266 Biochimica et Biophysica A cta, 1220(1994) 266-276 © 1994 Elsevier Science B.V. All rights reserved 0167-4889/94/$07.00 BBAMCR 13510 Irradiation of bovine aortic endothelial cells enhances the synthesis and secretion of sulphated glycosaminoglycans David A. Pye *, Shant Kumar, Min J. Wang and Robin D. Hunter Department of Clinical Research, Christie Hospital, Wilmslow Road, Manchester M20 9BX (UK) (Received 17 May 1993) Key words: Radiation; Heparan sulfate; Chondroitin sulfate; Dermatan sulfate; Glycosaminoglycan; Endothelium;(Bovineaorta) The effect of X-irradiation on the synthesis of heparan sulphate (HS) and chondroitin/dermatan sulphate (CS/DS) by bovine aortic endothelial cells (BAEC), was studied by measuring the incorporation of [35S]sulphate and [3H]glucosamine into newly synthesized glycosaminoglycan (GAG) chains. Medium extracts from irradiated cultures (5Gy) were found to contain approx. 130% more HS and 200% more CS/DS than unirradiated controls. Smaller increases were observed in cellular extracts, irradiated cultures (5Gy) containing approx. 60% more HS and 100% more CS/DS than unirradiated controls. Structural studies showed no significant changes occurred upon irradiation in either the amounts or distribution of N-and O-sulphate groups in the HS molecule. Values for N-sulphation of 41.1% control and 41.5% irradiated (5Gy) were obtained, the corresponding values for O-sulphation being 19.9% control and 20.2% irradiated. Isotope incorporation data indicated that sulphation of CS/DS may decrease after irradiation, however, analysis of chondroitin ABC lyase derived disaceharides showed no changes in the proportion of non-sulphated and O-sulphated disaccharides. The present study indicates that X-irradiation stimulates the synthesis and secretion of HS and CS/DS proteoglycans (PGs) by BAEC. This could be relevant to many features which are found to be indicative of radiation-induced damage. Introduction The risk of normal tissue damage imposes severe limitations on the radiotherapy of malignant tumours. Histopathological evidence shows that damage to the endothelium occurs during early vascular injury, the prominent characteristics of late damage being atrophy and fibrosis. Accumulating data suggest that vascular injury by therapeutic radiation is a mediator of damage to normal tissue and is perhaps the most consistent pathogenetic mechanism in delayed radiation injury [1,2]. A reduction in vascular density is seen to precede both tissue regeneration and dysfunction in chronic radiation damage to a variety of organs [3-5]. The endothelial cell is believed to be the most radiosensi- tive component of the vascular wall as determined by * Corresponding author. Fax:+ 44 614463109. Abbreviations: GIcNAc, N-acetylglucosamine; GIcNSO 3, N- sulphated glucosamine; GIc(6S)NSO 3, N-sulphated glucosamine 6- sulphate; GIc(3S)NSO 3, N-sulphated glueosamine 3-sulphate; GleA, glucuronic acid; GIcA(2S),glucuronicacid 2-sulphate; IdoA, iduronic acid; IdoA(2S) iduronic acid 2-sulphate; GalNAc, N-acetylgalac- tosamine; GaI(6S)NAc, N-aeetylgalactosamine 6-sulphate; GaI(4S)NAc, N-acetylgalactosamine 4-sulphate; GaI(4SX6S)NAc, N-acetylgalactosamine4,6 di-sulphate. ultrastructural studies. Capillary endothelial cells upon irradiation have shown acute changes such as plasma- membrane irregularities, increased cell permeability and cell swelling. These events lead to a narrowing or occlusion of the lumen, the development of platelet and fibrin thrombi, rupture of the capillary wall and microhaemorrhage [4]. The result is a decrease in blood flow to irradiated tissues thereby leading to necrosis and replacement of normal tissue elements by fibrotic tissue. Radiation has been found to influence some en- dothelial cell functions including events associated with blood coagulation and thrombosis, such as increased synthesis and release of Von Willebrand factor, a de- creased capacity to produce prostacyclin and a de. crease in the cell's ability to secrete plasminogen acti. vator [6-8]. Other effects include inhibition ol sodium-dependent amino-acid transport and promo. tion of endothelial cell mitogen release [9,10]. Studies on the sensitivity of endothelial cells tc radiation in vitro by clonogenic assays employ artificia substrata such as plastic and gelatin [11,12]. However endothelial cells in vivo are attached to a subendothe lial matrix which serves as a regulator for specific cel functions such as differentiation and proliferatiox [13,14]. It has been shown that the sensitivity of en SSDI 0167-4889(93)E0159-W