Anti-thrombin III deficiency primes the endothelial inflam matory response via upregulation of adhesion molecules

Thomas P. Shanley*, Neeti Vasi, James Stark, Christopher Morris

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Introduction: Our understanding of the relationship between the coagulation cascade and vascular inflammation remains incomplete. Anti-thrombin III (AT-III) deficiency appears to place the host at risk for increased morbidity and mortality from inflammatory challenges. We hypothesized that AT-III deficiency might lead to an enhanced inflammatory response by altering endothelial cell phenotype. In order to test this hypothesis, an in vitro model of AT-III depletion was developed using human umbilical vein endothelial cells (HUVEC). Both constitutive and thrombin-stimulated expression of HUVEC adhesion molecules (CAM's) were examined under conditions of normal culture medium and AT-III depleted medium. Methods: HUVEC (passage 3-9) were grown to confluency in either standard media with fetal calf serum (FCS) or AT-III depleted (AT3-) media. AT3- media was generated by adsorbing the FCS additive against a sepharose-heparin column to remove AT-III to undetectable antigenic levels and 0-20% functional levels (control=88-120%). Cell surface expression of ICAM-1 and VCAM-1 was determined by FACS analysis. Shed soluble ICAM-1 (sICAM-1) in culture media was measured by ELISA. mRNA expression was determined by Northern blot. Functional activity of ICAM-1 expression was confirmed by neutrophil adhesion assay. Results: HUVEC passaged in standard media and stimulated with thrombin (2U/ml) demonstrated a significant increase in cell surface expression of ICAM-1 (1 log-fold), and a modest increase in VCAM-1 expression. Cells first passaged in AT3-media (7 to 14 days) demonstrated increased baseline sICAM-1 and constitutive cell surface expression of ICAM-1 versus HUVEC passaged in normal media. Upon thrombin stimulation, HUVEC in AT- media had substantially increased expression of ICAM-1 (3-4 log-fold) and VCAM-1 (-1 log-fold) as compared to standard passaged HUVEC. Increased ICAM-1 expression may be regulated at the transcriptional level as constitutive ICAM-1 mRNA was significantly elevated in AT3- media as compared to control. Increased surface expression correlated to functional neutrophil adhesion. Conclusions: Depletion of AT-III initiates increased constitutive CAM expression and primes the endothelium for an augmented inflammatory response which may contribute to enhanced vascular injury and multiple organ failure.

Original languageEnglish (US)
Pages (from-to)A125
JournalCritical care medicine
Volume27
Issue number1 SUPPL.
DOIs
StatePublished - 1999

ASJC Scopus subject areas

  • Critical Care and Intensive Care Medicine

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