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Pentoxifylline Prevents Endothelial Damage Due to Ischemia and Reperfusion Injury
JOURNAL OF SURGICAL RESEARCH ARTICLE NO.
67, 21–25 (1997)
JR964940
Pentoxifylline Prevents Endothelial Damage Due to Ischemia and Reperfusion Injury DOUGLAS A. COE, M.D., JULIE A. FREISCHLAG, M.D., DAWN JOHNSON, M.D., JUNAID H. MUDALIAR, M.D., SCOTT A. KOSCIESZA, B.S., DAVID K. TRAUL, M.D., PHILLIP C. CHIANG, M.D., ROBERT A. CAMBRIA, M.D., GARY R. SEABROOK, M.D., AND JONATHAN B. TOWNE, M.D. Department of Vascular Surgery, Medical College of Wisconsin and Surgical Services, Zablocki VA Medical Center, Milwaukee, Wisconsin Presented at the Annual Symposium of the Association of Veterans Administration Surgeons, Detroit, Michigan, April 28–30, 1996
INTRODUCTION Background. Endothelial injury after ischemia and reperfusion is characterized by an increase in permeability, cellular edema, and loss of acetylcholine-mediated vasorelaxation. Three hours of ischemia followed by 2 hr of reperfusion in the New Zealand white rabbit hindlimb has been shown to result in loss of acetylcholine-induced superficial femoral artery vasorelaxation. The purpose of this study was to evaluate the effect of intraarterial pentoxyfylline (PTX) on this endothelial injury. Methods. New Zealand white rabbits underwent 3 hr of complete hindlimb ischemia followed by 2 hr of reperfusion. Twenty milliliters of either 100 mM PTX or normal saline was infused over 20 min via the circumflex iliac artery at initiation of reperfusion. Superficial femoral artery rings were then evaluated in vitro for endothelial cellmediated relaxation. Rings were exposed to standardized incremental doses of acetylcholine after norepinephrine-induced contraction and percentage relaxation was measured. Sections of arteries were also sent for hematoxylin and eosin staining. Results. Similar contraction responses following NE stimulation were observed between control and PTX-treated rings. Control rings relaxed a mean of 14.97 { 3.64, 23.17 { 5.61, and 31.84 { 8.43% in response to acetylcholine doses of 6 1 1008, 1 1 1007, and 1.5 1 1007 M, respectively. In contrast, PTX-treated segments relaxed a mean of 47.52 { 8.88, 62.32 { 6.83, and 76.73 { 4.91% to the same doses of acetylcholine. Differences in relaxation between control and PTX-treated vessels were significantly different at each dose (P õ 0.05, Student’s t test). Histologic examination of the PTX-treated and control arteries revealed an intact endothelium without morphologic differences between the two groups. Conclusion. In this model of rabbit hindlimb ischemia, endothelial cell-mediated vasorelaxation was preserved with the administration of intraarterial PTX during reperfusion compared to controls. The different relaxation responses could not be attributed to altered arterial contractility in response to norepinephrine, or explained by histologic changes in the arterial wall. These studies demonstrate a potential modality for therapeutic intervention to reduce reperfusion injury after acute limb ischemia. q 1997 Academic Press
A major consequence of acute ischemia continues to be reperfusion injury. Several pathophysiologic mechanisms invoked by reperfusion injury have become better defined. Interactions between the blood elements and the vascular endothelium are responsible for the reperfusion injury. Restoration of blood flow after acute ischemia leads to production of oxygen free radicals which are directly toxic to endothelial cells [1]. Manifestations of endothelial injury include cellular swelling and lysis due to loss of ionic and osmotic gradients and loss of vasoregulatory control. The contractile properties of the arterial wall are altered in response to ischemic insults. Loss of acetylcholine-induced relaxation, secondary to either diminished production or increased destruction of the endothelium-derived vasodilator nitric oxide, has been demonstrated [2]. Free radicals can interact with inflammatory cells leading to cytokine induction with subsequent activation of polymorphonuclear leukocytes (PMN) [3]. Endothelial response to certain cytokines results in the up-regulation of cellular adhesion molecules. PMN then attach to the endothelium and release lytic enzymes leading to tissue damage. Previous work in this laboratory [4] has demonstrated endothelial dysfunction in arteries subjected to 3 hr of ischemia followed by 2 hr of reperfusion. A potential ‘‘window of opportunity’’ exists during which therapeutic intervention could be initiated before severe effects from reperfusion become evident in the affected vascular bed. Multiple pharmacological agents could potentially limit reperfusion injury, including substances that target free radicals, limit initiation of the inflammatory response, or serve as substrate for compounds that preserve endothelial function. Pentoxifylline (PTX) is a methylxanthine derivative with multiple hemorheologic properties, but the exact mechanisms of its pharmacology are not understood. Clinically, PTX has been used to treat intermittent claudication. It is thought to increase red blood cell deformability by increasing intracellular cyclic AMP, thereby improving oxygen delivery to chronically isch0022-4804/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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emic tissues [5]. The effect of PTX on leukocytes in vitro include increases in PMN cyclic AMP and decreased oxygen free radical production [6]. PTX has been demonstrated to limit PMN response to inflammatory cytokines with reduction in cellular activation, phagocytosis, and endothelial adhesion [7]. In addition, evidence suggests PTX reduces nitric oxide destruction, resulting in improved acetylcholine-mediated vasorelaxation [8]. This study evaluated the effects of PTX administered at the time of reperfusion on endothelial-dependent vasomotor relaxation after rabbit hindlimb ischemia with reperfusion. MATERIALS AND METHODS Thirteen New Zealand white rabbits were divided into two groups: a control group (n Å 5), and a PTX group (n Å 8). The rabbits were anesthetized initially with intramuscular ketamine (44 mg/kg) and xylazine (5 mg/kg). Intravenous access was obtained using an ear vein, and continuous intravenous anesthesia was maintained using a-choralose (20 mg/kg/hr). A carotid artery cannula was inserted for continuous blood pressure monitoring and vascular access, and a tracheostomy was placed for mechanical ventilation. Heating pads were used to maintain body temperature. Exposure of the infrarenal abdominal aorta, bilateral iliac arteries, and the right femoral artery was obtained through a midline laparotomy and right groin dissection. The right circumflex femoral branch from the common iliac artery was cannulated for later intraarterial infusion. All aortic and iliac collaterals were ligated, and complete ischemia was induced by placing clamps on the right common iliac and femoral arteries. Complete ischemia was verified by the absence of arterial Doppler signals in the femoral artery distal to the clamps. Complete hindlimb ischemia using this model has been documented by angiography in previous studies [9]. After the 3 hr of ischemia, the clamps were removed, and reperfusion was documented by a return of Doppler signal in the distal femoral artery. At the initiation of reperfusion, intraarterial infusion through the iliac branch catheter of 20 ml of normal saline for the control group or 20 ml or 100 mM PTX for the experimental group was begun at a rate of 1 ml/ min. At the end of the 2-hr reperfusion period, the right femoral artery was atraumatically excised, and the rabbit was euthanized using high-dose pentobarbital. The procedures performed on the rabbits were approved by the animal research committee and conformed to guidelines outlined in the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, National Institutes of Health 85-23, Revised 1985). The harvested arteries were carefully dissected free from sur-
FIG. 1. Sample contraction/relaxation curves of a control and PTX-treated vessel showing the contraction response to KCl and norepinephrine, and the relaxation response to acetylcholine.
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FIG. 2. Maximum contraction of control and PTX-exposed rings to KCl and norepinephrine (NE) stimulation.
rounding tissue and cut into 3-mm segments while being bathed in oxygenated Kreb’s solution. Four rings from each vessel were immediately suspended on tungsten hooks connected to tension transducers, and bathed in organ chambers containing oxygenated Kreb’s solution (pH 7.4, 377C). Each ring was preloaded with 2 g tension and allowed to equilibrate for 90 min. The Kreb’s baths were changed frequently during this equilibration period. Tension data from the transducers were transferred to a signal amplifier. Data from the amplifier were then recorded by Codas software (DataQ Instruments, Akron, OH) on an IBM-compatible personal computer. Vessel viability was confirmed by twice inducing contraction with 80 mM potassium chloride, after which the baths were changed. After all vessels returned to baseline 2 g tension, one ring from each vessel was maximally contracted using 2 1 1004 M norepinephrine. Contraction was then induced in the other three rings from the same vessel to approximately 50% of maximum with incremental doses of norepinephrine. Once contraction had reached a stable state, the rings were then exposed to standardized incremental doses of acetylcholine. The decrease in tension induced by the acetylcholine was recorded and percentage of relaxation was determined. Contraction and relaxation data from the ring that reached plateau nearest to 50% of maximal contraction after norepinephrine stimulation were chosen for statistical analysis (Fig. 1). Histologic sections of vessels from both groups were stained with hematoxylin and eosin and examined under light microscopy to assess vessel architecture. Statistics were performed on SPSS software (SPSS Inc., Chicago, IL). Contraction and relaxation data were analyzed using Student’s t test, and data are presented as mean { standard error of the mean (SEM).
RESULTS
The mean contraction of superficial femoral arteries from control and PTX-treated animals to KCl stimulation was 6.84 { 0.58 and 7.41 { 0.27 g, respectively. Mean maximal contraction to norepinephrine in control rings was 10.04 { .054 and 10.35 { 0.35 g in PTXexposed vessels. There were no significant differences between the two groups in the amount of contraction to KCl or norepinephrine stimulation (Fig. 2). Relaxation response to acetylcholine is illustrated in Fig. 3. Percentage relaxation to 6 1 1008 M acetylcholine was 14.97 { 3.64% in control rings and 47.52 { 8.88% in PTX-treated rings. Relaxation response to 1 1 1007 M acetylcholine was 23.17 { 5.61 and 62.32 { 6.83% in saline and PTX-exposed segments, respectively. Finally, the degree of relaxation to 1.5 1 1007 M acetylcholine in control rings was 31.84 { 8.43%, and in PTX-treated vessels it was 76.73 { 4.91%. Differences in relaxation response to all three doses of
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COE ET AL.: PENTOXIFYLLINE PREVENTS ENDOTHELIAL DAMAGE
FIG. 3. Percentage relaxation of control and PTX-treated rings to incremental concentrations of acetylcholine.
acetylcholine was significant between saline and PTXtreated rings. Examination of histologic sections of superficial femoral artery segments revealed no morphologic differences between control and PTX-exposed vessels (Fig. 4). Medial and adventitial layers were easily differentiated, no inflammatory cellular infiltrate could be identified, and section from both groups retained an intact endothelial surface. DISCUSSION
Manifestations of ischemia and reperfusion have been well described. In this model of hindlimb isch-
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emia, reperfusion injury was demonstrated by a loss of arterial relaxation to acetylcholine administration. Control vessels in this study demonstrated percentage relaxation ranging between 15 and 30% to increasing doses of acetylcholine. This is similar to work previously reported from this laboratory which demonstrated loss of acetylcholine-induced relaxation between 6 and 35% after 3 hr of ischemia and 2 hr of reperfusion [4]. In addition, that report demonstrated no significant difference in contractile capability between sham arteries and arteries subjected to ischemia and reperfusion. Similar results were observed in this study as there was no difference in contraction to NE (or KCl) between control and PTX-treated rings. This suggests that vessel contractility had no impact on the ring’s ability to relax. A study previously published by this lab compared rabbit hindlimbs undergoing sham operation (no ischemia or reperfusion) to hindlimbs subjected to 3 hr of ischemia followed by 2 hr of reperfusion [4]. The technical aspects of that study regarding surgical preparation, vessel manipulation, and contraction/relaxation determination were identical to those of the current study. Sham vessels (n Å 5) relaxed 13.73 { 2.11, 47.88 { 7.23, and 72.44 { 9.00% to incremental doses of acetylcholine. In contrast, the relaxation responses to the same doses of acetylcholine in vessels subjected to 3 hr of ischemia and 2 hr of reperfusion (n Å 5) were 6.10
FIG. 4. Representative hematoxylin and eosin sections of control (left) and PTX-treated (right) superficial femoral arteries. No histologic differences are identified; intimal, medial, and adventitial layers are maintained in both sections.
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{ 1.02, 15.33 { 2.56, and 34.67 { 6.31% (P £ 0.05 when compared to control). Intraarterial administration of 100 mM PTX at initiation of reperfusion resulted in a significant increase in arterial relaxation to acetylcholine exposure compared to vessels infused with saline. PTX-perfused vessels relaxed between 48 and 77% to increasing doses of acetylcholine compared to control vessels which relaxed a mean of between 15 and 32% with the same acetylcholine doses. Differences in acetylcholine-induced relaxation are presumably due to variations in local nitric oxide levels, but whether the decreased relaxation in control vessels is due to reduced production or increased destruction of nitric oxide is unknown. Our results are consistent with those of Berkenboom and co-workers who reported a significant increase in relaxation of rat aortic rings after PTX exposure to acetylcholine, but not after exposure to indomethicin or methylene blue or mechanical removal of the endothelium. They suggest that a potential mechanism of action of PTX is reduced nitric oxide destruction by oxygen free radicals [8]. Pentoxifylline has a variety of physiologic effects which include increased red cell cyclic AMP levels resulting in stabilization of cellular membranes and enhanced red cell deformability [5]. Others believe PTX functions more by reducing plasma fibrinogen levels and blood viscosity [10]. In addition to its red cell effects, PTX has been found to have a potent influence on PMN function, particularly after PMN exposure to inflammatory cytokines [11]. PTX has been demonstrated to suppress lipopolysaccaride-induced TNF production at the level of mRNA expression [12] through a dose-dependent decrease in cytosolic calcium concentration [13]. Dauber and colleagues have demonstrated increased PMN cyclic AMP levels leading to diminished superoxide production, reduced PMN response to TNFa and interleukin-1 stimulation, and reduced PMN adhesion to the vascular endothelium [7]. Along with these effects, PMN and monocyte phagocytosis of latex particles has been shown to be reduced after incubation with PTX [6]. The role of leukocytes in ischemia and reperfusion has been well established. The central role of PMN in ischemia and reperfusion injury was demonstrated by Yokota et al. by near complete inhibition of their parameters of reperfusion injury after selective leukopenia was induced using dimethylmyleran [14]. PMN stimulation occurs in part secondary to exposure to locally produced oxygen free radicals and products of arachadonic acid metabolism [15]. Activated PMN in turn become a source for oxygen metabolites, thromboxanes, and proteolytic enzymes [15 – 17]. Once activated, these PMN exhibit increased chemotaxis and phagocytosis [18]. Evidence exists that interaction between endothelial cell-derived immunoglobulin-like cell adhesion molecules (ICAMs, VCAMs) and b2 integrins are required for leukocyte infiltration into postischemic tissues [19]. TNF is a major stimulus for adhesion molecule up-
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regulation [20], therefore inhibition of cytokine induction could indirectly limit reperfusion injury by decreasing leukocyte adhesion. Studies have utilized adenosine [21] or various monoclonal antibodies to limit reperfusion injury through reduced white cell adhesion [22–24]. Ma and associates investigated the impact of an anti-ICAM antibody (MAb RR1/1) on feline myocardial and coronary artery endothelial reperfusion injury. Administration of the MAb resulted in reduced tissue necrosis, myeloperoxidase activity, and improved coronary artery ring vasorelaxation to acetylcholine compared to cats receiving a nonbinding control antibody [25]. PTX has been shown to reduce both TNF induction and adhesion molecule expression [26, 27]. Others have demonstrated reduced venous effluent platelet activating factor levels after canine hindlimb ischemia following treatment PTX with reduction in reperfusion injury [28]. Although the precise mechanism of action of PTX in reducing loss of nitric oxide-mediated vasorelaxation remains elusive, several potential pathways have become apparent. Because of its inhibitory effect on superoxide production and enhancement of PMN cyclic AMP levels, leukocyte activation and phagocytosis are decreased, release of inflammatory cytokines is reduced, and local nitric oxide levels may be maintained. With reduction in TNF and platelet activating factor, endothelial cell expression of cell adhesion molecules is reduced, resulting in blunted PMN accumulation and infiltration with subsequent reduction in tissue destruction. Since these events occur in flowing blood, PTX needs to be present in this milieu to exert its effects. This may explain why our results were observed with PTX infusion at the time of reperfusion but not prior to reperfusion (unpublished data). Because reperfusion injury to the endothelium does not occur immediately [4], opportunity for therapeutic intervention exists prior to development of irreversible tissue damage. Pentoxifylline appears to have a role for use in clinically relevant skeletal muscle ischemia when it is administered at the beginning of reperfusion. REFERENCES 1. Parks, D. A., and Granger, D. N. Ischemia-induced vascular changes: Role of xanthine oxidase and hydroxyl radicals. Am. J. Physiol. 245: G285, 1983. 2. Gryglewski, R. J., Palmer, R. M. J., and Moncada, S. Superoxide anion is involved in the breakdown of endothelium-derived relaxing factor. Nature 320: 454, 1986. 3. Khwaja, A., Carver, J. E., and Linch, D. C. Interactions of granulocyte macrophage colony stimulating factor (CSF), granulocyte CSF, and tumor necrosis factor-a in the priming of the neutrophil respiratory burst. Blood 79: 745, 1992. 4. Chiang, P. C., Traul, D. K., Farooq, M. M., Lesniak, R. J., Seabrook, G. R., Towne, J. B., and Freischlag, J. A. Loss of superficial femoral artery relaxation following ischemia-reperfusion. J. Surg. Res. 60: 361, 1996. 5. Ehrly, A. M. The effect of pentoxifylline on the deformability of erythrocytes and on the muscular oxygen pressure in patients with chronic arterial disease. J. Med. 10: 331, 1979. 6. Bessler, H., Gilgal, R., Djaldetti, M., and Zahavi, I. Effect of
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COE ET AL.: PENTOXIFYLLINE PREVENTS ENDOTHELIAL DAMAGE pentoxifylline on phagocytic activity, cAMP levels, and superoxide anion production by monocytes and polymorphonuclear cells. J. Leukoc. Biol. 40: 747, 1986. 7. Dauber, I. M., Lesnefsky, E. J., Ashmore, R. C., Martel, D. M., Sheridan, F. M., Weil, J. V., and Horwitz, L. D. Coronary vascular injury due to ischemia-reperfusion is reduced by pentoxifylline. J. Pharmacol. Exp. Ther. 260: 1250, 1992.
19. 20.
8. Berkenboom, G., Fang, Z., Unger, P., Goldman, M., and Fontaine, J. Endothelium-dependent effects of pentoxifylline in rat aorta. Eur. J. Pharmacol. 193: 81, 1991.
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9. Freischlag, J. A., and Hanna, D. Neutrophil (PMN) phagocytosis and chemotaxis after 2 hours of ischemia. J. Surg. Res. 50: 648, 1991.
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10. Jarret, P. M., Moreland, M., and Browse, N. L. The effects of oxypentifylline (Trental) on fibrinolytic activity and plasma fibrinogen levels. Curr. Med. Res. Opin. 4: 492, 1977. 11. Mandel, G. L. ARDS, neutrophils, and pentoxiphylline. Am. Rev. Respir. Dis. 138: 1103, 1988. 12. Strieter, R. M., Remick, D. G., Ward, P. A., Spengler, R. N., Lynch III, J. P., Larrick, J., and Kunkel, S. L. Cellular and molecular regulation of tumor necrosis factor-alpha production by pentoxifylline. Biochem. Biophys. Res. Commun. 155: 1230, 1988. 13. Mandel, G. L. Cytokines, phagocytes, and pentoxifylline. J. Cardiovasc. Pharmacol. 25: S20, 1995.
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14. Yokota, J., Minei, J. P., Fantini, G. A., and Shires, G. T. Role of leukocytes in reperfusion injury of skeletal muscle after partial ischemia. Am. J. Physiol. 257: H1068, 1989. 15. Klausner, J. M., Paterson, I. S., Valeri, C. R., Shepro, D., and Hechtman, H. B. Limb ischemia-induced increase in permeability is mediated by leukocytes and leukotrienes. Ann. Surg. 208: 755, 1988. 16. Freischlag, J. A., and Hanna, D. Superoxide anion release (O0 2 ) after ischemia and reperfusion. J. Surg. Res. 50: 565, 1991. 17. Carden, D. L., and Korthuis, R. J. Role of neutrophil elastase in postischemic granulocyte extravasation and microvascular dysfunction in skeletal muscle. FASEB J. 49: A1248, 1990. 18. Freischlag, J. A., and Hanna, D. Neutrophil (PMN) phagocyto-
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27.
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sis and chemotaxis after reperfusion injury. J. Surg. Res. 52: 152, 1992. Bevilacqua, M. P. Endothelial-leukocyte adhesion molecules. Annu. Rev. Immunol. 11: 767, 1993. Gamble, J. R., Harlan, J. M., Klebanoff, S. J., and Vadas, M. A. Stimulation of the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor. Proc. Natl. Acad. Sci. USA 82: 8667, 1985. Grisham, M. B., Hernandez, L. A., and Granger, D. N. Adenosine inhibits ischemia-reperfusion-induced leukocyte adherence and extravasation. Am. J. Physiol. 257: H1334, 1989. Hartman, J. C., Anderson, D. C., Wiltse, A. L., Lane, C. L., Rosenbloom, C. L., Manning, A. M., Humphrey, W. R., Wall, T. M., and Shebuski, R. J. Protection of ischemic/reperfused canine myocardium by CL18/6, a monoclonal antibody to adhesion molecule ICAM-1. Cardiovasc. Res. 30: 47, 1995. Farhood, A., McGuire, G. M., Manning, A. M., Miyasaka, M., Smith, C. W., and Jaeschke, H. Intercellular adhesion molecule 1 (ICAM-1) expression and its role in neutrophil-induced ischemia-reperfusion injury in rat liver. J. Leukoc. Biol. 57: 368, 1995. Vollmar, B., Glasz, J., Menger, M. D., and Messmer, K. Leukocytes contribute to hepatic ischemia/reperfusion injury via intercellular adhesion molecule-1-mediated venular adherence. Surgery 117: 195, 1995. Ma, X., Lefer, D. J., Lefer, A. M., and Rothlein, R. Coronary endothelial and cardiac protective effects of a monoclonal antibody to intercellular adhesion molecule-1 in myocardial ischemia and reperfusion. Circulation 86: 937, 1992. Elbim, C., Lefebvre, J., Hakim, J., and Gougerot-Pocidalo, M. A. Effects of pentoxifylline on human polymorphonuclear neutrophil responses to TNF in wole blood. Eur. Cytokine Netw. 6: 113, 1995. Weill, D., Wautier, J., Dosquet, C., Wautier, M., Carreno, M., and Boval, B. Monocyte modulation of endothelial leukocyte adhesion molecules. J. Lab. Clin. Med. 125: 768, 1995. Adams, J. G., Dhar, A., Shukla, S. D., and Silver, D. Effect of pentoxifylline on tissue injury and platelet-activating factor production during ischemia-reperfusion injury. J. Vasc. Surg. 21: 742, 1995.
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67, 21–25 (1997)
JR964940
Pentoxifylline Prevents Endothelial Damage Due to Ischemia and Reperfusion Injury DOUGLAS A. COE, M.D., JULIE A. FREISCHLAG, M.D., DAWN JOHNSON, M.D., JUNAID H. MUDALIAR, M.D., SCOTT A. KOSCIESZA, B.S., DAVID K. TRAUL, M.D., PHILLIP C. CHIANG, M.D., ROBERT A. CAMBRIA, M.D., GARY R. SEABROOK, M.D., AND JONATHAN B. TOWNE, M.D. Department of Vascular Surgery, Medical College of Wisconsin and Surgical Services, Zablocki VA Medical Center, Milwaukee, Wisconsin Presented at the Annual Symposium of the Association of Veterans Administration Surgeons, Detroit, Michigan, April 28–30, 1996
INTRODUCTION Background. Endothelial injury after ischemia and reperfusion is characterized by an increase in permeability, cellular edema, and loss of acetylcholine-mediated vasorelaxation. Three hours of ischemia followed by 2 hr of reperfusion in the New Zealand white rabbit hindlimb has been shown to result in loss of acetylcholine-induced superficial femoral artery vasorelaxation. The purpose of this study was to evaluate the effect of intraarterial pentoxyfylline (PTX) on this endothelial injury. Methods. New Zealand white rabbits underwent 3 hr of complete hindlimb ischemia followed by 2 hr of reperfusion. Twenty milliliters of either 100 mM PTX or normal saline was infused over 20 min via the circumflex iliac artery at initiation of reperfusion. Superficial femoral artery rings were then evaluated in vitro for endothelial cellmediated relaxation. Rings were exposed to standardized incremental doses of acetylcholine after norepinephrine-induced contraction and percentage relaxation was measured. Sections of arteries were also sent for hematoxylin and eosin staining. Results. Similar contraction responses following NE stimulation were observed between control and PTX-treated rings. Control rings relaxed a mean of 14.97 { 3.64, 23.17 { 5.61, and 31.84 { 8.43% in response to acetylcholine doses of 6 1 1008, 1 1 1007, and 1.5 1 1007 M, respectively. In contrast, PTX-treated segments relaxed a mean of 47.52 { 8.88, 62.32 { 6.83, and 76.73 { 4.91% to the same doses of acetylcholine. Differences in relaxation between control and PTX-treated vessels were significantly different at each dose (P õ 0.05, Student’s t test). Histologic examination of the PTX-treated and control arteries revealed an intact endothelium without morphologic differences between the two groups. Conclusion. In this model of rabbit hindlimb ischemia, endothelial cell-mediated vasorelaxation was preserved with the administration of intraarterial PTX during reperfusion compared to controls. The different relaxation responses could not be attributed to altered arterial contractility in response to norepinephrine, or explained by histologic changes in the arterial wall. These studies demonstrate a potential modality for therapeutic intervention to reduce reperfusion injury after acute limb ischemia. q 1997 Academic Press
A major consequence of acute ischemia continues to be reperfusion injury. Several pathophysiologic mechanisms invoked by reperfusion injury have become better defined. Interactions between the blood elements and the vascular endothelium are responsible for the reperfusion injury. Restoration of blood flow after acute ischemia leads to production of oxygen free radicals which are directly toxic to endothelial cells [1]. Manifestations of endothelial injury include cellular swelling and lysis due to loss of ionic and osmotic gradients and loss of vasoregulatory control. The contractile properties of the arterial wall are altered in response to ischemic insults. Loss of acetylcholine-induced relaxation, secondary to either diminished production or increased destruction of the endothelium-derived vasodilator nitric oxide, has been demonstrated [2]. Free radicals can interact with inflammatory cells leading to cytokine induction with subsequent activation of polymorphonuclear leukocytes (PMN) [3]. Endothelial response to certain cytokines results in the up-regulation of cellular adhesion molecules. PMN then attach to the endothelium and release lytic enzymes leading to tissue damage. Previous work in this laboratory [4] has demonstrated endothelial dysfunction in arteries subjected to 3 hr of ischemia followed by 2 hr of reperfusion. A potential ‘‘window of opportunity’’ exists during which therapeutic intervention could be initiated before severe effects from reperfusion become evident in the affected vascular bed. Multiple pharmacological agents could potentially limit reperfusion injury, including substances that target free radicals, limit initiation of the inflammatory response, or serve as substrate for compounds that preserve endothelial function. Pentoxifylline (PTX) is a methylxanthine derivative with multiple hemorheologic properties, but the exact mechanisms of its pharmacology are not understood. Clinically, PTX has been used to treat intermittent claudication. It is thought to increase red blood cell deformability by increasing intracellular cyclic AMP, thereby improving oxygen delivery to chronically isch0022-4804/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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emic tissues [5]. The effect of PTX on leukocytes in vitro include increases in PMN cyclic AMP and decreased oxygen free radical production [6]. PTX has been demonstrated to limit PMN response to inflammatory cytokines with reduction in cellular activation, phagocytosis, and endothelial adhesion [7]. In addition, evidence suggests PTX reduces nitric oxide destruction, resulting in improved acetylcholine-mediated vasorelaxation [8]. This study evaluated the effects of PTX administered at the time of reperfusion on endothelial-dependent vasomotor relaxation after rabbit hindlimb ischemia with reperfusion. MATERIALS AND METHODS Thirteen New Zealand white rabbits were divided into two groups: a control group (n Å 5), and a PTX group (n Å 8). The rabbits were anesthetized initially with intramuscular ketamine (44 mg/kg) and xylazine (5 mg/kg). Intravenous access was obtained using an ear vein, and continuous intravenous anesthesia was maintained using a-choralose (20 mg/kg/hr). A carotid artery cannula was inserted for continuous blood pressure monitoring and vascular access, and a tracheostomy was placed for mechanical ventilation. Heating pads were used to maintain body temperature. Exposure of the infrarenal abdominal aorta, bilateral iliac arteries, and the right femoral artery was obtained through a midline laparotomy and right groin dissection. The right circumflex femoral branch from the common iliac artery was cannulated for later intraarterial infusion. All aortic and iliac collaterals were ligated, and complete ischemia was induced by placing clamps on the right common iliac and femoral arteries. Complete ischemia was verified by the absence of arterial Doppler signals in the femoral artery distal to the clamps. Complete hindlimb ischemia using this model has been documented by angiography in previous studies [9]. After the 3 hr of ischemia, the clamps were removed, and reperfusion was documented by a return of Doppler signal in the distal femoral artery. At the initiation of reperfusion, intraarterial infusion through the iliac branch catheter of 20 ml of normal saline for the control group or 20 ml or 100 mM PTX for the experimental group was begun at a rate of 1 ml/ min. At the end of the 2-hr reperfusion period, the right femoral artery was atraumatically excised, and the rabbit was euthanized using high-dose pentobarbital. The procedures performed on the rabbits were approved by the animal research committee and conformed to guidelines outlined in the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, National Institutes of Health 85-23, Revised 1985). The harvested arteries were carefully dissected free from sur-
FIG. 1. Sample contraction/relaxation curves of a control and PTX-treated vessel showing the contraction response to KCl and norepinephrine, and the relaxation response to acetylcholine.
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FIG. 2. Maximum contraction of control and PTX-exposed rings to KCl and norepinephrine (NE) stimulation.
rounding tissue and cut into 3-mm segments while being bathed in oxygenated Kreb’s solution. Four rings from each vessel were immediately suspended on tungsten hooks connected to tension transducers, and bathed in organ chambers containing oxygenated Kreb’s solution (pH 7.4, 377C). Each ring was preloaded with 2 g tension and allowed to equilibrate for 90 min. The Kreb’s baths were changed frequently during this equilibration period. Tension data from the transducers were transferred to a signal amplifier. Data from the amplifier were then recorded by Codas software (DataQ Instruments, Akron, OH) on an IBM-compatible personal computer. Vessel viability was confirmed by twice inducing contraction with 80 mM potassium chloride, after which the baths were changed. After all vessels returned to baseline 2 g tension, one ring from each vessel was maximally contracted using 2 1 1004 M norepinephrine. Contraction was then induced in the other three rings from the same vessel to approximately 50% of maximum with incremental doses of norepinephrine. Once contraction had reached a stable state, the rings were then exposed to standardized incremental doses of acetylcholine. The decrease in tension induced by the acetylcholine was recorded and percentage of relaxation was determined. Contraction and relaxation data from the ring that reached plateau nearest to 50% of maximal contraction after norepinephrine stimulation were chosen for statistical analysis (Fig. 1). Histologic sections of vessels from both groups were stained with hematoxylin and eosin and examined under light microscopy to assess vessel architecture. Statistics were performed on SPSS software (SPSS Inc., Chicago, IL). Contraction and relaxation data were analyzed using Student’s t test, and data are presented as mean { standard error of the mean (SEM).
RESULTS
The mean contraction of superficial femoral arteries from control and PTX-treated animals to KCl stimulation was 6.84 { 0.58 and 7.41 { 0.27 g, respectively. Mean maximal contraction to norepinephrine in control rings was 10.04 { .054 and 10.35 { 0.35 g in PTXexposed vessels. There were no significant differences between the two groups in the amount of contraction to KCl or norepinephrine stimulation (Fig. 2). Relaxation response to acetylcholine is illustrated in Fig. 3. Percentage relaxation to 6 1 1008 M acetylcholine was 14.97 { 3.64% in control rings and 47.52 { 8.88% in PTX-treated rings. Relaxation response to 1 1 1007 M acetylcholine was 23.17 { 5.61 and 62.32 { 6.83% in saline and PTX-exposed segments, respectively. Finally, the degree of relaxation to 1.5 1 1007 M acetylcholine in control rings was 31.84 { 8.43%, and in PTX-treated vessels it was 76.73 { 4.91%. Differences in relaxation response to all three doses of
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FIG. 3. Percentage relaxation of control and PTX-treated rings to incremental concentrations of acetylcholine.
acetylcholine was significant between saline and PTXtreated rings. Examination of histologic sections of superficial femoral artery segments revealed no morphologic differences between control and PTX-exposed vessels (Fig. 4). Medial and adventitial layers were easily differentiated, no inflammatory cellular infiltrate could be identified, and section from both groups retained an intact endothelial surface. DISCUSSION
Manifestations of ischemia and reperfusion have been well described. In this model of hindlimb isch-
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emia, reperfusion injury was demonstrated by a loss of arterial relaxation to acetylcholine administration. Control vessels in this study demonstrated percentage relaxation ranging between 15 and 30% to increasing doses of acetylcholine. This is similar to work previously reported from this laboratory which demonstrated loss of acetylcholine-induced relaxation between 6 and 35% after 3 hr of ischemia and 2 hr of reperfusion [4]. In addition, that report demonstrated no significant difference in contractile capability between sham arteries and arteries subjected to ischemia and reperfusion. Similar results were observed in this study as there was no difference in contraction to NE (or KCl) between control and PTX-treated rings. This suggests that vessel contractility had no impact on the ring’s ability to relax. A study previously published by this lab compared rabbit hindlimbs undergoing sham operation (no ischemia or reperfusion) to hindlimbs subjected to 3 hr of ischemia followed by 2 hr of reperfusion [4]. The technical aspects of that study regarding surgical preparation, vessel manipulation, and contraction/relaxation determination were identical to those of the current study. Sham vessels (n Å 5) relaxed 13.73 { 2.11, 47.88 { 7.23, and 72.44 { 9.00% to incremental doses of acetylcholine. In contrast, the relaxation responses to the same doses of acetylcholine in vessels subjected to 3 hr of ischemia and 2 hr of reperfusion (n Å 5) were 6.10
FIG. 4. Representative hematoxylin and eosin sections of control (left) and PTX-treated (right) superficial femoral arteries. No histologic differences are identified; intimal, medial, and adventitial layers are maintained in both sections.
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{ 1.02, 15.33 { 2.56, and 34.67 { 6.31% (P £ 0.05 when compared to control). Intraarterial administration of 100 mM PTX at initiation of reperfusion resulted in a significant increase in arterial relaxation to acetylcholine exposure compared to vessels infused with saline. PTX-perfused vessels relaxed between 48 and 77% to increasing doses of acetylcholine compared to control vessels which relaxed a mean of between 15 and 32% with the same acetylcholine doses. Differences in acetylcholine-induced relaxation are presumably due to variations in local nitric oxide levels, but whether the decreased relaxation in control vessels is due to reduced production or increased destruction of nitric oxide is unknown. Our results are consistent with those of Berkenboom and co-workers who reported a significant increase in relaxation of rat aortic rings after PTX exposure to acetylcholine, but not after exposure to indomethicin or methylene blue or mechanical removal of the endothelium. They suggest that a potential mechanism of action of PTX is reduced nitric oxide destruction by oxygen free radicals [8]. Pentoxifylline has a variety of physiologic effects which include increased red cell cyclic AMP levels resulting in stabilization of cellular membranes and enhanced red cell deformability [5]. Others believe PTX functions more by reducing plasma fibrinogen levels and blood viscosity [10]. In addition to its red cell effects, PTX has been found to have a potent influence on PMN function, particularly after PMN exposure to inflammatory cytokines [11]. PTX has been demonstrated to suppress lipopolysaccaride-induced TNF production at the level of mRNA expression [12] through a dose-dependent decrease in cytosolic calcium concentration [13]. Dauber and colleagues have demonstrated increased PMN cyclic AMP levels leading to diminished superoxide production, reduced PMN response to TNFa and interleukin-1 stimulation, and reduced PMN adhesion to the vascular endothelium [7]. Along with these effects, PMN and monocyte phagocytosis of latex particles has been shown to be reduced after incubation with PTX [6]. The role of leukocytes in ischemia and reperfusion has been well established. The central role of PMN in ischemia and reperfusion injury was demonstrated by Yokota et al. by near complete inhibition of their parameters of reperfusion injury after selective leukopenia was induced using dimethylmyleran [14]. PMN stimulation occurs in part secondary to exposure to locally produced oxygen free radicals and products of arachadonic acid metabolism [15]. Activated PMN in turn become a source for oxygen metabolites, thromboxanes, and proteolytic enzymes [15 – 17]. Once activated, these PMN exhibit increased chemotaxis and phagocytosis [18]. Evidence exists that interaction between endothelial cell-derived immunoglobulin-like cell adhesion molecules (ICAMs, VCAMs) and b2 integrins are required for leukocyte infiltration into postischemic tissues [19]. TNF is a major stimulus for adhesion molecule up-
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regulation [20], therefore inhibition of cytokine induction could indirectly limit reperfusion injury by decreasing leukocyte adhesion. Studies have utilized adenosine [21] or various monoclonal antibodies to limit reperfusion injury through reduced white cell adhesion [22–24]. Ma and associates investigated the impact of an anti-ICAM antibody (MAb RR1/1) on feline myocardial and coronary artery endothelial reperfusion injury. Administration of the MAb resulted in reduced tissue necrosis, myeloperoxidase activity, and improved coronary artery ring vasorelaxation to acetylcholine compared to cats receiving a nonbinding control antibody [25]. PTX has been shown to reduce both TNF induction and adhesion molecule expression [26, 27]. Others have demonstrated reduced venous effluent platelet activating factor levels after canine hindlimb ischemia following treatment PTX with reduction in reperfusion injury [28]. Although the precise mechanism of action of PTX in reducing loss of nitric oxide-mediated vasorelaxation remains elusive, several potential pathways have become apparent. Because of its inhibitory effect on superoxide production and enhancement of PMN cyclic AMP levels, leukocyte activation and phagocytosis are decreased, release of inflammatory cytokines is reduced, and local nitric oxide levels may be maintained. With reduction in TNF and platelet activating factor, endothelial cell expression of cell adhesion molecules is reduced, resulting in blunted PMN accumulation and infiltration with subsequent reduction in tissue destruction. Since these events occur in flowing blood, PTX needs to be present in this milieu to exert its effects. This may explain why our results were observed with PTX infusion at the time of reperfusion but not prior to reperfusion (unpublished data). Because reperfusion injury to the endothelium does not occur immediately [4], opportunity for therapeutic intervention exists prior to development of irreversible tissue damage. Pentoxifylline appears to have a role for use in clinically relevant skeletal muscle ischemia when it is administered at the beginning of reperfusion. REFERENCES 1. Parks, D. A., and Granger, D. N. Ischemia-induced vascular changes: Role of xanthine oxidase and hydroxyl radicals. Am. J. Physiol. 245: G285, 1983. 2. Gryglewski, R. J., Palmer, R. M. J., and Moncada, S. Superoxide anion is involved in the breakdown of endothelium-derived relaxing factor. Nature 320: 454, 1986. 3. Khwaja, A., Carver, J. E., and Linch, D. C. Interactions of granulocyte macrophage colony stimulating factor (CSF), granulocyte CSF, and tumor necrosis factor-a in the priming of the neutrophil respiratory burst. Blood 79: 745, 1992. 4. Chiang, P. C., Traul, D. K., Farooq, M. M., Lesniak, R. J., Seabrook, G. R., Towne, J. B., and Freischlag, J. A. Loss of superficial femoral artery relaxation following ischemia-reperfusion. J. Surg. Res. 60: 361, 1996. 5. Ehrly, A. M. The effect of pentoxifylline on the deformability of erythrocytes and on the muscular oxygen pressure in patients with chronic arterial disease. J. Med. 10: 331, 1979. 6. Bessler, H., Gilgal, R., Djaldetti, M., and Zahavi, I. Effect of
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