- Email: [email protected]
Inhibition of endothelium-dependent vasorelaxation by sickle erythrocytes
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rapid digital angiographic method. Circulation 1987;75:45260. 13. Rosner B. Analysis of variance. In: Rosner B. Fundamentals of biostatistics. Boston: PWS-Kent Publishing, 1989:494-5. 14. Vita JA, Treasure CB, Yeung AC, Vekshtein VI, Fantasia GM, Fish RD, Ganz P, Selwyn AP. Patients with evidence of coronary endothelial dysfunction as assessed by acetylcholine infusion demonstrate marked increase in sensitivity to constrictor effects of catecholamines. Circulation 1992;85:1390-7.
15. Werns SW, Walton JA, Hsia HH, Nabel EG, Sanz ML, Pitt B. Evidence of endothelial dysfunction in angiographically normal coronary arteries of patients with coronary artery disease. Circulation 1989;79:287-91. 16. Linsell CR, Lightman SL, Mullen PE, Brown MJ, Causon RC. Circadian rhythms of epinephrine and norepinephrine in man. J Clin Endocrinol Metab 1985;60:1210-5.
Inhibition of endothelium-dependent vasorelaxation by sickle erythrocytes Interactions between erythrocytes and vascular endothelium have been implicated in the pathogenesis of vaso-occlusion in sickle cell anemia. Sickle erythrocytes adhere to endothelial cells and facilitate trapping of rigid sickle cells in microvessels. Compensatory dilation of precapillary arterioles may mitigate the occlusion. The endothelium regulates vasoreactivity by elaborating endothelium-derived relaxing factor (EDRF), a small molecule that passes freely into vascular smooth muscle where it initiates vasorelaxation by activating soluble guanylate cyclase in the smooth muscle cell cytoplasm. Endothelial release of EDRF can be stimulated by agonists such as acetylcholine. It is highly sensitive to decomposition by superoxide anions and is rapidly bound and inactivated by oxyhemoglobin in solution. The purpose of this study was to determine whether sickle cell interaction with endothelium disrupts this mechanism of endothelial regulation of vasomotor tone. Transverse strips of rabbit aorta, mounted isometrically in organ baths, were contracted with norepinephrine, and relaxation responses to acetylcholine or other agonists were determined. Responses were measured under control conditions and again in the presence of oxyhemoglobin A or S, or erythrocytes or ghosts from normal control subjects or patients with homozygous sickle cell anemia. Sickle erythrocytes inhibited vasorelaxation to acetylcholine by 83%. Approximately half of the inhibition was attributable to a small amount of oxyhemoglobin S that was leaked into the buffer from the erythrocytes. Consistent with this, sickle erythrocyte ghosts inhibited vasorelaxation to acetylcholine by up to 45%. Ghosts from normal erythrocytes did not inhibit vasorelaxation to acetylcholine, and the inhibition seen with normal erythrocytes was entirely attributable to leakage of oxyhemoglobin A into the bath buffer. There was little or no inhibition of vasorelaxation to nitroglycerin, isoproterenol, or 8-bromocyclic guanosine monophosphate by sickle erythrocytes or ghosts, demonstrating that sickle erythrocytes predominantly inhibit endotheUum-dependent vasorelaxation. In addition, superoxide dismutase significantly diminished the inhibition of endothelium-dependent vasorelaxation as a result of sickle erythrocytes and ghosts. These findings indicate that sickle erythrocytes and their membranes interfere with endothelium-dependent vasorelaxation by inhibiting EDRF. Disruption of endothelial regulation of vasoreactivity may contribute to the development of vaso-occlusive phenomena in patients with sickle cell anemia. (AM HEARTJ 1993;126:338-346,) M o r r i s M o s s e r i , M D , A. N. B a r t l e t t - P a n d i t e , K a r e n W e n c , J e f f r e y M. I s n e r , M D , a n d R o b e r t W e i n s t e i n , M D Boston, Mass.
From the Departmentsof Medicine(Cardiologyand Hematology)and Biomedical Research, St. Elizabeth's Hospital, Tufts UniversitySchool of Medicine. Supported in part by a grant from Newman's Own, Inc., and grants HL-15157 and HL-40518 and an AcademicAward in Vascular Medicine (HL-02824) from the National Institutes of Health, Bethesda, Md. Received for publicationDec. 21, 1992;accepted Feb. 1, 1993. Reprint requests: Robert Weinstein,MD, St. Elizabeth's Hospital, 736 Cambridge St., Boston, MA 02135. Copyright © 1993 by Mosby-YearBook, Inc. 0002-8703/93/$1.00+ .10 4/1/47106
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Sickle cell a n e m i a is c h a r a c t e r i z e d b y v a s o - o c c l u s i v e e v e n t s , w h i c h m a y o c c u r i n a wide r a n g e of vessels f o r m t h e m i c r o v a s c u l a t u r e t o m u s c u l a r a r t e r i e s , 1-5 A l t h o u g h t h e m o s t severe m o r b i d i t y of t h e d i s e a s e is largely vascular, the p r i m a r y molecular defect results i n a n a l t e r e d h e m o g l o b i n ( h e m o g l o b i n S) a n d conseq u e n t c h a n g e s i n e r y t h r o c y t e s , t h u s r a i s i n g t h e possibility that vascular occlusion results from interaction between these abnormal erythrocytes and the
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blood vessel wall. 6 T h e first line of defense in the maintenance of vascular luminal integrity is the endothelium. 7 T h e endothelial cell actively regulates vasoreactivity, s platelet function, 9,10 the coagulation system,11 fibrinolysis,12,13 and other processes imp o r t a n t to maintenance of vascular patency. Sickle erythrocytes have been shown to interact with hum a n endothelial cells: they adhere to endothelial monolayers,14,15 stimulate prostacyclin synthesis and release, 16 and inhibit h u m a n venous and arterial endothelial cell D N A synthesis. 17 Hence it is i m p o r t a n t to know whether interaction between sickle erythrocytes and the endothelium affects the endothelial regulatory processes m e n t i o n e d previously. Detailed knowledge in this area would provide for a greater u n d e r s t a n d i n g of the pathogenesis of vaso-occlusion in patients with sickle cell anemia. Recent interest has focused on a novel endothelial regulator, referred to as endothelium-derived relaxing factor (EDRF), the major form of which is widely accepted to be identical to nitric oxide, is, 19 E D R F is formed during the conversion of L-arginine to citrulline by oxidation of a guanidonitrogen via a N A D P H and CA2+-dependent pathway. 2°, 21 This small molecule passes freely through cell m e m b r a n e s and can bind to and activate soluble guanylate cyclase producing an increase in intracellular cyclic guanosine monophosphate. 2224 E D R F participates in the regulation of several processes including vascular smooth muscle relaxation and contraction, 25 platelet aggregation and adherence to vascular endothelium, 9, 26, 27 and endothelial release of endothelin. 2s Its synthesis and release from endothelial cells is stimulated by a variety of mediators including acetylcholine, arachidonic acid, adenosine t r i p h o s p h a t e and adenosine monophosphate, bradykinin, and others. 25,29 It is rapidly inactivated by superoxide anions 3°-32 and is bound and inactivated by h e m o g l o b i n Y -35 T h e significance of E D R F in maintenance of vascular patency and its known sensitivity to hemoglobin and oxidation suggest t h a t interference with E D R F may be one mechanism by which erythrocyte-endothelial interaction promotes vascular occlusion in sickle cell anemia. T h e s e considerations p r o m p t e d us to determine whether sickle erythrocytes interfere with end o t h e l i u m - d e p e n d e n t vasorelaxation.
METHODS All salts and buffers were reagent grade and were obtained from Sigma Chemical Company (St. Louis, Mo). Human erythrocyte superoxide dismutase (3000 U/mg protein) and 8-bromoguanosine 3',5'-cyclic monophosphate (8-bromo-cGMP) were also from Sigma Chemical Company, as was acetylcholine chloride. Pharmaceutical grade nitroglycerin was obtained from Dupont Pharma-
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ceuticals (Wilmington, Del). Isoproterenol hydrochloric acid and norepinephrine bitartrate were from Sterling Drug (New York, N.Y.). All agonists were diluted to convenient working stock concentrations in muscle bath buffer (see below) on the day of the experiment.
Preparation of erythrocytes, ghosts, and oxyhemoglobin, Washed leukocyte-poor packed erythrocytes were prepared from adult volunteers. The patient population, method of obtaining blood samples, and preparation of washed erythrocytes have recently been describedF For these experiments, erythrocytes were packed to a hematocrit of 60 % to 80% and kept in an ice bucket in 12 × 75 ml plastic snap top tubes (Fisher Scientific, Pittsburgh, Pa.). The tops were kept loose to avoid an airtight seal. For preparation of hemolysate-free erythrocyte ghosts by hypotonic lysis, 361 volume of washed intact erythrocytes was added to 30 volumes of 3P8 buffer (3 mmol/L sodium phosphate buffer, 1 mmol/L ethylenediametetraacetic acid, and 100 mmol/L phenylmethylsulfonyl fluoride, pH 8.0). The 3P8 buffer was kept on ice for ghost preparation. After vigorous mixing, the preparation was centrifuged at 22,000 g for 15 minutes at 4 ° C. The supernate was carefully removed by aspiration, the tube was rotated horizontally, and the "cream pellet" at the bottom of the tube was removed by aspiration. The ghosts were resuspended in 30 volumes of ice-cold 3P8 buffer, mixed vigorously, and recentrifuged, and removal of the supernate and "cream pellet" was repeated. These steps were repeated until absorption of the supernate at 385,405, 560, 577, and 630 nm was nil. The white ghosts were kept on ice in 3P8 buffer at a ghost protein concentration of 1.2 to 1.5 mg/ml as determined colorimetrically37 during the course of each experiment. The heme content of AA and SS ghosts was less than 1.85 nmol/mg and 3.3 nmot/mg, respectively, as determined spectrophotometrically.17 Oxyhemoglobins A and S were prepared according to the method of Caughey and Watkins 3s as modified by Hebbel et al. 39 Briefly, washed buffy coat-poor erythrocytes were lysed in 5 volumes of distilled deionized water (DDH20) and centrifuged at 16,000 rpm in a Sorvall RC-5 centrifuge (DuPont Co., Wilmington, Del.) at 4 ° C for 15 minutes with an SS-34 rotor. The top 75 % of each tube was harvested and centrifuged at 35,000 rpm for 40 minutes in a Beckman L5-75 ultracentrifuge (Beckman Instruments, Inc., Fullerton, Calif.) with a SW 41 Ti rotor. The ghost-free lysate was then dialyzed overnight against 60 volumes of DDH20 (with three changes), with oxygen continuously bubbling through the dialysate, by means of Spectrapor membrane tubing of 6 to 8,000 molecular weight cut-off (Spectrum Medical Industries, Los Angeles, Calif.). The clear reddish solution was applied to diethylaminoethyl sepharose CL-6B in 5 mmol/L TRIS HC1, pH 8.6, at 5 ml/min at 4 ° C. The column was washed in the same buffer until absorption of the column effluent was 0 at 630, 577, and 560 nm. Hemoglobin was then eluted from the column with 10 mmol/L TRIS HC1, pH 7.1, containing 40 mmol/L sodium chloride. The column eluate was concentrated with Centriprep YM-10 membranes (Amicon, Danvers, Mass.) and applied to Sephadex G-100 in 5 mmol/L
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TRIS HC1, pH 8.1. The major hemoglobin peak was dialyzed against 5 mmol/L sodium phosphate buffer, pH 7.4, containing 0.15 mol/L NaC1. After membrane concentration as described previously, the preparations were determined to be >95% oxyhemoglobin (A or S) by analysis of absorption spectra at 700, 630, 577, and 560 nm according to Winterbourn. 4° Bioassay technique. Tranverse strips of descending thoracic aorta (2.5 mm wide) were obtained from anesthetized male New Zealand white rabbits (2 to 3 kg) and mounted isometrically in water-jacketed organ baths by means of small plastic clips. The lower clip was attached to a glass hook at the bottom of the bath with surgical silk suture. The upper clip was attached with a suture to an isometric force transducer (model FT 03, Grass Instrument Co., Quincy, Mass.). The bath buffer consisted of (in mmol/L): NaC1 118; KC1 4.8, CaC12 3.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 24, Ca-EDTA 0.026, and glucose 11.1. Temperature was maintained at 37 ° C by a recirculating water bath. A stream of 95 % oxygen/5 % carbon dioxide, continuously bubbling up from a scintered glass inlet at the bottom of the bath, maintained pH at 7.4 and pO2 at 500 torr. The strips were preloaded with 2 gm of isometric tension and permitted to relax and equilibrate for up to 1 hour before the experiments were begun. Isometric tension generated (positive or negative) in response to agonists was recorded continuously with a Grass model 5 PIB 3 amplifier and model 7 polygraph (Grass Instrument Co.). The arterial strips were precontracted with norepinephrine sufficient to generate half maximal tension (usually 10 -s to 5 x 10 -s mol/L as shown in the figures). After equilibration, acetylcholine (10 -s to 10 -6 mol/L) was added to the baths to determine the functional integrity of endothelium-dependent vasorelaxation, after which the bath buffer was rapidly drained, the tissue was washed three times with fresh bath buffer, and the experiments were performed. Vasoreactivity responses to agonists in the presence of erythrocytes, ghosts, or oxyhemoglobin were performed by incubating the tissue in the bath with erythrocytes (final hematocrit in the bath was 0.6% to 0.8% ), ghosts (11 to 15 ttg/ml of ghost protein in the baths), or oxyhemoglobin (10 -9 to 5 × 10 -6 mol/L in the bath) for at least 15 minutes. The arterial strips were then contracted with norepinephrine. When the contraction response reached a plateau, relaxation to agonists such as acetylcholine, nitroglycerin, isoproterenol, or 8-bromo-cGMP was assessed. Contraction and relaxation (in milligrams of tension) were read directly from a continuous polygraph recording. Vasoreactivity to acetylcholine was also monitored in the presence of ghosts prepared from erythrocytes pretreated with 2 mmol/L diisopropyl fluorophosphate to assure that inhibitory effects of ghosts (and erythrocytes) were not merely due to degradation of acetylcholine by cholinesterase. Visual inspection of the baths confirmed that erythrocytes and ghosts were maintained swirling in suspension throughout the entire volume of baths during experiments. The maintenance of erythrocytes and ghosts in suspension, and their continual swirling through the baths, was facilitated
AmericanHeartJournal
by the bubbling stream of gas from the bottom of the baths (see above). To control for the inhibition of endothelium-dependent vasorelaxation resulting from leakage of hemoglobin into the baths from lysed erythrocytes, the bath buffer was harvested with a large plastic pipette after experiments and centrifuged at 500 g for 10 minutes at 4 ° C. The oxyhemoglobin concentration in the buffer was then determined by analysis of absorption at 700, 630, 577, and 560 nm. 4° Statistical analysis. For each experiment the vascular strip served as its own control specimen. Therefore the effect of an individual treatment was analyzed by t test for independent samples if the control tests performed with the individual strips yielded comparable results. Statistical analysis was performed by means of the PC Statistician Human Systems Dynamics, Northridge, Calif. Graphic analysis was performed by means of Sigma Plot (Jandel Scientific, Sausolito, Calif.). Figures show means and standard errors of the various treatments represented. To normalize the results from similar experiments performed on separate days, the data for vasorelaxation are expressed relative to the tension generated with norepinephrine in each experiment. Thus a "percentage relaxation" refers to the percentage of norepinephrine-generated tension that is reversed with the use of a relaxing agonist in the control specimens or in the presence of erythrocytes, ghosts, or hemoglobin. In certain figures, relaxation in the presence of erythrocytes, ghosts, or hemoglobin is presented as the ratio of the percentage relaxation obtained in each experiment to the percentage relaxation obtained in the control specimen for the experiment. Hence a ratio of 1.0 indicates no inhibition of relaxation compared to the control value. RESULTS Inhibition of vasorelaxation. I n h i b i t i o n of vasorelax-
ation was achieved in vitro b y h u m a n hemoglobin, erythrocytes, a n d e r y t h r o c y t e ghosts. In free solution, h e m o g l o b i n of r a t or bovine origin inhibits vasorelaxation to acetylcholine in a p r e d i c t a b l e m a n n e r at m i c r o m o l a r concentrations. 3335 T o t a k e into acc o u n t the possibility t h a t , h e m o g l o b i n m i g h t be released into organ b a t h s as a result of m i n o r hemolysis during our e x p e r i m e n t s with erythrocytes, and t h a t this free h e m o g l o b i n m i g h t result in " a r t i f a c t u al" inhibition of vasorelaxation responses in our assays, we first d e t e r m i n e d the ability of h u m a n h e m o globins A a n d S to inhibit vasorelaxation to acetylcholine. As shown in Fig. 1, vasorelaxation to acetylcholine was unaffected b y 10 -7 m o l / L h u m a n h e m o g l o b i n b u t was c o m p l e t e l y inhibited at concent r a t i o n s greater t h a n 2 to 3 x 10 -6 mol/L. T h e c o n c e n t r a t i o n - d e p e n d e n t inhibition by hemoglobins S and A were similar, indicating t h a t neither of these h u m a n hemoglobins exerts an a priori superior inhibitory effect on vasorelaxation to acetylcholine. E x p e r i m e n t s were c o n d u c t e d in which vasorelax-
Volume 126, Number 2 American Heart Journal
Mosseri et al. 341
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[Hb02] (/~rnoles,/L) Fig. 1. Vasorelaxation to acetylcholine is inhibited by human hemoglobin and erythrocytes. Rabbit aortic strips were contracted with norepinephrine and then treated with acetylcholine to induce relaxation. Strips were then recontracted with norepinephrine in the presence of hemoglobin A (filled circles), hemoglobin S (filled triangles), AA erythrocytes (striped bar), or SS erythrocytes (cross-hatched bar), and relaxation to acetylcholine was observed again. Percentage relaxation of vascular strips under experimental conditions as compared with control conditions, for each individual strip, is shown as a ratio on the ordinate. Relax-. ation ratio of 1.0 indicates no inhibition (see Methods). Data points for hemoglobin represent at least five individual experiments, and bars for erythrocytes represent 19 experiments with S$ red blood cells (SSRBC) and 23 experiments with AA red blood cells (AARBC). Human hemoglobins A and S inhibited acetylcholine-induced vasorelaxation in a concentration-dependent manner. No inhibition was seen at 0.1 ttmol/L hemoglobin, and complete inhibition was seen at 2 to 3/~mol/L hemoglobin. Negative relaxation ratios at 5 #mol/L hemoglobin indicate that further vasoconstriction occurred in response to acetylcholine at that hemoglobin concentration. Bars for SS and AA erythrocytes are placed on the abscissa according to mean concentration of hemoglobin S or A (respectively) released into bath buffer during experiments (see text). Thus by comparison with hemoglobin curves, apparent inhibition of vasorelaxation by AA erythrocytes is easily attributed to hemoglobin A, which leaked into bath buffer during experiments. On the other hand, leakage of hemoglobin S into bath buffer cannot account for inhibition of vasorelaxation by SS erythrocytes. HbO2A, Oxyhemoglobin A; Hb02S, oxyhemoglobin S.
ation responses were d e t e r m i n e d with low concentrations (hematocrit 0.6% to 0.8%) of SS or AA erythrocytes in organ baths. B a t h buffer was periodically harvested at the end of the experiments, cleared of e r y t h r o c y t e suspension by centrifugation, and assayed for free hemoglobin concentration spect r o p h o t o m e t r i c a l l y to d e t e r m i n e whether sufficient hemolysis was occurring to account for the inhibition of vasorelaxation during the experiments. As shown in Fig. 1, vasorelaxation to acetylcholine in the presence of SS or AA erythrocytes was only approximately 17 % or 13 %, respectively, of vasorelaxation in the absence of erythrocytes. However, the average free hemoglobin concentration in the bath buffer was much greater in experiments with AA erythrocytes c o m p a r e d with SS erythrocytes (6.8 _+ 2.13 #mol/L 0.6_+ 0.27 #mol/L, AA vs SS; p = 0 . 0 1 5 , n = 7 ) . Hence, although the entire a p p a r e n t inhibitory effect
of AA erythrocytes could be explained on the basis of free hemoglobin release into the bath buffer, this did not account for the inhibitory effect of SS erythrocytes on vasorelaxation to acetylcholine. T h e data represented in Fig. 1 also suggest that, when corrected for free hemoglobin release, sickle erythrocytes inhibit acetylcholine-induced vasorelaxation by approximately 40 %. T o examine h u m a n erythrocyte inhibition of vasorelaxation to acetylcholine i n d e p e n d e n t of the hemoglobin in the bath buffer, ghosts were p r e p a r e d from SS and AA erythrocytes. These were washed thoroughly in lysis buffer (see Methods) and were used in vasorelaxation e x p e r i m e n t s in place of intact erythrocytes. As shown in Fig. 2, SS ghosts decreased vasorelaxation to 10 -s mol/L acetylcholine by 45 % (p = 0.006, n = 7) and decreased vasorelaxation to 5 × 10 -s mol/L acetylcholine by 27% (/9 = 0.001,
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3, B). These data suggest that the effects of SS erythrocytes and ghosts on acetylcholine-induced vasorelaxation is due to the inhibition of EDRF.
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Fig. 2. Sickle erythrocyte ghosts inhibit vasorelaxation to acetylcholine. Rabbit aortic strips were contracted with norepinephrine, and vasorelaxation was then induced with acetylcholine. Strips were recontracted with norepinephrine in the presence of SS ghosts (cross-hatched bars) or AA ghosts (striped bars). Vasorelaxation in the presence of ghosts was then induced with acetylcholine. As in Fig. 1, relaxation under experimental conditions is compared with relaxation under control conditions for each experiment as a relaxation ratio. Vertical bars for SS ghosts represent seven experiments each, and vertical bars for AA ghosts represent five experiments each. SS ghosts decreased vasorelaxation to acetylcholine by 45% at 10-s mol/L acetylcholine (p = 0.006) and by 27 % at 5 × 10-s mol/L acetylcholine (p = 0.001). AA ghosts did not inhibit vasorelaxation at either concentration of acetylcholine (relaxation ratio greater than 1.0 at both concentrations).
n = 7). In contrast, AA ghosts did not inhibit vasorelaxation at either concentration of acetylcholine. The oxyhemoglobin S concentration in the bath buffer in SS ghost experiments was less than 6 x 10 -s tool/L, not sufficient to inhibit acetylcholine-induced vasorelaxation (as shown in Fig. 1). These findings indicate that the inhibition of vasorelaxation seen with intact SS erythrocytes is not merely due to leakage of oxyhemoglobin S into the bath buffer and is not completely dependent on intracorpuscular oxyhemoglobin S. Acetylcholine induces vasorelaxation by stimulating synthesis and release of EDRF. s Superoxide dismutase enhances the in vitro and in vivo activity of E D R F by preventing its degradation by superoxide anions in solution. 3°, 31 As shown in Fig. 3, superoxide dismutase (1000 U/ml) decreased SS ghost inhibition of acetylcholine-induced vasorelaxation by 25 % (p = 0.025; Fig. 3, A) and reduced SS erythrocyte inhibition by approximately 50% (p < 0.01; Fig.
Vasorelaxation inhibited by SS erythrocytes and ghosts: Dependency on endothelium T h e preceding
experiments demonstrate that SS erythrocytes and ghosts inhibit endothelium-dependent vasorelaxation to acetylcholine. Additional experiments were performed to investigate whether endothelium-independent vasorelaxation is also inhibited. In Fig. 4, the effect of SS ghosts on vasorelaxation to acetylcholine (endothelium dependent) is compared with the effect of SS ghosts on vasorelaxation to nitroglycerin and to isoproterenol (both endothelium independent). Nitroglycerin directly generates nitric oxide and thus raises cyclic guanosine monophosphate (GMP) inside vascular smooth muscle cells, in effect acting pharmacologically similar to EDRF but bypassing the endothelium. Isoproterenol also works directly on vascular smooth muscle but a cyclic adenosine monophosphate p a t h w a y Y As shown in Fig. 4, A, the SS ghosts inhibited vasorelaxation to acetylcholine by 27% (p = 0.001), whereas relaxation to nitroglycerin was not significantly affected (7 % decrease, p > 0.5). Similarly, as shown in Fig. 4, B, concentration-dependent vasorelaxation to isoproterenol was the same in the presence and absence of SS ghosts. Analogous experiments performed in the presence and absence of SS erythrocytes demonstrated an 83 % decrease in vasorelaxation to acetylcholine, a 21% decrease in vasorelaxation to isoproterenol, and no decrease in vasorelaxation to nitroglycerin in the presence of SS erythrocytes. Additional evidence regarding the ability of vascular strips to respond to an endotheliumindependent rise in intracellular cyclic GMP was obtained using a non-hydrolyzable analogue, 8-bromocyclic GMP. As shown in Fig. 5, vasorelaxation to 8-bromocyclic GMP was unaffected by the presence of SS erythrocytes. Hence SS erythrocyte membranes and intact SS erythrocytes predominantly inhibit endothelium-dependent vasorelaxation. Furthermore, in the presence of SS erythrocytes and ghosts the guanylate cyclase pathway in vascular smooth muscle remains intact. However, the ability of the endothelium to activate this pathway by the stimulated release of EDRF is clearly impaired in the presence of SS erythrocytes or ghosts. DISCUSSION
The severity of vascular occlusive morbidity in sickle cell anemia has led investigators to consider that interaction between sickle erythrocytes and the endothelium may compromise the maintenance of
Volume 126, Number 2
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Fig. 3. Inhibition of endothelium-dependent vasorelaxation by SS ghosts and erythrocytes is reversed by superoxide dismutase (SOD). Rabbit aortic strips were precontracted with norepinephrine and then relaxed with acetylcholine. Sequence of contraction and relaxation was repeated for each strip with either SS ghosts (A) or SS erythrocytes (R) in organ baths (see Methods). Sequence of contraction and relaxation was then repeated in the presence of SS ghosts or erythrocytes (A and B, respectively), but with the addition of SOD (1000 U/ml) in organ baths. Strips were washed three times with bath buffer between each sequence of contraction and relaxation. Percentage relaxation in control specimens and under respective experimental conditions is presented. Each bar represents mean results of two experiments. As shown in A, control strips relaxed 72.8 -+ 3.07% in response to acetylcholine, and relaxation in the presence of SS ghosts was only 40.5 + 1.5% (p = 0.004 vs control). However, vasorelaxation in the presence of SS ghosts was 51 _+ 0.99% when SOD was also in bath buffer (p = 0.025 vs ghosts alone). Aortic strips represented in B relaxed 97.3 + 2.7% under control conditions and only relaxed 36.3 _+ 1.3% when sickle erythrocytes were in organ baths (p < 0.01 vs control). With SOD and sickle erythrocytes in baths together, relaxation of the same strips was 62.2 __ 2.4% (p < 0.01 vs SS erythrocytes alone).
vascular patency. Sickle erythrocytes may directly occlude microvessels by adhering to endothelium41 or may directly alter endothelial functions such as prostacyclin production 16 and DNA synthesis. 17 Anatomic studies suggest frank endothelial damage in patients with sickle cell anemia, 3' 4, 42 and there is an increase in circulating endothelial cells in these patients during vasocclusive crises, 43 further suggesting endothelial damage during these events. Hebbel 6 has recentlY observed that the potential for altered endothelial regulation of vasoreactivity in the pathogenesis of vaso-occlusive events in sickle cell anemia remains unexplored. Observations of periodic oscillatory microvascular flow in patients with sickle cell anemia, by means of intravital microscopy44 and laser Doppler velocimetry,45 suggest that regulation of vasoreactivity in these patients is altered. In the present study we have employed a wellcharacterized bioassays to determine whether interaction between sickle erythrocytes and endothelium directly alters endothelial regulation of vasoreactiv-
ity by interfering specifically with EDRF, the major endothelial mediator of vasoreactivity.25 Sickle erythrocytes markedly inhibited vasorelaxation to acetylcholine, a relaxing agent that functions by stimulating elaboration of EDRF from the endothelium. 22-25 The inhibition was significantly diminished by superoxide dismutase, further indicating that the effect seen with sickle erythrocytes is mediated by EDRF. 3°-32 In addition, sickle erythrocytes did not directly inhibit endothelium-independent vasorelaxation mediated via cyclic GMP (nitroglycerin or 8-bromocyclic-GMP) or cyclic adenosine monophosphate (isoproterenol). Sickle erythrocyte ghosts also inhibited vasorelaxation to acetylcholine, indicating that these inhibitory effects result from interaction between the endothelium and the sickle erythrocyte membrane and are not totally due to the scavenging of EDRF by oxyhemoglobin S. Inhibition of vasorelaxation to acetylcholine was also observed when normal (AA) erythrocytes were in the organ baths. However, spectrophotometric anal-
344
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[ISUpR,ul (moles/u) Fig. 4. Vasorelaxationresponsesinhibitedbysickleerythrocyte ghosts are endothelium dependent. Rabbit aortic strips were contracted with norepinephrine and then exposed to various vasorelaxant agonists. Each strip was then washed three times with bath buffer, sickle erythrocyte ghosts were added to organ baths, and the same sequence of contraction and vasorelaxation was repeated. A , VaSorelaxation to endothelium-dependent relaxing agent acetylcholine (ACH; 5 x 10-s mol/L) is compared with vasorelaxation to endothelium-independent relaxing agent nitroglycerin (NTG; 10-s tool/L). Relaxation to acetytcholine was 72,1 _+ 6.1% under control conditions and 52.3 _+ 6.1% with SS ghosts in baths (p = 0.001, n = 7). Relaxation to nitroglycerin was 77 _+ 6.3 % under control conditions and 71.1 + 2.9 % with SS ghosts in organ baths (p = 0.290, n = 5). B, Vasorelaxation to endothelium-independent relaxing agent (Isuprel), in the presence and absence of SS ghosts, is shown over isoproterenol concentrati0n range of 10-7 to 3 × 10-6 mol/L. No difference was obtained between control relaxation and relaxation with SS ghosts in organ baths. Each data point represents mean of five experiments.
ysis of s u p e r n a t a n t organ b a t h buffer revealed sufficient oxyhemoglobin A (but not S) to account for all of the a p p a r e n t inhibition of E D R F observed in exp e r i m e n t s with normal (but not sickle) e r y t h r o c y t e s despite the gross visual clarity of the buffer. This conclusion is consistent with the finding t h a t normal
i
--5
10
4x10
-5
(moles/)
Fig. 5. Sickle erythrocytes do not prevent vasorelaxation to cyclic guanosine monophosphate (GMP). Rabbit aortic strips were contracted with norepinephrine and then treated with varying concentrations of 8-bromocyclic G M P in the presence or absence of SS erythrocytes (see Methods). This nonhydrolyzable analogue of cyclic G M P induces endothelium-independent vasorelaxation by directly increasing smooth muscle cyclic G M P . Over the range of concentrations tested (from m i n i m u m to m a x i m u m vasorelaxation response), vasorelaxation was identical whether SS erythrocytes were present or absent from organ baths (p > 0.5 at each Concentration). Each data point on control curve represents mean of three experiments; each data point On the SS R B C curve represents mean of two
experiments.
e r y t h r o c y t e ghosts did not inhibit vasorelaxation to acetylcholine. In addition, normal h u m a n erythrocytes used at a 10 % h e m a t o c r i t have recently been found to not inhibit vasorelaxation to acetylcholine by canine femoral arteries in a flow system in which free hemoglobin did not accumulate in the b u f f e r s Our experiments were conducted with low concentrations (0.6% to 0.8% h e m a t o c r i t ) o f erythrocytes, representing a hemoglobin concentration of up to a p p r o x i m a t e l y 40 umol/L in the baths. H u m a n hemoglobin in free solution was found to completely inhibit vasorelaxation to acetylcholine at a concentration of 2 to 3/~mol/L. Despite the presence in the baths of more t h a n enough intracorpuscular hemoglobin to inhibit vasorelaxation completely, vasorelaxation was not 100 % inhibited by sickle erythrocytes. T h e s e findings suggest t h a t inhibition of E D R F by sickle erythrocytes is not simply on the basis of competitive binding of E D R F to the relatively large pool of intracorpuscular hemoglobin, b u t is also due to interaction between the sickle e r y t h r o c y t e membrane and the endothelium. T h e ability of sickle
Volume 126, Number 2 American Heart Journal
e r y t h r o c y t e g h o s t s to i n h i b i t E D R F - m e d i a t e d res p o n s e s s u p p o r t s t h i s c o n c l u s i o n . T h e a t t e n u a t i o n of t h e SS e r y t h r o c y t e effects b y s u p e r o x i d e d i s m u t a s e m a y suggest t h a t the i n h i b i t i o n involves the decomp o s i t i o n of E D R F ( n i t r i c oxide) b y excess o x i d a n t r a d i c a l s g e n e r a t e d b y t h e sickle e r y t h r o c y t e m e r e branes.39, 46 T h e d i s c r e p a n c y b e t w e e n t h e i n h i b i t o r y c a p a b i l i t y of i n t r a c o r p u s c u l a r h e m o g l o b i n a n d hem o g l o b i n i n free s o l u t i o n i n o u r s t u d i e s is c o n s i s t e n t w i t h r e c e n t s i m i l a r o b s e r v a t i o n s b y H o u s t o n et al. 27 T h e experimental conditions used in our studies, i n c l u d i n g low h e m a t o c r i t a n d t h e a b s e n c e of p l a s m a p r o t e i n s , w o u l d n o t b e e x p e c t e d to favor sickle e r y t h r o c y t e a d h e r e n c e to t h e e n d o t h e l i u m i n o r g a n bathsl~, 47 a n d are n o t p h y s i o l o g i c i n a clinical sense. H o w e v e r , sickle e r y t h r o c y t e s h a v e b e e n d e m o n s t r a t e d to i n t e r a c t w i t h v a s c u l a r e n d o t h e l i u m u n d e r p h y s i o l o g i c c o n d i t i o n s a n d i n t h e a b s e n c e of s t r o n g adherence:iT, 41, 47, 48 N i t r i c oxide ( E D R F ) is n o t o n l y t h e p r i n c i p a l e n d o t h e l i u m - d e p e n d e n t r e g u l a t o r of v a s o m o t o r t o n e 25 b u t m a y p l a y a s i g n i f i c a n t role i n t h e m o d u l a t i o n of p l a t e l e t a d h e s i o n a n d aggregat i o n 9, 19 a n d m a c r o p h a g e c y t o t o x i c activity. 49 T h e d y s r e g u l a t i o n of v a s o m o t o r , t h r o m b o t i c , a n d i n f l a m m a t o r y processes as a r e s u l t of i n t e r f e r e n c e w i t h n i tric o x i d e - d e p e n d e n t p a t h w a y s b y t h e sickle e r y t h r o c y t e m a y c o n t r i b u t e to t h e " s t o c h a s t i c ''6 n a t u r e of v a s o - o c c l u s i v e e v e n t s i n p a t i e n t s w i t h sickle cell a n e mia. We thank the Boston Sickle Cell Center and the hematology/ oncology nurses of St. Elizabeth's Hospital for help with procurement of blood specimens, and Patsy Bustos for preparation of the manuscript. REFERENCES
1. Diggs LW, Bruegge CFV. Vascular occlusive mechanisms in sickle cell disease. J Natl Med Assoc 1954;46:46-9. 2. Konotey-Ahulu FID. The sickle cell diseases: clinical manifestations including the "sickle crisis." Arch Intern Med 1974;133:611-9. 3. Boros L, Thomas C, Weiner WJ. Large cerebral vessel disease in sickle cell anemia. 3 Neurol Neurosurg Psychiatry 1976; 39:1236-9. 4. Merkel KHH, Ginsberg PL, Parker Jr JC, Post MJD. Cerebrovascular disease in sickle cell anemia: a clinical, pathological and radiological correlation. Stroke 1978;9:45-52. 5. Stockman JA, Nigro MA, Mishkin MM, Oski FA. Occlusion of large cerebral vessels in sickle-cell anemia. N Engl J Med 1972;287:846-9. 6. Hebbel RP. Beyond hemoglobin polymerization: the red blood cell membrane and sickle disease pathophysiology. Blood 1991;77:214-37. 7. Simionescu M, Simionescu N. Functions of the endothelial cell surface. In: Berne RM, ed. Annual review of physiology. Palo Alto: Annual Reviews Inc., 1986:279-93. 8. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288:373-6. 9. Bhardwaj R, Page CP, May GR, Moore PK. Endothelium-derived relaxing factor inhibits platelet aggregation in human
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40. Winterbourn CC. Reactions of superoxide with hemoglobin. In: Greenwald RA, ed. Handbook of methods for oxygen radical research. Boca Raton: CRC Press, 1985:137. 41. Kaul DK, Fabry ME, Nagel RL. Microvascular sites and characteristics of sickle cell adhesion to vascular endothelium in shear flow conditions: pathopbysiological implications. Proc Natl Acad Sci USA 1989;86:3356-60. 42. Klug PP, Kaye N, Jensen WN. Endothelial cell and vascular damage in the sickle cell disorders. Blood Cells 1982;8:175-84. 43. Sowemino-Coker SO, Meiselman HJ, Francis Jr RB. Increased circulating endothelial cells in sickle cell crisis. Am J Hematol 1989;31:263-5. 44. Lipowsky HH, Sheikh NU, Katz DM. Intravital microscopy of capillary hemodynamics in sickle cell disease. J Clin Invest 1987;80:117-27. 45. Rodgers GP, Schechuter AN, Noguchi CT, Klein HG, Nienhuis AW, Bonner RF. Periodic microcircnlatory flow in patients with sickle-cell disease. N Engl J Med 1984;311:1534-8. 46. Hebbe] RP, Eaton JW, Balasingam M, Steinberg MH. Spontaneous oxygen radical generation by sickle erythrocytes. J Clin Invest 1982;70:1253-9. 47. Mohandas N, Evans E. Adherence of sickle erythrocytes to vascular endothelial cells: requirement for both cell membrane changes and plasma actors. Blood 1984;64:282-7. 48. Barabino GM, McIntire LV, Eskin SG, Sears DA, Udden M. Endothelial cell interactions with sickle cell, sickle trait, mechanically injured and normal erythrocytes under controlled flow. Blood 1987;70:152-7. 49. Marletta MA, Yoon PS, Iyengar R, Leaqf CD, Wishnok JS. Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry 1988;27:8706-11.
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Inhibition of endothelium-dependent vasorelaxation by sickle erythrocytes Interactions between erythrocytes and vascular endothelium have been implicated in the pathogenesis of vaso-occlusion in sickle cell anemia. Sickle erythrocytes adhere to endothelial cells and facilitate trapping of rigid sickle cells in microvessels. Compensatory dilation of precapillary arterioles may mitigate the occlusion. The endothelium regulates vasoreactivity by elaborating endothelium-derived relaxing factor (EDRF), a small molecule that passes freely into vascular smooth muscle where it initiates vasorelaxation by activating soluble guanylate cyclase in the smooth muscle cell cytoplasm. Endothelial release of EDRF can be stimulated by agonists such as acetylcholine. It is highly sensitive to decomposition by superoxide anions and is rapidly bound and inactivated by oxyhemoglobin in solution. The purpose of this study was to determine whether sickle cell interaction with endothelium disrupts this mechanism of endothelial regulation of vasomotor tone. Transverse strips of rabbit aorta, mounted isometrically in organ baths, were contracted with norepinephrine, and relaxation responses to acetylcholine or other agonists were determined. Responses were measured under control conditions and again in the presence of oxyhemoglobin A or S, or erythrocytes or ghosts from normal control subjects or patients with homozygous sickle cell anemia. Sickle erythrocytes inhibited vasorelaxation to acetylcholine by 83%. Approximately half of the inhibition was attributable to a small amount of oxyhemoglobin S that was leaked into the buffer from the erythrocytes. Consistent with this, sickle erythrocyte ghosts inhibited vasorelaxation to acetylcholine by up to 45%. Ghosts from normal erythrocytes did not inhibit vasorelaxation to acetylcholine, and the inhibition seen with normal erythrocytes was entirely attributable to leakage of oxyhemoglobin A into the bath buffer. There was little or no inhibition of vasorelaxation to nitroglycerin, isoproterenol, or 8-bromocyclic guanosine monophosphate by sickle erythrocytes or ghosts, demonstrating that sickle erythrocytes predominantly inhibit endotheUum-dependent vasorelaxation. In addition, superoxide dismutase significantly diminished the inhibition of endothelium-dependent vasorelaxation as a result of sickle erythrocytes and ghosts. These findings indicate that sickle erythrocytes and their membranes interfere with endothelium-dependent vasorelaxation by inhibiting EDRF. Disruption of endothelial regulation of vasoreactivity may contribute to the development of vaso-occlusive phenomena in patients with sickle cell anemia. (AM HEARTJ 1993;126:338-346,) M o r r i s M o s s e r i , M D , A. N. B a r t l e t t - P a n d i t e , K a r e n W e n c , J e f f r e y M. I s n e r , M D , a n d R o b e r t W e i n s t e i n , M D Boston, Mass.
From the Departmentsof Medicine(Cardiologyand Hematology)and Biomedical Research, St. Elizabeth's Hospital, Tufts UniversitySchool of Medicine. Supported in part by a grant from Newman's Own, Inc., and grants HL-15157 and HL-40518 and an AcademicAward in Vascular Medicine (HL-02824) from the National Institutes of Health, Bethesda, Md. Received for publicationDec. 21, 1992;accepted Feb. 1, 1993. Reprint requests: Robert Weinstein,MD, St. Elizabeth's Hospital, 736 Cambridge St., Boston, MA 02135. Copyright © 1993 by Mosby-YearBook, Inc. 0002-8703/93/$1.00+ .10 4/1/47106
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Sickle cell a n e m i a is c h a r a c t e r i z e d b y v a s o - o c c l u s i v e e v e n t s , w h i c h m a y o c c u r i n a wide r a n g e of vessels f o r m t h e m i c r o v a s c u l a t u r e t o m u s c u l a r a r t e r i e s , 1-5 A l t h o u g h t h e m o s t severe m o r b i d i t y of t h e d i s e a s e is largely vascular, the p r i m a r y molecular defect results i n a n a l t e r e d h e m o g l o b i n ( h e m o g l o b i n S) a n d conseq u e n t c h a n g e s i n e r y t h r o c y t e s , t h u s r a i s i n g t h e possibility that vascular occlusion results from interaction between these abnormal erythrocytes and the
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blood vessel wall. 6 T h e first line of defense in the maintenance of vascular luminal integrity is the endothelium. 7 T h e endothelial cell actively regulates vasoreactivity, s platelet function, 9,10 the coagulation system,11 fibrinolysis,12,13 and other processes imp o r t a n t to maintenance of vascular patency. Sickle erythrocytes have been shown to interact with hum a n endothelial cells: they adhere to endothelial monolayers,14,15 stimulate prostacyclin synthesis and release, 16 and inhibit h u m a n venous and arterial endothelial cell D N A synthesis. 17 Hence it is i m p o r t a n t to know whether interaction between sickle erythrocytes and the endothelium affects the endothelial regulatory processes m e n t i o n e d previously. Detailed knowledge in this area would provide for a greater u n d e r s t a n d i n g of the pathogenesis of vaso-occlusion in patients with sickle cell anemia. Recent interest has focused on a novel endothelial regulator, referred to as endothelium-derived relaxing factor (EDRF), the major form of which is widely accepted to be identical to nitric oxide, is, 19 E D R F is formed during the conversion of L-arginine to citrulline by oxidation of a guanidonitrogen via a N A D P H and CA2+-dependent pathway. 2°, 21 This small molecule passes freely through cell m e m b r a n e s and can bind to and activate soluble guanylate cyclase producing an increase in intracellular cyclic guanosine monophosphate. 2224 E D R F participates in the regulation of several processes including vascular smooth muscle relaxation and contraction, 25 platelet aggregation and adherence to vascular endothelium, 9, 26, 27 and endothelial release of endothelin. 2s Its synthesis and release from endothelial cells is stimulated by a variety of mediators including acetylcholine, arachidonic acid, adenosine t r i p h o s p h a t e and adenosine monophosphate, bradykinin, and others. 25,29 It is rapidly inactivated by superoxide anions 3°-32 and is bound and inactivated by h e m o g l o b i n Y -35 T h e significance of E D R F in maintenance of vascular patency and its known sensitivity to hemoglobin and oxidation suggest t h a t interference with E D R F may be one mechanism by which erythrocyte-endothelial interaction promotes vascular occlusion in sickle cell anemia. T h e s e considerations p r o m p t e d us to determine whether sickle erythrocytes interfere with end o t h e l i u m - d e p e n d e n t vasorelaxation.
METHODS All salts and buffers were reagent grade and were obtained from Sigma Chemical Company (St. Louis, Mo). Human erythrocyte superoxide dismutase (3000 U/mg protein) and 8-bromoguanosine 3',5'-cyclic monophosphate (8-bromo-cGMP) were also from Sigma Chemical Company, as was acetylcholine chloride. Pharmaceutical grade nitroglycerin was obtained from Dupont Pharma-
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ceuticals (Wilmington, Del). Isoproterenol hydrochloric acid and norepinephrine bitartrate were from Sterling Drug (New York, N.Y.). All agonists were diluted to convenient working stock concentrations in muscle bath buffer (see below) on the day of the experiment.
Preparation of erythrocytes, ghosts, and oxyhemoglobin, Washed leukocyte-poor packed erythrocytes were prepared from adult volunteers. The patient population, method of obtaining blood samples, and preparation of washed erythrocytes have recently been describedF For these experiments, erythrocytes were packed to a hematocrit of 60 % to 80% and kept in an ice bucket in 12 × 75 ml plastic snap top tubes (Fisher Scientific, Pittsburgh, Pa.). The tops were kept loose to avoid an airtight seal. For preparation of hemolysate-free erythrocyte ghosts by hypotonic lysis, 361 volume of washed intact erythrocytes was added to 30 volumes of 3P8 buffer (3 mmol/L sodium phosphate buffer, 1 mmol/L ethylenediametetraacetic acid, and 100 mmol/L phenylmethylsulfonyl fluoride, pH 8.0). The 3P8 buffer was kept on ice for ghost preparation. After vigorous mixing, the preparation was centrifuged at 22,000 g for 15 minutes at 4 ° C. The supernate was carefully removed by aspiration, the tube was rotated horizontally, and the "cream pellet" at the bottom of the tube was removed by aspiration. The ghosts were resuspended in 30 volumes of ice-cold 3P8 buffer, mixed vigorously, and recentrifuged, and removal of the supernate and "cream pellet" was repeated. These steps were repeated until absorption of the supernate at 385,405, 560, 577, and 630 nm was nil. The white ghosts were kept on ice in 3P8 buffer at a ghost protein concentration of 1.2 to 1.5 mg/ml as determined colorimetrically37 during the course of each experiment. The heme content of AA and SS ghosts was less than 1.85 nmol/mg and 3.3 nmot/mg, respectively, as determined spectrophotometrically.17 Oxyhemoglobins A and S were prepared according to the method of Caughey and Watkins 3s as modified by Hebbel et al. 39 Briefly, washed buffy coat-poor erythrocytes were lysed in 5 volumes of distilled deionized water (DDH20) and centrifuged at 16,000 rpm in a Sorvall RC-5 centrifuge (DuPont Co., Wilmington, Del.) at 4 ° C for 15 minutes with an SS-34 rotor. The top 75 % of each tube was harvested and centrifuged at 35,000 rpm for 40 minutes in a Beckman L5-75 ultracentrifuge (Beckman Instruments, Inc., Fullerton, Calif.) with a SW 41 Ti rotor. The ghost-free lysate was then dialyzed overnight against 60 volumes of DDH20 (with three changes), with oxygen continuously bubbling through the dialysate, by means of Spectrapor membrane tubing of 6 to 8,000 molecular weight cut-off (Spectrum Medical Industries, Los Angeles, Calif.). The clear reddish solution was applied to diethylaminoethyl sepharose CL-6B in 5 mmol/L TRIS HC1, pH 8.6, at 5 ml/min at 4 ° C. The column was washed in the same buffer until absorption of the column effluent was 0 at 630, 577, and 560 nm. Hemoglobin was then eluted from the column with 10 mmol/L TRIS HC1, pH 7.1, containing 40 mmol/L sodium chloride. The column eluate was concentrated with Centriprep YM-10 membranes (Amicon, Danvers, Mass.) and applied to Sephadex G-100 in 5 mmol/L
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TRIS HC1, pH 8.1. The major hemoglobin peak was dialyzed against 5 mmol/L sodium phosphate buffer, pH 7.4, containing 0.15 mol/L NaC1. After membrane concentration as described previously, the preparations were determined to be >95% oxyhemoglobin (A or S) by analysis of absorption spectra at 700, 630, 577, and 560 nm according to Winterbourn. 4° Bioassay technique. Tranverse strips of descending thoracic aorta (2.5 mm wide) were obtained from anesthetized male New Zealand white rabbits (2 to 3 kg) and mounted isometrically in water-jacketed organ baths by means of small plastic clips. The lower clip was attached to a glass hook at the bottom of the bath with surgical silk suture. The upper clip was attached with a suture to an isometric force transducer (model FT 03, Grass Instrument Co., Quincy, Mass.). The bath buffer consisted of (in mmol/L): NaC1 118; KC1 4.8, CaC12 3.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 24, Ca-EDTA 0.026, and glucose 11.1. Temperature was maintained at 37 ° C by a recirculating water bath. A stream of 95 % oxygen/5 % carbon dioxide, continuously bubbling up from a scintered glass inlet at the bottom of the bath, maintained pH at 7.4 and pO2 at 500 torr. The strips were preloaded with 2 gm of isometric tension and permitted to relax and equilibrate for up to 1 hour before the experiments were begun. Isometric tension generated (positive or negative) in response to agonists was recorded continuously with a Grass model 5 PIB 3 amplifier and model 7 polygraph (Grass Instrument Co.). The arterial strips were precontracted with norepinephrine sufficient to generate half maximal tension (usually 10 -s to 5 x 10 -s mol/L as shown in the figures). After equilibration, acetylcholine (10 -s to 10 -6 mol/L) was added to the baths to determine the functional integrity of endothelium-dependent vasorelaxation, after which the bath buffer was rapidly drained, the tissue was washed three times with fresh bath buffer, and the experiments were performed. Vasoreactivity responses to agonists in the presence of erythrocytes, ghosts, or oxyhemoglobin were performed by incubating the tissue in the bath with erythrocytes (final hematocrit in the bath was 0.6% to 0.8% ), ghosts (11 to 15 ttg/ml of ghost protein in the baths), or oxyhemoglobin (10 -9 to 5 × 10 -6 mol/L in the bath) for at least 15 minutes. The arterial strips were then contracted with norepinephrine. When the contraction response reached a plateau, relaxation to agonists such as acetylcholine, nitroglycerin, isoproterenol, or 8-bromo-cGMP was assessed. Contraction and relaxation (in milligrams of tension) were read directly from a continuous polygraph recording. Vasoreactivity to acetylcholine was also monitored in the presence of ghosts prepared from erythrocytes pretreated with 2 mmol/L diisopropyl fluorophosphate to assure that inhibitory effects of ghosts (and erythrocytes) were not merely due to degradation of acetylcholine by cholinesterase. Visual inspection of the baths confirmed that erythrocytes and ghosts were maintained swirling in suspension throughout the entire volume of baths during experiments. The maintenance of erythrocytes and ghosts in suspension, and their continual swirling through the baths, was facilitated
AmericanHeartJournal
by the bubbling stream of gas from the bottom of the baths (see above). To control for the inhibition of endothelium-dependent vasorelaxation resulting from leakage of hemoglobin into the baths from lysed erythrocytes, the bath buffer was harvested with a large plastic pipette after experiments and centrifuged at 500 g for 10 minutes at 4 ° C. The oxyhemoglobin concentration in the buffer was then determined by analysis of absorption at 700, 630, 577, and 560 nm. 4° Statistical analysis. For each experiment the vascular strip served as its own control specimen. Therefore the effect of an individual treatment was analyzed by t test for independent samples if the control tests performed with the individual strips yielded comparable results. Statistical analysis was performed by means of the PC Statistician Human Systems Dynamics, Northridge, Calif. Graphic analysis was performed by means of Sigma Plot (Jandel Scientific, Sausolito, Calif.). Figures show means and standard errors of the various treatments represented. To normalize the results from similar experiments performed on separate days, the data for vasorelaxation are expressed relative to the tension generated with norepinephrine in each experiment. Thus a "percentage relaxation" refers to the percentage of norepinephrine-generated tension that is reversed with the use of a relaxing agonist in the control specimens or in the presence of erythrocytes, ghosts, or hemoglobin. In certain figures, relaxation in the presence of erythrocytes, ghosts, or hemoglobin is presented as the ratio of the percentage relaxation obtained in each experiment to the percentage relaxation obtained in the control specimen for the experiment. Hence a ratio of 1.0 indicates no inhibition of relaxation compared to the control value. RESULTS Inhibition of vasorelaxation. I n h i b i t i o n of vasorelax-
ation was achieved in vitro b y h u m a n hemoglobin, erythrocytes, a n d e r y t h r o c y t e ghosts. In free solution, h e m o g l o b i n of r a t or bovine origin inhibits vasorelaxation to acetylcholine in a p r e d i c t a b l e m a n n e r at m i c r o m o l a r concentrations. 3335 T o t a k e into acc o u n t the possibility t h a t , h e m o g l o b i n m i g h t be released into organ b a t h s as a result of m i n o r hemolysis during our e x p e r i m e n t s with erythrocytes, and t h a t this free h e m o g l o b i n m i g h t result in " a r t i f a c t u al" inhibition of vasorelaxation responses in our assays, we first d e t e r m i n e d the ability of h u m a n h e m o globins A a n d S to inhibit vasorelaxation to acetylcholine. As shown in Fig. 1, vasorelaxation to acetylcholine was unaffected b y 10 -7 m o l / L h u m a n h e m o g l o b i n b u t was c o m p l e t e l y inhibited at concent r a t i o n s greater t h a n 2 to 3 x 10 -6 mol/L. T h e c o n c e n t r a t i o n - d e p e n d e n t inhibition by hemoglobins S and A were similar, indicating t h a t neither of these h u m a n hemoglobins exerts an a priori superior inhibitory effect on vasorelaxation to acetylcholine. E x p e r i m e n t s were c o n d u c t e d in which vasorelax-
Volume 126, Number 2 American Heart Journal
Mosseri et al. 341
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[Hb02] (/~rnoles,/L) Fig. 1. Vasorelaxation to acetylcholine is inhibited by human hemoglobin and erythrocytes. Rabbit aortic strips were contracted with norepinephrine and then treated with acetylcholine to induce relaxation. Strips were then recontracted with norepinephrine in the presence of hemoglobin A (filled circles), hemoglobin S (filled triangles), AA erythrocytes (striped bar), or SS erythrocytes (cross-hatched bar), and relaxation to acetylcholine was observed again. Percentage relaxation of vascular strips under experimental conditions as compared with control conditions, for each individual strip, is shown as a ratio on the ordinate. Relax-. ation ratio of 1.0 indicates no inhibition (see Methods). Data points for hemoglobin represent at least five individual experiments, and bars for erythrocytes represent 19 experiments with S$ red blood cells (SSRBC) and 23 experiments with AA red blood cells (AARBC). Human hemoglobins A and S inhibited acetylcholine-induced vasorelaxation in a concentration-dependent manner. No inhibition was seen at 0.1 ttmol/L hemoglobin, and complete inhibition was seen at 2 to 3/~mol/L hemoglobin. Negative relaxation ratios at 5 #mol/L hemoglobin indicate that further vasoconstriction occurred in response to acetylcholine at that hemoglobin concentration. Bars for SS and AA erythrocytes are placed on the abscissa according to mean concentration of hemoglobin S or A (respectively) released into bath buffer during experiments (see text). Thus by comparison with hemoglobin curves, apparent inhibition of vasorelaxation by AA erythrocytes is easily attributed to hemoglobin A, which leaked into bath buffer during experiments. On the other hand, leakage of hemoglobin S into bath buffer cannot account for inhibition of vasorelaxation by SS erythrocytes. HbO2A, Oxyhemoglobin A; Hb02S, oxyhemoglobin S.
ation responses were d e t e r m i n e d with low concentrations (hematocrit 0.6% to 0.8%) of SS or AA erythrocytes in organ baths. B a t h buffer was periodically harvested at the end of the experiments, cleared of e r y t h r o c y t e suspension by centrifugation, and assayed for free hemoglobin concentration spect r o p h o t o m e t r i c a l l y to d e t e r m i n e whether sufficient hemolysis was occurring to account for the inhibition of vasorelaxation during the experiments. As shown in Fig. 1, vasorelaxation to acetylcholine in the presence of SS or AA erythrocytes was only approximately 17 % or 13 %, respectively, of vasorelaxation in the absence of erythrocytes. However, the average free hemoglobin concentration in the bath buffer was much greater in experiments with AA erythrocytes c o m p a r e d with SS erythrocytes (6.8 _+ 2.13 #mol/L 0.6_+ 0.27 #mol/L, AA vs SS; p = 0 . 0 1 5 , n = 7 ) . Hence, although the entire a p p a r e n t inhibitory effect
of AA erythrocytes could be explained on the basis of free hemoglobin release into the bath buffer, this did not account for the inhibitory effect of SS erythrocytes on vasorelaxation to acetylcholine. T h e data represented in Fig. 1 also suggest that, when corrected for free hemoglobin release, sickle erythrocytes inhibit acetylcholine-induced vasorelaxation by approximately 40 %. T o examine h u m a n erythrocyte inhibition of vasorelaxation to acetylcholine i n d e p e n d e n t of the hemoglobin in the bath buffer, ghosts were p r e p a r e d from SS and AA erythrocytes. These were washed thoroughly in lysis buffer (see Methods) and were used in vasorelaxation e x p e r i m e n t s in place of intact erythrocytes. As shown in Fig. 2, SS ghosts decreased vasorelaxation to 10 -s mol/L acetylcholine by 45 % (p = 0.006, n = 7) and decreased vasorelaxation to 5 × 10 -s mol/L acetylcholine by 27% (/9 = 0.001,
August 1993
342
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Mosseri et al.
3, B). These data suggest that the effects of SS erythrocytes and ghosts on acetylcholine-induced vasorelaxation is due to the inhibition of EDRF.
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Fig. 2. Sickle erythrocyte ghosts inhibit vasorelaxation to acetylcholine. Rabbit aortic strips were contracted with norepinephrine, and vasorelaxation was then induced with acetylcholine. Strips were recontracted with norepinephrine in the presence of SS ghosts (cross-hatched bars) or AA ghosts (striped bars). Vasorelaxation in the presence of ghosts was then induced with acetylcholine. As in Fig. 1, relaxation under experimental conditions is compared with relaxation under control conditions for each experiment as a relaxation ratio. Vertical bars for SS ghosts represent seven experiments each, and vertical bars for AA ghosts represent five experiments each. SS ghosts decreased vasorelaxation to acetylcholine by 45% at 10-s mol/L acetylcholine (p = 0.006) and by 27 % at 5 × 10-s mol/L acetylcholine (p = 0.001). AA ghosts did not inhibit vasorelaxation at either concentration of acetylcholine (relaxation ratio greater than 1.0 at both concentrations).
n = 7). In contrast, AA ghosts did not inhibit vasorelaxation at either concentration of acetylcholine. The oxyhemoglobin S concentration in the bath buffer in SS ghost experiments was less than 6 x 10 -s tool/L, not sufficient to inhibit acetylcholine-induced vasorelaxation (as shown in Fig. 1). These findings indicate that the inhibition of vasorelaxation seen with intact SS erythrocytes is not merely due to leakage of oxyhemoglobin S into the bath buffer and is not completely dependent on intracorpuscular oxyhemoglobin S. Acetylcholine induces vasorelaxation by stimulating synthesis and release of EDRF. s Superoxide dismutase enhances the in vitro and in vivo activity of E D R F by preventing its degradation by superoxide anions in solution. 3°, 31 As shown in Fig. 3, superoxide dismutase (1000 U/ml) decreased SS ghost inhibition of acetylcholine-induced vasorelaxation by 25 % (p = 0.025; Fig. 3, A) and reduced SS erythrocyte inhibition by approximately 50% (p < 0.01; Fig.
Vasorelaxation inhibited by SS erythrocytes and ghosts: Dependency on endothelium T h e preceding
experiments demonstrate that SS erythrocytes and ghosts inhibit endothelium-dependent vasorelaxation to acetylcholine. Additional experiments were performed to investigate whether endothelium-independent vasorelaxation is also inhibited. In Fig. 4, the effect of SS ghosts on vasorelaxation to acetylcholine (endothelium dependent) is compared with the effect of SS ghosts on vasorelaxation to nitroglycerin and to isoproterenol (both endothelium independent). Nitroglycerin directly generates nitric oxide and thus raises cyclic guanosine monophosphate (GMP) inside vascular smooth muscle cells, in effect acting pharmacologically similar to EDRF but bypassing the endothelium. Isoproterenol also works directly on vascular smooth muscle but a cyclic adenosine monophosphate p a t h w a y Y As shown in Fig. 4, A, the SS ghosts inhibited vasorelaxation to acetylcholine by 27% (p = 0.001), whereas relaxation to nitroglycerin was not significantly affected (7 % decrease, p > 0.5). Similarly, as shown in Fig. 4, B, concentration-dependent vasorelaxation to isoproterenol was the same in the presence and absence of SS ghosts. Analogous experiments performed in the presence and absence of SS erythrocytes demonstrated an 83 % decrease in vasorelaxation to acetylcholine, a 21% decrease in vasorelaxation to isoproterenol, and no decrease in vasorelaxation to nitroglycerin in the presence of SS erythrocytes. Additional evidence regarding the ability of vascular strips to respond to an endotheliumindependent rise in intracellular cyclic GMP was obtained using a non-hydrolyzable analogue, 8-bromocyclic GMP. As shown in Fig. 5, vasorelaxation to 8-bromocyclic GMP was unaffected by the presence of SS erythrocytes. Hence SS erythrocyte membranes and intact SS erythrocytes predominantly inhibit endothelium-dependent vasorelaxation. Furthermore, in the presence of SS erythrocytes and ghosts the guanylate cyclase pathway in vascular smooth muscle remains intact. However, the ability of the endothelium to activate this pathway by the stimulated release of EDRF is clearly impaired in the presence of SS erythrocytes or ghosts. DISCUSSION
The severity of vascular occlusive morbidity in sickle cell anemia has led investigators to consider that interaction between sickle erythrocytes and the endothelium may compromise the maintenance of
Volume 126, Number 2
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American Heart Journal
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Fig. 3. Inhibition of endothelium-dependent vasorelaxation by SS ghosts and erythrocytes is reversed by superoxide dismutase (SOD). Rabbit aortic strips were precontracted with norepinephrine and then relaxed with acetylcholine. Sequence of contraction and relaxation was repeated for each strip with either SS ghosts (A) or SS erythrocytes (R) in organ baths (see Methods). Sequence of contraction and relaxation was then repeated in the presence of SS ghosts or erythrocytes (A and B, respectively), but with the addition of SOD (1000 U/ml) in organ baths. Strips were washed three times with bath buffer between each sequence of contraction and relaxation. Percentage relaxation in control specimens and under respective experimental conditions is presented. Each bar represents mean results of two experiments. As shown in A, control strips relaxed 72.8 -+ 3.07% in response to acetylcholine, and relaxation in the presence of SS ghosts was only 40.5 + 1.5% (p = 0.004 vs control). However, vasorelaxation in the presence of SS ghosts was 51 _+ 0.99% when SOD was also in bath buffer (p = 0.025 vs ghosts alone). Aortic strips represented in B relaxed 97.3 + 2.7% under control conditions and only relaxed 36.3 _+ 1.3% when sickle erythrocytes were in organ baths (p < 0.01 vs control). With SOD and sickle erythrocytes in baths together, relaxation of the same strips was 62.2 __ 2.4% (p < 0.01 vs SS erythrocytes alone).
vascular patency. Sickle erythrocytes may directly occlude microvessels by adhering to endothelium41 or may directly alter endothelial functions such as prostacyclin production 16 and DNA synthesis. 17 Anatomic studies suggest frank endothelial damage in patients with sickle cell anemia, 3' 4, 42 and there is an increase in circulating endothelial cells in these patients during vasocclusive crises, 43 further suggesting endothelial damage during these events. Hebbel 6 has recentlY observed that the potential for altered endothelial regulation of vasoreactivity in the pathogenesis of vaso-occlusive events in sickle cell anemia remains unexplored. Observations of periodic oscillatory microvascular flow in patients with sickle cell anemia, by means of intravital microscopy44 and laser Doppler velocimetry,45 suggest that regulation of vasoreactivity in these patients is altered. In the present study we have employed a wellcharacterized bioassays to determine whether interaction between sickle erythrocytes and endothelium directly alters endothelial regulation of vasoreactiv-
ity by interfering specifically with EDRF, the major endothelial mediator of vasoreactivity.25 Sickle erythrocytes markedly inhibited vasorelaxation to acetylcholine, a relaxing agent that functions by stimulating elaboration of EDRF from the endothelium. 22-25 The inhibition was significantly diminished by superoxide dismutase, further indicating that the effect seen with sickle erythrocytes is mediated by EDRF. 3°-32 In addition, sickle erythrocytes did not directly inhibit endothelium-independent vasorelaxation mediated via cyclic GMP (nitroglycerin or 8-bromocyclic-GMP) or cyclic adenosine monophosphate (isoproterenol). Sickle erythrocyte ghosts also inhibited vasorelaxation to acetylcholine, indicating that these inhibitory effects result from interaction between the endothelium and the sickle erythrocyte membrane and are not totally due to the scavenging of EDRF by oxyhemoglobin S. Inhibition of vasorelaxation to acetylcholine was also observed when normal (AA) erythrocytes were in the organ baths. However, spectrophotometric anal-
344
August 1 9 9 3 American Heart Journal
Mosseri e~ al.
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[ISUpR,ul (moles/u) Fig. 4. Vasorelaxationresponsesinhibitedbysickleerythrocyte ghosts are endothelium dependent. Rabbit aortic strips were contracted with norepinephrine and then exposed to various vasorelaxant agonists. Each strip was then washed three times with bath buffer, sickle erythrocyte ghosts were added to organ baths, and the same sequence of contraction and vasorelaxation was repeated. A , VaSorelaxation to endothelium-dependent relaxing agent acetylcholine (ACH; 5 x 10-s mol/L) is compared with vasorelaxation to endothelium-independent relaxing agent nitroglycerin (NTG; 10-s tool/L). Relaxation to acetytcholine was 72,1 _+ 6.1% under control conditions and 52.3 _+ 6.1% with SS ghosts in baths (p = 0.001, n = 7). Relaxation to nitroglycerin was 77 _+ 6.3 % under control conditions and 71.1 + 2.9 % with SS ghosts in organ baths (p = 0.290, n = 5). B, Vasorelaxation to endothelium-independent relaxing agent (Isuprel), in the presence and absence of SS ghosts, is shown over isoproterenol concentrati0n range of 10-7 to 3 × 10-6 mol/L. No difference was obtained between control relaxation and relaxation with SS ghosts in organ baths. Each data point represents mean of five experiments.
ysis of s u p e r n a t a n t organ b a t h buffer revealed sufficient oxyhemoglobin A (but not S) to account for all of the a p p a r e n t inhibition of E D R F observed in exp e r i m e n t s with normal (but not sickle) e r y t h r o c y t e s despite the gross visual clarity of the buffer. This conclusion is consistent with the finding t h a t normal
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experiments.
e r y t h r o c y t e ghosts did not inhibit vasorelaxation to acetylcholine. In addition, normal h u m a n erythrocytes used at a 10 % h e m a t o c r i t have recently been found to not inhibit vasorelaxation to acetylcholine by canine femoral arteries in a flow system in which free hemoglobin did not accumulate in the b u f f e r s Our experiments were conducted with low concentrations (0.6% to 0.8% h e m a t o c r i t ) o f erythrocytes, representing a hemoglobin concentration of up to a p p r o x i m a t e l y 40 umol/L in the baths. H u m a n hemoglobin in free solution was found to completely inhibit vasorelaxation to acetylcholine at a concentration of 2 to 3/~mol/L. Despite the presence in the baths of more t h a n enough intracorpuscular hemoglobin to inhibit vasorelaxation completely, vasorelaxation was not 100 % inhibited by sickle erythrocytes. T h e s e findings suggest t h a t inhibition of E D R F by sickle erythrocytes is not simply on the basis of competitive binding of E D R F to the relatively large pool of intracorpuscular hemoglobin, b u t is also due to interaction between the sickle e r y t h r o c y t e membrane and the endothelium. T h e ability of sickle
Volume 126, Number 2 American Heart Journal
e r y t h r o c y t e g h o s t s to i n h i b i t E D R F - m e d i a t e d res p o n s e s s u p p o r t s t h i s c o n c l u s i o n . T h e a t t e n u a t i o n of t h e SS e r y t h r o c y t e effects b y s u p e r o x i d e d i s m u t a s e m a y suggest t h a t the i n h i b i t i o n involves the decomp o s i t i o n of E D R F ( n i t r i c oxide) b y excess o x i d a n t r a d i c a l s g e n e r a t e d b y t h e sickle e r y t h r o c y t e m e r e branes.39, 46 T h e d i s c r e p a n c y b e t w e e n t h e i n h i b i t o r y c a p a b i l i t y of i n t r a c o r p u s c u l a r h e m o g l o b i n a n d hem o g l o b i n i n free s o l u t i o n i n o u r s t u d i e s is c o n s i s t e n t w i t h r e c e n t s i m i l a r o b s e r v a t i o n s b y H o u s t o n et al. 27 T h e experimental conditions used in our studies, i n c l u d i n g low h e m a t o c r i t a n d t h e a b s e n c e of p l a s m a p r o t e i n s , w o u l d n o t b e e x p e c t e d to favor sickle e r y t h r o c y t e a d h e r e n c e to t h e e n d o t h e l i u m i n o r g a n bathsl~, 47 a n d are n o t p h y s i o l o g i c i n a clinical sense. H o w e v e r , sickle e r y t h r o c y t e s h a v e b e e n d e m o n s t r a t e d to i n t e r a c t w i t h v a s c u l a r e n d o t h e l i u m u n d e r p h y s i o l o g i c c o n d i t i o n s a n d i n t h e a b s e n c e of s t r o n g adherence:iT, 41, 47, 48 N i t r i c oxide ( E D R F ) is n o t o n l y t h e p r i n c i p a l e n d o t h e l i u m - d e p e n d e n t r e g u l a t o r of v a s o m o t o r t o n e 25 b u t m a y p l a y a s i g n i f i c a n t role i n t h e m o d u l a t i o n of p l a t e l e t a d h e s i o n a n d aggregat i o n 9, 19 a n d m a c r o p h a g e c y t o t o x i c activity. 49 T h e d y s r e g u l a t i o n of v a s o m o t o r , t h r o m b o t i c , a n d i n f l a m m a t o r y processes as a r e s u l t of i n t e r f e r e n c e w i t h n i tric o x i d e - d e p e n d e n t p a t h w a y s b y t h e sickle e r y t h r o c y t e m a y c o n t r i b u t e to t h e " s t o c h a s t i c ''6 n a t u r e of v a s o - o c c l u s i v e e v e n t s i n p a t i e n t s w i t h sickle cell a n e mia. We thank the Boston Sickle Cell Center and the hematology/ oncology nurses of St. Elizabeth's Hospital for help with procurement of blood specimens, and Patsy Bustos for preparation of the manuscript. REFERENCES
1. Diggs LW, Bruegge CFV. Vascular occlusive mechanisms in sickle cell disease. J Natl Med Assoc 1954;46:46-9. 2. Konotey-Ahulu FID. The sickle cell diseases: clinical manifestations including the "sickle crisis." Arch Intern Med 1974;133:611-9. 3. Boros L, Thomas C, Weiner WJ. Large cerebral vessel disease in sickle cell anemia. 3 Neurol Neurosurg Psychiatry 1976; 39:1236-9. 4. Merkel KHH, Ginsberg PL, Parker Jr JC, Post MJD. Cerebrovascular disease in sickle cell anemia: a clinical, pathological and radiological correlation. Stroke 1978;9:45-52. 5. Stockman JA, Nigro MA, Mishkin MM, Oski FA. Occlusion of large cerebral vessels in sickle-cell anemia. N Engl J Med 1972;287:846-9. 6. Hebbel RP. Beyond hemoglobin polymerization: the red blood cell membrane and sickle disease pathophysiology. Blood 1991;77:214-37. 7. Simionescu M, Simionescu N. Functions of the endothelial cell surface. In: Berne RM, ed. Annual review of physiology. Palo Alto: Annual Reviews Inc., 1986:279-93. 8. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288:373-6. 9. Bhardwaj R, Page CP, May GR, Moore PK. Endothelium-derived relaxing factor inhibits platelet aggregation in human
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