Reconciling VEGF With VPF: The Importance of Increased Vascular Permeability for Stroma Formation in Tumors, Healing Wounds, and Chronic Inflammation.

It is widely believed that vascular endothelial growth factor (VEGF) induces angiogenesis by its direct mitogenic and motogenic actions on vascular endothelial cells. However, these activities are only detected when endothelial cells are cultured at very low (0.1%) serum concentrations and would not be expected to take place at the much higher serum levels found in angiogenic sites in vivo. This conundrum can be resolved by recalling VEGF's original function, that of an extremely potent vascular permeability factor (VPF). In vivo VPF/VEGF increases microvascular permeability such that whole plasma leaks into the tissues where it undergoes clotting by tissue factor that is expressed on tumor and host connective tissue cells to deposit fibrin and generate serum. By providing tissue support and by reprogramming the gene expression patterns of cells locally, fibrin and serum can together account for the formation of vascular connective tissue stroma. In sum, by increasing vascular permeability, VPF/VEGF triggers the "wound healing response," setting in motion a fundamental pathophysiological process that induces the mature stroma that is found not only in healing wounds but also in solid tumors and chronic inflammatory diseases. Once initiated by increased vascular permeability, this response may be difficult to impede, perhaps contributing to the limited success of anti-VEGF therapies in treating cancer.

Tumors: Wounds That Do Not Heal-A Historical Perspective with a Focus on the Fundamental Roles of Increased Vascular Permeability and Clotting.

Similarities between solid tumor stroma generation, wound healing, chronic inflammation, and associated inflammatory diseases have prompted interest from the time of Virchow. However, it was not until the 1970s that these entities were shown to share important molecular mechanisms. Foundational to all of them is the initiating role of vascular endothelial growth factor (VEGF-A) in increasing vascular permeability to plasma and plasma proteins. Extravasated plasma activates the tissue factor clotting pathway, leading to extravascular deposition of a fibrin gel. Fibrin serves initially as a provisional stroma that provides a favorable substrate for the attachment and migration of tumor cells, as well as host fibroblasts, endothelial, and inflammatory cells. Fibrin and its degradation products have proangiogenic activity with important roles in the generation of new blood vessels and connective tissue stroma. Over time, fibrin is degraded and replaced by vascular and subsequently by dense, relatively avascular collagenous connective tissue, the end-product referred to as desmoplasia in tumors and scar in healed wounds. Fibrin and the mature stroma that replaces it provide a diffusion barrier to chemotherapy and a structural barrier that inflammatory cells must cross to reach tumor cells. Plasma solutes of varying size cross the endothelial cells lining capillaries and venules of normal tissues and "mother" vessels of tumors and wounds by different anatomical pathways. VEGF-A levels fall back to normal as wounds heal but remain perpetually elevated in solid tumors. Thus, tumors may heal centrally but continually initiate new healing activity as they grow and invade surrounding normal tissues.

Early Actions of Anti-Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor Drugs on Angiogenic Blood Vessels.

Tumors induce their heterogeneous vasculature by secreting vascular endothelial growth factor (VEGF)-A. Anti-VEGF/VEGF receptor (VEGFR) drugs treat cancer, but the underlying mechanisms remain unclear. An adenovirus expressing VEGF-A (Ad-VEGF-A164) replicates the tumor vasculature in mice without tumor cells. Mother vessels (MV) are the first angiogenic vessel type to form in tumors and after Ad-VEGF-A164. Multiday treatments with a VEGF trap reverted MV back to normal microvessels. We now show that, within hours, a single dose of several anti-VEGF drugs collapsed MV to form glomeruloid microvascular proliferations (GMP), accompanied by only modest endothelial cell death. GMP, common in many human cancers but of uncertain origin, served as an intermediary step in MV reversion to normal microvessels. The vasodisruptive drug combretastatin CA4 also targeted MV selectively but acted differently, extensively killing MV endothelium. Antivascular changes were quantified with a novel Evans blue dye assay that measured vascular volumes. As in tumors, Ad-VEGF-A164 strikingly increased endothelial nitric oxide synthase (eNOS) expression. The eNOS inhibitor N(G)-Nitro-l-arginine methyl ester mimicked anti-VEGF/VEGFR drugs, rapidly collapsing MV to GMP. Inhibition of eNOS reduces synthesis of its vasodilatory product, nitric oxide, leading to arterial contraction. Patients and mice receiving anti-VEGF/VEGFR drugs develop hypertension, reflecting systemic arterial contraction. Together, anti-VEGF/VEGFR drugs act in part by inhibiting eNOS, causing vasocontraction, MV collapse to GMP, and subsequent reversion of GMP to normal microvessels, all without extensive vascular killing.

Vessel co-option is common in human lung metastases and mediates resistance to anti-angiogenic therapy in preclinical lung metastasis models.

Anti-angiogenic therapies have shown limited efficacy in the clinical management of metastatic disease, including lung metastases. Moreover, the mechanisms via which tumours resist anti-angiogenic therapies are poorly understood. Importantly, rather than utilizing angiogenesis, some metastases may instead incorporate pre-existing vessels from surrounding tissue (vessel co-option). As anti-angiogenic therapies were designed to target only new blood vessel growth, vessel co-option has been proposed as a mechanism that could drive resistance to anti-angiogenic therapy. However, vessel co-option has not been extensively studied in lung metastases, and its potential to mediate resistance to anti-angiogenic therapy in lung metastases is not established. Here, we examined the mechanism of tumour vascularization in 164 human lung metastasis specimens (composed of breast, colorectal and renal cancer lung metastasis cases). We identified four distinct histopathological growth patterns (HGPs) of lung metastasis (alveolar, interstitial, perivascular cuffing, and pushing), each of which vascularized via a different mechanism. In the alveolar HGP, cancer cells invaded the alveolar air spaces, facilitating the co-option of alveolar capillaries. In the interstitial HGP, cancer cells invaded the alveolar walls to co-opt alveolar capillaries. In the perivascular cuffing HGP, cancer cells grew by co-opting larger vessels of the lung. Only in the pushing HGP did the tumours vascularize by angiogenesis. Importantly, vessel co-option occurred with high frequency, being present in >80% of the cases examined. Moreover, we provide evidence that vessel co-option mediates resistance to the anti-angiogenic drug sunitinib in preclinical lung metastasis models. Assuming that our interpretation of the data is correct, we conclude that vessel co-option in lung metastases occurs through at least three distinct mechanisms, that vessel co-option occurs frequently in lung metastases, and that vessel co-option could mediate resistance to anti-angiogenic therapy in lung metastases. Novel therapies designed to target both angiogenesis and vessel co-option are therefore warranted. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Stromal-Based Signatures for the Classification of Gastric Cancer.

Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomics-based systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification. Cancer Res; 76(9); 2573-86. ©2016 AACR.

Intracellular distribution of TM4SF1 and internalization of TM4SF1-antibody complex in vascular endothelial cells.

Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane-associated glycoprotein that is highly and selectively expressed on the plasma membranes of tumor cells, cultured endothelial cells, and, in vivo, on tumor-associated endothelium. Immunofluorescence microscopy also demonstrated TM4SF1 in cytoplasm and, tentatively, within nuclei. With monoclonal antibody 8G4, and the finer resolution afforded by immuno-nanogold transmission electron microscopy, we now demonstrate TM4SF1 in uncoated cytoplasmic vesicles, nuclear pores and nucleoplasm. Because of its prominent surface location on tumor cells and tumor-associated endothelium, TM4SF1 has potential as a dual therapeutic target using an antibody drug conjugate (ADC) approach. For ADC to be successful, antibodies reacting with cell surface antigens must be internalized for delivery of associated toxins to intracellular targets. We now report that 8G4 is efficiently taken up into cultured endothelial cells by uncoated vesicles in a dynamin-dependent, clathrin-independent manner. It is then transported along microtubules through the cytoplasm and passes through nuclear pores into the nucleus. These findings validate TM4SF1 as an attractive candidate for cancer therapy with antibody-bound toxins that have the capacity to react with either cytoplasmic or nuclear targets in tumor cells or tumor-associated vascular endothelium.

Tumor Stroma, Tumor Blood Vessels, and Antiangiogenesis Therapy.

Solid tumors generally require a vascularized connective tissue stroma if they are to grow beyond minimal size. They generate that stroma in part by secreting vascular endothelial growth factor (VEGF), a potent vascular permeability and angiogenic factor. Increased vascular permeability leads to deposition of a provisional fibrin stroma, which supports tumor, connective tissue, and inflammatory cell migration and plays an active role in the formation of mature vascularized stroma. Vascular endothelial growth factor-induced tumor blood vessels are heterogeneous, of at least 6 distinct types, and develop linearly over time. They include both angiogenic (mother vessels, glomeruloid microvascular proliferations, vascular malformations, capillaries) and arteriovenogenic (feeding arteries, draining veins) blood vessels. Attacking the tumor vasculature with drugs that target VEGF or its receptors (VEGFR) has come into vogue but has been less effective than had been hope for. One reason for this is that anti-VEGF/VEGFR therapy attacks only a subset of tumor blood vessels, the earliest to form. New targets on late-forming blood vessels such as feeding arteries would be useful in helping antivascular cancer therapy fulfill its promise.

Novel Anti-TM4SF1 Antibody-Drug Conjugates with Activity against Tumor Cells and Tumor Vasculature.

Antibody-drug conjugates (ADC) represent a promising therapeutic modality for managing cancer. Here, we report a novel humanized ADC that targets the tetraspanin-like protein TM4SF1. TM4SF1 is highly expressed on the plasma membranes of many human cancer cells and also on the endothelial cells lining tumor blood vessels. TM4SF1 is internalized upon interaction with antibodies. We hypothesized that an ADC against TM4SF1 would inhibit cancer growth directly by killing cancer cells and indirectly by attacking the tumor vasculature. We generated a humanized anti-human TM4SF1 monoclonal antibody, v1.10, and armed it with an auristatin cytotoxic agent LP2 (chemical name mc-3377). v1.10-LP2 selectively killed cultured human tumor cell lines and human endothelial cells that express TM4SF1. Acting as a single agent, v1.10-LP2 induced complete regression of several TM4SF1-expressing tumor xenografts in nude mice, including non-small cell lung cancer and pancreas, prostate, and colon cancers. As v1.10 did not react with mouse TM4SF1, it could not target the mouse tumor vasculature. Therefore, we generated a surrogate anti-mouse TM4SF1 antibody, 2A7A, and conjugated it to LP2. At 3 mpk, 2A7A-LP2 regressed several tumor xenografts without noticeable toxicity. Combination therapy with v1.10-LP2 and 2A7A-LP2 together was more effective than either ADC alone. These data provide proof-of-concept that TM4SF1-targeting ADCs have potential as anticancer agents with dual action against tumor cells and the tumor vasculature. Such agents could offer exceptional therapeutic value and warrant further investigation. Mol Cancer Ther; 14(8); 1868-76. ©2015 AACR.

Tumors: wounds that do not heal-redux.

Similarities between tumors and the inflammatory response associated with wound healing have been recognized for more than 150 years and continue to intrigue. Some years ago, based on our then recent discovery of vascular permeability factor (VPF)/VEGF, I suggested that tumors behaved as wounds that do not heal. More particularly, I proposed that tumors co-opted the wound-healing response to induce the stroma they required for maintenance and growth. Work over the past few decades has supported this hypothesis and has put it on a firmer molecular basis. In outline, VPF/VEGF initiates a sequence of events in both tumors and wounds that includes the following: increased vascular permeability; extravasation of plasma, fibrinogen and other plasma proteins; activation of the clotting system outside the vascular system; deposition of an extravascular fibrin gel that serves as a provisional stroma and a favorable matrix for cell migration; induction of angiogenesis and arterio-venogenesis; subsequent degradation of fibrin and its replacement by "granulation tissue" (highly vascular connective tissue); and, finally, vascular resorption and collagen synthesis, resulting in the formation of dense fibrous connective tissue (called "scar tissue" in wounds and "desmoplasia" in cancer). A similar sequence of events also takes place in a variety of important inflammatory diseases that involve cellular immunity.

Role of BRAFV600E in the first preclinical model of multifocal infiltrating myopericytoma development and microenvironment.

Myopericytoma (MPC) is a rare tumor with perivascular proliferation of pluripotent stem-cell-like pericytes. Although indolent, MPC may be locally aggressive with recurrent disease. The pathogenesis and diagnostic biomarkers of MPC are poorly understood. We discovered that 15% of benign MPCs (thyroid, skin; 3 of 20 samples) harbored BRAF(WT/V600E); 33.3% (1 of 3 samples) of BRAF(WT/V600E)-MPCs were multifocal/infiltrative/recurrent. Patient-MPC and primary MPC cells harbored BRAF(WT/V600E), were clonal and expressed pericytic-differentiation biomarkers crucial for its microenvironment. BRAF(WT/V600E)-positive thyroid MPC primary cells triggered in vitro (8.8-fold increase) and in vivo (3.6-fold increase) angiogenesis. Anti-BRAF(V600E) therapy with vemurafenib disrupted angiogenic and metabolic properties (~3-fold decrease) with down-regulation (~2.2-fold decrease) of some extracellular-matrix (ECM) factors and ECM-associated long non-coding RNA (LincRNA) expression, with no effects in BRAF(WT)-pericytes. Vemurafenib also inhibited (~3-fold decrease) cell viability in vitro and in BRAF(WT/V600E)-positive thyroid MPC patient-derived xenograft (PDX) mice (n = 5 mice per group). We established the first BRAF(WT/V600E)-dependent thyroid MPC cell culture. Our findings identify BRAF(WT/V600E) as a novel genetic aberration in MPC pathogenesis and MPC-associated biomarkers and imply that anti-BRAF(V600E) agents may be useful adjuvant therapy in BRAF(WT/V600E)-MPC patients. Patients with BRAF(WT/V600E)-MPC should be closely followed because of the risk for multifocality/recurrence.

TM4SF1: a new vascular therapeutic target in cancer.

Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane glycoprotein that regulates cell motility and proliferation. TM4SF1 is an attractive cancer target because of its high expression in both tumor cells and on the vascular endothelial cells lining tumor blood vessels. We generated mouse monoclonal antibodies against human TM4SF1 in order to evaluate their therapeutic potential; 13 of the antibodies we generated reacted with extracellular loop-2 (EL2), TM4SF1's larger extracellular, lumen-facing domain. However, none of these antibodies reacted with mouse TM4SF1, likely because the EL2 of mouse TM4SF1 differs significantly from that of its human counterpart. Therefore, to test our antibodies in vivo, we employed an established model of engineered human vessels in which human endothelial colony-forming cells (ECFC) and human mesenchymal stem cells (MSC) are incorporated into Matrigel plugs that are implanted subcutaneously in immunodeficient nude mice. We modified the original protocol by (1) preculturing human ECFC on laminin, fibronectin, and collagen-coated plates, and (2) increasing the ECFC/MSC ratio. These modifications significantly increased the human vascular network in Matrigel implants. Two injections of one of our anti-TM4SF1 EL2 monoclonal antibodies, 8G4, effectively eliminated the human vascular component present in these plugs; they also abrogated human PC3 prostate cancer cells that were incorporated into the ECFC/MSC Matrigel mix. Together, these studies provide a mouse model for assessing tumor xenografts that are supplied by a human vascular network and demonstrate that anti-TM4SF1 antibodies such as 8G4 hold promise for cancer therapy.

The vascular permeabilizing factors histamine and serotonin induce angiogenesis through TR3/Nur77 and subsequently truncate it through thrombospondin-1.

Angiogenesis plays an important role in cancer and in many other human diseases. Vascular endothelial growth factor-A (VEGF-A), the best known angiogenic factor, was originally discovered as a potent vascular permeability factor (VPF), suggesting that other vascular permeabilizing agents, such as histamine and serotonin, might also have angiogenic activity. We recently demonstrated that, like VEGF-A, histamine and serotonin up-regulate the orphan nuclear receptor and transcription factor TR3 (mouse homolog Nur77) and that TR3/Nur77 is essential for their vascular permeabilizing activities. We now report that histamine and serotonin are also angiogenic factors that, at low micromolar concentrations, induce endothelial cell proliferation, migration and tube formation in vitro, and angiogenesis in vivo. All of these responses are mediated through specific histamine and serotonin receptors, are independent of VEGF-A, and are directly dependent on TR3/Nur77. Initially, the angiogenic response closely resembled that induced by VEGF-A, with generation of "mother" vessels. However, after ~10 days, mother vessels began to regress as histamine and serotonin, unlike VEGF-A, up-regulated the potent angiogenesis inhibitor thrombospondin-1, thereby triggering a negative feedback loop. Thus, histamine and serotonin induce an angiogenic response that fits the time scale of acute inflammation.

Revealing the role of phospholipase Cß3 in the regulation of VEGF-induced vascular permeability.

VEGF induces vascular permeability (VP) in ischemic diseases and cancer, leading to many pathophysiological consequences. The molecular mechanisms by which VEGF acts to induce hyperpermeability are poorly understood and in vivo models that easily facilitate real-time, genetic studies of VP do not exist. In the present study, we report a heat-inducible VEGF transgenic zebrafish (Danio rerio) model through which VP can be monitored in real time. Using this approach with morpholino-mediated gene knock-down and knockout mice, we describe a novel role of phospholipase Cß3 as a negative regulator of VEGF-mediated VP by regulating intracellular Ca2+ release. Our results suggest an important effect of PLCß3 on VP and provide a new model with which to identify genetic regulators of VP crucial to several disease processes.

Heterogeneity of the tumor vasculature: the need for new tumor blood vessel type-specific targets.

Therapies directed against VEGF-A and its receptors are effective in treating many mouse tumors but have been less so in treating human cancer patients. To elucidate the reasons that might be responsible for this difference in response, we investigated the nature of the blood vessels that appear in human and mouse cancers and the tumor "surrogate" blood vessels that develop in immunodeficient mice in response to an adenovirus expressing VEGF-A(164). Both tumor and tumor surrogate blood vessels are heterogeneous and form by two distinct processes, angiogenesis and arterio-venogenesis. The first new angiogenic blood vessels to form are mother vessels (MV); MV arise from preexisting venules and capillaries and evolve over time into glomeruloid microvascular proliferations (GMP) and subsequently into capillaries and vascular malformations (VM). Arterio-venogenesis results from the remodeling and enlargement of preexisting arteries and veins, leading to the formation of feeder arteries (FA) and draining veins (DV) that supply and drain angiogenic vessels. Of these different blood vessel types, only the two that form first, MV and GMP, were highly responsive to anti-VEGF therapy, whereas "late"-formed capillaries, VM, FA and DV were relatively unresponsive. This finding may explain, at least in part, the relatively poor response of human cancers to anti-VEGF/VEGFR therapies, because human cancers, present for months or years prior to discovery, are expected to contain a large proportion of late-formed blood vessels. The future of anti-vascular cancer therapy may depend on finding new targets on "late" vessels, apart from those associated with the VEGF/VEGFR axis.

Anti-VEGF/VEGFR therapy for cancer: reassessing the target.

Judah Folkman recognized that new blood vessel formation is important for tumor growth and proposed antiangiogenesis as a novel approach to cancer therapy. Discovery of vascular permeability factor VEGF-A as the primary tumor angiogenesis factor prompted the development of a number of drugs that targeted it or its receptors. These agents have often been successful in halting tumor angiogenesis and in regressing rapidly growing mouse tumors. However, results in human cancer have been less impressive. A number of reasons have been offered for the lack of greater success, and, here, we call attention to the heterogeneity of the tumor vasculature as an important issue. Human and mouse tumors are supplied by at least 6 well-defined blood vessel types that arise by both angiogenesis and arterio-venogenesis. All 6 types can be generated in mouse tissues by an adenoviral vector expressing VEGF-A(164). Once formed, 4 of the 6 types lose their VEGF-A dependency, and so their responsiveness to anti-VEGF/VEGF receptor therapy. If therapies directed against the vasculature are to have a greater impact on human cancer, targets other than VEGF and its receptors will need to be identified on these resistant tumor vessels.

Vascular hyperpermeability, angiogenesis, and stroma generation.

It has been known for more than half a century that the tumor microvasculature is hyperpermeable to plasma proteins. However, the identity of the leaky vessels and the consequences of vascular hyperpermeability have received little attention. This article places tumor vascular hyperpermeability in a broader context, relating it to (1) the low-level "basal" permeability of the normal vasculature; (2) the "acute," short-term hyperpermeability induced by vascular permeability factor/vascular endothelial growth factor (VPF/VEGF-A) and other vascular permeabilizing agents; and (3) the "chronic" hyperpermeability associated with longer-term exposure to agents such as VPF/VEGF-A that accompanies many types of pathological angiogenesis. Leakage of plasma protein-rich fluids is important because it activates the clotting system, depositing an extravascular fibrin gel provisional matrix that serves as the first step in stroma generation.

Tumor-surrogate blood vessel subtypes exhibit differential susceptibility to anti-VEGF therapy.

Antivascular therapy directed against VEGF or its receptors (VEGFR) has been successful when administered at early stages of tumor vessel growth but is less effective when administered later. Tumor blood vessels are heterogeneous, so vessel subpopulations may differ in their requirements for tumor cell-secreted VEGF and in their susceptibility to anti-VEGF/VEGFR therapy. Human cancers contain several distinct blood vessel types, including mother vessels (MV), glomeruloid microvascular proliferations (GMP), vascular malformations (VM), feeding arteries (FA), and draining veins (DV), all of which can be generated in mice in the absence of tumor cells using expression vectors for VEGF-A(164). In this study, we investigated the sensitivity of each of these vessel types to anti-VEGF therapy with Aflibercept (VEGF Trap), a potent inhibitor of VEGF-A(164). Administering VEGF Trap treatment before or shortly after injection of a recombinant VEGF-A(164)-expressing adenovirus could prevent or regress tumor-free neovasculature, but it was progressively less effective if initiated at later times. Early-forming MVs and GMPs in which the lining endothelial cells expressed high levels of VEGFR-2 were highly susceptible to blockade by VEGF Trap. In contrast, late-forming VMs, FAs, and DVs that expressed low levels of VEGFR-2 were largely resistant. Together, our findings define the susceptibility of different blood vessel subtypes to anti-VEGF therapy, offering a possible explanation for the limited effectiveness of anti-VEGF-A/VEGFR treatment of human cancers, which are typically present for months to years before discovery and are largely populated by late-forming blood vessels.

Orphan nuclear transcription factor TR3/Nur77 regulates microvessel permeability by targeting endothelial nitric oxide synthase and destabilizing endothelial junctions.

Low-level basal vascular permeability (BVP) provides nutrients to normal tissues, and increased vascular permeability is characteristic of inflammation and cancer. We recently reported that VEGF-A, a potent vascular permeabilizing and angiogenic factor, exerts much of its angiogenic activity by up-regulating expression of TR3/Nur77, an orphan nuclear transcription factor, in vascular endothelial cells (EC). To determine whether TR3/Nur77 had a more general role in regulating vascular permeability, we found that histamine, serotonin, and platelet-activating factor, small molecule vascular permeabilizing agents, also increased TR3/Nur77 expression acutely in EC. BVP and the acute vascular hyperpermeability (AVH) induced by these vascular permeabilizing factors were greatly decreased in Nur77(-/-) mice, and both BVP and AVH correlated with Nur77 expression levels in several different mouse strains. BVP and AVH were enhanced in transgenic mice in which Nur77 was selectively overexpressed in vascular EC, whereas both were suppressed in mice overexpressing dominant-negative Nur77. Chronic vascular hyperpermeability (CVH) was induced long before the onset of angiogenesis in a modified, in vivo Matrigel assay that included PT67 cells packaging retroviruses expressing Nur77-sense, whereas inclusion of cells packaging viruses expressing Nur77-antisense prevented VEGF-A-induced CVH. TR3/Nur77 modulated vascular permeability by increasing endothelial nitric-oxide synthase expression and by downregulating several EC junction proteins that maintain vascular homeostasis. Both functions required TR3/Nur77 transcriptional activity. Taking these data together, TR3/Nur77 is up-regulated by several vascular permeabilizing agents and has critical roles in mediating BVP, AVH, and CVH.

TM4SF1: a tetraspanin-like protein necessary for nanopodia formation and endothelial cell migration.

Transmembrane-4-L-six-family-1 (TM4SF1) is a tetraspanin-like membrane protein that is highly and selectively expressed by cultured endothelial cells (EC) and, in vivo, by EC lining angiogenic tumor blood vessels. TM4SF1 is necessary for the formation of unusually long (up to a 50 µm), thin (~100-300 nm wide), F-actin-poor EC cell projections that we term 'nanopodia'. Immunostaining of nanopodia at both the light and electron microsopic levels localized TM4SF1 in a regularly spaced, banded pattern, forming TM4FS1-enriched domains. Live cell imaging of GFP-transduced HUVEC demonstrated that EC project nanopodia as they migrate and interact with neighboring cells. When TM4SF1 mRNA levels in EC were increased from the normal ~90 mRNA copies/cell to ~400 copies/cell through adenoviral transduction, EC projected more and longer nanopodia from the entire cell circumference but were unable to polarize or migrate effectively. When fibroblasts, which normally express TM4SF1 at ~5 copies/cell, were transduced to express TM4SF1 at EC-like levels, they formed typical TM4SF1-banded nanopodia, and broadened, EC-like lamellipodia. Mass-spectrometry demonstrated that TM4SF1 interacted with myosin-10 and ß-actin, proteins involved in filopodia formation and cell migration. In summary, TM4SF1, like genuine tetraspanins, serves as a molecular organizer that interacts with membrane and cytoskeleton-associated proteins and uniquely initiates the formation of nanopodia and facilitates cell polarization and migration.

Tumor microenvironment and progression.

Tumor blood vessels are heterogeneous, of at least six distinct types, are induced primarily by vascular endothelial growth factor-A (VEGF-A), and provide a potentially useful therapeutic target. Breast cancer is characterized by changes in the microenvironment that result in altered tensional homeostasis. Also, breast cancers arise as the result of epigenetic as well as genetic changes. Tumor blood vessel pericytes result, in part, from bone marrow precursor cells, and VEGF is a negative regulator of glioblastoma tumor cell invasion.