Monoclonal Antibodies Targeting Vascular Endothelial Growth Factor

The use of monoclonal antibodies targeting the vascular endothelial growth factor (VEGF) pathway has been a significant addition to cancer therapy. One of the VEGF family members, VEGF-A (commonly referred to as VEGF), has been demonstrated to be important in angiogenesis. Although the mechanism of action of these antibodies is still under study, the anti-VEGF antibody bevacizumab has been approved for treatment of various solid cancers including colorectal, lung, and breast cancers as well as glioblastoma and renal cell carcinoma. Addition of bevacizumab to chemotherapy as adjuvant therapy in colorectal cancer did not improve disease-free survival. Bevacizumab is being tested in other clinical settings such as adjuvant therapy, maintenance therapy, and in combination with both chemotherapy and other targeted agents such as the epidermal growth factor receptor kinase inhibitor erlotinib. In addition to bevacizumab, other antibody-based therapies targeting the VEGF pathway are being tested. Ramucirumab and IMC-18F1 are monoclonal antibodies that target the VEGF receptors VEGFR-2 and VEGFR-1, respectively. Aflibercept (VEGF-Trap), a peptide-antibody fusion targeting VEGF ligand, is being tested in clinical trials. Much research is focused on identifying biomarkers to predict which patients will benefit from anti-VEGF therapy. Recent results suggest that VEGF single nucleotide polymorphisms may be predictive of patient response to bevacizumab. Improved imaging modalities such as dynamic contrast-enhanced MRI (DCE-MRI) can better characterize the efficacy of anti-angiogenic agents. As anti-VEGF treatments such as bevacizumab have been integrated into the treatment of many different types of cancers, the development of bevacizumab-resistant tumors has become more common. Recent studies show that targeting other angiogenesis signaling pathways such as platelet-derived growth factor-C (PDGF-C), Bombina variagata peptide 8 (Bv8, also known as prokineticin-2), and VEGFR-3 may lead to enhanced response in anti-VEGF resistant tumors. In the future, tailored treatments consisting of combinations of chemotherapy, other targeted therapies, and anti-angiogenesis agents will hopefully result in better patient outcomes.     

Angiogenesis in gliomas: biology and molecular pathophysiology

Glioblastoma multiforme (GBM) is characterized by exuberant angiogenesis, a key event in tumor growth and progression. The pathologic mechanisms driving this change and the biological behavior of gliomas remain unclear. One mechanism may involve cooption of native blood vessels by glioma cells inducing expression of angiopoietin-2 by endothelial cells. Subsequently, vascular apoptosis and involution leads to necrosis and hypoxia. This in turn induces angiogenesis that is associated with expression of hypoxia-inducible factor (HIF)-1alpha and vascular endothelial growth factor (VEGF) in perinecrotic pseudopalisading glioma cells. Here we review the molecular and cellular mechanisms implicated in HIF-1-dependent and HIF-1-independent glioma-associated angiogenesis. In GBMs, both tumor hypoxia and genetic alterations commonly occur and act together to induce the expression of HIF-1. The angiogenic response of the tumor to HIF-1 is mediated by HIF-1-regulated target genes leading to the upregulation of several proangiogenic factors such as VEGF and other adaptive response molecules. Understanding the roles of these regulatory processes in tumor neovascularization, tumor growth and progression, and resistance to therapy will ultimately lead to the development of improved antiangiogenic therapies for GBMs.     

Normalization of the vasculature for treatment of cancer and other diseases

New vessel formation (angiogenesis) is an essential physiological process for embryologic development, normal growth, and tissue repair. Angiogenesis is tightly regulated at the molecular level. Dysregulation of angiogenesis occurs in various pathologies and is one of the hallmarks of cancer. The imbalance of pro- and anti-angiogenic signaling within tumors creates an abnormal vascular network that is characterized by dilated, tortuous, and hyperpermeable vessels. The physiological consequences of these vascular abnormalities include temporal and spatial heterogeneity in tumor blood flow and oxygenation and increased tumor interstitial fluid pressure. These abnormalities and the resultant microenvironment fuel tumor progression, and also lead to a reduction in the efficacy of chemotherapy, radiotherapy, and immunotherapy. With the discovery of vascular endothelial growth factor (VEGF) as a major driver of tumor angiogenesis, efforts have focused on novel therapeutics aimed at inhibiting VEGF activity, with the goal of regressing tumors by starvation. Unfortunately, clinical trials of anti-VEGF monotherapy in patients with solid tumors have been largely negative. Intriguingly, the combination of anti-VEGF therapy with conventional chemotherapy has improved survival in cancer patients compared with chemotherapy alone. These seemingly paradoxical results could be explained by a "normalization" of the tumor vasculature by anti-VEGF therapy. Preclinical studies have shown that anti-VEGF therapy changes tumor vasculature towards a more "mature" or "normal" phenotype. This "vascular normalization" is characterized by attenuation of hyperpermeability, increased vascular pericyte coverage, a more normal basement membrane, and a resultant reduction in tumor hypoxia and interstitial fluid pressure. These in turn can lead to an improvement in the metabolic profile of the tumor microenvironment, the delivery and efficacy of exogenously administered therapeutics, the efficacy of radiotherapy and of effector immune cells, and a reduction in number of metastatic cells shed by tumors into circulation in mice. These findings are consistent with data from clinical trials of anti-VEGF agents in patients with various solid tumors. More recently, genetic and pharmacological approaches have begun to unravel some other key regulators of vascular normalization such as proteins that regulate tissue oxygen sensing (PHD2) and vessel maturation (PDGFRβ, RGS5, Ang1/2, TGF-β). Here, we review the pathophysiology of tumor angiogenesis, the molecular underpinnings and functional consequences of vascular normalization, and the implications for treatment of cancer and nonmalignant diseases.     

Potential therapeutic implications of intracrine angiogenesis

Angiogenesis, in most cases, is a requirement for tumor growth beyond a diameter of a few millimeters and is, therefore, a major target for cancer therapy. The intracellular actions of certain extracellular signaling proteins (intracrines) have been reported, and it is clear that intracrines such as vascular endothelial growth factor, basic fibroblast growth factor, angiogenin, angiotensin, and endothelin, among others, are involved in angiogenesis. We have proposed that intracrine networks play an important role in angiogenesis, and have suggested that very similar intracrine networks exist in some tumor cells. These notions have implications for the development of anti-angiogenesis therapies because they suggest that the inhibition of intracellular intracrine trafficking pathways may be an effective therapeutic target. Here the participation and regulation of intracrines in angiogenesis is explored, as are the actions of various anti-angiogenic factors.     

抗血管生成药物在非小细胞肺癌中的应用及研究进展

新生血管的形成是肿瘤生长、进展、转移的基础,此过程涉及各类受体介导的细胞信号通路,其中刺激血管生成作用最强的生长因子是血管内皮生长因子（VEGF）。随着VEGF作用的深入认识和血管靶向治疗的临床实践,以抗新生血管为核心的治疗策略取得显著成效。现将抗血管药物的作用机制及其在治疗非小细胞肺癌中的研究进展作简要综述。     

The possible role of chemotherapy in antiangiogenic drug resistance

The use of antiangiogenic drugs for cancer treatment was welcomed because of the hypothesis that they would be much less likely to lose their therapeutic activity as a result of tumor-acquired resistance over time. Unfortunately, the clinical experience has shown that acquired resistance to antiangiogenic therapeutic strategies is possible since many patients whose tumors initially respond to drugs such as bevacizumab (a monoclonal antibody against VEGF), sorafenib, or sunitinib (tyrosine kinase inhibitors targeting VEGF receptors and PDGF receptors) or metronomic chemotherapy (e.g. low dose cyclophosphamide) become nonresponsive, often within months of therapy initiation. Indeed, the role of associated antineoplastic chemotherapy in antiangiogenic resistance seems to be ignored by the previous studies and the real part played by these drugs has to be written yet. The studies undertaken on antiangiogenic resistance mainly involved mechanisms directly related to the antiangiogenic drugs alone and as such lead one to ask whether the acquired resistance to angiogenesis pathway-targeting might also be mediated by the chemotherapeutic drugs usually associated (at least into the clinic) with these types of drugs. The proposed hypothesis is concerning the possibility that the acquired resistance to antiangiogenic therapy could be actively and heavily modulated by the choice of the associated chemotherapeutic drug. The chemotherapeutic compounds may delay or accelerate the process through the induction, upregulation or downregulation of pro-angiogenic or anti-angiogenic factors or their receptors in the tumor, endothelial and other type of cells of the tumor microenvironment. In conclusion, the consequences of our hypothesis could be promptly translated into the preclinical studies and verified in clinical trials, involving cancer patients resistant to chemotherapy plus antiangiogenic drug schedules.     

VEGF分泌量及分泌来源对肿瘤血管生长影响的数值模拟

目的 数值模拟研究血管内皮生长因子（vascular endothelial growth factor, VEGF）分泌量及分泌来源对肿瘤血管生成的影响。方法 建立肿瘤内外血管生成的二维离散数学模型。围绕VEGF的分泌及其诱导新生血管形成肿瘤富血管区的过程,考虑细胞外基质的旁分泌作用以及对内皮细胞运动的趋触作用,以微血管密度作为定量指标,探讨VEGF的分泌量及不同的分泌来源对血管生成的影响。结果 肿瘤增殖细胞区、VEGF高浓度区、富血管区三者统一,微血管密度与VEGF的表达有关,随着增殖细胞区域的扩大,即VEGF的表达越来越多,微血管密度也越来越大,但在不同类型的肿瘤中,VEGF不同分泌来源的比重与微血管密度无明显相关性。结论 模型探讨了VEGF分泌量及分泌来源对肿瘤血管生成的影响,其中对VEGF的不同分泌来源的考虑可作为研究靶向VEGF治疗肿瘤的模型基础。     

Molecular mechanisms underlying the onset of degenerative aortic valve disease

Morbidity from degenerative aortic valve disease is increasing worldwide, concomitant with the ageing of the general population and the habitual consumption of diets high in calories and cholesterol. Immunohistologic studies have suggested that the molecular mechanism occurring in the degenerate aortic valve resembles that of atherosclerosis, prompting the testing of HMG CoA reductase inhibitors (statins) for the prevention of progression of native and bioprosthetic aortic valve degeneration. However, the effects of these therapies remain controversial. Although the molecular mechanisms underlying the onset of aortic valve degeneration are largely unknown, research in this area is advancing rapidly. The signaling components involved in embryonic valvulogenesis, such as Wnt, TGF-β₁, BMP, and Notch, are also involved in the onset of aortic valve degeneration. Furthermore, investigations into extracellular matrix remodeling, angiogenesis, and osteogenesis in the aortic valve have been reported. Having noted avascularity of normal cardiac valves, we recently identified chondromodulin-I (chm-I) as a crucial anti-angiogenic factor. The expression of chm-I is restricted to cardiac valves from late embryogenesis to adulthood in the mouse, rat, and human. In human degenerate atherosclerotic valves, the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases and angiogenesis is observed in the area of chm-I downregulation. Gene targeting of chm-I resulted in VEGF expression, angiogenesis, and calcification in the aortic valves of aged mice, and aortic stenosis is detected by echocardiography, indicating that chm-I is a crucial factor for maintaining normal cardiac valvular function by preventing angiogenesis. The present review focuses on the animal models of aortic valve degeneration and recent studies on the molecular mechanisms underlying the onset of degenerative aortic valve disease.     

Differential drug class-specific metastatic effects following treatment with a panel of angiogenesis inhibitors

Inhibiting angiogenesis has become an important therapeutic strategy for cancer treatment but, like other current targeted therapies, benefits experienced for late-stage cancers can be curtailed by inherent refractoriness or by acquired drug resistance, requiring a need for better mechanistic understanding of such effects. Numerous preclinical studies have demonstrated that VEGF pathway inhibitors suppress primary tumour growth and metastasis. However, it has been recently reported that short-term VEGF and VEGFR inhibition can paradoxically accelerate tumour invasiveness and metastasis in certain models. Here we comprehensively compare the effects of both antibody and small molecule receptor tyrosine kinase (RTK) inhibitors targeting the VEGF-VEGFR pathway, using short-term therapy in various mouse models of metastasis. Our findings demonstrate that antibody inhibition of VEGF pathway molecules does not promote metastasis, in contrast to selected small molecule RTK inhibitors at elevated-therapeutic drug dosages. In particular, a multi-targeted RTK inhibitor, sunitinib, which most profoundly potentiated metastasis, also increased lung vascular permeability and promoted tumour cell extravasation. Mechanistically, sunitinib, but not anti-VEGF treatment, attenuated endothelial barrier function in culture and caused a global inhibition of protein tyrosine phosphorylation, including molecules important for maintaining endothelial cell-cell junctions. Together these findings indicate that, rather than a specific consequence of inhibiting the VEGF signalling pathway, pharmacological inhibitors of the VEGF pathway can have dose- and drug class-dependent side-effects on the host vasculature. These findings also advocate for the continued identification of mechanisms of resistance to anti-angiogenics and for therapy development to overcome it.     

Vascular endothelial growth factor-D: signaling mechanisms,biology, and clinical relevance

Vascular endothelial growth factor-D (VEGF-D) is a secreted glycoprotein that promotes growth of blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis), and can induce remodeling of large lymphatics. VEGF-D enhances solid tumor growth and metastatic spread in animal models of cancer, and in some human cancers VEGF-D correlates with metastatic spread, poor patient outcome, and, potentially, with resistance to anti-angiogenic drugs. Hence, VEGF-D signaling is a potential target for novel anti-cancer therapeutics designed to enhance anti-angiogenic approaches and to restrict metastasis. In the cardiovascular system, delivery of VEGF-D in animal models enhanced angiogenesis and tissue perfusion, findings which have led to a range of clinical trials testing this protein for therapeutic angiogenesis in cardiovascular diseases. Despite these experimental and clinical developments, our knowledge of the signaling mechanisms driven by VEGF-D is still evolving—here we explore the biology of VEGF-D, its signaling mechanisms, and the clinical relevance of this growth factor.     

Generation of a syngeneic mouse model to study the effects of vascular endothelial growth factor in ovarian carcinoma

Vascular endothelial growth factor (VEGF) performs multifaceted functions in the tumor microenvironment promoting angiogenesis, suppressing anti-tumor immune response, and possibly exerting autocrine functions on tumor cells. However, appropriate syngeneic animal models for in vivo studies are lacking. Using retroviral transfection and fluorescence-activated cell sorting, we generated a C57BL6 murine ovarian carcinoma cell line that stably overexpresses the murine VEGF164 isoform and the enhanced green fluorescent protein. VEGF164 overexpression dramatically accelerated tumor growth and ascites formation, significantly enhanced tumor angiogenesis, and substantially promoted the survival of tumor cells in vivo. In vitro, VEGF164 overexpression significantly enhanced cell survival after growth factor withdrawal and conferred resistance to apoptosis induced by cis-platin through an autocrine mechanism. VEGF/green fluorescent protein-expressing tumors were not recognized by the adaptive immune system. After vaccination, a specific anti-tumor T-cell response was detected, but tumor growth was not inhibited. This engineered murine carcinoma model should prove useful in the investigation of the role of VEGF in modulating the tumor microenvironment and affecting the complex interactions among angiogenesis mechanisms, anti-tumor immune mechanisms, and tumor cell behavior at the natural state or during therapy in ovarian carcinoma.     

Antiangiogenic properties of silver nanoparticles

Angiogenesis is an important phenomenon involved in normal growth and wound healing processes. An imbalance of the growth factors involved in this process, however, causes the acceleration of several diseases including malignant, ocular, and inflammatory diseases. Inhibiting angiogenesis through interfering in its pathway is a promising methodology to hinder the progression of these diseases. The function and mechanism of silver nanoparticles (Ag-NPs) in angiogenesis have not been elucidated to date. PEDF is suggested to be a potent anti-angiogenic agent. In this study, we postulated that Ag-NPs might have the ability to inhibit angiogenesis, the pivotal step in tumor growth, invasiveness, and metastasis. We have demonstrated that Ag-NPs could also inhibit vascular endothelial growth factor (VEGF) induced cell proliferation, migration, and capillary-like tube formation of bovine retinal endothelial cells like PEDF. In addition, Ag-NPs effectively inhibited the formation of new blood microvessels induced by VEGF in the mouse Matrigel plug assay. To understand the underlying mechanism of Ag-NPs on the inhibitory effect of angiogenesis, we showed that Ag-NPs could inhibit the activation of PI3K/Akt. Together, our results indicate that Ag-NPs can act as an anti-angiogenic molecule by targeting the activation of PI3K/Akt signaling pathways.     

Notch signaling in glioblastoma: a developmental drug target?

Malignant gliomas are among the most devastating tumors for which conventional therapies have not significantly improved patient outcome. Despite advances in imaging, surgery, chemotherapy and radiotherapy, survival is still less than 2 years from diagnosis and more targeted therapies are urgently needed. Notch signaling is central to the normal and neoplastic development of the central nervous system, playing important roles in proliferation, differentiation, apoptosis and cancer stem cell regulation. Notch is also involved in the regulation response to hypoxia and angiogenesis, which are typical tumor and more specifically glioblastoma multiforme (GBM) features. Targeting Notch signaling is therefore a promising strategy for developing future therapies for the treatment of GBM. In this review we give an overview of the mechanisms of Notch signaling, its networking pathways in gliomas, and discuss its potential for designing novel therapeutic approaches.     

Non-receptor protein-tyrosine kinases as molecular targets for antiangiogenic therapy (Review)

Antiangiogenic therapy, including blockade of vascular endothelial growth factor (VEGF) signaling, was highly anticipated to improve the prognosis for patients with advanced cancers following the success of preclinical animal models. However, antiangiogenic monotherapy with VEGF antagonists has produced disappointing results in clinical trials to date. One of the reasons for this poor outcome is that angiogenesis is not solely regulated by VEGF. Inhibition of VEGF signaling, therefore, may select for tumor cell populations that stimulate angiogenesis through VEGF-independent pathways. Successful antiangiogenic therapy, therefore, may require simultaneous blockade of signaling downstream from multiple proangiogenic factor receptors. Recently, we found that non-receptor protein-tyrosine kinases, including members of the Src and Fes families, play vital roles in the responses of cultured endothelial cells to several proangiogenic factors. In this review, we summarize the contributions of these kinase families to angiogenic pathways in endothelial cells, and discuss the potential of these kinases as new targets for antiangiogenic drug discovery.     

Correlation between vascular endothelial growth factor,angiogenesis, and tumor-associated macrophages in invasive ductal breast carcinoma

Angiogenic and anti-angiogenic factors, secreted by tumor, inflammatory, and stromal cells play an important role in regulation of neovascularization. Among the most important of these is vascular endothelial growth factor (VEGF), a specific mitogen for endothelium, which increases vascular permeability and induces proteolytic enzymes necessary for vascular remodeling. Tumor-associated macrophages (TAMs) can express complex functions related to tumor biology, including growth, proliferative rate, stroma formation and dissolution, and neovascularization. The aim of this study was to define, using immunohistochemical analysis, the microvessel density (MVD), VEGF expression, and TAMs level in 97 human invasive ductal breast carcinomas not otherwise specified (NOS), investigate a possible relationship between them and then correlate their values with tumor grade, mitotic activity index (MAI), tumor size and lymph-node status. Statistical analysis showed a strong positive relationship between MVD and VEGF expression ( P<0.001). Furthermore, both MVD and VEGF expression were significantly correlated with tumor grade and lymph-node status, and TAMs infiltration with MAI. TAM level showed a significant positive connection with VEGF expression and MVD. These in situ observations suggest that VEGF stimulates angiogenesis in human invasive ductal breast carcinoma NOS and attracts macrophages to the tumor locus, which then may be involved in angiogenesis promotion. The expression of this angiogenic molecule, and MVD and TAM level, can provide additional prognostic significance and help in the identification of patients who need postoperative adjuvant therapy.     

Correlation between tumor vascularity, vascular endothelial growth factor production by tumor cells, serum vascular endothelial growth factor levels, and serum angiogenic activity in patients with breast carcinoma.

Angiogenesis is an important prognostic factor in invasive breast carcinoma. We analyzed sera and tumor samples from 36 patients with primary breast carcinomas to determine the relationship between tumor vascularity, vascular endothelial growth factor (VEGF) production by tumor cells, levels of circulating VEGF (measured by ELISA assay), and levels of endothelial growth factors analyzed by a functional test of human umbilical vein endothelial cells (HUVEC) proliferation. Tumor vascularity was correlated directly with VEGF production by the tumor, indicating that VEGF production is a relevant factor in determining angiogenesis in primary tumor. No correlation was found either between the number of vessels in the tumor or the production of VEGF by tumor cells and the levels of serum angiogenic factors including VEGF. On the contrary, the two serum tests correlated together because a high serum level of VEGF is more frequent in cases with the presence of HUVEC-stimulating growth factors. These data indicate that the principal source of factors stimulating angiogenesis in the primary tumor is the tumor itself. This is an important issue in the context of anti-angiogenic therapeutic approaches, which should be planned to interfere with tumor production of angiogenic factors rather than with circulating angiogenic factors. In conclusion, whereas the vessel count and VEGF production by tumor cells are parameters that give direct information on tumor angiogenesis, long-term follow-up is necessary to determine the clinical significance of the determination of serum HUVEC-stimulating factors in the progression of breast carcinoma.     

Pro-Angiogenic Cytokines as Cardiovascular Therapeutics

Coronary artery and peripheral vascular disease are global health concerns with limited therapies. Currently available medical and surgical therapies for these disease processes are highly effective for only a fraction of patients. Extensive effort has been devoted to finding molecular therapies to enhance perfusion and function of ischemic myocardial and peripheral skeletal muscle. Angiogenic cytokines (fibroblast growth factor [FGF], vascular endothelial growth factor [VEGF], hepatocyte growth factor [HGF], placental growth factor, stromal cell-derived factor-1alpha) have shown theoretical and experimental promise in upregulating endogenous endothelial progenitor cell-mediated angiogenesis. Preliminary clinical trials have suggested improvements in myocardial and peripheral perfusion following therapy with FGF, VEGF, and HGF. Further studies on the efficacy of cytokine-mediated angiogenesis are required before widespread clinical application is possible. Investigation into adjunctive cytokine therapies for myocardial and peripheral muscle ischemia is warranted. Based on experimental evidence, appropriate angiogenic cytokine therapy should provide benefits in both perfusion and hemodynamic function.