Angiogenesis as a Novel Component of Inflammatory Bowel Disease Pathogenesis

Background & Aims: Angiogenesis is a critical component of neoplastic and chronic inflammatory disorders, but whether angiogenesis also occurs in inflammatory bowel disease (IBD) has yet to be established. We assessed mucosal vascularization, expression of endothelial αVβ3 integrin, angiogenic factors, and their bioactivity in Crohn’s disease (CD) and ulcerative colitis (UC) mucosa. Methods: Mucosal endothelium was immunostained for CD31 and factor VIII and quantified by digital morphometry. αVβ3 expression was studied in vivo by confocal microscopy and in vitro by flow cytometric analysis of human intestinal microvascular endothelial cells (HIMECs). Vascular endothelial growth factor (VEGF), interleukin (IL)-8, and bFGF levels were measured in mucosal extracts and cells and angiogenic bioactivity shown by induction of HIMEC migration and the corneal and chorioallantoic membrane angiogenesis assays. Results: Microvessel density was increased in IBD mucosa. Endothelial αVβ3 was strongly expressed in IBD but only sporadically in normal mucosa and was up-regulated in HIMECs by VEGF, tumor necrosis factor α, and bFGF. IBD mucosal extracts induced a significantly higher degree of HIMEC migration than control mucosa, and this response was mostly dependent on IL-8 and less on basic fibroblast growth factor or vascular endothelial growth factor. Compared with normal mucosa, IBD mucosal extracts induced a potent angiogenic response in both the corneal and chorioallantoic membrane assays. Conclusions: These results provide morphological, phenotypic and functional evidence of potent angiogenic activity in both CD and UC mucosa, indicating that the local microvasculature undergoes an intense process of inflammation-dependent angiogenesis. Thus, angiogenesis appears to be an integral component of IBD pathogenesis, providing the practical and conceptual framework for anti-angiogenic therapies in IBD. Background & Aims: Angiogenesis is a critical component of neoplastic and chronic inflammatory disorders, but whether angiogenesis also occurs in inflammatory bowel disease (IBD) has yet to be established. We assessed mucosal vascularization, expression of endothelial αVβ3 integrin, angiogenic factors, and their bioactivity in Crohn’s disease (CD) and ulcerative colitis (UC) mucosa. Methods: Mucosal endothelium was immunostained for CD31 and factor VIII and quantified by digital morphometry. αVβ3 expression was studied in vivo by confocal microscopy and in vitro by flow cytometric analysis of human intestinal microvascular endothelial cells (HIMECs). Vascular endothelial growth factor (VEGF), interleukin (IL)-8, and bFGF levels were measured in mucosal extracts and cells and angiogenic bioactivity shown by induction of HIMEC migration and the corneal and chorioallantoic membrane angiogenesis assays. Results: Microvessel density was increased in IBD mucosa. Endothelial αVβ3 was strongly expressed in IBD but only sporadically in normal mucosa and was up-regulated in HIMECs by VEGF, tumor necrosis factor α, and bFGF. IBD mucosal extracts induced a significantly higher degree of HIMEC migration than control mucosa, and this response was mostly dependent on IL-8 and less on basic fibroblast growth factor or vascular endothelial growth factor. Compared with normal mucosa, IBD mucosal extracts induced a potent angiogenic response in both the corneal and chorioallantoic membrane assays. Conclusions: These results provide morphological, phenotypic and functional evidence of potent angiogenic activity in both CD and UC mucosa, indicating that the local microvasculature undergoes an intense process of inflammation-dependent angiogenesis. Thus, angiogenesis appears to be an integral component of IBD pathogenesis, providing the practical and conceptual framework for anti-angiogenic therapies in IBD. See CME Quiz on page 2207. See CME Quiz on page 2207. Angiogenesis, also termed neoangiogenesis, is the process of new capillary formation from preexisting vasculature in adult tissues.1Folkman J. Shing Y. Angiogenesis.J Biol Chem. 1992; 267: 10931-10934Abstract Full Text PDF Google Scholar Angiogenesis is a fundamental constituent of many complex biological processes, including growth, development, and repair. In the last 3 decades, angiogenesis has emerged as a phenomenon essential for the growth of tumors, and its inhibition has been hailed as a cornerstone of cancer therapy.2Folkman J. Tumor angiogenesis therapeutic implications.N Engl J Med. 1971; 285: 1182-1186Google Scholar In reality, the importance of angiogenesis extends well beyond cancer biology and has been shown to be an integral component of nonneoplastic chronic inflammatory and autoimmune diseases as diverse as atherosclerosis, rheumatoid arthritis, diabetic retinopathy, psoriasis, airway inflammation, peptic ulcers, and Alzheimer’s disease.3Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease.Nat Med. 1995; 1: 27-31Google Scholar, 4Carmeliet P. Angiogenesis in health and disease.Nat Med. 2003; 9: 653-660Google Scholar, 5Gould V.E. Wagner B.M. Angiogenesis an expanding universe.Hum Pathol. 2002; 33: 1061-1063Google Scholar Growth of new blood vessels is intrinsic to inflammation and is associated with structural changes, including activation and proliferation of endothelial cells and capillary and venule remodeling, all of which result in an expansion of the tissue microvascular bed.6Majno G. Chronic inflammation links with angiogenesis and wound healing.Am J Pathol. 1998; 153: 1035-1039Google Scholar, 7Carmeliet P. Mechanisms of angiogenesis and arteriogenesis.Nat Med. 2000; 6: 389-395Google Scholar, 8Bagli E. Xagorari A. Papetropoulos A. Murphy C. Fotsis T. Angiogenesis in inflammation.Autoimmun Rev. 2004; 3: S26Google Scholar A potential functional consequence of this expansion is the promotion of inflammation through various correlated mechanisms. First, influx of inflammatory cells may increase; second, there is an increased nutrient supply that keeps feeding a metabolically active immune process; and third, the activated endothelium contributes to the local production of cytokines, chemokines, and matrix metalloproteinases.9Firestein G.S. Starving the synovium angiogenesis and inflammation in rheumatoid arthritis.J Clin Invest. 1999; 103: 3-4Google Scholar, 10Szekanecz Z. Koch A.E. Vascular endothelium and immune responses implications for inflammation and angiogenesis.Rheum Dis Clin North Am. 2004; 30: 97-114Google Scholar Therefore, the anatomic expansion of the microvascular bed combined with its increased functional activation can foster further recruitment of inflammatory cells, and angiogenesis and inflammation may become chronically codependent processes.6Majno G. Chronic inflammation links with angiogenesis and wound healing.Am J Pathol. 1998; 153: 1035-1039Google Scholar, 8Bagli E. Xagorari A. Papetropoulos A. Murphy C. Fotsis T. Angiogenesis in inflammation.Autoimmun Rev. 2004; 3: S26Google Scholar, 10Szekanecz Z. Koch A.E. Vascular endothelium and immune responses implications for inflammation and angiogenesis.Rheum Dis Clin North Am. 2004; 30: 97-114Google Scholar, 11Jackson J.R. Seed M.P. Kircher C.H. Willoughby D.A. Winkler J.D. The codependence of angiogenesis and chronic inflammation.FASEB J. 1997; 11: 457-465Crossref Scopus (622) Google Scholar A prime example of the process of immune-driven angiogenesis is seen in rheumatoid arthritis in which neovascularization is one of the earliest histopathological findings and a key contributor to pannus formation, in addition to playing an active proinflammatory role by being a source of cytokines, chemokines, and proteases.12Koch A.E. The role of angiogenesis in rheumatoid arthritis recent developments.Ann Rheum Dis. 2000; 59: i65-i71Google Scholar Similar phenomena are observed in psoriasis,13Creamer D. Sullivan D. Bicknell R. Barker J. Angiogenesis in psoriasis.Angiogenesis. 2002; 5: 231-236Google Scholar and in fact they are likely to occur in all chronic immune–mediated inflammatory conditions, including Crohn’s disease (CD) and ulcerative colitis (UC), the 2 major forms of inflammatory bowel disease (IBD).14Fiocchi C. Inflammatory bowel disease etiology and pathogenesis.Gastroenterology. 1998; 115: 182-205Google Scholar Very recently, IBD has been listed among the several conditions characterized or caused by abnormal or excessive angiogenesis,4Carmeliet P. Angiogenesis in health and disease.Nat Med. 2003; 9: 653-660Google Scholar although detailed information about the angiogenic status in inflamed mucosa is essentially nonexisting.15Laroux F.S. Grisham M.B. Immunological basis of inflammatory bowel disease role of the microcirculation.Microcirculation. 2001; 8: 283-301Google Scholar, 16Hatoum O.A. Binion D.G. The vasculature and inflammatory bowel disease contribution to pathogenesis and clinical pathology.Inflamm Bowel Dis. 2005; 11: 304-313Google Scholar At present, only a handful of reports suggest the presence of an abnormal vascular bed in IBD tissue. Based on resin casting, the CD mucosal microvasculature displays an aberrant morphology,17Wakefield A.J. Sawyerr A.M. Dhillon A.P. Pittilo R.M. Rowles P.M. Lewis A.A. Pounder R.E. Pathogenesis of Crohn’s disease multifocal gastrointestinal infarction.Lancet. 1989; 2: 1057-1062Google Scholar whereas Doppler ultrasound shows increased vessel density in the inflamed bowel loops18Spalinger J. Patriquin H. Miron M.C. Marx G. Herzog D. Dubois J. Dubinsky M. Seidman E.G. Doppler US in patients with Crohn disease vessel density in the diseased bowel reflects disease activity.Radiology. 2000; 217: 787-791Google Scholar and a recent histologic study indicates that vascularization in IBD is particularly prominent in areas of active inflammation.19Saito S. Tsuno N.H. Sunami E. Hori N. Kitayama J. Kazama S. Okaji Y. Kawai K. Kanazawa T. Watanabe T. Shibata Y. Nagawa H. Expression of platelet-derived endothelial cell growth factor in inflammatory bowel disease.J Gastroenterol. 2003; 38: 229-237Google Scholar However, direct evidence of vascular remodeling and functionally active angiogenesis in IBD is still missing. Therefore, the present study was designed to obtain objective and direct evidence of increased vascularization in IBD mucosa, show activation of the local microvasculature, assess the levels of angiogenic factors in the inflamed tissues, identify their cellular sources, and detect biologically active angiogenic activity in IBD mucosa. Surgical specimens were used for isolation of human intestinal microvascular endothelial cells (HIMECs), human intestinal fibroblasts (HIFs), and lamina propria mononuclear cells (LPMCs). All specimens were of colonic origin, and cells were isolated and cultured as previously reported.20Fiocchi C. Battisto J.R. Farmer R.G. Gut mucosal lymphocytes in inflammatory bowel disease isolation and preliminary functional characterization.Dig Dis Sci. 1979; 24: 705-717Google Scholar, 21Binion D.G. West G.A. Ina K. Ziats N.P. Emancipator S.N. Fiocchi C. Enhanced leukocyte binding by intestinal microvascular endothelial cells in inflammatory bowel disease.Gastroenterology. 1997; 112: 1895-1907Abstract Full Text Full Text PDF Scopus (184) Google Scholar, 22Strong S.A. Pizarro T.T. Klein J.S. Cominelli F. Fiocchi C. Proinflammatory cytokines differentially modulate their own expression in human intestinal mucosal mesenchymal cells.Gastroenterology. 1998; 114: 1244-1256Google Scholar, 23Vogel J.D. West G.A. Danese S. de la Motte C. Phillips M.H. Strong S.A. Willis J. Fiocchi C. CD40-mediated immune-nonimmune cell interactions induce mucosal fibroblast chemokines leading to T-cell transmigration.Gastroenterology. 2004; : 63-80Abstract Full Text Full Text PDF Scopus (67) Google Scholar Tissues were obtained from histologically normal large-bowel specimens from patients admitted for bowel resection because of malignant and nonmalignant conditions, including colon cancer, benign polyps, and diverticulosis. Involved and uninvolved CD and UC colonic tissues were also obtained. All diagnoses were confirmed by clinical, radiologic, endoscopic and histologic criteria. The study was approved by the Institutional Review Board of University Hospitals of Cleveland. Immunostaining was performed as previously described.23Vogel J.D. West G.A. Danese S. de la Motte C. Phillips M.H. Strong S.A. Willis J. Fiocchi C. CD40-mediated immune-nonimmune cell interactions induce mucosal fibroblast chemokines leading to T-cell transmigration.Gastroenterology. 2004; : 63-80Abstract Full Text Full Text PDF Scopus (67) Google Scholar Briefly, paraffin-embedded intestinal sections of histologically normal control and IBD-involved mucosa or paired uninvolved colonic mucosa were cut at 3 μm thickness, deparaffinized then hydrated, blocked for endogenous peroxidase using 3% H2O2/H2O, and subsequently subjected to microwave epitope enhancement by using a Dako Target retrieval solution (Dako, Carpenteria, CA) at pH 10.00. Incubation with a primary antibody cocktail containing anti-CD31 and anti–von Willebrand factor (Dako) was performed at 1:200 dilution for 30 minutes at room temperature. Detection was achieved by using a standard streptavidin-biotin system (Dako), and antigen localization was visualized with 3′-3-diamino benzidene (DAB; Sigma, St Louis, MO). Computerized morphometric analysis was performed by using an international consensus method for quantification of angiogenesis.24Weidner N. Current pathologic methods for measuring intratumoral microvessel density within breast carcinoma and other solid tumors.Breast Cancer Res Treat. 1995; 36: 169-180Google Scholar, 25Vermeulen P.B. Gasparini G. Fox S.B. Toi M. Martin L. McCulloch P. Pezzella F. Viale G. Weidner N. Harris A.L. Dirix L.Y. Quantification of angiogenesis in solid human tumours an international consensus on the methodology and criteria of evaluation.Eur J Cancer. 1996; 32A: 2474-2484Google Scholar Briefly, stained colonic sections were scanned at low power (×40) to detect the most vascularized area, after which at least 5 microphotographs at high (×200) magnification were taken of the mucosa, defined as the area above the muscularis mucosae (ie, the lamina propria), as well as the submucosa, defined as the area between the muscularis mucosae and muscularis propria. The following parameters were obtained by using an Optronics Color digital camera (Olympus Corporation, Tokyo, Japan): (1) number of vessels/field (vascular density), (2) blood vessel total area/tissue section, (3) mean blood vessel area (in μm2)/tissue section, and (4) larger diameter/vessel. Quantitative analysis of data was performed by using Image Pro Plus (Media Cybernetics, Silver Spring, MD). Staining, microscopy, and imaging were performed as previously reported26Danese S. de la Motte C. Sturm A. JD V. West G.A. Strong S.A. Katz J. Fiocchi C. Platelets trigger a CD40-dependent inflammatory response in the microvasculature of inflammatory bowel disease patients.Gastroenterology. 2003; 124: 1249-1264Google Scholar by using a Leica TCS-SP laser scanning confocal microscope (Leica, Heidelberg, Germany) equipped with 3 lasers and photodetectors that permit detection of 3 separate fluorochromes. Frozen sections of histologically normal control and IBD-involved and uninvolved colonic mucosa were fixed in cold acetone for 1 minute, blocked in Hanks’ balanced salt solution containing 2% fetal bovine serum (30 minutes at 25°C), and stained with a monoclonal antibody directed to αvβ3 (Chemicon International, Temecula, CA; 1 μg/mL), or a polyclonal anti-CD31 antibody (1:100) (Santa Cruz Biotecnology, Santa Cruz, CA), both in Hanks’ balanced salt solution with 2% fetal bovine serum overnight at 4°C. After incubation, the slides were rinsed 3 times and incubated with either biotinylated goat antimouse IgG (for αvβ3) or Alexa 488 (green) conjugated antigoat IgG (1:1000) (Molecular Probes, Eugene, OR) for 1 hour at room temperature. The anti-αvβ3–incubated sections were again rinsed 3 times and exposed to a solution of fluorescein isothiocyanate conjugated (FITC) streptavidin (1:500) for 15 minutes. All slides were rinsed 3 additional times, blotted, and cover slips placed by using Vectashield mounting medium containing 4′6-diamidino-2-phenylindole (Vector Labs, Inc, Burlingame, CA). The slides were sealed and stored at −20°C until examined by confocal microscopy. Quantification of αVβ3 integrin expression was performed on immunostained frozen sections by a semiquantitative method (scores from 0 to 3), as described by Vonlaufen et al.27Vonlaufen A. Wiedle G. Borisch B. Birrer S. Luder P. Imhof B.A. Integrin alpha(v)beta(3) expression in colon carcinoma correlates with survival.Mod Pathol. 2001; 14: 1126-1132Google Scholar Detection of HIMEC integrin αVβ3 expression was performed by flow cytometry as previously described.26Danese S. de la Motte C. Sturm A. JD V. West G.A. Strong S.A. Katz J. Fiocchi C. Platelets trigger a CD40-dependent inflammatory response in the microvasculature of inflammatory bowel disease patients.Gastroenterology. 2003; 124: 1249-1264Google Scholar Briefly, HIMECs were plated on plastic and starved for 48 hours and then exposed to 100 U/mL tumor necrosis factor α (TNF-α) 50 ng/mL of basic fibroblast growth factor (bFGF), or 50 ng/mL of vascular endothelial growth factor (VEGF). After 24 hours, HIMECs were quickly tripsinized, washed twice, and incubated with anti-αVβ3 monoclonal or isotype control antibody for 30 minutes in ice, washed again, and incubated with a mouse secondary-fluorescein isothiocyanate conjugated (FITC) antibody. After additional washing, HIMECs were analyzed by flow cytometry. Samples were analyzed by quantitative flow cytometry by using a Coulter Epics XL Flow Cytometer (Beckman Coulter, Inc, Fullerton, CA). Each analysis was performed on at least 10,000 events. Quantification of αvβ3 expression was obtained by using the Winlist software program (Verity Software House, Topsham, ME). In some experiments, vitronectin-grown HIMECs were incubated with 100 μg/mL Vitaxin (Medimmune, Gaithersburg, MD), a humanized αVβ3 specific antibody, or with 500 μg/mL of the αvβ3-blocking peptide RGDV, and then plated again on vitronectin-coated wells. HIMEC morphology was followed up by digital photography (Olympus), and, after 6 hours, adherent and nonadherent cells were collected, stained with propidium iodide, and apoptosis determined by flow cytometry.28Sturm A. Leite A.Z. Danese S. Krivacic K.A. West G.A. Mohr S. Jacobberger J.W. Fiocchi C. Divergent cell cycle kinetics underlie the distinct functional capacity of mucosal T cells in Crohn’s disease and ulcerative colitis.Gut. 2004; 53: 1624-1631Google Scholar Endoscopic biopsies were collected from the mucosa of UC and CD patients with active and inactive disease and from normal mucosa of control subjects. Biopsies were homogenized and sonicated in protein-lyses buffer containing protease inhibitors.29Koch A.E. Volin M.V. Woods J.M. Kunkel S.L. Connors M.A. Harlow L.A. Woodruff D.C. Burdick M.D. Strieter R.M. Regulation of angiogenesis by the C-X-C chemokines interleukin-8 and epithelial neutrophil activating peptide 78 in the rheumatoid joint.Arthritis Rheum. 2001; 44: 31-40Google Scholar, 30Keane M.P. Arenberg D.A. Lynch 3rd, J.P. Whyte R.I. Iannettoni M.D. Burdick M.D. Wilke C.A. Morris S.B. Glass M.C. DiGiovine B. Kunkel S.L. Strieter R.M. The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis.J Immunol. 1997; 159: 1437-1443Google Scholar Samples were centrifuged at 900 × g for 15 minutes; supernatants were collected, filter sterilized, their protein concentration measured (Bio-Rad Laboratories, Hercules, CA), and kept frozen until analysis.31Danese S. Katz J. Saibeni S. Papa A. Gasbarrini A. Vecchi M. Fiocchi C. Activated platelets are the source of elevated levels of soluble CD40 ligand in the circulation of inflammatory bowel disease patients.Gut. 2003; 52: 1435-1441Google Scholar For induction of angiogenic factor production by HIFs or HIMECs, cells were seeded in 24-well cluster plates at 5 × 104/well/mL of their respective medium and grown to subconfluence, when 100 U/mL TNF-α (R&D Systems, Minneapolis, MN) or fresh medium were added. After a 48-hour incubation period, the confluent monolayers were extensively washed and supernatants were harvested for storage at −20°C. For induction of angiogenic factor production by LPMCs, 2 × 106/well/mL cells were left untreated or stimulated with lipopolysaccharide (LPS) (1 μg/mL) or anti-CD3/CD28 (10 and 1 μg/mL, respectively). After 48 hours, supernatants were collected and stored at −20°C until analysis. Interleukin (IL)-8, VEGF, and bFGF were measured in the supernatants by enzyme-linked immunosorbent assay (ELISA, R&D Systems), as previously reported.32Danese S. de la Motte C. Rivera-Reyes B.M. Sans M. Levine A.D. Fiocchi C. Cutting edge: T cells trigger CD40-dependent platelet activation and granular RANTES release: a novel pathway for immune response amplification.J Immunol. 2004; 172: 2011-2015Google Scholar Content of VEGF in mucosal extracts was assessed by Western blotting using a primary rabbit anti-VEGF antibody (Santa Cruz) and analyzed by densitometry. Chemotaxis was assessed by using previously reported methods with some modifications.23Vogel J.D. West G.A. Danese S. de la Motte C. Phillips M.H. Strong S.A. Willis J. Fiocchi C. CD40-mediated immune-nonimmune cell interactions induce mucosal fibroblast chemokines leading to T-cell transmigration.Gastroenterology. 2004; : 63-80Abstract Full Text Full Text PDF Scopus (67) Google Scholar, 33Heidemann J. Ogawa H. Dwinell M.B. Rafiee P. Maaser C. Gockel H.R. Otterson M.F. Ota D.M. Lugering N. Domschke W. Binion D.G. Angiogenic effects of interleukin 8 (CXCL8) in human intestinal microvascular endothelial cells are mediated by CXCR2.J Biol Chem. 2003; 278: 8508-8515Google Scholar Briefly, fluorescence-blocked polycarbonate filters (8 μm pore size; BD Bioscience, Franklin Lakes, NJ) were coated with human fibronectin (10 μg/mL) for 1 hour at room temperature. By using a transwell system, 35 × 104 HIMECs were plated in the upper chamber in MCDB-131 medium, while the lower chamber contained chemotaxis buffer with phorbol myristate acetate (PMA) (10 ng/mL), bFGF (50 ng/mL), or VEGF (50 ng/mL) as positive controls or increasing concentrations (0.01–0.1 μg/mL) of control or IBD-derived mucosal extracts. In some wells, 25 μg/mL of blocking antibodies against IL-8, bFGF, or VEGF or control antibody were added to the chemotaxis buffer, as well as 100 μg/mL of Vitaxin or 500 μg/mL of the αvβ3-blocking peptide RGDV. After 4 hours, buffer was removed from both chambers, and HIMECs migrated onto the lower surface of the porous membrane were washed twice in phosphate-buffered saline (PBS) and stained with calcein for 15 minutes at 37°C. Duplicates of migrated cells were observed with an inverted fluorescence microscope and counted in 6 random high-power (×200) fields. Quantitative analysis of data was performed by using the Image Pro Plus software. The murine corneal angiogenesis model was performed as previously described34Pola R. Ling L.E. Silver M. Corbley M.J. Kearney M. Blake Pepinsky R. Shapiro R. Taylor F.R. Baker D.P. Asahara T. Isner J.M. The morphogen sonic hedgehog is an indirect angiogenic agent upregulating two families of angiogenic growth factors.Nat Med. 2001; 7: 706-711Google Scholar by using a protocol approved by the animal experimentation committee of the Catholic University of Rome. Briefly, a single corneal pocket was created in each eye by using a modified von Graefe cataract knife. Filter papers containing PBS, 50 ng/mL of VEGF, or 0.1 μg of control or IBD mucosal extracts were prepared and placed in each corneal pocket, positioned 1.0 mm from the corneal limbus. After surgery, erythromycin ophthalmic ointment was applied to each eye. Corneal neovascularization was evaluated 6 days after surgery by fluorescence microscopy, when mice received an intracardiac injection of 500 μg of the endothelial cell marker BS-1 lectin, conjugated to FITC (Vector Laboratories). Thirty minutes later, mice were sacrificed, and each eye was enucleated and fixed in 1% paraformaldehyde solution for 1 hour. Then, corneal hemispheres were prepared under the dissecting microscope, placed on glass slides, and analyzed by fluorescence microscopy. Six to 8 eyes were studied in each group. The chick embryo chorioallantoic membrane (CAM) assay was performed as described.35West D. Thompson W. Sells P. Burbridge M. Angiogenesis assays using chick chorioallantoic membrane.in: Murray C. Angiogenesis protocols. Methods Mol Med. 2001: 107-129Google Scholar Briefly, a 4-day-old shell-less CAM was plated in a petri dish at 37°C in a humidified incubator. On day 6, a 1-mm filter paper disk containing PBS, 50 ng/mL of VEGF, or 0.1 μg of control or IBD mucosal extracts was plated on the yolk sac membrane. After 2 days, the vascularization was observed under a fiberoptic light source and photographed. Data were analyzed by Graphpad software (San Diego, CA) and expressed as mean ± standard error of the mean. The analysis of variance followed by the appropriate post hoc test was used. Statistical significance was set at P < .05. To obtain objective evidence of whether mucosal vascularization is increased in IBD, we initially performed immunohistochemical staining of histologically normal control and active CD- and UC-involved colonic tissue with antibodies specifically recognizing surface CD31 and intracytoplasmic von Willebrand/factor VIII, 2 widely used endothelial markers.24Weidner N. Current pathologic methods for measuring intratumoral microvessel density within breast carcinoma and other solid tumors.Breast Cancer Res Treat. 1995; 36: 169-180Google Scholar, 25Vermeulen P.B. Gasparini G. Fox S.B. Toi M. Martin L. McCulloch P. Pezzella F. Viale G. Weidner N. Harris A.L. Dirix L.Y. Quantification of angiogenesis in solid human tumours an international consensus on the methodology and criteria of evaluation.Eur J Cancer. 1996; 32A: 2474-2484Google Scholar Normal colon showed sporadic thin vessels in the lamina propria and submucosa, whereas tissue involved by CD or UC contained numerous readily detectable dilated vessels in both the lamina propria and submucosa (Figure 1). The same sections were morphometrically analyzed by multiple parameters obtained with a computerized imaging system.25Vermeulen P.B. Gasparini G. Fox S.B. Toi M. Martin L. McCulloch P. Pezzella F. Viale G. Weidner N. Harris A.L. Dirix L.Y. Quantification of angiogenesis in solid human tumours an international consensus on the methodology and criteria of evaluation.Eur J Cancer. 1996; 32A: 2474-2484Google Scholar The microvascular density measured as total number of vessels/field, percentage section area, mean vessel area, and mean larger vessel diameter were all significantly (P < .05–.01) increased in the lamina propria of CD and UC compared with histologically normal control specimens (Figure 2). The same parameters were also increased in the submucosa of IBD compared with control specimens (not shown). When paired specimens of inflamed and noninflamed autologous IBD mucosa were analyzed, a significantly (P < .05) increased vascularization was detected in the inflamed segments, whereas no differences were found between uninvolved IBD and control mucosa (not shown).Figure 2Computerized morphometric analysis of the microvasculature in control and IBD mucosa. After immunohistochemical staining for CD31 and von Willebrand/factor VIII of the microvasculature of histologically normal control, CD and UC colonic mucosa, sections were analyzed for the total number of vessels/field (microvascular density), percentage section area, mean vessel area, and larger vessel diameter. Data derived from 8 control, 8 CD and 9 UC samples. *P < .05 for UC and CD compared with control; **P < .01 for UC and CD compared with control.View Large Image Figure ViewerDownload (PPT) Morphometric evidence of increased vascularization suggests that angiogenesis is present in both forms of IBD, but this must be confirmed by detection of integrins selectively expressed by proliferating endothelial cells.36Eliceiri B.P. Cheresh D.A. The role of alpha v integrins during angiogenesis insights into potential mechanisms of action and clinical development.J Clin Invest. 1999; 103: 1227-1230Google Scholar, 37Eliceiri B.P. Cheresh D.A. Role of alpha v integrins during angiogenesis.Cancer J. 2000; 6: S245-S249Google Scholar Among several integrins, αVβ3 is unique because this molecule is expressed exclusively by angiogenic endothelium. Resting endothelium does not display the integrin αVβ3, whose expression is indispensable for proliferation during angiogenesis.37Eliceiri B.P. Cheresh D.A. Role of alpha v integrins during angiogenesis.Cancer J. 2000; 6: S245-S249Google Scholar Vessels were identified by CD31 staining (not shown), and, by using antibodies against αVβ3 (red) and a nuclear staining (4′,6-diamidino-2-phenylindole, blue), abundant αVβ3-positive endothelial cells were readily discernible in both CD and UC mucosa (Figure 3A). The integrin αVβ3 was barely detectable in the resting microvasculature of normal control mucosa and uninvolved IBD mucosa (not shown). To validate this visual assessment, a semiquantitative analysis was performed confirming a significant (P < .01) increase of αVβ3-expressing vessels in IBD compared with normal control mucosa (Figure 3B). In view of the detection of αVβ3-positive endothelial cells in both UC and CD mucosa, we investigated 2 functional aspects of this molecule that are critical to angiogenesis: first, its up-regulation by inflammatory and angiogenesis mediators and, se