Helicobacter pylori infection induces gastric precancerous lesions and persistent expression of Angpt2, Vegf-A and Tnf-A in a mouse model

Abstract

Introduction: Helicobacter pylori colonizes the gastric mucosa and induces chronic inflammation.

Methods: Using a mouse model of H. pylori-induced gastritis, we evaluated the mRNA and protein expression levels of proinflammatory and proangiogenic factors, as well as the histopathological changes in gastric mucosa in response to infection. Five- to six-week-old female C57BL/6N mice were challenged with H. pylori SS1 strain. Animals were euthanized after 5-, 10-, 20-, 30-, 40- and 50-weeks post infection. mRNA and protein expression of Angpt1, Angpt2, VegfA, Tnf-α, bacterial colonization, inflammatory response and gastric lesions were evaluated.

Results: A robust bacterial colonization was observed in 30 to 50 weeks-infected mice, which was accompanied by immune cell infiltration in the gastric mucosa. Compared to non-infected animals, H. pylori-colonized animals showed an upregulation in the expression of Tnf-A, Angpt2 and VegfA at the mRNA and protein levels. In contrast, Angpt1 mRNA and protein expression was downregulated in H. pylori-colonized mice.

Conclusion: Our data show that H. pylori infection induces the expression of Angpt2, Tnf-A and Vegf-A in murine gastric epithelium. This may contribute to the pathogenesis of H. pylori-associated gastritis, however the significance of this should be further addressed.

Keywords: ANGPT1; ANGPT2; Helicobacter pylori; TNF-A; VEGFA; angiogenesis; mouse model.

Figure 1

Schematic view of the experimental groups of the mouse model of Helicobacter pylori SS1 infection used in the study. A total of 39 female C57BL/6N mice were divided into 7 groups (n per group indicated between parentheses), 6 of these groups were infected with H. pylori and one group received NaCl 0.9% only (control group, n=5). Mice were euthanized between 5-50 w.p.i. Control non-infected mice were sacrificed at 50 w.p.i. Created with BioRender.com.

Figure 2

Histopathology of the murine gastric corpus mucosa in unchallenged and H. pylori-challenged mice. Tissue sections from resected gastric mucosa of unchallenged and H. pylori-inoculated mice were stained by H&E. (A) Unchallenged mice show a normal mucosal architecture. (B) Five weeks after H. pylori inoculation, minor multifocal infiltration of immune cells is observed. (C) Minor infiltration and some metaplastic patches are observed ten weeks after challenge. (D) The pathology and infiltration are intensified after thirty weeks of infection, with hyperplasia and multiple foci of mucous metaplasia. (E) Animals infected for forty and fifty (F) weeks also showed an extensive infiltration of immune cells with areas of hyperplasia and metaplasia (black arrows). H. pylori colonization status and histopathological assessment of inflammation. (G) H. pylori score at different time-points. (H). Inflammation score of the murine gastric mucosa with H. pylori infection at different time-points (scored according to the scheme proposed by Wang et al. (22) and Rogers (23), one experiment; n = 5-7 mice per point; black line represents median values). Formalin-fixed, paraffin-embedded gastric tissue were stained with hematoxylin and eosin and examined. Scale bars: 50 μm (20x).

Figure 3

H. pylori-SS1 upregulates the expression of pro-angiogenic factors in murine gastric mucosa. mRNA expression was analyzed by real-time PCR, and represented as Delta (Δ) Ct values. The higher ΔCt values represents the lower expression of genes. (A) Tnf-A, (B) VegfA and (C) Angpt2 mRNAs show a concomitant upregulation in time, becoming more evident from 30 w.p.i, whereas (D) Angpt1 mRNA is downregulated at 40 and 50 w.p.i. When comparing not infected animals with the 50 w.p.i. group, a significant upregulation was observed for (E) Tnf-A, (F) VegfA and (G) Angpt2 mRNAs expression, but a significant deregulation for (H) Angpt1mRNA. (I-L) Western blot and concordant relative quantification of VegfA, Angpt1 and Angpt2 proteins in mouse gastric mucosa of H. pylori-SS1 infected and uninfected mice are shown. VegfA and Angpt2 are upregulated and Angpt1 is downregulated in time. Data presented as mean ± SD of each experimental group. ***p < 0.001.

Figure 4

H. pylori upregulates Angpt2 expression in murine gastric mucosa. (A, B) IHC staining in blood vessels is positive (brown color) for Angpt1 and negative for Angpt2 in unchallenged mice. (C, D) IHC staining of Angpt1 compared to Angpt2 in lamina propria and blood vessels of infected mice 50 w.p.i. IHC staining with Angpt2 in infected mice 30 w.p.i. compared to 50 w.p.i. mice. Scale bars: (A, B, E, G) ≈ 50 μm; (C, D, F, H) ≈ 100 μm.

Figure 5

Model for H. pylori-induced angiogenesis in gastric mucosa. In a CagA-independent pathway, H. pylori infection may trigger the activation of NF-κB (A), which in turn induces TNF-α production (B) in lymphocytes and macrophages. TNF-α activates endothelial cells, which promotes Angpt2 release (C) from Weibel-Palade bodies. H. pylori induces the expression of Angpt2 and VegfA from epithelial cells in a CagA-independent pathway, and also the activation and proliferation of endothelial cells (D). Created with BioRender.com.