Acquired microvascular dysfunction in inflammatory bowel disease: loss of nitric oxide- mediated vasodilation  Ossama A Hatoum, David G Binion, Mary F Otterson, David D Gutterman  Gastroenterology  Volume 125, Issue 1, Pages 58-69 (July 2003) DOI: 10.1016/S0016-5085(03)00699-1

Figure 1 Comparison of Ach-induced dilation of intestinal arterioles in patients with and without IBD. (A ) A segment of normal large bowel with partial trimming of the mucosa showing a dense layer of submucosal microvessels (arrows). (B) Responses to Ach in human submucosal intestinal microvessels from control patients and patients with IBD using in vitro videomicroscopy. Microvessels from control tissues dilate in response to increasing doses of Ach (n = 34), whereas microvessels from IBD tissues (n = 33) showed significantly attenuated dilation to Ach (∗P < 0.05). (C ) The response of microvessels from patients with CD and UC. Microvessels from patients with CD (n = 20) and UC (n = 13) showed similar attenuation in dilation to Ach. Vessels were preconstricted with endothelin 1. The y-axis indicates the percent change from preconstricted diameter. Values are presented as mean ± SEM. Gastroenterology 2003 125, 58-69DOI: (10.1016/S0016-5085(03)00699-1)

Figure 1 Comparison of Ach-induced dilation of intestinal arterioles in patients with and without IBD. (A ) A segment of normal large bowel with partial trimming of the mucosa showing a dense layer of submucosal microvessels (arrows). (B) Responses to Ach in human submucosal intestinal microvessels from control patients and patients with IBD using in vitro videomicroscopy. Microvessels from control tissues dilate in response to increasing doses of Ach (n = 34), whereas microvessels from IBD tissues (n = 33) showed significantly attenuated dilation to Ach (∗P < 0.05). (C ) The response of microvessels from patients with CD and UC. Microvessels from patients with CD (n = 20) and UC (n = 13) showed similar attenuation in dilation to Ach. Vessels were preconstricted with endothelin 1. The y-axis indicates the percent change from preconstricted diameter. Values are presented as mean ± SEM. Gastroenterology 2003 125, 58-69DOI: (10.1016/S0016-5085(03)00699-1)

Figure 1 Comparison of Ach-induced dilation of intestinal arterioles in patients with and without IBD. (A ) A segment of normal large bowel with partial trimming of the mucosa showing a dense layer of submucosal microvessels (arrows). (B) Responses to Ach in human submucosal intestinal microvessels from control patients and patients with IBD using in vitro videomicroscopy. Microvessels from control tissues dilate in response to increasing doses of Ach (n = 34), whereas microvessels from IBD tissues (n = 33) showed significantly attenuated dilation to Ach (∗P < 0.05). (C ) The response of microvessels from patients with CD and UC. Microvessels from patients with CD (n = 20) and UC (n = 13) showed similar attenuation in dilation to Ach. Vessels were preconstricted with endothelin 1. The y-axis indicates the percent change from preconstricted diameter. Values are presented as mean ± SEM. Gastroenterology 2003 125, 58-69DOI: (10.1016/S0016-5085(03)00699-1)

Figure 2 Effect of endothelial denudation on Ach-induced human intestinal arteriolar dilation. (A ) Mechanical endothelial denudation of submucosal intestinal microvessels from control tissues abolished the dilation to Ach (n = 6), resulting in a pronounced vasoconstriction. (B and C ) The effect of papaverine and nitroprusside, respectively, on human intestinal microvessel vasodilation. Dose-dependent dilation was observed in response to papaverine and nitroprusside in control patients and patients with IBD. Values represent mean ± SEM. Gastroenterology 2003 125, 58-69DOI: (10.1016/S0016-5085(03)00699-1)

Figure 3 The role of NO and vasodilator prostaglandins in Ach-induced vasodilation in IBD and control intestinal arterioles. Effect of inhibiting (A ) NO synthase by l-NAME (n = 7), (B) cyclooxygenase by indomethacin (n = 8), or (C ) both (n = 6) on the dilation of human control intestinal microvessels to Ach. Either inhibitor reduced dilation to Ach by 50%, whereas the combined treatment with indomethacin and l-NAME produced an almost 80% reduction in dilation. In D–F, microvessels from patients with IBD were incubated with both l-NAME (n = 5), indomethacin (n = 7), and combined treatment (l-NAME and indomethacin; n = 7), respectively. IBD vessels showed no effect with l-NAME alone but, in contrast to control vessels, with indomethacin or indomethacin plus l-NAME showed pronounced vasoconstriction in response to Ach (#P < 0.05 vs. control). Values represent mean ± SEM. Gastroenterology 2003 125, 58-69DOI: (10.1016/S0016-5085(03)00699-1)

Figure 3 The role of NO and vasodilator prostaglandins in Ach-induced vasodilation in IBD and control intestinal arterioles. Effect of inhibiting (A ) NO synthase by l-NAME (n = 7), (B) cyclooxygenase by indomethacin (n = 8), or (C ) both (n = 6) on the dilation of human control intestinal microvessels to Ach. Either inhibitor reduced dilation to Ach by 50%, whereas the combined treatment with indomethacin and l-NAME produced an almost 80% reduction in dilation. In D–F, microvessels from patients with IBD were incubated with both l-NAME (n = 5), indomethacin (n = 7), and combined treatment (l-NAME and indomethacin; n = 7), respectively. IBD vessels showed no effect with l-NAME alone but, in contrast to control vessels, with indomethacin or indomethacin plus l-NAME showed pronounced vasoconstriction in response to Ach (#P < 0.05 vs. control). Values represent mean ± SEM. Gastroenterology 2003 125, 58-69DOI: (10.1016/S0016-5085(03)00699-1)

Figure 4 Role of ROS in endothelium-dependent vasodilation. ROS production in human intestinal microvessels from control, uninvolved, and involved tissue from patients with IBD was assessed using fluorochromes and confocal fluorescence microscopy. Microvessels were incubated for 10 minutes in HE and DCF-DA before confocal fluorescence microscopy. Relative fluorescence intensity (red and green) corresponds with superoxide anion and ROS formation in the vessel wall, respectively. The relative fluorescence is measured in reference to freshly isolated vessels from the same surgical specimen pretreated with superoxide dismutase plus catalase. Pretreatment with these enzymes allows for a basal assessment of minimal ROS generation in these vessels. (C and D) Involved IBD microvessels showed increased HE and DCF fluorescence within the vessel wall compared with (A and B) vessels from uninvolved IBD and control tissues, which corresponds with increased generation of superoxide and ROS. (E ) Summary of the fluorescence intensity results using image analysis software showed significantly greater levels of O2· − and ROS production in involved vessels compared with adjacent uninvolved vessels from the same patients with IBD and from control subjects. (F ) Effects of the superoxide dismutase mimetic MnTBAP on endothelium-dependent dilation to Ach in chronically inflamed IBD microvessels. MnTBAP significantly improved dilation to Ach in microvessels from patients with IBD. Note that Ach-induced dilation was still impaired compared with vessel responses from control tissues. #P < 0.05 vs. control. Values represent mean ± SEM. Gastroenterology 2003 125, 58-69DOI: (10.1016/S0016-5085(03)00699-1)

Figure 4 Role of ROS in endothelium-dependent vasodilation. ROS production in human intestinal microvessels from control, uninvolved, and involved tissue from patients with IBD was assessed using fluorochromes and confocal fluorescence microscopy. Microvessels were incubated for 10 minutes in HE and DCF-DA before confocal fluorescence microscopy. Relative fluorescence intensity (red and green) corresponds with superoxide anion and ROS formation in the vessel wall, respectively. The relative fluorescence is measured in reference to freshly isolated vessels from the same surgical specimen pretreated with superoxide dismutase plus catalase. Pretreatment with these enzymes allows for a basal assessment of minimal ROS generation in these vessels. (C and D) Involved IBD microvessels showed increased HE and DCF fluorescence within the vessel wall compared with (A and B) vessels from uninvolved IBD and control tissues, which corresponds with increased generation of superoxide and ROS. (E ) Summary of the fluorescence intensity results using image analysis software showed significantly greater levels of O2· − and ROS production in involved vessels compared with adjacent uninvolved vessels from the same patients with IBD and from control subjects. (F ) Effects of the superoxide dismutase mimetic MnTBAP on endothelium-dependent dilation to Ach in chronically inflamed IBD microvessels. MnTBAP significantly improved dilation to Ach in microvessels from patients with IBD. Note that Ach-induced dilation was still impaired compared with vessel responses from control tissues. #P < 0.05 vs. control. Values represent mean ± SEM. Gastroenterology 2003 125, 58-69DOI: (10.1016/S0016-5085(03)00699-1)

Figure 5 Microvascular endothelial dysfunction in IBD is acquired and specific to areas of chronic inflammation. (A ) Vessels from patients with diverticulitis showed preservation of Ach-induced vasodilation compared with control vessels. (B) Paired specimens included vessels from unaffected segments (uninvolved IBD) and involved areas from 6 patients with IBD. Uninvolved IBD microvessels showed similar patterns of Ach-induced vasodilation compared with normal controls. (C ) Microvessels isolated from the mesentery of patients with IBD (n = 4) showed similar Ach-induced dilation compared with control mesenteric vessels. #P < 0.05 vs. control. Values represent mean ± SEM. Gastroenterology 2003 125, 58-69DOI: (10.1016/S0016-5085(03)00699-1)