Vascular Endothelial Growth factor-A Signaling Research Articles

WT1-Dependent Sulfatase Expression Maintains the Normal Glomerular Filtration Barrier

Paracrine signaling between podocytes and glomerular endothelial cells through vascular endothelial growth factor A (VEGFA) maintains a functional glomerular filtration barrier. Heparan sulfate proteoglycans (HSPGs), located on the cell surface or in the extracellular matrix, bind signaling molecules such as VEGFA and affect their local concentrations, but whether modulation of these moieties promotes normal crosstalk between podocytes and endothelial cells is unknown. Here, we found that the transcription factor Wilms' Tumor 1 (WT1) modulates VEGFA and FGF2 signaling by increasing the expression of the 6-O-endosulfatases Sulf1 and Sulf2, which remodel the heparan sulfate 6-O-sulfation pattern in the extracellular matrix. Mice deficient in both Sulf1 and Sulf2 developed age-dependent proteinuria as a result of ultrastructural abnormalities in podocytes and endothelial cells, a phenotype similar to that observed in children with WT1 mutations and in Wt1(+/-) mice. These kidney defects associated with a decreased distribution of VEGFA in the glomerular basement membrane and on endothelial cells. Collectively, these data suggest that WT1-dependent sulfatase expression plays a critical role in maintaining the glomerular filtration barrier by modulating the bioavailability of growth factors, thereby promoting normal crosstalk between podocytes and endothelial cells.

Complex receptor-ligand dynamics control the response of the VEGF system to protease injury

BackgroundVascular homeostasis and response to injury are dependent on the coordinated activity of growth factors such as vascular endothelial growth factor-A (VEGF). VEGF signaling is mediated by VEGF receptors 1 (VEGFR1) and 2 (VEGFR2). VEGF also binds to extracellular matrix (ECM) and neuropilin (NP), a cell surface glycoprotein that enhances VEGF binding to VEGFR2 while inhibiting VEGF-VEGFR1 interactions. Proteases such as neutrophil elastase release VEGF bound to ECM; however, this results in proteolytic processing of VEGF to a smaller species termed VEGF fragment (VEGFf). We hypothesized that the generation and presence of VEGFf would have significant effects on the binding distribution of VEGF.ResultsWe show that VEGFf, unlike VEGF, does not bind ECM, fibronectin, or NP-1. Using computational simulations, we find that excess VEGFf can lead to increased binding of VEGF to VEGFR2 through VEGFf binding to VEGFR1 and subsequent liberation of NP-1. We show experimentally that VEGF-induced migration has a biphasic response to conversion of VEGF to VEGFf. Simulations suggest that a simple change in VEGFR1 or VEGFR2 complexes are unlikely to be responsible and that a more complex integration of signals is more likely involved.ConclusionsThese findings suggest that proteolytic damage at sites of tissue injury and inflammation has the potential to modulate the VEGF system through a complex process and highlight the need for quantitative analysis to reveal mechanisms of growth factor control.

Epithelial: Endothelial cross-talk regulates exocrine differentiation in developing pancreas

Endothelial cells are required to initiate pancreas development from the endoderm. They also control the function of endocrine islets after birth. Here we investigate in developing pancreas how the endothelial cells become organized during branching morphogenesis and how their development affects pancreatic cell differentiation. We show that endothelial cells closely surround the epithelial bud at the onset of pancreas morphogenesis. During branching morphogenesis, the endothelial cells become preferentially located near the central (trunk) epithelial cells and remain at a distance from the branch tips where acinar cells differentiate. This correlates with predominant expression of the angiogenic factor vascular endothelial growth factor-A (VEGF-A) in trunk cells. In vivo ablation of VEGF-A expression by pancreas-specific inactivation of floxed Vegfa alleles results in reduced endothelial development and in excessive acinar differentiation. On the contrary, acinar differentiation is repressed when endothelial cells are recruited around tip cells that overexpress VEGF-A. Treatment of embryonic day 12.5 explants with VEGF-A or with VEGF receptor antagonists confirms that acinar development is tightly controlled by endothelial cells. We also provide evidence that endothelial cells repress the expression of Ptf1a, a transcription factor essential for acinar differentiation, and stimulate the expression of Hey-1 and Hey-2, two repressors of Ptf1a activity. In explants, we provide evidence that VEGF-A signaling is required, but not sufficient, to induce endocrine differentiation. In conclusion, our data suggest that, in developing pancreas, epithelial production of VEGF-A determines the spatial organization of endothelial cells which, in turn, limit acinar differentiation of the epithelium.

Intersegmental vessel formation in zebrafish: requirement for VEGF but not BMP signalling revealed by selective and non-selective BMP antagonists.

Bone morphogenetic proteins (BMPs) were first identified through their role in inducing bone and cartilage formation, but many other important functions have since been ascribed to BMPs, including dorsoventral patterning, angiogenesis and tissue homeostasis. Using dorsomorphin and LDN193189, selective small molecule inhibitors of BMP signalling, we investigated the role of BMP signalling in early vascular patterning in zebrafish. The effects of dorsomorphin and LDN193189 on vascular endothelial growth factor-a (VEGF) and BMP signalling in developing zebrafish and in human pulmonary artery endothelial cells were determined using confocal microscopy, Western blotting and quantitative PCR. We showed that dorsomorphin, similar to the VEGF inhibitor SU5416, strongly inhibits intersegmental vessel formation in zebrafish and that this is due to inhibition of VEGF activation of VEGF receptor 2 (VEGFR2), leading to reduced VEGF-induced phospho-ERK (extracellular regulated kinase) 1/2 and VEGF target gene transcription. These effects occurred at concentrations of dorsomorphin that block BMP signalling. We also showed that LDN193189, an analogue of dorsomorphin, more potently blocks BMP signalling but has no effect on VEGF signalling in zebrafish and does not disrupt early vascular patterning. Dorsomorphin inhibits both BMP and VEGF signalling, whereas LDN193189 is a more selective BMP antagonist. Results obtained in cardiovascular studies using dorsomorphin need to be interpreted with caution, and use of LDN193189 would be preferable due to its selectivity. Our data also suggest that BMP signalling is dispensable for early patterning of intersegmental vessels in zebrafish.

Global lymphoid tissue remodeling during a viral infection is orchestrated by a B cell–lymphotoxin-dependent pathway

AbstractAdaptive immune responses are characterized by substantial restructuring of secondary lymphoid organs. The molecular and cellular factors responsible for virus-induced lymphoid remodeling are not well known to date. Here we applied optical projection tomography, a mesoscopic imaging technique, for a global analysis of the entire 3-dimensional structure of mouse peripheral lymph nodes (PLNs), focusing on B-cell areas and high endothelial venule (HEV) networks. Structural homeostasis of PLNs was characterized by a strict correlation between total PLN volume, B-cell volume, B-cell follicle number, and HEV length. After infection with lymphocytic choriomeningitis virus, we observed a substantial, lymphotoxin (LT) β-receptor–dependent reorganization of the PLN microarchitecture, in which an initial B-cell influx was followed by 3-fold increases in PLN volume and HEV network length on day 8 after infection. Adoptive transfer experiments revealed that virus-induced PLN and HEV network remodeling required LTα1β2-expressing B cells, whereas the inhibition of vascular endothelial growth factor-A signaling pathways had no significant effect on PLN expansion. In summary, lymphocytic choriomeningitis virus-induced PLN growth depends on a vascular endothelial growth factor-A–independent, LT- and B cell–dependent morphogenic pathway, as revealed by an in-depth mesoscopic analysis of the global PLN structure.

VEGF and Pleiotrophin Modulate the Immune Profile of Breast Cancer

Angiogenesis, the sprouting of the existing vascular network to form new vessels, is required for the growth of solid tumors. For this reason, the primary stimulant of angiogenesis, vascular endothelial growth factor-A (VEGF), is an attractive target for tumor therapy. In fact, there are currently numerous anti-VEGF therapies in clinical development for the treatment of various cancers, including breast cancer. VEGF signals through two primary VEGF receptors, VEGFR1 and VEGFR2. VEGFR2 is the primary angiogenic receptor, and VEGFR1 has been implicated in macrophage chemotaxis and tumor cell survival and invasion. It has only been appreciated recently that the VEGFRs are expressed not only on endothelial cells and tumor cells but also on many host immune cells. Therefore, to better understand the effects of anti-VEGF therapy it is important to consider the effects of VEGF on all cells in the tumor microenvironment, including immune cells. Bevacizumab (Avastin®, Genetech), which binds VEGF and inhibits interaction with VEGFR1 and VEGFR2, was approved for the treatment of metastatic HER2/NEU-negative breast cancer in 2008, however, the majority of human mammary tumors are either innately resistant or will acquire resistance to anti-VEGF therapy. This suggests that these tumors activate alternate angiogenesis pathways. Pleiotrophin (PTN) is an important angiogenic cytokine in breast cancer and is expressed at high levels in approximately 60% of human breast tumors. PTN functions as an angiogenic factor and promotes remodeling of the tumor microenvironment as well as epithelial-mesenchymal transition (EMT). In addition, PTN can have profound effects on macrophage phenotype. The present review focuses on the functions of VEGF and PTN on immune cell infiltration and function in breast cancer. Furthermore, we will discuss how anti-VEGF therapy modulates the immune cell profile.

VEGF-A Links Angiogenesis and Inflammation in Inflammatory Bowel Disease Pathogenesis

Vascular endothelial growth factor A (VEGF-A) mediates angiogenesis and might also have a role in inflammation and immunity. We examined whether VEGF-A signaling has a role in inflammatory bowel disease (IBD). Expression levels of VEGF-A, and its receptors VEGFR-1 and VEGFR-2, were examined in samples from patients with IBD and compared with those of controls. The capacity of VEGF-A to induce angiogenesis was tested in human intestinal microvascular endothelial cells using cell-migration and matrigel tubule-formation assays. Levels of vascular cellular adhesion molecule-1 and intercellular adhesion molecule were measured by flow cytometry to determine induction of inflammation; neutrophil adhesion was also assayed. Expression patterns were determined in tissues from mice with dextran sulfate sodium (DSS)-induced colitis; the effects of VEGF-A overexpression and blockade were assessed in these mice by adenoviral transfer of VEGF-A and soluble VEGFR-1. Intestinal angiogenesis was measured by quantitative CD31 staining and leukocyte adhesion in vivo by intravital microscopy. Levels of VEGF-A and VEGFR-2 increased in samples from patients with IBD and colitic mice. VEGF-A induced angiogenesis of human intestinal microvascular endothelial cells in vitro as well as an inflammatory phenotype and adherence of neutrophils to intestinal endothelium. Overexpression of VEGF-A in mice with DSS-induced colitis worsened their condition, whereas overexpression of soluble VEGFR-1 had the opposite effect. Furthermore, overexpression of VEGF-A increased mucosal angiogenesis and stimulated leukocyte adhesion in vivo. VEGF-A appears to be a novel mediator of IBD by promoting intestinal angiogenesis and inflammation. Agents that block VEGF-A signaling might reduce intestinal inflammation in patients with IBD.

Inhibition of Vascular Endothelial Growth Factor-A Signaling Induces Hypertension: Examining the Effect of Cediranib (Recentin; AZD2171) Treatment on Blood Pressure in Rat and the Use of Concomitant Antihypertensive Therapy

Inhibition of vascular endothelial growth factor-A (VEGF) signaling is a key therapeutic approach in oncology given the role of VEGF in angiogenesis and vascular permeability in solid tumors. Clinical trials examining VEGF signaling inhibitors commonly report hypertension. We examined the effect of cediranib, a highly potent VEGF signaling inhibitor, on the blood pressure of rats and the ability of standard antihypertensive agents to modulate the consequences of VEGF signaling inhibition. The ability of cediranib to induce hypertensive changes and the effect of giving antihypertensive therapy were investigated in conscious, unrestrained telemetered rats. Two antihypertensive agents were studied: captopril, an angiotensin-converting enzyme inhibitor, and nifedipine, a dihydropyridine calcium channel blocker. The antitumor activity of cediranib, alone and in combination with nifedipine, was also evaluated in a LoVo human colorectal tumor xenograft model in nude rats. All treatments were given orally. Administration of 0.1 to 1.5 mg/kg/d of cediranib for 4 consecutive days induced a relatively mild hypertensive effect, elevating diastolic blood pressure by 10 to 14 mmHg. Dosing 3 mg/kg/d cediranib for 4 days induced a marked hypertension of 35 to 50 mmHg. Captopril (30 mg/kg, qd) was effective at lowering a 10 mmHg increase in blood pressure but not a 35 to 50 mmHg increase. However, the latter was rapidly reversed by administration of nifedipine (10 mg/kg, bd). Coadministration of nifedipine did not negatively affect the antitumor activity of cediranib (1.5 mg/kg/d). Hypertension is a direct consequence of inhibiting VEGF signaling but can be controlled with appropriately selected, standard antihypertensive medication.

An Open, Phase I Study of Cediranib in Patients with Acute Myeloid Leukemia (AML).

Prognosis is poor for the majority of AML patients, particularly the elderly and those with relapsed or refractory AML, and more effective treatment options are needed. Vascular endothelial growth factor-A (VEGF) and its receptors (VEGFRs) may contribute to the pathophysiology of AML and are considered as potential therapeutic targets. Cediranib (RECENTIN™; AZD2171) is an oral, highly potent and selective VEGF signaling inhibitor with activity versus VEGFR-1, -2 and -3, and additional activity against c-Kit. This two-part study evaluated the safety and tolerability of multiple once-daily oral doses of cediranib in patients with relapsed or refractory AML and elderly patients with de novo or secondary AML, with secondary assessments of pharmacokinetics (PK), pharmacodynamics (PD) and efficacy. Part A was a dose-escalation phase to determine the maximum tolerated dose (MTD); Part B was an expansion phase to explore the MTD and a lower, biologically active dose. A total of 35 patients (mean age, 68 years [range 50–81]; 15 male, 20 female) entered the study and received treatment with cediranib (Part A, n=23; Part B, n=12). Part A comprised 10mg (n=4), 20mg (n=6) and 30mg (n=13) dose cohorts, and intra-patient dose escalation was permitted. In Part B, patients received cediranib 20mg (n=3) or 30mg (n=9). Cediranib ≤30mg was generally well tolerated; diarrhea (n=19), hypertension (n=14) and fatigue (n=13) were the most common adverse events (AEs). Cediranib 30mg was the highest dose studied, and was declared the MTD based on dose-limiting toxicities of hypertension and diarrhea experienced by 3 patients receiving 30mg in Part A. Maximal plasma concentrations at steady-state were attained 0.83–4.1 h post-dosing. Steady-state plasma concentrations were attained after approximately 7 days of repeated once-daily dosing. Following multiple oral doses of cediranib 30mg in Parts A and B, the unbound geometric mean Css,min was 5x the IC50 value required to inhibit proliferation of human umbilical vein endothelial cells in preclinical assays, supporting the once-daily oral dosing regimen. The relationship between PK and two PD parameters (VEGF levels and blood pressure) were investigated and there appeared to be a positive correlation between exposure (Css,max and AUCss) and plasma VEGF levels, but not blood pressure. Time-dependent changes in soluble VEGFR-2 levels were observed. A best investigator assessment of AML objective response according to predefined criteria (Cheson et al, J Clin Oncol 2003; 21:4642–9) was observed for 6 patients (1 morphological complete remission with incomplete blood recovery [Cri], 4 partial responses and 1 minor response). However, none of the responses resulted in improvement in normal hemopoiesis. No responses were observed in 6 patients with Flt-3 ITD mutations. One patient remained on treatment for 455 days. Once-daily oral cediranib ≤30mg was generally well tolerated in patients with AML, with encouraging preliminary evidence of activity as a monotherapy. The AE and PK profiles of cediranib in AML are consistent with those seen in solid tumors.

Regulation of embryonic lung vascular development by vascular endothelial growth factor receptors, Flk‐1 and Flt‐1

The biological effects of vascular endothelial growth factor A (VEGF-A) are mediated by fetal liver kinase-1 (Flk-1) and fms-like tyrosine kinase-1 (Flt-1). In lung tissue, VEGF-A is diffusely expressed throughout the embryonic stages, whereas the development of vascular endothelial cells is not uniform. Noting the signaling properties of the two receptors, we hypothesized that Flk-1 and Flt-1 regulate the embryonic development of lung vasculature. We herein show the spatiotemporal expression and experimental inhibition of Flk-1 and Flt-1 of embryonic mouse lung tissue. When Flk-1 was predominantly expressed (embryonic day [E] 9.5-E13.5), then vascular endothelial cells actively proliferated. When Flt-1 was enhanced (E14.5-E16.5), these cells less actively proliferated, thereby constituting organized networks. The treatment of cultured lung buds (E11.5) with antisense oligonucleotides complementary to Flk-1 inhibited branching of capillaries and proliferation of endothelial cells. In contrast, the inhibition of Flt-1 promoted the branching of capillaries and enhanced proliferation of endothelial cells. Of interest, inhibition of Flt-1 promoted Flk-1 expression. These results suggest that the two VEGF-A receptors regulate pulmonary vascular development by modulating the VEGF-A signaling.

Nerve‐Vessel Interactions During Vertebrate Development

During vertebrate development, the nervous and vascular systems develop similar anatomical architecture and branching pattern but functionally distinct. We are interested in cellular and molecular mechanisms by which these two systems coordinate their development. We have found that in the embryonic skin, peripheral nerves control the patterns of blood vessel branching and arterial differentiation. As a result, the arteries run along the nerves. The facts that nerves secrete vascular endothelial growth factor-A (VEGF)-A which stimulates arterial differentiation in vitro, and neuropilin-1 (NRP1), a co-receptor for VEGF-A, is preferentially expressed by artery, suggest that nerve-derived VEGF-A may function to induce arteries via NRP1. Using a nerve-specific deletion of Vegf-A and endothelial cell specific deletion of Nrp1 in mice, we have shown that nerve-VEGF-A signal is required for the nerve-dependent arterial differentiation in vivo. Surprisingly, nerve-vessel alignment occurs normally in these conditional mutants, despite the defect in arterial differentiation. This genetic dissociation of arterial differentiation from nerve-vessel alignment, by inhibition of the VEGF signals, suggests that the alignment and patterning process may require nerve-derived signals distinct from VEGF.

Tetralogy of Fallot and Alterations in Vascular Endothelial Growth Factor-A Signaling and Notch Signaling in Mouse Embryos Solely Expressing the VEGF120 Isoform

The importance of vascular endothelial growth factor-A (VEGF) and subsequent Notch signaling in cardiac outflow tract development is generally recognized. Although genetic heterogeneity and mutations of these genes in both humans and mouse models relate to a high susceptibility to develop outflow tract malformations such as tetralogy of Fallot and peripheral pulmonary stenosis, no etiology has been proposed so far. Using immunohistochemistry, in situ hybridization, and quantitative RT-PCR on embryonic hearts, we have shown spatiotemporal increase and abnormal patterning of Vegf/VEGF/(phosphorylated) VEGFR-2, (cleaved) Notch1, and Jagged2 in the outflow tract of Vegf120/120 mouse embryos. This coincides with hyperplasia of specifically the outflow tract cushions and a high degree of subpulmonary myocardial apoptosis that, in later stages, manifest as pulmonary stenosis and ventricular septal defects. We postulate that increase of VEGF and Notch signaling during right ventricular outflow tract development can lead to abnormal development of both cushion and myocardial structures. Defective right ventricular outflow tract development as presented provides new insight in the etiology of tetralogy of Fallot.

Molecular mechanisms controlling the coupled development of myocardium and coronary vasculature

Cardiac failure affects 1.5% of the adult population and is predominantly caused by myocardial dysfunction secondary to coronary vascular insufficiency. Current therapeutic strategies improve prognosis only modestly, as the primary cause -- loss of normally functioning cardiac myocytes -- is not being corrected. Adult cardiac myocytes are unable to divide and regenerate to any significant extent following injury. New cardiac myocytes are, however, created during embryogenesis from progenitor cells and then by cell division from existing cardiac myocytes. This process is intimately linked to the development of coronary vasculature from progenitors originating in the endothelium, the proepicardial organ and neural crest. In this review, we systematically evaluate approx. 90 mouse mutations that impair heart muscle growth during development. These studies provide genetic evidence for interactions between myocytes, endothelium and cells derived from the proepicardial organ and the neural crest that co-ordinate myocardial and coronary vascular development. Conditional knockout and transgenic rescue experiments indicate that Vegfa, Bmpr1a (ALK3), Fgfr1/2, Mapk14 (p38), Hand1, Hand2, Gata4, Zfpm2 (FOG2), Srf and Txnrd2 in cardiac myocytes, Rxra and Wt1 in the proepicardial organ, EfnB2, Tek, Mapk7, Pten, Nf1 and Casp8 in the endothelium, and Bmpr1a and Pax3 in neural crest cells are key molecules controlling myocardial development. Coupling of myocardial and coronary development is mediated by BMP (bone morphogenetic protein), FGF (fibroblast growth factor) and VEGFA (vascular endothelial growth factor A) signalling, and also probably involves hypoxia. Pharmacological targeting of these molecules and pathways could, in principle, be used to recreate the embryonic state and achieve coupled myocardial and coronary vascular regeneration in failing hearts.

Inhibition of Platelet-Derived Growth Factor B Signaling Enhances the Efficacy of Anti-Vascular Endothelial Growth Factor Therapy in Multiple Models of Ocular Neovascularization

'Vascular endothelial growth factor-A (VEGF-A) blockade has been recently validated as an effective strategy for the inhibition of new blood vessel growth in cancer and ocular pathologies. However, several studies have also shown that anti-VEGF therapy may not be as effective in the treatment of established unwanted blood vessels, suggesting they may become less dependent on VEGF-A for survival. The VEGF-A dependence of vessels may be related to the presence of vascular mural cells (pericytes or smooth muscle cells). Mural cell recruitment to the growing endothelial tube is regulated by platelet-derived growth factor-B (PDGF-B) signaling, and interference with this pathway causes disruption of endothelial cell-mural cell interactions and loss of mural cells. We have investigated the basis of blood vessel dependence on VEGF-A in models of corneal and choroidal neovascularization using a combination of reagents (an anti-VEGF aptamer and an anti-PDGFR-beta antibody) to inhibit both the VEGF-A and PDGF-B signaling pathways. We demonstrate that neovessels become refractory to VEGF-A deprivation over time. We also show that inhibition of both VEGF-A and PDGF-B signaling is more effective than blocking VEGF-A alone at causing vessel regression in multiple models of neovascular growth. These findings provide insight into blood vessel growth factor dependency and validate a combination therapy strategy for enhancing the current treatments for ocular angiogenic disease.

VEGF-A signaling through Flk-1 is a critical facilitator of early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis

Vascular endothelial growth factor-A (VEGF-A) signaling directs both vasculogenesis and angiogenesis. However, the role of VEGF-A ligand signaling in the regulation of epithelial–mesenchymal interactions during early mouse lung morphogenesis remains incompletely characterized. Fetal liver kinase-1 (Flk-1) is a VEGF cognate receptor (VEGF-R2) expressed in the embryonic lung mesenchyme. VEGF-A, expressed in the epithelium, is a high affinity ligand for Flk-1. We have used both gain and loss of function approaches to investigate the role of this VEGF-A signaling pathway during lung morphogenesis. Herein, we demonstrate that exogenous VEGF 164, one of the 3 isoforms generated by alternative splicing of the Vegf-A gene, stimulates mouse embryonic lung branching morphogenesis in culture and increases the index of proliferation in both epithelium and mesenchyme. In addition, it induces differential gene and protein expression among several key lung morphogenetic genes, including up-regulation of BMP-4 and Sp-c expression as well as an increase in Flk-1-positive mesenchymal cells. Conversely, embryonic lung culture with an antisense oligodeoxynucleotide (ODN) to the Flk-1 receptor led to reduced epithelial branching, decreased epithelial and mesenchymal proliferation index as well as downregulating BMP-4 expression. These results demonstrate that the VEGF pathway is involved in driving epithelial to endothelial crosstalk in embryonic mouse lung morphogenesis.

Retinal blood vessels develop in response to local VEGF-A signals in the absence of blood flow

The role of hemodynamic forces and other signals from circulating blood in guiding the development of the retinal vasculature was examined by following the growth of these vessels in organ cultures. Retinal vascular development in organ cultures was monitored by immunofluorescent staining of retinal whole-mounts using antibodies against ICAM-2, a specific marker for endothelial cells and by vascular adenosine disphosphatase activity. Under culture conditions, the retinal vasculature from mice at postnatal day 3 (P3) grew from the optic nerve area to the edge of the retina in a manner similar to that observed in vivo. Both inner and outer vascular plexuses formed in retinal explants. Within the first few days of organ culture, the initial uniform meshwork of blood vessels was reorganized into arterioles, venules, and capillaries. As in animals, the initial retinal vascular plexus contained abundant vessels, and afterward some vessels regressed leading to the formation of a mature vascular bed. Changes in vascular density due to blood vessel growth and remodeling were confirmed by RT-PCR and Western blot analyses of ICAM-2 mRNA and protein levels, respectively. In addition, during in vitro retinal vascularization, arterioles acquired mural cell coverage, as shown by positive staining for α-smooth muscle actin. Thus, blood flow and blood-derived signals were not required for the development and maturation of retinal vessels. In contrast, stability of blood vessels in retinal explants was tightly regulated by endogenous levels of vascular endothelial growth factor-A (VEGF-A). VEGF-A was expressed in the explants throughout the culture period, and addition of neutralizing antibodies against VEGF-A to the organ culture caused a severe regression of blood vessels from the vascular front toward the optic nerve. In contrast, addition of anti-FGF-2 antibodies had no effect on the developing vasculature. Thus, retinal vascular development is dependent on local VEGF-A signals rather than systemic signals.

AZD2171: A Highly Potent, Orally Bioavailable, Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase Inhibitor for the Treatment of Cancer

Inhibition of vascular endothelial growth factor-A (VEGF) signaling is a promising therapeutic approach that aims to stabilize the progression of solid malignancies by abrogating tumor-induced angiogenesis. This may be accomplished by inhibiting the kinase activity of VEGF receptor-2 (KDR), which has a key role in mediating VEGF-induced responses. The novel indole-ether quinazoline AZD2171 is a highly potent (IC50 < 1 nmol/L) ATP-competitive inhibitor of recombinant KDR tyrosine kinase in vitro. Concordant with this activity, in human umbilical vein endothelial cells, AZD2171 inhibited VEGF-stimulated proliferation and KDR phosphorylation with IC50 values of 0.4 and 0.5 nmol/L, respectively. In a fibroblast/endothelial cell coculture model of vessel sprouting, AZD2171 also reduced vessel area, length, and branching at subnanomolar concentrations. Once-daily oral administration of AZD2171 ablated experimental (VEGF-induced) angiogenesis in vivo and inhibited endochondral ossification in bone or corpora luteal development in ovary; physiologic processes that are highly dependent upon neovascularization. The growth of established human tumor xenografts (colon, lung, prostate, breast, and ovary) in athymic mice was inhibited dose-dependently by AZD2171, with chronic administration of 1.5 mg per kg per day producing statistically significant inhibition in all models. A histologic analysis of Calu-6 lung tumors treated with AZD2171 revealed a reduction in microvessel density within 52 hours that became progressively greater with the duration of treatment. These changes are indicative of vascular regression within tumors. Collectively, the data obtained with AZD2171 are consistent with potent inhibition of VEGF signaling, angiogenesis, neovascular survival, and tumor growth. AZD2171 is being developed clinically as a once-daily oral therapy for the treatment of cancer.

Plasmalemmal vesicle-associated protein (PLVAP) is expressed by tumour endothelium and is upregulated by vascular endothelial growth factor-A (VEGF)

Vascular endothelial growth factor-A (VEGF) is an important regulator of vascular permeability. In preclinical studies, VEGF induces endothelial fenestrations in pre-existing and neo-vasculature, while inhibition of VEGF leads to a reduction in endothelial fenestrations. Recently, vascular regression in response to VEGF inhibition has been shown to correlate with the presence of endothelial fenestrations. Plasmalemmal vesicle-associated protein (PLVAP) is believed to be a component of diaphragmed endothelial fenestrations, but a direct relationship with VEGF signalling has not been established. The aim of this study was to characterize the expression pattern of PLVAP and investigate whether PLVAP is a transcriptional target of VEGF signal transduction. The expression pattern of PLVAP was characterized in normal and neoplastic human tissues by in situ hybridization and/or immunohistochemistry. The role of VEGF signal transduction in the regulation of PLVAP expression was investigated in vitro using receptor-selective engineered forms of VEGF, a neutralizing monoclonal antibody against VEGF, and inhibitors of downstream signalling pathways. PLVAP mRNA and protein were widely expressed in the endothelium of normal and neoplastic tissues. In cultured endothelial cells, VEGF signalling through receptor 2 stimulated expression of PLVAP total RNA and protein. This induction could be blocked with an anti-VEGF monoclonal antibody and by inhibitors of phosphatidylinositol 3-kinase (LY294002) or p38 mitogen-activated protein kinase (SB203580), but not by PD98059, a mitogen-activated protein/extracellular signal-regulated kinase 1 inhibitor. These data show that PLVAP is more widely expressed in the vasculature of normal tissues than previously thought and that it is expressed in the vasculature of most human tumours. We suggest that PLVAP is a downstream target of VEGF signalling. This work solidifies the association between VEGF and the appearance and maintenance of fenestrations by providing a potential mechanistic link.

VEGF‐A promotes tissue repair‐associated lymphatic vessel formation via VEGFR‐2 and the α1β1 and α2β1 integrins

Vascular endothelial growth factor-A (VEGF-A) is strongly up-regulated in wounded cutaneous tissue and promotes repair-associated angiogenesis. However, little is known about its role in lymphatic regeneration of the healing skin. We studied wound healing in transgenic mice that overexpress VEGF-A specifically in the epidermis and in wild-type mice in the absence or presence of inhibitors of VEGF-A signaling. Surprisingly, transgenic overexpression of VEGF-A in the skin promoted lymphangiogenesis at the wound healing site, whereas systemic blockade of VEGFR-2 prevented lymphatic vessel formation. Studies in cultured lymphatic endothelial cells revealed that VEGF-A induced expression of the alpha1 and alpha2 integrins, which promoted their in vitro tube formation and their haptotactic migration toward type I collagen. VEGF-A-induced lymphatic endothelial cord formation and haptotactic migration were suppressed by anti-alpha1 and anti-alpha2 integrin blocking antibodies, and systemic blockade of the alpha1 and alpha2 integrins inhibited VEGF-A-driven lymphangiogenesis in vivo. We propose that VEGF-A promotes lymphatic vasculature formation via activation of VEGFR-2 and that lineage-specific differences of integrin receptor expression contribute to the distinct dynamics of wound-associated angiogenesis and lymphangiogenesis.

THE NEURAL TUBE PATTERNS VESSELS DEVELOPMENTALLY USING THE VEGF SIGNALING PATHWAY

Embryonic blood vessels form in a reproducible pattern that interfaces with other embryonic structures and tissues, but the sources and identities of signals that pattern vessels are not well characterized. We hypothesized that the neural tube provides vascular patterning signal(s) that direct formation of the perineural vascular plexus (PNVP) that encompasses the neural tube at mid-gestation. Both surgically placed ectopic neural tubes and ectopic neural tubes engineered genetically were able to recruit a vascular plexus, showing that the neural tube is the source of a vascular patterning signal. In mouse-quail chimeras with the graft separated from the neural tube by a buffer of host cells, graft-derived vascular cells contributed to the PNVP, indicating that the neural tube signal(s) can act at a distance. Murine neural tube vascular endothelial growth factor A (VEGFA) expression was temporally and spatially correlated with PNVP formation, suggesting it is a component of the neural tube signal. A collagen explant model was developed in which presomitic mesoderm explants formed a vascular plexus in the presence of added VEGFA. Co-cultures between presomitic mesoderm and neural tube also supported vascular plexus formation, indicating that the neural tube could replace the requirement for VEGFA. Moreover, a combination of pharmacological and genetic perturbations showed that VEGFA signaling through FLK1 is a required component of the neural tube vascular patterning signal. Thus, the neural tube is the first structure identified as a midline signaling center for embryonic vascular pattern formation in higher vertebrates, and VEGFA is a necessary component of the neural tube vascular patterning signal. These data suggest a model whereby embryonic structures with little or no capacity for angioblast generation act as a nexus for vessel patterning.