Vascular Endothelial Growth Factor Overexpressing Research Articles

VEGF Overexpression Significantly Increases Nanoparticle-Mediated siRNA Delivery and Target-Gene Downregulation.

The availability of nanoparticles (NPs) to deliver small interfering RNA (siRNA) has significantly expanded the specificity and range of ‘druggable’ targets for precision medicine in cancer. This is especially important for cancers such as triple negative breast cancer (TNBC) for which there are no targeted treatments. Our purpose here was to understand the role of tumor vasculature and vascular endothelial growth factor (VEGF) overexpression in a TNBC xenograft in improving the delivery and function of siRNA NPs using in vivo as well as ex vivo imaging. We used triple negative MDA-MB-231 human breast cancer xenografts derived from cells engineered to overexpress VEGF to understand the role of VEGF and vascularization in NP delivery and function. We used polyethylene glycol (PEG) conjugated polyethylenimine (PEI) NPs to deliver siRNA that downregulates choline kinase alpha (Chkα), an enzyme that is associated with malignant transformation and tumor progression. Because Chkα converts choline to phosphocholine, effective delivery of Chkα siRNA NPs resulted in functional changes of a significant decrease in phosphocholine and total choline that was detected with 1H magnetic resonance spectroscopy (MRS). We observed a significant increase in NP delivery and a significant decrease in Chkα and phosphocholine in VEGF overexpressing xenografts. Our results demonstrated the importance of tumor vascularization in achieving effective siRNA delivery and downregulation of the target gene Chkα and its function.

A novel approach for the discrimination of culture medium from Vascular Endothelial Growth Factor (VEGF) overexpressing colorectal cancer cells

Abstract Objectives The expression level of Vascular Endothelial Growth Factor (VEGF) is assumed as a prognostic marker for several tumor types, including colorectal cancer. Therefore, the determination of pre- and post-therapy levels of VEGF appears to have great value in the assessment of tumor prognosis. Enzyme-Linked Immunosorbent Assay (ELISA) is commonly used for the determination of serum or plasma VEGF levels, but the method is costly and time-consuming. In this study, we aimed to describe a rapid and cost-effective analysis method to discriminate VEGF overexpressing colorectal cancer-derived conditioned medium (CM). Methods Attenuated Total Reflection (ATR)-Fourier Transform Infrared (FTIR) spectroscopy, combined with Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA), was used to differentiate VEGF overexpressing colorectal cancer cell line CM from CM obtained from the corresponding control cells which express and secrete relatively lower amount of VEGF. Results Samples belong to VEGF overexpressing colorectal cancer cells were clearly distinguished from the control group with very high PC scores as PC1 + PC2 = 96%. Besides, a 100% accurate distinction between these two groups was achieved by the LDA analysis. Conclusions ATR-FTIR spectroscopy combined with pattern recognition techniques was able to discriminate CM of VEGF overexpressing colorectal cancer cells with high efficiency and accuracy.

Dental pulp stem cells overexpressing stromal-derived factor-1α and vascular endothelial growth factor in dental pulp regeneration.

The current study aimed to investigate the effects of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α) overexpressing dental pulp stem cells (DPSCs) in vascularized dental pulp regeneration in vivo. Human DPSCs were transfected with VEGF or SDF-1α using premade lentiviral particles. Overexpression was verified by quantitative polymerase chain reaction (q-PCR), enzyme-linked immunosorbent assay (ELISA), and western blot analysis. Effects of SDF-1α and VEGF overexpressing DPSCs on their proliferation (CCK-8 and MTT assays) and endothelial vascular-tube formation (Matrigel assay) were investigated in vitro. Human tooth roots sectioned into 6-mm segments were injected with gene-modified DPSCs encapsulated in PuraMatrix hydrogel and implanted in the dorsum of severe-combined-immunodeficient (SCID) mice. Implants were retrieved after 4weeks and examined for regenerated pulp-like tissue and vascularization using histology and immunohistochemistry. p < 0.05 was considered statistically significant. Gene-modified DPSCs expressed significantly high levels (p < 0.05) of SDF-1α and VEGF mRNA and proteins, respectively. Transfected DPSCs showed a significantly higher cell proliferation compared to that of wild-type DPSCs. Furthermore, they enhanced endothelial cell migration and vascular-tube formation on Matrigel in vitro. When injected into tooth root canals and implanted in vivo, DPSCs/SDF-1α + DPSCs/VEGF-mixed group resulted in significantly increased length of regenerated pulp-like tissue within the root canals compared to that of wild-type DPSCs/VEGF and DPSCs/SDF-1α groups. Vessel area density was significantly higher in DPSCs/SDF-1α and mixed DPSCs/SDF-1α + DPSCs/VEGF groups than in DPSCs-VEGF alone or wild-type DPSCs groups. A combination of VEGF-overexpressing and SDF-1α-overexpressing DPSCs could enhance the area of vascularized dental pulp regeneration in vivo. Enhancing vascularization in pulp regeneration is crucial to overcome the clinical limitation of the limited blood supply to the root canals via a small apical foramen enclosed by hard dentin.

Bevacizumab and near infrared probe conjugated iron oxide nanoparticles for vascular endothelial growth factor targeted MR and optical imaging.

Vascular endothelial growth factor (VEGF) plays a pivotal role in the cascade of development and progression of cancers. Targeting this cancer hallmark is a logical strategy for imaging based cancer detection and monitoring the anti-angiogenesis treatment. Using Bevacizumab (Avastin®), which is a recombinant humanized monoclonal antibody directly against VEGF and an angiogenesis inhibitor, as a targeting ligand, a multimodal VEGF targeted molecular imaging probe was developed by conjugating near infrared dye (NIR830) labeled bevacizumab to magnetic iron oxide nanoparticles (IONP) for optical and magnetic resonance (MR) imaging of cancers over-expressing VEGF. The targeting effect of NIR830-bevacizumab-IONPs on VEGF over-expressing cells was investigated by receptor mediated cell uptake experiments and a blocking assay using VEGF over-expressing 4T1 breast cancer cells. Systemic administration of VEGF-targeted NIR830-bevacizumab-IONPs into mice bearing 4T1 breast tumors resulted in higher accumulation of targeting IONPs in tumors compared to non-targeted IONPs. Quantitative analysis of T2-weighted MRI at 48 h post-injection revealed that the averaged percentage of signal intensity change in tumors treated with NIR830-bevacizumab-IONPs was 52.4 ± 11.0% compared to 26.9 ± 12.4% in controls treated with non-targeted IONPs. The results demonstrated the feasibility and efficacy of NIR830-bevacizumab-IONPs as a VEGF targeting dual-modality molecular imaging probe that can be potentially used for imaging of cancers with VEGF over-expression and delivery of bevacizumab for imaging guided anti-cancer treatment.

Interleukin-17A neutralization alleviated ocular neovascularization by promoting M2 and mitigating M1 macrophage polarization.

Neovascularization (NV), as a cardinal complication of several ocular diseases, has been intensively studied, and research has shown its close association with inflammation and immune cells. In the present study, the role of interleukin-17A (IL-17A) in angiogenesis in the process of ocular NV both in vivo and in vitro was investigated. Also, a paracrine role of IL-17A was demonstrated in the crosstalk between endothelial cells and macrophages in angiogenesis. In the retinas of mice with retinopathy of prematurity, the IL-17A expression increased significantly at postnatal day 15 (P15) and P18 during retinal NV. Mice given IL-17A neutralizing antibody (NAb) developed significantly reduced choroidal NV and retinal NV. Studies on vascular endothelial growth factor (VEGF) over-expressing mice suggested that IL-17A modulated NV through the VEGF pathway. Furthermore, IL-17A deficiency shifted macrophage polarization toward an M2 phenotype during retinal NV with significantly reduced M1 cytokine expression compared with wild-type controls. In vitro assays revealed that IL-17A treated macrophage supernatant gave rise to elevated human umbilical vascular endothelial cell proliferation, tube formation and VEGF receptor 1 and receptor 2 expression. Therefore, IL-17A could potentially serve as a novel target for treating ocular NV diseases. The limitation of this study involved the potential mechanisms, such as which transcription accounted for macrophage polarization and how the subsequent cytokines were modulated when macrophages were polarized. Further studies need to be undertaken to definitively determine the extent to which IL-17A neutralizing anti-angiogenic activity depends on macrophage modulation compared with anti-VEGF treatment.

Hypoxia-specific, VEGF-expressing neural stem cell therapy for safe and effective treatment of neuropathic pain

Vascular endothelial growth factor (VEGF) is an angiogenic cytokine that stimulates the differentiation and function of vascular endothelial cells. VEGF has been implicated in improving nervous system function after injury. However, uncontrolled overexpression of VEGF increases the risk of tumor formation at the site of gene delivery. For this reason, VEGF expression needs to be strictly controlled. The goal of the present study was to understand the effects of hypoxia-induced gene expression system to control VEGF gene expression in neural stem cells (NSCs) on the regeneration of neural tissue after sciatic nerve injury. In this study, we used the erythropoietin (Epo) enhancer-SV40 promoter system (EpoSV-VEGF-NSCs) for hypoxia-specific VEGF expression. We used three types of NSCs: DsRed-NSCs as controls, SV-VEGF-NSCs as uncontrolled VEGF overexpressing NSCs, and EpoSV-VEGF-NSCs. For comparison of VEGF expression at normoxia and hypoxia, we measured the amount of VEGF secreted. VEGF expression decreased at normoxia and increased at hypoxia for EpoSV-VEGF-NSCs; thus, EpoSV-VEGF-NSCs controlled VEGF expression, dependent upon oxygenation condition. To demonstrate the therapeutic effect of EpoSV-VEGF-NSCs, we transplanted each cell line in a neuropathic pain sciatic nerve injury rat model. The transplanted EpoSV-VEGF-NSCs improved sciatic nerve functional index (SFI), mechanical allodynia, and re-myelination similar to the SV-VEGF-NSCs. Additionally, the number of blood vessels increased to a level similar to that of the SV-VEGF-NSCs. However, we did not observe tumor generation in the EpoSV-VEGF-NSC animals that were unlikely to have tumor formation in the SV-VEGF-NSCs. From our results, we determined that EpoSV-VEGF-NSCs safely regulate VEGF gene expression which is dependent upon oxygenation status. In addition, we found that they are therapeutically appropriate for treating sciatic nerve injury.

Role of Nitric Oxide Isoforms in Vascular and Alveolar Development and Lung Injury in Vascular Endothelial Growth Factor Overexpressing Neonatal Mice Lungs

BackgroundThe role of vascular endothelial growth factor (VEGF)-induced 3 different nitric oxide synthase (NOS) isoforms in lung development and injury in the newborn (NB) lung are not known. We hypothesized that VEGF-induced specific NOS pathways are critical regulators of lung development and injury.MethodologyWe studied NB wild type (WT), lung epithelial cell-targeted VEGF165 doxycycline-inducible overexpressing transgenic (VEGFTG), VEGFTG treated with a NOS1 inhibitor (L-NIO), VEGFTG x NOS2-/- and VEGFTG x NOS3+/- mice in room air (RA) for 7 postnatal (PN) days. Lung morphometry (chord length), vascular markers (Ang1, Ang2, Notch2, vWF, CD31 and VE-cadherin), cell proliferation (Ki67), vascular permeability, injury and oxidative stress markers (hemosiderin, nitrotyrosine and 8-OHdG) were evaluated.ResultsVEGF overexpression in RA led to increased chord length and vascular markers at PN7, which were significantly decreased to control values in VEGFTG x NOS2−/− and VEGFTG x NOS3+/- lungs. However, we found no noticeable effect on chord length and vascular markers in the VEGFTG / NOS1 inhibited group. In the NB VEGFTG mouse model, we found VEGF-induced vascular permeability in the NB murine lung was partially dependent on NOS2 and NOS3-signaling pathways. In addition, the inhibition of NOS2 and NOS3 resulted in a significant decrease in VEGF-induced hemosiderin, nitrotyrosine- and 8-OHdG positive cells at PN7. NOS1 inhibition had no significant effect.ConclusionOur data showed that the complete absence of NOS2 and partial deficiency of NOS3 confers protection against VEGF-induced pathologic lung vascular and alveolar developmental changes, as well as injury markers. Inhibition of NOS1 does not have any modulating role on VEGF-induced changes in the NB lung. Overall, our data suggests that there is a significant differential regulation in the NOS-mediated effects of VEGF overexpression in the developing mouse lung.

Detection of vascular endothelial growth factor in colon cancer xenografts using bevacizumab based near infrared fluorophore conjugate.

BackgroundThe aim of this study was to develop the near infrared fluorescence (NIRF)-based imaging agent for the visualization of vascular endothelial growth factor (VEGF) in colon cancer. AlexaFluor 750 conjugating with bevacizumab, and injected intravenously into nude mice bearing VEGF over-expressing HT29 human colorectal cancer. Optical imaging was performed at 15 min, 24 h and 48 h post injection. Immunofluorescences staining of the tumor sections were performed. HT29 colorectal cancer xenografts were clearly visualized with bevacizumab-AlexaFluor 750.ResultsEx vivo analysis showed 2.1 ± 0.4%, 37.6 ± 6.3% and 38.5 ± 6.2% injected dose/g accumulated in the tumors at 15 min, 24 h and 48 h respectively. Tumor uptake was significantly decreased in pretreated with excess of bevacizumab (p = 0.002). Immunofluorescence analysis showed strong staining of anti-CD 31 antibody around the blood vessels. Anti-VEGF-A and bevacizumab showed heterogeneous expression throughout the tumor.ConclusionsCurrent study successfully detected the VEGF expression in HT29 colorectal cancer xenografts, signifying as a potential agent for non-invasive imaging of VEGF expression, which may be applied in clinical practice.

Scaffold-Based Transplantation of Vascular Endothelial Growth Factor—Overexpressing Stem Cells Leads to Neovascularization in Ischemic Myocardium but Did Not Show a Functional Regenerative Effect

The transplantation of skeletal myoblasts (SkM) might improve cardiac function after myocardial infarction via paracrine action. We used scaffold-based cell transfer by using vascular endothelial growth factor (VEGF)-overexpressing myoblasts. Skeletal myoblasts were isolated and expanded from newborn Lewis rats. Cells were transfected with pCINeo-VEGF(121) and seeded on polyurethane (PU) scaffolds. The seeded scaffolds were epicardially implanted in rats 2 weeks after myocardial infarction (group: PU-VEGF-SkM). Before this intervention and 6 weeks later, pressure/volume loops were analyzed followed by histology. Additional study groups (n = 10 per group) were injected with VEGF-overexpressing myoblasts (Inj-VEGF-SkM) or unmodified myoblasts (Inj-SkM) or underwent a sham operation. Overexpression of VEGF was verified in vitro. The transplantation of growth factor producing myoblast-seeded scaffolds resulted in enhanced angiogenesis of ischemically damaged myocardium in vivo. However, the infarction size was not reduced. In group Inj-SkM, hemodynamics remained unchanged. Systolic function as measured by dP/dt(max) was not significantly altered in PU-VEGF-SkM (preinterventionally 2,156 ± 1,222 mmHg vs. postinterventionally 2,134 ± 850 mmHg). Other systolic function and diastolic function parameters as measured by dP/dt(min), tau, and pressure half-time were not restored in groups PU-VEGF-SkM and Inj-VEGF-SkM either. Transplantation of VEGF-overexpressing skeletal myoblasts leads to neovascularization in infarcted hearts. No functional myocardial recovery was observed. Scaffold-based transfer of genetically-modified cells remains a useful tool to study paracrine stem cell action.

Hepatocyte Growth Factor or Vascular Endothelial Growth Factor Gene Transfer Maximizes Mesenchymal Stem Cell–Based Myocardial Salvage After Acute Myocardial Infarction

Mesenchymal stem cell (MSC)-based regenerative strategies were investigated to treat acute myocardial infarction and improve left ventricular function. Murine AMI was induced by coronary ligation with subsequent injection of MSCs, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), or MSCs +HGF/VEGF into the border zone. Left ventricular ejection fraction was calculated using micro-computed tomography imaging after 6 months. HGF and VEGF protein injection (with or without concomitant MSC injection) significantly and similarly improved the left ventricular ejection fraction and reduced scar size compared with the MSC group, suggesting that myocardial recovery was due to the cytokines rather than myocardial regeneration. To provide sustained paracrine effects, HGF or VEGF overexpressing MSCs were generated (MSC-HGF, MSC-VEGF). MSC-HGF and MSC-VEGF showed significantly increased in vitro proliferation and increased in vivo proliferation within the border zone. Cytokine production correlated with MSC survival. MSC-HGF- and MSC-VEGF-treated animals showed smaller scar sizes, increased peri-infarct vessel densities, and better preserved left ventricular function when compared with MSCs transfected with empty vector. Murine cardiomyocytes were exposed to hypoxic in vitro conditions. The LDH release was reduced, fewer cardiomyocytes were apoptotic, and Akt activity was increased if cardiomyocytes were maintained in conditioned medium obtained from MSC-HGF or MSC-VEGF cultures. This study showed that (1) elevating the tissue levels of HGF and VEGF after acute myocardial infarction seems to be a promising reparative therapeutic approach, (2) HGF and VEGF are cardioprotective by increasing the tolerance of cardiomyocytes to ischemia, reducing cardiomyocyte apoptosis and increasing prosurvival Akt activation, and (3) MSC-HGF and MSC-VEGF are a valuable source for increased cytokine production and maximize the beneficial effect of MSC-based repair strategies.

VEGF‐induced heme oxygenase‐1 confers cytoprotection from lethal hyperoxia in vivo

Prolonged exposure to hyperoxia results in hyperoxic acute lung injury (HALI). Vascular endothelial growth factor (VEGF) has been shown to have cytoprotective effects and prolong survival in an in vivo model of HALI. Heme oxygenase-1 (HO-1) has protective effects in hyperoxia; therefore, we hypothesized that induction of HO-1 would be an important contributor to VEGF-induced cytoprotection. Using inducible, lung-specific VEGF overexpressing transgenic mice, we demonstrated that VEGF is a potent inducer of HO-1 mRNA and protein in mouse lung. To evaluate the effect of inhibition of HO-1 on injury, VEGF transgenic mice were treated with HO-1 short interfering RNA (HO-1 siRNA) and exposed to hyperoxia. Total lung lavage protein concentration, TUNEL staining, lipid peroxidation, and wet-to-dry ratio were all increased, consistent with increased injury. These findings were corroborated by survival studies in which inhibition of HO-1 function using tin-protoporphryin or silencing of HO-1 with lentiviral HO-1 short hairpin RNA (ShRNA) significantly decreased survival in hyperoxia. We conclude from these data that VEGF-induced HO-1 is an important contributor to cytoprotection in this in vivo model of acute lung injury and that alterations in VEGF function in the lung are likely to be important determinants of the outcome of acute lung injury.

Antiangiogenic activity of aplidine, a new agent of marine origin.

The antineoplastic compound aplidine, a new marine-derived depsipeptide, has shown preclinical activity in vitro on haematological and solid tumour cell lines. It is currently in early phase clinical trials. The exact mechanism of action of this anticancer agent still needs to be clarified. We have previously reported that aplidine blocks the secretion of the angiogenic factor vascular endothelial growth factor (VEGF) by the human leukaemia cells MOLT-4, suggesting a possible effect on tumour angiogenesis. This study was designed to investigate the antiangiogenic effect of aplidine. In vivo, in the chick embryo allantoic membrane (CAM) assay, aplidine inhibited spontaneous angiogenesis, angiogenesis elicited by exogenous VEGF and FGF-2, and induced by VEGF overexpressing 1A9 ovarian carcinoma cells. In vitro, at concentrations achievable in the plasma of patients, aplidine inhibited endothelial cell functions related to angiogenesis. It affected VEGF- and FGF-2-induced endothelial cell proliferation, inhibited cell migration and invasiveness assessed in the Boyden chamber and blocked the production of matrix metalloproteinases (MMP-2 and MMP-9) by endothelial cells. Finally, aplidine prevented the formation of capillary-like structures by endothelial cells on Matrigel. These findings indicate that aplidine has antiangiogenic activity in vivo and inhibits endothelial cell functional responses to angiogenic stimuli in vitro. This effect might contribute to the antineoplastic activity of aplidine.

Tumor growth enhancing effects of vascular endothelial growth factor are associated with increased nitric oxide synthase activity and inhibition of apoptosis in human breast carcinoma xenografts

Previously, we demonstrated the significance of vascular endothelial growth factor (VEGF) in promoting the growth of tetracycline-regulated human VEGF 165 retroviral vector transduced T47-D breast carcinoma cells, particularly at the early stages of tumor development (Cancer Res. 57 (1997) 3924). Here, we showed histologically that the VEGF overexpressing (VEGF (+)) T47-D cells formed a distinct tumor nodule at day 11, while control cells showed no evidence of replication. The VEGF (+) tumors contained large avascular cavities at days 11 and 21, which were replaced by basement membrane-lined channels at day 30. The number of proliferating tumor cells was not significantly different between the VEGF (+) and control tumors, but the number of apoptotic cells was significantly decreased in the VEGF (+) tumors. Increased nitric oxide synthase (NOS) activity was also observed in the VEGF (+) tumors. These findings indicate that VEGF contributes to tumor growth through inhibition of apoptosis and increased NOS activity, which may be critical during pre-vascular stages of tumor development.