Production, Exacerbating Effect, and EV-Mediated Transcription of Hepatic CCN2 in NASH: Implications for Diagnosis and Therapy of NASH Fibrosis

Non-alcoholic steatohepatitis (NASH) is characterized by steatosis, hepatocyte ballooning, and inflammation and may progress to include increasingly severe fibrosis, which portends more serious disease and is predictive of patient mortality. Diagnostic and therapeutic options for NASH fibrosis are limited, and the underlying fibrogenic pathways are under-explored. Cell communication network factor 2 (CCN2) is a well-characterized pro-fibrotic molecule, but its production in and contribution to NASH fibrosis requires further study. Hepatic CCN2 expression was significantly induced in NASH patients with F3–F4 fibrosis and was positively correlated with hepatic Col1A1, Col1A2, Col3A1, or αSMA expression. When wild-type (WT) or transgenic (TG) Swiss mice expressing enhanced green fluorescent protein (EGFP) under the control of the CCN2 promoter were fed up to 7 weeks with control or choline-deficient, amino-acid-defined diet with high (60%) fat (CDAA-HF), the resulting NASH-like hepatic pathology included a profound increase in CCN2 or EGFP immunoreactivity in activated hepatic stellate cells (HSC) and in fibroblasts and smooth muscle cells of the vasculature, with little or no induction of CCN2 in other liver cell types. In the context of CDAA-HF diet-induced NASH, Balb/c TG mice expressing human CCN2 under the control of the albumin promoter exhibited exacerbated deposition of interstitial hepatic collagen and activated HSC compared to WT mice. In vitro, palmitic acid-treated hepatocytes produced extracellular vesicles (EVs) that induced CCN2, Col1A1, and αSMA in HSC. Hepatic CCN2 may aid the assessment of NASH fibrosis severity and, together with pro-fibrogenic EVs, is a therapeutic target for reducing NASH fibrosis

Therapeutic effects of serum extracellular vesicles in liver fibrosis

The lack of approved therapies for hepatic fibrosis seriously limits medical management of patients with chronic liver disease. Since extracellular vesicles (EVs) function as conduits for intercellular molecular transfer, we investigated if EVs from healthy individuals have anti-fibrotic properties. Hepatic fibrogenesis or fibrosis in carbon tetrachloride (CCl4)- or thioacetic acid-induced liver injury models in male or female mice were suppressed by serum EVs from normal mice (EVN) but not from fibrotic mice (EVF). CCl4-treated mice undergoing EVN therapy also exhibited reduced levels of hepatocyte death, inflammatory infiltration, circulating AST/ALT levels and hepatic or circulating pro-inflammatory cytokines. Hepatic histology, liver function tests or circulating proinflammatory cytokine levels were unaltered in control mice receiving EVN. As determined using PKH26-labelled EVN, principal target cells included hepatic stellate cells (HSC; a normally quiescent fibroblastic cell that undergoes injury-induced activation and produces fibrosis during chronic injury) or hepatocytes which showed increased EVN binding after, respectively, activation or exposure to CCl4. In vitro, EVN decreased proliferation and fibrosis-associated molecule expression in activated HSC, while reversing the inhibitory effects of CCl4 or ethanol on hepatocyte proliferation. In mice, microRNA-34c, -151-3p, -483-5p, -532-5p and -687 were more highly expressed in EVN than EVF and mimics of these microRNAs (miRs) individually suppressed fibrogenic gene expression in activated HSC. A role for these miRs in contributing to EVN actions was shown by the ability of their corresponding antagomirs to individually and/or collectively block the therapeutic effects of EVN on activated HSC or injured hepatocytes. Similarly, the activated phenotype of human LX-2 HSC was attenuated by serum EVs from healthy human subjects and contained higher miR-34c, -151-3p, -483-5p or -532-5p than EVs from hepatic fibrosis patients. In conclusion, serum EVs from normal healthy individuals are inherently anti-fibrogenic and anti-fibrotic, and contain microRNAs that have therapeutic actions in activated HSC or injured hepatocytes. Abbreviations: ALT: alanine aminotransferase; AST: aspartate aminotransferase; CCl4: carbon tetrachloride; CCN2: connective tissue growth factor; E: eosin; EGFP: enhanced green fluorescent protein; EVs: extracellular vesicles; EVF: serum EVs from mice with experimental hepatic fibrosis; EVN: serum EVs from normal mice; H: hematoxylin; HSC: hepatic stellate cell; IHC: immunohistochemistry; IL: interleukin; MCP-1: monocyte chemotactic protein-1; miR: microRNA; mRNA: messenger RNA; NTA: nanoparticle tracking analysis; PCNA: proliferating cell nuclear antigen; qRT-PCR: quantitative real-time polymerase chain reaction; SDS-PAGE: sodium dodecyl sulphate – polyacrylamide gel electrophoresis; αSMA: alpha smooth muscle actin; TAA: thioacetic acid; TG: transgenic; TGF-β: transforming growth factor beta; TEM: transmission electron microscopy; TNFα: tumour necrosis factor alpha

Factors Associated With Surveillance Testing in Individuals With COVID-19 Symptoms During the Last Leg of the Pandemic: Multivariable Regression Analysis

Abstract BackgroundRural underserved areas facing health disparities have unequal access to health resources. By the third and fourth waves of SARS-CoV-2 infections in the United States, COVID-19 testing had reduced, with more reliance on home testing, and those seeking testing were mostly symptomatic. ObjectiveThis study identifies factors associated with COVID-19 testing among individuals who were symptomatic versus asymptomatic seen at a Rapid Acceleration of Diagnostics for Underserved Populations phase 2 (RADx-UP2) testing site in West Virginia. MethodsDemographic, clinical, and behavioral factors were collected via survey from tested individuals. Logistic regression was used to identify factors associated with the presence of individuals who were symptomatic seen at testing sites. Global tests for spatial autocorrelation were conducted to examine clustering in the proportion of symptomatic to total individuals tested by zip code. Bivariate maps were created to display geographic distributions between higher proportions of tested individuals who were symptomatic and social determinants of health. ResultsAmong predictors, the presence of a physical (adjusted odds ratio [aOR] 1.85, 95% CI 1.3-2.65) or mental (aOR 1.53, 95% CI 0.96-2.48) comorbid condition, challenges related to a place to stay/live (aOR 307.13, 95% CI 1.46-10,6372), no community socioeconomic distress (aOR 0.99, 95% CI 0.98-1.00), no challenges in getting needed medicine (aOR 0.01, 95% CI 0.00-0.82) or transportation (aOR 0.23, 95% CI 0.05-0.64), an interaction between community socioeconomic distress and not getting needed medicine (aOR 1.06, 95% CI 1.00-1.13), and having no community socioeconomic distress while not facing challenges related to a place to stay/live (aOR 0.93, 95% CI 0.87-0.99) were statistically associated with an individual being symptomatic at the first test visit. ConclusionsThis study addresses critical limitations to the current COVID-19 testing literature, which almost exclusively uses population-level disease screening data to inform public health responses