In this study, we demonstrated (I) distinct expression patterns of five genes encoding for proteins involved in the formation of tight junctions in esophageal mucosa. In particular Claudin-1 in ERD and to lesser extent Claudin-2 was expressed at higher levels in patients with GERD. In contrast, ZO-1, ZO-2, and Occludin were not affected by the presence of GERD. (II) In general, altered gene expression of Claudin-1/-2 did not correlate with the degree of histomorphological changes in the esophageal mucosa of patients with GERD.
Tight junctions are composed of transmembrane proteins such as Occludin, 24 Claudins, several junctional adhesion molecules (JAMs) with different isoforms, E-Cadherin as well as cytosolic binding partners
]. The selection of the five genes studied was based on functional aspects. Occludin is critical for the formation of tight junctions in most tissues
]. Claudin-1 is one of the numerous Claudins that seals intercellular space leading to higher barrier function
], while Claudin-2 is the only pore-forming member of this family resulting in increased permeability
]. Zonula occludens (ZO)-1 and-2 are cytosolic partners of tight junctions in most epithelial surfaces
]. The selected genes present important components of the tight junctional complex, and were considered to allow assessment about alterations of tight junctions in relation to GERD. A comprehensive analysis concerning the general expression pattern of other junctional proteins was not performed.
Recently, several studies demonstrated characteristic histopathological alterations in esophageal mucosa of patients with GERD and a proinflammatory response including the activation of related pathways such as NFκB, PAR-2, ROS and iNOS
]. Several in vitro
and animal studies have provided evidence that incubation of esophageal mucosa or squamous cell lines either with acidified media with/without bile acids or proinflammatory cytokines can provoke changes in transepithelial electric resistance and increased transepithelial permeability
]. Notably, several studies demonstrated a cytokine-mediated change of tight junction-related molecules in various cell models. For instance, IL-6 markedly induces Claudin-2 expression via
MEK and PI3K signaling leading to increased tight junction permeability
]. In a rabbit model of GERD, elevated IL-6 expression correlated with induction of several tight junction-related proteins (Claudin-1, Occludin, JAM-1, ZO-1)
] and altered the motogenic activity of smooth muscle cells
All together, there is sufficient data showing that the exposure of mixed gastric or gastroduodenal refluxate causes altered esophageal epithelial barrier function, inflammation and cellular damage, although the timely order of these processes is a matter of debate
]. As today, it is well accepted that impaired epithelial barrier function of the distal esophagus presents a major pathophysiological process in GERD.
This study shows an upregulation of tight junction-related proteins in relation to ERD and NERD in mucosal samples. In particular, Claudin-1 and Claudin-2, though mediating opposite functionally effects, were induced, while cytoplasmic adapters and Occludin were rather unchanged in relation to controls. The higher expression of Claudin-1 (both on transcript and protein level) was the only significant difference identified between patients with ERD and NERD. The fact that all other identified changes were similar between NERD and ERD supports the concept of similar pathophysiological mechanisms between both diseases. Unexpectedly, these changes did not correlate with histomorphological alterations, in particular with dilated ICS in esophageal mucosa. This finding is in contrast to the recently identified correlation between histopathological alterations, in particular basal cell hyperplasia, and elevated gene expression of desmosomal proteins
]. In this study, few borderline correlations were found for basal cell hyperplasia and some genes only, but notably these findings were mostly restricted to reflux-negative controls, whereas patients with GERD did not reveal significant correlations between histopathological alterations and transcript levels of the five genes. Since correlation analyses were performed in an explorative way (without adjustment for multiple comparison), the few significant correlations (with borderline significance) do not support a general role of these findings for the pathophysiology of GERD. Taken into consideration this limitation and the fact that that the overall majority of our comparisons (17 out of 20) revealed no correlations, we conclude that our data do not give evidence for an association between the gene expression of the five genes studied and the histopathological changes in our study groups. It is well known that extent of basal cell hyperplasia reflects proliferative status of esophageal mucosa
]. Since the identified correlations between gene expression levels and basal cell hyperplasia were mostly restricted to controls, it is unlikely that elevated Claudin-1 levels in ERD reflect tissue repair in context to mucosal damage caused by refluxate in these patients. Since we and others demonstrated more severe histomorphological alterations in ERD than NERD, the overall consistent changes of the 5 genes and their corresponding proteins in both diseases seem to be of limited relevance to the mucosal integrity and function. Furthermore, it is notable that some of the stainings revealed not the typical membrane-restricted expression pattern as demonstrated for these tight junction-related molecules im most gastrointestinal tissues
]. However, cytoplasmic or diffuse membranous expression patterns have been identified for Claudin-2
] and ZO-1
] in human gastrointestinal tissue and for Claudin-1 in esophageal mucosa of rat
]. Occludin staining pattern or expression in esophageal mucosa differs frequently also from those identified in gastric or intestinal mucosa
]. Overall, the subcellular distribution of the 5 tight junction-related proteins seems to differ partially from those identified in columnar-lined epithelium. However, the study was not aimed to analyze the subcellular distribution pattern of the molecules in esophageal mucosa on the subcellular level. The presence of appropriate negative and positive control stainings in other tissues, and the good concordance between expression data on transcript and protein level in general provide further indirect evidence for the specificity of immunohistochemical stainings.
Based on the descriptive study design, it remains open whether the altered gene expression levels of Claudin-1 and −2 contribute to GERD pathophysiology or merely are markers for the existing disease. Furthermore it is notable that the majority of patients received GERD medications (PPI, H2RA) in the past before entering study. Even a stop of at least 2 weeks was mandatory to enter the study, we can not exclude that the effects of long-term therapy in the past or the changes induced by the 2-week stop of medication (e.g. acid rebound)
] could have affect the expression of the five genes studied. Another limitation is the assessment of protein expression by an immunohistochemical score that can be done semiquantitatively at best. Besides this methodological aspect, posttranscriptional regulatory mechanisms can lead to different findings between gene expression analysis performed on transcript and protein levels. But as mentioned above, overall we observed a good concordance between both levels even not all significant findings were confirmed by both methodologies. Since we studied five selected components of tight junction complexes in GERD only, general conclusions can not be made. Assessment of other tight junction related molecules (e.g. Claudins, JAMs, Tricellulin)
] in regard to GERD needs to be performed.