Given the important role of angiogenesis in liver pathology, the current study investigated the role of Runt-related transcription factor 1 (RUNX1), a regulator of developmental angiogenesis, in the pathogenesis of non-alcoholic steatohepatitis (NASH). Quantitative RT-PCRs and a transcription factor analysis of angiogenesis-associated differentially expressed genes in liver tissues of healthy controls, patients with steatosis and NASH, indicated a potential role of RUNX1 in NASH. The gene expression of RUNX1 was correlated with histopathological attributes of patients. The protein expression of RUNX1 in liver was studied by immunohistochemistry. To explore the underlying mechanisms, in vitro studies using RUNX1 siRNA and overexpression plasmids were performed in endothelial cells (ECs). RUNX1 expression was significantly correlated with inflammation, fibrosis and NASH activity score in NASH patients. Its expression was conspicuous in liver non-parenchymal cells. In vitro, factors from steatotic hepatocytes and/or VEGF or TGF-β significantly induced the expression of RUNX1 in ECs. RUNX1 regulated the expression of angiogenic and adhesion molecules in ECs, including CCL2, PECAM1 and VCAM1, which was shown by silencing or over-expression of RUNX1. Furthermore, RUNX1 increased the angiogenic activity of ECs. This study reports that steatosis-induced RUNX1 augmented the expression of adhesion and angiogenic molecules and properties in ECs and may be involved in enhancing inflammation and disease severity in NASH.
Background: Runt-related transcription factor (RUNX1) regulates inflammation in non-alcoholic steatohepatitis (NASH). Methods: We performed in vivo targeted silencing of the RUNX1 gene in liver sinusoidal endothelial cells (LSECs) by using vegfr3 antibody tagged immunonano-lipocarriers encapsulated RUNX1 siRNA (RUNX1 siRNA) in murine models of methionine choline deficient (MCD) diet-induced NASH. MCD mice given nanolipocarriers-encapsulated negative siRNA were vehicle, and mice with standard diet were controls. Results: Liver RUNX1 expression was increased in the LSECs of MCD mice in comparison to controls. RUNX1 protein expression was decreased by 40% in CD31-positive LSECs of RUNX1 siRNA mice in comparison to vehicle, resulting in the downregulation of adhesion molecules, ICAM1 expression, and VCAM1 expression in LSECs. There was a marked decrease in infiltrated T cells and myeloid cells along with reduced inflammatory cytokines in the liver of RUNX1 siRNA mice as compared to that observed in the vehicle. Conclusions: In vivo LSEC-specific silencing of RUNX1 using immunonano-lipocarriers encapsulated siRNA effectively reduces its expression of adhesion molecules, infiltrate on of immune cells in liver, and inflammation in NASH.
Studies have demonstrated that aging is associated with a substantial decline in numbers and angiogenic activity of endothelial progenitor cells (EPCs). In view of senescence being an important regulator of age-related cell survival and function, in the current study, we correlated EPCs numbers and functions with their senescence status and mechanisms in young and elderly subjects. Healthy young subjects (n = 30, below 60 y) and old subjects (n = 30, equal to or above 60 y) participated in the study. Subjects had no significant disease or risk factors of disease and aging was the only risk factor in the aged subjects. Enumeration of CD34-vegfr2 dual positive EPCs was performed. The ex vivo culture of EPCs was done to study colony formation, migration, and senescence-associated beta-galactosidase activity. The expression of cell cycle and senescence regulatory proteins including, p53, p21, and sirtuin 1 (SIRT1), a deacetylase protein was studied in cultured EPCs by RT-PCR and immunofluorescence staining. In vivo proliferation, ex vivo colonies, migration, and secretory ability of EPCs was significantly higher in young subjects than that in elderly subjects. EPCs in old subjects showed enhanced senescence and decreased expression of SIRT1 in comparison to that observed in young subjects. An inhibition of SIRT1 in EPCs of young subjects led to significant increase in senescence and reduction of cell differentiation. The study suggests that EPCs have decreased proliferation and functions in aged subjects due to increased senescence which may be attributable to decreased expression of SIRT1.
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