kinetic expression HO and NOS genes in LPS-stimulated porcine aortic endothelial cells

It was well demonstrated that Lipopolysaccharide (LPS) induces apoptosis in many cellular types including Porcine Aortic Endothelial Cells (PAEC) (Bernardini et al, 2005. Cell Stress & Chaperones 10(4):340-8). Heme oxygenase (inducible HO-1 and constitutive HO-2) and nitric oxide synthase (inducible iNOS and endothelial eNOS) are in some way involved in LPS-induced apoptosis (Henningsson et al, 2001. Am J Physiol Cell Physiol 280:1242-1254). HO-1 protective effects (Zuckerbraun et al, 2003. J Exp Med 198(11):1707-1716; Sarady et al, 2004. FASEB J 18(7):854-6) and nitric oxide (NO) controversial role (Mehta, 2005. Vascul Pharmaco 43(6):390-403) as well as their relationship are not fully understood. The aim of the present study was to investigate the kinetic induction of HO-1, iNOS, HO-2 and eNOS mRNA by LPS treatment in a primary culture of PAEC. EXPERIMENT 1. PAEC were exposed to LPS 10μg/ml for 1, 7, 15 and 24 hours. To detect HO-1, iNOS, HO-2 and eNOS mRNA expression Real Time quantitative PCR was performed. Cell Death Detection Elisa kit (Roche, Germany) was used to evaluate the apoptosis rate. LPS induced apoptosis after 7 hours of treatment. At this time both HO-1 and iNOS reached their peak of expression, while eNOS mRNA level decreased. HO-2 mRNA expression was not affected by LPS treatment. EXPERIMENT 2. To better evaluate earlier differences in the HO-1, iNOS, HO-2 and eNOS mRNA expression, PAEC were exposed to LPS 10μg/ml for 1, 2, 3, 4, 5, 6 and 7 hours. Both iNOS and HO-1 mRNA level increased significantly at 2 hours, then iNOS reached the plateau at 3 hours, while HO-1 reached the maximum level at 4 hours. eNOS mRNA decrease is confirmed being significative at 5 hours. Taking together our data show a similar kinetic trend of HO-1 and iNOS mRNA expression in our model of LPS-induced apoptosis. Therefore, further studies are required to investigate the interactions among these enzymes and their relative products.