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.
Greco F., Bernardini C., Zannoni A., Ribeiro L., Bacci M.L., Forni M. (2006). kinetic expression HO and NOS genes in LPS-stimulated porcine aortic endothelial cells. s.l : s.n.
kinetic expression HO and NOS genes in LPS-stimulated porcine aortic endothelial cells
BERNARDINI, CHIARA;ZANNONI, AUGUSTA;DE ANDREA RIBEIRO, LUCIANA;BACCI, MARIA LAURA;FORNI, MONICA
2006
Abstract
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
