Altered liver sinusoidal endothelial cells in MASLD and their evolution following lanifibranor treatment

Abstract
Background and aims: The landscape of metabolic dysfunction-associated steatotic liver disease (MASLD), a burgeoning global health concern, is characterized by a complex interplay of metabolic dysregulation and liver pathology. Within this intricate context, comprehensive data regarding alterations in liver sinusoidal endothelial cells (LSECs), which are specialized endothelial cells lining the liver sinusoids, in patients afflicted with MASLD, and importantly, their responsiveness to therapeutic interventions, remain notably limited. Given the critical role of LSECs in maintaining hepatic microcirculation and liver function, understanding their changes in MASLD is paramount. Consequently, this study was meticulously designed with several key objectives: firstly, to precisely identify and characterize the specific features associated with LSEC capillarization in patients diagnosed with MASLD, a process where LSECs lose their fenestrations and become more like typical capillaries; secondly, to rigorously investigate whether these observed LSEC alterations are capable of regression following treatment with lanifibranor, a pan-peroxisome proliferator-activated receptor (PPAR) agonist; and thirdly, to delineate the specific and differential roles of the various PPAR isotypes (alpha, delta, and gamma) in mediating the observed LSEC changes within the context of MASLD.

Methods: To achieve these multifaceted aims, a dual-pronged approach combining human clinical data with robust preclinical animal models was employed. For the human component, liver biopsies were meticulously analyzed from a cohort of patients who were being considered for inclusion in the NATIVE clinical trial. These biopsies were collected at two critical time points: at baseline (from a total of 249 patients), providing a snapshot of LSEC status prior to intervention, and subsequently after a 24-week treatment period (from a subset of 173 patients) during which they received either placebo or lanifibranor. CD34 expression, a well-established immunohistochemical marker indicative of LSEC capillarization, was quantitatively assessed on these biopsy samples. For the preclinical investigation, two distinct rat models of MASLD were utilized. These animal models served as invaluable tools to systematically investigate not only the effects of lanifibranor on LSECs but also, more granularly, the specific contributions and mechanisms of action of individual mono-PPAR agonists on LSEC changes.

Results: The analysis of lobular CD34 staining, a direct indicator of LSEC capillarization within the liver lobules, revealed significant findings even at the earliest stages of MASLD. Specifically, lobular CD34 staining was found to be more intense in patients diagnosed with isolated steatosis (52% positive staining) compared to individuals with no MASLD (10% positive staining), with this difference being statistically significant (p = 0.03). This suggests that LSEC capillarization is an early event in the pathogenesis of MASLD. In the overall patient cohort, CD34 staining was notably more intense in patients with metabolic dysfunction-associated steatohepatitis (MASH) (63% positive staining) than in those without MASH (41% positive staining), a difference that was also statistically significant (p = 0.01). Furthermore, the intensity of CD34 staining demonstrated a strong positive correlation with the degree of liver fibrosis, indicating that LSEC capillarization worsens with advancing scarring of the liver. To a lesser but still significant extent, CD34 staining also correlated with the severity of liver inflammation.

The therapeutic intervention with lanifibranor yielded encouraging results. Treatment with lanifibranor was significantly associated with a more common improvement in CD34 periportal staining (p = 0.025), suggesting a positive effect on LSEC phenotype in the portal areas of the liver. Moreover, lanifibranor treatment led to less frequent worsening of lobular CD34 staining (p = 0.028), indicating a protective or ameliorative effect on LSEC capillarization within the lobules. Preclinical studies using rat models of MASLD further corroborated and expanded upon these human observations. Compared to healthy control rats, rats with MASLD exhibited significantly higher CD34 staining, reflecting increased capillarization. Concomitantly, these MASLD rats displayed elevated portal venous pressure and increased intrahepatic vascular resistance, both hallmarks of liver microcirculatory dysfunction, along with impaired liver endothelial function. Critically, treatment with lanifibranor in these MASLD rat models normalized or substantially improved all these abnormalities, including CD34 staining, portal venous pressure, intrahepatic vascular resistance, and endothelial function. In contrast, while individual mono-PPAR agonists did induce partial improvements in these parameters, their effects were not as comprehensive or robust as those observed with the pan-PPAR agonist lanifibranor, suggesting a synergistic or more complete therapeutic action of activating all three PPAR isotypes.

Conclusions: In patients with MASLD, the extent of LSEC capillarization, as indicated by CD34 expression, was already increased at the earliest stages of the disease, specifically at the stage of isolated steatosis, and progressively worsened with the severity of liver fibrosis and, to a lesser degree, with liver inflammation. Crucially, the findings from both human patients and preclinical rat models afflicted with MASLD consistently demonstrated that treatment with lanifibranor, the pan-PPAR agonist, was significantly associated with a marked improvement in the liver endothelial phenotype, indicating a reversal or amelioration of LSEC capillarization and associated vascular dysfunction.

Impact and implications: The current body of knowledge regarding changes in liver sinusoidal endothelial cells (LSECs) in patients with metabolic dysfunction-associated steatotic liver disease (MASLD) and, critically, their responsiveness to therapeutic interventions, has been notably limited. This study addresses a significant gap in this understanding by providing robust evidence regarding the dynamics of LSEC alterations in MASLD. A key finding of this research is the unequivocal demonstration that LSEC capillarization is an early pathological event, already present in both the lobular zone of the liver of human patients and in rat models at the stage of isolated steatosis, even before the overt onset of metabolic dysfunction-associated steatohepatitis (MASH). Furthermore, this capillarization process is shown to progress significantly with advancing liver fibrosis and, to a lesser but still important extent, with the severity of liver inflammation, underscoring its role in disease progression. The study also highlights the promising therapeutic potential of lanifibranor, a pan-peroxisome proliferator-activated receptor agonist that is currently undergoing evaluation in a pivotal Phase III clinical trial for MASLD. Treatment with lanifibranor was shown to notably improve LSEC capillarization, signifying a beneficial impact on endothelial health. Beyond this, lanifibranor also effectively mitigated associated intrahepatic vascular resistance and reduced portal pressure in both MASLD patients and animal models. These comprehensive improvements in LSEC phenotype and hepatic hemodynamics strongly suggest that directly targeting LSECs through agents like lanifibranor appears to be a highly promising and potentially transformative therapeutic approach for improving outcomes in patients suffering from MASH. This novel focus on endothelial dysfunction opens up new avenues for drug development in the challenging field of liver disease.

Keywords
CD34; Intrahepatic vascular resistance; LSECs; Liver fibrosis; Liver inflammation; MASH; PPAR; Vascular biology; lanifibranor.

Copyright
The authors retain the full copyright to this work, published in 2025.

Conflict of interest statement
P-ER has disclosed receiving research funding from Terrafirma and acting as a consultant for Mursla, Genfit, Boehringer Ingelheim, Cook, Jazz, and Abbelight. Additionally, P-ER has received speaker fees from AbbVie. SMF has a comprehensive list of disclosures, having served as a lecturer for AbbVie, Allergan, Bayer, Eisai, Genfit, Gilead Sciences, Janssens Cilag, Intercept, Inventiva, Merck Sharp & Dome, Novo Nordisk, Promethera, and Siemens. He has acted as a consultant for a wide array of companies, including AbbVie, Actelion, Aelin Therapeutics, AgomAb, Aligos Therapeutics, Allergan, Astellas, Astra Zeneca, Bayer, Boehringer Ingelheim, Bristoll-Meyers Squibb, CSL Behring, Coherus, Echosens, Eisai, Enyo, Galapagos, Galmed, Genetech, Genfit, Genflow Bio, Gilead Sciences, Intercept, Inventiva, Janssens Pharmaceutica, Julius Clinical, Madrigal, Medimmune, Merck Sharp & Dome, NGM Bio, Novartis, Novo Nordisk, PRO.MED.CS, Promethera, and Roche. Furthermore, his institution has received grants from Astellas, Falk Pharma, Genfit, Gilead Sciences, GlympsBio, Janssens Pharmaceutica, Inventiva, Merck Sharp & Dome, Pfizer, and Roche. WJK has received lecturer fees for the PanNASH initiative and has been granted travel support from Ipsen and Norgine. He is also a co-inventor of a patent related to the use of lipopigment imaging for disease, which was filed by MGH/MIT (US 20190307390). MFA has disclosed acting as an advisor for 89Bio, Boehringer Ingelheim, Hanmi, Intercept, Inventiva, Madrigal, and Novo Nordisk. She has received grants (paid directly to her institution) from 89Bio, Akero, Hamni, Inventiva, Madrigal, and Novo Nordisk. Additionally, MFA has served as a speaker for MedScape, the Chronic Liver Disease Foundation, Clinical Care Options, and Fishawack, Inc. For a complete and comprehensive understanding of all potential conflicts of interest, readers are directed to consult the accompanying ICMJE disclosure forms provided by the authors.