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Background. Renal artery stenosis (RAS) causes renal injury partly via microvascular (MV) endothelial dysfunction and damage. Vascular endothelial growth factor (VEGF) is crucial for preservation of microvasculature and promotes vascular proliferation and endothelial repair. We have previously shown that MV rarefaction is associated with decreased VEGF in the kidney exposed to chronic RAS, accompanied by deteriorated renal function and fibrosis. We hypothesized that preserving the renal microcirculation in the stenotic kidney will halt the progression of renal damage.

Methods. Unilateral RAS was induced in 16 pigs. In eight, VEGF (0.05 micrograms/kg) was infused intra-renally at the onset of RAS. After 6 weeks, single-kidney haemodynamics and function were assessed using in vivo multi-detector computed tomography (CT). Renal microvessels, angiogenic pathways and morphology were investigated ex vivo using micro-CT, real-time PCR and histology.

Results. Blood pressure and degree of RAS was similar in RAS and RAS + VEGF pigs. Single-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were reduced in RAS compared to Normal (221.1 ± 46.5 and 29.9 ± 3.8 vs. 522.5 ± 60.9 and 49.3 ± 3.4 mL/min, respectively, P < 0.05), accompanied by decreased cortical MV density and increased renal fibrosis. Pre-emptive administration of VEGF preserved MV architecture, attenuated fibrosis and normalized RBF and GFR (510.8 ± 50.9 and 39.9.1 ± 4.1 mL/min, P = not significant vs. Normal).

Conclusions. This study underscores the importance of the renal microcirculation in renovascular disease. Intra-renal administration of VEGF preserved renal MV architecture and function of the stenotic kidney, which in turn preserved renal haemodynamics and function and decreased renal fibrosis. These observations suggest that preventing renal MV loss may be a potential target for therapeutic approaches for patients with chronic renovascular disease.

VEGF is a secreted mitogen associated with angiogenesis and is also a potent vascular permeability factor. The biological role of VEGF in the ischemic brain remains unknown. This study was undertaken to investigate whether VEGF enhances cerebral microvascular perfusion and increases blood-brain barrier (BBB) leakage in the ischemic brain. Using magnetic resonance imaging (MRI), three-dimensional laser-scanning confocal microscope, and functional neurological tests, we measured the effects of administrating recombinant human VEGF165 (rhVEGF165) on angiogenesis, functional neurological outcome, and BBB leakage in a rat model of focal cerebral embolic ischemia. Late (48 hours) administration of rhVEGF165 to the ischemic rats enhanced angiogenesis in the ischemic penumbra and significantly improved neurological recovery. However, early postischemic (1 hour) administration of rhVEGF165 to ischemic rats significantly increased BBB leakage, hemorrhagic transformation, and ischemic lesions. Administration of rhVEGF165 to ischemic rats did not change BBB leakage and cerebral plasma perfusion in the contralateral hemisphere. Our results indicate that VEGF can markedly enhance angiogenesis in the ischemic brain and reduce neurological deficits during stroke recovery and that inhibition of VEGF at the acute stage of stroke may reduce the BBB permeability and the risk of hemorrhagic transformation after focal cerebral ischemia.

Objective

The endogenous role of the VEGF family member vascular endothelial growth factor-B (VEGF-B) in pathological angiogenesis remains unclear.

Methods and Results

We studied the role of VEGF-B in various models of pathological angiogenesis using mice lacking VEGF-B (VEGF-B−/−) or overexpressing VEGF-B167. After occlusion of the left coronary artery, VEGF-B deficiency impaired vessel growth in the ischemic myocardium whereas, in wild-type mice, VEGF-B167 overexpression enhanced revascularization of the infarct and ischemic border zone. By contrast, VEGF-B deficiency did not affect vessel growth in the wounded skin, hypoxic lung, ischemic retina, or ischemic limb. Moreover, VEGF-B167 overexpression failed to enhance vascular growth in the skin or ischemic limb.

Conclusion

VEGF-B appears to have a relatively restricted angiogenic activity in the ischemic heart. These insights might offer novel therapeutic opportunities.

Vascular endothelial growth factor (VEGF) plays an important role in tumour angiogenesis. VEGF binds to tyrosine kinase receptors, which are expressed almost exclusively on tumour endothelium. Therefore, VEGF can be used to target toxin molecules to tumour vessels for anti-angiogenic therapy. However, recent evidence suggests that VEGF can also bind in an isoform-specific fashion to a newly identified neuropilin-1 (NP-1) receptor. NP-1 is widely expressed in normal tissue and presents a potential target for unwanted toxicity. As a consequence, we investigated whether the VEGF121 isoform, which lacks the NP-1 binding domain, could be used to target toxin polypeptides to tumour vasculature. Treatment of endothelial cells with a VEGF121–diphtheria toxin (DT385) conjugate selectively inhibited proliferating endothelial cells, whereas confluent cultures were completely resistant to the construct. In addition, VEGF121–DT385 conjugate treatment completely prevented tumour cell induced angiogenesis in vivo. Most importantly, the conjugate inhibited tumour growth in athymic mice and induced tumour-specific vascular damage. There was also no apparent toxicity associated with the treatment. Our results suggest that proliferating endothelial cells are highly sensitive to VEGF121–toxin conjugates and that the binding to NP-1 receptors is not necessary for efficient inhibition of tumour growth. © 2000 Cancer Research Campaign

The vascular endothelial growth factor (VEGF) signaling pathway appears to be the dominant pathway involved in tumor angiogenesis, providing a rationale for targeting the VEGF receptors (VEGFR-1, -2, and -3) in the treatment of cancers. In particular, VEGF signaling is thought to be important in renal cell carcinoma (RCC) because of the deregulation of the pathway through nearly uniform loss of the von Hippel Lindau protein. The tyrosine kinase inhibitors (TKIs) sorafenib, sunitinib, and pazopanib are approved by the US Food and Drug Administration for the treatment of advanced RCC; however, these multitargeted agents inhibit a wide range of kinase targets in addition to the VEGFRs, resulting in a range of adverse effects unrelated to efficient VEGF blockade. This article reviews recent advances in the development of the second-generation VEGFR TKIs, including the more selective VEGFR TKIs tivozanib and axitinib, and focuses on the potential benefits of novel inhibitors with improved potency and selectivity.

Promoting angiogenesis via delivery of vascular endothelial growth factor (VEGF) and other angiogenic factors is both a potential therapy for cardiovascular diseases and a critical aspect for tissue regeneration. The recent demonstration that VEGF signaling is modulated by the Notch signaling pathway, however, suggests that inhibiting Notch signaling may enhance regional neovascularization, by altering the responsiveness of local endothelial cells to angiogenic stimuli. We tested this possibility with in vitro assays using human endothelial cells, as well as in a rodent hindlimb ischemia model. Treatment of cultured human endothelial cells with DAPT, a gamma secretase inhibitor, increased cell migration and sprout formation in response to VEGF stimulation with a biphasic dependence on DAPT concentration. Further, delivery of an appropriate combination of DAPT and VEGF from an injectable alginate hydrogel system into ischemic hindlimbs led to a faster recovery of blood flow than VEGF or DAPT alone; perfusion levels reached 80% of the normal level by week 4 with combined DAPT and VEGF delivery. Direct intramuscular or intraperitoneal injection of DAPT did not result in the same level of improvement, suggesting that appropriate presentation of DAPT (gel delivery) is important for its activity. DAPT delivery from the hydrogels also did not lead to any adverse side effects, in contrast to systemic introduction of DAPT. Altogether, these results suggest a new approach to promote angiogenesis by controlling Notch signaling, and may provide new options to treat patients with diseases that diminish angiogenic responsiveness.

Motivated by the increasing use of anti-VEGF therapies and the many nonvascular functions of VEGF, this study explores the effects of VEGF neutralization on RPE structure, survival, and gene expression both in vitro and in vivo.

Purpose.

Despite a lack of active angiogenesis, VEGF is expressed in nearly every adult tissue, and recent evidence suggests that VEGF may serve as a survival factor for both vascular and nonvascular tissues. VEGF blockade is a widely used treatment for neovascular diseases such as wet age-related macular degeneration (AMD). Therefore, it was sought in this study to evaluate the expression and role of endogenous VEGF in RPE.

Methods.

VEGF and VEGFR2 expression in the murine retina were assessed during development. Bevacizumab was used to neutralize VEGF in ARPE-19 cells, and the effects on cell survival and apical microvill were assessed by TUNEL and SEM, respectively. VEGF was systemically neutralized in vivo by adenoviral-mediated overexpression of soluble VEGFR1 (sFlt). RPE and choriocapillaris were analyzed by transmission electron microscopy (TEM). Changes in gene expression were evaluated by quantitative real-time PCR.

Results.

VEGF expression was detected in the developing RPE as early as embryonic day (E) 9.5, whereas VEGFR2 expression by RPE began nonuniformly between postnatal (P) day 6.5 and P8.5. VEGF neutralization in vitro led to increased apoptosis and reduced microvilli density and length. Systemic VEGF neutralization led to transient degenerative changes; RPE were vacuolated and separated from photoreceptor outer segments, and choriocapillaris fenestrations were decreased. VEGF levels were elevated in RPE of Ad-sFlt1 mice at day 4 postinfection, and there was increased expression of the neurotrophic factor CD59a at day 14.

Conclusions.

These results indicate that VEGF plays a critical role in survival and maintenance of RPE integrity. Potential undesired off-target effects should be considered with chronic use of anti-VEGF agents.

Background

Percutaneous trasluminal renal angioplasty (PTRA) is the most frequent therapeutic approach to resolve renal artery stenosis (RAS). However, renal function recovers in only 30% of the cases. The causes of these poor outcomes are still unknown. We hypothesize that preserving the renal microcirculation distal to RAS will improve the responses to PTRA.

Methods and Results

RAS was induced in 28 pigs. In 14, vascular endothelial growth factor (VEGF)-165 was infused intra-renally (RAS+VEGF, 0.05 µg/kg). Single-kidney function was assessed in all pigs in vivo using ultra-fast CT after 6 weeks. Half of the RAS/RAS+VEGF completed their observation, and the other half underwent PTRA, VEGF was repeated, and CT studies repeated 4 weeks later. Pigs were then euthanized, the stenotic kidney removed, renal microvascular (MV) architecture reconstructed ex-vivo using 3D micro-CT, and renal fibrosis quantified.

Degree of RAS and hypertension were similar in RAS and RAS+VEGF. Renal function and MV density were decreased in RAS but improved in RAS+VEGF. PTRA largely resolved RAS, but the improvements of hypertension and renal function were greater in RAS+VEGF+PTRA than in RAS+PTRA, accompanied by a 34% increase in MV density and decreased fibrosis.

Conclusion

Preservation of the MV architecture and function in the stenotic kidney improved the responses to PTRA, indicating that renal MV integrity plays a role in determining the responses to PTRA. This study indicates that damage and early loss of renal MV is an important determinant of the progression of renal injury in RAS and instigates often irreversible damage.

Angiogenesis (neovascularization) plays a crucial role in a variety of physiological and pathological conditions including cancer, cardiovascular disease, and wound healing. Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis. Multiple VEGF receptors are expressed on endothelial cells, including signaling receptor tyrosine kinases (VEGFR1 and VEGFR2) and the nonsignaling co-receptor Neuropilin-1. Neuropilin-1 binds only the isoform of VEGF responsible for pathological angiogenesis (VEGF165), and is thus a potential target for inhibiting VEGF signaling. Using the first molecularly detailed computational model of VEGF and its receptors, we have shown previously that the VEGFR–Neuropilin interactions explain the observed differential effects of VEGF isoforms on VEGF signaling in vitro, and demonstrated potent VEGF inhibition by an antibody to Neuropilin-1 that does not block ligand binding but blocks subsequent receptor coupling. In the present study, we extend that computational model to simulation of in vivo VEGF transport and binding, and predict the in vivo efficacy of several Neuropilin-targeted therapies in inhibiting VEGF signaling: (a) blocking Neuropilin-1 expression; (b) blocking VEGF binding to Neuropilin-1; (c) blocking Neuropilin–VEGFR coupling. The model predicts that blockade of Neuropilin–VEGFR coupling is significantly more effective than other approaches in decreasing VEGF–VEGFR2 signaling. In addition, tumor types with different receptor expression levels respond differently to each of these treatments. In designing human therapeutics, the mechanism of attacking the target plays a significant role in the outcome: of the strategies tested here, drugs with similar properties to the Neuropilin-1 antibody are predicted to be most effective. The tumor type and the microenvironment of the target tissue are also significant in determining therapeutic efficacy of each of the treatments studied.

Synopsis

Neuropilin is a co-receptor for some of the isoforms of the vascular endothelial growth factor (VEGF) family. The presence of Neuropilin on endothelial or other cells increases binding of these isoforms to their signaling receptor VEGFR2, thus increasing pro-angiogenesis signaling and stimulating vascular growth. Neuropilin is thus a suitable target for anti-angiogenesis therapy, which holds promise for the treatment of vasculature-dependent diseases such as cancer and diabetic retinopathy. In this study, Mac Gabhann and Popel perform computational simulations of VEGF transport in breast cancer, using a previously validated model of VEGF–VEGF receptor interactions, as well as geometrical information on the tumor itself—tumor cells, vasculature, and extracellular matrix. Three different molecular therapies targeting Neuropilin are tested in silico, and the simulations predict that one of these therapies will be effective at reducing VEGFR2 signaling in certain types (or subtypes) of tumors, while the others will not. Thus, we demonstrate that identification of a target molecule is not sufficient; different therapeutic strategies targeting the same molecule may result in different outcomes.

Vascular endothelial growth factor (VEGF) inhibits differentiation and maturation of dendritic cells (DC), suggesting a potential immunosuppressive role for this proangiogenic factor. Bevacizumab, sorafenib and sunitinib target VEGF-mediated angiogenesis and are active against several types of cancer, but their effects on the immune system are poorly understood. In this study, VEGF and supernatants of renal carcinoma cell lines cultured under hypoxia were found to alter the differentiation of human monocytes to DC. Resulting DC showed impaired activity, as assessed by the alloreactive mixed T-lymphocyte reaction. Bevacizumab and sorafenib, but not sunitinib, reversed the inhibitory effects of VEGF, but not of those mediated by tumour supernatants. Dendritic cells matured under the influence of VEGF expressed less human leukocyte antigen-DR (HLA-DR) and CD86, and this effect was restored by bevacizumab and sorafenib. Finally, tumour-cell supernatants decreased interleukin-12 (IL-12) production by mature DC, and such inhibition was not restored by any of the tested drugs, delivered either as single agents or in combination. The deleterious effects of tumour-cell supernatants were mainly mediated by thermostable molecules distinct from VEGF. These results indicate that inhibition of the differentiation of monocytes to DC is a multifactorial effect, and that they support the development of combinations of angiogenesis inhibitors with immunological modulators.

Background

Vascular endothelial growth factor (VEGF) is a potent regulator of angiogenesis, and its role in cancer biology has been widely studied. Many cancer therapies target angiogenesis, with a focus being on VEGF-mediated signaling such as antibodies to VEGF. However, it is difficult to predict the effects of VEGF-neutralizing agents. We have developed a whole-body model of VEGF kinetics and transport under pathological conditions (in the presence of breast tumor). The model includes two major VEGF isoforms VEGF121 and VEGF165, receptors VEGFR1, VEGFR2 and co-receptors Neuropilin-1 and Neuropilin-2. We have added receptors on parenchymal cells (muscle fibers and tumor cells), and incorporated experimental data for the cell surface density of receptors on the endothelial cells, myocytes, and tumor cells. The model is applied to investigate the action of VEGF-neutralizing agents (called "anti-VEGF") in the treatment of cancer.

Results

Through a sensitivity study, we examine how model parameters influence the level of free VEGF in the tumor, a measure of the response to VEGF-neutralizing drugs. We investigate the effects of systemic properties such as microvascular permeability and lymphatic flow, and of drug characteristics such as the clearance rate and binding affinity. We predict that increasing microvascular permeability in the tumor above 10-5 cm/s elicits the undesired effect of increasing tumor interstitial VEGF concentration beyond even the baseline level. We also examine the impact of the tumor microenvironment, including receptor expression and internalization, as well as VEGF secretion. We find that following anti-VEGF treatment, the concentration of free VEGF in the tumor can vary between 7 and 233 pM, with a dependence on both the density of VEGF receptors and co-receptors and the rate of neuropilin internalization on tumor cells. Finally, we predict that free VEGF in the tumor is reduced following anti-VEGF treatment when VEGF121 comprises at least 25% of the VEGF secreted by tumor cells.

Conclusions

This study explores the optimal drug characteristics required for an anti-VEGF agent to have a therapeutic effect and the tumor-specific properties that influence the response to therapy. Our model provides a framework for investigating the use of VEGF-neutralizing drugs for personalized medicine treatment strategies.

Vascular endothelial growth factor (VEGF) is a potent stimulator of vascular angiogenesis, permeability, and remodeling that also plays important roles in wound healing and tissue cytoprotection. To begin to define the roles of VEGF in diseases like asthma and COPD, we characterized the effects of lung-targeted transgenic VEGF165 and defined the innate immune pathways that regulate VEGF tissue responses. The former studies demonstrated that VEGF plays an important role in Th2 inflammation because, in addition to stimulating angiogenesis and edema, VEGF induced eosinophilic inflammation, mucus metaplasia, subepithelial fibrosis, myocyte hyperplasia, dendritic cell activation, and airways hyperresponsiveness via IL-13–dependent and -independent mechanisms. VEGF was also produced at sites of aeroallergen-induced Th2 inflammation, and VEGF receptor blockade ameliorated adaptive Th2 inflammation and Th2 cytokine elaboration. The latter studies demonstrated that activation of the RIG-like helicase (RLH) innate immune pathway using viral pathogen–associated molecular patterns such as Poly(I:C) or viruses ameliorated VEGF-induced tissue responses. In accord with these findings, Poly(I:C)-induced RLH activation also abrogated aeroallergen-induced Th2 inflammation. When viewed in combination, these studies suggest that VEGF excess can contribute to the pathogenesis of Th2 inflammatory disorders such as asthma and that abrogation of VEGF signaling via RLH activation can contribute to the pathogenesis of viral disorders such as virus-induced COPD exacerbations. They also suggest that RLH activation may be a useful therapeutic strategy in asthma and related disorders.

Background

Angiogenic therapy with vascular endothelial growth factor (VEGF) has been proposed as a treatment paradigm for patients suffering from an insufficiency of collateral vessels. Diabetes is associated with increase in the production of VEGF and therefore additional VEGF may not be beneficial. Accordingly, we sought to determine the efficacy of VEGF therapy to augment collateral formation and tissue perfusion in a diabetic mouse ischemic hindlimb model.

Methods

Diabetic and non-diabetic mice were studied in parallel for the efficacy of VEGF administration. Diabetes was induced with streptozotocin. Hindlimb ischemia was produced by severing the left iliac artery. An outlet tube from an osmotic infusion pump with placebo/ 500 micrograms of plasmid-DNA encoding VEGF was fenestrated and tunneled into the left quadriceps muscle.

Results

VEGF induced more rapid and complete restoration of blood flow in normal mice. However, in the setting of diabetes there was no difference between VEGF Vs. placebo in the rate or adequacy of flow restoration. There was a significant increase in smooth muscle actin and Factor-VIII antigen densities in diabetic animals and in animals which received VEGF.

Conclusions

Angiogenic therapy with VEGF in the setting of diabetes does not appear to have the beneficial effects seen in the absence of diabetes.

Vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis, which is required for tumor growth and metastasis. In this article, a review of the functional and biological roles of the VEGF pathway in driving angiogenesis and growth of gynecologic malignancies was performed. Based on the biological functions of VEGF, multiple approaches for targeting the VEGF/VEGF-receptor complex have been developed and many of these have demonstrated substantial activity in preclinical models. These promising data have led to rapid clinical development of VEGF-targeted agents. Therefore, we also assessed the status of VEGF-targeted therapies and associated toxicities in gynecologic malignancies. However, many questions remain related to optimal dosing, sequencing of therapies, management of toxicities, appropriate patient selection, and assessment of response, which will require further studies. Nevertheless, VEGF-targeted therapies offer hope for improving the outcome of cancer patients.

Proteins that promote angiogenesis, such as vascular endothelial growth factor (VEGF), are major targets for cancer therapy. Accordingly, proteins that specifically activate expression of factors like VEGF are potential alternative therapeutic targets and may help to combat evasive resistance to angiogenesis inhibitors. VEGF mRNA contains two internal ribosome entry sites (IRESs) that enable selective activation of VEGF protein synthesis under hypoxic conditions that trigger angiogenesis. To identify novel regulators of VEGF IRES-driven translation in human cells, we have developed a high-throughput screening approach that combines siRNA treatment with transfection of a VEGF-IRES reporter mRNA. We identified the kinase MAPK3 as a novel positive regulator of VEGF IRES-driven translation and have validated its regulatory effect on endogenous VEGF. Our automated method is scalable and readily adapted for use with other mRNA regulatory elements. Consequently, it should be a generally useful approach for high-throughput identification of novel regulators of mRNA translation.

Background: VEGF is central to cancer angiogenesis; however, we have a poor understanding of how VEGF is regulated in lung tumors.

Results: High levels of SP-1 transcription factor expression amplify basal and hypoxia-induced VEGF expression.

Conclusion: SP-1 plays a key role in both genetic and hypoxic microenvironment regulation of VEGF in cancer.

Significance: Targeting of both VEGF and SP-1 may provide a more effective cancer therapy.

VEGF plays a central role in angiogenesis in cancer. Non-small cell lung cancer (NSCLC) tumors have increased microvascular density, localized hypoxia, and high VEGF expression levels; however, there is a lack of understanding of how oncogenic and tumor microenvironment changes such as hypoxia lead to greater VEGF expression in lung and other cancers. We show that NSCLC cells secreted higher levels of VEGF than normal airway epithelial cells. Actinomycin D inhibited all NSCLC VEGF secretion, and VEGF minimal promoter-luciferase reporter constructs were constitutively active until the last 85 base pairs before the transcription start site containing three SP-1 transcription factor-binding sites; mutation of these VEGF promoter SP-1-binding sites eliminated VEGF promoter activity. Furthermore, dominant negative SP-1, mithramycin A, and SP-1 shRNA decreased VEGF promoter activity, whereas overexpression of SP-1 increased VEGF promoter activity. Chromatin immunoprecipitation assays demonstrated SP-1, p300, and PCA/F histone acetyltransferase binding and histone H4 hyperacetylation at the VEGF promoter in NSCLC cells. Cultured NSCLC cells expressed higher levels of SP-1 protein than normal airway epithelial cells, and double-fluorescence immunohistochemistry showed a strong correlation between SP-1 and VEGF in human NSCLC tumors. In addition, hypoxia-driven VEGF expression in NSCLC cells was SP-1-dependent, with hypoxia increasing SP-1 activity and binding to the VEGF promoter. These studies are the first to demonstrate that overexpression of SP-1 plays a central role in hypoxia-induced VEGF secretion.

Malignancy is a major problem in patients treated with immunosuppressive agents. We have demonstrated that treatment with calcineurin inhibitors (CNIs) can induce the activation of proto-oncogenic Ras, and may promote a rapid progression of human renal cancer through the overexpression of vascular endothelial growth factor (VEGF). Interestingly, we found that CNI-induced VEGF overexpression and cancer cell proliferation was inhibited by rapamycin treatment, indicating potential involvement of the mammalian target of rapamycin (mTOR) pathway in this tumorigenic process. Here, we examined the role of mTOR pathway in mediating CNI- and Ras-induced overexpression of VEGF in human renal cancer cells (786-0 and Caki-1). We found that the knockdown of raptor (using siRNA) significantly decreased CNI-induced VEGF promoter activity as observed by promoter-luciferase assay, suggesting the role of mTOR complex1 (mTORC1) in CNI-induced VEGF transcription. It is known that mTOR becomes activated following phosphorylation of its negative regulator PRAS40, which is a part of mTORC1. We observed that CNI treatment and activation of H-Ras (through transfection of an active H-Ras plasmid) markedly increased the phosphorylation of PRAS40, and the transfection of cells using a dominant-negative plasmid of Ras, significantly decreased PRAS40 phosphorylation. Protein kinase C (PKC)-ζ and PKC-δ, which are critical intermediary signaling molecules for CNI-induced tumorigenic pathway, formed complex with PRAS40; and we found that the CNI treatment increased the complex formation between PRAS40 and PKC, particularly (PKC)-ζ. Inhibition of PKC activity using pharmacological inhibitor markedly decreased H-Ras-induced phosphorylation of PRAS40. The overexpression of PRAS40 in renal cancer cells significantly down-regulated CNI- and H-Ras-induced VEGF transcriptional activation. Finally, it was observed that CNI treatment increased the expression of phosho-PRAS40 in renal tumor tissues in vivo. Together, the phosphorylation of PRAS40 is critical for the activation of mTOR in CNI-induced VEGF overexpression and renal cancer progression.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), increases microvascular permeability and is a specific mitogen for endothelial cells. Expression of VPF/VEGF previously was demonstrated in a variety of tumor cells, in cultures of pituitary-derived cells, and in corpus luteum. Here we present evidence, by Northern analysis and in situ hybridization, that the VPF/VEGF gene is expressed in many adult organs, including lung, kidney, adrenal gland, heart, liver, and stomach mucosa, as well as in elicited peritoneal macrophages. The highest levels of VPF/VEGF transcripts were found in epithelial cells of lung alveoli, renal glomeruli and adrenal cortex, and in cardiac myocytes. The prominence of VPF/VEGF mRNA in these tissues suggests a possible role for VPF/VEGF in regulating baseline microvascular permeability, which is essential for tissue nutrition and waste removal. We also demonstrate particularly high VPF/VEGF mRNA levels in several human tumors, where it may be involved in promoting tumor angiogenesis and stroma generation, both as an endothelial cell mitogen and indirectly by its permeability enhancing effect that leads to the deposition of a provisional fibrin gel matrix.

Images

Vascular endothelial growth factor (VEGF) and its receptors Flt-1 and KDR play important roles in physiological and pathological angiogenesis. Ribozymes that target the VEGF receptor mRNAs were developed and their biological activities in cell culture and an animal model were assessed. Ribozymes targeting Flt-1 or KDR mRNA sites reduced VEGF-induced proliferation of cultured human vascular endothelial cells and specifically lowered the level of Flt-1 or KDR mRNA present in the cells. Anti- Flt-1 and KDR ribozymes also exhibited anti-angiogenic activity in a rat corneal pocket assay of VEGF-induced angiogenesis. This report illustrates the anti-angiogenic potential of these ribozymes as well as their value in studying VEGF receptor function in normal and pathophysiologic states.

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising target for cancer therapy. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. The present study was designed to determine the role of VEGF in tumor growth and metastasis using RNA interference (RNAi) technology. Four small interfering RNA (siRNA) sequences for the VEGF gene were cloned into expression plasmids and transfected into human colorectal carcinoma (CRC) SW620 cells. Stable transfection of these plasmids decreased VEGF protein expression, leading to the potent suppression of tumor cell proliferation, migration, invasion, and angiogenesis in vitro. Furthermore, in subcutaneous and intrasplenic/portal injection models involving athymic nude mice, the tumor growth and metastasis of SW620 cells expressing VEGF siRNA were significantly inhibited compared with untransfected cells or cells transfected with control vector alone. Immunohistochemical analyses of tumor sections revealed a decreased vessel density and decreased VEGF expression in the animals where siRNA against VEGF were expressed. These results indicate that RNAi of VEGF can be an effective antiangiogenic strategy for CRC.

Despite setbacks, the clinical development of antiangiogenic agents has accelerated remarkably over the past 3–4 years. Consequently, there are currently three direct inhibitors of the VEGF pathway approved for use in cancer therapy. Other agents that block the VEGF pathway are in advanced stages of clinical development and have shown promising results. With these exciting developments come crucial questions regarding the use of these new molecular-targeted agents, alone or in combination with standard cytotoxic or targeted agents. Importantly, the mechanisms of action of anti-VEGF therapy remain unknown. Here, we discuss several potential mechanisms of action such as tumor vascular normalization, bone marrow-derived cell recruitment blockade and cytostatic effects of anti- VEGF therapy. We review the current progress, the major stumbling blocks and the future directions for anti-cancer therapy using anti-VEGF agents, emphasizing clarification of the underlying molecular mechanisms of action and biomarker identification and validation.

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis which drives endothelial cell survival, proliferation, and migration while increasing vascular permeability. Playing an important role in the physiology of normal ovaries, VEGF has also been implicated in the pathogenesis of ovarian cancer. Essentially by promoting tumor angiogenesis and enhancing vascular permeability, VEGF contributes to the development of peritoneal carcinomatosis associated with malignant ascites formation, the characteristic feature of advanced ovarian cancer at diagnosis. In both experimental and clinical studies, VEGF levels have been inversely correlated with survival. Moreover, VEGF inhibition has been shown to inhibit tumor growth and ascites production and to suppress tumor invasion and metastasis. These findings have laid the basis for the clinical evaluation of agents targeting VEGF signaling pathway in patients with ovarian cancer. In this review, we will focus on VEGF involvement in the pathophysiology of ovarian cancer and its contribution to the disease progression and dissemination.

Background

Angiogenesis is a process by which new capillaries are formed from pre-existing blood vessels in physiological (e.g., exercise, wound healing) or pathological (e.g., ischemic limb as in peripheral arterial disease, cancer) contexts. This neovascular mechanism is mediated by the vascular endothelial growth factor (VEGF) family of cytokines. Although VEGF is often targeted in anti-angiogenic therapies, there is little knowledge about how its concentration may vary between tissues and the vascular system. A compartment model is constructed to study the VEGF distribution in the tissue (including matrix-bound, cell surface receptor-bound and free VEGF isoforms) and in the blood. We analyze the sensitivity of this distribution to the secretion rate, clearance rate and vascular permeability of VEGF.

Results

We find that, in a physiological context, VEGF concentration varies approximately linearly with the VEGF secretion rate. VEGF concentration in blood but not in tissue is dependent on the vascular permeability of healthy tissue. Model simulations suggest that relative VEGF increases are similar in blood and tissue during exercise and return to baseline within several hours. In a pathological context (tumor), we find that blood VEGF concentration is relatively insensitive to increased vascular permeability in tumors, to the secretion rate of VEGF by tumors and to the clearance. However, it is sensitive to the vascular permeability in the healthy tissue. Finally, the VEGF distribution profile in healthy tissue reveals that about half of the VEGF is complexed with the receptor tyrosine kinase VEGFR2 and the co-receptor Neuropilin-1. In diseased tissues, this binding can be reduced to 15% while VEGF bound to the extracellular matrix and basement membranes increases.

Conclusion

The results are of importance for physiological conditions (e.g., exercise) and pathological conditions (e.g., peripheral arterial disease, coronary artery disease, cancer). This mathematical model can serve as a tool for understanding the VEGF distribution in physiological and pathological contexts as well as a foundation to investigate pro- or anti-angiogenic strategies.

This study showed that VEGF-C may potentially serve as an important target in corneal transplant by modulating corneal alloimmunity at three different levels. Anti-VEGF-C inhibits the ingrowth of lymphatic and blood vessel, reduces the trafficking of APC, and, interestingly, inhibits APC maturation.

Purpose.

To investigate the role of anti–vascular endothelial growth factor (VEGF)-C therapy in corneal graft survival and concomitant suppression of hem- and lymph-angiogenesis.

Methods.

Corneal suture model in BALB/c mice was placed and immunohistochemical staining was performed with CD31/PECAM-1 and LYVE-1 to quantify the level of blood and lymphatic vessels. Corneal transplants were done in BALB/c mice from C57BL/6 mice donors; grafts were subsequently scored for opacity. VEGF-C was blocked in the angiogenesis and transplant model using neutralizing monoclonal anti-VEGF-C (VGX-100) by intraperitoneal injection. To determine the function of VEGF-C in maturation of antigen-presenting cells (APCs), bone marrow–derived dendritic cells were generated and matured in the presence or absence of VEGF-C.

Results.

VEGF-C expression was demonstrated to be markedly upregulated in corneal graft rejection. VEGF-C blockade, through administration of a VEGF-C blocking monoclonal antibody, suppresses corneal angiogenic responses, inhibits trafficking and maturation of APCs, and significantly improves allotransplant survival.

Conclusions.

These data suggest VEGF-C as a potentially important target in corneal transplant pharmacotherapy and immunobiology.

The physiology of microvessels limits the growth and development of tumours. Tumours gain nutrients and excrete waste through growth-associated microvessels. New anticancer therapies target this microvasculature by inhibiting vascular endothelial growth factor A (VEGF-A) splice isoforms that promote microvessel growth. However, certain VEGF-A splice isoforms in normal tissues inhibit growth of microvessels. Thus, it is the VEGF-A isoform balance, which is controlled by mRNA splicing, that orchestrates angiogenesis. Here, we highlight the functional differences between the pro-angiogenic and the anti-angiogenic VEGF-A isoform families and the potential to harness the synthetic capacity of cancer cells to produce factors that inhibit, rather than aid, cancer growth.