MicroRNA‐34a Knockout Attenuates Endothelial Progenitor Dysfunction in Cholestatic Liver Injury

Abstract

BackgroundThe noncoding miRNA‐34a (miR‐34a) is involved in the pathogenesis of chronic liver disease and shows potential for application as a biomarker for early diagnosis and intervention. CD34+ endothelial progenitor cells (EPCs) have angiogenic potentials contributing to neovascularization within ischemic sites or to vascular repair after vessel wall injury. However, a clear mechanism of endothelial progenitor dysfunction in chronic liver disorders still remains elusive. The objective of current study is to define the microRNA regulated endothelial progenitor dysfunction during cholestatic liver injury.MethodsBile duct ligation (BDL) and miR‐34a knockout mice were used as the animal models. CD34+ cells were isolated from mouse liver using laser capture microdissection (LCM). A capture probe covalently bound to an oligonucleotide containing biotin and a color‐coded reporter probe were designed for 84 endothelial function‐related genes and analyzed with the nCounter Single Cell Gene Expression Assay. The mediators of endothelial injury were defined in BDL/miR‐34a knockout mice in vivo and cultured human liver sinusoidal endothelial cells (HLSECs) with miR‐34a modifications in vitro by real‐time PCR assay, immunohistochemistry and Western blot analysis.ResultsUsing BDL mouse model of cholestatic liver injury, results showed the significant increase in serum ALT, hepatic CD34 and miR‐34a, severe inflammation, pericellular fibrosis, and intensified nitrosative stress induced by a 16‐fold induction of nitric oxide synthase (NOS3). In CD34+ cells isolated from BDL mice liver by LCM, nCounter single cell analysis demonstrated the enhanced expressions of sinusoidal endothelial dysfunction (SED) markers ICAM1, endothelial marker vWF as well as the inflammatory cytokines TNFα, CCl2, IL‐1β, IFNβ and IL‐7. The upregulation of miR‐34a in HLSECs led to a time‐dependent repression of its target protein Sirt1 levels as shown by western blot analysis, and a significant increase of NOS3. SED marker ICAM‐1 and endothelial marker vWF were significantly up‐regulated in the progressive phases of CLI. Lack of miR‐34a in vivo reversed the serum ALT level, and restored the levels of Sirt1 coupled with decreased NOS3 expression as well as the reduced levels of TNFα, CCl2, IL‐1β, IFNβ and IL‐7 in LCM isolated CD34+ cells analyzed by nCounter single cell gene expression assay. Depletion of miR‐34a in vivo also induced a significant down‐regulation of profibrogenic genes such as α‐SMA, collagen A1 and MMPs in total liver tissues and LCM isolated CD34+ cells by single cell gene assay from BDL mice liver, along with significantly reduced Sirius red staining and liver angiogenesis.ConclusionOur discovery that CD34 associated endothelial progenitor dysfunction is regulated by miR‐34a during cholestatic liver injury implicates an exciting field in which the epigenomic microRNAs of endothelial progenitor cells may be manipulated with potential therapeutic benefits for the patients with chronic liver diseases.Support or Funding InformationNIH/NIDDK R01 award and VA Merit Review Award