血管内皮生成因子165诱导血管形成中镁离子作用的研究

目的探讨血管内皮生成因子165（VEGF165）对人脐带静脉内皮细胞（HUVEC）内游离镁离子浓度（[Mg^2＋];）的调节机制及镁离子（Mg^2＋）与血管形成的相关性。方法采用荧光指示剂mag-fura-2,运用PTi阳离子测定系统动态测HUVEC内的[Mg^2＋];。新鲜脐带内灌注胶原酶消化,获得内皮细胞,用含20％胎牛血清的M199液进行培养,当细胞外Mg^2＋浓度分别为0、1和2mmol/L时,观察VEGF。65促进HUVEC血管形成的能力。结果VEGF。65诱导的[Mg^2＋]i增加与细胞外Mg^2＋浓度无关。在细胞外无Mg^2＋时,VEGF165能剂量依赖性地增加[Mg^2＋]i。VEGF165诱导的[Mg^2＋];增加与细胞外Na^＋浓度和细胞内Ca^2＋浓度无关。经VEGF的受体亚型2（KDR）阻断剂SU1498预处理,能明显阻断VEGF165诱导的[Mg^2＋]i增加。当细胞外Mg^2＋为0mmol/L时,HUVEC血管形成作用受到明显抑制,VEGF165也不刺激血管形成。当细胞外Mg^2＋为1或2mmol/L时,HUVEC能形成血管,两组间差异无统计学意义,VEGF165则可促进HUVEC形成血管,两组间差异无统计学意义。当细胞外Mg^2＋为1或2mmol/L时,KDR阻断剂SU1498明显抑制VEGF165促进HUVEC血管形成的作用。结论VEGF165通过KDR信号传递途径使细胞内的Mg^2＋库释放Mg^2＋,从而增加HUVEC的[Mg^2＋]i,并对促进血管形成起重要作用。     

Impact of atorvastatin and omega-3 ethyl esters 90 on plasma plant sterol concentrations and cholesterol synthesis in type 2 diabetes: a randomised placebo controlled factorial trial

ObjectiveMetabolic syndrome (MetS) is associated with impaired angiogenesis. Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis through binding to its specific receptor, VEGF receptor-2 (VEGFR-2), whereas the expression of VEGF and VEGFR-2 in the myocardium of insulin-resistant rats is down-regulated. Soluble VEGF receptor-1 (sVEGFR-1) and -2 (sVEGFR-2) have been reported to inhibit angiogenesis both in vitro and in vivo. However, the balance between circulating levels of VEGF and its soluble receptors, which may reflect and/or affect cardiovascular VEGF signaling, in subjects with MetS is unknown.Methods and resultsWe carried out a cross-sectional study including 272 consecutive, apparently healthy subjects who were not receiving any drugs. Plasma levels of VEGF and serum levels of its soluble receptors were determined using enzyme-linked immunosorbent assays. VEGF and sVEGFR-1 levels did not differ between subjects with and those without MetS. However, sVEGFR-2 levels were significantly increased in MetS compared with non-MetS subjects. Stepwise regression analysis revealed that HOMA-IR was the strongest independent determinant of the sVEGFR-2 level. Accordingly, the mean sVEGFR-2 levels increased in proportion to both the accumulation of components of MetS and quartile of HOMA-IR. Interestingly, multiple regression analyses revealed that independent determinants of VEGF were the body mass index and blood pressure, whereas, in contrast, those of sVEGFR-2 were HOMA-IR and high-sensitivity C-reactive protein.ConclusionsThe correlation of sVEGFR-2 with insulin resistance supports the need for further investigations to define the clinical utility and predictive value of serum sVEGFR-2 levels in cardiovascular dysfunction in MetS.     

Dual effects of heparin on VEGF binding to VEGF receptor-1 and transduction of biological responses

Vascular endothelial growth factor (VEGF) regulates blood vessel formation by binding to the receptor tyrosine kinases VEGF receptor-1 (Flt-1) or VEGF receptor-2 (KDR) and to the structurally unrelated neuropilins. As exon 7-containing isoforms of VEGF bind to heparin, angiogenesis may be modulated by heparin/heparan sulfate. We analyzed the effect of heparin on VEGF₁₆₅-binding and activation of VEGF receptor-1 in porcine aortic endothelial cells, which lack expression of VEGF receptor-2 and neuropilins. Heparin decreased binding of ¹²⁵I-VEGF to 50% at 5 μg/ml and cross-linking of ¹²⁵I-VEGF to VEGF receptor-1 on intact cells was similarly decreased. Schatchard analyses showed that the affinity for binding of ¹²⁵I-VEGF to VEGF receptor-1 was decreased in the presence of heparin. In contrast, VEGF receptor-1 kinase activity was elevated when cells were treated simultaneously with VEGF and heparin. In accordance, VEGF-induced tyrosine phosphorylation of phospholipase Cγ (PLCγ) and DNA synthesis were augmented by heparin. However, basal PLCγ tyrosine phosphorylation and DNA synthesis levels were to some extent increased by incubation of cells with heparin alone. We conclude that although heparin decreases binding of VEGF to VEGF receptor-1, the remaining binding results in more efficient kinase activation. Taken together, there is no loss of VEGF/VEGF receptor-1 function in the presence of heparin. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Angiogenesis regulatory factors in the vitreous from patients with proliferative diabetic retinopathy

We determined the levels of the endogenous angiogenesis inhibitors soluble vascular endothelial growth factor receptor-1 (sVEGFR-1), thrombospondin (TSP)-1 and TSP-2 in the vitreous fluid from patients with proliferative diabetic retinopathy (PDR) and correlated their levels with clinical disease activity and the levels of vascular endothelial growth factor (VEGF). Vitreous samples from 30 PDR and 25 nondiabetic patients were studied by enzyme-linked immunosorbent assay. TSP-1 was not detected. VEGF and TSP-2 levels were significantly higher in PDR with active neovascularization compared with inactive PDR and nondiabetic patients (P < 0.001 for both comparisons). VEGF, sVEGFR-1 and TSP-2 levels were significantly higher in PDR with hemorrhage compared with PDR without hemorrhage and nondiabetic patients (P = 0.0063; 0.0144; <0.001, respectively). VEGF and sVEGFR-1 levels were significantly higher in PDR without traction retinal detachment (TRD) compared with PDR with TRD and nondiabetic patients (P = 0.038; 0.022, respectively). TSP-2 levels were significantly higher in PDR with TRD compared with PDR without TRD and nondiabetic patients (P < 0.001). There was a significant correlation between levels of VEGF and sVEGFR-1 (r = 0.427, P = 0.038). Our findings suggest that upregulation of sVEGFR-1 and TSP-2 may be a protective mechanism against progression of angiogenesis associated with PDR. TSP-2 might be associated with TRD.     

VEGF and its soluble receptor VEGFR-2 in hypertensive disorders during pregnancy: the Indian scenario

Hypertensive disorders are the most common medical problem encountered during pregnancy due to defective angiogenesis during placental development. Vascular endothelial growth factor (VEGF) is one of the angiogenic growth factors that stimulates angiogenesis. The recombinant form of its soluble receptor VEGF receptor-2 (sVEGFR-2) has anti-angiogenic activity. However, there is a paucity of information on serum VEGF and sVEGFR-2 concentrations in different sub-groups of hypertensive disorders during pregnancy. In this cross-sectional study, we evaluated the concentrations and the diagnostic utility of VEGF and sVEGFR-2 in gestational hypertension (GH, n=90), pre-eclampsia (PE, n=180), eclampsia (n=90) and control (n=180) pregnancy at different gestations. VEGF levels were significantly higher in PE and eclamptic (median=19.53?pg?ml(-1); 60.36?pg?ml(-1), P=0.0001) groups as compared with the control ones (median=18?pg?ml(-1)). But, the serum sVEGFR-2 levels were found to be significantly decreased from GH to eclampsia groups (median=5196; 3972?pg?ml(-1)) as compared with control groups (median=7417?pg?ml(-1)). As the gestation advanced, there was an inverse association in the serum concentrations of sVEGFR-2 among the control, GH, PE and eclampsia groups. At both 34 and >34 weeks of gestations, higher sensitivity and specificity were observed for sVEGFR-2 in differentiating GH (50.8, 50%; 76.6, 76.6%), PE (63, 63%; 90, 90%) and eclampsia (65, 66.6%; 90, 90%) from the control pregnancy. This upregulation of VEGF and downregulation of sVEGFR-2 concentrations in different study groups may be due to hypoxia and could be involved intimately in the pathogenesis of these disorders. This study may contribute in understanding etio-pathogenesis of different hypertensive disorders during pregnancy.     

Visfatin induces human endothelial VEGF and MMP-2/9 production via MAPK and PI3K/Akt signalling pathways: novel insights into visfatin-induced angiogenesis

AIMS: Visfatin is a novel adipokine whose plasma concentrations are altered in obesity and obesity-related disorders; these states are associated with an increased incidence of cardiovascular disease. We therefore investigated the effect of visfatin on vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP-2, MMP-9) production and the potential signalling cascades. METHODS AND RESULTS: In human umbilical vein endothelial cells (HUVECs), visfatin significantly and dose-dependently up-regulated gene expression and protein production of VEGF and MMPs and down-regulated expression of tissue inhibitors of MMPs (TIMP-1 and TIMP-2). The gelatinolytic activity of MMPs (analysed by zymography) correlated with mRNA and western blot findings. Interestingly, visfatin significantly up-regulated VEGF receptor 2 expression. Inhibition of VEGFR2 and VEGF [by soluble FMS-like tyrosine kinase-1 (sFlt1)] down-regulated visfatin-induced MMP induction. Visfatin induced dose- and time-dependent proliferation and capillary-like tube formation. Importantly, visfatin was noted to have anti-apoptotic effects. In HUVECs, visfatin dose-dependently activated PI3K/Akt (phosphatidylinositol 3-kinase/Akt) and ERK(1/2) (extracellular signal-regulated kinase) pathways. The functional effects and MMP/VEGF induction were shown to be dependent on the MAPK/PI3K-Akt/VEGF signalling pathways. Inhibition of PI3K/Akt and ERK(1/2) pathways led to significant decrease of visfatin-induced MMP and VEGF production and activation, along with significant reduction in endothelial proliferation and capillary tube formation. CONCLUSION: Our data provide the first evidence of visfatin-induced endothelial VEGF and MMP production and activity. Further, we show for the first time the involvement of the MAPK and PI3K/Akt signalling pathways in mediating these actions, as well as endothelial cell proliferation. Collectively, our findings provide novel insights into visfatin-induced endothelial angiogenesis.     

Regulation of vascular endothelial growth factor by melatonin in human breast cancer cells

Melatonin exerts oncostatic effects on breast cancer by interfering with the estrogen‐signaling pathways. Melatonin reduces estrogen biosynthesis in human breast cancer cells, surrounding fibroblasts and peritumoral endothelial cells by regulating cytokines that influence tumor microenvironment. This hormone also exerts antiangiogenic activity in tumoral tissue. In this work, our objective was to study the role of melatonin on the regulation of the vascular endothelial growth factor (VEGF) in breast cancer cells. To accomplish this, we cocultured human breast cancer cells (MCF‐7) with human umbilical vein endothelial cells (HUVECs). VEGF added to the cultures stimulated the proliferation of HUVECs and melatonin (1 mm ) counteracted this effect. Melatonin reduced VEGF production and VEGF mRNA expression in MCF‐7 cells. MCF‐7 cells cocultured with HUVECs stimulated the endothelial cells proliferation and increased VEGF levels in the culture media. Melatonin counteracted both stimulatory effects on HUVECs proliferation and on VEGF protein levels in the coculture media. Conditioned media from MCF‐7 cells increased HUVECs proliferation, and this effect was significantly counteracted by anti‐VEGF and 1 mm melatonin. All these findings suggest that melatonin may play a role in the paracrine interactions between malignant epithelial cells and proximal endothelial cells through a downregulatory action on VEGF expression in human breast cancer cells, which decrease the levels of VEGF around endothelial cells. Lower levels of VEGF could be important in reducing the number of estrogen‐producing cells proximal to malignant cells as well as decreasing tumoral angiogenesis.     

Protective Actions of Globular and Full-Length Adiponectin on Human Endothelial Cells: Novel Insights into Adiponectin-Induced Angiogenesis

Background/Aims: Adiponectin levels are decreased in diabetes and atherosclerosis. Coexisting hyperglycaemia and systemic inflammation predisposes to dysregulated angiogenesis and vascular disease. We investigated the effect of globular adiponectin (gAd) and full-length adiponectin (fAd) on angiogenesis and pro-angiogenic molecules, i.e. matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF), in human microvascular endothelial cells (HMEC-1). Methods: Angiogenesis was assessed by studying capillary tube formation in HMEC-1 on growth factor-reduced Matrigel. Endothelial cell migration assay was performed in a modified Boyden chamber. Results: Endothelial cell proliferation, in vitro migration and angiogenesis were significantly increased by gAd (mediated by AdipoR1, AMPK-Akt pathways), and gAd significantly increased MMP-2, MMP-9 and VEGF expression levels. The effect of gAd on VEGF appears to be mediated by AdipoR1, whilst the effect of gAd on MMP-2 and MMP-9 appears to be mediated by AdipoR1 and AdipoR2. Only endothelial cell proliferation was significantly increased by fAd in human microvascular endothelial cells and appears to be mediated by AdipoR2. No significant effects on MMP-2, MMP-9 and VEGF were observed. Importantly, gAd decreased glucose and C-reactive protein-induced angiogenesis with a concomitant reduction in MMP-2, MMP-9 and VEGF in HMEC-1 cells. Conclusion: We report novel insights into the mechanisms of adiponectin on angiogenesis.     

Berberine inhibits angiogenic potential of Hep G2 cell line through VEGF down-regulation in vitro

Background and Aim: Berberine, an herbal alkaloid, has been reported to have promotion potential of apoptosis and anticancer effect on a variety of human tumor cells. To obtain more specific understanding of those consequences of berberine on hepatocellular carcinoma (HCC) and the tumor microenvironment, we conducted in vitro experiments to investigate the inhibitory effect of berberine on tumor‐induced angiogenesis using HCC cells and human umbilical vein endothelial cells (HUVECs). Methods: Human umbilical vein endothelial cell growth was quantified with the CCK‐8 cell proliferation assay; cell migration was observed with a Boyden chamber (Transwell, Corning, Lowell, MA, USA), and angiogenesis was assessed by endothelial tube formation in Matrigel in vitro. In addition, VEGF level was determined by ELISA and VEGF mRNA expression by RT‐PCR. Results: Berberine inhibited the capacity of HCC to stimulate HUVEC's proliferation, migration and endothelial tube formation, suggesting that berberine could influence the cross‐talk between the HCC cell and vascular endothelial cells. These results demonstrate berberine's antiangiogenesis property and its clinical potential as an inhibitor of tumor angiogenesis. Subsequently analyses reveal that berberine prevents secretion of VEGF from HCC and down‐regulates VEGF mRNA expression. Conclusion: These findings strongly suggest that berberine is a potential antiangiogenic agent and a promising antitumor drug for HCC.     

Soluble VEGFR-1 secreted by endothelial cells and monocytes is present in human serum and plasma from healthy donors

It was shown before that the soluble form of VEGFR-1 (sVEGFR-1) is present in serum of pregnant women. The aim of the present study was to investigate the presence of this endogenous vascular endothelial growth factor-A (VEGF-A) antagonist in human serum in more detail. sVEGFR-1 was detected in human serum and plasma from normal healthy male and female donors by ELISA. sVEGFR-1 levels ranged from non-detectable up to 440 pg/ml, with no significant difference between male and female donors. In addition, vein endothelial cells (ECs) from an intact vascular bed, the umbilical cord, were shown to secrete sVEGFR-1. Furthermore, human peripheral blood monocytes, a non-EC type expressing VEGFR-1, were shown to contribute to the sVEGFR-1 detectable in human serum and plasma for the first time. EC- and monocyte-derived sVEGFR-1 proved capable of inhibiting the VEGF-induced proliferation and migration of ECs in vitro. Finally, secretion of sVEGFR-1 was increased by the angiogenic factor basic fibroblast growth factor (bFGF) in human ECs and was also enhanced in lipopolysaccharide-activated human monocytes. In human umbilical vein endothelial cells, both the membrane-bound and the sVEGFR-1 seem to be equally regulated on the mRNA as well as the protein level. The presence of an sVEGFR-1 in human serum and plasma of normal male and female donors strongly suggests that it plays an important role as a naturally occurring VEGF antagonist in the regulation and availability of VEGF-mediated biological activities in vivo. This revised version was published online in June 2006 with corrections to the Cover Date.     

Thalidomide-induced antiangiogenic action is mediated by ceramide through depletion of VEGF receptors, and is antagonized by sphingosine-1-phosphate

Thalidomide, which is clinically recognized as an efficient therapeutic agent for multiple myeloma, has been thought to exert antiangiogenic action through an unknown mechanism. We here show a novel mechanism of thalidomide-induced antiangiogenesis in zebrafish embryos. Thalidomide induces the defect of major blood vessels, which is demonstrated by their morphologic loss and confirmed by the depletion of vascular endothelial growth factor (VEGF) receptors such as neuropilin-1 and Flk-1. Transient increase of ceramide content through activation of neutral sphingomyelinase (nSMase) precedes thalidomide-induced vascular defect in the embryos. Synthetic cell permeable ceramide, N-acetylsphingosine (C2-ceramide) inhibits embryonic angiogenesis as well as thalidomide. The blockade of ceramide generation by antisense morpholino oligonucleotides for nSMase prevents thalidomide-induced ceramide generation and vascular defect. In contrast to ceramide, sphingosine-1-phosphate (S1P) inhibits nSMase-dependent ceramide generation and restores thalidomide-induced embryonic vascular defect with an increase of expression of VEGF receptors. In human umbilical vein endothelial cells (HUVECs), thalidomide-induced inhibition of cell growth, generation of ceramide through nSMase, and depletion of VEGF receptors are restored to the control levels by pretreatment with S1P. These results suggest that thalidomide-induced antiangiogenic action is regulated by the balance between ceramide and S1P signal.     

Functions of the VEGF/VEGF receptor system in the vascular system

The vascular endothelial growth factor (VEGF)/VEGF receptor system plays a central regulatory role in physiological and pathological angiogenesis. During embryogenesis, the VEGF/VEGF receptor system is critically involved in the formation of the vascular system by regulating both the growth and the survival of blood vessels. In the vasculature of the adult organism, the high-affinity signaling VEGF receptor-2 (VEGFR-2) is downregulated but is reinduced during transient phases of physiological angiogenesis. Moreover, a variety of pathological conditions are associated with the upregulation of VEGF and the VEGF receptors. VEGF stimulates angiogenesis and the survival of endothelial cells in tumors, thereby enabling tumor expansion and metastasis. VEGF is also upregulated in ischemic diseases, such as coronary heart disease or stroke, and is thought to stimulate the--often insufficient--compensatory formation of blood vessels. The implication of VEGF in these pathological processes has opened up promising new therapeutic strategies. In malignancies, attempts are made to inhibit VEGF-mediated signaling and angiogenesis. In ischemic disease, the exogenous application of VEGF may enhance the formation of collaterals. However, considering the complexity of the regulatory pathways involved in the formation of new blood vessels under physiological conditions, a treatment relying on VEGF as the sole angiogenic factor may be insufficient, and the combination with other factors may improve the functionality of newly formed blood vessels and the efficacy of therapeutic angiogenesis.     

TRAIL negatively regulates VEGF-induced angiogenesis via caspase-8-mediated enzymatic and non-enzymatic functions

Solid tumors supply oxygen and nutrients required for angiogenesis by producing vascular endothelial growth factor (VEGF). Thus, inhibitors of VEGF signaling abrogate tumor angiogenesis, resulting in the suppression of tumor growth and metastasis. We here investigated the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on VEGF-induced angiogenesis. TRAIL inhibited VEGF-induced in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs) and in vivo neovascularization in chicken embryos and mice. TRAIL blocked VEGF-induced angiogenic signaling by inhibiting ERK, Src, FAK, paxillin, Akt, and eNOS. Further, TRAIL blocked intracellular Ca²⁺ elevation and actin reorganization in HUVECs stimulated with VEGF, without inhibiting VEGF receptor-2 tyrosine phosphorylation. TRAIL increased caspase-8 activity, without inducing caspase-9/-3 activation and apoptosis. Moreover, TRAIL resulted in cleavage of FAK into FAK-related non-kinase-like fragments in VEGF-stimulated HUVECs, which was blocked by a caspase-8 inhibitor and cellular caspase-8-like inhibitory protein. Biochemical and pharmacological inhibition of caspase-8 and FAK blocked the inhibitory effects of TRAIL on VEGF-stimulated anti-angiogenic signaling and events. In addition, caspase-8 knockdown also suppressed VEGF-mediated signaling and angiogenesis, suggesting that procaspase-8 plays a role of a non-apoptotic modulator in VEGF-induced angiogenic signaling. These results suggest that TRAIL inhibits VEGF-induced angiogenesis by increasing caspase-8 activity and subsequently decreasing non-apoptotic signaling functions of procaspase-8, without inducing caspase-3 activation and endothelial cell cytotoxicity. These data indicate that caspase-8 may be used as an anti-angiogenic drug for solid tumors resistant to TRAIL and anti-tumor drugs.     

Localization of vascular endothelial growth factor (VEGF) receptors in normal and adenomatous pituitaries: detection of a non-endothelial function of VEGF in pituitary tumours

As for any solid tumour, pituitary adenoma expansion is dependent on neovascularization through angiogenesis. In this process, vascular endothelial growth factor (VEGF) and its receptors VEGFR-1, VEGFR-2 and neuropilin-1 (NRP-1) may play an outstanding role. The intention of this work was to study the expression/localization and possible function of VEGF receptors in pituitary adenomas. VEGF receptor mRNA and protein expression was studied by in situ hybridization, immunohistochemistry and RT-PCR in 6 normal human pituitaries, 39 human pituitary adenomas and 4 rodent pituitary adenoma cell lines. VEGFR-1 expressing somatotroph MtT-S cells were used as a model to study the role of VEGF on cell proliferation and to elucidate the underlying mechanism of action. In normal pituitaries, VEGFR-1 was detected in endocrine cells, whereas VEGFR-2 and NRP-1 were exclusively expressed in endothelial cells. In pituitary tumours, a heterogeneous VEGFR expression pattern was observed by IHC. VEGFR-1, VEGFR-2 and NRP-1 were detected in 24, 18 and 17 adenomas respectively. In the adenomas, VEGFR-1 was expressed in epithelial tumour cells and VEGFR-2/NRP-1 in vessel endothelial cells. Functional studies in VEGFR-1-positive MtT-S cells showed that the ligands of VEGFR-1 significantly stimulated cell proliferation. This effect was mediated through the phosphatidylinositol-3-kinase-signalling pathway and involves induction of cyclin D1 and Bcl-2. Based on our results, we speculate that the ligands of VEGF receptors, such as VEGF-A and placenta growth factor, not only play a role in angiogenesis in pituitary adenomas, but also affect the growth of pituitary tumour cells through VEGFR-1.     

From combinatorial peptide selection to drug prototype (I): Targeting the vascular endothelial growth factor receptor pathway

Inhibition of blood vessel formation is a viable therapeutic approach in angiogenesis-dependent diseases. We previously used a combinatorial screening on vascular endothelial growth factor (VEGF)-activated endothelial cells to select the sequence CPQPRPLC and showed that the motif Arg-Pro-Leu targets VEGF receptor-1 and neuropilin-1. Here, we evaluated and validated _D(LPR), a derivative molecule with strong antiangiogenesis attributes. This prototype drug markedly inhibits neovascularization in three mouse models: Matrigel-based assay, functional human/murine blood vessel formation, and retinopathy of prematurity. In addition to its systemic activity, _D(LPR) also inhibits retinal angiogenesis when administered in an eye-drop formulation. Finally, in preliminary studies, we have showed targeted drug activity in an experimental tumor-bearing mouse model. These results show that drugs targeting extracellular domains of VEGF receptors are active, affect signal transduction, and have potential for clinical application. On a larger context, this study illustrates the power of ligand-directed selection plus retro-inversion for rapid drug discovery and development.