Differential angiogenic regulation of experimental colitis

Abstract

Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders of the intestinal tract with unknown multifactorial etiology that, among other things, result in alteration and dysfunction of the intestinal microvasculature. Clinical observations of increased colon microvascular density during IBD have been made. However, there have been no reports investigating the physiological or pathological importance of angiogenic stimulation during the development of intestinal inflammation. Here we report that the dextran sodium sulfate and CD4+CD45RBhigh T-cell transfer models of colitis stimulate angiogenesis that results in increased blood vessel density concomitant with increased histopathology, suggesting that the neovasculature contributes to tissue damage during colitis. We also show that leukocyte infiltration is an obligatory requirement for the stimulation of angiogenesis. The angiogenic response during experimental colitis was differentially regulated in that the production of various angiogenic mediators was diverse between the two models with only a small group of molecules being similarly controlled. Importantly, treatment with the anti-angiogenic agent thalidomide or ATN-161 significantly reduced angiogenic activity and associated tissue histopathology during experimental colitis. Our findings identify a direct pathological link between angiogenesis and the development of experimental colitis, representing a novel therapeutic target for IBD.

Figure 1

PECAM-1 blood vessel density measurement during experimental colitis. Vascular density of normal and diseased colons was determined by staining frozen sections with anti-PECAM-1 antibody (red) followed by DAPI nuclear counterstaining (blue). A: PECAM-1 staining of normal DSS vehicle-treated colon tissue. B: Increased PECAM-1 staining of 3% DSS-treated colon tissue. C: Secondary antibody control staining of DSS-treated colon tissue. D: DAPI alone staining of DSS-treated colon tissue. E: PECAM-1 staining of CD4⁺CD45RB^low T-cell transfer control colon tissue. F: Enhanced PECAM-1 staining of CD4⁺CD45RB^high T-cell transfer colitis tissue. G: Secondary antibody control staining of CD4⁺CD45RB^high T-cell transfer colitis tissue. H: DAPI alone staining of CD4⁺CD45RB^high T-cell transfer colitis tissue. I: Quantitative measurement of vascular density in the tissue sections as determined by calculating and angiogenic index that is derived from the total PECAM-1 surface area divided by the total DAPI surface area. J: The amount of radiolabeled PECAM-1 antibody binding within the colon vasculature demonstrating a significant increase in vessel surface area during experimental colitis. n values for each condition are reported within the bar graph. *P < 0.05 versus control. Original magnifications, ×200. Scale bar = 100 μm.

Figure 2

FITC L. esculentum lectin analysis of angiogenesis during experimental colitis. Fluorescently labeled L. esculentum lectin was injected intravenously to specifically stain endothelial cells to independently validate PECAM-1 antibody data as well as evaluate the perfusion of colitis-induced neovascularization. Distal colon tissue was also stained with PECAM-1 antibody to determine whether these markers of neovascularization co-localized with one another. A: PECAM-1 staining (red) of DSS-induced angiogenesis; B: FITC L. esculentum lectin intravascular labeling (green) of the same section. Importantly, C demonstrates a large amount of PECAM-1/lectin co-localization throughout the section. D: Lectin angiogenic index, which was obtained by dividing the total lectin surface area by the total DAPI surface area. n values for each condition are reported within the bar graph. *P < 0.05 versus control. Original magnifications, ×200. Scale bar = 100 μm.

Figure 3

Relationships between angiogenic index and tissue pathology during experimental colitis. Serial tissue sections were obtained from both experimental colitis models to evaluate histopathological score and angiogenic index from identical specimens. Angiogenic index and pathological score data were plotted against one another and correlation indexes determined. A: Angiogenic index and tissue pathology scores closely correlate in the 3% DSS colitis model with an r² = 0.90. B: Angiogenic index and tissue pathology closely correlate in the CD4⁺CD45RB^high T-cell colitis model with an r² = 0.91. P values and n values for correlation determination are reported in each graph. C: Temporal nature of increased angiogenesis during DSS-mediated colitis. D: The temporal nature of tissue histopathology during DSS-mediated colitis. n values for each condition are reported within the bar graph. *P < 0.05 versus day 0.

Figure 4

Measurement of VEGF-A expression levels during experimental colitis. VEGF-A protein levels were examined between the two experimental colitis models using Western blot and enzyme-linked immunodetection assays. Fifty μg of total protein tissue extract from control or experimental colitis colons was separated using SDS-polyacrylamide gel electrophoresis. A: VEGF-A protein levels by Western blot analysis between control and experimental colitis. B: Relative scan density of VEGF-A Western blots demonstrating a significant increase in VEGF-A protein in CD4⁺CD45RB^high colitis tissue extract. C: Temporal ELISA measurement of VEGF-A during DSS-induced colitis. D: ELISA measurement of VEGF-A protein (pg/μg total protein) in tissue extracts from CD4⁺CD45RB^low control and CD4⁺CD45RB^high T-cell transfer colons. n values for each experimental condition are reported within the bar graph. *P < 0.05 versus control.

Figure 5

Leukocyte recruitment is required for angiogenic activity during experimental colitis. The importance of leukocyte recruitment for angiogenic stimulation during experimental colitis was examined using CD18-null mutant mice treated with 3% DSS. A: Illustrates 3% DSS histopathology of wild-type mice displaying prominent leukocyte infiltration, tissue edema, and epithelial cell damage. Conversely, B shows 3% DSS-mediated tissue pathology in CD18-null mutant mice. C: 3% DSS-mediated histopathological scores between wild-type and CD18-null mice. Likewise, D reports angiogenic index scores from 3% DSS-treated wild-type or CD18-null mice. n values for each condition are reported within the bar graph. Original magnifications, ×200. Scale bar = 100 μm. *P < 0.05 versus control.

Figure 6

Thalidomide anti-angiogenic therapy attenuates DSS experimental colitis tissue pathology and neovascularization. The anti-angiogenic agent thalidomide (200 mg/kg) was used to evaluate the pathological importance of angiogenic activity during experimental colitis. A: Representative histomorphology from dimethyl sulfoxide vehicle-treated mice subjected to 3% DSS colitis. B: The effect of thalidomide treatment on 3% DSS-induced colitis. C: The tissue histopathology score between vehicle and thalidomide-treated mice subjected to 3% DSS colitis. Likewise, D demonstrates the angiogenic index between vehicle and thalidomide-treated mice subjected to 3% DSS colitis. n values for each condition are reported within the bar graph. *P < 0.05 versus control. Original magnifications, ×200. Scale bar = 100 μm.

Figure 7

ATN-161 anti-angiogenic therapy attenuates CD4⁺CD45RB^high experimental colitis tissue damage and neovascularization. The α₅β₁ and α_Vβ₃ angiogenic integrin antagonist ATN-161 was used to determine the pathological importance of neovascularization during CD4⁺CD45RB^high colitis. A: Representative histopathology of mice treated with ATN-163 (1 mg/kg) control peptide. B: Representative histopathology of mice treated with active drug ATN-161 (1 mg/kg). Note the stark contrast in immune cell infiltrates and crypt architecture. C: The histopathology scores from mice treated with ATN-163 or ATN-161. D: The angiogenic index of mice treated with ATN-163 or ATN-161. E: The effect of 10 μg/ml ATN-161 on leukocyte adhesion to unstimulated and 10 ng/ml TNF-α-activated endothelial monolayers. n values for each group are reported within the bar graph. *P < 0.05. Original magnifications, ×200. Scale bar = 100 μm.