Impact of DICER1 and DROSHA on the Angiogenic Capacity of Human Endothelial Cells

RNAi-mediated knockdown of DICER1 and DROSHA, enzymes critically involved in miRNA biogenesis, has been postulated to affect the homeostasis and the angiogenic capacity of human endothelial cells. To re-evaluate this issue, we reduced the expression of DICER1 or DROSHA by RNAi-mediated knockdown and subsequently investigated the effect of these interventions on the angiogenic capacity of human umbilical vein endothelial cells (HUVEC) in vitro (proliferation, migration, tube formation, endothelial cell spheroid sprouting) and in a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. In contrast to previous reports, neither knockdown of DICER1 nor knockdown of DROSHA profoundly affected migration or tube formation of HUVEC or the angiogenic capacity of HUVEC in vivo. Furthermore, knockdown of DICER1 and the combined knockdown of DICER1 and DROSHA tended to increase VEGF-induced BrdU incorporation and induced angiogenic sprouting from HUVEC spheroids. Consistent with these observations, global proteomic analyses showed that knockdown of DICER1 or DROSHA only moderately altered HUVEC protein expression profiles but additively reduced, for example, expression of the angiogenesis inhibitor thrombospondin-1. In conclusion, global reduction of miRNA biogenesis by knockdown of DICER1 or DROSHA does not inhibit the angiogenic capacity of HUVEC. Further studies are therefore needed to elucidate the influence of these enzymes in the context of human endothelial cell-related angiogenesis.

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Abstract 116: Arterial Endothelial Cell Has Stronger Angiogenic Potential Compared To Venous Endothelial Cell Through Up-regulation Of Endogenous FGF2 And FGF5 Expression In 3D Microfluidic System

Circulation Research ◽

10.1161/res.115.suppl_1.116 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Ha-Rim Seo ◽

Hyo Eun Jeong ◽

Hyung Joon Joo ◽

Seung-Cheol Choi ◽

Jong-Ho Kim ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Molecular Mechanisms ◽

Umbilical Vein ◽

Assay System ◽

Vascular Density ◽

Angiogenic Potential ◽

Angiogenesis Assay

Background: Human body contains many kinds of different type of endothelial cells (EC). However, cellular difference of their angiogenic potential has been hardly understood. We compared in vitro angiogenic potential between arterial EC and venous EC and investigated its underlying molecular mechanisms. Method: Used human aortic endothelial cells (HAEC) which was indicated from arterial EC and human umbilical vein endothelial cells (HUVEC) indicated from venous EC. To explore angiogenic potential in detail, we adopted a novel 3D microfluidic angiogenesis assay system, which closely mimic in vivo angiogenesis. Results: In 3D microfluidic angiogenesis assay system, HAEC demonstrated stronger angiogenic potential compared to HUVEC. HAEC maintained its profound angiogenic property under different biophysical conditions. In mRNA microarray sorted on up- regulated or down-regulated genes, HAEC demonstrated significantly higher expression of gastrulation brain homeobox 2 (GBX2), fibroblast grow factor 2 (FGF2), FGF5 and collagen 8a1. Angiogenesis-related protein assay revealed that HAEC has higher secretion of endogenous FGF2 than HUVEC. HAEC has only up-regulated FGF2 and FGF5 in this part of FGF family. Furthermore, FGF5 expression under vascular endothelial growth factor-A (VEGF-A) stimulation was higher in HAEC compared to HUVEC although VEGF-A augmented FGF5 expression in both HAEC and HUVEC. Those data suggested that FGF5 expression in both HAEC and HUVEC is partially dependent to VEGF-A stimulate. HUVEC and HAEC reduced vascular density after FGF2 and FGF5 siRNA treat. Conclusion: HAEC has stronger angiogenic potential than HUVEC through up-regulation of endogenous FGF2 and FGF5 expression

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Accutin, a New Disintegrin, Inhibits Angiogenesis In Vitro and In Vivo by Acting as Integrin vβ3 Antagonist and Inducing Apoptosis

Blood ◽

10.1182/blood.v92.9.3268 ◽

1998 ◽

Vol 92 (9) ◽

pp. 3268-3276 ◽

Cited By ~ 90

Author(s):

Chia Hsin Yeh ◽

Hui-Chin Peng ◽

Tur-Fu Huang

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Fluorescein Isothiocyanate ◽

Adenosine Diphosphate ◽

Tube Formation ◽

Platelet Glycoprotein ◽

Dependent Manner ◽

Antiangiogenic Effect

Abstract Endothelial integrins play an essential role in angiogenesis and cell survival. Accutin, a new member of disintegrin family derived from venom of Agkistrodon acutus, potently inhibited human platelet aggregation caused by various agonists (eg, thrombin, collagen, and, adenosine diphosphate [ADP]) through the blockade of fibrinogen binding to platelet glycoprotein IIb/IIIa (ie, integrin IIbβ3). In this report, we describe that accutin specifically inhibited the binding of monoclonal antibody (MoAb) 7E3, which recognizes integrin vβ3, to human umbilical vein endothelial cells (HUVECs), but not those of other anti-integrin MoAbs such as 2β1, 3β1, and 5β1. Moreover, accutin, but not the control peptide GRGES, dose-dependently inhibited the 7E3 interaction with HUVECs. Both 7E3 and GRGDS, but not GRGES or Integrelin, significantly blocked fluorescein isothiocyanate-conjugated accutin binding to HUVEC. In functional studies, accutin exhibited inhibitory effects on HUVEC adhesion to immobilized fibrinogen, fibronectin and vitronectin, and the capillary-like tube formation on Matrigel in a dose- and RGD-dependent manner. In addition, it exhibited an effective antiangiogenic effect in vivo when assayed by using the 10-day-old embryo chick CAM model. Furthermore, it potently induced HUVEC apoptotic DNA fragmentation as examined by electrophoretic and flow cytometric assays. In conclusion, accutin inhibits angiogenesis in vivo and in vitro by blocking integrin vβ3 of endothelial cells and by inducing apoptosis. The antiangiogenic activity of disintegrins might be explored as the target of developing the potential antimetastatic agents. © 1998 by The American Society of Hematology.

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THE LUPUS ANTICOAGULANT DOES NOT INHIBIT THE RELEASE OF PROSTACYCLIN FROM HUMAN ENDOTHELIAL CELLS

10.1055/s-0038-1644228 ◽

1987 ◽

Author(s):

W Petraiuolo ◽

E Bovill ◽

J Hoak

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Causal Relationship ◽

Lupus Anticoagulant ◽

Umbilical Vein ◽

Human Endothelial Cells ◽

Human Umbilical Vein ◽

Blood Institute ◽

Specialized Center ◽

National Heart Lung

Decreased endothelial cell production of prostacyclin (PGI2) in response to the lupus anticoagulant has been previously demonstrated, and postulated to have a causal relationship to the thrombotic events associated with the lupus anticoagulant. Five patients who exhibited the anticoagulant were studied in an effort to determine if a relationship exists between exposure of endothelial cells to the lupus anticoagulant and decreased production of prostacyclin (PGI2). Human endothelial cells derived from human umbilical vein grown in culture were exposed to IgG fractions of patient plasmas containing the lupus anticoagulant. The amount of PGI2 released was determined by radioimmunoassay for 6-keto-PGF-l-alpha. The average PGI2 release in the controls was 20.6 picomol/500,000 endothelial cells, whereas those cells exposed to the lupus anticoagulant had a range of 25 to 114 picmol/500,000 cells. We were unable to demonstrate inhibition of the release of PGI2 by human endothelial cells, following exposure to the lupus anticoagulant.(Supported by NIH Grant HL 33723-2 and a Specialized Center of Research in Thrombosis Award HL 35058-01 from the National Heart, Lung and Blood Institute.)

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DEPTOR Deficiency-Mediated mTORc1 Hyperactivation in Vascular Endothelial Cells Promotes Angiogenesis

Cellular Physiology and Biochemistry ◽

10.1159/000488619 ◽

2018 ◽

Vol 46 (2) ◽

pp. 520-531 ◽

Cited By ~ 7

Author(s):

Yan Ding ◽

Lanlan Shan ◽

Wenqing Nai ◽

Xiaojun Lin ◽

Ling Zhou ◽

...

Keyword(s):

Endothelial Cells ◽

Signaling Pathway ◽

Molecular Mechanisms ◽

Gene Knockout ◽

Umbilical Vein ◽

Vascular Endothelial Cell ◽

Tube Formation ◽

Vascular Endothelial

Background/Aims: The mechanistic target of rapamycin (mTOR) signaling pathway is essential for angiogenesis and embryonic development. DEP domain-containing mTOR-interacting protein (DEPTOR) is an mTOR binding protein that functions to inhibit the mTOR pathway In vitro experiments suggest that DEPTOR is crucial for vascular endothelial cell (EC) activation and angiogenic responses. However, knowledge of the effects of DEPTOR on angiogenesis in vivo is limited. This study aimed to determine the role of DEPTOR in tissue angiogenesis and to elucidate the molecular mechanisms. Methods: Cre/loxP conditional gene knockout strategy was used to delete the Deptor gene in mouse vascular ECs. The expression or distribution of cluster of differentiation 31 (CD31), vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-1α) were detected by immunohistochemical staining or western blot. Tube formation assay was used to measure angiogenesis in vitro. Results: Deptor knockdown led to increased expression of CD31, VEGF and HIF-1α in heart, liver, kidney and aorta. After treatment with rapamycin, their expression was significantly down regulated. In vitro, human umbilical vein endothelial cells (HUVECs) were transfected with DEPTOR-specific small interfering RNA (siRNA), which resulted in a significant increase in endothelial tube formation and migration rates. In contrast, DEPTOR overexpression markedly reduced the expression of CD31, VEGF and HIF-1α. Conclusions: Our findings demonstrated that deletion of the Deptor gene in vascular ECs resulted in upregulated expression of CD31 and HIF-1α, and further stimulated the expression of VEGF which promoted angiogenesis, indicating that disruption of normal angiogenic pathways may occur through hyperactivation of the mTORC1/HIF-1α/VEGF signaling pathway.

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CYLD regulates angiogenesis by mediating vascular endothelial cell migration

Blood ◽

10.1182/blood-2009-10-248526 ◽

2010 ◽

Vol 115 (20) ◽

pp. 4130-4137 ◽

Cited By ~ 52

Author(s):

Jinmin Gao ◽

Lei Sun ◽

Lihong Huo ◽

Min Liu ◽

Dengwen Li ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Migration ◽

Endothelial Cell ◽

Vascular Endothelial Cells ◽

Vascular Endothelial Cell ◽

Tube Formation ◽

Endothelial Cell Migration ◽

Vascular Endothelial

Cylindromatosis (CYLD) is a deubiquitinase that was initially identified as a tumor suppressor and has recently been implicated in diverse normal physiologic processes. In this study, we have investigated the involvement of CYLD in angiogenesis, the formation of new blood vessels from preexisting ones. We find that knockdown of CYLD expression significantly impairs angiogenesis in vitro in both matrigel-based tube formation assay and collagen-based 3-dimensional capillary sprouting assay. Disruption of CYLD also remarkably inhibits angiogenic response in vivo, as evidenced by diminished blood vessel growth into the angioreactors implanted in mice. Mechanistic studies show that CYLD regulates angiogenesis by mediating the spreading and migration of vascular endothelial cells. Silencing of CYLD dramatically decreases microtubule dynamics in endothelial cells and inhibits endothelial cell migration by blocking the polarization process. Furthermore, we identify Rac1 activation as an important factor contributing to the action of CYLD in regulating endothelial cell migration and angiogenesis. Our findings thus uncover a previously unrecognized role for CYLD in the angiogenic process and provide a novel mechanism for Rac1 activation during endothelial cell migration and angiogenesis.

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Thrombospondin-4 (TSP4) gene-modified bone marrow stromal cells (BMSCs) promote the effect of therapeutic angiogenesis in critical limb ischemia (CLI) of diabetic rats

10.21203/rs.2.15779/v1 ◽

2019 ◽

Author(s):

Qian Zhang ◽

Tao Wang ◽

Xiangfeng Wu ◽

Ying Wang ◽

Xuanqin Wu ◽

...

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Endothelial Cell ◽

Growth Factor ◽

Motor Function ◽

Limb Ischemia ◽

Tube Formation ◽

Endothelial Cell Proliferation ◽

Thrombospondin 4

Abstract Background: Critical limb ischemia (CLI) is the leading cause of lower limb amputation. Traditional treatments for CLI have limitations. Studies have shown that thrombospondin-4 (TSP4) can promote the growth of neovascularization. Results: In this study, we observed the angiogenesis efficiency of TSP4-overexpressing BMSC transplantation in CLI treatment. The recombinant FT106-tsp4-gfp lentiviral vector plasmid was constructed and transfected into 293FT cells. Primary BMSCs were successfully infected with the tsp4 virus, and TSP4 overexpression was confirmed before TSP4-BMSCs infusion. In vitro, TSP4-BMSCs were co-cultured with human umbilical vein endothelial cells (HUVECs). Vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGF-β) secretion were measured in the co-culture supernatants by ELISA. The effect of TSP4-BMSCs on endothelial cell proliferation and migration was detected. Meanwhile, the effects of TSP4-BMSC on the angiogenesis of endothelial cells were tested by tube formation experiment and arterial ring test. In vivo, a rat CLI model was established, and 60 CLI rats were randomly divided into the CLI, BMSC + CLI and TSP4-BMSC + CLI groups. The effect of TSP4-BMSC on angiogenesis was detected by the motor function, immunohistochemistry and immunofluorescence staining assays. Neovascular density was detected by digital substraction angiography (DSA). Our results demonstrated that TSP4-BMSCs obviously increased TSP4, VEGF, Ang-1, MMP9, MMP2 and p-Cdc42/Rac1 expression in endothelial cells. TSP4-BMSCs treatment notably upregulated the TGF-β/smad2/3 signal pathway in HUVECs. In vivo, TSP4-BMSCs improved the motor function score of the CLI rats and increased MMP2, MMP9, Ang-1, VEGF and vWF protein expression in tissue of the ischaemic area. Meanwhile, new blood vessels can be observed around the ischemic area after TSP4-BMSCs treatment. Conclusion: Our data illustrate that TSP4-BMSCs can promote endothelial cell proliferation, migration, tube formation and the recovery of motor function in diabetic hind limb ischaemic rats. TSP4-BMSCs have better therapeutic effects than BMSCs.

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Thrombospondin-4 (TSP4) gene-modified bone marrow stromal cells (BMSCs) promote the effect of therapeutic angiogenesis in critical limb ischemia (CLI) of diabetic rats

10.21203/rs.3.rs-15860/v1 ◽

2020 ◽

Author(s):

Qian Zhang ◽

Tao Wang ◽

Xiangfeng Wu ◽

Ying Wang ◽

Xuanqin Wu ◽

...

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Endothelial Cell ◽

Growth Factor ◽

Motor Function ◽

Limb Ischemia ◽

Tube Formation ◽

Endothelial Cell Proliferation ◽

Thrombospondin 4

Abstract Background Critical limb ischemia (CLI) is the leading cause of lower limb amputation. Traditional treatments for CLI have limitations. Studies have shown that thrombospondin-4 (TSP4) can promote the growth of neovascularization. In this study, we observed the angiogenesis efficiency of TSP4-overexpressing BMSC transplantation in CLI treatment. Methods The recombinant FT106-tsp4-gfp lentiviral vector plasmid was constructed and transfected into 293FT cells. Primary BMSCs were successfully infected with the tsp4 virus, and TSP4 overexpression was confirmed before TSP4-BMSCs infusion. In vitro, TSP4-BMSCs were co-cultured with human umbilical vein endothelial cells (HUVECs). Vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGF-β) secretion were measured in the co-culture supernatants by ELISA. The effect of TSP4-BMSCs on endothelial cell proliferation and migration was detected. Meanwhile, the effects of TSP4-BMSC on the angiogenesis of endothelial cells were tested by tube formation experiment and arterial ring test. In vivo, a rat CLI model was established, and 60 CLI rats were randomly divided into the CLI, BMSC + CLI and TSP4-BMSC + CLI groups. The effect of TSP4-BMSC on angiogenesis was detected by the motor function, immunohistochemistry and immunofluorescence staining assays. Neovascular density was detected by digital substraction angiography (DSA). Results Our results demonstrated that TSP4-BMSCs obviously increased TSP4, VEGF, Ang-1, MMP9, MMP2 and p-Cdc42/Rac1 expression in endothelial cells. TSP4-BMSCs treatment notably upregulated the TGF-β/smad2/3 signal pathway in HUVECs. In vivo, TSP4-BMSCs improved the motor function score of the CLI rats and increased MMP2, MMP9, Ang-1, VEGF and vWF protein expression in tissue of the ischaemic area. Meanwhile, new blood vessels can be observed around the ischemic area after TSP4-BMSCs treatment. Conclusions Our data illustrate that TSP4-BMSCs can promote endothelial cell proliferation, migration, tube formation and the recovery of motor function in diabetic hind limb ischaemic rats. TSP4-BMSCs have better therapeutic effects than BMSCs.

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Adenosine stimulates proliferation of human endothelial cells in culture

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1993.265.1.h131 ◽

1993 ◽

Vol 265 (1) ◽

pp. H131-H138 ◽

Cited By ~ 9

Author(s):

M. F. Ethier ◽

V. Chander ◽

J. G. Dobson

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Growth ◽

Cell Cultures ◽

Umbilical Vein ◽

Fibroblast Cell ◽

Human Endothelial Cells ◽

Cell Counts ◽

Endothelial Cell Cultures ◽

Endothelial Cell Growth

The effect of adenosine on proliferation of human endothelial cells was investigated by adding adenosine to the medium of cultures derived from human umbilical veins. Cell counts on cultures grown in 10 microM adenosine for 4–7 days were 41–53% greater than counts from control cultures. In contrast, 10 microM adenosine had no effect on growth of a human fibroblast cell strain (IMR-90). Neither inosine nor 2',5'-dideoxyadenosine influenced endothelial cell growth at concentrations of 0.1 or 10 microM. Addition of adenosine deaminase abolished the proliferative effect of added adenosine and inhibited proliferation by 16% in control cultures, suggesting that endogenous adenosine may enhance proliferation in culture. The adenosine receptor antagonist, 8-phenyltheophylline, at 0.1 and 1.0 microM blocked the enhanced proliferation caused by 10 microM adenosine. Addition of 10 microM adenosine enhanced DNA synthesis in endothelial cell cultures as indicated by an increased incorporation of [3H]thymidine into acid-insoluble cell material. The results indicate that addition of physiological concentrations of adenosine to human umbilical vein endothelial cell cultures stimulates proliferation, possibly via a surface receptor, and suggest that adenosine may be a factor for human endothelial cell growth and possibly angiogenesis.

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Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis

AJP Cell Physiology ◽

10.1152/ajpcell.00230.2008 ◽

2008 ◽

Vol 295 (5) ◽

pp. C1292-C1301 ◽

Cited By ~ 74

Author(s):

Anke C. Webler ◽

U. Ruth Michaelis ◽

Rüdiger Popp ◽

Eduardo Barbosa-Sicard ◽

Andiappan Murugan ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Growth Factor ◽

Protein Expression ◽

Second Messengers ◽

Tube Formation ◽

Dominant Negative ◽

Endothelial Cell Proliferation ◽

Angiogenic Response

Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acid (EET) regioisomers, which activate several signaling pathways to promote endothelial cell proliferation, migration, and angiogenesis. Since vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, we assessed a possible role of EETs in the VEGF-activated signal transduction cascade. Stimulation with VEGF increased CYP2C promoter activity in endothelial cells and enhanced CYP2C8 mRNA and protein expression resulting in increased intracellular EET levels. VEGF-induced endothelial cell tube formation was inhibited by the EET antagonist 14,15-epoxyeicosa-5( Z)-enoicacid (14,15-EEZE), which did not affect the VEGF-induced phosphorylation of its receptor or basic fibroblast growth factor (bFGF)-stimulated tube formation. Moreover, VEGF-stimulated endothelial cell sprouting in a modified spheroid assay was reduced by CYP2C antisense oligonucleotides. Mechanistically, VEGF stimulated the phosphorylation of the AMP-activated protein kinase (AMPK), which has also been linked to CYP induction, and the overexpression of a constitutively active AMPK mutant increased CYP2C expression. On the other hand, a dominant-negative AMPK mutant prevented the VEGF-induced increase in CYP2C RNA and protein expression in human endothelial cells. In vivo (Matrigel plug assay) in mice, endothelial cells were recruited into VEGF-impregnated plugs; an effect that was sensitive to 14,15-EEZE and the inclusion of small interfering RNA directed against the AMPK. The EET antagonist did not affect responses observed in plugs containing bFGF. Taken together, our data indicate that CYP2C-derived EETs participate as second messengers in the angiogenic response initiated by VEGF and that preventing the increase in CYP expression curtails the angiogenic response to VEGF.

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P11.30 Special VEGF90kDa form in exosome from GBM cells activates TAZ in endothelial cells to promote angiogenesis and contributes bevacizumab resistance

Neuro-Oncology ◽

10.1093/neuonc/noz126.176 ◽

2019 ◽

Vol 21 (Supplement_3) ◽

pp. iii49-iii49

Author(s):

Z Wang ◽

Y Yuan ◽

C Xu ◽

Y Liu

Keyword(s):

Endothelial Cells ◽

Western Blotting ◽

Umbilical Vein ◽

Tube Formation ◽

Cellular Migration ◽

Vegf Expression ◽

Angiogenic Potential ◽

Huvec Cells

Abstract BACKGROUND Glioblastoma (GBM) has obvious blood vessels proliferation, which is one of the important histological diagnostic criteria. Bevacizumab, VEGF targeted inhibitor, is found inefficient in 2/3 cases after the clinical trial. Here, we found a specific 90kDa form of VEGF (VEGF90K) in exosomes of GBM could activate TAZ expression in endothelial cells to promote angiogenesis, which might also contribute to Bevacizumab treatment resistance. MATERIAL AND METHODS Exosomes were isolated from U87, LN229 GBM cell culture medium and characterized using transmission electron microscopy, nanoparticle analysis, and western blotting. VEGF expression in GBM and TAZ expression in human umbilical vein endothelial cells (HUVEC) are inhibited by shRNA. In vitro migration, proliferation, and tube formation assays were used to assess the angiogenic potential of HUVEC cells. Western blotting was used to analysis TAZ expression in HUVEC. Bevacizumab or exosome inhibitor GW4869 were used separately or together to evaluation their blocking effects on angiogenic functions of GBM cells. RESULTS we found a unique 90kDa form of VEGF (VEGF90K) from exosome of GBM cells could activate TAZ expression in HUVEC cells which correlated with higher levels of cellular migration, proliferation, and tube formation. Knockdown VEGF expression in GBM exosomes or TAZ expression in HUVEC cells could decrease the angiogenic function of GBM cells. GW4869 combine Bevacizumab had significantly inhibited the angiogenic ability of GBM cells in vivo and in vitro. CONCLUSION A specific exosome-associated VEGF from GBM can promote angiogenesis by activating TAZ expression in endothelial cells. Targeted inhibition of exosome secretion can significantly inhibit tumor angiogenesis and restore bevacizumab effect. Our study highlights the unique properties of VEGF in exosome which might help to explore new treatment strategies of GBM.

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