Simvastatin decreases myocardial tumor necrosis factor α content in heart transplant recipients

CLINICAL HEART TRANSPLANTATION

Simvastatin Decreases Myocardial Tumor Necrosis Factor ␣ Content in Heart Transplant Recipients Cynthia K. Wallace, MSPH,e Sonny J. Stetson, BS,a,c,d,e Seref A. Ku ¨ c¸u ¨ ker, MD,b Katy A. Becker, BS,a,c,d,e a,c a,c,d,e John A. Farmer, MD, Susan C. McRee, RN, BSN, Michael M. Koerner, MD, PhD,a,c,d,e b George P. Noon, MD, and Guillermo Torre-Amione, MD, PhDa,c,d,e Background: Statins improve patient survival and decrease rejection episodes in heart transplant recipients. We studied the effects of simvastatin treatment on myocardial tumor necrosis factor ␣ (TNF-␣) expression; TNF-␣ is a potent pro-inflammatory cytokine associated with hypertrophy and fibrosis in heart transplant recipients. Methods: We randomized 10 consecutive heart transplant recipients to receive either 20 mg/day simvastatin (n ⫽ 5) or placebo (n ⫽ 5) for 6 months after cardiac transplantation. Routine surveillance endomyocardial biopsy specimens were obtained from all patients. We analyzed tissues for myocardial TNF-␣ content, total collagen content, and myocyte size using semiquantitative immunohistochemistry. Results: Myocyte size and total collagen content of placebo and simvastatin groups did not show a statistically significant difference at any biopsy time point. Myocardium TNF-␣ content (% tissue area stained) at 1 week after transplantation was similar in the simvastatin and placebo groups. At the 24th week after transplantation, when compared with Week 1 values, we found a significant decrease in myocardium TNF-␣ content in the simvastatin group (15.0% ⫾ 2.3% vs 5.8% ⫾ 2.4%, p ⫽ 0.02) that was not observed in the placebo group (15.0% ⫾ 1.5% vs 12.0% ⫾ 2.6%, p ⫽ not significant). Conclusion: Simvastatin treatment in heart transplant recipients decreased myocardium TNF-␣ expression. This decrease did not translate into a difference in the markers of hypertrophy. However, decreased myocardial TNF-␣ may be a marker of a general statin-mediated decrease in inflammation in the transplanted heart that leads to improved graft and patient survival. J Heart Lung Transplant 2005; 24:46 –51. Copyright © 2005 by the International Society for Heart and Lung Transplantation.

Treatment with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) decreases plasma low-density lipoprotein (LDL) while increasing high-density lipoprotein (HDL). The use of statins decreases mortality and rejection episodes in cardiac transplant recipients;1–3 however, the mechanisms responsible for the beneficial effects in heart transplant recipients are not known. An increasing number of reports indicate that the effects of statins are achieved not only through direct decrease in atherosclerosis secondary to LDL cholesterol decrease, but also

From the Methodist DeBakey Heart Center, aThe M. E. DeBakey Department of Surgery, Division of Transplantation and Assist Devices, bMedicine (Section of Cardiology), cGene and Judy Campbell Lab for Cardiac Transplant Research, dWinters Center for Heart Failure Research, eBaylor College of Medicine, Houston, Texas. Reprint requests: Dr. Guillermo Torre-Amione, Baylor College of Medicine, 6550 Fannin, Suite 1901, Houston, Texas 77030. Telephone: 713-798-1404. Fax: 713-798-8744. E-mail: gtorre@ bcm.tmc.edu. Copyright © 2005 by the International Society for Heart and Lung Transplantation. 1053-2498/05/$–see front matter. doi:10.1016/ j.healun.2003.09.037

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through modulation of the immune response through down-regulation of intermediate products of the cholesterol biosynthetic pathway. For example, mevalonate, the product of HMG-CoA reduction, is necessary for lymphocyte proliferation;4,5 in vitro and in vivo evidence suggests that statins consequently may inhibit lymphocyte proliferation and functionality.6,7 Consistent with the hypothesis that statin therapy results in down-regulation of the inflammatory response, Calabresi et al8 have shown that increased HDL concentration may exert a direct cardioprotective effect through inflammatory modulation as manifested by dose-dependent decreases in cardiac tumor necrosis ␣ (TNF-␣).8 Studies of carotid plaques in patients treated with simvastatin showed significantly fewer macrophages, T lymphocytes, and HLA-DR⫹ cells than in untreated controls.9 In heart transplant recipients, treatment with pravastatin is associated with a decrease in natural-killer cell cytotoxicity,1 and a decrease in circulating concentrations of TNF-␣, a decrease that reversed when pravastatin therapy was discontinued.10 Myocardial TNF-␣ is a pro-inflammatory cytokine that, when overexpressed, may lead to cardiomyopa-

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thy, left ventricular hypertrophy, and decreased cardiac contractility.11 Tumor necrosis factor ␣ is not present in normal myocardium; however, in failing myocardium, TNF-␣ expression increases and the receptors for TNF-␣ are down-regulated.12 Furthermore, circulating concentrations of TNF-␣ also are increased in patients with heart failure, and the degree of increase correlates with worsening heart failure.13 Together, these data indicate that TNF-␣ may play an important pathogenetic role in conditions of cardiac TNF-␣ overexpression. In transplanted hearts, myocardial TNF-␣ concentrations increase rapidly after surgery and remain persistently increased. Interestingly, in association with neoexpression of TNF-␣, accelerated cardiac hypertrophy also occurs, a condition that may explain post-transplant cardiomyopathy and diastolic dysfunction.14 Thus, we suggest that TNF-␣, by virtue of its ability to induce cardiac hypertrophy may contribute to longterm cardiac dysfunction after heart transplantation. Because HMG-CoA reductase inhibitors improve outcomes in heart transplant recipients and exert immunomodulatory effects, we investigated whether the use of simvastatin, an HMG-CoA reductase inhibitor, decreases myocardial TNF-␣ content in heart transplant recipients. METHODS Patient Cohort We conducted the studies after written informed consent was obtained from each patient and with approval of the institutional review board of the Methodist Hospital and Baylor College of Medicine. The cohort was comprised of 10 consecutive patients who underwent cardiac transplantation at the Methodist Hospital/ Baylor College of Medicine Multi-Organ Transplant Center. We conducted these studies before the clinically accepted practice of routine statin use in our program (1997). Source of Human Myocardium Heart transplant recipients were observed with routine rejection surveillance and clinically indicated endomyocardial biopsies. According to our institution protocol, serial endomyocardial biopsy specimens were obtained from all transplant recipients every week for the 1st month, every other week for the 2nd and 3rd months, and then once a month through the 6th month. Myocardial biopsy specimens (size, 0.5–1 mg; 4 per patient) were obtained with a bioptome under fluoroscopic guidance from the right ventricular side of the septum. Myocardial Biopsies Endomyocardial biopsy samples were immediately immersed in 2% paraformaldehyde for 45 minutes, followed by 75% alcohol, then dehydrated into increasing

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concentrations of alcohols, then cleared through xylene, and subsequently embedded in paraffin. Total Collagen Content Human myocardial tissue samples were sectioned at 5 ␮m and stained for 1 hour in picrosirius red solution (0.1% solution Sirius Red F3B in saturated aqueous picric acid, Direct Red 80 obtained from Sigma-Aldrich Chemical Company; Milwaukee, WI), as previously described.15–17 The stained sections were then dehydrated rapidly in 3 changes of 95% and 100% alcohols, cleared in xylene, and mounted in xylene-based mounting medium. Total collagen content was the sum of all areas stained within the slide including interstitial, perivascular, and microscopic scars. Myocyte Size Endomyocardial biopsy samples were sectioned at 5 ␮m and stained with hematoxylin– eosin to measure myocyte size. At 40⫻ magnification, a point-to-point perpendicular line was drawn across the cross-sectional area of the myocytes at the level of the nucleus, and computer-imaging software (Image-Pro® Plus Version 4.1, Media Cybernetics; Silver Spring, MD) then measured this diameter length. We excluded transverse- or oblique-sectioned myocytes. We measured 50 myocytes per slide from each tissue specimen and expressed results as mean and standard error measured. Myocardial TNF-␣ Concentrations We performed immunohistochemistry using a standard immunoperoxidase technique on 5-␮m human tissue sections.18 Endomyocardial biopsy samples were immediately immersed in 2% paraformaldehyde for 45 minutes followed by 75% alcohol and then dehydrated into increasing concentrations of alcohols, cleared through xylene, and subsequently embedded in paraffin. To detect TNF-␣, deparaffinized sections were blocked for endogenous peroxidase activity and quenched by preincubating slides in 0.3% H2O2 in methanol for 20 minutes in a humidity chamber. Next, we flooded the slides with ⫺20°C acetone for 3 minutes. After washing in phosphate buffer saline (PBS), we incubated the slides for 30 minutes in 1% blocking solution (1 g 99% albumin, bovine fraction V and 10 ml PBS). A mouse monoclonal anti–TNF-␣ immunoglobulin G1(IgG1) antibody (dilution 1:10, Santa Cruz Biotechnology; Santa Cruz, CA) was applied before incubation for 2 hours in a humidity chamber. The tissues were then washed in PBS and incubated with biotinylated anti-mouse IgG, (dilution, 1:200; Vector Laboratories; Burlingame, CA) for 30 to 60 minutes. After another PBS washing, we treated tissue sections with streptavidin conjugated to horseradish peroxidase (Vector Laboratories; Burlingame, CA) for 30 minutes. After washes in

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PBS, tissue sections were incubated with 3,3⬘-diaminobenzidine (Vector Laboratories) as the substrate at room temperature until suitable stains developed, and then counterstained with Harris hematoxylin for 1 minute. Sections were then dehydrated in graded alcohols and cover slipped with Cytoseal XYL (Stephens Scientific; Kalmazoo, MI). For control tissues, we substituted primary antibodies with mouse IgG1 isotype antibody (R&D Systems; Minneapolis, MN). In preliminary experiments, we stained myocardial samples at varying concentrations of anti–TNF-␣ antibody, ranging from a 1-to-10 to a 1-to-1,000 dilution. Peak staining always occurred at a 1-to-10 dilution, and we used this concentration of antibody for all subsequent experiments. Quantitative Analysis of Stained Areas We photographed 6 microscopic fields per specimen slide, using a Diagnostic Instrument Spot II color camera (Diagnostic Instrument; Sterling Heights, MI) mounted on an AX70 fluorescence microscope (Olympus; Melville, NY). All fields were digitized to a computer database and stored for analysis. Staining was analyzed with Image-Pro Plus Version 4.1 analysis software (Media Cybernetics; Silver Spring, MD) using color cube-based selection criteria for positive staining. We analyzed intensity level (range) and area according to the method of Matsuo et al.19 Results in this study are based on areas of positive staining within the color spectrum for 3,3⬘-diaminobenzidine (DAB for TNF-␣) of all intensities greater than those found in mouse IgG1isotype-control–stained sections without correction for intensity. For total collagen content, all tissue specimens were obtained, processed, and analyzed in the same manner, with results expressed as mean and standard error measured. An investigator masked to the origin of the samples without knowledge of whether the slides originated from treated or from non-treated patients, analyzed the slides. However, because variation exists in the intensity of the staining from 1 experiment to another, comparisons between groups were performed only within the same experiment. Although absolute values varied from experiment to

Table 2. Effect of Simvastatin on Lipid Measurements at Week 24 Characteristic Total cholesterol LDL HDL AST ALT Total bilirubin

Units mg/dl mg/dl mg/dl ␮/liter ␮/liter mg/dl

Simvastatin 160.8 ⫾ 13.5 88.2 ⫾ 7.8 44.4 ⫾ 2.7 32.2 ⫾ 8.2 32.8 ⫾ 9.2 0.7 ⫾ 0.1

Placebo 181.2 ⫾ 20.4 91.8 ⫾ 18.4 54.6 ⫾ 10.2 16.8 ⫾ 2.7 18.2 ⫾ 4.6 0.9 ⫾ 0.05

Mean ⫾ SEM. ALT, alanine aminotransferase; AST, aspartate aminotransferase; HDL, high-density lipoprotein; LDL, low density lipoprotein.

experiment, the relative size of immunopositive areas did not change. Statistical Analysis We made statistical comparisons between the 2 groups using the 2-tailed t-test and the 1-way analysis of variance followed by the Tukey-Kramer multiple comparisons test corrections for myocyte size, total collagen, and TNF-␣ expression. A p value ⱕ 0.05 was considered significant. RESULTS Patient Characteristics Table 1 presents detailed characteristics of the patient cohort. All patients studied were maintained with a cyclosporine, prednisone, and mycophenolate mofetil immunosuppression protocol, with average serum concentrations of each drug similar in the 2 groups. No patient in the placebo group received an HMG-CoA

Table 1. Patient Demographics Characteristic Age Male/female Cause, ICM/NCM Donor ischemic time (minutes) Donor age

Simvastatin (n ⫽ 5) 55.6 ⫾ 4.1 5/0 2/3 140.4 ⫾ 22.7 27.6 ⫾ 2.7

Placebo (n ⫽ 5) 62.8 ⫾ 3.0 5/0 3/2 227.6 ⫾ 13.6 36.0 ⫾ 6.8

Mean ⫾ SEM. ICM, ischemic cardiomyopathy; NCM, non-ischemic cardiomyopathy.

Figure 1. Endomyocardial biopsy specimens of the right ventricular septum were obtained starting at Week 1 to 24 weeks. Percentage tissue area stained for tumor necrosis factor ␣ (TNF-␣) was plotted against time (weeks) for placebo and simvastatin groups. The difference between the 2 groups became apparent at the 10th week, representing significantly smaller concentrations of myocardial TNH-␣ for the simvastin group. The difference between groups increased with time. The figure insert shows placebo simvastin at the 24th week after transplantaion (p ⬍ 0.05).

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Figure 2. Control and transplant myocardium (placebo and simvastin) at the 24th week were stained for tumor necrosis factor ␣ (TNF-␣). Semiquantitative analysis of stained areas was performed. As shown, control myocardium does not contain TNF-␣; whereas the placebo group expresses increased concentrations, and the simvastin group expresses decreased concentrations of myocardial TNF-␣ (brown areas indicate positive immunostaining, original magnification ⫻40).

reductase inhibitor. The 10 patients studied were randomized to receive either 20 mg/day simvastatin (n ⫽ 5) or placebo (n ⫽ 5) for 6 months after cardiac transplantation. All patients survived the study duration. Table 2 summarizes serum lipid profiles and liver function parameters of both groups at 24 weeks after transplantation. Total cholesterol concentrations were slightly smaller for the simvastatin group (160.8 ⫾ 13.5 mg/dl vs 181.2 ⫾ 20.4 mg/dl), but lipid profiles of both groups were similar. Liver enzymes were increased in the simvastatin group but not at pathologic concentrations. Myocardial TNF-␣ Content Figure 1 shows myocardial TNF-␣ content over time in the simvastatin- and placebo-treated groups. Myocardial TNF-␣ concentrations at 1 week were equal in both groups (15.0% ⫾ 2.3% vs 15.0% ⫾ 1.5%, p ⫽ not significant). This increased TNF-␣ content decreased in parallel for both groups through Week 8 after transplantation. However, after Week 8, TNF-␣ concentrations in the placebo group plateaued, whereas those of the simvastatin group continued to decrease throughout the 24-week follow-up. In comparing the final measurement at the 24th week after transplantation with the initial value at the 1st week, we found a significant decrease in myocardial TNF-␣ content in the simvastatin group (15.0% ⫾ 2.3% vs 5.8% ⫾ 2.4%, p ⫽ 0.02), but

not in the placebo group (15.0% ⫾ 1.5% vs 12.0% ⫾ 2.6%, p ⫽ not significant), see insert in Figure 1. Figure 2 shows representative immunohistochemistry for myocardial TNF-␣ contents (brown) of right ventricular myocardial biopsy samples from healthy control and transplanted (placebo and simvastatin) hearts. Markers of Hypertrophy At 24 weeks, we found no significant difference in myocyte size between the simvastatin and placebo groups (21.0 ⫾ 3.6 ␮m vs 21.0 ⫾ 0.8 ␮m, p ⫽ not significant). Nor did we find a difference in total collagen content between the 2 groups at 24 weeks (25.0% ⫾ 4.7% vs 21.0% ⫾ 5.7%). Neither myocyte size nor total collagen content differed significantly between the simvastatin- and placebo-treated groups at any biopsy time point (data not shown). DISCUSSION This study demonstrates that simvastatin treatment in transplant recipients progressively decreases myocardial TNF-␣ expression compared with patients given placebo. Although this decrease did not result in a significant difference in markers of hypertrophy, such as myocyte size and collagen content, this finding may provide important evidence to support an immunomodulatory role of statins in heart transplant recipients

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and expands on previous observations by others that showed a decrease in serum TNF after statin therapy. The major cardiac complications of heart transplantation are the development of transplant-associated arteriopathy and cardiac allograft hypertrophy. Even in the absence of histologic rejection, the transplanted heart is exposed chronically to inflammatory cytokines that may be deleterious to cardiac function, perhaps by contributing to either arteriopathy or hypertrophy. In the current report, we confirm our previous finding that documented persistent expression of myocardial TNF-␣ in transplant myocardium. But more importantly, we provide a potential new mechanism of action for statins in heart transplant recipients, decreased myocardial TNF-␣ content. The lipid-decreasing effects of statins decrease transplant vasculopathy,3 but the data also suggest that statins exert a beneficial effect through immunomodulation. Recent work has shown that statins may exert anti-inflammatory and anti-proliferative effects in endothelial and vascular smooth muscle cells through inhibiting activation of transcription factors.20 Simvastatin also has been associated in vivo with decreased concentrations of the inflammatory marker C-reactive protein in hyperlipidemic patients.21 Increased concentrations of HDLs exert a dose-dependent influence on concentrations of cardiac TNF-␣ through both TNF-␣ binding and down-regulation of myocardial TNF-␣ mRNA.8 High-density lipoproteins also increase cardioprotective prostaglandin release, again in a dose-dependent manner.8 It is quite possible then that the prolonged survival seen in transplant recipients treated with simvastatin and other HMG-CoA reductase inhibitors is caused by mitigation of the pro-inflammatory milieu in which the transplanted heart exists, in addition to decreases in cholesterol-mediated vasculopathy. Certainly, the progressive decrease in myocardial TNF-␣ observed in our patients treated with simvastatin would lend credence to this hypothesis. Cardiac hypertrophy after transplantation is a welldescribed condition that may be responsible for the large number of patients who manifest diastolic dysfunction and impaired exercise tolerance. If TNF-␣, a cytokine known to promote cardiac hypertrophy,11 plays a role in the development of cardiac allograft hypertrophy, perhaps statins also influence long-term allograft function by decreasing allograft hypertrophy. Indeed, in this report, we measured various markers of hypertrophy at the histologic level; however, our results did not demonstrate a decrease in either myocyte size or collagen content. In summary, we demonstrated that the use of statins decreased myocardial TNF-␣ content in heart transplant recipients. These findings provide evidence for another potential immunomodulatory effect for statins, but also

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suggest that therapies directed against TNF-␣ may prevent long-term cardiac allograft dysfunction. This investigation involved a small number of patients and a relatively short period of follow-up. These factors may have influenced the lack of observed correlation between decreased myocardial TNF-␣ and decreases in either myocyte size or myocardial collagen content. In addition, the method used to determine TNF-␣ is only semi-quantitative and permits us to establish comparisons only within the groups tested. The small amount of tissue obtained during the biopsy limits our ability to perform other types of more precise and quantitative protein determinations. This research was supported by an unrestricted grant from Merck & Co., Inc.

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