Unfractionated Heparin and New Heparin Analogues from Ascidians (Chordate-Tunicate) Ameliorate Colitis in Rats

Abstract

The anti-inflammatory effect of mammalian heparin analogues, named dermatan sulfate and heparin, isolated from the ascidian Styela plicata was accessed in a TNBS-induced colitis model in rats. Subcutaneous administration of the invertebrate compounds during a 7-day period drastically reduced inflammation as observed by the normalization of the macroscopic and histological characteristics of the colon. At the molecular level, a decrease in the production of TNF-α, TGF-β, and VEGF was observed, as well as a reduction of NF-κB and MAPK kinase activation. At the cellular level, the heparin analogues attenuated lymphocyte and macrophage recruitment and epithelial cell apoptosis. A drastic reduction in collagen-mediated fibrosis was also observed. No hemorrhagic events were observed after glycan treatment. These results strongly indicate the potential therapeutic use of these compounds for the treatment of colonic inflammation with a lower risk of hemorrhage when compared with mammalian heparin. The anti-inflammatory effect of mammalian heparin analogues, named dermatan sulfate and heparin, isolated from the ascidian Styela plicata was accessed in a TNBS-induced colitis model in rats. Subcutaneous administration of the invertebrate compounds during a 7-day period drastically reduced inflammation as observed by the normalization of the macroscopic and histological characteristics of the colon. At the molecular level, a decrease in the production of TNF-α, TGF-β, and VEGF was observed, as well as a reduction of NF-κB and MAPK kinase activation. At the cellular level, the heparin analogues attenuated lymphocyte and macrophage recruitment and epithelial cell apoptosis. A drastic reduction in collagen-mediated fibrosis was also observed. No hemorrhagic events were observed after glycan treatment. These results strongly indicate the potential therapeutic use of these compounds for the treatment of colonic inflammation with a lower risk of hemorrhage when compared with mammalian heparin. Inflammatory bowel diseases (IBD) 3The abbreviations used are: IBD, inflammatory bowel diseases; CD, Crohn disease; TNF, tumor necrosis factor; IL, interleukin; IFN, interferon; GAG, glycosaminoglycan; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; VEGF, vascular endothelial growth factor; LPS, lipopolysaccharide; ANOVA, analysis of variance; TNBS, trinitrobenzene sulfonic acid; aPTT, activated partial thromboplastin time; DS, dermatan sulfate; MAP, mitogen-activated protein; HS, heparan sulfate; CS, chondroitin sulfate. comprise basically Crohn disease (CD) and ulcerative colitis and are characterized by chronic inflammation of the gastrointestinal tract. The etiology of IBD is complex and multifactorial, involving genetic predisposition and environmental triggers, as well as microbial and immune factors (1.Xavier R.J. Podolsky D.K. Nature. 2007; 448: 427-434Crossref PubMed Scopus (3303) Google Scholar). In CD, the chronic inflammatory process is a consequence of an imbalance in the production of proinflammatory and immunoregulatory cytokines, which results in a T helper cell type 1 (Th1) phenotype (2.Papadakis K. Targan S. Inflamm. Bowel Dis. 2000; 6: 303-313Crossref PubMed Scopus (105) Google Scholar, 3.Reinecker H. Steffen M. Witthoeft T. Pflueger I. Schreiber S. MacDermott R. Raedler A. Clin. Exp. Immunol. 1993; 94: 174-181Crossref PubMed Scopus (788) Google Scholar). Th1-type response is characterized by the production of tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-18, interferon (IFN)-γ, among other proinflammatory cytokines, and also involves the production of growth factors such as transforming growth factor (TGF)-β, and vascular endothelial growth factor (VEGF) (4.Fiocchi C. Gastroenterology. 1998; 115: 182-205Abstract Full Text Full Text PDF PubMed Scopus (1870) Google Scholar, 5.Podolsky D. N. Engl. J. Med. 2002; 347: 417-429Crossref PubMed Scopus (3184) Google Scholar, 6.Macdonald T.T. Monteleone G. Science. 2005; 307: 1920-1925Crossref PubMed Scopus (816) Google Scholar). In this context, the nuclear transcription factor κB (NF-κB) was identified as a key regulator of the expression of proinflammatory genes, determining the course of mucosal inflammation in IBD (7.Atreya I. Atreya R. Neurath M.F. J. Intern. Med. 2008; 263: 591-596Crossref PubMed Scopus (598) Google Scholar). The increased secretion of proinflammatory cytokines in IBD intestinal mucosa is accompanied by the over expression of adhesion molecules, including the selectins (8.Schurmann G.M. Bishop A.E. Facer P. Vecchio M. Lee J.C. Rampton D.S. Polak J.M. Gut. 1995; 36: 411-418Crossref PubMed Scopus (87) Google Scholar, 9.Andoh A. Tsujikawa T. Hata K. Araki Y. Kitoh K. Sasaki M. Yoshida T. Fujiyama Y. Am. J. Gastroenterol. 2005; 100: 2042-2048Crossref PubMed Scopus (68) Google Scholar). Animal models of mucosal inflammation have been utilized to investigate the pathogenesis of IBD and to evaluate possible new therapies. The trinitrobenzene sulfonic acid (TNBS)-induced colitis constitutes an established experimental model, which exhibits clinical and histological similarities to CD, and the course of colonic damage has been well characterized as a Th1-type immune response, with the resulting production of proinflammatory cytokines (10.Strober W. Fuss I.J. Blumberg R.S. Annu. Rev. Immunol. 2002; 20: 495-549Crossref PubMed Scopus (1154) Google Scholar). Heparin is a sulfated glycosaminoglycan (GAG) largely utilized in the clinical practice for anticoagulation and prevention and treatment of vascular thromboembolism (11.Roden L. Ananth S. Campbell P. Curenton T. Ekborg G. Manzella S. Pillion D. Meezan E. Adv. Exp. Med. Biol. 1992; 313: 1-20Crossref PubMed Scopus (27) Google Scholar, 12.Hirsh J. Warkentin T.E. Shaughnessy S.G. Anand S.S. Halperin J.L. Raschke R. Granger C. Ohman E.M. Dalen J.E. Chest. 2001; 119: 64S-94SAbstract Full Text Full Text PDF PubMed Scopus (1261) Google Scholar). Apart from its well-established anticoagulant and antithrombotic effects, heparin has anti-inflammatory properties such as inhibition of leukocyte adhesion and migration (13.Papa A. Danese S. Gasbarrini A. Gasbarrini G. Aliment Pharmacol. Ther. 2000; 14: 1403-1409Crossref PubMed Scopus (71) Google Scholar), and modulation of cytokine production (14.Elsayed E. Becker R.C. J. Thromb. Thrombolysis. 2003; 15: 11-18Crossref PubMed Scopus (59) Google Scholar). Based on these activities, and the suggested efficacy observed in several open clinical studies (15.Gaffney P.R. Doyle C.T. Gaffney A. Hogan J. Hayes D.P. Annis P. Am. J. Gastroenterol. 1995; 90: 220-223PubMed Google Scholar, 16.Folwaczny C. Wiebecke B. Loeschke K. Am. J. Gastroenterol. 1999; 94: 1551-1555Crossref PubMed Scopus (123) Google Scholar, 17.Ang Y.S. Mahmud N. White B. Byrne M. Kelly A. Lawler M. McDonald G.S. Smith O.P. Keeling P.W. Aliment Pharmacol. Ther. 2000; 14: 1015-1022Crossref PubMed Scopus (93) Google Scholar), heparin has been proposed as an alternative for the treatment of IBD. However, heparin therapy may cause hemorrhage and other adverse side effects (18.Schulman S. Beyth R. Kearon C. Levine M. Chest. 2008; 133: 257S-298SAbstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar). Our laboratory has isolated and characterized several heparin analogues from marine invertebrates (19.Santos J. Mesquita J. Belmiro C. da Silveira C. Viskov C. Mourier P. Pavao M. Thromb. Res. 2007; 121: 213-223Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 20.Cardilo-Reis L.C.M. Silveira C.B. Pavao M.S. Braz J. Med. Biol. Res. 2006; 39: 1409-1415Crossref PubMed Scopus (12) Google Scholar, 21.Pavao M. An. Acad. Bras. Cienc. 2002; 74: 105-112Crossref PubMed Scopus (26) Google Scholar, 22.Vicente C. Zancan P. Peixoto L. Alves-Sa R. Araujo F. Mourao P. Pavão M.S. Thromb. Haemost. 2001; 86: 1215-1220Crossref PubMed Scopus (37) Google Scholar, 23.Pavao M. Aiello K. Werneck C. Silva L. Valente A. Mulloy B. Colwell N. Tollefsen D. Mourao P. J. Biol. Chem. 1998; 273: 27848-27857Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 24.Pavao M. Mourao P. Mulloy B. Tollefsen D. J. Biol. Chem. 1995; 270: 31027-31036Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The study of the anticoagulant properties of the marine invertebrate glycans indicated that although they are capable of inhibiting venous and arterial thrombosis, they are less anticoagulant and have no bleeding effect after intravenous administration to experimental animals. Therefore, in the present work, we investigate the anti-inflammatory effect of heparin analogues obtained from the ascidian Styela plicata in a rat model of colon inflammation. Animals—Male Wistar rats (each weighing between 250 and 300 g) obtained from a local supplier were maintained on a 12-h/12-h light and dark cycle in a temperature-controlled room (24 °C). Animals were housed in rack-mounted, wire cages with 3 animals per cage. Standard laboratory pelleted formula and tap water were provided ad libitum. The care and use of animals, as well as procedures reported in this study were approved by the institutional care committee of the Federal University of Rio de Janeiro and are in accordance with the guidelines of the International Care and Use Committee of the National Institutes of Health, and Guide for the Care and Use of Laboratory Animals (25.Institute of Laboratory Animal Resources, Commission on Live Sciences, National Research Council (1996), National Academic Press, Scholar). of rats were with and and colitis was by of of a of sulfonic acid in a the animals were to and water ad libitum. such as and were observed during this an period of 1 animals were to of of animals and during 1 The colitis was to colitis and animals were on experimental were mammalian heparin DS, mammalian dermatan sulfate S. plicata and S. plicata These were to colitis by treatment with the indicated per by a over In the ascidian were with chondroitin rats to the which was after the the animals were as in the and to a The colon was with and were from the colon for histological A by was in animals. Inflammatory of the were in in with and by The histological were and inflammatory inflammatory and was and K. Y. T. S. S. H. J. S. Gut. 2001; PubMed Scopus Google Scholar). of the were in and in in a were and in a maintained were and for in a of of the intestinal mucosa was the to to to were in in for to in phosphate-buffered for were with for with the in a room from were with and as in for were for with a was by with a and and in histological and the were room with for The were with in for 1 in The used was the in for were for 1 with a of were with in phosphate-buffered and for in an and and observed with a At from were from were with and as of in the were in in and The was used to in P. M. PubMed Scopus (87) Google Scholar, P. M. J. 1993; PubMed Scopus Google Scholar). At per were light of colon were in for and in by in for were by the terminal nick the in to the of the were in a of analysis of light and was a for epithelial and from were as In the and the of the cell were by the of in the per in In the the of was as the of epithelial in the and in the of colonic The of was by the for in to intestinal per an analysis were of the experimental and and mucosal were in with and for in a for the was and were and the used for of the of cytokines TNF-α, TGF-β, and VEGF by a The of the was by the In was to with of The of rat TNF-α, TGF-β, and VEGF was less and of intestinal g) were in of of and and for The were for room of in of the and was to the The was for The from the were and the with a of by of and maintained for The was by for room and in of The of in the was by the of the T. PubMed Scopus Google Scholar). from the intestinal were by as M. Aiello K. Werneck C. Silva L. Valente A. Mulloy B. Colwell N. Tollefsen D. Mourao P. J. Biol. Chem. 1998; 273: 27848-27857Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). of the and a of chondroitin dermatan and heparan sulfate as acid of were to a in and for 1 the were with and with in The of the were by of the on a The of was by and after with with acid as M. Aiello K. Werneck C. Silva L. Valente A. Mulloy B. Colwell N. Tollefsen D. Mourao P. J. Biol. Chem. 1998; 273: 27848-27857Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). were obtained by the with of and for in a were by with were in a of per well with for in the of a of heparin and after heparin In was to the effect of heparin on of cell was from the of with heparin heparin The of was by to the of the by experimental animals from the were with an of of and of as The was isolated and with a for were for analysis of At animals were used per was as rat was with of of of were to the and the in a were on heparin from the during to colon The in the was on an are as of per were the for among the experimental were with the in which were the between inflammatory the of by and the cytokine were the The of was in the that animals to treatment colitis accompanied by a were observed in the colon of the animals. Inflammatory in the colon mucosal and of inflammation. of administration of ascidian mammalian heparin and dermatan sulfate with treatment and for A drastic of the inflammatory was observed in the animals after administration of mammalian ascidian the of No were observed the of analysis of intestinal an in the damage in animals, when compared with the mucosa of animals A and of mammalian ascidian reduced the inflammatory in the However, the obtained by ascidian heparin were lower that obtained by the mammalian of S. plicata heparin chondroitin S. plicata to the the effect of the in the colitis is characterized by a immune response with of and (10.Strober W. Fuss I.J. Blumberg R.S. Annu. Rev. Immunol. 2002; 20: 495-549Crossref PubMed Scopus (1154) Google Scholar). Therefore, we the cell in colonic The of and increased in the colonic of animals, when compared with that of the Subcutaneous administration of mammalian ascidian reduced the of and in animals. Heparin of its was in when compared with and However, ascidian heparin was in the mammalian ascidian reduced and the and of heparin analogues on lymphocyte the colon. from Wistar rats after TNBS-induced colitis with heparin ascidian heparin analogues administration were in in in a and to analysis Heparin and heparin analogues were to the animals as in the of The of per was in analysis of were light are of among the experimental were with the The of was and in the IBD, production is with damage S. S. J. J. I. B. H. Raedler A. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and the of the NF-κB in a of cell Therefore, we the administration of mammalian ascidian production and NF-κB in the colon. treatment a in the of in the colonic The of the cytokine was accompanied by a in NF-κB Subcutaneous administration of of its drastically reduced production to observed in the and no was observed on the effect of heparin. NF-κB drastically reduced in colon after administration No was observed in the effect of mammalian ascidian and heparin. However, ascidian heparin the reduction in NF-κB of heparin analogues on NF-κB in the colon. from Wistar rats after TNBS-induced colitis with heparin ascidian heparin analogues administration were in and to analysis Heparin and heparin analogues were to the animals as in the of analysis of were light The of with nuclear per was in are of among the experimental were with the The of was production is also by MAPK and of these J.M. J. Rev. 2001; PubMed Scopus Google Scholar). Therefore, we the of the MAPK in the colonic after treatment a of in the colonic reduced the of reduced in ascidian and heparin reduced the of in that of the No was observed on the effect of mammalian and heparin as well as on that of ascidian and heparin. is a key cytokine during of epithelial cell and after intestinal A. Podolsky D. Exp. Res. PubMed Scopus Google Scholar, J.L. Gut. 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