Angiogenesis In Human Endothelial Cells Research Articles

Abstract We102: Synthetic N-Acylurea Derivative Reduces Angiogenesis in Human Endothelial Cells by Talin Modulation

Talin is a focal adhesion protein that activates integrins and recruits other focal adhesion proteins. Talin regulates the interactions between integrins and the extracellular matrix, which are critical for endothelial cells during angiogenesis. In this study, we successfully synthesized a novel talin modulator, N-((2-(1H-indol-3-yl)ethyl)carbamoyl)-2-(benzo[d][1,3]dioxol-5-yloxy)acetamide, referred to as KCH-1521. KCH-1521 was determined to bind talin and modulate downstream signaling molecules of talin. After 24 hrs of treatment, KCH-1521 changed the cell morphology of HUVECs and reduced focal adhesion protein expression including vinculin and paxillin. Talin downstream signaling is regulated via FAK, AKT, and ERK pathways, however, treatment with KCH-1521 decreased phosphorylation of FAK, AKT, and ERK, leading to a reduction of cell proliferation, survival, and angiogenesis. Interestingly, the expression of various angiogenic genes was significantly decreased after treatment with KCH-1521. Also, in vitro tube forming assay revealed that KCH-1521 reduced angiogenic networks in a time-dependent manner. To investigate the reversibility of its effects, KCH-1521 was removed after treatment. HUVECs recovered their morphology through rearrangement of the cytoskeleton and the expression of angiogenic genes was also recovered. By further optimization and in vivo studies of KCH-1521, a novel drug of talin modulation could be used to achieve therapeutic anti-angiogenesis for vascular diseases and cancers.

Stem cell factor and cKIT modulate endothelial glycolysis in hypoxia.

In hypoxia, endothelial cells (ECs) proliferate, migrate, and form new vasculature in a process called angiogenesis. Recent studies have suggested that ECs rely on glycolysis to meet metabolic needs for angiogenesis in ischaemic tissues, and several studies have investigated the molecular mechanisms integrating angiogenesis and endothelial metabolism. Here, we investigated the role of stem cell factor (SCF) and its receptor, cKIT, in regulating endothelial glycolysis during hypoxia-driven angiogenesis. SCF and cKIT signalling increased the glucose uptake, lactate production, and glycolysis in human ECs under hypoxia. Mechanistically, SCF and cKIT signalling enhanced the expression of genes encoding glucose transporter 1 (GLUT1) and glycolytic enzymes via Akt- and ERK1/2-dependent increased translation of hypoxia inducible factor 1A (HIF1A). In hypoxic conditions, reduction of glycolysis and HIF-1α expression using chemical inhibitors significantly reduced the SCF-induced in vitro angiogenesis in human ECs. Compared with normal mice, mice with oxygen-induced retinopathy (OIR), characterized by ischaemia-driven pathological retinal neovascularization, displayed increased levels of SCF, cKIT, HIF-1α, GLUT1, and glycolytic enzymes in the retina. Moreover, cKIT-positive neovessels in the retina of mice with OIR showed elevated expression of GLUT1 and glycolytic enzymes. Further, blocking SCF and cKIT signalling using anti-SCF neutralizing IgG and cKIT mutant mice significantly reduced the expression of HIF-1α, GLUT1, and glycolytic enzymes and decreased the pathological neovascularization in the retina of mice with OIR. We demonstrated that SCF and cKIT signalling regulate angiogenesis by controlling endothelial glycolysis in hypoxia and elucidated the SCF/cKIT/HIF-1α axis as a novel metabolic regulation pathway during hypoxia-driven pathological angiogenesis.

Metachromin C, a marine-derived natural compound, shows potential in antitumor activity.

Metachromin C was first isolated from the marine sponge Hippospongia metachromia and has been reported to possess potent cytotoxicity against leukemia cells. However, its antitumor activity and possible mechanisms in pancreatic cancer remain unclear. The effects of Metachromin C on cell viability were estimated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The compound demonstrated a cytotoxic effect on four pancreatic cancer cell lines (PANC-1, BxPC-3, MiaPaCa-2, and AsPC-1). The significant S phase arrest observed with Metachromin C treatment suggests its impact on DNA replication machinery. Metachromin C might interfere with the binding of Topoisomerase I (TOPO I) to DNA, inhibit TOPO I activity, prevent DNA relaxation, cause DNA damage, and consequently activate the DNA repair pathway. Additionally, anti-migration and anti-invasion abilities of Metachromin C were confirmed using the transwell assay. It also inhibited angiogenesis in human endothelial cells by reducing cell proliferation, migration, and disrupting tube formation. Moreover, Metachromin C dose-dependently inhibited the growth of intersegmental vessels, subintestinal vessels, and the caudal vein plexus in a zebrafish embryo model, confirming its inhibitory effect on new vessel formation in vivo. Taken together, Metachromin C could not only inhibit the growth of pancreatic cancer cells but also act as an anti-angiogenic compound simultaneously.

Peptide Nanofiber System for Sustained Delivery of Anti-VEGF Proteins to the Eye Vitreous.

Ranibizumab is a recombinant VEGF-A antibody used to treat the wet form of age-related macular degeneration. It is intravitreally administered to ocular compartments, and the treatment requires frequent injections, which may cause complications and patient discomfort. To reduce the number of injections, alternative treatment strategies based on relatively non-invasive ranibizumab delivery are desired for more effective and sustained release in the eye vitreous than the current clinical practice. Here, we present self-assembled hydrogels composed of peptide amphiphile molecules for the sustained release of ranibizumab, enabling local high-dose treatment. Peptide amphiphile molecules self-assemble into biodegradable supramolecular filaments in the presence of electrolytes without the need for a curing agent and enable ease of use due to their injectable nature-a feature provided by shear thinning properties. In this study, the release profile of ranibizumab was evaluated by using different peptide-based hydrogels at varying concentrations for improved treatment of the wet form of age-related macular degeneration. We observed that the slow release of ranibizumab from the hydrogel system follows extended- and sustainable release patterns without any dose dumping. Moreover, the released drug was biologically functional and effective in blocking the angiogenesis of human endothelial cells in a dose-dependent manner. In addition, an in vivo study shows that the drug released from the hydrogel nanofiber system can stay in the rabbit eye's posterior chamber for longer than a control group that received only a drug injection. The tunable physiochemical characteristics, injectable nature, and biodegradable and biocompatible features of the peptide-based hydrogel nanofiber show that this delivery system has promising potential for intravitreal anti-VEGF drug delivery in clinics to treat the wet form age-related macular degeneration.

Ginkgo biloba Golden Leaf Extract (GGLE) Inhibits Melanoma Cell Invasion and Angiogenesis Through Inhibition of Angiogenin.

The popular dietary supplements of Ginkgo biloba (Ginkgo) products have been reported to have anti-cancer activities in multiple cellular and animal studies, with the benefits yet to be proven with clinical trials. The mechanisms of action are not clear, forming a barrier to investigation in Gingko-specific benefits to cancer patients, especially when combined with other therapies. Here we reported on the discovery of a novel mechanism by which a Ginkgo golden leaf extract (GGLE) inhibited melanoma cell invasion and angiogenesis. GGLE did not inhibit melanoma cells via direct cytotoxicity. Instead, GGLE significantly inhibited total RNase activities in melanoma cells under both normoxia and hypoxia conditions. The RNase angiogenin was induced twofolds by hypoxia, and the induction was significantly suppressed by GGLE treatment in a dose dependent manner. As a result of angiogenin inhibition, GGLE inhibited melanoma cell migration and invasion in a dose dependent manner. Conditioned media from melanoma cell culture sufficiently induced in vitro angiogenesis in human endothelial cells, whereas the conditioned media of GGLE-treated melanoma cells significantly inhibited this angiogenetic activity. This was accompanied with markedly reduced angiogenin concentrations in the GGLE-treated melanoma cell conditioned media. We concluded that, instead of direct cytotoxicity, GGLE inhibited angiogenin synthesis and secretion by melanoma cells, resulting in inhibition of tumor cell invasion and tumor-induced angiogenesis. This new mechanism opens the door for investigation in GGLE influencing tumor microenvironment, and warrants further investigation and validation in vivo.

The combination of mesoglycan and VEGF promotes skin wound repair by enhancing the activation of endothelial cells and fibroblasts and their cross-talk

Skin wound healing requires accurate therapeutic topical managements to accelerate tissue regeneration. Here, for the first time, we found that the association mesoglycan/VEGF has a strong pro-healing activity. In detail, this combination induces angiogenesis in human endothelial cells promoting in turn fibroblasts recruitment. These ones acquire a notable ability to invade the matrigel coating and to secrete an active form of metalloproteinase 2 in presence of endothelial cells treated with mesoglycan/VEGF. Next, by creating intrascapular lesions on the back of C57Bl6 mice, we observed that the topical treatments with the mesoglycan/VEGF promotes the closure of wounds more than the single substances beside the control represented by a saline solution. As revealed by eosin/hematoxylin staining of mice skin biopsies, treatment with the combination mesoglycan/VEGF allows the formation of a well-structured matrix with a significant number of new vessels. Immunofluorescence analyses have revealed the presence of endothelial cells at the closed region of wounds, as evaluated by CD31, VE-cadherin and fibronectin staining and of activated fibroblasts assessed by vimentin, col1A and FAP1α. These results encourage defining the association mesoglycan/VEGF to activate endothelial and fibroblast cell components in skin wound healing promoting the creation of new vessels and the deposition of granulation tissue.

NRASQ61R mutation in human endothelial cells causes vascular malformations.

Somatic mutations in NRAS drive the pathogenesis of melanoma and other cancers but their role in vascular anomalies and specifically human endothelial cells is unclear. The goals of this study were to determine whether the somatic-activating NRASQ61R mutation in human endothelial cells induces abnormal angiogenesis and to develop in vitro and in vivo models to identify disease-causing pathways and test inhibitors. Here, we used mutant NRASQ61R and wild-type NRAS (NRASWT) expressing human endothelial cells in in vitro and in vivo angiogenesis models. These studies demonstrated that expression of NRASQ61R in human endothelial cells caused a shift to an abnormal spindle-shaped morphology, increased proliferation, and migration. NRASQ61R endothelial cells had increased phosphorylation of ERK compared to NRASWT cells indicating hyperactivation of MAPK/ERK pathways. NRASQ61R mutant endothelial cells generated abnormal enlarged vascular channels in a 3D fibrin gel model and in vivo, in xenografts in nude mice. These studies demonstrate that NRASQ61R can drive abnormal angiogenesis in human endothelial cells. Treatment with MAP kinase inhibitor U0126 prevented the change to a spindle-shaped morphology in NRASQ61R endothelial cells, whereas mTOR inhibitor rapamycin did not.

Avian Reovirus P17 Suppresses Angiogenesis by Promoting DPP4 Secretion.

Avian reovirus p17 (ARV p17) is a non-structural protein known to activate autophagy, interfere with gene transcription and induce a significant tumor cell growth inhibition in vitro and in vivo. In this study, we show that ARV p17 is capable of exerting potent antiangiogenic properties. The viral protein significantly inhibited the physiological angiogenesis of human endothelial cells (ECs) by affecting migration, capillary-like structure and new vessel formation. ARV p17 was not only able to suppress the EC physiological angiogenesis but also rendered ECs insensitive to two different potent proangiogenic inducers, such as VEGF-A and FGF-2 in the three-dimensional (3D) Matrigel and spheroid assay. ARV p17 was found to exert its antiangiogenic activity by upregulating transcription and release of the well-known tumor suppressor molecule dipeptidyl peptidase 4 (DPP4). The ability of ARV p17 to impact on angiogenesis is completely new and highlights the “two compartments” activity of the viral protein that is expected to hamper the tumor parenchymal/stromal crosstalk. The complex antitumor activities of ARV p17 open the way to a new promising field of research aimed to develop new therapeutic approaches for treating tumor and cancer metastasis.

Abstract 16176: Newly Identified MiR-6770-3p Dramatically Induces Angiogenesis in Human Endothelial Cells While Uncommonly Decreasing Cell Proliferation

Micro-RNAs (miRs) are small non-coding RNAs that alter the expression of multiple mRNA targets. Although they participate in physiological processes, dysregulation of their expression is implicated in various diseases. We investigated whether miRs could be involved in the regulation of FXYD1 expression, a sub-unit of the Na+/K+-ATPase pump, that we previously described as both cardio- and vasculoprotective, under oxidative stress conditions. Using in silico analysis, we identified 3 potential miRs that could target FXYD1 mRNA: miR-3178, miR-3960 and miR-6770-3p. Using a FXYD1-3’UTR-luciferase reporter assay, we found that the overexpression of miR-6770-3p in HEK293T cells resulted in the largest reduction in luciferase activity, showing a strong targeting of FXYD1 mRNA. A mutation of the binding site in the 3’UTR of FXYD1 restored luciferase activity, confirming the binding site of miR-6770-3p. Mir-6770-3p is a novel miR that currently has no known function. MiR-6770-3p is the minor strand of its 5p/3p miR duplex in both the human endothelial cell (EC) line, EA.hy.926, and human dermal microvascular (HMVECs). Its overexpression dramatically increased endothelial tube formation in a Matrigel angiogenesis assay (>4 fold change vs. control transfection, p<0.05) and was also associated with a 2-3-fold increase of VEGFR2 and Endoglin mRNA levels, respectively, in both cell types. Conversely, it was also uncommonly associated with a decrease in cell proliferation, suggesting that the pro-angiogenic effect of miR-6770-3p was not due to increased proliferation. Interestingly and contrary to our previous findings, miR-6770-3p was the major strand in Human Umbilical Vein ECs (HUVECs). In this cell type, overexpression of the 3p strand in fact decreased tube formation, while retaining its negative effect on proliferation. The difference in miR major and minor strand may partly explain the differences in functional behaviours in HUVECs, as compared to HMVECs. In conclusion, we have identified a novel miR which has the potential to modulate vessel formation in vitro, and induce critical regulators of angiogenesis; however, further work is required to better understand how this miR performs these functions.

Design of a small molecule that stimulates vascular endothelial growth factor A enabled by screening RNA fold-small molecule interactions.

Vascular endothelial growth factor A (VEGFA) stimulates angiogenesis in human endothelial cells, and increasing its expression is a potential treatment for heart failure. Here, we report the design of a small molecule (TGP-377) that specifically and potently enhances VEGFA expression by the targeting of a non-coding microRNA that regulates its expression. A selection-based screen, named two-dimensional combinatorial screening, revealed preferences in small-molecule chemotypes that bind RNA and preferences in the RNA motifs that bind small molecules. The screening program increased the dataset of known RNA motif–small molecule binding partners by 20-fold. Analysis of this dataset against the RNA-mediated pathways that regulate VEGFA defined that the microRNA-377 precursor, which represses Vegfa messenger RNA translation, is druggable in a selective manner. We designed TGP-377 to potently and specifically upregulate VEGFA in human umbilical vein endothelial cells. These studies illustrate the power of two-dimensional combinatorial screening to define molecular recognition events between ‘undruggable’ biomolecules and small molecules, and the ability of sequence-based design to deliver efficacious structure-specific compounds.

Intra-pancreatic tissue-derived mesenchymal stromal cells: a promising therapeutic potential with anti-inflammatory and pro-angiogenic profiles

BackgroundHuman pancreata contain many types of cells, such as endocrine islets, acinar, ductal, fat, and mesenchymal stromal cells (MSCs). MSCs are important and shown to have a promising therapeutic potential to treat various disease conditions.MethodsWe investigated intra-pancreatic tissue-derived (IPTD) MSCs isolated from tissue fractions that are routinely discarded during pancreatic islet isolation of human cadaveric donors. Furthermore, whether pro-angiogenic and anti-inflammatory properties of these cells could be enhanced was investigated.ResultsIPTD-MSCs were expanded in GMP-compatible CMRL-1066 medium supplemented with 5% human platelet lysate (hPL). IPTD-MSCs were found to be highly pure, with > 95% positive for CD90, CD105, and CD73, and negative for CD45, CD34, CD14, and HLA-DR. Immunofluorescence staining of pancreas tissue demonstrated the presence of CD105+ cells in the vicinity of islets. IPTD-MSCs were capable of differentiation into adipocytes, chondrocytes, and osteoblasts in vitro, underscoring their multipotent features. When these cells were cultured in the presence of a low dose of TNF-α, gene expression of tumor necrosis factor alpha-stimulated gene-6 (TSG-6) was significantly increased, compared to control. In contrast, treating cells with dimethyloxallyl glycine (DMOG) (a prolyl 4-hydroxylase inhibitor) enhanced mRNA levels of nuclear factor erythroid 2-related factor 2 (NRF2) and vascular endothelial growth factor (VEGF). Interestingly, a combination of TNF-α and DMOG stimulated the optimal expression of all three genes in IPTD-MSCs. Conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-α contained higher levels of pro-angiogenic (VEGF, IL-6, and IL-8) compared to controls, promoting angiogenesis of human endothelial cells in vitro. In contrast, levels of MCP-1, a pro-inflammatory cytokine, were reduced in the conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-α.ConclusionsThe results demonstrate that IPTD-MSCs reside within the pancreas and can be separated as part of a standard islet-isolation protocol. These IPTD-MSCs can be expanded and potentiated ex vivo to enhance their anti-inflammatory and pro-angiogenic profiles. The fact that IPTD-MSCs are generated in a GMP-compatible procedure implicates a direct clinical application.

Crocetin promotes angiogenesis in human endothelial cells through PI3K-Akt-eNOS signaling pathway.

Previous studies proved the pro-angiogenic effect of Crocetin, a natural carotenoid dicarboxylic acid, in both in vivo and in vitro models. However, the exact mechanism of Crocetin action has not completely been elucidated yet. The current experiment was designed to find the activity of PI3K-Akt-eNOS axis after the treatment of endothelial cells with Crocetin in vitro. Human Umbilical Vein Endothelial Cells (HUVECs) were incubated with various concentrations of Crocetin (1, 5, 25, 50, and 100 µM) over a period of 72 h. Crocetin significantly increased HUVECs viability after 72 h as compared with the control group. We also found that Crocetin promoted the formation of the capillary-like structure compared to the control (p<0.05). Moreover, an improved migration rate and increased MMP-9 activity were observed in HUVECs that received 50 µM Crocetin (p<0.05). Crocetin enhanced the uptake of Ac-LDL which is correlated with increased lipid metabolism. Based on the data from the current experiment, protein level of VEGFR-1, -2 and p-Akt/Akt, p-eNOS/eNOS ratios were increased 72 h after the treatment of HUVECs with Crocetin (p<0.05). In contrast, the transcription level of VEGF was reduced in Crocetin-treated cells. These data demonstrated that Crocetin promotes HUVECs angiogenesis potential by the modulation of VEGF signaling pathway and increased cell viability. The PI3K/Akt/eNOS axis is required for a Crocetin-associated activity in endothelial cells.

Low‑intensity pulsed ultrasound promotes apoptosis and inhibits angiogenesis via p38 signaling‑mediated endoplasmic reticulum stress in human endothelial cells.

Aberrant increase in angiogenesis contributes to the progression of malignant solid tumors. An alternative anti-angiogenesis therapy is critical for cancer, since the current anti-angiogenesis drugs lack specificity for tumor cells. In the present study, the effects and mechanisms of low-intensity pulsed ultrasound (LIPUS) on human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMECs) were investigated, and the therapeutic potential of this technology was assessed. HUVECs and HMECs were treated with LIPUS (0.5 MHz; 210 mW/cm2) for 1 min and cultured for 24 h. Flow cytometry and Cell Counting Kit-8 assays demonstrated that LIPUS treatment at a dose of 210 mW/cm2 promoted apoptosis and decreased the viability in HUVECs and HMECs. Real-time cell analysis also revealed that LIPUS did not affect the proliferation or migration of HUVECs. An endothelial cell tube formation assay indicated that LIPUS treatment inhibited the angiogenic ability of HUVECs and HMECs. Furthermore, LIPUS increased the protein levels of the apoptosis-associated cleaved Caspase-3 and decreased the B-cell lymphoma-2 levels. LIPUS increased the phosphorylation of p38 mitogen-activated protein kinase (MAPK), and the levels of endoplasmic reticulum (ER) stress-associated markers, including activating transcription factor-4 (ATF-4) and phosphorylated eukaryotic initiation factor 2α (eIF2α). The p38 inhibitor SB203580 reversed the pro-apoptotic and anti-angiogenic effects of LIPUS in cells. Finally, inhibition of p38 decreased the LIPUS-induced elevation of p-eIF2α and ATF-4 levels. Taken together, these results suggested that LIPUS promoted apoptosis and inhibited angiogenesis in human endothelial cells via the activation of p38 MAPK-mediated ER stress signaling.

Andrographolide inhibits breast cancer through suppressing COX-2 expression and angiogenesis via inactivation of p300 signaling and VEGF pathway

BackgroundAndrographolide (Andro), a diterpenoid lactone, has been used for treatment of various cancers with less adverse effects. However, the underlying mechanisms regarding its anti-tumor mechanism still remain unclear.MethodsCell viability and proliferation were measured by CCK8 and CFSE dilution assay. The localization of p50/p65 or cytochrome c was determined using confocal immunofluorescence. Streptavidin-agarose pulldown or ChIP assays were used to detect the binding of multiple transactivators to COX-2 promoter. The promoter activity was examined by a dual-Luciferase reporter assay. The functions of Andro on COX-2-mediated angiogenesis were also investigated using human HUVEC cells through tube formation and spheroids sprouting assay. The in vivo anti-tumor efficacy of Andro was analyzed in xenografts nude mice.ResultsThe results indicated that Andro could significantly inhibit the proliferation of human breast cancers, and suppress COX-2 expression at both protein and mRNA levels. Furthermore, Andro could dose-dependently inhibit COX-2-mediated angiogenesis in human endothelial cells. We have also found that Andro significantly promoted the activation of cytochrome c and activated caspase-dependent apoptotic signaling pathway. Our further explorations demonstrated that Andro inhibited the binding of the transactivators CREB2, C-Fos and NF-κB and blocked the recruitment of coactivator p300 to COX-2 promoter. Moreover, Andro could effectively inhibit the activity of p300 histone acetyltransferase (HAT), thereby attenuating the p300-mediated acetylation of NF-κB. Besides, Andro could also dramatically inhibit the migration, invasion and tubulogenesis of HUVECs in vitro. In addition, Andro also exhibited effective anti-tumor efficacy as well as angiogenesis inhibition in vivo.ConclusionIn current study, we explore the potential effects of Andro in suppressing breast cancer growth and tumor angiogenesis, as well as the precise mechanisms. This work demonstrated the potential anti-cancer effects of Andro, indicating that Andro could inhibit COX-2 expression through attenuating p300 HAT activity and suppress angiogenesis via VEGF pathway, and thereby could be developed as an antitumor agent for the treatment of breast cancer.

Resveratrol inhibits VEGF‐induced angiogenesis in human endothelial cells associated with suppression of aerobic glycolysis via modulation of PKM2 nuclear translocation

Endothelial cells (ECs) mainly depend on aerobic glycolysis to generate angiogenesis. Deregulation of glycolysis is often observed in human endothelial cells during angiogenesis. In the present study, we first report that resveratrol (RST), which has been intensively studied in glucose metabolism of various cancer cells, has a profound inhibitory effect on tube formation and migration via suppression of glycolysis in human umbilical vein endothelial cells (HUVECs) induced by vascular endothelial growth factor (VEGF). Moreover, we further reveal that RST reduced the mRNA and protein level of glucose transporter-1(GLUT1), hexokinase II (HK2), phosphofructokinase-1(PFK1) and pyruvate kinase M2 (PKM2) through modulation of ERK-mediated PKM2 nuclear translocation. Our results provide a novel mechanism to account for the inhibition of RST on VEGF-mediated angiogenesis and suggest that targeting aerobic glycolysis or nuclear PKM2 may be a new approach for pathological angiogenesis prevention or treatment.

Activation of the complement system in an osteosarcoma cell line promotes angiogenesis through enhanced production of growth factors

There is increasing evidence that the complement system is activated in various cancer tissues. Besides being involved in innate immunity against pathogens, the complement system also participates in inflammation and the modulation of tumor microenvironment. Recent studies suggest that complement activation promotes tumor progression in various ways. Among some cancer cell lines, we found that human bone osteosarcoma epithelial cells (U2-OS) can activate the alternative pathway of the complement system by pooled normal human serum. Interestingly, U2-OS cells showed less expression of complement regulatory proteins, compared to other cancer cell lines. Furthermore, the activated complement system enhanced the production of growth factors, which promoted angiogenesis of human endothelial cells. Our results demonstrated a direct linkage between the complement system and angiogenesis using the in vitro model, which suggest the complement system and related mechanisms might be potential targets for cancer treatment.

Granulin Secreted by the Food-Borne Liver Fluke Opisthorchis viverrini Promotes Angiogenesis in Human Endothelial Cells.

The liver fluke Opisthorchis viverrini is a food-borne, zoonotic pathogen endemic to Thailand and adjacent countries in Southeast Asia. The adult developmental stage of the O. viverrini parasite excretes and secretes numerous proteins within the biliary tract including the gall bladder. Lesions caused by the feeding activities of the liver fluke represent wounds that undergo protracted cycles of healing and re-injury during chronic infection, which can last for decades. Components of the excretory/secretory (ES) complement released by the worms capably drive proliferation of bile duct epithelial cells and are implicated in establishing the oncogenic milieu that leads to bile duct cancer, cholangiocarcinoma. An ES protein, the secreted granulin-like growth factor termed Ov-GRN-1, accelerates wound resolution in mice and in vitro. To investigate angiogenesis (blood vessel development) that may contribute to wound healing promoted by liver fluke granulin and, by implication, to carcinogenesis during chronic opisthorchiasis, we employed an in vitro tubule formation assay (TFA) where human umbilical vein endothelial cells were grown on gelled basement matrix. Ten and 40 nM Ov-GRN-1 significantly stimulated angiogenesis as monitored by cellular proliferation and by TFA in real time. This demonstration of potent angiogenic property of Ov-GRN-1 bolsters earlier reports on the therapeutic potential for chronic non-healing wounds of diabetics, tobacco users, and the elderly and, in addition, showcases another of the hallmark of cancer characteristic of this carcinogenic liver fluke.

A redox-sensitive signaling pathway mediates pro-angiogenic effect of chlordecone via estrogen receptor activation.

Chlordecone is able to induce pro-angiogenic effect through an estrogen receptor (ERα) pathway involving NO release and VEGF. The present study aimed to determine the molecular mechanisms by which chlordecone promotes angiogenesis in human endothelial cells. High but not low concentration of chlordecone increased mitochondrial respiratory capacity and mitochondrial DNA content in endothelial cells. The ROS scavenger MnTMPyP was able to prevent the increase of both VEGF expression and capillary length induced by chlordecone. A significant increase of cytoplasmic O2- production was observed after 1 and 4 h incubation of chlordecone, but not after 2 h. The NADPH oxidase inhibitor apocynin or silencing p47phox prevented angiogenesis and tube formation but also the increase in production of O2- at 1 h. In addition, apocynin as well silencing p47phox prevented eNOS activation and the NO synthase inhibitor L-NAME inhibited mitochondrial O2-production. All the previous effects of chlordecone were prevented by fulvestrant. Our results indicate that an adaptation of the mitochondrial energy metabolism occurs in the chlordecone angiogenic response. Finally, we showed that chlordecone induces endothelial cells angiogenesis by a cross-talk involving NADPH oxidase and mitochondrial O2-via a NO sensitive pathways through activation of ERα. These findings propose that a molecular mechanism may partly explain the epidemiological evidence implicating chlordecone as risk factor carcinogenesis.

The novel brassinosteroid analog BR4848 inhibits angiogenesis in human endothelial cells and induces apoptosis in human cancer cells in vitro

We report the synthesis and detailed biological study of the synthetic brassinosteroid analog 2α,3α-dihydroxy-6-oxo-5α-androstan-17β-yl N-(tert-butoxycarbonyl)-D,L-valinate (BR4848). The panel of cancer cell lines was used for characterization of its antiproliferative activity, yet had no adverse effects in normal human fibroblasts. In HeLa cells, BR4848-induced apoptosis was accompanied by increase of apoptotic subG1 cells, PARP-1 and caspase-7 fragmentation, downregulation of Bcl-2 and Mcl-1, an increase in caspase activity and G2/M phase cell cycle arrest. Antiproliferative properties of BR4848 were exhibited by inhibition of phosphorylation of Akt, Erk1/2 and FAK. Furthermore, the developed analog exhibited in vitro antiangiogenic activity in human umbilical vein endothelial cells (HUVECs). BR4848-induced apoptosis accompanied with G2/M arrest was detected in endothelial cells. BR4848 also inhibited adhesion, tube formation and migration of endothelial cells by inhibition of FAK, Erk 1/2, CDK5, VEGFR2, TNFα-stimulated production of IL-6, angiopoietin-2 and Jagged1. Finally, BR4848 did not modulate the activity nor nuclear translocation of any of the steroid receptors (ERα, ERβ, AR, MR and PR) included in reporter cell-based assays, which excludes the genomic activity of steroid receptors as a contributing factor to the observed biological activities of BR4848.

Myocardial overexpression of TIMP3 after myocardial infarction exerts beneficial effects by promoting angiogenesis and suppressing early proteolysis.

Myocardial infarction (MI) results in loss of cardiomyocytes, adverse extracellular matrix (ECM) and structural remodeling, and left ventricular (LV) dilation and dysfunction. Tissue inhibitors of metalloproteinase (TIMPs) inhibit matrix metalloproteinases (MMPs), the main regulators of ECM turnover. TIMPs also have MMP-independent functions. TIMP3 levels are reduced in the heart within 24 h of MI in mice. We investigated if overexpression of TIMP3 post-MI limits adverse remodeling and LV dilation and dysfunction. MI was induced by left anterior descending coronary artery ligation in 10- to 12-wk-old male C57BL/6J mice, and adenoviral constructs expressing human (h)TIMP3 (Ad-hTIMP3) or no TIMP (Ad-Null) were injected in the peri-infarct zone (5.4 × 107 plaque-forming units/heart, 5 injections/heart). Cardiac function assessed by echocardiography showed improved LV physiology and reduced LV dilation after TIMP3 overexpression compared with the Ad-Null-MI group. Post-MI adverse remodeling was attenuated in the Ad-hTIMP3-MI group, as assessed by greater cardiomyocyte density, less infarct expansion, and ECM disruption. TIMP3 overexpression blunted the early rise in proteolytic activities post-MI. A higher density of coronary arteries and a greater number of proliferating endothelial cells were detected in the infarct and peri-infarct regions in the Ad-hTIMP3-MI group compared with the Ad-Null-MI group. In vitro three-dimensional angiogenesis assay confirmed that recombinant TIMP3 promotes angiogenesis in human endothelial cells, although biphasically and in a dose-dependent manner. Intriguingly, overexpression of Ad-hTIMP3 at 10-fold higher concentration had no beneficial effects, consistent with antiangiogenic effects of TIMP3 at higher doses. In conclusion, optimal overexpression of TIMP3 can be a promising therapeutic approach to limit adverse post-MI remodeling by dually inhibiting early proteolysis and promoting angiogenesis.NEW & NOTEWORTHY Here, we report that tissue inhibitor of metalloproteinase 3 overexpression after myocardial infarction improves myocardial structural remodeling and function by promoting angiogenesis and inhibiting early proteolysis. This demonstrates the therapeutic potential of preserving the local balance of tissue inhibitor of metalloproteinase 3 in the heart given its diverse functions in modulating different processes involved in the adverse postmyocardial infarction remodeling.