Expression of Vascular Endothelial Growth Factor (VEGF) in Macrophages, Fibroblasts, and Endothelial Cells in Pterygium Treated with 5-Fluorouracil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expression of VEGF and collagen using a latex biomembrane as bladder replacement in rabbits

Objective: To investigate the VEGF expression and collagen deposition using a latex biomembrane as bladder replacement in rabbits. Materials and Methods: After partial cystectomy, a patch of a non-vulcanized latex biomembrane (2 x 2 cm) was sewn to the bladder of rabbits with 5/0 monofilament polydioxanone sulfate sutures in a watertight manner. Groups of 5 animals were killed at 15, 45 and 90 days after surgery and the bladder was removed. Sections of 5 mu m were cut and stained with picrosirius-red in order to estimate the amount of extracellular matrix in the graft. To confirm the presence of VEGF in tissues, protein expression was determined by immunohistochemistry. Results: No death, urinary leakage or graft extrusion occurred in any group. All bladders showed a spherical shape. A progressive reduction in the amount of collagen occurred in the graft area and was negatively and linearly correlated with time (p < 0.001). VEGF expression was higher in grafted areas when compared to controls at 15 and 45 days after surgery and decreased with time (p < 0.001). Conclusion: The latex biomembrane as a matrix for partial bladder replacement in rabbits promotes temporary collagen deposition and stimulates the angiogenic process.

The relationship between lymphatic vascular density and vascular endothelial growth factor A (VEGF-A) expression with clinical-pathological features and survival in pancreatic adenocarcinomas

Abstract: Background Pancreatic cancer is a rare tumor with an extremely low survival rate. Its known risk factors include the chronic use of tobacco and excessive alcohol consumption and the presence of chronic inflammatory diseases, such as pancreatitis and type 2 diabetes. Angiogenesis and lymphangiogenesis, which have been the focus of recent research, are considered prognostic factors for cancer development. Knowing the angiogenic and lymphangiogenic profiles of a tumor may provide new insights for designing treatments according to the different properties of the tumor. The aim of this study was to evaluate the density of blood and lymphatic vessels, and the expression of VEGF-A, in pancreatic adenocarcinomas, as well as the relationship between blood and lymphatic vascular density and the prognostically important clinical-pathological features of pancreatic tumors. Methods Paraffin blocks containing tumor samples from 100 patients who were diagnosed with pancreatic cancer between 1990 and 2010 were used to construct a tissue microarray. VEGF expression was assessed in these samples by immunohistochemistry. To assess the lymphatic and vascular properties of the tumors, 63 cases that contained sufficient material were sectioned routinely. The sections were then stained with the D2-40 antibody to identify the lymphatic vessels and with a CD34 antibody to identify the blood vessels. The vessels were counted individually with the Leica Application Suite v4 program. All statistical analyses were performed using SPSS 18.0 (Chicago, IL, USA) software, and p values ≤ 0.05 were considered significant. Results In the Cox regression analysis, advanced age (p=0.03) and a history of type 2 diabetes (p=0.014) or chronic pancreatitis (p=0.02) were shown to be prognostic factors for pancreatic cancer. Blood vessel density (BVD) had no relationship with clinical-pathological features or death. Lymphatic vessel density (LVD) was inversely correlated with death (p=0.002), and by Kaplan-Meyer survival analysis, we found a significant association between low LVD (p=0.021), VEGF expression (p=0.023) and low patient survival. Conclusions Pancreatic carcinogenesis is related to a history of chronic inflammatory processes, such as type 2 diabetes and chronic pancreatitis. In pancreatic cancer development, lymphangiogenesis can be considered an early event that enables the dissemination of metastases. VEGF expression and low LVD can be considered as poor prognostic factors as tumors with this profile are fast growing and highly aggressive. Virtual slides. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5113892881028514

Vascular Endothelial Growth Factor Expression in Invasive Papillary Thyroid Carcinoma

Background: The vascular endothelial growth factor (VEGF) is a major promoter of endothelial growth and migration. Some studies have shown a correlation between expression of this growth factor and prognosis in several cancers, including well-differentiated thyroid cancer. Aim: We studied VEGF expression, local invasiveness, and other prognostic factors in papillary thyroid carcinoma (PTC) to test the hypothesis that the expression of VEGF is correlated with the degree of invasion of PTC. Patients and Methods: Clinical and pathological data of 76 patients with PTC were retrospectively reviewed. Group 1 consisted of patients with gross locally invasive tumors, group 2 consisted of patients with only invasion of the thyroid capsule, and group 3 consisted of patients with noninvasive PTC. Results: VEGF expression was noted within the tumor in all groups of PTC patients but was absent in the surrounding normal tissue. Older patients had higher expression of VEGF than younger patients. The age of patients with strong reaction to VEGF was 46 +/- 14 (mean +/- standard deviation), and that in patients with a weaker reaction was 39 +/- 16 (p<0.05). Only 20% of patients with a follicular variant of PTC had a strong reaction to VEGF compared with 68% of patients with classical PTC (p<0.01). Conclusions: VEGF expression appears to be an early event in the development of PTC. Whether VEGF expression promotes the progression of PTC is not known, but the answer to this question may be important in view of its greater expression in older patients, a group whose prognosis in PTC is worse.

Vascular endothelial growth factor expression and microvascular density in soft tissue sarcomas in dogs

The aim of the current study was to evaluate the expression of vascular endothelial growth factor (VEGF) and the microvascular density in canine soft-tissue sarcomas. Immunohistochemistry for VEGF expression was performed on 20 canine neoplasms by the streptavidin-biotin-peroxidase method using an anti-VEGF mouse monoclonal antibody (ab-119). The Volume fraction of microvessels in the sarcomas was quantified in hematoxylin and eosin-stained tissue sections. At least 10 fields of view (40x magnification) per neoplasm were analyzed by positioning a grid with 100 points and counting the microvessels that fell into the intersection points. This percentage was considered the volume fraction of these microvessels in the tumor section. VEGF expression was detected in 65% of the neoplasms. In 92.3% of the neoplasms, the expression occurred in the peritumor region; in 46.15%, in the intratumor region; and in 38.46%, the expression was present in both regions. The cells responsible for VEGF expression were fibroblasts and macrophages in the peritumor region or in the pseudocapsule and neoplastic cells in the intratumor region. Greater intratumoral VEGF was expressed in hemangiopericytomas (P = 0.04). No difference was present in the volume fraction of tumor microvessels between VEGF-positive and VEGF-negative neoplasms (P = 0.3416) or for the different types of neoplasms (P = 0.5). The results of this study suggest that VEGF participates in the angiogenesis of soft-tissue sat-coma in dogs. Additional research will be necessary to elucidate the contribution of VEGF to the progression of malignancy.

DNA binding-independent transcriptional activation of the vascular endothelial growth factor gene (VEGF) by the Myb oncoprotein

Myb is a key transcription factor that can regulate proliferation, differentiation, and apoptosis, predominantly in the haemopoietic system. Abnormal expression of Myb is associated with a number of cancers, both haemopoietic and non-haemopoietic. In order to better understand the role of Myb in normal and tumorigenic processes, we undertook a cDNA array screen to identify genes that are regulated by this factor. In this way, we identified the gene encoding vascular endothelial growth factor (VEGF) as being potentially regulated by the Myb oncoprotein in myeloid cells. To determine whether this was a direct effect on VEGF gene transcription, we examined the activity of the murine VEGF promoter in the presence of either wild-type (WT) or mutant forms of Myb. It was found that WT Myb was able to activate the VEGF promoter and that a minimal promoter region of 120 bp was sufficient to confer Myb responsiveness. Surprisingly, activation of the VEGF promoter was independent of DNA binding by Myb. This was shown by the use of DNA binding-defective Myb mutants and by mutagenesis of a potential Myb-binding site in the minimal promoter. Mutation of Sp1 sites within this region abolished Myb-mediated regulation of a reporter construct, suggesting that Myb DNA binding-independent activation of VEGF expression occurs via these Sp1 binding elements. Regulation of VEGF production by Myb has implications for the potential role of Myb in myeloid leukaemias and in solid tumours where VEGF may be functioning as an autocrine growth factor. (c) 2006 Elsevier Inc. All rights reserved.

Acute hyperglycemia rapidly stimulates VEGF mRNA translation in the kidney. Role of angiotensin type 2 receptor (AT2)

Angiotensin II (Ang II) and vascular endothelial growth factor (VEGF) are important mediators of kidney injury in diabetes. Acute hyperglycemia increased synthesis of intrarenal Ang I and Ang II and resulted in activation of both Ang II receptors, AT1 and AT2, in the kidney. Losartan (specific AT1 antagonist) or PD123319 (specific AT2 antagonist) did not affect hyperglycemia but prevented activation of renal AT1 and AT2, respectively. In murine renal cortex, acute hyperglycemia increased VEGF protein but not mRNA content after 24 h, which suggested translational regulation. Blockade of AT2, but not AT1, prevented increase in VEGF synthesis by inhibiting translation of VEGF mRNA in renal cortex. Acute hyperglycemia increased VEGF expression in wild type but not in AT2 knockout mice. Binding of heterogeneous nuclear ribonucleoprotein K to VEGF mRNA, which stimulates its translation, was prevented by blockade of AT2, but not AT1. The Akt-mTOR-p70(S6K) signaling pathway, involved in the activation of mRNA translation, was activated in hyperglycemic kidneys and was blocked by the AT2 antagonist. Elongation phase is an important step of mRNA translation that is controlled by elongation factor 1A (eEF1A) and 2 (eEF2). Expression of eEF1A and activity of eEF2 was higher in kidney cortex from hyperglycemic mice and only the AT2 antagonist prevented these changes. To assess selectivity of translational control of VEGF expression, we measured expression of fibronectin (FN) and laminin beta 1 (lam beta 1): acute hyperglycemia increased FN expression at both protein and mRNA levels, indicating transcriptional control, and did not affect the expression of lam beta 1. To confirm results obtained with PD123319, we induced hyperglycemia in AT2 knockout mice and found that in the absence of AT2, translational control of VEGF expression by hyperglycemia was abolished. Our data show that acute hyperglycemia stimulates Ang II synthesis in murine kidney cortex, this leads to AT2 activation and stimulation of VEGF mRNA translation, via the Akt-mTOR-p70(S6K) signaling pathway. Our data show that exclusive translational control of protein expression in the kidney by acute hyperglycemia is not a general phenomenon, but do not prove that it is restricted to VEGF. (C) 2010 Elsevier Inc. All rights reserved.

Differential regulation of vascular endothelial growth factor expression by peroxisome proliferator-activated receptors in bladder cancer cells.

The growth of any solid tumor depends on angiogenesis. Vascular endothelial growth factor (VEGF) plays a prominent role in vesical tumor angiogenesis regulation. Previous studies have shown that the peroxisome proliferator-activated receptor gamma (PPARgamma) was involved in the angiogenesis process. Here, we report for the first time that in two different human bladder cancer cell lines, RT4 (derived from grade I tumor) and T24 (derived from grade III tumor), VEGF (mRNA and protein) is differentially up-regulated by the three PPAR isotypes. Its expression is increased by PPARalpha, beta, and gamma in RT4 cells and only by PPARbeta in T24 cells via a transcriptional activation of the VEGF promoter through an indirect mechanism. This effect is potentiated by an RXR (retinoid-X-receptor), selective retinoid LG10068 providing support for a PPAR agonist-specific action on VEGF expression. While investigating the downstream signaling pathways involved in PPAR agonist-mediated up-regulation of VEGF, we found that only the MEK inhibitor PD98059 reduced PPAR ligand-induced expression of VEGF. These data contribute to a better understanding of the mechanisms by which PPARs regulate VEGF expression. They may lead to a new therapeutic approach to human bladder cancer in which excessive angiogenesis is a negative prognostic factor.

Deleterious Actions Of Vegf On The Blood Retinal Barrier And Photoreceptor Survival In The Light-damage Model

Purpose: The retinal balance between pro- and anti-angiogenic factors is critical for angiogenesis control, but is also involved in cell survival. We previously reported upregulation of VEGF and photoreceptor (PR) cell death in the Light-damage (LD) model. Preliminary results showed that anti-VEGF can rescue PR from cell death. Thus, we investigated the role of VEGF on the retina and we herein described the effect of anti-VEGF antibody delivered by lentiviral gene transfer in this model.Methods: To characterize the action of VEGF during the LD, we exposed Balb/c mice subretinally injected with LV-anti-VEGF, or not, to 5'000 lux for 1h. We next evaluated the retinal function, PR survival and protein expression (VEGF, VEGFR1/2, Src, PEDF, p38MAPK, Akt, Peripherin, SWL-opsin) after LD. We analyzed Blood retinal barrier (BRB) integrity on flat-mounted RPE and cryosections stained with β-catenin, ZO-1, N-cadherin and albumin.Results: Results indicate that the VEGF pathway is modulated after LD. LD leads to extravascular albumin leakage and BRB breakdown: β-catenin, ZO-1 and N-cadherin translocate to the cytoplasm of RPE cells showing loss of cell cohesion. This phenomenon is in adequacy with the VEGF time-course expression. Assessment of the retinal function reveals that PR rescue correlates with the level of LV-anti-VEGF expression. Rhodopsin content was higher in the LV-anti-VEGF group than in controls and measures of the ONL thickness indicate that LV-anti-VEGF preserves by 82% the outer nuclear layer from degeneration. Outer segments (OS) appeared well organized with an appropriate length in the LV-anti-VEGF group compared to controls, and the expression of SWL-opsin is maintained in the OS without being mislocalized as in the LV-GFP group. Finally, LV-anti-VEGF treatment prevents BRB breakdown and maintained RPE cell integrity.Conclusions: This study involves VEGF in LD and highlights the prime importance of the BRB integrity for PR survival. Taken together, these results show that anti-VEGF is neuroprotective in this model and maintains functional PR layer in LD-treated mice.

Multigenic lentiviral vectors for combined and tissue-specific expression of miRNA- and protein-based antiangiogenic factors.

Lentivirus-based gene delivery vectors carrying multiple gene cassettes are powerful tools in gene transfer studies and gene therapy, allowing coexpression of multiple therapeutic factors and, if desired, fluorescent reporters. Current strategies to express transgenes and microRNA (miRNA) clusters from a single vector have certain limitations that affect transgene expression levels and/or vector titers. In this study, we describe a novel vector design that facilitates combined expression of therapeutic RNA- and protein-based antiangiogenic factors as well as a fluorescent reporter from back-to-back RNApolII-driven expression cassettes. This configuration allows effective production of intron-embedded miRNAs that are released upon transduction of target cells. Exploiting such multigenic lentiviral vectors, we demonstrate robust miRNA-directed downregulation of vascular endothelial growth factor (VEGF) expression, leading to reduced angiogenesis, and parallel impairment of angiogenic pathways by codelivering the gene encoding pigment epithelium-derived factor (PEDF). Notably, subretinal injections of lentiviral vectors reveal efficient retinal pigment epithelium-specific gene expression driven by the VMD2 promoter, verifying that multigenic lentiviral vectors can be produced with high titers sufficient for in vivo applications. Altogether, our results suggest the potential applicability of combined miRNA- and protein-encoding lentiviral vectors in antiangiogenic gene therapy, including new combination therapies for amelioration of age-related macular degeneration.

Retinoic Acid and VEGF Delay Smooth Muscle Relative to Endothelial Differentiation to Coordinate Inner and Outer Coronary Vessel Wall Morphogenesis

Rationale: Major coronary vessels derive from the proepicardium, the cellular progenitor of the epicardium, coronary endothelium, and coronary smooth muscle cells (CoSMCs). CoSMCs are delayed in their differentiation relative to coronary endothelial cells (CoEs), such that CoSMCs mature only after CoEs have assembled into tubes. The mechanisms underlying this sequential CoE/CoSMC differentiation are unknown. Retinoic acid (RA) is crucial for vascular development and the main RA-synthesizing enzyme is progressively lost from epicardially derived cells as they differentiate into blood vessel types. In parallel, myocardial vascular endothelial growth factor (VEGF) expression also decreases along coronary vessel muscularization. Objective: We hypothesized that RA and VEGF act coordinately as physiological brakes to CoSMC differentiation. Methods and Results: In vitro assays (proepicardial cultures, cocultures, and RALDH2 [retinaldehyde dehydrogenase-2]/VEGF adenoviral overexpression) and in vivo inhibition of RA synthesis show that RA and VEGF act as repressors of CoSMC differentiation, whereas VEGF biases epicardially derived cell differentiation toward the endothelial phenotype. Conclusion: Experiments support a model in which early high levels of RA and VEGF prevent CoSMC differentiation from epicardially derived cells before RA and VEGF levels decline as an extensive endothelial network is established. We suggest this physiological delay guarantees the formation of a complex, hierarchical, tree of coronary vessels. (Circ Res. 2010;107:204-216.)

Expressão imuno-histoquímica das proteínas MMP-9, VEGF e FVW em lesões centrais e periféricas de células gigantes

Central giant cell lesion (CGCL) and peripheral giant cell lesion (PGCL) of the jaws have a distinct clinical behavior, although they share histopathologic features. It is still unclear whether these clinical differences are supported by a distinct pattern of immunoexpression of markers for multinucleated giant cells (GC) and mononuclear cells (MC). The purpose of this study was to compare the immunohistochemical expression of VEGF, MMP-9 in CG and MC and measure the vascularization by vWF to check whether there are differences in expression of these biomarkers between CGCL and PGCL. Paraffin wax blocks of 20 cases of LCCG and 20 LPCG were retrieved. MMP-9 immunoreactivity was greater in the CM of PGCL compared to VEGF (p<0.05). VEGF expression was greater in the CM of CGCL compared to PGCL (p<0.05) and it was greater in the overall expression of CGCL compared to PGCL (p<0.05). Vascularity was quantified by microvascular counting (MVC). MVC was greater in the PGCL compared CGCL (p<0.05). MMP-9 showed a greater tendency of expression in CGCL, though was not significant (p>0.05). We tested correlation between the proteins studied in each group and found a significant negative correlation between VEGF and vWF in CGCL (p<0.05). These results suggest that there are differences in the expression of VEGF in CM and overall expression between the lesions, although no statistically significant difference in the overall expression of the MMP-9. Then, there was a trend in increased expression of MMP-9 and VEGF in CGCL, possibly by the involvement of both proteins in osteoclastogenesis. Additionally, the results of this study indicate a higher degree of vascularization in PGCL compared to CGCL, fact that can be directly linked to the reactive nature of the PGCL, where the inflammatory process with its rich angiogenesis contributes significantly to these findings.

Low level laser therapy increases angiogenesis in a model of ischemic skin flap in rats mediated by VEGF, HIF-1α and MMP-2

It is known that low level laser therapy is able to improve skin flap viability by increasing angiogenesis. However, the mechanism for new blood vessel formation is not completely understood. Here, we investigated the effects of 660 nm and 780 nm lasers at fluences of 30 and 40 J/cm2 on three important mediators activated during angiogenesis. Sixty male Wistar rats were used and randomly divided into five groups with twelve animals each. Groups were distributed as follows: skin flap surgery non-irradiated group as a control; skin flap surgery irradiated with 660 nm laser at a fluence of 30 or 40 J/cm2 and skin flap surgery irradiated with 780 nm laser at a fluence of 30 or 40 J/cm2. The random skin flap was performed measuring 10 × 4 cm, with a plastic sheet interposed between the flap and the donor site. Laser irradiation was performed on 24 points covering the flap and surrounding skin immediately after the surgery and for 7 consecutive days thereafter. Tissues were collected, and the number of vessels, angiogenesis markers (vascular endothelial growth factor, VEGF and hypoxia inducible factor, HIF-1α) and a tissue remodeling marker (matrix metalloproteinase, MMP-2) were analyzed. LLLT increased an angiogenesis, HIF-1α and VEGF expression and decrease MMP-2 activity. These phenomena were dependent on the fluences, and wavelengths used. In this study we showed that LLLT may improve the healing of skin flaps by enhancing the amount of new vessels formed in the tissue. Both 660 nm and 780 nm lasers were able to modulate VEGF secretion, MMP-2 activity and HIF-1α expression in a dose dependent manner. © 2013 Published by Elsevier B.V.

Immunohistochemical investigation of the angiogenic proteins VEGF, HIF-1α and CD34 in invasive ductal carcinoma of the breast

The expression of prognostic markers in cancer has become important in diagnostic routine and research. A high mitotic rate compromises the individual cell access to oxygen and nutrients, due to reduced blood supply. Cells undertake adaptive measures such as vascular endothelial growth factor (VEGF), expressed under the control of hypoxia-inducible factor-1α (HIF-1α). CD34 is an endothelial marker which can show the presence and distribution of blood vessels. This study evaluated the presence and relative expression of VEGF, HIF-1α and CD34 using immunohistochemistry of 60 breast tumors and double staining, correlating the findings with clinical and pathological variables. High VEGF expression was correlated with low cell proliferation, lymph node-negative, smaller tumor size and patients not receiving hormone therapy. High HIF-1α expression predominated in younger (<50-year) patients, subjected to neo-adjuvant therapy and in p53-negative tumors. Absence of metastasis, radiotherapy or hormone treatment, and estrogen receptor (ER)-positive tumors showed high CD34 immunoreactivity. We suggest that the angiogenic factors VEGF, HIF-1α and CD34 are important in breast cancer progression and their abundance in breast tumors has prognostic and predictive value. Crown Copyright © 2013.

Immunohistochemical evaluation of vascular endothelial growth factor (VEGF) in splenic hemangiomas and hemangiosarcomas in dogs

Formation of new blood vessels is paramount for tumour growth and metastatic dissemination and vascular endothelial growth factor (VEGF) is one of the key regulators of this process. The purpose of this study was to evaluate the immunohistochemical expression of VEGF in 23 splenic hemangiosarcomas and 7 splenic hemangiomas in dogs. Blood tests performed previous to splenectomy were analysed for correlation with tumour VEGF expression. Results showed significantly higher VEGF expression in hemangiosarcomas than hemangiomas and lower hematocrit values and red cell count in dogs affected with malignant neoplasia (P < 0.05). These findings suggest the presence of high VEGF levels may be related to the malignant vascular proliferation seen in hemangiosarcomas.