1. Volume 144, Issue 3, Pages 613-623.e9 (March 2013)
Pro-Angiogenic Activity of TLRs and NLRs: A Novel Link Between Gut Microbiota and Intestinal Angiogenesis 
Anja Schirbel, Sean Kessler, Florian Rieder, Gail West, Nancy Rebert, Kewal Asosingh, Christine McDonald, Claudio Fiocchi 
Gastroenterology 
Volume 144, Issue 3, Pages e9 (March 2013)
DOI: /j.gastro
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2. Figure 1
(A) TLR and NLR activation-induced HIMEC migration. Wounded control and IBD HIMEC monolayers were exposed to optimal stimulatory doses of bFGF, TNF-α, and TLR2/6, TLR4, NOD1, and NOD2 ligands, and migrated cells were counted after 24 hours. *P < .04–.01 for stimulated vs unstimulated (medium) control HIMECs, **P < .02–.002 for stimulated vs unstimulated (medium) IBD HIMECs (n = 10 and 14 for control and IBD HIMECs, respectively). L, ligand; hpf, high-power field. (B) TLR and NLR activation induced HIMEC transmigration. Control and IBD HIMECs were placed in the upper compartment of a Boyden chamber, optimal stimulatory doses of bFGF, TNF-α, and TLR2/6, TLR4, NOD1, and NOD2 ligands were placed in the lower compartment, and the number of transmigrated cells was counted after 8 hours. *P < .03–.01 for stimulated vs unstimulated (medium) control HIMECs, **P < .01 for stimulated vs unstimulated (medium) IBD HIMECs (n = 8 for both control and IBD HIMECs). (C) TLR and NLR activation induced HIMEC proliferation. Control and IBD HIMEC monolayers were exposed to optimal stimulatory doses of bFGF, TNF-α, and TLR2/6, TLR4, NOD1, and NOD2 ligands, and proliferation was assessed by 3H thymidine uptake after 24 hours. *P < .001 for stimulated vs unstimulated (medium) control HIMECs, **P < .01–.001 for stimulated vs unstimulated IBD HIMECs (n = 10 and 11 for control and IBD HIMECs, respectively).
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3. Figure 2
(A) TLR and NLR activation-induced HIMEC tube formation. Control and IBD HIMECs were seeded on Matrigel in the presence and absence of optimal stimulatory doses of VEGF, TNF-α, and TLR2/6, TLR4, NOD1, and NOD2 ligands, and tube formation visually was assessed for up to 24 hours. Figure is representative of 9 and 8 experiments for control and IBD HIMECs, respectively, at 6 hours. (B) TLR- and NLR-induced HIMEC vessel sprouting in a murine aortic ring assay. Murine aortic rings were placed in Matrigel in the presence and absence of optimal stimulatory doses of VEGF, TNF-α, and TLR2/6, TLR4, NOD1, and NOD2 ligands, and vessel sprouting was visually assessed after 6 days. Figure is representative of 5 and 5 experiments for control and IBD HIMECs, respectively.
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4. Figure 3
VEGF-, TLR4L-, and NLRL-induced cell infiltration and vessel formation in vivo. Collagen gels individually containing medium alone (control), VEGF, TLR4 or NOD1 ligands were implanted surgically in the abdominal subcutaneous tissue of mice, harvested after 7 days, and stained for H&E and CD31 to visualize vessel growth. Upper panels were taken at 10× magnification, lower panels were taken at 40× magnification. Figure is representative of 5 experiments. TLR4L (UP), ultrapure LPS; TLR4L (C), crude LPS.
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5. Figure 4
(A) HIMEC proliferation in response to HIF-conditioned medium. HIMECs were cultured alone (medium), in the presence of TNF-α or optimal stimulatory doses of TLR and NLR ligands (black columns), or in HIF medium conditioned alone (medium), by TNF-α, or the same doses of TLR and NLR ligands (grey columns); after 24 hours proliferation was assessed by 3H thymidine uptake. *P < .05–.03 for TLR/NLR activation-conditioned HIF medium compared with direct stimulation with TLR and NLR ligands, **P < .03 for TLR4 activation-conditioned HIF medium compared with unstimulated HIF medium (n = 10 for both control and IBD HIMECs). HIFs (n = 3) also were cultured in the presence of 0%, 2%, 5%, 10%, and 20% FBS for 24, 48, and 72 hours, respectively, and proliferation was assessed as described earlier. None of the different concentrations of FBS induced a significant increase in HIMEC proliferation compared with HIMEC cultured alone (0% FBS) at any of the 3 times points (not shown). (B) IL-8 production by TLR- and NLR-activated HIMECs. Control and IBD HIMEC monolayers were exposed to optimal stimulatory doses of VEGF, TNF-α, TLR2/6, TLR4, NOD1, and NOD2 ligands for 24 hours and levels of IL-8 were measured by enzyme-linked immunosorbent assay in the culture supernatants. *P < .03–.001 for stimulated vs unstimulated (medium) control HIMECs, **P < .004–.001 for stimulated vs unstimulated (medium) IBD HIMECs (n = 5 for both control and IBD HIMECs). (C) IL-8 production by TLR- and NLR-activated HIFs. Control and IBD HIF monolayers were exposed to optimal stimulatory doses of bFGF, TNF-α, TLR2/6, TLR4, NOD1, and NOD2 ligands for 24 hours and levels of IL-8 were measured by enzyme-linked immunosorbent assay in the culture supernatants. *P < .05–.004 for stimulated vs unstimulated (medium) control HIFs, **P < .03–.004 for stimulated vs unstimulated IBD HIFs (n = 3 and 4 for control and IBD HIFs, respectively).
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6. Figure 5
(A) Effect of RIP2 knockdown on NLR activation induced HIMEC transmigration. Transmigration was performed as described earlier in Figure 1B, using cells transfected with scrambled or RIP2 siRNA and then exposed to medium alone, bFGF, TNF-α, or NOD1 and NOD2 ligands for 8 hours. *P < .01 for siRIP2 RNA vs scrambled siRNA (n = 3 and 3 for control and IBD HIMECs, respectively). hpf, high-power field. (B) Effect of RIP2 knockdown on NLR-induced HIMEC tube formation. The tube formation assay was performed as described earlier in Figure 2A, using cells transfected with scrambled or RIP2 siRNA and then exposed to medium alone, NOD1L, or NOD2L. Figure is representative of 3 and 2 experiments with control and IBD HIMECs, respectively, at 4 hours.
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7. Figure 6
(A) Effect of TRAF6 knockdown on TLR and NLR activation induced HIMEC transmigration. Transmigration was performed as described earlier in Figure 1B, using cells transfected with scrambled or TRAF6 siRNA and then exposed to medium alone, bFGF, or TLR2/6, TLR4, NOD1, and NOD2 ligands for 8 hours. *P < .04–.006 for siTRAF6 RNA vs scrambled RNA (n = 3 and 3 for control and IBD HIMECs, respectively). hpf, high-power field. (B) Effect of TRAF6 knockdown on TLR- and NLR-induced HIMEC tube formation. The tube formation assay was performed as described earlier in Figure 2A, using cells transfected with scrambled or TRAF6 siRNA and then exposed to medium alone, TLR2/6, TLRL4, NOD1 or NOD2 ligands. Figure is representative of 3 and 2 experiments with control and IBD HIMECs, respectively, at 5 hours.
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8. Figure 7
(A) TLR- and NLR-mediated MAPK and NF-κB activation. HIMEC monolayers were exposed to optimal stimulatory doses of TLR2/6, TLR4, NOD1, or NOD2 ligands for the indicated time points, harvested, lysed, and protein extracted for immunoblotting using antibodies against total and phosphorylated (p) p38, ERK, IKBa, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Figure is representative of 3 control and 3 IBD HIMECs, respectively. (B) TLR and NLR activation-induced VEGF-R2 phosphorylation. HIMEC monolayers were exposed to optimal stimulatory doses of TNF-α or TLR2/6, TLR4, NOD1, or NOD2 ligands for 48 hours, harvested, lysed, and protein extracted for immunoblotting using the respective antibody and GAPDH antibody. Lower panels: densitometric analysis of the ratio of p-VEGF-R2/VEGF-R2 under each condition. Figure is representative of 3 control and 5 IBD HIMECs. (C) TLR and NLR activation-induced FAK phosphorylation. HIMEC monolayers were exposed to optimal stimulatory doses of VEGF or TLR2/6, TLR4, NOD1, or NOD2 ligands for 48 hours, harvested, lysed, and protein extracted for immunoblotting using the respective antibody and GAPDH antibody. Lower panels: densitometric analysis of the ratio of p-FAK/total FAK under each condition. Figure is representative of 3 control and 5 IBD HIMECs.
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9. Supplementary Figure 1
(A) NLR and TLR activation-induced HIMEC migration. Confluent HIMEC monolayers were scraped with a sterile razor blade, washed with phosphate-buffered saline to remove cell debris, and then cultured for 24 hours in the absence (medium) and presence of bacterial ligands specific for TLR2/6, TLR4, NOD1, and NOD2 or the pro-angiogenic factor bFGF as a positive control. Migration was assessed by counting the total number of cells crossing below and above the wound (demarcated by the 2 red lines) in 4 random microscopic high-power fields at 100× magnification using an image analysis system. Figure shows results with an IBD HIMEC line at 24 hours and is representative of 10 control and 14 IBD HIMEC experiments. (B) Comparative effect of bFGF and VEGF on HIMEC migration. Control and IBD HIMEC monolayers were processed as described earlier in panel A. They were exposed to optimal stimulatory doses of bFGF, VEGF, and TNF-α, and TLR2/6, TLR4, NOD1, and NOD2 ligands, and migrated cells were counted after 24 hours. Because bFGF appeared to be slightly more potent than VEGF, it was selected for further migration, transmigration, and proliferation experiments. *P < .04–.01 for stimulated vs unstimulated (medium) control HIMECs. P = .63 was considered not significant. **P < .02–.002 for stimulated vs unstimulated (medium) IBD HIMECs (n = 6 and 6 for control and IBD HIMECs, respectively). L, ligand; hpf, high-power field.
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10. Supplementary Figure 2
VEGF- and bFGF-induced HIMEC tube formation. HIMECs were seeded on Matrigel in the presence and absence of optimal stimulatory doses of VEGF and bFGF and tube formation was assessed visually for up to 24 hours. Because VEGF was slightly more potent than bFGF, VEGF was used in all subsequent tube formation assays. Figure is representative of 2 experiments.
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11. Supplementary Figure 3
(A) TLR- and NLR-induced HIMEC vessel sprouting in murine aortic ring assay. Murine aortic rings were placed in Matrigel in the presence and absence of optimal stimulatory doses of VEGF, TNF-α, and TLR2/6, TLR4, NOD1, and NOD2 ligands, and vessel sprouting was assessed visually after 6 days. The upper panel and lower panels are magnifications (40× and 60×, respectively) of experimental results displayed in Figure 2B, and show that cells emerging from the aortic explants form typical microvascular structures with multiple sequential branching. (B) Sprouting of endothelial cells from aortic rings from Tie2 GFP mice. Aortic rings isolated from transgenic mice expressing GFP exclusively in endothelial cells were treated with the indicated ligand while embedded in Matrigel. Sprouting GFP-expressing endothelial cells that emigrated out of the aorta into the Matrigel were immunostained (green) with goat anti-GFP, rabbit anti-goat biotin, and streptavidin antibodies. Slides were mounted with Vectashield with blue 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. The expected green autofluorescence of the connective tissue (red arrows) is noted in all panels, but not in the (A) unstained cells (white arrows) sprouting from the aortic ring, confirming the lack of cell autofluorescence. (B–E) Green anti-GFP specific immunostaining of cells (yellow arrows) close to or sprouting from the aortic ring is noted. All images are at 40× magnification. Figure is representative of 3 experiments. (C) Endothelial cell marker expression by cells sprouting from aortic rings from Tie2 GFP mice. To confirm that the transgenic cells emigrating out of the murine Tie2-GFP aortas into the Matrigel in response to the indicated ligands are in fact endothelial cells, sequential sections to those shown in panel A were immunostained with both anti-CD31 (red) and anti-GFP (green), followed by the appropriate secondary Alexa-Fluor conjugate (Life Technologies) along with nuclei blue staining with DAPI. Co-localized staining for both markers appears yellow. All images are at 40× magnification. Figure is representative of 3 experiments.
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12. Supplementary Figure 3
(A) TLR- and NLR-induced HIMEC vessel sprouting in murine aortic ring assay. Murine aortic rings were placed in Matrigel in the presence and absence of optimal stimulatory doses of VEGF, TNF-α, and TLR2/6, TLR4, NOD1, and NOD2 ligands, and vessel sprouting was assessed visually after 6 days. The upper panel and lower panels are magnifications (40× and 60×, respectively) of experimental results displayed in Figure 2B, and show that cells emerging from the aortic explants form typical microvascular structures with multiple sequential branching. (B) Sprouting of endothelial cells from aortic rings from Tie2 GFP mice. Aortic rings isolated from transgenic mice expressing GFP exclusively in endothelial cells were treated with the indicated ligand while embedded in Matrigel. Sprouting GFP-expressing endothelial cells that emigrated out of the aorta into the Matrigel were immunostained (green) with goat anti-GFP, rabbit anti-goat biotin, and streptavidin antibodies. Slides were mounted with Vectashield with blue 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. The expected green autofluorescence of the connective tissue (red arrows) is noted in all panels, but not in the (A) unstained cells (white arrows) sprouting from the aortic ring, confirming the lack of cell autofluorescence. (B–E) Green anti-GFP specific immunostaining of cells (yellow arrows) close to or sprouting from the aortic ring is noted. All images are at 40× magnification. Figure is representative of 3 experiments. (C) Endothelial cell marker expression by cells sprouting from aortic rings from Tie2 GFP mice. To confirm that the transgenic cells emigrating out of the murine Tie2-GFP aortas into the Matrigel in response to the indicated ligands are in fact endothelial cells, sequential sections to those shown in panel A were immunostained with both anti-CD31 (red) and anti-GFP (green), followed by the appropriate secondary Alexa-Fluor conjugate (Life Technologies) along with nuclei blue staining with DAPI. Co-localized staining for both markers appears yellow. All images are at 40× magnification. Figure is representative of 3 experiments.
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13. Supplementary Figure 3
(A) TLR- and NLR-induced HIMEC vessel sprouting in murine aortic ring assay. Murine aortic rings were placed in Matrigel in the presence and absence of optimal stimulatory doses of VEGF, TNF-α, and TLR2/6, TLR4, NOD1, and NOD2 ligands, and vessel sprouting was assessed visually after 6 days. The upper panel and lower panels are magnifications (40× and 60×, respectively) of experimental results displayed in Figure 2B, and show that cells emerging from the aortic explants form typical microvascular structures with multiple sequential branching. (B) Sprouting of endothelial cells from aortic rings from Tie2 GFP mice. Aortic rings isolated from transgenic mice expressing GFP exclusively in endothelial cells were treated with the indicated ligand while embedded in Matrigel. Sprouting GFP-expressing endothelial cells that emigrated out of the aorta into the Matrigel were immunostained (green) with goat anti-GFP, rabbit anti-goat biotin, and streptavidin antibodies. Slides were mounted with Vectashield with blue 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. The expected green autofluorescence of the connective tissue (red arrows) is noted in all panels, but not in the (A) unstained cells (white arrows) sprouting from the aortic ring, confirming the lack of cell autofluorescence. (B–E) Green anti-GFP specific immunostaining of cells (yellow arrows) close to or sprouting from the aortic ring is noted. All images are at 40× magnification. Figure is representative of 3 experiments. (C) Endothelial cell marker expression by cells sprouting from aortic rings from Tie2 GFP mice. To confirm that the transgenic cells emigrating out of the murine Tie2-GFP aortas into the Matrigel in response to the indicated ligands are in fact endothelial cells, sequential sections to those shown in panel A were immunostained with both anti-CD31 (red) and anti-GFP (green), followed by the appropriate secondary Alexa-Fluor conjugate (Life Technologies) along with nuclei blue staining with DAPI. Co-localized staining for both markers appears yellow. All images are at 40× magnification. Figure is representative of 3 experiments.
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14. Supplementary Figure 4
(A) HIMEC proliferation in response to HIF-conditioned medium. HIMECs were cultured in medium alone (white column), TNF-α alone (light gray column), or in the presence of increasing (10%, 20%, and 50%) concentrations of medium conditioned by HIF alone (dark gray columns) or TNF-α–stimulated HIFs (black columns), and proliferation was assessed by 3H-thymidine uptake after 24 hours (n = 3). *P < .05 and **P < .01 compared with medium alone or TNF-α alone. (B) bFGF production by TLR- and NLR-activated HIMECs. Control and IBD HIMEC monolayers were exposed to optimal stimulatory doses of VEGF, TNF-α, or TLR2/6, TLR4, NOD1, and NOD2 ligands for 24 hours and levels of bFGF were measured by enzyme-linked immunosorbent assay (ELISA) in the culture supernatants (n = 4 and 5 for control and IBD HIMECs, respectively). *P < .05 for IBD HIMECs compared with medium alone.
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15. Supplementary Figure 5
(A) Effect of bFGF and IL-8 blockade on TLR and NLR activation-induced HIMEC migration. Wounded control and IBD HIMEC monolayers were exposed to optimal stimulatory doses of bFGF, TNF-α, TLR2/6, TLR4, NOD1, and NOD2 ligands, in the presence and absence of combined bFGF and IL-8 blocking antibodies, and migrated cells were counted after 24 hours. *P < .05–.001 for comparison of experiments with and without blocking antibodies (n = 3 for both control and IBD HIMECs). hpf, high-power field. (B) Effect of VEGF and IL-8 blockade on conditioned HIF medium-induced HIMEC proliferation. HIMECs were cultured alone (medium), in the presence of TNF-α or optimal stimulatory doses of TLR and NLR ligands (black columns), or in HIF medium conditioned alone (medium), conditioned by TNF-α, or conditioned by the same doses of TLR and NLR ligands (grey columns). bFGF was used as a positive control. Experiments were performed in the presence and absence of combined VEGF and IL-8 blocking antibodies, and proliferation was assessed by 3H-thymidine uptake after 24 hours. *P < .05–.01 for comparison of experiments with and without blocking antibodies (n = 3 for both control and IBD HIMECs).
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16. Supplementary Figure 6
Knockdown of RIP2 and TRAF6 protein by RNA interference. HIMECs were transfected by electroporation with (A) 0.02, 0.05, or 0.1 nmol/mL of RIP2 siRNA, (B) 0.2, 0.4, or 0.6 nmol/mL of TRAF6 siRNA, the same doses of the respective scrambled siRNA, or left alone in the Nucleofector solution without applying electroporation. After 24, 48, or 72 hours the cells were harvested, lysed, protein extracted, and used for immunoblotting with antibodies specific for human RIP2, TRAF6, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Figure is representative of 5 and 3 experiments for RIP2 and TRAF6, respectively, performed at 48 hours.
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17. Supplemental Figure 7
Lack of NOD1 or NOD2 stimulatory components in Matrigel used for tube formation assay. Enhanced green fluorescence protein fluorescence of HEK293 cells and HEK293 2F10 cells transfected with NOD1 or NOD2 expression plasmids and NF-κB–enhanced green fluorescence protein reporter were plated on Matrigel in medium alone or in the presence of NOD1 and NOD2 ligands for 22 hours. Cells plated in medium alone showed minimal fluorescence (left panels), indicating the absence of NOD1 or NOD2 stimulatory components in the Matrigel, whereas cells plated with added NOD1 and NOD2 ligands (right panels) displayed strong enhanced green fluorescence protein fluorescence.
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