Human Umbilical Vein Endothelial Cells Tube Formation Research Articles

454-P: Conditioned Medium from Dental Pulp Stem Cells Promotes Angiogenesis

Background/Aim: We previously reported that conditioned medium (CM) of stem cells from human exfoliated deciduous teeth (SHED) improved diabetic polyneuropathy in diabetic mice due to an increase of capillary blood flow in the vasa nervorum, and in in vitro study only secreted factors of less than 6 kDa fraction of SHED-CM promoted neurite outgrowth of mouse dorsal root ganglion neurons. In this study, we examined whether SHED-CM had beneficial effects on angiogenesis. Methods: SHED-CM was collected 48 hours after culturing in serum-free DMEM, and was separated into two fractions according to molecular weight (less than 6 kDa and more than 6 kDa). Exosomes were isolated from SHED-CM by ultracentrifugation. Human umbilical-vein endothelial cells (HUVECs) were cultured with six different media (DMEM, DMEM with VEGF, SHED-CM, less than 6 kDa, more than 6 kDa or DMEM with exosomes) for 12-48 hours, then MTT assay, wound healing assay, Boyden chamber assay and tube formation assay were performed to evaluate the cell viability, migration ability and tube formation ability in HUVECs. Rat aortic ring assay and mouse Matrigel plug assay were performed to assess neovascularization and endothelial cell migration. Results: SHED-CM, especially more than 6 kDa fraction, significantly promoted cell viability, migration and tube formation in HUVECs, compared with DMEM. Meanwhile, less than 6 kDa fraction promoted only tube formation, and the effects of exosomes were negligible. In aortic ring assay and Matrigel plug assay, SHED-CM accelerated neovascularization and increased endothelial cell migration in HUVECs, and the effects of more than 6 kDa fraction were stronger than that of less than 6 kDa fraction. These data suggested that the secreted factors in more than 6 kDa fraction played an essential role in promoting angiogenesis. Conclusion: Soluble factors from SHED might have angiogenesis-promoting effects and hold promise for comprehensive clinical applications in vascular diseases and diabetic complications. Disclosure M. Kato: None. S. Tsunekawa: None. N. Nakamura: None. E. Miura-Yura: None. Y. Yamada: None. Y. Hayashi: None. R. Inoue: None. M. Mohiuddin: Other Relationship; Self; Abbott Japan Co. Ltd, ARKRAY, Astellas Pharma Inc., Astellas Pharma Inc., AstraZeneca K.K., Boehlinger Ingelheim Japan Co. Ltd., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo, Daiichi Sankyo, Eli Lilly Japan K.K., Eli Lilly Japan K.K., Fukuda Denshi, Japan Tobacco Inc., Kaken Pharmaceutical Co., Ltd., Kissei Pharmaceutical Co., Ltd., Kowa pharmaceu. co. Ltd., Kowa Pharmaceu. Co., Ltd., Kyowa Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Mitsubishi Tanabe Pharma Corporation, MSD K.K., MSD K.K., Mylan N. V., Novartis Pharma K.K., Novartis Pharma K.K., Novo Nordisk Pharma Ltd., Novo Nordisk Pharma Ltd., Ono Pharmaceu. Co., Ltd., Ono Pharmaceutical Co., Ltd., Otsuka Pharmaceutical Co., Ltd., Pfizer Japan Inc., Sanofi K.K., Sanofi K.K., Sanwa Kagaku Kenkyusho, Sanwa Kagaku Kenkyusho, Shionogi & Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Takeda Pharmaceutical Company Limited, Terumo Co. Ltd. Y. Morishita: None. T. Himeno: None. M. Kondo: None. Y. Kato: None. H. Kamiya: Speaker’s Bureau; Self; Astellas Pharma Inc., AstraZeneca K.K., Boehringer Ingelheim K.K., Daiichi Sankyo, Eli Lilly Japan K.K., Fukuda Denshi, Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K.K., Novartis Pharma K.K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K.K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. K. Naruse: None. J. Nakamura: Research Support; Self; Astellas Pharma Inc., Boehlinger Ingelheim Japan Co., Ltd., Daiichi Sankyo, Eli Lilly Japan K.K., Japan Tobacco Inc., Kaken Pharmaceutical Co., Ltd., Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K.K., Novartis Pharma K.K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Otsuka Pharmaceutical Co., Ltd., Pfizer Japan Inc., Sanofi K.K., Sanwa Kagaku Kenkyusho, Shionogi & Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker’s Bureau; Self; Abbott Japan Co., Ltd., ARKRAY, Astellas Pharma Inc., AstraZeneca K.K., Boehlinger Ingelheim Japan Co., Ltd.,, Daiichi Sankyo, Eli Lilly Japan K.K., Fukuda Denshi, Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K.K., Mylan, Novartis Pharma K.K., Novo Nordisk Pharma Ltd, Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Terumo Medical Corporation.

AB0089 THE ANALYSIS FOR THE INHIBITION OF ANGIOGENESIS BY JAK INHIBITOR

Background:Many blood vessels are generated in the hyperplastic synovial tissue of patients with rheumatoid arthritis (RA), and lead to chronic tissue inflammation and joint destruction [1]. Janus kinase (JAK) family consisting of JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) are chain receptors which phosphorylate signal transducers and activators of transcription (STAT) and mediate inflammatory diseases including RA [2]. Nowadays, several JAK inhibitors such as Tofacitinib (TOF), Baricitinib (BAR) and Peficitinib (PEF) have been developed and demonstrated to have the inhibitory effects on inflammatory arthritis [3-5]. However, there were few reports concerning their effects on angiogenesis in vitro.Objectives:The purpose of the present study is to investigate the influence of JAK inhibitors on angiogenesis of human umbilical vein endothelial cell (HUVEC) activated by vascular endothelial growth factor (VEGF).Methods:The cell line of HUVECs were used for this study. The activity of proliferation and tube formation were analyzed by counting assay and tube formation assay, respectively.In counting assay, HUVECs (5 × 104cells/ml) were seeded onto 96-well cell culture plate with 20 ng/ml VEGF including various doses (0.1µM, 1µM, 5µM) of TOF, BAR or PEF. After 48 hours incubation at 37°C in a humidified atmosphere containing 5% CO2, cell proliferation of each groups was assessed using cell counting kit. In tube formation assay, HUVECs (5 × 104cells/ml) were treated with 20ng/ml VEGF including various dose (0.1µM, 1µM, 5µM) of TOF, BAR or PEF for 00 hours, then seeded onto 48-well plate applied with Matrigel. After 24 hours incubation on Matrigel, the capillary-like tube formation of each well was photographed using phase contrast microscopy. Tube formation were quantitated by measurement of the length of branch.Results:HUVECs were activated in proliferation and tube formation by VEGF treatment. And, the proliferation and tube formation of HUVECs activated by VEGF were suppressed by All of TOF, BAR and PEF. In particular, TOF and PEF could suppress them highly.Conclusion:This study showed the inhibitory effect of JAK inhibitors on proliferation and tube formation of HUVECs activated by VEGF. In particular, the angiogenesis of HUVECs activated by VEGF was highly suppressed by TOF and PEF. VEGF is reported to regulate the angiogenesis through multi JAK-STAT signaling pathways [6]. The inhibitory effects on angiogenesis of TOF, BAR and PEF might depend on the differences in their affinity for JAKs. VEGF has been shown to a have a central involvement in the angiogenic process in RA [7]. JAK inhibitors might suppress the angiogenesis in RA synovial tissues by inhibiting VEGF signaling.

The Inhibition of Protein Kinase C β Contributes to the Pathogenesis of Preeclampsia by Activating Autophagy

BackgroundPreeclampsia is a devastating hypertensive disorder of pregnancy with unknown mechanism. Recent studies have considered abnormal autophagy as a new cellular mechanism for this disorder, while little is known about how autophagy is specifically involved and what factors are implicated. Here, we report a previously unrecognized preeclampsia-associated autophagic regulator, PKCβ, that is involved in placental angiogenesis.MethodsPKCβ levels were evaluated by quantitative real-time PCR, western blotting, immunofluorescence and by the analysis of public data. The autophagy-regulating role of PKCβ inhibition in preeclampsia pathogenesis was studied in a mouse model, and in human umbilical vein endothelial cells (HUVECs) and human choriocarcinoma cells (JEG-3).FindingsPKCβ was significantly downregulated in human preeclamptic placentas. In a mouse model, the selective inhibition of PKCβ by Ruboxistaurin was sufficient to induce preeclampsia-like symptoms, accompanied by excessive autophagic flux and a disruption in the balance of pro- and anti-angiogenic factors in mouse placentas. In contrast, autophagic inhibition by 3-methyladenine partially normalized hypertension, proteinuria and placental angiogenic imbalance in PKCβ-inhibited mice. Our in vitro experiments demonstrated that PKCβ inhibition activated autophagy, thus blocking VEGFA-induced HUVEC tube formation and resulting in the significant upregulation of sFLT1 and downregulation of VEGFA in JEG-3 cells.InterpretationThese data support a novel model in which autophagic activation due to PKCβ inhibition leads to the impairment of angiogenesis and eventually results in preeclampsia.FundingShanghai Key Program of Clinical Science and Technology Innovation, National Natural Science Foundation of China and Shanghai Medical Center of Key Programs for Female Reproductive Diseases.

Cyperenoic acid, a sesquiterpene derivative from Croton crassifolius, inhibits tumor growth through anti-angiogenesis by attenuating VEGFR2 signal pathway in breast cancer

BackgroundCyperenoic acid, one of the main chemical constituents of the root of Croton crassifolius, exhibited potent anti-angiogenic property on the zebrafish embryo model with little cytotoxicity. Nevertheless, its anti-angiogenic mechanism and anti-tumor effect have not been investigated. PurposeTo investigate the anti-angiogenic mechanisms of cyperenoic acid and evaluate it whether could exert anti-tumor effect by inhibiting angiogenesis. Study designTargeting vascular endothelial growth factor receptor-2 (VEGFR2) pathway to inhibit tumor angiogenesis is a significant strategy for cancer treatment. Initially, the anti-angiogenic effect of cyperenoic acid as well as the mechanisms of the action was studied using both in-vitro and in-vivo methodologies. Then, its anti-tumor effect through anti-angiogenesis by attenuating VEGFR2 signaling pathway was evaluated. MethodsThe in-vitro inhibitory effect of cyperenoic acid on the vascular endothelial growth factor (VEGF)-induced angiogenesis was evaluated using human umbilical vein endothelial cells (HUVECs) model. Moreover, its ex-vivo and in-vivo effects were evaluated using the aortic ring assay and the matrigel plug assay. The influence of the cyperenoic acid on tyrosine phosphorylation of VEGFR2 was studied by western blotting assay and the influence on downstream signaling pathway of VEGFR2 also be detected. Computer-docking simulations were carried out to study the interaction between cyperenoic acid and VEGFR2. Finally, its inhibitory effect on tumor growth was studied using breast cancer xenograft model. ResultsCyperenoic acid possessed little toxicity to HUVECs, but it significantly inhibited VEGF-induced proliferation, invasion, migration and tube formation of HUVECs. Moreover, it inhibited VEGF-induced sprout formation ex vivo and vessel formation in vivo. Further mechanistic study showed that cyperenoic acid could suppress VEGFR2 tyrosine kinase activity and alter its downstream signaling pathways in VEGF-induced HUVECs. In addition, it could form two hydrogen bonds with the ATP binding pocket of the VEGFR2 kinase domain by docking. For breast cancer xenograft model, cyperenoic acid suppressed tumor growth, but no obvious toxic pathologic changes were observed in mice. Besides, it suppressed the phosphorylation of VEGFR2 in tumor, demonstrating its anti-angiogenic ability in vivo partly targeting the VEGFR2. ConlusionCyperenoic acid could exert anti-tumor effect in breast cancer by inhibiting angiogenesis via VEGFR2 signaling pathway.

The angiogenic effects of exosomes secreted from retinal pigment epithelial cells on endothelial cells

Exosomes are informative microvesicles associated with intercellular communication via the transfer of many molecular constituents such as proteins, lipids, and nucleic acids; environmental changes and the cellular status around cells greatly affect exosome components. Cells of the retinal pigment epithelium (RPE) are key players in retinal homeostasis. Transforming growth factor (TGF)-β and tumour necrosis factor (TNF)-α are increased in the vitreous and retina in several retinal diseases and activate and undergo epithelial-mesenchymal transition (EMT) in RPE cells. EMT is closely associated with mechanisms of wound healing, including fibrosis and related angiogenesis; however, whether exosome components depend on the cell status, epithelium or mesenchyme and whether these exosomes have pro- or anti-angiogenic roles in the retina are unknown. We performed this study to investigate whether these EMT inducers affect the kinds of components in exosomes secreted from RPE cells and to assess their angiogenic effects. Exosomes were collected from culture media supernatants of a human RPE cell line (ARPE-19) stimulated with or without 10 ng/ml TNF-α and/or 5 ng/ml TGF-β2. NanoSight tracking analysis and immunoblot analysis using exosome markers were used to qualify harvested vesicles. Angiogenic factor microarray analysis revealed that exosomes derived from ARPE-19 cells cultured with TNF-α alone (Exo-TNF) and co-stimulated with TNF-α and TGF-β2 (Exo-CO) contained more angiogenic factors than exosomes derived from control cells (Exo-CTL) or ARPE-19 cells cultured with TGF-β2 alone (Exo-TGF). To assess the effect on angiogenesis, we performed chemotaxis, tube formation, and proliferation assays of human umbilical vein endothelial cells (HUVECs) stimulated with or without exosomes. HUVECs migrated to RPE-derived exosomes, and exosomes derived from ARPE-19 cells accelerated HUVEC tube formation. In contrast, Exo-TNF and Exo-CO reduced HUVEC proliferation. Our findings provide insight into the mechanisms underlying the relation between angiogenesis and exosomes derived from RPE cells.

CM082, a novel angiogenesis inhibitor, enhances the antitumor activity of gefitinib on epidermal growth factor receptor mutant non-small cell lung cancer in vitro and in vivo.

BackgroundCM082 is a novel angiogenesis inhibitor targeting vascular endothelial growth factor receptor (VEGFR) and platelet‐derived growth factor receptor (PDGFR). The purpose of this research was to evaluate the antitumor activity of CM082 combined with gefitinib on epidermal growth factor receptor (EGFR) mutant non‐small cell lung cancer (NSCLC) in vitro and in vivo.MethodsThe effect of CM082 on human umbilical vein endothelial cells (HUVECs) was assessed. In vitro and in vivo efficacy of CM082 combined with gefitinib on EGFR NSCLC cell lines (HCC827 harboring E746_A750 deletion and H3255 harboring L858R) and a xenograft model was evaluated.ResultsCM082 inhibited VEGF‐induced cell growth, phosphorylation of VEGFR and downstream signaling molecules, tube formation, and cell migration of HUVECs. Furthermore, CM082 combined with gefitinib was more effective in inhibiting growth and colony formation and inducing apoptosis of H3255 and HCC827 cells in vitro than monotherapy. Moreover, tumor growth inhibition by the combination in a H3255 xenograft model was 107.7% more than that by gefitinib (93.6%) (P < 0.0001) and CM082 (51.4%) (P < 0.0001) alone. In addition, coadministration had a better effect on inhibiting cell proliferation, inducing apoptosis, and inhibiting the expression of CD31 and VEGF‐A. The combination therapy had a stronger inhibition effect on STAT3 phosphorylation than monotherapy.ConclusionsCM082, a novel angiogenesis inhibitor, enhances the antitumor activity of gefitinib on EGFR mutant NSCLC by inhibiting proliferation and angiogenesis and promoting apoptosis of tumor cells.Key points Significant findings of the study CM082, a novel angiogenesis inhibitor, enhances the antitumor activity of gefitinib on EGFR mutant NSCLC by inhibiting proliferation and angiogenesis and promoting apoptosis of tumor cells. What this study adds These findings justify evaluation of the efficacy of CM082 combined with gefitinib in patients with EGFR mutant advanced NSCLC in clinical trials.

Extracellular vesicles from human embryonic stem cell-derived cardiovascular progenitor cells promote cardiac infarct healing through reducing cardiomyocyte death and promoting angiogenesis

Human pluripotent stem cells (hPSCs)-derived cardiovascular progenitor cells (CVPCs) are a promising source for myocardial repair, while the mechanisms remain largely unknown. Extracellular vesicles (EVs) are known to mediate cell–cell communication, however, the efficacy and mechanisms of hPSC-CVPC-secreted EVs (hCVPC-EVs) in the infarct healing when given at the acute phase of myocardial infarction (MI) are unknown. Here, we report the cardioprotective effects of the EVs secreted from hESC-CVPCs under normoxic (EV-N) and hypoxic (EV-H) conditions in the infarcted heart and the long noncoding RNA (lncRNA)-related mechanisms. The hCVPC-EVs were confirmed by electron microscopy, nanoparticle tracking, and immunoblotting analysis. Injection of hCVPC-EVs into acutely infracted murine myocardium significantly improved cardiac function and reduced fibrosis at day 28 post MI, accompanied with the improved vascularization and cardiomyocyte survival at border zones. Consistently, hCVPC-EVs enhanced the tube formation and migration of human umbilical vein endothelial cells (HUVECs), improved the cell viability, and attenuated the lactate dehydrogenase release of neonatal rat cardiomyocytes (NRCMs) with oxygen glucose deprivation (OGD) injury. Moreover, the improvement of the EV-H in cardiomyocyte survival and tube formation of HUVECs was significantly better than these in the EV-N. RNA-seq analysis revealed a high abundance of the lncRNA MALAT1 in the EV-H. Its abundance was upregulated in the infarcted myocardium and cardiomyocytes treated with hCVPC-EVs. Overexpression of human MALAT1 improved the cell viability of NRCM with OGD injury, while knockdown of MALAT1 inhibited the hCVPC-EV-promoted tube formation of HUVECs. Furthermore, luciferase activity assay, RNA pull-down, and manipulation of miR-497 levels showed that MALAT1 improved NRCMs survival and HUVEC tube formation through targeting miR-497. These results reveal that hCVPC-EVs promote the infarct healing through improvement of cardiomyocyte survival and angiogenesis. The cardioprotective effects of hCVPC-EVs can be enhanced by hypoxia-conditioning of hCVPCs and are partially contributed by MALAT1 via targeting the miRNA.

Interfering Human Papillomavirus E6/E7 Oncogenes in Cervical Cancer Cells Inhibits the Angiogenesis of Vascular Endothelial Cells via Increasing miR-377 in Cervical Cancer Cell-Derived Microvesicles.

BackgroundThe dysregulation of the human papillomavirus 18 E6 and E7 oncogenes plays a critical role in the angiogenesis of cervical cancer (CC), including the proliferation, migration, and tube formation of vascular endothelial cells. Interfering E6/E7 increases the number of CC cell-derived microvesicles (CC-MVs). Additionally, microRNAs (miRNAs) can modulate CC angiogenesis and can be encapsulated in MVs.ObjectiveWe aim to investigate whether E6/E7 affects CC angiogenesis via regulating miRNAs in CC-MVs.MethodsCC-MVs were isolated from a CC cell line (HeLa) which were transfected with small interfering RNAs (siRNAs) against E6/E7 or co-transfected with miR-377 mimics/inhibitors. The expression of several miRNAs in CC-MVs was detected using quantitative real-time PCR. After co-incubating CC-MVs with human umbilical vein endothelial cells (HUVECs), cell proliferation, migration, and tube formation of HUVECs were determined using cell counting kit-8, transwell, and tube formation assays, respectively.ResultsMiR-377 was increased in E6/E7-interfering CC-MVs. Overexpressing miR-377 in CC-MVs suppressed HUVEC proliferation, migration, and tube formation. LPAR2, the cell surface G protein-coupled receptor, was the downstream target of miR-377 in HUVECs. The co-transfection of E6/E7 siRNAs and miR-377 inhibitors in CCs negated the effect of E6/E7 siRNAs on the elevation of miR-377 in CC-MVs. In HUVECs, the co-transfection of E6/E7 siRNAs and miR-377 inhibitors restored the LPAR2 expression which was reduced by the E6/E7 siRNA transfection. Meanwhile, miR-377 mimic reduced LPAR2 expression and inhibited HUVEC proliferation, migration, and tube formation, while such response was negated by LPAR2 overexpression.ConclusionInterfering E6/E7 increased miR-377 in CC-MVs, and overexpressing miR-377 in CC-MVs inhibited angiogenesis of HUVECs via reducing LPAR2.

The transcription factor ZFHX3 is crucial for the angiogenic function of hypoxia-inducible factor 1α in liver cancer cells

Angiogenesis is a hallmark of tumorigenesis, and hepatocellular carcinoma (HCC) is hypervascular and therefore very dependent on angiogenesis for tumor development and progression. Findings from previous studies suggest that in HCC cells, hypoxia-induced factor 1α (HIF1A) and zinc finger homeobox 3 (ZFHX3) transcription factors functionally interact in the regulation of genes in HCC cells. Here, we report that hypoxia increases the transcription of the ZFHX3 gene and enhances the binding of HIF1A to the ZFHX3 promoter in the HCC cell lines HepG2 and Huh-7. Moreover, ZFHX3, in turn, physically associated with and was functionally indispensable for HIF1A to exert its angiogenic activity, as indicated by in vitro migration and tube formation assays of human umbilical vein endothelial cells (HUVECs) and microvessel formation in xenograft tumors of HCC cells. Mechanistically, ZFHX3 was required for HIF1A to transcriptionally activate the vascular endothelial growth factor A (VEGFA) gene by binding to its promoter. Functionally, down-regulation of ZFHX3 in HCC cells slowed their tumor growth, and addition of VEGFA to conditioned medium from ZFHX3-silenced HCC cells partially rescued the inhibitory effect of this medium on HUVEC tube formation. In human HCC, ZFHX3 expression was up-regulated, and this up-regulation correlated with both HIF1A up-regulation and worse patient survival, confirming a functional association between ZFHX3 and HIF1A in human HCC. We conclude that ZFHX3 is an angiogenic transcription factor that is integral to the HIF1A/VEGFA signaling axis in HCC cells.

Polyhedral Oligomeric Silsesquioxane-Incorporated Gelatin Hydrogel Promotes Angiogenesis during Vascularized Bone Regeneration.

Many approaches have been made toward the development of scaffolds with good biocompatibility and appreciable physicochemical properties to facilitate stem cell adhesion, osteogenic differentiation, and vascularization in tissue engineering. Nowadays, vascularization is a main bottleneck in tissue engineering strategies that is needed to be overcome and developed. Herein, we construct a series of polyhedral oligomeric silsesquioxane (POSS)-modified porous gelatin hydrogels with different POSS concentrations from 0 to 5 wt %, defined as X% POSS hydrogels (X = 0, 1, 2, 3, 4, 5) to support vascularized bone repair. The introduction of POSS into gelatin effectively promoted adhesive protein adsorption and integrin α5β1 expression, subsequently leading to enhanced adhesion of both rat bone marrow mesenchymal stem cells and human umbilical vein endothelial cells (HUVECs). In vitro experiments further demonstrated that POSS-containing hybrid hydrogels more effectively support the angiogenic tube and network formation in HUVECs than the 0% POSS hydrogel. Besides, POSS-containing hybrid hydrogels showed desirable performance as a sustained release system of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2), and they further accelerated vascular network establishment and the formation of a new bone in defect regions. When the hydrogels were implanted into critical-sized rat calvarial defects in vivo, the VEGF/BMP-2-coupled 3% POSS group gained a higher blood vessel volume in the bone defect regions (5.49 ± 0.35 mm3) than the 3% POSS group (3.12 ± 0.20 mm3) and the 0% POSS group (1.57 ± 0.25 mm3), suggesting that the 3% POSS hydrogel with VEGF/BMP-2 would expedite vascularization. Based on these evaluations, our results indicated that the POSS-incorporated gelatin hydrogel would provide a promising bone graft scheme in potential clinical application of large bone defect repair.

Alternagin-C (ALT-C), a disintegrin-like protein, attenuates alpha2beta1 integrin and VEGF receptor 2 signaling resulting in angiogenesis inhibition

Angiogenesis, a crucial process in tumor progression, is mainly regulated by vascular endothelial growth factor (VEGF) and its receptor, VEGFR2. Studies have shown the interaction between α2β1 integrin, a collagen receptor, and VEGFR2 in VEGF-driven angiogenesis in vitro and in vivo. Alternagin-C (ALT-C), an ECD-disintegrin-like protein from Bothrops alternatus snake venom, has high affinity for α2β1 integrin and shows antiangiogenic activity in concentrations higher than 100 nM. Despite previous results, its mechanism of action on angiogenic signaling pathways has not been addressed. Here we evaluate the antiangiogenic activity of ALT-C in human umbilical vein endothelial cells (HUVECs) associated or not with VEGF, as well as its interference in the α2β1/VEGFR2 crosstalk. ALT-C (1000 nM) affected actin cytoskeleton, decreased the number of cell filopodia, and strongly inhibited HUVEC tube formation, adhesion to type I collagen and cell migration. Down-regulation of α2β1/VEGFR2 crosstalk by ALT-C decreased the protein content and phosphorylation of VEGFR2 and β1 integrin subunit, inhibited ERK 1/2 and PI3K signaling and regulated FAK/Src and paxillin pathways. Furthermore, ALT-C increased the content of the autophagic markers LC3B and Beclin-1 in the presence of VEGF, which is associated with decreased angiogenesis. In conclusion, we suggest that ALT-C, after binding to α2β1 integrin, inhibits VEGF/VEGFR2 signaling, which results in impaired angiogenesis. These results demonstrate that ALT-C may be a potential candidate for the development of antiangiogenic therapies for tumor and metastasis treatment and help to understand the complexity and fundamental role of integrin inhibition in the tumor microenvironment.

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Long non‑coding RNA NORAD regulates angiogenesis of human umbilical vein endothelial cells via miR‑590‑3p under hypoxic conditions.

Dysregulation of angiogenesis can be caused by hypoxia, which may result in severe diseases of the heart, including coronary artery disease. Hypoxia-inducible factor 1 (HIF-1) modulates angiogenesis via the regulation of several angiogenic factors. However, the underlying mechanism of hypoxia-induced angiogenesis remains unknown. In the present study, it was hypothesized that long non-coding RNA (lncRNA) non-coding RNA activated by DNA damage (NORAD) may serve a role in the process of angiogenesis via the regulation of microRNA(miR)-590-3p under hypoxic conditions. The effect of NORAD and miR-590-3p on cell viability and properties associated with angiogenesis, including cell migration and tube formation in human umbilical vein endothelial cells (HUVECs) under hypoxic conditions, were assessed. Potential downstream angiogenic factors of miR-590-3p were also determined by molecular experiments. It was identified that NORAD expression was upregulated and miR-590-3p expression was downregulated in hypoxia-exposed HUVECs, and also in myocardial infarction (MI) left ventricle tissues in mice. Moreover, downregulation of NORAD expression resulted in decreased cell viability and angiogenic capacity, but further knocking down miR-590-3p expression reversed these alterations, resulting in increased cell migration and tube formation in HUVECs under hypoxic conditions for 24 h. It was demonstrated that NORAD overexpression also increased cell vitality and tube-formation capacity. Furthermore, NORAD was identified to bind with miR-590-3p directly, and miR-590-3p was shown to target certain proangiogenic agents, such as vascular endothelial growth factor (VEGF)A, fibroblast growth factor (FGF)1 and FGF2 directly. Therefore, the present results suggested that lncRNA NORAD may bind with miR-590-3p to regulate the angiogenic ability of HUVECs via the regulation of several downstream proangiogenic factors under hypoxia. Thus, the lncRNA NORAD/miR-590-3p axis may be a novel regulatory pathway in the angiogenic mechanisms in HUVECs, which highlights a potentially novel perspective for treating ischemia/hypoxia-induced angiogenic diseases.

The Uremic Toxin Indoxyl Sulfate Impairs Angiogenesis Through Aryl Hydrocarbon Receptor Activation

Introduction The aryl hydrocarbon receptor (AHR) is a ligand‐activated transcription factor that coordinates expression of genes involved in xenobiotic metabolism of a variety of environmental and endogenous toxins. Several metabolites, including indoxyl sulfate (IS) and kynurenine, that are derived from tryptophan metabolism are known AHR ligands. Aberrant tryptophan metabolism in the intestinal microbiota combined with impaired kidney function results in accumulation of these metabolites, defined as uremia. Previous studies have suggested that kidney disease results in impaired angiogenesis, however the molecular mechanisms are unknown. The goal of this study was to examine the impact of AHR activation on endothelial cell biology.Methods and ResultsTo accomplish this goal, assessments of endothelial cell proliferation, migration, and tube formation assays in human umbilical vein endothelial cells (HUVECs) and assessment of endothelial cell sprouting in aortic rings from adult C57BL/6J mice (N = 8). HUVECs and aortic rings were exposed to 500μM IS, which did not induce cell death, but activated the AHR by significantly increasing CYP1A1 mRNA levels (~50‐fold in HUVECs and ~8‐fold in aortic rings). IS treatment significantly impaired tube formation in HUVECs (IS: 13.5 ± 1.76 vs. Vehicle: 24 ± 1.9; P &lt; 0.001) and endothelial sprouting in aortic rings (IS: 6.1 ± 0.9 vs. Vehicle: 20.7 ± 0.9; P &lt; 0.001). Additional experiments in HUVECs demonstrated that IS treatment did not impair endothelial cell migration at 24 hours (IS: 100 ± 0 vs Vehicle: 100 ± 0; P = 0.32) but completely ablated the cells ability to proliferate (IS: 1317.8 ± 67.5 vs. Vehicle: 4388.7 ± 371.8; P &lt; 0.001). To examine the role of the AHR, we used shRNA targeting the AHR (shAHR) and chemical AHR antagonists, resveratrol and CH223191. Both chemical antagonism and genetic knockdown of the AHR prevented AHR‐mediated increases in CYP1A1 mRNA with IS‐treatment. Moreover, chemical antagonism and shAHR treatments rescued cell proliferation (shAHR: 2910 ± 282.1 vs. Scramble: 1035 ± 41.7; P = 0.001), tube formation (shAHR: 10.6 ± 1.0 vs. Scramble: 1.1 ± 0.38; P &lt; 0.001), and sprouting (shAHR: 22.9 ± 0.86 vs. Scramble: 7.9 ± 1.26; P &lt; 0.001) following IS treatment. To isolate the AHR, we generated a plasmid encoding a constitutively active AHR (CAAHR) by deletion of the ligand binding domain. Transfection with CAAHR increased both AHR and CYP1A1 expression, confirming non‐ligand dependent AHR activity. Consistent with IS treatment, expression of the CAAHR impaired endothelial cell tube formation (CAAHR: 5.6 ± 1.5 vs. GFP: 20.2.8 ± 1.2; P &lt; 0.001) and sprouting (CAAHR: 18.5 ± 1.3 vs. GFP: 25.8 ± 2.9, P = 0.028). CAAHR impairments in angiogenesis were driven by significant decreases in endothelial cell proliferation, however endothelial cell migration was not affected.ConclusionAHR activation impairs angiogenesis by compromising endothelial cell proliferation. Genetic or pharmaceutical treatments to prevent AHR activation rescued endothelial cell proliferation and angiogenesis. Taken together, these findings establish the role of the AHR in vascular biology and highlight the potential for AHR antagonism to improve angiogenesis in ischemic cardiovascular diseases.Support or Funding InformationSupported by a seed grant from the UF Office of Research

Exosomal lncRNA RAMP2-AS1 Derived from Chondrosarcoma Cells Promotes Angiogenesis Through miR-2355-5p/VEGFR2 Axis.

ObjectiveExosomes derived from cancer cells can alter the microenvironment and enhance cancer malignancy through the regulation of peripheral cell functions. The present study focused on the crosstalk between chondrosarcoma cells and human umbilical vein endothelial cells (HUVECs) mediated by exosomes derived from chondrosarcoma cells and aimed to explore the potential molecular mechanism.Materials and MethodsChondrosarcoma cell-derived exosomes were isolated and characterized. Cell proliferation assay, tube formation assay and transwell migration assay were performed to characterize the effects of exosomes on HUVECs. The lncRNA microarray was used to select differentially expressed lncRNAs in HUVECs treated with or without exosomes. Serum samples of patients with chondrosarcoma were collected to analyze the correlation between the RAMP2-AS1 level and the clinicopathological features. Online databases were used to predict the target microRNA of RAMP2-AS1. Dual luciferase reporter assay, Western blotting and qRT-PCR assays were performed to verify the interactions among RAMP2-AS1, miR-2355-5p and VEGFR2. Rescue experiments were conducted to validate the existence of the RAMP2-AS1/miR-2355-5p/VEGFR2 axis.ResultsThe exosomes secreted by chondrosarcoma cells could enhance HUVECs proliferation, migration and tube formation. LncRNA microarray analysis revealed that exosomes carried lncRNA RAMP2-AS1, and further verification showed that the level of RAMP2-AS1 was increased in the serum of chondrosarcoma patients and was closely related to local invasiveness, distant metastasis and poor prognosis. Subsequent experiments demonstrated that RAMP2-AS1 knockdown could partly abrogate the promoting effects on angiogenesis induced by exosomes derived from chondrosarcoma cells. Moreover, dual luciferase reporter assay and rescue experiments suggested that the RAMP2-AS1/miR-2355-5p/VEGFR2 axis was responsible for exosome-induced angiogenesis of HUVECs.ConclusionChondrosarcoma cell-derived exosomes carry lncRNA RAMP2-AS1, which acts as a ceRNA of miR-2355-5p to regulate VEGFR2 expression, thereby positively regulating the angiogenic ability of HUVECs. Thus, exosomal RAMP2-AS1 has the potential as a novel biomarker and therapeutic target for chondrosarcoma.

The role and mechanism of asymmetric dimethylarginine in fetal growth restriction via interference with endothelial function and angiogenesis.

Fetal growth restriction (FGR) is a high-risk pregnancy, and placental dysfunction is the main cause of FGR. The upregulation of asymmetric dimethylarginine (ADMA) is linked to FGR pathology, but the mechanism needs to be investigated. The levels of ADMA and other related molecules were measured in human biological samples. We further used human umbilical vein endothelial cells (HUVECs) to reveal the mechanism of ADMA-induced FGR in vitro. Compared with the control group, FGR patients had higher placental resistance, and ADMA levels were increased in the maternal blood, cord blood, and placenta; additionally, nitric oxide (NO) production decreased, accompanied by a decreased expression of endogenous NO synthase (eNOS). The expression of vascular growth factor (VEGF) and placental growth factor (PLGF) in the maternal blood during the third trimester and umbilical cord of the FGR group was lower than the control group. The PLGF levels in the placentas of the FGR group were also reduced, while the expression of soluble fms-like tyrosine kinase-1 (sFlt-1) increased. In in vitro cell experiments, NO production was obviously lower when the cells were exposed to 100μM of ADMA, with no difference in eNOS expression. There was a dose-dependent decrease in PLGF expression with increasing doses of ADMA, and the levels of sFlt-1 increased. Moreover, we confirmed that tube formation in HUVECs was lower after ADMA treatment compared with the control group. The accumulation of ADMA during pregnancy has an adverse effect on fetal development via interference with placental endothelial function and angiogenesis.

Silica nanomaterials induce organ injuries by Ca2+-ROS-initiated disruption of the endothelial barrier and triggering intravascular coagulation

BackgroundThe growing use of silica nanoparticles (SiNPs) in many fields raises human toxicity concerns. We studied the toxicity of SiNP-20 (particle diameter 20 nm) and SiNP-100 (100 nm) and the underlying mechanisms with a focus on the endothelium both in vitro and in vivo.MethodsThe study was conducted in cultured human umbilical vein endothelial cells (HUVECs) and adult female Balb/c mice using several techniques.ResultsIn vitro, both SiNP-20 and SiNP-100 decreased the viability and damaged the plasma membrane of cultured HUVECs. The nanoparticles also inhibited HUVECs migration and tube formation in a concentration-dependent manner. Both SiNPs induced significant calcium mobilization and generation of reactive oxygen species (ROS), increased the phosphorylation of vascular endothelial (VE)-cadherin at the site of tyrosine 731 residue (pY731-VEC), decreased the expression of VE-cadherin expression, disrupted the junctional VE-cadherin continuity and induced F-actin re-assembly in HUVECs. The injuries were reversed by blocking Ca2+ release activated Ca2+ (CRAC) channels with YM58483 or by eliminating ROS with N-acetyl cysteine (NAC). In vivo, both SiNP-20 and SiNP-100 (i.v.) induced multiple organ injuries of Balb/c mice in a dose (range 7–35 mg/kg), particle size, and exposure time (4–72 h)-dependent manner. Heart injuries included coronary endothelial damage, erythrocyte adhesion to coronary intima and coronary coagulation. Abdominal aorta injury exhibited intimal neoplasm formation. Lung injuries were smaller pulmonary vein coagulation, bronchiolar epithelial edema and lumen oozing and narrowing. Liver injuries included multifocal necrosis and smaller hepatic vein congestion and coagulation. Kidney injuries involved glomerular congestion and swelling. Macrophage infiltration occurred in all of the observed organ tissues after SiNPs exposure. SiNPs also decreased VE-cadherin expression and altered VE-cadherin spatial distribution in multiple organ tissues in vivo. The largest SiNP (SiNP-100) and longest exposure time exerted the greatest toxicity both in vitro and in vivo.ConclusionsSiNPs, administrated in vivo, induced multiple organ injuries, including endothelial damage, intravascular coagulation, and secondary inflammation. The injuries are likely caused by upstream Ca2+-ROS signaling and downstream VE-cadherin phosphorylation and destruction and F-actin remodeling. These changes led to endothelial barrier disruption and triggering of the contact coagulation pathway.

Human mesenchymal stromal cell source and culture conditions influence extracellular vesicle angiogenic and metabolic effects on human endothelial cells in vitro.

Mesenchymal stem/stromal cell (MSC)-derived extracellular vesicles (EVs) are a possible cell-free alternative to MSCs because they retain the regenerative potential of MSCs, while still mitigating some of their limitations (such as the possible elicitation of host immune responses). The promotion and restoration of angiogenesis, however, is an important component in treating trauma-related injuries, and has not been fully explored with EVs. Herein, we describe the effects of monolayer adipose-derived EVs, spheroid adipose-derived EVs (SAd-EVs), monolayer bone marrow-derived EVs (MBM-EVs), and spheroid bone marrow-derived EVs (SBM-EVs) on human umbilical vein endothelial cell (HUVEC) tube formation and mitochondrial respiration. The successful isolation of EVs derived from adipose MSCs or bone marrow MSCs in monolayer or spheroid cultures was confirmed by NanoSight (particle size distribution) and Western blot (surface marker expression). The EV angiogenic potential was measured using a 24-hour HUVEC tube formation assay. The EV effects on HUVEC mitochondrial function were evaluated using the Seahorse respirometer machine. The number of junctions, branches, and the average length of branches formed at 24 hours of tube formation were significantly affected by cell and culture type; overall adipose-derived EVs outperformed bone marrow-derived EVs, and spheroid-derived EVs outperformed monolayer-derived EVs. Additionally, adipose-derived EVs resulted in significantly increased HUVEC mitochondrial maximal respiration and adenosine triphosphate (ATP) production, while only MBM-EVs negatively impacted HUVEC proton leak. Adipose-derived EVs promoted HUVEC tube formation significantly more than bone marrow-derived EVs, while also maximizing HUVEC mitochondria function. Results demonstrate that, as with MSC therapies, it is possible to tailor EV culture and production to optimize therapeutic potential. Basic or Foundational Research.

Pro-angiogenic activity of Tongnao decoction on HUVECs in vitro and zebrafish in vivo

Ethnopharmacological relevanceTongnao Decoction (TND) is a Chinese decoction approved and used in Jiangsu Province Hospital for the treatment of ischemic stroke. It shows conclusive efficiency in the improvement of neurologic impairment and activities of daily living of the patients. Aim of the studyRecently, angiogenesis has been recognized as a potential therapeutic strategy for treating cerebral ischemia. This study was aimed to provide comprehensive evidence for the pro-angiogenic effect of TND and characterize the underlying mechanism. Materials and methodsWe firstly established the chemical fingerprinting of TND. Then, the in vitro pro-angiogenic activities of TND were tested on human umbilical vein endothelial cells (HUVECs) through cell viability, wound healing and tube formation assays. The in vivo pro-angiogenic effects were evaluated on transgenic zebrafish embryos [Tg (fli-1: EGFP)] through the formation of intersegmental vessels (ISVs), subintestinal vessels (SIVs) and central arteries (CtAs). Lastly, the potential mechanisms of TND were analyzed by a blocking assay with eight pathways-specific kinase inhibitors. ResultsTND promoted the proliferation, migration and tube formation of HUVECs. TND also rescued the impairment of ISVs, SIVs and CtAs caused by VRI in a dose-dependent manner in zebrafish embryos. TND could activate vascular endothelial growth factor receptor-2 (VEGFR-2), phosphoinositide 3-kinase (PI3K) – protein kinase B (Akt) and Raf – mitogen-activated protein kinase1/2 (MEK1/2) – extracellular regulated kinase 1/2 (ERK1/2) signaling pathways. ConclusionOur study firstly demonstrated the pro-angiogenic activities of TND. Our work provided evidences for the clinical usage of TND in restoring neurovascular function through promoting angiogenesis in the ischemic cerebral microvascular.

Tanshinone IIA reduces secretion of pro‑angiogenic factors and inhibits angiogenesis in human colorectal cancer.

Tumor angiogenesis is an important factor which precipitates recurrence and metastasis of colorectal cancer (CRC). Angiogenesis is also a significant feature which accompanies invasion and metastasis of CRC. Tumor hypoxia activates hypoxia inducible factor (HIF), which promotes angiogenesis in CRC. HIF significantly promotes cell proliferation and angiogenesis in CRC, facilitating invasion and metastasis. Tanshinone IIA (Tan IIA) has been revealed to effectively inhibit angiogenesis in CRC, although the underlying mechanism remains to be determined. The aim of the present study was to determine the effects of HIF-1α on hypoxia induced angiogenesis in CRC cells, the effects of Tan IIA on the expression of pro-angiogenic factors in CRC cells, and on human umbilical vein endothelial cell (HUVEC) tube formation in normal and hypoxic conditions. The results of the present study revealed that Tan IIA not only decreased HIF-1α expression and inhibited the secretion level of vascular endothelial growth factor and basic fibroblast growth factor, but also efficiently decreased proliferation, tube formation and metastasis of HUVECs. The results highlight the potential of Tan IIA-mediated targeting of HIF-1α as a potential therapeutic option for treatment of patients with CRC.