intravascular adenosine at reperfusion reduces infarct size and neutrophil adherence

Intravascular Adenosine at Reperfusion Reduces Infarct Size and Neutrophil Adherence James Todd, MD, Zhi-Qing Zhao, MD, PhD, Mark W. Williams, BS, Hiroki Sato, MD, PhD, David G. L. Van Wylen, PhD, and Jakob Vinten-Johansen, PhD Department of Cardiothoracic Surgery Research Laboratory, Bowman Gray School of Medicine, Winston-Salem, North Carolina

Background. Adenosine has been shown to reduce infarct size predominantly during reperfusion by adenosine A2-receptor-mediated processes. This cardioprotection may involve inhibition of events in the vascular compartment, such as adherence-independent and adherence-dependent actions of neutrophils. This study tested the hypothesis that adenosine exerts its cardioprotection during reperfusion by targeting effectors in the vascular compartment.

Methods. Polyadenylic acid (molecular weight, 230,000 daltons) was used as an intravascularly confined adenosine mimetic. In anesthetized New Zealand white rabbits, the left coronary artery was occluded for 30 minutes and reperfused for 120 minutes.

Results. Polyadenylic acid (1 mg/kg bolus, 0.5 mg • kg -1 • h -1) given 5 minutes before reperfusion significantly (p < 0.05) reduced infarct size compared with

vehicle (23% + 2% versus 37% ± 2% area at risk). The Al-antagonist KW-3902 had no effect on this polyadenylic acid-induced protection (17% + 3%), whereas the A1-A 2 antagonist sulfophenytheophyll ine blocked this infarct size reduction (41% ± 2%). In vitro adherence of platelet-activating factor-activated neutrophils to thoracic aortic endothelium was significantly diminished by polyadenylic acid (185 --- 12 neutrophils/mm 2 versus 36 + 4 neutrophils/mm 2 endothelial surface). Sulfophenytheo- phylline inhibited this effect (280 ± 6 neutrophils/mm2), whereas KW-3902 did not (31 ± 7 neutrophils/mm2).

Conclusions. An intravascular adenosine mimetic agent exerts cardioprotection during reperfusion by targeting receptor-mediated mechanisms in the intravascular compartment, possibly involving inhibition of neutrophil-related processes.

(Ann Thorac Surg 1996;62:1364-72)

A denosine (ADO) has been shown to have potent cardioprotective effects in nonlethal (stunning) and

lethal (necrosis) models of ischemia reperfusion [1, 2]. In surgical ischemia reperfusion, ADO as an adjunct to blood cardioplegia reversed global postischemic contractile dysfunction [3]. Adenosine-mediated cardioprotection induced by ischemic preconditioning [4] and exogenous ADO in models of nonlethal injury (ie, contractile dysfunction, stunning), have been shown to be mediated primarily through Al-receptor mechanisms [5, 6]. In this regard, ADO must be administered before or during ischemia to exert this protection, as administration during reperfusion has been without effect in a number of studies [2, 7]. In contrast, the predominant (75% of total) cardioprotection observed in models of irreversible injury (ie, infarction) may be exerted during reperfusion, and may be mediated by A2-receptor mechanisms [8, 9], whereas Al-mediated cardioprotection confers compara- tively little protection [10]. Key A2-mediated mechanisms are inhibition of neutrophil activation, superoxide generation, and adherence to the endothelium [11, 12]. The

Accepted for publication May 29, 1996.

Address reprint requests to Dr Vinten-Johansen, Department of Cardio- thoracic Surgery., Cardiothoracic Research Laboratory, Emory University• Crawford Long Hospital, 550 Peachtree St, NE, Atlanta, GA 30365-2225.

actions of neutrophils occur principally during the early moments of reperfusion [13, 14]. The similarity in chro- nology (ie, during reperfusion) between ADO-mediated cardioprotection and the intravascular activity of neutrophils would suggest that the vascular compartment, rather than the interstitial or myocyte compartments, may be primary targets for inhibition of neutrophil- mediated reperfusion injury by ADO. Adenosine is released into both the vascular and interstitial compartments.

This study tests the hypothesis that ADO exerts its cardiac protection during reperfusion by activating receptors within the intravascular compartment, resulting in an a t tenuat ion of po lymorphonuc lea r leukocyte (PMN) adherence and diminished myocardial necrosis in an i schemia / repe r fus ion model . Polyadenyl ic acid (Poly-A) was used in this study as a high molecular weight ADO mimetic that was confined to the intravascular space.

Material and M e t h o d s

In Vitro Studies The experimental procedures complied with the "Guid- ing Principles in the Use and Care of Animals" approved by the Council of the American Physiological Society as well as with state and federal regulations. The institu- tional Animal Care and Use Committee at Bowman Gray School of Medicine approved the experimental protocol.

© 1996 by The Society of Thoracic Surgeons 0003-4975/96]$15.00 Published by Elsevier Science Inc PII S0003-4975(96)00495-X

Ann Thorac Surg TODD ET AL 1365 1996;62:1364-72 INTRAVASCULAR ADENOSINE AND REPERFUSION INJURY

FEMORAL ARTERY RINGS. The direct vascular responses to Poly-A in comparison with ADO were determined in rabbit-isolated femoral artery rings using the organ bath technique. Rabbit femoral artery segments were carefully isolated, trimmed of adipose tissue, cut into 2-mm rings, and mounted on stainless steel hooks in a temperature- regulated organ chamber containing 37°C Krebs- Henseleit solution with the following composition (in mmol/L): NaC1, 118; KC1, 4.7; KH2PO 4, 1.2; MgSO4, 1.2; CaC12, 2.5; NaHCO3, 12.5; and glucose, 10. The Krebs- Henseleit solution was bubbled with 95% 02-5% CO 2 and pH was adjusted to 7.4. The vascular rings were connected to isometric force transducers, and changes in isometric force were digitized at 2 Hz using an analog to digital converter (Data Translation, Marlboro, MA). The rings were equilibrated for 1 hour at an optimal tension of 2 g, and precontracted with the thromboxane A 2 mimetic U46619 (Upjohn Pharmaceuticals, Inc, Kalama- zoo, MI). Once a stable contraction was observed, cumulative concentration-relaxation responses to ADO and Poly-A were obtained in the presence and the absence of the A1-A 2 antagonist sulfophenyltheophylline (SPT, 500 gmol/L) and the specific Al-receptor antagonist KW-3902 (50 gmol/L) [15]. The in vitro concentrations spanned the range of calculated plasma concentrations of the respective drugs as described previously [15]. Cumulative drug concentrations refer to final organ chamber concentrations. Responses to ADO and Poly-A are calculated as percent cumulative relaxation from the precontraction level.

NEUTROPHIL ISOLATION. Twenty-milliliter samples of pe- ripheral blood collected from donor rabbits were mixed with 3.0 mL of anticoagulating agents, which included 1.6% citric acid and 2.5% sodium citrate at pH 5.4, and 17 mL of 6% Hespan solution. Neutrophils were isolated as described previously [16]. Using this procedure, final suspensions contained more than 98% neutrophils (he- matoxylin/eosin staining) and cell viability was more than 99% as determined by trypan blue exclusion.

NEUTROPHIL ADHERENCE ASSAY. Alterations in the endothelial cell adherence component of neutrophil activity by Poly-A were assessed using neutrophils labeled with Zynaxis PKH-26 vital fluorescent dye (Zynaxis Cell Sci- ence, Inc, Malvern, PA) as described previously [17] for canine neutrophils. The labeling procedure yields cells possessing normal viability and function. Thoracic aorta rings 2 to 3 mm in length were carefully opened without disturbing the endothelium, and placed in 5-mL round cell culture dishes containing 3 mL of Krebs-Henseleit buffer at 37°C. The labeled PMNs (4×106 cells/mL) were randomly divided into the following groups: (1) unstimu- lated neutrophils (PMNs) alone; (2) PMNs stimulated with platelet-activating factor (PAF, 100 nmol; Biomol, Plymouth Meeting, PA); (3) PMNs, PAF (100 nmol), and Poly-A (1 nmol, 10 nmol, 100 nmol, and 1 /xmol in separate groups); (4) PMNs, PAF (100 nmol), Poly-A (100 nmol), and A1-A 2 antagonist SPT (100 /xmol/L, 200 ~zmol/L, 500/xmol/L, and 1 mmol/L in separate groups);

and (5) PMNs, PAF (100 nmol), Poly-A (100 nmol), and KW-3902 (1 /xmol/L, 5 /~mol/L, 10 ~mol/L, 50 /xmol/L). The PMNs were then allowed to incubate for 20 minutes. After incubation, coronary segments were removed and dipped in fresh Krebs-Henseleit solution three times with the endothelium side up to wash off nonadherent PMNs. Adherence was determined by the number of PMNs adhering to the endothelial surface in six separate fields of each arterial segment under epifluorescence microscopy (490 nm excitation, 504 nm emission) using a calibrated grid to measure surface area of the field. The adherent PMNs were summed for each segment and are expressed as numbers of PMN/mm 2 of segment.

In Vivo Studies

SURGICAL PREPARATION OF ANIMALS. Male New Zealand White rabbits weighing 4 to 5 kg were anesthetized with an intramuscular injection of ketamine HC1 (35 mg/kg) and xylazine (6 mg/kg). The right femoral artery and vein were cannulated for intravenous administration and arterial pressure monitoring. A continuous infusion of anesthesia (25 mg/mL of ketamine and 25 mg/mL of xylazine) was administered throughout the experiment by a Harvard infusion pump at 1.0 mL/h through a double-lumen femoral vein catheter. The trachea was exposed through a midline incision in the ventral neck and intubated. The rabbit was ventilated with oxygen- enriched room air to maintain arterial oxygen tension more than 100 mm Hg. Arterial carbon dioxide tension was maintained near 35 mm Hg, and acidemia was corrected with sodium bicarbonate as needed. The heart was exposed through a median sternotomy, and the pericardium was opened. A 4-0 Prolene suture with a tapered needle (Ethicon Somerville, NJ) was passed around a branch of the left coronary artery, and the ends of the sutures were passed through a short polyvinyl (PE90) tube to form a snare. A bolus of sodium heparin (300 U/kg) was given intravenously and the rabbit was allowed to stabilize hemodynamically for 10 to 20 minutes.

PREPARATION AND DOSAGE OF DRUGS. T h e A D O A1-A 2- receptor antagonist 8-p-sulfophenyltheophylline (SPT; Research Biochemical, Inc, Natick, MA) and the large ADO moiety, Poly-A (molecular weight >200,000 [aver- age for lot used = 230,000] Sigma, St. Louis, MO) were prepared by mixing the powder with 5 mL of 0.9% saline solution at 37°C just before injection. Each Poly-A mole- cule has one ADO moiety that can interact with ADO receptors. Poly-A was given as an initial bolus of I mg/kg over 5 minutes followed by an infusion of 0.5 mg• kg -1 • h 1. To select a dosage of Poly-A, we compared its effects with ADO on segments of vascular rings as well as the in vivo hemodynamic changes induced in the dose- response study. A dose of 20 mg/kg body weight SPT was used to block ADO receptors. This dose of SPT was sufficient to completely abolish the in vivo hypotensive effects of Poly-A. The A1 selective ADO receptor antagonist, 8-(3-noradamantyl)-l,3-dipropylxanthine (KW- 3902, Kyowa Hakko Kogyo, Japan), was dissolved in

1366 TODD ET AL Ann Thorac Surg INTRAVASCULAR ADENOSINE AND REPERFUSION INJURY 1996;62:1364-72

100 /zL of ethyl alcohol and 50 /~L of 1.0 N NaOH, and then di luted with 2 mL of 0.9% saline solution at 37°C to achieve a final concentrat ion of 2 mg/mL. The amount of KW-3902 used (50 /~mol/L, approximat ing the in vivo p lasma concentrat ion of 1 mg/kg) was selected from an earl ier s tudy where this amount complete ly blocked the negative inotropic effects of R-PIA (a p redomina te ly A 1 agonist) up to concentrat ions of 100/zmol[L in both an in vivo and in vitro model [15]. The ethyl a l coho l -NaOH vehicle used with KW-3902 had no effect on infarct size compared with saline solution.

Experimental Protocol After a 10- to 20-minute postsurgical stabil ization period, animals were r andomized to one of four groups: (1) saline vehicle (n = 14): rabbi ts received saline infusion 5 minutes before reperfusion; (2) Poly-A t rea tment (n = 15): a subhypotens ive dose of Poly-A (1 mg/kg bolus over 5 minutes, then 0.5 m g - k g 1 h 1) was infused intravenously 5 minutes before coronary reperfus ion and dis- cont inued 60 minutes after initiation of reperfusion; (3) Poly-A t rea tment plus SPT blockade (SPTPA, n = 10): SPT (20 mg/kg bolus) was infused 2 minutes before init iation of Poly-A treatment; (4) Poly-A t rea tment plus Al - recep tor b lockade (KWPA, n - 8): KW-3902 (1 mg/kg bolus) was infused 2 minutes before init iation of Poly-A treatment . A subhypotens ive dose of Poly-A was determined in a pilot s tudy using mean arterial pressure as the p r imary end-point .

In all groups, s teady-s ta te hemodynamic data were acquired at basel ine as well as before and after infusion of SPT and Poly-A to de te rmine any drug effects on hemodynamic variables (heart rate [HR], mean arterial pressure [MAP], and systolic b lood pressure). The left coronary ar tery was then reversibly occluded by t ighten- ing the snare to produce a zone of regional ischemia in the left ventricle. Ischemia was confirmed visually by cyanosis and dyskinesis in the area at risk. Previous b lood flow analysis wi th rad io labe led mic rospheres showed that regional coronary artery occlusion p roduced a 98% reduct ion in b lood flow to the area at risk in the rabbit [8]. Hemodynamic data were again collected after 25 (predrug) and 30 minutes (postdrug) of ischemia. After 30 minutes of coronary occlusion, the left coronary artery snare was re leased to allow reperfusion for a total of 120 minutes. Hemodynamic data were measured at 15, 30, 60, and 120 minutes of reperfusion.

Determination of Area at Risk and Infarct Size Upon complet ion of 120 minutes of reperfusion, the coronary artery snare was re t ightened and 4 to 6 mL of 20% Unisperse Blue dye (Ciba-Geigy, Summit, NJ) was injected into the left main atr ium through a 25-gauge needle. This procedure s ta ined the normal ly per fused area and thereby demarca ted the area at risk by dye exclusion. After the blue dye had circulated sufficiently to stain the per fused myocard ium homogeneously , 2 mg of sod ium pentobarbi ta l was injected into the left a tr ium and al lowed to circulate briefly, and the hear t was rapid ly excised. The left ventricle was isolated from the rest of

the hear t and was cut into t ransverse slices approxi- mate ly 2 m m thick. The normal area (stained blue) was separa ted from the area at risk. The area at risk was then placed in a 37°C solution of 1% t r iphenyl te t razol ium chloride (Sigma Chemical , St. Louis, MO) for 10 minutes. The t r iphenyl te t razol ium chlor ide-s ta ined (noninfarcted) tissue was separa ted from the pale (necrotic) t issue and each respective area was weighed. The area at risk was calculated as the sum of the noninfarcted and necrotic weight of the t issue per fused by the occluded vessel, d iv ided by the weight of the left ventricle and expressed as a percentage. The area of necrosis was calculated as the weight of necrotic tissue d iv ided by the weight of the left ventricle and expressed as a percentage. Area of necrosis /area at risk was calculated by dividing the weight of the necrotic tissue by the weight of the total area at risk.

Data Acquisition Femoral arterial pressures were digi t ized at 250 Hz using a 12-bit analog to digital converter (Data Translation, Marlboro, MA) and s tored on hard disk using a video- graphics p rogram deve loped in our laboratory. Pressure waveforms were visually displayed, and dysrhythmic beats were excluded. The HR, peak systolic pressure , end-diastol ic pressure , and MAP were averaged from no less than 15 beats. Pressure- ra te product , used as an index of myocardia l oxygen demand, was calculated as the product of HR and peak systolic pressure .

Microdialysis Studies Intersti t ial pur ines were measu red in a separa te series of exper iments by microdialysis [18] as descr ibed previously [19]. The microdialysis probe was implan ted into the myocardia l area at risk to a midmyocard ia l depth and immed ia t e ly connec ted to a gas- t ight glass syr inge moun ted on a microprocessor-control led precis ion sy- ringe pump (Pump 22; Harvard Appara tus , South Natick, MA). The dialysis cannula was cont inuously per fused with deaera ted Krebs-Hensele i t buffer at a rate of 2.0 /zL/min. An addi t ional dialysis catheter was placed so that the dialysis membrane was posi t ioned in the cavity of the right ventricle to s imul taneous ly compare right ventr icular chamber (mixed venous) b lood pur ine levels, including the re turn from the coronary sinus. The dialysis catheters were al lowed to stabilize for 70 minutes before the exper imenta l protocol was begun. At the end of the exper iment , the heart was dissected to verify the posi t ion of the dialysis probes. Purine levels were measured in three of the groups as previously defined: saline solution (n - 6), Poly-A (n = 6), and SPTPA (n = 6) where n = rabbi t used in these microdialysis experiments .

Dialysate samples were assayed for ADO, inosine, hypoxanthine, and xanthine by high performance l iquid chromatography, using a Supelcosil LC-18S column (Su- pelcosil, Inc., Bellefonte, PA) and a 1% (pH 5.3) to 25% (pH 5.58) methanol in 100 mmol /L KH2PO 4 gradient . Purine nucleosides were de t e rmined using external stan-

Ann Thorac Surg TODD ET AL 1367 1996;62:1364-72 INTRAVASCULAR ADENOSINE AND REPERFUSION INJURY

| --@-- Control * I00~- [ ] KWPA T

l a SPTPA ~ | + PA //A /I

0.001 0.01 0.1 1 10 100

Concentration (pM) Fig 1. Concentrah'on-relaxah'on response curves to incremental concentrah'ons of adenosine (ADO) or Poly-A (PA) in isolated rabbit femoral artery rings precontracted with U46619 (10 nmol/L). Re- sponses are expressed as percent relaxation from precontracted tension. Values are mean ÷ standard error of the mean. (Control - response in untreated rings; KWPA - KW-3902 pretreatment [50 tzmol/L] before Poly-A incubation; SPTPA - SPT pretreatment [500 txmol/L] before Poly-A incubation; *p < 0.05 versus control and SPTPA; +p < 0.05 versus control.)

dards to quantify the compounds of interest. Assays were per formed by Dr Van Wylen at the Depar tmen t of Biology, St. Olaf College, Northfield, Minnesota.

Criteria f o r Exclus ion

Standard exclusion criteria were (1) poor ischemia observed as lack of cyanosis and dyskinesia and /o r poor reperfus ion by lack of hyperemia or appearance of cya- notic banding, (2) poor demarca t ion of the area at risk dur ing Unisperse blue staining, (3) ventr icular fibrillation that did not convert spontaneous ly within 30 seconds or after three cardioversion shocks, (4) failure to complete the entire protocol, (5) extremely large (more than 50% area at risk producing hemodynamic instabili ty) or small (less than 15% area at risk) areas at risk, and (6) misplace- men t of the microdialysis catheter in the left ventr icular free wall or cavity of the right ventricle in the microdialysis studies.

Statist ical A n a l y s i s

Analysis of variance for repea ted measures was used to de te rmine if t ime- and group- re la ted differences oc- curred in the hemodynamic and pur ine data. If significant g roup/ t ime interactions were found, Duncan ' s mul- t iple range test was appl ied to locate the sources of differences. One-way analysis of variance was used to analyze overall group differences in infarct size variables followed by Duncan ' s post hoc test to de te rmine group differences. A p value of less than 0.05 was accepted as statistically different. Results are repor ted as mean and s tandard error of the mean.

Resul ts

Vascular R i n g Data

Vasodilator responses to Poly-A and ADO in isolated femoral ar tery rings are summar i zed in Figure 1. Poly-A relaxed the vascular r ings in a cumulat ive concentrat ion- dependen t manner . Comple te relaxation was achieved at 1.11 /~mol/L Poly-A and the concentrat ion of Poly-A requ i red to achieve 50% of maximal relaxation (ECso) was 0.11/~mol/L. Maximum relaxation was achieved at lower doses of Poly-A compared with ADO, the lat ter of which showed a r ight shift in the concent ra t ion- response curve relative to Poly-A. SPT (approximat ing the in vivo concentrat ions used in the study) significantly inhibi ted the max imum vasodi la tor response of Poly-A, with the maximum relaxation averaging 39% ± 5% at 1.1 ~mol /L Poly-A (p < 0.05% versus ADO). KW-3902 at a concentrat ion that also approx imated the calculated p lasma concentrat ion (50/~mol/L), d id not antagonize the vasodi lator effect of Poly-A, thereby inferr ing an A2-media ted vasodi la tor response to Poly-A.

Neu t roph i l A d h e r e n c e to Vascular E n d o t h e l i u m

When uns t imula ted PMNs were coincubated with aortic segments ( represent ing nonspecific adherence) , very little adherence was observed on the endothel ia l surface (Fig 2). However , s t imulat ion of the PMNs with PAF resul ted in a significant fourfold increase in adherence. Poly-A t rea tment resul ted in a dose -dependen t a t tenua- tion of PMN adherence to the endo the l ium al though even the lowest dose (1 nmol/L) resul ted in 80% at tenu- ation of adherence of PAF-s t imula ted PMNs relative to the PMN + PAF group (Fig 2, top). A 1 antagonism with KW-3902 had no effect on the a t t enua ted PMN adherence in Poly-A-t rea ted segments (Fig 2, middle) . SPT reversed the inhibi tory effect of Poly-A in a dose -dependen t manner up to 500 ~mol /L (Fig 2, bottom). Concentra t ion of SPT (1 mmol/L) d id not resul t in any further increase in adherence compared with 500 /~mol/L. Both the 500 /~mol/L and the 1 mmol /L SPTPA groups showed a significant increase not only over uns t imula ted PMNs but also over PAF-stimulated PMNs (PMN + PAF), suggest ing inhibi t ion of basal ly re leased endogenous ADO.

H e m o d y n a m i c s

The results repor ted in this section of the s tudy represen t data for 44 rabbits from a total of 55 comple ted experiments: 12 in the control group, 14 in the Poly-A group, 10 in the SPTPA group, and 8 in the KWPA group. Four exper iments were excluded because of failure to complete the protocol, one for an excessively small area at risk and three for failure to reperfuse after snare release. In addit ion, dialysis data from three rabbits were ex- c luded due to poor catheter p lacement .

Hemodynamic data in all groups are summar i zed in Table 1. H e m o d y n a m i c va r iab les were c ompa rab l e among the groups at basel ine and at 25 minutes of ischemia before any drugs were adminis tered . However, in all groups HR significantly increased dur ing this t ime (p < 0.05). The HR rema ined relat ively constant dur ing


250

200 E E 150

z 100 13.

5O

250

PMN

+ + -k

PMN PA PA PA PA + (lnM) (10nM) (100nM) (IuM)

PAF = P M N + P A F I

200 (Xl

E 150

100 Z

50

400

PMN

"k "k "k "k

PMN KW KW KW KW + (lpM) (Spa) (10pa) (50pM)

PAF ~---'- PMN + PAF + PA (100nM)-~l

300 E

"~ 200 Z

o.. 100

PMN PMN SPT SPT SPT SPT + (100pM)(200pM) (500pM) (1raM)

PAF ~-- PMN + PAF + PA (100nM)~

Fig 2. Neutrophil (PMN) adherence to coronary endothelium, repre- sented by the number of adherent PMN/mm 2 of coronary endothelium. (Top) Adherent population of PMN with incremental concentrations of Poly-A (PA). (Middle) The effect of incremental concentrations orAl-antagonist KW-3902 (KW) on Poly-A-treated (100 t~mol/L) activated PMNs. (Bottom) The ~:ffect of incremental concentrations of SPT in the presence qf 100 I~mol/L Poly-A. (*p < 0.05 versus PMN ~ platelet-activating factor [PAF].)

reperfus ion in all groups. There was a tendency for HR to decrease (p > 0.05) after 15 minutes of reperfusion. After 120 minutes of reperfusion, HR was significantly lower in the Poly-A group compared with the SPTPA group. There were no differences at any t ime per iod in the other groups. There were no differences in the MAP among groups at basel ine or through the end of ischemia. The bolus of Poly-A just before reperfusion significantly low- ered MAP, which r ema ined at this lower level for the r ema inde r of the exper iment , whereas the MAP of the remain ing groups showed more of a gradual decline dur ing reperfusion. By the end of reperfusion, MAP was comparab le among groups. The p ressu re - ra t e product at basel ine and dur ing ischemia was similar among the groups. Poly-A infusion resul ted in a significantly lower p res su re - ra t e product than SPTPA, but not when com- pa red with any of the other groups. This difference was no longer present by the end of reperfus ion as the p ressu re - ra t e product of the SPTPA group progress ively

diminished. No significant difference existed be tween Poly-A and the vehicle group at any t ime point, a l though p ressu re - ra t e product in the Poly-A group t ended to be lower. Changes in hemodynamic pa ramete r s were not predict ive of group differences in infarct size.

Myocard ia l Infarct Size

The masses of the left ventricle, the area at r i sk and the area of necrosis in grams are summar ized in Table 2. Left ventr icular mass was similar in all groups with the exception of the KWPA group in which the mass was slightly, but significantly, less than in the other groups. The area p laced at risk by coronary occlusion ranged be tween 36.4% ± 3.1% and 29.4% ± 2.0% of the left vent r icular mass; there was no statistical difference among groups in the area at risk whe ther expressed as absolute mass (Table 2) or as a percentage of the left ventr icular mass (Fig 3, top). When necrosis was ex- p ressed as absolute mass, infarct size was significantly lower in the Poly-A and KWPA groups compared with vehicle, whereas SPT reversed the infarct size reduct ion observed in ei ther group receiving Poly-A (Fig 3, middle). Al though the SPT-treated group t ended to have a h igher area of necrosis than the vehicle, this did not reach significance.

The area of necrosis, expressed as a percentage of the area at risk is shown in Figure 3, bottom. Poly-A treatment significantly reduced infarct size compared with vehicle. This reduct ion in myocard ia l necrosis with Poly-A was unaffected by Al - recep tor blockade. SPT t rea tment complete ly reversed the observed protect ion with Poly-A. Al though the area of necrosis /area at risk was slightly higher for the SPT group compared with control, this did not reach significance.

Microdialys is Data

Dialysate values repor ted are absolute concentrat ions in the dialysate not corrected for efficiency of recovery. Previous work in our labora tory using a flow rate of 2 /~L/min demons t ra t ed a 28.6% recovery of ADO, 26.6% recovery of inosine, 42.3% recovery of hypoxanthine, and 36.8% recovery of xanthine. Transmural p lacement of the microdialysis probe was consistent among the three groups in which the technique was used (vehicle, Poly-A, and SPTPA), averaging 47.5% ± 4.8% of epicardial - to- endocardia l distance for Poly-A, 49.0% ± 6.0% for vehicle, and 51.0% _+ 4.0% for SPTPA. Figure 4 shows pur ine concentrat ions in the interst i t ium of the area at risk dur ing baseline, ischemia, and reperfusion per iods for the three groups. In the vehicle group interst i t ial ADO levels significantly increased from basel ine dur ing ischemia, and by 30 minutes of reperfus ion had re tu rned to basel ine values. There were no significant group differences dur ing ischemia as drugs were not given until reperfusion. Poly-A infusion at reperfus ion did not significantly alter the ADO recovered from the inters t i t ium at any t ime point compared with vehicle. Al though the SPTPA group had a somewhat lower total amount of dialysate ADO, this was never significantly different from either of the other two groups. Similar t empora l and

Ann Thorac Surg TODD ET AL 1369 1996;62:1364-72 IN~fRAVASCULAR ADENOSINE AND REPERFUSION INJURY

Table 1. H e m o d y n a m i c Variables Before and D u r i n g Coronary Occlus ion and Reper fus ion

Index Control I25 R15 R60 R120

HR (bea t s /min)

Vehicle 151 ÷ 8 174 ± 8 ~ 171 + 7 174 + 7 174 ÷ 7

PA 148 ÷ 6 163 ± 7 ~ 169 + 5 161 ÷ 4 159 + 4

KWPA 151 ÷ 5 165 _+ 8 '~ 168 ± 7 167 + 9 165 + 8

SPTPA 168 ÷ 9 180 + 7 "~ 180 + 8 184 + 7 L1 185 + 8 b

MAP (ram Hg)

Vehic le 75 ÷ 3 66 ± 3 ~ 63 _+ 3 61 + 4 59 _+ 3

PA 75 _- 3 68 + 2 ~ 58 + 2 ~ 61 ± 2 61 -- 2

KWPA 84 _- 3 73 + 2 a 72 + 1 ~" 71 + 3* 67 _+ 4

SPTPA 79 - 5 72 + 3 ~ 71 + 3 ~ 62 + 3 ~' 58 ± 3

PRP (ram H g / m i n × 1,000)

Vehicle 14.6 z 0.5 14.5 -- 0.7 14.0 + 0.7 13.8 " 0.7 ~ 13.5 +- 0.9

PA 14.6 ÷ 0.5 14.1 ÷ 0.6 12.9 + 0.5 ~ 12.6 + 0.4 12.4 ÷ 0.5

KWPA 15.0 " 0.5 15.1 -- 0.6 15.7 + 0.5 b 13.4 + 0.5 ~'~" 13.4 ± 0.4

SPTPA 16.1 " 0.7 16.2 -~ 0.8 16.1 ± 0.8 b'~ 14.7 -+ 0.8 ~'b 13.6 - 0.8 '~

Values are mean _+ standard error of the mean. .1 p < 0.05 versus previous value; b p < 0.05 versus PA; ~ p < 0.05 versus vehicle.

HR = heart rate; I25 = ischemia 25 minutes into occlusion; KWPA = KW-3902 pretreatment (50 ~mol/L) before Poly-A incubation; MAP = mean arterial pressure; PA = Poly-A; PRP = pressure-rate product; SPTPA = SPT pretreatment (500 ktmol/L) before Poly-A incubation.

g r o u p p r o f i l e s w e r e o b s e r v e d for h y p o x a n t h i n e , i n o s i n e ,

a n d t o t a l p u r i n e s . T i m e - r e l a t e d c h a n g e s w e r e n o t o b -

s e r v e d fo r x a n t h i n e o r u r i c ac id . T h e p u r i n e c o n c e n t r a -

t i o n s m e a s u r e d i n t h e p l a s m a (F ig 5) w e r e g e n e r a l l y

l o w e r t h a n t h o s e s e e n i n t h e i n t e r s t i t i u m a t b a s e l i n e a n d

e n d of r e p e r f u s i o n (F ig 4), e s p e c i a l l y fo r A D O , h y p o x a n -

t h i n e , i n o s i n e , a n d t o t a l p u r i n e s . A l t h o u g h t h e p l a s m a

A D O c o n c e n t r a t i o n s r e m a i n e d r e l a t i v e l y s t a b l e t h r o u g h -

o u t i s c h e m i a a n d r e p e r f u s i o n , t h e r e w a s a s i g n i f i c a n t

e l e v a t i o n in t h e p l a s m a c o n c e n t r a t i o n m e a s u r e d a t 120

m i n u t e s of r e p e r f u s i o n in e a c h g r o u p (Fig 5). H o w e v e r ,

n e i t h e r P o l y - A i n f u s i o n n o r SPT t r e a t m e n t a l t e r e d t h e

p l a s m a A D O c o n c e n t r a t i o n s c o m p a r e d w i t h v e h i c l e .

G r o u p o r t i m e - r e l a t e d d i f f e r e n c e s w e r e n o t o b s e r v e d i n

t h e o t h e r v a r i a b l e s .

C o m m e n t

I n t h e p r e s e n t s t u d y , P o l y - A , a h i g h m o l e c u l a r w e i g h t

A D O c o n g e n e r , w a s u s e d to a c t i v a t e A D O r e c e p t o r s

s p e c i f i c a l l y i n t h e i n t r a v a s c u l a r c o m p a r t m e n t . P o l y - A ,

w h e n g i v e n a t t h e s t a r t of r e p e r f u s i o n , s i g n i f i c a n t l y

r e d u c e d i n f a r c t s i z e b y 41% c o m p a r e d w i t h v e h i c l e i n d e -

p e n d e n t of l i d o c a i n e ; t h e d o s e of P o l y - A u s e d w a s i n t e n -

Table 2. M y o c a r d i a l Tissue W e i g h t s (g)

Area Vehicle PA SPTPA KWPA

LV 3.61 + 0.09 3.97 + 0.15 3.84 _- 0.09 3.56 _- 0.11 ~

AR 1.07 ± 0.09 1.30 + 0.11 1.31 - 0.06 1.31 _- 0.13

AN 0.44 ÷ 0.05 0.31 + 0.04 b 0.54 _~ 0.05 a 0.24 - 0.06 b

Values are mean _+ standard error of the mean. .1 p < 0.05 versus PA and KWPA; b p < 0.05 versus vehicle.

AN - area of necrosis: AR area at risk; KWPA KW-3902 pretreatment (50 /~mol/L) before Poly-A incubation; LV left ventricle; PA - Poly-A; SPTPA SPT pretreatment (500 ~mol/L) before Poly-A incubation.

t i o n a l l y c h o s e n to c a u s e m i n i m u m h y p o t e n s i o n ( l e s s t h a n

10 m m H g ) to a v o i d t h e c o n f o u n d i n g e f fec t of d e c r e a s e d

c a r d i a c w o r k l o a d . T h i s i n f a r c t s i z e r e d u c t i o n w a s r e -

v e r s e d b y a n o n s e l e c t i v e A 1-A 2 r e c e p t o r a n t a g o n i s t (SPT),

b u t w a s u n o p p o s e d b y t h e A l - a n t a g o n i s t , i n f e r r i n g a n

A 2 - m e d i a t e d m e c h a n i s m . I n f a r c t s i z e s o b s e r v e d w i t h

t h e s e a n t a g o n i s t s g i v e n i n c o m b i n a t i o n w i t h P o l y - A w e r e

s i m i l a r to t h o s e o b s e r v e d in p r e v i o u s s t u d i e s i n w h i c h t h e

a n t a g o n i s t s w e r e g i v e n a l o n e ( w i t h o u t P o l y - A ) [8, 10].

6O

so • --., 40

30

• ='/ 20

o

2O

lO ..J

e- 5

o

VEH PA SPTPA KWPA

VEH PA SPTPA KWPA

6O so 40

30 20

c=

0 VEH PA SPTPA KWPA

Fig ,3. The size of the area at risk relative to the left ventricle (Ar/ LV, top) and infarct size data expressed as a percent of the left ventricular mass (An/LV, middle) or as the area of necrosis (An/Ar, bottom). Columns represent group means + standard error of the mean. (KWPA - KW-3902 pretreatment (50 ktmol/L) before Poly-A incubation; *p < 0.05 versus vehicle (VEH) and SIYE pretreatment [500 I~mol/L] before Poly-A incubation [SPTPA].)


Fig 4. Time-related profiles of interstitial dialysate concentrations of purines from the left ventricular area at risk. Time is shown in minutes of A . ischemia (ISCH) or reperfusion (REP). Values are mean +_ standard 1 5

error of the mean. (CNTL = control ~ 1 2 concentrations after 60 minutes of .~ 0.9 stabilization; *p < 0.05 Poly-A [PA] and SI~ pretreatment (500 tzmol/L) ~ 06 before Poly-A incubation /SPTPA] *~ versus vehicle.)

o3, 0 . 0 , T , T : -- "I ~

CNTL 10 25 10 50 120

F - ,scH I ,REP I

C.

a .

100

8O v = 60

'~o 40

2 0

0

CNTL

o V e h i c l e

T PA S P T P A

A _ _ .

10 25 10 50 120

} -~ ISCH I REP t

D.

6 0 -

'-- 40

20

0

CNTL i i i i i

10 25 10 50 120

I -~ ISCH I REP I

E.

5

3

,~. 2

1

0 CNTL

i i i i i

10 25 10 50 120

I ~ ,SCH I REP [

m x

F.

150

120

D.

m O

0.6 -

0.4

0.2

I 0 . 0 , , , ,

;NTL 10 25 10 50 120

~--" ISCH REP J

9°¢K__ 6 O

3O

0 , i CNTL 10 25 10 50 120

~'~ ISCH I REP I

Time (minutes)

Finally, Poly-A inhibited PMN adherence to the vascular endothelium by an A2-mediated mechanism. Taken to- gether, these data suggest that the cardioprotective actions of ADO during reperfusion are exerted in large part in the intravascular compartment, and may involve inhibition of neutrophil adherence and the subsequent neutrophil-related pathologic cascade of injury. In addition, these data further support the overall hypothesis that ADO exerts cardioprotection by A2-receptor-mediated mechanisms, perhaps through neutrophil inhibition, in models of irreversible injury.

Poly-A has a molecular weight of more than 230,000 daltons, and contains a single vasoactive moiety at its 3' end. Using a macrornolecular ADO congener avoids the spillover of ADO into the interstitial compartment. The ADO mimetic actions of Poly-A were confirmed in the present study by dose-dependent relaxation of femoral artery in agreement with a study by Schrader and col-

leagues [20]. This vasodilation was antagonized by SPT, but not by the Al-specific antagonist KW-3902, consistent with the known A2-mediated vasodilation by ADO. The interstitial and plasma microdialysis data from the present study confirm that Poly-A was not degraded to ADO in vivo, and did not alter the release pattern of ADO during ischemia and reperfusion, and is consistent with earlier reports demonstrating that Poly-A remains in the vascular space. However, the large molecular weight of Poly-A prevented sampling of the macromol- ecule by dialysis, particularly in the area at r isk to rule out transendothelial migration in this area of microvas- cular damage. In addition, the probe measuring mixed venous plasma purines may have underestimated ADO levels in the coronary microcirculation because of rapid dearnination.

Infusion of Poly-A 5 minutes before reperfusion caused a small but significant decrease in mean arterial

Ann Thorac Surg TODD ET AL 1371 1996;62:1364-72 1NTRAVASCULAR ADENOSINE AND REPERFUSION INJURY

&.

0.20

0.15

0.10

0.05

0.00 CNTL 20 15 120

I - - ISCH I R E P - - t

B.

0.4

0.3"

c¢ 0 ,2

-- 0.1

0.0

o V e h i c l e

- - o - - P A

s, S P T P A

i [ i CNTL 20 15 120

I---- ISCH I REP I

Fig 5. Time-related profiles qf plasma dialysate concentrations of purines from within the right ven- tHcular lumen as an estimate of coronary sinus and mixed venous concentrations. Time is shown in minutes of ischemia (ISCH) or repe~usion (REP). Values are mean ++_ standard error qf the mean. (CNTL = control levels after 60 minutes qf stabilization; *p < 0.05 versus previous value in each group [there were no significant differences among groups].)

C.

A 2.0

.~ 1.5

~ 1.0

o o. 0.5

0.0

CNTL 20 15 120

~'~ ISCH I R E P - - t

D, 1.0

i " o.s

0.6 ,E

0.4

x 0.2

0.0 CNTL 20 15 120

I ~ ISCH I R E P - - I

E l l

8

,~ 4

2

o

CNTL 20 15 120

I - - ISCH I R E P - - t

F.

3

= 2

n

I--

0

CNTL 20 15 120

~ " ISCH I REP I

Time (minutes)

pressure (approximately 10 mm Hg), which was not seen in the group cotreated with Poly-A and the antagonist SPT. However, hemodynamic variables, including the pressure-rate product were comparable in all groups during ischemia. This suggests that the severity of ischemia may have been similar among groups, barring differences in collateral blood flow. However, our previous studies demonstrate little variability in collateral blood flow in rabbits, and collateral blood flow shows an insensitivity to antagonists of endogenously released ADO (ie, SPT) [8, 15, 21]. Although the pressure-rate product was significantly less in the Poly-A-treated group at 15 minutes of reperfusion, there has been no correlation between levels of pressure-rate product or its components during reperfusion and infarct size as there has been for these variables during ischemia. Therefore, it is highly unlikely that a decrease in the pressure-rate

product during reperfusion would contribute to infarct size reduction in the Poly-A group.

Neutrophils play an important role in ischemic reperfusion injury in the myocardium [22]. Adenosine inhibits superoxide anion production directly by activated neutrophils [12, 23, 24], and inhibits both adherence to endothel ium [11, 24] and endothelial adherence- dependent superoxide anion production [24]. This inhibition of neutrophil function has been attributed to ADO A2-receptor-mediated processes [24]. In agreement with these observations, Poly-A reduced PAF-stimulated adherence to coronary artery endothelium by receptor- mediated processes. The lack of antagonism to ADO's inhibition of PMN adherence by a specific Al-antagonist (at doses that inhibited catecholamine-stimulated posi- tive inotropy in papillary muscle preparations [15]) suggests principally an A2-mediated effect. Therefore,


Poly-A had properties similar to those of ADO regarding inhibit ion of neutrophil adherence to coronary vascular.

In summary, in light of the confinement of Poly-A to the intravascular compartment, and the lack of change in interstitial ADO concentrations accompanying t reatment with Poly-A, the data suggest that the reduction of infarct size by the macromolecular ADO congener may be due to its effects in the intravascular compartment. Further- more, blockade of this protection by SPT suggests receptor-mediated effects, specifically of the A 2 subtype. Direct confirmation of this inference is hampered by the lack of a water-soluble A2-antagonist suitable for in vivo use. This study adds further support to the concept that ADO exerts infarct size-reducing effects by inhibit ion of receptor-mediated events during the early reperfusion period. This cardioprotection may involve inhibi t ion of neutrophils and possibly the vascular endothelium. Further- more, this study highlights the vascular compartment as a pr imary site of these cardioprotective mechanisms of ADO. In vivo, the vascular endothel ium is uniquely positioned to play a pivotal role in this protection in the vascular compar tment by being a significant source of ADO [25], as well as being an efficient barrier in the transport of ADO between vascular and interstitial compartments. The concentration of ADO achieved at the margins of the blood laminar flow pattern (ie, the un- stirred layer) or in the microenvironment created by closely juxtaposed neutrophils and endothelial cells is unknown, but may exceed measured plasma or interstitial values. More important, pharmacologic augmenta- tion of endothelially derived ADO by regulating agents or other modulators of ADO concentrations may represent an important therapeutic approach in targeting nonsurgical and surgical reperfusion injury in a compartment that participates dramatically in its genesis and perpetration. The direct clinical application of Poly-A may be limited by its relatively potent vasodilator effect and its prolonged half-life compared with ADO.

We express our thanks to Sharon Ireland and Martha Oldland for preparation of the manuscript, to Akira Karasawa, PhD, from Kyowa Hakko Kogyo Co, Ltd, for the generous gift of KW-3902, and to Ciba-Geigy (Summit, NJ) for the gift of CGS-21680.

References

1. Mullane K, Bullough D. Harnessing an endogenous cardioprotective mechanism: cellular sources and sites of action of adenosine. J Mol Cell Cardiol 1994;27:1041-54.

2. Lasley RD, Mentzer RM Jr. Protective effects of adenosine in the reversibly injured heart. Ann Thorac Surg 1995;60:843-6.

3. Hudspeth DA, Nakanishi K, Vinten-Johansen J, et al. Aden- osine in blood cardioplegia prevents postischemic dysfunction in ischemically injured hearts. Ann Thorac Surg 1994;58: 1637-44.

4. Liu GS, Thornton J, Van Winkle DM, Stanley AW, Olsson RA, Downey JM. Protection against infarction afforded by preconditioning is mediated by A1 adenosine receptors in rabbit hearts. Circulation 1991;84:350-6.

5. Lasley RD, Rhee JW, Van Wylen DGL, Mentzer RM Jr. Adenosine A1 receptor mediated protection of the globally ischemic isolated rat heart. J Mol Cell Cardiol 1990;22:39-47.

6. Mentzer RM Jr, Bunger R, Lasley RD. Adenosine enhanced preservation of myocardial function and energetics. Possible involvement of the adenosine A1 receptor system. Cardio- vasc Res 1993;27:28-35.

7. Randhawa MPS Jr, Lasley RD, Mentzer RM Jr. Salutary effects of exogenous adenosine administration on in vivo myocardial stunning. J Thorac Cardiovasc Surg 1995;110: 63-74.

8. Zhao Z-Q, McGee DS, Nakanishi K, et al. Receptor- mediated cardioprotective effects of endogenous adenosine are exerted primarily during reperfusion after coronary occlusion in the rabbit. Circulation 1993;88:709-19.

9. Zhao Z-Q, Nakanishi K, Vinten-Johansen J. Al-receptor- mediated infarct size reduction by endogenous adenosine is exerted primarily during myocardial ischemia. FASEB J 1993;7:A419.

10. Vinten-Johansen J, Zhao Z-Q. Cardioprotection from iseh- ernic-reperfusion injury by adenosine. In: Abd-Elfattah AS, Wechsler AS, eds. Purines and mycardial protection. Boston: Kluwer Academic Publishers, 1995:315-44.

11. Cronstein BN, Levin RI, Belanoff J, Weissrnann G, Hirsch- horn R. Adenosine: an endogenous inhibitor of neutrophil- mediated injury to endothelial cells. J Clin Invest 1986;78: 760-70.

12. Cronstein BN, Kramer SB, Weissmann G, Hirschhorn R. Adenosine: a physiological modulator of superoxide anion generation by human neutrophils. J Exp Med 1983;158: 1160-77.

13. Dreyer WJ, Michael LH, West MW, et al. Neutrophil accu- mulation in ischernic canine myocardium: insights into time course, distribution, and mechanism of localization during early reperfusion. Circulation 1991;84:400-11.

14. Lefer AM, Tsao PS, Lefer DJ, Ma X. Role of endothelial dysfunction in the pathogenesis of reperfusion injury after myocardial ischemia. FASEB J 1991;5:2029-34.

15. Zhao Z-Q, Nakanishi K, McGee DS, Tan P, Vinten-Johansen J. Al-receptor mediated myocardial infarct size reduction by endogenous adenosine is exerted primarily during isch- aemia. Cardiovasc Res 1994;28:270-9.

16. Doerschuk CM, Allard MF, Hogg JC. Neutrophil kinetics in rabbits during infusion of zyrnosan-activated plasma. J Appl Physiol 1989;67:88-95.

17. Sato H, Zhao Z-Q, Vinten-Johansen J. L-arginine inhibits neutrophil adherence and coronary artery dysfunction. Car- diovasc Res 1996;31:63-72.

18. Van Wylen DGL, Willis J, Sodhi J, Weiss RJ, Lasley RD, Mentzer RM Jr. Cardiac microdialysis to estimate interstitial adenosine and coronary blood flow. Am J Physiol 1990;258: H1642-9.

19. Zhao Z-Q, Williams MW, Sato H, et al. Acadesine reduces myocardial infarct size by an adenosine mediated mechanism. Cardiovasc Res 1995;29:495-505.

20. Schrader J, Nees S, Gerlach E. Evidence for a cell surface adenosine receptor on coronary myocytes and atrial muscle cells: studies with an adenosine derivative of high molecular weight. Pflug Archiv 1977;369:251-7.

21. Toombs CF, McGee DS, Johnston WE, Vinten-Johansen J. Myocardial protective effects of adenosine. Infarct size reduction with pretreatrnent and continued receptor stimula- tion during ischemia. Circulation 1992;86:986-94.

22. Mehta JL, Nichols WW, Mehta P. Neutrophils as potential participants in acute myocardial ischemia: relevance to reperfusion. J Am Coll Cardiol 1988;11:1309-16.

23. Gunther GR, Herring MB. Inhibition of neutrophil superoxide production by adenosine released from vascular endothelial cells. Ann Vasc Surg 1991;5:325-30.

24. Zhao Z-Q, Sato H, Vinten-Johansen J. Adenosine A 2- receptor activation inhibits neutrophil-mediated injury to coronary endothelium by attenuating superoxide radical generation and adherence. Am J Physiol (in press)..

25. Srnolenski RT, Kochan Z, McDouall R, Page C, Seymour AL, Yacoub MH. Endothelial nucleotide catabolism and adenosine production. Cardiovasc Res 1994;28:100-4.

intravascular adenosine at reperfusion reduces infarct size and neutrophil adherence

Documents