Fig 2: Synthetic NPY fragments derived from NEP proteolysis induce fibrogenesis in HSC(A) Nep−/− mice were used to isolate primary HSCs and were treated with full-length NPY (1 nM) or their respective cleaved fragments (cNPY) (30 μM). After 24-h treatment, cells were used for further analysis.(B) mRNA expression levels of Col1A1 and Acta2 were analyzed after 24-h treatment, ∗∗p < 0.01. Data were normalized to the expression of 18S RNA.(C) Western blot analysis and quantifications of Col1a1 and α-SMA protein expression using GAPDH as a loading control.(D–G) Nep−/− HSCs were fixed after treatment for immunofluorescence with collagen I and αSMA antibodies. Confocal microscopy was used to detect these proteins. Changes in collagen I protein were detected in cells treated with the cNPY fragments as well as the changes detected in αSMA protein in the cells treated with full-length NPY; ∗p < 0.05 and ∗∗p < 0.01 compared to control. Representative images and fluorescence quantification from three independent experiments are shown. Images were taken with the 20x confocal objective that corresponds to a scale bar of 50 μm.

Fig 3: Nep−/− shows more contraction when treated with NPY than in WT HSC(A) Hepatic Nep mRNA expression in hepatocytes (HEPs), Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs) from healthy (Ctrl) and BDL-treated WT mice. qPCR data were referred to HEPs and normalized to the expression of 18S RNA.(B) NPY ELISA from fetal calf serum (FCS, n = 3), human serum control and cirrhosis (n = 5, n = 8), fibroblast-conditioned medium (FCM, n = 3), and supernatants of conditioned hepatic stellate cells (HSC, n = 3). Dulbecco’s Modified Eagle Medium (DMEM, 0% FCS) was used as negative control (Neg. Ctrl). Results are expressed as mean ± standard deviation (SD).(C–F) Nep−/− and WT (C57BL/6) mice were used to isolate HSCs, and cells were treated with NPY 1nM. mRNA expression levels of Tgfβ1, Col1A1, and Acta2 were analyzed after 24 h treatment. Results are expressed as mean ± SEM; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.(G) Contraction assay of primary HSCs cultured on collagen gels and treated with and without recombinant NPY 0–100 nM for 0–48 h. The relative area was calculated by recording the diameter change of gels at several time points with a digital camera at a fixed distance above the gels. Results are expressed in percentage of initial gel surface and as mean ± SEM; n = 3–6 per group. ∗p < 0.05 compared to control.(H) Migration assay of WT and Nep−/− HSCs. Nep−/− migrated faster than the WT HSCs after 24 h. Results are expressed as mean ± SEM; ∗∗p < 0.01, ∗∗∗∗p < 0.0001.(I) In situ liver perfusion of cirrhotic animals. For pre-contraction of the livers, methoxamine (100 μM) was used, and then, increasing concentrations of full-length NPY and cleaved C-terminal fragments of NPY (cNPY) (as indicated) were used to measure the perfusion pressure of the livers (n = 3 per group). ∗p < 0.05 compared to MTX.

Fig 4: NPY levels correlate with liver disease in mice and in human hepatic cirrhosis(A) Serum NPY ELISA from control and cirrhotic patients undergoing transjugular intrahepatic portosystemic shunt (TIPS) treatment, n = 7 (TIPS-group, cirrhotic patients) and n = 6 (controls), ∗∗p < 0.02 for cirrhotic patients vs. healthy individuals (absolute values).(B) Serum NPY ELISA in cirrhotic patients shows increased NPY in Child-Pugh class C compared with Child-Pugh class A patients, n = 5 per group, ∗p < 0.05 for Child-Pugh class C vs. Child-Pugh class A cirrhotic patients. Results are expressed as mean ± standard error of the mean (SEM).(C) Serum NPY ELISA from portal vein (PV, hepatic inflow) and hepatic vein (HV, hepatic outflow) in TIPS patients, n = 16 per group, ∗∗p < 0.02 for PV vs. HV.(D) Serum NPY ELISA from hepatic vein (HV) and hepatic artery (HA) in n = 21 per group, n.s. (not significant).(E) NPY levels compared with portal vein and hepatic artery, ∗∗∗p < 0.001.(F) In silico analysis of hepatic mRNA NPY levels compared with TGFB1 expression in human liver tissues.(G) Hepatic Nep and Col1A1 mRNA expression in patients with chronic liver disease; number of XY pairs = 17, r = 0.7472, p = 0.0006.(H and I) Hepatic Nep mRNA expression from bile duct ligation (BDL)-treated and CCl4-treated WT (C57BL/6) mice for 2 and 4 weeks, respectively, ∗∗p < 0.02 for BDL- or CCl4-treated vs. corresponding control mice. Data were normalized to the expression of 18S RNA.(J) Liver homogenates NPY ELISA from WT and Nep−/− mice, control vs. CCl4, ∗p < 0.05.(K) Morphometric analysis of Sirius red staining of livers from WT mice compared with Nep−/− with and without NPYFL (left panel) and WT BDL mice vs. Nep−/− BDL with and without NPYFL (right panel). n = 5 per group, ∗∗p < 0.01, ∗∗∗p < 0.001, and n.s. (non-significant).(L) Liver sections stain with Sirius red. Scale bar: 200 μm.(M) Col1A1 mRNA expression of WT mice comparing non-treated controls vs. animals that were injected with full-length NPY, ∗∗p < 0.01. Col1A1 mRNA expression of Nep−/− mice after profibrotic stimuli (BDL) comparing control vs. Nep−/− after injecting full-length NPY, ∗∗p < 0.01. Data were normalized to the expression of 18S RNA.

Fig 5: Nep deletion, AT1R blockage, or angiotensin-converting enzyme inhibition reduces portal hypertension(A) Portal pressure is significantly increased in Nep−/− mice under basal conditions further enhanced in CCl4- and BDL-treated Nep−/−mice compared with WT mice. Results are expressed as mean ± SEM; n = 6 per group, ∗p < 0.05, ∗∗p < 0.01 for BDL- or CCl4-treated mice vs. corresponding control mice. All data were normalized to the expression of 18S RNA.(B) During liver fibrosis, NPY is cleaved by NEP producing two different cNPY peptides, which induce a fibrogenic response mediated by Y1R (red arrows). Full-length NPY enhances contraction through activation of the Y1R and Gαi-adenylate cyclase (AD) (green arrows). Ang II, agonist of AT1R, results in contraction, further increased by the activation of Y1R. In the absence of NEP (gray) and AT1R blockage (losartan) or ACE inhibition (captopril) (red), full-length NPY will decrease fibrosis. ANG II, which results in contraction (gray), will not exert its function due to the administration of either captopril (red) or losartan (red). In the absence of NEP, full-length NPY will activate Y1R and decrease the fibrogenic response previously mediated by cNPY (green arrows). Deletion of NEP in combination with ACE inhibitor or AT1R blockage reduces portal pressure and fibrosis.(C and D) Sirius red staining in captopril- and losartan-treated CCl4Nep−/− mice and their respective morphometric analysis. Scale bar: 200 μm.(E and F) Portal pressure measurement (mm Hg) in captopril- and losartan-treated CCl4Nep−/− mice and hepatic Col1a1 mRNA levels. Results are expressed as mean ± SEM; n = 6 per group, ∗∗p < 0.01 for CCl4-treated mice vs. corresponding control mice and n = 6 per group, ∗p < 0.05, and ∗∗∗p < 0.001 for CCl4-treated mice vs. corresponding control mice.