Malnutrition and sarcopenia have been shown to impact morbidity and mortality in liver cirrhosis in retrospective studies in patients evaluated for liver transplantation. The first part of the study will examine, in a prospective multicenter investigation, the role of sarcopenia on 1 year mortality in a large national cohort of patients with liver cirrhosis of different origin and severity. Sarcopenia will be evaluated by imaging CT scan. The CT scan transverse L3 images will be analyzed centrally by two trained observers by SliceOmatic Software V4.2. Skeletal muscle will be identified and quantified by HU thresholds of -29 to +150. Patients will be classified as having sarcopenia using cut-off values proposed by Carey et al.
Patients will be followed for a 12 months period. Clinical, functional and biochemical evaluation will be performed during the study at different time points (3, 6, and 12 months). The occurrence of complications (ascites, hepatic encephalopathy, GI bleeding,..), episodes of hospitalization or any relevant clinical event will be recorded at each visit. The study has been approved by the Ethical Committee Review Board. A sample size of 277 patients has been planned.
The second part of the study will investigate liver-muscle crosstalk in an in vitro model of human skeletal muscle cells. Exosomes, that are small vesicles (40¿100 nm) possess the capability of `communicating¿ with neighboring or distant cells by fusing with the plasma membrane and delivering their cargo, including proteins, mRNAs, and miRNAs. Exosomes will be extracted from the plasma of patients with liver cirrhosis of different origin and severity. Nutritional status and sarcopenia will also be recorded. The in vitro model we¿ll allow us to evaluate whether liver-derived exosomes, and their miRNA cargo, can interfere with muscle cell proliferation and differentiation and with protein homeostasis inducing the sarcopenic process.
In patients with liver cirrhosis, malnutrition is associated with an increased risk of mortality, higher prevalence of complications and bacterial infections, as well as more frequent hospitalizations and longer hospital stay. The main component of malnutrition in liver cirrhosis is sarcopenia, a condition characterized by loss of muscle mass and strength.
As highlighted by a recent systematic review and meta-analysis all the studies on the impact of sarcopenia/sarcopenic obesity and myosteatosis in cirrhotic patients are retrospective studies, mostly involving non-consecutive patients investigated before liver transplantation. Moreover, most of the studies were produced by non-European centers (Canadians, Americans, and Japanese) that published multiple reports utilizing the same patient series. All these factors have caused a possible selection bias. Furthermore, the methods used to evaluate sarcopenia and myosteatosis were not homogeneous (the entire muscle area, or area of the psoas or psoas diameter) as well as the cut-offs used.
WHAT IS NEW IN OUR STUDY (Part 1)
At variance with previous investigations, we propose a prospective study aimed at analyzing the impact of sarcopenia in patients with liver cirrhosis. The study will enroll an large national cohort of patients with cirrhosis. To avoid the selection bias of considering only patients with advanced stage of liver cirrhosis the patients waiting for liver transplantation will not be included. Patients will be enrolled in several hepatology centers in Italy following the same protocol to obtain a multicenter investigation. The patients will be followed for 12 months to obtain all information about morbidity and mortality. The collection and analysis of the data will be centralized.
The results of the study will give the opportunity to increase knowledge about the prevalence and characteristics of sarcopenia in a large cohort of patients with cirrhosis ranging from mild to severe liver damage. The prospective design of the study and the information obtained after 12 months follow up, will give the opportunity to investigate original cut off values for the skeletal muscle index, obtained at TC imaging, fitting with the prediction of patients¿ morbidity and survival.
Several factors are involved in the pathogenesis of malnutrition in the patient with liver cirrhosis as inadequate dietary intake, altered nutrient absorption and modification in the use of energy substrates due to liver disease [Tandon P 2017, Plauth M 2002]. The origin of sarcopenia in liver cirrhosis is multifactorial. Increased proteolysis, systemic inflammation, disturbances in major muscle growth regulatory proteins including increased myostatin decreased testosterone and insulin-like growth factor-1, chronic hyperammoniemia and physical inactivity have been reported as possible pathogenetic factors.
To our knowledge study exploring the crosstalk between liver and muscle in patients with liver cirrhosis are lacking.
Extracellular vesicles (EVs), are intercellular mediators of cell-to-cell communication; Evs sustain cellular cross-talk within liver and between different organs and tissues. Exosomes are small vesicles (40¿100 nm) matured inside multivesicular bodies, are the most studied EVs. EVs from injured hepatocytes can target several cell types and modulate their behavior.
In skeletal muscle several biological processes are mediated by specific miRNAs; they play key roles in regulating satellite cells and inducing myogenesis, by their reciprocal regulatory relationship with multiple transcription factors. miRNAs that are specifically, or highly expressed in skeletal muscle tissue are called myomiRs and include miR-1, miR-133a, miR-133b, miR-206, miR-208b, miR-486 and miR-499 (29). Given the close metabolic interplay between muscle and liver, we can hypothesize that these tissues could crosstalk via extracellular vesicles and that induction or progression of sarcopenia in liver disease could be mediated by hepatic EVs. In particular we suppose that exosomes released in circulation from injured and/or cirrhotic liver, could vehicle to skeletal muscle molecules, especially miRNA, able to trigger or contribute to sarcopenia.
WHAT IS NEW IN OUR STUDY (Part 2)
To our knowledge no studies have previously examined exosomes as possible mediators of liver-muscle crosstalk. We will explore weather exosomes in cirrhotic patients may interact and regulate muscle trophism in an in vitro model of muscle cells culture.