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.
ASJC Scopus subject areas
- Critical Care and Intensive Care Medicine