Throughout this investigation, it was noted that certain gastric epithelial cell islands in culture developed intracellular vacuoles (Figure 8.2). The association between vacuolation and the presence of Helicobacter spp. in the tissue from which the cells were isolated was investigated. Cell vacuolation was scored on a scale of 0-5, where 0 indicates no vacuolation and 5 indicates severe vacuolation, such as that seen in Figure 8.2. Cells isolated from Helicobacter-positive mucosal tissue samples, were found to have significantly higher vacuolation scores than cells isolated from
Helicobacter-negative samples (Figure 8.3). However, no differences in spreading
Figure 8.2- Severe vacuolation (score of 5) in a gastric epithelial cell island isolated from Helicobacter-positive biopsy-derived tissue. Arrows indicate intracellular vacuoles. Images taken at magnification of 20X; scale bar: 10 µm
Figure 8.3- Effects of Helicobacter status on vacuolation in cultured gastric epithelial cell islands; Vacuolation was scored on a scale of 0-5, where 0 = no vacuolation and 5 = severe vacuolation. Data (presented as mean +/- SEM) was analysed using one-way analysis of variance.
Cells isolated from Helicobacter-positive tissue samples showed significantly higher levels of
vacuolation than cells isolated from Helicobacter-negative tissue samples (p<0.05, indicated by *).
0 0.5 1 1.5 2 2.5 3 Positive Negative A ve rag e v ac u o lation sc o re Helicobacter status *
8.4.Discussion
The data presented in this chapter provides evidence that COX-2 protein expression is up-regulated in the canine gastric mucosa during infection with spiral bacteria, while EP3 and EP4 protein expression is unaffected. The majority of current work has focused on the effects of H. pylori infection, given that it is a highly prevalent human pathogen (Khalifa et al., 2010). Although H. pylori has not been identified in dogs (Neiger & Simpson, 2000), Helicobacter species reported in the canine stomach include H. felis, H. bilis, Flexispira rappini, H. bizzozeronii, H. salomonis
and H. Heilmannii (Eaton et al., 1996; Jalava et al., 1998; Neiger et al., 1999). In
previous studies, COX-2 mRNA and protein expression have been shown to be markedly up-regulated in the gastric mucosa of human patients with H. pylori- positive gastritis when compared with normal mucosa (Fu et al., 1999). Additionally, COX-2 expression is higher in tissue samples from patients with H. pylori-positive gastritis than in H. pylori-negative gastritis (Fu et al., 1999), thus induced COX-2 expression may be a direct response to H. pylori infection, as opposed to the presence of gastritis. Furthermore, increased COX-2 protein expression is reduced following successful eradication of H. pylori (McCarthy et al., 1999). In vitro studies have confirmed the effects of Helicobacter on COX-2 expression, for instance, culturing H. pylori with a normal gastric epithelial cell line for 24 h, was shown to cause a 6-fold increase in COX-2 protein expression and a subsequent increase in PGE2 production (Shen et al., 2006). Similarly, H. pylori up-regulated COX-2
mRNA expression and PGE2 release in the human adenocarcinoma cell line, MKN-
28 in vitro (Romano et al., 1998).
H. pylori infection has been shown to activate NF-κB in human gastric epithelial
cells both in vitro and in vivo (Keates et al., 1997), as the COX-2 gene contains an NF-κB binding site in its promoter region (Tanabe & Tohnai, 2002), this could be a potential mechanism for COX-2 induction. Such a mechanism has been described for the induction of COX-2 expression by H. pylori in human gastric epithelial cells in
vitro (Chang et al., 2004). H. pylori was reported to act through the toll-like
receptors, TLR2 and TLR9 to activate PI/PLCɣ, which induces PKCα and c-Src activation, leading to the tyrosine phosphorylation of IKKα/β. The NIK/IKKα/β pathway is also activated and both pathways converge, resulting in the
phosphorylation and degradation of IκBα and activation of NF-κB in the COX-2 promoter region, leading to the induction of COX-2 gene expression (Chang et al., 2004). Exposure of the AGS gastric cancer cell line to Helicobacter pylori has been shown to activate NF-kB signaling, COX-2 expression and paracrine activation of cell migration and invasion (Varro et al., 2004). Furthermore, COX-2 is a potentially important paracrine regulator of PAI-2, a key factor in the regulation of epithelial cell apoptosis and cell migration (Varro et al., 2004).
While it is clear that Helicobacter infection induces the expression of COX-2 in vivo
and in vitro, the data obtained during this investigation suggests that other factors
may play a role in this. While COX-2 protein expression was markedly increased in some samples positive for spiral bacteria, certain samples expressed similar levels of COX-2 to samples negative for spiral bacteria, thus suggesting that other factors may influence the effects of spiral bacteria on protein expression. No correlation was found between the samples expressing high COX-2 and any of the other clinical parameters, however, due to the small sample size, the effects of breed on protein expression could not be meaningfully investigated. Thus, future studies using a larger sample size are needed to further investigate the association between COX-2 expression and breed. The presence of spiral bacteria was further categorised as colonising deep within the gastric glands, however the presence of deep spiral bacteria was found to have no significant effect on COX-2, EP3 or EP4 protein expression. However, it is interesting that in the small number of cases where deep
Helicobacter infection was noted, COX-2 and EP4 expression was low.
In this study, cultured gastric epithelial cell islands isolated from mucosal tissue samples positive for Helicobacter were shown to have increased cytoplasmic vacuolation. Approximately 50% of H. pylori isolates in Western countries produce the cytotoxin, VacA, which induces cytoplasmic vacuolation in eukaryotic cells (Maeda et al., 1998). A previous study reported similar cytoplasmic vacuolation to that seen here, in primary cultures of human gastric epithelial cells, incubated with
the H. pylori vacuolating cytotoxin and primary cells were significantly more
sensitive to the effects of the cytotoxin than cell lines (Smoot et al., 1996). The VacA cytotoxin appears to be unique to the H. pylori species (Beswick et al., 2006) and much less is known about the virulence of non-H. pylori Helicobacters. However H.
trogontum has been shown to cause similar vacuolation in ileal epithelial cells (Moura et al., 1998). Clearly, more work needs to be done in order to characterise virulence factors associated with other Helicobacter species. Given the association between Helicobacter status and cultured gastric epithelial cells described in this chapter, it seems possible that the Helicobacter are colonising intracellularly and as such survive antibiotic treatment in the culture medium. H. bizzozeronii and H. felis have recently been reported to localise intracellularly in parietal cells and macrophages in the fundic mucosa of Beagle dogs (Lanzoni et al., 2011).
The relationship between protein expression and gastric inflammation, as indicated by various markers, was also investigated. The WSAVA Gastrointestinal Standardization Group has produced a set of standards for the characterisation of inflammatory changes in endoscopic biopsy samples from the gastrointestinal mucosa of small companion animals (Day et al., 2008). These reporting guidelines were used to evaluate inflammatory changes in the biopsy samples used in this investigation. The inflammatory changes reported include the presence of intraepithelial lymphocytes, lymphoplasmacytic, eosinophillic and neutrophillic infiltration and lymphofollicular hyperplasia. No association was found between EP3, EP4 and COX-2 protein expression and gastric mucosal inflammation. Increased COX-2 expression in H. pylori gastritis and in tissue adjacent to gastric ulceration has been previously described (Jackson et al., 2000). Additionally, a study comparing PGE2 receptor expression in normal and inflamed human colonic mucosa,
reported a significant increase in EP4 expression in the mucosal T-lymphocytes of inflamed tissue (Cosme et al., 2000) and EP4 epithelial expression in the inflamed mucosa was more intermittent compared with the even distribution seen in the normal mucosa (Cosme et al., 2000). Furthermore, during inflammation non-surface epithelial cells newly and significantly express EP2 and EP3 (Takafuji et al., 2000). As such, the lack of relationship found between protein expression and markers of inflammation is surprising.
The most common neoplasm to affect the canine stomach is gastric carcinoma (Carrasco et al., 2011). Particular breeds show an increased risk for developing gastric carcinoma (Willard, 2012) and little is known about its pathogenesis, but it is assumed to be similar to that of human gastric carcinoma. Many of the markers of
malignancy that are associated with increased COX-2 expression, for instance, invasion and metastasis (Han, 2003) are related to increased cell migration. As only 1 of the 36 patients studied was diagnosed with gastric adenocarcinoma, no meaningful conclusions could be made with regards to its influence on protein expression. Furthermore, none of the patients studied were diagnosed with gastric lymphoma. The over-expression of COX-2 in gastric cancer tissue has been previously reported (Lim et al., 2000; Mao et al., 2007) and was significantly related to metastasis and the depth of invasion (Mao et al., 2007). Increased COX-2 expression has been identified as an independent prognostic marker for poor outcome in gastric cancer and COX-2 is considered an important treatment target in a number of veterinary tumours, particularly transitional cell carcinoma (Doré, 2011). COX-2 over-expression is related to advanced tumour penetration depth, lymph node metastases and non-curative operation (Mrena et al., 2005). COX-2 over-expression is more prevalent in larger tumours and in more invasive cancers, i.e. cancers with more metastatic nodes and a greater invasion depth (Han, 2003). A previous study comparing COX-2 protein expression in gastric carcinoma tissue samples and paired samples from adjacent normal mucosal tissue, found that the carcinoma tissue expressed significantly higher COX-2 levels (Murata et al., 1999). In this investigation, the tissue sample obtained from the patient diagnosed with gastric adenocarcinoma, expressed relatively low levels of EP3, EP4 and COX-2 protein.
Little is known about EP receptor expression in gastric cancer. In veterinary patients, COX-2 and EP1 and EP2 expression are markedly increased in bone tumours (Millanta et al., 2012). In human GI tract tumours, EP3 expression is known to be markedly decreased in colon cancer tissue and its down-regulation is thought to contribute to colon carcinogenesis (Shoji et al., 2004). In contrast, EP4 protein expression has been shown to be increased in colorectal cancers (Chell et al., 2006). Thus, EP3 and EP4 clearly have a role in carcinogenesis and more work needs to be carried out in order to determine the nature of this role in gastric cancer. As samples used for this investigation are obtained from routine endoscopic biopsies and gastric carcinoma is relatively uncommon, the study size was very small. A retrospective evaluation of COX-2, EP3 and EP4 immunoreactivity in canine gastric tumours
using archived material would allow for a larger sample size and would be a valuable staring point to evaluate a potential role for these proteins in canine gastric cancer.
Chapter 9 – Final discussion