Emily S Koeck1, Tatiana Iordanskaia1, Samantha Sevilla1, Sarah C Ferrante2, Monica J Hubal3, Robert J Freishtat4, Evan P Nadler5. 1. Sheikh Zayed Institute for Pediatric Surgical Innovation, Children's National Medical Center, Washington, DC. 2. Department of Integrative Systems Biology, George Washington University School of Medicine and Health Sciences, Washington, DC; Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC. 3. Sheikh Zayed Institute for Pediatric Surgical Innovation, Children's National Medical Center, Washington, DC; Department of Integrative Systems Biology, George Washington University School of Medicine and Health Sciences, Washington, DC; Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC. 4. Department of Integrative Systems Biology, George Washington University School of Medicine and Health Sciences, Washington, DC; Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC; Division of Emergency Medicine, Children's National Medical Center, Washington, DC. 5. Sheikh Zayed Institute for Pediatric Surgical Innovation, Children's National Medical Center, Washington, DC; Department of Integrative Systems Biology, George Washington University School of Medicine and Health Sciences, Washington, DC. Electronic address: enadler@childrensnational.org.
Abstract
BACKGROUND: The pathogenesis of nonalcoholic fatty liver disease (NAFLD) has been attributed to increased systemic inflammation and insulin resistance mediated by visceral adipose tissue (VAT), although the exact mechanisms are undefined. Exosomes are membrane-derived vesicles containing messenger RNA, microRNA, and proteins, which have been implicated in cancer, neurodegenerative, and autoimmune diseases, which we postulated may be involved in obesity-related diseases. We isolated exosomes from VAT, characterized their content, and identified their potential targets. Targets included the transforming growth factor beta (TGF-β) pathway, which has been linked to NAFLD. We hypothesized that adipocyte exosomes would integrate into HepG2 and hepatic stellate cell lines and cause dysregulation of the TGF-β pathway. METHODS: Exosomes from VAT from obese and lean patients were isolated and fluorescently labeled, then applied to cultured hepatic cell lines. After incubation, culture slides were imaged to detect exosome uptake. In separate experiments, exosomes were applied to cultured cells and incubated 48-h. Gene expression of TGF-β pathway mediators was analyzed by polymerase chain reaction, and compared with cells, which were not exposed to exosomes. RESULTS: Fluorescent-labeled exosomes integrated into both cell types and deposited in a perinuclear distribution. Exosome exposure caused increased tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and integrin ανβ-5 expression and decreased matrix metalloproteinase-7 and plasminogen activator inhibitor-1 expression in to HepG2 cells and increased expression of TIMP-1, TIMP-4, Smad-3, integrins ανβ-5 and ανβ-8, and matrix metalloproteinase-9 in hepatic stellate cells. CONCLUSIONS: Exosomes from VAT integrate into liver cells and induce dysregulation of TGF-β pathway members in vitro and offers an intriguing possibility for the pathogenesis of NAFLD.
BACKGROUND: The pathogenesis of nonalcoholic fatty liver disease (NAFLD) has been attributed to increased systemic inflammation and insulin resistance mediated by visceral adipose tissue (VAT), although the exact mechanisms are undefined. Exosomes are membrane-derived vesicles containing messenger RNA, microRNA, and proteins, which have been implicated in cancer, neurodegenerative, and autoimmune diseases, which we postulated may be involved in obesity-related diseases. We isolated exosomes from VAT, characterized their content, and identified their potential targets. Targets included the transforming growth factor beta (TGF-β) pathway, which has been linked to NAFLD. We hypothesized that adipocyte exosomes would integrate into HepG2 and hepatic stellate cell lines and cause dysregulation of the TGF-β pathway. METHODS: Exosomes from VAT from obese and lean patients were isolated and fluorescently labeled, then applied to cultured hepatic cell lines. After incubation, culture slides were imaged to detect exosome uptake. In separate experiments, exosomes were applied to cultured cells and incubated 48-h. Gene expression of TGF-β pathway mediators was analyzed by polymerase chain reaction, and compared with cells, which were not exposed to exosomes. RESULTS: Fluorescent-labeled exosomes integrated into both cell types and deposited in a perinuclear distribution. Exosome exposure caused increased tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and integrin ανβ-5 expression and decreased matrix metalloproteinase-7 and plasminogen activator inhibitor-1 expression in to HepG2 cells and increased expression of TIMP-1, TIMP-4, Smad-3, integrins ανβ-5 and ανβ-8, and matrix metalloproteinase-9 in hepatic stellate cells. CONCLUSIONS: Exosomes from VAT integrate into liver cells and induce dysregulation of TGF-β pathway members in vitro and offers an intriguing possibility for the pathogenesis of NAFLD.
Authors: Maleeha F Ahmad; David Ferland; Nadia Ayala-Lopez; G Andres Contreras; Emma Darios; Janice Thompson; Alexander Ismail; Kyan Thelen; Adam J Moeser; Robert Burnett; Arun Anantharam; Stephanie W Watts Journal: Arterioscler Thromb Vasc Biol Date: 2019-02 Impact factor: 8.311
Authors: Marcelo A Mori; Raissa G Ludwig; Ruben Garcia-Martin; Bruna B Brandão; C Ronald Kahn Journal: Cell Metab Date: 2019-08-22 Impact factor: 27.287