Vascular Endothelial Growth Factor Vegf Research Articles

Determination of Endothelial Stalk versus Tip Cell Potential during Angiogenesis by H2.0-like Homeobox-1

SummaryTissue branching morphogenesis requires the hierarchical organization of sprouting cells into leading “tip” and trailing “stalk” cells [1, 2]. During new blood vessel branching (angiogenesis), endothelial tip cells (TCs) lead sprouting vessels, extend filopodia, and migrate in response to gradients of the secreted ligand, vascular endothelial growth factor (Vegf) [3]. In contrast, adjacent stalk cells (SCs) trail TCs, generate the trunk of new vessels, and critically maintain connectivity with parental vessels. Here, we establish that h2.0-like homeobox-1 (Hlx1) determines SC potential, which is critical for angiogenesis during zebrafish development. By combining a novel pharmacological strategy for the manipulation of angiogenic cell behavior in vivo with transcriptomic analyses of sprouting cells, we identify the uniquely sprouting-associated gene, hlx1. Expression of hlx1 is almost entirely restricted to sprouting endothelial cells and is excluded from adjacent nonangiogenic cells. Furthermore, Hlx1 knockdown reveals its essential role in angiogenesis. Importantly, mosaic analyses uncover a cell-autonomous role for Hlx1 in the maintenance of SC identity in sprouting vessels. Hence, Hlx1-mediated maintenance of SC potential regulates angiogenesis, a finding that may have novel implications for sprouting morphogenesis of other tissues.

The attenuation of restenosis following arterial gene transfer using carbon nanotube coated stent incorporating TAT/DNAAng1+Vegf nanoparticles

This study report the development of a nanobiohybrid hydrogel based endovascular stent device capable of preventing postangioplasty in-stent restenosis (ISR) by promoting significant vascular endothelial recovery in a site-specific manner. The hydrogel is comprised of fibrin matrices, assembled layer-by-layer (LbL) on stent surface with alternate layers carrying endosomolytic Tat peptide/DNA nanoparticles (NPs) or NPs hybridized to polyacrylic acid (PAA) wrapped single-walled carbon nanotubes (NP–CNT). Here, the hydrogel works as a reservoir to carry, protect, and simultaneously deliver pro-angiogenic, vascular endothelial growth factor (Vegf) and Angiopoietin-1(Ang1), gene carrying NPs to the target site. In vitro results demonstrated that CNTs incorporated in the hydrogel layers play a major role in tuning the bioactivity of the stent. In addition, the developed stent formulation can significantly reduce the loss of therapeutics while traversing through the vessel and during deployment. In vivo experiments in balloon-injured canine femoral artery demonstrated that the NCS (+) group, carrying NPvegf+Ang1, can significantly enhance re-endothelialization of injured artery compared to control NCS (−), carrying NPNull, and bare metal stent (BMS) groups, attenuate stenosis (18.5±9.03% vs 39.56±13.8 vs 45.34±8.3%; n=8, p<0.05) and prevent neointima formation (1.53±0.36mm2 vs 2.51±0.27mm2 vs 2.66±0.14mm2; n=8, p<0.05) as analyzed angiography and histomorphometric analysis. These data collectively implicate that this new technology can be useful for stent and other biomedical devices through controlled delivery of multiple biotherapeutics.

Pathological neoangiogenesis depends on oxidative stress regulation by ATM

The ataxia telangiectasia mutated (ATM) kinase, a master regulator of the DNA damage response (DDR), acts as a barrier to cellular senescence and tumorigenesis. Aside from DDR signaling, ATM also functions in oxidative defense. Here we show that Atm in mice is activated specifically in immature vessels in response to the accumulation of reactive oxygen species (ROS). Global or endothelial-specific Atm deficiency in mice blocked pathological neoangiogenesis in the retina. This block resulted from increased amounts of ROS and excessive activation of the mitogen activated kinase p38α rather than from defects in the canonical DDR pathway. Atm deficiency also lowered tumor angiogenesis and enhanced the antiangiogenic action of vascular endothelial growth factor (Vegf) blockade. These data suggest that pathological neoangiogenesis requires ATM-mediated oxidative defense and that agents that promote excessive ROS generation may have beneficial effects in the treatment of neovascular disease.

Nanoantibodies for detection and blocking of bioactivity of human vascular endothelial growth factor a165

Nanoantibodies (single-domain antibodies, nanobodies) derived from noncanonical single-chain immunoglobulins provide an attractive tool for in vitro and in vivo diagnostics as well as for development of targeted drugs for clinical use. Nanoantibodies against several clinically important targets have been developed and are actively investigated. However, no development of nanoantibodies against vascular endothelial growth factor VEGF-A(165) has been reported. We describe here the generation of nanoantibodies derived from single-chain Bactrian camel immunoglobulins directed against VEGF-A(165). We demonstrate that these nanoantibodies are suitable for enzyme-linked immunoassay to quantify human VEGF-A(165) as well as for blocking its activity. Our results provide a basis for diagnostic kit development for quantification of VEGF-A(165), which emerges as a biomarker useful in various pathological conditions. In addition, the nanoantibodies might be used for development of therapeutic molecules targeting VEGF-A(165)-dependent pathological neoangiogenesis.

P-0267 Correlation Between Serum Vascular Endothelial Growth Factor (Vegf) and Vegf-1 Expression in Patients with Colorectal Cancer. Preliminary Results

ABSTRACT Introduction In patients with colorectal cancer (CRC), several growth factors have been identified as regulator of angiogenesis, including vascular endothelial growth factor (VEGF), a pro-angiogenic factor which can be safely measured in the blood. Angiogenesis plays an important role in the progression of CRC, as well as in the development of regional lymph node and distant metastasis. VEGF is minimally expressed or not expressed in colorectal adenomas and in normal colonic mucosa, while it is overexpressed in inflammatory bowel diseases as well as in CRCs. It has been shown that high microvessels density, an indirect measure of the degree of angiogenesis, correlates with increased tumor aggressiveness and risk of metastasis development, and that overexpression of VEGF in cancer tissues represents a sensitive marker of angiogenesis. Because undetected metastases can contribute to the failure of primary treatment, their early identification has a substantial impact on adequate therapy. In this setting, several serum tumor markers and tissue-extracting prognostic factors have been tested, including endothelial cell growth factor, and angiogenic cytokines, such as VEGF. In several studies, preoperative serum VEGF was increased in patients with CRC compared to controls, and high VEGF serum concentration tended to occur with more advanced disease. However, these data are not confirmed, and exclusively VEGF overexpression in cancer tissues significantly correlates with tumor biology. The aim of this study was to evaluate the relationship between serum VEGF, and the expression of VEGF-1 in a group of patients with CRC. Download : Download full-size image Methods Archival paraffin-embedded tumor samples from 38 patients (26 men, 12 women, median age 64 years, range 39-76) with stage I-II colorectal adenocarcinoma who underwent intentionally curative surgical resection, were reviewed. None of the patients received preoperative adjuvant chemotherapy. Written informed consent was obtained from all the participants. Serum VEGF was measured with a commercially available human VEGF quantitative enzyme-linked immunosorbent assay (ELISA) kit, according to the manufacturer's instructions. All analyses were made in duplicate, and the mean value was used for statistical calculations. Tissue microarray blocks for immunohistochemistry staining was obtained, serial 4 µm sections were cut, and then assessed using a commercial anti-human VEGF (clone VG-1). The Pearson's correlation coefficient calculation was used to evaluate results. Results The expression of VEGF was tested using a 4-grade scoring system, according to the degree of cytoplasmic staining, ranging from negative to intense staining. Overall, the mean serum VEGF concentration was 272.7±140.5 pg/mL (range 41-670 pg/mL), while the VEGF-1 grade was 2.8±1.0. There was a significant relationship between the stage of the disease and VEGF (R=0.43, p=0.006), and between serum VEGF and VEGF-1 grade (R=0.64, t=5.06, p Conclusion Tumor growth is dependent on angiogenesis and VEGF is potent growth factor with angiogenic activity. It can safely be tested by measurements of both serum VEGF levels and grade of tumor tissue expression of VEGF-1.

Enhancement of endothelial cell migration by constitutively active LPA1-expressing tumor cells

Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors (LPA receptors; LPA1 to LPA6). They indicate a variety of cellular response by the interaction with LPA, including cell proliferation, migration and differentiation. Recently, we have reported that constitutive active mutated LPA1 induced the strong biological effects of rat neuroblastoma B103 cells. In the present study, we examined the effects of mutated LPA1 on the interaction between B103 cells and endothelial F-2 cells. Each LPA receptor expressing B103 cells were maintained in serum-free DMEM and cell motility assay was performed with a Cell Culture Insert. When F-2 cells were cultured with conditioned medium from Lpar1 and Lpar3-expressing cells, the cell motility of F-2 cells was significantly higher than control cells. Interestingly, the motile activity of F-2 cells was strongly induced by mutated LPA1 than other cells, correlating with the expression levels of vascular endothelial growth factor (Vegf)-A and Vegf-C. Pretreatment of LPA signaling inhibitors inhibited F-2 cell motility stimulated by mutated LPA1. These results suggest that activation of LPA signaling via mutated LPA1 may play an important role in the promotion of angiogenesis in rat neuroblastoma cells.

In Vivo Delivery of Plasmid DNA into Rabbit Myocardium Using Polylysine Copolymers

The terplex system for delivering DNA to targeted cells comprises three components-stearyl polylysine (PLL), low-density lipoprotein (LDL), and DNA. This article describes the use of a terplex complex to deliver plasmid DNA carrying the vascular endothelial growth factor VEGF165 into rabbit myocardium. Transfection with VEGF is assessed by comparing tissue pathology and pumping function of the heart in control and experimental animals after induced myocardial infarction.

Abstract 4253: In vivo activity of small molecule HIF2α inhibitors

Abstract Hypoxia-inducible factor (HIF) is a central regulator of the cellular response to hypoxia. Hypoxia promotes HIF activity by increasing its stability, transcriptional activity and translation. HIF target genes involved in several cellular processes including proliferation, angiogenesis, metabolism and promotion of the “stem-cell” phenotype. In normoxic conditions, HIF-α is recognized for degradation by the von Hippel-Lindau tumor suppressor protein (VHL). Loss-of-VHL function leads to constitutive expression of HIF regulatory subunits and consequent over-expression of HIF-target genes. We and others have shown that HIF2a is necessary and sufficient for the development of VHL-related renal cell carcinoma. We have developed small molecules that repress HIF-2α translation, in vitro, by enhancing the binding of Iron-Regulatory Protein 1 (IRP1) to the Iron-Responsive Element (IRE) within the 5′-UTR of HIF-2α mRNA (Zimmer M. et al. Molecular Cell 2008;32: 828-848). We showed that these small molecules down-regulate HIF2a expression in vitro, in all renal cell carcinoma cell lines. To validate these small molecules in vivo, we tested their efficacy on wild-type zebrafish embryos. Treatment of 3 days post fertilization zebrafish embryos with 100microM of the hypoxic mimetic DMOG or hypoxia dramatically upregulated the expression of HIF-target genes, such as Vascular Endothelial Growth Factor (Vegf), erythropoietin (Epo) and prolyl-hydroxylase 3 (Phd3). Pre-treatment of embryos with the small molecule HIF2a inhibitor 76 potently decreased the DMOG-induced upregulation of HIF target genes. The IC50 values of these small molecules were significantly less in vivo than in vitro. We are currently testing the efficacy of HIF2a inhibitors in rescuing the VHL zebrafish phenotype and prolonging survival of VHL mutant zebrafish embryos. In addition, data on the efficacy of these inhibitors on the growth of human renal cell carcinoma cells as tumors in the nude mouse xenograft assay will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4253. doi:1538-7445.AM2012-4253

Lysophosphatidic Acid Induces Lymphangiogenesis and IL-8 Production in Vitro in Human Lymphatic Endothelial Cells

The bioactive phospholipid lysophosphatidic acid (LPA) and its receptors LPA(1-3) are aberrantly expressed in many types of human cancer. LPA has been reported to induce tumor cell proliferation, migration, and cytokine production. However, whether LPA exerts an effect on lymphatic endothelial cells (LECs) or on lymphangiogenesis, a process of new lymphatic vessel formation that is associated with increased metastasis and poor prognosis in cancer patients, has been unknown. Here, we show that LPA induces cell proliferation, survival, migration, and tube formation, and promotes lymphangiogenesis in vitro in human dermal LECs. In addition, LPA induces IL-8 expression by enhancing IL-8 promoter activity via activation of the NF-κB pathway in LECs. Using IL-8 siRNA and IL-8 neutralizing antibody, we revealed that IL-8 plays an important role in LPA-induced lymphangiogenesis in vitro. Moreover, using siRNA inhibition, we discovered that LPA-induced lymphangiogenesis in vitro and IL-8 production are mediated via the LPA(2) receptor in LECs. Finally, using human sentinel afferent lymphatic vessel explants, we demonstrated that LPA up-regulates IL-8 production in the LECs of lymphatic endothelia. These studies provide the first evidence that LPA promotes lymphangiogenesis and induces IL-8 production in LECs; we also reveal a possible new role of LPA in the promotion of tumor progression, as well as metastasis, in different cancer types.

MiR-221 Is Required for Endothelial Tip Cell Behaviors during Vascular Development

Angiogenesis requires coordination of distinct cell behaviors between tip and stalk cells. Although this process is governed by regulatory interactions between the vascular endothelial growth factor (Vegf) and Notch signaling pathways, little is known about the potential role of microRNAs. Through deep sequencing and functional screening in zebrafish, we find that miR-221 is essential for angiogenesis. miR-221 knockdown phenocopied defects associated with loss of the tip cell-expressed Flt4 receptor. Furthermore, miR-221 was required for tip cell proliferation and migration, as well as tip cell potential in mosaic blood vessels. miR-221 knockdown also prevented "hyper-angiogenesis" defects associated with Notch deficiency and miR-221 expression was inhibited by Notch signaling. Finally, miR-221 promoted tip cell behavior through repression of two targets: cyclin dependent kinase inhibitor 1b (cdkn1b) and phosphoinositide-3-kinase regulatory subunit 1 (pik3r1). These results identify miR-221 as an important regulatory node through which tip cell migration and proliferation are controlled during angiogenesis.

Pigment Epithelium-Derived Factor: Chemistry, Structure, Biology, and Applications

Pigment epithelium-derived factor (PEDF) is a multifunctional serpin protein with demonstrable neurotrophic, antiangiogenic, antitumorigenic, antimetastatic, anti-inflammatory, antioxidative properties among others. PEDF exists naturally in most organs of the human body, and it is released from most cell types as an extracellular diffusible and circulating glycoprotein. It has been studied mostly in the eye, where its levels are altered in diseases characterized by retinopathies, such as age-related macular degeneration and diabetic retinopathy. The importance of PEDF in the development, maintenance, and function of the retina and CNS is evident in animal models for inherited and light-induced retinal degeneration, as well as for degeneration of spinal cord motor neurons. Pathological ocular neovascularization- and retinal degeneration-related animal models have prompted clinical development. Clinical trials to assess the safety of a viral expression vector for PEDF in the context of age-related macular degeneration have been performed. Interestingly, PEDF has multiple biological effects against tumors, and its efficacy has been demonstrated in several animal models for tumor growth and progression. The mechanisms of PEDF action on tumors have been associated to inhibition of tumor angiogenesis, and also negative effects directed on tumor cells. Recently, the involvement of PEDF in stem cell biology has been revealed. Moreover, PEDF is a potential diagnostic tool for several diseases triggered by pathological neovascularization, retinal degenerations, or tumors. The present issue includes three original research and four review articles. In one article, V. Chandolu and C. R. Dass review the biological functions of PEDF against cancer, with a focus on a particular type of bone cancer called osteosarcoma. They summarize the progress in understanding the function of PEDF in antiangiogenesis, tumor cell differentiation, and direct tumor cell inhibition. In another review article, J.-T. Liu et al. address the role of PEDF in stem/progenitor cell-associated neovascularization, in particular in cardiovascular and neurovascular biology. M. Elahy et al. review the promising significance of PEDF in stem cell biology, specifically in human embryonic stem cells, mesenchymal stem cells (MSCs), neural stem sells (NSCs), and stem cells overexpressing the PEDF gene. X.-F. Zhu et al. summarize the advances of PEDF in diabetic retinopathy, its protective effect on oxidative stress, which is the main trigger for the pathology, and the potential application of PEDF in diabetic retinopathy. In a research article, P. Subramanian et al. report the identification of biochemical forms of PEDF with distinct biological effects on tumor cells, which may explain the multifunctional modality of this protein. In a second research article, M. L. Broadhead et al. report the effects of two PEDF-derived peptides on a clinically relevant murine model of osteosarcoma with spontaneous metastasis. Finally, T. Falk et al. report that human retinal pigment epithelial cells grown on plastic-based microcarriers retain the ability to produce both PEDF and vascular endothelial growth factor VEGF-A. This constitutes a novel candidate delivery system for neurotrophic factors with potential application in neurodegenerative diseases, such as Parkinson's. In summary, given the multimodal nature of PEDF, the present issue aims to enhance our understanding of the chemistry, structure, biology, and application of PEDF in biomedicine. It gives an overview of the current status of research on PEDF and will prove useful as source of reference for students and researchers. S. Patricia Becerra Crispin R. Dass Takeshi Yabe Susan E. Crawford

Current Trends and Future Directions in Clinical Trials for Malignant Melanoma Treatment Using Anti-Angiogenic Strategies

Melanoma is the most lethal skin cancer with a high propensity to metastasis and conventionally is poorly responsive to non-surgical treatments including chemotherapy and radiotherapy. Considerable advances have been made recently targeting BRAF mutations and immune regulation and, for the first time, credible options exist for patients with metastatic disease. Angiogenesis, the growth of new blood vessels, is an absolute prerequisite for tumour growth beyond a few millimetres in size. Melanoma neovascularisation is correlated with poor prognosis, reduced overall survival, ulceration and increased rate of relapse. Melanoma cells secrete several proangiogenic cytokines including Vascular Endothelial Growth Factor VEGF-A and raised levels of expression are associated with the switch from indolent radial, to invasive vertical and then metastatic growth phases. Understanding the processes underlying angiogenesis and how it relates to tumour growth broadly and to melanoma specifically is instrumental in the current drive to develop new treatments that target a range of tumour cell receptors and intracellular processes from receptor antagonism to monoclonal antibodies aimed at the disruption of the process of tumour angiogenesis. We discuss recent and current trials for metastatic melanoma therapy, and discuss potential directions of future treatment scheduling considering different treatment scheduling approaches beyond the parameters of standard drug trials.

Phase I evaluation of telatinib, a VEGF receptor tyrosine kinase inhibitor, in combination with bevacizumab in subjects with advanced solid tumors

BackgroundBlocking both receptor and ligand of the vascular endothelial growth factor (receptor) VEGF(R) pathway might be feasible and increase antitumor activity. This phase I study investigated telatinib, an oral tyrosine kinase inhibitor targeting VEGFR-2, combined with bevacizumab, in adults with solid tumors. Patients and methodsTwenty-six patients were treated in successive cohorts with telatinib (twice-daily continuously, 450–900 mg) or bevacizumab (bi-weekly, starting dose 5 mg/kg). Safety, pharmacokinetics, endothelial (progenitor) cell (E(P)C)/growth factor kinetics and efficacy were assessed. ResultsMost frequent adverse events were pain, nausea, voice changes and fatigue. Five dose-limiting toxicities (DLTs) occurred: hypertension (cohort I and II), bowel perforation, lipase increase and atrial flutter (cohort III). Cumulative toxicity resulted in a bevacizumab dose reduction to 1 mg/kg (cohort III). Due to three DLTs (n = 14), this cohort represented the best-tolerated dose level. Bevacizumab effectively neutralized plasma VEGF even at 1 mg/kg. Twelve patients had stable disease (clinical benefit 46%). EPC and SDF-1α levels increased during monotherapy telatinib. ConclusionsTelatinib (450 mg b.i.d.) combined with bevacizumab (1 mg/kg bi-weekly) shows antitumor activity, but accumulating constitutional toxicity impedes long-term treatment of patients. Therefore, this combination will not be pursued in a phase II setting.

Effects of Raspberry Phytochemical Extract on Cell Proliferation, Apoptosis, and Serum Proteomics in a Rat Model

The red raspberry extract possesses potent antioxidant capacity and anticancerous activity in vitro and in vivo. The objective of this study was to determine whether red raspberry extract affected the cell cycle, angiogenesis, and apoptosis in hepatic lesion tissues from a rat model induced by diethylnitrosamine (DEN) as well as changes of serum proteomics. Rats were treated with red raspberry extract (0.75, 1.5, or 3.0 g/kg of body weight) by gavage starting 2 h after DEN administration and continued for 20 wk. Red raspberry extract inhibited cell proliferation, vascular endothelial growth factor VEGF expression, and induced apoptosis in the hepatic lesion tissues. In addition, 2 protein peaks (2597.93 and 4513.88 m/z) were identified to differentially express in the 3.0 g/kg body weight and positive control groups by serum proteomics. These results suggest that a dietary supplement with red raspberry effectively protects against chemically induced hepatic lesions in rats.

Do Atherosclerosis and Obesity-Associated Susceptibility to Cancer Share Causative Link to oxLDL and LOX-1?

Abbreviations 4-HNE 4-Hydroxynonenal APC Antigen presenting cell CHO Chinese hamster ovary cells COX-2 Cyclooxygenase-2 FASN Fatty acid synthase HFD High fat diet HSPs Heat shock proteins ICAM Inter-cellular adhesion molecule LDL Low density lipoproteins LDLR LDL receptor LOX-1 Lectin-type oxidized LDL receptor 1 LPC Lysophosphatidyl choline Lp-PLA2 Liporpotein-associated phospholipase A 2 LRP LDL receptor related protein MCP-1 Monocyte chemoattractant protein MPO Myeloperoxidase MUFA Monounsaturated fatty acids NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells oxLDL Oxidized low density lipoproteins oxPAPC 1-palmitoyl-2-arachidonoyl-sn-glycero3-phosphocholine PAPC 1-palmitoyl-2-arachidonoyl-sn-glycero3-phosphocholine PPAR γ Peroxisome proliferator-activated receptor gamma PPRE PPARA response element PUFA Polyunsaturated fatty acids ROS Reactive oxygen species RPE Retinal pigment epithelium SCD1 Stearoyl-CoA desaturase-1 SMCs Smooth muscle cells SOD Superoxide dismutase TNFα Tumor necrosis factor-alpha VCAM Vascular cellular adhesion molecule VEGF Vascular endothelial growth factor

HIF-1α in Epidermis: Oxygen Sensing, Cutaneous Angiogenesis, Cancer, and Non-Cancer Disorders

Besides lung, postnatal human epidermis is the only epithelium in direct contact with atmospheric oxygen. Skin epidermal oxygenation occurs mostly through atmospheric oxygen rather than tissue vasculature, resulting in a mildly hypoxic microenvironment that favors increased expression of hypoxia-inducible factor-1α (HIF-1α). Considering the wide spectrum of biological processes, such as angiogenesis, inflammation, bioenergetics, proliferation, motility, and apoptosis, that are regulated by this transcription factor, its high expression level in the epidermis might be important to HIF-1α in skin physiology and pathophysiology. Here, we review the role of HIF-1α in cutaneous angiogenesis, skin tumorigenesis, and several skin disorders.

Genetic background determines mouse strain differences in inflammatory angiogenesis

Inflammation and angiogenesis are key components of fibrovascular tissue growth, a biological event underlying both physiological (wound healing) and pathological conditions (tumor development, chronic inflammation). We investigated these components in three frequently used mouse strains (Swiss, Balb/c and C57BL/6J) to verify the influence of genetic background on the kinetics of inflammatory cell recruitment/activation, neovascularization, extracellular matrix deposition, and cytokine production in polyether-polyurethane sponge implanted subcutaneously in male mice of these strains. The kinetics of neutrophil recruitment/activation as assessed by myeloperoxidase (MPO) activity was 2- and 3-fold higher in Balb/c implants at day 1 compared with Swiss and C57BL/6J implants, respectively. Macrophage accumulation/activation as NAG (n-acetyl β-glucosaminidase) activity was higher in Swiss implants. The levels the monocyte chemoattractant protein 1 (CCL2(MCP-1)) peaked at day 10 in the three types of implants but was produced more by C57BL/6J mice. Angiogenesis (hemoglobin, vascular endothelial growth factor-VEGF, and number of vessels) differed among the strains. Swiss implants had the highest hemoglobin content but the lowest VEGF levels. In contrast, Balb/c implants had higher VEGF levels but lower hemoglobin. Collagen deposition and transforming growth factor β-1; TGFβ-1 levels also varied among the groups. Swiss and Balb/c implants had progressive increase in TGFβ-1 from 4 to 14days, while C57BL/6J implants achieved the peak at day 10 and fell at day 14. These findings emphasize the major contribution of genetic background in the temporal pattern and intensity of inflammatory angiogenesis components that may have functional consequences in physiological and pathological conditions where these processes co-exist.

Inhibitory effects of zoledronic acid on cell proliferation and invasion in human nasopharyngeal carcinoma cell line HNE1

To explore the in vitro anti-tumor effects of zoledronic acid on cell proliferation and invasion in human nasopharyngeal carcinoma cell line HNE1. The cytotoxic effects of zoledronic acid on HNE1 cells were detected by MTT assay, invasion of HNE1 cells by Transwell assay, secretion of (vascular endothelial growth factor)VEGF by (enzyme-linked immunosorbent assay) ELISA and the activities of MMP (matrix metalloproteinase) 2 and MMP9 by gelatine zymography. And the expressions of mRNA and proteins of MMP2, MMP9 and VEGF were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. After a treatment of zoledronic acid at 2.5, 5, 10, 20 and 40 mol/L for 48 h or 72 h, the highest inhibition rate of proliferation at approximately 50% was observed in the 40 mol/L group after 72 h. The inhibitory effect was not in a dose/time-dependent manner. After a 24-hour treatment of zoledronic acid at different concentrations (0, 10, 20 and 40 mol/L), the numbers of membrane-invading cells were 75.8 ± 2.6, 54.8 ± 5.4, 44.6 ± 6.4 and 38.6 ± 8.2 respectively (all P < 0.01). Gelatinase zymography demonstrated that the activities of MMP2 and MMP9 were inhibited significantly only in cells treated at 0 µmol/L. After a 24-hour exposure to zoledronic acid at 0, 10, 20 and 40 µmol/L, the concentrations of VEGF in supernatant were (5264 ± 89), (4626 ± 30), (4155 ± 40) and (1908 ± 171) g/L respectively (all P < 0.01). The expressions of mRNA and protein of MMP2, MMP9 and VEGF were down-regulated. Zoledronic acid can inhibit the in vitro proliferation and invasion of HNE1 cell through suppressing the secretion of VEGF, the activities of MMP2 and MMP9 and the expressions of VEGF, MMP2 and MMP9.

Levels of VEGF but not VEGF 165b are Increased in the Vitreous of Patients With Retinal Vein Occlusion

To determine the concentration of the pro-angiogenic vascular endothelial growth factor VEGF(165) (VEGF) and the anti-angiogenic VEGF(165b) in vitreous samples of patients with branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO) in comparison to patients without retinal occlusive disease. Experimental laboratory investigation. Vitreous samples were collected from patients undergoing surgery for arteriovenous dissection after BRVO, radial optic neurotomy after CRVO in the occlusion group, or macular pucker or macular hole in the control group. Concentrations of VEGF and VEGF(165b) were determined by ELISA and an ELISA-type antibody microarray. Average vitreal concentration of VEGF was 8.6 ng/mL in the CRVO group and 2.0 ng/mL in the BRVO group as compared to 0.26 ng/mL in the control group. Average vitreal concentration of VEGF(165b) was 27 pg/mL in the CRVO group, 42 pg/mL in the BRVO group, and 49 pg/mL in the control group. In patients with CRVO and BRVO, the angiogenic balance was shifted towards angiogenic stimulation. The severity of RVO from BRVO to CRVO correlates with an increase of VEGF and the decrease of VEGF(165b), indicating a pro-angiogenic shift. Altering the ratio of VEGF(165b)/VEGF(165) might be a feasible approach for treating retinal occlusive diseases.

Flt1 acts as a negative regulator of tip cell formation and branching morphogenesis in the zebrafish embryo

Endothelial tip cells guide angiogenic sprouts by exploring the local environment for guidance cues such as vascular endothelial growth factor (VegfA). Here we present Flt1 (Vegf receptor 1) loss- and gain-of-function data in zebrafish showing that Flt1 regulates tip cell formation and arterial branching morphogenesis. Zebrafish embryos expressed soluble Flt1 (sFlt1) and membrane-bound Flt1 (mFlt1). In Tg(flt1(BAC):yfp) × Tg(kdrl:ras-cherry)(s916) embryos, flt1:yfp was expressed in tip, stalk and base cells of segmental artery sprouts and overlapped with kdrl:cherry expression in these domains. flt1 morphants showed increased tip cell numbers, enhanced angiogenic behavior and hyperbranching of segmental artery sprouts. The additional arterial branches developed into functional vessels carrying blood flow. In support of a functional role for the extracellular VEGF-binding domain of Flt1, overexpression of sflt1 or mflt1 rescued aberrant branching in flt1 morphants, and overexpression of sflt1 or mflt1 in controls resulted in short arterial sprouts with reduced numbers of filopodia. flt1 morphants showed reduced expression of Notch receptors and of the Notch downstream target efnb2a, and ectopic expression of flt4 in arteries, consistent with loss of Notch signaling. Conditional overexpression of the notch1a intracellular cleaved domain in flt1 morphants restored segmental artery patterning. The developing nervous system of the trunk contributed to the distribution of Flt1, and the loss of flt1 affected neurons. Thus, Flt1 acts in a Notch-dependent manner as a negative regulator of tip cell differentiation and branching. Flt1 distribution may be fine-tuned, involving interactions with the developing nervous system.