Endothelial Cell Growth Supplement Research Articles

Type of culture medium determines properties of cultivated retinal endothelial cells: induction of substantial phenotypic conversion by standard DMEM

Contradictory behavior of microvascular retinal endothelial cells (REC) - a reliable in vitro model to study retinal diseases - have recently been reported which might result from cultivating the cells in standard DMEM not optimized for this cell type. Therefore, we studied DMEM's effects on phenotype and behavior of immortalized bovine REC. Cells were cultivated in endothelial cell growth medium (ECGM) until a confluent monolayer was reached and then further kept for 1–4 days in ECGM, DMEM, or mixes thereof all supplemented with 5% fetal bovine serum, endothelial cell growth supplement, 90 μg/ml heparin, and 100 nM hydrocortisone. Within hours of cultivation in DMEM, the cell index – measured to assess the cell layer's barrier function - dropped to ~5% of the initial value and only slowly recovered, not only accompanied by stronger expression of HSP70 mRNA and secretion of interleukin-6, but also by lower expressions of tight junction proteins claudin-5, claudin-1 or of the marker of cell type conversion caveolin-1. Altered subcellular localizations of EC-typic claudin-5, vascular endothelial cadherin and von Willebrand factor were also observed. Taken together, all experiments with (retinal) EC cultivated in common DMEM need to be interpreted very cautiously and should at least include phenotypic validation.

The VALVE REGEN project. Developing an innovative tissue engineered heart valve, in vitro and in vivo testing – preliminary results

Abstract Background Regenerative Medicine and Tissue Engineering are the grounds on which multidisciplinary teams aspire to obtain the perfect valvular substitute, which overcomes shortcomings of the present prostheses. Purpose Was to obtain a tissue engineered heart valve (TEHV) by repopulating with valvular resident cells – endothelial (EC) and fibroblasts (FB) a decellularized heart valve scaffold. Then their functionality and behavior was assessed in vitro and in vivo. Methods This study is part of a research grant approved by the Ethics Committee of the University. Six ovine pulmonary valves underwent a perfusion based decellularization protocol. Using a sequence of chemical and enzymatic treatment under a pressure gradient, cell removal was achieved and attested by histological investigations (DAPI nuclear staining –4',6-diamidino-2-phenylindol and haematoxylin-eosin) and DNA extraction. Ovine sub-dermal adipose tissue was harvested followed by stem cells isolation and culture. Using Endothelial Cell Growth Supplement and mechanical stimuli EC were differentiated and with Transforming Growth Factor-B1, FB were obtained. FB were internally seeded into cuspis bases using a 22 gauge needle and externally on the adventitia by using a rotator allowing a uniform distribution and seeding of cells. EC were seeded into leaflets pockets and intra-luminal also using the rotator. The repopulated valves were preconditionated in a bioreactor by gradually exposing them to the pulmonary hemodynamic regimen. By using a high speed camera, their behavior was examined when exposed to in vivo conditions. The in vivo testing was performed by surgical implantation in the gold model considered animal – the sheep. By transesophageal ultrasound (TEE US) and epicardic US, their intra-operatory function was evaluated. Post-procedure, evaluation was performed by periodic trans-thoracic (TTE US). Results Six TEHV were obtained. The decellularization histology assessment revealed acellular scaffolds and non-detectable nucleic material at the DNA extraction. Six adipose derived stem cells cultures were obtained and subsequently specialized towards EC and FB lines. The repopulation procedures underwent without incidents. During the bioreactor preconditioning, the TEHV showed complete opening and competent central coaptation. Leaflets presented physiological movement and absence of damage of valvular apparatus. The TEE US evaluation in vivo revealed normal valvular function without signs of stenosis or regurgitation. The periodic TTE US showed preserved valvular function. Conclusions Our preliminary results point out a manufactured TEHV with physiological behavior when tested in vitro and in vivo. Their interaction with a living body will be pointed out only in the explant phase, after histology analysis. The present results appear optimistic but only extended studies and follow-ups will certify their superiority in terms of performances and behavior. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): This paper was financed by a grant from the Competitiveness Operational Programme 2014-2020, Tissue engineering technologies for cardiac valve regeneration, valve-regen, id P_37_673, Mysmis code: 103431, contract 50/05.09.2016.

Mortality From Coronavirus Disease 2019 Increases With Unsaturated Fat and May Be Reduced by Early Calcium and Albumin Supplementation

Mortality From Coronavirus Disease 2019 Increases With Unsaturated Fat and May Be Reduced by Early Calcium and Albumin Supplementation

Photobiomodulation alters the viability of HUVECs cells

The aim of the present study was to investigate the influence of low-level red (660nm) and infrared (780nm) laser with four different radiance exposures on human umbilical vein endothelial cells (HUVECs) in vitro. HUVECs (1.5 × 104) were incubated in 96-well culture plates. The cells were maintained in M199 medium supplemented with 20% fetal bovine serum, 1% antibiotic (penicillin), 1% anti-mycotic (Fungizone), and 1% endothelial cell growth supplement. After centrifugation, irradiations (660/780nm, 40mW, 1, 5, 10, and 20J/cm2, 1s, 5s, 10s, and 20s, respectively, total energy 0.4J, 2J, 4J, and 8J, and beam spot size at target 0.04cm2) were performed at the bottom of Falcon tubes such that the laser beam directly reached the cell without passing through the culture medium. The cells were divided into groups based on radiant exposures. Cell viability and protein concentration were verified after 1, 2, 3, 6, 8, and 10days. Red laser increased the cell viability and protein concentration in all groups (three-way ANOVA, p < 0.05) beginning on the second day. The greatest peak compared with the control was found when the radiant exposure was 5J/cm2 and 10J/cm2. Infrared laser inhibited cell viability and modulated the protein concentration in the cells, with the highest peak protein concentration found on the second day in the group with radiant exposure of 1J/cm2 and 10J/cm2 (three-way ANOVA, p < 0.05). Red laser increased the viability and concentration of total proteins in HUVECs, whereas infrared laser had an inhibitory effect on cell viability, while maintaining the total protein concentration similar to that found in the control group.

Outgrowth, proliferation, viability, angiogenesis and phenotype of primary human endothelial cells in different purchasable endothelial culture media: feed wisely

Function and dysfunction of endothelial cells are regulated by a multitude of factors. Endothelial cell research often requires in vitro cell culture experiments. Hence, various culture media specifically designed to promote endothelial cell growth are available. These strikingly differ in their composition: complex media contain endothelial cell growth supplement (ECGS), an extract produced of bovine brain with undefined amounts of biologically active compounds, whilst defined media contain selected growth factors in defined concentrations. We here compared the effect of seven purchasable endothelial cell culture media on colony outgrowth, proliferation, viability, in vitro angiogenesis and phenotype of mature primary human endothelial cells using feto-placental endothelial cells isolated from chorionic arteries (fpEC). The effect of media on colony outgrowth was additionally tested on umbilical cord blood-derived endothelial progenitor cells (ECFCs). Outgrowth, purity, proliferation and viability differed between media. Outgrowth of fpEC and ECFCs was best in a defined medium containing EGF, FGF2 and VEGF. By contrast, established fpEC isolations proliferated best in complex media containing ECGS, heparin and ascorbic acid. Also viability of cells was higher in complex media. In vitro angiogenesis was most intense in a defined medium containing the highest number of individual growth factors. FACS analysis of surface markers for endothelial cell subtypes revealed that endothelial phenotype of fpEC was unaffected by media composition. Our data demonstrate the fundamental effect of endothelial cell culture media on primary cell isolation success and behaviour. Whether the composition of supplements is suitable also for individual experiments needs to be tested specifically.

Isolation and culture of endothelial cells from embryonic rat yolk sac

Abstract Yolk sac blood islands are the first morphologic evidence of hematopoietic development during mammalian embryogenesis, and visseral yolk sac mesoderm gives rise to the first embryonic blood cells within a rich endothelial network. Present study reports the isolation and culture of endothelial cells from 11.5 days old embryonic rat yolk sac. The embryos were dissected from 11.5 days pregnant Wistar rat (Rattus norvegicus) and the external yolk sac membrane and embryos were removed under aseptic condition. After washing three times with Calcium-Magnesium free Hank’s balanced salt solution (CMF-HBSS), the tissue was minced, and fragments were incubated in CMF-HBSS containing 2mg/ml Trypsin, 100mg/ml collagenase I and 40mg/ml DNAse at 37°C until the tissue was completely dispersed. The digestion effect was then neutralized by fetal bovine serum at 1:3 (v/v). The cell suspension was centrifuged at 1000 rpm for 10 min., the supernatants were discarded and the cell pellets resuspended in Dulbecco modified Eagle medium containing 15% fetal bovine serum, 1.25mg/ml amphotericin B, 25mg/ml gentamycin sulphate and 100mg/ml endothelial cell growth supplement. The resuspended cells were plated in two diverse 25cm2 culture flasks for overnight differential adherence at 37°C. The non-adherent cells were removed by gentle aspiration and adherent cells refed with fresh medium. The cells were transferred using 1ml of 0.2% Trypsin when cultures reached near-confluence. The cultured yolk sac endothelial cells had characteristic cobblestone appearence and positive immunofluorescent staining for von Willebrand Factor (vWF). Weibel-Palade bodies, the major ultrastructural marker for endothelium, were also detected in cultured cells by electron microscopy.

Levistilide A inhibits angiogenesis in liver fibrosis via vascular endothelial growth factor signaling pathway.

Levistilide A (C24H28O4, molecular weight = 380.48) derived from Angelica sinensis (Danggui) has been reported to inhibit hepatic stellate cell proliferation. This study investigated the effects of levistilide A on liver fibrosis relating to angiogenesis, particularly on the characteristic change in liver sinusoidal endothelial cells. LX-2 cells were activated by TGF-β1, and the human hepatic sinusoidal endothelial cells (HHSECs) were induced by endothelial cell growth supplement. Cell viability was detected using a methylthiazoldiphenyl-tetrazolium bromide assay; F-actin was visualized through the fluorescence probe method; cell proliferation was examined using the EdU kit; antiangiogenesis activity was assessed using the tube formation assay and transgenic zebrafish model. To verify the results invivo, rats were subcutaneously injected with CCl4 twice a week for six weeks to duplicate the liver fibrosis model and then treated with 10 mL/kg of normal saline, 4 mg/kg of sorafenib, and 3 and 6 mg/kg of levistilide A for three weeks from the fourth week. Collagen deposition was detected through Sirius Red staining; liver microvasculature was examined through vWF labeling and X-ray 2D imaging; sinusoidal fenestrations were observed through scanning electron microscopy; collagen I, α-SMA, CD31, vascular endothelial growth factor (VEGF), and VEGF-R2 were detected through Western blotting. Our results indicated that levistilide A attenuated LX-2 cell activation and HHSEC proliferation. The ability of HHSECs to form tubelike structures in Matrigel was inhibited, and the number of functional vessels in transgenic zebrafish decreased. In invivo experiments, levistilide A reduced collagen deposition and the number of new microvessels; ameliorated sinusoid capillarization; and downregulated the expression of CD31, VEGF, and VEGF-R2. These findings suggest that levistilide A can inhibit liver fibrosis through antiangiogenesis by alleviating sinusoid capillarization via the VEGF signaling pathway. Impact statement Levistilide A has been reported to inhibit hepatic stellate cell (HSC) proliferation. In this study, we further investigated the mechanisms of levistilide A on liver fibrosis relating to angiogenesis, particularly on the characteristic change in liver sinusoidal endothelial cells. The cell models of HSC and liver sinusoidal endothelial cell and CCl4 induced liver fibrosis model were used. These results suggest that levistilide A can inhibit liver fibrosis through antiangiogenesis by alleviating sinusoid capillarization via the vascular endothelial growth factor signaling pathway. The effect of levistilide A on liver fibrosis was confirmed, and its detailed mechanism was also discussed. These findings suggest that levistilide A may be a great potential drug for treating liver fibrosis through antiangiogenesis, and this effect will be verified in other fibrotic animal model studies or by clinical trials.

PP.31.10] ENDOTHELIAL CELL GROWTH SUPPLEMENT IMPROVES CARDIAC FUNCTION FOLLOWING MOUSE MYOCARDIAL INFARCTION

Objective: Myocardial infarction (MI) and subsequent heart failure is a leading cause of death in high or middle income countries. Development of treatments that stimulate heart tissue regeneration and revascularization/angiogenesis is important to improve the prognosis of these patients. Our recent work demonstrated that endothelial cell growth supplement (ECGS)-containing medium stimulates endothelial differentiation of c-kit positive cardiac progenitor cells in vitro. The objective of the current work was to test whether ECGS can activate endogenous cardiac progenitor cells and improve cardiac function in a mouse MI model. Design and method: Ligation of the left anterior descending (LAD) artery was performed on mice at 2 months of age under anesthetization and intubation. After displacing the pericardium, a 7–0 silk suture was passed under the LAD artery 2 mm below the left atrium and permanently tied, to eliminate blood flow to the area distal to the suture. ECGS or saline (as control) were injected intramyocardially to the ligation area through a sterile Hamilton syringe with a 30 gauge needle immediately after the ligation. Mice that survived the procedure were maintained for 3 weeks. Echocardiography was performed to monitor the cardiac function prior to organ collection. Results: The results showed that MI increased c-kit positive cells and blood vessel formation as well as massive fibrosis in the infarcted area 3 weeks post-surgery. ECGS treatment significantly reduced cardiac fibrosis, especially in the non-infarcted area and border zone (1.96 ± 0.28 % in ECGS treated group vs 4.54 ± 1.08 % in saline treated control group, p < 0.05). ECGS also improved ejection fraction in these animals compared to the saline treated control group (49.9 ± 2.9 % ECGS treated vs 37.4 ± 4.6 % saline treated, p < 0.05). However, ECGS had no significant effect on infarct size in these animals. Conclusions: The current study demonstrates a reduction in cardiac fibrosis and improvement of cardiac function with ECGS in a mouse MI model, which may link to our in vitro observation that ECGS can activate endothelial differentiation of cardiac progenitor cells.

Isolation and in vitro culture of vascular endothelial cells from mice.

In cardiovascular disorders, understanding of endothelial cell (EC) function is essential to elucidate the disease mechanism. Although the mouse model has many advantages for in vivo and in vitro research, efficient procedures for the isolation and propagation of primary mouse EC have been problematic. We describe a high yield process for isolation and in vitro culture of primary EC from mouse arteries (aorta, braches of superior mesenteric artery, and cerebral arteries from the circle of Willis). Mouse arteries were carefully dissected without damage under a light microscope, and small pieces of the vessels were transferred on/in a Matrigel matrix enriched with endothelial growth supplement. Primary cells that proliferated in Matrigel were propagated in advanced DMEM with fetal calf serum or platelet-derived serum, EC growth supplement, and heparin. To improve the purity of the cell culture, we applied shearing stress and anti-fibroblast antibody. EC were characterized by a monolayer cobble stone appearance, positive staining with acetylated low density lipoprotein labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate, RT-PCR using primers for von-Willebrand factor, and determination of the protein level endothelial nitric oxide synthase. Our simple, efficient method would facilitate in vitro functional investigations of EC from mouse vessels.

Inhibition and mechanism of 15 (S)-hydroxyeicosate traenoic acid on proliferation of hypoxic retinal microvascular endothelial cells in vitro

Background Retninal neovascular diseases caused by hypoxia has become a major blinding disease,which is lack of effective chemical treatment currently,it's important to study the molecuar mechanism of the disease,so as to guide the clinical medication.Objective This study was to explore the effect of 15 (S)-hydroxyeicosate traenoic acid (15-HETE) on the proliferation of hypoxic retinal microvascular endothelial cells (RMVECs) and its probable mechanism.Methods RMVECs were isolated from C57BL/6J mice and incubated and then identified with anti-Ⅷ factor antibody by immunochemistry and immunofluorescence.The cells were divided into the normoxia group and the hypoxia group.The hypoxia cell models were established by treated with 125 μmol/L CoCl2.The cells were cultured with serum-free DMEM containing endothelial cell growth supplement (ECGS)and high glucose for 48 hours,and then different concentrations of 15-HETE (0.0,0.1,1.0,5.0 μmol/L) were added in the medium for 48 hours respectively to subgroup the groups.The proliferation of the cells (absorbance,A) was detected using MTT.The relative expression levels of protein and mRNA of hypoxia inducible factor-1α (HIF-1α),bcl-2 and caspase-3 were assayed by reverse transcription PCR (RT-RCR)and Western blot.Results The cells showed the positive response for anti-Ⅷ factor antibody with the positive rate of (94.38 ±4.25)％.No significant difference was found in the cell proliferation of various groups under the normoxia condition (F =0.283,P =0.837),but under the hypoxia condition,the proliferation values were significantly different among various groups (F =702.582,P＜0.001).The cell proliferation value in the 1.0 μmol/L 15-HETE group and 5.0 μmol/L 15-HETE group was lower than that of the simple hypoxia group respectively(both at P＜0.05).The inhibitory rates in the 0.1,1.0,5.0 μ mol/L 15-HETE groups were (1.09±0.31) ％,(21.09± 3.53) ％ and (49.86 ±4.15) ％,showing a dosedependent manner.No significant difference was seen in the expression levels of bcl-2,caspase-3 and HIF-1α mRNA in various groups under the normoxia conditions.However,compared with the simple normoxia group,the relative expressions of bcl-2 mRNA and HIF-1α mRNA in the cells were increased by 1.53 folds and 1.7 folds in the simple hypoxia group respectively,and caspasse-3 mRNA expression decreased by 70％ (all at P ＜ 0.05).Under the normoxia condition,the expression of bcl-2 and pro-caspase-3 protein in the cells were not significantly different among the various groups (P＞0.05),however,the expressions of bcl-2 and pro-caspase-3 proteins were elevated by 1.6 folds and 1.9 folds in the hypoxia group in compared with the normoxia group (P＜0.05).Compared with the simple hypoxia group,the expressions of bcl-2 and pro-caspase-3 were lowed by 40.4％ and 42.5％ in the 5.0 μmol/L 15-HETE group (P＜0.05).Conclusions 15-HETE inhibits the proliferation of RMVECs and therefore suppresses neovascularization by down-regulating the expressions of HIF-1α and bcl-2 and the activation of caspase-3 in a dose-dependent manner. Key words: Retinal neovascularization; Retinal microvascular endothelial cell; Hypoxia inducible factor1α ; Bcl-2; Caspase-3

P184Optimalization of isolation and culture conditions of endothelial cells from human heart

Purpose: Endothelial cells have an essential role in the maintenance of cardiac function. Cardiac endothelial cells in response of different cardiovascular risk factors become activated and this activation is often followed by endothelial dysfunction. The change of endothelial phenotype may influence interactions with surrounding cardiac fibroblasts, myocytes, pericytes. The aim of our work is to produce in vitro culture system with high cell purity to investigate endothelial cells in different heart diseases. Poor quality of these tissue samples indicates the optimalization of isolation and culture conditions for both cardiac microvascular and endocardial endothelial cells (CMVE and EE cells). Methods: Human heart samples were obtained from valve surgery or explanted hearts during transplantation. CMVE cells were isolated from left ventricle with collagenase and trypsin digestion. EE cells were digested with collagenase from mitral valve. After separation the pellets were seeded to culture dishes and grown in a medium containing endothelial cell growth supplement. These basic methods would be changed as follows. The time of enzymatic digestion was changed. The collected cell suspensions were preincubated to allow fibroblasts to attach to the bottom of culture dish. The decanting medium was centrifuged repeatedly and collected cells were cultured in specific endothelial medium completed with sera from different origins. CMVE cells were treated with puromycin to eliminate pericytes. Cells were seeded on different artificial extracellular surface components (collagen, laminin, gelatin, fibronectin). The CMVE and EE cells were characterized by uptake of acetylated LDL and immunostaining of vonWillebrand factor, VE-cadherin and CD31. Fibroblasts were identified by vimentin staining. Results: Primary cells isolated with basic methods exhibited different cell morfologies, they were multilayered and only the 4% and 10% of the cells were endothelial cells, CMVE and EE respectively, separated by flourescent-activated cell sorting (FACS). The attachment of fibroblasts and the puromycin treatment were effective in the primary cell cultures. Numerous cell colonies were observed within 2-3 days on gelatin coated surface and confluence was achieved within 10-14 days. Conclusion: With use of these culture conditions the endothelial cell rate is increased in primery cultures and further separation by FACS or immunomagnetic methods will be more effective. These optimalized cell culture systems provide possibility to investigate endothelial functions in different heart diseases.

Distinct Renal Pathology and a Chemotactic Phenotype after Enterohemorrhagic Escherichia coli Shiga Toxins in Non-Human Primate Models of Hemolytic Uremic Syndrome

Enterohemorrhagic Escherichia coli cause approximately 1.5 million infections globally with 176,000 cases occurring in the United States annually from ingesting contaminated food, most frequently E. coli O157:H7 in ground beef or fresh produce. In severe cases, the painful prodromal hemorrhagic colitis is complicated by potentially lethal hemolytic uremic syndrome (HUS), particularly in children. Bacterial Shiga-like toxins (Stx1, Stx2) are primarily responsible for HUS and the kidney and neurologic damage that ensue. Small animal models are hampered by the inability to reproduce HUS with thrombotic microangiopathy, hemolytic anemia, and acute kidney injury. Earlier, we showed that nonhuman primates (Papio) recapitulated clinical HUS after Stx challenge and that novel therapeutic intervention rescued the animals. Here, we present detailed light and electron microscopic pathology examination of the kidneys from these Stx studies. Stx1 challenge resulted in more severe glomerular endothelial injury, whereas the glomerular injury after Stx2 also included prominent mesangiolysis and an eosinophilic inflammatory infiltration. Both toxins induced glomerular platelet-rich thrombi, interstitial hemorrhage, and tubular injury. Analysis of kidney and other organs for inflammation biomarkers showed a striking chemotactic profile, with extremely high mRNA levels for IL-8, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1α and elevated urine chemokines at 48hours after challenge. These observations give unique insight into the pathologic consequences of each toxin in a near human setting and present potential pathways for therapeutic intervention.

Methyl tert butyl ether is anti‐angiogenic in both in vitro and in vivo mammalian model systems

Methyl-tertiary butyl ether (MTBE), a well known gasoline oxygenate, and US Food and Drug Administration approved gallstone treatment, has been previously shown to specifically target teleost embryonic angiogenesis. The studies reported here were to determine whether similar vascular disrupting effects occur in higher vertebrate models. Rat brain endothelial cells were isolated and allowed to form microcapillary-like tubes on Matrigel. MTBE (0.34-34.0 mm) exposure resulted in a dose-dependent reduction of tube formation, with the LOAEL at 0.34 mm, while MTBE's primary metabolite, tertiary butyl alcohol had no effect on tube formation. HUVECs, a primary cell line representing macrovascular cells, were able to form tubes on Matrigel in the presence of MTBE (1.25-80 mm), but the tubes were narrower than those formed in the absence of MTBE. In a mouse Matrigel plug implantation assay, 34.0 mm MTBE completely inhibited vessel invasion into plugs containing endothelial cell growth supplement (ECGS) compared with control plugs with ECGS alone. When timed-pregnant Fisher 344 rats were gavaged with MTBE (500-1500 mg kg(-1) ) from day 6 of organogenesis through 10 days post-parturition, no organ toxicity or histological changes in pup vasculature were observed. Results of the in vitro cell culture studies show that MTBE is anti-angiogenic at mm concentrations and has potential use as an anti-angiogenic treatment for solid tumors with minimal toxicity.

ENOS Phosphorylation Under Flow-Mediated Shear Stress: Effects of Flow Pattern and Ambient Oxygen Concentration

Vascular endothelial cells (EC) are continuously exposed to flow-mediated shear stress, a friction force generated by flowing blood, which is increased during aerobic exercise. The flow-mediated shear stress is the most important physiological stimulus for the formation of nitric oxide (NO) via activation of endothelial nitric oxide synthase (eNOS). It has been demonstrated that flow-mediated shear stress modulates vascular structure and function, and is a major determinant for vascular remodeling and arterial tone, which depends primarily on NO produced by EC in response to shear stress. NO plays many essential roles including vessel relaxation, inhibition of apoptosis, and anti-inflammation. However, effects of flow patterns and ambient oxygen concentration on the eNOS activation and NO production are largely unknown. PURPOSE: To determine the effects of flow patterns (low and high) and ambient oxygen concentration (5%, 10% and 20% O2) on eNOS activation and NO production in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were grown in M199 supplemented with 20% FBS, Endothelial Cell Growth Supplement, Heparin, P/S and Fungizone. At 100% confluency, HUVECs were subjected to physiological level of low (l-flow) or high (h-flow) shear stress (5 or 20 dyne/cm2) using a cone-and-plate shear apparatus under 21% O2 (atmospheric oxygen), 10% O2 (arterial blood level), or 5% O2 (venous blood level). Protein samples were analyzed by western blotting. RESULTS: The morphology of HUVECs was not different among the ECs grown under different oxygen concentrations. However, the growth rate was slightly higher at 10% O2 at low confluency (< 30%) while it was similar at high confluency (60-100%). During 5-60 mins of h-flow, eNOS phosphorylation at the Ser633 residue was similar. However, under 24 hrs of h-flow, the levels of eNOS phosphorylation at both Ser1177 and Ser633 were 2-3 fold higher in 10% O2 compared to that of 21% O2. NO production level was also significantly higher in 10% O2 than in 21% O2 under 24 hrs of h-flow. CONCLUSION: Physiological level of h-flow under 10% O2 increases eNOS phosphorylation and NO production greater than that of under 21% O2 in human endothelial cells. The present study may provide a better in vitro model for the area of vascular mechanotransduction research.

Fibulin-5 Is Up-regulated by Hypoxia in Endothelial Cells through a Hypoxia-inducible Factor-1 (HIF-1α)-dependent Mechanism

Hypoxia modulates gene expression and affects multiple aspects of endothelial cell biology. Fibulin-5 (FBLN5) is an extracellular matrix protein essential for elastic fiber assembly and vasculogenesis that participates in vascular remodeling and controls endothelial cell adhesion, motility, and proliferation. In this context, we aimed to analyze FBLN5 regulation by hypoxia in endothelial cells. Hypoxia (1% O(2)) increased FBLN5 mRNA levels in endothelial cells in a time-dependent manner. Maximal induction (∼2.5-fold) was achieved after 24 h of hypoxia. This effect paralleled an increase in both intracellular and extracellular FBLN5 protein levels. The increase in FBLN5 mRNA levels observed in hypoxic cells was blocked by inhibitors of the PI3K/Akt/mTOR pathway (LY294002 and rapamycin) and mimicked by dimethyl oxal glycine, which prevents proline hydroxylase-mediated degradation of HIF-1α. Silencing of HIF-1α completely prevented hypoxia-induced FBLN5 up-regulation. Accordingly, both hypoxia and HIF-1α overexpression increased FBLN5 transcriptional activity. Serial promoter deletion and mutagenesis studies revealed the involvement of a putative hypoxia response element (HRE) located at -78 bp. In fact, EMSA and ChIP assays demonstrated increased HIF-1 binding to this site in hypoxic cells. Interestingly, the rate of endothelial cells undergoing apoptosis in cultures exposed to hypoxia increased in FBLN5 knockdown cells, suggesting that hypoxia-induced FBLN5 expression contributes to preserve cell survival. These results provide evidence that HIF-1 signaling underlies the increase of FBLN5 expression elicited by hypoxia in endothelial cells and suggest that FBLN5 induction could be involved in the adaptive survival response of endothelial cells to hypoxia.

Induction of SENP1 in Endothelial Cells Contributes to Hypoxia-driven VEGF Expression and Angiogenesis

SENP1 (SUMO-specific protease 1) has been shown to be essential for the stability and activity of hypoxia-inducible factor 1 (HIF-1α) under hypoxia conditions. However, it is unknown how SENP1 activation and hypoxia signaling are coordinated in the cellular response to hypoxia. Here, we report the essential role of SENP1 in endothelial cells as a positive regulator of hypoxia-driven VEGF production and angiogenesis. SENP1 expression is increased in endothelial cells following exposure to hypoxia. Silencing of HIF-1α blocks SENP1 expression in cell response to hypoxia. Mutation of the hypoxia response element (HRE) on the Senp1 promoter abolishes its transactivation in response to hypoxia. Moreover, silencing of SENP1 expression decreases VEGF production and abrogates the angiogenic functions of endothelial cell. We also find that the elongated endothelial cells in embryonic brain section and vascular endothelial cells in embryonic renal glomeruli in Senp1(-/-) mice are markedly reduced than those in wild-type. Thus, these results show that hypoxia implies a positive feedback loop mediated by SENP1. This feedback loop is important in VEGF production, which is essential for angiogenesis in endothelial cells.

Nobiletin, a citrus polymethoxyflavonoid, suppresses multiple angiogenesis‐related endothelial cell functions and angiogenesis in vivo

Nobiletin is a citrus polymethoxyflavonoid that suppresses tumor growth and metastasis, both of which depend on angiogenesis. We recently identified nobiletin as a cell differentiation modulator. Because cell differentiation is a critical event in angiogenesis, it might be possible that nobiletin could exhibit antiangiogenic activity, resulting in suppression of these tumor malignant properties. To verify this possibility, we examined the antiangiogenic effects of nobiletin in vitro and in vivo. Nobiletin had concentration-dependent inhibitory effects on multiple functions of angiogenesis-related endothelial cells (EC); it suppressed the proliferation, migration and tube formation on matrigel of human umbilical vein EC (HUVEC) stimulated with endothelial cell growth supplement (ECGS), a mixture of acidic and basic fibroblast growth factors (FGFs). Gelatin zymography and northern blotting revealed that nobiletin suppressed pro-matrix metalloproteinase-2 (proMMP-2) production and MMP-2 mRNA expression in ECGS-stimulated HUVEC. Nobiletin also downregulated cell-associated plasminogen activator (PA) activity and urokinase-type PA mRNA expression. Furthermore, nobiletin inhibited angiogenic differentiation induced by vascular endothelial growth factor and FGF, an in vitro angiogenesis model. This inhibition was accompanied by downregulation of angiogenesis-related signaling molecules, such as extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase, and transcriptional factors (c-Jun and signal transducer and activator of transcription 3), and activation of the caspase pathway. In a chick embryo chorioallantoic membrane assay, nobiletin showed an antiangiogenic activity, the ID(50) value being 10μg (24.9nmol) per egg. These results indicate that nobiletin is a novel antiangiogenic compound that exhibits its activity through combined inhibition of multiple angiogenic EC functions.

Inhibition of Glycogen Synthase Kinase 3β (GSK3β) Decreases Inflammatory Responses in Brain Endothelial Cells

Immune mediators and leukocyte engagement of brain microvascular endothelial cells (BMVECs) contribute to blood-brain barrier impairment during neuroinflammation. Glycogen synthase kinase 3beta (GSK3beta) was recently identified as a potent regulator of immune responses in in vitro systems and animal models. However, the role of GSK3beta in regulation of immune endothelial functions remains undetermined. Here we evaluated the effect of GSK3beta inhibition on the regulation of inflammatory responses in BMVECs. A focused PCR gene array of 84 genes was performed to identify the cytokine and chemokine gene expression profile in tumor necrosis factor (TNF) alpha-stimulated BMVECs after GSK3beta inactivation by specific inhibitors. Fifteen of 39 genes induced by TNFalpha stimulation were down-regulated after GSK3beta inhibition. Genes known to contribute to neuroinflammation that were most negatively affected by GSK3beta inactivation included IP-10/CXCL10, MCP-1/CCL2, IL-8/CXCL8, RANTES/CCL5, and Groalpha/CXCL1. GSK3beta suppression resulted in diminished secretion of these proinflammatory mediators by inflamed BMVECs detected by ELISA. GSK3beta inhibition in BMVECs reduced adhesion molecule expression as well as monocyte adhesion to and migration across cytokine stimulated BMVEC monolayers. Interactions of monocytes with TNFalpha-activated BMVECs led to barrier disruption, and GSK3beta suppression in the endothelium restored barrier integrity. GSK3beta inhibition in vivo substantially decreased leukocyte adhesion to brain endothelium under inflammatory conditions. In summary, inhibition of GSK3beta emerges as an important target for stabilization of the blood-brain barrier in neuroinflammation.

Annexin A2 Is a Molecular Target for TM601, a Peptide with Tumor-targeting and Anti-angiogenic Effects

TM601 is a synthetic form of chlorotoxin, a 36-amino acid peptide derived from the venom of the Israeli scorpion, Leirius quinquestriatus, initially found to specifically bind and inhibit the migration of glioma cells in culture. Subsequent studies demonstrated specific in vitro binding to additional tumor cell lines. Recently, we demonstrated that proliferating human vascular endothelial cells are the only normal cell line tested that exhibits specific binding to TM601. Here, we identify annexin A2 as a novel binding partner for TM601 in multiple human tumor cell lines and human umbilical vein endothelial cell (HUVEC). We demonstrate that the surface binding of TM601 to the pancreatic tumor cell line Panc-1 is dependent on the expression of annexin A2. Identification of annexin A2 as a binding partner for TM601 is also consistent with the anti-angiogenic effects of TM601. Annexin A2 functions in angiogenesis by binding to tissue plasminogen activator and regulating plasminogen activation on vascular endothelial cells. We demonstrate that in HUVECs, TM601 inhibits both vascular endothelial growth factor- and basic fibroblast growth factor-induced tissue plasminogen activator activation, which is required for activation of plasminogen to plasmin. Consistent with inhibition of cell surface protease activity, TM601 also inhibits platelet-derived growth factor-C induced trans-well migration of both HUVEC and U373-MG glioma cells.

Interleukin-25 Expressed by Brain Capillary Endothelial Cells Maintains Blood-Brain Barrier Function in a Protein Kinase Cϵ-dependent Manner

Interleukin (IL)-25, a member of the IL-17 family of cytokines, is expressed in the brains of normal mice. However, the cellular source of IL-25 and its function in the brain remain to be elucidated. Here, we show that IL-25 plays an important role in preventing infiltration of the inflammatory cells into the central nervous system. Brain capillary endothelial cells (BCECs) express IL-25. However, it is down-regulated by inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, IL-17, interferon-gamma, IL-1beta, and IL-6 in vitro, and is also reduced in active multiple sclerosis (MS) lesions and in the inflamed spinal cord of experimental autoimmune encephalomyelitis, an animal model of MS. Furthermore, IL-25 restores the reduced expression of tight junction proteins, occludin, junction adhesion molecule, and claudin-5, induced by TNF-alpha in BCECs and consequently repairs TNF-alpha-induced blood-brain barrier (BBB) permeability. IL-25 induces protein kinase Cepsilon (PKCepsilon) phosphorylation, and up-regulation of claudin-5 is suppressed by PKCepsilon inhibitor peptide in the IL-25-stimulated BCECs. These results suggest that IL-25 is produced by BCECs and protects against inflammatory cytokine-induced excessive BBB collapse through a PKCepsilon-dependent pathway. These novel functions of IL-25 in maintaining BBB integrity may help us understand the pathophysiology of inflammatory brain diseases such as MS.