Paracrine or virus-mediated induction of decorin expression by endothelial cells contributes to tube formation and prevention of apoptosis in collagen lattices

Resting endothelial cells express the small proteoglycan biglycan, whereas sprouting endothelial cells also synthesize decorin, a related proteoglycan. Here we show that decorin is expressed in endothelial cells in human granulomatous tissue. For in vitro investigations, the human endothelium-derived cell line, EA.hy 926, was cultured for 6 or more days in the presence of 1% fetal calf serum on top of or within floating collagen lattices which were also populated by a small number of rat fibroblasts. Endothelial cells aligned in cord-like structures and developed cavities that were surrounded by human decorin. About 14% and 20% of endothelial cells became apoptotic after 6 and 12 days of co-culture, respectively. In the absence of fibroblasts, however, the extent of apoptosis was about 60% after 12 days, and cord-like structures were not formed nor could decorin production be induced. This was also the case when lattices populated by EA.hy 926 cells were maintained under one of the following conditions: 1) 10% fetal calf serum; 2) fibroblast-conditioned media; 3) exogenous decorin; or 4) treatment with individual growth factors known to be involved in angiogenesis. The mechanism(s) by which fibroblasts induce an angiogenic phenotype in EA.hy 926 cells is (are) not known, but a causal relationship between decorin expression and endothelial cell phenotype was suggested by transducing human decorin cDNA into EA.hy 926 cells using a replication-deficient adenovirus. When the transduced cells were cultured in collagen lattices, there was no requirement of fibroblasts for the formation of capillary-like structures and apoptosis was reduced. Thus, decorin expression seems to be of special importance for the survival of EA.hy 926 cells as well as for cord and tube formation in this angiogenesis model.     

Variability in E-selectin expression, mRNA levels and sE-selectin release between endothelial cell lines and primary endothelial cells

Endothelial cell lines express markers and are assumed to exhibit other endothelial cell responses. We investigated E-selectin expression from human umbilical vein endothelial cells, the spontaneously transformed ECV304 line and the hybrid line EA.hy926 by flow cytometry and immunofluorescence, mRNA and soluble E-selectin release. In cells exposed to tumour necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta), median (range) percentage of E-selectin-positive HUVECs increased from 1.6(0.9-6. 2)% to 91.4(83.0-96.1)%, (P=0.001) using flow cytometry. In contrast, E-selectin expression by ECV304 and EA.hy926 cell lines was 100-fold lower. E-selectin mRNA was detectable after 2 h, maximal at 6 h in HUVECs and undetectable in EA.hy926 and ECV304 cell lines after exposure to TNF-alpha/IL-1beta. sE-selectin accumulation increased (P=0.004) in HUVECs only. Neutrophil adherence to ECV304 and EA.hy926 cells was poor compared to HUVECs (P=0.004). The cell lines ECV304 and EA.hy926 do not exhibit normal endothelium expression of E-selectin, and may not be appropriate for studies of adhesion.     

Regulation of endothelial cells functions by ultrasonic supernatant of Streptococcus pyogenes

Angiogenesis and vascular remodeling are vital components of inflammation. As an inflammation evolves, vessels expand to supply nutrients and inflammatory mediators, sustaining the accumulation of activated immune cells in the affected tissues. This study demonstrates that ultrasonic supernatant of Streptoccocus pyogenes has anti-angiogenic properties: inhibit EA.hy 926 human endothelial cells metabolism, adhesion, migration, proliferation. At the same time Streptococcal components inhibit signaling pathways that involve FAK and ERK1/2. These effects are not associated with necrosis or apoptosis in cell culture. Taking together, our results suggest that impairing angiogenic function of endothelial cells might contribute to the reduced tissue perfusion, hypoxia, and subsequent regional tissue necrosis caused by Streptococci group A.     

Lysophosphatidic acid‐triggered pathways promote the acquisition of trophoblast endovascular phenotype in vitro

Successful implantation and placentation requires that extravillous cytotrophoblast acquires an endovascular phenotype and remodels uterine spiral arteries. Defects in this mechanism correlate with severe obstetric complications as implantation failure and preeclampsia. Lysophosphatidic acid (LPA) participates in embryo implantation and contributes to vascular physiology in different biological systems. However, the role of LPA on trophoblast endovascular transformation has not been studied. Due to difficulties in studying human pregnancy in vivo, we adopted a pharmacological approach in vitro to investigate LPA action in various aspects of trophoblast endovascular response, such as the formation of endothelial capillary‐like structures, migration, and proliferation. The HTR‐8/SVneo cell line established from human first trimester cytotrophoblast was used to model the acquisition of the endovascular phenotype by the invading trophoblast. LPA increased HTR‐8/SVneo tube formation, migration (wound healing assay and phalloidin staining) and proliferation (MTT assay). LPA G protein‐coupled receptors, LPA₁ and LPA₃, were expressed in HTR‐8/SVneo. By using selective antagonists, we showed that enhanced tubulogenesis was mediated by LPA₃. In addition, cyclooxygenase‐2 and inducible nitric oxide synthase pathways participated in LPA‐stimulated tubulogenesis. Inducible nitric oxide synthase was activated downstream cyclooxygenase‐2. Furthermore, prostaglandin E2 and a nitric oxide donor (SNAP) increased trophoblast tube formation in a concentration‐dependent manner. Finally, we observed that cyclooxygenase‐2 and inducible nitric oxide synthase were localized in the nucleus, and LPA did not modify their cellular distribution. Our results show that LPA‐triggered regulatory pathways promote trophoblast endovascular response in vitro, suggesting a new role for LPA during spiral artery remodeling at the maternal‐fetal interface.     

Interleukin-1β enhances cell adhesion in human endothelial cells via microRNA-1914–5p suppression

Atherosclerosis is a chronic inflammatory disease and the underlying cause of most cardiovascular diseases. Interleukin (IL)-1β facilitates early atherogenic lesion formation by increasing monocyte adhesion to endothelial cells via upregulation of adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1). MicroRNAs (miRNAs) have been shown to be associated with inflammatory conditions in the vascular system. The expression of circulating miR-1914–5p is reportedly downregulated in patients with cardiovascular diseases. However, the role of miR-1914–5p downregulation in IL-1β–induced endothelial cell dysfunction and the effect of miR-1914–5p on lesion formation remain unclear. Therefore, we investigated whether miR-1914–5p is associated with monocyte adhesion in human endothelial cells. IL-1β decreased miR-1914–5p expression in EA.hy926 cells. In addition, miR-1914–5p depletion enhanced ICAM-1 expression and monocyte adhesion in EA.hy926 cells. Moreover, miR-1914–5p mimic suppressed monocyte adhesion and ICAM-1 expression induced by IL-1β in endothelial cells. These results suggest that suppression of miR-1914–5p expression by IL-1β may be an important regulator in mediating monocyte adhesion in endothelial cells. Further investigation of miR-1914–5p may lead to the development of novel therapeutic strategies for atherosclerosis.     

源自天蚕素A和爪蟾素2的杂合肽P18体外抑制内皮细胞血管生成

目的探讨杂合肽P18体外对内皮细胞EA.hy926血管生成的抑制作用.方法采用MTT法检测P18对EA.hy926细胞增殖的影响;应用Matrigel实验检测P18对内皮细胞形成管状结构的影响;利用流式细胞术分析P18对内皮细胞的损伤作用.结果 MTT结果显示P18可明显抑制EA.hy926细胞的增殖,且抑制率存在剂量依赖性;Matrigel实验表明P18具有抑制EA.hy926细胞体外分化成管状结构的作用;流式结果显示15 μM P18作用内皮细胞6 h后,所诱导的细胞坏死比例达到81.4%.结论体外实验结果表明,杂合肽P18具有体外抑制EA.hy926细胞血管生成的作用.     

HOXA-AS2靶向miR-17对人脐静脉内皮细胞生物学功能以及炎症因子的影响

目的探讨HOXA-AS2对动脉粥样硬化（AS）模型细胞生物学功能以及炎症因子的影响及分子机制。 方法本实验共分为4个实验;实验1：用100 μg/mL的ox-LDL处理EA.hy926细胞作为ox-LDL组,正常培养的细胞作为对照组;实验2：将pcDNA3.1、pcDNA3.1-HOXA-AS2转染至EA.hy926细胞中再用100 μg/mL的ox-LDL处理,记为ox-LDL+pcDNA3.1组、ox-LDL+pcDNA3.1-HOXA-AS2组;实验3：将pcDNA3.1、pcDNA3.1-HOXA-AS2、si-NC、si-HOXA-AS2分别转染至EA.hy926细胞中,记为pcDNA3.1组、pcDNA3.1-HOXA-AS2组、si-NC组、si-HOXA-AS2组;实验4：将pcDNA3.1-HOXA-AS2与miR-NC、miR-17分别共转染至EA.hy926细胞中再用100 μg/mL的ox-LDL处理,记为ox-LDL+pcDNA3.1-HOXA-AS2+miR-NC组、ox-LDL+pcDNA3.1-HOXA-AS2+miR-17组。实时荧光定量PCR （RT-qPCR）检测HOXA-AS2和miR-17的表达水平;蛋白质印迹（Western blot）法检测细胞周期蛋白依赖性激酶抑制剂1A （P21）、cleaved caspase 3蛋白表达;MTT检测细胞增殖情况;流式细胞术检测细胞凋亡;ELISA法检测白细胞介素-1 （IL-1）、白细胞介素-6 （IL-6）水平;荧光素酶报告实验检测HOXA-AS2和miR-17的靶向关系。两组间比较采用独立样本t检验,多组间比较采用方差分析,组间两两比较采用LSD-t检验。 结果与对照组比较,ox-LDL组EA.hy926细胞中HOXA-AS2表达水平（0.23±0.02比1.02±0.10）,细胞增殖率[（47.83±5.01）﹪比（100.06±10.20）﹪]均降低,细胞凋亡率[（26.81±2.47）﹪比（8.23±0.80）﹪]、P21 （0.82±0.08比0.20±0.02）、cleaved caspase 3 （0.67±0.06比0.14±0.01）、IL-1[（792.34±59.37）ng/L比（326.14±34.59） ng/ L]和IL-6表达水平[（53.67±4.65）ng/L比（19.25±2.11）ng/L]均升高,差异具有统计学意义（P均< 0.05）。与ox-LDL+pcDNA3.1组比较,ox-LDL+pcDNA3.1-HOXA-AS2组EA.hy926细胞中HOXA-AS2表达水平（0.87±0.09比0.22±0.02）、细胞增殖率[（89.94±8.34）﹪比（48.21±4.86）﹪]均升高,细胞凋亡率[（12.33±1.18）﹪比（26.83±2.48）﹪]、P21 （0.33±0.03比0.81±0.08）、cleaved caspase 3 （0.24±0.02比0.69±0.06）、IL-1[ （446.25±46.84）ng/L比（802.21±60.18）ng/L]和IL-6表达水平[（25.64±2.65）ng/L比（55.21±5.10）ng/L]均降低,差异具有统计学意义（P均< 0.001）。与ox-LDL+pcDNA3.1-HOXA-AS2+miR-NC组比较,ox-LDL+pcDNA3.1-HOXA-AS2+miR-17组EA.hy926细胞中miR-17表达水平（2.14±0.21比1.05±0.10）、细胞凋亡率[（23.31±2.33）﹪比（13.75±1.44）﹪]、IL-1水平[（684.26±62.38）ng/L比（451.21±43.58）ng/L]、IL-6水平[（41.29±4.37）ng/L比（26.11±2.39）ng/L]均升高,细胞增殖率[（53.67±5.46）﹪比（90.21±9.16）﹪]降低,差异具有统计学意义（P均< 0.001）。HOXA-AS2与miR-17存在结合位点,荧光素酶报告实验显示,与miR-NC组比较,miR-17组中转染WT-HOXA-AS2的EA.hy926细胞荧光素酶活性降低（0.33±0.03比1.01±0.10,P < 0.05）,而转染MUT-HOXA-AS2的EA.hy926细胞荧光素酶活性差异无统计学意义（P > 0.05）;与anti-miR-NC组比较,anti-miR-17组中转染WT-HOXA-AS2的EA.hy926细胞荧光素酶活性升高（2.29±0.21比1.00±0.10,P < 0.05）,而转染MUT-HOXA-AS2的EA.hy926细胞荧光素酶活性差异无统计学意义（P > 0.05）。 结论过表达HOXA-AS2促进细胞增殖,抑制ox-LDL引起的细胞凋亡和炎症因子的释放,其机制可能与miR-17有关。     

G1对EA.hy926内皮细胞内质网应激的抑制作用

目的观察GPR30受体激动剂G1对高糖诱导的EA.hy926内皮细胞内质网应激(endoplasmic reticulum stress,ERS)的影响。方法选用EA.hy926内皮细胞为研究对象,分为3组:正常对照组(Con,17.51mmol/L葡萄糖)、高糖组(HG,33.3mmol/L葡萄糖)、高糖+G1组(HG+G1,HG+1umol/L G1),利用流式细胞术检测3组细胞凋亡率,Western blot法检测ERS相关分子Bip、IRE1、PERK及凋亡分子Bax、Bcl-2的表达变化,RT-PCR法检测Bip和CHOP的mRNA表达变化。结果 HG组与Con组比较,细胞凋亡率明显升高(P0.01),Bip、IRE1、PERK及凋亡分子Bax表达上调(P0.01,P0.05或P0.001),Bcl-2的表达下调(P0.01),Bip mRNA、CHOP mRNA表达上调(P0.001及P0.01);HG+G1组与HG组比较,细胞凋亡率明显降低(P0.05),Bip、IRE1、PERK及凋亡分子Bax表达下调(P0.05或P0.01),Bcl-2的表达上调(P0.05),Bip mRNA、CHOP mRNA表达下调(P0.001及P0.01)。结论 GPR30受体激动剂G1可抑制EA.hy926内皮细胞内质网应激。     

Endothelin-1 is expressed and released by a human endothelial hybrid cell line (EA.hy 926).

A permanent vascular endothelial cell line, EA.hy 926, was shown to express endothelin-1 (ET-1) mRNA and to secrete big ET-1 and ET-1 into culture medium. The concentration of both big ET-1 and ET-1 was significantly increased in EA.hy 926 culture medium by phosphoramidon, a metalloproteinase inhibitor, suggesting that phosphoramidon sensitive protease(s) may be responsible for the degradation of ET-1 and big ET-1. EA.hy 926 cells responded to various regulators of ET-1 similarly as primary human vascular endothelial cells. The production of ET-1 was increased by thrombin and decreased by vasodilators such as atrial natriuretic peptide, brain natriuretic peptide and nitroprusside, and by 8-bromo cyclic GMP and papaverine. This continuous human endothelial hybrid cell line could facilitate studies of regulation of ET-1 production in human endothelial cells, which in primary cultures have limited replication potential.     

Effect of thalidomide affecting VEGF secretion, cell migration, adhesion and capillary tube formation of human endothelial EA.hy 926 cells

Angiogenesis, new blood vessel formation, is a multistep process, precisely regulated by pro-angiogenic cytokines, which stimulate endothelial cells to migrate, proliferate and differentiate to form new capillary microvessels. Excessive vascular development and blood vessel remodeling appears in psoriasis, rheumatoid arthritis, diabetic retinopathy and solid tumors formation. Thalidomide [alpha-(N-phthalimido)-glutarimide] is known to be a potent inhibitor of angiogenesis, but the mechanism of its inhibitory action remains unclear. The aim of the study was to investigate the potential influence of thalidomide on the several steps of angiogenesis, using in vitro models. We have evaluated the effect of thalidomide on VEGF secretion, cell migration, adhesion as well as in capillary formation of human endothelial cell line EA.hy 926. Thalidomide at the concentrations of 0.01 microM and 10 microM inhibited VEGF secretion into supernatants, decreased the number of formed capillary tubes and increased cell adhesion to collagen. Administration of thalidomide at the concentration of 0.01 microM increased cell migration, while at 10 microM, it decreased cell migration. Thalidomide in concentrations from 0.1 microM to 10 microM did not change cell proliferation of 72-h cell cultures. We conclude that anti-angiogenic action of thalidomide is due to direct inhibitory action on VEGF secretion and capillary microvessel formation as well as immunomodulatory influence on EA.hy 926 cells migration and adhesion.     

7, 8-Dihydroxyflavone Protects an Endothelial Cell Line from H2O2 Damage

7, 8-dihydroxyflavone (7, 8-DHF), a selective agonist for TrkB receptors, has been well studied for its neurotrophic functions. However, its roles outside the neural tissues have scarcely been studied as yet. In this study, we investigated the protecting roles of 7, 8-DHF in EA.hy926 cells, a human umbilic vein endothelial cell line which was exposed to hydrogen peroxide (H₂O₂). We found that 7, 8-DHF significantly protected the cells from being damaged by H₂O₂ through suppression of apoptosis, attenuation of inflammatory factor releasing and inhibition of reactive oxygen species generation. The potent biological effects of 7, 8-DHF were probably executed via its binding to TrkB receptors because the receptor specific antagonist ANA-12 significantly blocked its protecting effects. The protecting roles of 7, 8-DHF in EA.hy926 cells suggest that it will be a promising compound to be developed into a health product that definitely benefits endothelial functions and prevents cardiovascular diseases.     

Production of prostacyclin and prostaglandin E2 in resting and IL-1beta-stimulated A549, HUVEC and hybrid EA.HY 926 cells.

Production of arachidonic acid (AA) metabolites - prostacyclin (PGI(2)) in large vessels and prostaglandin E(2) (PGE(2)) in microcirculation is intrinsically involved in maintenance of vascular wall homeostasis. EA.hy 926 is a hybrid cell line, is derived by fusion of HUVEC with A549 cells. The aim of this study was to examine the production of prostacyclin and PGE2 in resting and IL-1beta-stimulated EA.ha 926 cells, in comparison with its progenitor cells. Non-stimulated EA.hy 926 cells has been found to produce much lower amounts of prostacyclin than resting HUVEC. Resting hybrid cells produced more PGE(2) than prostacyclin, despite they expressed high levels of COX-1 and PGI(2) synthase. On the contrary to HUVEC and A549, EA.hy 926 cells did not respond to IL-1beta with COX-2 induction and increase of prostaglandin production, however they did it in response to lysophosphatidylcholine (LPC). The characteristics of EA.hy 926 cells in terms of the pattern of prostanoid formation could facilitate studies on endothelial metabolism and role of these important lipid mediators.     

No upregulation of lectin-like oxidized low-density lipoprotein receptor-1 in serum-deprived EA.hy926 endothelial cells under oxLDL exposure, but increase in autophagy

The oxidized low-density lipoprotein (oxLDL)-dependent activation of the lectin-like oxLDL receptor-1 (LOX-1) triggers apoptosis in vascular cells and appears to be involved in atherosclerosis. Autophagy might be an alternate to apoptosis in endothelial cells. The EA.hy926 endothelial cell line has been reported to undergo necrosis under oxLDL stimulation. For this reason, we studied the expression of LOX-1 and its oxLDL-dependent function in EA.hy926 cells under serum starvation. Untreated and oxLDL-treated cells expressed the LOX-1 protein at similar levels 6h after starvation. After 24h without oxLDL and with native LDL (nLDL), statistically significant higher levels were found in LOX-1 than in the oxLDL-treated probes. The oxLDL cultures with low LOX-1 expression displayed stronger features of autophagy than those with nLDL as there were remodelling of actin filaments, disrupture of adherens junctions (immunofluorescence staining), and autophagosomes with the characteristic double membrane at the ultrastructural level. For the advanced oxLDL exposure times (18 and 24 h), autophagic vacuoles/autophagolysosomes were morphologically identified accompanied by a decrease in lysosomes. The autophagosome marker protein MAP LC3-II (Western blotting) was significantly augmented 6 and 18 h after oxLDL treatment compared with cultures treated with nLDL and medium alone. Signs of apoptosis were undetectable in cultures under oxLDL exposure, yet present under staurosporin (apoptosis inducer), i.e. presence of apoptotic bodies and cleaved caspase 3. We conclude that serum starvation upregulates LOX-1 in EA.hy926 cells, whereas the additional oxLDL treatment downregulates the receptor and intensifies autophagy probably by increase in oxidative stress.     

Trophoblast–endothelium signaling involves angiogenesis and apoptosis in a dynamic bioprinted placenta model

Trophoblast invasion and remodeling of the maternal spiral arteries are required for pregnancy success. Aberrant endothelium–trophoblast crosstalk may lead to preeclampsia, a pregnancy complication that has serious effects on both the mother and the baby. However, our understanding of the mechanisms involved in this pathology remains elementary because the current in vitro models cannot describe trophoblast–endothelium interactions under dynamic culture. In this study, we developed a dynamic three-dimensional (3D) placenta model by bioprinting trophoblasts and an endothelialized lumen in a perfusion bioreactor. We found the 3D printed perfusion bioreactor system significantly augmented responses of endothelial cells by encouraging network formations and expressions of angiogenic markers, cluster of differentiation 31 (CD31), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), and vascular endothelial growth factor A (VEGFA). Bioprinting favored colocalization of trophoblasts with endothelial cells, similar to in vivo observations. Additional analysis revealed that trophoblasts reduced the angiogenic responses by reducing network formation and motility rates while inducing apoptosis of endothelial cells. Moreover, the presence of endothelial cells appeared to inhibit trophoblast invasion rates. These results clearly demonstrated the utility and potential of bioprinting and perfusion bioreactor system to model trophoblast–endothelium interactions in vitro. Our bioprinted placenta model represents a crucial step to develop advanced research approach that will expand our understanding and treatment options of preeclampsia and other pregnancy-related pathologies.     

Alteration in the endothelial cell functional activity by the <Emphasis Type="Italic">Streptoccocus pyogenes</Emphasis> ultrasonic lysate

Angiogenesis and vascular remodeling are vital inflammation components. As an inflammation evolves, vessels expand to supply nutrients and inflammatory mediators, sustaining the accumulation of activated immune cells in the affected tissues. Here, we examined angiogenic properties of EA.hy 926 human endothelial cells treated with ultrasonic lysate of Streptoccocus pyogenes. It was found that the lysate inhibited the cell adhesion, migration, metabolism and proliferation in a dose-dependent manner but even in the highest dose it did not affect the cells viability. Streptococcal components inhibit signaling pathways that involve FAK and ERK1/2. Taking together, our results suggest that impaired angiogenic function of endothelial cells may contribute to the tissue perfusion reduction, hypoxia development and subsequent regional tissue necrosis caused by Streptococci group A.     

厄贝沙坦对人脐静脉内皮细胞株EA.hy926增殖、凋亡、VEGF mRNA表达的影响

目的:应用不同浓度厄贝沙坦对人脐静脉内皮细胞株EA.hy 926的增殖、凋亡生物学效应及血管发生主要基因VEGFmRNA的表达进行体外研究,探讨厄贝沙坦对内皮细胞的血管生成效应。方法:各种浓度厄贝沙坦对人脐静脉内皮细胞株EA.hy926共同孵育24 h。细胞增殖采用CCK8法分析,Annexin V/PI双染法检测细胞凋亡。RT-PCR验证VEGFmRNA的表达。结果:厄贝沙坦各浓度干预组细胞形态无明显变化,CCK8结果提示厄贝沙坦各干预组相比对照组细胞增殖活力增高(P<0.05),呈浓度非依赖性。流式细胞仪分析厄贝沙坦各浓度干预组细胞无明显凋亡。RT-PCR发现厄贝沙坦1×10-4,1×10-5,1×10-6mol/L浓度组VEGFmRNA表达增高(P<0.05)。结论:厄贝沙坦促进EA.hy926细胞株细胞增殖,上调VEGFmRNA的表达。这提示除了降压效应,血管紧张素受体拮抗剂在缺血性心脏病如慢性心力衰竭治疗中具有一定作用。     

Proteomic analysis of anti-angiogenic effects by a combined treatment with vinblastine and rapamycin in an endothelial cell line

Angiogenesis controls the new blood supply routes into the tumor mass via the host endothelial cells (ECs). In this study, the EA.hy926 endothelial cell line has been treated with vinblastine (VBL) and rapamycin (RAP), both separately and in combination at low doses. Recently, we demonstrated the synergistic antiangiogenic effects of a combination of VBL and RAP at very low doses in vitro and in vivo. Herein, we confirm the ability of this combined treatment to statistically inhibit the proliferation of ECs, in a synergistic manner, by inducing apoptosis. The aim of this study was to substantiate these findings at the protein level. Differential proteomic analysis was performed on untreated control cells, treated with VBL, incubated with RAP, or subjected to a drug combination. Differentially expressed 113 polypeptide chains were visualized and 65 were identified via MALDI-TOF analysis. Some of the regulated proteins are involved in the processes of angiogenesis, proliferation, migration, and apoptosis. The down-modulation of ATP synthase, annexin A2, heat shock p70, glucose-6-phosphate dehydrogenase, vasodilator-stimulated phosphoprotein, proteasome 26S, tryptophanyl-tRNA synthetase, and stathmin/OP18, as well as the up-modulation of carbonyl reductase, Rho-GDI, and histone H1.0 correlates with the synergistic antiangiogenic activity of VBL and RAP.