VEGF-A promotes angiogenesis after acute myocardial infarction through increasing ROS production and enhancing ER stress-mediated autophagy

Proangiogenesis is generally regarded as an effective approach for treating ischemic heart disease. Vascular endothelial growth factor (VEGF)-A is a strong and essential proangiogenic factor. Reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, and autophagy are implicated in the process of angiogenesis. This study is designed to clarify the regulatory mechanisms underlying VEGF-A, ROS, ER stress, autophagy, and angiogenesis in acute myocardial infarction (AMI). A mouse model of AMI was successfully established by occluding the left anterior descending coronary artery. Compared with the sham-operated mice, the microvessel density, VEGF-A content, ROS production, expression of vascular endothelial cadherin, positive expression of 78 kDa glucose-regulated protein/binding immunoglobulin protein (GRP78/Bip), and LC3 puncta in CD31-positive endothelial cells of the ischemic myocardium were overtly elevated. Moreover, VEGF-A exposure predominantly increased the expression of beclin-1, autophagy-related gene (ATG) 4, ATG5, inositol-requiring enzyme-1 (IRE-1), GRP78/Bip, and LC3-II/LC3-I as well as ROS production in the human umbilical vein endothelial cells (HUVECs) in a dose and time-dependent manner. Both beclin-1 small interfering RNA and 3-methyladenine treatment predominantly mitigated VEGF-A-induced tube formation and migration of HUVECs, but they failed to elicit any notable effect on VEGF-A-increased expression of GRP78/Bip. Tauroursodeoxycholic acid not only obviously abolished VEGF-A-induced increase of IRE-1, GRP78/Bip, beclin-1 expression, and LC3-II/LC3-I, but also negated VEGF-A-induced tube formation and migration of HUVECs. Furthermore, N-acetyl- l -cysteine markedly abrogated VEGF-A-increased ROS production, IRE-1, GRP78/Bip, beclin-1 expression, and LC3-II/LC3-I in the HUVECs. Taken together, our data demonstrated that increased spontaneous production of VEGF-A may induce angiogenesis after AMI through initiating ROS–ER stress-autophagy axis in the vascular endothelial cells.     

Enhanced autophagy blocks angiogenesis via degradation of gastrin-releasing peptide in neuroblastoma cells

Neuroblastoma is characterized by florid vascularization leading to rapid tumor dissemination to distant organs; angiogenesis contributes to tumor progression and poor clinical outcomes. We have previously demonstrated an increased expression of gastrin-releasing peptide (GRP) and its receptor, GRPR, in neuroblastoma and that GRP activates the PI3K-AKT pathway as a proangiogenic factor during tumor progression. Interestingly, AKT activation phosphorylates MTOR, a critical negative regulator of autophagy, a cellular process involved in the degradation of key proteins. We hypothesize that inhibition of GRPR enhances autophagy-mediated degradation of GRP and subsequent inhibition of angiogenesis in neuroblastoma. Here, we demonstrated a novel phenomenon where targeting GRPR using shRNA or a specific antagonist, RC-3095, decreased GRP secretion by neuroblastoma cells and tubule formation by endothelial cells in vitro. Furthermore, shGRPR or RC-3095 treatment enhanced expression of proautophagic proteins in human neuroblastoma cell lines, BE(2)-C, and BE(2)-M17. Interestingly, rapamycin, an inhibitor of MTOR, enhanced the expression of the autophagosomal marker LC3-II and GRP was localized within LC3-II-marked autophagosomes in vitro as well as in vivo, indicating autophagy-mediated degradation of GRP. Moreover, overexpression of ATG5 or BECN1 attenuated GRP secretion and tubule formation, whereas opposite effects were observed with siRNA silencing of ATG5 and BECN1. Our data supported the role of autophagy in the degradation of GRP and subsequent inhibition of angiogenesis. Therefore, activation of autophagy may lead to novel antivascular therapeutic strategies in the treatment of highly vascular neuroblastomas.     

Induction of autophagic cell death in human ovarian carcinoma cells by Antrodia salmonea through increased reactive oxygen species generation

We reported in our previously executed studies that the fermented culture broth of Antrodia salmonea (AS), a mushroom used in Taiwanese folk medicine induced reactive oxygen species (ROS)-mediated apoptosis in human ovarian carcinoma cells. In this study, we studied the anticancer efficacies of AS (0–240 μg/ml) by examining the key molecular events implicated in cell death associated with autophagy in SKOV-3 and A2780 human ovarian carcinoma cells and clarified the fundamental molecular mechanisms. Treatment of ovarian carcinoma cells with AS-induced autophagic cell death mediated by increased microtubule-associated protein LC3-II, GFP-LC3 puncta, and acidic vesicular organelle (AVO) formation. These events are linked with the activation of p62/SQSTM1, the inhibition of ATG4B, the expression of ATG7, and the dysregulation of Beclin-1/Bcl-2 (i.e., B-cell lymphoma 2). N-acetylcysteine inhibited AS-induced ROS generation, which in turn constricted AS-induced LC3 conversion, AVO formation, and ATG4B inhibition, indicating ROS-mediated autophagy cell death. In addition, the 3-methyladenine (3-MA) or chloroquine (CQ)-induced autophagy inhibition decreased AS-induced apoptosis. Additionally, apoptosis inhibition by Z-VAD-FMK, a pan-caspase inhibitor, substantially suppressed AS-induced autophagy. Furthermore, AS-inhibited HER-2/ neu and PI3K/AKT signaling pathways which were reversed by autophagy inhibitors 3-MA and CQ. Thus, A. salmonea is a potential chemopreventive agent that is capable of activating ROS-mediated autophagic cell death in ovarian carcinoma cells.     

Role and mechanism of arsenic in regulating angiogenesis

Arsenic is a wide spread carcinogen associated with several kinds of cancers including skin, lung, bladder, and liver cancers. Lung is one of the major targets of arsenic exposure. Angiogenesis is the pivotal process during carcinogenesis and chronic pulmonary diseases, but the role and mechanism of arsenic in regulating angiogenesis remain to be elucidated. In this study we show that short time exposure of arsenic induces angiogenesis in both human immortalized lung epithelial cells BEAS-2B and adenocarcinoma cells A549. To study the molecular mechanism of arsenic-inducing angiogenesis, we find that arsenic induces reactive oxygen species (ROS) generation, which activates AKT and ERK1/2 signaling pathways and increases the expression of hypoxia-inducible factor 1 (HIF-1) and vascular endothelial growth factor (VEGF). Inhibition of ROS production suppresses angiogenesis by decreasing AKT and ERK activation and HIF-1 expression. Inhibition of ROS, AKT and ERK1/2 signaling pathways is sufficient to attenuate arsenic-inducing angiogenesis. HIF-1 and VEGF are downstream effectors of AKT and ERK1/2 that are required for arsenic-inducing angiogenesis. These results shed light on the mechanism of arsenic in regulating angiogenesis, and are helpful to develop mechanism-based intervention to prevent arsenic-induced carcinogenesis and angiogenesis in the future.     

Proper autophagy is indispensable for angiogenesis during chick embryo development

People have known that autophagy plays a very important role in many physiological and pathological events. But the role of autophagy on embryonic angiogenesis still remains obscure. In this study, we demonstrated that Atg7, Atg8 and Beclin1 were expressed in the plexus vessels of angiogenesis at chick yolk sac membrane and chorioallantoic membrane. Interfering in autophagy with autophagy inducer or inhibitor could restrict the angiogenesis in vivo, which might be driven by the disorder of angiogenesis-related gene expressions, and also lead to embryonic hemorrhage, which was due to imperfection cell junctions in endothelial cells including abnormal expressions of tight junction, adheren junction and desmosome genes. Using HUVECs, we revealed that cell viability and migration ability changed with the alteration of cell autophagy exposed to RAPA or 3-MA. Interestingly, tube formation assay showed that HUVECs ability of tube formation altered with the change of Atg5, Atg7 and Atg8 manipulated by the transfection of their corresponding siRNA or plasmids. Moreover, the lost cell polarity labeled by F-actin and the absenced β-catenin in RAPA-treated and 3-MA-treated cell membrane implied intracellular cytoskeleton alteration was induced by the activation and depression of autophagy. Taken together, our current experimental data reveal that autophagy is really involved in regulating angiogenesis during embryo development.     

Autophagy triggered by magnolol derivative negatively regulates angiogenesis

Angiogenesis has a key role in the tumor progression and metastasis; targeting endothelial cell proliferation has emerged as a promising therapeutic strategy for the prevention of cancer. Previous studies have revealed a complex association between the process of angiogenesis and autophagy and its outcome on tumorigenesis. Autophagy, also known as type-II cell death, has been identified as an alternative way of cell killing in apoptotic-resistant cancer cells. However, its involvement in chemoresistance and tumor promotion is also well known. In this study, we used a derivate of natural product magnolol (Ery5), a potent autophagy inducer, to study the association between the autophagy and angiogenesis in both in vitro and in vivo model system. We found that the robust autophagy triggered by Ery5, inhibited angiogenesis and caused cell death independent of the apoptosis in human umbilical cord vein endothelial cells and PC-3 cells. Ery5 induced autophagy effectively inhibited cell proliferation, migration, invasion and tube formation. We further demonstrated that Ery5-mediated autophagy and subsequent inhibition of angiogenesis was reversed when autophagy was inhibited through 3-methyl adenine and knocking down of key autophagy proteins ATG7 and microtubule-associated protein light chain 3. While evaluating the negative regulation of autophagy on angiogenesis, it was interesting to find that angiogenic environment produced by the treatment of VEGF and CoCl2 remarkably downregulated the autophagy and autophagic cell death induced by Ery5. These studies, while disclosing the vital role of autophagy in the regulation of angiogenesis, also suggest that the potent modulators of autophagy can lead to the development of effective therapeutics in apoptosis-resistant cancer.     

Autophagy protects against necrotic renal epithelial cell-induced death of renal interstitial fibroblasts

We recently reported that necrotic renal proximal tubular cells (RPTC) can induce the death of renal interstitial fibroblasts. Since autophagy plays either cytoprotective or cytodestructive roles depending on the experimental condition, the present study was carried out to investigate whether necrotic RPTC would induce autophagy of renal interstitial fibroblasts and, if so, whether autophagy would contribute to cell death or exert a protective effect. Exposure of necrotic RPTC supernatant (RPTC-Sup) induced autophagy in renal interstitial fibroblast cells (NRK-49F) in a time- and dose-dependent manner, and its induction was earlier than caspase-3 activation. Inhibition of autophagy with 3-methyladenine (3-MA) or knockdown of Beclin-1, a molecule involved in the initiation of autophagosome formation, with small interference RNA (siRNA) significantly enhanced necrotic RPTC-Sup-induced cell death. Necrotic RPTC-Sup induced phosphorylation of extracellular signal-regulated kinases (ERK1/2), p38, c-Jun NH(2)-terminal kinases (JNKs), and AKT. Treatment with an ERK1/2 pathway inhibitor, but not with specific inhibitors for p38, JNKs, or AKT pathways, blocked NRK-49F autophagy and cell death upon exposure to necrotic RPTC-Sup. Furthermore, knockdown of MEK1 with siRNA also reduced autophagy along with cell death in NRK-49F exposed to necrotic RPTC-Sup. In contrast, overexpression of MEK1/2 increased RPTC-Sup-induced fibroblast cell death without enhancing autophagy. Collectively, this study demonstrates that necrotic RPTC induce both autophagy and cell death and that autophagy plays a cytoprotective or prosurvival role in renal fibroblasts. Furthermore, necrotic RPTC-induced autophagy and cell death in renal fibroblasts is mediated by the activation of the MEK1-ERK1/2 signaling pathway.     

Adiponectin inhibits high glucose-induced angiogenesis via inhibiting autophagy in RF/6A cells

Adiponectin, one of the adipose-derived hormone with metabolic activity, has been reported to conversely affect angiogenesis of endothelial cells in vitro. The previous study in animal models has demonstrated that adiponectin has a protective role in retinal vascular injury following pathological stimuli. However, clinical research regarding the relationship between plasma adiponectin level and diabetic retinopathy (DR) are inconclusive. The aim of this study was to investigate the effect of adiponectin on high glucose-induced retinal angiogenesis and its association with autophagy by using rhesus choroid-retinal endothelial (RF-6A) cells as a model. We found that cell vitality decreased and cell migration and tube formation increased in the high-glucose group. Treatment with adiponectin or 3-methyladenine (3-MA, an autophagy inhibitor) increased cell viability and inhibited cell migration and tube formation. In the high-glucose group, the protein expression of Bax and apoptosis rate of cells increased and the expression of Bcl-2 decreased, whereas treatment with adiponectin or 3-MA reversed these results. Autophagy was activated in the high-glucose group to present as more LC3B fluorescent dots and higher expressions of LC3B, Atg5 proteins as well as lower expression of p62. Treatment with adiponectin or 3-MA inhibited autophagy by promoting the expression of p-PI3K, p-AKT, and p-mTOR when compared with the high-glucose group. The results of this study suggested that adiponectin inhibits high glucose-induced angiogenesis of RF/6A cells by inhibiting autophagy, and promotion of the PI3K/AKT/mTOR pathway might be involved in the anti-autophagy activities of adiponectin.     

Inhibition of miR-21-5p suppresses high glucose-induced proliferation and angiogenesis of human retinal microvascular endothelial cells by the regulation of AKT and ERK pathways via maspin

The aim of the present study is to investigate the role of miR-21-5p in angiogenesis of human retinal microvascular endothelial cells (HRMECs). HRMECs were incubated with 5 mM glucose, 30 mM glucose or 30 mM mannitol for 24 h, 48 h or 72 h. Then, HRMECs exposed to 30 mM glucose were transfected with miR-21-5p inhibitor. We found that high glucose increased the expression of miR-21-5p, VEGF, VEGFR2 and cell proliferation activity. Inhibition of miR-21-5p reduced high glucose-induced proliferation, migration, tube formation of HRMECs, and reversed the decreased expression of maspin as well as the abnormal activation of PI3K/AKT and ERK pathways. Down-regulation of maspin by siRNA significantly increased the activities of PI3K/AKT and ERK pathways. In conclusion, inhibition of miR-21-5p could suppress high glucose-induced proliferation and angiogenesis of HRMECs, and these effects may partly dependent on the regulation of PI3K/AKT and ERK pathways via its target protein maspin.     

Antiangiogenic activity of 4-O-methylgallic acid from Canavalia gladiata, a dietary legume

Development of nontoxic and biologically safe antiangiogenic agent has been highlighted as a promising way to treat angiogenesis related diseases including cancer. Herein, we isolated 4-O-methylgallic acid (4-OMGA) from the seed of Canavalia gladiata, a dietary legume, on the basis of the growth inhibitory activity for bovine aortic endothelial cells (BAECs). The compound potently inhibits endothelial cell invasion and tube formation stimulated with basic fibroblast growth factor (bFGF) at low micromolar concentrations where it shows no cytotoxicity to the cells. In addition, 4-OMGA inhibits vascular endothelial cell growth factor (VEGF) production under hypoxic condition and the production of reactive oxygen species (ROS) in the endothelial cells stimulated with VEGF. These results demonstrate that 4-OMGA is a compound having potential for an antiangiogenic agent.     

3,4‐dimethoxystilbene,a resveratrol derivative with anti‐angiogenic effect,induces both macroautophagy and apoptosis in endothelial cells

Angiogenesis plays an important role in many pathological processes. Identification of novel anti‐angiogenic agents will provide new insights into the mechanisms for angiogenesis as well as potential lead compounds for developing new drugs. In the present study, a series of resveratrol methylated derivatives have been synthesized and screened. We found trans‐3,4‐dimethoxystilbene (3,4‐DMS) with the fullest potential to develop as an anti‐angiogenic agent. In vitro and in vivo analyses suggested that 3,4‐DMS could effectively inhibit endothelial cell proliferation, migration, tube formation, and endogenous neovascularization. Our results showed that 3,4‐DMS exerted its anti‐angiogenic effect likely through induction of endothelial cell apoptosis via a pathway involving p53, Bax, cytochrome c, and caspase proteases. Moreover, 3,4‐DMS also induced macroautophagy in endothelial cells through activation of AMPK and the downstream inhibition of mTOR signaling pathway. Further studies indicated that intracellular calcium ([Ca²⁺]_i) might bridge the 3,4‐DMS‐induced apoptosis and macroautophagy through modulating reactive oxygen species (ROS) levels in endothelial cells. Combination of 3,4‐DMS with inhibitor of autophagy, such as 3‐methyladenine (3‐MA) and autophagy‐related gene (ATG) 5 small interfering RNA (siRNA), potentiated the pro‐apoptotic and anti‐angiogenic effects of 3,4‐DMS. Our study provides a novel angiogenic inhibitor and a useful tool in exploring the molecular mechanisms for the crosstalk between apoptosis and macroautophagy in endothelial cells. 3,4‐DMS could be served as a potential lead compound for developing a class of new drugs targeting angiogenesis‐related diseases. J. Cell. Biochem. 114: 697–707, 2013. © 2012 Wiley Periodicals, Inc.     

A novel effect of polymorphonuclear leukocytes in the facilitation of angiogenesis

The purpose of this study was to examine whether the adhesion of polymorphonuclear leukocytes (PMNs) to endothelial cells and/or reactive oxygen species (ROS) released from PMNs are responsible for inducing angiogenesis. Angiogenesis was assessed by tube formation using endothelial cells obtained from bovine thoracic aorta (BAECs) grown on a layer of collagen type I. Addition of PMNs to BAECs weakly induced angiogenesis. The angiogenesis induced by PMNs alone was further enhanced by treatment of the PMNs with N-formyl-methionyl-leucyl-phenylalanine (FMLP), a selective activator of PMN. The involvement of PMN adhesion to BAECs via adhesion molecules in angiogenesis was investigated by using monoclonal antibodies against E-selectin and intercellular adhesion molecule-1 (ICAM-1). These antibodies blocked both the PMN adhesion to BAECs and the enhancement of angiogenesis induced by FMLP-treated PMNs. Furthermore, the enhancement of angiogenesis by FMLP-treated PMNs was blocked by catalase, a scavenging enzyme of H2O2, but not by superoxide dismutase (SOD). These results suggest that PMNs induce angiogenesis in vitro, and that the mechanism of stimulation of angiogenesis by PMNs may involve the adherence of PMNs to endothelial cells via E-selectin and ICAM-1, and H2O2, but not superoxide. Thus, activated PMNs in pathological states may not only induce tissue injury, but may also function as regulators of angiogenesis.     

NADPH oxidase subunit p22phox-mediated reactive oxygen species contribute to angiogenesis and tumor growth through AKT and ERK1/2 signaling pathways in prostate cancer

Excessive generation of reactive oxygen species (ROS) in cancer cells is associated with cancer development, but the underlying mechanisms and therapeutic significance remain elusive. In this study, we reported that levels of ROS and p22^phox expression are greatly increased in human prostate cancer tissues, and knockdown of p22^phox by specific small interfering RNA (siRNA) decreased ROS levels in prostate cancer cells. We also showed that stable downregulation of p22^phox in prostate cancer cells inhibited cell proliferation and colony formation, which was mediated by AKT and extracellular signal-regulated kinase (ERK)1/2 signaling pathways and their downstream molecules hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF). The NADPH oxidase subunit NOX1 was also elevated in prostate cancer cells, and was involved in activation of AKT/ERK/HIF-1/VEGF pathway and regulation of cell proliferation. Knockdown of p22^phox resulted in inhibition of tumor angiogenesis and tumor growth in nude mice. These findings reveal a new function of p22^phox in tumor angiogenesis and tumor growth, and suggest that p22^phox is a potential novel target for prostate cancer treatment.     

Autophagy induction by SIRT6 is involved in oxidative stress-induced neuronal damage

SIRT6 is a NAD+-dependent histone deacetylase and has been implicated in the regulation of genomic stability, DNA repair, metabolic homeostasis and several diseases. The effect of SIRT6 in cerebral ischemia and oxygen/glucose deprivation (OGD) has been reported, however the role of SIRT6 in oxidative stress damage remains unclear. Here we used SH-SY5Y neuronal cells and found that overexpression of SIRT6 led to decreased cell viability and increased necrotic cell death and reactive oxygen species (ROS) production under oxidative stress. Mechanistic study revealed that SIRT6 induced autophagy via attenuation of AKT signaling and treatment with autophagy inhibitor 3-MA or knockdown of autophagy-related protein Atg5 rescued H2O2-induced neuronal injury. Conversely, SIRT6 inhibition suppressed autophagy and reduced oxidative stressinduced neuronal damage. These results suggest that SIRT6 might be a potential therapeutic target for neuroprotection.     

Resveratrol depletes mitochondrial DNA and inhibition of autophagy enhances resveratrol-induced caspase activation

We recently demonstrated that resveratrol induces caspase-dependent apoptosis in multiple cancer cell types. Whether apoptosis is also regulated by other cell death mechanisms such as autophagy is not clearly defined. Here we show that inhibition of autophagy enhanced resveratrol-induced caspase activation and apoptosis. Resveratrol inhibited colony formation and cell proliferation in multiple cancer cell types. Resveratrol treatment induced accumulation of LC3-II, which is a key marker for autophagy. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased resveratrol-mediated caspase activation and cell death in breast and colon cancer cells. Inhibition of autophagy by silencing key autophagy regulators such as ATG5 and Beclin-1 enhanced resveratrol-induced caspase activation. Mechanistic analysis revealed that Beclin-1 did not interact with proapoptotic proteins Bax and Bak; however, Beclin-1 was found to interact with p53 in the cytosol and mitochondria upon resveratrol treatment. Importantly, resveratrol depleted ATPase 8 gene, and thus, reduced mitochondrial DNA (mtDNA) content, suggesting that resveratrol induces damage to mtDNA causing accumulation of dysfunctional mitochondria triggering autophagy induction. Together, our findings indicate that induction of autophagy during resveratrol-induced apoptosis is an adaptive response.     

A critical role of autophagy in antileukemia/lymphoma effects of APO866, an inhibitor of NAD biosynthesis

APO866, an inhibitor of NAD biosynthesis, exhibits potent antitumor properties in various malignancies. Recently, it has been shown that APO866 induces apoptosis and autophagy in human hematological cancer cells, but the role of autophagy in APO866-induced cell death remains unclear. Here, we report studies on the molecular mechanisms underlying APO866-induced cell death with emphasis on autophagy. Treatment of leukemia and lymphoma cells with APO866 induced both autophagy, as evidenced by an increase in autophagosome formation and in SQSTM1/p62 degradation, but also increased caspase activation as revealed by CASP3/caspase 3 cleavage. As an underlying mechanism, APO866-mediated autophagy was found to deplete CAT/catalase, a reactive oxygen species (ROS) scavenger, thus promoting ROS production and cell death. Inhibition of autophagy by ATG5 or ATG7 silencing prevented CAT degradation, ROS production, caspase activation, and APO866-induced cell death. Finally, supplementation with exogenous CAT also abolished APO866 cytotoxic activity. Altogether, our results indicated that autophagy is essential for APO866 cytotoxic activity on cells from hematological malignancies and also indicate an autophagy-dependent CAT degradation, a novel mechanism for APO866-mediated cell killing. Autophagy-modulating approaches could be a new way to enhance the antitumor activity of APO866 and related agents.