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Protective Effect of Defibrotide on Perfusion Induced Endothelial Damage
Thrombosis Research 99 (2000) 335–341
ORIGINAL ARTICLE
Protective Effect of Defibrotide on Perfusion Induced Endothelial Damage Tangu¨l S¸an, Hadi Moini1, Kaya Emerk1, Serpil Bilsel1 Departments of Histology-Embryology and 1Biochemistry Faculty of Medicine, Marmara University, I˙stanbul, Turkey (Received 11 October 1999 by Editor O.N. Ulutin; revised/accepted 11 April 2000)
Abstract
Key Words: Endothelium; Defibrotide; Ultrastructure
In the present study, in vitro effects of Defibrotide (D) on perfusion-induced changes in the morphology of endothelium were investigated by scanning (SEM) and transmission (TEM) electron microscope. Human umbilical cord veins were incubated or perfused with platelet-rich plasma alone (PRP) or platelet-rich plasma with Defibrotide (PRP⫹D) at 3ml/min or 14ml/min and the changes observed were compared. SEM examination of luminal surfaces demonstrated that perfusion with high flow rates may damage endothelial cells and lead to morphological changes which may be prevented by the presence of Defibrotide in the perfusate. Also, the marked reduction in the number of adhered platelets on luminal surface of veins incubated or perfused with Defibrotide compared to veins treated with platelet-rich plasma only revealed that Defibrotide has anti-thrombotic effects. TEM examination of ruthenium red (RR) stained thin sections of veins demonstrated that perfusion disrupts the glycosaminoglcan (GAG) coat on endothelial cells. But the presence of D in the perfusate preserves the integrity of GAG, indicating further cytoprotective effects of the drug on endothelium. 2000 Elsevier Science Ltd. All rights reserved.
efibrotide is the sodium salt of singlestranded DNA isolated from porcine tissues by controlled depolymerization [1]. Defibrotide is a drug used in the prevention of deep vein thrombosis and in the treatment of some vascular disorders like peripheral obliterative arterial diseases and acute thrombophlebitis [2,3,4]. Experimental studies demonstrate that Defibrotide decreases the amount of cholesterol in hypercholesterolemic rabbit aorta with no modification of plasma or lipoprotein cholesterol [5]. Administration of Defibrotide decreases the endothelial surface area involved in the atherosclerotic process [6,7]. Moreover, protective activity of Defibrotide against damage induced by ischemia and postischemic reperfusion was reported in animal models [8,9]. All the suggested vascular protection by Defibrotide could be due to its effect on endothelium. Very recently, this point of view was resumed and the beneficial effects of Defibrotide on cardiovascular function of hypercholesterolemic rabbits and normal rats was attributed to the endothelium protection exerted by Defibrotide [10,11]. As the morphological studies on endothelial protection potential of Defibrotide are limited in number (amounting to just one [12]), we planned to study the effect of Defibrotide on perfusioninduced endothelial damage in human umbilical cord veins. These veins are known to be low pressure systems and can be damaged by high flow rates with consequent morphological changes in their luminal surfaces [13].
¨ niversitesi, Corresponding author: Dr. Tangu¨l S¸an, Marmara U Tıp Faku¨ltesi Histoloji-Embriyoloji 81326 Haydarpas¸a, I˙stanbul, Turkey. Tel: ⫹90 (216) 330 8806; fax: ⫹90 (216) 363 5378 and ⫹90 (216) 414 4731; E-mail: ⬍[email protected]⬎.
D
0049-3848/00 $–see front matter 2000 Elsevier Science Ltd. All rights reserved. PII S0049-3848(00)00256-5
336
T. S¸an et al./Thrombosis Research 99 (2000) 335–341
1. Materials and Methods
2. Results
Human umbilical cord veins were washed with phosphate buffered saline (PBS) and cut into several segments. One segment was used as control while the other segments were either incubated or perfused with platelet-rich plasma with or without Defibrotide (1mg/ml) by using a peristaltic pomp (P-1Pharmacia) at different flow rates (3ml/min, 14ml/min), at 37⬚C for one hour. To obtain the PRP, blood from healthy volunteers was drawn into tubes containing 3.8% citrate (1/10, citrate/ blood ratio) and centrifuged at 180 g for 15 min. After collecting PRP, the remaining portion of blood was centrifuged at 1800 g for 15 min to obtain platelet-poor plasma (PPP). The final platelet number in PRP was standardized to 300.000 /l using autologous PPP.
2.1. SEM Investigation
1.1. SEM Method
2.1.2. 3ml/min Perfusion Experiments SEM appearance of luminal surface of the control vein segments revealed orderly populated hexagonal endothelial cells (Fig. 2a). In contrast to this appearance, the umbilical cord veins perfused with PRP only were characterized by rigid, disrupted appearance. Diffuse surface swelling of the endothelium was evident on the luminal surface. Numerous scattered platelets on endothelial cells were observed in this group (Fig. 2b). In contrast, the surface endothelial cells in the group perfused with PRP⫹D maintained their integrity. Along with this finding, a marked reduction in the number of surface platelets as compared to the above-mentioned perfused goup was evident (Fig. 2c).
All samples were fixed at 4⬚C with 2% glutaraldehyde in cacodylate buffer (0.1M, pH 7.3), washed with the same buffer and then post-fixed with 1% osmium tetroxide (OsO4) in the same buffer. Samples were dehydrated in graded series of ethanol and dried with CO2 in a critical point dryer (E3000 BIO-RAD), then sputtered under vacuum with gold (SC502 BIO-RAD). Morphological changes and interaction of platelets with endothelium were evaluated by SEM (Jeol CSM-5200).
1.2. TEM Method Vein segments that were perfused with PRP and PRP⫹D at 3ml/min were fixed at 4⬚C with 2% glutaraldehyde in cacodylate buffer (0.1M, pH 7.3). Post-fixation was carried out for one hour in 1% osmium tetroxide in the same buffer to which a stock solution of ruthenium red (RR) 0.003% was added in the ratio of 1:5 [14]. Following these procedures, specimens were washed with cacodylate buffer solution and the segments were stained en block with 2% aqueous uranyl acetate which also contained the stock solution of RR in a ratio of 1:5. Ultrathin sections of RR stained vessel walls were evaluated by TEM (Jeol - EM1200).
2.1.1. Incubation Experiments Luminal surface of the control umbilical veins were characterized by intact longitudinal relief with elongated endothelial cells, with few platelets sticking to the luminal surface (Fig. 1a). The endothelium of the umbilical veins incubated with PRP only exhibited normal longitudinal relief with numerous platelets attached to the luminal surface (Fig. 1b). The cord veins which were incubated with PRP⫹D showed no detectable surface changes compared to the control and to the ones incubated with PRP. But decreased platelet adhesion and well-ordered endothelial surface was noticed in this group (Fig. 1c).
2.1.3. 14ml/min Perfusion Experiments Luminal topography of the control cord vein demonstrated characteristic homogeneous population of endothelial cells (Fig. 3a). Perfusion with PRP resulted in a disorganized surface appearance. Furthermore, numerous platelets stuck to the surface were observed (Fig. 3b). Luminal surface of the cord vein perfused with PRP⫹D showed characteristic regular cobblestone appearance of the endothelium. Fewer platelets were observed in this group as compared to the Defibrotide-free group (Fig. 3c).
T. S¸an et al./Thrombosis Research 99 (2000) 335–341
Fig. 1. Scanning electron micrographs of the luminal surface of umbilical cord veins. (a) The control segment characterized by intact endothelial surface and few platelets on luminal surface. (b) The segment incubated with PRP showing numerous platelets attached to surface. (c) The segment incubated with PRP⫹D showing no surface relief changes and no thrombus formation.
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Fig. 2. Scanning electron micrographs showing the luminal surface of the umbilical cord veins. (a) The control segment revealing regular organized hexagonal endothelial cells. (b) The segment perfused with PRP at 3ml/min flow rate, showing disrupted surface apearence, swollen endothelial cells and numerous scattered platelets. (c) The segment perfused with PRP⫹D at 3 ml/min flow rate showing intact endothelial cells. Further, marked reduction in the number of surface platelets compared to control groups.
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T. S¸an et al./Thrombosis Research 99 (2000) 335–341
2.2. TEM Investigation TEM observation of luminal surface of the control veins showed an intact and thick GAG layer on the endothelial cells by RR staining (Fig. 4a). The investigation of segments perfused with PRP (3 ml/ min) revealed a partially disrupted GAG coat (Fig. 4b), while veins perfused with PRP⫹D revealed an intact continuous GAG layer over the endothelial cells, with characteristic dense vesicles (Fig. 4c).
3. Discussion
Fig. 3. Scanning electron micrographs showing the luminal surface of the umbilical cord veins. (a) The control segment showing homogenously organized endothelial cells. (b) The segment perfused with PRP at 14 ml/min flow rate showing disorganized endothelial surface and increased number of platelets. (c) The segment perfused with PRP⫹D at 14ml/min flow rate showing cobblestone apperence of endothelial cells with scarce number of platelets.
In the present experiments, incubation of the vessels with PRP or PRP⫹D did not cause any significant change in the morphology of the luminal surface. However, the number of adhering platelets was less in the vessels incubated with PRP⫹D. When the vessels were perfused with PRP 3 ml/ min or 14 ml/min, the regular topography of the endothelial layer was disrupted. These changes were more prominent at the higher flow rate. Umbilical veins perfused with PRP revealed disorganized luminal surface appearance with occasionally partially-detached endothelial cells. Numerous platelets were stuck to the luminal surface. The presence of Defibrotide in the perfusate prevented the flow-induced morphological changes indicating endothelial damage. In the presence of Defibrotide, less platelets were stuck to the luminal surface of the vessel. In the experimental conditions here reported, Defibrotide may preserve the endothelium by a direct action on endothelial cells [9]. This “endothelial sparing” effect would have the dual benefit of maintaining both PGI2 and endothelium derived relaxing factor (NO) release [9], since Defibrotide has been shown by others to promote PGI2 [15] and NO release [16]. The increased generation of PGI2 causes, as a consequence, a rise in platelet cAMP [17,18] which is a signal for platelets to deaggregate. NO causes, as well, inhibition of platelet aggregation [9]. Platelets are inflamatory cells [19]; when the endothelium is damaged platelets adhere to it, thereby becoming activated. Activated platelets release from their granules a number of substances such as seratonin, ADP, PAF, PDGF, TGF, cationic proteins and proteolitic enzymes
T. S¸an et al./Thrombosis Research 99 (2000) 335–341
Fig. 4. Transmission electron micrographic appearance of the luminal surface of the umbilical cord vein with an intact GAG layer (→) in the control group (a); disrupted GAG coat (→) in the PRP perfused group (b) and intact GAG coat (→) in the PRP⫹D perfused group at 3 ml/min flow rate (c). Two platelets (➤) are on luminal surface of PRP perfused segments (b).
(collagenase and elastase) that modify tissue integrity [19]. The antiplatelet activity of Defibrotide is well known and described in a number of publications [6,15,17,20]; hence it is no surprise that in the
339
presence of Defibrotide less platelets stick to the luminal surface of umbilical veins. In the present study, the luminal surface of veins perfused with PRP was characterized by a discontinuous glycosaminoglycan coat which, presumably, indicates a degenerated morphology. At variance, the veins perfused with PRP⫹D exhibited a more intense and homogeneous coat with vesicles characteristic of endothelial cells. Many glycosaminoglycans (in particular heparansulfate and heparin, products of endothelium and smooth muscle) can inhibit both migration and proliferation of smooth muscle cells [21]. Heparin can bind to and sequester PDGF and bFGF, can release TGF, form a carrier protein, and can modulate the phenotype of the smooth muscle cells [21]. Recent observations showed that toxic oxygen metabolites can contribute to endothelial damage and alterations in function [22]. Heparin (and related compounds) can protect endothelium from free radical damage [23]. Heparin and heparinoids (with low anticoagulant potency) attenuate postischemic endothelial cell dysfunction [24,25]. Standard porcine mucosal heparin attenuates postischemic endothelial cell dysfunction as measured by release of nitric oxide [24]. An increasing number of clinical studies report the beneficial effects of D in treatment of various vascular diseases and support the findings of protective effect of D on vascular functions [4,26,27]. Due to its endothelium sparing effect, antiplatelet activity and profibrinolytic potential with lack of anticoagulant effect and toxicity, the drug may have advantages over other available treatments [28]. The observed response to D in treatment of severe veno-occlusive disease with high risk of hemorrhage (accompanying stem cell transplantation and liver disease) are compelling for further studies [29]. In conclusion, Defibrotide appears to exert a direct endothelium protective activity (“endothelium sparing” effect), by preserving the endothelial ability to generate PGI2, NO [9], and conserving the endothelial glycosaminoglycan layer, while deactivating platelets. By these mechanisms of action, besides others described in a number of papers, Defibrotide could have preserved the morphology of the lumen of umbilical veins in the present experimental conditions.
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We are grateful to Dr. O.N. Ulutin for his help in editing the manuscript.
11.
References 1. United States Patent No. 5,223,609 of June 29, 1993. 2. Ulutin ON. Clinical effectiveness of Defibrotide in vaso-occlusive disorders and its mode of action. Semin Thromb Hemost 1988; 14(Suppl.):58–63. 3. Ulutin ON, Balkuv-Ulutin S, Ug˘ur MS, Ulutin ¨ zsoy Y, C¸izmeci G. The pharmacology and T, O clinical pharmacology of Defibrotide. A new profibrinolitic, antithrombotic and antiplatelet substance. In: Liu CY, Chien S, editors. Fibrinogen, Thrombosis, Coagulation and Fibrinolysis. Advances In Experimental Medicine And Biology Series. New York: Plenum Press, 1990. pp. 429–38. 4. Ulutin ON. Antithrombotic effect and clinical potential of Defibrotide. Semin Thromb Hemost 1993;19(Suppl.1):186–91. 5. Pescador R, Porta R, Conz A, Mantovani M, Prino G. Defibrotide decreases cholesterol amount in hypercholesterolemic rabbit aorta, with no modification of plasma or lipoprotein cholesterol. Life Sci 1989;44:789–97. 6. Lo¨bel P, Schro¨r K. Stimulation of vascular prostacyclin and inhibition of platelet function by oral Defibrotide in cholesterol-fed rabbits. Atherosclerosis 1989;80:69–79 7. Pescador R, Tettamanti R, Salvetti L, Canto A, Barone D, Porta R, Mantovani M, Ferro L. Effects of Defibrotide on leukocytosis in rabbits with diet-induced atherosclerosis. Life Sci 1995;57:579–89. 8. Schro¨r K, Ackermann G, Hohlfeld T, Lo¨bel P, Ney P, Schro¨der H, Strobach H. Endothelial protection by Defibrotide—a new strategy for treatment of myocardial infarction? Z Kardiol 1989;78(Suppl.6):35–41. 9. Lefer AM, Aoki N, Mulloy D. Coronary-endothelium protective effects of Defibrotide in ischaemia and reperfusion. Naunyn-Schmiedeberg’s Arch Pharmacol 1990;341:246–50. 10. Shin YK, Champbell B, Lefer AM. Novel beneficial effects of Defibrotide, an endothelium protecting agent, following ischaemia and re-
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a perspective for the 1990s. Nature 1993;362: 801–9. Ryan US. Activation of endothelial cells. Ann NY Acad Sci 1987;516:22–50. Heibert LM, Liu J. Heparin protects cultured arterial endothelial cells from damage by toxic oxygen metabolites. Atherosclerosis. 1990; 83:47–51. Stenberg WC III, Makhoul RG, Adelman B. Heparin prevents postischemic endothelial cell dysfunction by a mechanism independent of its anticoagulant activity. J Vasc Surg 1993; 17:318–27. Stenberg WC III, Sobel M, Makhoul RG. Heperinoids with low anticoagulant potency attenuate postischemic endothelial cell dysfunction. J Vasc Surg 1995;21:477–83. Bonomini V, Vangelista A, Frasc’a GM. A new antithrombotic agent in the treatment of acute renal failure due to hemolytic-uremic syn-
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drome and thrombotic thrombocytopenic purpura letter. Nephron 1984;37:144. 27. Vangelista A, Frasc’a GM, Raimondi C, Liviano-D’Arcangelo G, Bonomini V. Effect of the defibrotide in acute renal failure due to thrombotic microangiopathy. Hemost 1986; 16(Suppl.1):51–4. 28. Palmer KJ, Goa KL. Defibrotide: A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in vascular disorders. Drug 1993;45:259–94. 29. Richardson PG, Elias AD, Krishnan A, Wheeler C, Nath R, Hoppensteadt D, Kinchla NM, Neuberg D, Waller EK, Antin JH, Soiffer R, Vredenburgh J, Lill M, Woolfrey AE, Bearman SI, Lacobelli M, Fareed J, Guinan EC. Treatment of severe veno-occlusive disease with Defibrotide: Compassionate use results in response without significant toxicity in a highrisk population. Blood 1998;92:737–44.
ORIGINAL ARTICLE
Protective Effect of Defibrotide on Perfusion Induced Endothelial Damage Tangu¨l S¸an, Hadi Moini1, Kaya Emerk1, Serpil Bilsel1 Departments of Histology-Embryology and 1Biochemistry Faculty of Medicine, Marmara University, I˙stanbul, Turkey (Received 11 October 1999 by Editor O.N. Ulutin; revised/accepted 11 April 2000)
Abstract
Key Words: Endothelium; Defibrotide; Ultrastructure
In the present study, in vitro effects of Defibrotide (D) on perfusion-induced changes in the morphology of endothelium were investigated by scanning (SEM) and transmission (TEM) electron microscope. Human umbilical cord veins were incubated or perfused with platelet-rich plasma alone (PRP) or platelet-rich plasma with Defibrotide (PRP⫹D) at 3ml/min or 14ml/min and the changes observed were compared. SEM examination of luminal surfaces demonstrated that perfusion with high flow rates may damage endothelial cells and lead to morphological changes which may be prevented by the presence of Defibrotide in the perfusate. Also, the marked reduction in the number of adhered platelets on luminal surface of veins incubated or perfused with Defibrotide compared to veins treated with platelet-rich plasma only revealed that Defibrotide has anti-thrombotic effects. TEM examination of ruthenium red (RR) stained thin sections of veins demonstrated that perfusion disrupts the glycosaminoglcan (GAG) coat on endothelial cells. But the presence of D in the perfusate preserves the integrity of GAG, indicating further cytoprotective effects of the drug on endothelium. 2000 Elsevier Science Ltd. All rights reserved.
efibrotide is the sodium salt of singlestranded DNA isolated from porcine tissues by controlled depolymerization [1]. Defibrotide is a drug used in the prevention of deep vein thrombosis and in the treatment of some vascular disorders like peripheral obliterative arterial diseases and acute thrombophlebitis [2,3,4]. Experimental studies demonstrate that Defibrotide decreases the amount of cholesterol in hypercholesterolemic rabbit aorta with no modification of plasma or lipoprotein cholesterol [5]. Administration of Defibrotide decreases the endothelial surface area involved in the atherosclerotic process [6,7]. Moreover, protective activity of Defibrotide against damage induced by ischemia and postischemic reperfusion was reported in animal models [8,9]. All the suggested vascular protection by Defibrotide could be due to its effect on endothelium. Very recently, this point of view was resumed and the beneficial effects of Defibrotide on cardiovascular function of hypercholesterolemic rabbits and normal rats was attributed to the endothelium protection exerted by Defibrotide [10,11]. As the morphological studies on endothelial protection potential of Defibrotide are limited in number (amounting to just one [12]), we planned to study the effect of Defibrotide on perfusioninduced endothelial damage in human umbilical cord veins. These veins are known to be low pressure systems and can be damaged by high flow rates with consequent morphological changes in their luminal surfaces [13].
¨ niversitesi, Corresponding author: Dr. Tangu¨l S¸an, Marmara U Tıp Faku¨ltesi Histoloji-Embriyoloji 81326 Haydarpas¸a, I˙stanbul, Turkey. Tel: ⫹90 (216) 330 8806; fax: ⫹90 (216) 363 5378 and ⫹90 (216) 414 4731; E-mail: ⬍[email protected]⬎.
D
0049-3848/00 $–see front matter 2000 Elsevier Science Ltd. All rights reserved. PII S0049-3848(00)00256-5
336
T. S¸an et al./Thrombosis Research 99 (2000) 335–341
1. Materials and Methods
2. Results
Human umbilical cord veins were washed with phosphate buffered saline (PBS) and cut into several segments. One segment was used as control while the other segments were either incubated or perfused with platelet-rich plasma with or without Defibrotide (1mg/ml) by using a peristaltic pomp (P-1Pharmacia) at different flow rates (3ml/min, 14ml/min), at 37⬚C for one hour. To obtain the PRP, blood from healthy volunteers was drawn into tubes containing 3.8% citrate (1/10, citrate/ blood ratio) and centrifuged at 180 g for 15 min. After collecting PRP, the remaining portion of blood was centrifuged at 1800 g for 15 min to obtain platelet-poor plasma (PPP). The final platelet number in PRP was standardized to 300.000 /l using autologous PPP.
2.1. SEM Investigation
1.1. SEM Method
2.1.2. 3ml/min Perfusion Experiments SEM appearance of luminal surface of the control vein segments revealed orderly populated hexagonal endothelial cells (Fig. 2a). In contrast to this appearance, the umbilical cord veins perfused with PRP only were characterized by rigid, disrupted appearance. Diffuse surface swelling of the endothelium was evident on the luminal surface. Numerous scattered platelets on endothelial cells were observed in this group (Fig. 2b). In contrast, the surface endothelial cells in the group perfused with PRP⫹D maintained their integrity. Along with this finding, a marked reduction in the number of surface platelets as compared to the above-mentioned perfused goup was evident (Fig. 2c).
All samples were fixed at 4⬚C with 2% glutaraldehyde in cacodylate buffer (0.1M, pH 7.3), washed with the same buffer and then post-fixed with 1% osmium tetroxide (OsO4) in the same buffer. Samples were dehydrated in graded series of ethanol and dried with CO2 in a critical point dryer (E3000 BIO-RAD), then sputtered under vacuum with gold (SC502 BIO-RAD). Morphological changes and interaction of platelets with endothelium were evaluated by SEM (Jeol CSM-5200).
1.2. TEM Method Vein segments that were perfused with PRP and PRP⫹D at 3ml/min were fixed at 4⬚C with 2% glutaraldehyde in cacodylate buffer (0.1M, pH 7.3). Post-fixation was carried out for one hour in 1% osmium tetroxide in the same buffer to which a stock solution of ruthenium red (RR) 0.003% was added in the ratio of 1:5 [14]. Following these procedures, specimens were washed with cacodylate buffer solution and the segments were stained en block with 2% aqueous uranyl acetate which also contained the stock solution of RR in a ratio of 1:5. Ultrathin sections of RR stained vessel walls were evaluated by TEM (Jeol - EM1200).
2.1.1. Incubation Experiments Luminal surface of the control umbilical veins were characterized by intact longitudinal relief with elongated endothelial cells, with few platelets sticking to the luminal surface (Fig. 1a). The endothelium of the umbilical veins incubated with PRP only exhibited normal longitudinal relief with numerous platelets attached to the luminal surface (Fig. 1b). The cord veins which were incubated with PRP⫹D showed no detectable surface changes compared to the control and to the ones incubated with PRP. But decreased platelet adhesion and well-ordered endothelial surface was noticed in this group (Fig. 1c).
2.1.3. 14ml/min Perfusion Experiments Luminal topography of the control cord vein demonstrated characteristic homogeneous population of endothelial cells (Fig. 3a). Perfusion with PRP resulted in a disorganized surface appearance. Furthermore, numerous platelets stuck to the surface were observed (Fig. 3b). Luminal surface of the cord vein perfused with PRP⫹D showed characteristic regular cobblestone appearance of the endothelium. Fewer platelets were observed in this group as compared to the Defibrotide-free group (Fig. 3c).
T. S¸an et al./Thrombosis Research 99 (2000) 335–341
Fig. 1. Scanning electron micrographs of the luminal surface of umbilical cord veins. (a) The control segment characterized by intact endothelial surface and few platelets on luminal surface. (b) The segment incubated with PRP showing numerous platelets attached to surface. (c) The segment incubated with PRP⫹D showing no surface relief changes and no thrombus formation.
337
Fig. 2. Scanning electron micrographs showing the luminal surface of the umbilical cord veins. (a) The control segment revealing regular organized hexagonal endothelial cells. (b) The segment perfused with PRP at 3ml/min flow rate, showing disrupted surface apearence, swollen endothelial cells and numerous scattered platelets. (c) The segment perfused with PRP⫹D at 3 ml/min flow rate showing intact endothelial cells. Further, marked reduction in the number of surface platelets compared to control groups.
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T. S¸an et al./Thrombosis Research 99 (2000) 335–341
2.2. TEM Investigation TEM observation of luminal surface of the control veins showed an intact and thick GAG layer on the endothelial cells by RR staining (Fig. 4a). The investigation of segments perfused with PRP (3 ml/ min) revealed a partially disrupted GAG coat (Fig. 4b), while veins perfused with PRP⫹D revealed an intact continuous GAG layer over the endothelial cells, with characteristic dense vesicles (Fig. 4c).
3. Discussion
Fig. 3. Scanning electron micrographs showing the luminal surface of the umbilical cord veins. (a) The control segment showing homogenously organized endothelial cells. (b) The segment perfused with PRP at 14 ml/min flow rate showing disorganized endothelial surface and increased number of platelets. (c) The segment perfused with PRP⫹D at 14ml/min flow rate showing cobblestone apperence of endothelial cells with scarce number of platelets.
In the present experiments, incubation of the vessels with PRP or PRP⫹D did not cause any significant change in the morphology of the luminal surface. However, the number of adhering platelets was less in the vessels incubated with PRP⫹D. When the vessels were perfused with PRP 3 ml/ min or 14 ml/min, the regular topography of the endothelial layer was disrupted. These changes were more prominent at the higher flow rate. Umbilical veins perfused with PRP revealed disorganized luminal surface appearance with occasionally partially-detached endothelial cells. Numerous platelets were stuck to the luminal surface. The presence of Defibrotide in the perfusate prevented the flow-induced morphological changes indicating endothelial damage. In the presence of Defibrotide, less platelets were stuck to the luminal surface of the vessel. In the experimental conditions here reported, Defibrotide may preserve the endothelium by a direct action on endothelial cells [9]. This “endothelial sparing” effect would have the dual benefit of maintaining both PGI2 and endothelium derived relaxing factor (NO) release [9], since Defibrotide has been shown by others to promote PGI2 [15] and NO release [16]. The increased generation of PGI2 causes, as a consequence, a rise in platelet cAMP [17,18] which is a signal for platelets to deaggregate. NO causes, as well, inhibition of platelet aggregation [9]. Platelets are inflamatory cells [19]; when the endothelium is damaged platelets adhere to it, thereby becoming activated. Activated platelets release from their granules a number of substances such as seratonin, ADP, PAF, PDGF, TGF, cationic proteins and proteolitic enzymes
T. S¸an et al./Thrombosis Research 99 (2000) 335–341
Fig. 4. Transmission electron micrographic appearance of the luminal surface of the umbilical cord vein with an intact GAG layer (→) in the control group (a); disrupted GAG coat (→) in the PRP perfused group (b) and intact GAG coat (→) in the PRP⫹D perfused group at 3 ml/min flow rate (c). Two platelets (➤) are on luminal surface of PRP perfused segments (b).
(collagenase and elastase) that modify tissue integrity [19]. The antiplatelet activity of Defibrotide is well known and described in a number of publications [6,15,17,20]; hence it is no surprise that in the
339
presence of Defibrotide less platelets stick to the luminal surface of umbilical veins. In the present study, the luminal surface of veins perfused with PRP was characterized by a discontinuous glycosaminoglycan coat which, presumably, indicates a degenerated morphology. At variance, the veins perfused with PRP⫹D exhibited a more intense and homogeneous coat with vesicles characteristic of endothelial cells. Many glycosaminoglycans (in particular heparansulfate and heparin, products of endothelium and smooth muscle) can inhibit both migration and proliferation of smooth muscle cells [21]. Heparin can bind to and sequester PDGF and bFGF, can release TGF, form a carrier protein, and can modulate the phenotype of the smooth muscle cells [21]. Recent observations showed that toxic oxygen metabolites can contribute to endothelial damage and alterations in function [22]. Heparin (and related compounds) can protect endothelium from free radical damage [23]. Heparin and heparinoids (with low anticoagulant potency) attenuate postischemic endothelial cell dysfunction [24,25]. Standard porcine mucosal heparin attenuates postischemic endothelial cell dysfunction as measured by release of nitric oxide [24]. An increasing number of clinical studies report the beneficial effects of D in treatment of various vascular diseases and support the findings of protective effect of D on vascular functions [4,26,27]. Due to its endothelium sparing effect, antiplatelet activity and profibrinolytic potential with lack of anticoagulant effect and toxicity, the drug may have advantages over other available treatments [28]. The observed response to D in treatment of severe veno-occlusive disease with high risk of hemorrhage (accompanying stem cell transplantation and liver disease) are compelling for further studies [29]. In conclusion, Defibrotide appears to exert a direct endothelium protective activity (“endothelium sparing” effect), by preserving the endothelial ability to generate PGI2, NO [9], and conserving the endothelial glycosaminoglycan layer, while deactivating platelets. By these mechanisms of action, besides others described in a number of papers, Defibrotide could have preserved the morphology of the lumen of umbilical veins in the present experimental conditions.
340
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We are grateful to Dr. O.N. Ulutin for his help in editing the manuscript.
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