Decrease In VEGF-A Research Articles

Impact of neonatal hyperoxia on the development of the coronary vascular bed: an experimental model for the study of cardiomyopathy associated with premature birth

Abstract Introduction Very preterm birth (PT; <32 weeks of gestation), nearly 2% of all births, leads to short-term complications such as bronchopulmonary dysplasia and retinopathy of prematurity that are driven by compromised organ vascular development. Adults born preterm display an increased risk of ischaemic cardiomyopathy and heart failure. Our group has shown that neonatal rats exposed to hyperoxia, simulating the PT neonatal conditions, develop cardiomyocytes hypertrophy, fibrosis and cardiac dysfunction, or Oxygen-Induced Cardiomyopathy (OIC). Whether cardiac changes also associate with alterations in vascular development in the left ventricle (LV) remains unknown. Purpose We hypothesized that neonatal exposure to hyperoxia impact the development of the LV vascular network. Methods Male pups were exposed to 80% O2 (OIC) or room air (CTRL) from day 3 (P3) to P10 of life. Hearts were collected at P10, 4- and 16 weeks. Arteriolar length and capillary density per cardiomyocyte were measured using stereology (elastin) and immunofluorescence (WGA, α-SMA, CD31). High-resolution 3D reconstruction of the vascular network was achieved by combining fluorescent labelling of coronaries (anti-α-SMA) and capillaries (anti-CD31) with a tissue-cleaning protocol (iDisco+), allowing detailed morphometric characterization of the vascular network. The expression of vascular endothelial growth factors and receptors (VEGFA, VEGFB, VEGFR 1 and 2, NRP1, and PIGF) were quantified by RT-qPCR and Western blots. Differences between groups were analyzed with Student's t-test (P < 0.05) (N = 6 per group). Results At P10, we observed a notable rise in the number of capillaries per cardiomyocyte (0.88 ± 0.05 vs. 0.62 ± 0.02 mm2) and a significant increase in VEGFA (+76%) and NRP1 (+38%) expression in the LV from OIC vs. CTRL rats. By 4 weeks, the OIC group showed a significant reduction in arteriolar length vs. CTRL (6.51 ± 0.76 vs. 9.77 ± 1.12 m/cm3), coupled with an increase in the number of capillaries per cardiomyocyte (1.11 ± 0.05 vs. 0.85 ± 0.03 mm2). VEGFA was decreased (-14%) and PLGF increased (+24%) in the LV of OIC rats. At 16 weeks, the OIC condition displayed a significant increase in the number of capillaries per cardiomyocyte (1.37 ± 0.03 vs. 0.98 ± 0.07 mm2) and a decrease in VEGFA (-23%), along with reduction in its receptors VEGFR1 (-27%), VEGFR2 (-20%) and NRP1 (-17%). Conclusion OIC resulting from neonatal hyperoxia exposure associates with vascular changes in the LV that persist to adulthood. These changes are characterized by a reduction in arteriolar length and an increase in the number of capillaries, accompanied by modulation of the expression of pro-angiogenic VEGFA and receptors. The impact of these vascular alterations on the susceptibility of the LV to ischemic or hypertension-related damage is to be explored. These findings suggest a crucial mechanistic pathway for understanding the risk of cardiac pathologies associated with preterm birth.

The effects of anti-PD-L1 monoclonal antibody on the expression of angiogenesis and invasion-related genes.

The role of PD-L1 in regulating the immunosuppressive tumor microenvironment via its binding on PD-1 receptors is extensively studied. The PD-1/PD-L1 axis is a significant way of cancer immune escape, and PD-L1 expression on tumor cells is suggested as a predictive marker for anti-PD-1/PD-L1 monoclonal antibodies (MoAbs). However, the tumor-intrinsic role of PD-L1 is not known well. Therefore, we aimed to investigate the effects of anti-PD-L1 antibodies on the expression of angiogenesis and metastasis-related genes in tumor cells. The experiments were done with prostate cancer and melanoma cells with low PD-L1 expression (<5%) and prostate and breast cancer cells with high PD-L1 expression (>50%). The gene and protein expressions of VEGFA, E-cadherin, TGFβ1, EGFR, and bFGF in tumor cells were assayed at the 3 different doses of the anti-PD-L1 antibody. We found that VEGFA, E-cadherin and TGFβ1 expressions increased in PD-L1 high cells but decreased in PD-L1 low cells after anti-PD-L1 treatment. EGFR expression levels were variable in PD-L1 high cells, while decreased in PD-L1 low cells upon treatment. Also, the anti-PD-L1 antibody was found to increase bFGF expression in the prostate cancer cell line with high PD-L1 expression. Our results suggest that the binding of PD-L1 on tumor cells by an anti-PD-L1 monoclonal antibody may affect tumor-intrinsic mechanisms. The activation of angiogenesis and metastasis-related pathways by anti-PD-L1 treatment in PD-L1 high tumors might be a tumor-promoting mechanism. The decrease of VEGFA, TGFβ1 and EGFR upon anti-PD-L1 treatment in PD-L1 low tumor cells provides a rationale for the use of those antibodies in PD-L1 low tumors.

Change of angiogenesis in the nodular form of benign mammary dysplasia during treatment with alkaloids, flavonoids and glycosides

Introduction. The formation of new blood vessels is called angiogenesis. As the tumor grows, the oxygen and nutrient requirements of the abnormal cells continuously increase, and new blood vessels are formed. These processes are disrupted by angiogenesis inhibitors. Therefore, it is warranted to investigate ways to enhance the capabilities of angiogenesis inhibitors alkaloids, flavonoids, and glycosides.&#x0D; Aim. To justify the extension of clinical use of alkaloids, flavonoids and glycosides (Conium maculatum and Hydrastis canadensis, Thuja occidentalis), being a part of the complex medical product Mastopol, taking into account the obtained data on the angiogenic balance in the nodular form of benign mammary dysplasia (BMD).&#x0D; Materials and methods. The study included 69 volunteers divided into two groups: Group 1 included 27 patients with medical history of surgery for nodular BMD who received antiproliferative preoperative therapy with Mastopol for 12 weeks. Group 2 (comparison group), n=42, also included patients with medical history of surgery for nodular BMD but without preoperative therapy with the herbal medicine Mastopol. We studied angiogenesis markers (VEGF-A [vascular endothelial growth factor] and pVEGF-1) and immunohistochemical marker CD34 (cluster of differentiation CD).&#x0D; Results. We found a significantly higher (p0.001) rate of CD34 detection on medium and small caliber vessels in the comparison group, i.e., in the patients who were not treated preoperatively with alkaloids, flavonoids, and glycosides (Mastopol), while blood supply to the nodules by the medium and small vessels was 1.4 and 1.8 times lower in the Group 1, respectively. Analysis of VEGF expression shows an increase of angiogenic promoters VEGF-A and pVEGF-1 in serum before treatment with biologically active herbal components of Mastopol in both groups. There were no statistically significant differences in vascular markers. However, a statistically significant decrease in VEGF-A and pVEGF-1 (p0.001) was noted in Group 1 after Mastopol therapy. Note the presence of a moderate direct correlation (rs=0.49) of CD34 expression with VEGF-A expression in Group 1 and rs=0.41 in Group 2, which indicated a direct relationship between angiogenesis and proangiogenic factors.&#x0D; Conclusion. In addition to the proven antiproliferative, anti-inflammatory, cytokine stabilizing, analgesic, and anti-edema properties of Mastopol, the study also showed the antiangiogenic effect of the herbal drug components. Considering the decreased vascular growth in fibroadenomas during Mastopol treatment, the indications for its use in patients with fibroadenomas before the surgery can be extended. Specific immunological test to measure VEGF can be proposed as a noninvasive screening method to evaluate the effectiveness of conservative treatment in general clinical practice.

IGF-I combined with exercise improve diabetes-induced vascular dysfunction in heart of male Wistar rats.

Introduction: This research investigates the impact of insulin-like growth factor-I (IGF -I)and exercise on mediators associated with angiogenesis (VEGF-A, TSP-1, and NF-кβ) and capillarization status of the diabetic rats’ hearts. Methods: Splitting of forty Wistar male rats into five groups occurred as following: control,diabetes, diabetes+IGF-I, diabetes+exercise, and diabetes+exercise+IGF-I.Through intraperitoneal administration of 60 mg/kg streptozotocin, the condition of Type 1diabetes was escalated. After four weeks of treatment with IGF-I (2 mg/kg/day) or treadmill exercise (17 m/min, zero degrees slope, 30 min/day), in the heart, microvascular density and protein levels of VEGF-A, TSP-1, and NF-кβ were determined by H&E staining and ELISA,respectively. Results: Within the diabetic group, observations present a significant decrease in VEGF-A and MVD levels, whereas an increase in the TSP-1 and NF-Κb levels. While these impacts were reversed by either IGF-I or exercise treatments, simultaneous treatment had synergistic effects. Moreover, among diabetic rats, undesirable histologic alterations of the heart were demonstrated, including myonecrosis, interstitial edema, hemorrhage, and mononuclear immune cell infiltration, whereas treatments improved these changes. Conclusion: These data manifest that IGF-I and exercise can increase the cardiac angiogenesis of diabetic rats through increasing expression of VEGF-A, and decreasing TSP-1 and NF-кβproteins level, also can improve myocardial tissue damages.

MiR-106a-5p Functions as a Tumor Suppressor by Targeting VEGFA in Renal Cell Carcinoma

MicroRNAs (miRNAs) regulate progression of different cancers. Nevertheless, there is limited information regarding the role of miR-106a-5p in renal cell carcinoma (RCC). Herein, we demonstrate that miR-106a-5p levels are drastically decreased in clear cell RCC (ccRCC) tissues and cell lines, which subsequently contribute to a poor patient overall survival and a high tumor stage. By screening and analyzing, we found that miR-106a-5p directly targets the 3′-UTR of the VEGFA mRNA and led to a decrease in VEGFA. This process is important for tumor cells' growth and colony formation, and overexpression of miR-106a-5p can especially kill kidney tumor cells. Therefore, our data reveal that miR-106a-5p functions as a tumor suppressor by regulating VEGFA and ccRCC may be susceptible to miR-106a-5p therapy.

Sildenafil ameliorates Alzheimer disease via the modulation of vascular endothelial growth factor and vascular cell adhesion molecule-1 in rats.

Alzheimer disease (AD) is a chronic neurodegenerative disease with multi-pathways pathogenesis. Sildenafil is a selective phosphodiesterase-5 inhibitor with a potential benefit in the treatment of AD. This study investigated the possible mechanisms underlying the effect of sildenafil in AD with emphasis on vascular endothelial growth factor (VEGF), and vascular cell adhesion molecule-1 (VCAM-1). Twenty-four adult male rats were classified into four groups; control group: received vehicles, sildenafil-control: received sildenafil (15 mg/kg/day, p.o.), AD group received Aluminum (25 mg/kg/day, p.o.), AD-treated group: received sildenafil (15 mg/kg/day, p.o.) for 6 weeks. AD was assessed by memory performance test and confirmed by histopathological examination and immunostaining of, neurogenesis marker nestin and α-synuclein. The levels of VEGF-A, VCAM-1, oxidative stress markers and TNF-α in brain tissue were evaluated. AD rats showed histopathological evidences of AD; along with increased latency time in the memory test. There was a decrease in VEGF-A, and an increase in VCAM-1, TNF-α, and oxidative stress markers. Immunohistochemical study showed a significant increase in α-synuclein and a significant decrease in nestin expressions in brain tissues. Sildenafil administration ameliorated the histopathological changes and decreased latency time. Such effect was associated with a decrease in VCAM-1, TNF-α and oxidative stress as well as an increase in VEGF-A. Sildenafil caused a significant increase in nestin and a decrease in α-synuclein immunostaining. These findings suggested a protective effect of sildenafil via modulation of VEGF-A, and VCAM-1.

The Role of VEGFA, COX2, HUR and CUGBP2 in Predicting the Response to Neoadjuvant Therapy in Rectal Cancer Patients.

Background and objectives: The effectiveness of neoadjuvant therapy, which is commonly used for stage II-III rectal cancer (RC) treatment, is limited. Genes associated with the pathogenesis of RC could determine response to this treatment. Therefore, the aim of this study was to investigate the potential predictive value of VEGFA, COX2, HUR and CUGBP2 genes and the associations between post-treatment changes in gene expression and the efficacy of neoadjuvant therapy. Materials and Methods: Biopsies from RC and healthy rectal tissue of 28 RC patients were collected before neoadjuvant therapy and 6-8 weeks after neoadjuvant therapy. The expression levels of VEGFA, COX2, HUR, CUGBP2 genes were evaluated using a quantitative real-time polymerase chain reaction. Results: The results reveal a significantly higher expression of VEGFA, COX2 and HUR mRNA in RC tissue compared to healthy rectal tissue (p < 0.05), and elevated VEGFA gene expression in pre-treatment tissues was associated with a better response to neoadjuvant therapy based on T-stage downstaging (p < 0.05). The expression of VEGFA, HUR and CUGBP2 genes significantly decreased after neoadjuvant therapy (p < 0.05). Responders to treatment demonstrated a significantly stronger decrease of VEGFA and COX2 expression after neoadjuvant therapy than non-responders (p < 0.05). Conclusions: The findings of this study suggest that the pre-treatment VEGFA gene expression might have predictive value for the response to neoadjuvant therapy, while the post-treatment decrease in VEGFA and COX2 gene expression could indicate the effectiveness of neoadjuvant therapy in RC patients.

Tumor Angiogenesis Is Differentially Regulated by Phosphorylation of Endothelial Cell Focal Adhesion Kinase Tyrosines-397 and -861.

Expression of focal adhesion kinase (FAK) in endothelial cells (EC) is essential for angiogenesis, but how FAK phosphorylation at tyrosine-(Y)397 and Y861 regulate tumor angiogenesis in vivo is unknown. Here, we show that tumor growth and angiogenesis are constitutively reduced in inducible, ECCre+;FAKY397F/Y397F -mutant mice. Conversely, ECCre+;FAKY861F/Y861F mice exhibit normal tumor growth with an initial reduction in angiogenesis that recovered in end-stage tumors. Mechanistically, FAK-Y397F ECs exhibit increased Tie2 expression, reduced Vegfr2 expression, decreased β1 integrin activation, and disrupted downstream FAK/Src/PI3K(p55)/Akt signaling. In contrast, FAK-Y861F ECs showed decreased Vegfr2 and Tie2 expression with an enhancement in β1 integrin activation. This corresponds with a decrease in Vegfa-stimulated response, but an increase in Vegfa+Ang2- or conditioned medium from tumor cell-stimulated cellular/angiogenic responses, mimicking responses in end-stage tumors with elevated Ang2 levels. Mechanistically, FAK-Y861F, but not FAK-Y397F ECs showed enhanced p190RhoGEF/P130Cas-dependent signaling that is required for the elevated responses to Vegfa+Ang2. This study establishes the differential requirements of EC-FAK-Y397 and EC-FAK-Y861 phosphorylation in the regulation of EC signaling and tumor angiogenesis in vivo. SIGNIFICANCE: Distinct motifs of the focal adhesion kinase differentially regulate tumor blood vessel formation and remodeling.

The rhenium(I)-diselenoether anticancer drug targets ROS, TGF-β1, VEGF-A, and IGF-1 in an in vitro experimental model of triple-negative breast cancers.

The rhenium(I)-diselenoether complex (Re-diSe) is a rhenium tricarbonyl-based drug chelated by a diselenoether ligand. In this work, we compared its inhibitory effects on the hormone-independent MDA-MB231cancer line and other different cancer cell lines after an exposure time of 72h by MTT assays. The sensitivity of MDA-MB231 was in the same range than the hormone-dependent MCF-7 breast cancer, the PC-3 prostate and HT-29 colon cancer cells, while the A549 lung and the HeLa uterine cancer cells were less sensitive. We compared the inhibitory effects of Re-diSe and of its diselenide ligand (di-Se) on MDA-MB231 and a normal HEK-293 human embryonic cell line, after 72h and 120h of exposure. The cytotoxicity was also studied by flow cytometry using ethidium bromide assays, as well as the effects on the ROS production by DFCA-test, while the levels of TGF-β1, VEGF-A, IGF-1 were addressed by ELISA tests. The dose required to inhibit 50% of the proliferation (IC50) of MDA-MB231 breast cancer cells decreased with the time of exposure to 120h, while the free ligand (di-Se) was found poorly active, demonstrating the important role of Re in this Re-diSe combination. The cytotoxic effects of Re-diSe were highly selective for cancer cells, with a significant increase of the number of dead cancer cells at 5μM for an exposure time of 120h, while normal cells were not affected. A remarkable and significant decrease of the production of ROS together with a decrease of VEGF-A, TGF-β1, and IGF-1 by the cancer cells were also observed when cancer cells were exposed to Re-diSe.

Abstract 4400: MicroRNA loaded circular dumbbell RNAs, a novel antiangiogenic approach

Abstract Angiogenesis is a tightly regulated biological process where new blood vessels are formed from pre-existing blood vessels. This process is also critical in diseases such as cancer; therefore, angiogenesis has been actively explored as a drug target for cancer therapy. Many patients treated with endogenous angiogenesis inhibitors show increased survival; however, eventual resistance to therapies is associated with disease progression and death. The future of effective anti-angiogenic therapy lies in the intelligent combination of multiple targeting agents with novel modes of delivery to maximize therapeutic effects. We therefore propose a novel and ‘out of the box' approach that utilizes dumbbell-RNA (dbRNA) to target pathological angiogenesis by simultaneously targeting multiple molecules and processes that contribute to angiogenesis. Dumbbell RNAs are a stable and safe alternative to viral, non-viral, and naked plasmid-based gene-transfer systems. They integrate the advantages of durable expression that is achievable with integrating viral vectors and short-term effects triggered by naked RNA. In this study, we have successfully constructed a plasmid expressing miR-34a dbRNA using the permuted intron-exon (PIE) method. We have also modified and optimized a cost effective protocol to purifiy dbRNA from bacterial culture with high purity. To test the efficacy of miR-34a dbRNA, we used the pancreatic cancer cell line MIA-PaCa2. When transfected into MIA-PaCa2 cell line we observed increased expression of miR-34a. Further, miR-34a overexpression caused a 3- fold and 2.5-fold decrease in VEGFA and VEGFB RNA expression respectively. Functional validation on the effect of miR-34a on angiogenesis was performed on Human umbilical vein endothelial cells (HUVEC) using the tube formation assay, where miR-34a dbRNA showed significant decrease in tube formation when compared to cells treated with scrambled dbRNA. We will further validate these results in vivo using Zebrafish angiogenesis model. In conclusion, our study demonstrate for the first time a novel approach to block angiogenesis using miR-34a loaded dbRNA. Our data also show that this approach may be used in targeting multiple molecules/pathways, and when used in combination with existing therapies can improve the efficacy of current treatment modalities. Citation Format: Manu Gnanamony, Sajani S. lakka, Jaime Libes, Julian Lin, Pushpa A. Joseph, Christopher S. Gondi. MicroRNA loaded circular dumbbell RNAs, a novel antiangiogenic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4400.

Endurance Training Attenuates Angiogenesis Following Breast Cancer by Regulation of MiR-126 and MiR-296 in Breast Cancer Bearing Mice

Background: In recent decades, various studies have shown a relationship between exercise and decreased mortality in patients with breast cancer. However, potentially involved molecular mechanisms are yet to be detected. Objectives: The goal of this study was to investigate the effect of endurance training on gene expression of miR-126, miR-296, HGS and protein expression of VEGF-A, in the tumor tissues of breast cancer bearing mice. Methods: Twelve laboratory female BALB/c mice, weighing 19 ± 1.05 g, were randomly divided into two groups of 6 each; that is, endurance training and control, after inducing cancer by subcutaneous injection with MC4-L2 into their right sides. Each session of endurance training involves 60-min of running on a treadmill at an intensity of 60% - 65% VO2max, five days a week for 10 weeks. Twenty-four hours after the last exercise session, the mice are sacrificed and the genes expression is measured. Results: Endurance training caused a significant decrease in miR-296 (P < 0.05) and an increase in the HGS (P < 0.05) and miR-126 (P < 0.05) at mRNA level, and a decrease in VEGF-A (P < 0.05) at protein level in tumor tissue compared to the controls. Decrease in proangiogenic factors and induction of anti-angiogenic factors are associated with decreased growth of tumor volume (P < 0.05) in the endurance training group compared to the control group. Conclusions: Data from this study demonstrate that endurance training can significantly decreases tumor growth in BALB/c mice. Endurance training mediates its effects through reduction of level of the vascular endothelial growth factors (VEGF) and VEGF receptors (VEGFR) in tumor tissue.

Antitumor immunity induced by VE-cadherin modified DC vaccine.

Dendritic cells (DCs) are the most potent antigen-presenting cells. A strong interest has been developed in DC vaccines for cancer immunotherapy. Besides, angiogenesis is essential for tumor growth. VE-cadherin has a crucial function in various aspects of vascular biological functions. Here, we produced the full VE-cadherin gene modified DC vaccine (DC-VEC). Its antitumor immunity and chief mechanism driving antitumor effect was evaluated. Analyses were performed including test of antitumor antibody, CTL-mediated cytotoxicity experiment, vascular density, evaluation of the variation of cells and cytokines in immunoregulation. Its damage to the major organs was also evaluated. DC-VEC vaccine resulted in retarded tumor progression and prolonged survival in mice. In DC-VEC group, large amount of immunoglobulin was generated, T cells exhibited greater cytotoxicity against VE-cadherin, and tumor angiogenesis was suppressed. Besides, a decrease of VEGF-A and TGF-β1, and an increase of IL-4 and IFN-γ were observed. CD4+ and CD8+ T cells were higher, with increased IFN-γ secretion. The percentage of myeloid-derived suppressor cells and regulatory T cells decreased mildly. Also, it had no pathologic changes in major organs. DC-VEC vaccine represents a promising antitumor immunotherapy. The main mechanism is associated with its anti-angiogenesis and immunoregulation response.

Bevacizumab Injection in Patients with Neovascular Age-Related Macular Degeneration Increases Angiogenic Biomarkers

To evaluate the expression of 19 angiogenic biomarkers in the aqueous humor before and after intravitreal bevacizumab injection (IVB) in eyes with neovascular age-related macular degeneration (AMD). Prospective, noncomparative, interventional case series. Twenty-three eyes of 23 treatment-naïve patients with choroidal neovascularization (CNV) secondary to neovascular AMD. Eyes were diagnosed with CNV secondary to neovascular AMD and were treated with 3 monthly IVBs. Aqueous humor samples were obtained by anterior chamber paracentesis at baseline and immediately before each intravitreal bevacizumab injection. Aqueous humor levels of 19 angiogenic biomarkers (angiopoietin 2, bone morphogenetic protein 9 [BMP-9], epidermal growth factor [EGF], endoglin, endothelin 1, fibroblast growth factor [FGF]-1 and FGF-2, follistatin, granulocyte colony-stimulating factor [GCSF], heparin-binding EGF-like growth factor [HB-EGF], hepatocyte growth factor [HGF], interleukin 8, leptin, placental growth factor [PLGF], vascular endothelial growth factor [VEGF]-A, VEGF-C, VEGF-D, and tissue inhibitor of metalloproteinases [TIMP]-1 and TIMP-2) were measured. Best-corrected visual acuity (BCVA), spectral-domain OCT parameters, and intraocular pressure also were evaluated. Baseline aqueous VEGF-A expression was elevated in all study eyes before treatment initiation. A statistically significant decrease of VEGF-A was observed at the 1- and 2-month follow-ups. A statistically significant increased concentration was observed in 7 biomarkers: VEGF-C, angiopoietin 2, endothelin 1, follistatin, HB-EGF, HGF, and interleukin 8. The other 11 study biomarker levels (VEGF-D, BMP-9, EGF, endoglin, FGF-1, FGF-2, GCSF, leptin, PLGF, TIMP-1, and TIMP-2) did not show any significant difference during follow-up. The BCVA statistically improved significantly at 2 months. Spectral-domain OCT parameters improved significantly at all follow-ups. Mean intraocular pressure values were not statistically different during the study period. Despite a decrease in VEGF-A, the aqueous levels of VEGF-C, angiopoietin 2, endothelin 1, follistatin, HB-EGF, HGF, and interleukin 8 increased significantly after intravitreal injection of bevacizumab. These upregulated angiogenic biomarkers may represent new therapeutic targets in exudative AMD.

Decrease of VEGF-A in myeloid cells attenuates glioma progression and prolongs survival in an experimental glioma model.

Glioblastomas are highly vascularized tumors with a prominent infiltration of macrophages/microglia whose role in promoting glioma growth, invasion, and angiogenesis has not been fully elucidated. The contribution of myeloid-derived vascular endothelial growth factor (VEGF) to glioma growth was analyzed in vivo in a syngeneic intracranial GL261 glioma model using a Cre/loxP system to knock out the expression of VEGF-A in CD11b + myeloid cells. Changes in angiogenesis-related gene expression profile were analyzed in mutant bone marrow-derived (BMD) macrophages in vitro. Furthermore, we studied the influence of macrophages on GL261 growth, invasiveness, and protein expression profile of angiogenic molecules as well as the paracrine effect of mutant macrophages on angiogenesis in vitro. Myeloid cell-restricted VEGF-A deficiency leads to a growth delay of intracranial tumors and prolonged survival. The tumor vasculature in mutant mice was more regular, with increased pericyte coverage. Expression analysis revealed significant downregulation of VEGF-A and slight upregulation of TGFβ-1 in BMD macrophages from mutant mice. Endothelial tube formation was significantly decreased by conditioned media from mutant macrophages. The expression of angiogenesis-related proteins in GL261 glioma cells in co-culture experiments either with wild-type or mutant macrophages remained unchanged, indicating that effects observed in vivo are due to myeloid-derived VEGF-A deficiency. Our results highlight the importance of VEGF derived from tumor-infiltrating myeloid cells for initiating vascularization in gliomas. The combination of antiangiogenic agents with myeloid cell-targeting strategies might provide a new therapeutic approach for glioblastoma patients.

Trastuzumab improves tumor perfusion and vascular delivery of cytotoxic therapy in a murine model of HER2+ breast cancer: preliminary results.

To employ in vivo imaging and histological techniques to identify and quantify vascular changes early in the course of treatment with trastuzumab in a murine model of HER2+ breast cancer. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to quantitatively characterize vessel perfusion/permeability (via the parameter K (trans) ) and the extravascular extracellular volume fraction (v e ) in the BT474 mouse model of HER2+ breast cancer (N = 20) at baseline, day one, and day four following trastuzumab treatment (10 mg/kg). Additional cohorts of mice were used to quantify proliferation (Ki67), microvessel density (CD31), pericyte coverage (α-SMA) by immunohistochemistry (N = 44), and to quantify human VEGF-A expression (N = 29) throughout the course of therapy. Longitudinal assessment of combination doxorubicin ± trastuzumab (N = 42) tested the hypothesis that prior treatment with trastuzumab will increase the efficacy of subsequent doxorubicin therapy. Compared to control tumors, trastuzumab-treated tumors exhibited a significant increase in K (trans) (P = 0.035) on day four, indicating increased perfusion and/or vessel permeability and a simultaneous significant increase in v e (P = 0.01), indicating increased cell death. Immunohistochemical and ELISA analyses revealed that by day four the trastuzumab-treated tumors had a significant increase in vessel maturation index (i.e., the ratio of α-SMA to CD31 staining) compared to controls (P < 0.001) and a significant decrease in VEGF-A (P = 0.03). Additionally, trastuzumab dosing prior to doxorubicin improved the overall effectiveness of the therapies (P < 0.001). This study identifies and validates improved perfusion characteristics following trastuzumab therapy, resulting in an improvement in trastuzumab-doxorubicin combination therapy in a murine model of HER2+ breast cancer. This data suggests properties of vessel maturation. In particular, the use of DCE-MRI, a clinically available imaging method, following treatment with trastuzumab may provide an opportunity to optimize the scheduling and improve delivery of subsequent cytotoxic therapy.

Abstract A02: Effect of curcumin on the tumor growth and angiogenesis of breast cancer

Abstract Background: Angiogenesis plays an important role in the pathogenesis of breast cancer. For growth, tumor requires the formation of new blood vessels, which are stimulated by angiogenic factors to initiate the proliferation of endothelial cells. The major contributors to angiogenesis are the vascular endothelial growth factor (VEGF) and its receptors, which promote cell proliferation, migration, permeability and survival. Controlling tumor-associated angiogenesis and metastasis are promising tactics in limiting cancer progression. Curcumin, a polyphenolic compound derived from the spice plant Curcuma longa, displays multiple actions on solid tumours including anti-angiogenic effects. In this study we evaluated the benefits of curcumin treatment on tumor growth and angiogenesis in breast cancer. Methods: MDA-MB-231 breast cancer cell line was cultured and cell viability was measured by MTT assay after incubation with different concentrations of curcumin (25 uM, 50 uM, 100 uM). We performed an in vivo study implanting cells in athymic nude mice. Mice were treated with 300 mg/kg of curcumin (n = 5) or vehicle (n = 8) daily, starting in the same day as tumor implantation and continued for 21 days. Tumors were measured with a digital caliper on day 7, 14 and 21 and the animals underwent SPECT scanning with Tc-99m tagged VEGF-c to detect in vivo angiogenesis (expression of VEGFR2/3). In addition, the markers of angiogenesis (VEGF-A, VEGF-C, VEGFR2, VEGFR3 and von Willebrand Factor (vWF)) and cell proliferation (Ki-67) were evaluated by immunohistochemistry in mammary tumor tissues. Results: Curcumin treatment in vitro was able to significantly decrease cell viability, after 4 hours of exposure, maintaining its efficacy at higher doses after 24 hours exposure (p&amp;lt;0.05). After 21 days of treatment, curcumin retards the breast tumor growth, showed by the slower tumoral development of the treated group compared to the control group (p&amp;lt;0.05). The mean tumor volume of control and treated animals were 282.00 ± 88.53 mm3 232.5± 53.2 mm3, respectively. Animals treated with curcumin showed lower activity of Tc-99-VEGF-C in the tumors compared to that of the animals that were treated with vehicle. The intensity of radioactivity in the control animals was 183.55 ± 20.92%, while the intensity of radioactivity in animals treated with curcumin was 134.9 ± 10.3%. Although there was difference in the radioactivity in the tumors between the two group, statistically significant difference was not achieved (p&amp;gt;0.05). However, decrease of VEGF-A, VEGF-C and VEGFR2 in curcumin treated mice was confirmed by immunohistochemistry (p&amp;lt;0.05). In addition, there was a decrease of micro-vessel density (vWF) and cell proliferation (Ki-67) in curcumin treated tumors (p&amp;lt;0.05). Conclusion: In summary, our results demonstrate that curcumin affect the viability of the breast cancer cells in vitro. In addition, in vivo study, curcumin is able to retards the breast tumor growth when compared to the control group. Both SPECT and histochemical analysis indicate decrease in angiogenesis following treatment with curcumin. Citation Format: Lívia Carvalho Ferreira, Ali Syed Arbab, Bruna Victorasso Jardim-Perassi, Thaiz Ferraz Borin, Naiane Nascimento Gonçalves, Ravi Sankara Varma Nadimpalli, Debora Aparecida Pires de Campos Zuccari. Effect of curcumin on the tumor growth and angiogenesis of breast cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A02. doi:10.1158/1538-7445.CHTME14-A02

Abstract 15674: Notch3/rbpjk Signaling Pathway in the Coronary Arteries is Involved in the Development of Heart Failure in Response to Hypertension

Background: Notch3, a receptor expressed in vascular smooth muscle cells (VSMC), plays a key role in the integrity of resistance arteries by controlling the maturation of VSMC through RBPJk, a canonical Notch transcriptional factor. We hypothesized that Notch3 signaling pathway in the VSMCs of the coronary arteries might play a key role in the cardiac adaptation to pressure-overload. Methods: To eliminate congenital defect biases, we used mice in which conditional tamoxifen- selective deletion of RBPJk in VSMC was induced at the age of 4 weeks (SMMHC-CRE ER T2, RBP-JK loxp/loxp). Seven weeks later, hypertension was induced in control (C) mice (n= 10) and those with RBPJk deletion (SM-RBPJ-KO, n = 13) by infusion of Angiotensin 2 (Ang2) (1μg/kg/min) using Alzet minipumps after surgery. Results: In response to Ang2, SM-RBPJ-KO and control mice showed similar hypertension (159 ±7 mmHg vs 150±10 mmHg). By day 8 after surgery, SM-RBPJ-KO mice only developed a severe heart failure with anarsaca. These mice showed a decrease in shortening fraction (-42%, p&lt;0.01), a left ventricular dilatation (+15%, p&lt;0.01), and a ventricular hypertrophy (+12%, p&lt;0.05) when compared to C + Ang2. As expected, C + Ang 2 hearts showed an increase in ANP, B-MHC and Gal-3 (+76%, +55%, +46%, p&lt;0.05 respectively vs C) mRNA expressions, whereas SM-RBPJ-KO hearts exhibited even higher induction of ANP (+48%, p&lt;0.01), Gal-3 (+74%, p&lt;0.01), CD68 (+55%, p&lt;0.05) and collagen-3 (+78%, p&lt;0.01) when compared to C + Ang2. This cardiac phenotype confirmed the severe heart failure. Interestingly, immuno-morphometric analysis showed an arteriolar rarefaction in SM-RBPJ-KO + Ang2 mice associated with a lack of angiogenic signaling pathway revealed by a decrease in VEGFa and angiopoietin 2 mRNAs (-50%, and -52%, p&lt;0.05, respectively vs. C + Ang2). Conclusion: We provide evidence that in adulthood, dysfunction of the Notch3/RBPJk signaling pathway in the coronary arteries contributes in the aggravation of heart failure in response to rapid increase in blood pressure.

The role of Iex-1 in the pathogenesis of venous neointimal hyperplasia associated with hemodialysis arteriovenous fistula.

Arteriovenous fistulas (AVFs) used for hemodialysis fail because of venous neointimal hyperplasia (VNH). There are 1,500,000 patients that have end stage renal disease worldwide and the majority requires hemodialysis. In the present study, the role of the intermediate early response gene X-1 (IEX-1), also known as IER-3 in the pathogenesis of VNH was evaluated. In human samples removed from failed AVF, there was a significant increase in IEX-1 expression localized to the adventitia. In Iex-1 −/− mice and wild type (WT) controls, chronic kidney disease was induced and an AVF placed 28 days later by connecting the carotid artery to jugular vein. The outflow vein was removed three days following the creation of the AVF and gene expression analysis demonstrated a significant decrease in vascular endothelial growth factor-A (Vegf-A) and monocyte chemoattractant protein-1 (Mcp-1) gene expression in Iex-1 −/− mice when compared to WT mice (P<0.05). At 28 days after AVF placement, histomorphometric and immune-histochemical analyses of the outflow vein demonstrated a significant decrease in neointimal hyperplasia with an increase in average lumen vessel area associated with a decrease in fibroblast, myofibroblast, and Ly6C staining. There was a decrease in proliferation (Ki-67) and an increase in the TUNEL staining in Iex-1 KO mice compared to WT. In addition, there was a decrease in Vegf-A, Mcp-1, and matrix metalloproteiniase-9 (Mmp-9) staining. Iex-1 expression was reduced in vivo and in vitro using nanoparticles coated with calcitriol, an inhibitor of Iex-1 that demonstrated that Iex-1 reduction results in decrease in Vegf-A. In aggregate, these results indicate that the absence of IEX-1 gene results in reduced VNH accompanied with a decrease in proliferation, reduced fibroblast, myofibroblast, and Ly6C staining accompanied with increased apoptosis mediated through a reduction in Vegf-A/Mcp-1 axis and Mmp-9. Adventitial delivery of nanoparticles coated with calcitriol reduced Iex-1 and VNH.

Regulation of ovarian angiogenesis and apoptosis by GnRH‐I analogs

An adequate vascular supply is important to provide endocrine and paracrine signals during follicular development. We evaluated the direct in vivo effects of both the GnRH-agonist Leuprolide acetate (LA) and the GnRH-antagonist Antide (Ant) on the expression of VEGF-A and ANPT-1 and their receptors in ovarian follicles from prepubertal eCG-treated rats. We also examined whether the changes observed in apoptosis by GnRH-I analogs have an effect on the caspase cascade. LA significantly decreased the levels of VEGF-A, its receptor Flk-1, and ANPT-1 when compared to controls, while the co-injection of Ant interfered with this effect. No changes were observed in the levels of Tie-2 after treatment with these analogs. When we measured the follicular content of caspase-3 protein, we observed that LA significantly increased the level of the active form. The co-injection of Ant interfered with this effect and Ant alone significantly decreased caspase-3 cleavage. IHC analyses corroborated these data. Notably, while LA increased caspase-3 activity levels, Ant decreased them when compared to controls. In follicles obtained from LA-treated rats, cleavage of PARP (a substrate of caspase-3) from the intact 113-kDa protein showed a significant enhancement in an 85-kDa fragment. The co-injection of Ant interfered with this effect. Ant alone significantly decreased PARP cleavage as compared to controls. We conclude that the decrease in VEGF-A, its receptor Flk-1/KDR, and ANPT-1 produced by the administration of GnRH-I agonist is one of the mechanisms involved in ovarian cell apoptosis. This suggests an intraovarian role of an endogenous GnRH-like peptide in gonadotropin-induced follicular development.

Fgf10 dosage is critical for the amplification of epithelial cell progenitors and for the formation of multiple mesenchymal lineages during lung development

The key role played by Fgf10 during early lung development is clearly illustrated in Fgf10 knockout mice, which exhibit lung agenesis. However, Fgf10 is continuously expressed throughout lung development suggesting extended as well as additional roles for FGF10 at later stages of lung organogenesis. We previously reported that the enhancer trap Mlcv1v-nLacZ-24 transgenic mouse strain functions as a reporter for Fgf10 expression and displays decreased endogenous Fgf10 expression. In this paper, we have generated an allelic series to determine the impact of Fgf10 dosage on lung development. We report that 80% of the newborn Fgf10 hypomorphic mice die within 24 h of birth due to respiratory failure. These mutant mouse lungs display severe hypoplasia, dilation of the distal airways and large hemorrhagic areas. Epithelial differentiation and proliferation studies indicate a specific decrease in TTF1 and SP-B expressing cells correlating with reduced epithelial cell proliferation and associated with a decrease in activation of the canonical Wnt signaling in the epithelium. Analysis of vascular development shows a reduction in PECAM expression at E14.5, which is associated with a simplification of the vascular tree at E18.5. We also show a decrease in α-SMA expression in the respiratory airway suggesting defective smooth muscle cell formation. At the molecular level, these defects are associated with decrease in Vegfa and Pdgfa expression likely resulting from the decrease of the epithelial/mesenchymal ratio in the Fgf10 hypomorphic lungs. Thus, our results indicate that FGF10 plays a pivotal role in maintaining epithelial progenitor cell proliferation as well as coordinating alveolar smooth muscle cell formation and vascular development.