Adipocyte-derived exosomal miRNAs: a novel mechanism for obesity-related disease.

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/m(2)) and five lean (age: 11-19 y, BMI: 22-25 kg/m(2)) 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.

Adipocyte exosomes induce transforming growth factor beta pathway dysregulation in hepatocytes: a novel paradigm for obesity-related liver disease.

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-ß) 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-ß 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-ß 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 ανß-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 ανß-5 and ανß-8, and matrix metalloproteinase-9 in hepatic stellate cells. CONCLUSIONS: Exosomes from VAT integrate into liver cells and induce dysregulation of TGF-ß pathway members in vitro and offers an intriguing possibility for the pathogenesis of NAFLD.

Integrin ß-8, but not ß-5 or -6, protein expression is increased in livers of children with biliary atresia.

OBJECTIVES: 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. METHODS: 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 t test with P < 0.01 considered significant because of the multiple comparisons. Interobserver variability was assessed by intraclass correlation (ICC). RESULTS: 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 ανß8 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 ανß5 (1.93 ± 0.84 vs 1.50 ± 0.90, P = 0.23) or integrin ανß6 (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 ανß5 (ICC 0.52), good for integrin ανß6 (ICC 0.72), and excellent for integrin ανß8 (ICC 0.79) and fibrosis (ICC 0.89). CONCLUSIONS: Our data show that integrin ανß8, but not integrin ανß5 or integrin ανß6, 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 ανß8 or more global integrin blocking strategies may be therapeutic options in BA, but further work is clearly needed.

Predicting sleep apnea in morbidly obese adolescents undergoing bariatric surgery.

INTRODUCTION: In adults, the association between obesity and obstructive sleep apnea (OSA) is established, and many are concerned OSA increases surgical risk. Pre-operative screening for OSA is standard, and this is also the case in adolescent bariatric surgery. We noted many of our patients were without significant OSA, despite being obese. We reviewed our experience with screening polysomnography (PSG) to determine any predictive variables or complications. METHODS: All bariatric surgery patients from our hospital who had undergone PSG were included, and were stratified into 'OSA' or 'no OSA' by obstructive apnea-hypopnea index (OAHI), as well as by sex. RESULTS: A total of 49 adolescents enrolled during the study period: 10 males and 39 females. OSA prevalence was 42.9 %; males 80 %, females 33.3 %. Height, weight, body mass index (BMI), and prevalence of hypertension were significantly higher in patients with OSA. By sex, females also had more metabolic syndrome and witnessed apneas, while only weight and BMI remained significant in males. There were no peri-operative complications. CONCLUSIONS: Despite uniform obesity, less than half our adolescents had significant OSA on PSG. As no modeling exists to predict OSA in morbidly obese adolescents, we continue to recommend routine PSG, especially in higher weight and BMI patients, and those with hypertension.

Prevalence of Chronic Gastritis or Helicobacter pylori Infection in Adolescent Sleeve Gastrectomy Patients Does Not Correlate with Symptoms or Surgical Outcomes.

BACKGROUND: In adults undergoing gastric bypass surgery, it is routine practice to perform pre-operative testing for Helicobacter pylori infection. Evidence suggests that infection impairs anastomotic healing and contributes to complications. There currently are no data for adolescents undergoing bariatric procedures. Despite few patients with pre-operative symptoms, we noted occasional patients with H. pylori detected after sleeve gastrectomy. We reviewed our experience with our adolescent sleeve gastrectomy cohort to determine the prevalence of H. pylori infection, its predictive factors, and association with outcomes. We hypothesized that H. pylori infection would be associated with pre-operative symptoms, but not surgical outcomes. METHODS: All patients undergoing sleeve gastrectomy at our hospital were included. We conducted a chart review to determine pre- or post-operative symptoms of gastroesophageal reflux disease GERD or gastritis, operative complications, and long-term anti-reflux therapy after surgery. Pathology reports were reviewed for evidence of gastritis and H. pylori infection. RESULTS: 78 adolescents had laparoscopic sleeve gastrectomy from January 2010 through July 2014. The prevalence of chronic gastritis was 44.9% (35/78) and 11.4% of those patients had H. pylori (4/35). Only one patient with H. pylori had pre-operative symptoms, and only 25.7% (9/35) of patients with pathology-proven gastritis had symptoms. One staple line leak occurred but this patient did not have H. pylori or gastritis. Mean patient follow-up was 10 (3-26) mos. CONCLUSIONS: There is a moderate prevalence of gastritis among adolescents undergoing sleeve gastrectomy, but only a small number of these patients had H. pylori infection. Neither the presence of chronic gastritis nor H. pylori infection correlated with symptoms or outcomes. Thus, in the absence of predictive symptomology or adverse outcome in those who are infected, we advocate for continued routine pathologic evaluation without the required need for pre-operative determination unless or until H. pylori infection is associated with adverse surgical outcomes.

Inpatient weight loss as a precursor to bariatric surgery for adolescents with extreme obesity: optimizing bariatric surgery.

BACKGROUND: As the obesity epidemic takes its toll on patients stricken with the disease and our health care system, debate continues regarding the use of weight loss surgery and its long-term consequences, especially for adolescents. One subset of patients regarding whom there is increased controversy is adolescents with extreme obesity (BMI > 60 kg/m(2)) because the risk of complications in this weight category is higher than for others undergoing bariatric surgery. Several strategies have been suggested for this patient group, including staged operations, combined operations, intragastric balloon use, and endoluminal sleeve placement. However, the device options are often not available to adolescents, and there are no data regarding staged or combined procedures in this age group. METHODS: All adolescents with BMI >60 kg/m(2) referred to our program were evaluated for inpatient medical weight loss prior to laparoscopic sleeve gastrectomy. The program utilizes a multidisciplinary approach with a protein-sparing modified fast diet, exercise, and behavioral modification. RESULTS: Three patients completed the program, and each achieved significant preoperative weight loss through the inpatient program and successfully underwent bariatric surgery. CONCLUSIONS: Presurgical weight loss via an inpatient program for adolescents with a BMI >60 kg/m(2) results in total weight loss comparable to a primary surgical procedure alone, with the benefit of decreasing the perioperative risk.

Dysregulation of upstream and downstream transforming growth factor-ß transcripts in livers of children with biliary atresia and fibrogenic gene signatures.

INTRODUCTION: Our previous work demonstrated that the transforming-growth factor (TGF) ß pathway plays a central role in the liver fibrosis associated with experimental biliary atresia (BA). To confirm these findings in humans, we performed an in silico analysis of publicly available microarray data from liver specimens from children with BA, with the hypothesis that the TGF-ß pathway would be dysregulated. METHODS: We analyzed publicly available liver gene expression microarray data from 47 infants with BA. We re-analyzed the microarray image files and clinical data to compare gene expression differences between the fibrogenic and inflammatory cohorts identified in the initial study. Targets from the microarray analysis were confirmed using the animal model of BA. RESULTS: Analysis of variance (ANOVA) detected 6903 transcripts (2822 distinct genes) differentially regulated between groups (p < 0.01; fold change >1.2). We used a targeted approach to identified a subgroup of 24 TGF-ß-related transcripts. Expressions for procollagen transcripts were increased in the fibrogenic group (1.2-fold to 1.4-fold); expression of matrix metalloproteinase (MMP)-7 was similarly increased 2-fold, while MMP-9 and plasminogen activator inhibitor-1 were decreased 2-fold and 3-fold respectively. Integrins ß5 (1.18-fold) and ß8 (1.84-fold) also demonstrated increased expression in the fibrogenic group. Increased expression of ß5 (3-fold) and ß8 (5-fold) as well as Smad-3 (4-fold) and Smad interacting protein (SIP)-1 (3.5-fold) mRNA was confirmed in experimental BA. Phosphorylated Smad-3 protein in the experimental group was also nearly twice that of the control group, further implicating the TGF-ß pathway. CONCLUSION: Gene transcripts for known upstream and downstream TGF-ß mediators are differentially expressed in liver specimens from children with BA and a fibrogenic gene signature. The same integrins that were dysregulated in the human specimens were also found to be up-regulated in our animal BA model, as were other intermediaries in the TGF-ß pathway. Further investigation into whether these mediators may be attractive targets for future therapy in children with BA is warranted.