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doi:10.1111/jgh.12748
O R I G I N A L A RT I C L E
Confocal laser endomicroscopy: A new gold standard for the assessment of mucosal healing in ulcerative colitis Vincent Macé,*,1 Amrita Ahluwalia,†,1 Emmanuel Coron,* Marc Le Rhun,* Arnaud Boureille,* Céline Bossard,‡ Jean-François Mosnier,‡ Tamara Matysiak-Budnik* and Andrzej S Tarnawski† *Institut des Maladies de l’Appareil Digestif, CIC INSERM 04 et Service d’Hépato-Gastroentérologie, and ‡Service d’Anatomie Pathologique et EA-4273 BIOMETADYS, CHU de Nantes, France; and †VALBHS and the University of California, Irvine, California, USA
Key words angiogenesis, confocal laser endomicroscopy (CLE), COX2, mitochondrial DNA (mtDNA) mutation, mucosal healing, ulcerative colitis. Correspondence Andrzej S Tarnawski, Veterans Administration Long Beach Healthcare System, and the University of California, Irvine, 5901 E. 7th Street (09/151), Long Beach, CA 90822-5201, USA. Email: [email protected]; [email protected] 1
These authors contributed equally.
Supportive foundations: Southern California Institute for Research and Education, and the VA Merit Review grant to Tarnawski, AS. Conflict of interest: The funding for this study was provided by Southern California Institute for Research and Education, and the VA Merit Review Award to AST.
Abstract Background and Aim: Endoscopic assessment of mucosal healing in ulcerative colitis (UC) is increasingly accepted as a measure of disease activity, therapeutic goal, and the key prognostic indicator. While regular endoscopy evaluates appearance of the mucosal surface, confocal laser endomicroscopy (CLE) enables in vivo visualization of subepithelial mucosa at 1000× magnification during ongoing endoscopy. Our aims were to determine using CLE whether endoscopically normal appearing colonic mucosa in patients with UC in remission (UC-IR) has fully regenerated mucosal structures, resolved inflammation, and to identify the mechanisms. Methods: Twelve patients (six controls and six with UC-IR) underwent colonoscopy using CLE and intravenous fluorescein infusion. During colonoscopy, CLE images of colonic mucosa and conventional mucosal biopsies were obtained and evaluated using image-analysis systems. We quantified; (i) regeneration of colonic crypts and blood microvessels; (ii) cyclooxygenase 2 (COX2) expression; (iii) mitochondrial DNA (mtDNA) mutations; (iv) inflammatory infiltration; and (v) vascular permeability (VP). Results: In control subjects, CLE demonstrated normal colonic crypts and microvasculature. COX2 expression was minimal, and < 7% crypts showed mtDNA mutations. Colonic mucosa of UC-IR patients had impaired and distorted crypt regeneration, increased COX2, 69% crypts with mtDNA mutations, persistent inflammation, and abnormal vascular architecture with increased VP (all P < 0.001 vs normal mucosa). Conclusions: (i) Endoscopically normal appearing colonic mucosa of patients with UC-IR remains abnormal: CLE demonstrates impaired crypt regeneration, persistent inflammation, distinct abnormalities in angioarchitecture and increased vascular permeability; molecular imaging showed increased COX2 and mtDNA mutations; (ii) CLE may serve as a new gold standard for the assessment of mucosal healing in UC.
Introduction During the second International Shimoda Symposium in 1998, we reported that the subepithelial mucosa of macroscopically healed gastric ulcer displays impaired and disorganized restoration of glandular and vascular structures and remains histologically and ultrastructurally abnormal.1 We postulated that these abnormalities may interfere with oxygenation, nutrient supply, and with mucosal defense, and therefore could be the basis for ulcer recurrence.1 These studies were basis for formulating a new concept of quality of gastric ulcer healing.1,2 Arakawa and coworkers demonstrated the relevance of this concept to healing of human gastric ulcers,3,4 and more recently showed that this concept also applies to ulcers in inflammatory bowel diseases (IBD).4
Endoscopic assessment of colonic mucosal healing in ulcerative colitis (UC) and Crohn’s disease is increasingly accepted as an important measure of disease activity, therapeutic goal, a key prognostic indicator, and an endpoint in clinical trials.5–14 The main contention supporting importance of mucosal healing in UC is that achieving mucosal healing may reduce or prevent relapses and complications, and improve quality of life. These issues were extensively reviewed and discussed in several recent publications.5–14 Indeed, complete mucosal healing is associated with sustained clinical remission and reduces the risk of colectomy in patients with UC.11–14 Mucosal healing in UC is defined mainly by endoscopy, based on macroscopic appearance of mucosal surface and the absence of ulcerations, vascular congestion, erythema,
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swelling, and nodularity during endoscopic examination.11–14 The quality of mucosal healing assessed by endoscopy has been proposed as the main predictor of recurrence in UC.12–14 Subsequent studies utilizing magnifying chromoendoscopy and narrow band imaging further improved endoscopic assessment of mucosa in UC and IBD.4,15 However, even in patients with UC and endoscopically “healed” colonic mucosa, the disease recurs in a significant proportion of patients, suggesting that macroscopic healing assessed endoscopically is not an equivalent of a completely normal mucosa. This is not surprising since a standard endoscopy visualizes appearance of mucosal surface only, and subepithelial abnormalities cannot be detected. Confocal laser endomicroscopy (CLE) is a new state-of-the-art technology that enables, real-time, in vivo visualization of subepithelial mucosal structures and cells at 1000× magnification (virtual biopsy) during ongoing endoscopy.15 Several studies have shown that CLE images of colonic mucosa correspond well to a standard histology and might even replace conventional histological diagnosis.15–21 CLE has been shown to reliably assess activity of the disease in inflammatory bowel diseases, both in UC15–17 and in Crohn’s disease,18 and to predict relapse by the detection of cell shedding and mucosal barrier loss.19 CLE has also been used for the detection of neoplastic lesions in patients with UC.20 CLE with intravenous fluorescein infusion allows a direct visualization of blood vessels in the mucosa, which are filled with fluorescein, and therefore may be used for evaluation of the mucosal vasculature in vivo.21 Most of the studies concerning CLE evaluation of UC included patients with active disease15–19 and only one clinical CLE imaging report focused exclusively on UC patients during remission.22 However, none of these studies examined in depth and quantitatively colonic mucosal crypts regeneration, regeneration of blood microvessels—their number, size, and characteristics nor did they measure and quantify in real-time and in vivo microvascular permeability, vessel size, and inflammatory cell infiltration. None of these studies did examine the expression COX2 and mitochondrial DNA (mtDNA) mutation. The aim of this pilot study was to test feasibility of in vivo, real-time measurements of mucosal microvascular permeability and to determine quantitatively using CLE whether and to what extent endoscopically normal appearing colonic mucosa of patients with UC in remission (UC-IR) has regenerated mucosal structures both epithelial and vasculature, resolved inflammation, and to identify the underlying mechanisms. Since COX2-generated prostaglandins may stimulate crypt regeneration and re-epithelialization via transactivating epidermal growth factor (EGF) receptor23,24 and promote neovascularization, we examined the expression of COX2 in colonic mucosa of control and UC-IR patients. In addition, we also examined whether regenerated crypts of colonic mucosa have somatic mitochondrial DNA mutations. The latter may have significant implications for disease recurrence and carcinogenesis.25–27
Methods These studies were approved by the Ethics Committee CHU de Nantes, France. All patients gave their written, informed consent to participate in the study in accordance with the Treaty of Helsinki. Patients included in this study were adult aged 36–72, with long-term UC (> 10 years after diagnosis) in complete clinical 86
remission, referred for surveillance colonoscopy to the Department of Gastroenterology of the University Hospital of Nantes from February 2008 until March 2010. Additionally, control patients in whom colonoscopy was indicated for other reasons (colorectal cancer screening, anemia) were also studied. For all the patients, the following information was collected: demographic data, duration of the disease, clinical disease activity index (CAI), and current treatment. Colonoscopy and CLE procedure. Colonoscopy combined with CLE (confocal endomicroscope, Pentax, Tokyo, Japan) was performed in all the patients. In this endomicroscope, a confocal laser microscope is integrated into the distal tip of a conventional video endoscope. After intravenous (i.v.) infusion of 5 mL of 10% fluorescein over 10 min, confocal imaging of colonic mucosa was performed in a standardized manner, and “virtual biopsies” were obtained after gently placing the distal end of the confocal laser endoscope at the mucosal surface. In addition, conventional biopsies were obtained at adjacent areas for histological examination. In each patient, at least 10 “virtual biopsies” and four conventional biopsies were obtained. All procedures were performed by the same experienced endoscopist. Endoscopic evaluation of the entire colonic mucosa was performed using white light followed by indigo carmine chromoendoscopy. The presence of macroscopic lesions as well as endoscopic signs of inflammation was recorded. Analysis of CLE images. Coded CLE images were analyzed by three investigators (AST, TMB, and AA) who were blinded to the results of standard histology. For each patient, 10 CLE images obtained from colon were analyzed, and the following parameters were examined: 1. Distribution, pattern, and CLE features of crypt regeneration in CLE images, including measurements of major (MA) and minor (MI) crypt lumen axis and MA/MI ratio, using the Image J system (National Institutes of Health – NIH), as described previously.28 This method allows quantitative analysis of colonic pit and crypt structure and the detection of residual inflammation.28 2. CLE features of mucosal microvessel: shape, size (maximal width), and characteristics, for example, elongation and tortuosity were assessed and measured (maximal width) using the Image J system (NIH). 3. Distribution and intensity of fluorescence inside and outside the mucosal microvessels. It was measured using the Image J system in 10 standardized mucosal areas and expressed as fluorescence signal intensity (FSI) on a scale from 0–255, which is the measure of microvascular permeability. FSI was measured inside the vessel and outside the vessel lamina propria in six standardized fields per image on 10 separate images, and the mean values were calculated for each patient. The FSI gradient between vessels and lamina propria was calculated; a lower FSI gradient indicates increased fluorescein leakage into the extra vascular space and reflects increased vascular permeability. To assure appropriate, standardized comparisons, the vascular permeability measurements were performed on CLE images obtained always at 8–10 min after initiation of the i.v. infusion of fluorescein.
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4. CLE features of inflammation. The number of inflammatory cells (black dots) in five standardized areas of lamina propria per each CLE image in normal controls and UC-IR patients were counted. Histology and immunohistochemistry. Four oriented mucosal biopsies from each patient were fixed in 10% formalin and embedded in paraffin. The sections were stained with HE and used for conventional histology. Unstained sections were used for immunohistochemistry. Histological activity of the disease was evaluated according to the Geboes score.29 The immunostaining for COX2 and mitochondrial DNA encoded cytochrome C oxidase subunit 1 (CCO) was performed using paraffin-embedded mucosal sections. Tissue sections were de-paraffinized and incubated with specific primary antibodies for COX2 (1:100; sc-1745 goat polyclonal, Santa Cruz Biotechnology, CA) and mitochondrial DNA-encoded cytochrome c oxidize subunit 1 (CCO) (1:100; #459600 mouse monoclonal antibody, Invitrogen, CA) overnight at 4°C. After washing, the sections were then incubated with a biotin-conjugated appropriate anti-goat or antimouse secondary antibodies (1:500; E0466, Dako, CA) at room temperature for 30 min and finally with peroxidase-conjugated streptavidin (1:500; P0397, Dako, CA) at room temperature for 30 min. Peroxidase activity was detected with AEC substrate-chromogen (K3464, Dako, CA). Immunostaining was evaluated independently by two investigators using a Nikon Optiphot microscope. The staining signal intensity was quantified using MetaMorph 7.0 (Molecular Devices, Downington, PA) in five randomly selected fields and expressed as arbitrary units. Statistical analysis. The quantitative values for crypt lumen axis and size of microvessels were expressed as median (MinMax) and the comparison between UC patients and control patients was performed using a nonparametric Mann–Whitney test. The differences were considered significant for P value less than 0.05. FSI gradient between outside and inside the microvessels, and COX2 and CCO staining intensity are expressed as the mean ± standard deviation (SD). Student’s t-test was used to determine statistical significance, and a P value less than 0.05 was considered statistically significant.
Results Patients and regular endoscopy. Six patients with UC-IR and six control patients were included. Demographic and clinical characteristics of all the patients are presented in Table 1. All patients with UC-IR had a macroscopically normal colonic mucosa on regular endoscopy, except 1 with minimal inflammation. All control patients had endoscopically normal mucosal appearance. Analysis of CLE images. In all control patients, detailed evaluation CLE images showed that colonic mucosal crypts had regular distribution pattern and had mostly round, regular lumen (Fig. 1c,d). In patients with UC-IR, crypts had distorted distribution pattern, with increased spaces between crypts indicating expansion of the lamina propria, and the crypt lumina were irregu-
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Table 1
Demographic and clinical data of all patients
Age (years), median (min-max) Sex (male/female) Duration of the disease (years), median (min-max) Clinical activity index, median (min-max) Current treatment
UC (n = 6)
Control (n = 6)
57 (36–72) 4/2 18 (15–26)
59 (46–67) 4/2 —
2.5 (1–7)
—
Imurel 3 5-ASA 1 Dipentum 1 or Combined 1†
—
†
5-ASA + Corticoids + Methotrexate. UC, ulcerative colitis.
Figure 1 Representative images of (a) standard endoscopy, (b) standard histology, and (c and d) confocal laser endomicroscopy (CLE) of normal colonic mucosa in a control patient. CLE shows a normal mucosal structure, with regular, round crypts (*), surrounded by normal size microvessels filled with fluorescein (arrows).
lar (Fig. 2c,d). The quantitative analysis of pit structure, performed similar as in our previous study in relation to Crohn’s colitis,28 demonstrated that the median major: minor (MA/MI) crypt lumen axis ratio was significantly higher in UC-IR patients (3.02, range: 1.98–4.1) than in control patients (1.45, range: 1.2–1.7) (P < 0.001), indicating impaired and distorted crypt regeneration of colonic mucosa in UC-IR patients. Expression of COX2 in colonic mucosa of control patients was absent or minimal and limited to few mononuclear cells in the lamina propria; COX2 expression was absent or minimal in epithelial cells lining mucosal surface and regenerated crypts (Fig. 3a). In contrast, in UC-IR patients COX2 expression was significantly increased in both mononuclear inflammatory cells in
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Figure 2 Representative images of colonic mucosa in patient with ulcerative colitis in remission (UC-IR). (a) Endoscopic view shows normal appearing mucosa. (b) Standard histology shows deformed crypts and enlarged blood microvessels between crypts (arrows). In patients with UC-IR (c and d), despite a normal macroscopic picture of colonic mucosa at endoscopy, CLE shows distinct abnormalities of mucosal structures: irregular and distorted crypts (*), with increased spaces between the crypts reflecting persistent inflammation, impaired crypt regeneration, and leakage of fluorescein into the extravascular space (arrows).
the lamina propria (which were significantly increased in number) as well in the epithelial cells lining mucosal surface and regenerated crypts (Fig. 3b,c). COX2 expression was 6.9-fold higher in epithelial cells of colonic crypts in UC-IR patients versus normal controls (P < 0.001) (Fig. 3d). The mtDNA mutation was assessed using immunostaining for mitochondrial DNA-encoded cytochrome c oxidase (CCO) subunit 1 in normal (Fig. 4a) and UC-IR patients (Fig. 4b,c). In colonic mucosa of UC-IR patients, CCO expression (signal intensity) was significantly decreased (1.6-fold) compared to control patients (P < 0.001) (Fig. 4d). Furthermore, the number of CCO-deficient colonic crypts (crypts with mtDNA mutations) was significantly increased patients with UC-IR (69% vs 7% in normal controls) (Fig. 4e). CLE image analysis showed signs of inflammation – infiltration of the mucosa with inflammatory cells (Fig. 5) in all six patients with UC, and this result correlated with the results of standard histology (Table 2). In UC-IR patients, the number of inflammatory cells in the lamina propria was significantly increased (6.6fold, P < 0.001) compared to control patients (Fig. 5). CLE assessment of the mucosal blood microvessels demonstrated that in control patients, mucosal blood microvessels were regular, thin, and distributed regularly around the normal crypts 88
Figure 3 Expression of COX2 in colonic mucosa in control patients (a) and in patients with ulcerative colitis in remission (UC-IR) (b and c). COX2 expression (brown staining) in colonic mucosa of control patients is limited to few mononuclear cells in the lamina propria (arrowheads). In contrast, in UC-IR patients, COX2 expression is significantly increased not only in mononuclear inflammatory cells in the lamina propria (arrowheads), which are significantly increased in number but also in the epithelial cells (arrows) lining mucosal surface and regenerated crypts. Quantitative analysis of COX2 expression (d) demonstrates that COX2 expression in colonic mucosa of patients with UC-IR is significantly increased (6.9 fold) compared to control patients (P < 0.001). □, Inflammatory Cells; ■, Epithelial Cells.
(Fig. 1c,d). In contrast, in colonic mucosa of UC-IR patients, mucosal blood microvessels were increased in number, and were tortuous and enlarged reflecting their abnormal regeneration and pathological angiogenesis. (Fig. 6c–e). The median width of the vessels was significantly larger in UC patients (17 μm, range: 16–22 μm) than in control patients (8 μm, range 6–10 μm) (P < 0.001). In vivo assessment of mucosal microvessels permeability using CLE. The FSI gradient between fluorescein fluorescence inside and outside the vessels (lamina propria) in colonic mucosa was used as a measure of vascular permeability. In UC-IR patients, this gradient was significantly lower than in control patients, indicating an increased vascular permeability in UC-IR patients (Fig. 7).
Discussion The present study shows that in patients with UC-IR, despite normally appearing colonic mucosa at regular endoscopy, distinct
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Figure 4 Mitochondrial DNA (mtDNA) mutations in colonic mucosa of control and patients with ulcerative colitis in remission (UC-IR) visualized using immunostaining for expression of mtDNA-encoded cytochrome C oxidase (CCO) subunit 1. MtDNA mutations result in deficiency of CCO enzyme. Brown staining in epithelial cells represents expression in colonic mucosa of CCO enzyme and reflects normal, nonmutated protein. Some epithelial cells lining crypts lack positive (brown) stain reflecting mtDNA mutations. In control patients, (a) all epithelial cells lining crypts have positive (brown) staining. In patients with UC in remission (UC-IR) (b, c), crypts lacking CCO staining are marked with *. (d) Quantitative analysis of CCO expression (signal intensity) in colonic mucosa of UC-IR patients was significantly decreased (1.6 fold) compared to control patients (P < 0.001). (e) Quantitative analysis of mtDNA mutations (which result in CCO deficiency) showed that in colonic mucosa of patients with UC-IR, mtDNA mutations were significantly increased; 69% of the colonic crypts in UC-IR patients showed mtDNA mutations, while only 7% of the colonic crypts in normal controls showed mtDNA mutations.
Table 2 CLE and histological evaluation of colonic mucosa in patients with UC in remission and in control patients
CLE diagnosis
Histological grade of disease activity†
Normal Moderate inflammation Severe inflammation Grade 0 Grade 1 Grade 4
UC (n = 6)
Control (n = 6)
0 4 2 0 5 1
6 0 0 6 0 0
† According to the score of Goebes et al. [29]. CLE, confocal laser endomicroscopy; UC, ulcerative colitis.
Figure 5 Confocal laser endomicroscopy (CLE) features of inflammation. CLE shows increased number of infiltrating inflammatory cells (black dots—indicated by arrows) in expanded lamina propria of colonic mucosa in patient with ulcerative colitis in remission (UC-IR). The number of infiltrating mononuclear cells is 6.6-fold increased in colonic mucosa of UC-IR patients versus control patients.
abnormalities of mucosal epithelial structures and microvessels are present. Importantly, these abnormalities were demonstrated by CLE and diagnosed in vivo and in real time. They include the presence of irregular, distorted crypt pattern, with deformed crypt lumina indicating impaired and distorted crypt regeneration, and
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Figure 6 (a–c). Confocal laser endomicroscopy images showing abnormal, enlarged and tortuous vessels in colonic mucosa of patients with ulcerative colitis in remission (UC-IR). Distorted and irregular-shaped crypts in colonic mucosa of UC-IR patients are surrounded by abnormal, tortuous, enlarged mucosal blood microvessels (indicated by arrows), reflecting their abnormal regeneration and pathological angiogenesis. Compared to normal mucosa of control patients, the vessels in colonic mucosa in UC-IR patients were significantly wider versus normal control patients (P < 0.001).
Figure 7 Colonic mucosal microvascular permeability determined by measurement of fluorescein fluorescence signal intensity (FSI) inside and outside the vessels (lamina propria) of colonic mucosa in patients with ulcerative colitis in remission (UC-IR) and in control patients. g.: gradient between FSI inside the vessels and in lamina propria outside the vessels. A significantly lower (fourfold) FSI gradient between vessels and lamina propria in UC-IR versus normal controls (P < 0.001) indicates increased leakage of fluorescein from the vessels and reflects increased vascular permeability. □, FSI inside the vessels; ■, FSI in lamina propria outside the vessels.
enlarged spaces between the crypts likely reflecting the presence of inflammatory infiltrates, expanded lamina propria and thus persistent inflammation. The mucosal vessels are enlarged and tortuous, and the mean size of the vessels is significantly increased, all reflecting pathological angiogenesis in patients with UC-IR. These findings are important and indicate that the evaluation criteria of mucosal healing in UC using regular endoscopy, currently accepted and widely used in clinical studies8–14 may not be optimal, and that CLE by allowing evaluation of subepithelial mucosal structures represents a more precise and in depth method for such evaluation. This has been suggested by some previous studies also indicating that in a subset of patients with UC-IR or Crohn’s disease with complete endoscopic remission, some changes in crypt and microvessel architecture are present.16–18,22 90
Gheorghe and coworkers found in UC patients with a normal endoscopic appearance of colonic mucosa subtle changes in crypt architecture in form of focal distortions of crypts and the presence of fluorescein leakage in the luminal openings of the crypts.22 However, they did not quantify crypts structure deformation. Regarding the process of colonic crypt regeneration, Wright et al. demonstrated that during healing of ulceration in human gastrointestinal tract, there is development of a novel cell lineage, which forms buds at the ulcer margin that expands through proliferation into tubules then ramifying to and ultimately forming new crypts.30 That study showed that this novel cell lineage secretes epidermal growth factor (EGF), which stimulates proliferation of these cells and induces crypt regeneration and ulcer healing.30 Our previous study demonstrated that the same process occurs during experimental gastric ulcer healing; regenerated gastric glands are deformed and all cells lining these glands strongly express EGF receptor.31 In the present study, we found increased COX2 expression in epithelial cells of regenerating colonic crypts. This may represent a novel mechanism underlying crypt regeneration since COX2generated prostaglandins are able to transactivate EGF receptor, as shown in our previous study23 and via this mechanism stimulate epithelial cell proliferation and migration.23,24 Moreover, COX2generated prostaglandin E2 may induce angiogenesis, new blood vessel formation.32–34 Another entirely novel finding of this study is that a significant proportion (69%) of regenerated mucosal crypts in UC-IR patients has mutated mtDNA as evidenced by a lack of cytochrome c oxidase expression. Cytochrome c oxidase reduces oxygen to water and is central to oxidative phosphorylation and the generation of ATP.35 Somatic mtDNA mutations resulting in CCO deficiency are present in normal colon and in colon cancer.26 Taylor et al. showed that in some colonic crypt cells, the mitochondrial CCO genes are mutated resulting in decreased CCO enzyme.27 That study showed that such colon crypt cells clonally expand to occupy first part and then the whole crypt by a process known as monoclonal conversion.27 Greaves et al. showed that these mutations spread in the normal human colon by crypt fission.26 Mutation of mtDNA and CCO deficiency can occur in noncancerous tissues and may induce ulcer recurrence in UC-IR via a similar mechanism and may also have implications for carcinogenesis in UC. In our study, all patients with clinically quiescent UC and macroscopically normal colonic mucosa (UC-IR) had distinct abnormalities not only in colonic mucosal epithelial crypts but also in mucosal blood microvessels. The mucosal vessels were enlarged and tortuous, and the mean size of the vessels is significantly increased, all these reflecting pathological angiogenesis. Angiogenesis plays an important role not only in normal tissue injury healing, but as shown, more recently also in inflammation.36,37 Vascular endothelial growth factor-A (VEGF-A) and its receptor (VEGFR2) are considered important mediators of pathological angiogenesis.36 In active IBD, increased levels of VEGF-A and VEGFR2 in colonic mucosa have been demonstrated.38 This finding confirmed the contention that abnormal angiogenesis is not only a consequence of inflammation but also may be a driving force inducing and promoting inflammatory process. It is thus conceivable that persistent pathological angiogenesis in spite of macroscopically healed mucosa may contribute to the disease recurrence in UC.
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In this study, we also demonstrated impaired function of endothelial “barrier” function of mucosal blood vessels in UC-IR patients, reflected by increased microvascular permeability to plasma containing fluorescein. For these studies, we used measurements of extravasation of fluorescein reflected by fluorescence gradient between outside and inside the vessels on CLE images as a marker of in vivo microvascular permeability. In our previous study, we have demonstrated that after i.v. passage, fluorescein crosses the endothelial microvascular “barrier” and penetrates into the extravascular space as well into the epithelial cells.39 The increased microvascular permeability in colonic mucosa of UC-IR patients may be an important mechanism for sustained inflammation by enabling the flux of inflammatory cells into extravascular space.40,41 This pilot study was aimed to test the feasibility of using CLE to quantify crypt regeneration and deformation, vessel size, vascular permeability and to use standard biopsy specimens to determine quantitatively the expression of COX2 and mtDNA mutation. In this regard, these aims were fully achieved. Naturally, limitation of such pilot study is that because of a small patient number, it cannot specifically evaluate the effect of treatment, predict recurrence, etc. However, once the feasibility of CLE quantitative assessment of mucosal healing in UC has been established, future studies can answer these questions. In summary, this study shows that in patients with UC in remission and with normal endoscopic appearance of colonic mucosa, CLE allows in vivo, real-time detection of structural and microvascular changes of the colonic mucosa, not detectable by standard endoscopy, which may contribute to the recurrence of the disease. In addition, CLE allows in vivo measurements of mucosal microvascular permeability and molecular imaging. CLE might be thus considered as the technique of choice and a new gold standard to evaluate mucosal healing in UC patients.
References 1 Tarnawski A, Hollander D, Krause WJ, Dabros W, Stachura J, Gergely H. “Healed” experimental gastric ulcers remain histologically and ultrastructurally abnormal. J. Clin. Gastroenterol. 1990; 12 (Suppl. 1): S139–47. 2 Tarnawski A, Stachura J, Krause WJ, Douglass TG, Gergely H. Quality of gastric ulcer healing: a new, emerging concept. J. Clin. Gastroenterol. 1991; 13 (Suppl. 1): S42–7. 3 Arakawa T, Kobayashi K. Quality of ulcer healing—a new concept to rank healed peptic ulcers. Gastroenterol. Jpn. 1993; 28 (Suppl. 5): 158–62. 4 Arakawa T, Watanabe T, Tanigawa T, Tominaga K, Fujiwara Y, Morimoto K. Quality of ulcer healing in gastrointestinal tract: Its pathophysiology and clinical relevance. World J. Gastroenterol. 2012; 18: 4811–22. 5 Lichtenstein GR, Rutgeerts P. Importance of mucosal healing in ulcerative colitis. Inflamm. Bowel Dis. 2010; 16: 338–46. 6 Mazzuoli S, Guglielmi FW, Antonelli E, Salemme M, Bassotti G, Villanacci V. Definition and evaluation of mucosal healing in clinical practice. Dig. Liver Dis. 2013; 45: 969–77. 7 Peyrin-Biroulet L, Bressenot A, Kampman W. Histologic Remission: The Ultimate Therapeutic Goal in Ulcerative Colitis? Clin. Gastroenterol. Hepatol. 2014; 12: 929–34.e2. 8 Yokoyama K, Kobayashi K, Mukae M, Sada M, Koizumi W. Clinical Study of the Relation between Mucosal Healing and
Confocal laser endomicroscopy & colitis
9
10
11
12 13
14
15
16
17
18
19
20
21
22
23
24
25
26
Long-Term Outcomes in Ulcerative Colitis. Gastroenterol. Res. Pract. 2013; doi: 10.1155/2013/192794. Seidelin JB, Coskun M, Nielsen OH. Mucosal healing in ulcerative colitis: pathophysiology and pharmacology. Adv. Clin. Chem. 2013; 59: 101–23. Dave M, Loftus EV Jr. Mucosal healing in inflammatory bowel disease-a true paradigm of success? Gastroenterol. Hepatol. (N Y) 2012; 8: 29–38. Pineton de Chambrun G, Peyrin-Biroulet L, Lémann M, Colombel JF. Clinical implications of mucosal healing for the management of IBD. Nat. Rev. Gastroenterol. Hepatol. 2010; 7: 15–29. Neurath MF, Travis SP. Mucosal healing in inflammatory bowel diseases: a systematic review. Gut 2012; 61: 1619–35. Rutgeerts P, Sandborn WJ, Feagan BG, Reinisch W, Olson A, Johanns J et al. Infliximab for induction and maintenance therapy for ulcerative colitis. N. Engl. J. Med. 2005; 353: 2462–76. Colombel JF, Rutgeerts P, Reinisch W, Esser D, Wang Y, Lang Y et al. Early mucosal healing with infliximab is associated with improved long-term clinical outcomes in ulcerative colitis. Gastroenterology 2011; 141: 1194–201. Kiesslich R, Goetz M, Lammersdorf K, Schneider C, Burg J, Stolte M et al. Chromoscopy-guided endomicroscopy increases the diagnostic yield of intraepithelial neoplasia in ulcerative colitis. Gastroenterology 2007; 132: 874–82. Watanabe O, Ando T, Maeda O, Hasegawa M, Ishikawa D, Ishiguro K et al. Confocal endomicroscopy in patients with ulcerative colitis. J. Gastroenterol. Hepatol. 2008; 23 (Suppl 2): S286–90. Li CQ, Xie XJ, Yu T, Gu XM, Zuo XL, Zhou CJ et al. Classification of inflammation activity in ulcerative colitis by confocal laser endomicroscopy. Am. J. Gastroenterol. 2010; 105: 1391–6. Neumann H, Vieth M, Atreya R, Grauer M, Siebler J, Bernatik T et al. Assessment of Crohn’s disease activity by confocal laser endomicroscopy. Inflamm. Bowel. Dis. 2012; 18: 2261–9. Kiesslich R, Duckworth CA, Moussata D, Gloeckner A, Lim LG, Goetz M et al. Local barrier dysfunction identified by confocal laser endomicroscopy predicts relapse in inflammatory bowel disease. Gut 2012; 61: 1146–53. Kiesslich R, Burg J, Vieth M, Gnaendiger J, Enders M, Delaney P et al. Confocal laser endoscopy for diagnosing intraepithelial neoplasias and colorectal cancer in vivo. Gastroenterology 2004; 127: 706–13. Gheonea DI, Cârt¸âna˘ T, Ciurea T, Popescu C, Ba˘da˘ra˘u A, Sa˘ftoiu A. Confocal laser endomicroscopy and immunoendoscopy for real-time assessment of vascularization in gastrointestinal malignancies. World J. Gastroenterol. 2011; 17: 21–7. Gheorghe C, Cotruta B, Iacob R, Becheanu G, Dumbrava M, Gheorghe L. Endomicroscopy for assessing mucosal healing in patients with ulcerative colitis. J. Gastrointestin. Liver Dis. 2011; 20: 423–6. Pai R, Soreghan B, Szabo IL, Pavelka M, Baatar D, Tarnawski AS. Prostaglandin E2 transactivates EGF receptor: a novel mechanism for promoting colon cancer growth and gastrointestinal hypertrophy. Nat. Med. 2002; 8: 289–93. Pai R, Nakamura T, Moon WS, Tarnawski AS. Prostaglandins promote colon cancer cell invasion; signaling by cross-talk between two distinct growth factor receptors. FASEB J. 2003; 17: 1640–7. Ussakli CH, Ebaee A, Binkley J, Brentnall TA, Emond MJ, Rabinovitch PS et al. Mitochondria and tumor progression in ulcerative colitis. J. Natl. Cancer Inst. 2013; 105: 1239–48. Greaves LC, Preston SL, Tadrous PJ, Taylor RW, Barron MJ, Oukrif D et al. Mitochondrial DNA mutations are established in human colonic stem cells, and mutated clones expand by crypt fission. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 714–9.
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27 Taylor RW, Barron MJ, Borthwick GM, Gospel A, Chinnery PF, Samuels DC et al. Mitochondrial DNA mutations in human colonic crypt stem cells. J. Clin. Invest. 2003; 112: 1351–60. 28 Musquer N, Coquenlorge S, Bourreille A, Aubert P, Matysiak-Budnik T, des Varannes SB et al. Probe-based confocal laser endomicroscopy: a new method for quantitative analysis of pit structure in healthy and Crohn’s disease patients. Dig. Liver Dis. 2013; 45: 487–92. 29 Geboes K, Riddell R, Ost A, Jensfelt B, Persson T, Löfberg R. A reproducible grading scale for histological assessment of inflammation in ulcerative colitis. Gut 2000; 47: 404–9. 30 Wright NA, Pike C, Elia G. Induction of a novel epidermal growth factor-secreting cell lineage by mucosal ulceration in human gastrointestinal stem cells. Nature 1990; 343: 82–5. 31 Tarnawski A, Stachura J, Durbin T, Sarfeh IJ, Gergely H. Increased expression of epidermal growth factor receptor during gastric ulcer healing in rats. Gastroenterology 1992; 102: 695–8. 32 Harada S, Nagy JA, Sullivan KA, Thomas KA, Endo N, Rodan GA et al. Induction of vascular endothelial growth factor expression by prostaglandin E2 and E1 in osteoblasts. J. Clin. Invest. 1994; 93: 2490–6. 33 Wang D, Wang H, Brown J, Daikoku T, Ning W, Shi Q et al. CXCL1 induced by prostaglandin E2 promotes angiogenesis in colorectal cancer. J. Exp. Med. 2006; 203: 941–51. 34 Pai R, Szabo IL, Soreghan BA, Atay S, Kawanaka H, Tarnawski AS. PGE(2) stimulates VEGF expression in endothelial cells via
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38
39
40
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ERK2/JNK1 signaling pathways. Biochem. Biophys. Res. Commun. 2001; 286: 923–8. Capaldi RA. Structure and function of cytochrome c oxidase. Annu. Rev. Biochem. 1990; 59: 569–96. Danese S. Inflammation and the mucosal microcirculation in inflammatory bowel disease: the ebb and flow. Curr. Opin. Gastroenterol. 2007; 23: 384–9. Dvorak HF, Detmar M, Claffey KP, Nagy JA, van de Water L, Senger DR. Vascular permeability factor/vascular endothelial growth factor: an important mediator of angiogenesis in malignancy and inflammation. Int. Arch. Allergy Immunol. 1995; 107: 233–5. Scaldaferri F, Vetrano S, Sans M, Arena V, Straface G, Stigliano E et al. VEGF-A links angiogenesis and inflammation in inflammatory bowel disease pathogen-esis. Gastroenterology 2009; 136: 585–95. Coron E, Mosnier JF, Ahluwalia A, Le Rhun M, Galmiche JP, Tarnawski AS, Matysiak-Budnik T. Colonic mucosal biopsies obtained during confocal endomicroscopy are pre-stained with fluorescein in vivo and are suitable for histologic evaluation. Endoscopy 2012; 44: 148–53. Munjal C, Tyagi N, Lominadze D, Tyagi SC. Matrix metalloproteinase-9 in homocysteine-induced intestinal microvascular endothelial paracellular and transcellular permeability. J. Cell. Biochem. 2012; 113: 1159–69. Bardin N, Reumaux D, Geboes K, Colombel JF, Blot-Chabaud M, Sampol J et al. Increased expression of CD146, a new marker of the endothelial junction in active inflammatory bowel disease. Inflamm. Bowel Dis. 2006; 12: 16–21.
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doi:10.1111/jgh.12748
O R I G I N A L A RT I C L E
Confocal laser endomicroscopy: A new gold standard for the assessment of mucosal healing in ulcerative colitis Vincent Macé,*,1 Amrita Ahluwalia,†,1 Emmanuel Coron,* Marc Le Rhun,* Arnaud Boureille,* Céline Bossard,‡ Jean-François Mosnier,‡ Tamara Matysiak-Budnik* and Andrzej S Tarnawski† *Institut des Maladies de l’Appareil Digestif, CIC INSERM 04 et Service d’Hépato-Gastroentérologie, and ‡Service d’Anatomie Pathologique et EA-4273 BIOMETADYS, CHU de Nantes, France; and †VALBHS and the University of California, Irvine, California, USA
Key words angiogenesis, confocal laser endomicroscopy (CLE), COX2, mitochondrial DNA (mtDNA) mutation, mucosal healing, ulcerative colitis. Correspondence Andrzej S Tarnawski, Veterans Administration Long Beach Healthcare System, and the University of California, Irvine, 5901 E. 7th Street (09/151), Long Beach, CA 90822-5201, USA. Email: [email protected]; [email protected] 1
These authors contributed equally.
Supportive foundations: Southern California Institute for Research and Education, and the VA Merit Review grant to Tarnawski, AS. Conflict of interest: The funding for this study was provided by Southern California Institute for Research and Education, and the VA Merit Review Award to AST.
Abstract Background and Aim: Endoscopic assessment of mucosal healing in ulcerative colitis (UC) is increasingly accepted as a measure of disease activity, therapeutic goal, and the key prognostic indicator. While regular endoscopy evaluates appearance of the mucosal surface, confocal laser endomicroscopy (CLE) enables in vivo visualization of subepithelial mucosa at 1000× magnification during ongoing endoscopy. Our aims were to determine using CLE whether endoscopically normal appearing colonic mucosa in patients with UC in remission (UC-IR) has fully regenerated mucosal structures, resolved inflammation, and to identify the mechanisms. Methods: Twelve patients (six controls and six with UC-IR) underwent colonoscopy using CLE and intravenous fluorescein infusion. During colonoscopy, CLE images of colonic mucosa and conventional mucosal biopsies were obtained and evaluated using image-analysis systems. We quantified; (i) regeneration of colonic crypts and blood microvessels; (ii) cyclooxygenase 2 (COX2) expression; (iii) mitochondrial DNA (mtDNA) mutations; (iv) inflammatory infiltration; and (v) vascular permeability (VP). Results: In control subjects, CLE demonstrated normal colonic crypts and microvasculature. COX2 expression was minimal, and < 7% crypts showed mtDNA mutations. Colonic mucosa of UC-IR patients had impaired and distorted crypt regeneration, increased COX2, 69% crypts with mtDNA mutations, persistent inflammation, and abnormal vascular architecture with increased VP (all P < 0.001 vs normal mucosa). Conclusions: (i) Endoscopically normal appearing colonic mucosa of patients with UC-IR remains abnormal: CLE demonstrates impaired crypt regeneration, persistent inflammation, distinct abnormalities in angioarchitecture and increased vascular permeability; molecular imaging showed increased COX2 and mtDNA mutations; (ii) CLE may serve as a new gold standard for the assessment of mucosal healing in UC.
Introduction During the second International Shimoda Symposium in 1998, we reported that the subepithelial mucosa of macroscopically healed gastric ulcer displays impaired and disorganized restoration of glandular and vascular structures and remains histologically and ultrastructurally abnormal.1 We postulated that these abnormalities may interfere with oxygenation, nutrient supply, and with mucosal defense, and therefore could be the basis for ulcer recurrence.1 These studies were basis for formulating a new concept of quality of gastric ulcer healing.1,2 Arakawa and coworkers demonstrated the relevance of this concept to healing of human gastric ulcers,3,4 and more recently showed that this concept also applies to ulcers in inflammatory bowel diseases (IBD).4
Endoscopic assessment of colonic mucosal healing in ulcerative colitis (UC) and Crohn’s disease is increasingly accepted as an important measure of disease activity, therapeutic goal, a key prognostic indicator, and an endpoint in clinical trials.5–14 The main contention supporting importance of mucosal healing in UC is that achieving mucosal healing may reduce or prevent relapses and complications, and improve quality of life. These issues were extensively reviewed and discussed in several recent publications.5–14 Indeed, complete mucosal healing is associated with sustained clinical remission and reduces the risk of colectomy in patients with UC.11–14 Mucosal healing in UC is defined mainly by endoscopy, based on macroscopic appearance of mucosal surface and the absence of ulcerations, vascular congestion, erythema,
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swelling, and nodularity during endoscopic examination.11–14 The quality of mucosal healing assessed by endoscopy has been proposed as the main predictor of recurrence in UC.12–14 Subsequent studies utilizing magnifying chromoendoscopy and narrow band imaging further improved endoscopic assessment of mucosa in UC and IBD.4,15 However, even in patients with UC and endoscopically “healed” colonic mucosa, the disease recurs in a significant proportion of patients, suggesting that macroscopic healing assessed endoscopically is not an equivalent of a completely normal mucosa. This is not surprising since a standard endoscopy visualizes appearance of mucosal surface only, and subepithelial abnormalities cannot be detected. Confocal laser endomicroscopy (CLE) is a new state-of-the-art technology that enables, real-time, in vivo visualization of subepithelial mucosal structures and cells at 1000× magnification (virtual biopsy) during ongoing endoscopy.15 Several studies have shown that CLE images of colonic mucosa correspond well to a standard histology and might even replace conventional histological diagnosis.15–21 CLE has been shown to reliably assess activity of the disease in inflammatory bowel diseases, both in UC15–17 and in Crohn’s disease,18 and to predict relapse by the detection of cell shedding and mucosal barrier loss.19 CLE has also been used for the detection of neoplastic lesions in patients with UC.20 CLE with intravenous fluorescein infusion allows a direct visualization of blood vessels in the mucosa, which are filled with fluorescein, and therefore may be used for evaluation of the mucosal vasculature in vivo.21 Most of the studies concerning CLE evaluation of UC included patients with active disease15–19 and only one clinical CLE imaging report focused exclusively on UC patients during remission.22 However, none of these studies examined in depth and quantitatively colonic mucosal crypts regeneration, regeneration of blood microvessels—their number, size, and characteristics nor did they measure and quantify in real-time and in vivo microvascular permeability, vessel size, and inflammatory cell infiltration. None of these studies did examine the expression COX2 and mitochondrial DNA (mtDNA) mutation. The aim of this pilot study was to test feasibility of in vivo, real-time measurements of mucosal microvascular permeability and to determine quantitatively using CLE whether and to what extent endoscopically normal appearing colonic mucosa of patients with UC in remission (UC-IR) has regenerated mucosal structures both epithelial and vasculature, resolved inflammation, and to identify the underlying mechanisms. Since COX2-generated prostaglandins may stimulate crypt regeneration and re-epithelialization via transactivating epidermal growth factor (EGF) receptor23,24 and promote neovascularization, we examined the expression of COX2 in colonic mucosa of control and UC-IR patients. In addition, we also examined whether regenerated crypts of colonic mucosa have somatic mitochondrial DNA mutations. The latter may have significant implications for disease recurrence and carcinogenesis.25–27
Methods These studies were approved by the Ethics Committee CHU de Nantes, France. All patients gave their written, informed consent to participate in the study in accordance with the Treaty of Helsinki. Patients included in this study were adult aged 36–72, with long-term UC (> 10 years after diagnosis) in complete clinical 86
remission, referred for surveillance colonoscopy to the Department of Gastroenterology of the University Hospital of Nantes from February 2008 until March 2010. Additionally, control patients in whom colonoscopy was indicated for other reasons (colorectal cancer screening, anemia) were also studied. For all the patients, the following information was collected: demographic data, duration of the disease, clinical disease activity index (CAI), and current treatment. Colonoscopy and CLE procedure. Colonoscopy combined with CLE (confocal endomicroscope, Pentax, Tokyo, Japan) was performed in all the patients. In this endomicroscope, a confocal laser microscope is integrated into the distal tip of a conventional video endoscope. After intravenous (i.v.) infusion of 5 mL of 10% fluorescein over 10 min, confocal imaging of colonic mucosa was performed in a standardized manner, and “virtual biopsies” were obtained after gently placing the distal end of the confocal laser endoscope at the mucosal surface. In addition, conventional biopsies were obtained at adjacent areas for histological examination. In each patient, at least 10 “virtual biopsies” and four conventional biopsies were obtained. All procedures were performed by the same experienced endoscopist. Endoscopic evaluation of the entire colonic mucosa was performed using white light followed by indigo carmine chromoendoscopy. The presence of macroscopic lesions as well as endoscopic signs of inflammation was recorded. Analysis of CLE images. Coded CLE images were analyzed by three investigators (AST, TMB, and AA) who were blinded to the results of standard histology. For each patient, 10 CLE images obtained from colon were analyzed, and the following parameters were examined: 1. Distribution, pattern, and CLE features of crypt regeneration in CLE images, including measurements of major (MA) and minor (MI) crypt lumen axis and MA/MI ratio, using the Image J system (National Institutes of Health – NIH), as described previously.28 This method allows quantitative analysis of colonic pit and crypt structure and the detection of residual inflammation.28 2. CLE features of mucosal microvessel: shape, size (maximal width), and characteristics, for example, elongation and tortuosity were assessed and measured (maximal width) using the Image J system (NIH). 3. Distribution and intensity of fluorescence inside and outside the mucosal microvessels. It was measured using the Image J system in 10 standardized mucosal areas and expressed as fluorescence signal intensity (FSI) on a scale from 0–255, which is the measure of microvascular permeability. FSI was measured inside the vessel and outside the vessel lamina propria in six standardized fields per image on 10 separate images, and the mean values were calculated for each patient. The FSI gradient between vessels and lamina propria was calculated; a lower FSI gradient indicates increased fluorescein leakage into the extra vascular space and reflects increased vascular permeability. To assure appropriate, standardized comparisons, the vascular permeability measurements were performed on CLE images obtained always at 8–10 min after initiation of the i.v. infusion of fluorescein.
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4. CLE features of inflammation. The number of inflammatory cells (black dots) in five standardized areas of lamina propria per each CLE image in normal controls and UC-IR patients were counted. Histology and immunohistochemistry. Four oriented mucosal biopsies from each patient were fixed in 10% formalin and embedded in paraffin. The sections were stained with HE and used for conventional histology. Unstained sections were used for immunohistochemistry. Histological activity of the disease was evaluated according to the Geboes score.29 The immunostaining for COX2 and mitochondrial DNA encoded cytochrome C oxidase subunit 1 (CCO) was performed using paraffin-embedded mucosal sections. Tissue sections were de-paraffinized and incubated with specific primary antibodies for COX2 (1:100; sc-1745 goat polyclonal, Santa Cruz Biotechnology, CA) and mitochondrial DNA-encoded cytochrome c oxidize subunit 1 (CCO) (1:100; #459600 mouse monoclonal antibody, Invitrogen, CA) overnight at 4°C. After washing, the sections were then incubated with a biotin-conjugated appropriate anti-goat or antimouse secondary antibodies (1:500; E0466, Dako, CA) at room temperature for 30 min and finally with peroxidase-conjugated streptavidin (1:500; P0397, Dako, CA) at room temperature for 30 min. Peroxidase activity was detected with AEC substrate-chromogen (K3464, Dako, CA). Immunostaining was evaluated independently by two investigators using a Nikon Optiphot microscope. The staining signal intensity was quantified using MetaMorph 7.0 (Molecular Devices, Downington, PA) in five randomly selected fields and expressed as arbitrary units. Statistical analysis. The quantitative values for crypt lumen axis and size of microvessels were expressed as median (MinMax) and the comparison between UC patients and control patients was performed using a nonparametric Mann–Whitney test. The differences were considered significant for P value less than 0.05. FSI gradient between outside and inside the microvessels, and COX2 and CCO staining intensity are expressed as the mean ± standard deviation (SD). Student’s t-test was used to determine statistical significance, and a P value less than 0.05 was considered statistically significant.
Results Patients and regular endoscopy. Six patients with UC-IR and six control patients were included. Demographic and clinical characteristics of all the patients are presented in Table 1. All patients with UC-IR had a macroscopically normal colonic mucosa on regular endoscopy, except 1 with minimal inflammation. All control patients had endoscopically normal mucosal appearance. Analysis of CLE images. In all control patients, detailed evaluation CLE images showed that colonic mucosal crypts had regular distribution pattern and had mostly round, regular lumen (Fig. 1c,d). In patients with UC-IR, crypts had distorted distribution pattern, with increased spaces between crypts indicating expansion of the lamina propria, and the crypt lumina were irregu-
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Table 1
Demographic and clinical data of all patients
Age (years), median (min-max) Sex (male/female) Duration of the disease (years), median (min-max) Clinical activity index, median (min-max) Current treatment
UC (n = 6)
Control (n = 6)
57 (36–72) 4/2 18 (15–26)
59 (46–67) 4/2 —
2.5 (1–7)
—
Imurel 3 5-ASA 1 Dipentum 1 or Combined 1†
—
†
5-ASA + Corticoids + Methotrexate. UC, ulcerative colitis.
Figure 1 Representative images of (a) standard endoscopy, (b) standard histology, and (c and d) confocal laser endomicroscopy (CLE) of normal colonic mucosa in a control patient. CLE shows a normal mucosal structure, with regular, round crypts (*), surrounded by normal size microvessels filled with fluorescein (arrows).
lar (Fig. 2c,d). The quantitative analysis of pit structure, performed similar as in our previous study in relation to Crohn’s colitis,28 demonstrated that the median major: minor (MA/MI) crypt lumen axis ratio was significantly higher in UC-IR patients (3.02, range: 1.98–4.1) than in control patients (1.45, range: 1.2–1.7) (P < 0.001), indicating impaired and distorted crypt regeneration of colonic mucosa in UC-IR patients. Expression of COX2 in colonic mucosa of control patients was absent or minimal and limited to few mononuclear cells in the lamina propria; COX2 expression was absent or minimal in epithelial cells lining mucosal surface and regenerated crypts (Fig. 3a). In contrast, in UC-IR patients COX2 expression was significantly increased in both mononuclear inflammatory cells in
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Figure 2 Representative images of colonic mucosa in patient with ulcerative colitis in remission (UC-IR). (a) Endoscopic view shows normal appearing mucosa. (b) Standard histology shows deformed crypts and enlarged blood microvessels between crypts (arrows). In patients with UC-IR (c and d), despite a normal macroscopic picture of colonic mucosa at endoscopy, CLE shows distinct abnormalities of mucosal structures: irregular and distorted crypts (*), with increased spaces between the crypts reflecting persistent inflammation, impaired crypt regeneration, and leakage of fluorescein into the extravascular space (arrows).
the lamina propria (which were significantly increased in number) as well in the epithelial cells lining mucosal surface and regenerated crypts (Fig. 3b,c). COX2 expression was 6.9-fold higher in epithelial cells of colonic crypts in UC-IR patients versus normal controls (P < 0.001) (Fig. 3d). The mtDNA mutation was assessed using immunostaining for mitochondrial DNA-encoded cytochrome c oxidase (CCO) subunit 1 in normal (Fig. 4a) and UC-IR patients (Fig. 4b,c). In colonic mucosa of UC-IR patients, CCO expression (signal intensity) was significantly decreased (1.6-fold) compared to control patients (P < 0.001) (Fig. 4d). Furthermore, the number of CCO-deficient colonic crypts (crypts with mtDNA mutations) was significantly increased patients with UC-IR (69% vs 7% in normal controls) (Fig. 4e). CLE image analysis showed signs of inflammation – infiltration of the mucosa with inflammatory cells (Fig. 5) in all six patients with UC, and this result correlated with the results of standard histology (Table 2). In UC-IR patients, the number of inflammatory cells in the lamina propria was significantly increased (6.6fold, P < 0.001) compared to control patients (Fig. 5). CLE assessment of the mucosal blood microvessels demonstrated that in control patients, mucosal blood microvessels were regular, thin, and distributed regularly around the normal crypts 88
Figure 3 Expression of COX2 in colonic mucosa in control patients (a) and in patients with ulcerative colitis in remission (UC-IR) (b and c). COX2 expression (brown staining) in colonic mucosa of control patients is limited to few mononuclear cells in the lamina propria (arrowheads). In contrast, in UC-IR patients, COX2 expression is significantly increased not only in mononuclear inflammatory cells in the lamina propria (arrowheads), which are significantly increased in number but also in the epithelial cells (arrows) lining mucosal surface and regenerated crypts. Quantitative analysis of COX2 expression (d) demonstrates that COX2 expression in colonic mucosa of patients with UC-IR is significantly increased (6.9 fold) compared to control patients (P < 0.001). □, Inflammatory Cells; ■, Epithelial Cells.
(Fig. 1c,d). In contrast, in colonic mucosa of UC-IR patients, mucosal blood microvessels were increased in number, and were tortuous and enlarged reflecting their abnormal regeneration and pathological angiogenesis. (Fig. 6c–e). The median width of the vessels was significantly larger in UC patients (17 μm, range: 16–22 μm) than in control patients (8 μm, range 6–10 μm) (P < 0.001). In vivo assessment of mucosal microvessels permeability using CLE. The FSI gradient between fluorescein fluorescence inside and outside the vessels (lamina propria) in colonic mucosa was used as a measure of vascular permeability. In UC-IR patients, this gradient was significantly lower than in control patients, indicating an increased vascular permeability in UC-IR patients (Fig. 7).
Discussion The present study shows that in patients with UC-IR, despite normally appearing colonic mucosa at regular endoscopy, distinct
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Figure 4 Mitochondrial DNA (mtDNA) mutations in colonic mucosa of control and patients with ulcerative colitis in remission (UC-IR) visualized using immunostaining for expression of mtDNA-encoded cytochrome C oxidase (CCO) subunit 1. MtDNA mutations result in deficiency of CCO enzyme. Brown staining in epithelial cells represents expression in colonic mucosa of CCO enzyme and reflects normal, nonmutated protein. Some epithelial cells lining crypts lack positive (brown) stain reflecting mtDNA mutations. In control patients, (a) all epithelial cells lining crypts have positive (brown) staining. In patients with UC in remission (UC-IR) (b, c), crypts lacking CCO staining are marked with *. (d) Quantitative analysis of CCO expression (signal intensity) in colonic mucosa of UC-IR patients was significantly decreased (1.6 fold) compared to control patients (P < 0.001). (e) Quantitative analysis of mtDNA mutations (which result in CCO deficiency) showed that in colonic mucosa of patients with UC-IR, mtDNA mutations were significantly increased; 69% of the colonic crypts in UC-IR patients showed mtDNA mutations, while only 7% of the colonic crypts in normal controls showed mtDNA mutations.
Table 2 CLE and histological evaluation of colonic mucosa in patients with UC in remission and in control patients
CLE diagnosis
Histological grade of disease activity†
Normal Moderate inflammation Severe inflammation Grade 0 Grade 1 Grade 4
UC (n = 6)
Control (n = 6)
0 4 2 0 5 1
6 0 0 6 0 0
† According to the score of Goebes et al. [29]. CLE, confocal laser endomicroscopy; UC, ulcerative colitis.
Figure 5 Confocal laser endomicroscopy (CLE) features of inflammation. CLE shows increased number of infiltrating inflammatory cells (black dots—indicated by arrows) in expanded lamina propria of colonic mucosa in patient with ulcerative colitis in remission (UC-IR). The number of infiltrating mononuclear cells is 6.6-fold increased in colonic mucosa of UC-IR patients versus control patients.
abnormalities of mucosal epithelial structures and microvessels are present. Importantly, these abnormalities were demonstrated by CLE and diagnosed in vivo and in real time. They include the presence of irregular, distorted crypt pattern, with deformed crypt lumina indicating impaired and distorted crypt regeneration, and
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Figure 6 (a–c). Confocal laser endomicroscopy images showing abnormal, enlarged and tortuous vessels in colonic mucosa of patients with ulcerative colitis in remission (UC-IR). Distorted and irregular-shaped crypts in colonic mucosa of UC-IR patients are surrounded by abnormal, tortuous, enlarged mucosal blood microvessels (indicated by arrows), reflecting their abnormal regeneration and pathological angiogenesis. Compared to normal mucosa of control patients, the vessels in colonic mucosa in UC-IR patients were significantly wider versus normal control patients (P < 0.001).
Figure 7 Colonic mucosal microvascular permeability determined by measurement of fluorescein fluorescence signal intensity (FSI) inside and outside the vessels (lamina propria) of colonic mucosa in patients with ulcerative colitis in remission (UC-IR) and in control patients. g.: gradient between FSI inside the vessels and in lamina propria outside the vessels. A significantly lower (fourfold) FSI gradient between vessels and lamina propria in UC-IR versus normal controls (P < 0.001) indicates increased leakage of fluorescein from the vessels and reflects increased vascular permeability. □, FSI inside the vessels; ■, FSI in lamina propria outside the vessels.
enlarged spaces between the crypts likely reflecting the presence of inflammatory infiltrates, expanded lamina propria and thus persistent inflammation. The mucosal vessels are enlarged and tortuous, and the mean size of the vessels is significantly increased, all reflecting pathological angiogenesis in patients with UC-IR. These findings are important and indicate that the evaluation criteria of mucosal healing in UC using regular endoscopy, currently accepted and widely used in clinical studies8–14 may not be optimal, and that CLE by allowing evaluation of subepithelial mucosal structures represents a more precise and in depth method for such evaluation. This has been suggested by some previous studies also indicating that in a subset of patients with UC-IR or Crohn’s disease with complete endoscopic remission, some changes in crypt and microvessel architecture are present.16–18,22 90
Gheorghe and coworkers found in UC patients with a normal endoscopic appearance of colonic mucosa subtle changes in crypt architecture in form of focal distortions of crypts and the presence of fluorescein leakage in the luminal openings of the crypts.22 However, they did not quantify crypts structure deformation. Regarding the process of colonic crypt regeneration, Wright et al. demonstrated that during healing of ulceration in human gastrointestinal tract, there is development of a novel cell lineage, which forms buds at the ulcer margin that expands through proliferation into tubules then ramifying to and ultimately forming new crypts.30 That study showed that this novel cell lineage secretes epidermal growth factor (EGF), which stimulates proliferation of these cells and induces crypt regeneration and ulcer healing.30 Our previous study demonstrated that the same process occurs during experimental gastric ulcer healing; regenerated gastric glands are deformed and all cells lining these glands strongly express EGF receptor.31 In the present study, we found increased COX2 expression in epithelial cells of regenerating colonic crypts. This may represent a novel mechanism underlying crypt regeneration since COX2generated prostaglandins are able to transactivate EGF receptor, as shown in our previous study23 and via this mechanism stimulate epithelial cell proliferation and migration.23,24 Moreover, COX2generated prostaglandin E2 may induce angiogenesis, new blood vessel formation.32–34 Another entirely novel finding of this study is that a significant proportion (69%) of regenerated mucosal crypts in UC-IR patients has mutated mtDNA as evidenced by a lack of cytochrome c oxidase expression. Cytochrome c oxidase reduces oxygen to water and is central to oxidative phosphorylation and the generation of ATP.35 Somatic mtDNA mutations resulting in CCO deficiency are present in normal colon and in colon cancer.26 Taylor et al. showed that in some colonic crypt cells, the mitochondrial CCO genes are mutated resulting in decreased CCO enzyme.27 That study showed that such colon crypt cells clonally expand to occupy first part and then the whole crypt by a process known as monoclonal conversion.27 Greaves et al. showed that these mutations spread in the normal human colon by crypt fission.26 Mutation of mtDNA and CCO deficiency can occur in noncancerous tissues and may induce ulcer recurrence in UC-IR via a similar mechanism and may also have implications for carcinogenesis in UC. In our study, all patients with clinically quiescent UC and macroscopically normal colonic mucosa (UC-IR) had distinct abnormalities not only in colonic mucosal epithelial crypts but also in mucosal blood microvessels. The mucosal vessels were enlarged and tortuous, and the mean size of the vessels is significantly increased, all these reflecting pathological angiogenesis. Angiogenesis plays an important role not only in normal tissue injury healing, but as shown, more recently also in inflammation.36,37 Vascular endothelial growth factor-A (VEGF-A) and its receptor (VEGFR2) are considered important mediators of pathological angiogenesis.36 In active IBD, increased levels of VEGF-A and VEGFR2 in colonic mucosa have been demonstrated.38 This finding confirmed the contention that abnormal angiogenesis is not only a consequence of inflammation but also may be a driving force inducing and promoting inflammatory process. It is thus conceivable that persistent pathological angiogenesis in spite of macroscopically healed mucosa may contribute to the disease recurrence in UC.
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In this study, we also demonstrated impaired function of endothelial “barrier” function of mucosal blood vessels in UC-IR patients, reflected by increased microvascular permeability to plasma containing fluorescein. For these studies, we used measurements of extravasation of fluorescein reflected by fluorescence gradient between outside and inside the vessels on CLE images as a marker of in vivo microvascular permeability. In our previous study, we have demonstrated that after i.v. passage, fluorescein crosses the endothelial microvascular “barrier” and penetrates into the extravascular space as well into the epithelial cells.39 The increased microvascular permeability in colonic mucosa of UC-IR patients may be an important mechanism for sustained inflammation by enabling the flux of inflammatory cells into extravascular space.40,41 This pilot study was aimed to test the feasibility of using CLE to quantify crypt regeneration and deformation, vessel size, vascular permeability and to use standard biopsy specimens to determine quantitatively the expression of COX2 and mtDNA mutation. In this regard, these aims were fully achieved. Naturally, limitation of such pilot study is that because of a small patient number, it cannot specifically evaluate the effect of treatment, predict recurrence, etc. However, once the feasibility of CLE quantitative assessment of mucosal healing in UC has been established, future studies can answer these questions. In summary, this study shows that in patients with UC in remission and with normal endoscopic appearance of colonic mucosa, CLE allows in vivo, real-time detection of structural and microvascular changes of the colonic mucosa, not detectable by standard endoscopy, which may contribute to the recurrence of the disease. In addition, CLE allows in vivo measurements of mucosal microvascular permeability and molecular imaging. CLE might be thus considered as the technique of choice and a new gold standard to evaluate mucosal healing in UC patients.
References 1 Tarnawski A, Hollander D, Krause WJ, Dabros W, Stachura J, Gergely H. “Healed” experimental gastric ulcers remain histologically and ultrastructurally abnormal. J. Clin. Gastroenterol. 1990; 12 (Suppl. 1): S139–47. 2 Tarnawski A, Stachura J, Krause WJ, Douglass TG, Gergely H. Quality of gastric ulcer healing: a new, emerging concept. J. Clin. Gastroenterol. 1991; 13 (Suppl. 1): S42–7. 3 Arakawa T, Kobayashi K. Quality of ulcer healing—a new concept to rank healed peptic ulcers. Gastroenterol. Jpn. 1993; 28 (Suppl. 5): 158–62. 4 Arakawa T, Watanabe T, Tanigawa T, Tominaga K, Fujiwara Y, Morimoto K. Quality of ulcer healing in gastrointestinal tract: Its pathophysiology and clinical relevance. World J. Gastroenterol. 2012; 18: 4811–22. 5 Lichtenstein GR, Rutgeerts P. Importance of mucosal healing in ulcerative colitis. Inflamm. Bowel Dis. 2010; 16: 338–46. 6 Mazzuoli S, Guglielmi FW, Antonelli E, Salemme M, Bassotti G, Villanacci V. Definition and evaluation of mucosal healing in clinical practice. Dig. Liver Dis. 2013; 45: 969–77. 7 Peyrin-Biroulet L, Bressenot A, Kampman W. Histologic Remission: The Ultimate Therapeutic Goal in Ulcerative Colitis? Clin. Gastroenterol. Hepatol. 2014; 12: 929–34.e2. 8 Yokoyama K, Kobayashi K, Mukae M, Sada M, Koizumi W. Clinical Study of the Relation between Mucosal Healing and
Confocal laser endomicroscopy & colitis
9
10
11
12 13
14
15
16
17
18
19
20
21
22
23
24
25
26
Long-Term Outcomes in Ulcerative Colitis. Gastroenterol. Res. Pract. 2013; doi: 10.1155/2013/192794. Seidelin JB, Coskun M, Nielsen OH. Mucosal healing in ulcerative colitis: pathophysiology and pharmacology. Adv. Clin. Chem. 2013; 59: 101–23. Dave M, Loftus EV Jr. Mucosal healing in inflammatory bowel disease-a true paradigm of success? Gastroenterol. Hepatol. (N Y) 2012; 8: 29–38. Pineton de Chambrun G, Peyrin-Biroulet L, Lémann M, Colombel JF. Clinical implications of mucosal healing for the management of IBD. Nat. Rev. Gastroenterol. Hepatol. 2010; 7: 15–29. Neurath MF, Travis SP. Mucosal healing in inflammatory bowel diseases: a systematic review. Gut 2012; 61: 1619–35. Rutgeerts P, Sandborn WJ, Feagan BG, Reinisch W, Olson A, Johanns J et al. Infliximab for induction and maintenance therapy for ulcerative colitis. N. Engl. J. Med. 2005; 353: 2462–76. Colombel JF, Rutgeerts P, Reinisch W, Esser D, Wang Y, Lang Y et al. Early mucosal healing with infliximab is associated with improved long-term clinical outcomes in ulcerative colitis. Gastroenterology 2011; 141: 1194–201. Kiesslich R, Goetz M, Lammersdorf K, Schneider C, Burg J, Stolte M et al. Chromoscopy-guided endomicroscopy increases the diagnostic yield of intraepithelial neoplasia in ulcerative colitis. Gastroenterology 2007; 132: 874–82. Watanabe O, Ando T, Maeda O, Hasegawa M, Ishikawa D, Ishiguro K et al. Confocal endomicroscopy in patients with ulcerative colitis. J. Gastroenterol. Hepatol. 2008; 23 (Suppl 2): S286–90. Li CQ, Xie XJ, Yu T, Gu XM, Zuo XL, Zhou CJ et al. Classification of inflammation activity in ulcerative colitis by confocal laser endomicroscopy. Am. J. Gastroenterol. 2010; 105: 1391–6. Neumann H, Vieth M, Atreya R, Grauer M, Siebler J, Bernatik T et al. Assessment of Crohn’s disease activity by confocal laser endomicroscopy. Inflamm. Bowel. Dis. 2012; 18: 2261–9. Kiesslich R, Duckworth CA, Moussata D, Gloeckner A, Lim LG, Goetz M et al. Local barrier dysfunction identified by confocal laser endomicroscopy predicts relapse in inflammatory bowel disease. Gut 2012; 61: 1146–53. Kiesslich R, Burg J, Vieth M, Gnaendiger J, Enders M, Delaney P et al. Confocal laser endoscopy for diagnosing intraepithelial neoplasias and colorectal cancer in vivo. Gastroenterology 2004; 127: 706–13. Gheonea DI, Cârt¸âna˘ T, Ciurea T, Popescu C, Ba˘da˘ra˘u A, Sa˘ftoiu A. Confocal laser endomicroscopy and immunoendoscopy for real-time assessment of vascularization in gastrointestinal malignancies. World J. Gastroenterol. 2011; 17: 21–7. Gheorghe C, Cotruta B, Iacob R, Becheanu G, Dumbrava M, Gheorghe L. Endomicroscopy for assessing mucosal healing in patients with ulcerative colitis. J. Gastrointestin. Liver Dis. 2011; 20: 423–6. Pai R, Soreghan B, Szabo IL, Pavelka M, Baatar D, Tarnawski AS. Prostaglandin E2 transactivates EGF receptor: a novel mechanism for promoting colon cancer growth and gastrointestinal hypertrophy. Nat. Med. 2002; 8: 289–93. Pai R, Nakamura T, Moon WS, Tarnawski AS. Prostaglandins promote colon cancer cell invasion; signaling by cross-talk between two distinct growth factor receptors. FASEB J. 2003; 17: 1640–7. Ussakli CH, Ebaee A, Binkley J, Brentnall TA, Emond MJ, Rabinovitch PS et al. Mitochondria and tumor progression in ulcerative colitis. J. Natl. Cancer Inst. 2013; 105: 1239–48. Greaves LC, Preston SL, Tadrous PJ, Taylor RW, Barron MJ, Oukrif D et al. Mitochondrial DNA mutations are established in human colonic stem cells, and mutated clones expand by crypt fission. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 714–9.
Journal of Gastroenterology and Hepatology 2015; 30 (Suppl. 1): 85–92 © 2015 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
91
Confocal laser endomicroscopy & colitis
V Macé et al.
27 Taylor RW, Barron MJ, Borthwick GM, Gospel A, Chinnery PF, Samuels DC et al. Mitochondrial DNA mutations in human colonic crypt stem cells. J. Clin. Invest. 2003; 112: 1351–60. 28 Musquer N, Coquenlorge S, Bourreille A, Aubert P, Matysiak-Budnik T, des Varannes SB et al. Probe-based confocal laser endomicroscopy: a new method for quantitative analysis of pit structure in healthy and Crohn’s disease patients. Dig. Liver Dis. 2013; 45: 487–92. 29 Geboes K, Riddell R, Ost A, Jensfelt B, Persson T, Löfberg R. A reproducible grading scale for histological assessment of inflammation in ulcerative colitis. Gut 2000; 47: 404–9. 30 Wright NA, Pike C, Elia G. Induction of a novel epidermal growth factor-secreting cell lineage by mucosal ulceration in human gastrointestinal stem cells. Nature 1990; 343: 82–5. 31 Tarnawski A, Stachura J, Durbin T, Sarfeh IJ, Gergely H. Increased expression of epidermal growth factor receptor during gastric ulcer healing in rats. Gastroenterology 1992; 102: 695–8. 32 Harada S, Nagy JA, Sullivan KA, Thomas KA, Endo N, Rodan GA et al. Induction of vascular endothelial growth factor expression by prostaglandin E2 and E1 in osteoblasts. J. Clin. Invest. 1994; 93: 2490–6. 33 Wang D, Wang H, Brown J, Daikoku T, Ning W, Shi Q et al. CXCL1 induced by prostaglandin E2 promotes angiogenesis in colorectal cancer. J. Exp. Med. 2006; 203: 941–51. 34 Pai R, Szabo IL, Soreghan BA, Atay S, Kawanaka H, Tarnawski AS. PGE(2) stimulates VEGF expression in endothelial cells via
92
35 36
37
38
39
40
41
ERK2/JNK1 signaling pathways. Biochem. Biophys. Res. Commun. 2001; 286: 923–8. Capaldi RA. Structure and function of cytochrome c oxidase. Annu. Rev. Biochem. 1990; 59: 569–96. Danese S. Inflammation and the mucosal microcirculation in inflammatory bowel disease: the ebb and flow. Curr. Opin. Gastroenterol. 2007; 23: 384–9. Dvorak HF, Detmar M, Claffey KP, Nagy JA, van de Water L, Senger DR. Vascular permeability factor/vascular endothelial growth factor: an important mediator of angiogenesis in malignancy and inflammation. Int. Arch. Allergy Immunol. 1995; 107: 233–5. Scaldaferri F, Vetrano S, Sans M, Arena V, Straface G, Stigliano E et al. VEGF-A links angiogenesis and inflammation in inflammatory bowel disease pathogen-esis. Gastroenterology 2009; 136: 585–95. Coron E, Mosnier JF, Ahluwalia A, Le Rhun M, Galmiche JP, Tarnawski AS, Matysiak-Budnik T. Colonic mucosal biopsies obtained during confocal endomicroscopy are pre-stained with fluorescein in vivo and are suitable for histologic evaluation. Endoscopy 2012; 44: 148–53. Munjal C, Tyagi N, Lominadze D, Tyagi SC. Matrix metalloproteinase-9 in homocysteine-induced intestinal microvascular endothelial paracellular and transcellular permeability. J. Cell. Biochem. 2012; 113: 1159–69. Bardin N, Reumaux D, Geboes K, Colombel JF, Blot-Chabaud M, Sampol J et al. Increased expression of CD146, a new marker of the endothelial junction in active inflammatory bowel disease. Inflamm. Bowel Dis. 2006; 12: 16–21.
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