Renal distribution of chymase.

<p>Panel A: Indirect immunofluorescence staining of kidney sections from cirrhotic rats (G3 group). Panel B: Immunohistochemical staining for chymase in kidney sections from either control animals (G1) or cirrhotic rats (G3). In the kidney of cirrhotic rats, chymase was found in the wall of cortical arterioles (Panel A), in the wall of proximal convoluted tubules (Panel B, green arrows), in distal convoluted tubules (Panel B, red arrows), and at the vascular pole of the glomerulus (Panel B, black arrow).</p

Chymase immunohistochemical localization in resected samples of normal and cirrhotic human liver.

<p>Original magnification as indicated.</p

Morphological analysis of chronic liver disease progression through αSMA immunohistochemistry.

<p>Administration of chymase inhibitor to healthy rats (G2) did not affect normal liver morphology. 13 weeks of CCl₄ (G3): development of liver cirrhosis (and ascites). The liver of rats receiving both CCl₄ and the chymase inhibitor was characterized by a significant prevention of fibrosis progression towards cirrhosis. This, already appreciable in G4, was maximal in animals treated with 20 mg/kg b.w. of the chymase inhibitor (G5).</p

Morphological analysis of chronic liver disease progression.

<p>13 weeks of CCl₄ (G3): development of liver cirrhosis (and ascites). The liver of rats receiving both CCl₄ and the chymase inhibitor was characterized by a significant prevention of fibrosis progression towards cirrhosis. This was maximal in animals treated with 20 mg/kg b.w. of the chymase inhibitor (G5). Prevention of fibrosis progression is clearly documented by either Gomori trichrome staining or Sirius Red.</p

Liver weight and function data, portal pressure, and hepatic tissue levels of Ang II and ET-1 in the different rat groups.

<p>Liver weight and function data, portal pressure, and hepatic tissue levels of Ang II and ET-1 in the different rat groups.</p

Microvesicles released from fat-laden cells promote activation of hepatocellular NLRP3 inflammasome: A pro-inflammatory link between lipotoxicity and non-alcoholic steatohepatitis

<div><p>Non-Alcoholic Fatty Liver Disease (NAFLD) is a major form of chronic liver disease in the general population in relation to its high prevalence among overweight/obese individuals and patients with diabetes type II or metabolic syndrome. NAFLD can progress to steatohepatitis (NASH), fibrosis and cirrhosis and end-stage of liver disease but mechanisms involved are still incompletely characterized. Within the mechanisms proposed to mediate the progression of NAFLD, lipotoxicity is believed to play a major role. In the present study we provide data suggesting that microvesicles (MVs) released by fat-laden cells undergoing lipotoxicity can activate NLRP3 inflammasome following internalization by either cells of hepatocellular origin or macrophages. Inflammasome activation involves NF-kB-mediated up-regulation of NLRP3, pro-caspase-1 and pro-Interleukin-1, then inflammasome complex formation and Caspase-1 activation leading finally to an increased release of IL-1β. Since the release of MVs from lipotoxic cells and the activation of NLRP3 inflammasome have been reported to occur in vivo in either clinical or experimental NASH, these data suggest a novel rational link between lipotoxicity and increased inflammatory response.</p></div

In vivo time-dependent analyses of inflammasome components, inflammatory cytokines and necrosis in MCD fed mice.

<p>“In vivo” analysis by quantitative real-time PCR (qPCR) of transcripts of inflammasome components (NLRP3 and CASP-1 <b>A, C</b>) and related cytokine (IL-1β, <b>B</b>) as well as of TNF-α (<b>E</b>) in WT mice fed with MCS diet or MCD diet for 4d, 2wks, 4wks and 8wks. <b>D</b>. Analysis of serum ALT in WT mice fed with MCS diet or MCD diet for 2wks, 4wks and 8 wks. Data in graphs are expressed as means ± SEM (n = 6 mice for any experimental group); p values are indicated and referred to mice fed the MCS control diet.</p

In vitro experimental model of lipotoxicity.

<p><b>A.</b> Red Oil-O staining in control HepG2 cells, HepG2 cells treated with BSA 1% or HepG2 cells exposed to palmitic acid 0.25mM in BSA 1% (BSA + PA) for 24hrs. <b>B.</b> Detection of Caspase-3/7 Activity in HepG2 cells treated with BSA 1% or PA 0.25mM for indicated times, analyzed by using Apo-ONE Caspase-3/7 Homogeneous Assay. <b>C.</b> Viability of HepG2 cells treated with BSA 1% or PA 0.25mM for indicated times, evaluated by MTT assay. <b>D,E.</b> Analysis of internalization of MVs in HepG2 cells by (<b>D</b>) flow cytometry or by (<b>E</b>) confocal microscopy: nuclei (blue fluorescence), MVs (red fluorescence) and cytoskeleton (F-actin, green fluorescence).</p

NLRP3 inflammasome activation is prevented by blocking the caspase 3 –dependent release of MVs.

<p><b>A.</b> Western blot analysis of caspase-3 in total extracts of HepG2 cells not transfected (C), transfected with non-silencing siRNA (NsC) or specific siRNA for caspase-3 (siCASP3) and treated with palmitic acid 0,25mM for 24hrs. Equal loading was confirm by re- probing the same membrane with monoclonal antibody against the house-keeping β-actin. <b>B.</b> Protein quantification of preparation of MVs as obtained from control HepG2 cells (C), from HepG2 cells transfected with the non silencing RNA and then exposed to PA (NsC) and HepG2 cells silenced for caspase 3 and then exposed to PA (siCASP3). Data are expressed as μg/μl and as mean ± SD being (n = 12 for any condition). <b>C.</b> Flow cytometric analysis of internalization of PKH26-fluorescent die-MVs in HepG2 naïve cells. Color legend: i) black trace is related to HepG2 naïve cells treated with ultracentrifugated solution (also marked with PKH26-fluorescent die) derived from cells just exposed to BSA (used as control), ii) pink trace is related to HepG2 naïve cells treated with MVs derived from HepG2 cells transfected with non-silencing treated with palmitic acid 0,25mM for 24hrs; iii) green trace is related to HepG2 naïve cells treated with MVs derived from HepG2 cells transfected with specific siRNA for caspase-3 and treated with palmitic acid 0,25mM for 24hrs. <b>D.</b> Western blot analysis of NF-Kb/p65 protein levels in cytosolic and nuclear extract obtained from HepG2 naïve cells exposed or not (C) to MVs derived from HepG2 cells transfected with non-silencing siRNA (NsC) or specific siRNA for caspase-3 (siCASP3) and treated with palmitic acid 0,25mM for 24hrs. Equal loading was confirmed by re-probing membrane with α-tubulin and Lamin-A (cytosolic and nuclear house-keeping, respectively). <b>E.</b> Western blot analysis of NLRP3 protein levels as well as of cleaved caspase-1 (p-10-CASP-1) and IL-1β in total extracts obtained by HepG2 naïve cells exposed or not (C) to MVs derived from HepG2 cells transfected with non-silencing siRNA (NsC) or specific siRNA for caspase-3 (siCASP3) and treated with palmitic acid 0,25mM for 24hrs. LPS 1mg/ml was used as positive control. Equal loading was confirmed by re-probing membrane with β-actin.</p