How PEDF prevents angiogenesis: a hypothesized pathway

Pigment epithelium-derived factor (PEDF) is a multiple functional protein, coded by the serine proteinase inhibitor, clade F, member 1 (SERPINF1) gene, which has both anti-angiogenic activity and neurotrophic activity at the same time. Its antiangiogenic activity in the mammalian eye is the most potent known at this time. However, the mechanism(s) by which PEDF works in vivo is still uncertain. Some observations suggest that PEDF can simultaneously inhibit the migration and proliferation induced by vascular endothelial growth factor (VEGF), and then further inhibits angiogenesis by interacting with specific cell surface receptors, but no such receptor has been reported to date. Here we propose a hypothesis that PEDF exerts its function by binding with intergrins. Intergrin can therefore serve as the receptor of PEDF.     

The proangiogenic phenotype of human tumor-derived endothelial cells depends on thrombospondin-1 downregulation via phosphatidylinositol 3-kinase/Akt pathway

umor-derived endothelial cells (TEC) display increased survival and angiogenic properties in respect to normal endothelial cells. The aim of this study was to investigate the mechanism potentially involved in TEC proangiogenic phenotype. We found that thrombospondin-1 (TSP-1), a potent physiological inhibitor of angiogenesis, was significantly reduced in TEC in respect to normal endothelial cells. This reduction was confirmed by immunofluorescence in the intratumor vessels of clear cell renal carcinomas. As TEC were shown to display a basal upregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, we evaluated the possible regulation of TSP-1 by this pathway by using LY294002 and wortmannin, the PI3K inhibitors, and rapamycin, the mammalian target of rapamycin (mTOR) inhibitor. In addition, we developed negative dominant TEC for Akt. TSP-1 production by TEC was enhanced by the treatment with LY294002 and wortmannin and with rapamycin, suggesting a negative regulation of TSP-1 expression by the PI3K/Akt/mTOR pathway. In addition, downregulation of Akt activation in negative dominant Akt TEC enhanced TSP-1 expression and release. Administration of exogenous TSP-1 to TEC reduced their proangiogenic properties in vitro and in vivo. In parallel, blockade of TSP-1 with an anti-TSP-1 antibody in negative dominant Akt TEC restored their proangiogenic phenotype to levels similar to wild-type TEC. In conclusion, these results indicate that the upregulation of the PI3K/Akt/mTOR pathway is responsible for the inhibition of TSP-1 synthesis which is critical in determining the proangiogenic phenotype of TEC. Strategies aimed to inhibit the PI3K/Akt/mTOR pathway may restore a normal quiescent endothelial phenotype in TEC by promoting TSP-1 production.     

Overexpression of pigment epithelium-derived factor decreases angiogenesis and inhibits the growth of human malignant melanoma cells in vivo

Pigment epithelium-derived factor (PEDF) has recently been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, and is involved in the pathogenesis of angiogenic eye disease such as proliferative diabetic retinopathy. However, a functional role for PEDF in tumor growth and angiogenesis remains to be determined. In this study, we have investigated both the in vitro and in vivo growth characteristics of human malignant melanoma G361 cell lines, stably transfected to overexpress human PEDF. Expression levels of PEDF proteins in melanoma cell lines G361 and A375 were comparable with that of human cultured melanocytes, whereas vascular endothelial growth factor levels in two tumor cell lines were much stronger than that in normal melanocytes. Overexpression of PEDF was found to significantly inhibit tumor growth and vessel formation in G361 nude mice xenografts. Furthermore, in vitro proliferation rates of G361 cells were decreased in PEDF-transfected cells. PEDF proteins showed dose-dependent induced growth retardation and apoptotic cell death in nontransfected G361 cells, which were completely prevented by treatment with antibodies against the Fas ligand. Our present study highlights two beneficial effects of PEDF treatment on melanoma growth and expansion; one is the suppression of tumor angiogenesis, and the other is induction of Fas ligand-dependent apoptosis in tumor cells. PEDF therefore might be a promising novel therapeutic agent for treatment of patients with melanoma.     

Angiopoietin-1 inhibits doxorubicin-induced human umbilical vein endothelial cell death by modulating fas expression and via the PI3K/Akt pathway.

Angiopoietin-1 (Ang-1) is essential for the maturation of blood vessels during vasculogenesis. Besides angiogenesis, recent publications indicate that Ang-1 is also a potent survival factor for endothelial cells; however, the mechanisms by which pathways remain elusive. Doxorubicin (DOX) is a powerful anticancer drug, but its use is severely restricted by its cardiotoxicity. The authors report here that Ang-1 inhibits DOX-induced cell death in human umbilical vein endothelial cells (HUVECs). Interestingly, the DOX-induced up-regulation in Fas (CD95/APO-1) and Fas ligand expression could be blocked by Ang-1, indicating a pivotal role of Ang-1 in DOX-induced Fas and Fas ligand expression. In addition, the prevention of cell death in this model system seems to be dependent on the activation of phosphatidylinositol 3-kinase (PI3K)/Akt, as Ang-1 fails to inhibit DOX-induced cell death while PI3K/Akt pathway was blocked by the PI3K inhibitor LY294002. Moreover, Ang-1 inhibits DOX-induced up-regulation of p53 through PI3K/Akt. Therefore, Ang-1 is a potent inhibitor for DOX-induced cell death through Fas and PI3K/Akt-mediated pathways.     

Up-regulation of vascular endothelial growth factor and down-regulation of pigment epithelium-derived factor messenger ribonucleic acid levels in leptin-exposed cultured retinal pericytes

Leptin, a circulating hormone secreted mainly from adipose tissues, is involved in the control of body weight. Recently, leptin was found to be an angiogenic factor and its vitreous levels were shown to be elevated in patients with angiogenic eye diseases such as proliferative diabetic retinopathy. However, the role of leptin in diabetic retinopathy is not fully understood. Since pericyte loss and dysfunction have been considered to be one of the characteristic changes of the early phases of diabetic retinopathy, we investigated the effects of leptin on the growth and function of bovine cultured retinal pericytes. Although it did not affect cell growth, leptin significantly up-regulated pericyte messenger ribonucleic acid levels of an endogenous angiogenic stimulator, vascular endothelial growth factor (VEGF). Leptin was also found to significantly inhibit gene expression of pigment epithelium-derived factor (PEDF), the most potent angiogenesis inhibitor in the mammalian eye, in pericytes. The present study suggests that leptin might elicit angiogenesis through VEGF induction as well as PEDF suppression in pericytes and could thus be involved in the development and progression of diabetic retinopathy, especially in obese insulin-resistant patients.     

PI3K/Akt信号通路及其抑制剂的研究进展

磷脂酰肌醇3激酶（PI3K）/丝氨酸-苏氨酸激酶（Akt）信号通路在信号转导的调控中扮演着重要角色,能调节细胞增殖、凋亡、代谢、运动、血管生成等生物过程。与其他信号通路相比,PI3K/Akt信号通路的组成部分更庞大,在肿瘤中更多见。目前已证实多种肿瘤中存在PI3K/Akt信号通路的超活化,对肿瘤细胞的存活、生长、运动、血管生成和代谢意义重大。因此,抑制PI3K和与通路相关的成分可能会使肿瘤生长受抑,使患者预后改善。PI3K/Akt信号通路抑制剂包括针对单一成分的抑制剂和双重抑制剂。目前大量的PI3K抑制剂已在临床前期研究中取得良好结果,有些已经在血液恶性肿瘤和实体肿瘤中进行了临床试验。在此综述中,我们简单的总结了PI3K-AKt通路的研究成果,讨论了PI3K抑制剂从临床前研究到临床研究的发展前景。     

HB-EGF promotes angiogenesis in endothelial cells via PI3-kinase and MAPK signaling pathways

OBJECTIVE: Heparin-binding EGF-like growth factor (HB-EGF) belongs to the epidermal growth factor (EGF) superfamily of ligands. It has been implicated as a regulator of angiogenesis. However, the mechanisms by which HB-EGF promotes angiogenesis are unknown. The goal of the present study was to define the pathways by which HB-EGF stimulates angiogenesis in endothelial cells. METHODS: To characterize the angiogenic activity of HB-EGF, we treated human umbilical vein endothelial cells (HUVEC) with HB-EGF and analyzed the effects on cell proliferation, migration and tube formation. Side-by-side assays with EGF were used for comparison. RESULTS: Both HB-EGF and EGF stimulated HUVEC migration in scratch assays and promoted vascular tube formation in 2D-angiogenesis assays, without inducing cell proliferation. HB-EGF- and EGF-induced HUVEC migration and capillary tube formation were dependent upon activation of PI3K, MAPK and eNOS. Importantly, HB-EGF-and EGF-induced tube formation was comparable to, but were independent of tube formation induced by VEGF. CONCLUSIONS: We have demonstrated that HB-EGF and EGF induce angiogenesis via activation of PI3K, MAPK and eNOS in a VEGF-independent fashion. Thus, the role played by HB-EGF in stimulating physiologic processes such as wound healing in vivo may be dependent, in part, on its ability to promote angiogenesis.     

姜黄素抑制HGF诱导血管内皮细胞迁移和VEGF表达的体内外研究

目的:探索姜黄素抑制肝细胞生长因子(HGF)诱导血管生成的分子机制。方法:利用管腔形成实验、划痕实验、Western blot实验和动物实验观察姜黄素、c-Met抑制剂SU11274、磷脂酰肌醇3-激酶(PI3K)抑制剂LY294002和m TOR抑制剂rapamycin对HGF诱导的内皮细胞迁移、管腔形成能力、血管内皮生长因子(VEGF)表达、相关信号通路和瘤体内血管密度的影响。结果:姜黄素可显著抑制HGF诱导内皮细胞发生迁移、小管形成及VEGF的表达,同时抑制c-Met/AKT/m TOR/S6通路的磷酸化,并可减少瘤体内VEGF的表达和微血管密度。使用c-Met抑制剂SU11274、PI3K抑制剂LY294002或m TOR抑制剂rapamycin能得到和姜黄素相似的效应。结论:姜黄素抑制HGF诱导的血管生成可能是通过抑制c-Met/AKT/m TOR/S6信号通路活化实现的。     

Akt、ERK1/2活化在脑源性神经营养因子促血管新生中的作用

目的： 探讨脑源性神经营养因子(BDNF) 促进血管新生的作用及其参与的信号通路,为抗肿瘤血管生成的研究提供新的实验依据。 方法: 以人脐静脉内皮细胞为对象,采用Western 印迹方法检测细胞内磷酸化Akt、ERK1/2蛋白质的表达； 采用Transwell小室迁移实验、管腔形成实验评价体外内皮细胞血管新生的能力，MTT法检测内皮细胞增殖 活性，FITC-Annexin-Ⅴ/PI双染流式细胞术分析细胞凋亡。 结果: BDNF以时间依赖性方式激活PI3K/Akt 和MEK1/ERK信号通路。分别应用PI3K激酶抑制剂Ly294002、 MEK1激酶抑制剂PD98059可以明显阻断BDNF对PI3K/Akt、MEK1/ERK信号通路的激活。100 μg/L 的BDNF体 外促内皮细胞血管新生能力与 25 μg/L 血管内皮生长因子（VEGF）相当， 其中BDNF诱导的细胞迁移分 别被Ly294002和PD98059阻断，其抑制率分别约为74%和36%；同样，Ly294002、PD98059可部分阻断BDNF诱 导的小管形成效应，其阻断率分别约57%和37%；而BDNF的促增殖效应仅被PD98059拮抗，抑制凋亡效应仅受Ly294002影响。 结论: BDNF在体外有促血管新生的作用。PI3K/Akt 和MEK1/ERK信号通路以不同机制共同调节这一过程,其中PI3K/Akt信号通路起着更为重要的调节作用。     

Remnant like particles may induce atherosclerosis via accelerating endothelial progenitor cells senescence

Remnant like particles (RLPs) are closely associated with coronary heart disease, whereas the underlying mechanisms are complex and have not been fully elucidated. Studies show that maintenance of endothelial cells layer is essential for normal function of vessel. Endothelial progenitor cells (EPCs) were shown to incorporate into sites of neovascularization and home to sites of endothelial denudation, thus provide an endogenous repair mechanism. Risk factors of coronary heart disease can impair EPCs repairing function by inducing EPCs senescence. EPCs senescence is associated with telomerase inactivation, which is regulated via phosphatidylinositol-3-kinase/Akt kinase (PI3K/Akt) signaling pathway. RLPs are triglyceride rich lipoproteins reflecting chylomicron remnants and very-low-density lipoprotein remnants. RLPs can impair endothelial function via inhibiting endothelial NO synthase (eNOS) activity and nitric oxide (NO) production by inducing intracellular oxidant levels. However, there is no research about effect of RLPs on EPCs. Evidence shows that RLPs can induce focal adhesion kinase (FAK) activation in monocytic U937 cells. Therefore, it can be hypothesized that RLPs could inhibit eNOS and telomerase activities, thus induce atherosclerosis by promoting EPCs senescence via FAK and its downstream PI3K/Akt pathway through an oxidative mechanism.     

Induction of macrophage migration inhibitory factor by lysophosphatidic acid: relevance to tumor growth and angiogenesis

Macrophage migration inhibitory factor (MIF) plays an important role not only in the immune system, but also in tumorigenesis. Lysophosphatidic acid (LPA), a unique lipid mediator, shares several biological functions with MIF, including promotion of tumor cell growth and associated angiogenesis. In this study, we investigated the signaling cross-talk between these two molecules during tumorigenesis and angiogenesis. We first examined the expression of MIF mRNA on a murine colon cancer cell line, colon 26, by LPA. We found that LPA enhanced the expression of MIF mRNA in a dose-dependent manner in vitro. In parallel, LPA stimulated cell growth and up-regulated the vascular endothelial growth factor (VEGF). These effects were dramatically blocked by 21 base double strand (ds) RNA specific for mouse MIF mRNA (RNAi). In vivo, colon 26 cells treated with MIF dsRNA were injected into the backs of mice. The size of tumor volumes became significantly smaller than that of controls. Angiogenesis examined by a Millipore chamber method was also suppressed by the MIF dsRNA. Next, we evaluated the signal transduction pathway relevant to the mitogen-activated protein kinase (MAPK) and Akt/PI3K pathways in response to LPA by RNAi. Ras activation and phosphorylation of Akt and ERK1/2 were strongly suppressed by the dsRNA. On the other hand, tyrosine phosphorylation was minimally changed by the treatment. Taken together, these results suggest that MIF could promote both tumor cell growth and angiogenesis induced by LPA via both the Ras-MAPK and Ras-Akt/PI3K signaling pathways.     

Ghrelin对棕榈酸诱导的内皮细胞凋亡的影响及机制

 目的 探讨ghrelin能否抑制棕榈酸诱导的大鼠主动脉内皮细胞凋亡及其与PI3K/Akt通路的关系。 方法 大鼠主动脉内皮细胞在分别在含或不含0.3mM棕榈酸的DMEM培养基中孵育,培养基中加或不加ghrelin及PI3K/Akt的阻断剂LY294002,流式细胞仪Annexin Ⅴ/PI法检测凋亡,分光光度计检测caspase-3活性,Western blot检测总Akt及磷酸化Akt。 结果0.3mM棕榈酸作用24小时增加大鼠主动脉内皮细胞凋亡,Ghrelin抑制棕榈酸诱导的内皮细胞凋亡。棕榈酸干预内皮细胞24小时能显著抑制Akt的磷酸化,加入ghrelin可引起Akt的活化。Ghrelin引起的Akt的活化能够显著地被PI3K/Akt阻断剂LY294002所阻断,而且LY294002能够阻断ghrelin对棕榈酸诱导的内皮细胞凋亡的保护作用。 结论 Ghrelin能够抑制棕榈酸诱导的大鼠主动脉内皮细胞凋亡,ghrelin的抗凋亡作用至少是部分通过PI3K/Akt通路起作用的。     

雷公藤甲素对血管生成的抑制作用

利用体外培养人脐静脉内皮细胞,经不同浓度的雷公藤甲素(0、5、10、20、30μg/L)处理后,MTT法显示雷公藤甲素可抑制内皮细胞的增殖,5μg/L雷公藤甲素的抑制率达29.15%;琼脂凝胶立体细胞培养系统检测发现内皮细胞经雷公藤甲素作用后,其游走能力降低;鸡胚尿囊膜试验观察到雷公藤甲素可有效抑制血管的生成;荧光定量RT-PCR检测发现雷公藤甲素可下调内皮细胞u-PAmRNA的表达。因此认为,雷公藤甲素可能在基因水平上干扰内皮细胞u-PAmRNA的表达,减少u-PA蛋白的生成,从而有效地抑制血管内皮细胞的增殖和移行,这可能是雷公藤甲素抑制血管生成的主要机制之一。     

Pigment epithelium-derived factor inhibits neointimal hyperplasia after vascular injury by blocking NADPH oxidase-mediated reactive oxygen species generation

Pigment epithelium-derived factor (PEDF) inhibits cytokine-induced endothelial cell activation through its antioxidative properties. However, the effect of PEDF on restenosis remains to be elucidated. Because the pathophysiological feature of restenosis is characterized by increased superoxide formation and accumulation of smooth muscle cells (SMCs), PEDF may inhibit this process via suppression of reactive oxygen species generation. We investigated here whether PEDF could prevent neointimal formation after balloon injury. PEDF levels were decreased in balloon-injured arteries. Adenoviral vector encoding human PEDF (Ad-PEDF) prevented neointimal formation. Expression and superoxide generation of the membrane components of NADPH oxidase, p22(phox) and gp91(phox), in the neointima were also suppressed by Ad-PEDF. Ad-PEDF reduced G(1) cyclin (cyclin D1 and E) expression and increased p27, a cyclin-dependent kinase inhibitor. In vitro, PEDF inhibited platelet-derived growth factor-BB-induced SMC proliferation and migration by blocking reactive oxygen species generation through suppression of NADPH oxidase activity via down-regulation of p22(PHOX) and gp91(PHOX). PEDF down-regulated G(1) cyclins and up-regulated p27 levels in platelet-derived growth factor-BB-exposed SMCs as well. These results demonstrate that PEDF could inhibit neointimal formation via suppression of NADPH oxidase-mediated reactive oxygen species generation. Our present study suggests that substitution of PEDF may be a novel therapeutic strategy for restenosis after balloon angioplasty.     

Ginsenoside Rg3 inhibits HIF-1α and VEGF expression in patient with acute leukemia via inhibiting the activation of PI3K/Akt and ERK1/2 pathways

Aberrant angiogenesis is essential to the development and progression of leukemia. Ginsenoside Rg3 has been commonly used in anti-angiogenic therapy of solid tumors. This study aimed to investigate the anti-angiogenic effects of Rg3 in patients with acute leukemia. Bone marrow stromal cells derived from patients with acute leukemia were treated with Rg3 and the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1α (HIF-1α) was detected by RT-PCR and western blot analysis. The results showed that Rg3 inhibited VEGF and HIF-1α expression at both mRNA and protein levels in bone marrow stromal cells. In addition, Rg3 treatment led to reduced serum levels of HIF-1α and VEGF in patients with acute leukemia. Mechanistically, we demonstrated that Rg3 downregulated the phosphorylation of Akt and ERK1/2 in BMSCs. In conclusion, Rg3 exhibits anti-leukemia effect in part due to its anti-angiogenic activity via inhibiting PI3K/Akt and ERK1/2 pathways, which act to regulate the expression of HIF-1α and VEGF.     

Inhibition of tumor endothelial ERK activation, angiogenesis, and tumor growth by sorafenib (BAY43-9006)

Activation of the Raf-MEK-ERK signal transduction pathway in endothelial cells is required for angiogenesis. Raf is the kinase most efficiently inhibited by the multikinase inhibitor sorafenib, which has shown activity against certain human cancers in clinical trials. To understand the mechanisms underlying this activity, we studied how it controlled growth of K1735 murine melanomas. Therapy caused massive regional tumor cell death accompanied by severe tumor hypoxia, decreased microvessel density, increased percentage of pericyte-covered vessels, and increased caliber and decreased arborization of vessels. These signs of K1735 angiogenesis inhibition, along with its ability to inhibit Matrigel neovascularization, showed that sorafenib is an effective anti-angiogenic agent. Extracellular signal-regulated kinase (ERK) activation in tumor endothelial cells, revealed by immunostaining for phospho-ERK and CD34, was inhibited, whereas AKT activation, revealed by phospho-AKT immunostaining, was not inhibited in K1735 and two other tumor types treated with sorafenib. Treatment decreased endothelial but not tumor cell proliferation and increased both endothelial cell and tumor cell apoptosis. These data indicate that sorafenib's anti-tumor efficacy may be primarily attributable to angiogenesis inhibition resulting from its inhibition of Raf-MEK-ERK signaling in endothelial cells. Assessing endothelial cell ERK activation in tumor bio-psies may provide mechanistic insights into and allow monitoring of sorafenib's activity in patients in clinical trials.     

Ginsenoside-Rg1 enhances angiogenesis and ameliorates ventricular remodeling in a rat model of myocardial infarction

Ginsenoside-Rg1 (Rg1) has been used in the traditional Chinese medicine for over 2,000?years. The present study was performed to test our hypothesis that Rg1 provides pro-angiogenic and anti-fibrotic benefits in the ischemic myocardium in a rat model of myocardial infarction. The expression of vascular endothelial growth factor (VEGF) and phosphorylation/activation of PI3K, Akt, and p38 MAPK signaling pathways were examined in human umbilical vein endothelial cells and in the myocardial samples of rats. In addition, the expression levels of TNF-?? and collagen I level, the number of newly formed blood vessels, the extent of myocardial fibrosis, and left ventricular function were measured in vivo. Our results demonstrated that administration of Rg1 increased VEGF expression levels, activated PI3K/Akt, and inhibited p38 MAPK in vitro and in vivo. Furthermore, Rg1 increased the density of newly formed vessels, decreased TNF-?? and collagen I expression levels and area of myocardial fibrosis, and improved left ventricle function in vivo. PI3K inhibitor LY294002 significantly attenuated Rg1-enhanced VEGF expression and capillary density. As well, inhibition of p38 MAPK slightly increased VEGF expression in vitro and in vivo, increased capillary density, and decreased TNF-?? and collagen I expression levels and area of myocardial fibrosis in vivo. Rg1-induced activation of PI3K/Akt also contributed to the downregulation of p38 MAPK. Thus, Rg1 is effective in promoting angiogenesis and attenuating myocardial fibrosis, resulting in ameliorated left ventricular function. The possible mechanisms may involve activation of PI3K/Akt, inhibition of p38 MAPK, and cross talk between the two signaling pathways.