Proliferation Of Microvascular Endothelial Cells Research Articles

Corosolic Acid Exhibits Anti-angiogenic and Anti-lymphangiogenic Effects on In Vitro Endothelial Cells and on an In Vivo CT-26 Colon Carcinoma Animal Model.

We describe the anti-angiogenic and anti-lymphangiogenic effects of corosolic acid, a pentacyclic triterpenoid isolated from Cornus kousa Burg. A mouse colon carcinoma CT-26 animal model was employed to determine the in vivo anti-angiogenic and anti-lymphangiogenic effects of corosolic acid. Corosolic acid induced apoptosis in CT-26 cells, mediated by the activation of caspase-3. In addition, it reduced the final tumor volume and the blood and lymphatic vessel densities of tumors, indicating that it suppresses in vivo angiogenesis and lymphangiogenesis. Corosolic acid inhibited the proliferation and tube formation of human umbilical vein endothelial cells and human dermal lymphatic microvascular endothelial cells. In addition, corosolic acid decreased the proliferation and migration of human umbilical vein endothelial cells stimulated by angiopoietin-1. Pretreatment with corosolic acid decreased the phosphorylation of focal adhesion kinase (FAK) and ERK1/2, suggesting that corosolic acid contains anti-angiogenic activity that can suppress FAK signaling induced by angiopoietin-1.

Recombinant human vascular endothelial growth factor receptor 1 effectively inhibits angiogenesis in vivo.

Vascular endothelial growth factor (VEGF) plays an important role in both physiological and pathological angiogenesis. VEGF receptor‑1 (VEGFR‑1) acts as a decoy VEGF receptor that enables the regulation of VEGF on the vascular endothelium. In the present study, the recombinant human VEGFR1D1‑3/Fc (rhVEGFR‑1), which contains key domains for VEGF binding, was cloned and expressed in Chinese hamster ovary (CHO) cells. The rhVEGFR‑1 protein was purified using protein‑A affinity chromatography. The molecular weight of rhVEGFR‑1 was found to be ~162 and 81 kD in non‑reducing and reducing SDS‑PAGE, respectively. The majority of the final protein products were in the dimeric conformation. Western blot analysis revealed that rhVEGFR‑1 was only capable of binding to the full glycan form of rhVEGF‑165 and rhVEGF‑121. The dissociation constant for the binding of rhVEGFR‑1 to VEGF‑165, detected using Biacore, was 285 pM. In addition, rhVEGFR‑1 inhibited the proliferation and migration of human microvascular endothelial cells. In vivo experiments also demonstrated that rhVEGFR‑1 inhibited chicken chorioallantoic membrane neovascularization and angiogenesis in nude mice. In conclusion, an anti‑angiogenic recombinant soluble VEGFR was expressed (up to 5 mg/l) in CHO cells and was shown to be capable of inhibiting neovascularization in vivo and in vitro.

Effect of trivalent arsenicals on cell proliferation in mouse and human microvascular endothelial cells

Chronic exposure to high levels of inorganic arsenic (iAs) has been associated with cancerous and non-cancerous health effects, including cardiovascular effects. However, the mechanism for a presumed toxic effect of arsenic on vascular tissue is not clear. Our working hypothesis is that inorganic trivalent arsenic and its methylated metabolites react with cysteine-containing cellular proteins and alter their function leading to adverse events such as cytotoxicity or proliferation. In this study, human microvascular endothelial cells (HMEC1) and mouse microvascular endothelial cells (MFP-MVEC) were exposed to arsenite (iAsIII), monomethylarsonous acid (MMAIII), or dimethylarsinous acid (DMAIII) for 72h to evaluate cytotoxicity, and for 24, 48 or 72h to evaluate cell proliferation. Both cell lines showed similar LC50 values, from 0.1 to 2.4μM, for all three trivalent arsenicals. The endothelial cells treated with1nM to 1μM concentrations of the three trivalent arsenicals did not show increased cell proliferation at 24, 48 or 72h or increased rate of proliferation at 72h of exposure. Overall, cytotoxicity of trivalent arsenicals to microvascular endothelial cells is similar to their cytotoxicity to epithelial cells, and that these compounds are not mitogenic.

MicroRNA-199a-5p Regulates the Proliferation of Pulmonary Microvascular Endothelial Cells in Hepatopulmonary Syndrome

Background/Aims: Pulmonary microvascular endothelial cell (PMVEC) proliferation and angiogenesis contribute to the development of hepatopulmonary syndrome (HPS). MicroRNA-199a-5p (miR-199a-5p) has emerged as a potent regulator of angiogenesis, and its expression levels significantly decrease in the serum of patients with hepatopathy. However, it has not been reported about whether miR-199a-5p might control PMVEC proliferation. Here, we described the miR-199a-5p governing PMVEC proliferation in HPS. Methods: PMVECs were treated with rat serum from common bile duct ligation (CBDL) or sham. MiR-199a-5p mimic or inhibitor was used to change the miR-199a-5p expression. Knockdown of caveolin-1 (Cav-1) was performed using siRNA. NSC-23766 was used to inhibit Rac1 activity. Gene and protein expressions were quantified by qRT-PCR and western blot. Cell proliferation was analyzed by ³H-TdR incorporation and CCK-8 assays. Stress fibers were detected by immunofluorescence. Results: CBDL rat serum induced the down-regulation of miR-199a-5p. Delivery of miR-199a-5p suppressed the CBDL rat serum-induced PMVEC proliferation whereas knockdown of miR-199a-5p promoted PMVEC proliferation. This was accompanied by a decrease and an increase in Cav-1 expression, respectively. Cav-1 siRNA abolished the enhancement of PMVEC proliferation induced by the miR-199a-5p inhibition. Although stress fibers were disrupted in Cav-1 deficient cells, NSC-23766 increased stress fibers and contributed to cell proliferation. Conclusions: CBDL rat serum induced down-regulation of miR-199a-5p in PMVECs, which led to an increase of Cav-1 gene expression. Increased Cav-1 expression, by inhibiting Rac1 activity, led to the formation of stress fibers, which contribute to PMVEC proliferation and thus the pathogenesis of HPS.

A simple method to assess in vivo proliferation in lung vasculature with EdU: the case of MMC-induced PVOD in rat.

5-Ethynyl-2′-deoxyuridine (EdU) incorporation is becoming the gold standard method for in vitro and in vivo visualization of proliferating cells. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration. It can be used to easily detect DNA replication in large tissue samples or organ explants with low proliferation and turnover of cells formerly believed to be in a “terminal” state of differentiation. Here we describe a protocol for the localization and identification of proliferating cells in quiescent or injured pulmonary vasculature, in a model of pulmonary veno-occlusive disease (PVOD). PVOD is an uncommon form of pulmonary hypertension characterized by progressive obstruction of small pulmonary veins. We previously reported that mitomycin-C (MMC) therapy is associated with PVOD in human. We demonstrated that MMC can induce PVOD in rats, which currently represents the sole animal model that recapitulates human PVOD lesions. Using the EdU assay, we demonstrated that MMC-exposed lungs displayed areas of exuberant microvascular endothelial cell proliferation which mimics pulmonary capillary hemangiomatosis, one of the pathologic hallmarks of human PVOD. In vivo pulmonary cell proliferation measurement represents an interesting methodology to investigate the potential efficacy of therapies aimed at normalizing pathologic angioproliferation.

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

Adhesion molecule-mediated hippo pathway modulates hemangioendothelioma cell behavior.

Hemangioendotheliomas are categorized as intermediate-grade vascular tumors that are commonly localized in the lungs and livers. The regulation of this tumor cell's proliferative and apoptotic mechanisms is ill defined. We recently documented an important role for Hippo pathway signaling via endothelial cell adhesion molecules in brain microvascular endothelial cell proliferation and apoptosis. We found that endothelial cells lacking cell adhesion molecules escaped from contact inhibition and exhibited abnormal proliferation and apoptosis. Here we report on the roles of adherens junction molecule modulation of survivin and the Hippo pathway in the proliferation and apoptosis of a murine hemangioendothelioma (EOMA) cell. We demonstrated reduced adherens junction molecule (CD31 and VE-cadherin) expression, increased survivin and Ajuba expression, and a reduction in Hippo pathway signaling resulting in increased proliferation and decreased activation of effector caspase 3 in postconfluent EOMA cell cultures. Furthermore, we confirmed that YM155, an antisurvivin drug that interferes with Sp1-survivin promoter interactions, and survivin small interference RNA (siRNA) transfection elicited induction of VE-cadherin, decreased Ajuba expression, increased Hippo pathway and caspase activation and apoptosis, and decreased cell proliferation. These findings support the importance of the Hippo pathway in hemangioendothelioma cell proliferation and survival and YM155 as a potential therapeutic agent in this category of vascular tumors.

Smad-independent pathway involved in transforming growth factor β1-induced Nox4 expression and proliferation of endothelial cells.

NADPH oxidase-derived reactive oxygen species are important for various cellular functions, including proliferation. Endothelial cells predominantly express the Nox4 isoform of NADPH oxidase, but it is not entirely clear how it is regulated. In this study, we investigated the signalling pathways involved in transforming growth factor-β1 (TGF-β1)-induced Nox4 expression and the proliferation of human microvascular endothelial cells (HMECs). TGF-β1 stimulated Nox4 messenger RNA and protein expression in HMECs. TGF-β1-induced Nox4 also increased hydrogen peroxide production, which was inhibited by diphenyleneiodonium and EUK134. The acute treatment of HMECs with TGF-β1 enhanced the phosphorylation of Smad2 and extracellular signal-regulated kinase (ERK) 1/2, without affecting p38MAPK, Akt, or Jun N-terminal kinase 1/2 (JNK1/2) pathways. Further, inhibition of Smad2 signalling using an inhibitor of activin receptor-linked kinase 5 SB431542 reduced TGF-β1-induced Nox4 expression, while inhibition of ERK1/2 with the inhibitor of mitogen-activated protein kinase kinase 1/2 U0126 decreased both basal and TGF-β1-induced Nox4 expression. Inhibition of ERK1/2 phosphorylation with U0126 did not affect Smad2 phosphorylation. Finally, TGF-β1 enhanced endothelial cell proliferation, which was reduced by U0126 but not by SB431542. These findings suggest that the non-canonical pathway ERK1/2 regulates Nox4 expression and may be involved in TGF-β1-induced proliferation of endothelial cells, which is vital during angiogenesis and vascular development.

Modulation of VEGF-induced retinal vascular permeability by peroxisome proliferator-activated receptor-β/δ.

Vascular endothelial growth factor (VEGF)-induced retinal vascular permeability contributes to diabetic macular edema (DME), a serious vision-threatening condition. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) antagonist/reverse agonist, GSK0660, inhibits VEGF-induced human retinal microvascular endothelial cell (HRMEC) proliferation, tubulogenesis, and oxygen-induced retinal vasculopathy in newborn rats. These VEGF-induced HRMEC behaviors and VEGF-induced disruption of endothelial cell junctional complexes may well share molecular signaling events. Thus, we sought to examine the role of PPARβ/δ in VEGF-induced retinal hyperpermeability. Transendothelial electrical resistance (TEER) measurements were performed on HRMEC monolayers to assess permeability. Claudin-1/Claudin-5 localization in HRMEC monolayers was determined by immunocytochemistry. Extracellular signal-regulated protein kinases 1 and 2 (Erk 1/2) phosphorylation, VEGF receptor 1 (VEGFR1) and R2 were assayed by Western blot analysis. Expression of VEGFR1 and R2 was measured by quantitative RT-PCR. Last, retinal vascular permeability was assayed in vivo by Evans blue extravasation. Human retinal microvascular endothelial cell monolayers treated with VEGF for 24 hours showed decreased TEER values that were completely reversed by the highest concentration of GSK0660 (10 μM) and PPARβ/δ-directed siRNA (20 μM). In HRMEC treated with VEGF, GSK0660 stabilized tight-junctions as evidenced by Claudin-1 staining, reduced phosphorylation of Erk1/2, and reduced VEGFR1/2 expression. Peroxisome proliferator-activated receptor β/δ siRNA had a similar effect on VEGFR expression and Claudin-1, supporting the specificity of GSK0660 in our experiments. Last, GSK0660 significantly inhibited VEGF-induced retinal vascular permeability and reduced retinal VEGFR1and R2 levels in C57BL/6 mice. These data suggest a protective effect for PPARβ/δ antagonism against VEGF-induced vascular permeability, possibly through reduced VEGFR expression. Therefore, antagonism/reverse agonism of PPARβ/δ siRNA may represent a novel therapeutic methodology against retinal hyperpermeability and is worthy of future investigation.

Protein tyrosine phosphatase 1B impairs diabetic wound healing through vascular endothelial growth factor receptor 2 dephosphorylation.

Impaired wound healing is a major complication of diabetes mellitus. The mechanisms that govern wound healing, however, are complex and incompletely understood. In the present study, we determined the inhibitory role of protein tyrosine phosphatase 1B (PTP1B) in the process of diabetic wound healing. First, by comparing the wound healing process in PTP1B knockout (PTP1B(-/-)) mice, ob/ob mice and their wild-type littermates in the presence or absence of streptozotocin treatment, we showed that the inhibition of mouse wound healing in streptozotocin-induced diabetic conditions is because of the upregulation and activation of PTP1B. Second, the impaired wound healing in ob/ob mice and streptozotocin-treated wild-type mice was rescued by a PTP1B inhibitor. Third, PTP1B, which is upregulated under hyperglycemic condition, inhibited the tube formation, proliferation, and migration of human microvascular endothelial cells induced by vascular endothelial growth factor, whereas this inhibition was largely abolished by the PTP1B inhibitor. Finally, mechanism study further indicated that PTP1B likely suppressed the proliferation, migration, and tube formation of vascular endothelial cells through dephosphorylation of vascular endothelial growth factor receptor 2. Our study demonstrated that PTP1B negatively modulated the diabetic wound healing process by dephosphorylating the endothelial cell vascular endothelial growth factor receptor 2 and that the specific inhibitor of PTP1B might serve as a potential novel therapeutic tool for diabetic wound healing.

Pulsed magnetic field accelerate proliferation and migration of cardiac microvascular endothelial cells.

Heart failure is a disease with multifactorial causes. Recently it was established that reduction in vascular density promoted the progression from adaptive cardiac hypertrophy to heart failure, therefore, therapeutic angiogenesis may be a promising method for treating heart failure. Cardiac microvascular endothelial cells (CMECs) play a major role in cardiac angiogenesis. In the present study, we investigated the direct and indirect effect of pulsed magnetic field (PMF) on the proliferation and migration of CMECs. CMECs were isolated from adult Sprague-Dawley (SD) rat hearts. We found PMF with a frequency of 15 Hz and an intensity of 1.8 mT accelerated the proliferation and migration of CMECs and cardiac myocytes (CMs). Moreover, CMECs treated with PMF released 1.5-fold higher vascular endothelial growth factor (VEGF) and 2-fold higher fibroblast growth factor-2 (FGF-2) when compared with PMF-free cells. In addition, CMs treated with PMF released twofold higher FGF-2 compared with PMF-free cells, but there was no change in VEGF levels. Those results suggested PMF has both a direct autocrine mitogenic and an indirect paracrine effect on CMECs proliferation and migration, and the effect of PMF on intercellular communication between CMECs and CMs was partially dependent on FGF-2, but independent on VEGF.

PP-092 CYTOTOXIC AND CYTOSTATIC EFFECTS OF PONATINIB ON IMATINIB-SENSITIVE AND -RESISTANT CHRONIC MYELOID LEUKEMIA CELLS

Retinal neovascularization is the most common cause of moderate to severe vision loss in all age groups. Despite the use of anti-VEGFA therapies, this complication continues to cause blindness, suggesting a role for additional molecules in retinal neovascularization. Besides VEGFA and VEGFB, hypoxia induced VEGFC expression robustly. Based on this finding, we tested the role of VEGFC in pathological retinal angiogenesis. VEGFC induced proliferation, migration, sprouting and tube formation of human retinal microvascular endothelial cells (HRMVECs) and these responses require CREB-mediated DLL4 expression and NOTCH1 activation. Furthermore, down regulation of VEGFC levels substantially reduced tip cell formation and retinal neovascularization in vivo. In addition, we observed that CREB via modulating the DLL4-NOTCH1 signaling mediates VEGFC-induced tip cell formation and retinal neovascularization. In regard to upstream mechanism, we found that down regulation of p38β levels inhibited hypoxia-induced CREB-DLL4-NOTCH1 activation, tip cell formation, sprouting and retinal neovascularization. Based on these findings, it may be suggested that VEGFC besides its role in the regulation of lymphangiogenesis also plays a role in pathological retinal angiogenesis and this effect depends on p38β and CREB-mediated activation of DLL4-NOTCH1 signaling.

Effect of bilirubin on proliferation of pulmonary microvascular endothelial cells of rats

Objective To evaluate the effect of bilirubin on proliferation of pulmonary microvascular endothelial cells (PMVECs) of rats.Methods Primary PMVECs harvested from 10 healthy male Sprague-Dawleyrats,weighing 120-150 g,aged 2-3 months,were cultured,purified and identified.PMVECs were seeded in low-glucose DMEM culture medium or 96-well culture plates,and divided into 5 groups according to the random number table:control group (group C) and different concentrations of bilirubin groups (B1-4 groups) with 30 wells or48 flasks in each group.In group C,low-glucose DMEM 1 ml was added.In B1-4 groups,5,10,20 and 50 μmol/L bilirubin 1 ml were added,respectively.At 24,48 and 72 h of incubation,proliferation of PMVECs was measured using CCK-8 assay and 3 H-TdR incorporation assay,Akt1 mRNA and Smad3 mRNA expression was measured by RT-PCR,and phosphor-Akt1 (p-Akt1) protein and Smad3 protein expression was detected using Western blot.Results Compared with group C,no significant changes were found in each parameter mentioned above at each time point in B1 and B2 groups,the proliferation of PMVECs was weakened,and Akt1 mRNA,p-Akt1 protein and Smad3 protein and mRNA expression was down-regulated at 48 h of incubation in B3 group,and the proliferation of PMVECs was weakened,and the expression of Akt1 mRNA,p-Akt1 protein and Smad3 mRNA and protein was down-regulated at 48 and 72 h of incubation in B4 group.Compared with group B3,the proliferation of PMVECs was weakened,and the expression of Akt1 mRNA,p-Akt1 protein and Smad3 mRNA and protein was down-regulated at 72 h of incubation in B4 group.Conclusion High concentration of bilirubin can inhibit proliferation of PMVECs and down-regulated expression of Akt1 and Smad3 is involved in the mechanism. Key words: Bilirubin; Cell proliferation; Endothelial cells

Comparison of binding characteristics and in vitro activities of three inhibitors of vascular endothelial growth factor A.

The objectives of this study were to evaluate the relative binding and potencies of three inhibitors of vascular endothelial growth factor A (VEGF), used to treat neovascular age-related macular degeneration, and assess their relevance in the context of clinical outcome. Ranibizumab is a 48 kDa antigen binding fragment, which lacks a fragment crystallizable (Fc) region and is rapidly cleared from systemic circulation. Aflibercept, a 110 kDa fusion protein, and bevacizumab, a 150 kDa monoclonal antibody, each contain an Fc region. Binding affinities were determined using Biacore analysis. Competitive binding by sedimentation velocity analytical ultracentrifugation (SV-AUC) was used to support the binding affinities determined by Biacore of ranibizumab and aflibercept to VEGF. A bovine retinal microvascular endothelial cell (BREC) proliferation assay was used to measure potency. Biacore measurements were format dependent, especially for aflibercept, suggesting that biologically relevant, true affinities of recombinant VEGF (rhVEGF) and its inhibitors are yet to be determined. Despite this assay format dependency, ranibizumab appeared to be a very tight VEGF binder in all three formats. The results are also very comparable to those reported previously.1-3 At equivalent molar ratios, ranibizumab was able to displace aflibercept from preformed aflibercept/VEGF complexes in solution as assessed by SV-AUC, whereas aflibercept was not able to significantly displace ranibizumab from preformed ranibizumab/VEGF complexes. Ranibizumab, aflibercept, and bevacizumab showed dose-dependent inhibition of BREC proliferation induced by 6 ng/mL VEGF, with average IC50 values of 0.088 ± 0.032, 0.090 ± 0.009, and 0.500 ± 0.091 nM, respectively. Similar results were obtained with 3 ng/mL VEGF. In summary Biacore studies and SV-AUC solution studies show that aflibercept does not bind with higher affinity than ranibizumab to VEGF as recently reported,4 and both inhibitors appeared to be equipotent with respect to their ability to inhibit VEGF function.

Endoglin promotes angiogenesis in cell- and animal-based models of retinal neovascularization.

We investigated endoglin expression in hypoxic microvascular endothelial cells and retinal endoglin expression in rats that develop experimental oxygen-induced retinopathy (OIR). We also tested neutralizing antibodies (Abs) against endoglin (anti-CD105 Ab) and VEGF (anti-VEGF Ab) either alone or in combination for efficacy against serum-induced retinal microvascular endothelial cell proliferation and retinal neovascularization (NV) in OIR rats. To our knowledge, this marks the first time that a biologic agent has been used to target retinal endoglin and modulate retinal neovascularization. Induction of endoglin by hypoxia was measured by immunohistochemical analysis and ELISA. Proliferation was quantified using a colorimetric 5-bromo-2-deoxyuridine ELISA. Western blots were used to measure endoglin levels in retinas of OIR rats. Immunohistochemical staining was also preformed in OIR rats using anti-CD105 and fluorescein isothiocyanate-conjugated isolectin B4 antibodies. Anti-CD105 Ab and Anti-VEGF Ab, administered alone or in combination, reduced serum-induced retinal microvascular endothelial cell proliferation. Additionally, in a rat model of oxygen-induced retinopathy, retinal endoglin was significantly increased at 14(2), 14(3), 14(4) and 14(6) compared with retinal levels in control rats. At 14(2), immunohistochemical analysis demonstrated that endoglin was elevated in newly developed vessels at the peripheral extent of major veins, precisely where NV is expected to develop in OIR rats. Neutralizing anti-CD105 reduced retinal NV in OIR rats. Our data support other studies showing that reduction of endoglin expression inhibits retinal NV. Our findings demonstrate that retinal endoglin immunolocalization overlaps with nascent neovascular structures in OIR rats. Therefore, endoglin may serve as a useful predictor of incipient neovascular disease.

Rho Kinase Inhibitor AMA0526 improves surgical outcome in a rabbit model of glaucoma filtration surgery

AbstractPurpose To elucidate the effect of the ROCK inhibitor AMA0526 on the wound healing process and surgical outcome of glaucoma filtration surgery.Methods The in vitro effect of ROCK inhibitor AMA0526 on human brain microvascular endothelial cells (HBMEC) and human Tenon fibroblasts (HTF) was determined using a proliferation assay. Secondly the in vivo effect of topical AMA0526 0.3% TID was investigated in a rabbit model of glaucoma filtration surgery (n=5/time point). Treatment outcome was studied by clinical investigation of the bleb area as well as immunohistological analyses for inflammation (CD45), angiogenesis (CD31) and collagen deposition at day 8, 14 and 30 after surgery. Contralateral eyes were used as control and were treated with vehicle.Results A dose‐dependent reduction of HBMEC and HTF proliferation was measured after incubation with AMA0526. Incubation with the highest concentration of AMA0526 reduced proliferation of HBMEC and HTF to 22 and 35%. In the surgery model, AMA0526 significantly improved bleb area and survival compared to vehicle treated eyes. Immunohistological analyses showed significant reduction of inflammation, angiogenesis and collagen deposition after treatment with the ROCK inhibitor. Compared to vehicle, inflammation was decreased by 33% at 8 days and angiogenesis by 52% on day 8 and by 29% at 14 days. Collagen deposition was significantly reduced by 11 and 42% on day 14 and 30.Conclusion This study shows that AMA0526 is able to inhibit proliferation of microvascular endothelial cells and Tenon fibroblasts in vitro, and to improve glaucoma surgery outcome in rabbits. In addition to improved bleb area, AMA0526 led to decreased inflammation, angiogenesis and fibrosis.

Epigalloccatechin-3-gallate Inhibits Ocular Neovascularization and Vascular Permeability in Human Retinal Pigment Epithelial and Human Retinal Microvascular Endothelial Cells via Suppression of MMP-9 and VEGF Activation

Epigalloccatechin-3-gallate (EGCG) is the main polyphenol component of green tea (leaves of Camellia sinensis). EGCG is known for its antioxidant, anti-inflammatory, antiviral, and anti-carcinogenic properties. Here, we identify EGCG as a new inhibitor of ocular angiogenesis and its vascular permeability. Matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) play a key role in the processes of extracellular matrix (ECM) remodeling and microvascular permeability during angiogenesis. We investigated the inhibitory effects of EGCG on ocular neovascularization and vascular permeability using the retina oriented cells and animal models induced by VEGF and alkaline burn. EGCG treatment significantly decreased mRNA and protein expression levels of MMP-9 in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) and tumor necrosis factor alpha (TNF-α) in human retinal pigment epithelial cells (HRPECs). EGCG also effectively protected ARPE-19 cells from cell death and attenuated mRNA expressions of key angiogenic factors (MMP-9, VEGF, VEGF Receptor-2) by inhibiting generation of reactive oxygen species (ROS). EGCG significantly inhibited proliferation, vascular permeability, and tube formation in VEGF-induced human retinal microvascular endothelial cells (HRMECs). Furthermore, EGCG significantly reduced vascular leakage and permeability by blood-retinal barrier breakdown in VEGF-induced animal models. In addition, EGCG effectively limited upregulation of MMP-9 and platelet endothelial cell adhesion molecule (PECAM/CD31) on corneal neovascularization (CNV) induced by alkaline burn. Our data suggest that MMP-9 and VEGF are key therapeutic targets of EGCG for treatment and prevention of ocular angiogenic diseases such as age-related macular degeneration, diabetic retinopathy, and corneal neovascularization.

MiR-7-5p is frequently downregulated in glioblastoma microvasculature and inhibits vascular endothelial cell proliferation by targeting RAF1.

The aberrant expression of microRNAs (miRNAs) is always associated with tumor development and progression. Microvascular proliferation is one of the unique pathologic features of glioblastoma (GBM) . In this study, the microvasculature from GBM or normal brain tissue derived from neurosurgeries was purified and total RNA was isolated from purified microvasculature. The difference of miRNA expression profiles between glioblastoma microvasculature and normal brain capillaries was investigated. It was found that miR-7-5p in GBM microvessels was significantly reduced compared with that in normal brain capillaries. In the in vitro experiments, overexpression of miR-7-5p significantly inhibited human umbilical vein endothelial cell proliferation. Forced expression of miR-7-5p in human umbilical vein endothelial cells in vitro significantly reduced the protein level of RAF1 and repressed the activity of the luciferase, a reporter vector carrying the 3'-untranslated region of RAF1. These findings indicate that RAF1 is one of the miR-7-5p target genes. Furthermore, a significant inverse correlation between miR-7-5p expression and RAF1 protein level in GBM microvasculature was found. These data suggest that miR-7-5p functions as a tumor suppressor gene to regulate GBM microvascular endothelial cell proliferation potentially by targeting the RAF1 oncogene, implicating an important role for miR-7-5p in the pathogenesis of GBM. It may serve as a guide for the antitumor angiogenesis drug development.

Effect of glutahione S-transferases on cardiac microvascular endothelial cells

Objective To detect the effect of glutahione S-transferases (GSTP1) expression on the migration,proliferation and apoptosis of cardiac microvascular endothelial cells (CMECs).Methods After obtaining CMECs from SD rats by enzymatic digestion method,the special plasmid of GSTP was transfected.The ability of migration,proliferation and apoptosis of CMECs were examined by using Transwell assay,methyl thiazol tetrazolium (MTT) assay and flow cytometry,respectively.Results The ability of migration of CMECs was significantly reduced in GSTP1 over-expression group as compared with control group (9.0 ±2.7 vs.12.8 ±2.5,P ＜0.05),and the ability of proliferation of CMECs was also obviously reduced in comparison with control group (0.26 ± 0.02 vs.0.05 ± 0.01,P ＜ 0.05).But the apoptotic index was increased significantly (P ＜ 0.05).Conclusion GSTP1 expression can effectively inhibit migration and proliferation,and promote apoptosis of CMECs. Key words: Glutahione S-transferases; Cardiac microvascular endothelial cells; Migration; Proliferation

NAP reduces murine microvascular endothelial cells proliferation induced by hyperglycemia.

Hyperglycemia has been identified as a risk factor responsible for micro- and macrovascular complications in diabetes. NAP (Davunetide) is a peptide whose neuroprotective actions are widely demonstrated, although its biological role on endothelial dysfunctions induced by hyperglycemia remains uninvestigated. In the present study we hypothesized that NAP could play a protective role on hyperglycemia-induced endothelial cell proliferation. To this end we investigated the effects of NAP on an in vitro model of murine microvascular endothelial cells grown in high glucose for 7 days. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and cyclin D1 protein expression analysis revealed that NAP treatment significantly reduces viability and proliferation of the cells. Hyperglycemia induced the activation of mitogen-activated protein kinase/extracellular signal-regulated protein kinase and/or phosphatidylinositol-3 kinase/Akt pathways in a time-dependent manner. NAP treatment reduced the phosphorylation levels of ERK and AKT in cells grown in high glucose. These evidences suggest that NAP might be effective in the regulation of endothelial dysfunction induced by hyperglycemia.