Induction Of Endothelial Cell Apoptosis Research Articles

Mitochondria as a possible target for cerebral amyloid angiopathy-mediated endothelial dysfunction in the presence of hyperhomocysteinemia.

Cardiovascular (CV) risk factors may potentiate cerebral amyloid angiopathy (CAA) pathology and neurovascular dysfunction, worsening neurodegeneration. The role of the mitochondria, key regulators of cell survival and death, in mediating cerebral endothelial cell (EC) dysfunction during CAA complicated with CV risk factors is unknown. Our lab has demonstrated that carbonic anhydrase inhibitors (CAi) reduce amyloid-induced cell death and mitochondrial dysfunction in EC. Here, we aim to understand the role of the mitochondria in the pathogenesis of mixed AD and vascular dementias. Furthermore, we seek to understand the effects of pan-CAi and CAi with high selectivity for mitochondria-localized isoforms (mCAi) in preventing the toxic effects of amyloid β (Aβ) and hyperhomocysteinemia (HHcy). Human ECs were challenged with Aβ, Hcy, or the combination in the presence or absence of CAi. Cell death, mitochondrial function, and blood-brain barrier (BBB) resistance effects were evaluated. The presence of Aβ and/or HHcy induced EC apoptosis, mediated by death receptors activation and mitochondrial dysfunction, and attenuated by CAi and mCAi. BBB permeability was increased by both Aβ and HHcy, and HHcy worsened amyloid-induced barrier permeability. These effects were prevented by CAi and mCAi. Both HHcy and Aβ had detrimental effects on EC mitochondrial function and BBB permeability, and HHcy worsened the effects of Aβ. These effects were prevented by CAi and mCAi. The ability of mCAi to protect against Aβ and/or HHcy suggests a central role of the mitochondria in mediating vascular dysfunction in the presence of Aβ and/or HHcy. Our studies support the importance of better understanding mitochondrial and EC pathways responsible for cerebrovascular dysfunction in CAA and mixed dementias.

Zearalenone Induces Endothelial Cell Apoptosis through Activation of a Cytosolic Ca2+/ERK1/2/p53/Caspase 3 Signaling Pathway.

Zearalenone (ZEN) is a mycotoxin that has been reported to damage various types of cells/tissues, yet its effects on endothelial cells (ECs) have never been investigated. Therefore, this study investigates the potential effects of ZEN using bovine aortic ECs (BAECs). In this study, we found that ZEN induced apoptosis of BAECs through increased cleavage of caspase 3 and poly ADP-ribose polymerase (PARP). ZEN also increased phosphorylation of ERK1/2 and p53, and treatment with the ERK1/2 or p53 inhibitor reversed ZEN-induced EC apoptosis. Transfection of BAECs with small interfering RNA against ERK1/2 or p53 revealed ERK1/2 as an upstream target of p53 in ZEN-stimulated apoptosis. ZEN increased the production of reactive oxygen species (ROS), yet treatment with the antioxidant did not prevent EC apoptosis. Similarly, blocking of estrogen receptors by specific inhibitors also did not prevent ZEN-induced apoptosis. Finally, chelation of cytosolic calcium (Ca2+) using BAPTA-AM or inhibition of endoplasmic reticulum (ER) Ca2+ channel using 2-APB reversed ZEN-induced EC apoptosis, but not by inhibiting ER stress using 4-PBA. Together, our findings demonstrate that ZEN induces EC apoptosis through an ERK1/2/p53/caspase 3 signaling pathway activated by Ca2+ release from the ER, and this pathway is independent of ROS production and estrogen receptor activation.

MiR-155 induces endothelial cell apoptosis and inflammatory response in atherosclerosis by regulating Bmal1.

Atherosclerosis is the leading cause of death from vascular diseases worldwide, and endothelial cell (EC) dysfunction is the key cause of atherosclerosis. miR-155 was found to induce endothelial injury and to trigger atherosclerosis. In addition, brain and muscle ARNT-like protein-1 (Bmal1) has been found to be closely related to EC function. Therefore, the present study aimed to explore the mechanism underlying the regulation of Bmal1 by miR-155 in the induction of EC apoptosis and inflammatory response in atherosclerosis. The atherosclerosis model in apolipoprotein E (ApoE)-/- mice was established. miR-155 and Bmal1 expression was quantified by RT-qPCR and western blot analysis, respectively. The role of miR-155 and Bmal1 in atherosclerosis was evaluated through changes in cardiac function, plaque area, cardiomyocyte apoptosis, and inflammatory factor levels in mice. Moreover, the regulatory relationship between them was identified by dual-luciferase reporter gene assay to explore the mechanism of action of miR-155. After the modeling, the expression of miR-155 was upregulated and Bmal1 was downregulated in aorta, and there was a significant linear correlation between them. Upregulation of miR-155 increased the atherosclerotic plaque area, cell apoptosis, total cholesterol (TC) and triglyceride (TG), as well as weakened aortic diastolic function. However, opposite changes occurred after downregulation of miR-155 or an increase in Bmal1. In addition, the microRNA.org website predicted that there were targeted binding sites between miR-155 and Bmal1, which was verified with a dual-luciferase reporter gene assay. miR-155 was able to inhibit the expression by targeting Bmal1. Moreover, a rescue experiment showed that Bmal1 hindered the promotion of miR-155 in regards to atherosclerosis. In conclusion, miR-155 induces EC apoptosis and inflammatory response, weakens aortic diastolic function, and promotes the progression of atherosclerosis through targeted inhibition of Bmal1.

CD137 Signaling Promotes Endothelial Apoptosis by Inhibiting Nrf2 Pathway, and Upregulating NF-κB Pathway.

Background Endothelial dysfunction and apoptosis resulting from oxidative stress can lead to the development of atherosclerosis. Our group has previously showed that CD137 signaling contributes to the progression of atherosclerosis and the vulnerability of plaques. The aim of this study is to investigate the effects of CD137 signaling in atherosclerosis on endothelial cells (ECs) apoptosis and to explore the underlying mechanisms. Methods Serum samples were collected from 11 patients with acute myocardial infarction and 4 controls. Peritoneal injection of agonist-CD137 recombinant protein in ApoE−/− mice was used to determine whether CD137 signaling can promote apoptosis in vivo, and human umbilical vein endothelial cells treated with agonist-CD137 recombinant protein, M5580 (a Nrf2 pathway agonist) and CAPE (a NF-κB pathway inhibitor) were used to explore the effect of Nrf2 and NF-κB pathway in CD137 signaling-induced ECs apoptosis in vitro. Results ELISA showed that Bcl-2 in the serum of AMI patients was lower than that of the control group, while TNF-α and sCD137 were higher than that of the control group. Confocal microscopy and Western blot analysis showed that the nuclear translocation of Nrf2 in the agonist-CD137 group was significantly inhibited, and the expression of its downstream antioxidant enzymes was also decreased when compared with control. Immunofluorescence and Western blot results showed that the nuclear translocation of NF-κB in the agonist-CD137 group was enhanced, and ELISA results showed that the secretion of proinflammatory cytokines in the agonist-CD137 group was increased. Immunofluorescence results revealed that ROS production in the agonist-CD137 group was higher than that in control, M5580 (a Nrf2 pathway agonist) and CAPE (a NF-κB pathway inhibitor) groups. In vitro studies using HUVECs and in vivo studies using high-fat-fed ApoE−/− mice showed that the number of apoptotic endothelial cells was the highest in the agonist-CD137 group. By contrast, both M5580 and CAPE treatments were able to reduce CD137 induced ECs apoptosis. Conclusions Our results showed that CD137 signaling promotes ECs apoptosis through prooxidative and proinflammatory mechanisms, mediated by Nrf2 and NF-κB pathways, respectively.

Downregulation of microRNA‑106a‑5p alleviates ox‑LDL‑mediated endothelial cell injury by targeting STAT3.

The apoptosis of endothelial cells (ECs) induced by oxidized low-density lipoprotein (ox-LDL) is an important contributing factor in the pathogenesis of atherosclerosis. It has been reported that microRNA (miR)-106a-5p is overexpressed in atherosclerotic plaques and involved in angiogenesis. However, its role and underlying mechanisms in ox-LDL induced EC apoptosis remain to be fully understood. In the present study the expression of miR-106a-5p in human umbilical vein ECs (HUVECs) stimulated with ox-LDL was investigated using reverse transcription-quantitative PCR analysis. Cell viability and apoptosis were assessed by MTT assay and flow cytometry, respectively. Caspase-3 activity and reactive oxygen species (ROS) levels were determined by commercial kits. The interaction between miR-106a-5p and signal transducer and activator of transcription 3 (STAT3) mRNA was examined by luciferase reporter assay. It was found that ox-LDL treatment significantly increased the levels of miR-106a-5p in a dose-dependent manner in HUVECs. Moreover, these results demonstrated that ox-LDL treatment inhibited cell viability, promoted cell apoptosis, increased caspase-3 activity and ROS levels, whereas inhibition of miR-106a-5p reversed the effects of ox-LDL on HUVECs. In addition, it was shown that STAT3 is a direct target of miR-106a-5p in HUVECs, and silencing of STAT3 impaired the protective effects of miR-106a-5p inhibition on cell apoptosis and oxidative injury induced by ox-LDL. Collectively, these results indicated that miR-106a-5p participated in ox-LDL-stimulated apoptosis and oxidative injury in HUVECs by regulating STAT3. Thus, suggesting that miR-106a-5p/STAT3 may serve as a novel therapeutic target for atherosclerosis in the future.

Rosuvastatin protects against endothelial cell apoptosis in vitro and alleviates atherosclerosis in ApoE-/- mice by suppressing endoplasmic reticulum stress.

The development of abnormal lipid-induced atherosclerosis is initiated with endothelial cell apoptosis. Vascular endothelial cells possess highly developed endoplasmic reticulum (ER), which is involved in lipid metabolism, indicating that ER stress may contribute chiefly to the induction of endothelial cell apoptosis. Based on its ability to reduce cholesterol levels, rosuvastatin may play an endothelial and vascular protective role by regulating ER stress. In the present study, the involvement of the inhibition of the ER stress-induced endothelial injury was investigated in combination with the lipid lowering effects of rosuvastatin. This compound can be used to inhibit cholesterol synthesis in atherosclerosis. Rosuvastatin decreased the apoptotic rates of human umbilical vascular endothelial cells (HUVECs) that had been stimulated with ox-low density lipoprotein (LDL) in vitro and repressed the mRNA levels of CHOP, sXBP1 and caspase-12, and decreased caspase-12 activity, as well as the content of glucose-regulated protein 78 (GRP78), phosphorylated (p)-protein kinase RNA-like ER kinase (PERK), p-inositol-requiring protein 1α (IRE1α) and p-eIF2α proteins. In addition, ApoE-/- mice were fed with atherogenic chow for 8 weeks for atherosclerosis induction and rosuvastatin was provided by intragastric administration for an additional 4 weeks. Subsequently, the atherosclerotic plaque formation in the aorta was evaluated by Oil Red O and hematoxylin and eosin staining, and the serum LDL, high-density lipoprotein, total cholesterol (TC) and triacylglycerol (TG) levels were measured. In addition, the induction of apoptosis of endothelial cells and the expression levels of GRP78, p-PERK, p-IRE1α and p-eIF2α were assessed in the aorta. Rosuvastatin repressed atherosclerotic plaque formation and endothelial apoptosis in the aorta and decreased LDL and TG levels in the serum, as determined by in vivo results. Furthermore, it downregulated the expression levels of protein chaperone GRP78, p-PERK, p-IRE1α and p-eIF2α in the aortic intima. The data indicated that rosuvastatin could protect HUVECs from ER stress-induced apoptosis triggered by oxidized LDL. It could also inhibit atherosclerosis formation in ApoE-/- mice aorta by regulating the PERK/eIF2α/C/EBPα-homologous protein and IRE1α/sXBP1 signaling pathways. Taken collectively, the present study demonstrated the preventive and therapeutic effects of rosuvastatin in protecting from the development of endothelial cell dysfunction diseases.

Thrombospondin-5 and fluvastatin promote angiogenesis and are protective against endothelial cell apoptosis.

The thrombospondins (TSPs), multifunctional matricellular proteins, are known mediators of endothelial cell (EC) angiogenesis and apoptosis. TSP-1, an antiangiogenic molecule, is important in the progression of vascular disease, in part by inducing EC apoptosis. TSP-2, although less studied, also induces EC apoptosis and inhibits angiogenesis. The effects of TSP-5 are largely unexplored in ECs, but TSP-5 is believed to be protective against arterial disease. Statin drugs have been shown to have beneficial pleiotropic effects, including decreasing EC apoptosis, increasing angiogenesis, and blocking TSP signaling. We hypothesized TSP-5 will be proangiogenic and antiapoptotic, and statin pretreatment would reverse the proapoptotic and antiangiogenic phenotype of TSP-1 and TSP-2. ECs were exposed to serum-free medium, TSP-1, TSP-2, or TSP-5 with or without fluvastatin pretreatment. Quantitative real-time polymerase chain reaction was performed on 96 apoptosis and 96 angiogenesis-related genes using microfluidic card assays. Angiogenesis was measured using Matrigel assays, while apoptosis was measured by fluorescent caspase assay. TSP-5 suppressed apoptotic genes and had a mixed effect on the angiogenic genes; however, TSP-5 did not alter apoptois but was proangiogenic. Pretreatment with fluvastatin downregulated proapoptotic genes and apoptosis and upregulated proangiogenic genes and angiogenesis. Findings indicate TSP-5 and fluvastatin have a protective effect on ECs, being proangiogenic and reversing the antiangiogenic effects of TSP-1 and TSP-2. In conclusion, TSP-5 and fluvastatin may be beneficial for inducing angiogenesis in the setting of ischemia.

Low shear stress induces endothelial cell apoptosis and monocyte adhesion by upregulating PECAM‑1 expression.

Low shear stress serves an important role in the initiation and progression of atherosclerotic lesions, with an impact on progression, but its detailed mechanisms are .not yet fully known. The present study aimed to investigate endothelial cell (EC) apoptosis, as well as monocyte adhesion induced by low shear stress and the potential underlying mechanisms. The expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) was demonstrated to be enhanced in human umbilical vascular ECs with a trend that was associated with time when stimulated by low shear stress compared with unstimulated cells. EC apoptosis was increased under low shear stress compared with unstimulated cells, and knockdown of PECAM-1 inhibited this process. Furthermore, downregulation of PECAM-1 reduced monocyte adhesion induced by low shear stress compared with that in the negative control cells. Mechanistically, PECAM-1 small interfering RNA transfection increased Akt and forkhead box O1 phosphorylation under low shear stress conditions compared with that in the negative control cells. Collectively, the findings of the present study revealed that low shear stress induced EC apoptosis and monocyte adhesion by upregulating PECAM-1 expression, which suggested that PECAM-1 may be a potential therapeutic target for atherosclerosis.

C1q/TNF-related protein 5 contributes to diabetic vascular endothelium dysfunction through promoting Nox-1 signaling

ObjectiveDysregulated adipokine profiles contribute to the pathogenesis of diabetic cardiovascular complications. Endothelial cell (EC) dysfunction, a common pathological alteration in cardiovascular disorders, is exaggerated in diabetes. However, it is unclear whether and how dysregulated adipokines may contribute to diabetic EC dysfunction. Methods and resultsSerum C1q/TNF-Related Protein 5 (CTRP5) were determined in control/diabetes patients, and control/diabetic mice (high-fat diet, HFD). We observed for the first time that serum total CTRP5 was increased, high molecular weight (HMW) form was decreased, but the globular form (gCTRP5) was significantly increased in diabetic patients. These pathological alterations were reproduced in diabetic mice. To determine the pathological significance of increased gCTRP5 in diabetes, in vivo, ex vivo and in vitro experiments were performed. Diabetic atherosclerosis and EC dysfunction were significantly attenuated by the in vivo administration of CTRP5 neutralization antibody (CTRP5Ab). EC apoptosis was significantly increased in diabetic EC (isolated from HFD animal aorta) or high glucose high lipid (HGHL) cultured HUVECs. These pathological alterations were further potentiated by gCTRP5 and attenuated by CTRP5Ab. Pathway specific discovery-driven approach revealed that Nox1 expression was one of the signaling molecules commonly activated by HFD, HGHL, and gCTRP5. Treatment with CTRP5Ab reversed HFD-induced Nox1 upregulation. Finally, Nox1siRNA was used to determine the causative role of Nox1 in gCTRP5 induced EC apoptosis in diabetes. Results showed that gCTRP5 activated the mitochondrial apoptotic signal of EC in diabetes, which was blocked by the silencing Nox1 gene. ConclusionWe demonstrated for the first time that gCTRP5 is a novel molecule contributing to diabetic vascular EC dysfunction through Nox1-mediated mitochondrial apoptosis, suggesting that interventions blocking gCTRP5 may protect diabetic EC function, ultimately attenuate diabetic cardiovascular complications.

Abstract 16591: Exosome-Mediated Endothelial Cell Apoptosis and Network Disruption by Lymphangioleiomyomatosis-Smooth Muscle Cells (LAM-SMCs) Derived from Human Induced Pluripotent Stem Cell (iPSC)

Introduction: In LAM, SMC-like cells invade the lung thus compromising respiratory function which is dependent on normal vascular and alveoli structure. We posit that while healthy mural SMCs stabilize fragile neovessels and maintain vascular homeostasis, LAM-SMCs promote disruption of lung microvasculature by inducing endothelial cell (EC) apoptosis. Methods/Results: iPSCs derived from LAM patients or healthy subjects were differentiated into SMCs using a teratoma protocol. Co-culture of healthy iSMCs with HUVECs on Matrigel improved EC network persistence for ≥72Hrs, while LAM-SMC co-culture led to rapid network collapse in <15Hrs. Culture of ECs with LAM-SMC conditioned media (24-72Hrs) increased EC apoptosis as assessed by Annexin V/PI flow cytometry, confirmed by increases in cleaved Caspase-3 protein expression. To further elucidate mechanism and kinetics of EC apoptosis, Annexin V/PI flow cytometry and Western Blot with Caspases 8,9,12 were performed in parallel at 3hr intervals from 0-12 Hrs. Increase in EC apoptosis at 9hrs, corresponded with a 6-fold increase in Cas-12 protein expression, followed by a 3-fold increase in Cas-9 at 12hrs with little change in Cas-8. This is in line with our RNAseq study showing dysregulation in endoplasmic reticulum and mitochondria related genes in LAM-SMC. Culture of HUVECs with LAM-SMC exosomes similarly induced EC apoptosis, with a profound decrease in EC gene expression (eNOS,TIE2,TAL1; 5-7 fold; p<0.05) but increase expression of vessel-destabilizing, ANGPT2 (6-fold; p<0.05). Scratch-wound assay also showed ~60% reduction (p<0.05) in EC migratory capacity. Intriguingly, apoptotic ECs released exosomes containing translationally controlled tumor protein, TCTP, which were taken up by LAM-SMCs, as evidenced by immunofluorescence studies, associated with increases in the levels of proliferation-associated phospho-mTOR-and-S6k protein. Conclusions: LAM-SMCs derived exosomes resulted in EC apoptosis and network destabilization. Conversely, apoptotic ECs enhanced LAM-SMC growth, possibly by Caspase-dependent exosome release of TCTP, a positive regulator of mTOR signaling. These novel mechanisms may promote both microvasculature disruption and uncontrolled growth of LAM-SMCs.

MiR21 sensitized B-lymphoma cells to ABT-199 via ICOS/ICOSL-mediated interaction of Treg cells with endothelial cells

BackgroundMicroRNAs (miRs) are involved in tumor progression by regulating tumor cells and tumor microenvironment. MiR21 is overexpressed in diffuse large B-cell lymphoma (DLBCL) and its biological impact on tumor microenvironment remains unclear.MethodsMiR21 was assessed by quantitative RT-PCR in patients with newly diagnosed DLBCL. The mechanism of action of miR21 on lymphoma progression and tumor angiogenesis was examined in vitro in B-lymphoma cell lines and in vivo in a murine xenograft model.ResultsSerum miR21 was significantly elevated in patients and associated with advanced disease stage, International Prognostic Index indicating intermediate-high and high-risk, and increased tumor angiogenesis. When co-cultured with immune cells and endothelial cells, miR21-overexpressing B-lymphoma cells were resistant to chemotherapeutic agents, but sensitive to Bcl-2 inhibitor ABT-199, irrespective of Bcl-2 expression on lymphoma cells. In both co-culture systems of Bcl-2positive and Bcl-2negative B-lymphoma cells, miR21 induced inducible co-stimulator (ICOS) expression on regulatory T (Treg) cells. Through crosstalking with Treg cells by ICOS ligand (ICOSL), endothelial cells were activated, resulting in stimulation of Bcl-2 expression and vessel formation. ABT-199 directly targeted Bcl-2 on endothelial cells, induced endothelial cell apoptosis and inhibited tumor angiogenesis. In a murine xenograft model established with subcutaneous injection of B-lymphoma cells, ABT-199 particularly retarded the growth of miR21-overexpressing tumors, consistent with the induction of endothelial cell apoptosis and inhibition of tumor angiogenesis.ConclusionsAs a serum oncogenic biomarker of B-cell lymphoma, miR21 indicated B-lymphoma cell sensitivity to ABT-199 via ICOS/ICOSL-mediated interaction of Treg cells with endothelial cells.

Dose dependent effects of cadmium on tumor angiogenesis

Angiogenesis is crucial for tumor growth and metastasis. Cadmium (Cd) exposure is associated with elevated cancer risk and mortality. Such association is, at least in part, attributable to Cd-induced tumor angiogenesis. Nevertheless, the reported effects of Cd on tumor angiogenesis appear to be either stimulatory or inhibitory, depending on the concentrations. Ultra-low concentrations of Cd (<0.5 μM) inhibit endothelial nitric oxide synthase activation, leading to reduced endothelial nitric oxide production and attenuated tumor angiogenesis. In contrast, low-lose Cd (1-10 μM) up-regulates vascular endothelial growth factor (VEGF)-mediated tumor angiogenesis by exerting sub-apoptotic levels of oxidative stress on both tumor cells and endothelial cells (ECs). The consequent activation of protein kinase B/Akt, nuclear factor-κB, and mitogen-activated protein kinase signaling cascades mediate the increased secretion of VEGF by tumor cells and the up-regulated VEGF receptor-2 expression in ECs. Furthermore, Cd in high concentrations (>10 μM) induces EC apoptosis via the activation of caspase-3, resulting in destruction of tumor vasculature. In this review, we summarize the current knowledge concerning the roles of Cd in tumor angiogenesis, with a focus on molecular mechanisms underlying the dose dependent effects of Cd on various EC phenotypes.

Antiendothelial αvβ3 Antibodies Are a Major Cause of Intracranial Bleeding in Fetal/Neonatal Alloimmune Thrombocytopenia

Fetal/neonatal alloimmune thrombocytopenia is a severe bleeding disorder, which can result in intracranial hemorrhage (ICH), leading to death or neurological sequelae. In whites, maternal anti-human platelet antigen-1a (HPA-1a) antibodies are responsible for the majority of cases. No predictive factors for ICH are available to guide prophylactic treatment during pregnancy. In this study, we investigated antibodies from mothers with ICH-positive fetal/neonatal alloimmune thrombocytopenia and with ICH-negative fetal/neonatal alloimmune thrombocytopenia to identify serological and functional differences between the groups. In an antigen capture assay, we observed a stronger binding of +ICH antibodies to endothelial cell (EC)-derived αvβ3. By absorption experiments, we subsequently identified anti-HPA-1a antibodies of anti-αvβ3 specificity in the +ICH but not in the -ICH cohort. Only the anti-αvβ3 subtype, but not the anti-β3 subtype, induced EC apoptosis of HPA-1a-positive ECs by caspase-3/7 activation, and mediated by reactive oxygen species. In addition, only the anti-αvβ3 subtype, but not the anti-β3 subtype, interfered with EC adhesion to vitronectin and with EC tube formation. We conclude that the composition of the anti-HPA-1a antibody subtype(s) of the mother may determine whether ICH occurs. Analysis of anti-HPA-1a antibodies of the anti-αvβ3 subtype in maternal serum has potential in the diagnostic prediction of ICH development and may allow for modification of prophylactic treatment in fetal/neonatal alloimmune thrombocytopenia.

Pterostilbene carboxaldehyde thiosemicarbazone, a resveratrol derivative inhibits 17β-Estradiol induced cell migration and proliferation in HUVECs

Angiogenesis plays important roles in tumor growth and metastasis, thus development of a novel angiogenesis inhibitor is essential for the improvement of therapeutics against cancer. Thrombospondins-1 (TSP-1) is a potent endogenous inhibitor of angiogenesis that acts through direct effects on endothelial cell migration, proliferation, survival, and activating apoptotic pathways. TSP-1 has been shown to disrupt estrogen-induced endothelial cell proliferation and migration. Here we investigated the potential of pterostilbene carboxaldehyde thiosemicarbazone (PTERC-T), a novel resveratrol (RESV) derivative, to inhibit angiogenesis induced by female sex steroids, particularly 17β-Estradiol (E2), on Human umbilical vein endothelial cells (HUVECs) and to elucidate the involvement of TSP-1 in PTERC-T action. Our results showed that PTERC-T significantly inhibited 17β-E2-stimulated proliferation of HUVECs and induced apoptosis as determined by annexin V/propidium iodide staining and cleaved caspase-3 expression. Furthermore, PTERC-T also inhibited endothelial cell migration, and invasion in chick chorioallantoic membrane (CAM) assay. In contrast, RESV failed to inhibit 17β-E2 induced HUVECs proliferation and invasion at similar dose. PTERC-T was also found to increase TSP-1 protein expression levels in a dose-dependent manner which, however, was counteracted by co-incubation with p38MAPK or JNK inhibitors, suggesting involvement of these pathways in PTERC-T action. These results suggest that the inhibitory effect of PTERC-T on 17β-E2 induced angiogenesis is associated, at least in part, with its induction of endothelial cell apoptosis and inhibition of cell migration through targeting TSP-1. Thus, PTERC-T could be considered as a potential lead compound for developing a class of new drugs targeting angiogenesis-related diseases.

PEDF and 34-mer inhibit angiogenesis in the heart by inducing tip cells apoptosis via up-regulating PPAR-γ to increase surface FasL.

Pigment epithelial-derived factor (PEDF) is a potent anti-angiogenic factor whose effects are partially mediated through the induction of endothelial cell apoptosis. However, the underlying mechanism for PEDF and the functional PEDF peptides 34-mer and 44-mer to inhibit angiogenesis in the heart has not been fully established. In the present study, by constructing adult Sprague-Dawley rat models of acute myocardial infarction (AMI) and in vitro myocardial angiogenesis, we showed that PEDF and 34-mer markedly inhibits angiogenesis by selectively inducing tip cells apoptosis rather than quiescent cells. Peptide 44-mer on the other hand exhibits no such effects. Next, we identified Fas death pathway as essential downstream regulators of PEDF and 34-mer activities in inhibiting angiogenesis. By using peroxisome proliferator-activated receptor γ (PPAR-γ) siRNA and PPAR-γ inhibitor, GW9662, we found the effects of PEDF and 34-mer were extensively blocked. These data suggest that PEDF and 34-mer inhibit angiogenesis via inducing tip cells apoptosis at least by means of up-regulating PPAR-γ to increase surface FasL in the ischemic heart, which might be a novel mechanism to understanding cardiac angiogenesis after AMI.

Role of JNK and NF-κB in mediating the effect of combretastatin A-4 and brimamin on endothelial and carcinoma cells.

The 4,5-diarylimidazole brimamin is an analog of the natural vascular-disrupting agent combretastatin A-4 (CA-4) with improved water solubility, tolerance by animals and efficacy in multidrug-resistant tumors. Here, we aimed at identifying the major mechanisms underlying the in vitro and in vivo actions of brimamin on endothelial and carcinoma cells, including vascularization. The contribution of specific signaling kinases to the effects of brimamin on cytoskeleton organization and the viability and differentiation of endothelial cells was assessed by MTT and tube formation assays in the presence or absence of specific kinase inhibitors. Changes in DNA affinity and expression of NF-κB in endothelial and carcinoma-derived cells and their solid tumors (xenografts) treated with brimamin were ascertained by electrophoretic mobility shift assays and Western blotting. The anti-vascular effect of brimamin in solid tumors was verified by CD31 immunostaining. We found that brimamin can inhibit tubulin polymerization and cause a reorganization of F-actin in Ea.hy926 endothelial cells. Its inhibitory effect on tube formation was found to depend on functional Rho kinase and JNK. JNK inhibition was found to suppress the induction of endothelial cell apoptosis by brimamin. In CA-4-refractory human BxPC-3 pancreas carcinoma-derived and triple-negative MDA-MB-231 breast carcinoma-derived cells brimamin was found to inhibit growth and to induce apoptosis at low nanomolar concentrations by blocking NF-κB activation in a dose-dependent manner. Brimamin was also found to reduce the in vivo growth rate and vascularization of MDA-MB-231 xenografts in mice. Residual tumor cells of these treated xenografts showed a relatively low expression of the p65 subunit of NF-κB. Our data indicate that cellular JNK and Rho kinase activities are crucial for the cytotoxic and cytoskeleton reorganizing effects of brimamin on endothelial cells. In addition, we found that in resistant carcinoma cells and xenografts brimamin can induce down-regulation of anti-apoptotic NF-κB expression and signaling. Its chemical properties and efficacy against clinically relevant cancer entities make brimamin a promising candidate vascular-disrupting agent.

TLR2 and neutrophils potentiate endothelial stress, apoptosis and detachment: implications for superficial erosion.

Superficial erosion of atheromata causes many acute coronary syndromes, but arises from unknown mechanisms. This study tested the hypothesis that Toll-like receptor-2 (TLR2) activation contributes to endothelial apoptosis and denudation and thus contributes to the pathogenesis of superficial erosion. Toll-like receptor-2 and neutrophils localized at sites of superficially eroded human plaques. In vitro, TLR2 ligands (including hyaluronan, a matrix macromolecule abundant in eroded lesions) induced endothelial stress, characterized by reactive oxygen species production, endoplasmic reticulum (ER) stress, and apoptosis. Co-incubation of neutrophils with endothelial cells (ECs) potentiated these effects and induced EC apoptosis and detachment. We then categorized human atherosclerotic plaques (n = 56) based on morphologic features associated with superficial erosion, 'stable' fibrotic, or 'vulnerable' lesions. Morphometric analyses of the human atheromata localized neutrophils and neutrophil extracellular traps (NETs) near clusters of apoptotic ECs in smooth muscle cell (SMC)-rich plaques. The number of luminal apoptotic ECs correlated with neutrophil accumulation, amount of NETs, and TLR2 staining in SMC-rich plaques, but not in 'vulnerable' atheromata. These in vitro observations and analyses of human plaques indicate that TLR2 stimulation followed by neutrophil participation may render smooth muscle cell-rich plaques susceptible to superficial erosion and thrombotic complications by inducing ER stress, apoptosis, and favouring detachment of EC.

Silencing p75NTR prevents proNGF-induced endothelial cell death and development of acellular capillaries in rat retina

Accumulation of the nerve growth factor precursor (proNGF) and its receptor p75NTR have been associated with several neurodegenerative diseases in both brain and retina. However, whether proNGF contributes to microvascular degeneration remain unexplored. This study seeks to investigate the mechanism by which proNGF/p75NTR induce endothelial cell (EC) death and development of acellular capillaries, a surrogate marker of retinal ischemia. Stable overexpression of the cleavage-resistant proNGF and molecular silencing of p75NTR were utilized in human retinal EC and rat retinas in vivo. Stable overexpression of proNGF decreased NGF levels and induced retinal vascular cell death evident by 1.9-fold increase in acellular capillaries and activation of JNK and cleaved-PARP that were mitigated by p75NTRshRNA. In vitro, overexpression of proNGF did not alter TNF-α level, reduced NGF, however induced EC apoptosis evident by activation of JNK and p38 MAPK, cleaved-PARP. Silencing p75NTR using siRNA restored expression of NGF and TrkA activation and prevented EC apoptosis. Treatment of EC with human-mutant proNGF induced apoptosis that coincided with marked protein interaction and nuclear translocation of p75NTR and the neurotrophin receptor interacting factor. These effects were abolished by a selective p75NTR antagonist. Therefore, targeting p75NTR represents a potential therapeutic strategy for diseases associated with aberrant expression of proNGF.

Abstract 1024: T11TS impedes glioma angiogenesis by attenuating brain endothelial Angiopoietin-1/Tie2 signaling and inducing apoptosis of glioma associated brain endothelial cells

Abstract Malignant gliomas are highly invasive primary brain tumors resistant to conventional treatments primarily because of sustained angiogenesis and infiltrative nature. Despite multimodal therapeutic interventions high grade gliomas have a propensity for recurrence. We recently reported that T11-target structure (T11TS), a membrane glycopeptide isolated from sheep erythrocyte membrane inhibits VEGF signaling and prosurvival PI3K/Akt pathway in glioma associated brain endothelial cells. Angiopoietin signaling mediated through endothelial Tie2 receptors is a potent mediator of glioma angiogenesis. Apoptosis of tumor endothelial cells acts as a crucial regulator of tumor angiogenesis and some angiogenesis inhibitors mediate their anti-angiogenic effect through induction of endothelial cell apoptosis. The effect of T11TS on brain endothelial cell Angiopoietin-1/Tie2 signaling and endothelial cell apoptosis has not been investigated so far. The goal of this study is to determine the effects of T11TS administration on endothelial Angiopoietin-1/Tie 2 signaling and on apoptosis of brain endothelial cells in a chemically induced glioma model. Furthermore, this study investigated the mechanistic details of brain endothelial cell apoptosis induction caused by in vivo T11TS therapy. Expression of angiopoietin-1, Tie2, phospho-Tie2 proteins, apoptotic pathway regulator proteins and caspases were determined by flowcytometry, immunoblotting, immunofluorescence imaging and in situ immunofluorescent staining. Apoptosis in brain endothelial cells was directly evaluated by flowcytomeric Annexin V-PI assay and mitochondrial membrane JC-1 assay. T11TS administration in glioma induced rats significantly downregulate Angiopoietin-1 expression and Tie2 receptor activation in brain endothelial cells thereby blocking Angiopoietin-1/Tie2 signaling. T11TS therapy triggered dose-dependent externalization of membrane phosphatidylserine, reduction in mitochondrial membrane potential and cleavage of caspase 3 which strongly indicated induction of apoptosis in glioma associated brain endothelial cells. T11TS administration in glioma was found to activate both intrinsic and extrinsic pathways of apoptosis by modulating expression of apoptotic regulator proteins like p53, Bax, Bcl-2, Fas and FasL, triggering cytochorome c release into cytosol and activating caspases 8, 3 and 9 in brain endothelial cells. T11TS administration also stimulated cleavage of Bid in glioma associated brain endothelial cells. This preclinical study demonstrated that blockage of Angiopoietin-1/Tie2 signaling in glioma associated brain endothelial cells and induction of apoptosis in brain endothelial cells constitute the underlying mechanistic events that contribute to the anti-angiogenic efficacy of T11TS in malignant glioma. Citation Format: Debanjan Bhattacharya, Suhnrita Chaudhuri, Swapna Chaudhuri. T11TS impedes glioma angiogenesis by attenuating brain endothelial Angiopoietin-1/Tie2 signaling and inducing apoptosis of glioma associated brain endothelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1024. doi:10.1158/1538-7445.AM2014-1024

Abstract 5447: Targeting vascular endothelial growth factor A and tumor hypoxia combined with radiation eradicates sarcomas through destruction of tumor vasculature and thwarting of the hypoxic response

Abstract Introduction: In a previous clinical trial, we demonstrated that bevacizumab, an anti-vascular endothelial growth factor A (VEGF-A) antibody, combined with radiation leads to &amp;gt;80% tumor necrosis in about one-half of patients with soft tissue sarcomas. Sarcomas with a poor response to this therapy had upregulation of hypoxia inducible factor 1α (HIF-1α) and HIF-1α target genes. Here, we describe two trimodality approaches to this problem: (1) blockade of HIF-1α using genetic or pharmacologic inhibition and (2) destruction of hypoxic regions of tumors using the hypoxia-activated chemotherapeutic, TH-302. Methods: Trimodality therapies were used in a HT1080 fibrosarcoma xenograft model and the LSL-KrasG12D/+/Trp53fl/fl genetically engineered mouse model of sarcoma. Treated tumors were examined for effects on cancer cells and tumor vasculature. Trimodality therapies were also studied in vitro on four sarcoma cell lines and on two types of endothelial cells. Results: In both mouse models, trimodality therapy with HIF-1α inhibition using low dose doxorubicin or HIF-1α shRNA was significantly better than any bimodality therapy in blocking tumor growth, with tumors growing to only 13-18% size of controls. When hypoxic areas of tumors were targeted with TH-302, this alternative trimodality therapy was again better than any bimodality therapy in blocking sarcoma tumor growth (tumor size 9% of controls), and tumors failed to grow after stopping treatment for more than 2 months. Analysis of treated tumors demonstrated the predominant effect with both trimodality therapies was through induction of endothelial cell apoptosis (2.6-3.3 fold more than the best bimodality therapy) and destruction of tumor vasculature (86-89% less microvessel density than controls). When the effects of trimodality therapy were examined in vitro, decreases in proliferation and colony formation and induction of apoptosis from trimodality therapy were much more pronounced in tumor-derived endothelial cells than in sarcoma cell lines. Conclusion: HIF-1α inhibition or hypoxia-activated chemotherapy is effective when combined VEGF-A inhibition and radiation in controlling sarcomas by maximizing destruction of tumor vasculature and blocking the hypoxic response. Clinical trials are currently in development using both these trimodality strategies. Citation Format: Changhwan Yoon, Hae-June Lee, Do Joong Park, William D. Tap, T. S. Karin Eisinger-Mathason, David G. Kirsch, M. Celeste Simon, Sam S. Yoon. Targeting vascular endothelial growth factor A and tumor hypoxia combined with radiation eradicates sarcomas through destruction of tumor vasculature and thwarting of the hypoxic response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5447. doi:10.1158/1538-7445.AM2014-5447