Abstract 978: Novel Endothelial-protective Effect of High Density Lipoprotein: Down-regulation of Toll-like Receptor 4
Introduction: Endothelium-derived Toll-like receptor (TLR) 4 is known to be the key molecule in lipopolysaccharide (LPS)-induced neutrophil sequestration into lungs. The aim of our study was to investigate whether the endothelial-protective effects of HDL also include regulation of Toll-like receptor (TLR) 4. Therefore, we analyzed the effect of HDL supplementation on TLR4 expression in human aortic endothelial cells (HAEC) and the effect of increased HDL following adenoviral apo AI (Ad.AI) gene transfer (GT) on lung TLR4 expression in an experimental model of LPS-induced endotoxic shock.
Methods: HDL (50 μg/ml) was supplemented to HAEC in the presence/absence of LPS (100 ng/ml). Surface-TLR4 expression on HAEC was analyzed by FACS. C57BL/6 mice were i.v. injected with 5 x 1010 total particles of Ad.AI or with Ad.Null. Fourteen days hereafter, mice were i.p. injected with LPS (80 mg/kg) or with saline and 20 hours afterwards sacrificed. For survival studies, endotoxic shock was induced in 25 mice per group. Lung myeloperoxidase activity (MPO), as a marker of neutrophil infiltration was analysed. Lung TLR4-mRNA expression was determined by real-time PCR, plasma interferon-gamma (INFγ) levels by ELISA.
Results: HDL supplementation significantly reduced TLR4 expression on HAEC. Ad.AI resulted in human apo AI expression levels of 80 ± 10 mg/dl at day 14. By 24 hours after LPS injection, 81% of LPS-treated Ad.Null and saline mice died, in contrast to 50% of Ad.AI LPS-treated mice. This was associated with a 1.6- and 1.7-fold (p<0.05) lower lung/body weight ratio in Ad.AI compared to Ad.Null and saline LPS-injected mice. LPS-induced lung TLR4 mRNA expression was 2.0-fold (p<0.05) reduced after human apo AI-GT compared to LPS controls. Moreover, human apo AI-GT significantly reduced neutrophil infiltration in LPS-treated mice, as indicated by 1.8-fold (p<0.05) and 1.9-fold (p<0.05) reduced MPO activity compared to controls, respectively. Plasma INFγ-levels were 2.8-fold and 2.2-fold (p<0.05) lower versus LPS-treated Ad.Null and saline mice, respectively.
Conclusion: The HDL-mediated reduction in lung TLR4 expression contributes for the reduced neutrophil infiltration and subsequently may account for improved mortality rate.