Kang, K. W. | Search bioRxiv

bioRxiv2021

Disease-specific eQTL screening reveals an anti-fibrotic effect of AGXT2 in nonalcoholic fatty liver disease

Background & AimsNonalcoholic fatty liver disease (NAFLD) poses an impending clinical burden. Genome-wide association studies have revealed a limited contribution of genomic variants to the disease, requiring alternative but robust approaches to identify disease-associated variants and genes. We carried out a disease-specific expression quantitative trait loci (eQTL) screen to identify novel genetic factors that specifically act on NAFLD progression on the basis of genotype. MethodsWe recruited 125 Korean biopsy-proven NAFLD patients and healthy individuals and performed eQTL analyses using 21,272 transcripts and 3,234,941 genotyped and imputed SNPs. We then selected eQTLs that were detected only in the NAFLD group, but not in the control group (i.e., NAFLD-eQTLs). An additional cohort of 162 Korean NAFLD individuals was used for replication. The function of the selected eQTL toward NAFLD development was validated using HepG2, primary hepatocytes and NAFLD mouse models. ResultsThe NAFLD-specific eQTL screening yielded 242 loci. Among them, AGXT2, encoding alanine-glyoxylate aminotransferase 2, displayed decreased expression in NAFLD patients homozygous for the non-reference allele of rs2291702, compared to no-NAFLD subjects with the same genotype (P = 4.79 x 10-6). This change was replicated in an additional 162 individuals, yielding a combined P-value of 8.05 x 10-8 from a total of 245 NAFLD patients and 48 controls. Knockdown of AGXT2 induced palmitate-overloaded hepatocyte death by increasing ER stress, and exacerbated NAFLD diet-induced liver fibrosis in mice. However, overexpression of AGXT2 reversely attenuated liver fibrosis and steatosis as well. ConclusionsWe implicate a new molecular role of AGXT2 in NAFLD. Our overall approach will serve as an efficient tool for uncovering novel genetic factors that contribute to liver steatosis and fibrosis in patients with NAFLD. Lay summaryElucidating causal genes for NAFLD has been challenging due to limited tissue availability and the polygenic nature of the disease. Using liver and blood samples from 125 biopsy-proven NAFLD and no-NAFLD Korean individuals and an additional 162 individuals for replication, we devised a new analytic method to identify causal genes. Among the candidates, we found that AGXT2-rs2291702 protects against liver fibrosis in a genotype-dependent manner with the potential for therapeutic interventions. Our approach enables the discovery of NAFLD causal genes that act on the basis of genotype.

genetics↗

bioRxiv2020

Highly Sensitive Identification of Lymphatic and Hematogenous Metastasis Routes of Novel Radiolabeled Exosomes Using Non-invasive PET Imaging

Clinically, there has been significant interest in the use of exosomes for diagnostic applications as promising biomarkers and therapeutic applications as therapeutic vehicles. However, knowledge of in vivo physiological biodistribution of exosomes was difficult to assess until now. Physiological distribution of exosomes in the body must be elucidated for clinical application. In this study, we aimed to develop reliable and novel methods to monitor biodistribution of exosomes using in vivo PET and optical imaging. MethodsExosomes were isolated from cultured medium of 4T1, mouse breast cancer cells. Exosomes were labeled with Cy7 and 64Cu (or 68Ga). In mice, radio/fluorescent dye-labeled exosomes were injected through the lymphatic routes (footpad injection) and hematogenous metastatic routes (tail vein injection). Fluorescence and PET images were obtained and quantified. Radio-activity of ex vivo organs was measured by gamma counter. ResultsPET signals from exosomes in the lymphatic metastatic route were observed in the draining lymph nodes, which are not distinguishable with optical imaging. Immunohistochemistry revealed greater uptake of exosomes in brachial and axillary lymph nodes than inguinal lymph node. After administration through the hematogenous metastasis pathway, accumulation of exosomes was clearly observed in PET images in the lungs, liver, and spleen, showing results similar to ex vivo gamma counter data. ConclusionExosomes from tumor cells were successfully labeled with 64Cu (or 68Ga) and visualized by PET imaging. These results suggest that this cell type-independent, quick, and easy exosome labeling method using PET isotopes could provide valuable information for further application of exosomes in the clinic.

cancer biology↗