Showing papers on "Vascular endothelial growth factor A published in 2017"

Combined antiangiogenic and anti–PD-L1 therapy stimulates tumor immunity through HEV formation

Abstract: Inhibitors of VEGF (vascular endothelial growth factor)/VEGFR2 (vascular endothelial growth factor receptor 2) are commonly used in the clinic, but their beneficial effects are only observed in a subset of patients and limited by induction of diverse relapse mechanisms. We describe the up-regulation of an adaptive immunosuppressive pathway during antiangiogenic therapy, by which PD-L1 (programmed cell death ligand 1), the ligand of the negative immune checkpoint regulator PD-1 (programmed cell death protein 1), is enhanced by interferon-γ–expressing T cells in distinct intratumoral cell types in refractory pancreatic, breast, and brain tumor mouse models. Successful treatment with a combination of anti-VEGFR2 and anti–PD-L1 antibodies induced high endothelial venules (HEVs) in PyMT (polyoma middle T oncoprotein) breast cancer and RT2-PNET (Rip1-Tag2 pancreatic neuroendocrine tumors), but not in glioblastoma (GBM). These HEVs promoted lymphocyte infiltration and activity through activation of lymphotoxin β receptor (LTβR) signaling. Further activation of LTβR signaling in tumor vessels using an agonistic antibody enhanced HEV formation, immunity, and subsequent apoptosis and necrosis in pancreatic and mammary tumors. Finally, LTβR agonists induced HEVs in recalcitrant GBM, enhanced cytotoxic T cell (CTL) activity, and thereby sensitized tumors to antiangiogenic/anti–PD-L1 therapy. Together, our preclinical studies provide evidence that anti–PD-L1 therapy can sensitize tumors to antiangiogenic therapy and prolong its efficacy, and conversely, antiangiogenic therapy can improve anti–PD-L1 treatment specifically when it generates intratumoral HEVs that facilitate enhanced CTL infiltration, activity, and tumor cell destruction.

Dual angiopoietin-2 and VEGFA inhibition elicits antitumor immunity that is enhanced by PD-1 checkpoint blockade

Abstract: Pathological angiogenesis is a hallmark of cancer and a therapeutic target. Vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2; also known as ANG2) are proangiogenic cytokines that sustain tumor angiogenesis and limit antitumor immunity. We show that combined ANGPT2 and VEGFA blockade by a bispecific antibody (A2V) provided superior therapeutic benefits, as compared to the single agents, in both genetically engineered and transplant tumor models, including metastatic breast cancer (MMTV-PyMT), pancreatic neuroendocrine tumor (RIP1-Tag2), and melanoma. Mechanistically, A2V promoted vascular regression, tumor necrosis, and antigen presentation by intratumoral phagocytes. A2V also normalized the remaining blood vessels and facilitated the extravasation and perivascular accumulation of activated, interferon-γ (IFNγ)–expressing CD8+ cytotoxic T lymphocytes (CTLs). Whereas the antitumoral activity of A2V was, at least partly, CTL-dependent, perivascular T cells concurrently up-regulated the expression of the immune checkpoint ligand programmed cell death ligand 1 (PD-L1) in tumor endothelial cells. IFNγ neutralization blunted this adaptive response, and PD-1 blockade improved tumor control by A2V in different cancer models. These findings position immune cells as key effectors of antiangiogenic therapy and support the rationale for cotargeting angiogenesis and immune checkpoints in cancer therapy.

Exercise induces cerebral VEGF and angiogenesis via the lactate receptor HCAR1

Abstract: Physical exercise can improve brain function and delay neurodegeneration; however, the initial signal from muscle to brain is unknown. Here we show that the lactate receptor (HCAR1) is highly enriched in pial fibroblast-like cells that line the vessels supplying blood to the brain, and in pericyte-like cells along intracerebral microvessels. Activation of HCAR1 enhances cerebral vascular endothelial growth factor A (VEGFA) and cerebral angiogenesis. High-intensity interval exercise (5 days weekly for 7 weeks), as well as L-lactate subcutaneous injection that leads to an increase in blood lactate levels similar to exercise, increases brain VEGFA protein and capillary density in wild-type mice, but not in knockout mice lacking HCAR1. In contrast, skeletal muscle shows no vascular HCAR1 expression and no HCAR1-dependent change in vascularization induced by exercise or lactate. Thus, we demonstrate that a substance released by exercising skeletal muscle induces supportive effects in brain through an identified receptor.

FGF-dependent metabolic control of vascular development

Abstract: Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are important to these processes. Although much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism, little is understood about the role of fibroblast growth factors (FGFs) in this context. Here we identify FGF receptor (FGFR) signalling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2). A decrease in HK2 levels in the absence of FGF signalling inputs results in decreased glycolysis, leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/Fgfr3 double mutant mice, while HK2 overexpression partly rescues the defects caused by suppression of FGF signalling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development.

Transdifferentiated Human Vascular Smooth Muscle Cells are a New Potential Cell Source for Endothelial Regeneration.

Abstract: Endothelial dysfunction is widely implicated in cardiovascular pathological changes and development of vascular disease. In view of the fact that the spontaneous endothelial cell (EC) regeneration is a slow and insufficient process, it is of great interest to explore alternative cell sources capable of generating functional ECs. Vascular smooth muscle cell (SMC) composes the majority of the vascular wall and retains phenotypic plasticity in response to various stimuli. The aim of this study is to test the feasibility of the conversion of SMC into functional EC through the use of reprogramming factors. Human SMCs are first dedifferentiated for 4 days to achieve a vascular progenitor state expressing CD34, by introducing transcription factors OCT4, SOX2, KLF4 and c-MYC. These SMC-derived progenitors are then differentiated along the endothelial lineage. The SMC-converted ECs exhibit typical endothelial markers expression and endothelial functions in vitro, in vivo and in disease model. Further comprehensive analysis indicates that mesenchymal-to-epithelial transition is requisite to initiate SMCs reprogramming into vascular progenitors and that members of the Notch signalling pathway regulate further differentiation of the progenitors into endothelial lineage. Together, we provide the first evidence of the feasibility of the conversion of human SMCs towards endothelial lineage through an intermediate vascular progenitor state induced by reprogramming.

Sirt1 Inhibits Oxidative Stress in Vascular Endothelial Cells.

Abstract: The vascular endothelium is a layer of cells lining the inner surface of vessels, serving as a barrier that mediates microenvironment homeostasis. Deterioration of either the structure or function of endothelial cells (ECs) results in a variety of cardiovascular diseases. Previous studies have shown that reactive oxygen species (ROS) is a key factor that contributes to the impairment of ECs and the subsequent endothelial dysfunction. The longevity regulator Sirt1 is a NAD+-dependent deacetylase that has a potential antioxidative stress activity in vascular ECs. The mechanisms underlying the protective effects involve Sirt1/FOXOs, Sirt1/NF-κB, Sirt1/NOX, Sirt1/SOD, and Sirt1/eNOs pathways. In this review, we summarize the most recent reports in this field to recapitulate the potent mechanisms involving the protective role of Sirt1 in oxidative stress and to highlight the beneficial effects of Sirt1 on cardiovascular functions.

Pericytes regulate VEGF-induced endothelial sprouting through VEGFR1.

Abstract: Pericytes adhere to the abluminal surface of endothelial tubules and are required for the formation of stable vascular networks. Defective endothelial cell-pericyte interactions are frequently observed in diseases characterized by compromised vascular integrity such as diabetic retinopathy. Many functional properties of pericytes and their exact role in the regulation of angiogenic blood vessel growth remain elusive. Here we show that pericytes promote endothelial sprouting in the postnatal retinal vasculature. Using genetic and pharmacological approaches, we show that the expression of vascular endothelial growth factor receptor 1 (VEGFR1) by pericytes spatially restricts VEGF signalling. Angiogenic defects caused by pericyte depletion are phenocopied by intraocular injection of VEGF-A or pericyte-specific inactivation of the murine gene encoding VEGFR1. Our findings establish that pericytes promote endothelial sprouting, which results in the loss of side branches and the enlargement of vessels when pericyte function is impaired or lost.

Long non-coding RNA taurine upregulated 1 enhances tumor-induced angiogenesis through inhibiting microRNA-299 in human glioblastoma.

Abstract: Angiogenesis is one of the critical biological elements affecting the development and progression of cancer. Long non-coding RNAs (lncRNAs) are important regulators and aberrantly expressed in various types of human cancer. Our previous studies indicated that lncRNA taurine upregulated 1 (TUG1) implicated in the regulation of blood-tumor barrier permeability; however, its role in glioblastoma angiogenesis still unclear. Here we demonstrated that TUG1 was up-expressed in human glioblastoma tissues and glioblastoma cell lines. Knockdown of TUG1 remarkably suppressed tumor-induced endothelial cell proliferation, migration and tube formation as well as reducing spheroid-based angiogenesis ability in vitro, which are the critical steps for tumor angiogenesis. Besides, knockdown of TUG1 significantly increased the expression of mircroRNA-299 (miR-299), which was down-expressed in glioblastoma tissues and glioblastoma cell lines. Bioinformatics analysis and luciferase reporter assay revealed that TUG1 influenced tumor angiogenesis via directly binding to the miR-299 and there was a reciprocal repression between TUG1 and miR-299 in the same RNA-induced silencing complex. Moreover, knockdown of TUG1 reduced the expression of vascular endothelial growth factor A (VEGFA), which was defined as a functional downstream target of miR-299. In addition, knockdown of TUG1, shown in the in vivo studies, has effects on suppressing tumor growth, reducing tumor microvessel density and decreasing the VEGFA expression by upregulating miR-299 in xenograft glioblastoma model. Overall, the results demonstrated that TUG1 enhances tumor-induced angiogenesis and VEGF expression through inhibiting miR-299. Also, the inhibition of TUG1 could provide a novel therapeutic target for glioblastoma treatment.

Glioma cells promote angiogenesis through the release of exosomes containing long non-coding RNA POU3F3.

Abstract: Objective Angiogenesis is a key event in the progression of gliomas, and emerging evidence suggests that exosomes are signaling extracellular organelles that modulate the tumor microenvironment and promote angiogenesis and tumor progression. This study aimed to explore the mechanism by which glioma-derived exosomes affect angiogenesis. Materials and methods qRT-PCR was used to determine the expression level of linc-POU3F3 in glioma tissue as well as glioma cell lines. Ultrafiltration combined with a purification method was used to isolate exosomes derived from A172 cells (A172-Exo) and linc-POU3F3 shRNA-treated A172 cells (shA172-Exo). Transmission electron microscopy, Western blot and tunable resistive pulse sensing (TRPS) were used to identify exosomes. In vitro migration, proliferation, and tube formation experiments, as well as in vivo CAM assays, were used to analyze the pro-angiogenesis ability of exosomes. qRT-PCR and Western blot were used to identify expression levels of angiogenesis-related genes and proteins in human brain microvascular endothelial cells (HBMECs) after being cultured with exosomes. Results The levels of linc-POU3F3 were upregulated in glioma tissue and significantly correlated with the advanced tumor stage. A172 cells exhibited the highest expression level. A172-Exo was similar to shA172-Exo (50-100 nm in diameter) and expressed Alix, Tsg101 and CD9, while the expression level of linc-POU3F3 in A172-Exo was significantly higher than that in shA172-Exo. HBMECs rapidly internalized A172-Exo and shA172-Exo, and the linc-POU3F3 expression level in HBMECs treated with A172-Exo was significantly higher than the level in HBMECs treated with shA172-Exo. A172-Exo exhibited better function in promoting HBMECs migration, proliferation, tubular-like structure formation in vitro and arteriole formation in vivo. The gene and protein expression level of bFGF, bFGFR, VEGFA, and Angio in HBMECs treated with A172-Exo was much higher than that of HBMECs treated with shA172-Exo. Conclusions These results indicated that gliomas can induce angiogenesis by secreting exosomes enriched in linc-POU3F3. Exosomes and lncRNA-POU3F3 may, therefore, function as a putative therapeutic target in glioma.

Glioma cells enhance angiogenesis and inhibit endothelial cell apoptosis through the release of exosomes that contain long non-coding RNA CCAT2.

Abstract: Angiogenesis is a key event in the progression of gliomas. Exosomes, as signaling extracellular organelles, modulate the tumor microenvironment and promote angiogenesis and tumor progression. We previously demonstrated that long intergenic non-coding RNA CCAT2 (linc-CCAT2) was overexpressed in glioma tissues and functioned to promote glioma progression. Therefore, this study aimed to explore an underlying mechanism of glioma cell-affected angiogenesis. First, qRT-PCR was used to determine the expression level of linc-CCAT2 in 4 glioma cell lines and 293T cells, and the results revealed that the U87-MG cells exhibited the highest expression level. Subsequently, the pro-angiogenesis function of exosomes that were derived from negative control shRNA-treated U87-MG cells (ncU87-Exo) and linc-CCAT2 shRNA-treated U87-MG cells (shU87-Exo) was evaluated in vitro and in vivo. We found that ncU87-Exo, which was enriched in linc-CCAT2, could be taken up by HUVECs. ncU87-Exo improved the linc-CCAT2 expression level in HUVECs and more strongly promoted HUVEC migration, proliferation, tubular-like structure formation in vitro and arteriole formation in vivo as well as inhibited HUVEC apoptosis induced by hypoxia. Further mechanistic studies revealed that ncU87-Exo could upregulate VEGFA and TGFβ expression in HUVECs as well as promote Bcl-2 expression and inhibit Bax and caspase-3 expression. Finally, gain-/loss-of-function studies revealed that the overexpression of linc-CCAT2 in HUVECs activated VEGFA and TGFβ, promoted angiogenesis, promoted Bcl-2 expression and inhibited Bax and caspase-3 expression, thus decreasing apoptosis. Downregulation of linc-CCAT2 revealed the opposite effect. Thus, our results revealed a new exosome‑mediated mechanism by which glioma cells could promote angiogenesis through the transfer of linc-CCAT2 by exosomes to endothelial cells. Moreover, we suggest that exosomes and linc-CCAT2 are putative therapeutic targets in glioma.

ANRIL: A Regulator of VEGF in Diabetic Retinopathy.

Abstract: Purpose Long noncoding RNAs (lncRNAs) previously thought to be "dark matter" of the genome, play key roles in various biological processes. The lncRNA ANRIL is located at a genetic susceptibility locus for coronary artery diseases and type 2 diabetes. We examined the role of ANRIL in diabetic retinopathy, through study of its regulation of VEGF both in vitro and in vivo. Methods Human retinal endothelial cells (HRECs) were subjected to incubation in high glucose to mimic diabetes. ANRIL expression was measured with or without small interfering RNA (siRNA) knockdown in HRECs. ANRIL knockout mice, with or without streptozotocin-induced diabetes, were also investigated. Cell and tissues were measured for VEGF mRNA and protein expression. Functional alterations in VEGF were determined through tube formation, cell proliferation, and retinal vascular permeability assays. Vascular endothelial growth factor regulation through ANRIL's interactions with polycomb repressive complex 2 (PRC2) components and p300 were studied thorough PRC2 blocker, siRNA, and RNA immunoprecipitation (RNA-IP) assays. Results High glucose and diabetes caused ANRIL upregulation in HRECs and in the retina. Glucose-mediated elevation of ANRIL, on silencing, prevented VEGF expression. Such regulation involved ANRIL-mediated control of the PRC2 components p300 and miR200b. Direct binding of ANRIL to p300 and enhancer of zeste homolog 2 (EZH2; a PRC2 component) were elevated following exposure to high glucose levels. Conclusions Our results demonstrate for the first time that ANRIL regulates VEGF expression and function in diabetic retinopathy. This regulation is mediated by p300, miR200b, and EZH2 of the PRC2 complex.

Vascular Endothelial Growth Factor (VEGF) Signaling in Tumour Vascularization: Potential and Challenges

Abstract: Angiogenesis is defined as the physiological process by which new blood vessels develop from pre-existing vessels; either by sprouting or intussusception. Inhibition of angiogenesis is one of the most encouraging strategies to manage the growth and metastasis of cancers. The functional and proliferative status of blood vessels is regulated by the balance between various key molecules that either stimulate or inhibit angiogenesis. During quiescence, the "angiogenic switch" is "off". However, during tumour development pro-angiogenic factors such as vascular endothelial growth factor (VEGF), basic and acidic fibroblast growth factor, tumour necrosis factor-α and interleukin-1 are pathologically enhanced. Persistent growth of tumour directed capillary networks creates a favourable microenvironment, promoting cancer growth, progression and metastasis. VEGF, particularly VEGF-A, is a key angiogenic factor. Targeting VEGF, its receptors and the downstream signaling cascade, is a viable strategy to prevent tumour growth and metastasis. The present review discusses the role of VEGF in tumour angiogenesis and the current understanding of anti-VEGF therapies as well as refractoriness of anti-angiogenesis cancer therapy.

Overexpression of lncRNA ANRIL up-regulates VEGF expression and promotes angiogenesis of diabetes mellitus combined with cerebral infarction by activating NF-κB signaling pathway in a rat model.

Abstract: // Bo Zhang 1,* , Dan Wang 2,* , Tie-Feng Ji 3 , Lei Shi 1 and Jin-Lu Yu 1 1 Department of Neurosurgery, The First Hospital of Jilin University, Changchun, P.R. China 2 Department of Ophthalmology, The First Hospital of Jilin University, Changchun, P.R. China 3 Department of Radiology, The First Hospital of Jilin University, Changchun, P.R. China * These authors have contributed equally to the manuscript Correspondence to: Jin-Lu Yu, email: // Keywords : LncRNA ANRIL; diabetes mellitus; cerebral infarction; NF-κB signaling pathway; vascular endothelial growth factor Received : July 27, 2016 Accepted : December 07, 2016 Published : January 03, 2017 Abstract Objective: This study aimed to explore the effects of lncRNA ANRIL on vascular endothelial growth factor (VEGF) and angiogenesis in diabetes mellitus (DM) combined with cerebral infarction (CI) through NF-κB signaling pathway. Methods: Adult male Wistar rats were randomly divided into control group and DM + CI group, and the DM + CI group were subdivided into Vector, shANRIL, PDTC, pcDNA-ANRIL, and pcDNA-ANRIL + PDTC groups. VEGF and FMS-like tyrosine kinase (FLT-1) expressions were measured by immunohistochemistry and endothelium dependent microvessel density (MVD) was detected by differentiation 31 (CD31) and para-amiuosalicylic acid (PAS) double staining. The qRT-PCR was applied to measure mRNA expressions of VEGF, FLT-1, Kinase insert domain protein receptor (FLK-1) and NF-κB, and Western blotting was conducted to detected expressions of VEGF, NF-κB and p-IκB/IκB. Results: Compared with the control group, protein expressions of VEGF, NF-κB, p-IκB/IκB, expression of ANRIL, and mRNA expressions of VEGF, FLT-1 and NF-κB were increased in the DM + CI group. Compared with the Vector group, protein expressions of VEGF, NF-κB, p-IκB/IκB, expression of ANRIL, mRNA expressions of VEGF, FLT-1 and NF-κB, and endothelium dependent MVD were increased in the pcDNA-ANRIL group, while decreased in the shANRIL group and PDTC group. Compared with the pcDNA-ANRIL group, protein expressions of VEGF, NF-κB, p-IκB/IκB, expression of ANRIL, mRNA expressions of VEGF, FLT-1 and NF-κB, and endothelium dependent MVD were decreased in the pcDNA-ANRIL + PDTC group. Conclusion: Overexpressed lncRNA ANRIL upregulates VEGF and promotes angiogenesis by activating NF-κB signaling pathway in DM + CI rats. .

The p38 pathway, a major pleiotropic cascade that transduces stress and metastatic signals in endothelial cells

Abstract: By gating the traffic of molecules and cells across the vessel wall, endothelial cells play a central role in regulating cardiovascular functions and systemic homeostasis and in modulating pathophysiological processes such as inflammation and immunity Accordingly, the loss of endothelial cell integrity is associated with pathological disorders that include atherosclerosis and cancer The p38 mitogen-activated protein kinase (MAPK) cascades are major signaling pathways that regulate several functions of endothelial cells in response to exogenous and endogenous stimuli including growth factors, stress and cytokines The p38 MAPK family contains four isoforms p38α, p38β, p38γ and p38δ that are encoded by four different genes They are all widely expressed although to different levels in almost all human tissues p38α/MAPK14, that is ubiquitously expressed is the prototype member of the family and is referred here as p38 It regulates the production of inflammatory mediators, and controls cell proliferation, differentiation, migration and survival Its activation in endothelial cells leads to actin remodeling, angiogenesis, DNA damage response and thereby has major impact on cardiovascular homeostasis, and on cancer progression In this manuscript, we review the biology of p38 in regulating endothelial functions especially in response to oxidative stress and during the metastatic process

Delivery of Small Interfering RNA to Inhibit Vascular Endothelial Growth Factor in Zebrafish Using Natural Brain Endothelia Cell-Secreted Exosome Nanovesicles for the Treatment of Brain Cancer

Abstract: Although small interfering RNA (siRNA) holds great therapeutic promise, its delivery to the disease site remains a paramount obstacle. In this study, we tested whether brain endothelial cell-derived exosomes could deliver siRNA across the blood–brain barrier (BBB) in zebrafish. Natural exosomes were isolated from brain endothelial bEND.3 cell culture media and vascular endothelial growth factor (VEGF) siRNA was loaded in exosomes with the assistance of a transfection reagent. While fluorescence-activated cell flow cytometry and immunocytochemistry staining studies indicated that wild-type exosomes significantly increased the uptake of fluorescence-labeled siRNA in the autologous brain endothelial cells, decreased fluorescence intensity was observed in the cells treated with the tetraspanin CD63 antibody-blocked exosome-delivered formulation (p < 0.05). In the transport study, exosomes also enhanced the permeability of rhodamine 123 in a co-cultured monolayer of brain endothelial bEND.3 cell and astrocyte. Inhibition at the expression of VEGF RNA and protein levels was observed in glioblastoma-astrocytoma U-87 MG cells treated with exosome-delivered siRNAs. Imaging results showed that exosome delivered more siRNAs across the BBB in Tg(fli1:GFP) zebrafish. In a xenotransplanted brain tumor model, exosome-delivered VEGF siRNAs decreased the fluorescence intensity of labeled cancer cells in the brain of zebrafish. Brain endothelial cell-derived exosomes could be potentially used as a natural carrier for the brain delivery of exogenous siRNA.

Loss of HIF-1α in natural killer cells inhibits tumour growth by stimulating non-productive angiogenesis

Abstract: Productive angiogenesis, a prerequisite for tumour growth, depends on the balanced release of angiogenic and angiostatic factors by different cell types within hypoxic tumours. Natural killer (NK) cells kill cancer cells and infiltrate hypoxic tumour areas. Cellular adaptation to low oxygen is mediated by Hypoxia-inducible factors (HIFs). We found that deletion of HIF-1α in NK cells inhibited tumour growth despite impaired tumour cell killing. Tumours developing in these conditions were characterised by a high-density network of immature vessels, severe haemorrhage, increased hypoxia, and facilitated metastasis due to non-productive angiogenesis. Loss of HIF-1α in NK cells increased the bioavailability of the major angiogenic cytokine vascular endothelial growth factor (VEGF) by decreasing the infiltration of NK cells that express angiostatic soluble VEGFR-1. In summary, this identifies the hypoxic response in NK cells as an inhibitor of VEGF-driven angiogenesis, yet, this promotes tumour growth by allowing the formation of functionally improved vessels.

MiRNA199a-3p suppresses tumor growth, migration, invasion and angiogenesis in hepatocellular carcinoma by targeting VEGFA, VEGFR1, VEGFR2, HGF and MMP2

Abstract: Increasing significance of tumor–stromal interaction in development and progression of cancer implies that signaling molecules in the tumor microenvironment (TME) might be the effective therapeutic targets for hepatocellular carcinoma (HCC). Here, the role of microRNA miR-199a-3p in the regulation of TME and development of HCC has been investigated by several in vitro and in vivo assays. Expression of miR-199a-3p was observed significantly low in HCC tissues and its overexpression remarkably inhibited in vivo tumor growth and metastasis to lung in NOD-SCID mice. In vitro restoration of miR-199a-3p expression either in endothelial cells (ECs) or in cancer cells (CACs) significantly diminished migration of ECs in co-culture assay. Again incubation of miR-199a-3p transfected ECs with either conditioned media (CM) of CACs or recombinant VEGF has reduced tube formation, in ECs and it was also dropped upon growth in CM of either anti-VEGF antibody-treated or miR-199a-3p-transfected CACs. In addition, bioinformatics and luciferase-reporter assays revealed that miR-199a-3p inhibited VEGF secretion from CACs and VEGFR1 and VEGFR2 expression on ECs and thus restricted cross talk between CACs and ECs. Again, restoration of miR-199a-3p in hepatic stellate cells (HSCs) reduced migration and invasion of CACs in co-culture assay, while it was enhanced by the overexpression of HGF suggesting miR-199a-3p has hindered HSC-CACs cross talk probably by inhibiting HGF and regulating matrix metalloproteinase MMP2, which were found as targets of miR-199a-3p subsequently by luciferase-reporter assay and gelatin zymography, respectively. Thus, these findings collectively highlight that miR-199a-3p restricts metastasis, invasion and angiogenesis in HCC and hence it may be considered as one of the powerful effective therapeutics for management of HCC patients.

Ly6Clo monocytes drive immunosuppression and confer resistance to anti-VEGFR2 cancer therapy

Abstract: Current anti-VEGF therapies for colorectal cancer (CRC) provide limited survival benefit, as tumors rapidly develop resistance to these agents. Here, we have uncovered an immunosuppressive role for nonclassical Ly6Clo monocytes that mediates resistance to anti-VEGFR2 treatment. We found that the chemokine CX3CL1 was upregulated in both human and murine tumors following VEGF signaling blockade, resulting in recruitment of CX3CR1+Ly6Clo monocytes into the tumor. We also found that treatment with VEGFA reduced expression of CX3CL1 in endothelial cells in vitro. Intravital microscopy revealed that CX3CR1 is critical for Ly6Clo monocyte transmigration across the endothelium in murine CRC tumors. Moreover, Ly6Clo monocytes recruit Ly6G+ neutrophils via CXCL5 and produce IL-10, which inhibits adaptive immunity. Preventing Ly6Clo monocyte or Ly6G+ neutrophil infiltration into tumors enhanced inhibition of tumor growth with anti-VEGFR2 therapy. Furthermore, a gene therapy using a nanoparticle formulated with an siRNA against CX3CL1 reduced Ly6Clo monocyte recruitment and improved outcome of anti-VEGFR2 therapy in mouse CRCs. Our study unveils an immunosuppressive function of Ly6Clo monocytes that, to our knowledge, has yet to be reported in any context. We also reveal molecular mechanisms underlying antiangiogenic treatment resistance, suggesting potential immunomodulatory strategies to enhance the long-term clinical outcome of anti-VEGF therapies.

The BET inhibitor JQ1 selectively impairs tumour response to hypoxia and downregulates CA9 and angiogenesis in triple negative breast cancer

Abstract: The availability of bromodomain and extra-terminal inhibitors (BETi) has enabled translational epigenetic studies in cancer. BET proteins regulate transcription by selectively recognizing acetylated lysine residues on chromatin. BETi compete with this process leading to both downregulation and upregulation of gene expression. Hypoxia enables progression of triple negative breast cancer (TNBC), the most aggressive form of breast cancer, partly by driving metabolic adaptation, angiogenesis and metastasis through upregulation of hypoxia-regulated genes (for example, carbonic anhydrase 9 (CA9) and vascular endothelial growth factor A (VEGF-A). Responses to hypoxia can be mediated epigenetically, thus we investigated whether BETi JQ1 could impair the TNBC response induced by hypoxia and exert anti-tumour effects. JQ1 significantly modulated 44% of hypoxia-induced genes, of which two-thirds were downregulated including CA9 and VEGF-A. JQ1 prevented HIF binding to the hypoxia response element in CA9 promoter, but did not alter HIF expression or activity, suggesting some HIF targets are BET-dependent. JQ1 reduced TNBC growth in vitro and in vivo and inhibited xenograft vascularization. These findings identify that BETi dually targets angiogenesis and the hypoxic response, an effective combination at reducing tumour growth in preclinical studies.

A polymer nanoparticle with engineered affinity for a vascular endothelial growth factor (VEGF165).

Abstract: Protein affinity reagents are widely used in basic research, diagnostics and separations and for clinical applications, the most common of which are antibodies. However, they often suffer from high cost, and difficulties in their development, production and storage. Here we show that a synthetic polymer nanoparticle (NP) can be engineered to have many of the functions of a protein affinity reagent. Polymer NPs with nM affinity to a key vascular endothelial growth factor (VEGF165) inhibit binding of the signalling protein to its receptor VEGFR-2, preventing receptor phosphorylation and downstream VEGF165-dependent endothelial cell migration and invasion into the extracellular matrix. In addition, the NPs inhibit VEGF-mediated new blood vessel formation in Matrigel plugs in vivo. Importantly, the non-toxic NPs were not found to exhibit off-target activity. These results support the assertion that synthetic polymers offer a new paradigm in the search for abiotic protein affinity reagents by providing many of the functions of their protein counterparts.

VEGFR3 Modulates Vascular Permeability by Controlling VEGF/VEGFR2 Signaling.

Abstract: Rationale: Vascular endothelial growth factor (VEGF) is the main driver of angiogenesis and vascular permeability via VEGF receptor 2 (VEGFR2), whereas lymphangiogenesis signals are transduced by VEGFC/D via VEGFR3. VEGFR3 also regulates sprouting angiogenesis and blood vessel growth, but to what extent VEGFR3 signaling controls blood vessel permeability remains unknown. Objective: To investigate the role of VEGFR3 in the regulation of VEGF-induced vascular permeability. Methods and Results: Long-term global Vegfr3 gene deletion in adult mice resulted in increased fibrinogen deposition in lungs and kidneys, indicating enhanced vascular leakage at the steady state. Short-term deletion of Vegfr3 in blood vascular endothelial cells increased baseline leakage in various tissues, as well as in tumors, and exacerbated vascular permeability in response to VEGF, administered via intradermal adenoviral delivery or through systemic injection of recombinant protein. VEGFR3 gene silencing upregulated VEGFR2 protein levels and phosphorylation in cultured endothelial cells. Consistent with elevated VEGFR2 activity, vascular endothelial cadherin showed reduced localization at endothelial cell–cell junctions in postnatal retinas after Vegfr3 deletion, or after VEGFR3 silencing in cultured endothelial cells. Furthermore, concurrent deletion of Vegfr2 prevented VEGF-induced excessive vascular leakage in mice lacking Vegfr3 . Conclusions: VEGFR3 limits VEGFR2 expression and VEGF/VEGFR2 pathway activity in quiescent and angiogenic blood vascular endothelial cells, thereby preventing excessive vascular permeability.

MicroRNA-29b Inhibits Angiogenesis by Targeting VEGFA through the MAPK/ERK and PI3K/Akt Signaling Pathways in Endometrial Carcinoma

Abstract: Objective: The purpose of this study is to explore the effects of microRNA-29b (miR-29b) regulating MAPK/ERK and PI3K/Akt signaling pathways on angiogenesis in endometrial carcinoma (EC) by targeting VEGFA. Methods: Between February 2013 and April 2015, 126 EC patients admitted to the Second Affiliated Hospital of Nanchang University were randomly selected, with 126 EC tissues and the corresponding adjacent normal tissues collected after surgery. The human EC cell lines RL-95-2 and HEC-1-B and human endometrial cells were assigned to the normal group (human endometrial cells), the blank group (untransfected RL-95-2 or HEC-1-B cells), the pMIR-control group (RL-95-2 or HEC-1-B cells transfected with an empty vector), the pMIR-miR-29b group (RL-95-2 or HEC-1-B cells transfected with the miR-29b plasmid), LNA-control group (RL-95-2 or HEC-1-B cells transfected with an oligonucleotide inhibitors control), the LNA-miR-29b inhibitors group (RL-95-2 or HEC-1-B cells transfected with miRCURY LNATM miR-29b inhibitors), the LNA-miR-29b inhibitors + PD98059 group (RL-95-2 or HEC-1-B cells transfected with miRCURY LNATM miR-29b inhibitors and PD98059, an inhibitor of the MAPK/ERK signaling pathway) and the LNA-miR-29b inhibitors + wortmannin group (RL-95-2 or HEC-1-B cells transfected with miRCURY LNATM miR-29b inhibitors and wortmannin, an inhibitor of the PI3K/Akt signaling pathway). qRT-PCR and Western blotting were conducted to detect the miR-29b expression and the mRNA and protein expressions of VEGFA, ERK, Akt, mTOR and Bcl-2. Immunohistochemistry (IHC) was performed to determine the microvessel density (MVD) expression in the EC tissues, adjacent normal tissues and nude-mice. Results: Compared with the adjacent normal tissues, miR-29b expression was down-regulated, the mRNA and protein expressions of VEGFA, ERK, Akt, mTOR and Bcl-2 were up-regulated, and MVD expression was increased in the EC tissues. Compared with the normal group, miR-29b expression was down-regulated, while the mRNA and protein expressions of VEGFA, ERK, Akt, mTOR and Bcl-2 were up-regulated in the other groups. Compared with the blank, pMIR-control and LNA-control groups, miR-29b expression was increased, while mRNA and protein expressions of VEGFA, ERK, Akt, mTOR and Bcl-2 were decreased in the pMIR-miR-29b group. The LNA-miR-29b inhibitors group exhibited elevated miR-29b expression and decreased mRNA and protein expressions of VEGFA, ERK, Akt, mTOR and Bcl-2 (All P < 0.05). Additionally, miR-29b expression was reduced in the LNA-miR-29b inhibitors + PD98059 and LNA-miR-29b inhibitors + wortmannin groups. In comparison to the normal group, MVD expression was elevated in the other groups. Compared with the blank, pMIR-control, LNA-control, LNA-miR-29b inhibitors + PD98059 and LNA-miR-29b inhibitors + wortmannin groups, MVD expression was decreased in the pMIR-miR-29b group but increased in the LNA-miR-29b inhibitors group. Conclusion: Our results indicate that miR-29b negatively modulates the MAPK/ERK and PI3K/Akt signaling pathways to inhibit angiogenesis in EC by targeting VEGFA.

Protective Effects of MicroRNA-126 on Human Cardiac Microvascular Endothelial Cells Against Hypoxia/Reoxygenation-Induced Injury and Inflammatory Response by Activating PI3K/Akt/eNOS Signaling Pathway.

Abstract: Objective: This study explored the protective effects of the microRNA-126 (miR-126)-mediated PI3K/Akt/eNOS signaling pathway on human cardiac microvascular endothelial cells (HCMECs) against hypoxia/reoxygenation (H/R)-induced injury and the inflammatory response Methods: Untreated HCMECs were selected for the control group After H/R treatment and cell transfection, the HCMECs were assigned to the H/R, miR-126 mimic, mimic-negative control (NC), miR-126 inhibitor, inhibitor-NC, wortmannin (an inhibitor of PI3K) and miR-126 mimic + wortmannin groups Super oxide dismutase (SOD), nitric oxide (NO), vascular endothelial growth factor (VEGF) and reactive oxygen species (ROS) were measured utilizing commercial kits Quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were performed to detect miR-126 expression and the mRNA and protein expression of inflammatory factors Western blotting was used to determine the expression of key members in the PI3K/Akt/eNOS signaling pathway ACCK-8 assay and flow cytometry were employed to examine cell proliferation and apoptosis, respectively The angiogenic ability in each group was detected by the lumen formation test Results: Compared to the control group, p/t-PI3K, p/t-Akt and p/t-eNOS expression, NO, VEGF and SOD levels, cell proliferation and in vitro lumen formation ability were decreased, while the ROS content, interleukin (IL)-6, IL-10 and tumor necrosis factor (TNF)-α expression and cell apoptosis were significantly increased in the H/R, mimic-NC, miR-126 inhibitor, inhibitor-NC, wortmannin and miR-126 mimic + wortmannin groups Additionally, in comparison with the H/R group, the miR-126 mimic group had elevated p/t-PI3K, p/t-Akt and p/t-eNOS expression, increased NO, VEGF and SOD contents, and strengthened cell proliferation and lumen formation abilities but also exhibited decreased ROS content, reduced IL-6, IL-10 and TNF-α expressions, and weakened cell apoptosis, while the miR-126 inhibitor and wortmannin group exhibited the opposite results Furthermore, decreased p/t-PI3K, p/t-Akt and p/t-eNOS expressions, decreased NO, VEGF and SOD contents, cell proliferation and lumen formation abilities, as well as increased ROS content, increased IL-6, IL-10 and TNF-α expression, and increased cell apoptosis were observed in the miR-126 mimic + wortmannin group compared to themiR-126 mimic group Conclusions: These findings indicated that miR-126 protects HCMECs from H/R-induced injury and inflammatory response by activating the PI3K/Akt/ eNOS signaling pathway

Vascular endothelial growth factor control mechanisms in skeletal growth and repair.

Abstract: Vascular endothelial growth factor A (VEGF) is a critical regulator of vascular development and postnatal angiogenesis and homeostasis, and it is essential for bone development and repair. Blood vessels serve both as structural templates for bone formation and they provide essential cells, growth factors and minerals needed for synthesis and mineralization, as well as turnover, of the extracellular matrix in bone. Through its regulation of angiogenesis, VEGF contributes to coupling of osteogenesis to angiogenesis, and it directly controls the differentiation and function of osteoblasts and osteoclasts. In this review, we summarize the properties of VEGF and its receptors that are relevant to bone formation and repair; the roles of VEGF during development of endochondral and membranous bones; and the contributions of VEGF to bone healing during different phases of bone repair. Finally, we discuss contributions of altered VEGF function in inherited disorders with bone defects as part of their phenotypes, and we speculate on what will be required before therapeutic strategies based on VEGF modulation can be developed for clinical use to treat patients with bone growth disorders and/or compromised bone repair. Developmental Dynamics 246:227-234, 2017. © 2016 Wiley Periodicals, Inc.

The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway

Abstract: Microvascular networks in the adventitia of large arteries control access of inflammatory cells to the inner wall layers (media and intima) and thus protect the immune privilege of the aorta and its major branches. In autoimmune vasculitis giant cell arteritis (GCA), CD4 T helper 1 (T H 1) and T H 17 cells invade into the wall of the aorta and large elastic arteries to form tissue-destructive granulomas. Whether the disease microenvironment provides instructive cues for vasculitogenic T cells is unknown. We report that adventitial microvascular endothelial cells (mvECs) perform immunoregulatory functions by up-regulating the expression of the Notch ligand Jagged1. Vascular endothelial growth factor (VEGF), abundantly present in GCA patients’ blood, induced Jagged1 expression, allowing mvECs to regulate effector T cell induction via the Notch–mTORC1 (mammalian target of rapamycin complex 1) pathway. We found that circulating CD4 T cells in GCA patients have left the quiescent state, actively signal through the Notch pathway, and differentiate into T H 1 and T H 17 effector cells. In an in vivo model of large vessel vasculitis, exogenous VEGF functioned as an effective amplifier to recruit and activate vasculitogenic T cells. Thus, systemic VEGF co-opts endothelial Jagged1 to trigger aberrant Notch signaling, biases responsiveness of CD4 T cells, and induces pathogenic effector functions. Adventitial microvascular networks function as an instructive tissue niche, which can be exploited to target vasculitogenic immunity in large vessel vasculitis.