VEGF Treatment Research Articles

Hairy/enhancer of Split Homologue-1 Suppresses Vascular Endothelial Growth Factor-induced Angiogenesis via Downregulation of Osteopontin Expression

Angiogenesis plays a critical role in the progression and vulnerability of atherosclerotic plaques; however, the orchestration of angiogenesis in atherosclerotic plaque formation remains unclear. The results of microarray analysis, real-time PCR and immunohistochemical analyses showed that Hairy/enhancer of split homologue-1 (Hes-1) expression was significantly decreased, while that of osteopontin (OPN) was increased, in atherosclerotic plaques. Meanwhile, immunofluorescence results demonstrated that both Hes-1 and OPN were expressed in endothelial cells (ECs) of neovessels in atherosclerotic plaques. The results of an in vitro study showed that Hes-1 was downregulated, while OPN was upregulated, in a time- and dose-dependent manner in human umbilical vein endothelial cells (HUVECs) by VEGF treatment. In addition, Hes-1 knockdown was found to have transcriptional promotion effect on OPN expression in HUVECs and enhance OPN-induced angiogenesis in response to VEGF. On the contrary, Hes-1 overexpression inhibited OPN expression in HUVECs and reduced angiogenesis in vitro and in vivo. The results of this study suggest that decreased Hes-1 expression in atherosclerotic plaques exaggerate VEGF-induced angiogenesis by upregulating OPN. Therefore, restoring Hes-1 expression and inhibiting OPN expression may be a promising strategy to prevent vulnerable plaque formation in patients with atherosclerosis.

Characterization of a Vascular Endothelial Growth Factor–loaded Bioresorbable Delivery System for Pulp Regeneration

IntroductionVascular endothelial growth factor (VEGF) is a signal protein that stimulates angiogenesis and vasculogenesis and has been used in tissue regeneration and pulp regeneration experimental models. The purpose of this study was to develop a delivery system composed of a biodegradable fiber and controlled release of VEGF to promote cell viability and secure an adequate blood supply for the survival of human stem cells of the apical papilla (SCAP) favoring endodontic regenerative procedures. MethodsWe developed a polydioxanone fiber, 50 μm in diameter, loaded with VEGF at a linear concentration of 12.2 ng/cm. Cytotoxic effects of the VEGF-loaded fiber (VF) on SCAP and mouse fibroblasts were assessed by using a multiparametric assay kit (XTT-NR-CVDE [Xenometrix, Allschwil, Switzerland]). We evaluated VF-induced mRNA expression of downstream growth factors by using a human growth factor Taqman array in real-time polymerase chain reaction. We also assessed the in vivo subcutaneous reaction of C57BL/6 mice to implants of VF alone and human root fragments (10 mm in length) filled with VF after 10, 20, and 45 days. Statistical analyses were performed by using analysis of variance and Student t tests or non-parametric alternatives. ResultsEnzyme-linked immunosorbent assay verified detectable concentrations of released VEGF in solution for 25 days. No cytotoxicity was observed on SCAP and mouse fibroblasts treated with VEGF. In addition, VEGF treatment also induced the expression of additional growth factors with roles in tissue and blood vessel formation and neuroprotective function. Implantation of VF and root fragments filled with VF showed biocompatibility in vivo, promoting new blood vessels and connective tissue formation into the root canal space with negligible inflammation. ConclusionsOur results show that the VF used in this study is biocompatible and may be a promising scaffold for additional optimization and use in endodontic regenerative procedures.

Abstract 436: Mfn2 is a Nodal Point Regulatory Step That Coordinates Angiogenic Phenotype in the Vascular Endothelium

Recent published data suggests that endothelial metabolism regulates angiogenesis. The key regulators of mitochondrial dynamics, mitofusins (Mfn1 and Mfn2) have been linked to cellular metabolic profile in tissues with high energetic requirements but their role in endothelial cells, were mitochondria function as signaling organelles is unknown. Here, we found that Mfn2 modulates postnatal neovascularization. Endothelial deletion of Mfn2 (Mfn2 ecko ) impaired, whereas, endothelial Mfn1 deletion (Mfn1 ecko ) did not affect clinical recovery, limb blood flow reperfusion, and capillary density compared with wild-type mice. These effects were associated with reduced eNOS activation in the ischemic leg of Mfn2 ecko mice compared with wild-type mice. Transplantation of bone marrow cells from WT to Mfn2ecko mice did not significantly improve blood perfusion in the ischemic limb of Mfn2 ecko mice. In agreement with these results, salient features of angiogenesis such as eNOS phosphorylation, cell migration, proliferation and capillary sprout formation were significantly inhibited in Mfn2 null primary mouse lung endothelial cells (Mfn2 -/- MLECs), whereas ablation of Mfn1 did not affect angiogenic signaling following VEGF stimulation. Because Mfn2 modulates the unfolded protein response (UPR) and the UPR machinery has been shown to generate angiomodulatory signals, we ascertained the effects of Mfn2 loss on UPR-mediated VEGF angiogenic signal transduction as potential mechanism. Mfn2 ablation sensitized Mfn2 -/- MLECs to proapototic UPR activation following VEGF treatment according to CHOP levels. Augmented CHOP levels corresponded with reduced eNOS phosphorylation. Inhibition of the UPR PERK signaling axis with PERK siRNA or reconstitution of Mfn2 (AdMfn2) reduced CHOP levels and significantly improved eNOS activation, EC proliferation and migration in Mfn2 -/- MLECs. Moreover, PERK siRNA or reconstitution of Mfn2 rescued the defect in capillary sprout formation in Mfn2 ecko mice. These data suggest that Mfn2 regulate angiogenic signaling in a UPR PERK axis-dependent manner and extend the role of Mfn2 beyond mitochondrial dynamics, metabolism and ER stress to angiogenesis and reparative vascular remodeling.

Abstract 565: Vascular Endothelial Growth Factor - An Important Factor in HDL Binding and Transcytosis Through Aortic Endothelial Cells

In the reverse cholesterol transport pathway cholesterol acceptors like high density lipoproteins (HDL) and apolipoprotein A-I (apoA-I), must cross the endothelium to get access to the donor cells in the arterial initima. However, it is unknown how they get translocated from the blood stream into the vascular wall. Previously, we showed that transendothelial HDL transport is modulated by scavenger receptor B I (SR-BI), ATP binding cassette transporter G1, endothelial lipase (EL), and the ectopic beta-ATPase/purinergic receptor axis. None of these proteins is a good candidate to directly mediate holoparticle uptake. They appear rather to indirectly modulate transendothelial transport of HDL by signalling or altering the structure of HDL. To unravel HDL transcytosis, a high-throughput screening was performed, incubating aortic endothelial cells (ECs) with kinase inhibiting drugs and fluorescent labelled HDL. Fixed cells were imaged using fluorescence wide-field microscope. Processing of microscopy acquired data using cell profiler and principle component analysis yielded, only inhibitors of the vascular endothelial growth factor receptor (VEGFR) and its down-stream signaling kinases. To verify and further characterize the screening hits towards their impact on binding, internalization and transport of 125I-HDL, we applied RNA interference and pharmacological means to ECs. Binding, internalization and transport of 125I-HDL through ECs were increased by VEGF treatment but decreased by inhibitors of VEGFR2 as well as knock-down of VEGFR2. RNA interference with VEGFR1 or VEGFR3 had no effect. The stimulatory effect of VEGF on 125I-HDL binding and internalization was decreased by knocking-down SR-BI or PDZK1. The identification of VEGF as a regulatory factor of transendothelial transport supports the concept that the endothelium is a specific and hence druggable barrier for the entry of HDL into the vascular wall.

Vascular endothelial growth factor induces anti‑Müllerian hormone receptor2 overexpression in ovarian granulosa cells of invitro fertilization/intracytoplasmic sperm injection patients.

Misregulation of vascular endothelial growth factorA (VEGF‑A) has been implicated in numerous types of ovarian disease, such as polycystic ovarian syndrome, ovarian hyperstimulation syndrome, endometriosis and ovarian cancer. VEGF regulates blood vessel permeability and angiogenesis. In our previous study, VEGF‑regulated gene expression was profiled in the uterus of a transgenic mouse model with repressed VEGF expression, which indicated that VEGF is an important regulator in controlling gene expression in the uterus. The anti‑Müllerian hormone (AMH) is expressed by ovarian granulosa cells (GCs) and acts through its type2 receptor, AMH receptor2 (AMHR2). Serum AMH levels are used to predict ovarian reserves and the small antral follicles contribute markedly to the serum AMH level. AMH recruits primordial follicles and inhibits excessive follicular development by follicular stimulating hormone (FSH). However, AMH may be influenced by suppression of gonadotrophin secretion and VEGF inhibition. In the current study, human primary ovarian GCs were isolated from ovarian follicle fluid of invitro fertilization/intracytoplasmic sperm injection cycles (IVF/ICSI). It was identified that the FSH receptor was consistently expressed in the isolated cells. VEGF‑A treatment stimulated AMHR2 overexpression at the gene and protein levels. In addition, VEGF induced AMHR2 expression on the surface of the isolated GCs from mature follicles. The VEGF treatment was also performed in an ovarian granulosa‑like cell line, KGN. AMH and AMHR2 are co‑expressed in normal GCs; however, as a result of VEGF misregulation, AMHR2 overexpression increases AMH binding, which may attenuate follicular or oocyte maturation. However, the associated function and underlying mechanism requires further investigation.

VEGF-A acts via neuropilin-1 to enhance epidermal cancer stem cell survival and formation of aggressive and highly vascularized tumors.

We identify a limited subpopulation of epidermal cancer stem cells (ECS cells), in squamous cell carcinoma, that form rapidly growing, invasive and highly vascularized tumors, as compared with non-stem cancer cells. These ECS cells grow as non-attached spheroids, and display enhanced migration and invasion. We show that ECS cell-produced vascular endothelial growth factor (VEGF)-A is required for the maintenance of this phenotype, as knockdown of VEGF-A gene expression or treatment with VEGF-A-inactivating antibody reduces these responses. In addition, treatment with bevacizumab reduces tumor vascularity and growth. Surprisingly, the classical mechanism of VEGF-A action via interaction with VEGF receptors does not mediate these events, as these cells lack VEGFR1 and VEGFR2. Instead, VEGF-A acts via the neuropilin-1 (NRP-1) co-receptor. Knockdown of NRP-1 inhibits ECS cell spheroid formation, invasion and migration, and attenuates tumor formation. These studies suggest that VEGF-A acts via interaction with NRP-1 to trigger intracellular events leading to ECS cell survival and formation of aggressive, invasive and highly vascularized tumors.

Multiple Growth Factors, But Not VEGF, Stimulate Glycosaminoglycan Hyperelongation in Retinal Choroidal Endothelial Cells.

A major feature of early age-related macular degeneration (AMD) is the thickening of Bruch's membrane in the retina and an alteration in its composition with increased lipid deposition. In certain pathological conditions proteoglycans are responsible for lipid retention in tissues. Growth factors are known to increase the length of glycosaminoglycan chains and this can lead to a large increase in the interaction between proteoglycans and lipids. Using choroidal endothelial cells, we investigated the effects of a number of AMD relevant growth factors TGFβ, thrombin, PDGF, IGF and VEGF on proteoglycan synthesis. Cells were characterized as of endothelial origin using the specific cell markers endothelial nitric oxide synthesis and von Willebrand factor and imaged using confocal microscopy. Cells were treated with growth factors in the presence and absence of the appropriate inhibitors and were radiolabeled with [35S]-SO4. Proteoglycans were isolated by ion exchange chromatography and sized using SDS-PAGE. Radiosulfate incorporation was determined by the cetylpyridinium chloride (CPC) precipitation technique. To measure cellular glycosaminoglycan synthesizing capacity we added xyloside and assessed the xyloside-GAGs by SDS-PAGE. TGFβ, thrombin, PDGF & IGF dose-dependently stimulated radiosulfate incorporation and GAG elongation as well as xyloside-GAG synthesis, however VEGF treatment did not stimulate any changes in proteoglycan synthesis. VEGF did not increase pAKT but caused a large increase in pERK relative to the response to PDGF. Thus, AMD relevant agonists cause glycosaminoglycan hyperelongation of proteoglycans synthesised and secreted by retinal choroidal endothelial cells. The absence of a response to VEGF is intriguing and identifies proteoglycans as a novel potential target in AMD. Future studies will examine the relevance of these changes to enhanced lipid binding and the development of AMD.

The placental microbiome; microbial presence in nature’s transplant

The placental microbiome; microbial presence in nature’s transplant

Lipid droplets as a novel cargo of tunnelling nanotubes in endothelial cells.

Intercellular communication is a fundamental process in the development and functioning of multicellular organisms. Recently, an essentially new type of intercellular communication, based on thin membrane channels between cells, has been reported. These structures, termed intercellular or tunnelling nanotubes (TNTs), permit the direct exchange of various components or signals (e.g., ions, proteins, or organelles) between non-adjacent cells at distances over 100 μm. Our studies revealed the presence of tunnelling nanotubes in microvascular endothelial cells (HMEC-1). The TNTs were studied with live cell imaging, environmental scanning electron microscopy (ESEM), and coherent anti-Stokes Raman scattering spectroscopy (CARS). Tunneling nanotubes showed marked persistence: the TNTs could connect cells over long distances (up to 150 μm) for several hours. Several cellular organelles were present in TNTs, such as lysosomes and mitochondria. Moreover, we could identify lipid droplets as a novel type of cargo in the TNTs. Under angiogenic conditions (VEGF treatment) the number of lipid droplets increased significantly. Arachidonic acid application not only increased the number of lipid droplets but also tripled the extent of TNT formation. Taken together, our results provide the first demonstration of lipid droplets as a cargo of TNTs and thereby open a new field in intercellular communication research.

NCOA1 promotes angiogenesis in breast tumors by simultaneously enhancing both HIF1α- and AP-1-mediated VEGFa transcription

Nuclear receptor coactivator 1 (NCOA1) is overexpressed in a subset of breast cancer and its increased expression positively correlates with disease recurrence and metastasis. Although NCOA1 is known to promote breast cancer metastasis through working with multiple transcription factors to upregulate the expression of Twist1, ITGA5, CSF-1, SDF1 and CXCR4, the role of NCOA1 in breast tumor angiogenesis has not been investigated. In this study, we found that the microvascular density (MVD) was significantly decreased and increased in Ncoa1-knockout and NCOA1-overexpressing mammary tumors, respectively, in several breast cancer mouse models. Knockout or knockdown of NCOA1 in breast cancer cell lines also markedly compromised their capability to induce angiogenesis in Matrigel plugs embedded subcutaneously in mice, while this compromised capability could be rescued by VEGFa treatment. At the molecular level, NCOA1 upregulates VEGFa expression in both mouse mammary tumors and cultured breast cancer cells, and it does so by associating with both c-Fos, which is recruited to the AP-1 site at bp -938 of the VEGFa promoter, and HIF1α, which is recruited to the HIF1α-binding element at bp -979 of the VEGFa promoter, to enhance VEGFa transcription. In 140 human breast tumors, high NCOA1 protein correlates with high MVD and patients with both high NCOA1 and high MVD showed significantly shorter survival time. In summary, this study revealed a novel mechanism that NCOA1 potentiates breast cancer angiogenesis through upregulating HIF1α and AP-1-mediated VEGFa expression, which reinforces the rational of targeting NCOA1 in controlling breast cancer progression and metastasis.

Evidence of Th2 polarization of the sentinel lymph node (SLN) in melanoma

Melanoma has a propensity for lymphatogenous metastasis. Improved understanding of the sentinel lymph node (SLN) immunological environment may improve outcomes. The immune phenotype of fresh melanoma SLNs (n = 13) were compared to fresh control lymph nodes (n = 13) using flow cytometry. RNA was isolated from CD4+ T cells of the SLN and control lymph node and assessed for Th1/Th2 gene expression pathways using qRT-PCR. In addition, VEGF expression was compared between primary melanoma (n = 6) and benign nevi (n = 6) using immunohistochemistry. Melanoma SLNs had fewer CD8+ T cells compared to controls (9.2% vs. 19.5%, p = 0.0005). The CD8+ T cells within the SLN appeared to have an exhausted phenotype demonstrated by increased PD-1 mRNA expression (2.2% vs. 0.8%, p = 0.004) and a five-fold increase in CTLA-4 mRNA expression. The SLN also contained an increased number of CD14 (22.7% vs. 7.7%, p = 0.009) and CD68 (9.3% vs. 2.7%, p = 0.001) macrophages, and CD20 B cells (31.1% vs. 20.7%, p = 0.008), suggesting chronic inflammation. RT-PCR demonstrated a significant Th2 bias within the SLN. In vitro studies demonstrated a similar Th2 polarization with VEGF treatment of control lymph nodes. The primary melanoma demonstrated strong VEGF expression and an increase in VEGFR1 within the SLN. Melanoma is associated with Th2-mediated “chronic inflammation,” fewer cytotoxic T cells, and an exhausted T cell phenotype within the SLN combined with VEGF overproduction by the primary melanoma. These immunologic changes precede nodal metastasis and suggests consideration of VEGF inhibitors in future immunotherapy studies.

A “Histodilutional Barrier” Formed in Gastric Mucosa by VEGF Pretreatment Protects Ethanol‐Induced Hemorrhagic Gastric Erosions in Rats

Since VEGF increases vascular permeability (VP), we tested the hypothesis that it might create a perivascular “histodilutional barrier”, like histamine, hence delay the absorption of damaging chemicals.MethodsFasted Sprague‐Dawley rats were given VEGF164 (0.01, 0.04, or 0.2 ug/100g) by gavage either alone or 30 min before administration of 75% ethanol (1 ml/kg). Evans blue was injected i.v. 15 min before autopsy. Rats were euthanized 30 min after VEGF alone or 1 hr after ethanol administration. Scrapings of gastric mucosa were harvested for VP measurement spectrophotometrically (560 nm) of Evans blueextravasation in the gastric mucosa. Hemorrhagic gastric mucosal lesions were measured by an Imaging Analysis System and calculated as % of glandular stomach, and also examined by histology.Results0.04ug/100g VEGF treatment significantly increased gastric VP compared to the controls (p < 0.05). Pretreatment with 0.01, 0.04, or 0.2 ug/100g VEGF reduced ethanol‐induced lesions from 10.6±1.7% (controls) to 8.9±1.8, 0.9±0.2 (p <0.01), or 2.8±0.7% (p <0.05), respectively. Histologic examination showed mild or moderate gastric edema in lamina propria or submucosa after VEGF alone, and no or only superficial gastric erosions in the rats treated with VEGF + ethanol.Conclusions1) Pretreatment with VEGF offered dose‐dependent gastroprotection in rats exposed to ethanol. 2) The likely mechanisms might be related to a histodilutional barrier induced by VEGF. 3) Further studies are needed to elucidate the molecular mechanisms of this new form of gastroprotection.(Supported by VA Merit Review Grants to S. Szabo).

Influence of mouse strain on ovarian tissue recovery after engraftment with angiogenic factor.

BackgroundFor women facing gonadotoxic treatment, cryopreservation of ovarian tissue with subsequent retransplantation during remission is a promising technique for fertility preservation. However, follicle loss within grafted ovarian tissue can be caused by ischemia and progressive revascularization. Several xenograft models using different immunodeficient rodent lines are suitable for studying ovarian tissue survival and follicular viability after frozen-thawed ovarian cortex transplantation. SCID mice, which are deficient for functional B and T cells, are the most commonly used mice for ovarian xenograft studies. However, due to incomplete immunosuppression, NOD-SCID mice displaying low NK cell function and an absence of circulating complement might be more appropriate. The present study aims to define the most appropriate immunodeficient mouse strain for ovarian tissue xenotransplantation by comparing ovarian graft recovery in SCID and NOD-SCID mice following engraftment in the presence of isoform 111 of vascular endothelial growth factor.MethodsSheep ovarian cortex fragments were embedded in a collagen matrix, with or without VEGF111, before being stitched onto the ovaries of SCID and NOD-SCID mice. Transplants were recovered after 3 days to study early revascularization or after 3 weeks to evaluate follicle preservation and tissue fibrosis through histological analyses.ResultsAt day 3, vessels were largely reorganized in the ovarian grafts of both mouse strains. After 3 weeks, the cortical tissue was clearly identifiable in SCID mice but not in NOD-SCID mice. Upon VEGF111 treatment, vascularization was significantly improved 3 days after transplantation in SCID mice. This increase in vessel density was correlated with better follicular preservation in SCID mice 3 weeks after transplantation. Fibrosis was not decreased by VEGF treatment in either mouse strain.ConclusionsTissue architecture and follicular morphology were better preserved in ovarian tissues grafted in SCID mice in comparison with NOD-SCID mice. Moreover, tissue revascularization was improved in SCID mice by VEGF111 graft treatment. Thus, we consider SCID mice to be the best murine model for studying ovarian tissue xenografts.

The Proangiogenic Effect of Iroquois Homeobox Transcription Factor Irx3 in Human Microvascular Endothelial Cells

Angiogenesis is a dynamic process required for embryonic development. However, postnatal vascular growth is characteristic of multiple disease states. Despite insights into the multistep process in which adhesion molecules, extracellular matrix proteins, growth factors, and their receptors work in concert to form new vessels from the preexisting vasculature, there remains a lack of insight of the nuclear transcriptional mechanisms that occur within endothelial cells (ECs) in response to VEGF. Iroquois homeobox gene 3 (Irx3) is a transcription factor of the Iroquois family of homeobox genes. Irx homeodomain transcription factors are involved in the patterning and development of several tissues. Irx3 is known for its role during embryogenesis in multiple organisms. However, the expression and function of Irx3 in human postnatal vasculature remains to be investigated. Here we show that Irx3 is expressed in human microvascular endothelial cells, and expression is elevated by VEGF stimulation. Genetic Irx3 gain and loss of function studies in human microvascular endothelial cells resulted in the modulation of EC migration during wound healing, chemotaxis and invasion, and tubulogenesis. Additionally, we observed increased delta-like ligand 4 (Dll4) expression, which suggests an increase in EC tip cell population. Finally, siRNA screening studies revealed that transient knockdown of Hey1, a downstream Notch signaling mediator, resulted in increased Irx3 expression in response to VEGF treatment. Strategies to pharmacologically regulate Irx3 function in adult endothelial cells may provide new therapies for angiogenesis.

Lipopolysaccharide (LPS)-mediated Angiopoietin-2-dependent Autocrine Angiogenesis Is Regulated by NADPH Oxidase 2 (Nox2) in Human Pulmonary Microvascular Endothelial Cells

Sepsis-mediated endothelial Angiopoeitin-2 (Ang2) signaling may contribute to microvascular remodeling in the developing lung. The mechanisms by which bacterial cell wall components such as LPS mediate Ang2 signaling in human pulmonary microvascular endothelial cells (HPMECs) remain understudied. In HPMEC, LPS-induced Ang2, Tie2, and VEGF-A protein expression was preceded by increased superoxide formation. NADPH oxidase 2 (Nox2) inhibition, but not Nox4 or Nox1 inhibition, attenuated LPS-induced superoxide formation and Ang2, Tie2, and VEGF-A expression. Nox2 silencing, but not Nox4 or Nox1 silencing, inhibited LPS-mediated inhibitor of κ-B kinase β (IKKβ) and p38 phosphorylation and nuclear translocation of NF-κB and AP-1. In HPMECs, LPS increased the number of angiogenic tube and network formations in Matrigel by >3-fold. Conditioned media from LPS-treated cells also induced angiogenic tube and network formation in the presence of Toll-like receptor 4 blockade but not in the presence of Ang2 and VEGF blockade. Nox2 inhibition or conditioned media from Nox2-silenced cells attenuated LPS-induced tube and network formation. Ang2 and VEGF-A treatment rescued angiogenesis in Nox2-silenced cells. We propose that Nox2 regulates LPS-mediated Ang2-dependent autocrine angiogenesis in HPMECs through the IKKβ/NF-κB and MAPK/AP-1 pathways.

Migration mechanism of mesenchymal stem cells studied by QD/NSOM

The migration of mesenchymal stem cells (MSCs) plays a key role in tumor-targeted delivery vehicles and tumor-related stroma formation. However, there so far has been no report on the distribution of cell surface molecules during the VEGF-induced migration of MSCs. Here, we have utilized near-field scanning optical microscopy (NSOM) combined with fluorescent quantum dot (QD)-based nano-technology to capture the functional relationship between CD44 and CD29 adhesion molecules on MSCs and the effect of their spatial rearrangements. Before VEGF-induced migration of MSCs, both CD44 and CD29 formed 200–220nm nano-domains respectively, with little co-localization between the two types of domains. Surprisingly, the size of the CD44 nano-domain rapidly increased in size to 295nm and apparently larger aggregates were formed following MSC treatment with VEGF for 10min, while the area of co-localization increased to 0.327μm2. Compared with CD44, CD29 was activated obviously later, for the fact that CD29 aggregation didn't appear until 30min after VEGF treatment. Consistently, its co-localization area increased to 0.917μm2. The CD44 and CD29 nano-domains further aggregated into larger nano-domains or even formed micro-domains on the membrane of activated MSCs. The aggregation and co-localization of these molecules promoted FAK formation and cytoskeleton rearrangement. All of the above changes induced by VEGF contributed to MSC migration. Taken together, our data of NSOM-based dual color fluorescent imaging demonstrated for the first time that CD44, together with CD29, involved in VEGF-induced migration of MSCs through the interaction between CD44 and its co-receptor of VEGFR-2.

Influence of osteogenic stimulation and VEGF treatment on in vivo bone formation in hMSC-seeded cancellous bone scaffolds.

BackgroundTissue engineering approaches for reconstruction of large bone defects are still technically immature, especially in regard to sufficient blood supply. Therefore, the aim of the present study was to investigate the influence of osteogenic stimulation and treatment with VEGF on new bone formation and neovascularization in hMSC-loaded cancellous bone scaffolds in vivo.MethodsCubic scaffolds were seeded with hMSC and either cultured in stem cell medium or osteogenic stimulation medium. One osteogenically stimulated group was additionally treated with 0.8 μg VEGF prior to subcutaneous implantation in athymic mice. After 2 and 12 weeks in vivo, constructs and selected organs were harvested for histological and molecular analysis.ResultsHistological analysis revealed similar vascularization of the constructs with and without VEGF treatment and absence of new bone formation in any group. Human DNA was detected in all inoculated scaffolds, but a significant decrease in cells was observed after 2 weeks with no further decrease after 12 weeks in vivo.ConclusionUnder the chosen conditions, osteogenic stimulation and treatment with VEGF does not have any influence on the new bone formation and neovascularization in hMSC-seeded cancellous bone scaffolds.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2474-15-350) contains supplementary material, which is available to authorized users.

Abstract 2682: ELTD1 as a target for anti-cancer therapy in rodent gliomas

Abstract Despite current therapies, glioblastoma multiforme (GBM) is a devastating cancer with a very poor prognosis. Specific protein biomarkers for GBM enable not only new prognostic avenues to tailor treatment, but valuable therapeutic targets to interfere with GBM growth. We recently discovered a new biomarker for high-grade gliomas, ELTD1 (epidermal growth factor, latrophilin, and 7 transmembrane domain-containing protein 1 on chromosome 1), via a novel bioinformatics approach, GAMMA (Global microarray meta-analysis). ELTD1 was found to be significantly higher (p<0.05) in high-grade gliomas (50 patients) compared with low-grade gliomas (21 patients). ELTD1 gene expression was found to be associated with tumor grade, survival across grade, and an increase in the mesenchymal subtype. From our previous results we hypothesized that ELTD1 may be a potential therapeutic target in high-grade gliomas. We tested the hypothesis in an orthotopic GL261 mouse glioma model in animals treated with an anrti-ELTD1 antibody, compared to those that were treated with an anti-VEGF antibody or an anti-c-Met antibody, and untreated tumors. Tumor volumes were measured by morphological magnetic resonance imaging (MRI). Our data in orthotopic GL261 glioma-bearing mice shows that injection of anti-ELTD1 antibodies lead to a significant decrease in tumor volumes (p<0.0001) and a significant increase in animal survival (p<0.05), superior to that achieved by a mouse anti-VEGF antibody or an antibody against c-Met, when compared to untreated mice. Tumor volumes were obtained at various time-points, and compared at 21 days following intracerebral implantation of GL261 cells in male C57BL6 mice. These tumor volumes at 21 days were 86.03+/-41.54 mm3 for untreated mice (n=5); 13.04+/-5.35 mm3 for anti-ELTD1 treated mice (n=5); 33.16+/-12.48 mm3 for anti-VEGF treated mice (n=4); and 28.49+/-11.11 mm3 for anti-c-Met treated mice (n=5). The mean days of survival for untreated, ELTD1, VEGF or c-Met treatment groups were 23.8+/-1.79, 28.2+/-2.39, 27+/-1.15 or 27+/-1.87 days, respectively. These pre-clinical results indicate that an additional therapy in the form of an anti-ELTD1 antibody either as a sole agent or in combination with current or new therapies could potentially provide high-grade glioma patients with a better prognostic outcome. Citation Format: Rheal A. Towner, Richard Pody, Nataliya Smith, Debra Saunders, Charity Njoku, Jonathan D. Wren. ELTD1 as a target for anti-cancer therapy in rodent gliomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2682. doi:10.1158/1538-7445.AM2014-2682

Gene profiling of human VEGF signaling pathways in human endothelial and retinal pigment epithelial cells after anti VEGF treatment.

BackgroundRanibizumab (Lucentis®) is a Fab-antibody fragment developed from Bevacizumab, a full-length anti-VEGF antibody. Both compounds are used for treating age-related macular degeneration (AMD). The influence of bevacizumab and ranibizumab on genes involved in signal transduction and cell signaling downstream of VEGF were compared in order to detect possible differences in their mode of action, which are not related to their Fab-antibody fragments.MethodsHuman umbilical vein cell lines (EA.hy926) and retinal pigment epithelial cells (ARP-19) were exposed to oxidative stress. The cells were treated with therapeutic concentrations of bevacizumab (0.25 mg/mL) and ranibizumab (125 mg/mL) for 24 hours prior to all experiments, and their effects on gene expressions were determined by RT- PCR.ResultsAfter exposure to bevacizumab, more genes in the endothelial cells were up-regulated (KDR, NFATc2) and down-regulated (Pla2g12a, Rac2, HgdC, PRKCG) compared to non-treated controls. After exposure to ranibizumab, fewer genes were up-regulated (PTGS2) and down-regulated (NOS3) compared to controls. In comparison between drugs, more genes were up-regulated (NFATc2 and KDR) and more were down-regulated (Pla2g12a, Pla2g1b, Ppp3r2, Rac2) by bevacizumab than by ranibizumab. In RPE cells, NOS3 and PGF were up-regulated and Pla2g12b was down-regulated after exposure to ranibizumab, while PIK3CG was up-regulated and FIGF was down-regulated after exposure to bevacizumab, but the differences in gene expression were minor between drugs (PIK3CGand PGF were down-regulated more by ranibizumab than by bevacizumab).ConclusionsThe different gene expressions after exposure to ranibizumab and bevacizumab in endothelial and RPE cells may indicate a somewhat different biological activity of the two compounds.

Anti VEGF treatment in myopic CNV

AbstractAbstract not provided