Abstract
Introduction:
We assessed if obesity perturbs the esophageal epithelial barrier function independent of promotion of gastroesophageal reflux (GER).
Methods:
38 participants were divided into four groups: Obesity-/GER-, Obesity+/GER-, Obesity-/GER+, Obesity+/GER+. Esophageal intercellular space (ICS) and desmosome density (structural integrity) and fluorescein leak (functional integrity) were measured.
Results:
The Obesity+/GER- group demonstrated increased ICS, reduced desmosome density, and increased fluorescein leak compared to control subjects. These changes were similar but not additive to findings seen in Obesity-/GER + and Obesity+/GER+ patients.
Discussion:
Central obesity impairs structural and functional integrity of the esophageal barrier independent of GER, likely predisposing to esophageal injury.
Introduction
Symptoms of gastroesophageal reflux disease (GERD), including heart burn and regurgitation, are the leading GI complaints in outpatient practice.1 There are two entities of GERD: erosive reflux (ERD) and non-erosive reflux disease (NERD).1 ERD is differentiated from NERD by the presence of visible esophageal erosions on endoscopy. One similarity between the two entities is the presence of dilated intercellular spaces (ICS) in the esophageal epithelium, which is considered an early sign of reflux injury.2–4
While ICS dilation was thought to be induced by pathologic acid exposure, it has also been observed in patients with normal esophageal pH measurement.2,3 This observation suggests that ICS dilation may lead to reflux perception even with physiologic reflux conditions.5–7 It also raises the possibility of non-reflux medicated mechanisms regulating ICS structure and function. While central obesity is independently associated with Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC), the precise mechanisms of this association remain unknown.8 We have previously reported widened ICS in patients with central obesity but physiologic levels of acid reflux on pH monitoring, suggesting that obesity may play a role in modulating ICS diameter.9 In addition, obesity has also been associated with impaired mucosal impedance.10 We therefore aimed to further investigate the effect of central obesity on both epithelial barrier morphology and function in this larger prospective study.
Methods
This study (NCT02776982) was approved by the Mayo Clinic IRB. A total of 38 individuals were enrolled. Details on inclusion and exclusion criteria, definition of GERD and central obesity, endoscopic and Bravo procedures (done off PPIs) can be found in supplemental methods. Based on the anthropometric and ambulatory pH data, study subjects were divided into Obesity-/GER-, Obesity+/GER-, Obesity-/ GER+, and Obesity+/GER+ groups.
The stratum spinosum layer of endoscopic biopsies taken 5 cm above the GEJ was imaged using Transmission Electron Microscopy (TEM) at 10,000x power. The mean ICS was obtained by randomly measuring 10 unique membrane segments using ImageJ (open-source NIH software). The density of desmosomes seen as linear and hyperdense epithelial structures (Supplemental figure 1) was calculated by dividing the number of desmosomes by the length of membrane as described previously11 and the mean was obtained by averaging 10 unique membrane segments. Probe based confocal laser endomicroscopy (p-CLE, Mauna Kea Technologies, Paris) was used to quantify paracellular fluorescein intensity reflecting epithelial leak.12 pCLE captured images at 5 cm above the GEJ, 30 seconds after the administration of intravenous fluorescein. Fluorescence leak intensity was measured by averaging the intensity of 10 randomly chosen square areas using ImageJ (Supplemental Figure 1).
Baseline characteristics were compared by using T-Test and ANOVA for continuous variables and Chi-squared Test for categorical variables using BlueSky statistics. Linear regression models were used to test associations between the variables.
Results
Baseline characteristics were comparable among the 4 groups, except that the Obesity-/GER+ group had exclusively female participants (Table 1). Reflux symptoms were significantly more common in the GER+ groups (p=0.03). Over half of the Obesity+/GER- group reported heartburn compared to 33% in the Obesity-/GER- group. None of the participants had BE on EGD.
Table 1.
Demographic and clinical characteristics of study participants.
| Obesity−/GER− (N=9) | Obesity+/GER− (N=13) | p Values (Obesity−/GER− vs Obesity+/GER−) | Obesity−/GER+ (N=7) | Obesity+/GER+ (N=9) | p Values (All groups) | |
|---|---|---|---|---|---|---|
| Age Mean (SD) | 48.11 (13.60) | 51.77 (15.99) | 0.81 | 51.71 (21.82) | 55.22 (12.30) | 0.83 |
| Male gender (%) | 33.33 | 38.46 | 0.58 | 0 | 33.33 | 0.31 |
| BMI Mean (SD) | 23.74 (3.77) | 29.82 (5.61) | <0.001** | 24.28 (3.17) | 28.31 (4.17) | 0.01* |
| WHR Mean (SD) | 0.83 (0.03) | 0.96 (0.08) | 0.01* | 0.81 (0.05) | 0.95 (0.06) | <0.001** |
| Current or prior smoking (%) | 0 | 0 | NA | 0 | 22.22 | 0.08 |
| Current alcohol use (%) | 88.89 | 84.62 | 0.77 | 100 | 66.67 | 0.31 |
| NSAID or ASA use (%) | 11.11 | 23.08 | 0.47 | 14.29 | 11.11 | 0.84 |
| Prior PPI use (%) | 55.56 | 61.54 | 0.78 | 42.86 | 77.78 | 0.55 |
| Esophagitis (LA Grade A) (%) | 11.11 | 15.38 | 0.77 | 14.29 | 22.22 | 0.93 |
| Esophagitis (LA Grade B-D) (%) | 11.11 | 0 | 0.22 | 42.86 | 22.22 | 0.08 |
| Heart Burn > once/week (%) | 33.33 | 53.85 | 0.34 | 100 | 77.78 | 0.03* |
| Regurgitation > once/week (%) | 22.22 | 30.77 | 0.66 | 57.14 | 33.33 | 0.5 |
| Dysphagia > once/week (%) | 22.22 | 23.08 | 0.96 | 42.86 | 0 | 0.2 |
Current alcohol use is defined as consumption of any amount of alcohol beverages at the time of the enrollment. PPI use is defined as being on therapeutic dose of PPI prior to the study. PPI was discontinued at the enrollment of the study. Grading of esophagitis is based on the Los Angeles grading system. Some p values were designated as NA due to the values for the corresponding category were 0. Abbreviations. WHR: waist to hip ratio. PPI: Proton pump inhibitors.
Quantitative and qualitative analyses demonstrated significantly greater ICS diameter in the Obesity+/GER- group compared to the Obesity-/GER- group (1.79±0.27 μm vs. 1.04±0.30 μm, p<0.001, Figure 1A, B). The ICS diameter was also significantly increased in the Obesity-/GER+ (1.63±0.23 μm, p= 0.001) and Obesity+/GER+ groups (1.65±0.27 μm, p<0.001) (Figure 1B).
Figure 1.
Central obesity is associated with ICS dilation, decreased desmosome density and increased permeability in the esophageal epithelium. (A). TEM images of esophageal epithelium at 5cm above the GEJ (magnification 10K). Purple arrows indicate dilated ICS. Red arrows indicate regions of ICS devoid of desmosomes. (B). The ICS in the Obesity+/GER- group is statistically increased as compared to the Obesity-/GER- control subjects. Obesity-/GER+ and Obesity+/GER+ groups also exhibit statistically significant ICS dilation. (C). Desmosomes are evenly distributed in Obesity-/GER- group (yellow arrow) but more sparsely found in the Obesity+/GER-, Obesity-/GER+ and Obesity+/GER+ groups (red arrows). (D). The desmosome density (as expressed as number of desmosomes per μm of membrane) is significantly reduced in the Obesity+/GER-, Obesity-/GER+ and Obesity+/GER+ groups. (E). Images captured from the confocal microscopy video of esophageal epithelium at 5cm above GEJ. The fluorescein leak intensity at the intercellular space appears to be higher in. (F). Quantitative analysis shows that fluorescein leak intensity in the Obesity+/GER-, Obesity-/GER+ and Obesity+/GER+ groups (which is normalized to that of Obesity-/GER- group) is significantly elevated.
The Obesity+/GER- group had a 40% reduction in desmosome density compared to the Obesity-/GER- group (p<0.001, Figure 1C, D). A similar 40% reduction was observed in the Obesity-/GER+ (p=0.002) and Obesity+/GER+ (p<0.001) groups (Figure 1D). Linear regression analysis demonstrated a significant correlation between desmosome density and ICS dilation (R2=0.2365, p=0.002, Figure 2A).
Figure 2.
Desmosome density and increase membrane permeability are strongly associated with ICS dilation. (A). Linear regression analysis demonstrated a significant association between reduced desmosome density and the ICS dilation. (B). Linear regression analysis showed a significant association between the fluorescein leak intensity and the ICS dilation.
The Obesity+/GER- group had a 19% increase in fluorescein leak intensity compared to the Obesity-/GERD- group (p= 0.007, Figure 1E, F). Similarly, the Obesity-/GER+ and Obesity+/GER+ groups exhibited significantly increased fluorescein leak intensity (20% and 24%, respectively, Figure 1F). A strong correlation was found between fluorescein leak intensity and ICS dilation (R2=0.3774, p<0.001, Figure 2B).
Multivariate linear regression analyses for ICS dilation, desmosome density, fluorescein leak demonstrated significant correlations with obesity and GERD (Supplemental Table 1). Age and sex did not appear to have a significant impact on these outcomes.
Discussion
In this prospective cohort study, we demonstrated that central obesity impairs both the structure and function of the esophageal epithelial barrier independent of increased esophageal acid exposure. We first showed that Obesity+/GER- patients have dilated ICS and reduced desmosome density in the distal esophagus. We also demonstrated that these patients exhibited increased fluorescein leak consistent with perturbed barrier function. The strong correlation between ICS and the desmosome density suggests a potential cellular mechanism for ICS dilation. Fluorescein leak intensity was significantly correlated with ICS dilation, indicating a possible causative relationship between the structural and functional abnormalities. The mechanism of how central obesity leads to these abnormalities remains elusive, but could be mediated by inflammatory cytokines released from visceral adipocytes.13
The degree of ICS dilation was comparable between the Obesity+/GER- and Obesity+/GER+ group. The lack of additive effect suggests that central obesity and GERD may impact a common pathway that influences ICS and/or that there is a low threshold for maximal disturbance of ICS structure. Indeed, similar to obesity, GERD effects may be mediated by cytokines, in addition to direct caustic injury from acid.14
This study has potential clinical implications. For example, the association of obesity with BE and EAC has been attributed to induction of GERD. These findings suggest that the independent effect of obesity on esophageal permeability whether to refluxate with noxious agents and/or carcinogens also needs to be considered. Furthermore, GERD symptoms are most attributed to increased acid reflux despite many GERD patients not responding to acid suppression therapy.6,15 Our observation of a higher percentage of Obesity+/GER- patients with heartburn suggests increased permeability to physiologic refluxate as a cause of heartburn in these patients. Future pathological and biochemical studies on esophageal inflammation are needed to elucidate the role of obesity in esophageal diseases.
Supplementary Material
Acknowledgments
Funding:
Clinical Research Award from the American College of Gastroenterology (to PGI); Bravo pH probes were provided by Medtronic, via an investigator-initiated grant.
This project was also supported by Grant Number UL1 TR002377 from the National Center for Advancing Translational Sciences (NCATS). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
Prasad Iyer: Research funding from Exact Sciences and Pentax Medical; Consultant: Medtronic
Footnotes
Disclosures:
Ying Gibbens: None
Ramona Lansing: None
Michele Johnson: None
Christopher Blevins: None
David Katzka: Consultant Shire
References
- 1.Savarino E V, de Bortoli N, de Cassan C, et al. The natural history of gastro-esophageal reflux disease: A comprehensive review. Dis Esophagus. 2017;30(2). doi: 10.1111/dote.12511 [DOI] [PubMed] [Google Scholar]
- 2.Caviglia R, Ribolsi M, Maggiano N, et al. Dilated intercellular spaces of esophageal epithelium in nonerosive reflux disease patients with physiological esophageal acid exposure. Am J Gastroenterol. 2005;100(3):543–548. doi: 10.1111/j.1572-0241.2005.40978.x [DOI] [PubMed] [Google Scholar]
- 3.Dent J Microscopic Esophageal Mucosal Injury in Nonerosive Reflux Disease. Clin Gastroenterol Hepatol. 2007;5(1). doi: 10.1016/j.cgh.2006.08.006 [DOI] [PubMed] [Google Scholar]
- 4.Vela MF, Craft BM, Sharma N, Freeman J, Hazen-Martin D. Refractory heartburn: Comparison of intercellular space diameter in documented GERD vs. Functional heartburn. Am J Gastroenterol. 2011;106(5):844–850. doi: 10.1038/ajg.2010.476 [DOI] [PubMed] [Google Scholar]
- 5.Caviglia R, Ribolsi M, Gentile M, et al. Dilated intercellular spaces and acid reflux at the distal and proximal oesophagus in patients with non-erosive gastro-oesophageal reflux disease. Aliment Pharmacol Ther. 2007;25(5):629–636. doi: 10.1111/j.1365-2036.2006.03237.x [DOI] [PubMed] [Google Scholar]
- 6.Van Malenstein H, Farré R, Sifrim D. Esophageal dilated intercellular spaces (DIS) and nonerosive reflux disease. Am J Gastroenterol. 2008;103(4):1021–1028. doi: 10.1111/j.1572-0241.2007.01688.x [DOI] [PubMed] [Google Scholar]
- 7.Calabrese C, Bortolotti M, Fabbri A, et al. Reversibility of GERD ultrastructural alterations and relief of symptoms after omeprazole treatment. Am J Gastroenterol. 2005;100(3):537–542. doi: 10.1111/j.1572-0241.2005.40476.x [DOI] [PubMed] [Google Scholar]
- 8.Singh S, Sharma AN, Murad MH, et al. Central adiposity is associated with increased risk of esophageal inflammation, metaplasia, and adenocarcinoma: A systematic review and meta-analysis. Clin Gastroenterol Hepatol. 2013;11(11):1–23. doi: 10.1016/j.cgh.2013.05.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Blevins CH, Sharma AN, Johnson ML, et al. Influence of reflux and central obesity on intercellular space diameter of esophageal squamous epithelium. United Eur Gastroenterol J. 2016;4(2):177–183. doi: 10.1177/2050640615598426 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Blevins CH, Dierkhising RA, Geno DM, et al. Obesity and GERD impair esophageal epithelial permeability through 2 distinct mechanisms. Neurogastroenterol Motil. 2018. doi: 10.1111/nmo.13403 [DOI] [PubMed] [Google Scholar]
- 11.Egu DT, Walter E, Spindler V, Waschke J. Inhibition of p38MAPK signalling prevents epidermal blistering and alterations of desmosome structure induced by pemphigus autoantibodies in human epidermis. Br J Dermatol. 2017;177(6):1612–1618. doi: 10.1111/bjd.15721 [DOI] [PubMed] [Google Scholar]
- 12.Dunbar KB. Endomicroscopy in the evaluation of Barrett’s esophagus. Curr Opin Gastroenterol. 2011;27(4):374–382. doi: 10.1097/MOG.0b013e328347bac4 [DOI] [PubMed] [Google Scholar]
- 13.Tilg H, Moschen AR. Adipocytokines: Mediators linking adipose tissue, inflammation and immunity. Nat Rev Immunol. 2006;6(10):772–783. doi: 10.1038/nri1937 [DOI] [PubMed] [Google Scholar]
- 14.Dunbar KB, Agoston AT, Odze RD, et al. Association of acute gastroesophageal reflux disease with esophageal histologic changes. JAMA - J Am Med Assoc. 2016;315(19):2104–2112. doi: 10.1001/jama.2016.5657 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Bredenoord AJ, Smout AJ. Refractory gastrooesophageal reflux disease. Eur J Gastroenterol Hepatol. 2008;20(3). doi: 10.1097/MEG.0b013e3282f1cc39 [DOI] [PubMed] [Google Scholar]
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