VEGF-induced Angiogenesis Research Articles

Pro-angiogenic Ginsenosides F1 and Rh1 Inhibit Vascular Leakage by Modulating NR4A1

Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, but VEGF-induced angiogenesis is often accompanied by a vascular permeability response. Ginsenosides are triterpenoid saponins from the well-known medicinal plant, ginseng, and have been considered a candidate for modulating angiogenesis. Here, we systemically investigated the effects of 10 different ginsenosides on human umbilical vein endothelial cells and newly identified that two PPT-type ginsenosides, F1 and Rh1 induce the migration and proliferation of endothelial cells. Interestingly, RNA transcriptome analysis showed that gene regulation induced by VEGF in endothelial cells is distinct from that of ginsenoside F1 and Rh1. In addition, F1 and Rh1 significantly inhibited vascular leakage both in vitro and in vivo, which are induced by vascular endothelial growth factor. Furthermore, comparative transcriptome analysis revealed that these effects of F1 and Rh1 on vascular leakage restoration are mainly caused by changes in VEGF-mediated TNFα signaling via NFκB, particularly by the suppression of expression and transcriptional activity of NR4A1 by F1 and Rh1, even in the presence of VEGF. These findings demonstrate that ginsenosides F1 and Rh1 can be a promising herbal remedy for vessel normalization in ischemic disease and cancer and that NR4A1 is the key target.

Anlotinib, a novel small molecular tyrosine kinase inhibitor, suppresses growth and metastasis via dual blockade of VEGFR2 and MET in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents, with highly aggressive behavior and early systemic metastasis. The survival rates for osteosarcoma remain unchanged over the past two decades. Studies aiming to find new or alternative therapies for patients with refractory osteosarcoma are urgently needed. Anlotinib, a novel multi-targeted tyrosine kinase inhibitor (TKI), has exhibited encouraging clinical activity in NSLCC and soft tissue sarcoma, whereas its effect on osteosarcoma has not been studied. In our study, we investigated the anti-tumor activity and underlying mechanism of anlotinib in osteosarcoma. Various in vitro and in vivo models of human osteosarcoma were used to determine the anti-proliferative, anti-angiogenesis and anti-metastasis efficacy of anlotinib. Our results showed that anlotinib suppressed tumor growth and increased the chemo-sensitivity of osteosarcoma. In addition, anlotinib inhibited migration and invasion in osteosarcoma cells. Furthermore, in order to explore the anti-tumor mechanism of anlotinib, phospho-RTK antibody arrays were performed. These analyses confirmed that anlotinib suppressed the phosphorylation of MET, VEGFR2 and the downstream signaling pathway activation. Moreover, we demonstrated that anlotinib blocked hepatocyte growth factor (HGF)-induced cell migration, invasion and VEGF-induced angiogenesis. Notably, a 143B-Luc orthotopic osteosarcoma model further showed that anlotinib significantly inhibited growth and lung metastasis of implanted tumor cells. Our preclinical work indicates that anlotinib acts as a novel inhibitor of VEGFR2 and MET that blocks tumorigenesis in osteosarcoma, which could be translated into future clinical trials.

Activator of G-protein signaling 8 is involved in VEGF-induced choroidal neovascularization

Choroidal neovascularization (CNV) is associated with age-related macular degeneration (AMD), a major cause of vision loss among elderly people. Vascular endothelial cell growth factor (VEGF) is essential for the development and progression of AMD, and VEGF signaling molecules are effective targets for the treatment of AMD. We recently reported that activator of G-protein signaling 8 (AGS8), a receptor-independent Gβγ regulator, is involved in VEGF-induced angiogenesis in cultured endothelial cells (EC); however, the role of AGS8 in CNV is not yet understood. This study aimed to explore the role of AGS8 in CNV in cultured cells, explanted choroid tissue, and laser-induced CNV in a mouse AMD model. AGS8 knockdown in cultured choroidal EC inhibited VEGF-induced VEGFR-2 phosphorylation, cell proliferation, and migration. AGS8 knockdown also downregulated cell sprouting from mouse choroidal tissue in ex vivo culture. A mouse model of laser-induced CNV, created to analyze the roles of AGS8 in vivo, demonstrated that AGS8 mRNA was significantly upregulated in choroidal lesions and AGS8 was specifically expressed in the neovasculature. Local AGS8 knockdown in intravitreal tissue significantly inhibited laser-induced AGS8 upregulation and suppressed CNV, suggesting that AGS8 knockdown in the choroid has therapeutic potential for AMD. Together, these results demonstrate that AGS8 plays critical roles in VEGF-induced CNV.

Effect of anacardic acid against echinococcosis through inhibition of VEGF-induced angiogenesis

Echinococcosis is a zoonotic infection caused by cestode species of the genus Echinococcus, with limited treatment options. It is urgent to develop new anti-hydatid agent. In this paper, we reported anacardic acid (AA), a natural product isolated from the Brazilian cashew-nut shell liquid, which presented a high activity against metacestodes of Echinococcus multilocularis (E. multilocularis) and Echinococcus granulosus sensu stricto (E. granulosus s.s.) in vitro and in vivo. AA exerted a better efficacy on E. granulosus s.s. protoscoleces and E. multilocularis metacestodes than that of albendazole (ABZ) and dihydroartemisinin (DHA) in vitro, and an inhibition on the growth of Echinococcus metacestode as effective as ABZ in vivo. Moreover, we also found that one of the mechanisms of AA against Echinococcus could be the suppression of angiogenesis on/in the metacestode mass through inhibiting vascular endothelial growth factor (VEGF)—induced signalling pathways. This work finds that AA is a new promising potential candidate drug for echinococcosis treatment.

Didymin by suppressing NF-κB activation prevents VEGF-induced angiogenesis in vitro and in vivo

Although didymin, a dietary flavonoid glycoside from citrus fruits, known to be a potent antioxidant with anti-cancer activities, its role in angiogenesis is not known. In this study, we examined the effect of didymin on VEGF-induced angiogenesis in vitro and in vivo models. Our results suggest that treatment of human umbilical vein endothelial cell (HUVECs) with didymin significantly prevented the VEGF-induced cell proliferation, migration, and invasion. Further, didymin significantly prevented the VEGF-induced endothelial tube formation in culture. Didymin also attenuated the VEGF-induced generation of ROS, activation of NF-κB and the expression of adhesion molecules such as VCAM-1, ICAM-1, and E-selectin in HUVECs. Further, didymin also prevented the VEGF-induced microvessel sprouting in ex vivo mouse aortic rings. Most importantly, didymin significantly prevented the invasion of endothelial cells and formation of blood capillary-like structures in Matrigel plug model of angiogenesis in mice. Thus, our results suggest a novel antiangiogenic efficacy of didymin in addition to its reported anti-cancer properties, which warrant further development of this agent for cancer therapy.

High levels of HtrA4 detected in preeclamptic circulation may disrupt endothelial cell function by cleaving the main VEGFA receptor KDR.

Systemic endothelial dysfunction is a key characteristic of preeclampsia (PE), which is a serious disorder of human pregnancy. We have previously reported that high-temperature requirement factor (Htr)A4 is a placenta-specific protease that is secreted into the maternal circulation and significantly up-regulated in PE, especially early-onset PE. We have also demonstrated that high levels of HtrA4 detected in the early onset PE circulation induce endothelial dysfunction in HUVECs. In the current study, we investigated whether HtrA4 could cleave the main receptor of VEGFA, the kinase domain receptor (KDR), thereby inhibiting VEGFA signaling. We first demonstrated that HtrA4 cleaved recombinant KDR in vitro. We then confirmed that HtrA4 reduced the level of KDR in HUVECs and inhibited the VEGFA-induced phosphorylation of Akt kinase, which is essential for downstream signaling. Further functional studies demonstrated that HtrA4 prevented the VEGFA-induced tube formation in HUVECs and dose-dependently inhibited the VEGFA-induced angiogenesis in explants of mouse aortic rings. These data strongly suggest that high levels of HtrA4 in the maternal circulation could cleave the main receptor of VEGFA in endothelial cells to induce a wide-spread impairment of angiogenesis. Our studies therefore suggest that HtrA4 is a potential causal factor of early onset PE.-Wang, Y., La, M., Pham, T., Lovrecz, G. O., Nie, G. High levels of HtrA4 detected in preeclamptic circulation may disrupt endothelial cell function by cleaving the main VEGFA receptor KDR.

Endothelial cell mineralocorticoid receptors oppose VEGF-induced gene expression and angiogenesis.

Aldosterone is a key factor in adverse cardiovascular remodeling by acting on the mineralocorticoid receptor (MR) in different cell types. Endothelial MR activation mediates hypertrophy, inflammation and fibrosis. Cardiovascular remodeling is often accompanied by impaired angiogenesis, which is a risk factor for the development of heart failure. In this study, we evaluated the impact of MR in endothelial cells on angiogenesis. Deoxycorticosterone acetate (DOCA)-induced hypertension was associated with capillary rarefaction in the heart of WT mice but not of mice with cell type-specific MR deletion in endothelial cells. Consistently, endothelial MR deletion prevented the inhibitory effect of aldosterone on the capillarization of subcutaneously implanted silicon tubes and on capillary sprouting from aortic ring segments. We examined MR-dependent gene expression in cultured endothelial cells by RNA-seq and identified a cluster of differentially regulated genes related to angiogenesis. We found opposing effects on gene expression when comparing activation of the mineralocorticoid receptor in ECs to treatment with vascular endothelial growth factor (VEGF), a potent activator of angiogenesis. In conclusion, we demonstrate here that activation of endothelial cell MR impaired angiogenic capacity and lead to capillary rarefaction in a mouse model of MR-driven hypertension. MR activation opposed VEGF-induced gene expression leading to the dysregulation of angiogenesis-related gene networks in endothelial cells. Our findings underscore the pivotal role of endothelial cell MR in the pathophysiology of hypertension and related heart disease.

Therapeutic Effects of Synthetic Antimicrobial Peptides, TRAIL and NRP1 Blocking Peptides in Psoriatic Keratinocytes.

Psoriasis is a chronic, recurrent, heterogeneous, cutaneous inflammatory skin disease for which there is no cure. It affects approximately 7.5 million people in the United States. Currently, several biologic agents that target different molecules implicated in the pathogenic processes of psoriasis are being assessed in diverse clinical studies. However, relapse usually occurs within weeks or months, meaning there is currently no cure for psoriasis. Therefore, recent studies have discovered diverse new potential treatments for psoriasis: inhibitors of bacteria such as Staphylococcus aureus, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and neuropilin 1 (NRP1). A promising approach that has recently been described involves modifying antimicrobial peptides to develop new cutaneous anti-bacterial agents that target inflammatory skin disease induced by Staphylococcus. Increased expression of TRAIL and its death receptors DR4 and DR5 has been implicated in the pathogenesis of plaque psoriasis. In addition, TRAIL has the ability to inhibit angiogenesis by inducing endothelial cell death and by negative regulation of VEGF-induced angiogenesis via caspase-8-mediated enzymatic and non-enzymatic functions. Since NRP1 regulates angiogenesis induced by multiple signals, including VEGF, ECM and semaphorins, and also initiates proliferation of keratinocytes through NF-κB signaling pathway in involved psoriatic skin, targeting NRP1 pathways may offer numerous windows for intervention in psoriasis. In this review, we will focus on the current knowledge about the emerging role of synthetic antimicrobial peptides, TRAIL and NRP1 blocking peptides in the pathogenesis and treatment of psoriasis.

MicroRNA-34a attenuates VEGF-mediated retinal angiogenesis via targeting Notch1

Pathological angiogenesis in the retina is one of the main ocular diseases closely associated with vision loss. This work investigated the roles of microRNA-34a (miR-34a) and its potential target Notch1, in retinal angiogenesis. For this we used oxygen-induced retinopathy (OIR) rats and human retinal microvascular endothelial cells (HRMECs) stimulated with vascular endothelial growth factor (VEGF). We performed hematoxylin-eosin staining, Western blot for VEGF, and immunofluorescence staining for CD31 to verify the establishment of our OIR model. We observed down-regulation of miR-34a, and up-regulation of Notch1 and Hey1 in retinas from OIR rats. We found similar results with the VEGF-stimulated HRMECs. By performing MTT assay, cell scratch assay, tube formation assay, and by detecting the expression of matrix-metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitors of metalloproteinases-1 (TIMP-1), and TIMP-2, we found that transfection of miR-34a ameliorated VEGF-mediated angiogenesis of HRMECs. We further observed that siRNA-induced gene silencing of Notch1 prevented VEGF-induced angiogenesis via regulating cell proliferation, cell migration, and tube formation of HRMECs. Additionally, activation of Notch1 by transfection of Notch1 plasmid attenuated the inhibitory effects of miR-34a on tube formation, in the present of VEGF. Results from our dual-luciferase reporter gene assay suggested that miR-34a targets Notch1. In summary, our data demonstrate that miR-34a attenuates retinal angiogenesis via targeting Notch1.

Decrease in VEGF-Induced Pericardial Adhesion Formation Using Bevacizumab After Surgery.

Some degrees of postoperative cardiac adhesions occur in response to the first cardiac surgery in patients that may limit surgeons for subsequent operations and increase the risk of heart injury. In this article, we established a model of postoperative pericardial adhesions, and because vascular endothelial growth factor (VEGF) seems to initiate adhesion formation through inflammatory responses, we used an anti-VEGF antibody, that is, bevacizumab, to examine its effects on postoperative adhesion formation. Twenty Wistar rats were divided in 2 groups: control and bevacizumab. After chest opening, pericardial sac was opened and the heart was fully exposed. In the bevacizumab group, bevacizumab (2.5 mg/kg) was applied locally on the heart and then the chest was closed. The control group received saline solution as placebo. After 42 days, high-sensitivity C-reactive protein in peripheral blood was measured, and re-sternotomy was performed to measure severity of pericardial adhesions. Then, the hearts were collected from all rats to evaluate percentage of CD-31-positive cells (as a marker of angiogenesis) using immunohistochemical staining. When the bevacizumab group was compared with the control group, we found that the mean score of adhesion (0.89 ± 0.38 vs 2.56 ± 0.41) and CD-31 expression (27.45 ± 3.75% vs 56.26 ± 1.98%) was decreased significantly after bevacizumab administration. However, we did not find any difference in high-sensitivity C-reactive protein levels of control and bevacizumab animals. In the current study, bevacizumab administration could effectively reduce adhesion formation after first sternotomy by preventing VEGF-induced angiogenesis through CD-31 downregulation.

YAP via interacting with STAT3 regulates VEGF-induced angiogenesis in human retinal microvascular endothelial cells

Endothelial dysfunction is a main feature of retinal neovascular diseases which are the leading cause of blindness in developed countries. Yes-associated protein (YAP) and signal transducer and activator of transcription factor 3 (STAT3) participate in angiogenesis via vascular endothelial growth factor (VEGF) signaling. Additionally, YAP can bind STAT3 in endothelial cells. In the study, dimethyloxalylglycine (DMOG) stimulated human retinal microvascular endothelial cells (HRMECs) was used as retinal endothelial hypoxia model. The proliferation of HRMECs, as well as t-YAP, p-STAT3 (Tyr705) increased, while p-YAP (Ser127), p-YAP (Ser397) decreased following hypoxia. Meanwhile, YAP and STAT3 translocated to the nucleus. YAP knockdown inhibited the proliferation, migration and tube formation of HRMECs. YAP overexpression up-regulated phosphorylation of STAT3. The YAP overexpression-induced HRMECs proliferation, migration and tube formation were reversed by S3I-201, a selective STAT3 inhibitor. YAP interacted with STAT3 to promote STAT3 nuclear translocation. Additionally, YAP and STAT3 promoted the transcription of VEGF synergistically. Finally, inhibition of YAP alleviated retinal pathological neovascularization in mouse oxygen-induced retinopathy (OIR) model. In summary, activated YAP interacted with STAT3 to promote the activation and nuclear translocation of STAT3, hence boosted the proliferation, migration and tube formation of HRMECs via VEGF signaling following hypoxia. The data will further elucidate the mechanisms of retinal neovascular diseases.

SUMOylation of VEGFR2 regulates its intracellular trafficking and pathological angiogenesis

Regulation of VEGFR2 represents an important mechanism for the control of angiogenesis. VEGFR2 activity can be regulated by post-translational modifications such as ubiquitination and acetylation. However, whether VEGFR2 can be regulated by SUMOylation has not been investigated. Here we show that endothelial-specific deletion of the SUMO endopeptidase SENP1 reduces pathological angiogenesis and tissue repair during hindlimb ischemia, and VEGF-induced angiogenesis in the cornea, retina, and ear. SENP1-deficient endothelial cells show increased SUMOylation of VEGFR2 and impaired VEGFR2 signalling. SUMOylation at lysine 1270 retains VEGFR2 in the Golgi and reduces its surface expression, attenuating VEGFR2-dependent signalling. Moreover, we find that SENP1 is downregulated and VEGFR2 hyper-SUMOylated in diabetic settings and that expression of a non-SUMOylated form of VEGFR2 rescues angiogenic defects in diabetic mice. These results show that VEGFR2 is regulated by deSUMOylation during pathological angiogenesis, and propose SENP1 as a potential therapeutic target for the treatment of diabetes-associated angiogenesis.

Oleanolic Acids Inhibit Vascular Endothelial Growth Factor Receptor 2 Signaling in Endothelial Cells: Implication for Anti-Angiogenic Therapy.

Angiogenesis must be precisely controlled because uncontrolled angiogenesis is involved in aggravation of disease symptoms. Vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR-2) signaling is a key pathway leading to angiogenic responses in vascular endothelial cells (ECs). Therefore, targeting VEGF/VEGFR-2 signaling may be effective at modulating angiogenesis to alleviate various disease symptoms. Oleanolic acid was verified as a VEGFR-2 binding chemical from anticancer herbs with similar binding affinity as a reference drug in the Protein Data Bank (PDB) entry 3CJG of model A coordination. Oleanolic acid effectively inhibited VEGF-induced VEGFR-2 activation and angiogenesis in HU-VECs without cytotoxicity. We also verified that oleanolic acid inhibits in vivo angiogenesis during the development and the course of the retinopathy of prematurity (ROP) model in the mouse retina. Taken together, our results suggest a potential therapeutic benefit of oleanolic acid for inhibiting angiogenesis in proangiogenic diseases, including retinopathy.

PDGF-BB regulates splitting angiogenesis in skeletal muscle by limiting VEGF-induced endothelial proliferation

VEGF induces normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo. This transition depends on the balance between VEGF-induced endothelial stimulation and PDGF-BB-mediated pericyte recruitment, and co-expression of PDGF-BB normalizes aberrant angiogenesis despite high VEGF doses. We recently found that VEGF over-expression induces angiogenesis in skeletal muscle through an initial circumferential vascular enlargement followed by longitudinal splitting, rather than sprouting. Here we investigated the cellular mechanism by which PDGF-BB co-expression normalizes VEGF-induced aberrant angiogenesis. Monoclonal populations of transduced myoblasts, expressing similarly high levels of VEGF alone or with PDGF-BB, were implanted in mouse skeletal muscles. PDGF-BB co-expression did not promote sprouting and angiogenesis that occurred through vascular enlargement and splitting. However, enlargements were significantly smaller in diameter, due to a significant reduction in endothelial proliferation, and retained pericytes, which were otherwise lost with high VEGF alone. A time-course of histological analyses and repetitive intravital imaging showed that PDGF-BB co-expression anticipated the initiation of vascular enlargement and markedly accelerated the splitting process. Interestingly, quantification during in vivo imaging suggested that a global reduction in shear stress favored the initiation of transluminal pillar formation during VEGF-induced splitting angiogenesis. Quantification of target gene expression showed that VEGF-R2 signaling output was significantly reduced by PDGF-BB co-expression compared to VEGF alone. In conclusion, PDGF-BB co-expression prevents VEGF-induced aberrant angiogenesis by modulating VEGF-R2 signaling and endothelial proliferation, thereby limiting the degree of circumferential enlargement and enabling efficient completion of vascular splitting into normal capillary networks despite high VEGF doses.

Polydatin suppresses VEGF-induced angiogenesis through binding with VEGF and inhibiting its receptor signaling.

Polydatin, also called piceid, is a stilbenoid glucoside of a resveratrol derivative. It derives mainly from the root and rhizome of Polygonum cuspidatum Sieb. et Zucc. Although the role of P. cuspidatum root in angiogenesis has been reported, the active chemical or chemicals responsible for such function is not known. Here, polydatin was proposed to bind VEGF, which therefore altered the functions of VEGF in angiogenesis. Several lines of evidence supported the pharmaceutical effects of polydatin in VEGF-induced angiogenesis. In human umbilical vein endothelial cells, polydatin inhibited VEGF-stimulated cell proliferation, cell migration, and tube formation. Moreover, polydatin showed suppressive effects on the subintestinal vessel formation in zebrafish embryos. In signaling cascades, polydatin application attenuated VEGF-induced phosphorylations of VEGF receptor 2 and JNK. Moreover, the VEGF-induced phosphorylations of Akt, eNOS, and Erk were significantly decreased in the presence of polydatin. In parallel, the formation of reactive oxygen species, triggered by VEGF, was markedly decreased under polydatin application. Thus, our results supported the angiogenic roles of polydatin, as well as its signaling mechanism in blocking VEGF-mediated responses. The current study provides support for the possible development of polydatin as a potential therapeutic agent for treatment and prevention of angiogenesis-related diseases.-Hu, W.-H., Wang, H.-Y., Kong, X.-P., Xiong, Q.-P., Poon, K. K.-M., Xu, L., Duan, R., Chan, G. K.-L., Dong, T. T.-X., Tsim, K. W.-K. Polydatin suppresses VEGF-induced angiogenesis through binding with VEGF and inhibiting its receptor signaling.

Mechanistic Study of the In Vitro and In Vivo Inhibitory Effects of Protocatechuic Acid and Syringic Acid on VEGF-Induced Angiogenesis.

The antiangiogenic activities of two structurally similar phenolics, protocatechuic acid (PA) and syringic acid (SA), were investigated. In vitro study using HUVECs demonstrated that both PA and SA (at 25 μM) significantly ( p < 0.05) inhibited VEGF-induced cell proliferation by 22.68 ± 5.6% and 21.93 ± 2.0%, respectively; cell migration by 50.04 ± 3.3% and 39.72 ± 4.7%, respectively; cell invasion by 44.16 ± 4.23% and 51.90 ± 2.73%, respectively; and cellular ROS generation by 11.48 ± 6.32% and 21.17 ± 9.10%, respectively. Our mechanistic study revealed that PA and SA blocked the VEGFR2-dependent Akt/ MMP2 and ERK pathways in HUVECs. These inhibitory effects were further confirmed by a decrease of endogenous alkaline phosphatase activity for PA and SA (21.47 ± 1.77% and 10.37 ± 1.27%, respectively) and the suppression of subintestinal vessel plexus formation in Tg (fli1a:EGFP) y1-type transgenic zebrafish embryos. PA and SA down-regulated the angiogenesis-related signal transduction pathway of VEGFα-VEGFR2 or Ang2- Tie2 in zebrafish. Moreover, it was also found that PA demonstrated a better inhibition on VEGF-induced migration of HUVEC and zebrafish vasculature. This might be due to the different number of hydroxyl and methoxy substituents possessed by PA and SA. Taken together, these findings indicate that phenolics PA and SA have potent antiangiogenic activities and are potential targets for the design and development of anticancer agents.

New insights into hypoxia-related mechanisms involved in different microvascular patterns of bronchopulmonary carcinoids and poorly differentiated neuroendocrine carcinomas. Role of ribonuclease T2 (RNASET2) and HIF-1α

Ribonuclease T2 (RNASET2) is a pleiotropic and polyfunctional protein, which exerts several different activities in neoplastic cells since the early steps of tumor development. Besides having an antitumorigenic activity, RNASET2 inhibits both bFGF-induced and VEGF-induced angiogenesis and has a role as a stress-response, alarmin-like, protein. In this study, we investigated RNASET2 expression in well-differentiated and poorly differentiated neuroendocrine neoplasms of the lung (Lu-NENs), which are known to show clear-cut differences in morphology, biology and clinical behavior. In addition, we explored possible relationships between RNASET2 expression and a series of immunohistochemical markers related to hypoxic stress, apoptosis, proliferation and angiogenesis. Our results showed a significantly higher expression of RNASET2, HIF-1α, and its target CA IX in poorly differentiated than in well-differentiated Lu-NENs, the former also showing higher proliferation and apoptotic rates, as well as a lower microvessel density (MVD) than the latter. Moreover, we were able to demonstrate in vitro an overexpression of RNASET2 in consequence of the activation of HIF-1α. In conclusion, we suggest that in poorly differentiated Lu-NENs, RNASET2 expression may be induced by HIF-1α, behaving as an alarmin-like molecule. In this aggressive group of cancers, which have highly deregulated proliferation pathways, RNASET2 fails to exert the growth-inhibiting effects described in other types of neoplasms. Its increased expression, however, may contribute to the typical phenotypic alterations seen in poorly differentiated Lu-NENs, such as the high apoptotic rate and the extensive necrosis, and may also enhance the low MVD observed in these neoplasms.

Inhibition of autophagy potentiated the anti-tumor effects of VEGF and CD47 bispecific therapy in glioblastoma.

Glioblastoma, characterized by extensive microvascular proliferation and invasive tumor growth, is one of the most common and lethal malignancies in adults. Benefits of the conventional anti-angiogenic therapy were only observed in a subset of patients and limited by diverse relapse mechanism. Fortunately, recent advances in cancer immunotherapy have offered new hope for patients with glioblastoma. Herein, we reported a novel dual-targeting therapy for glioblastoma through simultaneous blockade of VEGF and CD47 signaling. Our results showed that VEGFR1D2-SIRPαD1, a VEGF and CD47 bispecific fusion protein, exerted potent anti-tumor effects via suppressing VEGF-induced angiogenesis and activating macrophage-mediated phagocytosis. Meanwhile, autophagy was activated by VEGFR1D2-SIRPαD1 through inactivating Akt/mTOR and Erk pathways in glioblastoma cells. Importantly, autophagy inhibitor or knockdown of autophagy-related protein 5 potentiated VEGFR1D2-SIRPαD1-induced macrophage phagocytosis and cytotoxicity against glioblastoma cells. Moreover, suppression of autophagy led to increased macrophage infiltration, angiogenesis inhibition, and tumor cell apoptosis triggered by VEGF and CD47 dual-targeting therapy, thus eliciting enhanced anti-tumor effects in glioblastoma. Our data revealed that VEGFR1D2-SIRPαD1 alone or in combination with autophagy inhibitor could effectively elicit potent anti-tumor effects, highlighting potential therapeutic strategies for glioblastoma through disrupting angiogenetic axis and CD47-SIRPα anti-phagocytic axis alone or in combination with autophagy inhibition.

Selenadiazole Derivatives Inhibit Angiogenesis-Mediated Human Breast Tumor Growth by Suppressing the VEGFR2-Mediated ERK and AKT Signaling Pathways.

Selenadiazole derivatives (SeDs) have been found to show promise in chemo-/radiotherapy applications by activating various downstream signaling pathways. However, the functional role of SeDs on angiogenesis, which is pivotal for tumor progression and metastasis, has not yet been elucidated. In the present study, we have examined the antiangiogenic activities of SeDs and elucidated their underlying mechanisms. The results showed that the as-synthesized SeDs not only enhanced their anticancer activities against several human cancer cells but also showed more potent inhibition on human umbilical vein endothelial cells (HUVECs). The in vitro results suggested that SeDs, especially 1 a, dose-dependently inhibited the vascular endothelial growth factor (VEGF)-induced cell migration, invasion, and capillary-like structure formation of HUVECs. Compound 1 a also significantly suppressed VEGF-induced angiogenesis in a Matrigel plug assay as part of a C57/BL6 mice assay by means of down regulation of VEGF. Furthermore, we found that 1 a significantly inhibited MCF-7 human breast tumor growth in nude mice without severe systematic cytotoxicity. Compound 1 a was more effective in inhibiting cell proliferation and induced a much more pronounced apoptosis effect in endothelial cells than MCF-7 cells, which implies that endothelial cells might be the primary target of 1 a. Further mechanistic studies on tumor growth inhibition effects and neovessel formation suppression demonstrated that 1 a inhibited cell viability of MCF-7 and HUVECs by induction of cell apoptosis, accompanied by poly(adenosine diphosphate ribose)polymerase (PARP) cleavage and caspase activation. Additionally, the 1 a-induced antiangiogenesis effect was achieved by abolishing the VEGF-VEGFR2-ERK/AKT (ERK=extracellular signal-regulated kinases; AKT=protein kinease B) signal axis and enhanced the apoptosis effect by triggering reactive oxygen species (ROS)-mediated DNA damage. Taken together, these results clearly demonstrate the antiangiogenic potency of SeDs and the underlying molecular mechanisms.

Manipulation of autophagy: a novelly potential therapeutic strategy for retinal neovascularization

BackgroundThe relationship between the role of VEGF and autophagy in the process of retinal angiogenesis is still unclear. In this study, we explored this issue by using the mouse retinal vascular endothelial cell (RVEC) as a model.MethodsRVECs were divided into the following groups: control, hypoxia (H), 3-methyladenine (3-MA) + H, VEGF + H, 3-MA + VEGF+H, anti-VEGF antibody + H, 3-MA+ anti-VEGF antibody + H. We then examined activation of autophagy by detecting formation of autophagosomes with transmission electron microscopy (TEM) and by counting the number of green fluorescent protein-positive (GFP+) puncta in RVECs. The turnover of microtubule associated protein 1 light chain 3 B (LC3B) and VEGF were examined by western blot. Cell migratory capacity was measured with wound healing assay and transwell assay. The capillary formation assay was performed to investigate the angiogenic capacity.ResultsHypoxia led to an increased number of autophagosome and of the GFP+ puncta, an increased ratio of LC3B-II/I and enhanced migratory and capillary-formation capacities of RVECs. Pre-treatment with 3-MA attenuated activation of autophagy and abrogated the enhanced cell migration and capillary formation under hypoxia. Exposure to VEGF significantly increased migratory and capillary formation capacities of RVECs under hypoxia and 3-MA decreased VEGF-induced angiogenesis without its expression. Formation of autophagosome, the number of GFP+ puncta of RVECs and expression of LC3B-II/I were both elevated in cells treated with anti-VEGF antibody and these effects were partially inhibited by 3-MA pretreatment.ConclusionOur present data may identify autophagic response as a novel target for enhancing the therapeutic efficacy of angiogenesis inhibitors.