Tissue Factor Promoter Genotype Alters Inflammatory Response in Neonatal Compared to Adult Human Microvascular Endothelial Cells.

Age- and tissue-dependent diversity of the vascular endothelium may contribute to the devastating sequelae of sepsis in newborn infants, who often develop multi-organ failure from excessive coagulation-related tissue necrosis. Our previous studies have demonstrated that cytokine and cytoadhesion molecule expression by IL-1- or TNF-α-activated macrovascular endothelial cells (EC) is altered depending upon their vascular origin. Since the microvasculature is especially prone to thrombotic complications of sepsis, we have compared expression of coagulation factors between neonatal and adult human microvascular EC (HMVEC) in response to IL-1, and found that only tissue factor (TF) was expressed differentially.

In addition to the distinct differences in TF expression by neonatal compared to adult HMVEC, we also found a high variability in TF expression between individual neonatal HMVEC. This finding led us to investigate the possible contribution of two polymorphic TF promoter haplotypes believed to play a role in TF regulation. Genotyping of the nine individual HMVEC in which TF expression was analyzed, using a Nanogen Workstation, revealed that the neonatal HMVEC homozygous for an 18 base pair (bp) deletion at -1208 (D/D) expressed much higher levels of TF mRNA and protein in response to IL-1 than those homozygous (I/I) or heterozygous (I/D) for the 18 bp insertion (see Figure), as had also been previously found in human umbilical vein endothelial cells (HUVEC). However, when IL-1-stimulated HMVEC of the three possible TF promoter genotypes were compared to I/D adult HMVEC, both the I/I (19.7±12.1%, 12.4%) and I/D (18.9±6.2%, 33.5%) neonatal HMVEC expressed significantly less, whereas the D/D (252±61%, 117%) neonatal HMVEC expressed significantly more TF mRNA and protein, respectively (see Figure). These results suggested that there is a unique regulatory mechanism limiting the induction of TF by IL-1 in neonatal HMVEC with at least one I-allele. This attenuation is neutralized in the D/D neonatal HMVEC, which express significantly more TF than the adult I/D HMVEC in response to IL-1.

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Using signal transduction pathway macroarray studies (SuperArray), we have also found that IL-1 specifically activates signaling through the Jak-STAT and a stress response pathway in neonatal HMVEC, whereas the six other signaling pathways represented were preferentially up-regulated in adult HMVEC. We are further characterizing the response of neonatal and adult microvascular, arterial, and venous EC to inflammation by systematically comparing their global gene expression patterns using microarray (SFGF) and multiplex protein (Luminex) analysis technology. In parallel studies, we are assessing the TF genotype of critically ill children to determine if the D/D TF promoter haplotype is associated with coagulopathy or multi-organ failure. From a developmental standpoint, TF may play a central role in coordinating the inflammatory and coagulation responses site-specifically on the surface of the vascular endothelium. The unique behavior of neonatal HMVEC appears to represent a critical transition between the fetal and adult states of gene regulation and expression, resulting in an altered response to inflammation and injury.