Inhibition Of Vascular Endothelial Growth Factor Expression Research Articles

FBXW7 inhibits invasion, migration and angiogenesis in ovarian cancer cells by suppressing VEGF expression through inactivation of β-catenin signaling.

F-box and WD repeat domain containing 7 (FBXW7) is a tumor suppressor gene frequently inactivated in several human malignancies. The present study aimed to investigate the role of FBXW7 in the invasion, migration and angiogenesis of ovarian cancer (OC) cells, and to identify its potential molecular mechanisms. First, the expression levels of FBXW7 and vascular endothelial growth factor (VEGF) were detected in several human OC cell lines using western blotting. Subsequently, FBXW7 was overexpressed to determine VEGF expression in SKOV3 cells. Transwell, wound healing and tube formation assays were performed following transfection with FBXW7 and VEGF overexpression plasmids to assess invasion, migration and angiogenesis in SKOV3 cells, respectively. Western blot analysis was performed to detect the expression levels of epithelial-to-mesenchymal transition and angiogenesis-associated proteins. In addition, the expression levels of β-catenin and c-Myc were assessed, and lithium chloride (LiCl), an agonist of β-catenin signaling, was used to elucidate the molecular mechanisms by which FBXW7 mediates its antitumor activity in OC. The results demonstrated that FBXW7 expression was markedly downregulated, whilst VEGF expression was markedly upregulated in OC cell lines compared with that in normal ovarian cells. Overexpression of FBXW7 significantly decreased VEGF expression in SKOV3 cells. Notably, overexpression of VEGF reversed the inhibitory effects of FBXW7 overexpression on the invasion, migration and angiogenesis of OC cells, accompanied by upregulated expression levels of N-cadherin, slug, CD31, VEGF receptor 1 (VEGFR1) and VEGFR2, and downregulated expression levels of E-cadherin. Furthermore, overexpression of FBXW7 markedly suppressed β-catenin and c-Myc expression, whereas the decreased expression levels of VEGF, VEGFR1 and VEGFR2 following overexpression of FBXW7 were increased after treatment of SKOV3 cells with LiCl. Overall, the results of the present study suggested that FBXW7 inhibited invasion, migration and angiogenesis of OC cells by suppressing VEGF expression through inactivation of β-catenin signaling. Thus, FBXW7 may be used as a novel therapeutic target for the treatment of OC.

Effects of Botulinum Toxin Type A on Microvessels in Hypertrophic Scar Models on Rabbit Ears.

Background Although Botulinum Toxin Type A (BTXA) has been applied to scar prevention and treatment, the mechanisms still require further exploration. Objective To investigate the effects of BTXA on microvessels in the hypertrophic scar models on rabbit ears. Methods Eight big-eared New Zealand rabbits (males or females) were selected to establish scar models. One ear of each rabbit (4 models in each ear) was selected randomly to be injected with BTXA immediately after modeling and included in the treated group, while the opposite ear was untreated and included in the control group. The growth of scars in each group was observed and recorded, and 4 rabbits were sacrificed on days 30 and 45 after modeling. Then, scar height was measured by hematoxylin-eosin (HE) staining, vascular endothelial growth factor (VEGF) expression was detected by immunohistochemical (IHC) testing, and microvessel density (MVD) was calculated based on CD34 (human hematopoietic progenitor cell antigen). Results The wounds in each group were well healed and free from infection or necrosis. On days 30 and 45, the scar height, MVD value, and VEGF expression in the treated group were lower than those in the control group (P < 0.05). For the treated group, the above indicators on day 45 were lower than on day 30 (P > 0.05). Besides, there was a positive correlation between the MVD value and the VEGF expression in the treated group (P < 0.05). Conclusion The injection of BTXA immediately after modeling inhibits VEGF expression and reduces angiogenesis, thereby inhibiting hypertrophic scar formation.

“Real life” polycyclic aromatic hydrocarbon (PAH) mixtures modulate hCG, hPL and hPLGF levels and disrupt the physiological ratio of MMP-2 to MMP-9 and VEGF expression in human placenta cell lines

Using JEG-3 and BeWo cells, we examined the effect of “real life” mixtures of polycyclic aromatic hydrocarbons (PAHs), at doses reported in maternal blood (Mix I) and in placental tissue (Mix II), on human chorionic gonadotropin (hCG), placental lactogen (hPL) and placental growth factor (hPLGF) secretion, protein expression and immunolocalization. Additionally, the action of PAH mixtures on basal and hormone-stimulated matrix metalloproteinase-2 (MMP-2), MMP-9 and vascular endothelial growth factor (VEGF) protein expression was evaluated. Under basal conditions, the PAH mixtures increased hCG and decreased hPLGF levels in both cell lines, while hPL expression was stimulated in JEG-3 and inhibited in BeWo. There was no effect on the MMP-2/MMP-9 ratio or VEGF expression. In hormone-stimulated cells, PAH mixtures changed the MMP-2/MMP-9 ratio in JEG-3 cells in favor of MMP-9, while in BeWo MMP-2 was favored. The effect on VEGF expression was cell specific and dependent on the mixture. In hCG-treated cells, only Mix II inhibited VEGF expression in JEG-3 cells. Neither PAH mixtures affected this protein in BeWo cells. In hPL-treated cells, Mix I had a stimulatory effect in JEG-3 cells, while Mix II exerted an inhibitory effect in BeWo cells. In hPLGF-treated cells, Mix II decreased in JEG-3 cells, but in BeWo cells, both mixtures increased VEGF expression. Considering that the evaluated protein hormones play crucial roles in angiogenesis and neovascularization in the placenta, “real life” PAH mixtures by disrupting protein hormones levels, the MMP-2/MMP-9 ratio and VEGF expression can lead to insufficiency and many pregnancy-related disorders.

Growth differentiation factor 9 inhibits vascular endothelial growth factor expression in human granulosa cells

In aortic endothelial cells, the TGFβ signaling pathway is involved in the regulation of vascular endothelial growth factor (VEGF), which encodes a potent angiogenic factor crucial for the development of ovarian hyperstimulation syndrome. Growth differentiation factor 9 (GDF9) is a member of the TGFβ family and its effect on VEGF expression in human granulosa cells is unknown. In this study, human granulosa cells were collected from patients during the course of oocyte retrieval for in vitro fertilization and were cultured in vitro. After the first 48 h of culture, cells were treated with GDF9 with or without SB431542 (an ALK5 inhibitor) at various doses. The medium was then collected to determine the concentration of VEGF by ELISA. Cellular RNA was collected and extracted for quantification by real-time quantitative fluorescence PCR. Our study showed that GDF9 suppressed VEGF release from human granulosa cells in a dose-dependent manner and also downregulated VEGF mRNA levels in these cells. Furthermore, SB431542 antagonized the suppression of VEGF mRNA by GDF9 and diminished the inhibitory effect of GDF9 on VEGF release by human granulosa cells. Our results indicated that GDF9 can inhibit VEGF expression in human granulosa cells and ALK5 might mediate this process.

ADAMTS-1 inhibits angiogenesis via the PI3K/Akt-eNOS-VEGF pathway in lung cancer cells.

BackgroundADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin repeats-1) is a recently characterized protein containing a metalloproteinase domain, a disintegrin-like domain and a thrombospondin type 1 motif, which is involved in angiogenesis. However, the roles of ADAMTS-1 in angiogenesis of lung cancer (LC) remain unclear.MethodsThe mRNA expression of ADAMTS-1 and VEGF was examined by qRT-PCR. Western blots were used to detect the protein expression of ADAMTS-1 and vascular endothelial growth factor (VEGF) in A549 cells and to analyse the cellular effect of a PI3K/Akt activator and an endothelial nitric oxide synthase (eNOS) activator. ADAMTS-1 and VEGF contents in cell culture supernatants were measured by ELISA. Cell viability, cell cycle, migration, and angiogenesis of HUVECs were evaluated by MTT assay, flow cytometry, scratch assay and tube formation assay, respectively.ResultsOur data revealed that the expression of ADAMTS-1 was downregulated, while the expression of VEGF was upregulated in A549 cells. Decreased ADAMTS-1 content was also detected in A549 cell culture supernatant. Overexpression of ADAMTS-1 inhibited VEGF expression and A549 cell proliferation. Moreover, ADAMTS-1 overexpression repressed proliferation, migration and angiogenesis of HUVECs. Mechanistically, ADAMTS-1 suppressed the expression of VEGF in HUVECs by inhibiting PI3K/Akt-eNOS, while a PI3K activator and an eNOS activator each partly reversed the expression of VEGF. In addition, activation of the PI3K/Akt pathway or VEGF overexpression reversed the inhibitory effect of ADAMTS-1 overexpression on HUVECs angiogenesis.ConclusionsThese results indicated that ADAMTS-1 inhibited angiogenesis of LC cells via regulation of the PI3K/Akt-eNOS/VEGF axis, which shed light on LC pathogenesis and provided potential targets for LC therapy.

Knockdown of TUG1 by shRNA inhibited renal cell carcinoma formation by miR-299–3p/VEGF axis in vitro and in vivo

Renal cell carcinoma (RCC) is one of the top ten deadly malignancies in the world. The long non-coding RNA taurine up-regulated gene 1 (TUG1) is a transcript that is up-regulated by taurine. There is ample evidence that TUG1 plays a crucial role in the progression of various cancers. This study aimed to investigate the role of TUG1 in RCC and its underlying molecular mechanisms. In the current study, knockdown of TUG1 by shRNA (sh-TUG1) significantly inhibited proliferation, invasion, migration and EMT processes of ACHN cells and OS-RC-2 cells, and induced apoptosis. Besides, bioinformatics analysis revealed that miR-299–3p is a target of TUG1. TUG1 overexpression (LV-TUG1) significantly inhibited the expression of miR-299–3p, whereas sh-TUG1 showed the opposite effect. Dual luciferase reporter assay further confirmed the targeting relationship between TUG1 and miR-299–3p. In addition, vascular endothelial growth factor (VEGFA) is a target of miR-299–3p. Knockdown of VEGFA (si-VEGFA) significantly inhibited the proliferation and motility of ACHN cells, and induced apoptosis. RT-qPCR results showed that sh-TUG1 similarly inhibited VEGFA expression. Further functional analysis indicated that sh-TUG1 inhibited tumorigenesis by down-regulating VEGFA levels. However, LV-TUG1 showed the opposite effects. Furthermore, animal experiments have shown that sh-TUG1 inhibited tumor growth and metastasis and induces apoptosis in vivo. These results indicate that sh-TUG1 inhibited renal cell carcinoma formation by miR-299–3p/VEGF axis in vitro and in vivo. Taken together, all of these results reveal a novel mechanism of TUG1 in RCC tumorigenesis, suggesting that targeted drugs for TUG1 provides a new direction for the treatment of RCC.

BNIP3-mediated Autophagy Induced Inflammatory Response and Inhibited VEGF Expression in Cultured Retinal Pigment Epithelium Cells Under Hypoxia.

Bcl-2/adenovirus E1B-19kDa-interacting protein (BNIP3), an important target of hypoxia-inducible factors-1 alpha (HIF-1α), was reported to be overexpressed under hypoxic condition. Our previous study demonstrated the protective effect on detached retina by BNIP3-mediated autophagy. The study investigated the role of BNIP3-mediated autophagy in retinal pigment epithelial (RPE) cells under hypoxia, and observed the relationship between BNIP3, vascular endothelial growth factor (VEGF) and inflammatory response in hypoxic RPE cells. BNIP3 knock down in retinal pigment epithelial cells was performed by small interfering RNA (siRNA) technology in ARPE-19 cells, a human RPE cell line. Both control and BNIP3-knockdown ARPE-19 cells were then subjected to a hypoxic challenge using cobalt (II) chloride (CoCl2). The expression of autophagy-related genes, VEGF and inflammatory factors (IL-18, IL-8, MMP-2, MMP-9, NLRP3, TNF-α) in RPE cells was examined using quantitative Polymerase Chain Reaction (qPCR). The protein levels of HIF-1α, BNIP3, the maker proteins (ATG5, LC3,p62, Beclin-1) of autophagy and the component proteins (p-p70S6K, p70S6K, mTOR, p-mTOR) of the mTORC1 pathway were analyzed by Western blot. BNIP3 subcellualr localization was detected by immunofluorescence. Cell viability was measured with Cell Counting kit-8. Cell apoptosis was examined by TUNEL staining and caspase-3 activity assay. The expression levels of BNIP3, HIF-1α and marker genes of autophagy were upregulated in ARPE-19 cells in response to hypoxia. Importantly, hypoxia-induced autophagy was mediated by the mTORC1 pathway, and was blocked upon BNIP3 knockdown. Additionally, hypoxia reduced cell viability, which was relieved by an mTORC1 inhibitor. Also, autophagy protected ARPE-19 cells from CoCl2-induced cell apoptosis. Moreover, inhibition of autophagy upregulated the expression of VEGF and IL-18, and downregulated the expression of other inflammatory factors in the hypoxic ARPE-19 cells. BNIP3-mediated autophagy under hypoxia is involved in regulating inflammatory response and VEGF expression, which consequently affects the cell viability of RPE cells.

Nrf2 Activator RS9 Suppresses Pathological Ocular Angiogenesis and Hyperpermeability.

Ocular angiogenesis, including retinopathy of prematurity, diabetic retinopathy, and exudative age-related macular degeneration, are closely related to oxidative stress. Many reports have shown that the cellular protective mechanism against oxidative stress and inflammatory response has nuclear factor-erythroid 2-related factor-2 (Nrf2) activity. The aim of this study was to investigate the effectiveness and mechanism of Nrf2 activation in treating the ocular diseases with abnormal vessels. The effects of Nrf2 activators, bardoxolone methyl (BARD) and RS9, were evaluated against vascular endothelial growth factor (VEGF)-induced cell migration in human retinal microvascular endothelial cells (HRMECs). We measured the expression of the Nrf2 target genes, Ho-1 and Nqo-1 mRNA, in mouse retinas after a single injection of BARD and RS9. The effects and mechanisms of RS9 against retinal angiogenesis were evaluated using an oxygen-induced retinopathy (OIR) model in mice. Moreover, the effect of RS9 against choroidal neovascularization (CNV) was evaluated in a laser-induced CNV monkey model. Both BARD and RS9 decreased VEGF-induced cell migration, and significantly increased Ho-1 mRNA expression; however, only RS9 significantly increased Nqo-1 mRNA. RS9 decreased retinal neovascularization through suppressing VEGF expression and increasing Nrf2, HO-1, platelet-derived growth factor receptor (PDGFR)-β, and tight junction proteins in OIR murine retinas. Furthermore, RS9 showed a tendency toward decreasing CNV lesions, and improved vascular leakage in a CNV monkey model. These data indicate that a Nrf2 activator might be a candidate for treatment of ocular diseases characterized by pathophysiological angiogenesis and hyperpermeability.

Litchi (Litchi chinensis Sonn.) flower proanthocyanidin fraction exhibited protective efficacy to suppress nickel-induced expression for vascular endothelial growth factor in HepG2 cells.

The protective efficacy of litchi (Litchi chinensis Sonn.) flower proanthocyanidin fraction (LFPF) composed of (-)-epicatechin and proanthocyanidin A2 against vascular endothelial growth factor (VEGF) generation induced by nickel (Ni) in hepatocellular carcinoma (Hep G2) cells was studied. VEGF is an angiogenic inducer, which promotes tumor angiogenesis, leading to rapid tumor growth and metastasis. VEGF could be substantially induced in the Ni-mediated Hep G2 cells. Through LFPF treatment, the Ni-induced VEGF generation could be suppressed significantly. The inhibition of HIF-1α expression by blocking phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathways, and the suppression of Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT 3), and Raf-1 proto-oncogene, serine/threonine kinase (RAF1)/mitogen-activated protein kinase (MEK1/2)/extracellular-signal-regulated kinase (ERK1/2) pathways are important molecular mechanisms for the LFPF action. LFPF should probably reduce the risk of liver cancer in Ni-contaminated environments by inhibiting VEGF expression. PRACTICAL APPLICATIONS: LFPF mainly contained (-)-epicatechin and proanthocyanidin A2. Our results demonstrated that LFPF considerably suppressed the Ni-induced VEGF expression through inhibition of JAK2/STAT 3 and RAF1/MEK1/2/ERK1/2 pathways and prohibited HIF-1α expression through blocking PI3K/AKT/mTOR pathway. Litchi flowers might have the potential to diminish the liver cancer risk in a Ni-contaminated environment through suitable treatment.

Ablation of aryl hydrocarbon receptor promotes angiotensin II-induced cardiac fibrosis through enhanced c-Jun/HIF-1α signaling.

Aryl hydrocarbon receptor (AHR) is a transcription factor that binds to DNA as a heterodimer with the AHR nuclear translocator (ARNT) after interaction with ligands, such as polycyclic and halogenated aromatic hydrocarbons and other xenobiotics. The endogenous ligands and functions of AHR have been the subject of many investigations. In the present study, the potential role of AHR signaling in the development of left ventricular hypertrophy and cardiac fibrosis by angiotensin II (Ang II) infusion was investigated in mice lacking the AHR gene (Ahr-/-). We also assessed the hypothesis that fenofibrate, a peroxisome proliferator-activated receptor-α (PPARα) activator, reduces cardiac fibrosis through the c-Jun signaling. Male Ahr-/- and age-matched wild-type mice (n = 8 per group) were infused with Ang II at 100ng/kg/min daily for 2weeks. Treatment with Ang II increased systolic blood pressure to comparable levels in Ahr-/- and wild-type mice. However, Ahr-/- mice developed severe cardiac fibrosis after Ang II infusion compared with wild-type mice. Ang II infusion also significantly increased the expression of endothelin in the left ventricles of Ahr-/- mice, but not in wild-type mice, and significantly increased the c-Jun signaling in Ahr-/- mice. Ang II infusion also significantly enhanced the expression of hypoxia-inducible factor-1α (HIF-1α) and the downstream target vascular endothelial growth factor (VEGF) in the left ventricles of Ahr-/- mice. These results suggested pathogenic roles for the AHR signaling pathway in the development of cardiac fibrosis. Treatment with fenofibrate reduced cardiac fibrosis and abrogated the effects of Ang II on the expression of endothelin, HIF-1α, and VEGF. The inhibitory effect of fenofibrate on cardiac fibrosis was mediated by suppression of VEGF expression through modulation of c-Jun/HIF-1α signaling.

β‑elemene inhibits oxygen‑induced retinal neovascularization via promoting miR‑27a and reducing VEGF expression.

The present study aimed to investigate the significant role of β-elemene in mouse models of oxygen-induced retinopathy (OIR). C57BL/6J neonatal mice were used to establish OIR models. They were divided into four groups: Normoxia, OIR, OIR control and OIR-treated. Mice in the OIR group were exposed to 75±5% oxygen for 5 days and returned to a normal oxygen environment on postnatal day 12 (P12). The OIR treated group was intravitreally injected with 1 µl β-elemene on P12 and subsequently returned to a normal oxygen environment for 5 days (P12-P17). Retinas were obtained on P17. Retinal neovascularization (RNV) was detected using adenosine diphosphatase staining and analyzed by counting the nuclei of neovascular endothelial cells. Vascular endothelial growth factor (VEGF) expression was determined by reverse transcription-quantitative polymerase chain reaction, immunohistochemistry and western blot analysis. MicroRNA (miRNA/miR) microarrays were used to screen out differentially expressed miRNAs between the OIR and β-elemene-treated groups. Binding the 3′-untranslated region (UTR) of VEGF and miR-27a was confirmed using luciferase assays. It was found that high oxygen concentrations accelerated RNV and increased the number of preretinal neovascular cells; β-elemene treatment reduced these effects. VEGF mRNA and protein expression was higher in the OIR and OIR control groups, compared with the normoxia and OIR-treated groups. Further, it was shown that miR-22, miR-181a-1, miR-335-5p, miR-669n, miR-190b, miR-27a and miR-93 were upregulated in the OIR-treated group, and downregulated in the OIR group. The prediction websites TargetScan and miRanda revealed that VEGF contained a potential miR-27a binding site in its 3′-untranslated region (UTR). Luciferase assays demonstrated that miR-27a directly bound to the 3′-UTR of VEGF. In vitro experiments demonstrated that miR-27a inhibited VEGF expression. In addition, β-elemene treatment upregulate miR-27a expression in vivo and in vitro. When miR-27a expression was depleted by miR-27a inhibitor, the protective effect of β-elemene on RNV was eliminated. The present study demonstrated that β-elemene reduced RNV in mouse OIR models via miR-27a upregulation, leading to reduced VEGF expression. This finding may contribute to the development of novel therapeutic strategies for human retinopathy.

Antiangiogenesis activity test of tin leaf (Ficus carica L.) on the number of blood vessels and VEGF expression of chorioallantoic membrane of embryonated chicken eggs

Abstract This research has been conducted on the antiangiogenesis activity test of the tin leaf (Ficus carica L.) on the number of blood vessels and vascular endothelial growth factor (VEGF) expression of chorioallantoic membrane (CAM) of embryonated chicken eggs. The general objective of this study is the use of natural products from the F. carica L. as chemopreventive in cancer through the mechanism of inhibition of angiogenesis. Inhibition of angiogenesis, supply of nutrients and oxygen to cancer cells will also be inhibited, so that indirectly cancer cells will be stunted. This study used 25 embryonated chicken eggs that divided into 5 treatments and each treatment has 5 repetitions. This treatment includes placebo negative controls, celecoxib-positive controls, and 3 treatment groups were given a water extract of F. carica L. which was divided into several doses of 75, 90, and 110 μg. The result found a significant difference (P &lt; .05) in the macroscopic observation of the number of blood vessels in the CAM loaded with the paper dish. Observation of VEGF expression in vascular endothelial cells in the CAM also had a significant difference (P &lt; .05). The optimal dose of water extract of F. carica L. of 90 μg has been able to inhibit the formation of new blood vessels by 65.51% and reduce VEGF expression by 45% in the chorioallantoic membrane. Thus, it can be concluded that the administration of water extract of F. carica L. can inhibit the formation of new blood vessels and inhibit VEGF expression.

Aflibercept Nanoformulation Inhibits VEGF Expression in Ocular In Vitro Model: A Preliminary Report.

Age-related macular degeneration (AMD) is one of the leading causes of blindness in the United States, affecting approximately 11 million patients. AMD is caused primarily by an upregulation of vascular endothelial growth factor (VEGF). In recent years, aflibercept injections have been used to combat VEGF. However, this treatment requires frequent intravitreal injections, leading to low patient compliance and several adverse side effects including scarring, increased intraocular pressure, and retinal detachment. Polymeric nanoparticles have demonstrated the ability to deliver a sustained release of drug, thereby reducing the necessary injection frequency. Aflibercept (AFL) was encapsulated in poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) via double emulsion diffusion. Scanning electron microscopy showed the NPs were spherical and dynamic light scattering demonstrated that they were uniformly distributed (PDI < 1). The encapsulation efficiency and drug loading were 75.76% and 7.76% respectively. In vitro release studies showed a sustained release of drug; 75% of drug was released by the NPs in seven days compared to the full payload released in 24 h by the AFL solution. Future ocular in vivo studies are needed to confirm the biological effects of the NPs. Preliminary studies of the proposed aflibercept NPs demonstrated high encapsulation efficiency, a sustained drug release profile, and ideal physical characteristics for AMD treatment. This drug delivery system is an excellent candidate for further characterization using an ocular neovascularization in vivo model.

Propranolol inhibits proliferation and invasion of hemangioma-derived endothelial cells by suppressing the DLL4/Notch1/Akt pathway

Infantile hemangioma (IH) is one of the most common benign vascular tumors of infancy. Propranolol has been recently introduced for the treatment of IH. However, the mechanism of protective effect has not been fully understood. In this study, hemangioma-derived endothelial cells (HemECs) were isolated and treated with propranolol. The cell viability was measured by MTT assay, and the cell cycle arrest was detected using flow cytometry. Cell invasion was determined using transwell assay. The expressions of matrix metalloproteinase (MMP)-2, MMP-9, Delta-like 4 (DLL4), Notch1, Akt, p-Akt, and vascular endothelial growth factor (VEGF) were detected using western blot. HemECs were incubated with recombinant human DLL4 (rhDLL4) to investigate the role of DLL4/Notch1 in the effect of propranolol. The results showed that propranolol inhibited cell viability of HemECs in a time-dependent manner. Propranolol suppressed cell proliferation of HemECs by arresting cell progression at G0/G1 phase. Propranolol inhibited the invasion ability of HemECs and reduced the expression levels of MMP-2 and MMP-9 in HemECs. Besides, propranolol treatment blocked the DLL4/Notch1 and Akt signaling and inhibited VEGF expression in HemECs. Treatment with rhDLL4 activated the Akt signaling and attenuated the effect of propranolol on HemECs. Our data indicated that propranolol inhibited the cell proliferation and invasion of HemECs. The effect was possibly involved in the DLL4/Notch1/Akt signaling pathway.

Effects of HIF-1α on diabetic retinopathy angiogenesis and VEGF expression.

To investigate the effect of hypoxia inducing factor (HIF)-1α on the expression of vascular endothelial growth factor (VEGF) and angiogenesis in diabetic retinopathy. 8-week healthy SD rats were used for the experiments. Under systemic anesthesia condition, control rats received a saline injection into the left ocular body (control A group), and 2 μl antisense oligonucleotides (ASODN) (10 μmol/L) into right eye (control B group). Model rats received a saline injection into the left eye (model A group), and 2 μl ASODN (10 μmol/L) into the right eye (model B group). Rats received an intraocular injection of HIF-1α ASODN for 2, 4, and 6 weeks (A1, A2, A3, B1, B2, B3, respectively). Retinal vessel development was observed by ADP staining. Vascular endothelial cells penetrating retinal inner membrane were counted. Immunohistochemistry was used to detect expressions of VEGF and HIF-1α proteins in the retina. Prominent angiogenesis and hyperplasia were found in model A group. Relatively fewer newly formed vessels were shown in model group B. However, no significant change of retinal vascular morphology was presented in control group. Of note, the vascular endothelial cell counts, VEGF and HIF-1α contents were significantly increased in model group (p < 0.05). After treatment with HIF-1α ASODN, lower endothelial cell counts was found in model B group (p < 0.05 comparing to model A). VEGF expression in model B group was significantly decreased, among which, model B3 was observed with lower cell counts than model B1 or B2 (p < 0.05 comparing to model A). Injection of HIF-1α ASODN significantly suppressed HIF-1α level in model B in a time-dependent manner. Retinal angiogenesis is closely related with increasing level of HIF-1α. Inhibition of HIF-1α suppressed VEGF expression and deterred angiogenesis in a time-dependent manner. This provided novel insights for treating diabetic retinopathy.

Protective effect and mechanism of Muntingia calabura Linn. fruit ethanolic extract against vascular endothelial growth factor production in nickel-stimulated hepatocellular carcinoma cells

The protective effect of the M. calabura Linn. fruit ethanolic extract (MFEE) against nickel (Ni)-induced vascular endothelial growth factor (VEGF) expression in hepatocellular carcinoma (HepG2) cells was investigated. MFEE contained 4 flavonoids: epicatechin, rutin, diosmin and luteolin, and 11 phenolic acids: gallic acid, gentisic acid, p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, ferulic acid, sinapic acid, syringic acid, p-anisic acid and rosmarinic acid. Substantial VEGF expression was assayed in Ni-stimulated HepG2 cells, which was significantly suppressed through MFEE treatment via down-regulating Raf-1 proto-oncogene, serine/threonine kinase (RAF1) /mitogen-activated protein kinase 1/2 (MEK1/2)/extracellular-signal-regulated kinase 1/2 (ERK1/2), and Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) pathways as well as inhibiting hypoxia-inducible factor (HIF)-1α expression down-regulated by phosphatidylinositol-3-kinase (PI3K)/ protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway. The results showed that MFEE should have the potential to lower liver cancer risk in the Ni-polluted environment by suppressing VEGF expression.

Intraperitoneal injection of thalidomide alleviates early osteoarthritis development by suppressing vascular endothelial growth factor expression in mice.

Vascular endothelial growth factor (VEGF) is expressed in articular cartilage and increases in expression levels have been associated with the progression of osteoarthritis (OA). Thalidomide is a drug that has been reported to inhibit angiogenesis and reduce VEGF production by downregulating VEGF expression. The objective of the present study was to determine whether intraperitoneal administration of thalidomide may attenuate early OA development in mice. Male C57BL/6 mice (10‑weeks‑old) were randomly assigned into the destabilization of the medial meniscus (Dmm) with thalidomide treatment (Dmm+Th), Dmm and Sham groups equally. An OA model was induced surgically in Dmm+Th and Dmm groups, and mice of the Dmm+Th group were subsequently treated with an intraperitoneal injection of thalidomide (200mg/kg/day). At 2 and 4weeks following surgery, the pathological alterations in cartilage samples were assessed qualitatively by hematoxylin and eosin staining and Safranin O/Fast green staining, and quantitatively by the Osteoarthritis Research Society International scoring system. The mRNA expression levels of matrix metalloproteinase‑13 (MMP‑13) and VEGF were measured by reverse transcription‑quantitative polymerase chain reaction. The protein expression levels of MMP‑13 and VEGF were detected by immunofluorescence and immunohistochemistry, respectively. The production of VEGF in serum was evaluated via an ELISA assay. Pathological scores were significantly higher in the Dmm and the Dmm+Th groups than those in the Sham group; however, the Dmm+Th group exhibited markedly less severe pathological changes compared with the Dmm group. Compared with the Sham group, the mRNA and protein expression levels of VEGF and MMP‑13 in the Dmm and the Dmm+Th groups were significantly increased. The Dmm+Th group exhibited significantly decreased expression levels of VEGF and MMP‑13, as well as significantly decreased serum VEGF concentration compared with the Dmm group. Thus, the results of the present study demonstrated that intraperitoneal administration of thalidomide may alleviate the development of early OA by suppressing VEGF expression in mice and may have potential as a novel therapy for the treatment of OA.

The effect of sevoflurane on retinal angiogenesis in a mouse model of oxygen-induced retinopathy.

Sevoflurane is commonly used in general anesthesia for premature neonates. The main mechanism of retinopathy of prematurity (ROP) is increased levels of vascular endothelial growth factor (VEGF). For the investigation of sevoflurane's effect on angiogenesis, the angiogenesis and VEGF expression in the retina were measured after administering sevoflurane in an oxygen-induced retinopathy mice model. The mice were divided into the normoxic group (Nc and Ns group; n=6) and the ROP group (C, Rc, and Rs group; n=6). Rc group were exposed to 75% oxygen for 5days beginning on postnatal day (P) 7, and then returned to room air. Age-matched mice in the C group were exposed to room air. To observe angiogenesis of the retina, the mice were sacrificed on P16. The Rs group was exposed to 2vol% sevoflurane for 2h on P12, P13, and P14 with 40% oxygen. The angiogenic area and the spreading distance of vessels on P4 were statistically decreased in the Ns group, compared to the Nc group. The avascular area on P16 was significantly increased and the expression of VEGF was suppressed in the Rs group compared to the Rc group. Sevoflurane can inhibit retinal angiogenesis via suppressing VEGF expression in an OIR mice model with exposure to relative hypoxia. Nevertheless, it is still difficult to apply the results of this study immediately to humans because of the heterogeneity of responses to sevoflurane.

Oestrogen Inhibits VEGF Expression And Angiogenesis In Triple-Negative Breast Cancer By Activating GPER-1.

Triple-negative breast cancer (TNBC) is the most malignant type of breast cancer with ample vascularisation and high vascular endothelial growth factor (VEGF) expression. The sex steroid hormone oestrogen is involved in several cellular activities associated with TNBC regulation. However, the role of oestrogen in VEGF expression and angiogenesis in TNBC remains unclear. In this study, we found that treatment with 17β-oestradiol (E2) inhibited VEGF mRNA and protein expression in the TNBC cell lines MDA-MB-468 and MDA-MB-436. To further elaborate on the phenomenon of E2-regulated angiogenesis, we showed that conditioned medium from the TNBC cell line MDA-MB-468 treated with E2 inhibits the tube formation ability of human umbilical vein endothelial cells (HUVECs). Additionally, the G-protein-coupled oestrogen receptor-1 (GPER-1)-specific agonist G-1 has a function similar to that of E2. While G-15, the selective antagonist of GPER-1, notably reversed the inhibitory effects of E2 and G-1 on VEGF expression and tube formation, suggesting that GPER-1 is involved in the E2-induced angiogenesis suppression in TNBC cells. Moreover, E2 inhibited in vivo tumour growth and angiogenesis and reduced the expression levels of VEGF, NF-κB/p65, STAT3, and the endothelial marker CD34 in MDA-MB-468 xenograft tumours. Our findings provide important evidence that E2 can inhibit VEGF expression and angiogenesis in TNBC by activating GPER-1, offering additional insight into tumour angiogenesis and targets for drug intervention in TNBC.

Abstract B124: A novel alkannin derivative suppresses breast cancer proliferation and angiogenesis through modulation of STAT3 signaling

Abstract Background: Angiogenesis is a cancer hallmark essential for establishment of tumor and metastasis. The elevated expression of the vascular endothelial growth factor (VEGF) in breast cancer is directly regulated by activated STAT-3 protein, and thus it represents a common molecular target for blocking angiogenesis induced by multiple signaling pathways in breast cancer. Therefore STAT3 inhibition is perceived as a promising anti-angiogenic therapy. The natural naphthoquinone compounds like shikonin/alkannin exhibit potent anticancer activities. However, poor solubility, low bioavailability, and unfavorable toxicity profiles limit their clinical efficacy and underscore the need for synthetic napthoquinone derivative-based therapeutic strategies. In the present back drop, we have analysed the anti-angiogenic effects of a novel alkannin derivative, 1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 3-(pyridine-2-yl)propanoate (ALKP), and explored its mechanism of action in breast cancer cells. Methods: We studied the effects of the alkannin derivative in vitro and in vivo. The effects of ALKP were evaluated in vitro using HUVEC and breast cancer cells (MDA-MB231, MDA-MB468) to assess proliferation, invasion, migration, and tube formation. Immunoblotting and gelatin zymography were done to measure the MMP activity, protein expression, and phosphorylation of signaling kinases. Fluorescent microscopy was used to study subcellular localization of proteins. The in vivo effects were analysed by using the matrigel plug assay and 4T1 induced syngeneic mice breast cancer model. Results: ALKP significantly inhibited proliferation, invasion, migration, MMP activity, and tube formation in MDA-MB231 as compared to the parent compound. A significant attenuation in microvessel sprouting from ex vivo rat aortic ring cultures was observed in ALKP-treated rings compared with alkannin. Our mechanistic studies revealed that ALKP inhibited phosphorylation of STAT3, Src, and FAK in MDAMB-231cells. ALKP exhibited potent inhibition of VEGF expression and nuclear localization of phospho-STAT3 in comparison to alkannin. A significant inhibition of in vivo angiogenesis in matrigel plug assay, tumor growth, and lung metastasis in 4T1-induced breast cancer model was shown by ALKP compared with alkannin, with lower toxicity in terms of body weight and other parameters. Immunohistochemical analysis of tumor and matrigel revealed marked decrease in PCNA, CD31, and VEGF-A markers. Conclusion: Collectively our findings demonstrate that ALKP inhibits tumor angiogenesis through inhibiting STAT3 and VEGF signaling. Citation Format: Akanchha Shukla, Ravi Thakur, Sukanya Pandeti, Sabbu Sathish Reddy, Narender Tadigoppula, Durga Prasad Mishra. A novel alkannin derivative suppresses breast cancer proliferation and angiogenesis through modulation of STAT3 signaling [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B124.