VEGF Family Of Growth Factors Research Articles

Stem Cells in Translational Cancer Research.

Since the seminal studies by Fialkow et al. in the seventies [1] and by Bonnet and Dick in the nineties [2], where an organized tumor cell hierarchy originating from transformed hematopoietic stem cells was found in myeloid leukemias, the cancer stem cell (CSC) model of tumor development drew the attention of the scientific community and became the focus of extensive discussion. This theme gained momentum over the past decade, with the identification of stemlike cells as drivers of tumor initiation, recurrence, and metastasis spread in a variety of nonhematopoietic human cancers. These characteristics postulated them as novel and promising therapeutic targets for cancer, especially in the context of drug-resistant tumors [3]. The origin of these stemlike cells is being extensively studied. In some types of tumors, such as in blood, brain, colon, and skin cancer, it has been shown that adult stem cells are prone to transformation due to a longer half-life within tissues compared to their cell progenies, facilitating the accumulation of oncogenic mutations as a result of prolonged exposure to genotoxic stresses [4]. In other cases, de novo acquisition of stem cell properties may occur in either normal or neoplastic cells due to genetic/epigenetic aberrations [5]. Finally, the CSC model has been put forward to explain why tumors like breast cancer might return years after surgery. In this situation, lower proliferation rate might allow cells to escape from aggressive therapy and allow for genetic/epigenetic evolution. Advances in the field, however, point to varied and more complex models of tumor development than previously envisioned, in which the stemlike phenotype may also be dynamically acquired by cancer cells through interaction with stromal cells and soluble factors [6]. A relevant question timely addressed in this special issue is the understanding of the contribution of resident stem cells to the tumor microenvironment (TME). This is of major relevance since they may significantly influence tumor progression, aggressiveness, and response to therapy. The contribution of pericytes to tumor growth, angiogenesis, metastasis, and evasion of immune destruction, which are classic hallmarks of cancer, is comprehensively reviewed by A. L. Ribeiro and O. K. Okamoto. In their article, the authors also discuss a pericyte-mediated regulation of stemness properties in cancer cells and argue in favor of pericytes as cellular targets for new cancer therapies aiming at the TME. Indeed, many new anticancer therapies targeting the TME are under development, with currently approved antiangiogenic drugs as examples of such strategy. However, events of tumor recurrence and poor response to antiangiogenic therapy still puzzle researchers and emphasize the need for continuous studies. In the article by M. Marcola and C. E. Rodrigues, the involvement of endothelial progenitor cells in tumor angiogenesis is critically reviewed. In their article, the specific roles of different members of the VEGF family of growth factors, as well as the relevance of the vascular niche to stemness in cancer cells, are also carefully discussed. Another practical issue of clinical relevance is the rigorous evaluation of potential oncogenic risks associated with stem cell therapy. Long-term safety issues must be properly addressed before stem cell-based therapies enter clinical trials, but such studies are outnumbered in the literature by studies evaluating therapeutic effects based on restoration of tissue integrity and physiological balance. While the original article by T. Jazedje et al. addresses this issue at the preclinical stage, showing in a murine breast adenocarcinoma model that mesenchymal stromal cells (MSC) may exert either pro- or antitumorigenic effects depending on the experimental condition, R. Schweizer et al. bring a more clinical perspective to this issue, presenting an interesting discussion about the possible oncogenic hazards of adipose-derived MSC in breast cancer patients subjected to breast reconstruction after mastectomy. Finally, due to their tumor homing properties, the use of stem cells as vehicles to deliver suicide genes is a strategy highly pursued in cancer gene therapy. However, low delivery efficiency and off-target effects are common limitations of current expression systems. In the original article by Y. Luo et al., the authors elegantly characterize a novel inducible transgene expression system based on the action of neural stem cells that could be further explored to treat malignant gliomas. In summary, the articles compiled in this special issue highlight how the growing investigation of tumor development through the lens of stem cell biologists is significantly impacting basic and translational cancer research. This interplay between stem cell and tumor biology offers an exceptional opportunity to improve our knowledge about cancer, one of the leading causes of death worldwide. Advances in this field are expected to bring significant impacts in cancer diagnosis and therapy. Oswaldo Keith Okamoto Ander Matheu Luca Magnani

Epidermal Expression of Neuropilin 1 Protects Murine keratinocytes from UVB-induced apoptosis

BackgroundNeuropilin 1 (NRP1) is expressed on several cell types including neurons and endothelial cells, where it functions as an important regulator in development and during angiogenesis. As a cell surface receptor, NRP1 is able to bind to members of the VEGF family of growth factors and to secreted class 3 semaphorins. Neuropilin 1 is also highly expressed in keratinocytes, but the function of NRP1 in epidermal physiology and pathology is still unclear.Methods and ResultsTo elucidate the role of NRP1 in skin in vivo we generated an epidermis-specific neuropilin 1 knock out mouse model by using the Cre-LoxP-System. Mice were viable and fertile and did not display any obvious skin or hair defects. After challenge with UVB irradiation, we found that deletion of epidermal NRP1 leads to increased rates of apoptosis both in vitro and in vivo. NRP1-deficient primary keratinocytes cultured in vitro showed significantly higher rates of apoptosis 24 hours after UVB. Likewise, there is a significant increase of active caspase 3 positive cells in the epidermis of Keratin 14-Cre-NRP1 (−/−) mice 24 hours after UVB irradiation. By Western Blot analysis we could show that NRP1 influences the cytosolic levels of Bcl-2, a pro-survival member of the Bcl-2 family. After UVB irradiation the amounts of Bcl-2 decrease in both protein extracts from murine epidermis and in NRP1-deficient keratinocytes in vitro, whereas wild type cells retain their Bcl-2 levels. Likewise, levels of phospho-Erk and Rac1 were lower in NRP1-knock out keratinocytes, whereas levels of pro-apoptotic p53 were higher.ConclusionNRP1 expression in keratinocytes is dispensable for normal skin development. Upon UVB challenge, NRP1 contributes to the prevention of keratinocyte apoptosis. This pro-survival function of NRP1 is accompanied by the maintenance of high levels of the antiapoptotic regulator Bcl-2 and by lower levels of pro-apoptotic p53.

The VD1 Neutralizing Antibody to Vascular Endothelial Growth Factor-D: Binding Epitope and Relationship to Receptor Binding

Vascular endothelial growth factor-D (VEGF-D) is a secreted protein that promotes tumor growth and metastatic spread in animal models of cancer. Expression of VEGF-D in prevalent human cancers was reported to correlate with lymph node metastasis and patient outcome—hence, this protein is a potential target for novel anticancer therapeutics designed to restrict tumor growth and spread. Here, we define the binding site in VEGF-D of a neutralizing antibody, designated VD1, which blocks the interaction of VEGF-D with its cell surface receptors vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 and is being used for the development of therapeutic antibodies. We show by peptide-based mapping and site-directed mutagenesis that the VD1 binding site includes the five residues 147NEESL 151 and that immunization with a synthetic peptide containing this motif generates antibodies that neutralize VEGF-D. The tertiary structure of VEGF-D indicates that the 147NEESL 151 epitope is located in the L2 loop of the growth factor, which is important for receptor binding. Mutation of any of these five residues influences receptor binding; for example, mutations to E148, which abolished binding to VD1, impaired the interaction with VEGFR-2 but enhanced binding to VEGFR-3. This structure/function study indicates that the VD1 binding epitope is part of the receptor binding site of VEGF-D, identifies a region of VEGF-D critical for binding of receptors and explains why VD1 does not bind other members of the VEGF family of growth factors.

Crystal Structure of Human Vascular Endothelial Growth Factor-B: Identification of Amino Acids Important for Receptor Binding

The development of blood vessels (angiogenesis) is critical throughout embryogenesis and in some normal postnatal physiological processes. Pathological angiogenesis has a pivotal role in sustaining tumour growth and chronic inflammation. Vascular endothelial growth factor-B (VEGF-B) is a member of the VEGF family of growth factors that regulate blood vessel and lymphatic angiogenesis. VEGF-B is closely related to VEGF-A and placenta growth factor (PlGF), but unlike VEGF-A, which binds to two receptor tyrosine kinases VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), VEGF-B and PlGF bind to VEGFR-1 and not VEGFR-2. There is growing evidence of a role for VEGF-B in physiological and pathological blood vessel angiogenesis. VEGF-B may provide novel therapeutic strategies for the treatment of vascular disease and be a potential therapeutic target in aberrant vessel formation. To help understand at the molecular level the differential receptor binding profile of the VEGF family of growth factors we have determined the crystal structure of human VEGF-B(10-108) at 2.48 Angstroms resolution. The overall structure is very similar to that of the previously determined cysteine-knot motif growth factors: VEGF-A, PlGF and platelet-derived growth factor-B (PDGF-B). We also present a predicted model for the association of VEGF-B with the second domain of its receptor, VEGFR-1. Based on this interaction and the present structural data of the native protein, we have identified several putative residues that could play an important role in receptor recognition and specificity.

The biology of vascular endothelial growth factor-B (VEGF-B)

The formation of new blood vessels (angiogenesis) is critical for both embryonic development and a variety of normal postnatal physiological processes. Various pathological processes, most notably tumour growth and chronic inflammation, are also known to be dependent on the new vessel formation. Amongst the variety of factors that contribute to the regulation of this complex process, vascular endothelial growth factor (VEGF or VEGF-A) is arguably the most well characterised. The VEGF family of growth factors is now known to comprise of VEGF-A plus four additional members, including VEGF-B. In contrast to VEGF-A, surprisingly little is known about the precise biological role of VEGF-B. Unlike VEGF-A, which binds to the two receptor tyrosine kinases VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), VEGF-B binds only to VEGFR-1 and the functional significance of VEGFR-1 signalling has remained problematic. More recently, however, evidence has emerged suggesting a key role for VEGFR-1 signalling in pathological angiogenesis and this has raised the possibility that, like VEGF-A, VEGFR-1 specific ligands such as VEGF-B may provide for novel therapeutic strategies and/or represent new therapeutic targets. Here we review current knowledge of the biology of VEGF-B. We note that although analysis to date, including expression profiling and the generation of gene targetted mice, has provided only limited insights, future studies using recently generated recombinant proteins and antagonist monoclonal antibodies should provide for a more comprehensive understanding.

Akt-1 Regulates Angiogenesis in Skin.

The major mechanism to adapt to ischemic conditions is the development of neovascularization, i.e. angiogenesis, a process driven by members of VEGF family of growth factors. Phosphoinositide 3-kinase/Akt pathway is a critical component of the signaling network that regulates endothelial cell function related to angiogenesis. VEGF treatment of endothelial cells results in rapid phosphorylation of Akt. Our studies demonstrated that Akt kinase activity is necessary for VEGF-induced and integrin-mediated endothelial cell adhesion and migration. Moreover, cell transfection with a constitutive active form of Akt (myr-Akt) leads to increased function of integrin receptors. Using Akt-1 null mice we found that Akt-1 controls VEGF-induced and integrin-dependent endothelial cell responses in vitro. Impaired endothelial cell migration and adhesion to extracellular matrix and a reduced rate of cell proliferation were observed in Akt-1 (−/−) endothelial cells compared to WT. There are three Akt isoforms with different tissue distribution, however, it appears that Akt-1 is a predominant isoform in skin and in skin microvasculature. This observation prompted us to perform series of in vivo experiments designed to assess the angiogenic response in skin in the absence of Akt-1. Angiogenesis assay using matrigel plugs revealed that the weight and hemoglobin content of matrigel plugs is about two fold higher in Akt (−/−) mice compared to WT mice. Tumor angiogenesis also appears to be enhanced in Akt(−/−) mice, resulting in the significantly lower degree of tumor necrosis. Blood vessels in Akt (−/−) mice appear to be smaller in diameter and have reduced laminin content. Our analysis revealed significant changes in blood vessel wall matrix composition of Akt (−/−) mice as compared to WT animals. These changes resulted in increased vascular permeability in skin of Akt (−/−) mice. Akt-1 is known to target multiple cellular processes including adhesive properties, cell survival, transcription and translation. It appears that the phenotype of Akt-1 (−/−) mice depends on the equilibrium between pro-angiogenic and anti-angiogenic roles of Akt-1 and reveals a central role for Akt-1 in the regulation of matrix production and maturation of blood vessels.

The role of tumor lymphangiogenesis in metastatic spread.

The high mortality rates associated with cancer can be attributed to the metastatic spread of tumor cells from the site of their origin. Tumor cells invade either the blood or lymphatic vessels to access the general circulation and then establish themselves in other tissues. Clinicopathological data suggest that the lymphatics are an initial route for the spread of solid tumors. Detection of sentinel lymph nodes by biopsy provides significant information for staging and designing therapeutic regimens. The role of angiogenesis in facilitating the growth of solid tumors has been well established, but the presence of lymphatic vessels and the relevance of lymphangiogenesis to tumor spread are less clear. Recently, the molecular pathway that signals for lymphangiogenesis and relatively specific markers for lymphatic endothelium have been described allowing analyses of tumor lymphangiogenesis to be performed in animal models. These studies demonstrate that tumor lymphangiogenesis is a major component of the metastatic process and implicate members of the VEGF family of growth factors as key mediators of lymphangiogenesis in both normal biology and tumors.

Proteolytic processing regulates receptor specificity and activity of VEGF-C.

The recently identified vascular endothelial growth factor C (VEGF-C) belongs to the platelet-derived growth factor (PDGF)/VEGF family of growth factors and is a ligand for the endothelial-specific receptor tyrosine kinases VEGFR-3 and VEGFR-2. The VEGF homology domain spans only about one-third of the cysteine-rich VEGF-C precursor. Here we have analysed the role of post-translational processing in VEGF-C secretion and function, as well as the structure of the mature VEGF-C. The stepwise proteolytic processing of VEGF-C generated several VEGF-C forms with increased activity towards VEGFR-3, but only the fully processed VEGF-C could activate VEGFR-2. Recombinant 'mature' VEGF-C made in yeast bound VEGFR-3 (K[D] = 135 pM) and VEGFR-2 (K[D] = 410 pM) and activated these receptors. Like VEGF, mature VEGF-C increased vascular permeability, as well as the migration and proliferation of endothelial cells. Unlike other members of the PDGF/VEGF family, mature VEGF-C formed mostly non-covalent homodimers. These data implicate proteolytic processing as a regulator of VEGF-C activity, and reveal novel structure-function relationships in the PDGF/VEGF family.