Developmental changes in lipopolysaccharide activation of rat aortic endothelial cells

Introduction: The etiology and pathophysiology of sepsis changes with age and we anticipate that the vascular system of young children will respond differently to sepsis when compared to adults. We are testing fee hypothesis that eodothelial cell activation by lipopolysaccharide (LPS) is different in rat aortic endothelial cells (RAEC) isolated from young versus adult animals. Methods: RAECs were isolated from pups (<1 week old) and adult Wistar male rats by immuno-isolation using magnetic beads. RAECs in passage 3-12 were treated with or without LPS (E. coli 055:B5, 10 μg/ml) for 24 hours. Endothelial cell viability after treatment was assessed by the dimethylthiazole tetrazolium (MTT) cytotoxicity assay. DNA synthesis was measured by determining [3H]-thymidine incorporation into DNA during each treatment. Intercellular coupling (a measure of cellular function) was assessed as intercellular resistance (Ri) and determined by the degree of spatial decay in a cell monolayer of a current injected into one cell. Results: Pup RAECs but not adult RAECs demonstrated a decline in DNA synthesis from control conditions when treatad with LPS. There were no changes in cell viability with LPS treatment and between adult and pup RAECs. % Change in [3H]- thymidine incorporation % Change in MTT cytotoxicity assay Pup RAEC 66 ± 17*(n=8) 92 ± 10 (n=10) Adult RAEC 87 ± 26 (n=10) 103 ± 13 (n=9) (All values % change from control ± SD;*p<0.05 compared to adult RAEC.) Ri was increased in both cell lines after exposure to LPS. Pup RAECs had significantly delated Ri compared to adult RAECs. The increase in Ri in response to LPS was proportionally higher in adult RAECs (54%) than in pup RAECs (29%). Control Ri (megaΩ) LPS treated Ri (magaΩ) Pup RAEC 5.6 ± 0.7 a 7.2 ± 1.6*, a Adult RAEC 3.7 ± 0.5 5.7±0.9*(*p<0.05 compared to control; a p<0.05 compared to adult RAEC.) Conclusions: Endothelial cell activation by LPS is different in pup RAECs when compared to adult RAECs, suggesting a developmental change in response to LPS.