Thesis title: Impact of platelet phenotype on liver fibrosis progression in metabolic dysfunction-associated steatotic liver disease
Introduction: During the last decades, lifestyle changes have deeply influenced human metabolism, giving rise to a global silent epidemic of chronic metabolic diseases worldwide, encompassing obesity, type 2 diabetes (T2D), and non-alcoholic fatty liver disease (NAFLD). NAFLD, now referred to as metabolic dysfunction-associated steatotic liver disease (MASLD), has become the most common cause of chronic liver disease. Robust scientific evidence closely links it with metabolic syndrome features, especially obesity and type 2 diabetes. MASLD results from the accumulation of liver fat, occurring in the absence of significant alcohol consumption and within the context of at least one metabolic dysfunction. This condition encompasses a spectrum of histopathological changes from simple steatosis (metabolic dysfunction-associated steatotic liver, MASL) to steatohepatitis (metabolic dysfunction-associated steatohepatitis, MASH) and may further lead to significant fibrosis and, eventually, MASH-related cirrhosis. The progression of MASL to MASH is not fully elucidated. While platelets have a well-established role in hemostasis, recent studies have revealed their active involvement in the inflammatory response and tissue remodeling. However, their role in MASLD progression remains unclear and subject to ongoing debate. This study aims to identify specific features of platelet phenotype across different stages of MASLD and to explore potential biomarkers linked to platelet phenotype that may predict significant fibrosis in affected patients.
Methods: Subjects medical history, demographics, imaging, and laboratory findings were collected. The diagnosis of MASLD followed the guidelines outlined by EASL-EASD-EASO and the latest consensus statement. Citrated venous whole blood, processed within one hour of collection, was utilized for quantifying platelet phenotype and assessing immune interactions through multi-parametric flow cytometry. Similarly, plasmatic cytokine concentrations were assessed using commercial ELISA kits. Statistical analysis was conducted to determine significant differences between the various groups.
Results: This study enrolled 87 individuals (44M:43F, median age 58.5), with 62 diagnosed with MASLD (28 MASL, 22 MASH, 12 cirrhosis) and 25 controls. Age and sex distributions were similar among groups (p = 0.420 and p = 0.672). Significant differences among groups were found for body mass index, waist circumference, systolic blood pressure, fasting plasma glucose, HDL cholesterol, triglycerides, AST, ALT, platelet count, and FIB-4 (all p < 0.05). The prevalence of T2D, hypertension (HTN), and MetS varied significantly (p < 0.001 for T2D and MetS; p = 0.014 for HTN), while dyslipidemia showed no difference (p = 0.496). Notable differences in platelet phenotypes were observed, with MASH patients exhibiting lower CD40 ligand expression (p = 0.027) compared to controls and higher CD42b levels compared to those with MASL (p = 0.009). In contrast, PAC1 levels were significantly decreased in MASH patients (p < 0.001). Additionally, soluble glycoprotein V (sGPV) and soluble P-selectin (sP-selectin) levels increased with disease progression (p = 0.004 and p = 0.038, respectively). Multiple linear regression indicated sGPV could be predicted by sP-selectin, sCD40L, and sTLT-1 (adjusted R² = 76%), with sP-selectin (p < 0.0001) and sTLT-1 (p = 0.0140) showing significant associations. Pro-inflammatory cytokines, particularly interleukin (IL)-1β (IL-1β) and IL-6, were elevated in the MASLD spectrum (p < 0.001), along with TNF-α in MASL and MASH (p = 0.011). IL-8 and IL-18 levels were higher in MASH and cirrhosis (p = 0.002 and p = 0.019, respectively), while no significant changes were noted for several other cytokines (p > 0.05). To identify biomarkers for predicting significant fibrosis in MASLD patients, we analyzed 34 patients with F0-1 (non-significant fibrosis) and 28 with F2-4 (significant fibrosis). Notable differences in P-selectin and PAC-1 expression were observed (p = 0.043 and 0.005), while other receptors showed no significant changes (p > 0.05). Elevated sP-selectin, sGPV, and sTLT-1 correlated with significant fibrosis (p = 0.001, 0.002, 0.023). Univariable analysis linked sP-selectin (OR = 1.154, p = 0.021) and sGPV (OR = 1.250, p = 0.038) to significant fibrosis. In multivariable logistic regression analysis, sP-selectin remained significant (OR = 1.200, p = 0.048) after adjusting for comorbidities. ROC analysis showed sP-selectin had an AUC of 0.746 (p = 0.019), indicating strong predictive value for significant fibrosis.
Conclusions: This study highlights the crucial role of platelets in the pathogenesis of liver damage, suggesting their potential as targets for therapeutic interventions and non-invasive diagnostic tests. By exploring both platelet involvement and non-invasive biomarkers, this research enhances our understanding of MASLD and paves the way for more effective diagnostic and treatment strategies.