Tissue Factor Promoter Haplotype Amplifies Inflammatory Response Via p38-MAPK in Neonatal Compared to Adult Human Microvascular Endothelial Cells

Abstract 1150

Age- and tissue-dependent diversity of the vascular endothelium may contribute to the devastating sequelae of inflammation and thrombosis 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 inflammation, we compared expression of several coagulation factors between neonatal and adult human microvascular EC (HMVEC; Lonza) in response to IL-1, and found only tissue factor (TF) was expressed differentially.

In addition to the differences in TF expression between neonatal vs. adult HMVEC, we also found high variability among individual neonatal HMVEC. This finding led us to investigate the possible contribution of two polymorphic TF promoter haplotypes reported to play a role in TF gene regulation. Genotyping revealed that neonatal HMVEC homozygous for an 18 base pair (bp) deletion at −1208 (D/D) expressed much higher levels of TF mRNA in response to IL-1 than those homozygous (I/I) or heterozygous (I/D) for the 18 bp insertion, as had also been previously reported for human umbilical vein EC. Moreover, IL-1-stimulated TF mRNA, protein, and activity levels were significantly (p<0.05) higher (4-fold, 2-fold, and 4-fold) in D/D neonatal than D/D adult HMVEC. Importantly, these results suggested that the D/D haplotype induces a uniquely robust response to IL-1 by the TF promoter in neonatal HMVEC.

Signal transduction pathway macroarray studies (SABiosciences), to compare IL-1 response, showed a striking Jak-STAT pathway activation in neonatal compared to adult HMVEC. Further analysis found that IL-1 induced a 5.8-fold increase in phosphorylation of p38-MAPK by neonatal HMVEC, significantly greater than the 1.9-fold increase by adult HMVEC (p<0.05). Inhibition of the p38-MAPK pathway with SB203580 eliminated the differential Jak-STAT activation and significantly reduced TF mRNA by 58% vs. 20% (p<0.01) in IL-1-stimulated D/D neonatal vs. adult HMVEC. These results suggest that IL-1 hyperactivation of p38-MAPK, possibly regulated by the Jak-STAT pathway, plays a role in up-regulating D/D neonatal HMVEC TF expression.

The following schematic model summarizes the hypothetical response of the neonatal HMVEC TF gene to IL-1:

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This model highlights key components of both the p38-MAPK and NF-κB regulatory pathways. It also illustrates hyperactivation of the p38 pathway (shown as thicker arrows) and its potential up-regulation of one, or more, known, or yet-to-be characterized, D/D promoter haplotype-specific transcription factors (shown as hatched arrows) in neonatal HMVEC.

This amplified inflammatory response of neonatal HMVEC appears to represent a critical developmental transition between the fetal and adult states of vascular gene regulation and expression, providing a potential basis for the increased frequency and morbidity associated with microvascular thrombosis in susceptible newborns.