Species-specific kinetics and zonation of hepatic DNA synthesis induced by ligands of PPARα

PPARα ligands evoke a profound mitogenic response in rodent liver, and the aim of this study was to characterise the kinetics of induction of DNA synthesis. The CAR ligand, 1,4-bis[2-(3,5- dichoropyridyloxy)]benzene, caused induction of hepatocyte DNA synthesis within 48 hours in 129S4/SvJae mice, but the potent PPARα ligand, ciprofibrate, induced hepatocyte DNA synthesis only after 3 or 4 days dosing; higher or lower doses did not hasten the DNA synthesis response. This contrasted with the rapid induction (24 hours) reported by Styles et al. (Carcinogenesis 9:1647-1655). C57BL/6 and DBA/2J mice showed significant induction of DNA synthesis after 4, but not 2, days ciprofibrate treatment. Alderley Park and 129S4/SvJae mice dosed with methylclofenapate induced hepatocyte DNA synthesis at 4, but not 2, days after dosing, and proved that inconsistency with prior work was not due to a difference in mouse strain or PPARα ligand. Ciprofibrate-induced liver DNA synthesis and growth was absent in PPARα- null mice, and are PPARα-dependent. In the Fisher344 rat, hepatocyte DNA synthesis was induced at 24 hours after dosing, with a second peak at 48 hours. Lobular localisation of hepatocyte DNA synthesis showed preferential periportal induction of DNA synthesis in rat, but panlobular zonation of hepatocyte DNA synthesis in mouse. These results characterise a markedly later hepatic induction of panlobular DNA synthesis by PPARα ligands in mouse, compared to rapid induction of periportal DNA synthesis in rat.

Nitric oxide synthase and NAD(P)H oxidase modulate coronary endothelial cell growth

Reactive oxygen species (ROS) including nitric oxide (NO) and superoxide anion (O2-) are associated with cell migration, proliferation and many growth-related diseases. The objective of this study was to determine whether there was a reciprocal relationship between rat coronary microvascular endothelial cell (CMEC) growth and activity/expressions (mRNA and protein) of endothelial NO synthase (eNOS) and NAD(P)H oxidase enzymes. Proliferating namely, 50% confluent CMEC possessed approximately three-fold increased activity and expression of both enzymes compared to 100% confluent cells. Treatment of CMEC with an inhibitor of eNOS (L-NAME, 100M) increased cell proliferation as assessed via three independent methods i.e. cell counting, determination of total cellular protein levels and [3H]thymidine incorporation. Similarly, treatment of CMEC with pyrogallol (0.3-3 mM), a superoxide anion (O2-)- generator, also increased CMEC growth while spermine NONOate (SpNO), a NO donor, significantly reduced cell growth. Co-incubation of CMEC with a cell permeable superoxide dismutase mimetic (Mn-III-tetrakis-4-benzoic acid-porphyrin; MnTBAP) plus either pyrogallol or NO did not alter cell number and DNA synthesis thereby dismissing the involvement of peroxynitrite (OONO-) in CMEC proliferation. Specific inhibitors of NAD(P)H oxidase but not other ROS-generating enzymes including cyclooxygenase and xanthine oxidase, attenuated cell growth. Transfection of CMEC with antisense p22-phox cDNA, a membrane-bound component of NAD(P)H oxidase, resulted in substantial reduction in [3H]thymidine incorporation, total cellular protein levels and expression of p22-phox protein. These data demonstrate a cross-talk between CMEC growth and eNOS and NAD(P)H oxidase enzyme activity and expression, thus suggesting that the regulation of these enzymes may be critical in preventing the initiation and/or progression of coronary atherosclerosis.