The Ameliorative Effect of C-Kit (+) Hepatic Endothelial Cells With Mertk Deficiency on Nonalcoholic Steatohepatitis

The prevalence of non-alcoholic fatty liver disease (NAFLD) has been steadily increasing, with 10-20% of affected individuals progressing to non-alcoholic steatohepatitis (NASH). NASH is pathologically characterized by hepatic inflammation, steatosis, and hepatocyte injury. Furthermore, this condition carries a significant risk of progression to advanced hepatic fibrosis (pathological Scheuer fibrosis stage F≥3), cirrhosis, and even hepatocellular carcinoma (HCC). In recent years, NASH has emerged as the leading contributor to the growing burden of cirrhosis worldwide, representing a major public health challenge. Despite the high incidence and clinical severity of NAFLD, there are currently no FDA-approved therapeutic agents for its management. Therefore, elucidating the molecular mechanisms underlying NAFLD-associated NASH progression is critical for developing targeted pharmacological interventions capable of preventing, ameliorating, or potentially reversing disease progression.

Study Overview

• Status: Active, not recruiting
• Conditions: Non-alcoholic Fatty Liver Disease (NAFLD)
• Intervention / Treatment: Procedure: Ultrasonund-guided percutaneous liver biopsy

Detailed Description

I. Study Background Non-alcoholic fatty liver disease (NAFLD) has become one of the most prevalent liver disorders globally. Despite its high incidence and severity, no effective therapeutic agents currently exist. Existing treatments focus on symptomatic relief, such as hepatoprotection and enzyme reduction, rather than addressing the underlying pathogenesis. Therefore, elucidating the mechanisms driving the progression of NAFLD-related non-alcoholic steatohepatitis (NASH) and hepatic fibrosis is critical for developing preventive, ameliorative, or even curative strategies, holding significant strategic importance.

1. Role of LSECs in NAFLD Pathogenesis:

   Liver sinusoidal endothelial cells (LSECs), the primary components of the hepatic sinusoidal endothelium, form a physical barrier regulating substance exchange between the hepatic parenchyma and circulation. As highly specialized endothelial cells (ECs) and the most abundant non-parenchymal cells (NPCs) in the liver, LSECs play pivotal roles in hepatic pathophysiology. Targeting LSEC function represents a promising approach to mitigate NAFLD progression and complications.

2. Heterogeneous Transcriptomic Features of LSEC Subpopulations in NAFLD:

   Single-cell RNA sequencing (scRNA-seq) enables genome-wide amplification and sequencing at the single-cell level. In the liver, hepatocytes (HCs) and NPCs exhibit marked transcriptomic heterogeneity, underpinning metabolic and functional zonation. This highlights the potential of single-cell analysis in understanding liver diseases. However, scRNA-seq studies on LSECs in NAFLD remain unexplored.

3. Hypothesis: C-Kit(+)/Gas6(-)-LSEC Subpopulation Exacerbates NAFLD via Gas6/Mertk-Mediated Regulation of PINK1 Mitophagy:

Bone marrow-derived endothelial progenitor cells (EPCs), marked by C-Kit (CD117), may express C-Kit in ECs under pathological conditions. Gas6, a ligand for TAM receptors (Tyro3, Axl, Mertk), activates downstream signaling cascades regulating cell migration, inflammation, survival, and mitophagy. In NAFLD, Gas6/Mertk exhibits cell-specific roles : Anti-inflammatory in hepatic macrophages; Pro-fibrotic in hepatic stellate cells (HSCs); Protective in hepatocytes (HCs). However, the role of Gas6/Mertk in LSECs during NAFLD remains unknown. Mitochondrial dysfunction is a hallmark of NAFLD, and impaired mitophagy (e.g., PINK1 deficiency) exacerbates hepatic inflammation, steatosis, and fibrosis. Emerging evidence suggests Gas6/Mertk enhances mitophagy to preserve mitochondrial function. For instance, Mertk-/- mice exhibit cardiomyocyte mitochondrial dysfunction, and Gas6 promotes mitophagy.

Our preliminary scRNA-seq data from HFD/ND mice identified a unique C-Kit(+)/Gas6(-)-LSEC subpopulation. These LSECs induced mitophagy impairment in co-cultured HCs/HSCs, manifesting as mitochondrial senescence, oxidative stress, HSC activation, HC lipid deposition, and inflammation. Investigating how this subpopulation drives NAFLD progression will unveil novel pathophysiological mechanisms and therapeutic targets, forming the basis of this study.

II. Study Objectives To elucidate the expression levels of C-Kit and Gas6/Mertk ligand-receptor pairs in liver tissues of NAFLD patients.

III. Study Procedures

(1) Inclusion and Exclusion Criteria

1. Inclusion Criteria: NAFLD diagnosis per Guidelines for the Prevention and Treatment of Non-Alcoholic Fatty Liver Disease (2018 Edition). Age 18-65 years.
2. Exclusion Criteria: Cirrhosis (compensated or decompensated), hepatocellular carcinoma, active viral hepatitis (HBV, HCV), primary biliary cholangitis, primary sclerosing cholangitis, recent history of illicit drug use, alcohol intake≥20 g/day for women and ≥30 g/day for men, current pregnancy or breastfeeding, severe comorbidities.

(2) Study Design

1. Liver Biopsy and Tissue Collection:

   Ultrasound-guided liver biopsy in 6 NAFLD patients. Histopathological evaluation (HE, Masson staining) for NASH and fibrosis (NAS and Scheuer scoring). Clinical data collection and liver tissue storage (-80°C).

2. Expression Analysis of C-Kit and Mertk:

Immunofluorescence co-staining of liver tissues. 3. Statistical Analysis: Compare C-Kit and Mertk expression levels in mild and severe NAFLD patients.

(3) Enrollment and Workflow

1. Baseline Data Collection:

   ①Demographic and clinical parameters: name, sex, age, ethnicity, birthdate, height, weight, disease onset, contact information.

   ② Laboratory tests: CBC, ALT, AST, TBIL, TG, cholesterol, glucose, total protein, albumin.

   ③Clinical assessments: blood pressure, BMI, abdominal ultrasound, FibroScan.

2. Biopsy Protocol:

   • Ultrasound-guided percutaneous liver biopsy within 1 month of enrollment. ②Tissue specimen requirements: ≥3 mm diameter, 5-8 mm length. ③Storage: -80°C for C-Kit and Mertk gene analysis.

IV. Expected Outcomes Clarify the regulatory relationship between C-Kit (+) LSECs and the Gas6-Mertk signaling pathway in NAFLD patient liver tissues.

• Study Type: Observational
• Enrollment (Estimated): 6

Contacts and Locations

Study Locations

• China
  • Shanghai
    • Shanghai, Shanghai, China, 200120
      • Shanghai East Hospital (East Hospital Affiliated To Tongji University)

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: No

• Sampling Method: Probability Sample

Study Population

The study population comprised adults with NAFLD confirmed by imaging (ultrasound, CT, or MRI), excluding secondary causes (e.g., alcohol consumption >30/20 g/day for men/women, viral hepatitis, or steatogenic medications). . Exclusion criteria included Cirrhosis (compensated or decompensated), hepatocellular carcinoma, primary biliary cholangitis, primary sclerosing cholangitis, recent history of illicit drug use, current pregnancy or breastfeeding, severe comorbidities. NAFLD severity was categorized using non-invasive fibrosis scores (NFS) or liver biopsy (steatosis, inflammation, fibrosis staging).

Description

Inclusion Criteria:

• Clinical diagnosis of Non-Alcoholic Fatty Liver Disease
• Age 18-65 years

Exclusion Criteria:

• Cirrhosis (compensated or decompensated)
• Hepatocellular carcinoma
• Active viral hepatitis (HBV, HCV)
• Primary biliary cholangitis
• Primary sclerosing cholangitis
• Recent history of illicit drug use
• Alcohol intake≥20 g/day for women and ≥30 g/day for men
• Current pregnancy or breastfeeding
• Severe comorbidities.

Study Plan

How is the study designed?

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Mertk expression levels of liver tissues	Liver Biopsy and Tissue Collection:Ultrasound-guided liver biopsy in 6 NAFLD patients. Histopathological evaluation (HE, Masson staining) for NASH and fibrosis (NAS and Scheuer scoring). Clinical data collection and liver tissue storage (-80°C).; Expression Analysis of Mertk:Immunofluorescence staining of liver tissues.; Statistical Analysis: Compare Mertk expression levels between mild and severe NAFLD patients.	From enrollment to the end of treatment at 4 weeks
C-Kit expression levels of liver tissues	Liver Biopsy and Tissue Collection:Ultrasound-guided liver biopsy in 6 NAFLD patients. Histopathological evaluation (HE, Masson staining) for NASH and fibrosis (NAS and Scheuer scoring). Clinical data collection and liver tissue storage (-80°C).; Expression Analysis of C-Kit:Immunofluorescence staining of liver tissues.; Statistical Analysis: Compare C-Kit expression levels between mild and severe NAFLD patients.	From enrollment to the end of treatment at 4 weeks

Collaborators and Investigators

• Sponsor: Shanghai East Hospital
• Collaborators: National Natural Science Foundation of China

Study record dates

Study Major Dates

• Study Start (Actual): April 1, 2024
• Primary Completion (Estimated): April 30, 2026
• Study Completion (Estimated): May 31, 2026

Study Registration Dates

• First Submitted: March 30, 2025
• First Submitted That Met QC Criteria: March 30, 2025
• First Posted (Actual): April 6, 2025

Study Record Updates

• Last Update Posted (Actual): April 8, 2025
• Last Update Submitted That Met QC Criteria: April 6, 2025
• Last Verified: March 1, 2025

More Information

Terms related to this study

• Additional Relevant MeSH Terms: Digestive System Diseases; Liver Diseases; Fatty Liver; Non-alcoholic Fatty Liver Disease
• Other Study ID Numbers: Approval No.2024YS-050

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No