Pigment epithelium-derived factor inhibits TNF-alpha-induced interleukin-6 expression in endothelial cells by suppressing NADPH oxidase-mediated reactive oxygen species generation

Pigment epithelium-derived factor (PEDF) has recently been shown to be involved in the pathogenesis of proliferative diabetic retinopathy. Atherosclerosis is an inflammatory-fibroproliferative disease as well. Oxidative stress plays a major role in retinopathy and atherosclerosis. Accordingly, we investigated effects of PEDF on reactive oxygen species (ROS) generation, NF-kappaB activation and interleukin (IL)-6 expression in TNF-alpha-exposed HUVEC. TNF-alpha significantly increased intracellular ROS generation, which was completely blocked by PEDF or diphenylene iodonium, an inhibitor of NADPH oxidase. Further, PEDF completely prevented the TNF-alpha-induced increase in NADPH oxidase activity. PEDF or an antioxidant, N-acetylcysteine, significantly inhibited the TNF-alpha-induced NF-kappaB activation. PEDF inhibited TNF-alpha-induced expression of IL-6 at both mRNA and protein levels. Moreover, TNF-alpha downregulated PEDF mRNA levels. Ligand blot analysis revealed that HUVEC possessed a membrane protein with binding affinity for PEDF. The results demonstrated that PEDF inhibited TNF-alpha-induced NF-kappaB activation and subsequent IL-6 overexpression in HUVEC by suppressing NADPH oxidase-mediated ROS generation. Our present study suggests that PEDF may play an important role in the development and progression of atherosclerosis.     

Pigment-epithelium-derived factor (PEDF) inhibits angiotensin-II-induced vascular endothelial growth factor (VEGF) expression in MOLT-3 T cells through anti-oxidative properties

Angiotensin II (Ang II), the dominant effector of the renin-angiotensin system, elicits numerous inflammatory-proliferative responses, thereby being involved in angiogenesis. T cells play an important role in angiogenesis as well by delivering vascular endothelial growth factor (VEGF) to inflammatory sites. Since we have previously shown that pigment-epithelium-derived factor (PEDF) blocks the Ang II signaling in endothelial cells, we studied here whether PEDF could inhibit the Ang-II-induced VEGF expression in MOLT-3 T and examined the potential mechanism of PEDF inhibitory effects. Ang II significantly up-regulated VEGF mRNA levels in MOLT-3 T cells, which was inhibited by PEDF or olmesartan, an Ang II type 1 receptor blocker. PEDF blocked the Ang-II-induced reactive oxygen species (ROS) generation in MOLT-3 T cells. Furthermore, H(2)O(2) was found to up-regulate VEGF mRNA levels in MOLT-3 T cells in a dose-dependent manner. These results demonstrate that PEDF could inhibit the Ang-II-induced VEGF expression in MOLT-3 T cells via suppression of ROS generation. Blockade by PEDF of VEGF expression in T cells may become a novel therapeutic target for pathological angiogenesis.     

Simvastatin enhances myocardial angiogenesis induced by vascular endothelial growth factor gene transfer

Statins have cardioprotective roles. We explored the cardiac angiogenic effects of simvastatin in combination with transient overexpression of vascular endothelial growth factor (VEGF). Compared with normal mice, 1-year-old ApoE(-/-) mice fed on a high-fat diet (HFD) had about 30% less myocardial capillary (P < 0.001) and arteriolar (P < 0.03) densities, associated with decreased VEGF (55%), VEGFR-1 (56%) and VEGFR-2 (78%) mRNA expressions and myocardial endothelial nitric oxide synthase (eNOS) production (58%). By contrast, angiopoietin-1 and angiopoietin-2 mRNA expressions were increased (500% P < 0.02, and 400% P < 0.01, respectively) in the ApoE(-/-) hearts. No change was observed in Tie-2 gene expression. Phosphorylation of antiapoptotic Akt was lower and proapoptotic p38 mitogen-activated protein kinase (MAPK) was higher in the ApoE(-/-) mice compared with controls. Intramyocardial VEGF gene transfer increased capillary and arteriolar densities in the ApoE(-/-) mice, and simvastatin treatment further enhanced capillary density (P < 0.03) to a level similar to that of normal mice. Simvastatin did not change the lipid profile but blocked p38 MAPK phosphorylation in the ApoE(-/-) myocardium. Concurrent with these changes, there were increased levels of expression of mVEGF (P < 0.04) and VEGFR-2 (P < 0.03) mRNAs and increased production of eNOS (P < 0.05) in the ApoE(-/-) mice, while no changes were detected in the angiopoietin system. Thus, increased myocardial angiogenesis in the ApoE(-/-) mice following transient overexpression of VEGF is further increased by additional simvastatin treatment. These effects occurred concurrently with simvastatin-induced stimulation of the VEGF system, increased eNOS production and reduction in p38 MAPK phosphorylation.     

Pigment epithelium-derived factor is involved in the vascular endothelial cells dysfunction induced by arsenic

<正>Objective To explore the role of pigment epithelium-derived factor (PEDF) in vascular endothelial cells dysfunction induced by sodium arsenite (Na As O2).Methods Human umbilical vein endothelial cells (EA.Hy926 cells) were treated with different levels of     

Therapeutic angiogenesis using vascular endothelial growth factor

Therapeutic angiogenesis using vascular endothelial growth factor can reduce tissue ischemia by simulating the natural process of angiogenesis. Vascular endothelial growth factor not only stimulates endothelial cells to proliferate and migrate, but also mobilizes endothelial progenitor cells and achieves vascular protection. Besides direct administration of angiogenic proteins, plasmids and viral vectors carrying angiogenic genes have been used. Animal experiments have shown promise with evidence of neovascularization and improved perfusion in the target myocardium. Initial phase I and II clinical trials results are encouraging and reflect the potential success of therapeutic angiogenesis as a clinical modality for the treatment of ischemic heart disease. This review discusses the role of vascular endothelial growth factor in therapeutic angiogenesis, along with the problems and considerations of this approach as a treatment strategy.     

色素上皮衍生因子抑制人肝癌裸鼠移植瘤生长

目的 观察人色素上皮衍生因子(PEDF)对血管生成的抑制作用,探讨PEDF在肝癌基因治疗中的应用前景. 方法构建色素上皮衍生因子慢病毒表达质粒pLenti-PEDF,经293T细胞包装,收集病毒上清液,感染肝癌细胞株HepG2.收集各组细胞的条件培养基,通过Westernblot分析各组细胞PEDF的表达情况,并通过人脐静脉血管内皮细胞增殖及迁移试验研究其体外生物学活性.人肝癌细胞HepG2移植到裸鼠皮下,研究Lenti-PEDF病毒对肝癌移植瘤生长的抑制作用,逆转录聚合酶链反应检测肿瘤组织中PEDF mRNA表达.多组间均数比较采用单因素方差分析,组间两两比较采用LSD法.结果 限制性内切酶酶切分析和测序结果均表明成功构建了PEDF慢病毒表达载体,以293T细胞包装的重组慢病毒能够高效感染肝癌细胞,肝痛细胞感染重组慢病毒后可高效表达PEDF并分泌到培养上清液中.体外研究结果表明,重组PEDF可明显抑制人脐静脉血管内皮细胞的增殖及迁移,抑制率分别达29%和48%,与空白对照组相比,差异有统计学意义(P值均<0.01).Lenti-PEDF病毒能明显抑制人肝癌裸鼠皮下移植瘤的生长(P<0.01).瘤内注射Lenti-PEDF病毒21 d后,逆转录聚合酶链反应在肿瘤组织检测到PEDF mRNA的过表达.结论 本研究初步提示PEDF能有效遏制肿瘤的血管生成和肿瘤生长,为肝癌的基因治疗提供了一条新的思路.     

Endothelin inhibits cholangiocarcinoma growth by a decrease in the vascular endothelial growth factor expression

Background: Endothelins (ET‐1, ET‐2, ET‐3) are peptides with vasoactive properties interacting with ET_A and ET_B receptors. ET‐1 inhibits secretin‐stimulated ductal secretion (hallmark of cholangiocyte growth) of cholestatic rats by interaction with ET receptors. Aim: The aims of the studies were to evaluate (i) the effect of ET‐1 on cholangiocarcinoma growth in Mz‐ChA‐1 cells and nude mice and (ii) whether ET‐1 regulation of cholangiocarcinoma growth is associated with changes in the expression of vascular endothelial growth factor‐A (VEGF‐A), VEGF‐C, VEGF receptor‐2 (VEGFR‐2) and VEGFR‐3. Methods: We determined the expression of ET_A and ET_B receptors on normal and malignant (Mz‐ChA‐1) cholangiocytes and human cholangiocarcinoma tissue and the effect of ET‐1 on the proliferation and expression of VEGF‐A, VEGF‐C (regulators of tumour angiogenesis) and its receptors, VEGFR‐2 and VEGFR‐3, in Mz‐ChA‐1 cells. In vivo, Mz‐ChA‐1 cells were injected into the flanks of athymic mice and injections of ET‐1 or saline into the tumours were performed daily. The effect of ET‐1 on tumour size, cell proliferation, apoptosis, collagen quantity and the expression of VEGF‐A and VEGF‐C and VEGFR‐2 and VEGFR‐3 were measured after 73 days. Results: Higher expression of ET_A and ET_B was observed in malignant compared with normal cholangiocytes. ET‐1 inhibited proliferation and VEGF‐A, VEGF‐C, VEGFR‐2 and VEGFR‐3 expression of Mz‐ChA‐1 cells. Chronic ET‐1 treatment decreased tumour volume, tumour cell proliferation and VEGF‐A and VEGF‐C expression but increased apoptosis and collagen tissue deposition compared with controls. Conclusions: Modulation of VEGF‐A and VEGF‐C (by ET‐1) may be important for managing cholangiocarcinoma growth.     

Elevated placental soluble vascular endothelial growth factor receptor-1 inhibits angiogenesis in preeclampsia

Preeclampsia is an inflammatory disorder in which serum levels of vascular endothelial growth factor (VEGF) and its soluble receptor-1 (sVEGFR-1, also known as sFlt-1) are elevated. We hypothesize that VEGF and placenta growth factor (PlGF) are dysregulated in preeclampsia due to high levels of sVEGFR-1, which leads to impaired placental angiogenesis. Analysis of supernatants taken from preeclamptic placental villous explants showed a four-fold increase in sVEGFR-1 than normal pregnancies, suggesting that villous explants in vitro retain a hypoxia memory reflecting long-term fetal programming. The relative ratios of VEGF to sVEGFR-1 and PlGF to sVEGFR-1 released from explants decreased by 53% and 70%, respectively, in preeclampsia compared with normal pregnancies. Exposure of normal villous explants to hypoxia increased sVEGFR-1 release compared with tissue normoxia (P<0.001), as did stimulation with tumor necrosis factor-alpha (P<0.01). Conditioned medium (CM) from normal villous explants induced endothelial cell migration and in vitro tube formation, which were both attenuated by pre-incubation with exogenous sVEGFR-1 (P<0.001). In contrast, endothelial cells treated with preeclamptic CM showed substantially reduced angiogenesis compared with normal CM (P<0.001), which was not further decreased by the addition of exogenous sVEGFR-1, indicating a saturation of the soluble receptor. Removal of sVEGFR-1 by immunoprecipitation from preeclamptic CM significantly restored migration (P<0.001) and tube formation (P<0.001) to levels comparable to that induced by normal CM, demonstrating that elevated levels of sVEGFR-1 in preeclampsia are responsible for inhibiting angiogenesis. Our finding demonstrates the dysregulation of the VEGF/PlGF axis in preeclampsia and offers an entirely new therapeutic approach to its treatment.     

Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor A

During bone formation and fracture healing there is a cross-talk between endothelial cells and osteoblasts. We previously showed that vascular endothelial growth factor A (VEGF-A) might be an important factor in this cross-talk, as osteoblast-like cells produce this angiogenic factor in a differentiation-dependent manner. Moreover, exogenously added VEGF-A enhances osteoblast differentiation. In the present study we investigated, given the coupling between angiogenesis and bone formation, whether bone morphogenetic proteins (BMPs) stimulate osteoblastogenesis and angiogenesis through the production of VEGF-A. For this we used the murine preosteoblast-like cell line KS483, which forms mineralized nodules in vitro, and an angiogenesis assay comprising 17-d-old fetal mouse bone explants that have the ability to form tube-like structures in vitro. Treatment of KS483 cells with BMP-2, -4, and -6 enhanced nodule formation, osteocalcin mRNA expression, and subsequent mineralization after 18 d of culture. This was accompanied by a dose-dependent increase in VEGF-A protein levels throughout the culture period. BMP-induced osteoblast differentiation, however, was independent of VEGF-A, as blocking VEGF-A activity by a VEGF-A antibody or a VEGF receptor 2 tyrosine kinase inhibitor did not affect BMP-induced mineralization. To investigate whether BMPs stimulate angiogenesis through VEGF-A, BMPs were assayed for their angiogenic activity. Treatment of bone explants with BMPs enhanced angiogenesis. This was inhibited by soluble BMP receptor 1A or noggin. In the presence of a VEGF-A antibody, both unstimulated and BMP-stimulated angiogenesis were arrested. Conditioned media of KS483 cells treated with BMPs also induced a strong angiogenic response, which was blocked by antimouse VEGF-A but not by noggin. These effects were specific for BMPs, as TGF beta inhibited osteoblast differentiation and angiogenesis while stimulating VEGF-A production. These findings indicate that BMPs stimulate angiogenesis through the production of VEGF-A by osteoblasts. In conclusion, VEGF-A produced by osteoblasts in response to BMPs is not involved in osteoblast differentiation, but couples angiogenesis to bone formation.     

PEDF-derived peptide inhibits corneal angiogenesis by suppressing VEGF expression

Pigment epithelium-derived factor (PEDF) a glycoprotein that belongs to the superfamily of serine protease inhibitors, has been recently shown to be the most potent inhibitor of angiogenesis in the mammalian eye. However, which active domain of PEDF protein could be involved in its anti-angiogenic properties remains unknown. Therefore, in this study, we examined which PEDF-derived synthetic peptides could inhibit corneal neovascularization induced by chemical cauterization in vivo. Rats treated with topical application of PEDF protein had 31% less corneal neovascularization at day 7 after the injury than phosphate-buffered saline (PBS)-treated rats. P5-2 and P5-3 peptides (residues 388-393 and 394-400 of PEDF protein, respectively) significantly suppressed the corneal neovascularization after chemical cauterization at day 7, and its anti-angiogenic potential was almost equal to that of full-length PEDF protein. Further, full-length PEDF protein and P5-3 peptide significantly decreased 8-hydroxy-2'-deoxyguanosine and vascular endothelial growth factor (VEGF) levels in the corneal. Our present study suggests that PEDF-derived synthetic peptide, P5-3 could inhibit the corneal neovascularization induced by chemical cauterization in rats by suppressing VEGF expression via its anti-oxidative properties.     

Oroxylin A inhibits angiogenesis through blocking vascular endothelial growth factor-induced KDR/Flk-1 phosphorylation

Purpose

In this study, we examined the antiangiogenic effect of oroxylin A in vitro and in vivo and explored the potential mechanisms for this effect.

Methods

Transwell assay and tube formation assay were used to evaluate the effects of oroxylin A on vascular endothelial growth factor (VEGF)-induced migration and tube formation of human umbilical vein endothelial cells (HUVECs). Rat aortic ring assay was also employed to assess the effect of oroxylin A on microvessel outgrowth from rat aorta. Human tumor xenografts model in nude mice was further used to investigate the antiangiogenic activity of oroxylin A in vivo. Western blot analysis was used to investigate the related mechanism.

Results

Oroxylin A remarkably suppressed the VEGF-stimulated migration and tube formation of HUVECs. It also inhibited microvessel sprouting from rat aortic ring in vitro. In addition, it suppressed the angiogenesis of xenograft tumor in nude mice, which concurred with the inhibition of tumor growth. Moreover, oroxylin A blocked VEGF-induced phosphorylation of KDR/Flk-1 and related downstream signaling molecules, including p38 mitogen-activated protein kinase, extracellular signal-regulated kinase and Akt.

Conclusion

Oroxylin A possessed antiangiogenic activities in vitro and in vivo, which could be an underlying mechanism of its anticancer effect.     

The role of vascular endothelial growth factor in angiogenesis

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and an angiogenic inducer as well as a mediator of vascular permeability. The biological effects of VEGF are mediated by two tyrosine kinase receptors, Flt-1 (VEGFr-1) and KDR (VEGFR-2). VEGF is essential for developmental angiogenesis and is also required for female reproductive functions and endochondral bone formation. Substantial evidence also implicates VEGF in tumors and intraocular neovascular syndromes. Currently, several clinical trials are ongoing to test the hypothesis that inhibition of VEGF activity may be beneficial for these conditions.     

Acidosis inhibits endothelial cell apoptosis and function and induces basic fibroblast growth factor and vascular endothelial growth factor expression

Endothelial cells are exposed to an acidotic environment in a variety of pathological and physiological conditions. However, the effect of acidosis on endothelial cell function is still largely unknown, and it was evaluated in the present study. Bovine aortic endothelial cells (BAECs) were grown in bicarbonate buffer equilibrated either with 20% CO(2) (pH 7.0, acidosis) or 5% CO(2) (pH 7.4, control). Acidosis inhibited BAEC proliferation in 10% FCS, whereas by day 7 in serum-free medium, cell number was 3-fold higher in acidotic cells than in control cells. Serum deprivation enhanced BAEC apoptosis, and apoptotic cell death was markedly inhibited by acidosis. Additionally, acidosis inhibited FCS-stimulated migration in a modified Boyden chamber assay and FCS-stimulated differentiation into capillary-like structures on reconstituted basement membrane proteins. Conditioned media from BAECs cultured for 48 hours either at pH 7.0 or pH 7.4 enhanced BAEC proliferation and migration at pH 7.4, and both effects were more marked with conditioned medium from BAECs grown in acidotic than in control conditions. Acidosis enhanced vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) mRNA expression as well as bFGF secretion, and a blocking bFGF antibody inhibited enhanced BAEC migration in response to conditioned medium from acidotic cells. These results show that acidosis protects endothelial cells from apoptosis and inhibits their proangiogenic behavior despite enhanced VEGF and bFGF mRNA expression and bFGF secretion.     

血管内皮生长因子基因转染骨髓间充质干细胞移植促进缺血心肌血管生成的研究

目的研究血管内皮生长因子(vascularendothelial growth factor,VEGF)基因转染骨髓间充质干细胞(mesenchymalstem cells,MSCs)移植对缺血心肌的血管生成作用。方法于2004年5月至2005年8月取第四军医大学西京医院分离、培养Wistar大鼠的MSCs,用真核表达载体pcDNA3.1(-)/hVEGF165转染MSCs。45只近交系Wistar大鼠随机均分为转染组(MSCs/VEGF组)、对照组(MSCs组)、无血清培养基组(DMEM组),结扎前降支建立急性心肌梗死模型后在梗死区边缘区行5×106细胞移植,DMEM组行等量培养基注射。细胞移植前行CM-DiI标记。移植1个月后行心脏B超测量射血分数值,组织化学染色评价新生血管密度。结果培养的MSCs呈典型贴壁生长成纤维样外观,pcDNA3.1(-)/hVEGF165能有效转染大鼠MSCs,移植1个月后MSCs/VEGF组较其余各组左室射血分数(LVEF),再生血管密度明显增加,差异均有显著性(P<0.01)。结论VEGF基因转染MSCs移植能显著促进缺血心肌血管再生,进而改善心脏功能。     

Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells

Phosphatidylinositol 3-kinase (PI 3-kinase) is a signaling molecule that controls numerous cellular properties and activities. The oncogene v-p3k is a homolog of the gene coding for the catalytic subunit of PI 3-kinase, p110alpha. P3k induces transformation of cells in culture, formation of hemangiosarcomas in young chickens, and myogenic differentiation in myoblasts. Here, we describe a role of PI 3-kinase in angiogenesis. Overexpression of the v-P3k protein or of cellular PI 3-kinase equipped with a myristylation signal, Myr-P3k, can induce angiogenesis in the chorioallantoic membrane (CAM) of the chicken embryo. This process is characterized by extensive sprouting of new blood vessels and enlargement of preexisting vessels. Overexpression of the myristylated form of the PI 3-kinase target Akt, Myr-Akt, also induces angiogenesis. Overexpression of the tumor suppressor PTEN or of dominant-negative constructs of PI 3-kinase inhibits angiogenesis in the yolk sac of chicken embryos, suggesting that PI 3-kinase and Akt signaling is required for normal embryonal angiogenesis. The levels of mRNA for vascular endothelial growth factor (VEGF) are elevated in cells expressing activated PI 3-kinase or Myr-Akt. VEGF mRNA levels are also increased by insulin treatment through the PI 3-kinase-dependent pathway. VEGF mRNA levels are decreased in cells treated with the PI 3-kinase inhibitor LY294002 and restored by overexpression of v-P3k or Myr-Akt. Overexpression of VEGF by the RCAS vector induces angiogenesis in chicken embryos. These results suggest that PI 3-kinase plays an important role in angiogenesis and regulates VEGF expression.     

Pigment epithelium-derived factor inhibits advanced glycation end-products-induced cytotoxicity in retinal pericytes

Aim

This study investigated the effects of pigment epithelium-derived factor (PEDF) on advanced glycation end-product (AGE)-induced cytotoxicity in porcine retinal pericytes and the signalling mechanism involved.

Methods

Retinal pericytes were isolated from porcine eyes and characterized by immunocytochemistry. The effect of AGEs and PEDF on cell proliferation was determined by bromodeoxyuridine (BrdU) assay. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was analyzed by luminescence assay. Reactive oxygen species (ROS), nitric oxide (NO), superoxide dismutase (SOD) and glutathione peroxidase (GSH) were determined by biochemical assays. Induction of apoptosis was determined by caspase-3 colorimetric assay and DNA fragmentation analysis. Src activity was assessed by transient transfection analysis, and the status of Src phosphorylation at Y419 was analyzed by a competitive ELISA method.

Results

AGEs significantly increased intracellular ROS generation in pericytes via NADPH oxidase and induced cell death via caspase-3 enzyme activation, whereas PEDF increased cell proliferation in a dose-dependent manner. In addition, PEDF inhibited AGE-induced ROS generation by increasing levels of SOD and GSH, and also blocked the activation of caspase-3. Furthermore, PEDF induced cell survival via the Src pathway by Src phosphorylation at Y419, as evidenced by a pharmacological inhibitor and Src mutants.

Conclusion

These results suggest that PEDF abrogates AGE-induced oxidative stress and apoptosis in retinal pericytes via the Src pathway, thereby suggesting that PEDF is an effective therapeutic agent for the treatment of loss of pericytes in early diabetic retinopathy.     

Therapeutic angiogenesis: Theoretic problems using vascular endothelial growth factor

Aniogenic growth factors constitute a potentially novel form of therapy for patients with ischemic vascular disease. In case of vascular endothelial growth factor (VEGF), a cytokine secreted from intact cells, bioavailability and meaningful angiogenic bioactivity was shown to be achievable by intramuscular gene transfer in patients with chronic critical limb ischemia. Angiogenesis, however, is a two-sided coin with detrimental consequences in non-target tissues. In particular, the theoretic risk of tumor or plaque angiogenesis must not be ignored, though based on experimental and clinical data there is every reason to believe that a short-term increase of circulating VEGF is safe. More sophisticated remains the controversy concerning mechanisms involved in apparent clinical benefits of growth factors (or growth factor genes). This article argues some theoretic problems using naked plasmid DNA encoding VEGF for the purpose of therapeutic angiogenesis.