OBJECTIVE

Based on rodent studies, we examined the hypothesis that increased adipose tissue (AT) mass in obesity without an adequate support of vascularization might lead to hypoxia, macrophage infiltration, and inflammation.

METHODS

Oxygen partial pressure (AT pO2) and AT temperature in abdominal AT (9 lean and 12 overweight/obese men and women) was measured by direct insertion of a polarographic Clark electrode. Body composition was measured by dual-energy X-ray absorptiometry, and insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp. Abdominal subcutaneous tissue was used for staining, quantitative RT-PCR, and chemokine secretion assay.

RESULTS

AT pO2 was lower in overweight/obese subjects than lean subjects (47 +/- 10.6 vs. 55 +/- 9.1 mmHg); however, this level of pO2 did not activate the classic hypoxia targets (pyruvate dehydrogenase kinase and vascular endothelial growth factor [VEGF]). AT pO2 was negatively correlated with percent body fat (R = -0.50, P < 0.05). Compared with lean subjects, overweight/obese subjects had 44% lower capillary density and 58% lower VEGF, suggesting AT rarefaction (capillary drop out). This might be due to lower peroxisome proliferator-activated receptor gamma1 and higher collagen VI mRNA expression, which correlated with AT pO2 (P < 0.05). Of clinical importance, AT pO2 negatively correlated with CD68 mRNA and macrophage inflammatory protein 1alpha secretion (R = -0.58, R = -0.79, P < 0.05), suggesting that lower AT pO2 could drive AT inflammation in obesity.

CONCLUSIONS

Adipose tissue rarefaction might lie upstream of both low AT pO2 and inflammation in obesity. These results suggest novel approaches to treat the dysfunctional AT found in obesity.

During embryogenesis, endothelial cells induce organogenesis before the development of circulation. These findings suggest that endothelial cells not only form passive conduits to deliver nutrients and oxygen, but also establish an instructive vascular niche, which through elaboration of paracrine trophogens stimulates organ regeneration, in a manner similar to endothelial-cell-derived angiocrine factors that support haematopoiesis. However, the precise mechanism by which tissue-specific subsets of endothelial cells promote organogenesis in adults is unknown. Here we demonstrate that liver sinusoidal endothelial cells (LSECs) constitute a unique population of phenotypically and functionally defined VEGFR3(+)CD34(-)VEGFR2(+)VE-cadherin(+)FactorVIII(+)CD45(-) endothelial cells, which through the release of angiocrine trophogens initiate and sustain liver regeneration induced by 70% partial hepatectomy. After partial hepatectomy, residual liver vasculature remains intact without experiencing hypoxia or structural damage, which allows study of physiological liver regeneration. Using this model, we show that inducible genetic ablation of vascular endothelial growth factor (VEGF)-A receptor-2 (VEGFR2) in the LSECs impairs the initial burst of hepatocyte proliferation (days 1-3 after partial hepatectomy) and subsequent reconstitution of the hepatovascular mass (days 4-8 after partial hepatectomy) by inhibiting upregulation of the endothelial-cell-specific transcription factor Id1. Accordingly, Id1-deficient mice also manifest defects throughout liver regeneration, owing to diminished expression of LSEC-derived angiocrine factors, including hepatocyte growth factor (HGF) and Wnt2. Notably, in in vitro co-cultures, VEGFR2-Id1 activation in LSECs stimulates hepatocyte proliferation. Indeed, intrasplenic transplantation of Id1(+/+) or Id1(-/-) LSECs transduced with Wnt2 and HGF (Id1(-/-)Wnt2(+)HGF(+) LSECs) re-establishes an inductive vascular niche in the liver sinusoids of the Id1(-/-) mice, initiating and restoring hepatovascular regeneration. Therefore, in the early phases of physiological liver regeneration, VEGFR2-Id1-mediated inductive angiogenesis in LSECs through release of angiocrine factors Wnt2 and HGF provokes hepatic proliferation. Subsequently, VEGFR2-Id1-dependent proliferative angiogenesis reconstitutes liver mass. Therapeutic co-transplantation of inductive VEGFR2(+)Id1(+)Wnt2(+)HGF(+) LSECs with hepatocytes provides an effective strategy to achieve durable liver regeneration.

Neuropilin-1 (NRP1) guides the development of the nervous and vascular systems. Binding to either semaphorins or VEGF, NRP1 acts with plexins to regulate neuronal guidance, or with VEGFR2 to mediate vascular development. We have generated two monoclonal antibodies that bind to the Sema- and VEGF-binding domains of NRP1, respectively. Both antibodies reduce angiogenesis and vascular remodeling, while having little effect on other VEGFR2-mediated events. Importantly, anti-NRP1 antibodies have an additive effect with anti-VEGF therapy in reducing tumor growth. Vessels from tumors treated with anti-VEGF show a close association with pericytes, while tumors treated with both anti-NRP1 and anti-VEGF lack this organization. We propose that blocking NRP1 function inhibits vascular remodeling, rendering vessels more susceptible to anti-VEGF therapy.

OBJECTIVE

To compare the efficacy and safety of ranibizumab and bevacizumab intravitreal injections to treat neovascular age-related macular degeneration (nAMD).

METHODS

Multicenter, noninferiority factorial trial with equal allocation to groups. The noninferiority limit was 3.5 letters. This trial is registered (ISRCTN92166560).

METHODS

People >50 years of age with untreated nAMD in the study eye who read ≥ 25 letters on the Early Treatment Diabetic Retinopathy Study chart.

METHODS

We randomized participants to 4 groups: ranibizumab or bevacizumab, given either every month (continuous) or as needed (discontinuous), with monthly review.

METHODS

The primary outcome is at 2 years; this paper reports a prespecified interim analysis at 1 year. The primary efficacy and safety outcome measures are distance visual acuity and arteriothrombotic events or heart failure. Other outcome measures are health-related quality of life, contrast sensitivity, near visual acuity, reading index, lesion morphology, serum vascular endothelial growth factor (VEGF) levels, and costs.

RESULTS

Between March 27, 2008 and October 15, 2010, we randomized and treated 610 participants. One year after randomization, the comparison between bevacizumab and ranibizumab was inconclusive (bevacizumab minus ranibizumab -1.99 letters, 95% confidence interval [CI], -4.04 to 0.06). Discontinuous treatment was equivalent to continuous treatment (discontinuous minus continuous -0.35 letters; 95% CI, -2.40 to 1.70). Foveal total thickness did not differ by drug, but was 9% less with continuous treatment (geometric mean ratio [GMR], 0.91; 95% CI, 0.86 to 0.97; P = 0.005). Fewer participants receiving bevacizumab had an arteriothrombotic event or heart failure (odds ratio [OR], 0.23; 95% CI, 0.05 to 1.07; P = 0.03). There was no difference between drugs in the proportion experiencing a serious systemic adverse event (OR, 1.35; 95% CI, 0.80 to 2.27; P = 0.25). Serum VEGF was lower with bevacizumab (GMR, 0.47; 95% CI, 0.41 to 0.54; P<0.0001) and higher with discontinuous treatment (GMR, 1.23; 95% CI, 1.07 to 1.42; P = 0.004). Continuous and discontinuous treatment costs were £9656 and £6398 per patient per year for ranibizumab and £1654 and £1509 for bevacizumab; bevacizumab was less costly for both treatment regimens (P<0.0001).

CONCLUSIONS

The comparison of visual acuity at 1 year between bevacizumab and ranibizumab was inconclusive. Visual acuities with continuous and discontinuous treatment were equivalent. Other outcomes are consistent with the drugs and treatment regimens having similar efficacy and safety.

BACKGROUND

Proprietary or commercial disclosures may be found after the references.

Activated EGF receptor (EGFR) plays an oncogenic role in several human malignancies. Although the intracellular effects of EGFR are well studied, its ability to induce and modulate tumor angiogenesis is less understood. We found previously that oncogenic EGFR can be shed from cancer cells as cargo of membrane microvesicles (MVs), which can interact with surfaces of other cells. Here we report that MVs produced by human cancer cells harboring activated EGFR (A431, A549, DLD-1) can be taken up by cultured endothelial cells, in which they elicit EGFR-dependent responses, including activation of MAPK and Akt pathways. These responses can be blocked by annexin V and its homodimer, Diannexin, both of which cloak phosphatidylserine residues on the surfaces of MVs. Interestingly, the intercellular EGFR transfer is also accompanied by the onset of VEGF expression in endothelial cells and by autocrine activation of its key signaling receptor (VEGF receptor-2). In A431 human tumor xenografts in mice, angiogenic endothelial cells stain positively for human EGFR and phospho-EGFR, while treatment with Diannexin leads to a reduction of tumor growth rate and microvascular density. Thus, we propose that oncogene-containing tumor cell-derived MVs could act as a unique form of angiogenesis-modulating stimuli and are capable of switching endothelial cells to act in an autocrine mode.

Angiogenesis does not only depend on endothelial cell invasion and proliferation: it also requires pericyte coverage of vascular sprouts for vessel stabilization. These processes are coordinated by vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) through their cognate receptors on endothelial cells and vascular smooth muscle cells (VSMCs), respectively. PDGF induces neovascularization by priming VSMCs/pericytes to release pro-angiogenic mediators. Although VEGF directly stimulates endothelial cell proliferation and migration, its role in pericyte biology is less clear. Here we define a role for VEGF as an inhibitor of neovascularization on the basis of its capacity to disrupt VSMC function. Specifically, under conditions of PDGF-mediated angiogenesis, VEGF ablates pericyte coverage of nascent vascular sprouts, leading to vessel destabilization. At the molecular level, VEGF-mediated activation of VEGF-R2 suppresses PDGF-Rbeta signalling in VSMCs through the assembly of a previously undescribed receptor complex consisting of PDGF-Rbeta and VEGF-R2. Inhibition of VEGF-R2 not only prevents assembly of this receptor complex but also restores angiogenesis in tissues exposed to both VEGF and PDGF. Finally, genetic deletion of tumour cell VEGF disrupts PDGF-Rbeta/VEGF-R2 complex formation and increases tumour vessel maturation. These findings underscore the importance of VSMCs/pericytes in neovascularization and reveal a dichotomous role for VEGF and VEGF-R2 signalling as both a promoter of endothelial cell function and a negative regulator of VSMCs and vessel maturation.

Inhibition of alphavbeta3 or alphavbeta5 integrin function has been reported to suppress neovascularization and tumor growth, suggesting that these integrins are critical modulators of angiogenesis. Here we report that mice lacking beta3 integrins or both beta3 and beta5 integrins not only support tumorigenesis, but have enhanced tumor growth as well. Moreover, the tumors in these integrin-deficient mice display enhanced angiogenesis, strongly suggesting that neither beta3 nor beta5 integrins are essential for neovascularization. We also observed that angiogenic responses to hypoxia and vascular endothelial growth factor (VEGF) are augmented significantly in the absence of beta3 integrins. We found no evidence that the expression or functions of other integrins were altered as a consequence of the beta3 deficiency, but we did observe elevated levels of VEGF receptor-2 (also called Flk-1) in beta3-null endothelial cells. These data indicate that alphavbeta3 and alphavbeta5 integrins are not essential for vascular development or pathological angiogenesis and highlight the need for further evaluation of the mechanisms of action of alphav-integrin antagonists in anti-angiogenic therapeutics.

Inhibition of tumor angiogenesis through blockade of the vascular endothelial growth factor (VEGF) signaling pathway is a novel treatment modality in oncology. Preclinical findings suggest that long-term clinical outcomes may improve with blockade of additional proangiogenic receptor tyrosine kinases: platelet-derived growth factor receptors (PDGFR) and fibroblast growth factor receptors (FGFR). BIBF 1120 is an indolinone derivative potently blocking VEGF receptor (VEGFR), PDGFR and FGFR kinase activity in enzymatic assays (IC(50), 20-100 nmol/L). BIBF 1120 inhibits mitogen-activated protein kinase and Akt signaling pathways in three cell types contributing to angiogenesis, endothelial cells, pericytes, and smooth muscle cells, resulting in inhibition of cell proliferation (EC(50), 10-80 nmol/L) and apoptosis. In all tumor models tested thus far, including human tumor xenografts growing in nude mice and a syngeneic rat tumor model, BIBF 1120 is highly active at well-tolerated doses (25-100 mg/kg daily p.o.), as measured by magnetic resonance imaging of tumor perfusion after 3 days, reducing vessel density and vessel integrity after 5 days, and inducing profound growth inhibition. A distinct pharmacodynamic feature of BIBF 1120 in cell culture is sustained pathway inhibition (up to 32 hours after 1-hour treatment), suggesting slow receptor off-kinetics. Although BIBF 1120 is rapidly metabolized in vivo by methylester cleavage, resulting in a short mean residence time, once daily oral dosing is fully efficacious in xenograft models. These distinctive pharmacokinetic and pharmacodynamic properties may help explain clinical observations with BIBF 1120, currently entering phase III clinical development.

Vascular endothelial growth factor (VEGF) is a potent mitogen specific for endothelial cells. Its expression is dramatically induced by low oxygen tension in a variety of cell types, and it has been suggested to be a key mediator of hypoxia-induced angiogenesis. Although VEGF action is targeted to endothelial cells, it is generally believed that these cells do not express VEGF. In addition, the mechanisms by which hypoxia regulates VEGF production remain unclear. We report in the present study that pulmonary artery endothelial cells do not express VEGF under basal conditions; however, significant VEGF mRNA levels accumulate when these cells are exposed to hypoxia. Using a DNA fragment containing human VEGF promoter sequence, we identified a 28-bp element that is necessary and sufficient to upregulate transcription in response to hypoxia. This element can act as a hypoxia-specific enhancer when placed upstream or downstream from a heterologous promoter. The enhancer includes, in addition to an octamer homologous to the hypoxia-inducible factor-1 (HIF-1) consensus, a sequence that resides 3' to the consensus. Although this sequence may not be involved in the binding of HIF-1, it is absolutely required for the enhancer activity and may be the binding site for certain constitutive binding proteins. The expression of VEGF by endothelial cells in response to hypoxia may provide an important mechanism by which endothelial cell permeability and proliferation is regulated in an autocrine manner.

The family of vascular endothelial growth factor (VEGF) proteins include potent and specific mitogens for vascular endothelial cells that function in the lation of angiogenesis Inhibition of VEGF-induced angiogenesis either by neutralizing antibodies or dominant-negative soluble receptor, blocks the growth of primary and metastatic experimental tumors Here we report that VEGF expression is induced in Lewis lung carcinomas (LLCs) both in vitro and vivo after exposure to ionizing radiation (IR) and in human tumor cell lines (Seg-1 esophageal adenocarcinoma, SQ20B squamous cell carcinoma, T98 and U87 glioblastomas, and U1 melanoma) in vitro. The biological significance of IR-induced VEGF production is supported by our finding that treatment of tumor-bearing mice (LLC, Seg-1, SQ20B, and U87) with a neutralizing antibody to VEGF-165 before irradiation is associated with a greater than additive antitumor effect. In vitro, the addition of VEGF decreases IR-induced killing of human umbilical vein endothelial cells, and the anti-VEGF treatment potentiates IR-induced lethality of human umbilical vein endothelial cells. Neither recombinant VEGF protein nor neutralizing antibody to VEGF affects the radiosensitivity of tumor cells These findings support a model in which induction of VEGF by IR contributes to the protection of tumor blood vessels from radiation-mediated cytotoxicity and thereby to tumor radioresistance.

The formation and guidance of specialized endothelial tip cells is essential for both developmental and pathological angiogenesis. Notch-1 signalling regulates the generation of tip cells, which respond to gradients of vascular endothelial growth factor (VEGF-A). The molecular cues and signalling pathways that control the guidance of tip cells are poorly understood. Bidirectional signalling by Eph receptors and ephrin ligands represents one of the most important guidance cues involved in axon path finding. Here we show that ephrin-B2 reverse signalling involving PDZ interactions regulates endothelial tip cell guidance to control angiogenic sprouting and branching in physiological and pathological angiogenesis. In vivo, ephrin-B2 PDZ-signalling-deficient mice (ephrin-B2DeltaV) exhibit a reduced number of tip cells with fewer filopodial extensions at the vascular front in the mouse retina. In pathological settings, impaired PDZ signalling decreases tumour vascularization and growth. Mechanistically, we show that ephrin-B2 controls VEGF receptor (VEGFR)-2 internalization and signalling. Importantly, internalization of VEGFR2 is necessary for activation and downstream signalling of the receptor and is required for VEGF-induced tip cell filopodial extension. Together, our results suggest that ephrin-B2 at the tip cell filopodia regulates the proper spatial activation of VEGFR2 endocytosis and signalling to direct filopodial extension. Blocking ephrin-B2 reverse signalling may be an attractive alternative or combinatorial anti-angiogenic therapy strategy to disrupt VEGFR2 function in tumour angiogenesis.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrialized nations, affecting 30-50 million people worldwide. The earliest clinical hallmark of AMD is the presence of drusen, extracellular deposits that accumulate beneath the retinal pigmented epithelium. Although drusen nearly always precede and increase the risk of choroidal neovascularization (CNV), the late vision-threatening stage of AMD, it is unknown whether drusen contribute to the development of CNV. Both in patients with AMD and in a recently described mouse model of AMD, early subretinal pigmented epithelium deposition of complement components C3 and C5 occurs, suggesting a contributing role for these inflammatory proteins in the development of AMD. Here we provide evidence that bioactive fragments of these complement components (C3a and C5a) are present in drusen of patients with AMD, and that C3a and C5a induce VEGF expression in vitro and in vivo. Further, we demonstrate that C3a and C5a are generated early in the course of laser-induced CNV, an accelerated model of neovascular AMD driven by VEGF and recruitment of leukocytes into the choroid. We also show that genetic ablation of receptors for C3a or C5a reduces VEGF expression, leukocyte recruitment, and CNV formation after laser injury, and that antibody-mediated neutralization of C3a or C5a or pharmacological blockade of their receptors also reduces CNV. Collectively, these findings establish a mechanistic basis for the clinical observation that drusen predispose to CNV, revealing a role for immunological phenomena in angiogenesis and providing therapeutic targets for AMD.

Angiogenesis, the formation of new blood vessels, is induced by various growth factors and cytokines that act either directly or indirectly. Vascular endothelial growth factor (VEGF) is a specific mitogen for vascular endothelial cells and therefore has a central role in physiological events of angiogenesis. Interleukin-6 (IL-6) expression on the other hand is elevated in tissues that undergo active angiogenesis but does not induce proliferation of endothelial cells. We demonstrate using Northern analysis that treatment of various cell lines with IL-6 for 6-48 h results in a significant induction of VEGF mRNA. The level of induction is comparable to the documented induction of VEGF mRNA by hypoxia or cobalt chloride, an activator of hypoxia-induced genes. In addition, it is demonstrated by transient transfection assays that the effect of IL-6 is mediated not only by DNA elements at the promoter region but also through specific motif(s) located in the 5'-untranslated region (5'-UTR) of VEGF mRNA. Our results imply that IL-6 may induce angiogenesis indirectly by inducing VEGF expression. It is also shown that the 5'-UTR is important for the expression of VEGF. The 5'-UTR of VEGF is exceptionally long (1038 base pairs) and very rich in G + C. This suggests that secondary structures in the 5'-UTR might be essential for VEGF expression through transcriptional and post-transcriptional control mechanisms.

Vascular endothelial growth factor (VEGF) is a secreted angiogenic mitogen whose target cell specificity appears to be restricted to vascular endothelial cells. Such factors are likely candidates for regulatory molecules involved in endothelial growth control. We have characterized the murine VEGF gene and have analysed its expression pattern in embryogenesis, particularly during brain angiogenesis. Analysis of cDNA clones predicted the existence of three molecular forms of VEGF which differ in size due to heterogeneity at the carboxy terminus of the protein. The predicted mature proteins consist of 120, 164 or 188 amino acid residues. Homodimers of the two lower molecular weight forms, but not of the higher molecular weight form, were secreted by COS cells transfected with the corresponding cDNAs and were equally potent in stimulating the growth of endothelial cells. During brain development, VEGF transcript levels were abundant in the ventricular neuroectoderm of embryonic and postnatal brain when endothelial cells proliferate rapidly but were reduced in the adult when endothelial cell proliferation has ceased. The temporal and spatial expression of VEGF is consistent with the hypothesis that VEGF is synthesized and released by the ventricular neuroectoderm and may induce the ingrowth of capillaries from the perineural vascular plexus. In addition to the transient expression during brain development, a persistent expression of VEGF was observed in epithelial cells adjacent to fenestrated endothelium, e.g. in choroid plexus and in kidney glomeruli. The data are consistent with a role of VEGF as a multifunctional regulator of endothelial cell growth and differentiation.

Inactivation of the von Hippel-Lindau protein (pVHL) has been implicated in the pathogenesis of renal carcinomas and central nervous system hemangioblastomas. These are highly vascular tumors which overproduce angiogenic peptides such as vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). Renal carcinoma cells lacking wild-type pVHL were found to produce mRNAs encoding VEGF/VPF, the glucose transporter GLUT1, and the platelet-derived growth factor B chain under both normoxic and hypoxic conditions. Reintroduction of wild-type, but not mutant, pVHL into these cells specifically inhibited the production of these mRNAs under normoxic conditions, thus restoring their previously described hypoxia-inducible profile. Thus, pVHL appears to play a critical role in the transduction of signals generated by changes in ambient oxygen tension.

OBJECTIVE

To assess long-term outcomes 7 to 8 years after initiation of intensive ranibizumab therapy in exudative age-related macular degeneration (AMD) patients.

METHODS

Multicenter, noninterventional cohort study.

METHODS

Sixty-five AMD patients originally treated with ranibizumab in the phase 3 Anti-VEGF Antibody for the Treatment of Predominantly Classic Choroidal Neovascularization in AMD (ANCHOR) trial, Minimally Classic/Occult Trial of the Anti-VEGF Antibody Ranibizumab in the Treatment of Neovascular AMD (MARINA) trial, and Open-Label Extension Trial of Ranibizumab for Choroidal Neovascularization Secondary to Age-Related Macular Degeneration (HORIZON).

METHODS

Fourteen clinical trial sites recruited their original subjects for a return evaluation. Individual subject comparisons were obtained from the ANCHOR, MARINA, and HORIZON databases.

METHODS

The primary end point was percentage with best-corrected visual acuity (BCVA) of 20/70 or better; secondary outcomes included mean change in letter score compared with previous time points and anatomic results on fluorescein angiography, spectral-domain ocular coherence tomography (OCT), and fundus autofluorescence (FAF).

RESULTS

At a mean of 7.3 years (range, 6.3-8.5 years) after entry into ANCHOR or MARINA, 37% of study eyes met the primary end point of 20/70 or better BCVA, with 23% achieving a BCVA of 20/40 or better. Thirty-seven percent of study eyes had BCVA of 20/200 or worse. Forty-three percent of study eyes had a stable or improved letter score (≥0-letter gain) compared with ANCHOR or MARINA baseline measurements, whereas 34% declined by 15 letters or more, with overall a mean decline of 8.6 letters (P<0.005). Since exit from the HORIZON study, study eyes had received a mean of 6.8 anti-vascular endothelial growth factor (VEGF) injections during the mean 3.4-year interval; a subgroup of patients who received 11 or more anti-VEGF injections had a significantly better mean gain in letter score since HORIZON exit (P<0.05). Active exudative disease was detected by spectral-domain OCT in 68% of study eyes, and 46% were receiving ongoing ocular anti-VEGF treatments. Macular atrophy was detected by FAF in 98% of eyes, with a mean area of 9.4 mm(2); the area of atrophy correlated significantly with poor visual outcome (P<0.0001).

CONCLUSIONS

Approximately 7 years after ranibizumab therapy in the ANCHOR or MARINA trials, one third of patients demonstrated good visual outcomes, whereas another third had poor outcomes. Compared with baseline, almost half of eyes were stable, whereas one third declined by 15 letters or more. Even at this late stage in the therapeutic course, exudative AMD patients remain at risk for substantial visual decline.

No validated biological markers (or biomarkers) currently exist for appropriately selecting patients with cancer for antiangiogenic therapy. Nor are there biomarkers identifying escape pathways that should be targeted after tumors develop resistance to a given antiangiogenic agent. A number of potential systemic, circulating, tissue and imaging biomarkers have emerged from recently completed phase I-III studies. Some of these are measured at baseline (for example VEGF polymorphisms), others are measured during treatment (such as hypertension, MRI-measured K(trans), circulating angiogenic molecules or collagen IV), and all are mechanistically based. Some of these biomarkers might be pharmacodynamic (for example, increase in circulating VEGF, placental growth factor) while others have potential for predicting clinical benefit or identifying the escape pathways (for example, stromal-cell-derived factor 1alpha, interleukin-6). Most biomarkers are disease and/or agent specific and all of them need to be validated prospectively. We discuss the current challenges in establishing biomarkers of antiangiogenic therapy, define systemic, circulating, tissue and imaging biomarkers and their advantages and disadvantages, and comment on the future opportunities for validating biomarkers of antiangiogenic therapy.

A mouse model involving the human papillomavirus type-16 oncogenes develops cervical cancers by lesional stages analogous to those in humans. In this study the angiogenic phenotype was characterized, revealing intense angiogenesis in high-grade cervical intraepithelial neoplasias (CIN-3) and carcinomas. MMP-9, a proangiogenic protease implicated in mobilization of VEGF, appeared in the stroma concomitant with the angiogenic switch, expressed by infiltrating macrophages, similar to what has been observed in humans. Preclinical trials sought to target MMP-9 and angiogenesis with a prototypical MMP inhibitor and with a bisphosphonate, zoledronic acid (ZA), revealing both to be antiangiogenic, producing effects comparable to a Mmp9 gene KO in impairing angiogenic switching, progression of premalignant lesions, and tumor growth. ZA therapy increased neoplastic epithelial and endothelial cell apoptosis without affecting hyperproliferation, indicating that ZA was not antimitotic. The analyses implicated cellular and molecular targets of ZA's actions: ZA suppressed MMP-9 expression by infiltrating macrophages and inhibited metalloprotease activity, reducing association of VEGF with its receptor on angiogenic endothelial cells. Given its track record in clinical use with limited toxicity, ZA holds promise as an "unconventional" MMP-9 inhibitor for antiangiogenic therapy of cervical cancer and potentially for additional cancers and other diseases where MMP-9 expression by infiltrating macrophages is evident.

KDR/Flk-1 tyrosine kinase, one of the two vascular endothelial growth factor (VEGF) receptors, induces mitogenesis and differentiation of vascular endothelial cells. To understand the mechanisms underlying the VEGF-A-induced growth signaling pathway, we constructed a series of human KDR mutants and examined their biological properties. An in vitro kinase assay and subsequent tryptic peptide mapping revealed that Y1175 and Y1214 are the two major VEGF-A-dependent autophosphorylation sites. Using an antibody highly specific to the phosphoY1175 region, we demonstrated that Y1175 is phosphorylated rapidly in vivo in primary endothelial cells. When the mutated KDRs were introduced into the endothelial cell lines by adenoviral vectors, only the Y1175F KDR, Tyr1175 to phenylalanine mutant, lost the ability to tyrosine phosphorylate phospholipase C-gamma and, significantly, reduced MAP kinase phosphorylation and DNA synthesis in response to VEGF-A. Furthermore, primary endothelial cells microinjected with anti-phosphoY1175 antibody clearly decreased DNA synthesis compared with control cells. These findings strongly suggest that autophosphorylation of Y1175 on KDR is crucial for endothelial cell proliferation, and that this region is a new target for anti-angiogenic reagents.

Patients with recurrent malignant glioma treated with bevacizumab, a monoclonal antibody to vascular endothelial growth factor (VEGF), alone or in combination with irinotecan have had impressive reductions in MRI contrast enhancement and vasogenic edema. Responses to this regimen, as defined by a decrease in contrast enhancement, have led to significant improvements in progression-free survival rates but not in overall survival duration. Some patients for whom this treatment regimen fails have an uncharacteristic pattern of tumor progression, which can be observed radiographically as an increase in hyperintensity on T2-weighted or fluid-attenuated inverse recovery (FLAIR) MRI. To date, there have been no reports of paired correlations between radiographic results and histopathologic findings describing the features of this aggressive tumor phenotype. In this study, we correlate such findings for 3 illustrative cases of gliomas that demonstrated an apparent phenotypic shift to a predominantly infiltrative pattern of tumor progression after treatment with bevacizumab. Pathologic examination of abnormal FLAIR areas on MRI revealed infiltrative tumor with areas of thin-walled blood vessels, suggesting vascular "normalization," which was uncharacteristically adjacent to regions of necrosis. High levels of insulin-like growth factor binding protein-2 and matrix metalloprotease-2 expression were seen within the infiltrating tumor. In an attempt to better understand this infiltrative phenotype associated with anti-VEGF therapy, we forced a highly angiogenic, noninvasive orthotopic U87 xenograft tumor to become infiltrative by treating the mice with bevacizumab. This model mimicked many of the histopathologic findings from the human cases and will augment the discovery of alternative or additive therapies to prevent this type of tumor recurrence in clinical practice.

Vascular development depends on the highly coordinated actions of a variety of angiogenic regulators, most of which apparently act downstream of vascular endothelial growth factor (VEGF). One potential such regulator is delta-like 4 ligand (Dll4), a recently identified partner for the Notch receptors. We generated mice in which the Dll4 gene was replaced with a reporter gene, and found that Dll4 expression is initially restricted to large arteries in the embryo, whereas in adult mice and tumor models, Dll4 is specifically expressed in smaller arteries and microvessels, with a striking break in expression just as capillaries merge into venules. Consistent with these arterial-specific expression patterns, heterozygous deletion of Dll4 resulted in prominent albeit variable defects in arterial development (reminiscent of those in Notch knockouts), including abnormal stenosis and atresia of the aorta, defective arterial branching from the aorta, and even arterial regression, with occasional extension of the defects to the venous circulation; also noted was gross enlargement of the pericardial sac and failure to remodel the yolk sac vasculature. These striking phenotypes resulting from heterozygous deletion of Dll4 indicate that vascular development may be as sensitive to subtle changes in Dll4 dosage as it is to subtle changes in VEGF dosage, because VEGF accounts for the only other example of haploid insufficiency, resulting in obvious vascular abnormalities. In summary, Dll4 appears to be a major trigger of Notch receptor activities previously implicated in arterial and vascular development, and it may represent a new opportunity for pro- and anti-angiogenic therapies.

Vascular endothelial growth factor receptor-3 (VEGFR-3/Flt4) binds two known members of the VEGF ligand family, VEGF-C and VEGF-D, and has a critical function in the remodelling of the primary capillary vasculature of midgestation embryos. Later during development, VEGFR-3 regulates the growth and maintenance of the lymphatic vessels. In the present study, we have isolated and cultured stable lineages of blood vascular and lymphatic endothelial cells from human primary microvascular endothelium by using antibodies against the extracellular domain of VEGFR-3. We show that VEGFR-3 stimulation alone protects the lymphatic endothelial cells from serum deprivation-induced apoptosis and induces their growth and migration. At least some of these signals are transduced via a protein kinase C-dependent activation of the p42/p44 MAPK signalling cascade and via a wortmannin-sensitive induction of Akt phosphorylation. These results define the critical role of VEGF-C/VEGFR-3 signalling in the growth and survival of lymphatic endothelial cells. The culture of isolated lymphatic endothelial cells should now allow further studies of the molecular properties of these cells.

Evidence accumulating over the last decade has established the fundamental role of vascular endothelial growth factor (VEGF) as a key regulator of normal and abnormal angiogenesis. The biological effects of VEGF are mediated by two tyrosine kinase receptors, Flt-1 (VEGFR-1) and KDR (VEGFR-2). The signaling and biological properties of these two receptors are strikingly different. VEGF is essential for early development of the vasculature to the extent that inactivation of even a single allele of the VEGF gene results in embryonic lethality. VEGF is also required for female reproductive functions and endochondral bone formation. Substantial evidence also implicates VEGF as an angiogenic mediator in tumors and intraocular neovascular syndromes, and numerous clinical trials are presently testing the hypothesis that inhibition of VEGF may have therapeutic value.