Tumor angiogenesis is an essential process for supplying rapidly growing malignant tissues with essential nutrients and oxygen. An angiogenic switch allows tumor cells to survive and grow, and provides them access to vasculature resulting in metastatic disease. Monocyte-derived macrophages recruited and reprogrammed by tumor cells serve as a major source of angiogenic factors boosting the angiogenic switch. Tumor endothelium releases angiopoietin-2 and further facilitates recruitment of TIE2 receptor expressing monocytes (TEM) into tumor sites. Tumor-associated macrophages (TAM) sense hypoxia in avascular areas of tumors, and react by production of angiogenic factors such as VEGFA. VEGFA stimulates chemotaxis of endothelial cells (EC) and macrophages. In some tumors, TAM appeared to be a major source of MMP9. Elevated expression of MMP9 by TAM mediates extracellular matrix (ECM) degradation and the release of bioactive VEGFA. Other angiogenic factors released by TAM include basic fibroblast growth factor (bFGF), thymidine phosphorylase (TP), urokinase-type plasminogen activator (uPA), and adrenomedullin (ADM). The same factors used by macrophages for the induction of angiogenesis [like vascular endothelial growth factor A (VEGF-A) and MMP9] support lymphangiogenesis. TAM can express LYVE-1, one of the established markers of lymphatic endothelium. TAM support tumor lymphangiogenesis not only by secretion of pro-lymphangiogenic factors but also by trans-differentiation into lymphatic EC. New pro-angiogenic factor YKL-40 belongs to a family of mammalian chitinase-like proteins (CLP) that act as cytokines or growth factors. Human CLP family comprises YKL-40, YKL-39, and SI-CLP. Production of all three CLP in macrophages is antagonistically regulated by cytokines. It was recently established that YKL-40 induces angiogenesis in vitro and in animal tumor models. YKL-40-neutralizing monoclonal antibody blocks tumor angiogenesis and progression. The role of YKL-39 and SI-CLP in tumor angiogenesis and lymphangiogenesis remains to be investigated.

Endothelial nitric oxide synthase (eNOS) is an important modulator of angiogenesis and vascular tone [1]. It is stimulated by treatment of endothelial cells in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent fashion by insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) [2] [3] and is activated by phosphorylation at Ser1177 in the sequence RIRTQS(1177)F (in the single-letter amino acid code) [4]. The protein kinase Akt is an important downstream target of PI 3-kinase [5] [6], regulating VEGF-stimulated endothelial cell survival [7]. Akt phosphorylates substrates within a defined motif [8], which is present in the sequence surrounding Ser1177 in eNOS. Both Akt [5] [6] and eNOS [9] are localized to, and activated at, the plasma membrane. We found that purified Akt phosphorylated cardiac eNOS at Ser1177, resulting in activation of eNOS. Phosphorylation at this site was stimulated by treatment of bovine aortic endothelial cells (BAECs) with VEGF or IGF-1, and Akt was activated in parallel. Preincubation with wortmannin, an inhibitor of Akt signalling, reduced VEGF- or IGF-1-induced Akt activity and eNOS phosphorylation. Akt was detected in immunoprecipitates of eNOS from BAECs, and eNOS in immunoprecipitates of Akt, indicating that the two enzymes associate in vivo. It is thus apparent that Akt directly activates eNOS in endothelial cells. These results strongly suggest that Akt has an important role in the regulation of normal angiogenesis and raise the possibility that the enhanced activity of this kinase that occurs in carcinomas may contribute to tumor vascularization and survival.

Studies with embryonic explants and embryonic stem cells have suggested a role for Hedgehog (Hh) signaling in hematopoiesis. However, targeted deletion of Hh pathway components in the mouse has so far failed to provide in vivo evidence. Here we show that zebrafish embryos mutant in the Hh pathway or treated with the Hh signaling inhibitor cyclopamine display defects in adult hematopoietic stem cell (HSC) formation but not in primitive hematopoiesis. Hh is required in the trunk at three consecutive stages during vascular development: for the medial migration of endothelial progenitors of the dorsal aorta (DA), for arterial gene expression, and for the formation of intersomitic vessel sprouts. Interference with Hh signaling during the first two stages also interferes with HSC formation. Furthermore, HSC and DA formation also share Vegf and Notch requirements, which further distinguishes them from primitive hematopoiesis and underlines their close relationship during vertebrate development.

Monocytes/macrophages are critical in orchestrating the tissue-repair response. However, the mechanisms that govern macrophage regenerative activities during the sequential phases of repair are largely unknown. In the present study, we examined the dynamics and functions of diverse monocyte/macrophage phenotypes during the sequential stages of skin repair. By combining the analysis of a new CCR2-eGFP reporter mouse model with conditional mouse mutants defective in myeloid cell-restricted CCR2 signaling or VEGF-A synthesis, we show herein that among the large number of inflammatory CCR2(+)Ly6C(+) macrophages that dominate the early stage of repair, only a small fraction strongly expresses VEGF-A that has nonredundant functions for the induction of vascular sprouts. The switch of macrophage-derived VEGF-A during the early stage of tissue growth toward epidermal-derived VEGF-A during the late stage of tissue maturation was critical to achieving physiologic tissue vascularization and healing progression. The results of the present study provide new mechanistic insights into CCR2-mediated recruitment of blood monocyte subsets into damaged tissue, the dynamics and functional consequences of macrophage plasticity during the sequential repair phases, and the complementary role of macrophage-derived VEGF-A in coordinating effective tissue growth and vascularization in the context of tissue-resident wound cells. Our findings may be relevant for novel monocyte-based therapies to promote tissue vascularization.

Intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents is increasingly used for the treatment of a wide variety of retinal diseases, including age-related macular degeneration, diabetic retinopathy and retinal vascular occlusions, and retinopathy of prematurity. Despite encouraging results in halting the disease and improving the vision, intravitreal injection of anti-VEGF agents may be associated with systemic adverse events and devastating ocular complications. In this review, we provide an overview of safety data for intravitreal injection of common anti-VEGF agents.

BACKGROUND

Previous studies have established that bone marrow-derived endothelial progenitor cells (EPCs) are present in the systemic circulation. In the current study, we investigated the hypothesis that gene transfer can be used to achieve phenotypic modulation of EPCs.

RESULTS

In vitro, ex vivo murine vascular endothelial growth factor (VEGF) 164 gene transfer augmented EPC proliferative activity and enhanced adhesion and incorporation of EPCs into quiescent as well as activated endothelial cell monolayers. To determine if such phenotypic modulation may facilitate therapeutic neovascularization, heterologous EPCs transduced with adenovirus encoding VEGF were administered to athymic nude mice with hindlimb ischemia; neovascularization and blood flow recovery were both improved, and limb necrosis/autoamputation were reduced by 63.7% in comparison with control animals. The dose of EPCs used for the in vivo experiments was 30 times less than that required in previous trials of EPC transplantation to improve ischemic limb salvage. Necropsy analysis of animals that received DiI-labeled VEGF-transduced EPCs confirmed that enhanced EPC incorporation demonstrated in vitro contributed to in vivo neovascularization as well.

CONCLUSIONS

In vitro, VEGF EPC gene transfer enhances EPC proliferation, adhesion, and incorporation into endothelial cell monolayers. In vivo, gene-modified EPCs facilitate the strategy of cell transplantation to augment naturally impaired neovascularization in an animal model of experimentally induced limb ischemia.

When and where to make or break new blood vessel connections is the key to understanding guided vascular patterning. VEGF-A stimulation and Dll4/Notch signaling cooperatively control the number of new connections by regulating endothelial tip cell formation. Here, we show that the Notch-regulated ankyrin repeat protein (Nrarp) acts as a molecular link between Notch- and Lef1-dependent Wnt signaling in endothelial cells to control stability of new vessel connections in mouse and zebrafish. Dll4/Notch-induced expression of Nrarp limits Notch signaling and promotes Wnt/Ctnnb1 signaling in endothelial stalk cells through interactions with Lef1. BATgal-reporter expression confirms Wnt signaling activity in endothelial stalk cells. Ex vivo, combined Wnt3a and Dll4 stimulation of endothelial cells enhances Wnt-reporter activity, which is abrogated by loss of Nrarp. In vivo, loss of Nrarp, Lef1, or endothelial Ctnnb1 causes vessel regression. We suggest that the balance between Notch and Wnt signaling determines whether to make or break new vessel connections.

Preclinical and clinical studies positively correlate the expression of vascular endothelial growth factor (VEGF)-C in tumors and the incidence of lymph node metastases. However, how VEGF-C regulates individual steps in the transport of tumor cells from the primary tumor to the draining lymph nodes is poorly understood. Here, we image and quantify these steps in tumors growing in the tip of the mouse ear using intravital microscopy of the draining lymphatic vessels and lymph node, which receives spontaneously shed tumor cells. We show that VEGF-C overexpression in cancer cells induces hyperplasia in peritumor lymphatic vessels and increases the volumetric flow rate in lymphatics at the base of the ear by 40%. The increases in lymph flow rate and peritumor lymphatic surface area enhance the rate of tumor cell delivery to lymph nodes, leading to a 200-fold increase in cancer cell accumulation in the lymph node and a 4-fold increase in lymph node metastasis. In our model, VEGF-C overexpression does not confer any survival or growth advantage on cancer cells. We also show that an anti-VEGF receptor (VEGFR)-3 antibody reduces both lymphatic hyperplasia and the delivery of tumor cells to the draining lymph node, leading to a reduction in lymph node metastasis. However, this treatment is unable to prevent the growth of tumor cells already seeded in lymph nodes. Collectively, our results indicate that VEGF-C facilitates lymphatic metastasis by increasing the delivery of cancer cells to lymph nodes and therapies directed against VEGF-C/VEGFR-3 signaling target the initial steps of lymphatic metastasis.

OBJECTIVE

Although the Milan criteria (MC) have been used to select liver transplantation candidates among patients with hepatocellular carcinoma (HCC), many patients exceeding the MC have shown good prognosis. Preoperative neutrophil-lymphocyte ratio (NLR) is a predictor of patient prognosis, but its mechanism has never been clarified.

METHODS

We assessed outcomes in 158 patients who had undergone living-donor liver transplantation (LDLT) for HCC. Recurrence-free survival (RFS) was determined in patients with high (≥ 4) and low (<4) NLR. Levels of expression of vascular endothelial growth factor (VEGF), interleukin (IL)-8, IL-17, CD68, and CD163 were measured.

RESULTS

The 5-year RFS rate was significantly lower in patients with high (n=26) than with low (n=132) NLR (30.3% vs. 89.0%, p<0.0001), in patients with high (n=15) than with low (n=79) NLR who met the MC (73.6% vs. 100%, p=0.0008) and in patients with high (n=11) than with low (n=53) NLR who exceeded the MC (0% vs. 76.1%, p=0.0002). Tumor expression of VEGF, IL8, IL-17, CD68, and CD163 was similar in the high and low NLR groups, but serum and peritumoral IL-17 levels were significantly higher in the high-NLR group (p=0.01 each). The density of peritumoral CD163 correlated with the density of peritumoral IL-17-producing cells (p=0.04) and was significantly higher in the high-NLR group (p=0.005).

CONCLUSIONS

NLR predicts outcomes after LDLT for HCC via the inflammatory tumor microenvironment. Combined with the MC, NLR may be a new criterion for LDLT candidates with HCC.

Tumor expression of the lymphangiogenic factor VEGF-C is correlated with metastasis and poor prognosis, and although VEGF-C enhances transport to the draining lymph node (dLN) and antigen exposure to the adaptive immune system, its role in tumor immunity remains unexplored. Here, we demonstrate that VEGF-C promotes immune tolerance in murine melanoma. In B16 F10 melanomas expressing a foreign antigen (OVA), VEGF-C protected tumors against preexisting antitumor immunity and promoted local deletion of OVA-specific CD8(+) T cells. Naive OVA-specific CD8(+) T cells, transferred into tumor-bearing mice, were dysfunctionally activated and apoptotic. Lymphatic endothelial cells (LECs) in dLNs cross-presented OVA, and naive LECs scavenge and cross-present OVA in vitro. Cross-presenting LECs drove the proliferation and apoptosis of OVA-specific CD8(+) T cells ex vivo. Our findings introduce a tumor-promoting role for lymphatics in the tumor and dLN and suggest that lymphatic endothelium in the local microenvironment may be a target for immunomodulation.

OBJECTIVE

To investigate the efficacy and safety of axitinib, an oral, potent, and selective inhibitor of vascular endothelial growth factor (VEGF) receptors 1, 2, and 3 in patients with metastatic renal cell carcinoma (mRCC) refractory to prior therapies that included, but were not limited to, sorafenib.

METHODS

In this multicenter, open-label, phase II study, patients with sorafenib-refractory mRCC received a starting dose of axitinib 5 mg orally twice daily. A one-arm, single-stage design was used to estimate the primary end point of objective response rate (ORR), defined by RECIST (Response Evaluation Criteria in Solid Tumors). Secondary end points included safety, duration of response, progression-free survival (PFS), overall survival (OS), and patient-reported outcomes.

RESULTS

Of 62 patients recruited, 100% had received prior sorafenib, and 74.2% had received two or more prior systemic treatments. The axitinib dose was titrated to greater than 5 mg twice daily in 53.2% of patients, and 35.5% of patients had the dose modified to less than 5 mg twice daily. In 62 patients evaluable for response, the ORR was 22.6%, and the median duration of response was 17.5 months. Median PFS and OS times were 7.4 months (95% CI, 6.7 to 11.0 months) and 13.6 months (95% CI, 8.4 to 18.8 months), respectively. All-causality grade 3 to 4 adverse events included hand-foot syndrome (16.1%), fatigue (16.1%), hypertension (16.1%), dyspnea (14.5%), diarrhea (14.5%), dehydration (8.1%), and hypotension (6.5%).

CONCLUSIONS

Axitinib has antitumor activity in patients with mRCC refractory to prior VEGF-targeted therapy, including sorafenib. Toxicities were mild to moderate and were manageable. A randomized, phase III trial to compare axitinib with sorafenib in patients who have mRCC refractory to one prior first-line therapy regimen is underway.

Vascular endothelial growth factor (VEGF) is an endothelial specific angiogenic mitogen secreted from various cell types including tumor cells. Increasing evidence suggests that VEGF is a major regulator of physiological and pathological angiogenesis, and the VEGF/VEGF receptor system has been shown to be necessary for glioma angiogenesis. Hypoxia seems to play a critical role in the induction of VEGF expression during glioma progression. C6 glioma cells provide an in vivo glioma model for the study of tumor angiogenesis, and the expression of VEGF in C6 cells has been shown to be up-regulated by hypoxia in vitro. However, little is known about the molecular mechanism of hypoxic induction of VEGF. Here, we demonstrate that hypoxic induction of VEGF in C6 cells is due to both transcriptional activation and increased stability of mRNA. Nuclear run-on assays revealed a fast and lasting transcriptional activation, whereas the determination of mRNA half-life showed a slower increase of mRNA stability during hypoxia. Reporter gene studies revealed that hypoxia responsive transcription-activating elements were present in the 5'-flanking region of the VEGF gene. These results suggested that several distinct molecular mechanisms were involved in hypoxia-induced gene expression and were activated in a biphasic manner.

Glioblastoma multiforme (GBM) is characterized by exuberant angiogenesis, a key event in tumor growth and progression. The pathologic mechanisms driving this change and the biological behavior of gliomas remain unclear. One mechanism may involve cooption of native blood vessels by glioma cells inducing expression of angiopoietin-2 by endothelial cells. Subsequently, vascular apoptosis and involution leads to necrosis and hypoxia. This in turn induces angiogenesis that is associated with expression of hypoxia-inducible factor (HIF)-1alpha and vascular endothelial growth factor (VEGF) in perinecrotic pseudopalisading glioma cells. Here we review the molecular and cellular mechanisms implicated in HIF-1-dependent and HIF-1-independent glioma-associated angiogenesis. In GBMs, both tumor hypoxia and genetic alterations commonly occur and act together to induce the expression of HIF-1. The angiogenic response of the tumor to HIF-1 is mediated by HIF-1-regulated target genes leading to the upregulation of several proangiogenic factors such as VEGF and other adaptive response molecules. Understanding the roles of these regulatory processes in tumor neovascularization, tumor growth and progression, and resistance to therapy will ultimately lead to the development of improved antiangiogenic therapies for GBMs.

Endothelial cells (ECs) form cell-cell adhesive junctional structures maintaining vascular integrity. This barrier is dynamically regulated by vascular endothelial growth factor (VEGF) receptor signaling. We created an inducible knockin mouse model to study the contribution of the integrin-associated focal adhesion tyrosine kinase (FAK) signaling on vascular function. Here we show that genetic or pharmacological FAK inhibition in ECs prevents VEGF-stimulated permeability downstream of VEGF receptor or Src tyrosine kinase activation in vivo. VEGF promotes tension-independent FAK activation, rapid FAK localization to cell-cell junctions, binding of the FAK FERM domain to the vascular endothelial cadherin (VE-cadherin) cytoplasmic tail, and direct FAK phosphorylation of β-catenin at tyrosine-142 (Y142) facilitating VE-cadherin-β-catenin dissociation and EC junctional breakdown. Kinase inhibited FAK is in a closed conformation that prevents VE-cadherin association and limits VEGF-stimulated β-catenin Y142 phosphorylation. Our studies establish a role for FAK as an essential signaling switch within ECs regulating adherens junction dynamics.

OBJECTIVE

Angiogenesis, a process fundamental to tumor growth, is regulated by angiogenic factors. This article reviews prognostic and other clinical implications of circulating angiogenic factors in cancer patients.

METHODS

A MEDLINE search of literature was performed using the names of various angiogenic factors as the key words. Studies pertaining to circulating angiogenic factors in cancer patients were reviewed. Pertinent literature regarding tumor expression of common angiogenic factors and their prognostic relevance in human cancers were also examined.

RESULTS

A substantial number of studies have demonstrated a strong association between elevated tumor expression of vascular endothelial growth factor (VEGF) and advanced disease or poor prognosis in various cancers. This supports the pivotal role of VEGF in regulating tumor angiogenesis. More recently, there is mounting evidence that the level of circulating VEGF in patients with different types of cancer may be predictive of tumor status and prognosis. Preliminary data also suggest that circulating VEGF may be useful in predicting and monitoring tumor response to anticancer therapies and in follow-up surveillance for tumor relapse. There are reports supporting the prognostic value of other circulating angiogenic factors such as basic fibroblast growth factor, platelet-derived endothelial cell growth factor, transforming growth factor-beta, and angiogenin, but their clinical significance is less conclusive because of limited data.

CONCLUSIONS

Circulating VEGF seems to be a reliable surrogate marker of angiogenic activity and tumor progression in cancer patients. Evaluation of circulating angiogenic factors is a promising novel approach of prognostication in cancer patients that has the advantages of being convenient and noninvasive, and it may provide new prognostic information that is not afforded by conventional clinicopathologic prognostic indicators.

Platelet-rich fibrin (PRF) belongs to a new generation of platelet concentrates, with simplified processing and without biochemical blood handling. In this third article, we investigate the immune features of this biomaterial. During PRF processing, leucocytes could also secrete cytokines in reaction to the hemostatic and inflammatory phenomena artificially induced in the centrifuged tube. We therefore undertook to quantify 5 significant cell mediators within platelet poor plasma supernatant and PRF clot exudate serum: 3 proinflammatory cytokines (IL-1beta, IL-6, and TNF-alpha), an antiinflammatory cytokine (IL-4), and a key growth promoter of angiogenesis (VEGF). Our data are correlated with that obtained in plasma (nonactivated blood) and in sera (activated blood). These initial analyses revealed that PRF could be an immune regulation node with inflammation retrocontrol abilities. This concept could explain the reduction of postoperative infections when PRF is used as surgical additive.

Dysregulation of Axl and its ligand growth arrest-specific 6 is implicated in the pathogenesis of several human cancers. In this study, we have used RNAi and monoclonal antibodies to assess further the oncogenic potential of Axl. Here we show that Axl knockdown reduces growth of lung and breast cancer xenograft tumors. Inhibition of Axl expression attenuates breast cancer cell migration and inhibits metastasis to the lung in an orthotopic model, providing the first in vivo evidence that links Axl directly to cancer metastasis. Axl knockdown in endothelial cells impaired tube formation and this effect was additive with anti-vascular endothelial growth factor (VEGF). Further analysis demonstrated that Axl regulates endothelial cell functions by modulation of signaling through angiopoietin/Tie2 and Dickkopf (DKK3) pathways. We have developed and characterized Axl monoclonal antibodies that attenuate non-small cell lung carcinoma xenograft growth by downregulation of receptor expression, reducing tumor cell proliferation and inducing apoptosis. Our data demonstrate that Axl plays multiple roles in tumorigenesis and that therapeutic antibodies against Axl may block Axl functions not only in malignant tumor cells but also in the tumor stroma. The additive effect of Axl inhibition with anti-VEGF suggests that blocking Axl function could be an effective approach for enhancing antiangiogenic therapy.

OBJECTIVE

The purpose of the study is to determine the effect of exogenous vascular endothelial growth factor (VEGF) on the primate retina and its vasculature.

METHODS

Ten eyes of five animals were studied. Physiologically relevant amounts of the 165 amino acid isoform of human recombinant VEGF were injected into the vitreous of six healthy cynomolgus monkey eyes. Inactivated human recombinant VEGF or vehicle was injected into four contralateral control subject eyes. Eyes were assessed by slit-lamp biomicroscopy, tonometry, fundus color photography, fundus fluorescein angiography, light microscopy, and immunostaining with antibodies against proliferating cell nuclear antigen and factor VIII antigen.

RESULTS

All six bioactive VEGF-injected eyes developed dilated, tortuous retinal vessels that leaked fluorescein. Eyes receiving multiple injections of VEGF developed progressively dilated and tortuous vessels, venous beading, edema, microaneurysms, intraretinal hemorrhages and capillary closure with ischemia. The severity of the retinopathy correlated with the number of VEGF injections. None of the four control eyes exhibited any abnormal retinal vascular changes. The endothelial cells of retinal blood vessels were proliferating cell nuclear antigen positive only in the bioactive VEGF-injected eyes.

CONCLUSIONS

Vascular endothelial growth factor is sufficient to produce many of the vascular abnormalities common to diabetic retinopathy and other ischemic retinopathies, such as hemorrhage, edema, venous beading, capillary occlusion with ischemia, microaneurysm formation, and intraretinal vascular proliferation.

We have identified several mechanisms by which the angiogenic cytokine vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) likely regulates endothelial cells (EC) migration. VPF/VEGF induced dermal microvascular EC expression of mRNAs encoding the alphav and beta3 integrin subunits resulting in increased levels of the alphavbeta3 heterodimer at the cell surface, and VPF/VEGF also induced mRNA encoding osteopontin (OPN), an alphavbeta3 ligand. OPN promoted EC migration in vitro; and VPF/VEGF induction of alphavbeta3 was accompanied by increased EC migration toward OPN. Because thrombin cleavage of OPN results in substantial enhancement of OPN's adhesive properties, and because VPF/VEGF promotes increased microvascular permeability leading to activation of the extrinsic coagulation pathway, we also investigated whether VPF/VEGF facilitates thrombin cleavage of OPN in vivo. Consistent with this hypothesis, co-injection of VPF/VEGF together with OPN resulted in rapid cleavage of OPN by endogenous thrombin. Furthermore, in comparison with native OPN, thrombin-cleaved OPN stimulated a greater rate of EC migration in vitro, which was additive to the increased migration associated with induction of alpha v beta 3. Thus, these data demonstrate cooperative mechanisms for VPF/VEGF regulation of EC migration involving the alphavbeta3 integrin, the alphavbeta3 ligand OPN, and thrombin cleavage of OPN. These findings also illustrate an operational link between VPF/VEGF induction of EC gene expression and VPF/VEGF enhancement of microvascular permeability, suggesting that these distinct biological activities may act accordingly to stimulate EC migration during angiogenesis.

Angiogenesis is a complex biological process involving the coordinated modulation of many genes. Histone deacetylases (HDAC) are a growing family of enzymes that mediate the availability of chromatin to the transcriptional machinery. Trichostatin-A (TSA) and suberoylanilide hydroxamic acid (SAHA), two HDAC inhibitors known to relieve gene silencing, were evaluated as potential antiangiogenic agents. TSA and SAHA were shown to prevent vascular endothelial growth factor (VEGF)-stimulated human umbilical cord endothelial cells (HUVEC) from invading a type I collagen gel and forming capillary-like structures. SAHA and TSA inhibited the VEGF-induced formation of a CD31-positive capillary-like network in embryoid bodies and inhibited the VEGF-induced angiogenesis in the CAM assay. TSA also prevented, in a dose-response relationship, the sprouting of capillaries from rat aortic rings. TSA inhibited in a dose-dependent and reversible fashion the VEGF-induced expression of VEGF receptors, VEGFR1, VEGFR2, and neuropilin-1. TSA and SAHA upregulated the expression by HUVEC of semaphorin III, a recently described VEGF competitor, at both mRNA and protein levels. This effect was specific to endothelial cells and was not observed in human fibroblasts neither in vascular smooth muscle cells. These observations provide a conspicuous demonstration that HDAC inhibitors are potent anti-angiogenic factors altering VEGF signaling.

Endochondral bone formation is regulated by systemically and locally acting growth factors. A role for vascular endothelial growth factor (VEGF) in this process has recently been proposed, because inactivation of VEGF inhibits endochondral bone formation via inhibition of angiogenesis. Despite the known effect of VEGF as specific endothelial growth factor, its effects on osteoblast differentiation have not been studied. We, therefore, examined the expression of VEGF-A, -B, -C, and -D and their receptors in a model of osteoblast differentiation using the mouse preosteoblast-like cell line KS483. Early in differentiation, KS483 cells express low levels VEGF-A, -B, and -D messenger RNA, whereas during mineralization, KS483 cells express high levels. In addition, expression of the VEGF receptors, VEGFR1, VEGFR2, and VEGFVEGF-A production during osteoblast differentiation was stimulated by insulin-like growth factor I that enhances osteoblast differentiation and was inhibited by PTH-related peptide that inhibits osteoblast differentiation. Furthermore, continuous treatment of KS483 cells with recombinant human VEGF-A stimulated nodule formation. Although treatment of KS483 cells with soluble FLT1, an agent that blocks binding of VEGF-A and -B to VEGFR1, did not inhibit nodule formation, this observation does not exclude involvement of VEGFR2 in the regulation of osteoblast differentiation. As it is known that VEGF-A, -C, and -D can act through activation of VEGFR2, other isoforms might compensate for VEGF-A loss. The expression pattern of VEGFs and their receptors shown here suggests that VEGFs play an important role in the regulation of bone remodeling by attracting endothelial cells and osteoclasts and by stimulating osteoblast differentiation.

The development of the nervous and vascular systems constitutes primary events in the evolution of the animal kingdom; the former provides electrical stimuli and coordination, while the latter supplies oxygen and nutrients. Both systems have more in common than originally anticipated. Perhaps the most striking observation is that angiogenic factors, when deregulated, contribute to various neurological disorders, such as neurodegeneration, and might be useful for the treatment of some of these pathologies. The prototypic example of this cross-talk between nerves and vessels is the vascular endothelial growth factor or VEGF. Although originally described as a key angiogenic factor, it is now well established that VEGF also plays a crucial role in the nervous system. We describe the molecular properties of VEGF and its receptors and review the current knowledge of its different functions and therapeutic potential in the nervous system during development, health, disease and in medicine.

The ability to adapt successfully to periods of relative hypoxia is crucial to the survival of all higher life forms. Several genes have previously been identified which are up-regulated in response to hypoxia; these include the genes encoding erythropoietin (Epo), platelet-derived growth factor B chain, endothelin, interleukin-1 alpha, ornithine decarboxylase, and vascular endothelial growth factor (VEGF). However, the molecular mechanisms by which hypoxia is sensed remain enigmatic. In addition, it is unknown whether the genes mentioned share a common oxygen-sensing signal transduction pathway. In this report we demonstrate multiple similarities between the oxygen-sensing mechanisms regulating the expression of VEGF and Epo. The expression of both mRNAs is significantly up-regulated by hypoxia and cobalt chloride (CoCl2), and the half-life of both mRNAs is markedly prolonged by cycloheximide. In addition, hypoxic induction of both Epo and VEGF is inhibited by carbon monoxide. As part of our investigation into the signal transduction pathway responsible for the hypoxia and cobalt induction of these genes, we discovered that the expression of members of the jun and fos protooncogene families is also up-regulated early after exposure to either of these stimuli. These findings provide support for the hypothesis that the mechanism(s) by which hypoxia is sensed at a molecular level may be highly conserved and tightly regulated.

Although directed migration is a feature of both individual cells and cell groups, guided migration has been studied most extensively for single cells in simple environments. Collective guidance of cell groups remains poorly understood, despite its relevance for development and metastasis. Neural crest cells and neuronal precursors migrate as loosely organized streams of individual cells, whereas cells of the fish lateral line, Drosophila tracheal tubes and border-cell clusters migrate as more coherent groups. Here we use Drosophila border cells to examine how collective guidance is performed. We report that border cells migrate in two phases using distinct mechanisms. Genetic analysis combined with live imaging shows that polarized cell behaviour is critical for the initial phase of migration, whereas dynamic collective behaviour dominates later. PDGF- and VEGF-related receptor and epidermal growth factor receptor act in both phases, but use different effector pathways in each. The myoblast city (Mbc, also known as DOCK180) and engulfment and cell motility (ELMO, also known as Ced-12) pathway is required for the early phase, in which guidance depends on subcellular localization of signalling within a leading cell. During the later phase, mitogen-activated protein kinase and phospholipase Cgamma are used redundantly, and we find that the cluster makes use of the difference in signal levels between cells to guide migration. Thus, information processing at the multicellular level is used to guide collective behaviour of a cell group.