Short-Term Inhibition of ADP-Induced Platelet Aggregation by Clopidogrel Ameliorates Radiation-Induced Toxicity in Rat Small Intestine

Summary

Endothelial dysfunction and increased platelet aggregation may be involved in the pathogenesis of normal tissue radiation toxicity. This study assessed clopidogrel, an inhibitor of ADP-induced platelet aggregation, as a modulator of intestinal radiation injury (radiation enteropathy).

Rat small intestine was exposed to 21 Gy X-radiation. Clopidogrel (20 mg/kg/day) or vehicle was administered from 2 days before to 10 days after irradiation. Structural radiation injury, neutrophil infiltration, smooth muscle cell proliferation, collagen content, and TGF-β1 expression were assessed 2 weeks (early phase) and 26 weeks (delayed phase) after irradiation, using quantitative histology and immunohistochemistry, morphometry, and real-time fluorogenic probe RT-PCR.

Irradiated intestine exhibited significant histopathologic injury, reduced mucosal surface area, vascular sclerosis, intestinal wall fibrosis, increased collagen content, and increased TGF-β1 expression. Clopidogrel reduced ADP-induced platelet aggregation by 93% and significantly attenuated the severity of post-radiation vascular sclerosis (p = 0.004 and p = 0.02) and the loss of mucosal surface area (p = 0.0008 and p = 0.003) at both 2 and 26 weeks. Clopidogrel also ameliorated overall histopathologic injury (p = 0.02), relative intestinal collagen content (p = 0.03), and collagen III immunoreactivity levels 2 weeks after irradiation, and caused a borderline reduction in the radiation-induced increase in extracellular matrix-associated TGF-β immunoreactivity at 26 weeks (p = 0.04). The effects of clopidogrel on steady-state TGF-β1 mRNA levels and neutrophil infiltration were not statistically significant.

Short-term clopidogrel administration affords protection against early and, to a lesser extent, delayed radiation enteropathy. Modulation of platelet aggregation should be subject to further studies as a potential method to increase safety and efficacy of radiation therapy.

Keywords

Platelet aggregation - clopidogrel - radiation therapy - intestines - radiation injuries - experimental

• References

• 1 Langley RE, Bump EA, Quartuccio SG, Medeiros D, Braunhut SJ. Radiation-induced apoptosis in microvascular endothelial cells. Br J Cancer 1997; 75: 666-72.
• 2 Paris F, Fuks Z, Kang A, Capodieci P, Juan G, Ehleiter D, Haimovitz-Friedman A, Cordon-Cardo C, Kolesnick R. Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice. Science 2001; 293: 293-7.
• 3 Law MP. Vascular permeability and late radiation fibrosis in mouse lung. Radiat Res 1985; 103: 60-76.
• 4 Dunn MM, Drab EA, Rubin DB. Effects of irradiation on endothelial cell-polymorphonuclear leukocyte interactions. J Appl Physiol 1986; 60: 1932-7.
• 5 Hallahan D, Clark ET, Kuchibhotla J, Gewertz BL, Collins T. E-selectin gene induction by ionizing radiation is independent of cytokine induction. Biochem Biophys Res Commun 1995; 217: 784-95.
• 6 Svanberg L, Åstedt B, Kullander S. On radiation-decreased fibrinolytic activity of vessel walls. Acta Obstet Gynecol Scand 1976; 55: 49-51.
• 7 Verheij M, Dewit LGH, van Mourik JA. The effect of ionizing radiation on endothelial tissue factor activity and its cellular localization. Thromb Haemost 1995; 73: 894-5.
• 8 Jahroudi N, Ardekani AM, Greenberger JS. Ionizing radiation increases transcription of the von Willebrand factor gene in endothelial cells. Blood 1996; 88: 3801-14.
• 9 Zhou Q, Zhao Y, Li P, Bai X, Ruan C. Thrombomodulin as a marker of radiation-induced endothelial cell injury. Radiat Res 1992; 131: 285-9.
• 10 Verheij M, Dewit LGH, Boomgard MN, Brinkman HJM, van Mourik JA. Ionizing radiation enhances platelet adhesion to the extracellular matrix of human endothelial cells by an increase in the release of von Willebrand factor. Radiat Res 1994; 137: 202-7.
• 11 te Poele JAM, van Kleef EM, van der Wal A, Dewit LGH, Stewart FA. Radiation-induced glomerular thrombus formation and nephropathy are not prevented by the ADP receptor antagonist clopidogrel. Int J Radiat Oncol Biol Phys 2001; 50: 1332-8.
• 12 Roy AF, Casey TT, Wilcox JN, Pelton RW, Moses HL. Synthesis of transforming growth factor-b1 by megacaryocytes and its localization to megacaryocyte and platelet a-granules. Blood 1990; 76: 1946-55.
• 13 Zheng H, Wang J, Koteliansky VE, Gotwals PJ, Hauer-Jensen M. Recombinant soluble transforming growth factor-β type II receptor ameliorates radiation enteropathy in the mouse. Gastroenterology 2000; 119: 1286-96.
• 14 Mills DC. ADP receptors on platelets. Thromb Haemost 1996; 76: 835-56.
• 15 Hauer-Jensen M, Poulakos L, Osborne JW. Effects of accelerated fractionation on radiation injury of the small intestine: A new rat model. Int J Radiat Oncol Biol Phys 1988; 14: 1205-12.
• 16 Berthrong M, Fajardo LF. Radiation injury in surgical pathology. Part II. Alimentary tract. Am J Surg Pathol 1981; 05: 153-78.
• 17 Langberg CW, Sauer T, Reitan JB, Hauer-Jensen M. Tolerance of rat small intestine to localized single dose and fractionated irradiation. Acta Oncol 1992; 31: 781-7.
• 18 Langberg CW, Waldron JA, Baker ML, Hauer-Jensen M. Significance of overall treatment time for the development of radiation-induced intestinal complications. An experimental study in the rat. Cancer 1994; 73: 2663-8.
• 19 Hauer-Jensen M, Sauer T, Devik F, Nygaard K. Late changes following single dose roentgen irradiation of rat small intestine. Acta Radiol Oncol 1983; 22: 299-303.
• 20 Baddeley AJ, Gundersen HJG, Cruz-Orive LM. Estimation of surface area from vertical sections. J Microsc 1986; 142: 259-76.
• 21 Langberg CW, Sauer T, Reitan JB, Hauer-Jensen M. Relationship between intestinal fibrosis and histopathologic and morphometric changes in consequential and late radiation enteropathy. Acta Oncol 1996; 35: 81-7.
• 22 Richter KK, Sung C-C, Langberg CW, Hauer-Jensen M. Association of transforming growth factor β (TGF-β) immunoreactivity with specific histopathologic lesions in subacute and chronic experimental radiation enteropathy. Radiother Oncol 1996; 39: 201-302.
• 23 Lopez-de leon A, Rojkind M. A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded sections. J Histochem Cytochem 1985; 33: 737-47.
• 24 Raviv G, Kiss R, Vanegas JP, Petein M, Danguy A, Schulman C, Wespes E. Objective measurement of the different collagen types in the corpus cavernosum of potent and impotent men: an immunohistochemical staining with computerized-image analysis. World J Urol 1997; 15: 50-5.
• 25 Wang J, Zheng H, Hauer-Jensen M. Influence of short-term octreotide administration on chronic tissue injury, transforming growth factor β (TGF-β) overexpression, and collagen accumulation in irradiated rat intestine. J Pharmacol Exp Ther 2001; 297: 35-42.
• 26 Wang J, Zheng H, Sung C-C, Richter KK, Hauer-Jensen M. Cellular sources of transforming growth factor β (TGF-β) isoforms in early and chronic radiation enteropathy. Am J Pathol 1998; 153: 1531-40.
• 27 Langberg CW, Hauer-Jensen M, Sung C-C, Kane CJM. Expression of fibrogenic cytokines in rat small intestine after fractionated irradiation. Radiother Oncol 1994; 32: 29-36.
• 28 Gassmann A. Zur Histologie der Röntgenulcera. Fortschr a d Geb d Roentgenstr 1899; 02: 199-207.
• 29 Fajardo LF. The unique physiology of endothelial cells and its implication in radiobiology. In: Meyer JMVJL. eds. Radiation Tolerance of Normal Tissues. Front Ther Oncol. Karger: Basel; 1989: 96-112.
• 30 Rubin DB, Drab EA, Ts’ao C, Gardner D, Ward WF. Prostacyclin synthesis in irradiated endothelial cells cultured from bovine aorta. J Appl Physiol 1985; 58: 592-7.
• 31 Verheij M, Dewit L. Radiation-induced von Willebrand factor release. Blood 1997; 90: 2109-10.
• 32 Richter KK, Fink LM, Hughes BM, Sung C-C, Hauer-Jensen M. Is the loss of endothelial thrombomodulin involved in the mechanism of chronicity in late radiation enteropathy?. Radiother Oncol 1997; 44: 65-71.
• 33 Bicher HI, D’Agostino L, Doss LL, Kaufman N, Amigone J. Prevention of ionizing radiation-induced liver microcirculation changes by the use of flow improvers. Adv Exp Med Biol 1977; 94: 383-9.
• 34 Mennie AT, Dalley VM, Dinneen LC, Collier HOJ. Treatment of radiation-induced gastrointestinal distress with acetylsalicylate. Lancet 1975; 02: 942-3.
• 35 Verheij M, Stewart FA, Oussoren Y, Weening JJ, Dewit L. Amelioration of radiation nephropathy by acetylsalicylic acid. Int J Radiat Biol 1995; 67: 587-96.
• 36 Freund U, Scholmerich J, Siems H, Kluge F, Schafer HE, Wannenmacher M. Unwanted side-effects in using mesalazine (5-aminosalicylic acid) during radiotherapy. Strahlenther Onkol 1987; 163: 678-80.
• 37 Baughan CA, Canney PA, Buchanan RB, Pickering RM. A randomized trial to assess the efficacy of 5-aminosalicylic acid for the prevention of radiation enteritis. Clin Oncol 1993; 05: 19-24.
• 38 Sharis PJ, Cannon CP, Loscalzo J. The antiplatelet effects of ticlopidine and clopidogrel. Ann Intern Med 1998; 129: 394-405.
• 39 Hollopeter G, Jantzen H-M, Vincent D, Li G, England L, Ramakrishnan V, Yang R-B, Nurden P, Nurden A, Julius D, Conley P. Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 2001; 409: 202-7.
• 40 Schafer AI. Antiplatelet therapy. Am J Med 1996; 101: 199-209.
• 41 Harker LA. Therapeutic inhibition of platelet function in stroke. Cerebrovasc Dis 1998; 08 (Suppl. 05) 8-18.
• 42 Harker LA, Marzec UM, Kelly AB, Chronos NR, Sundell IB, Hanson SR, Herbert JM. Clopidogrel inhibition of stent, graft, and vascular thrombogenesis with antithrombotic enhancement by aspirin in nonhuman primates. Circulation 1998; 98: 2461-9.
• 43 Moshfegh K, Redondo M, Julmy F, Wuillemin WA, Gebauer MU, Haeberli A, Meyer BJ. Antiplatelet effects of clopidogrel compared with aspirin after myocardial infarction: enhanced inhibitory effects of combination therapy. J Am Coll Cardiol 2000; 36: 699-705.
• 44 Savi P, Labouret C, Delesque N, Guette F, Lupker J, Herbert J-M. P2Y(12), a new platelet ADP receptor, target of clopidogrel. Biochem Biophys Res Commun 2001; 283: 379-83.
• 45 Zhang FL, Luo L, Gustafson E, Lachowicz J, Smith M, Qiao X, Liu Y-H, Chen G, Pramanik B, Laz TM, Palmer K, Bayne M, Monsma FJ. ADP is the cognate ligand for the orphan G protein-coupled receptor SP1999. J Biol Chem 2001; 276: 8608-15.
• 46 Schultz-Cherry S, Murphy-Ullrich JE. Thrombospondin causes activation of latent transforming growth factor-β secreted by endothelial cells by a novel mechanism. J Cell Biol 1993; 122: 923-32.