0383 : In vitro 3D model of in vitro angiogenesis using human endothelial cells and pericytes

ABSTRACT Mouse capillary endothelial cells (1G11 cell line) embedded in type I collagen gels undergo in vitro angiogenesis. Cells rapidly reorganize and form capillary-like structures when stimulated with serum. Transforming growth factor β1 (TGF-β1) alone can substitute for serum and induce cell survival and tubular network formation. This TGF-β1-mediated angiogenic activity depends on phosphatidylinositol 3-kinase (PI3K) and p42/p44 mitogen-activated protein kinase (MAPK) signaling. We showed that specific inhibitors of either pathway (wortmannin, LY-294002, and PD-98059) all suppressed TGF-β1-induced angiogenesis mainly by compromising cell survival. We established that TGF-β1 stimulated the expression of TGF-α mRNA and protein, the tyrosine phosphorylation of a 170-kDa membrane protein representing the epidermal growth factor (EGF) receptor, and the delayed activation of PI3K/Akt and p42/p44 MAPK. Moreover, we showed that all these TGF-β1-mediated signaling events, including tubular network formation, were suppressed by incubating TGF-β1-stimulated endothelial cells with a soluble form of an EGF receptor (ErbB-1) or tyrphostin AG1478, a specific blocker of EGF receptor tyrosine kinase. Finally, addition of TGF-α alone poorly stimulated angiogenesis; however, by reducing cell death, it strongly potentiated the action of TGF-β1. We therefore propose that TGF-β1 promotes angiogenesis at least in part via the autocrine secretion of TGF-α, a cell survival growth factor, activating PI3K/Akt and p42/p44 MAPK.

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Vascular repair utilising immobilised heparin conjugate for protection against early activation of inflammation and coagulation

Thrombosis and Haemostasis ◽

10.1160/th14-09-0724 ◽

2015 ◽

Vol 113 (06) ◽

pp. 1312-1322 ◽

Cited By ~ 18

Author(s):

Sofia Nordling ◽

Jaan Hong ◽

Karin Fromell ◽

Fredrik Edin ◽

Johan Brännström ◽

...

Keyword(s):

Endothelial Cells ◽

Thrombus Formation ◽

Innate Immune ◽

Human Endothelial Cells ◽

Coagulation Activation ◽

Ischaemia Reperfusion Injury ◽

Novel Approach ◽

One Step ◽

Chamber Model

SummaryIschaemia-reperfusion injury (IRI) poses a major challenge in many thrombotic conditions and in whole organ transplantation. Activation of the endothelial cells and shedding of the protective vascular glycocalyx during IRI increase the risk of innate immune activation, cell infiltration and severe thrombus formation, promoting damage to the tissue. Here, we present a novel one-step strategy to protect the vasculature by immobilisation of a unique multi-arm heparin conjugate to the endothelium. Applying a new in vitro blood endothelial cell chamber model, the heparin conjugate was found to bind not only to primary human endothelial cells but also directly to the collagen to which the cells adhered. Incubation of hypoxic endothelial cells with freshly drawn human blood in the blood chambers elicited coagulation activation reflected by thrombin anti-thrombin formation and binding of platelets and neutrophils. Immobilisation of the heparin conjugate to the hypoxic endothelial cells created a protective coating, leading to a significant reduction of the recruitment of blood cells and coagulation activation compared to untreated hypoxic endothelial cells. This novel approach of immobilising multi-arm heparin conjugates on the endothelial cells and collagen of the basement membrane ensures to protect the endothelium against IRI in thrombotic disorders and in transplantation.

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Differential roles of ICAM-1 and E-selectin in polymorphonuclear leukocyte-induced angiogenesis

AJP Cell Physiology ◽

10.1152/ajpcell.00223.2001 ◽

2002 ◽

Vol 282 (4) ◽

pp. C917-C925 ◽

Cited By ~ 39

Author(s):

Masako Yasuda ◽

Shunichi Shimizu ◽

Kyoko Ohhinata ◽

Shinji Naito ◽

Shogo Tokuyama ◽

...

Keyword(s):

Monoclonal Antibody ◽

Endothelial Cells ◽

Polymorphonuclear Leukocytes ◽

Vascular Endothelial Cell ◽

Collagen Gel ◽

Intercellular Adhesion Molecule ◽

Vascular Endothelial ◽

Endothelial Cell Cultures ◽

In Vitro Angiogenesis

Ets-1, which stimulates metalloproteinase gene transcription, has a key role in angiogenesis. We first examined whether activated polymorphonuclear leukocytes (PMNs) enhanced angiogenesis through the induction of Ets-1. Addition of activated PMNs to endothelial cells stimulated both in vitro angiogenesis in collagen gel and Ets-1 expression. Both angiogenesis and Ets-1 expression induced by PMNs were reduced by ets-1 antisense oligonucleotide, suggesting that Ets-1 is an important factor in PMN-induced angiogenesis. Although intercellular adhesion molecule (ICAM)-1 and E-selectin are involved in PMN-induced angiogenesis, the mechanisms underlying their roles in angiogenesis have yet to be elucidated. PMN-induced Ets-1 expression was reduced by a monoclonal antibody against ICAM-1 but not E-selectin despite the inhibition of PMN-induced angiogenesis by both antibodies. Moreover, the stimulation of angiogenesis by H2O2without PMNs was inhibited by a monoclonal antibody to E-selectin but not ICAM-1. These findings suggested that ICAM-1 in endothelial cells may act as a signaling receptor to induce Ets-1 expression, whereas E-selectin seems to function in the formation of tubelike structures in vascular endothelial cell cultures.

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Centrifugal enhancement of Kaposi's sarcoma-associated virus infection of human endothelial cells in vitro

Journal of Virological Methods ◽

10.1016/j.jviromet.2008.07.026 ◽

2008 ◽

Vol 154 (1-2) ◽

pp. 160-166 ◽

Cited By ~ 24

Author(s):

Seung-Min Yoo ◽

Ae-Kyung Ahn ◽

Taegun Seo ◽

Hyo Bong Hong ◽

Myung-Ae Chung ◽

...

Keyword(s):

Endothelial Cells ◽

Virus Infection ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Human Endothelial Cells

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The Effects of Geometry and Static Boundary Conditions on Microvessel Outgrowth in a 3D Model of Angiogenesis

ASME 2010 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2010-19587 ◽

2010 ◽

Cited By ~ 1

Author(s):

Clayton J. Underwood ◽

Laxminarayanan Krishnan ◽

Lowell T. Edgar ◽

Steve Maas ◽

James B. Hoying ◽

...

Keyword(s):

Boundary Condition ◽

Boundary Conditions ◽

Direct Relationship ◽

3D Model ◽

Mechanical Strain ◽

Matrix Stiffness ◽

Collagen Gels ◽

3D Collagen ◽

In Vitro Angiogenesis

We reported previously that, in addition to mechanical strain, a constrained boundary condition alone can alter the organization of microvessel outgrowth during in vitro angiogenesis [1]. After 6 days of culture in vitro, microvessels aligned parallel to the long axis of rectangular 3D collagen gels that had constrained edges on the ends. However, unconstrained cultures did not show any alignment of microvessels. The ability to direct microvessel outgrowth during angiogenesis has significant implications for engineering prevascularized grafts and tissues in vitro, therefore an understanding of this process is important. Since there is direct relationship between the ability of endothelial cells to contract 3D gels and matrix stiffness [2], we hypothesize that some constrained boundary conditions will increase the apparent matrix stiffness and in turn will limit gel contraction, prevent microvessel alignment, and reduce microvessel outgrowth. The objective of this study was to compare microvessel growth and alignment under several different static boundary conditions.

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