Nonalcoholic fatty liver disease or metabolic dysfunction-associated fatty liver disease diagnoses and cardiovascular diseases: From epidemiology to drug approaches.

BACKGROUND: A consensus of experts has proposed to replace the term nonalcoholic fatty liver disease (NAFLD), whose global prevalence is 25%, with metabolic dysfunction-associated fatty liver disease (MAFLD), to describe more appropriately the liver disease related to metabolic derangements. MAFLD is closely intertwined with type 2 diabetes, obesity, dyslipidaemia, all linked to a rise in the risk of cardiovascular disease (CVDs). Since controversy still stands on whether or not NAFLD/MAFLD raises the odds of CVD, the present review aims to evaluate the impact of NAFLD/MAFLD aetiologies on CV health and the potential correction by dietary and drug approaches. RESULTS: Epidemiological studies indicate that NAFLD raises risk of fatal or non-fatal CVD events. NAFLD patients have a higher prevalence of arterial plaques and stiffness, coronary calcification, and endothelial dysfunction. Although genetic and environmental factors strongly contribute to NAFLD pathogenesis, a Mendelian randomization analysis indicated that the PNPLA3 genetic variant leading to NAFLD may not be causally associated with CVD risk. Among other genetic variants related to NAFLD, TM6SF2 appears to be protective, whereas MBOAT7 may favour venous thromboembolism. CONCLUSIONS: NAFLD is correlated to a higher CVD risk which may be ameliorated by dietary interventions. This is not surprising, since new criteria defining MAFLD include other metabolic risk abnormalities fuelling development of serious adverse extrahepatic outcomes, for example CVD. The present lack of a targeted pharmacological approach makes the identification of patients with liver disease at higher CVD risk (eg diabetes, hypertension, obesity or high levels of C-reactive protein) of major clinical interest.

Proprotein convertase 7 rs236918 associated with liver fibrosis in Italian patients with HFE-related hemochromatosis.

BACKGROUND AND AIM: p.Cys282Tyr homozygosity is the prevalent genotype in (HFE)-related Hereditary Hemochromatosis with low penetrance and variable expression. However, liver cirrhosis and hepatocellular carcinoma remain the main causes of mortality in these patients. Detection of genetic modifiers identifying patients at risk for liver damage would be relevant for their clinical management. We evaluated proprotein convertase 7 (PCSK7) rs236918 as genetic marker of risk of liver fibrosis in an Italian cohort of p.Cys282Tyr homozygotes. METHODS: Liver fibrosis was histologically assessed by Ishak score. We evaluated PCSK7 alleles and genotypes frequencies according to single or grouped staging scores: absent/mild fibrosis (stage: 0-2), moderate (stage: 3-4), and severe fibrosis/cirrhosis (stage: 5-6). Single nucleotide polymorphism genotyping was performed by restriction fragment length polymorphism or Taqman 5'-nuclease assays. RESULTS: The rs236918 allele C frequency increased from stages 0-2 to 5-6 (7.1% vs 13.6%, vs 21.9%, P = 0.003). The wild-type genotype was significantly more frequent in the absent/mild fibrosis group (54.2%) compared with only 17% in patients with severe fibrosis/cirrhosis. At univariate proportional odds model, patients with GC + CC genotypes were 2.77 times (P = 0.0018) more likely to have worse liver staging scores than wild-type patients. In the adjusted analysis, odds ratio was 2.37 (P = 0.0218), and 2.56 (P = 0.0233) when the analysis was restricted to males. An exploratory mediation analysis suggested a direct effect of genotype on severe fibrosis/cirrhosis (odds ratio = 3.11, P = 0.0157), and a mild non-significant indirect effect mediated through iron accounting for 28%. CONCLUSIONS: These findings confirm that PCSK7 rs236918 C allele is a risk factor for cirrhosis development in Italian patients with HFE-Hemochromatosis.

Proprotein convertase subtilisin/kexin type 7 (PCSK7) is essential for the zebrafish development and bioavailability of transforming growth factor ß1a (TGFß1a).

Proprotein convertase subtilisin/kexin (PCSK) enzymes convert proproteins into bioactive end products. Although other PCSK enzymes are known to be essential for biological processes ranging from cholesterol metabolism to host defense, the in vivo importance of the evolutionarily ancient PCSK7 has remained enigmatic. Here, we quantified the expressions of all pcsk genes during the 1st week of fish development and in several tissues. pcsk7 expression was ubiquitous and evident already during the early development. To compare mammalian and zebrafish PCSK7, we prepared homology models, which demonstrated remarkable structural conservation. When the PCSK7 function in developing larvae was inhibited, we found that PCSK7-deficient fish have defects in various organs, including the brain, eye, and otic vesicle, and these result in mortality within 7 days postfertilization. A genome-wide analysis of PCSK7-dependent gene expression showed that, in addition to developmental processes, several immune system-related pathways are also regulated by PCSK7. Specifically, the PCSK7 contributed to the mRNA expression and proteolytic cleavage of the cytokine TGFß1a. Consequently, tgfß1a morphant fish displayed phenotypical similarities with pcsk7 morphants, underscoring the importance of this cytokine in the zebrafish development. Targeting PCSK activity has emerged as a strategy for treating human diseases. Our results suggest that inhibiting PCSK7 might interfere with normal vertebrate development.

Identification of novel non-HFE mutations in Chinese patients with hereditary hemochromatosis.

BACKGROUNDS: Hereditary hemochromatosis (HH) is mainly caused by homozygous p.C282Y mutations in HFE in the Caucasians. We recently reported non-HFE mutations constitute the major cause of HH in Chinese. However, there is still a relatively high proportion of cases with primary iron overload from unexplained causes. We aimed to explore novel non-HFE mutations in Chinese patients with primary iron overload. METHODS: Whole exome sequence was conducted to screen mutations in novel HH-related genes in the 9 cases with unexplained primary iron overload. Then the representative candidate genes were screened for mutations in another cohort of 18 HH cases. The biological function of the selected genes and variants were analyzed in vitro. RESULTS: Whole exome sequencing of 9 cases with unexplained primary iron overload identified 42 missense variants in 40 genes associated with iron metabolism pathway genes such as UBE2O p.K689R and PCSK7 p.R711W. Subsequent Sanger sequencing of the UBE2O and PCSK7 genes in the 27 cases with primary iron overload identified p.K689R in UBE2O, p.R711W and p.V143F in PCSK7 at frequency of 2/27,1/27 and 2/27 respectively. In vitro siRNA interference of UBE2O and PCSK7 resulted in down-regulated HAMP mRNA expression. Adenovirus generation of UBE2O p.K689R in cell lines resulted in increased expression of SMAD6 and SMAD7 and downregulation of p-SMAD1/5 and HAMP expression, and the reduction of hepcidin level. CONCLUSIONS: Our study identified a series of novel candidate non-HFE mutations in Chinese patients with HH. These may provide insights into the genetic basis of unexplained primary iron overload.

Post-Transcriptional Effects of miRNAs on PCSK7 Expression and Function: miR-125a-5p, miR-143-3p, and miR-409-3p as Negative Regulators.

The regulatory mechanism of PCSK7 gene is still unknown, although its encoded protein PC7 is the most ancient and highly conserved of all proprotein convertases and exhibits enzymatic and non-enzymatic functions in liver triglyceride regulation. Bioinformatics algorithms were used to predict regulatory microRNAs (miRNAs) of PCSK7 expression. This led to the identification of four miRNAs, namely miR-125a-5p, miR-143-3p, miR-409-3p, and miR-320a-3p, with potential binding sites on the 3'-untranslated region (3'-UTR) of human PCSK7 mRNA. The expression patterns of these miRNAs and PCSK7 mRNA were assessed in three different cell lines with quantitative polymerase chain reaction (qPCR), which revealed reciprocal expression patterns between the expression levels of the four selected miRNAs and PCSK7. Next, the interactions and effects of these miRNAs on PCSK7 expression levels were investigated via cell-based expression analysis, dual-luciferase assay, and Western blot analysis. The data revealed that PCSK7 mRNA levels decreased in cells transfected with vectors overexpressing miR-125a-5p, miR-143-3p, and miR-409-3p, but not miR-320a-3p. The dual-luciferase assay demonstrated that the above three miRNAs could directly interact with putative target sites in PCSK7 3'-UTR and regulate its expression, whereas miR-320-3p exhibited no interaction. Western blot analysis further revealed that the overexpression of miR-125a-5p in Huh7 cells inhibits the expression and ability of PC7 to cleave human transferrin receptor 1. Our results support a regulatory role of these miRNAs on PCSK7 expression and function and open the way to assess their roles in the regulation of PC7 activity in vivo in the development of hepatic steatosis.

Proprotein convertase 7 (PCSK7) reduces apoA-V levels.

The locus of the human proprotein convertase subtilisin-kexin type-7 (PC7) gene (PCSK7) is on chromosome 11q23.3 close to the gene cluster APOA5/APOA4/APOC3/APOA1, a region implicated in the regulation of lipoprotein metabolism. A GWAS reported the association of PCSK7 SNPs with plasma triglyceride (TG), and exome sequencing of African Americans revealed the association of a low-frequency coding variant of PC7 (R504H; SNP rs142953140) with a ~ 30% TG reduction. Another PCSK7 SNP rs508487 is in linkage disequilibrium with a promoter variant of the liver-derived apolipoprotein A-V (apoA-V), an indirect activator of the lipoprotein lipase (LpL), and is associated with elevated TG levels. We thus hypothesized that PC7 regulates the levels/activity of apoA-V. Studies in the human hepatic cell line HuH7 revealed that wild-type (WT) PC7 and its endoplasmic reticulum (ER)-retained forms bind to and enhance the degradation of human apoA-V in acidic lysosomes in a nonenzymatic fashion. PC7-induced degradation of apoA-V is inhibited by bafilomycin A1 and the alkalinizing agents: chloroquine and NH4 Cl. Thus, the PC7-induced apoA-V degradation implicates an ER-lysosomal communication inhibited by bafilomycin A1. In vitro, the natural R504H mutant enhances PC7 Ser505 phosphorylation at the structurally exposed Ser-X-Glu507 motif recognized by the secretory kinase Fam20C. Co-expression of the phosphomimetic PC7-S505E with apoA-V resulted in lower degradation compared to WT, suggesting that Ser505 phosphorylation of PC7 lowers TG levels via reduced apoA-V degradation. In agreement, in Pcsk7-/- mice fed high-fat diet, plasma apoA-V levels and adipocyte LpL activity are increased, providing an in vivo mechanistic link for a role of liver PC7 in enhanced TG storage in adipocytes.

Enhanced Proteolytic Processing of Recombinant Human Coagulation Factor VIII B-Domain Variants by Recombinant Furins.

Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg(1648) and Glu(1649). By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg(1648) FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg(1648) FVIII processing site by PCSK6.