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Google Scholar OpenURL Placeholder Text. Dominant-negative inhibition of Flk-1 suppresses the growth of many tumor types in vivo. Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant.

Viral vector-mediated transduction of a modified platelet factor 4 cDNA inhibits angiogenesis and tumor growth. Retroviral and adenoviral mediated transduction of angiostatin cDNA inhibits angiogenesis and tumor growth.

Transfection of thrombospondin 1 complementary DNA into a human breast carcinoma cell line reduces primary tumor growth, metastatic potential, and angiogenesis. Expression of vascular permeability factor vascular endothelial growth factor by epidermal keratinocytes during wound healing.

Vascular endothelial growth factor messenger ribonucleic acid expression in the primate ovary. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis.

Upregulation of vascular endothelial growth factor expression induced by myocardial ischaemia: implications for coronary angiogenesis. Vascular permeability factor gene expression in normal and neoplastic human ovaries. Regulation of vascular endothelial growth factor expression in cultured keratinocytes.

Implications for normal and impaired wound healing. VEGF gene expression is upregulated in electrically stimulated rat skeletal muscle. Hypoxia-induced paracrine regulation of vascular endothelial growth factor receptor expression. Modulation of gene expression by nitric oxide.

Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets.

Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells. Critical components of the female reproductive pathway are suppressed by the angiogenesis inhibitor AGM In vivo adenovirus-mediated gene transfer of the Escherichia coli cytosine deaminase gene to human colon carcinoma-derived tumors induces chemosensitivity to 5-fluorocytosine.

Regional delivery of an adenovirus vector containing the Escherichia coli cytosine deaminase gene to provide local activation of 5-fluorocytosine to suppress the growth of colon carcinoma metastatic to liver.

Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase.

Tumors expressing the cytosine deaminase suicide gene can be eliminated in vivo with 5-fluorocytosine and induce protective immunity to wild type tumor. Parenteral gene therapy with p53 inhibits human breast tumors in vivo through a bystander mechanism without evidence of toxicity.

Molecular and biological properties of the vascular endothelial growth factor family of proteins. Vascular endothelial growth factor, a potent and selective angiogenic agent. The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA.

The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. The vascular endothelial growth factor VEGF isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF.

Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Expression of vascular permeability factor vascular endothelial growth factor and its receptors in adenocarcinomas of the gastrointestinal tract.

Expression of vascular permeability factor vascular endothelial growth factor and its receptors in breast cancer. Similarities between the oxygen-sensing mechanisms regulating the expression of vascular endothelial growth factor and erythropoietin. Hypoxic stimulation of vascular endothelial growth factor expression in vitro and in vivo.

Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene flt closely related to the fms family. A receptor tyrosine kinase cDNA isolated from a population of enriched primitive hematopoietic cells and exhibiting close genetic linkage to c-kit.

The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. The binding of vascular endothelial growth factor to its receptors is dependent on cell surfaceassociated heparin-like molecules.

Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor. High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis.

Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor.

Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Selective binding of VEGF to one of the three vascular endothelial growth factor receptors of vascular endothelial cells.

Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Vascular permeability factor vascular endothelial growth factor in guinea pig and human tumor and inflammatory effusions. The fibroblast growth factor family: structural and biological properties.

Localization of basic fibroblast growth factor and vascular endothelial growth factor in human glial neoplasms. Human basic fibroblast growth factor gene encodes four polypeptides: three initiate translation from non-AUG codons.

Increased levels of fibroblast growth factor-like activity in urine from patients with bladder or kidney cancer. Increased serum levels of basic fibroblast growth factor in patients with renal cell carcinoma. Release of basic fibroblast growth factor, an angiogenic factor devoid of secretory signal sequence: a trivial phenomenon or a novel secretion mechanism?

Endothelial cell-derived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix.

Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene.

Platelet factor-4 inhibits the mitogenic activity of VEGF and VEGF using several concurrent mechanisms. Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides.

Growth inhibition of murine melanoma and human colon carcinoma by recombinant human platelet factor 4. Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor.

Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR. Angiostatin induces and sustains dormancy of human primary tumors in mice. A recombinant human angiostatin protein inhibits experimental primary and metastatic cancer.

In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase. Successful retroviral mediated transduction of a reporter gene in human dendritic cells: feasibility of therapy with gene-modified antigen presenting cells.

Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo. Virus-mediated delivery of antigenic epitopes into dendritic cells as a means to induce CTL.

Therapeutic antitumor response after immunization with a recombinant adenovirus encoding a model tumor-associated antigen.

Human dendritic cells genetically engineered to express high levels of the human epithelial tumor antigen mucin MUC Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells. Retroviral transduction of human dendritic cells with a tumor-associated antigen gene.

Active immunotherapy of cancer with a nonreplicating recombinant fowlpox virus encoding a model tumor-associated antigen.

Identification and characterization of the blood vessels of solid tumors that are leaky to circulating macromolecules. Adenovirus interaction with distinct integrins mediates separate events in cell entry and gene delivery to hematopoietic cells.

Upregulation of integrins alpha v beta 3 and alpha v beta 5 on human monocytes and T lymphocytes facilitates adenovirus-mediated gene delivery. Integrin alpha v beta 5 selectively promotes adenovirus mediated cell membrane permeabilization.

Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment. E-selectin is present in proliferating endothelial cells in human hemangiomas. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor VEGF using soluble VEGF-receptor chimeric proteins.

Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Regulation by vascular endothelial growth factor of human colon cancer tumorigenesis in a mouse model of experimental liver metastasis.

Suppression of solid tumor growth by immunoneutralizing monoclonal antibody against human basic fibroblast growth factor. Suramin: a novel growth factor antagonist with activity in hormone-refractory metastatic prostate cancer.

Suramin, an anticancer and angiosuppressive agent, inhibits endothelial cell binding of basic fibroblast growth factor, migration, proliferation, and induction of urokinase-type plasminogen activator. Antitumor effects of an antiangiogenic polysaccharide from an Arthrobacter species with or without a steroid.

Administration of pentosan polysulfate to patients with human immunodeficiency virus-associated Kaposi's sarcoma. A synthetic matrix metalloproteinase inhibitor decreases tumor burden and prolongs survival of mice bearing human ovarian carcinoma xenografts. A new class of steroids inhibits angiogenesis in the presence of heparin or a heparin fragment.

Tumor infarction in mice by antibody-directed targeting of tissue factor to tumor vasculature. Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumour growth. Potent anti-angiogenic action of AGM comparison to the fumagillin parent.

Antiangiogenic therapy of transgenic mice impairs de novo tumor growth. Prevention of hepatic metastasis of human colon cancer by angiogenesis inhibitor TNP Angiogenesis inhibitor TNP AGM potently inhibits the tumor growth of hormone-independent human breast and prostate carcinoma cell lines.

Inhibition of angiogenesis by interferons: effects on tumor- and lymphocyte-induced vascular responses. Interferons alpha and beta down-regulate the expression of basic fibroblast growth factor in human carcinomas. Treatment of pulmonary hemangiomatosis with recombinant interferon alfa-2a.

Interleukin inhibits angiogenesis induced by human tumor cell lines in vivo. In vivo efficacy of a novel inhibitor of selected signal transduction pathways including calcium, arachidonate, and inositol phosphates.

Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Inhibition of squamous cell carcinoma angiogenesis by direct interaction of retinoic acid with endothelial cells.

Inhibition of human endothelial cell proliferation in vitro and neovascularization in vivo by D-penicillamine. Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P.

Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells. Identification of EGF as an angiogenic factor present in conditioned medium from human salivary gland adenocarcinoma cell clones with varying degrees of metastatic potential.

Transforming growth factoralpha: a more potent angiogenic mediator than epidermal growth factor. Estrogen promotes angiogenic activity in human umbilical vein endothelial cells in vitro and in a murine model. In vitro and in vivo activation of endothelial cells by colony-stimulating factors.

Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. Inhibition of interleukin 8 attenuates angiogenesis in bronchogenic carcinoma. Leukotrienes C4 and D4 promote angiogenesis via a receptor-mediated interaction. Placenta growth factor.

Identification of angiogenic activity and the cloning and expression of platelet-derived endothelial cell growth factor. Migratory and proliferative effect of platelet-derived growth factor in rabbit retinal endothelial cells: evidence of an autocrine pathway of platelet-derived growth factor.

Induction of vascular endothelial growth factor expression by prostaglandin E2 and E1 in osteoblasts. Transforming growth factor type beta: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro.

Tumor necrosis factor type alpha, a potent inhibitor of endothelial cell growth in vitro , is angiogenic in vivo. Macrophage-induced angiogenesis is mediated by tumour necrosis factor-alpha.

Interferon-gamma-inducible protein 10 IP is an angiostatic factor that inhibits human non-small cell lung cancer NSCLC tumorigenesis and spontaneous metastases.

Human interferon-inducible protein 10 is a potent inhibitor of angiogenesis in vivo. The kilodalton N-terminal fragment of human prolactin is a potent inhibitor of angiogenesis. The 16K fragment of prolactin specifically inhibits basal or fibroblast growth factor stimulated growth of capillary endothelial cells.

The role of matrix metalloproteases and their inhibitors in tumour invasion, metastasis and angiogenesis. Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Phase I clinical and pharmacokinetic study of oral carboxyamidotriazole, a signal transduction inhibitor.

The endogenous oestrogen metabolite 2-methoxyoestradiol inhibits angiogenesis and suppresses tumour growth. Download all slides.

Views More metrics information. Total Views Email alerts Article activity alert. Advance article alerts. New issue alert. Receive exclusive offers and updates from Oxford Academic. Citing articles via Google Scholar. Latest Most Read Most Cited Cancer risks among first-degree relatives of women with a genetic predisposition to breast cancer.

Outdoor air pollution exposure and uterine cancer incidence in the sister study. Cancer cells begin the angiogenesis process by sending signals to nearby tissue and activating growth factors that allow the tumor to form new blood vessels.

One such molecule is called vascular endothelial growth factor, or VEGF. Researchers developed drugs called angiogenesis inhibitors, or anti-angiogenic therapy, to disrupt the growth process. These drugs search out and bind themselves to VEGF molecules, which prohibits them from activating receptors on endothelial cells inside blood vessels.

Bevacizumab Avastin ® works in this manner. It is used to treat glioblastoma and cancers of the lung , kidney , breast , colon and rectum. Other angiogenesis inhibitor drugs work on a different part of the process, by stopping VEGF receptors from sending signals to blood vessel cells.

These drugs are known as tyrosine kinase inhibitors TKI. Sunitinib Sutent ® is an example of a tyrosine kinase inhibitor. For this reason, these drugs are typically used in combination with chemotherapy or other treatments. Angiogenesis inhibitors are particularly effective for treating liver cancer , kidney cancer and neuroendocrine tumors.

Since they act on blood vessel formation and not the tumor itself, the side effects of angiogenesis inhibitors are different than traditional chemotherapy drugs. My Chart. Donate Today. Request an Appointment Request an Appointment New Patients Current Patients Referring Physicians.

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Approved angiogenesis inhibitors include:. Side effects of treatment with VEGF-targeting angiogenesis inhibitors can include hemorrhage , clots in the arteries with resultant stroke or heart attack , hypertension , impaired wound healing, reversible posterior leukoencephalopathy syndrome a brain disorder , and protein in the urine.

Gastrointestinal perforation and fistulas also appear to be rare side effects of some angiogenesis inhibitors. Antiangiogenesis agents that target the VEGF receptor have additional side effects, including fatigue, diarrhea, biochemical hypothyroidism , hand-foot syndrome , cardiac failure, and hair changes.

Home About Cancer Cancer Treatment Types of Cancer Treatment Immunotherapy Angiogenesis Inhibitors. Angiogenesis Inhibitors On This Page What is angiogenesis? Why is angiogenesis important in cancer? How do angiogenesis inhibitors work?

What angiogenesis inhibitors are being used to treat cancer in humans? Do angiogenesis inhibitors have side effects? What is angiogenesis? Approved angiogenesis inhibitors include: Axitinib Inlyta® Bevacizumab Avastin® Cabozantinib Cometriq® Everolimus Afinitor® Lenalidomide Revlimid® Lenvatinib mesylate Lenvima® Pazopanib Votrient® Ramucirumab Cyramza® Regorafenib Stivarga® Sorafenib Nexavar® Sunitinib Sutent® Thalidomide Synovir, Thalomid® Vandetanib Caprelsa® Ziv-aflibercept Zaltrap®.

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Download Free PDF. Anti-angiogenic gene therapy of cancer: Current status and future prospects. Marika Crescenzi. See Full PDF Download PDF. Related Papers. Molecular Therapy Transcription Controlled Gene Therapy Against Tumor Angiogenesis. Download Free PDF View PDF.

Current Genomics Antiangiogenic Gene Therapy in Cancer. Journal of Clinical Investigation Transcription-controlled gene therapy against tumor angiogenesis. The Journal of Gene Medicine Lethality in an anti-angiogenic tumor gene therapy model upon constitutive but not inducible expression of the soluble vascular endothelial growth factor receptor 1.

Molecular Genetics and Metabolism Targeting Tumor Angiogenesis with Gene Therapy. Gene Therapy A gene therapy for cancer based on the angiogenesis inhibitor, vasostatin. British journal of cancer Systemic inhibition of tumour angiogenesis by endothelial cell-based gene therapy.

Tumori Journal Rationale, Problems and Perspectives in Anti-angiogenic Therapy. Cancer Gene Therapy Blood outgrowth endothelial cell-based systemic delivery of antiangiogenic gene therapy for solid tumors. Cancer Gene Therapy Evaluation of endostatin antiangiogenesis gene therapy in vitro and in vivo.

Molecular Aspects of Medicine 28 87— www. Pre-clinical studies have clearly indicated that these factors are essentially cytostatic and that they need long-term administration in order to obtain pro- longed anti-tumor effects, representing a rational basis for their delivery by a gene therapy approach.

E-mail address: stefano. indraccolo unipd. All rights reserved. Persano et al. Keywords: Angiogenesis; Cancer; Gene therapy; Interferon; Angiostatin; Endostatin Contents 1. Angiogenesis and gene therapy. Normal and tumor angiogenesis.

Innovative gene therapy approaches designed around molecular features of tumor angiogenesis. Potential advantages of anti-angiogenesis gene therapy approaches. Delivery of angiogenesis inhibitors by gene therapy.

Which vector for optimal treatment? Angiogenesis dependency of tumors: a critical issue to choose the appropriate therapeutic approach. Angiogenic therapy and metastasis inhibition. Future perspectives of anti-angiogenic gene therapy. Angiogenesis and gene therapy 1. Normal and tumor angiogenesis A prerequisite for tumour development, local invasion and metastatic dissemina- tion, angiogenesis, is a multistep process of new blood vessel formation from pre- existing vasculature that includes the activation, proliferation and migration of endothelial cells ECs , disruption of vascular basement membranes, remodelling of the extracellular matrix of tissues, formation of vascular tubes and networks, recruitment of supporting cells, including smooth muscle cells and pericytes, and connection to the pre-existing vascular network Kong and Crystal, Most physiological angiogenesis occurs in the embryo.

In adults the endothelium is in a quiescent state with only 0. Angiogenesis also underlies other pathological conditions such as rheumatoid arthritis, macular degeneration, psoriasis, atherosclerosis, restenosis, and diabetic retinopathy Webb and Vande Woude, Under normal circumstances, angiogenesis is a highly ordered process under tight regulation by both angiogenesis-inducing and angiogenesis-inhibiting factors Table 1.

These factors include soluble growth factors, such as vascular endothelial growth factor VEGF , angiopoietins Ang , fibroblast growth factor FGF , platelet- derived growth factor PDGF , transforming growth factor-b TGF-b , metallopro- teinases MMPs , and other membrane-associated molecules, such as integrins and cadherins Liu and Deisseroth, Other factors, such as interleukin-8 IL-8 , have a less established role in normal angiogenesis but are nevertheless important in tumor angiogenesis Yuan et al.

In addition to these molecules, mechanical forces mediated by blood flow on vascular endothelium contribute to the angiogen- esis process: in fact, vessels that are not perfused generally undergo regression Papetti and Herman, In Judah Folkman was the first to suggest that tumor growth is dependent on new blood vessel formation.

Meanwhile, it is widely recognized that angiogenesis is a critical process required by solid tumors to support their growth. Angiogenic factors produced by tumor cells are able to stimulate proliferation and migration of resident endothelial cells ECs ; they also recruit inflammatory cells that secrete proteases that degrade the extracellular matrix and mobilize circulating progenitor endothelial cells, that can contribute to the vascular network formation.

First, many tumors initially profit from cooption of the existing vasculature. However, tumor growth around blood vessels is generally followed by ECs apoptosis and loosening of vessel integrity mediated by the up-regulation of Ang2. At that point, almost invariably, the tumor induces neovascular sprouts from neighbouring vessels Holash et al.

Second, circulating haematopoietic precursors may contribute to the regulation of angiogenesis in tumors Asahara et al. Third, certain tumors may also attract differentiated haematopoietic cells, includ- ing mast cells, monocytes, macrophages, and inflammatory cells Coussens et al.

In general, most experimental attempts to interfere with the angiogenic switch have tried to reduce activation and recruitment of intratumoral EC, i. by increasing the production of anti-angiogenic factors which affect the response of EC to pro- angiogenic molecules or neutralizing biologically active pro-angiogenic factors.

Some recent strategies, however, aim to contrast recruitment of circulating precursor cells, or exploit the selective activation of angiogenesis-inducible promoters to target therapeutic genes into the tumor microenvironment.

Innovative gene therapy approaches designed around molecular features of tumor angiogenesis The therapeutic promise of angiogenesis research is based on the assumption that ECs are essentially a genetically stable cell population irrespective of the heteroge- neous mutational events affecting tumor cells Scappaticci, Moreover, since a single vessel can support the growth of many tumor cells, its destruction would impair the viability of many tumor cells, and, even more importantly, the same anti-angiogenic molecule could possibly be used to treat different tumors.

Finally, the ECs are easily accessible by the therapeutic agents injected intravenously Liu and Deisseroth, On the other hand, the tumor vasculature is abnormal and ECs that form tumor blood vessels differ structurally and phenotypically from the normal vasculature: tumor vessels have a great permeability and lack functional pericytes in many areas; EC proliferation is detected.

These vessels are also characterized by the presence of fenestrae, transcellular holes and the loss of the basement membrane Carmeliet et al. Some known features of tumor-associated blood vessels have been evaluated to target anti-angiogenic drugs Sedlacek, ; Tandle et al.

Among the peptides that are known to home to the tumor vasculature, many contain RGD and NGR sequence motifs, whose receptors have been identified.

For example, RGD-contain- ing peptides bind to avb3 and avb5 integrins, whereas peptides with NGR sequence motifs bind to aminopeptidase N protein. Both receptors were shown to be critical for angiogenesis Brooks et al. Meanwhile, some progress has been made at the level of the transcriptional targeting of the vectors and we will shortly comment two innovative approaches which exploit either hypoxia-response elements or the cell type-specific Tie2 gene reg- ulatory sequences to switch on expression of therapeutic genes in the tumor microenvironment.

Hypoxia is an intrinsic feature of solid tumors and is a common cause of treat- ment failure. Since the first observation of Thomlinson and Gray , hypoxia in tumors has been consistently documented. Low oxygen levels stimulate a range of pro-angiogenic factors, thus suggesting that hypoxia may contribute to tumor vas- cularization and metastatic spread.

Rather than designing therapeutic strategies to overcome hypoxic resistance, a number of groups have chosen to exploit the hypoxic nature of tumors to gain a therapeutic advantage.

This has led to the development of hypoxia-selective agents i. Hypoxia inducible factor 1 HIF-1 has demonstrated oxygen-responsiveness and is responsible for the regulation of oxy- gen-responsive genes.

HIF-1 is a heterodimer consisting of a and b subunits, with the resulting protein being post-translationally stabilized in response to hypoxia Jaakkola et al. The most common genes up-regulated include: erythropoietin Epo Goldberg et al. For example, Shibata and colleagues created constructs with five copies of HREs derived from the promoter sequence of the VEGF gene in com- bination with a CMV minimal promoter Shibata et al.

These promoters have been used in vectors containing a bacterial nitroreductase gene NTR able to activate an anticancer pro-drug.

Significant anti-tumor effects were achieved by the intra-peritoneal injection of the pro-drug in a model of human fibrosar- coma HT Shibata et al. Finally, some of these hypoxia-specific expression systems have been tested on brain tumors; Ruan et al.

produced a recombinant adeno- associated virus rAAV in which the pro-apoptotic gene bax can be regulated in hypoxic conditions by inserting nine HREs from the Epo gene in the promoter sequence Ruan et al. Tie2-based vectors work in a different way.

Tie2 is a vascular EC-specific receptor tyrosine kinase essential for the regulation of vascular network formation and the remodelling for its binding to the angiopoietins. In this study, tumors recruited a population of bone marrow-derived mononuclear cells that expressed a distinctive combination of surface markers CD45, CD11b, Tie2, Sca-1, and CD34 , and were closely associated, albeit not directly incorporated, with blood vessels.

Moreover, the finding that selective elimination of these cells dramatically impaired tumor growth and angiogenesis demonstrated their involvement in the reg- ulation of tumor angiogenesis De Palma et al. Potential advantages of anti-angiogenesis gene therapy approaches Gene therapy based strategies of angiogenesis inhibition have also attracted inter- est due to their potential advantages compared with conventional modalities of administration of anti-angiogenic drugs.

First of all, since effective suppression of pathological angiogenesis may eventually require chronic treatment, gene therapy strategies could be useful to achieve selective delivery to affected tissues and pro- longed expression of the therapeutic agents Scappaticci, Gene therapy also represents a method for circumventing the production problems of many recombi- nant proteins including their stability and solubility; adequate production of anti-angiogenic factors by recombinant engineering methods has been sometimes problematic see angiostatin and may limit their clinical application.

Gene transfer usage allows the correct folding of proteic agents and their stability in vivo since they are assembled in their physiologic environment Sedlacek, ; Tanaka et al.

A particularly attractive feature of gene transfer is the possibility of targeting gene delivery to selective tissues, thus achieving localized gene expression and high regio- nal drug concentrations without increasing the systemic levels of the therapeutic agents. Although this approach is valuable only for treatment of localized tumors, such as SNC tumors and possibly ovarian cancer, the confinement of the anti-angio- genic effects may avoid undesirable suppression of essential physiologic functions Kong and Crystal, and thus reduce side effects.

Delivery of angiogenesis inhibitors by gene therapy Extensive research has led to the identification of several negative regulators of angiogenesis see Table 1 , which may act either directly to inhibit cellular functions required for the angiogenic process, including EC proliferation or migration, or, alternatively, behave as indirect inhibitors through suppression of the production of angiogenic factors by tumor cells Abdollahi et al.

Identified in very elegant studies, they exert pivotal angiostatic activities in the regulation of tumor angiogenesis. In general, these proteins are good candidates for gene delivery, since they are produced physiologically, probably being not immunogenic, and are effective extracellularly.

A comprehensive list of endogenous inhibitors of angiogenesis delivered by gene trans- fer is given in Table 2. As a detailed review of all these factors is outside the aims of this review, we will comment in detail only the results obtained with representative inhibitors, which have often been delivered by gene therapy.

Interferons The discovery in of a link between interferon alpha IFN-a and endothelial cell damage was the first step to its subsequent characterization as an angiogenesis inhibitor Folkman, The IFN family consists of three major glycoproteins, IFN-a, IFN-b, and IFN-c.

Although IFNs were first known as antiviral agents, it was subsequently clear that they modulate various cellular functions, including cell growth and differentiation, oncogene expression, and that they regulate various aspects of the immune response.

Since , IFN-a has been used to treat both hae- matological malignancies and certain solid tumors, with contrasting evidence of ther- apeutic activity. Overall, the mechanisms underlying the therapeutic effects are complex and the reasons why only certain tumors are sensitive to IFN-a treatment are not yet fully understood.

In particular, IFN-a angiostatic activity has been asso- ciated to both down-regulation of bFGF and VEGF levels Singh et al. Angiostatin inhibits EC migration and proliferation most likely through its ability to bind a cell surface ATP synthase, which may act as a receptor Moser et al.

Other studies revealed that angiostatin may induce apoptosis in EC, albeit at relatively high concentrations Claesson-Welsh et al. Several approaches have been developed to translate the basic science behind angiostatin into therapeutic efficacy.

Initial efforts aimed at the generation of recom- binant angiostatin using the Pichia pastoris expression system Sim et al. Moreover, production of large amounts of active angiostatin turn out to be expen- sive and technical problems were frequently encountered.

An attractive alternative has been represented by gene delivery. Expression of angiostatin has been obtained in tumor models both by transfecting angiostatin- expression plasmids Matsumoto et al.

All these studies report significant inhibition of tumor growth and micro- vessel density in various tumor models, as discussed in detail later on. However, Joseph et al. These data may underscore that angiostatin monotherapy could show efficacy only on certain tumor types, although factors which could predict therapeutic response remain unknown.

Endostatin Endostatin is an endogenous angiogenesis inhibitor identified as a 20 kDa C-ter- minal fragment of collagen XVIII, and isolated from conditioned media of cultured hemangioendothelioma EOMA cells; for endostatin a functional receptor on vas- cular endothelium is integrin a5b1 Sudhakar et al.

Endostatin is stored in platelets and is released, for example, after administration of COX-2 inhibitors to animals. The affinity of endostatin for platelets could have clinically relevant consequences; in fact, if exog- enous endostatin is also sequestered in platelets after administration, and circulating platelets have to be saturated before plasma endostatin reaches therapeutic levels, than it may be useful to start treatment with a loading dose of endostatin or to add COX-2 inhibitors to the regimen Folkman, Presently, phase I clinical tri- als of endostatin as a fusion protein infusion for various cancers are underway.

The gene encoding endostatin is somehow an ideal candidate for gene therapy because the recombinant protein appears to be safe when delivered to the patient for a long time and yet is difficult to produce in large amounts.

A comprehensive descrip- tion of the results of endostatin therapy was recently published by Folkman One study Jin et al. In the first case, a block in tumor growth by suppression of tumor angiogenesis in vivo was obtained.

The second type of tumor, however, was not influ- enced by endostatin gene therapy, probably due to the different sensitivity of blood vessels to endostatin itself; thus tumor-associated blood vessels could differ among different tumors, but which features may render them responsive to endostatin remain largely elusive.

Our group investigated the efficacy of endostatin gene therapy in the treatment of breast cancer using retroviral vectors Indraccolo et al.

Again, heteroge- neous therapeutic effects were noticed, albeit comparable expression levels were obtained; a possible explanation for these findings was that endostatin may not be enough to starve rapidly growing tumors, although this hypothesis was challenged by Folkman subsequently Kerbel and Folkman, All these studies addressed the effects of endostatin gene therapy mainly on the prevention of tumor growth, but did not focus on its effects on established tumors.

Li et al. They showed a significant reduction of tumor growth and microvessel formation, resulting in a prolonged median survival rate of NPC metas- tasis-bearing mice.

These results, however, represent the exception rather than the rule. A second series of studies analyzed the combined effect of angiostatin and endo- statin and found a strong anti-proliferative effect of rAAV-mediated angiostatin and endostatin gene transfer in EC in vitro and significant anti-tumor effects in ovarian cancer models Isayeva et al.

Raikwar et al. Treatment resulted in enhanced inhibition and complete regression of both the treated and untreated tumors on the other flank of the mice in a vast majority of animals. Although these studies suggest that long-term expression of both inhibitors may be effective as a preventive approach against recurrent and metastasis cancer, regression of established tumors even by the combined treatment remains elusive.

Vasostatin has been delivered by gene therapy in sev- eral studies. Initially, it was observed that multiple intramuscular injections of plas- mid DNA delayed tumor growth in fibrosarcoma and Lewis lung carcinoma models Pike et al.

Interestingly, plasma levels of vasostatin in these experiments were in the nanomolar range, which may indicate an unusual potency of this inhibitor. More recently, these results were confirmed and extended by Li et al. However, the finding that gene transfer of vasostatin into neuroendocrine tumor cells resulted in enhanced malignant behaviour Liu et al.

Most of the currently available viral vectors, including adenoviral, adeno-associ- ated, retroviral, lentiviral, and herpes simplex viral vectors, have been exploited for tumour vascular targeting approaches. All these vector types can be routinely man- ufactured and purified to high titres, remaining stable and storable for long periods of time.

Adenoviral vectors Ads are characterized by high gene transfer efficiency and high levels of transgene expression; furthermore, they can infect both dividing and non-dividing cells. However, some drawbacks include the transient expression of the transgene, the existence or rapid induction of neutralizing antibody in humans and the inflammatory and immune response elicited by the vector itself Liu and Deisseroth, Beyond their exploitation to deliver single anti-angiogenic factors such as angiostatin or endostatin, summarized below, it is worthwhile to consider two recent developments which may lead to an improvement of therapeutic efficacy.

Jin et al. Their novel strategy combines tumor cell-specific killing using a conditionally replicating oncolytic virus, termed Ad-hOC-E1, with tumor endothelium-specific targeting using an Ad vector secreting Flk1-Fc, a soluble form of the VEGF receptor Flk1 fused to the Fc domain of immu- noglobulins, which behaves as a VEGF-neutralizing agent.

In other studies, Ads were used to obtain sustained expression of the early growth response protein-1 EGR-1 in EC Lucerna et al. EGR-1 is involved in responses to many different growth factors and environmental stresses, such as hypoxia.

It is broadly expressed and it controls the expression of a wide variety of genes involved in angiogenesis and tumorigenesis Fahmy et al.

In their work, Lucerna et al. showed that EGR-1 induces potent feedback inhibitory mechanisms, including strong up-regulation of transcriptional repressors, negative cell cycle check point effectors and proteins with anti-angiogenic activities.

In conclusion, gene therapy-based expression of EGR-1 may represent a new approach in cancer treatment since it seems to induce multiple pathways of anti-angiogenesis, growth arrest and induction of apoptosis in proliferating cells Lucerna et al.

This integrated effect could possibly skip resistance to therapy, which is increasingly encountered when a single angiogenic factor is blocked, due to the emergence of clones which utilize redundant pathways to induce angiogenesis.

Adeno-associated viral vectors AAV have also been considered for anti-angio- genic gene therapy approaches. These viral vectors are single-strand DNA, replica- tion-defective viruses.

In the absence of a helper virus like the adenovirus, the wild-type AAV genome tends to integrate into the host-cell chromosomal DNA to maintain a latent state.

In the presence of the helper virus, however, the AAV gen- ome replicates episomally. Similarly to Ads, AAV can also infect both dividing and non dividing cells, however they generate low-level humoral immune responses. Xu et al. engineered AAV to express mouse angiostatin. After intraportal delivery of the vector, high-level and stable expression of angiostatin lasting 6 months was observed in hepatocytes.

Furthermore, treatment led to significant suppression of the growth of EL-4 lymphoma tumors in the liver and prolonged survival time of the mice. The growth of neovessels was inhibited significantly and extensive apoptosis of tumor cells was observed.

The AAV-angiostatin viruses did not appear to be toxic to mice, and there was no detectable apoptosis of hepatocytes. These results warrant future investigation especially as a means to target otherwise unresectable liver metastases after surgical removal of primary tumors Xu et al.

Production of large amounts of clinical grade AAV has remained elusive so far, thus limiting their use in patients. Lentiviral vectors LV have also been used to deliver genes involved in the con- trol of angiogenesis, because they can achieve permanent integration of the therapeu- tic gene into non-dividing cells.

LV can be pseudotyped with a variety of envelopes, achieving a broad trophism Shichinohe et al. However, transduction effi- ciency for ECs is relatively low and significant vector-associated cytotoxicity has been noticed, possibly due to the VSV-G envelope Pfeifer et al.

Shichinohe and coworkers constructed recombinant LV expressing angio- statin and endostatin and stably transduced T24 human bladder cancer cells, a tumor model relatively resistant to adenoviruses infection due to loss of the coxsac- kievirus-adenovirus receptor expression.

Long-term expression and secretion of these angiogenesis inhibitors resulted in significant inhibition of cellular proliferation of co-cultured EC. In our experience, IFN-a-encoding lentiviral vectors injected i. in ovarian can- cer-bearing mice provided long-term local production of IFN-a associated with remarkable anti-tumor activity.

Therapeutic effects, however, were only seen in microscopic tumors, thus indicating that angiogenesis inhibition is likely to repre- sent a valuable early intervention strategy in ovarian cancer Indraccolo et al. Clearly, arresting tumor angiogenesis without interfering with physiological angi- ogenesis represents a formidable challenge for current gene transfer vectors.

Angiogenesis dependency of tumors: a critical issue to choose the appropriate therapeutic approach Although it is recognized that angiogenesis is involved in the progressive growth of virtually all tumor types, marked variations in the intensity of the angiogenic response among different histotypes have been observed Eberhard et al.

Malignant gliomas, the most common type of primary brain tumors Louis et al. In these tumors, microvessels characteristically loose their normal blood—brain barrier properties and leak fluid into the brain.

A cerebral oedema is the common conse- quence. The degree of vascularization has been correlated with their prognosis, which remains dismal, a fact which has remained unchanged over the last decades despite the employment of multimodal therapeutic approaches.

Due to the dramatic activation of angiogenesis in these tumors, angiogenesis inhibitors may have therapeutic efficacy, especially in combination with other estab- lished drugs. Several studies attempted to apply viral vector-mediated gene delivery of angiogenic inhibitors to malignant gliomas in pre-clinical models.

Studies using Ad carrying the angiostatin gene demonstrated a strong inhibition of tumor growth after a single injection into small tumors established from C6 rat glioma cells Gris- celli et al. AAV have also been considered for their ability to penetrate solid tumors better than Ad.

Ma et al. A single i. injec- tion of vector before implantation of malignant glioma cells prevented tumor growth in nude mice: angiostatin appeared to force tumor cells into a dormant state by inducing apoptosis both in endothelial and in glioma cells.

Peroulis et al. Their results showed that locally produced endostatin had an anti-prolifera- tive effect on ECs and inhibited both angiogenesis and tumor growth in vivo. These promising results were extended by Ohlfest et al. who delivered an angiostatin—endo- statin fusion gene statin-AE and a soluble vascular endothelial growth factor recep- tor sFlt-1 into human glioblastoma xenografts by non viral gene transfer Ohlfest et al.

Therapeutic effects of VEGF neutralization have also been shown by Davidoff, who evaluated gene therapy-mediated expression of soluble flk-1 from NXS2 neuroblastoma cells Davidoff et al.

On the other hand, when considering survival, other groups have failed to show anti-tumor effects following angiostatin or endostatin gene therapy in glioma models De Bouard et al. The reasons for this discrepancy between results obtained in different preclinical studies could be related to differences in the features of the vasculature among experimentally induced tumors in animals or in the quantity of the anti-angiogenic factors produced.

These findings, which remark the anti- angiogenic activity of type I interferons, were subsequently reinforced by Streck et al. Finally, although pre-clinical work on CNS tumors has been useful for highlight- ing the most potent angiogenesis inhibitors, it has also raised a serious concern.

Although the extension in the survival of treated animals is an unquestionable indi- cator of benefit from anti-angiogenic therapy, further work will certainly be required to investigate this issue in detail, in order to establish whether selection of an invasive phenotype may represent an escape mechanism from angiogenesis inhibition.

Angiogenic therapy and metastasis inhibition In addition to its pivotal role in primary tumors, experimental and clinical evi- dence suggest that angiogenesis intervenes in the process of metastasis as well.

Increased microvessel density MVD correlates with progression, severity and prog- nosis of many tumor types. For example, prostate cancer progression from prostatic intraductal neoplasia PIN to poorly differentiated adenocarcinoma is associated with increased MVD Huss et al.

Therefore, angiogenesis appears to be necessary at the beginning as well as at the completion of the metastatic cascade. In experimental animals, tumor cells are rarely shed into the circulation before a primary tumor is vascularized, but they can appear in the circulation continuously thereafter.

The number of cells spread from the primary tumor as well as the number of lung metastasis seem to correlate with blood vessel density. Tumor cells can enter the circulation by penetrating through proliferating leaky capillaries that have frag- mented basement membranes.

Further, angiogenic factors, such as bFGF and VEGF, induce increased production of plasminogen activator PA and collagenases in proliferating endothelial cells, contributing further to degradation of basement membranes and facilitating the entry of tumor cells into the circulation.

However, therapeutic efficacy can be affected by differences in metastatic sites and by the presence or absence of the primary tumor. Moreover, therapeutic interventions targeted at met- astatic spread appear most feasible at the stage involving the appearance of mobile cancer cells or at the stage involving growth initiation of dormant cells or microme- tastasis Dimitroff et al.

Among the pre-clinical attempts in the metastatic setting, Sacco et al. The authors investigated the possibility of delivering anti-angiogenic genes via non-viral vectors represented by liposomes carrying DNAs coding for murine angiostatin, human angiostatin, human endo- statin, and the MMPs inhibitor human TIMP2, under the control of various promot- ers.

The vectors were injected i. in MMTV-neu females; subsequently their ability to inhibit tumor growth and metastasis was evaluated. The results showed that angiostatin delayed both primary tumor growth and metastatic dissemination. In a study of Mae et al. In fact unilateral pneumonectomy initiates a rapid and diffusive hyperplasia of the remaining lung cells.

In particular, there is indirect evidence that lung metastasis may grow more rapidly after pneumonectomy. An anti-angiogenic therapy could impair the growth of these metastasis. An Ad vec- tor expressing the extracellular portion of the flt-1 vascular endothelial growth factor receptor was used to achieve angiogenesis inhibition.

The vector was administered intratracheally after pneumonectomy; the results showed that the vector markedly suppressed metastasis outgrowth after pneumonectomy. Sun et al. The effect of intramuscular gene delivery of angio- statin, endostatin or their combination in suppressing secondary tumors after removal of primary tumors was evaluated.

Intramuscular delivery of angiostatin and endostatin, especially in combination, resulted in elevated expression levels of both anti-angiogenic factors in the circulation and suppressed secondary tumor growth by inhibiting tumors angiogenesis and inducing cell apoptosis.

Future perspectives of anti-angiogenic gene therapy Angiogenesis has been shown to play a pivotal role in the development and pro- gression of human malignancies and it may contribute to other molecular mecha- nisms involved in tumor progression, including regulation of cancer stem cell behaviour.

Selection of patients who could best profit from anti-angiogenic treatments is highly desirable but at the moment the field lacks robust and validated predictive markers. Moreover, while focusing therapeutic efforts on certain types of cancer known to be highly angiogenic, including glioma and renal cell carcinoma, is certainly valuable, it is currently not possible to discriminate among individuals with a given tumor type in order to select those who could attain prolonged benefit from anti-angiogenic treatments, and, yet, results from recent clinical trials with Avastin in colorectal cancer patients strongly indicate that a subset of good responders may in fact exists.

Thus, whatever the means of administration of anti-angiogenic factors — i. recombinant proteins versus gene therapy — a major challenge in the field will be to identify, beginning at the pre-clinical level, which molecular or perhaps metabolic features render tumor cells resistant or, more rather, sensitive to angiogenesis inhibition.

Moreover, as far as gene therapy is concerned, new studies will be required 1 to address the relative potency of the different anti-angiogenic factors available and 2 to examine in detail possible synergistic effects between various combinations of genes and between anti-angiogenic genes and conventional therapies, including radiotherapy, chemotherapy and, possibly, hormonal therapy.

In the meantime, there are lessons to be learned from experiments in mice as far as angiogenesis inhibition is concerned.

First, in general, anti-angiogenic factors are far more effective in preventing tumor growth than causing regression of established tumors; therefore, clinical trials with angiogenesis-interfering drugs in patients should be designed to take these results into consideration and possibly be per- formed in patients with minimal residual disease, rather than advanced patients.

Intriguingly, rapid, albeit transient, tumor regression is commonly observed when vasculotoxic agents, such as combretastatins or vascular targeting immunotoxins, are used Neri and Bicknell, ; Tozer et al.

Thus, it will certainly be inter- esting in the near future to combine a vasculotoxic drug with anti-angiogenic ther- apy, in order to achieve a durable control of established tumors. In fact, first reports on this matter are highly encouraging Kerbel, Is anti- angiogenic gene therapy by any means more effective than the apparently simple injection of anti-angiogenic factors?

Controversial questions, but what is certain is potential advantages of gene delivery systems for anti-angiogenic proteins, which include bypassing the production problems associated with large-scale production of anti-angiogenic factors and the possibility of obtaining sustained local production of a given factor.

On the other hand, information about comparative efficacy is lim- ited so far, and the few results available may diverge because of differences in distri- bution, half-life and specificity.

In conclusion, inhibition of angiogenesis presents an attractive possibility for treating cancer, also in view of its safety, but further preclin- ical studies are demanded to turn it into a successful therapeutic approach in patients.

Acknowledgements We are grateful to C. Case for help in the preparation of this manuscript. The Authors are supported in part by grants from Italian Association for Research on Cancer AIRC ; FIRB; Ministero della Salute, Ricerca Finalizzata e Programma Straordinario per la Ricerca Oncologica; Fondazione Cassa di Risparmio di Padova e Rovigo.

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Citation: Lupo G, Caporarello N, Olivieri M, Cristaldi M, Motta C, Bramanti V, Avola R, Salmeri M, Nicoletti F and Anfuso CD Anti-angiogenic Therapy in Cancer: Downsides and New Pivots for Precision Medicine.

Received: 04 October ; Accepted: 14 December ; Published: 06 January Copyright © Lupo, Caporarello, Olivieri, Cristaldi, Motta, Bramanti, Avola, Salmeri, Nicoletti and Anfuso. This is an open-access article distributed under the terms of the Creative Commons Attribution License CC BY.

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Experimental Pharmacology and Drug Discovery. This article is part of the Research Topic Precision Medicine: an Approach of Multiple Targeted Therapy in Cancer View all 15 articles. Anti-angiogenic Therapy in Cancer: Downsides and New Pivots for Precision Medicine. Gabriella Lupo Nunzia Caporarello Melania Olivieri Martina Cristaldi Carla Motta Vincenzo Bramanti Roberto Avola Mario Salmeri Ferdinando Nicoletti Carmelina D.

Cancer-Related Angiogenesis and Anti-Angiogenic Therapy The blood vessels supplying tumors are permeable, tortuous, heterogeneous in their morphological structure and efficiency of perfusion, and greatly different from those composing the normal vasculature.

TABLE 1. Tumor response to anti-angiogenic therapy. x PubMed Abstract CrossRef Full Text Google Scholar. CAN PubMed Abstract CrossRef Full Text Google Scholar. MCT PubMed Abstract CrossRef Full Text Google Scholar. CCR PubMed Abstract CrossRef Full Text Google Scholar.

An interesting concept in anti-angiogenic therapy is vascular normalization and re-distribution of flow in tumour vascular bed when anti-angiogenics are combined with the conventional chemotherapy regimen [ 32 ].

It has been suggested that normalising the tumour vasculature would diminish endothelial and perivascular cells, decrease the high interstitial pressures in solid tumours, enhance oxygenation and chemotherapy delivery into tumour cells [ 11 ].

Antiangiogenic agents do not achieve enough efficacy when they destroy tumour vascular networks as monotherapy but rather, by pruning tumour vascular networks when administered with other chemotherapeutics, they reduce vascular hydrostatic pressure, tumour-associated oedema and temporarily improve tumour hypoxia, thus improving delivery and activity of chemotherapeutics which can then effectively destroy tumour cells.

This has been demonstrated in colorectal cancers and glioblastoma multiforme [ 32 , 33 ]. Recently, a combination of bevacizumab with paclitaxel and carboplatin in patients with non-small cell lung cancer NSCLC has also shown improved survival [ 11 ]. In tumours, molecules involved in immune checkpoint e.

PD-1 interacts with its ligand, PD-L1 in immune and cancer stromal cells to inhibit the proliferation and survival of T cells which are important in immune surveillance of tumours [ 33 ]. Hijacking of PD-PD-L1 pathway activation by solid tumours leads to T cell exhaustion and increased expression of FoxP3 by regulatory T cells Tregs with resultant immunosuppression and tumour resistance.

The combination of low-dose VEGFR2 blockade and a cancer vaccine also led to an increased immune response to tumour cells, vascular normalisation and improved survival in mice models of breast cancer and colon cancer [ 34 , 35 ]. There are now ongoing trials investigating the role of dual anti-angiogenic therapy and immunotherapy using bevacizumab with atezolizumab e.

in advanced renal cell cancers NCT [ 33 ]. Triple therapy using a combination of anti-angiogenic agents, immunotherapy and conventional chemotherapy are also being trialed in metastatic solid tumours NCT, NCT [ 33 ]. These trials have a high potential for overcoming of tumour resistance to anti-angiogenic molecules in future.

Reliable biomarkers of tumour response to antiangiogenic therapy have become a focus of attention given the risk of tumour resistance and adverse events. However, most of the studies have been inconsistent.

Circulating VEGF levels have been investigated as a predictive biomarker of response to anti-VEGF therapy. In the study by Hillan et al. In the TARGET trial which investigated sorafenib in advanced renal cell carcinoma, serum VEGF levels had an inverse relationship with progression-free survival and overall survival [ 37 ].

Taken together, it seems that while VEGF has prognostic value, it is not a reliable predictor of response to therapy. Vascular endothelial cadherin is another potential biomarker [ 38 ]. It is important in maintaining EC contact. It also plays important role in regulating cell proliferation, apoptosis and modulates VEGFR2 function.

In the same vein, integrins that mediate cell-cell and cell-extracellular matrix interactions may be important biomarkers because of their roles in tumour invasion and metastasis.

Nanoparticles bearing αvβ3 integrins are being investigated for molecular tumour imaging. Circulating levels of HGF, IL-6, IL-8, osteopontin and TIMP1 have been shown to identify patients who had greater overall survival benefit from treatment in pazopanib-treated patients with metastatic renal cell cancers in one study [ 41 ].

Challenges with the use of circulating biomarkers include the absence of standardization of measurements across centres and the absence of accepted cut-off levels for these circulating biomarkers. Moreover, circulating factors tend to fluctuate in disease settings and disease stage.

Mast cells and miRNAs are increasingly being investigated as diagnostic and prognostic biomarkers in tumours like colorectal cancers and are potential therapeutic targets [ 42 ].

High mast cells density is correlated with the advanced stage of colorectal cancer and tumour progression. Recently, mast cell tryptase inhibitors e. gabexalate mesylate and nafamostat mesylate have been studied in metastatic gastric cancers with encouraging result [ 43 ]. There has been an interest in non-coding miRNAs in colorectal cancer progression.

miRNA and miRNA are oncogenic miRNAs seen at all stages of colorectal cancer progression [ 42 ]. Their levels in tumour tissues have been correlated with survival in individuals with colorectal cancers.

miRNA has been shown to confer tumour resistance to 5-fluoro uracil by downregulating MutS homologue-2 while high levels of miRNA have been correlated with oxaliplatin resistance [ 42 ].

The development of drugs which target the secretion or action of these miRNAs holds great promise for the prevention and treatment of tumour resistance in patients on anti-angiogenic treatment and conventional chemotherapy.

Microvascular density in serial tumour biopsies has been proposed as a reliable biomarker of response along with the measurement of circulating angiogenic markers and adhesion molecules [ 44 ]. A meta-analysis showed that micro-vessel density predicted survival in non-small cell lung cancer NSCLC [ 45 ].

Anti-angiogenics may not only affect tumour vessels but also the normal vasculature; thus, healthy tissue in tumours may be used to monitor antiangiogenic therapy in tumours.

Vessel density and intra-tumour blood supply may be estimated using imaging methods like contrast-enhanced MRI or PET.

In one clinical trial of metastatic colon cancer, epithelial and stromal VEGF expression and micro-vessel density were not predictive of the benefit of the addition of bevacizumab to 5-fluorouracil based therapy [ 46 ].

Vascular imaging using ultrasound, CT, MRI or PET is another predictive marker that can be used to assess response to treatment as shown by the use of MRI in monitoring response to antiangiogenic therapy in patients with glioblastoma multiforme GBM [ 47 ].

High levels of vascular perfusion on vascular imaging predicted response and outcome in patients with metastatic renal cell cancers who were treated with TKIs [ 48 ]. A recent study by Rojas et al. Challenges with using these imaging modalities include marked variability in methodologies used to assess imaging biomarkers across studies and the need for standardization of tumour molecular imaging.

Different types of biomarkers e. circulating and imaging may have to be combined to yield a composite biomarker for more robust predictors of response to antiangiogenic therapy.

The cardiovascular adverse effects of antiangiogenic therapy are worthy of mention. Some of the reported side effects are hypertension, cardiac dysfunction and myocardial ischaemia. These agents act by reducing nitric oxide expression which leads to vasoconstriction and elevation of blood pressure [ 50 ].

Other pathophysiologic pathways for hypertension include increased expression of endothelin-1, microvascular rarefaction, activation of the renin-angiotensin-aldosterone axis, oxidative stress, pressure natriuresis and arterial stiffness.

VEGF signaling pathway inhibitors cause an increase in blood pressure with 7. Blood pressure elevation occurs rapidly within hours or days of starting anti-VEGF therapy and is commensurate with effective VEGF signaling inhibition. It remains unclear whether blood pressure goals in such patients should be the same as for the general population even though current hypertension guidelines do not discriminate between these patients and the general population.

The risk of hypertensive target organ damage is increased in these patients. The National Cancer Institute recommends formal cardiovascular assessment before commencing anti-angiogenic therapy, and antihypertensives should be commenced in such patients once there is a more than 20mmHg rise in diastolic blood pressure from baseline even if blood pressure remains in the normotensive range [ 51 ].

There is a need to clarify the blood pressure threshold at which anti-angiogenic dose reduction or termination should be considered.

The preferred classes of antihypertensives in such instances are also a matter of debate. It is better to avoid non-dihydropyridine calcium channel blockers since they inhibit the CYP3A4 which is responsible for the metabolism of antiangiogenic medications and can thus elevate plasma levels of anti-angiogenics with resultant worsening of hypertension.

Anti-angiogenic therapy has been implicated in cardiotoxicity. The risk is particularly high in those who develop hypertension. Moreover, the risk of left ventricular LV dysfunction remains high among patients whose blood pressure has been controlled while on medications like sunitinib.

Such capillary density may not match the increase in myocardial area or hypertrophy. This mismatch causes reduced fractional shortening and increased LV end-diastolic pressure [ 50 ]. In mice treated with TKIs like sunitinib and also in patients on anti-angiogenic therapy, there is capillary rarefaction and myocyte mitochondrial swelling and degenerative changes which are compounded by apoptosis in those with high blood pressure [ 50 ].

It appears that increased afterload accelerates this capillary rarefaction and may underlie the development of LV dysfunction. Cardiotoxicity also involves alteration in myocardial energetics via AMP-kinase inhibition and resultant mitochondrial dysfunction.

Such changes lead to reduced contractility and increase the susceptibility of the heart to other insults.

Such cardiotoxicity may be due to both on-target and off-target effects of TKIs on the heart which leads to adverse remodeling and cardiac dilatation. This underscores the need to monitor left ventricular function in patients on anti-angiogenic therapy. Myocardial ischaemia has been observed with some antiangiogenic agents including bevacizumab, sunitinib, sorafenib and regorafenib [ 50 ].

This LV dysfunction is usually asymptomatic and is reversible on early withdrawal of such therapy. Risk factors for such arterial thrombotic events are unclear but background heart disease, hypertension, older age and use of other cardiotoxic drugs likely play important roles.

The strong link between coronary ischaemia and cardiotoxicity with the use of anti-angiogenic therapy appears to be related to perfusion contraction mismatch [ 50 ].

Reduction in nitric oxide signaling and endothelial dysfunction that occur following acute VEGF therapy accelerates coronary vasoconstriction, arterial inflammation, atherosclerosis and platelet reactivity. This is particularly important for those molecules which also affect PDGF signaling where there is decoupling of the pericyte-endothelial myocardial interaction.

Theoretical concerns exist for small molecule receptor tyrosine kinase inhibitors about cardiotoxicity and heart failure risk especially in those with pre-existing cardiac diseases due to disruption of AMP-kinase activity [ 52 ].

The risk of the left ventricular systolic dysfunction during anti-angiogenic therapy is difficult to predict. Many of the patients in reported studies had been treated with radiotherapy and chemotherapy which may also cause cardiotoxicity.

Stress echocardiography may play a role in the evaluation of those with an intermediate or high pre-test probability of coronary artery disease who are being placed on anti-VEGF therapy. Additionally, PET and cardiac MRI may be used to determine myocardial blood flow reserve in these situations.

The clinical approach to anti-angiogenic therapy in the setting of cardiovascular risk is presented in Fig. Nanoparticles allow absorption of a large quantity of a drug due to the large surface area to volume ratio [ 53 ].

Small molecules, proteins, DNA and miRNAs can be loaded into nanoparticles for delivery into tumours. Nanoparticles have advantages over conventional chemotherapy because of their multifunctional targeted roles in the tumour environment.

Potential approaches include tissue reoxygenation, either through in situ oxygen supply or increasing intra-tumour hydrogen peroxide metabolism. Organic liposomes, polymers and inorganic gold, silver and silicate based nanoparticles have been developed for use in experimental tumour models.

Some nanoparticles have been designed to silence the expression of HIF-1α gene by antisense oligonucleotides or by miRNAs. Some liposomes carrying camptothecin or topotecan inhibit topoisomerase I [ 53 ]. The flow of nanomedicines into tumours may be negatively influenced by hypoxia of tumour microenvironment despite the existence of enhanced permeability and retention effect EPR [ 53 ].

EPR in solid tumours is due to their vascular abnormalities which lead to extravasation of nanometric molecules in tumours which may thus reach a higher concentration than in normal tissue. The intense hypoxic environment of tumours may be a barrier to the EPR effect.

Nanotechnology have circumvented this and can enhance EPRs by using hyperthermia to mediate vascular permeability in solid tumours, ultrasound-induced cavitation to modify tumour tissue, application of nitric oxide-releasing agents to expand blood vessels or administration of antihypertensive to normalize blood flow [ 53 ].

These have been achieved in tumours to promote tumour heating using photo-stimulation, magnetism, radiofrequency waves or ultrasound. Tumour vessel normalization has also been attempted using gold nanoparticles to provide human recombinant endostatin rhEs in tumours by EPR to facilitate transient vessel normalization and improve anti-tumour therapeutic efficacy.

Some have also developed nanoparticles of combination therapy of antiangiogenic and conventional chemotherapy e. lipid derivative conjugates LGCs containing gemcitabine and paclitaxel to simultaneously restore tumour vasculature and deliver cytotoxic drugs [ 53 ].

There is however a need to evaluate the safety and toxicity of nanoparticles. Safety concerns include direct toxicity, nanoparticle aggregate long-term accumulation and immunogenicity.

There is also a need to improve drug loading capacity and capability of sustained release of the cargo of nanoparticles in vivo. This will minimize the risk of accumulation of nanoparticles in healthy tissues and facilitate effective delivery to the target tumours.

This is important because vascular permeability, oncotic pressure, interstitial pressure and complex nature of tumour stroma affect the movement of nanoparticles in and out of tumour microenvironment.

There is a need to stratify patients according to their EPR release to define those patients who can benefit from nanoparticles. There are different delivery methods for nanoparticles.

These include exosomes, plasma membrane coating, use of chitosan and even the use of mesenchymal stem cells. Exosomes allow intracellular delivery of their cargo by fusion of membranes. They can cross biological barriers like the blood-brain barrier easily.

Undesired effects of the exosome components and lack of standardized production protocols are limitations to their use. Plasma membrane coating with nanoparticles is another delivery technique for nanoparticles as anti-angiogenics.

Examples of nanoparticles delivered this way include tungsten oxide which has been used in lymphoma models [ 53 ]. Platelet membranes provide immune evasion and active adhesion to tumour cells due to their P-selectin interaction with ligands expressed on tumour cells.

Some have used red cell membranes which are very abundant in the circulation and have immune escape and long circulation time. Chitosan is another carrier derived from chitin. It is less cytotoxic and is biodegradable and metabolized easily by the kidneys.

In mice models of breast cancer, chitosan nanoparticles containing anti-Rho small interfering RNA siRNA showed tumour anti-angiogenesis [ 56 ]. The binding of αvβ3 integrin to chitosan nanoparticles is an important development. The receptor for αvβ3 integrin is widely expressed in tumours and has shown potentials in ovarian cancer models.

Encapsulation of paclitaxel with chitosan nanoparticles has shown efficacy in breast cancer [ 57 ]. There is now interest in the use of mesenchymal stem cells MSCs to deliver nanoparticles.

Hypoxic conditioning of such MSCs used as cell-based therapy can be used for aggressive tumours like glioblastoma multiforme since MSCs can traffic across the blood-brain barrier [ 53 ].

Blocking tumour stem cells via anti-angiogenic therapies is another theoretical approach since the tumour stem cell sub-population in some tumours like breast cancers may be more adept at promoting angiogenesis than their non-stem cell counterparts.

The different delivery methods for nanoparticles are compared in Table 2. Anti-angiogenic therapy in cancers has enormous potentials using VEGF signaling pathways.

Cardiovascular toxicity and off-target effects of anti-angiogenic drugs are impediments to their long-term use in those at high cardiovascular risk. Continued research into effective nanoparticle-based delivery methods is an exciting and developing field in cancer therapeutics.

Understanding of the molecular and cellular mechanisms of tumour angiogenesis will facilitate the development of newer effective anti-angiogenic molecules. GBD Disease and Injury Incidence and Prevalence Collaborators.

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Lancet Oncol. Maj E, Papiernik D, Wietrzyk J. As the cellular and molecular events that underlie tumor angiogenesis become better defined, rational strategies can be derived to apply this molecular tourniquet.

An ideal antiangiogenesis strategy should be targeted to only the organs that contain the tumors and should not interfere with normal angiogenesis; it must achieve a high ratio of regional-to-systemic concentrations to minimize systemic toxicity; it must have a biologic half-life sufficient to counter the proangiogenesis phenotype of the tumor; and its antiangiogenesis effects should be regulatable.

Gene transfer strategies potentially satisfy many of these requirements. Critical to the success of antiangiogenesis gene transfer is the fact that endothelial cells are activated or suppressed in trans , depending on the composition of the extracellular milieu.

In this context, gene therapy for angiogenesis does not have to transduce all or any specific populations of cells in the target organs to achieve a high, local concentration of the antiangiogenesis proteins.

Antiangiogenesis treatment is likely to be most effective in a low tumor burden state. In such a setting, therapeutic antiangiogenesis can be expected to prolong the state of tumor dormancy by suppressing micrometastases that remain despite successful treatment of the primary tumors.

One appropriate clinical approach to using antiangiogenesis therapy in cancer is to combine it with conventional therapy to reduce the initial tumor burden, followed by its use in an adjuvant setting to prolong disease-free survival.

Google Scholar. Google Preview. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Navbar Search Filter JNCI: Journal of the National Cancer Institute This issue JNCI Portfolio Medicine and Health Books Journals Oxford Academic Mobile Enter search term Search.

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Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Why Use Gene Therapy to Deliver Antiangiogenesis Agents? Biology of Tumor Angiogenesis Relevant to Antiangiogenesis Gene Therapy. Antiangiogenesis With the Use of Gene Therapy.

Challenges to Successful Antiangiogenesis Gene Therapy. Journal Article. Gene Therapy Strategies for Tumor Antiangiogenesis. Hwai-Loong Kong , Hwai-Loong Kong.

Oxford Academic. Ronald G. Revision received:. Split View Views. Cite Cite Hwai-Loong Kong, Ronald G. Select Format Select format. ris Mendeley, Papers, Zotero.

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Abstract Based on the concept that solid tumors cannot grow without the generation of new blood vessels, there is growing interest in the use of antiangiogenesis agents to inhibit tumor growth. Open in new tab Download slide.

Google Scholar Crossref. Search ADS. Google Scholar PubMed. OpenURL Placeholder Text. Review article: angiogenesis, neovascular proliferation and vascular pathophysiology as targets for cancer therapy. Google Scholar Google Preview OpenURL Placeholder Text.

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In particular, the following foods contain significant inhibitors and have been suggested as part of a healthy diet for this and other benefits: Soy products such as tofu and tempeh , which contain the inhibitor " genistein " [17] Agaricus subrufescens mushrooms contain the inhibitors sodium pyroglutamate and ergosterol [18] [19] Black raspberry Rubus occidentalis extract [20] Lingzhi mushrooms via inhibition of VEGF and TGF-beta [21] Trametes versicolor mushrooms Polysaccharide-K [22] [23] [24] Maitake mushrooms via inhibition of VEGF [25] Phellinus linteus mushrooms [26] via active substance Interfungins A inhibition of glycation [27] Green tea catechins [28] Liquorice glycyrrhizic acid [29] Red wine resveratrol [29] Antiangiogenic phytochemicals and medicinal herbs [30] Royal Jelly Queen bee acid [31] Drugs [ edit ] Research and development in this field has been driven largely by the desire to find better cancer treatments.

Bevacizumab binds to VEGF inhibiting its ability to bind to and activate VEGF receptors. Sunitinib and Sorafenib inhibit VEGF receptors. Sorafenib also acts downstream. Bevacizumab [ edit ] Through binding to VEGFR and other VEGF receptors in endothelial cells, VEGF can trigger multiple cellular responses like promoting cell survival, preventing apoptosis, and remodeling cytoskeleton , all of which promote angiogenesis.

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