Inhibition of Endothelial Cell Migration, Intercellular Communication, and Vascular Tube Formation by Thromboxane A2

doi:10.1074/jbc.274.50.35562

Inhibition of Endothelial Cell Migration, Intercellular Communication, and Vascular Tube Formation by Thromboxane A₂*

From the Departments of ^‡Medicine (Cardiology),^‡Molecular Pharmacology, ^¶Pathology, and ^‖Neuroscience, the Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461 and the ^**University Sao Judas Tadeu, Sao Paulo 03166-000, Brazil

Abstract

The eicosanoid thromboxane A₂ (TXA₂) is released by activated platelets, monocytes, and the vessel wall and interacts with high affinity receptors expressed in several tissues including endothelium. Whether TXA₂ might alter endothelial migration and tube formation, two determinants of angiogenesis, is unknown. Thus, we investigated the effect of the TXA₂ mimetic [1S-(1α,2β(5Z),3α(1E,3R),4α]-7-[3-(3-hydroxy-4-(4′-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5′-heptenoic acid (IBOP) on human endothelial cell (HEC) migration and angiogenesis in vitro. IBOP stimulation inhibited HEC migration by 50% and in vitro capillary formation by 75%. These effects of IBOP were time- and concentration-dependent with an IC₅₀ of 25 nm. IBOP did not affect integrin expression or cytoskeletal morphology of HEC. Since gap junction-mediated intercellular communication increases in migrating HEC, we determined whether IBOP might inhibit coupling or connexin expression in HEC. IBOP reduced the passage of microinjected dyes between HEC by 50%, and the effects of IBOP on migration and tube formation were mimicked by the gap junction inhibitor 18β-glycyrrhetinic acid (1 μm) with a similar time course and efficacy. IBOP (24 h) did not affect the expression or phosphorylation of connexin 43 in whole HEC lysates. Immunohistologic examination of HEC suggested that IBOP may impair functional coupling by altering the cellular distribution of gap junctions, leading to increased connexin 43 internalization. Thus, this finding that TXA₂ mimetics can prevent HEC migration and tube formation, possibly by impairing intercellular communication, suggests that antagonizing TXA₂ signaling might enhance vascularization of ischemic tissue.

Footnotes

↵* This work was supported by National Institutes of Health Grants HL47032-05 and HL38449 and Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Brazil, Grant J997/2379-2.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ To whom correspondence should be addressed: Rm. G42, Forchheimer Bldg., Dept. of Cardiology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2366; Fax: 718-430-8989; E-mail: ashton@aecom.yu.edu.
↵2 Y. Gao and J. A. Ware, unpublished observations.
Abbreviations:

TXA₂

thromboxane A₂

TP

thromboxane A₂ receptor

IBOP

[1S-(1α,2β(5Z),3α(1E,3R),4α]-7-[3-(3-hydroxy-4-(4′-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5′-heptenoic acid

HEC

human endothelial cells

PBS

phosphate-buffered saline

Cx43

connexin 43

GA

glycyrrhetinic acid

SQ29548

[[1S]1α,2β(5Z),3β,4α]-7-[3[[2-[(phenylamino)carbonyl]-hydrazino] methyl]-7-oxabicyclo[2.2.1]-hept-2-yl]

PBS-B

phosphate buffered saline with 1% (w/v) bovine serum albumin

FITC

fluorescein isothiocyanate

PIPES

1,4-piperazinediethanesulfonic acid
- Received August 11, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.