Cyclosporine treatment preserves coronary resistance artery function in rat cardiac allografts

CARDIAC REJECTION

Cyclosporine Treatment Preserves Coronary Resistance Artery Function in Rat Cardiac Allografts Farzad Moien-Afshari, MD,a Jonathan C. Choy, BS,b Bruce M. McManus, MD, PhD,b and Ismail Laher, PhDa Background: A marked decline in vascular myogenic response occurs during the course of rat cardiac allograft rejection. Two important contributory features are an inducible nitrous oxide synthase (iNOS)-catalyzed, NO-mediated vasodilation and a loss of smooth muscle function. In this study, we examine the effect of cyclosporine immunosuppressive therapy on the alleviation of arterial dysfunction of coronary resistance arteries in allografts using pressure myography. Methods: Rats receiving heterotopic abdominal cardiac transplantation were treated with cyclosporine (5 mg/kg), Cremophore or distilled water. Coronary septal arteries (internal diameter 200 ␮m) were dissected from isograft (Lewis to Lewis) and allograft (Fisher to Lewis) rat hearts at Day 21 post-transplantation and mounted on a pressure myograph. Pressure-induced vasoconstriction was measured before and after iNOS inhibition with aminoguanidine (AG; 100 ␮mol/liter). Both endothelium-based (AChinduced) and endothelium-independent (sodium nitroprusside–induced) vasorelaxation were also recorded in each group. Results: Pressure-induced myogenic contraction was reduced in allograft coronary arteries at Day 21 post-transplantation compared with matched isografts (p ⬍ 0.05). AG potentiated myogenic tone in allograft arteries, but had no effect on untreated Day 21 isograft vessels, indicating the presence of iNOS-based relaxation only in allograft vessels. Depolarization-induced vasoconstriction was lower in allograft compared with isograft arteries (p ⬍ 0.05). Cyclosporine therapy also improved depolarization-induced constriction in allograft vessels compared with untreated groups (p ⬍ 0.05). Furthermore, cyclosporine therapy preserved endothelium-based and endotheliumindependent vasorelaxation in allograft arteries at Day 21 post-transplantation. Conclusions: Cyclosporine immunosuppressive therapy has a significant effect on the alleviation of early endothelial and smooth muscle dysfunction in coronary allograft arteries. J Heart Lung Transplant 2004;23:193–203. From the aDepartment of Pharmacology and Therapeutics, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia; and bDepartment of Clinical Pathology, McDonald Research Laboratories, The iCAPTURE Centre, St. Paul’s Hospital, Vancouver, British Columbia, Canada. Submitted September 11, 2002; revised January 5, 2003; accepted February 8, 2003. Supported by grants from the Canadian Heart and Stroke Foundation of British Columbia and Yukon, (I.L., B.M.M.), fellowships from the UBC UGF Fund and Michael Smith Foundation (J.C.), and UBC UGF Fund (F.M-A.).

Reprint requests: Ismail Laher, PhD, Department of Pharmacology and Therapeutics, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. Telephone: 604-822-5882. Fax: 604-224-5142. E-mail: [email protected] Copyright © 2004 by the International Society for Heart and Lung Transplantation. 1053-2498/04/$–see front matter doi:10.1016/S1053-2498(03)00113-X

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ore than 2,000 North Americans benefit from cardiac transplantation each year1,2; however, longterm allograft survival is limited by transplant vascular disease (TVD), a severe and progressive form of atherosclerosis.3 Early TVD manifests as a malfunction of endothelial and arterial smooth muscle cells (SMC), so that changes in arterial function are apparent4,5 and can be monitored to assess the progression of TVD. It is assumed that these early arterial malfunctions are secondary to immune mechanisms and cytotoxic materials released by immune cells infiltrating the allograft tissue.6 The therapeutic goals for immunosuppressive therapy are to inhibit these secondary events of TVD mediated by immune cells. The mediators released by immune cells during the course of allograft rejection have a number of effects, including increasing the inducible isoform of nitric oxide synthase (iNOS) in vascular SMC.7,8 The activity of this sub-type of NOS is independent of intracellular calcium levels and is directly related to the amount of enzyme present in the cytoplasm.9 Therefore, once induced, iNOS can produce sufficient amounts of NO to cause sustained vasodilation of resistance arteries and increased coronary flow, which can lead to deterioration of coronary circulatory homeostasis and cause interstitial edema. The resulting edema decreases cardiac muscle compliance and contractile ability enough to cause ventricular failure.10 Increased levels of NO in arteries can also cause contractile dysfunction indirectly by decreasing the number of viable SMC through induction of apoptosis.11 The host’s immune system also damages SMC in allograft tissue directly. For example, neutrophils and T-cytotoxic lymphocytes in vitro can lyse myocytes or alter their function. Similarly, macrophages and T-helper lymphocytes infiltrating allograft interstitial space can produce cytokines that hamper SMC contractility.12 Cyclosporine (CsA) is an immunosuppressive agent currently used for prevention of allograft rejection. CsA inhibits the production of interleukin-2 and other lymphokines by T-helper lymphocytes. If not reduced in number, these lymphokines further activate Tcytotoxic and B lymphocytes and macrophages and together they can destroy allograft tissue.13 Furthermore, lymphokines are strong iNOS inducers, and therefore preventing their production decreases excessive NO production in transplant tissue.14 On the basis of this evidence, we hypothesized that treating heart allograft recipient rats with CsA would prevent early transplant vasculopathy by preventing tissue damage caused by host cell–mediated immunity. Our data indicate that CsA (5 mg/kg) reduces the

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effect of immune-mediated and iNOS-induced injury and possibly other damaging mechanisms on cardiac allograft vasculature in rats. Therefore, the net result of CsA therapy is preservation of vascular smooth muscle and endothelial cell function, which may in turn contribute to graft vascular integrity, thereby limiting long-term graft injury due to TVD.

MATERIALS AND METHODS Animal Groups All protocols were designed according to the guidelines of the animal care committee of the University of British Columbia. Male rats, weighing 200 to 250 g at the time of surgery, were used for transplantation. Isograft (Lewis to Lewis) and allograft (Lewis to Fisher) hearts were heterotopically transplanted by suturing the ascending aorta of donor hearts to the abdominal aorta and the pulmonary artery to the inferior vena cava of the recipient animals. Venous connections to the donor atria were ligated.15 Both isograft and allograft groups were then divided into 3 sub-groups according to the treatment schedule and received either (a) cyclosporine (CsA) or (b) CsA solvent/vehicle Cremophor or (c) no treatment (distilled water) for 14 days. CsA was administered subcutaneously (SC) at a dose of 5 mg/kg and the vehicletreated group received the same volume of solvent (Cremophor) as the CsA-treated group. The nontreated group was injected with distilled water (SC). Rats received heparin (50 U/kg, intraperitoneally [IP]) and were killed by an overdose of sodium pentobarbital (40 mg/kg, IP) at Day 21 post-surgery.

Morphologic Analysis Transverse sections of allograft and isograft hearts, untreated or treated with CsA, were fixed in formalin and embedded in paraffin, and stained with Verhoff’s elastin to visualize the internal and external arterial elastic laminae. Digital photographs of the arteries, with diameters between 50 and 200 ␮m, were taken using a Spot digital camera. The external (EEL) and internal elastic laminae (IEL) were outlined and their lengths measured using imageanalysis software (IMAGEPROPLUS [IPP]). Medial thickness was measured using the following formula: EEL/IEL ⫺ 1. Because an increase in medial thickness would result in a greater difference in the length of the EEL and IEL, this ratio provides an accurate assessment of the medial thickness.

Quantitation of In Situ TUNEL Terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining was

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performed using the protocol suggested by the manufacturer (Intergen, Purchase, NY), with some minor modifications, to detect DNA fragmentation suggestive of apoptosis. Formalin-fixed, paraffinembedded sections were de-paraffinized in xylene and re-hydrated in a series of ethanol washes. Sections were then incubated with 20 ␮g/ml proteinase K for 20 minutes, TdT enzyme for 1 hour and alkaline phosphatase– conjugated anti-digoxigenin for 30 minutes. Chromagen Vector Red (Vector Laboratories, Burlingame, CA) was used to visualize the staining and hematoxylin was used for counterstaining. At the end of alternate steps, washings with TBST were performed. IPP was then used, which is a color segmentation file that recognizes either TUNEL-positivity or nuclear staining with hematoxylin on the basis of hue, saturation and intensity. TUNEL-positive cells and total nuclei were then counted automatically in the medial layer by IPP, and results were expressed as the percentage of apoptotic cells in the media of the arteries studied.

Tissue Preparation and Diameter Measurement After animals were killed, donor hearts were isolated and dissected while maintained in ice-cold physiologic salt solution (PSS; see Chemicals and Buffers sub-section). Coronary septal arteries were located through a right ventricular wall opening and dissected and cleaned of adherent cardiac muscle tissue. For all functional studies, a 0.8- to 1.2-mm segment of the artery (inner diameter ⬇200 ␮m) at the level of the superior papillary muscle was excised and mounted at both ends onto glass cannulae in a pressure myograph chamber (Danish Myo Technology, Aarhus, Denmark). Both ends of the artery were tied using a single strand teased from 4-0 surgical silk thread and the chamber was placed on an inverted microscope stage to measure the arterial diameter. One of cannula was close-ended and the other connected through a pressure transducer to a peristaltic feedback pump to maintain constant pressure and also to monitor transmural pressure. The vessels were superfused continuously with bubbled (95% O2, 5% CO2) PSS (pH 7.35 to 7.4) at 37°C. Transmural pressure was then gradually increased to induce myogenic tone.

Myogenic and Depolarization-Induced Tone Pressure– constriction curves were determined in all artery segments. Intravascular pressure was gradually increased in a stepwise fashion to 80 mm Hg. Vessels that did not develop a leak were equilibrated for 1 hour at this pressure to develop myogenic tone (vessels

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that did not develop spontaneous constriction were excluded). After development of pressure-induced constriction, transmural pressure was then reduced to 10 mm Hg and increased in a stepwise manner at 5-minute intervals to 120 mm Hg. The internal diameter was recorded at each step and then compared with diameters occurring in calcium-free PSS at the end of the experiment to determine the degree of myogenic tone at each pressure. The artery was incubated for 1 hour with aminoguanidine (AG; 100 ␮mol/liter), and the protocol was repeated to determine the effect of Ca2⫹-independent production of NO on the myogenic response.4 Potassium chloride depolarization-induced constriction was also measured in arteries incubated with AG. These experiments were made at a low pressure (20 mm Hg) to eliminate pressure-induced vasoconstriction. A series of increasing concentrations of KCl solutions were prepared by mixing PSS with KCl (127 mmol/liter) solution (KCl; see Chemicals and Buffers sub-section). Finally, the vessel chamber was perfused with calcium-free PSS and vessel diameters were measured with stepwise increases in pressure to determine the passive diameter at each pressure. Myogenic and depolarization induced tones were then calculated (see Statistical Analysis and Calculations sub-section).

Vasodilation Induced by Acetylcholine and Sodium Nitropusside A separate segment of septal artery was used to determine acetylcholine (ACh) or sodium nitroprusside (SNP) concentration–response curves. After developing maximum myogenic tone (80 mm Hg), cumulative concentrations of ACh (10 nmol/liter to 10 ␮mol/liter) or SNP (10 nmol/liter to 1 ␮mol/liter) were added and the new steady-state internal diameters of the artery were registered.

Chemicals and Buffers All buffer reagents were purchased from BDH (Mississauga, Ontario, Canada) and drugs were purchased from Sigma (St. Louis, MO). The composition of PSS (millimoles per liter) was: NaCl, 119; KCl, 4.7; KH2PO4, 1.18; MgSO4, 1.17; NaHCO3, 24.9; ethylene-diamine tetraacetic acid (EDTA), 0.023; CaCl2, 1.6; and dextrose, 11.1. Isotonic substitutions (replacement of Na with equimolar concentrations of K) were used when using PSS solutions with increased K concentrations.

Statistical Analysis and Calculations Results are expressed as mean ⫾ SEM. Data were analyzed with either analysis of variance (ANOVA)

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and/or repeated-measures ANOVA with multiple comparisons using Bonferroni’s test when appropriate. The results of statistical tests were considered statistically significant at p ⬍ 0.05; n represents the number of rats. Myogenic tone was calculated as percentage of arterial constriction at each pressure step using the formula: % constriction ⫽ 100 ⫻ (DCa-free, P ⫺ Dp)/DCa-free, P, where DCa-free, P is the arterial diameter at pressure P in calcium-free PSS, and Dp is the diameter in PSS at pressure P. Depolarization-induced constriction was measured as: 100 ⫻ (DCa-free, 20 ⫺ D[KCl])/DCa-free, 20, where DCa-free, 20 is the arterial diameter in calcium-free PSS at the pressure of 20 mm Hg and D[KCl] is the vessel diameter at any concentration of potassium chloride. Percentage of relaxation/dilation of the arteries in response to ACh and SNP was calculated as: 100 ⫻ (D[Drug], 80 ⫺ D80)/(DCa-free, 80 ⫺ D80), where D[Drug], 80 is the diameter in PSS in the presence of a particular concentration of drug (ACh or SNP) at a pressure of 80 mm Hg, D80 is the diameter at PSS and pressure 80 mm Hg, and DCa-free, 80 is the passive diameter of vessel in calcium-free PSS at pressure 80 mm Hg.

RESULTS In all experimental groups, coronary arterial function of the transplanted heart in vehicle-treated recipient rats was the same as in untreated animals (data not shown).

Vascular Diameter and Distensibility The mean diameter for all septal arteries was 218 ⫾ 2.7 mm at the intraluminal pressure of 10 mm Hg in calcium-free PSS (n ⫽ 45). Pressure– diameter data in calcium-free PSS, indicating vascular compliance, was similar in all CsA-treated and untreated isograft and allograft arteries (not indicated).

Medial Thickness There were marked morphologic differences in the media of arteries from non-treated allograft hearts in comparison to allograft arteries from CsA-treated recipients and isograft hearts. The media of vessels from allograft hearts were thinner than their isograft counterparts (Figure 1A,B). Treatment of recipient rats with CsA completely preserved medial thickness in allograft arteries similar to isograft controls, suggesting that the observed morphologic changes were the result of the activated immune response (Figure 1A,B).

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TUNEL Staining There were a limited number of TUNEL-positive cells in coronary arteries of untreated allograft hearts and no TUNEL staining in vessels from isograft and CsA-treated allograft hearts. TUNEL staining was localized to cellular nuclei, and the number of TUNEL-positive cells in the media was very small. As assessed by quantitation of TUNELpositivity by IPP, there were significantly more TUNEL-positive cells in the medial layer of vessels from non-treated allograft hearts (n ⫽ 12) as compared with their isograft (n ⫽ 7) counterparts and animals treated with CsA only (n ⫽ 13) (Table I and Figure 2).

Effect of CsA Treatment on Myogenic Tone Figure 2 demonstrates the relationship between arterial diameters in response to increasing transmural pressure in an artery from a 21-day posttransplant isograft heart in comparison to an artery from a CsA-treated allograft and an untreated allograft recipient rat. Pressure-induced myogenic tone was lower in coronary arteries from untreated allograft recipient rats when compared with tone from non-treated isograft tissue (p ⬍ 0.05, n ⫽ 8 to 9; Figure 3A). CsA immunosuppressive therapy in graft recipients markedly improved myogenic tone in allograft vessels compared with untreated vessels (p ⬍ 0.05, n ⫽ 6 to 8; Figure 3A,B). As can be seen in Figure 3A,B, CsA abolished the differences in myogenic tone between arteries from allograft and isograft cardiac transplant rats. Coronary septal arteries of allografts in both CsA-treated and untreated recipients tended to have an increased myogenic tone in the presence of AG. This difference was statistically significant only in the latter group. In fact, vascular tone of untreated allograft recipients increased after incubation with AG, indicating that iNOS-produced NO may be responsible for part of the loss of tone in these arteries (Figure 3A,B).

Effect of CsA Therapy on Depolarization-Induced Tone by KCl Solution In untreated allograft recipients, there was decreased constriction of coronary septal arteries at high concentrations of K⫹ as compared with responses in isograft hearts (p ⬍ 0.05, [KCl] ⱖ66 mmol/liter, n ⫽ 8 or 9; Figure 4). CsA therapy of allograft recipient rats restored arterial constrictor responses to K⫹ to values similar to those in isograft rat hearts (n ⫽ 6 to 8; Figure 4). Treatment with

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FIGURE 1 (A) A comparison in medial thickness at Day 21 post-transplantation among the

coronary arteries of untreated or CsA-treated allograft and isograft hearts. Coronary artery medial thickness of allografts in untreated recipients was significantly lower than that of the other 3 groups (*p ⬍ 0.01, ANOVA), indicating the destruction of vascular SMC in the media of these arteries. (B) Verhoff’s elastin stain showing arterial internal and external elastic laminae. The media of untreated allograft artery was thinner than in the other 3 arteries. N, no treatment; C, CsA-treated; A, allograft; S, isograft; 21, Day 21 post-transplantation.

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TABLE I Numbers of TUNEL-positive cells (mean ⫾ SEM) in the medial layers of coronary septal arteries Section

N21A (n ⴝ 12)

C21A (n ⴝ 13)

N21S (n ⴝ 7)

C21S (n ⴝ 10)

TUNEL positivity

1.6 ⫾ 0.5*

0.0 ⫾ 0.0%

0.0 ⫾ 0.0%

0.0 ⫾ 0.0%

N, untreated; C, CsA-treated; A, allograft; S, isograft; 21, Day 21 post-transplantation. *p ⬍ 0.01.

CsA thus abolished the differences in response to K⫹ of arteries from isografts and allografts. Treatment of isograft animals did not change the constrictor response of coronary arteries to K⫹ (Figure 4). The reduced response to K⫹ (a non–receptormediated constriction) and diminished myogenic constriction to pressure suggest a possible decrease in the number of viable or functionally active smooth muscle cells, or that there may be a defect in

the common portion of the smooth muscle contraction pathway.4

Effect of CsA on Endothelium-Based and Endothelium-Independent Vasorelaxation Vasodilation to ACh occurs through endothelial production of NO. We evaluated the function of endothelial cells by comparing the effect of ACh in CsA-treated vs untreated allograft arteries. The

FIGURE 2 TUNEL staining of transplant arteries. A comparison in the number of

TUNEL-positive cells at Day 21 post-transplantation among the coronary arteries of untreated or CsA-treated hosts receiving allograft or isograft hearts. The arrow represents an apoptotic cell nucleus in the media of an allograft artery in an untreated recipient. N, no treatment; C, CsA-treated; A, allograft; S, isograft; 21, Day 21 post-transplantation.

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tion-induced tone in allograft septal coronary arteries compared when with age- and size-matched isograft vessels. CsA therapy prevented loss of tone in allograft arteries. (2) There was a significant decline in both endothelium-dependent and -independent vasodilation in arteries of untreated allograft hearts. Immunosuppression with CsA restored vasorelaxation in allograft arteries.

Inhibition of Myogenic Tone in Allograft Arteries and Effect of CsA Therapy FIGURE 3 Pressure– diameter traces from coronary

arteries harvested from CsA-treated and untreated allograft or isograft recipients. There was a profound inhibition of myogenic tone in untreated allograft arteries compared with untreated isograft vessels, which developed strong constriction in response to pressure. This tone reduction in allograft arteries was reflected by the large diameter increase as pressure increased. CsA treatment preserved the tone in allograft arteries. N, no treatment; C, CsA-treated; A, allograft; S, isograft; 21, Day 21 post-transplantation.

dilation to ACh was then compared with that of SNP (a direct NO-releasing agent) to determine whether there was also a smooth muscle component for the weaker response to ACh. The vasodilation induced by ACh was significantly greater in isograft arteries than in allograft vessels (p ⬍ 0.01), so that the maximal relaxation (at 5 ␮mol/liter ACh) was 83.4 ⫾ 5.2% vs 27.0 ⫾ 4.9%, respectively. Treating allograft recipients with CsA markedly improved vasodilation to ACh (p ⬍ 0.01, n ⫽ 6 to 8) and the maximum response was increased to 80 ⫾ 4.3%. CsA treatment did not alter the responses to ACh in isograft arteries (Figure 5). The magnitude of SNP-induced relaxation was significantly lower in non-treated allografts in comparison to responses in isografts (p ⬍ 0.01, n ⫽ 8 or 9). The maximal response to SNP (5 ␮mol/liter) in non-treated allografts was 41.3 ⫾ 5.5% (n ⫽ 8) compared with 75.7 ⫾ 4.4% (n ⫽ 9) in isografts (p ⬍ 0.05). Treatment with CsA improved vasorelaxation to SNP in allograft arteries (p ⬍ 0.01, n ⫽ 6 to 8), so that the maximum was increased to 67.6 ⫾ 8.2% (n ⫽ 6). The responses to SNP were unaffected by CsA treatment of isograft rats (Figure 6).

DISCUSSION In this study of 21-day-old post-cardiac transplant rats, we report the following: (1) There was a profound inhibition of myogenic and depolariza-

iNOS-induced NO and CsA. There was a significant decline in myogenic tone in allograft arteries when compared with isograft vessels. Reduced myogenic tone in allograft arteries was significantly improved by blocking iNOS with AG; however, the magnitude of tone in these arteries in the presence of AG was still lower than that of their isograft counterparts. This finding indicates that part of the weaker myogenic tone in arteries of rejected allografts could be due to an excessive production of iNOS-based NO. In this study we have demonstrated for the first time that when allograft recipient rats are treated with CsA (5 mg/kg), myogenic tone in graft arteries at Day 21 post-transplantation is preserved, similar to responses in isograft vessels. Moreover, we have shown that incubation with AG does not significantly increase the magnitude of myogenic tone in CsA-treated allograft arteries. Therefore, treating cardiac transplant recipients with CsA (5 mg/kg) prevents iNOS induction. Smooth muscle intrinsic contractile defect and CsA. Depolarization-induced constriction by KCl solution was also significantly lower in coronary arteries of allografts when compared with isografts. This weaker response to KCl could not be due to the effect of increased level of iNOS produced NO in the arterial wall, because these experiments were performed after iNOS blockade. Profound inhibition of depolarization-induced tone and myogenic tone, as discussed earlier, suggests that the SMC contractile defect is not limited to specific mechanisms responsible for initiation of myogenic tone, but that it also affects the common pathway involved in SMC constriction. Alternatively, the population of viable and functional SMC could be lower in rejected allograft arteries. The lower number of viable cells in arteries of rejected allografts could be the result of either cell destruction by host cellular and humoral immunity,12 or the effect of cytokine-induced NO-mediated apoptosis.11 The latter mechanism is related to the host immune system, but indirectly through the upregu-

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FIGURE 4 Myogenic tone in coronary arteries of CsA-treated or untreated recipients of

allograft or isograft hearts in the presence or absence of AG. (A) In the absence of AG, there was a significant decline in myogenic tone in untreated allograft arteries compared with isograft vessels (°p ⬍ 0.05, n ⫽ 8 or 9, repeated-measures ANOVA). Treating allograft vessels with AG improved the tone (*p ⬍ 0.05, repeated-measures ANOVA), indicating iNOS-based NO-mediated tone suppression in these arteries. (B) CsA treatment in a graft recipient had no effect on isograft arteries, but improved the myogenic tone reduction in allografts arteries compared with their untreated counterparts (**p ⬍ 0.05, n ⫽ 6, repeated-measures ANOVA), so that the response was not different from that of the isograft tissue. In both CsA-treated isograft and allograft arteries, incubation with AG potentiated the tone, but not significantly. C, CsA-treated; N, no treatment; 21, Day 21 post-transplantation; AG, aminoguanidine.

lation of iNOS by cytokines released from the attacking immune cells.16 CsA preservation of vascular SMC in allograft arteries. In accordance with the functional data, we observed that arteries of allograft hearts had a

thinner media compared with isograft arteries. This loss of medial thickness in allograft arteries was a direct result of immune-mediated damage, because immunosuppression with CsA could prevent this development, similar to the manner in which it can

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FIGURE 5 KCl depolarization-induced tone. There was a significant inhibition in KCl-

induced tone in untreated allograft arteries compared with isograft vessels (**p ⬍ 0.05, n ⫽ 8 or 9, repeated-measures ANOVA). CsA therapy markedly improved this tone reduction (*p ⬍ 0.05, n ⫽ 6 to 8 repeated-measures ANOVA). N, no treatment; C, CsAtreated; 21, Day 21 post-transplantation.

prevent medial and perivascular immune cell infiltration.17 In contrast to the results of the present study, Hamano et al demonstrated that, despite CsA therapy (10 mg/kg/day), intimal thickness in allograft arteries increased by vascular SMC proliferation, whereas medial thickness decreased due to SMC apoptosis and collagen atrophy at Days 30 and 60 post-transplantation.18 This contrast is probably attributable to harvesting the vessels at a later timepoint post-transplantation. In another study of a dog model of arterial transplantation, medial thickness at 3 months post-transplantation was well preserved in carotid allografts in recipients treated

with 25 mg/kg/day CsA. Thus, CsA at low doses (5 mg/kg/day) was shown to preserve allograft artery wall thickness in the early phases of transplantation19; however, over time (⬎8 weeks), even 10 mg/kg/day CsA was not effective in the prevention of graft arterial changes and higher doses of CsA (e.g., 25 mg/kg/day) were needed to prevent arterial medial changes. Finally, we found that the number of apoptotic cells in the medial layer of allograft arteries was significantly higher than in isograft arteries. CsA treatment in allograft recipients may inhibit programmed cell death in the graft arteries. However,

FIGURE 6 ACh-induced endothelium-dependent arterial relaxation. The response in

allograft arteries of untreated recipient rats was markedly lower compared with isograft vessels (**p ⬍ 0.01, n ⫽ 6 to 8, repeated-measures ANOVA). CsA therapy of the graft recipient rat had no effect on isograft tissue, but significantly improved response in the arteries of allograft hearts (*p ⬍ 0.01). N, no treatment; C, CsA-treated; 21, Day 21 post-transplantation.

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FIGURE 7 SNP-induced endothelium-independent vascular relaxation. The response of

the arteries in allograft hearts was significantly lower than that of isograft vessels (**p ⬍ 0.05, n ⫽ 8 or 9, repeated- measures ANOVA). The graphs also indicate that CsA therapy in allograft recipient rats had a significant effect on improving vascular relaxatory response to SNP when compared with untreated animals (*p ⬍ 0.05, n ⫽ 8 or 9, repeated-measures ANOVA). N, no treatment; C, CsA-treated; 21, Day 21 post-transplantation.

we examined the grafts for only 21 days and longterm results of CsA therapy on apoptosis in allograft arteries may be different. For example, in a similar model of cardiac allograft rejection, CsA therapy decreased apoptosis only in the early (ⱕ14 days) stages of post-transplantation, but at later timepoints (ⱖ28 days) the numbers of TUNEL-positive cells were similar in coronary arteries from CsAtreated and untreated recipients.20

Endothelial Cell Function and CsA The results of our study indicate that the endothelium-based vasodilatory response to ACh was significantly lower in untreated allograft coronary arteries. This finding is in accordance with previous studies demonstrating the presence of endothelial cell malfunction in allograft arteries that appears as an attenuated vasorelaxatory response to ACh.5,6 In fact, endothelial cell malfunction is the first event in the development of transplant vasculopathy, and therefore may be used to predict graft vasculopathy before the clinical end-point.5,6,21 Allograft arteries from CsA-treated recipient rats were able to dilate to ACh to the same extent as isograft vessels. ACh vasorelaxation is mediated by endothelial nitric oxide synthase (eNOS).22 Endothelium-released NO relaxes SMC by increasing the intracellular cyclic guanosine monophosphate (cGMP) content and opening of potassium channels. Moreover, there is an NO-independent relaxatory mechanism for ACh in resistance arteries through the release of endothelium-derived hyperpolarizing factor.23 Therefore, in our study, the weaker ACh-induced vasore-

laxation in allograft arteries could be due to a defect in endothelial cell activity or reduced SMC responsiveness to NO. Another possibility is that ACh induces vasoconstriction by direct stimulation of receptors on vascular SMC.24,25 In allograft tissue, ACh has the opportunity to affect the sub-endothelial smooth muscle cell layer directly and cause constriction. Electron micrographs and silver-staining methods have indicated some degree of endothelial cell denudation at Day 20 post-transplantation.6,26 This may cause a constrictor effect of ACh in allograft arteries, which would counter-balance its NO-mediated dilatory effects. Our results indicate that allograft arteries have a reduced response to cumulative doses of SNP. However, this defective response was relieved by CsA treatment, so that no difference in relaxatory response was observed between coronary arteries of CsA-treated allograft and untreated isograft recipients. SNP releases NO after its metabolization by arterial SMC.27 Because both ACh and SNP use common NO-mediated vasorelaxation pathways, the weak relaxatory response to both compounds in allograft heart arteries suggests a defect in the cGMP-mediated pathway of smooth muscle relaxation. However, we cannot conclude that there is a specific defect in endothelial cell production of NO in allograft arteries, because the magnitude of endothelium-based vasorelaxation (ACh-induced) was not significantly lower than the endothelium-independent (SNP-induced) dilation in these vessels.

The Journal of Heart and Lung Transplantation Volume 23, Number 2

Moien-Afshari et al.

Clinical Significance and Conclusions Poor myogenic tone in allograft-resistant arteries increases coronary blood flow and hydrostatic pressure surpasses oncotic pressure in end-arterioles. The net result of this pressure inequilibrium is the formation of cardiac interstitial edema. Furthermore, endothelial cell damage, which occurs in allograft arteries, increases permeability to plasma proteins and further aggravates edema.4 This generalized edema decreases ventricular compliance and leads to heart failure in the course of acute immune rejection of the graft.28 CsA, by preventing the loss of coronary artery myogenic tone and preserving endothelial cell integrity in an aortic allograft model,6 can prevent interstitial edema formation and, as a result, can delay or prevent acute transplant heart failure. In conclusion, our study indicates that CsA treatment in allograft recipients preserves SMC function and may also prevent endothelial cell dysfunction in the transplant coronary artery and, as a result, is expected to prevent ventricular failure secondary to myocardial edema, which retards the development of TVD. REFERENCES 1. Canada’s National Organ, and Tissue Information Site, Facts, and FAQs. http://www.hc-sc.gc.ca/english/organandtissue/facts_faqs/ (accessed 21 July 2002). 2. Transplant Patient Data Source (2000, February 16). Richmond, VA: United Network for Organ Sharing. http:// 207.239.150.13/tpd/ (accessed 21 July 2002). 3. Young JB. Allograft vasculopathy: diagnosing the nemesis of heart transplantation. Circulation 1999;100:458 –60. 4. Skarsgard PL, Wang X, McDonald P, et al. Profound inhibition of myogenic tone in rat cardiac allografts is due to eNOS- and iNOS-based nitric oxide and an intrinsic defect in vascular smooth muscle contraction. Circulation 2000;101: 1303–10. 5. Hollenberg SM, Klein LW, Parrillo JE, et al. Coronary endothelial dysfunction after heart transplantation predicts allograft vasculopathy and cardiac death. Circulation 2001; 104:3091–6. 6. Andriambeloson E, Pally C, Hengerer B, et al. Transplantation-induced endothelial dysfunction as studied in rat aorta allografts. Transplantation 2001;72:1881–9. 7. Yang X, Chowdhury N, Cai B, et al. Induction of myocardial nitric oxide synthase by cardiac allograft rejection. J Clin Invest 1994;94:714 –21. 8. Russell ME, Wallace AF, Wyner LR, Newell JB, Karnovsky MJ. Upregulation and modulation of inducible nitric oxide synthase in rat cardiac allografts with chronic rejection and transplant arteriosclerosis. Circulation 1995;92:457–64. 9. Loscalzo J, Welch G. Nitric oxide and its role in the cardiovascular system. Progr Cardiovasc Dis 1995;38:87–104. 10. Szabo G, Batkai S, Dengler TJ, et al. Systolic and diastolic properties and myocardial blood flow in the heterotopically

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