Tea catechins inhibit angiogenesis in vitro,measured by human endothelial cell growth,migration and tube formation,through inhibition of VEGF receptor binding

We have investigated whether tea catechins (EC, ECg, EGC, EGCg) have any inhibitory effects on angiogenesis and which step they affect during the process. The effects of catechins were tested on in vitro models of angiogenesis, namely, growth, migration and tube formation of human umbilical vein endothelial cells. All four catechins inhibited angiogenesis in vitro in the three different bioassays with concentrations ranging from 1.56 to 100 μM. Among the four catechins tested, epigallocatechin gallate (EGCg) was the most effective in inhibiting angiogenesis in all three assays. When these four catechins were tested on VEGF binding assay, only EGCg inhibited the binding of VEGF, a major angiogenesis inducing factor, to endothelial cells in a concentration dependent manner. These results indicate that while all four tea catechins inhibit the process of angiogenesis, EGCg alone can reduce the binding of VEGF to its receptors and thus affects the downstream signaling.     

Selective inhibition of vascular endothelial growth factor (VEGF) receptor 2 (KDR/Flk-1) activity by a monoclonal anti-VEGF antibody blocks tumor growth in mice

Vascular endothelial growth factor (VEGF) is a multifunctional angiogenic growth factor that is a primary stimulant of the development and maintenance of a vascular network in embryogenesis and the vascularization of solid tumors. At the present time there are two well-characterized receptors for VEGF that are selectively expressed on endothelium. VEGF receptor 2 [VEGFR2 (KDR/Flk-1)] mediates endothelial cell mitogenesis and permeability increases, whereas the role of VEGF receptor 1 [VEGFR1 (Flt-1)] has not been clearly defined. In the present study, a monoclonal antibody, 2C3, is shown to block the interaction of VEGF with VEGFR2 but not with VEGFR1 through ELISA, receptor binding assays, and receptor activation assays. 2C3 blocks the VEGF-induced vascular permeability increase in guinea pig skin. 2C3 has potent antitumor activity, inhibiting the growth of newly injected and established human tumor xenografts in mice. These findings demonstrate the usefulness of 2C3 in dissecting the pathways that are activated by VEGF in cells that express both VEGFR1 and VEGFR2, as well as highlighting the dominant role of VEGFR2 in mediating VEGF-induced vascular permeability increase and tumor angiogenesis.     

Sunitinib inhibits lymphatic endothelial cell functions and lymph node metastasis in a breast cancer model through inhibition of vascular endothelial growth factor receptor 3

Introduction  

Metastasis is a common event and the main cause of death in cancer patients. Lymphangiogenesis refers to the formation of new lymphatic vessels and is thought to be involved in the development of metastasis. Sunitinib is a multi-kinase inhibitor that blocks receptor tyrosine kinase activity, including that of vascular endothelial growth factor receptors (VEGFRs). Although sunitinib is a clinically available angiogenesis inhibitor, its effects on lymphangiogenesis and lymph node metastasis remain unclear. The purpose of this study was to investigate the effects of sunitinib on vascular endothelial growth factor receptor 3 (VEGFR-3) and a related event, lymphangiogenesis.     

Induction of tube formation by angiopoietin-1 in endothelial cell/fibroblast co-culture is dependent on endogenous VEGF

The angiopoietin-1 (Ang1)/Tie2 receptor system is known to be important for angiogenesis and vascular remodeling. However, its contribution to the survival and morphogenesis of endothelial cells is still not well elucidated. In this study, we analyzed the role of the Ang1/Tie2 pathway in cell survival and tube formation using a human umbilical vein endothelial (HUVE) cell and fibroblast co-culture system. In this system, which mimics angiogenesis in vivo , fibroblasts secrete a basal level of vascular endothelial growth factor (VEGF), and Ang1 stimulated tube formation. However, anti-VEGF or anti-VEGF receptor-2 neutralizing antibody blocked the Ang1-induced tube formation. Furthermore, other angiogenic factors such as hepatic growth factor (HGF) and basic fibroblast growth factor (bFGF) showed the same pheno-type as Ang1, i.e., a stimulatory effect only in the presence of endogenous VEGF. The Ang1-promoted tube formation was mainly due to suppression of HUVE cell apoptosis in a PI3-kinase-dependent manner. These findings suggest that Ang1 stimulates tube formation in vivo via the PI3-kinase/Akt pathway, but this effect takes place only in a VEGF-dependent manner.     

RRIG1 mediates effects of retinoic acid receptor beta2 on tumor cell growth and gene expression through binding to and inhibition of RhoA

The expression of retinoic acid receptor beta2 (RAR-beta2) is frequently lost in various cancers and their premalignant lesions. However, the restoration of RAR-beta2 expression inhibits tumor cell growth and suppresses cancer development. To understand the molecular mechanisms responsible for this RAR-beta2-mediated antitumor activity, we did restriction fragment differential display-PCR and cloned a novel retinoid receptor-induced gene 1 (RRIG1), which is differentially expressed in RAR-beta2-positive and RAR-beta2-negative tumor cells. RRIG1 cDNA contains 2,851 bp and encodes a protein with 276 amino acids; the gene is localized at chromosome 9q34. Expressed in a broad range of normal tissues, RRIG1 is also lost in various cancer specimens. RRIG1 mediates the effect of RAR-beta2 on cell growth and gene expression (e.g., extracellular signal-regulated kinase 1/2 and cyclooxygenase-2). The RRIG1 protein is expressed in the cell membrane and binds to and inhibits the activity of a small GTPase RhoA. Whereas induction of RRIG1 expression inhibits RhoA activation and f-actin formation and consequently reduces colony formation, invasion, and proliferation of esophageal cancer cells, antisense RRIG1 increases RhoA activity and f-actin formation and thus induces the colony formation, invasion, and proliferation of these cells. Our findings therefore show a novel molecular pathway involving RAR-beta2 regulation of RRIG1 expression and RRIG1-RhoA interaction. An understanding of this pathway may translate into better control of human cancer.     

Vascular endothelial growth factor (VEGF) inhibition by small molecules

Angiogenesis is essential for primary tumours to grow and metastasise, and is driven by the production of positive angiogenic factors. The Vascular Endothelial Growth Factor (VEGF) family is central to the process of angiogenesis and comprises 5 molecules designated A, B, C, D and E. VEGF is overexpressed in several solid malignancies. The actions of VEGF are mediated through receptors possessing tyrosine kinase activity: VEGFR-1 (Flt-1), VEGFR-2 (Kdr/Flk-1) and VEGFR-3 (Flt-4). Anti-VEGF strategies include the use of antibodies to VEGF or its receptors, the use of ribozymes to decrease receptor expression, and the use of inhibitors of tyrosine kinase to reduce receptor activation and downstream signalling. The focus of this review is small molecule inhibitors of VEGF receptors which target their intrinsic tyrosine kinase activity. The clinical development of the following agents is discussed: SU5416, SU11248, SU6668, PTK/ZK, ZD6474.     

Anti-angiogenic effect of Biophytum sensitivum is exerted through its cytokine modulation activity and inhibitory activity against VEGF mRNA expression, endothelial cell migration and capillary tube formation

Angiogenesis is a crucial step essential for the growth, progression and metastasis of solid tumors. Substances produced by inflammatory cells, such as cytokines play an important role in the stimulation and progression of angiogenesis. In this study we investigated the anti-angiogenic effect of Biophytum sensitivum, using in vivo as well as in vitro models. In vitro antiangiogenic activity was studied using B16-F10 melanoma cell-induced capillary formation in C57BL/6 mice. Intraperitoneal administration of the extract at a concentration of 50 mg/kg significantly inhibited the tumor directed capillary formation induced by melanoma cells. The cytokine profile in the serum of these animals showed a drastically increased level of proinflammatory cytokines such as IL-1beta, IL-6, TNF-alpha, GM-CSF and the direct endothelial cell proliferating agent, VEGF. Administration of Biophytum extract could differentially regulate these cytokine's elevation. The differential elevation is further evidenced by the increased production of IL-2 and tissue inhibitor of metalloprotease-1 (TIMP-1) in the B16-F10 injected, extract treated animals. The extract of B. sensitivum at non-toxic concentrations (1 microg/ml, 5 microg/ml and 10 microg/ml) inhibited the VEGF-induced vessel sprouting in rat aortic ring assay. Moreover, B. sensitivum was able to inhibit the VEGF-induced proliferation, cell migration and capillary-like tube formation of primary cultured human endothelial cells. Furthermore B. sensitivum showed inhibitory effect on VEGF mRNA levels in B16-F10 melanoma cells. Hence the observed antiangiogenic activity of the plant B. sensitivum is exerted through its cytokine modulation activity and inhibitory activity against VEGF mRNA expression.     

Use of soluble recombinant decoy receptor vascular endothelial growth factor trap (VEGF Trap) to inhibit vascular endothelial growth factor activity

Primary tumors and metastases require blood vessel formation to support their continued growth and eventual metastasis. They use existing vasculature during initial growth but eventually must orchestrate the development and maintenance of new vessels--a process termed angiogenesis--to grow beyond a small size and spread. Angiogenesis is regulated by a number of soluble factors, the relative proportions of which can exacerbate or inhibit the process. Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis, produced by the majority of human solid tumors. Inhibitors of VEGF might have an impact on the growth and metastasis of these cancers. The relevance of this strategy to the treatment of colorectal cancer was first successfully demonstrated in human clinical trials using a monoclonal antibody against VEGF. A potent antiangiogenic soluble recombinant decoy, VEGF Trap is a protein constructed from VEGF receptor-binding domains linked to an immunoglobulin G(1) constant region. It possesses an affinity for VEGF that is significantly higher than that of the monoclonal antibody. VEGF Trap has demonstrated marked efficacy in halting angiogenesis and shrinking tumors in preclinical animal models and is currently being studied in phase I clinical trials in humans with advanced solid malignancies.     

Hedgehog signaling inhibition blocks growth of resistant tumors through effects on tumor microenvironment

Hedgehog (Hh) signaling is implicated in bone development and cellular transformation. Here we show that inhibition of Hh pathway activity inhibits tumor growth through effects on the microenvironment. Pharmacologic inhibition of the Hh effector Smoothened (Smo) increased trabecular bone in vivo and inhibited osteoclastogenesis in vitro. In addition, enhanced Hh signaling due to heterozygosity of the Hh inhibitory receptor Patched (Ptch1(+/-)) increased bone resorption, suggesting direct regulation of osteoclast (OC) activity by the Hh pathway. Ptch1(+/-) mice had increased bone metastatic and subcutaneous tumor growth, suggesting that increased Hh activation in host cells promoted tumor growth. Subcutaneous growth of Hh-resistant tumor cells was inhibited by LDE225, a novel orally bioavailable SMO antagonist, consistent with effects on tumor microenvironment. Knockdown of the Hh ligand Sonic Hh (SHH) in these cells decreased subcutaneous tumor growth and decreased stromal cell production of interleukin-6, indicating that tumor-derived Hh ligands stimulated tumor growth in a paracrine fashion. Together our findings show that inhibition of the Hh pathway can reduce tumor burden, regardless of tumor Hh responsiveness, through effects on tumor cells, OCs, and stromal cells within the tumor microenvironment. Hh may be a promising therapeutic target for solid cancers and bone metastases.     

Complete inhibition of vascular endothelial growth factor (VEGF) activities with a bifunctional diabody directed against both VEGF kinase receptors, fms-like tyrosine kinase receptor and kinase insert domain-containing receptor

Vascular endothelial growth factor (VEGF) binds to and mediates its activity mainly through two tyrosine kinase receptors, VEGF receptor 1 [or fms-like tyrosine kinase receptor (Flt-1)] and VEGF receptor 2 [or kinase insert domain-containing receptor (KDR)]. Numerous studies have shown that overexpression of VEGF and its receptor plays an important role in tumor-associated angiogenesis and hence in both tumor growth and metastasis. We demonstrated previously that antagonistic antibodies to KDR specifically inhibited VEGF-stimulated receptor activation, cell migration, and endothelial cell mitogenesis. Here we constructed a recombinant bifunctional diabody that is capable of blocking both Flt-1 and KDR from binding to their ligands, including VEGF and placenta growth factor (PlGF). The diabody was expressed in Escherichia coli and purified by single-step affinity chromatography. The diabody retained the capacity to bind both KDR and Flt-1 and effectively blocked interaction between KDR and VEGF, Flt-1 and VEGF, and Flt-1 and PlGF. Furthermore, the diabody is a stronger inhibitor than its parent antibodies to VEGF-stimulated mitogenesis of human endothelial cells, as well as both VEGF- and PlGF-induced migration of human leukemia cells. Taken together, our results suggest that dual receptor blockade with the bifunctional diabody may prove to be a more efficient approach in inhibiting VEGF-stimulated angiogenesis.     

Correlation between human cell growth response to interleukin 1 and receptor binding

Human recombinant interleukin 1 alpha (IL-1 alpha) and IL-1 beta inhibited the replication of the mammary tumor cell line, MDA-MB-415; stimulated division in the colon carcinoma, SW-48; and had no effect on the growth of the milk mammary line, HBL-100. Inhibition of growth was reflected in a significant decrease in DNA synthesis accompanying a transient increase in RNA synthesis. Specific binding of 125I-labeled recombinant IL-1 beta by MDA-MB-415 and SW-48 reached a maximum by 2 h of incubation, and an equivalent amount was bound by each cell type. Binding was inhibited in a dose-dependent manner by unlabeled IL-1 alpha or IL-1 beta. Scatchard plot analysis revealed that MDA-MB-415 cells expressed approximately 700 binding sites with an apparent dissociation constant of 8.8 x 10(-10) M. Reversibility of growth inhibition was independent of dose or time of incubation, but DNA synthesis did not return to control values. Flow cytometric analysis of DNA content showed that growth inhibition was cell cycle phase nonspecific with a slight reduction in the proportion of cells in S phase. The major conclusion from these studies was that inhibition or stimulation of malignant cell growth by IL-1 was related to the presence of receptor sites.     

Differential behavior of VEGF receptor expression and response to TNP-470 in two immortalized human endothelial cell lines

Angiogenesis consists of endothelial cell proliferation, migration and tube formation. It is useful to investigate endothelial cell behavior using immortalized endothelial cell lines. We characterized cell growth property, growth factor dependency and response to angioinhibitory drugs; TNP-470, staurosporine, radicicol and genistein, using human umbilical vein endothelial cells (HUVECs) immortalized by human papilloma virus (HPV)-16 E6-E7, named HUVECs/E6-E7, and HUVECs/E6-E7 transformed by v-Ki-ras gene, named HUVECs/E6-E7/ras. The dependency to vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) for cell proliferation decreased in HUVECs/E6-E7, but were restored in HUVECs/E6-E7/ras. Flow cytometric analysis demonstrated that a VEGF receptor KDR/flk-1 was down-regulated in HUVECs/E6-E7 but not in HUVECs/E6-E7/ras. Expression of another VEGF receptor flt-1 was consistent in all cells including HUVECs, HUVECs/E6-E7 and HUVECs/E6-E7/ras. According to the analysis of the angioinhibitory drugs, HUVECs/E6-E7 was obviously resistant to TNP-470, but HUVECs/E6-E7/ras showed similar response compared to HUVECs which suggests that v-Ki-ras signaling pathway is associated with VEGF receptor expression and make HUVECs/E6-E7 sensitive to TNP-470 by modulating the signal transduction cascade. In conclusion, HPV-16 E6-E7 and v-Ki-ras genes have unique growth properties and these immortalized cells are useful for investigating signal transduction pathways of endothelial cells, and for screening of angioinhibitory drugs.     

Vascular endothelial growth factor trap blocks tumor growth, metastasis formation, and vascular leakage in an orthotopic murine renal cell cancer model.

PURPOSE: Angiogenesis inhibitors have shown clinical benefit in patients with advanced renal cell cancer, but further therapeutic improvement is needed. Vascular endothelial growth factor (VEGF) Trap is a newly developed VEGF-blocking agent with stronger affinity and broader activity than the anti-VEGF antibody bevacizumab. In this study, we tested the activity of VEGF Trap in an orthotopic murine model of renal cancer with spontaneous lung metastases. EXPERIMENTAL DESIGN: Murine syngeneic renal cell carcinoma cells (RENCA) transfected with a luciferase-expressing vector were injected into the renal capsule of BALB/c mice. I.p. treatment with VEGF Trap or control protein (10 or 25 mg/kg twice weekly) was started shortly after tumor injection to prevent tumor development (prevention model) or after established tumors were formed to inhibit tumor growth and metastasis formation (intervention model). RESULTS: In the prevention model, VEGF Trap inhibited tumor growth by 87 +/- 14% compared with control (P=0.007) and significantly prolonged survival. In the intervention model, VEGF Trap inhibited tumor growth by 74 +/- 9% (P<0.001) and the formation of lung metastases was inhibited by 98% (P<0.004). Microvascular density was reduced by 66% due to VEGF Trap treatment (P<0.001). In addition, VEGF Trap prevented fibrinogen leakage into the tumor microenvironment representative for reduced vascular leaking as shown by immunohistochemical staining. CONCLUSIONS: VEGF Trap is a potent inhibitor of RENCA tumor growth and metastasis formation and blocks the biological function of VEGF in vivo. These results support further clinical development of VEGF Trap for renal cell cancer and other cancer types.     

Enhanced susceptibility of irradiated tumor vessels to vascular endothelial growth factor receptor tyrosine kinase inhibition

Previous experiments with PTK787/ZK222584, a specific inhibitor of vascular endothelial growth factor receptor (VEGFR) tyrosine kinases, using irradiated human FaDu squamous cell carcinoma in nude mice, suggested that radiation-damaged tumor vessels are more sensitive to VEGFR inhibition. To test this hypothesis, the tumor transplantation site (i.e., the right hind leg of nude mice) was irradiated 10 days before transplantation of FaDu to induce radiation damage in the host tissue. FaDu tumors vascularized by radiation-damaged blood vessels appeared later, grew at a slower rate, and showed more necrosis and a smaller vessel area per central tumor section than controls. PTK787/ZK222584 at a daily dose of 50 mg/kg body weight had no impact on growth of control tumors. In contrast, tumors vascularized by radiation-damaged vessels responded to PTK787/ZK222584 with longer latency and slower growth rate than controls, and a trend toward further increase in necrosis, indicating that irradiated tumor vessels are more susceptible to VEGFR inhibition than unirradiated vessels. Although not proving causality, expression analysis of VEGF and VEGFR2 shows that enhanced sensitivity of irradiated vessels to a specific inhibitor of VEGFR tyrosine kinases correlates with increased expression of the molecular target.     

Porphyrin analogues as novel antagonists of fibroblast growth factor and vascular endothelial growth factor receptor binding that inhibit endothelial cell proliferation, tumor progression, and metastasis

Fibroblast growth factors (FGFs) and vascular endothelial growth factor (VEGF) play a pivotal role in the multistep pathway of tumor progression, metastasis, and angiogenesis. We have identified a porphyrin analogue, 5,10,15,20-tetrakis(methyl-4-pyridyl)-21H,23H-porphine-tetra -p-tosylate salt (TMPP), as a potent inhibitor of FGF2 and VEGF receptor binding and activation. TMPP demonstrated potent inhibition of binding of soluble FGF receptor 1 (FGFR1) to FGF2 immobilized on heparin at submicromolar concentrations. TMPP inhibits binding of radiolabeled FGF2 to FGFR in a cell-free system as well as to cells genetically engineered to express FGFR1. Furthermore, TMPP also inhibits the binding of VEGF to its tyrosine kinase receptor in a dose-dependent manner. In an in vitro angiogenic assay measuring the extent of endothelial cell growth, tube formation, and sprouting, TMPP dramatically reduced the extent of the FGF2-induced endothelial cell outgrowth and differentiation. In a Lewis lung carcinoma model, mice receiving TMPP showed a marked inhibition of both primary tumor progression and lung metastases development, with nearly total inhibition of the metastatic phenotype upon alternate daily injections of TMPP at 25 microg/g of body mass. Finally, novel meso-pyridylium-substituted, nonsymmetric porphyrins, as well as a novel corrole-based derivative, with >50-fold increase in activity in vitro, had a significantly improved efficacy in blocking tumor progression and metastasis in vivo.     

Estrogen receptor alpha mediates breast cancer cell resistance to paclitaxel through inhibition of apoptotic cell death

Estrogen receptors (ER) are expressed in approximately 65% of human breast cancer. Cumulative data from clinical trials and retrospective analyses suggest that some chemotherapeutic agents may be less effective in patients with ER-positive (ER+) tumors than those with ER-negative (ER-) tumors. Paclitaxel is an active agent used in breast cancer chemotherapy. To investigate the possible influence of ER on the therapeutic efficacy of paclitaxel and its underlying mechanism, we established several isogenic ER+ cell lines by stable transfection of ERalpha expression vectors into ER- breast cancer BCap37 cells. We showed that 17-beta estradiol significantly reduces the overall cytotoxicity of paclitaxel in BCap37-expressing ERalpha but has no influence on the ER- parental cells. Further analyses indicate that expression of ERalpha in BCap37 cells mainly interferes with paclitaxel-induced apoptotic cell death, without affecting paclitaxel-induced microtubule bundling and mitotic arrest. Moreover, we found that the addition of ICI 182,780 (Fulvestrant), a selective ER down-regulator, could completely reverse the resistance of ER+ BCap37 cells to paclitaxel. These findings showed that ERalpha-mediated breast tumor cell resistance to paclitaxel was through selective inhibition of paclitaxel-induced tumor cell apoptosis. Additionally, the combination of ICI 182,780 also sensitizes MCF-7 and T47D cell lines to the treatment of paclitaxel, which further confirmed the correlation between ERalpha and drug resistance in ER+ tumor cells. The results obtained from this study provide useful information for understanding ER-mediated resistance to paclitaxel and possibly other antineoplastic agents.     

Colorectal carcinoma cell production of transforming growth factor beta decreases expression of endothelial cell vascular endothelial growth factor receptor 2

BACKGROUND:

Vascular endothelial growth factor (VEGF) signaling is a target for antiangiogenic cancer therapy. The authors have previously observed that up to 40% of vessels in colorectal carcinoma (CRC) tumors are negative for VEGF receptor 2 (VEGFR2) expression. Differential activity of transforming growth factor beta (TGF‐β) is a potential contributor to this receptor heterogeneity because TGF‐β contributes to both angiogenesis and CRC tumor progression.

METHODS:

The authors analyzed VEGFR2 expression by Western blotting, and TGF‐β expression in endothelial and CRC cell lines, respectively. In addition, they immunostained endothelial cells in CRC xenografts to find an association between VEGFR2 and TGF‐β levels or activity.

RESULTS:

In bovine aortic endothelial cells (BAECs), TGF‐β1 significantly repressed VEGFR2 protein in a time‐dependent and dose‐dependent fashion (P < .05). Serum‐free conditioned media from various malignant human CRC cell lines (HCT116, 379.2, Dks8, and DLD1) induced down‐regulation of VEGFR2 in BAECs. This effect was proportional to the total levels of TGF‐β1 and TGF‐β2 and was blocked by SB‐431542 and SD‐208, TGF‐β receptor I inhibitors. Immunofluorescence staining of subcutaneous mouse xenografts of HCT116, 379.2, Dks8, and SW480 cells revealed vessels with an inverse relationship between TGF‐β activity and VEGFR2 expression. Oxygen and bone morphogenetic protein 9 levels were shown to modulate TGF‐β–induced VEGFR2 down‐regulation.

CONCLUSIONS:

In combination with other factors, TGF‐β may contribute to the vascular heterogeneity in human colorectal tumors. Cancer 2011;. © 2011 American Cancer Society.     

Effect of VEGF and VEGF Trap on vascular endothelial cell signaling in tumors

Vascular endothelial growth factor (VEGF) A is a major promoter of tumor angiogenesis and a prime target of antiangiogenic cancer therapy. To examine whether endothelial cell signaling might provide histological biomarkers of angiogenesis and VEGF activity in vivo, normal mouse organs and multiple tumor models were studied immunohistochemically for endothelial expression of activated ERK, STAT3 and AKT. Phospho(p)-ERK and p-STAT3 expression was negligible in the endothelia of normal organs but was significantly elevated in tumor endothelium. p-AKT was present at significant and comparable levels in both tumor and normal endothelia. In K1735 tumors induced to express more VEGF, endothelial p-ERK, p-STAT3 and p-AKT increased accompanied by signs of accelerated angiogenesis. Treatment of K1735 and Colo-205 tumors with the VEGF inhibitor, VEGF Trap (aflibercept), decreased tumor endothelial p-ERK, p-STAT3 and p-AKT expression accompanied by signs of antiangiogenic effect. These results show that endothelial p-ERK and p-STAT3 (but not p-AKT) distinguish tumor from normal vessels and that the presence of these two signaling intermediates may be useful indicators of tumor angiogenic activity and angiogenesis inhibition by VEGF antagonists.Key words: VEGF, VEGF Trap, endothelial cells, signal transduction, angiogenesis, biomarker, p-ERK, p-STAT3, p-AKT     

Expression of vascular endothelial growth factor (VEGF)-D and its receptor, VEGF receptor 3, as a prognostic factor in endometrial carcinoma.

PURPOSE: To evaluate the prognostic value of vascular endothelial growth factor (VEGF)-D and VEGF receptor (VEGFR)-3 in endometrial carcinoma. EXPERIMENTAL DESIGN: We assessed the levels of immunoreactivity for VEGF-D and VEGFR-3 in 71 endometrial carcinomas, 14 complex atypical endometrial hyperplasias, and 16 normal endometria by immunohistochemistry. RESULTS: VEGF-D was stained in both tumor cells and adjacent stromal cells. VEGFR-3 was stained in both tumor cells and adjacent endothelial cells. Immunoreactivity for VEGF-D in tumor cells and adjacent stromal cells became significantly stronger as lesions progressed from normal endometrium to advanced carcinoma. Similarly, immunoreactivity for VEGFR-3 in tumor cells and adjacent endothelial cells was significantly greater as lesions progressed from normal endometrium to advanced carcinoma. A strong correlation was found between high levels of VEGF-D immunoreactivity in carcinoma cells and VEGFR-3 in both carcinoma cells and adjacent endothelial cells. Similarly, high levels of VEGF-D immunoreactivity in stromal cells were significantly correlated with those of VEGFR-3 in both carcinoma cells and endothelial cells. High levels of VEGF-D in carcinoma cells and stromal cells, as well as those of VEGFR-3 in carcinoma cells and endothelial cells, were significantly related to myometrial invasion and lymph node metastasis. A strong correlation was found between poor survival and high levels of VEGF-D in both carcinoma cells and stromal cells and between poor survival and high levels of VEGFR-3 in carcinoma cells. Moreover, the high levels of VEGF-D in stromal cells and VEGFR-3 in carcinoma cells were independent prognostic factors in endometrial carcinoma. CONCLUSIONS: The presence of VEGF-D and VEGFR-3 in endometrial carcinoma may predict myometrial invasion and lymph node metastasis and may prospectively identify patients who are at increased risk for poor outcome. In addition, VEGF-D and VEGFR-3 may be promising targets for new therapeutic strategies in endometrial carcinoma.     

可溶性VEGF受体基因sflt-1与反义VEGF核苷酸对新生血管形成的影响

背景与目的:肿瘤组织中新生血管提供的大量营养物质和生长因子是肿瘤快速生长的关键,因此如何抑制肿瘤组织中新生血管的形成、促使肿瘤组织坏死也是肿瘤治疗中的一条值得探讨的途径。本文拟研究和观察可溶性血管内皮细胞生长因子(vascularendothelialgrowthfactor,VEGF)受体基因sflt-1与反义VEGF对新生血管形成的影响。方法:用Ad-反义VEGF感染肝癌细胞株MM45T.Li后,观察反义VEGF对MM45T.Li分泌VEGF的影响;将人工重组的3'ΔFlt-1蛋白(C末端缺失的Flt-1蛋白)加入条件培养液中,观察3'ΔFlt-1对VEGF刺激的脐静脉血管内皮细胞(humanumbilicalvascularendotheliumcell,HUVEC)增殖的影响;并将Ad-反义VEGF、Ad-sflt-1注射于鸡胚尿囊绒毛膜中,观察sflt-1、反义VEGF对鸡胚新生血管形成的影响。结果:反义VEGF重组腺病毒感染MM45T.Li细胞后,细胞培养上清中VEGF浓度仅为对照组中VEGF浓度的15%(P<0.01);在含有3'ΔFlt-1蛋白的调价培养液中,HUVEC的增殖明显减慢,在一定的范围内与剂量呈负相关;两者都能有效地抑制鸡胚新生血管的形成。结论:sflt-1与反义VEGF均能有效抑制新生血管的形成,两者联合能增强抑制效果,但其作用机制不同。