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. 2013 May 13;8(5):e64227.
doi: 10.1371/journal.pone.0064227. Print 2013.

Probiotic yeast inhibits VEGFR signaling and angiogenesis in intestinal inflammation

Xinhua Chen  1 , Guoxun Yang, Joo-Hye Song, Hua Xu, Dan Li, Jeffrey Goldsmith, Huiyan Zeng, Patricia A Parsons-Wingerter, Hans-Christian Reinecker, Ciaran P Kelly
Affiliations

Probiotic yeast inhibits VEGFR signaling and angiogenesis in intestinal inflammation

Xinhua Chen et al. PLoS One. .

Abstract

Background and aims: Saccharomyces boulardii (Sb) can protect against intestinal injury and tumor formation, but how this probiotic yeast controls protective mucosal host responses is unclear. Angiogenesis is an integral process of inflammatory responses in inflammatory bowel diseases (IBD) and required for mucosal remodeling during restitution. The aim of this study was to determine whether Sb alters VEGFR (vascular endothelial growth factor receptor) signaling, a central regulator of angiogenesis.

Methods: HUVEC were used to examine the effects of Sb on signaling and on capillary tube formation (using the ECMatrix™ system). The effects of Sb on VEGF-mediated angiogenesis were examined in vivo using an adenovirus expressing VEGF-A(164) in the ears of adult nude mice (NuNu). The effects of Sb on blood vessel volume branching and density in DSS-induced colitis was quantified using VESsel GENeration (VESGEN) software.

Results: 1) Sb treatment attenuated weight-loss (p<0.01) and histological damage (p<0.01) in DSS colitis. VESGEN analysis of angiogenesis showed significantly increased blood vessel density and volume in DSS-treated mice compared to control. Sb treatment significantly reduced the neo-vascularization associated with acute DSS colitis and accelerated mucosal recovery restoration of the lamina propria capillary network to a normal morphology. 2) Sb inhibited VEGF-induced angiogenesis in vivo in the mouse ear model. 3) Sb also significantly inhibited angiogenesis in vitro in the capillary tube assay in a dose-dependent manner (p<0.01). 4) In HUVEC, Sb reduced basal VEGFR-2 phosphorylation, VEGFR-2 phosphorylation in response to VEGF as well as activation of the downstream kinases PLCγ and Erk1/2.

Conclusions: Our findings indicate that the probiotic yeast S boulardii can modulate angiogenesis to limit intestinal inflammation and promote mucosal tissue repair by regulating VEGFR signaling.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist. The patent application entitled “Vessel Generation Analysis” has been filed with the United States Patent Office by NASA and Dr. Patricia Parsons-Wingerter as Lead Innovator. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors by PLOS ONE.

Figures

Figure 1
Figure 1. Oral administration of Sb reduced DSS-colitis induced weight loss, histological damage and neo-vascularization in mouse colon.
We used the murine DSS-colitis model to assess the effect of Sb on neo-vascularization in acute colitis. We administered DSS (4% for 5 days) to mice to induce colitis. 1A) Daily administration of Sb by gavage significantly attenuated weight-loss (N = 5, *p<0.05, **p<0.01). Data points represent mean relative weight ± standard error. 1B) Histological scores for several parameters of colonic inflammation on Day 5 were evaluated [control (N = 5), DSS alone (N = 5), DSS plus Sb (N = 5)]. Data points represent mean score ± standard error, **, p<0.01. Representative H/E stained colonic images are listed. 1C) Confocal fluorescence images of the colonic microvasculature were taken 10 minutes after i.v injection of Alexa 647 WGA. Three dimensional image reconstructions were translated into two dimensional grayscale images and binarized to black-and-white vascular patterns for analysis using VESGEN software. Representative images are shown. 1D) Quantitative data are presented in Tables 1 and 2. Comparisons of vascular network and avascular space indices among control, DSS and DSS+ Sb groups. Compared to control mice, the DSS group had significantly increased numbers of branch points and density of vessel endpoints (p = 0.004, p = 0.02 respectively). Sb treatment significantly reduced these DSS effects (p = 0.045, p = 0.02 respectively). The mean area per AVS (µm2) in the lamina propria was reduced in the DSS group compared to the control group (p = 0.01). Sb treatment normalized this effect (p = 0.02).
Figure 2
Figure 2. Sb inhibits VEGF-induced angiogenesis in the mouse ear vasculature model.
The effects of Sb on VEGF-induced angiogenesis in vivo was assessed using an adenovirus expressing VEGF-A(164) (Ad-VEGF-A(164)) in the ears of adult nude mice (NuNu). Ad-VEGF-A164 (5×106 pfu) was injected into both ears. One hour later the right ears of mice (N = 5) were injected s.c. with 10 µl SbS (ion-exchange chromatography enriched and DMEM diluted) and the left ears were injected s.c. with 10 µl vehicle (DMEM) as control. A second injection of SbS or vehicle was administered 24 hours later. Each of the 5 mice showed an angiogenesis response to Ad-VEGF-A164 in the control (left) ear. The inhibitory effect of SbS was evident with reduced new vessel formation in the right ear compared to the left. The images shown were obtained on day 16 after Ad-VEGF-A164 administration.
Figure 3
Figure 3. Sb inhibits HUVEC capillary tube formation.
The ECMatrix™ in vitro angiogenesis assay was used to examine the effects of Sb on capillary tube formation. HUVEC (∼1×104 cells) were cultured in 96-well plates previously coated with Matrigel and incubated for 16 hours at 37°C in the absence or presence of different doses of SbS. Representative images of tubule formation in the control and SbS-treated groups are shown in the lower panel. Tubular structures were counted from 10 randomly selected images. Percentage (%) of control is the mean number of tubules expressed as a proportion of that in the control group. SbS significantly inhibited HUVEC tubule formation in a dose-dependent manner (*p<0.05 for 1/32 dilution vs control; **p<0.01 for 1/16, 1/8, 1/4 vs control). Bars represent mean ± SE.
Figure 4
Figure 4. Sb inhibits VEGFR signaling in HUVEC.
4A) HUVEC grown normally in complete medium were exposed to SbS for varying time periods (0–180 minutes). Cell extracts were prepared for Western blotting using antibodies against total and phopho-specific VEGFR-2. The band of phosphorylated VEGFR-2 immediately disappeared (within 1 minute) upon exposure to SbS. In contrast, the amount of total VEGFR2 remained stable. 4B) HUVEC were starved overnight and then treated with 10 ng/ml VEGF in the presence or absence of SbS for 0 to 30 minutes. Cell extracts were prepared for Western blotting with antibodies against total or phopho-specific VEGFR-2, PLCγ, or Erk1/2. VEGF induced activation of both VEGFR-2 phosphorylation sites as recognized by p-Tyr1175 and p-Tyr996 phospho-site specific antibodies. The downstream mediators PLCγ and Erk1/2 are also activated by VEGF. The presence of SbS inhibited the phosphorylation of VEGFR-2 in response to VEGF and also reduced activation of the downstream kinases PLCγ and Erk1/2. Total protein levels were not affected.
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