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Background:Obesity is frequently complicated by comorbid conditions, yet how excess adipose contributes is poorly understood. Although adipocytes in obese individuals induce systemic inflammation via secreted cytokines, another potential mediator has recently been identified (i.e., adipocyte-derived exosomes). We hypothesized that adipocyte-derived exosomes contain mediators capable of activating end-organ inflammatory and fibrotic signaling pathways.Methods:We developed techniques to quantify and characterize exosomes shed by adipocytes from seven obese (age: 12–17.5 y, BMI: 33–50?kg/m2) and five lean (age: 11–19 y, BMI: 22–25?kg/m2) subjects.Results:Abundant exosomal miRNAs, but no mRNAs, were detected. Comparison of obese vs. lean visceral adipose donors detected 55 differentially expressed miRNAs (P < 0.05; fold change =|1.2|). qRT-PCR confirmed downregulation of miR-148b (ratio = 0.2 (95% confidence interval = 0.1, 0.6)) and miR-4269 (0.3 (0.1, 0.8)), and upregulation of miR-23b (6.2 (2.2, 17.8)) and miR-4429 (3.8 (1.1-13.4)). Pathways analysis identified TGF-ß signaling and Wnt/ß-catenin signaling among the top canonical pathways expected to be altered with visceral adiposity based on projected mRNA targets for the 55 differentially expressed miRNAs. A select mRNA target was validated in vitro.Conclusion:These data show that visceral adipocytes shed exosomal-mediators predicted to regulate key end-organ inflammatory and fibrotic signaling pathways.

Our previous work demonstrated altered messenger RNA expression of integrin ß-5 and -8, using an in silico analysis of publically available data from patients with biliary atresia (BA); however, we were unable to demonstrate statistically significant differences in protein expression because of sample size. In the present study, we repeated the analysis of liver fibrosis and protein expression of the integrins in a larger cohort of patients with BA and compared them with patients undergoing liver biopsy for other diagnoses, with the hypothesis that =1 of the integrins would be differentially expressed. Liver specimens were obtained at 2 collaborating institutions. Samples from infants with BA (n = 23) were compared with samples from those who underwent liver biopsy for neonatal hepatitis (n = 9). All of the specimens were analyzed by 2 pathologists (C.R. and R.A.), who were blinded to the diagnoses. Standard Ishak scoring was performed to evaluate fibrosis and inflammation, and immunohistochemical (IHC) positivity was graded from 0 to 4. Comparisons between the IHC positivity and Ishak scoring for the BA and control groups were performed using the Student ttest with P< 0.01 considered significant because of the multiple comparisons. Interobserver variability was assessed by intraclass correlation (ICC). Pooled analysis from specimens from patients with BA showed significantly more fibrosis than controls based on Ishak scores (3.21 ± 1.82 vs 1.17 ± 1.00, P< 0.005). IHC evaluation showed increased integrin a?ß8 protein expression when compared with controls (2.67 ± 0.81 vs 1.72 ± 0.62, P< 0.005); however, there were no significant differences in integrin a?ß5 (1.93 ± 0.84 vs 1.50 ± 0.90, P= 0.23) or integrin a?ß6 (0.85 ± 1.20 vs 0.94 ± 0.85, P= 0.82) expression. These data were confirmed on individual analysis. Interobserver agreement was fair for integrin a?ß5 (ICC 0.52), good for integrin a?ß6 (ICC 0.72), and excellent for integrin a?ß8 (ICC 0.79) and fibrosis (ICC 0.89). Our data show that integrin a?ß8, but not integrin a?ß5 or integrin a?ß6, protein expression is increased in liver specimens of patients with BA. These data support the mounting evidence that transforming growth factor-ß (TGF-ß) activation is responsible for the fibrosis found in BA. Anti-integrin a?ß8 or more global integrin blocking strategies may be therapeutic options in BA, but further work is clearly needed.

Our previous work demonstrated altered messenger RNA expression of integrin -5 and -8, using an in silico analysis of publically available data from patients with biliary atresia (BA); however, we were unable to demonstrate statistically significant differences in protein expression because of sample size. In the present study, we repeated the analysis of liver fibrosis and protein expression of the integrins in a larger cohort of patients with BA and compared them with patients undergoing liver biopsy for other diagnoses, with the hypothesis that ≥1 of the integrins would be differentially expressed.