In cirrhosis, liver microvascular dysfunction is a key factor increasing hepatic vascular resistance to portal blood flow, which leads to portal hypertension. De‐regulated inflammatory and pro‐apoptotic processes due to chronic injury play important roles in the dysfunction of liver sinusoidal cells. The present study aimed at characterizing the effects of the pan‐caspase inhibitor emricasan on systemic and hepatic hemodynamics, hepatic cells phenotype, and underlying mechanisms in preclinical models of advanced chronic liver disease. We investigated the effects of 7‐day emricasan on hepatic and systemic hemodynamics, liver function, hepatic microcirculatory function, inflammation, fibrosis, hepatic cells phenotype, and paracrine interactions in rats with advanced cirrhosis due to chronic CCl₄ administration. The hepato‐protective effects of emricasan were additionally investigated in cells isolated from human cirrhotic livers. Cirrhotic rats receiving emricasan showed significantly lower portal pressure than vehicle‐treated animals with no changes in portal blood flow, indicating improved vascular resistance. Hemodynamic improvement was associated with significantly better liver function, reduced hepatic inflammation, improved phenotype of hepatocytes, liver sinusoidal endothelial cells, hepatic stellate cells and macrophages, and reduced fibrosis. In vitro experiments demonstrated that emricasan exerted its benefits directly improving hepatocytes’ expression of specific markers and synthetic capacity, and ameliorated nonparenchymal cells through a paracrine mechanism mediated by small extracellular vesicles released by hepatocytes. Conclusion: This study demonstrates that emricasan improves liver sinusoidal microvascular dysfunction in cirrhosis, which leads to marked amelioration in fibrosis, portal hypertension and liver function, and therefore encourages its clinical evaluation in the treatment of advanced chronic liver disease.

The poor prognosis of chronic liver disease (CLD) generates the need to investigate the evolving mechanisms of disease progression, thus disclosing therapeutic targets before development of clinical complications. Considering the central role of liver sinusoidal endothelial cells (LSECs) in pre-neoplastic advanced CLD, the present study aimed at investigating the progression of CLD from an endothelial holistic perspective. RNAseq defined the transcriptome of primary LSECs isolated from three pre-clinical models of advanced CLD, during the progression of the disease, and from fresh human cirrhotic tissue. At each stage of the disease, the effects of LSECs secretome on neighboring cells and proteomic analysis of LSECs-derived extracellular vesicles (EVs) were also determined. CLD was associated with deep common modifications in the transcriptome of LSECs in the pre-clinical models. Pathway enrichment analysis showed predominance of genes related with pro-oncogenic, cellular communication processes, and EVs biogenesis during CLD progression. Crosstalk experiments revealed endothelial EVs as potent angiocrine effectors. The proteome of LSECs EVs showed stage-specific signatures, including over-expression of tropomyosin-1. Proof-of-principle experiments treating cirrhotic HSCs with recombinant tropomyosin-1 suggested de-activating effects. Our data provide the basis for discovering novel biomarkers and therapeutic targets for new disease-modifying treatments for patients with advanced CLD.

Refractory ascites is characterized by severe circulatory dysfunction and increased sensitivity to renal impairment by non-selective beta-blockers (NSBB). A novel serum microRNA signature, identified in the secretome of human cirrhotic hepatocytes, allows for the molecular characterization of patients with decompensated cirrhosis more prone to develop significant cardiac impairment after NSBB, as well as those more likely to respond to NSBB in terms of HVPG reduction. Noninvasive staging of decompensated cirrhosis is an unmet clinical need. The aims of this study were to characterize and validate a novel microRNA (miRNA) signature to stage decompensated cirrhosis and predict the portal pressure and systolic cardiac response to nonselective beta-blockers (NSBBs). Serum samples from patients with decompensated cirrhosis (n = 36) and healthy controls (n = 36) were tested for a novel signature of five miRNAs (miR-452-5p, miR-429, miR-885-5p, miR-181b-5p, and miR-122-5p) identified in the secretome of primary human hepatocytes and for three miRNAs (miR-192-5p, miR-34a-5p, and miR-29a-5p) previously discovered as biomarkers of chronic liver disease. All patients had ascites, which was refractory in 18 (50%), and were placed on NSBBs for variceal bleeding prophylaxis. In all patients, serum miRNAs, hepatic venous pressure gradient, and an echocardiogram study were performed before and 1 month after NSBBs. Patients with cirrhosis had lower serum levels of miR-429, miR-885-5p, miR-181b-5p, miR-122-5p, miR-192-5p, and miR-29a-5p (P < 0.05). Baseline serum miR-452-5p and miR-429 levels were lower in NSBB responders (P = 0.006). miR-181b-5p levels were greater in refractory ascites than in diuretic-sensitive ascites (P = 0.008) and correlated with serum creatinine. miR-452-5p and miR-885-5p were inversely correlated with baseline systemic vascular resistance (ρ = −0.46, P = 0.007; and ρ = −0.41, P = 0.01, respectively) and with diminished systolic contractility (ρ = −0.55, P = 0.02; and ρ = −0.55, P = 0.02, respectively) in patients with refractory ascites after NSBBs. Conclusion: Analysis of a miRNA signature in serum discriminates between patients with decompensated cirrhosis who show more severe systemic circulatory dysfunction and compromised systolic function after beta-blockade and those more likely to benefit from NSBBs.

Inflammation and oxidative stress play a key role in the pathophysiology of advanced chronic liver disease (ACLD) and portal hypertension (PH). Considering the current lack of effective treatments, we evaluated an anti-inflammatory and antioxidant nutraceutical rich in docosahexaenoic acid (DHA) as a possible therapy for ACLD. We investigated the effects of two-week DHA supplementation (500 mg/kg) on hepatic fatty acids, PH, oxidative stress, inflammation, and hepatic stellate cell (HSC) phenotype in rats with ACLD. Additionally, the effects of DHA were evaluated in murine macrophages and human HSC. In contrast to vehicle-treated animals, cirrhotic rats receiving DHA reestablished a healthy hepatic fatty acid profile, which was associated with an improvement in PH. The mechanisms underlying this hemodynamic improvement included a reduction in oxidative stress and inflammation, as well as a marked HSC deactivation, confirmed in human HSC. Experiments with cultured macrophages showed that treatment with DHA protects against pro-inflammatory insults. The present preclinical study demonstrates that a nutraceutical rich in DHA significantly improves PH in chronic liver disease mainly by suppressing inflammation and oxidative stress-driven HSC activation, encouraging its evaluation as a new treatment for PH and cirrhosis.