Procollagen C-Proteinase Enhancer-1 (PCPE-1) deficiency in mice reduces liver fibrosis but not NASH progression.

BACKGROUND AND AIMS: Nonalcoholic Steatohepatitis (NASH) is a major cause of end-stage liver diseases such as cirrhosis and hepatocellular carcinoma resulting ultimately in increased liver-related mortality. Fibrosis is the main driver of mortality in NASH. Procollagen C-Proteinase Enhancer-1 (PCPE-1) plays a key role in procollagen maturation and collagen fibril formation. To assess its role in liver fibrosis and NASH progression, knock-out mice were evaluated in a dietary NASH model. METHODS: Global constitutive Pcolce-/- and WT male mice were fed with a Choline Deficient Amino acid defined High Fat Diet (CDA HFD) for 8 weeks. Liver triglycerides, steatosis, inflammation and fibrosis were assessed at histological, biochemical and gene expression levels. In addition, human liver samples from control and NASH patients were used to evaluate the expression of PCPE-1 at both mRNA and protein levels. RESULTS: Pcolce gene deficiency prevented diet-induced liver enlargement but not liver dysfunction. Furthermore, liver triglycerides, steatosis and inflammation were not modified in Pcolce-/- male mice compared to WT under CDA HFD. However, a significant decrease in liver fibrosis was observed in Pcolce-/- mice compared to WT under NASH diet, associated with a decrease in total and insoluble collagen content without any significant modifications in the expression of genes involved in fibrosis and extracellular matrix remodeling. Finally, PCPE-1 protein expression was increased in cirrhotic liver samples from both NASH and Hepatitis C patients. CONCLUSIONS: Pcolce deficiency limits fibrosis but not NASH progression in CDA HFD fed mice.

Deficiency of Procollagen C-Proteinase Enhancer 1 in Mice has No Major Impact on Cardiac Collagen and Function Under Basal Conditions.

ABSTRACT: Maturation of fibrillar collagen is known to play a crucial role in the pathophysiology of myocardial fibrosis. Procollagen C-proteinase enhancer 1 (PCPE1) has a key role in procollagen maturation and collagen fibril formation. The phenotype of both male and female PCPE1 knock-out mice was investigated under basal conditions to explore the potential of PCPE1 as a therapeutic target in heart failure. Global constitutive PCPE1-/- mice were generated. Serum procollagen I C-terminal propeptide, organ histology, and cutaneous wound healing were assessed in both wild type (WT) and PCPE1-/- mice. In addition, the cardiac expression of genes involved in collagen metabolism was investigated and the total and insoluble cardiac collagen contents determined. Cardiac function was evaluated by echocardiography. No differences in survival, clinical chemistry, or organ histology were observed in PCPE1-/- mice compared with WT. Serum procollagen I C-terminal propeptide was lower in PCPE1-/- mice. Cardiac mRNA expression of Bmp1, Col1a1, Col3a1, and Loxl2 was similar, whereas Tgfb and Loxl1 mRNA levels were decreased in PCPE1-/- mice compared with sex-matched WT. No modification of total or insoluble cardiac collagen content was observed between the 2 strains. Ejection fraction was slightly decreased in PCPE1-/- male mice, but not in females. Finally, wound healing was not altered in PCPE1-/- mice. PCPE1 deficiency does not trigger any major liabilities and does not affect cardiac collagen content nor its function under basal conditions. Further studies are required to evaluate its role under stressed conditions and determine its suitability as a therapeutic target for heart failure.

S62798, a potent TAFIa inhibitor, accelerates endogenous fibrinolysis in a murine model of pulmonary thromboembolism.

Enhancement of fibrinolysis constitutes a promising approach to treat thrombotic diseases. Venous thrombosis and thromboembolism risks are associated with increased plasma levels of TAFI (Thrombin Activatable Fibrinolysis Inhibitor) as well as its active form TAFIa. A new TAFIa inhibitor, namely S62798 has been identified. Its ability to enhance fibrinolysis was investigated both in vitro and in vivo in a mouse model of pulmonary thromboembolism, as well as its effect on bleeding. S62798 is a highly selective human, mouse and rat TAFIa inhibitor (IC50 = 11; 270; 178 nmol/L, respectively). It accelerates lysis of a human clot in vitro, evaluated by thromboelastometry (EC50 = 27 nmol/L). In a rat tail bleeding model, no effect of S62798 treatment was observed up to 20 mg/kg. Enhancement of endogenous fibrinolysis by S62798 was investigated in a mouse model of Tissue Factor-induced pulmonary thromboembolism. Intravenous administration of S62798 decreased pulmonary fibrin clots with a minimal effective dose of 0.03 mg/kg. Finally, effect of S62798 in combination with heparin was evaluated. When treatment of heparin was done in a curative setting, no effect was observed whereas a significantly decreased pulmonary fibrin deposition was observed in response to S62798 alone or in combination with heparin. This study demonstrates that S62798 is a potent TAFIa inhibitor with minimal risk of bleeding. In vivo, curative S62798 intravenous treatment, alone or associated with heparin, accelerated clot lysis by potentiating endogenous fibrinolysis and thus decreased pulmonary fibrin clots. S62798 is expected to be a therapeutic option for pulmonary embolism patients on top of anticoagulants.

Preserved regulation of renal perfusion pressure by small and intermediate conductance KCa channels in hypertensive mice with or without renal failure.

The purpose of this study was to assess, in the murine kidney, the mechanisms underlying the endothelium-dependent control of vascular tone and whether or not, in a severe model of hypertension and renal failure, KCa channels contribute to its regulation. Wild-type (BL) and double-transgenic female mice expressing human angiotensinogen and renin (AR) genes received either control or a high-salt diet associated to a nitric oxide (NO) synthase inhibitor treatment (BLSL and ARSL). Changes in renal perfusion pressure (RPP) were measured in isolated perfused kidneys. BLSL and AR were moderately hypertensive without kidney disease while ARSL developed severe hypertension and renal failure. In the four groups, methacholine induced biphasic endothelium-dependent responses, a transient decrease in RPP followed by a cyclooxygenase-dependent increase in RPP. In the presence or not of indomethacin, the vasodilatations were poorly sensitive to NO synthase inhibition. However, in the presence of cyclooxygenase and NO synthase inhibitors, apamin, and/or TRAM-34, blockers of KCa2.3 and KCa3.1, respectively, abolished the decrease in RPP in response to either methacholine or the two activators of KCa2.3/KCa3.1, NS309, and SKA-31. Thus, KCa2/3 channels play a major role in the regulation of murine kidney perfusion and this mechanism is maintained in hypertension, even when severe and associated with kidney damage.

Altered TP receptor function in isolated, perfused kidneys of nondiabetic and diabetic ApoE-deficient mice.

Early manifestations of kidney disease occur in atherosclerosis and activation of TP (thromboxane A(2)) receptors is implicated in atherosclerotic, diabetes, and renal diseases. The purpose of the present study was to analyze, in isolated, perfused mouse kidneys, the participation of TP receptors in renal vasoconstrictions and vasodilatations. In kidneys, taken from wild-type C57BL6, apolipoprotein E-deficient (ApoE-KO) and diabetic ApoE-KO mice, changes in perfusion pressure were recorded. Constrictions to TP receptor ligands U 46619, arachidonic acid, PGH(2), and 8-iso-PGF(2alpha), but not those to angiotensin II, endothelin, or norepinephrine, were inhibited by the selective TP receptor antagonist Triplion (S 18886; 10 nM). Acetylcholine and prostacyclin evoked biphasic responses during methoxamine constrictions; the constrictor part was blocked by Triplion. In ApoE-KO mouse kidneys, compared with C57BL6, a specific decrease in norepinephrine response and no modification in dilator responses were observed. In diabetic ApoE-KO mouse kidneys, constrictions to U 46619 and those to 8-iso-PGF(2alpha) were significantly and selectively augmented, without modification in the expression of the TP receptor, and again without any significant change in vasodilator activity. Thus TP receptors are functional, and their activation is not involved in norepinephrine, endothelin, and angiotensin II vasoconstrictions but is implicated in the unusual vasoconstrictions to acetylcholine and prostacyclin. Increased responsiveness of TP receptors occurs in diabetic ApoE-KO mouse kidneys. Thus early changes in TP receptor-mediated vasoconstrictor activity may participate in the development of kidney disease in atherosclerosis and diabetes.

Eya1 and Eya2 proteins are required for hypaxial somitic myogenesis in the mouse embryo.

In mammals, Pax3, Six4, Six1 and Six5 genes are co-expressed with Eya1, Eya2 and Eya4 genes during mouse somitogenesis. To unravel the functions of Eya genes during muscle development, we analyzed myogenesis in Eya2-/- and in Eya1-/- embryos. A delay in limb myogenesis was observed between E10 and E13 in Eya1-/- embryos only, that is later compensated. Compound E18 Eya1-/-Eya2-/+ fetuses present a muscle phenotype comparable with that of Six1-/- fetuses; lacking a diaphragm and with a specific absence of limb muscles, suggesting either genetic epistasis between Six and Eya genes, or biochemical interactions between Six and Eya proteins. We tested these two non-exclusive possibilities. First, we show that Six proteins recruit Eya proteins to drive transcription during embryogenesis in the dermomyotomal epaxial and hypaxial lips of the somites by binding MEF3 DNA sites. Second, we show that Pax3 expression is lost in the ventrolateral (hypaxial) dermomyotomes of the somite in both Eya1-/-Eya2-/- embryos and in Six1-/-Six4-/- embryos, precluding hypaxial lip formation. This structure, from which myogenic cells delaminate to invade the limb does not form in these double mutant embryos, leading to limb buds without myogenic progenitor cells. Eya1 and Eya2, however, are still expressed in the somites of Six1Six4 double mutant and in splotch embryos, and Six1 is expressed in the somites of Eya1Eya2 double mutant embryos and in splotch embryos. Altogether these results show that Six and Eya genes lie genetically upstream of Pax3 gene in the formation of ventrolateral dermomyotome hypaxial lips. No genetic links have been characterized between Six and Eya genes, but corresponding proteins activate key muscle determination genes (Myod, Myogenin and Mrf4). These results establish a new hierarchy of genes controlling early steps of hypaxial myogenic commitment in the mouse embryo.