Vascular Endothelial Growth Factor A Secretion Research Articles

Differential response to hypoxia in leiomyoma and myometrial cells

AimsRecent evidence suggests that repetitive hypoxia occurs during menstrual cycles due to vasoconstriction and myometrial contraction. It is unknown if hypoxia contributes to the development of uterine leiomyoma, the most common tumor of the female reproductive system. This study aims to characterize the response to hypoxia in leiomyoma and myometrial cells; and determine if an aberrant leiomyoma response to hypoxia may contribute to leiomyomatogenesis. Main methodsPrimary and immortalized leiomyoma and myometrial cells were cultured under normoxic and hypoxic conditions. Expression levels of vascular endothelial growth factor-A (VEGF-A), adrenomedullin (ADM), endothelin-1 (ET-1), and hypoxia-inducible factor-1 alpha (HIF-1α) were measured by qRT-PCR, western blotting and ELISA. Cell proliferation was assessed using MTT assay and proliferating-cell-nuclear-antigen (PCNA) expression. KC7F2 (HIF-1α inhibitor) was used to examine the regulating mechanisms. Key findingsAs expected, hypoxia induced HIF-1α expression in both leiomyoma and myometrial cells. However, hypoxia induced VEGF-A, ET-1 and ADM expression and VEGF-A secretion into the culture media in leiomyoma but not myometrial cells. MTT assay and PCNA expression showed that hypoxia induces proliferation in leiomyoma, but not myometrial cells. HIF-1α inhibitor abrogated the hypoxia-induced VEGF-A, ET-1, ADM, and PCNA expression in leiomyoma cells. SignificanceThis study suggests an aberrant leiomyoma cellular response to hypoxia compared to myometrium. This differential response to menstruation-related repetitive hypoxia episodes may lead to selective proliferation of hypoxia-adaptive leiomyoma cells and contribute to leiomyoma growth. Thus, in addition to adding to our understanding of leiomyoma pathobiology, the study proposes angiogenic factors as a potential leiomyoma therapeutic target.

Cancer-Associated Fibroblasts Promote Migration and Invasion of Non-Small Cell Lung Cancer Cells via miR-101-3p Mediated VEGFA Secretion and AKT/eNOS Pathway.

Cancer-associated fibroblasts (CAFs) are major component of tumor microenvironment (TME), which plays crucial roles in tumor growth, invasion and metastasis; however, the underling mechanism is not fully elucidated. Despite many studies are focused on the tumor promoting effect of CAFs-derived cytokines, the upstream regulators of cytokine release in CAFs is largely unknown. Here we found that miR-101-3p was downregulated in primary lung cancer-associated CAFs compared to normal fibroblasts (NFs). Ectopic overexpression of miR-101-3p suppressed CAFs activation, and abrogated the promoting effect of CAFs on migration and invasion of non-small cell lung cancer cells (NSCLC), through attenuating CAFs’ effect on epithelial mesenchymal transition (EMT) process, metastasis-related genes (MMP9, TWIST1) and AKT/endothelial nitric oxide synthase (eNOS) signaling pathway. Further study indicated that vascular endothelial growth factor A (VEGFA) was a novel target of miR-101-3p, and CAFs-derived VEGFA mediated the effect of miR-101-3p on migration and invasion of lung cancer cells, demonstrated by using recombinant VEGFA and VEGFA neutralizing antibody. Interestingly, the analysis of the Cancer Genome Atlas (TCGA) database revealed that lung cancer tissues expressed lower level of miR-101-3p than non-cancerous tissues, and low/medium-expression of miR-101-3p was associated with poor overall survival (OS) rate. Moreover, the mouse xenograft experiment also showed that CAFs accelerated tumor growth whereas miR-101-3p diminished CAFs’ effect. These findings revealed a novel mechanism that CAFs facilitated lung cancer metastasis potential via miR-101-3p/VEGFA/AKT signaling pathway, suggesting miR-101-3p as a potential candidate for metastasis therapy.

Knockdown of growth factor receptor bound protein 7 suppresses angiogenesis by inhibiting the secretion of vascular endothelial growth factor A in ovarian cancer cells

ABSTRACT Growth factor receptor bound protein 7 (GRB7) plays an important role in regulating the growth and metastasis of ovarian cancer. Angiogenesis is the basis for the growth, invasion, and metastasis of malignant tumors. In the current study, we aimed to determine whether GRB7 plays a role in regulating angiogenesis in ovarian cancer. Immunohistochemistry on tissue microarray showed that GRB7 and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) protein expression were positively correlated in ovarian cancer tissues. GRB7 knockdown suppressed vascular endothelial growth factor A (VEGFA) expression and reduced VEGFA secretion. The effects of GRB7-silenced SKOV-3 cells on human umbilical vein endothelial cells (HUVECs) were evaluated using a transwell cell co-culture model, which showed that knockdown of GRB7 in SKOV-3 cells suppressed HUVEC proliferation, migration, invasion, and tube formation. Moreover, knockdown of GRB7 in SKOV-3 cells downregulated the expression of proteins associated with angiogenesis, including vascular endothelial growth factor receptor-2 (VEGFR2), mitogen-activated protein kinase kinase 1 (MAP2K1/MEK1), extracellular signal-regulated kinases 1 and 2 (ERK1/2), notch receptor 1 (NOTCH1), and delta-like canonical Notch ligand 4 (DLL4) in HUVECs. In conclusion, knockdown of GRB7 in ovarian cancer cells is an attractive potential therapeutic target for the suppression of angiogenesis in ovarian cancer. GRB7 may regulate angiogenesis through VEGFA/VEGFR2 signaling and its downstream pathways.

The protein 4.1R downregulates VEGFA in M2 macrophages to inhibit colon cancer metastasis

M2 macrophages are crucial components of the tumour microenvironment and have been shown to be closely related to tumour progression. Co-culture with 4.1R−/− M2 macrophages enhances the malignancy of colon cancer (CC), but the mechanism remains unclear. Here, we report that protein 4.1R knockout reduced the phagocytosis of M2 macrophages (M-CSF/IL-4-treated bone marrow cells) and promoted MC38 colon cancer cell proliferation, migration, invasion, tumour formation and epithelial-mesenchymal transition (EMT), which are regulated by M2 macrophages. Further mechanistic dissection revealed that the 4.1R knockout upregulated vascular endothelial growth factor A (VEGFA) secreted by M2 macrophages and promoted colon cancer progression by activating the PI3K/AKT signalling pathway. In summary, our present study identified that 4.1R downregulates VEGFA secretion in M2 macrophages and delays the malignant potential of colon cancer by inhibiting the PI3K/AKT signalling pathway.

Toll-Like Receptor Signalling Pathways Regulate Hypoxic Stress Induced Fibroblast Growth Factor but Not Vascular Endothelial Growth Factor-A in Human Microvascular Endothelial Cells

Retinal diseases are the leading causes of irreversible blindness worldwide. The role of toll-like receptor (TLR) signalling mechanisms (MyD88 and TRIF) in the production of pro-angiogenic growth factors from human microvascular endothelial cells (HMEC-1) under hypoxic stress remains unexplored. HMEC-1 was incubated under normoxic (5% CO2 at 37 °C) and hypoxic (1% O2, 5% CO2, and 94% N2; at 37 °C) conditions for 2, 6, 24, and 48 h, respectively. For TLR pathway analysis, HMEC-1 was pre-treated with pharmacological inhibitors (Pepinh-MyD88 and Pepinh-TRIF) and subjected to normoxia and hypoxia conditions. Gene and protein expressions of vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor (FGF-2), hypoxia inducible factor 1-alpha (HIF1-α) were performed using quantitative polymerase chain reaction (qPCR), ELISA, and Western blot methodologies. Levels of TLR3 and TLR4 were analysed by flow cytometry. Under hypoxia, levels of VEGF-A and FGF-2 were elevated in a time-dependent fashion. Inhibition of MyD88 and TRIF signalling pathways decreased FGF-2 levels but failed to modulate the secretion of VEGF-A from HMEC-1. Blocking a known regulator, endothelin receptor (ETR), also had no effect on VEGF-A secretion from HMEC-1. Overall, this study provides the proof-of-concept to target TLR signalling pathways for the management of blinding retinal diseases.

MiR-126 Mimic Counteracts the Increased Secretion of VEGF-A Induced by High Glucose in ARPE-19 Cells.

Vascular endothelial growth factor-A (VEGF-A) has a pathologic role in microvascular diabetic complication, such as diabetic retinopathy (DR). miR-126 plays an important role in vascular development and angiogenesis by regulating the expression of VEGF-A. Since levels of miR-126 have been found downregulated in diabetes, this study is aimed at investigating whether hyperglycemia affects expression of miR-126 in a retinal pigment epithelium cell line. ARPE-19 cells were transfected with miR-126 inhibitor or with miR-126 mimic and the respective scramble negative control. After 24 hours, medium was replaced and cells were cultured for 24 hours in normal (CTR) or diabetic condition (HG). Then, we analyzed mRNA levels of miR-126, VEGF-A, PI3KR2, and SPRED1. We also evaluated protein amount of HIF-1α, PI3KR2, and SPRED1 and VEGF-A secretion. The results showed that exposure of ARPE-19 cells to HG significantly decreased miR-126 levels; mRNA levels of VEGF-A and PI3KR2 were inversely correlated with those of miR-126. Overexpression of miR-126 under HG restored HIF-1α expression and VEGF-A secretion to the level of CTR cells. These results indicate that reduced levels of miR-126 may contribute to DR progression by increasing expression of VEGF-A in RPE cells. In addition, we provide evidence that upregulation of miR-126 in RPE cells counteracts the rise of VEGF-A secretion induced by hyperglycemia. In conclusion, our data support a role of miR-126 mimic-approach in counteracting proangiogenic effects of hyperglycemia.

In vitro biological response of human osteoblasts in 3D chitosan sponges with controlled degree of deacetylation and molecular weight

We have studied the effect of chitosan sponges, produced from chitosan batches with distinct degree of deacetylation (DDA) and molecular weight (Mw), on the adhesion, growth and differentiation of primary human osteoblasts with an aim to offer a suitable tool for guided bone regeneration. All the chitosan sponges revealed similar microstructure, irrespective of the DDA (58, 73, 82, 88, and 91 %) and Mw (749, 547, 263, 215, and 170 kDa, respectively). Cell spreading was higher on sponges having a higher DDA. Higher DDA induced a more pronounced increase in alkaline phosphatase activity, osteopontin (OPN), vascular endothelial growth factor-A (VEGF), interleukin-6 (IL-6), and reduction in monocyte chemoattractant protein-1 (MCP-1), sclerostin (SOST) and dickkopf related protein-1 as compared to lower DDA. Lower DDA induced the increased secretion of osteoprotegerin and SOST as compared to higher DDA. The combination of higher DDA and Mw induced an increased secretion of VEGF and IL-6, however reduced the secretion of OPN as compared to chitosan with similar DDA but with lower Mw. In summary, the variations in cellular responses to the different chitosan sponges indicate a potential for individual tailoring of desired responses in guided bone regeneration.

Activating CD137 Signaling Promotes Sprouting Angiogenesis via Increased VEGFA Secretion and the VEGFR2/Akt/eNOS Pathway.

Combination of antiangiogenesis and immunotherapy may be an effective strategy for treatment of solid tumors. Our previous work reported that activation of CD137 signaling promotes intraplaque angiogenesis. A number of studies have demonstrated that vascular endothelial growth factor receptor 2 (VEGFR2) is a key target for angiogenesis. However, it is unknown whether CD137-mediated angiogenesis is related to VEGFR2. In this study, we investigated the effect of CD137 on the VEGFR2 expression and explored the underlying mechanisms of CD137-mediated angiogenesis. Knock-out of CD137 in ApoE−/− mice significantly decreased neovessel density in atherosclerotic plaques. CD137 silencing or inhibition attenuated endothelial cell (ECs) proliferation, migration, and tube formation. We found activation of CD137 signaling for increased VEGFR2 transcription and translation steadily. Moreover, CD137 signaling activated phosphorylated VEGFR2 (Tyr1175) and the downstream Akt/eNOS pathway, whereas neutralizing CD137 signaling weakened the activation of VEGFR2 and the downstream Akt/eNOS pathway. The aortic ring assay further demonstrated that CD137 signaling promoted ECc sprouting. Inhibition of VEGFR2 by siRNA or XL184 (cabozantinib) and inhibition of downstream signaling by LY294002 (inhibits AKT activation) and L-NAME (eNOS inhibitor) remarkably abolished proangiogenic effects of CD137 signaling both in vitro and ex vivo. In addition, the condition medium from CD137-activated ECs and vascular endothelial growth factor A (VEGFA) had similar effects on ECs that expressed high VEGFR2. Additionally, activating CD137 signaling promoted endothelial secretion of VEGFA, while blocking CD137 signaling attenuated VEGFA secretion. In conclusion, activation of CD137 signaling promoted sprouting angiogenesis by increased VEGFA secretion and the VEGFR2/Akt/eNOS pathway. These findings provide a basis for stabilizing intraplaque angiogenesis through VEGFR2 intervatioin, as well as cancer treatment via combination of CD137 agonists and specific VEGFR2 inhibitors.

Synergistic effects of multiple myeloma cells and tumor-associated macrophages on vascular endothelial cells in vitro.

Angiogenesis is a prerequisite for multiple myeloma development. Tumor cells can stimulate angiogenesis by secreting vascular endothelial growth factor A (VEGFA), but we previously reported that tumor angiogenesis was not significantly reduced when VEGFA expression was inhibited in myeloma cells. Tumor-associated macrophages (TAMs) are important components of the tumor microenvironment and have been reported to be involved in the regulation of angiogenesis. In this study, we performed in vitro macrophage coculture studies and studies with RPMI 8226 and TAMs cell-conditioned media to explore their effects on the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). Our results showed that M2 macrophages and RPMI 8226 cells could synergistically promote HUVEC proliferation, migration, and tube formation, and that VEGFA depletion in both cell types suppressed HUVEC tube formation ability. Conversely, M1 macrophages inhibited the tube formation in HUVECs. Mechanistically, M2 macrophage secretion of VEGFA may affect vascular endothelial growth factor receptor 1 signaling to regulate angiogenesis. In summary, our results suggest that macrophage clearance or inducing of transformation of M2 macrophages into M1 macrophages are potential treatment strategies for multiple myeloma.

Amelioration of Large Bile Duct Damage by Histamine-2 Receptor Vivo-Morpholino Treatment

Histamine binds to one of the four G-protein-coupled receptors expressed by large cholangiocytes and increases large cholangiocyte proliferation via histamine-2 receptor (H2HR), which is increased in patients with primary sclerosing cholangitis (PSC). Ranitidine decreases liver damage in Mdr2-/- (ATP binding cassette subfamily B member 4 null) mice. We targeted hepatic H2HR in Mdr2-/- mice using vivo-morpholino. Wild-type and Mdr2-/- mice were treated with mismatch or H2HR vivo-morpholino by tail vein injection for 1 week. Liver damage, mast cell (MC) activation, biliary H2HR, and histamine serum levels were studied. MC markers were determined by quantitative real-time PCR for chymase and c-kit. Intrahepatic biliary mass was detected by cytokeratin-19 and F4/80 to evaluate inflammation. Biliary senescence was determined by immunofluorescence and senescence-associated β-galactosidase staining. Hepatic fibrosis was evaluated by staining for desmin, Sirius Red/Fast Green, and vimentin. Immunofluorescence for transforming growth factor-β1, vascular endothelial growth factor-A/C, and cAMP/ERK expression was performed. Transforming growth factor-β1 and vascular endothelial growth factor-A secretion was measured in serum and/or cholangiocyte supernatant. Treatment with H2HR vivo-morpholino in Mdr2-/--mice decreased hepatic damage; H2HR protein expression and MC presence or activation; large intrahepatic bile duct mass, inflammation and senescence; and fibrosis, angiogenesis, and cAMP/phospho-ERK expression. Inhibition of H2HR signaling ameliorates large ductal PSC-induced damage. The H2HR axis may be targeted in treating PSC.

Increased number of intratumoral IL-17+ cells, a harbinger of the adverse prognosis of triple-negative breast cancer.

Triple negative breast cancer (TNBC) is an aggressive cancer subtype and lack of effective targeted therapies. It has been recently reported that Interleukin 17 (IL-17), a family of cytokines secreted in tumor microenvironment, affects tumor progression through a variety of molecular pathways. Its role in TNBC is so far still poorly explored. We employed immunohistochemistry to evaluate the distribution of IL-17+ cells in TNBC with no special type features (TNBC-NST), their association with tumor microangiogenesis, as well as their impact on prognosis of the patients. In comparison to medullary carcinoma with triple-negative molecular features (TNBC-MC), we found a significant increase in IL-17+ cell infiltrates in intratumoral stroma and extratumoral stroma of TNBC-NST. Similarly, stromal cells with co-expression of CD4 and IL-17 were noted in intratumoral and extratumoral stroma in both TNBC-NST and TNBC-MC. In addition, intratumoral IL-17+ cells were positively associated with tumor cell expression of vascular endothelial growth factor A (VEGFA) and with intratumoral tumor microvascular density (MVD). Multivariate analysis identified that intratumoral IL-17+ cells (P = 0.018), MVD (P = 0.039), and TNM stage (P = 0.002) were independent prognostic factors for predicting poor PFS. The study indicates that IL-17 is overexpressed in intratumoral stromal cells of TNBC-NST. The overexpression of IL-17 might engage in active tumor microangiogenesis through its signal transduction pathways resulting in increased tumor secretion of VEGFA, and then promote tumor progression. IL-17 might serve as a potential new target for individualized therapy to TNBC-NST patients by development of specific antibodies. Additional study is deemed to further explore the role of IL-17+ stromal cells in breast cancer.

Effect of ovarian steroids on vascular endothelial growth factor a expression in bovine uterine endothelial cells during adenomyosis

BackgroundAdenomyosis is a uterine dysfunction defined as the presence of endometrial glands within the myometrium. There is evidence that proangiogenic factors may play a role during the development of adenomyosis; however, exact mechanism remains unknown. The aim of the study was to determine the action of vascular endothelial growth factor A (VEGFA) in uterine tissue and uterine vascular endothelial cells during adenomyosis.ResultsUterine tissues were collected and examined for the presence and extent of adenomyosis. Gene and protein expression of VEGFA and its two receptors (VEGFR1 and VEGFR2) was evaluated with quantitative polymerase chain reaction and Western blotting, respectively, in endometrium and myometrium during adenomyosis. Immunolocalization of VEGFA and its receptors within uterine tissues during adenomyosis was also determined. In an in vitro experiment, endothelial cells from non-adenomyotic bovine uteri were treated with media conditioned by non-adenomyotic or adenomyotic uterine slices treated with 17-beta-oestradiol (E2) or progesterone (P4). Both gene and protein expression of VEGFR2 were elevated in endometrium in stages 3–4 of adenomyosis. Protein expression of VEGFA and VEGFR2 as well as VEGFA secretion were increased in endothelial cells treated with media conditioned by adenomyotic uterine slices after E2 treatment.ConclusionsResults suggest that VEGFA signalling is an important component, next to E2, that enhances VEGFA action and participates in adenomyosis development in cows.

Human CCR6+ Th17 Lymphocytes Are Highly Sensitive to Radiation-Induced Senescence and Are a Potential Target for Prevention of Radiation-Induced Toxicity

This study addresses the sensitivity of different peripheral CD4+ T-lymphocyte subsets to irradiation (IR) and identifies potential targets for the prevention or treatment of radiation-induced toxicity. This study was performed on peripheral blood mononuclear cells or sorted peripheral memory lymphocytes of CCR6+ mucosa-homing Th17/CCR6negTh and regulatory T subtypes of healthy volunteers. Cells were irradiated with a 2 Gy with or without pharmacologic inhibitors of different signaling pathways. Senescence of irradiated cells was assessed by resistance to apoptosis and determination of various senescence-associated biomarkers (senescence associated b-galactosidase activity, p16Ink4a-, p21Cdkn1a-, gH2A.X-, H2A.J expression). Cytokine production was measured in supernatants of irradiated cells by Luminex technology. Not all CD4+ memory T lymphocyte subsets were equally radiosensitive. High sensitivity of CCR6+Th17 lymphocytes to IR-induced senescence was shown by expression of the histone variant H2A.J, higher SA-b-Gal activity, and upregulation of p16Ink4a and p21Cdkn1a expression. Lower Annexin V staining and cleaved caspase-3, and higher expression of antiapoptotic genes Bcl-2 and Bcl-xL LF, showed that CCR6+Th17 lymphocytes were more resistant to IR-induced apoptosis than CCR6neg memory Th and regulatory T lymphocytes. After a 2 Gy IR, both CCR6+Th17 and CCR6neg cells acquired a moderate senescence-associated secretory phenotype, but only CCR6+Th17 cells secreted interleukin 8 (IL-8) and vascular endothelial growth factor-A (VEGF-A). Pharmacologic targeting of reactive oxygen species (ROS), mitogen-activated protein kinases (MAPKs), and mammalian target of rapamycin (mTOR) signaling pathways prevented the expression of senescent markers and IL-8 and VEGF-A expression by CCR6+Th17 cells after IR. This study suggests that IR induces senescence of CCR6+Th17 lymphocytes associated with secretion of IL-8 and VEGF-A that may be detrimental to the irradiated tissue. ROS-MAPKs signaling pathways are candidate targets to prevent this CCR6+Th17-dependent radiation-induced potential toxicity. Finally, the ratio of circulating H2A.J+ senescent CCR6+ Th17/CD4+ T lymphocytes may be a candidate marker of individual intrinsic radiosensitivity.

Ni-induced TGF-β signaling promotes VEGF-a secretion via integrin β3 upregulation.

Nickel compounds are associated with lung and skin cancer incidence increase and accumulation of nickel in the body contributes to carcinogenesis. Upregulation of certain integrins in the primary tumor is associated with cancer metastasis and poor prognosis. However, the molecular mechanisms of nickel-induced cancer metastasis are still unclear. The purpose of the present study was to investigate the effects of nickel chloride (NiCl2 ) on the progression of cancer during metastasis. The results of showed that NiCl2 induces the expression of integrin β3 mRNA and protein in a dose- and time-dependent manner. Inhibition of integrin αvβ3 activation by ITGB3 ligand mimetics and GR144053, as well as downregulation of ITGB3 by lentiviral shRNA gene silencing, diminished NiCl2 -induced secretion of vascular endothelial growth factor-a (VEGF-a). Furthermore, pretreatment with type I TGF-β receptor inhibitor, SB525334, suppressed the expression of ITGB3 at cell surface and secretion of VEGF-a in NiCl2 -treated cells. In conclusion, NiCl2 induces the expression of ITGB3 through TGF-β signaling activation, followed by increasing VEGF-a secretion, revealing a novel role for ITGB3 in nickel compound-induced cancer metastasis and tumor angiogenesis.

MART-10, a 1α,25(OH)2D3 Analog, Potently Represses Metastasis of ER+ Breast Cancer Cells with VEGF-A Overexpression.

Breast cancer ranks second in the list of cancer-related deaths for women. Even under multidisciplinary treatment, 25-50% of patients with breast cancer still ultimately develop metastasis, leading to poor prognosis. In addition to inducing angiogenesis, vascular endothelial growth factor-A (VEGF-A) is believed to directly increase cancer cell metastatic potential and overexpression of VEGF-A is associated with higher invasiveness of breast cancer. 1α,25(OH)2D3, the active form of vitamin D, and its analogs have been widely applied as anticancer agents in the past. Western blot, migration and invasion assays, enzyme-linked immunosorbent assay, and immunofluorescent stain were applied in this study. VEGF-A increased cell migration and invasion in estrogen receptor-positive (ER+) breast cancer MCF-7 cells. VEGF-A induced an autocrine loop in MCF-7 cells as VEGF-A treatment increased both VEGF-A expression and secretion. The expression of VEGF receptor type 2 (VEGFR2) and neuropilin 1 was also up-regulated by VEGF-A in MCF-7 cells. In addition, F-actin synthesis and LIM domain kinase 1 (LIMK-1) phosphorylation were increased by VEGF-A. VEGF-A also increased β-catenin expression and nuclear translocation of both β-catenin and nuclear factor-ĸB (NF-ĸB), indicating increased β-catenin and NF-ĸB activity. 1α,25(OH)2D3 and MART-10, an analog of 1α,25(OH)2D3, effectively repressed VEGF-A-induced MCF-7 cell migration and invasion and other VEGF-A-induced effects on MCF-7 cells, with MART-10 being more potent than 1α,25(OH)2D3 Conclusion: MART-10 can be deemed as a promising agent for prevention and treatment of metastasis of ER+ breast cancer with VEGF-A overexpression.

Abstract 2042: Hypoxia induces miR-153 through the IRE1α-XBP1 pathway to fine-tune the HIF1α/VEGFA axis in breast cancer angiogenesis

Abstract It is well documented that hypoxia activates the hypoxia-inducible factor (HIF1α)/vascular endothelial growth factor A (VEGFA) axis to promote angiogenesis in breast cancer. However, it is unclear how this axis is negatively regulated. A number of studies suggest that miR-153 inhibits tumor growth and metastasis by targeting SNAI1, ZEB2, MTDH, ADAM19, AKT1, HECTD3, and MCL1. We recently reported that miR-153 was induced by mifepristone and inhibited the breast cancer stem cells by down-regulating KLF5. In this study, we demonstrated that miR-153 directly inhibits expression of HIF1α by binding to the 3'UTR of HIF1A mRNA, as well as suppresses tube formation of primary human umbilical vein endothelial cells (HUVECs) and breast cancer angiogenesis by decreasing the secretion of VEGFA. Importantly, expression of miR-153 was induced by hypoxia-stimulated ER stress, which activates IRE1α and its downstream transcription factor XBP1. XBP1 directly binds to the promoter of the miR-153 host gene PTPRN and activates transcription. These results indicate that hypoxia induces miR-153 to fine tune the HIF1α/VEGFA axis in breast cancer angiogenesis and miR-153 could be used for breast cancer anti-angiogenesis therapy. Citation Format: Huichun Liang, Zhongmei Zhou, Ceshi Chen. Hypoxia induces miR-153 through the IRE1α-XBP1 pathway to fine-tune the HIF1α/VEGFA axis in breast cancer angiogenesis [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 2042.

ATAD2 silencing decreases VEGFA secretion through targeting has-miR-520a to inhibit angiogenesis in colorectal cancer.

ATPase family AAA domain-containing protein 2 (ATAD2) is involved in various types of cancers, including colorectal cancer. This study aimed to determine the role of ATAD2 in angiogenesis in colorectal cancer. Here, we downregulated ATAD2 expression in HCT116 and SW480 cells, and collected the conditioned medium (CM) from control and ATAD2-silenced cells. The effect of CM on human umbilical vein endothelial cells (HUVEC) was evaluated by using CCK-8, wound healing, tube formation, Western blot, and dual-luciferase reporter assays. Our results showed that the proliferation, migration, and tube formation of HUVEC were reduced in presence of ATAD2-silenced CM, and the levels of phosphorylated vascular endothelial growth factor receptor 2 (P-VEGFR2), CD31, and CD34 were downregulated. Mechanism studies showed that ATAD2 silencing regulated the expression of vascular endothelial growth factor A (VEGFA) and miR-520a. Moreover, we found that miR-520a could bind to ATAD2, and its inhibitor partly reversed the alterations in HUVEC induced by CM from ATAD2-silenced cells. In addition, we demonstrated that miR-520a directly bound to 3'-UTR of VEGFA and inhibited its expression. Collectively, our results indicate that ATAD2 inhibition suppresses VEGFA secretion by increasing miR-520a levels. Our study suggests ATAD2 as a potential therapeutic target for angiogenesis in colorectal cancer.

Cyclic stretch induces VEGFA alternative splicing via Serine/Arginine-Rich Splicing Factor 1

Abnormal proliferation of vascular smooth muscle cells (VSMCs) induced by high cyclic stretch is crucial in the vascular remodeling during hypertension. Vascular endothelial growth factor A (VEGFA) alternative splicing plays important roles in the pathological process of vascular diseases and remodeling. However, the roles of VEGFA isoforms in modulating VSMC functions in response to cyclic stretch remain unclear. We hypothesize that high cyclic stretch may induce VEGFA alternative splicing via Serine/arginine-rich splicing factor 1 (SRSF1) which subsequently induce VSMC proliferation. In the present research, hypertensive rat model was established using the abdominal aortic constriction method. In comparison with sham-operated group, immunohistology staining showed translocation of SRSF1 into nuclei in hypertensive rat thoracic aorta, and RT-PCR detected a shift of VEGFA expression pattern, including the increased expression of VEGFA120 and VEGFA164, but not VEGFA188.Then VSMCs were subjected to cyclic stretch in vitro using a Flexercell strain unit. VEGFA ELISA assay showed 15% cyclic stretch increased the secretion of VEGFA which significantly increased proliferation of VSMCs. Western blot and immunofluorescence detected accumulation of SRSF1 in nuclei after 15% cyclic stretch application. Furthermore, SRSF1-specific siRNA transfection reversed the VEGFA secretion induced by pathological high cyclic stretch. Our present results suggested that pathologically high cyclic stretch induces the shuttling of SRSF1 which results in the secretive pattern splicing of VEGFA and finally contributes to the proliferation of VSMCs.

Hypoxia induces miR-153 through the IRE1\u03b1-XBP1 pathway to fine tune the HIF1\u03b1/VEGFA axis in breast cancer angiogenesis

It is well documented that hypoxia activates the hypoxia-inducible factor 1-alpha (HIF1α)/vascular endothelial growth factor A (VEGFA) axis to promote angiogenesis in breast cancer. However, it is unclear how this axis is negatively regulated. In this study, we demonstrated that miR-153 directly inhibits expression of HIF1α by binding to the 3′UTR of HIF1A mRNA, as well as suppresses tube formation of primary human umbilical vein endothelial cells (HUVECs) and breast cancer angiogenesis by decreasing the secretion of VEGFA. Importantly, expression of miR-153 was induced by hypoxia-stimulated ER stress, which activates IRE1α and its downstream transcription factor X-box binding protein 1 (XBP1). X-box binding protein 1 directly binds to the promoter of the miR-153 host gene PTPRN and activates transcription. These results indicate that hypoxia induces miR-153 to fine tune the HIF1α/VEGFA axis in breast cancer angiogenesis and miR-153 could be used for breast cancer anti-angiogenesis therapy.

LncRNA-AK131850 Sponges MiR-93-5p in Newborn and Mature Osteoclasts to Enhance the Secretion of Vascular Endothelial Growth Factor a Promoting Vasculogenesis of Endothelial Progenitor Cells

Background/Aims: In the process of bone development and remodeling, the vasculature is regarded as the communicative network between the bone and neighboring tissues. Recently, it has been reported that the processes of angiogenesis and osteogenesis are coupled temporally and spatially. However, few studies reported the relationship and relevant mechanism between osteoclastogenesis and vasculogenesis. Methods: Arraystar Mouse lncRNA microarray V3.0 was firstly used to analyze the differentially expressed lncRNA genes in osteoclast different stages during osteoclastogenesis. Cell counting kit 8 (CCK-8) analysis, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, migration and tube formation assays were used to detect impact of osteoclast different stages on the proliferation, differentiation, migration and tube formation of endothelial progenitor cells (EPCs), respectively. Finally, transfection of AK131850 shRNA, miR-93-5p mimic and miR-93-5p inhibitor, qRT-PCR, western blotting, enzyme-linked immunosorbent assay (ELISA), fluorescence in situ hybridization (FISH) and luciferase reporter assay were carried out to dissect molecular mechanisms. Results: In this study, we found that newborn OCs (N-OC) and mature OCs (M-OC) during osteoclastogenesis significantly promoted proliferation, differentiation, migration and tube formation of endothelial progenitor cells (EPCs). Through lncRNA microarray and GO&pathway analysis, we found that AK131850 and co-expressed gene, vascular endothelial growth factor a (VEGFa), were significantly up-regulated in N-OC and M-OC. After inhibition of AK131850 the promoting effect of N-OC and M-OC on EPCs was reversed. Furthermore, we found that AK131850 directly competed miR-93-5p in N-OC and M-OC through sponge, thereby increasing VEGFa transcription, expression and secretion through derepressing of miR-93-5p on VEGFa. Conclusion: Our results provided the first finding that lncRNA-AK131850 sponged miR-93-5p in N-OC and M-OC during osteoclastogenesis to enhance the secretion of VEGFa, thus promoting vasculogenesis of EPCs.