Vascular Endothelial Growth Factor A mRNA Levels Research Articles

Abstract 1096: Investigating the role of the adipocyte-macrophage interaction in breast cancer metastasis

Abstract Obesity is linked with a higher incidence of hormone-receptor positive breast cancer in post-menopausal women. Obesity is also associated with a worse outcome after the diagnosis of breast cancer, including greater tumor burden and higher grade tumors. Although several theories exist to explain these relationships, the proposed mechanisms are still being investigated. Obesity and the resulting expansion of adipose tissue is associated with aberrant cytokine production and a chronic inflammatory state, characterized by the recruitment of macrophages. Macrophages have been shown elsewhere to promote breast cancer progression. Therefore, we hypothesize that the interaction between adipocytes and macrophages promotes metastasis of breast cancer. Our data suggest that oxidized lipids released from adipocytes may have a role in mediating this interaction. We used a co-culture system to determine whether adipocytes could promote a pro-tumor phenotype in the macrophages. THP-1 macrophages were co-cultured with primary human visceral adipocytes using a Transwell system for 8 hours, then macrophage mRNA was analyzed for changes in transcription of markers for increased metastatic potential. Vascular Endothelial Growth Factor A (VEGF-A) mRNA levels were significantly increased in macrophages after exposure to adipocytes in comparison to control macrophages that were cultured alone. Co-culture experiments were repeated using human breast adipocytes to more directly model the conditions in the breast microenvironment. Again VEGF-A mRNA levels were significantly increased in macrophages after co-culture with adipocytes. Obesity is associated with stressed and necrotic adipose tissue in the breast. Therefore, to mimic obesity related dysfunction and cell death, we treated breast adipocytes with Tumor Necrosis Factor-alpha and Interleukin 6 prior to co-culturing with macrophages. The cytokine treatment enhanced the effects of adipocyte co-culture on macrophage VEGF-A mRNA levels. The increased inflammation and adipocyte dysfunction seen in obesity has been proposed to favor the generation of biologically active oxidized lipid mediators such as 4-hydroxynonenal (4-HNE). We therefore treated THP-1 macrophages with 4-HNE as a model oxidized lipid, hypothesizing that 4-HNE may convey some of the effects on macrophage gene expression seen in co-culture. We found that transcription of VEGF-D and platelet derived growth factor (PGF), both members of the VEGF family of cytokines, were significantly enhanced in 4-HNE-treated THP-1 cells. Together, our data show that the interaction of adipocytes and macrophages in obese breast tissue can stimulate pro-angiogenic macrophage signaling. This is a novel mechanism whereby the obese microenvironment can promote breast cancer metastasis. Citation Format: Nalini V.S. Yadav, Aaron T. Jacobs, Linda Connelly. Investigating the role of the adipocyte-macrophage interaction in breast cancer metastasis. [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 1096. doi:10.1158/1538-7445.AM2014-1096

Peritumoral brain edema in angiomatous supratentorial meningiomas: an investigation of the vascular endothelial growth factor A pathway

The aim of this work was to study the vascular endothelial growth factor A (VEGF-A) pathway and peritumoral brain edema (PTBE) through comparison of non-angiomatous and angiomatous meningiomas. Meningiomas are common intracranial tumors, which often have PTBE. VEGF-A is an integral part of PTBE formation and angiogenesis, and the capillary-rich angiomatous meningiomas are known for their PTBE. The VEGF-A receptor VEGFR-2 is responsible for the angiogenic effect of VEGF-A on endothelial cells, which is enhanced by the co-receptor neuropilin-1. Forty non-angiomatous, 22 angiomatous meningiomas, and 10 control tissue samples were collected for the study. Magnetic resonance images were available for 40 non-angiomatous and 10 angiomatous meningiomas. Tissue sections were immunostained for CD34, MIB-1, estrogen- and progesterone receptors. ELISA, chemiluminescence, and RT-qPCR were used for VEGF-A, VEGFR-2, and neuropilin-1 protein and mRNA quantification. Angiomatous meningiomas had larger PTBE (695 vs 218 cm(3) , p = 0.0045) and longer capillary length (3614 vs 605 mm/mm(3) , p < 0.0001). VEGF-A mRNA, neuropilin-1 mRNA, and VEGFR-2 protein levels were higher in angiomatous meningiomas independently of the capillary length (p < 0.05). Neuropilin-1 protein levels were lower in angiomatous meningiomas (p < 0.0001). The VEGF-A pathway and tumor capillary length may be essential for PTBE-formation in meningiomas. Further investigations of this pathway could lead to earlier therapy and targeted pharmacological treatment options.

The wound healing effects of vitamin A eye drops after a corneal alkali burn in rats

We investigated the wound healing effect of retinyl palmitate eyedrops following a corneal alkali burn in rats. A total of 160 Sprague-Dawley male rats were divided into two groups and central corneas were injured by contacting eyes with filter paper saturated with 0.01 m NaOH for 45 seconds. Vitamin A group was treated with retinyl palmitate and antibiotic (Cravit(®) : 0.5% levofloxacin) eye drops four times daily for 3 days and the control group with vehicle and antibiotic eye drops. Corneal wound healing by fluorescein staining and impression cytologic analysis were conducted at 0, 24, 48 and 72 hr after injury. Vascular endothelial growth factor A (VEGF-A), thrombospondin 2, matrix metalloproteinase 9 (MMP 9) and transforming growth factor-β (TGF-β) were measured in corneas by ELISA, immunofluorescent staining and real-time PCR. Corneal wound healing was better in the vitamin A group than in the control group. Early sprouting of new vessel was observed in the control group at 72 hr, but not in the vitamin A group. Corneal thrombospondin 2 proteins in ELISA were higher in the vitamin A group, but VEGF-A, MMP 9 and TGF-β proteins were higher in the control group (p < 0.05). Similarly, thrombospondin 2 immunofluorescent staining was stronger, whereas VEGF-A, MMP 9 and TGF-β staining were weaker in the vitamin A group (p < 0.05). In addition, thrombospondin 2 mRNA levels were higher, whereas VEGF-A, MMP 9 and TGF-β mRNA levels were lower in the vitamin A group (p < 0.05). Retinyl palmitate eye drops can inhibit VEGF-A and activate thrombospondin 2 and improve conjunctival impression cytologic findings. Furthermore, retinyl palmitate eye drops were found to promote corneal healing after an alkali burn in rats.

Role of the angiogenic components, VEGFA, FGF2, OPN and RHOC, in urothelial cell carcinoma of the urinary bladder

The objective of this study was to analyze the expression profile of the angiogenic components, vascular endothelial growth factor-A (VEGFA), basic fibroblast growth factor-2 (FGF2), osteopontin (OPN) and ras homolog gene family, member C (RHOC), in urothelial cell carcinoma (UCC) of the urinary bladder and to examine their role as candidate diagnostic biomarkers. Using qPCR, 77 samples of UCC of the urinary bladder and 77 matched tumor-associated normal samples were investigated to determine the expression of the four angiogenic components. The correlation between gene expression, patient survival and pathological features of the tumors was also examined. The VEGFA and OPN transcript levels were greater in the bladder cancer tissue than in the normal urothelium (P<0.001). Patients with higher VEGFA mRNA levels showed a tendency towards shorter cancer-specific survival. OPN levels showed a gradual increase, the lowest levels being found in non-invasive carcinoma and the highest in muscle invasive tumors. Elevated OPN levels indicated poor prognosis in connection with advanced disease stage (P<0.001). Both superficially invasive and muscle invasive tumors had significantly higher FGF2 levels compared to the control tissues (P=0.018 and P=0.050, respectively). Moreover, FGF2 was significantly higher in the metastatic vs. the non-metastatic tumors (P=0.0097). FGF2 levels exhibited a trend towards a correlation with worse patient survival. RHOC mRNA levels were higher in muscle invasive compared to superficially invasive tumors, as well as in grade III vs. grade I/II tumors. Furthermore, we detected worse overall survival for patients with high RHOC expression levels. VEGFA and FGF2 exhibited the best linear combination in the ROC curves for specificity and sensitivity. Thus, VEGFA and FGF2 may serve as candidate biomarkers for diagnostic purposes. Higher OPN expression may be used as a potential biomarker to predict patient survival relative to advanced tumor stage. However, further studies are required to investigate its role in urinary bladder carcinogenesis.

Inhibition of STAT3 by RNA interference suppresses angiogenesis in colorectal carcinoma

In order to investigate signal transduction and activation of transcription 3 (STAT3) signaling on angiogenesis in colorectal carcinoma (CRC) after inhibiting STAT3 expression, we constructed the HT-29-shSTAT3 cell line by lentivirus-mediated RNAi. Cell growth was assessed with MTT and the cell cycle distribution by flow cytometry. CRC nude mouse models were established and tumor growth was monitored periodically. On day 30, all mice were killed and tumor tissues were removed. Microvessel density (MVD) was determined according to CD34-positive staining. The expression of vascular endothelial growth factor A (VEGFA), matrix metalloproteinase-2 (MMP2) and basic fibroblast growth factor (FGF2) was monitored by quantitative real-time PCR and Western blot analysis. Knockdown of STAT3 expression significantly inhibited cell growth in HT-29 cells, with a significantly higher proportion of cells at G0/G1 (P < 0.01). Consistently, in vivo data also demonstrated that tumor growth was significantly inhibited in mice injected with HT-29-shSTAT3 cells. MVD was 9.80 ± 3.02 in the HT-29-shSTAT3 group, significantly less than that of the control group (P < 0.01). mRNA and protein levels of VEGFA and MMP2 in the HT-29-shSTAT3 group were significantly lower than in the control group (P < 0.05), but no significant difference was observed in the mRNA or protein level of FGF2 (P > 0.05). Taken together, these results demonstrate that STAT3 signaling is important to the growth of CRC and promotes angiogenesis by regulating VEGFA and MMP2 expression.

Vascular endothelial growth factor-A and chemokine ligand (CCL2) genes are upregulated in peripheral blood mononuclear cells in Indian amyotrophic lateral sclerosis patients

BackgroundWe have earlier shown that protein levels of vascular endothelial growth factor-A (VEGF-A) and chemokine ligand-2 (CCL2) were elevated in Indian amyotrophic lateral sclerosis (ALS) patients. Here, we report the mRNA levels of VEGF-A and CCL2 in Indian ALS patients since they display extended survival after disease onset.MethodsVEGF-A and CCL2 mRNA levels were measured in peripheral blood mononuclear cells (PBMCs) of 50 sporadic Indian ALS patients using Real Time Polymerase Chain Reaction (PCR) and compared with normal controls (n = 50). Their levels were adjusted for possible confounders like cigarette smoking, alcohol and meat consumption.ResultsVEGF-A and CCL2 mRNA levels were found to be significantly elevated in PBMCs in ALS patients as compared to controls. PBMCs from definite ALS revealed higher VEGF-A mRNA expression as compared to probable and possible ALS. CCL2 mRNA levels were found to be unaltered when definite, probable and possible ALS were compared. PBMCs from patients with respiratory dysfunction showed much higher VEGF-A and CCL2 elevation when compared to patients without respiratory dysfunction. No association of smoking, alcohol and meat consumption with VEGF-A and CCL2 was observed after analyzing the data with univariate and multivariate analysis.ConclusionVEGF-A and CCL2 mRNA upregulation in PBMCs may have a clinico-pathological/etiological/epidemiological association with ALS pathogenesis. The cross-cultural and cross-ethnic investigations of these molecules could determine if they have any role in enhancing the mean survival time unique to Indian ALS patients.

Early Atheroma-Derived Agonists of Peroxisome Proliferator–Activated Receptor-γ Trigger Intramedial Angiogenesis in a Smooth Muscle Cell–Dependent Manner

Neovascularization favors intraplaque hemorrhage and plaque rupture. Development of therapeutic strategies against atheromatous angiogenesis requires elucidation of its initiating factors. We investigated the contribution of smooth muscle cells (SMCs) and atheroma-derived lipids to the initiation of atheroma-associated neoangiogenesis. Forty human aortic segments, each harvested from a different donor, were classified as healthy or as bearing early atheromatous lesions, including fatty streaks and fibrolipidic atheroma, according to their histological features. Immunostaining for blood vessels and vascular endothelial growth factor-A (VEGF-A), as well as measurement of VEGF-A protein and mRNA levels by ELISA and real-time PCR, revealed that angiogenesis and VEGF-A production were enhanced in the medial layer of atheromatous aortas. The intramedial vessel density and invasiveness and the production of VEGF-A by medial SMCs were indeed increased in atheromatous aortas compared with healthy aortas. Furthermore, intimal layers of atheromatous aortas were enriched in soluble lipid mediators capable of inducing a sustained increase in VEGF-A production by medial SMCs, turning these cells into potent inducers of angiogenesis when incorporated into mouse Matrigel implants. Both effects were inhibited by the peroxisome proliferator-activated receptor-γ inhibitor GW9662 and mimicked by its agonist, rosiglitazone. We show that VEGF-A production is upregulated in medial SMCs of human atheromatous aortas and that peroxisome proliferator-activated receptor-γ agonists derived from early intimal lesions are likely to contribute to this phenotypic change. Our findings suggest that medial SMCs are central organizers of an angiogenic response initiated by the subendothelial accumulation of atherogenic lipids.

Expression of vascular endothelial growth factor A during ligand-induced down-regulation of luteinizing hormone receptor in the ovary

Vascular endothelial growth factor A (VEGF-A) is one of the most important regulators of ovarian angiogenesis. In this study, we examined the temporal relationship between VEGF-A and luteinizing hormone receptor (LHR) mRNA expression during ligand-induced down-regulation of LHR. Immature female rats were treated with pregnant mare's serum gonadotropin followed by 25 IU hCG 56 h later (day 0). On day 5, treatment with hCG (50 IU) to down-regulate LHR showed a temporal decrease in VEGF-A mRNA and protein levels in parallel with decreasing LHR mRNA. This effect was specific since the expression of CYP11A1 mRNA showed no decline. Examination of VEGF-A mRNA expression, using in situ hybridization histochemistry with 35S-labeled antisense VEGF-A mRNA probe, showed intense signal in the corpora lutea on day 5. Treatment with 50 IU hCG to down-regulate LHR mRNA showed a decline in the intensity of VEGF-A mRNA in the corpora lutea. VEGF-A mRNA expression returned to control level 53 h later when the expression of LHR mRNA also recovered. These results show that the transient down-regulation of VEGF-A mRNA and protein closely parallels the ligand-induced down-regulation of LHR mRNA. The present study establishes a close association between VEGF-A and LHR mRNA expression, suggesting the possibility that VEGF-A-induced vascularization of the ovary is dictated by the expression of LHR and this might play a regulatory role in ovarian physiology.

Influence of peritoneal fluid on the expression of angiogenic and proteolytic factors in cultures of endometrial cells from women with endometriosis

Endometriosis, defined as the presence of endometrium outside the uterus, is one of the most frequent benign gynaecological diseases. It has been suggested that both endometrial and peritoneal factors, related to angiogenesis and proteolysis, can be implicated in this disease. The aim of this study was to evaluate the influence of peritoneal fluid on the expression of angiogenic and proteolytic factors in cultures of endometrial cells from women with and without endometriosis. Endometrial cells were isolated, cultured and treated with endometriotic or normal peritoneal fluid. Vascular endothelial growth factor-A (VEGF-A), urokinase plasminogen activator (uPA), matrix metalloproteinase-3 (MMP-3) and their inhibitors including thrombospondin-1, plasminogen activator inhibitor-1 and MMP inhibitor type 1 (TIMP-1) mRNA levels were evaluated by quantitative RT-PCR, and protein levels were quantified by ELISA. Peritoneal fluid from women with endometriosis induced an increase in VEGF-A and uPA protein and VEGF-A mRNA and uPA mRNA levels in endometrial cell culture from women with (P < 0.01) and without endometriosis (P < 0.05). The highest levels of VEGF-A and uPA were observed in endometrial cell cultures from patients with endometriosis and treated with peritoneal fluid from women with endometriosis. Peritoneal fluid from women with endometriosis induced more VEGF and uPA expression in endometrial cell culture from women with endometriosis than did normal peritoneal fluid. Endometrial-peritoneal interactions increased angiogenic and proteolytic factors in endometrial cells, which could contribute to the development of endometriotic lesions.

DNA microarray analysis of dental pulp fibroblasts exfoliated from deciduous teeth

Dental pulp plays an important role in tooth vitality. Previous studies have indicated that stem cells can be isolated from dental pulp, and dental pulp exfoliated from deciduous teeth has become a useful alternative for dental tissue engineering because of its higher proliferation rate. In the present study, we analyzed the differences in gene expressions between human dental pulps exfoliated from deciduous and permanent teeth by DNA microarray assays. A scatter plot of mRNA levels based on fluorescent signals in human dental pulps from deciduous and permanent teeth indicated a dispersed distribution pattern. In a scatter plot of the genes, 2,573 genes were expressed at >2-fold higher levels in dental pulp from deciduous teeth, compared with permanent teeth. To confirm the microarray results, insulin-like growth factorbinding protein 5 (IGFBP5) and vascular endothelial growth factor A (VEGFA) showing higher mRNA expression levels were selected and analyzed for their mRNA levels by RT-PCR. Both the IGFBP5 and VEGFA mRNA levels were upregulated by about 3-fold in dental pulp from deciduous teeth compared with permanent teeth. Thus, the RT-PCR results for these genes were consistent with the microarray data. The dental pulps in deciduous and permanent teeth differ significantly with regard to their developmental processes, tissue structures and functions. The present findings using DNA microarray analyses to detect differences in the gene expressions of deciduous and permanent teeth may be useful for dental pulp tissue engineering.

Reduction of VEGF-A and CTGF expression in diabetic nephropathy is associated with podocyte loss

Micro-vascular and renal complications in diabetic patients are a considerable clinical challenge. In a previous study, we found a significant decrease in vascular endothelial growth factor A (VEGF-A) mRNA levels in glomeruli from patients with diabetic nephropathy (DN). We now set out to investigate the relationship between reduced VEGF-A and connective tissue growth factor (CTGF) expression levels, the number of podocytes, and the extent of interstitial fibrosis. Laser capture microdissection was applied to obtain glomerular RNA from 28 patients with DN and 22 controls. mRNA levels of VEGF-A, CTGF, nephrin, podocin, and Wilms tumor1 (WT1) were measured using real-time polymerase chain reaction. Protein expression was evaluated using immuno-stainings for VEGF-A and CTGF, as well as markers for podocytes (WT1) and endothelial cells (CD31). We found a significant decrease in glomerular mRNA levels for VEGF-A (2.5 times), CTGF (1.6), nephrin (2.8), podocin (3.3), and WT1 (1.7) in patients with DN. There was a significant correlation between expression of podocyte markers and VEGF-A mRNA levels, and an inverse correlation between podocin message and the extent of interstitial fibrosis. CD31-positive area was significantly decreased (3.2 times) in patients with DN. Reduction of angiogenic factors correlated with the extent of interstitial fibrosis. This downregulation was related to a reduction of podocytes in DN. The results may suggest that downregulation of VEGF-A and CTGF in DN is a result of podocyte loss.

Signaling pathways for arsenic-stimulated vascular endothelial growth factor-a expression in primary vascular smooth muscle cells.

Chronic arsenic exposure is associated with an increased risk for cancer, cardiovascular disease (including ischemic heart disease and hypertension), peripheral vascular disease, and diabetes. Arsenic causes blood vessel growth and remodeling in vivo and cell specific, dose-dependent induction vascular endothelial growth factor-A (VEGF), which is essential for both processes. The current study examined the hypothesis that low, environmentally relevant levels of trivalent arsenic (AsIII) activate discrete signaling pathways in vascular smooth muscle cells (SMC) to induce expression of VEGF. AsIII caused a progressive increase in VEGF mRNA levels over a 48 h period in primary porcine SMC with a threshold of 1-2.5 microM. VEGF protein levels increased with a similar concentration dependence and time course. Hypoxia inducible factor-1alpha (HIF-1alpha) protein and mRNA levels also increased in response to AsIII. However, unlike the response to an iron chelator, AsIII-induced VEGF was not inhibited by siRNA directed toward HIF-1alpha. Instead, a novel protein kinase C, PKCdelta, was activated by AsIII to induce VEGF and stabilize HIF-1alpha. Consistent with this activation, AsIII caused coordinate increases in the levels of the intracellular second messenger diacyglycerol (DAG). These data suggest that AsIII induced divergent signaling pathways in SMCs that lead to independent increases in VEGF expression and HIF-1alpha signaling. However, these pathways both require initial increases in DAG levels and PKC activity.

Oxidative Stress Regulates Vascular Endothelial Growth Factor-A Gene Transcription through Sp1- and Sp3-dependent Activation of Two Proximal GC-rich Promoter Elements

Enhanced VEGF-A (vascular endothelial growth factor A) gene expression is associated with increased tumor growth and metastatic spread of solid malignancies including gastric cancer. Oxidative stress has been linked to tumor-associated neoangiogenesis; underlying mechanisms, however, remained poorly understood. Therefore, we studied the effect of oxidative stress on VEGF-A gene expression in gastric cancer cells. Oxidative stress generated by H(2)O(2) application potently stimulated VEGF-A protein and mRNA levels as determined by enzyme-linked immunosorbent assay and real-time PCR techniques, respectively, and elevated the activity of a transfected (-2018) VEGF-A promoter reporter gene construct in a time- and dose-dependent manner (4-8-fold). These effects were abolished by the antioxidant N-acetylcysteine, demonstrating specificity of oxidative stress responses. Functional 5' deletion analysis mapped the oxidative stress response element of the human VEGF-A promoter to the sequence -88/-50, and a single copy of this element was sufficient to confer basal promoter activity as well as oxidative stress responsiveness to a heterologous promoter system. Combination of EMSA studies, Sp1/Sp3 overexpression experiments in Drosophila SL-2 cells, and systematic promoter mutagenesis identified enhanced Sp1 and Sp3 binding to two GC-boxes at -73/-66 and -58/-52 as the core mechanism of oxidative stress-triggered VEGF-A transactivation. Additionally, in Gal4-Sp1/-Sp3-Gal4-luciferase assays, oxidative stress increased Sp1 but not Sp3 transactivating capacity, indicating additional mechanism(s) of VEGF-A gene regulation. Signaling studies identified a cascade comprising Ras --> Raf --> MEK1 --> ERK1/2 as the main pathway mediating oxidative stress-stimulated VEGF-A transcription. This study for the first time delineates the mechanisms underlying regulation of VEGF-A gene transcription by oxidative stress and thereby further elucidates potential pathways underlying redox control of neoangiogenesis.

Differential expression of vascular endothelial growth factor-A (VEGF-A) and VEGF-B after brain injury.

Our previous study demonstrated that vascular endothelial growth factor (VEGF), now referred to as VEGF-A, plays a significant role in blood-brain barrier (BBB) breakdown and angiogenesis after brain injury. In this study, VEGF-A expression was compared with that of VEGF-B in the rat cortical cold injury model over a period of 6 hours to 6 days post-injury. VEGF-A and VEGF-B mRNA were detected by in situ hybridization and their protein was detected by immunohistochemistry. The presence of VEGF-A and VEGF-B proteins in endothelium of lesion vessels was related to BBB breakdown by double labeling for either of these growth factors and fibronectin, which was used as a marker of BBB breakdown. Significant induction of both VEGF-A and VEGF-B mRNA occurred at the lesion site during the period of maximal endothelial proliferation. VEGF-A mRNA levels peaked at 3 and 4 days post-injury and returned to basal expression by day 6, while VEGF-B mRNA levels remained elevated up to day 6. VEGF-B protein was constitutively expressed in endothelium of all cerebral vessels. After brain injury, there was increased immunoreactivity for VEGF-B at the lesion site, this protein being present in the endothelium and vascular smooth muscle cells of pial vessels, in inflammatory cells, and later in proliferating endothelial cells, endothelium of neovessels, and astrocytes. Lesion vessels showing BBB breakdown to fibronectin showed endothelial VEGF-A protein but not VEGF-B protein. Constitutive expression of VEGF-B in normal endothelium suggests that it may have a role in maintenance of the BBB in steady states, while its induction at both the gene and protein level post-injury indicates that it has an essential role in angiogenesis and the repair processes after brain injury.