Simvastatin and dehydroepiandrosterone sulfate effects against hypoxic pulmonary hypertension are not additive.

Pulmonary hypertension is a group of disorders characterized by elevated mean pulmonary artery pressure (mPAP) and pulmonary vascular resistance. To test our hypothesis that combining two drugs useful in experimental pulmonary hypertension, statins and dehydroepiandrosterone sulfate (DHEA S), is more effective than either agent alone, we induced pulmonary hypertension in adult male rats by exposing them to hypoxia (10%O2) for 3 weeks. We treated them with simvastatin (60 mg/l) and DHEA S (100 mg/l) in drinking water, either alone or in combination. Both simvastatin and DHEA S reduced mPAP (froma mean±s.d. of 34.4±4.4 to 27.6±5.9 and 26.7±4.8 mmHg, respectively), yet their combination was not more effective (26.7±7.9 mmHg). Differences in the degree of oxidative stress (indicated by malondialdehydeplasma concentration),the rate of superoxide production (electron paramagnetic resonance), or blood nitric oxide levels (chemiluminescence) did not explain the lack of additivity of the effect of DHEA S and simvastatin on pulmonary hypertension. We propose that the main mechanism of both drugs on pulmonary hypertension could be their inhibitory effect on 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, which could explain their lack of additivity.

MMP-19 deficiency causes aggravation of colitis due to defects in innate immune cell function.

Matrix metalloproteinases (MMPs) are potential biomarkers for disease activity in inflammatory bowel disease (IBD). However, clinical trials targeting MMPs have not succeeded, likely due to poor understanding of the biological functions of individual MMPs. Here, we explore the role of MMP-19 in IBD pathology. Using a DSS-induced model of colitis, we show evidence for increased susceptibility of Mmp-19-deficient (Mmp-19(-/-)) mice to colitis. Absence of MMP-19 leads to significant disease progression, with reduced survival rates, severe tissue destruction, and elevated levels of pro-inflammatory modulators in the colon and plasma, and failure to resolve inflammation. There was a striking delay in neutrophil infiltration into the colon of Mmp-19(-/-) mice during the acute colitis, leading to persistent inflammation and poor recovery; this was rescued by reconstitution of irradiated Mmp-19(-/-) mice with wild-type bone marrow. Additionally, Mmp-19-deficient macrophages exhibited decreased migration in vivo and in vitro and the mucosal barrier appeared compromised. Finally, chemokine fractalkine (CX3CL1) was identified as a novel substrate of MMP-19, suggesting a link between insufficient processing of CX3CL1 and cell recruitment in the Mmp-19(-/-) mice. MMP-19 proves to be a critical factor in balanced host response to colonic pathogens, and for orchestrating appropriate innate immune response in colitis.

ADAM10/17-dependent release of soluble c-Met correlates with hepatocellular damage.

The signalling pathway elicited by hepatocyte growth factor (HGF) and its receptor c-Met is indispensable for liver development and regeneration. It has been described that c-Met is released from the cell surface by a disintegrin and metalloprotease 10 (ADAM10) resulting in a soluble c-Met form known as sMet. Using the human hepatocellular HepG2 and hepatic stellate cell LX2 lines we show that sMet is released from the cell surface of liver cells by both ADAM17 and ADAM10, with ADAM17 appearing to be the major proteinase. Moreover, using a mouse model of 3,5-diethoxycarbonyl- 1,4-dihydroxycollidine (DDC)-induced hepatobiliary obstruction we show that serum levels of sMet correlate well with the liver damage state and consecutive regeneration as well as with established markers of liver damage such as alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and total bilirubin. However, sMet exhibited remarkably better correlation with liver damage and inflammation than did serum tumour necrosis factor α (TNF-α), whose shedding is also mediated by ADAM proteolytic activity. Our results indicate that the proteolytic activity of ADAM10/17 is essential for regulating HGF/c-Met signalling during acute liver damage and following regeneration and that the differential serum levels of sMet together with expression of c-Met/HGF might be a useful indicator not only for damage, but also for ongoing liver regeneration.

Delphinidin and cyanidin inhibit PDGF(AB)-induced VEGF release in vascular smooth muscle cells by preventing activation of p38 MAPK and JNK.

BACKGROUND AND PURPOSE: Red wine polyphenols (RWPs) inhibit the expression of vascular endothelial growth factor (VEGF), a major pro-angiogenic and pro-atherosclerotic factor, in vascular smooth muscle cells (VSMCs). The aim of this study was to identify which red wine polyphenols were inhibitory and to determine the mechanism underlying the inhibitory effects. EXPERIMENTAL APPROACH: Release of VEGF stimulated by platelet derived growth factor(AB) (PDGF(AB)), from human aortic VSMCs was measured by immunoassay and phosphorylation of kinases by Western blot analysis. The direct antioxidant properties of polyphenols were determined by electron paramagnetic resonance and the cellular formation of reactive oxygen species (ROS) by dichlorofluorescein. KEY RESULTS: The inhibitory effect of RWPs on PDGF(AB)-induced release of VEGF was mimicked by delphinidin but not by quercetin, catechins, resveratrol, gallic acid or caffeic acid. In the anthocyanin class, not only delphinidin but also cyanidin prevented VEGF release whereas malvidin and peonidin were without effect. RWPs, delphinidin and cyanidin directly scavenged ROS and prevented the PDGF(AB)-induced formation of ROS in VSMCs. Malvidin and peonidin did not scavenge ROS but prevented the cellular formation of ROS. Although the p38 MAPK, ERK1/2 and JNK pathways have been involved in the PDGF(AB)-induced expression of VEGF, in our experiments, only phosphorylation of p38 MAPK and JNK was inhibited by RWPs, delphinidin and cyanidin. CONCLUSIONS AND IMPLICATIONS: Anthocyanins presenting a hydroxyl residue at position 3' are able to inhibit PDGF(AB)-induced VEGF expression by preventing activation of p38 MAPK and JNK in VSMCs.

Diaphorase can metabolize some vasorelaxants to NO and eliminate NO scavenging effect of 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO).

Diaphorase was studied as a possible oxidoreductase participating in NO production from some vasorelaxants. In the presence of NADH or NADPH, diaphorase can convert selected NO donors, glycerol trinitrate (GTN) and formaldoxime (FAL) to nitrites and nitrates with NO as an intermediate. This activity of diaphorase was inhibited by diphenyleneiodonium (DPI) (inhibitor of some NADPH-dependent flavoprotein oxidoreductases), while it remained uninhibited by NG-nitro-L-arginine methyl ester (inhibitor of NO synthase) 7-Ethoxyresorufin (inhibitor of cytochrome P-450 1A1 and cytochrome P-450 NADPH-dependent reductase) inhibited the conversion of GTN only. Existence of NO as an intermediate of the reaction was supported by results of electron paramagnetic resonance spectroscopy. In addition to its ability to affect the above mentioned NO donors, diaphorase was able to reduce 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) and thus to eliminate its NO scavenging effect. This activity of diaphorase could also be inhibited by DPI. The reaction of diaphorase with GTN and PTIO was not affected by superoxide dismutase (SOD) or catalase. Reaction of FAL with diaphorase was lowered with SOD by 38 % indicating the partial participation of superoxide anion probably generated by the reaction of diaphorase with NADH or NADPH. Catalase had no effect. Diaphorase could apparently be one of the enzymes participating in the metabolism of studied NO donors to NO. The easy reduction and consequent elimination of PTIO by diaphorase could affect its use as an NO scavenger in biological tissues.

Possible metabolic pathways of conversion of formaldoxime and glyceryl trinitrate to NO.

The oxidation of N-hydroxylated compounds may result in production of nitrogen oxides, including nitric oxide (NO). Oxidation may be independent on NO-synthase. Production of nitrites and nitrates via NO from formaldoxime and glyceryl trinitrate was studied and compared. Superoxide ion, ions Fe2+ and Fe3+, methemoglobin and methemoglobin + NADPH + methylene blue, oxyhemoglobin and oxyhemoglobin + NADPH + methylene blue in the presence of atmospheric oxygen were used as oxidoreductive agents. Formaldoxime (triformaxime) was chosen as a newly recognized atypical cyclic oxime which can be converted to NO and glyceryl trinitrate as a well-known NO donor of quite different structure. From the oxidoreductive agents used, glyceryl trinitrate was not converted to nitrites or nitrates by Fe2+ or Fe3+ and by methemoglobin alone. Formaldoxime was resistant to the action of superoxide ion and methemoglobin alone. Importance of these possible metabolic pathways for production of NO from examined vasodilators is discussed.