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Up-regulation of intestinal vascular endothelial growth factor by Afa/Dr diffusely adhering Escherichia coli.

Cane G, Moal VL, Pagès G, Servin AL, Hofman P, Vouret-Craviari V - PLoS ONE (2007)

Bottom Line: This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells.Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways.Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR 6543, Université de Nice-Sophia Antipolis, Nice, France.

ABSTRACT

Background: Angiogenesis has been recently described as a novel component of inflammatory bowel disease pathogenesis. The level of vascular endothelial growth factor (VEGF) has been found increased in Crohn's disease and ulcerative colitis mucosa. To question whether a pro-inflammatory Escherichia coli could regulate the expression of VEGF in human intestinal epithelial cells, we examine the response of cultured human colonic T84 cells to infection by E. coli strain C1845 that belongs to the typical Afa/Dr diffusely adhering E. coli family (Afa/Dr DAEC).

Methodology: VEGF mRNA expression was examined by Northern blotting and q-PCR. VEGF protein levels were assayed by ELISA and its bioactivity was analysed in endothelial cells. The bacterial factor involved in VEGF induction was identified using recombinant E. coli expressing Dr adhesin, purified Dr adhesin and lipopolysaccharide. The signaling pathway activated for the up-regulation of VEGF was identified using a blocking monoclonal anti-DAF antibody, Western blot analysis and specific pharmacological inhibitors.

Principal findings: C1845 bacteria induce the production of VEGF protein which is bioactive. VEGF is induced by adhering C1845 in both a time- and bacteria concentration-dependent manner. This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells. Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways.

Conclusions: Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells. Thus, these results suggest a link between an entero-adherent, pro-inflammatory E. coli strain and angiogenesis which appeared recently as a novel component of IBD pathogenesis.

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C1845 bacteria increase VEGF expression in cultured intestinal epithelial cells.In A, increase in VEGF mRNA expression. Confluent serum-starved T84 cells (5×106 cells/well) were infected with wild-type C1845 bacteria and total RNA was prepared as indicated in the Materials and Methods section. Results for non-infected (NI) or cells infected with 5×107 CFU/ml C1845 bacteria for the indicated time are shown on the left panel. The dose response effect is presented on the right. Cells were infected for four hours with the indicated number of bacteria. Results of Northern blots are presented in the upper part of the Figure and q-PCR shown in the lower part. These results are representative of three independent experiments. In B, confluent serum-starved LS174, Caco-2/TC7 or INT407 cells were infected with 5×107 CFU/ml wild-type C1845 bacteria for four hours. VEGF mRNA expression was assayed by q-PCR. The signal corresponding to VEGF and 36B4 transcripts was quantified using a phosphoImager. Under each condition the signal was normalized to the 36B4 probe. Results are expressed as arbitrary units corresponding to the fold stimulation of treated versus non-treated conditions. In C, increase in the VEGF protein in the culture medium of wild-type C1845-infected T84 cells. Cells were infected with 5×107 CFU/ml wild-type C1845 bacteria for four hours and the supernatant were collected as indicated in the Materials and Methods section. The VEGF protein level was then quantified using an ELISA. Quantification of the results from two independent experiments (means±SD) is shown. *, p<0,01
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pone-0001359-g001: C1845 bacteria increase VEGF expression in cultured intestinal epithelial cells.In A, increase in VEGF mRNA expression. Confluent serum-starved T84 cells (5×106 cells/well) were infected with wild-type C1845 bacteria and total RNA was prepared as indicated in the Materials and Methods section. Results for non-infected (NI) or cells infected with 5×107 CFU/ml C1845 bacteria for the indicated time are shown on the left panel. The dose response effect is presented on the right. Cells were infected for four hours with the indicated number of bacteria. Results of Northern blots are presented in the upper part of the Figure and q-PCR shown in the lower part. These results are representative of three independent experiments. In B, confluent serum-starved LS174, Caco-2/TC7 or INT407 cells were infected with 5×107 CFU/ml wild-type C1845 bacteria for four hours. VEGF mRNA expression was assayed by q-PCR. The signal corresponding to VEGF and 36B4 transcripts was quantified using a phosphoImager. Under each condition the signal was normalized to the 36B4 probe. Results are expressed as arbitrary units corresponding to the fold stimulation of treated versus non-treated conditions. In C, increase in the VEGF protein in the culture medium of wild-type C1845-infected T84 cells. Cells were infected with 5×107 CFU/ml wild-type C1845 bacteria for four hours and the supernatant were collected as indicated in the Materials and Methods section. The VEGF protein level was then quantified using an ELISA. Quantification of the results from two independent experiments (means±SD) is shown. *, p<0,01

Mentions: Northern blot analysis of total RNA using a specific probe for the VEGF gene shows a very low level of VEGF mRNA expression in control, non-infected T84 cells while a time and dose-dependent increase in VEGF mRNA expression was observed in T84 infected cells (Figure 1A, upper panel). The transcript of 4 kb is preferentially expressed, however the expression of the three other spliced variants, ranging from 1.5 to 4.0 kb, is revealed with a longer exposure of the autoradiogram (data not shown). VEGF mRNA expression in non-infected and wild-type C1845-infected T84 cells was also quantified by q-PCR (Figure 1A, lower panel). The maximum increase in VEGF mRNA expression (3.6-fold increase) was achieved when cells were infected with 5×107 CFU/ml of wild-type C1845 bacteria for four hours. This increase in VEGF mRNA expression in response to wild-type C1845 bacteria is neither cell line-dependent, since we observed the same effect in the human LS174 colon cell line and the fully-differentiated enterocyte-like Caco-2/TC7 cells nor transformation-dependent since bacteria increase the expression of VEGF in INT407, a non-transformed cell line (Figure 1B).


Up-regulation of intestinal vascular endothelial growth factor by Afa/Dr diffusely adhering Escherichia coli.

Cane G, Moal VL, Pagès G, Servin AL, Hofman P, Vouret-Craviari V - PLoS ONE (2007)

C1845 bacteria increase VEGF expression in cultured intestinal epithelial cells.In A, increase in VEGF mRNA expression. Confluent serum-starved T84 cells (5×106 cells/well) were infected with wild-type C1845 bacteria and total RNA was prepared as indicated in the Materials and Methods section. Results for non-infected (NI) or cells infected with 5×107 CFU/ml C1845 bacteria for the indicated time are shown on the left panel. The dose response effect is presented on the right. Cells were infected for four hours with the indicated number of bacteria. Results of Northern blots are presented in the upper part of the Figure and q-PCR shown in the lower part. These results are representative of three independent experiments. In B, confluent serum-starved LS174, Caco-2/TC7 or INT407 cells were infected with 5×107 CFU/ml wild-type C1845 bacteria for four hours. VEGF mRNA expression was assayed by q-PCR. The signal corresponding to VEGF and 36B4 transcripts was quantified using a phosphoImager. Under each condition the signal was normalized to the 36B4 probe. Results are expressed as arbitrary units corresponding to the fold stimulation of treated versus non-treated conditions. In C, increase in the VEGF protein in the culture medium of wild-type C1845-infected T84 cells. Cells were infected with 5×107 CFU/ml wild-type C1845 bacteria for four hours and the supernatant were collected as indicated in the Materials and Methods section. The VEGF protein level was then quantified using an ELISA. Quantification of the results from two independent experiments (means±SD) is shown. *, p<0,01
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pone-0001359-g001: C1845 bacteria increase VEGF expression in cultured intestinal epithelial cells.In A, increase in VEGF mRNA expression. Confluent serum-starved T84 cells (5×106 cells/well) were infected with wild-type C1845 bacteria and total RNA was prepared as indicated in the Materials and Methods section. Results for non-infected (NI) or cells infected with 5×107 CFU/ml C1845 bacteria for the indicated time are shown on the left panel. The dose response effect is presented on the right. Cells were infected for four hours with the indicated number of bacteria. Results of Northern blots are presented in the upper part of the Figure and q-PCR shown in the lower part. These results are representative of three independent experiments. In B, confluent serum-starved LS174, Caco-2/TC7 or INT407 cells were infected with 5×107 CFU/ml wild-type C1845 bacteria for four hours. VEGF mRNA expression was assayed by q-PCR. The signal corresponding to VEGF and 36B4 transcripts was quantified using a phosphoImager. Under each condition the signal was normalized to the 36B4 probe. Results are expressed as arbitrary units corresponding to the fold stimulation of treated versus non-treated conditions. In C, increase in the VEGF protein in the culture medium of wild-type C1845-infected T84 cells. Cells were infected with 5×107 CFU/ml wild-type C1845 bacteria for four hours and the supernatant were collected as indicated in the Materials and Methods section. The VEGF protein level was then quantified using an ELISA. Quantification of the results from two independent experiments (means±SD) is shown. *, p<0,01
Mentions: Northern blot analysis of total RNA using a specific probe for the VEGF gene shows a very low level of VEGF mRNA expression in control, non-infected T84 cells while a time and dose-dependent increase in VEGF mRNA expression was observed in T84 infected cells (Figure 1A, upper panel). The transcript of 4 kb is preferentially expressed, however the expression of the three other spliced variants, ranging from 1.5 to 4.0 kb, is revealed with a longer exposure of the autoradiogram (data not shown). VEGF mRNA expression in non-infected and wild-type C1845-infected T84 cells was also quantified by q-PCR (Figure 1A, lower panel). The maximum increase in VEGF mRNA expression (3.6-fold increase) was achieved when cells were infected with 5×107 CFU/ml of wild-type C1845 bacteria for four hours. This increase in VEGF mRNA expression in response to wild-type C1845 bacteria is neither cell line-dependent, since we observed the same effect in the human LS174 colon cell line and the fully-differentiated enterocyte-like Caco-2/TC7 cells nor transformation-dependent since bacteria increase the expression of VEGF in INT407, a non-transformed cell line (Figure 1B).

Bottom Line: This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells.Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways.Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR 6543, Université de Nice-Sophia Antipolis, Nice, France.

ABSTRACT

Background: Angiogenesis has been recently described as a novel component of inflammatory bowel disease pathogenesis. The level of vascular endothelial growth factor (VEGF) has been found increased in Crohn's disease and ulcerative colitis mucosa. To question whether a pro-inflammatory Escherichia coli could regulate the expression of VEGF in human intestinal epithelial cells, we examine the response of cultured human colonic T84 cells to infection by E. coli strain C1845 that belongs to the typical Afa/Dr diffusely adhering E. coli family (Afa/Dr DAEC).

Methodology: VEGF mRNA expression was examined by Northern blotting and q-PCR. VEGF protein levels were assayed by ELISA and its bioactivity was analysed in endothelial cells. The bacterial factor involved in VEGF induction was identified using recombinant E. coli expressing Dr adhesin, purified Dr adhesin and lipopolysaccharide. The signaling pathway activated for the up-regulation of VEGF was identified using a blocking monoclonal anti-DAF antibody, Western blot analysis and specific pharmacological inhibitors.

Principal findings: C1845 bacteria induce the production of VEGF protein which is bioactive. VEGF is induced by adhering C1845 in both a time- and bacteria concentration-dependent manner. This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells. Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways.

Conclusions: Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells. Thus, these results suggest a link between an entero-adherent, pro-inflammatory E. coli strain and angiogenesis which appeared recently as a novel component of IBD pathogenesis.

Show MeSH
Related in: MedlinePlus