Abstract 20152: Multimerin2 Influences Vascularization Through Modulation of the Extracellular Matrix

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Petra E Burgisser ◽  
Dennie Tempel ◽  
Caroline Cheng ◽  
Gerard Pasterkamp ◽  
Henricus Duckers
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tube Formation ◽  
Endothelial Cell Proliferation ◽  
Mouse Development ◽  
New Genes ◽  
A Genome ◽  
Extracellular Matrix Components

Background: Vascular development is crucial in normal physiology and pathophysiology, and is in part orchestrated by a dynamic balance between extracellular matrix (ECM) and endothelial cells. The molecular mechanisms that govern this balance and as such play a role in the formation of new blood vessels remain largely unknown. In order to identify new genes involved in blood vessel formation, we performed a genome-wide gene expression array analysis in Flk1+ angioblasts during mouse development using a commercial platform (Affymetrix). Multimerin2 (Mmrn2) expression was found to be upregulated in angioblasts and endothelial cells and in the developing vasculature of zebrafish and mice. Methods & Results: Subsequent in vitro studies using an adenoviral construct to induce Mmrn2 overexpression showed a negative effect on angiogenesis. Mmrn2 overexpression resulted in a reduction of 2D tube formation capacity of human aortic endothelial cells (HAECs), as seen in a decrease in the number of junctions and tubules (P<0.05, N=3). Another observed effect after Mmrn2 overexpression was a G0/G1 cell cycle arrest in HAECs (P<0.001, N=8) and decreased migration of the cells in a Boyden chamber assay (P<0.05, N=10). Further in vitro experiments, using immunofluorescence, showed Mmrn2 was detected outside of HAECs, were it co-localized with the extracellular matrix components perlecan, laminin and fibronectin. Direct interaction with these proteins was confirmed using a combination of co-immunoprecipitation followed by proteomics (LC-MS), and western blot to identify possible binding partners. Interestingly, overexpression of Mmrn2 also resulted in enhanced expression of these extracellular components, suggesting that Mmrn2 changes the phenotype from a more proliferative endothelial cell towards a more productive endothelial cell. Conclusions: These findings suggest that Mmrn2 is an abundant and important extracellular matrix protein, secreted by endothelial cells, which influences the expression of other ECM proteins such as fibronectin, laminin and perlecan. We hypothesize that Mmrn2 switches the phenotype of the endothelial cell and subsequently diminishes endothelial cell proliferation, migration and overall tube formation.

scholarly journals Roundabout4 Suppresses Glioma-Induced Endothelial Cell Proliferation, Migration and Tube Formation in Vitro by Inhibiting VEGR2-Mediated PI3K/AKT and FAK Signaling Pathways

2015 ◽  
Vol 35 (5) ◽  
pp. 1689-1705 ◽  
Author(s):  
Heng Cai ◽  
Yixue Xue ◽  
Zhen Li ◽  
Yi Hu ◽  
Zhenhua Wang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Signaling Pathways ◽  
Conditioned Medium ◽  
Tube Formation ◽  
Endothelial Cell Proliferation ◽  
Akt Inhibitor ◽  
Over Expression

Background and Aims: Endothelial cell (EC) proliferation, migration, and tube formation are the critical steps for tumor angiogenesis, which is involved in the formation of new tumor blood vessels. Roundabout4 (Robo4), a new member of Robo proteins family, is specifically expressed in endothelial cells. This study aimed to investigate the effects of Robo4 on glioma-induced endothelial cell proliferation, migration and tube formation in vitro. Methods and Results: We found that Robo4 was endogenously expressed in Human Brain Microvascular Endothelial Cells (HBMECs), while Robo4 was significantly down-regulated in endothelial cells cultured in glioma conditioned medium. Robo4 over-expression remarkably suppressed glioma-induced endothelial cell proliferation, migration and tube formation in vitro. In addition, Robo4 influenced the glioma-induced angiogenesis via binding to its ligand Slit2. Further studies demonstrated that the knockdown of Robo4 up-regulated the phosphorylation of VEGFR2, PI3K, AKT and FAK in EC cultured in glioma conditioned medium. VEGFR2 inhibitor SU-1498, AKT inhibitor LY294002 and FAK inhibitor 14 (FAK inhibitor) blocked the Robo4 knockdown-mediated alteration in glioma angiogenesis in vitro. Conclusion: Our results proved that Robo4 suppressed glioma-induced endothelial cell proliferation, migration and tube formation in vitro by inhibiting VEGR2-mediated activation of PI3K/AKT and FAK signaling pathways.

scholarly journals Exosomal CircHIPK3 Released from Hypoxia-Induced Cardiomyocytes Regulates Cardiac Angiogenesis after Myocardial Infarction

2020 ◽  
Vol 2020 ◽  
pp. 1-19 ◽  
Author(s):  
Yan Wang ◽  
Ranzun Zhao ◽  
Changyin Shen ◽  
Weiwei Liu ◽  
Jinson Yuan ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Cycle Progression ◽  
Cell Activity ◽  
Cell Communication ◽  
Tube Formation ◽  
Endothelial Cell Migration ◽  
Endothelial Cell Proliferation

Exosomes play critical roles in mediating cell-to-cell communication by delivering noncoding RNAs (including miRNAs, lncRNAs, and circRNAs). Our previous study found that cardiomyocytes (CMs) subjected to hypoxia released circHIPK3-rich exosomes to regulate oxidative stress damage in cardiac endothelial cells. However, the role of exosomes in regulating angiogenesis after myocardial infarction (MI) remains unknown. The aim of this study was to establish the effects of exosomes derived from hypoxia-induced CMs on the migration and angiogenic tube formation of cardiac endothelial cells. Here, we reported that hypoxic exosomes (HPC-exos) can effectively reduce the infarct area and promote angiogenesis in the border surrounding the infarcted area. HPC-exos can also promote cardiac endothelial cell migration, proliferation, and tube formation in vitro. However, these effects were weakened after silencing circHIPK3 in hypoxia-induced CMs. We further verified that silencing and overexpressing circHIPK3 changed cardiac endothelial cell proliferation, migration, and tube formation in vitro by regulating the miR-29a expression. In addition, exosomal circHIPK3 derived from hypoxia-induced CMs first led to increased VEGFA expression by inhibiting miR-29a activity and then promoted accelerated cell cycle progression and proliferation in cardiac endothelial cells. Overexpression of miR-29a mimicked the effect of silencing circHIPK3 on cardiac endothelial cell activity in vitro. Thus, our study provides a novel mechanism by which exosomal circRNAs are involved in the communication between CMs and cardiac endothelial cells.

Naturally Produced Extracellular Matrix Is an Excellent Substrate for Canine Endothelial Cell Proliferation and Resistance to Shear Stress on PTFE Vascular Grafts

1997 ◽  
Vol 78 (05) ◽  
pp. 1392-1398 ◽  
Author(s):  
A Schneider ◽  
M Chandra ◽  
G Lazarovici ◽  
I Vlodavsky ◽  
G Merin ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Cell Attachment ◽  
Thrombus Formation ◽  
Endothelial Cell Proliferation ◽  
Ptfe Graft ◽  
Vascular Prostheses ◽  
Endothelial Cell Attachment

SummaryPurpose: Successful development of a vascular prosthesis lined with endothelial cells (EC) may depend on the ability of the attached cells to resist shear forces after implantation. The present study was designed to investigate EC detachment from extracellular matrix (ECM) precoated vascular prostheses, caused by shear stress in vitro and to test the performance of these grafts in vivo. Methods: Bovine aortic endothelial cells were seeded inside untreated polytetrafluoro-ethylene (PTFE) vascular graft (10 X 0.6 cm), PTFE graft precoated with fibronectin (FN), or PTFE precoated with FN and a naturally produced ECM (106 cells/graft). Sixteen hours after seeding the medium was replaced and unattached cells counted. The strength of endothelial cell attachment was evaluated by subjecting the grafts to a physiologic shear stress of 15 dynes/cm2 for 1 h. The detached cells were collected and quantitated. PTFE or EC preseeded ECM coated grafts were implanted in the common carotid arteries of dogs. Results: While little or no differences were found in the extent of endothelial cell attachment to the various grafts (79%, 87% and 94% of the cells attached to PTFE, FN precoated PTFE, or FN+ECM precoated PTFE, respectively), the number of cells retained after a shear stress was significanly increased on ECM coated PTFE (20%, 54% and 85% on PTFE, FN coated PTFE, and FN+ECM coated PTFE, respectively, p <0.01). Implantation experiments in dogs revealed a significant increase in EC coverage and a reduced incidence of thrombus formation on ECM coated grafts that were seeded with autologous saphenous vein endothelial cells prior to implantation. Conclusion: ECM coating significantly increased the strength of endothelial cell attachment to vascular prostheses subjected to shear stress. The presence of adhesive macromolecules and potent endothelial cell growth promoting factors may render the ECM a promising substrate for vascular prostheses.

The role of matrix metalloproteinase activity in the maturation of human capillary endothelial cells in vitro

1999 ◽  
Vol 112 (10) ◽  
pp. 1599-1609 ◽  
Author(s):  
B.M. Kraling ◽  
D.G. Wiederschain ◽  
T. Boehm ◽  
M. Rehn ◽  
J.B. Mulliken ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Proliferation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Endothelial Cell Proliferation ◽  
Protein Levels ◽  
Matrix Deposition ◽  
Capillary Endothelial Cells ◽  
Vessel Maturation

Vessel maturation during angiogenesis (the formation of new blood vessels) is characterized by the deposition of new basement membrane and the downregulation of endothelial cell proliferation in the new vessels. Matrix remodeling plays a crucial, but still poorly understood role, in angiogenesis regulation. We present here a novel assay system with which to study the maturation of human capillary endothelial cells in vitro. When human dermal microvascular endothelial cells (HDMEC) were cultured in the presence of dibutyryl cAMP (Bt2) and hydrocortisone (HC), the deposition of a fibrous lattice of matrix molecules consisting of collagens type IV, type XVIII, laminin and thrombospondin was induced. In basal medium (without Bt2 and HC), HDMEC released active matrix metalloproteinases (MMPs) into the culture medium. However, MMP protein levels were significantly reduced by treatment with Bt2 and HC, while protein levels and activity of endogenous tissue inhibitor of MMPs (TIMP) increased. This shift in the proteolytic balance and matrix deposition was inhibited by the specific protein kinase A inhibitors RpcAMP and KT5720 or by substituting analogues without reported glucocorticoid activity for HC. The addition of MMP inhibitors human recombinant TIMP-1 or 1,10-phenanthroline to cultures under basal conditions induced matrix deposition in a dose-dependent manner, which was not observed with the serine protease inhibitor epsilon-amino-n-caproic acid (ACA). The deposited basement membrane-type of matrix reproducibly suppressed HDMEC proliferation and increased HDMEC adhesion to the substratum. These processes of matrix deposition and downregulation of endothelial cell proliferation, hallmarks of differentiating new capillaries in the end of angiogenesis, were recapitulated in our cell culture system by decreasing the matrix-degrading activity. These data suggest that our cell culture assay provides a simple and feasible model system for the study of capillary endothelial cell differentiation and vessel maturation in vitro.

CYLD regulates angiogenesis by mediating vascular endothelial cell migration

Blood ◽  
2010 ◽  
Vol 115 (20) ◽  
pp. 4130-4137 ◽  
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.

Induction of endothelial proliferation and angiogenesis through activating the ERK1/2/EGF pathway mediate by CXC chemokine receptor 2 by chemokine (C-X-C motif) ligand 1.

2013 ◽  
Vol 31 (6_suppl) ◽  
pp. 138-138 ◽  
Author(s):  
Makito Miyake ◽  
Steve Goodison ◽  
Evan Gomes ◽  
Wasia Rizwani ◽  
Shanti Ross ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Chemokine Receptor ◽  
Cellular Proliferation ◽  
Human Endothelial Cell ◽  
Endothelial Cell Proliferation ◽  
Cxc Chemokine ◽  
Inhibition Of Angiogenesis

138 Background: Endothelial cell growth and proliferation are critical for tumoral angiogenesis. We report here that blockade of Chemokine (C-X-C motif) ligand 1 (CXCL1) results in reduction of human endothelial cell proliferation and its ability to induce angiogenesis. Methods: Two human endothelial cell lines, HUVEC and HDMEC, were used in the in vitro assays. Proliferation assay and matrigel tube formation assay were performed to test the inhibitory effect of anti-CXCL antibody on the activity of endothelial cells in vitro. Matrigel plug assay in nude mice was performed to test the in vivo angiogenic activity of CXCL1. Results: CXCL1 interacts with its receptor CXC chemokine Receptor 2 and induces endothelial cell proliferation, whereas blockade of CXCL1 is associated with reduction in cellular proliferation through a decrease in levels of cyclin D and cdk4 and inhibition of angiogenesis through EGF and ERK 1/2. Targeting CXCL1 inhibits neoangiogenesis but has no effect on disrupting established vasculature. Furthermore targeting CXCL1 is associated with reduction in migration of human endothelial cells in an in vitro model. Additionally, neutralizing antibody against CXCL1 in a xenograft angiogenesis model resulted in inhibition of angiogenesis. Conclusions: CXCL1-induced regulation of angiogenesis has not been studied extensively in human cancers, thus these findings illustrate a novel contribution of CXCL1 interactions in pathological angiogenesis. Therefore, the ability to selectively modulate CXCL1, specifically in tumoral angiogenesis, may promote the development of novel oncologic therapeutic strategies.

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

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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