase C (PKC) pathways. In the present study, we examined the effects of high glucose and PKC activation on ECE-1 expression in primary human umbilical vein endothelial cells (HUVECs) and in HUVEC line (EA.hy926). Increasing glucose concentration, but not mannitol, from 5.5-22.2 mmol/liter for 3 d, enhanced prepro endothelin-1 (ET-1) mRNA expression, ET-1 levels, ECE-1 protein, and mRNA expressions by 7, 4, 20, and 2.6-fold, respectively. High glucose increased ECE-1 protein expression dose and time dependently. By Western blot analysis, PKC-beta1, -beta2, and -delta isoform levels were significantly increased relative to other isoforms when glucose level was increased. Treatment with Rottlerin, a PKC-delta isoform inhibitor, reduced significantly the glucose-induced ET-1 secretion, and ECE-1 protein expression, but (S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16,21-dimetheno 1H,1(3)H-dibenzo[e,k]pyrrolo[3,4-h] (1, 4, 3) oxadiaza-cyclohexadecene-1,3(2H)-dione or Gö6976, specific PKC-beta and -alpha inhibitors, respectively, did not. Overexpression of PKC-delta but not PKC-alpha or -beta1 isoforms by adenovirus vector containing the respective cDNA in HUVECs incubated with 5.5 mmol/liter glucose, increased in parallel PKC proteins, and glucose-induced endothein-1 and ECE-1 protein expression by 4- to 6-fold. These results show that enhanced ECE-1 expression induced by hyperglycemia is partly due to activation of the PKC-delta isoform. Thus, inhibition of this PKC isoform may prevent diabetes-related increase in ET-1.