CCN2): A protagonist in cardiac allograft vasculopathy development?

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Connective tissue growth factor (CTGF/CCN2): A protagonist in cardiac allograft vasculopathy development? Malena P. Pantou, PhD,a Athanasios Manginas, MD,b Peter A. Alivizatos, MD,c and Dimitrios Degiannis, MD, PhDa From the aMolecular Immunopathology and Histocompatibility Laboratory, the bFirst Department of Cardiology, and the cFirst Department of Cardiac Surgery & Thoracic Transplantation Unit, Onassis Cardiac Surgery Center, Athens, Greece.

KEYWORDS: cardiac allograft vasculopathy; CCN2; CTGF; everolimus; SNP; serum; cardiac allograft transplantation

BACKGROUND: Connective tissue growth factor (CTGF) has been reported to be upregulated in experimental models of chronic cardiac allograft rejection. We investigated the contribution of CTGF to the development of cardiac allograft vasculopathy (CAV), a surrogate marker for chronic rejection. METHODS: This prospective study included 72 adult heart allograft recipients. Genotyping of the rs6918698 polymorphism was performed by sequence-specific primer polymerase chain reaction (PCR). CTGF protein levels were measured in serum. CTGF messenger RNA (mRNA) from myocardial biopsy specimens was quantified by quantitative real-time PCR. RESULTS: Recipient genotype was associated with the development of CAV (p ⫽ 0.014) and the carriers of the C allele (CC and CG genotype) were high-risk recipients for the development of CAV (odds ratio, 3.30; 95% confidence interval, 1.12–9.74; p ⫽ 0.044). Serum CTGF protein levels could not be associated with the presence of the C allele but were significantly lower in the patients that had developed CAV (p ⫽ 0.038). This was attributed to the addition of everolimus to their immunosuppression scheme. Myocardial relative CTGF mRNA expression was estimated to be approximately twice as much in the CAV patients than in the patients without CAV (p ⫽ 0.013). CONCLUSIONS: The important role of CTGF during the development of CAV in heart transplantation was supported by the association of CAV with the recipient CTGF-945 CC/CG genotypes. The CAV patients, who were all receiving everolimus treatment, displayed elevated myocardial CTGF mRNA transcription levels, while everolimus has been observed to reduce serum CTGF protein levels. J Heart Lung Transplant 2012;31:881–7 © 2012 International Society for Heart and Lung Transplantation. All rights reserved.

In cardiac transplantation, allografts that undergo chronic rejection manifest a progressive loss of function, interstitial fibrosis, and the occlusion of luminal structures. In detail, graft arterial vessels are characterized by gradual sub-endothelial tissue development, a process that is referred to as cardiac allograft vasculopathy (CAV).1 Hence, CAV and interstitial fibrosis are commonly viewed as surrogate markers for chronic rejection, and the changes observed during Reprint requests: Malena P. Pantou, PhD, Molecular Immunopathology and Histocompatibility Laboratory, Onassis Cardiac Surgery Center, 356 Sygrou Ave, Athens TK 176 74, Greece. Telephone: 0030-210-949-3023. Fax: 0030-210-949-3018. E-mail address: [email protected]

the development of this pathologic condition resemble chronic tissue remodeling and/or wound repair processes that follow tissue injury.2 CTGF, which has recently risen as a potent mediator of these phenomena,3 is a matricellular protein member of the CCN (abbreviation for “connective-tissue-growth-factor, cysteine-rich-protein-61, and nephroblastoma-over-expressed”) family. Like all other members of this family, CTGF displays a modular protein structure. The gene expressing CTGF consists of 5 exons and encodes for a 38-kDa cysteine-rich molecule that is secreted in the extracellular matrix (ECM). In the promoter region of the gene, a Smad-binding site and a signal transducer and activator of transcription (STAT) 3

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response element have been shown to be important in the regulation of CTGF expression.4 – 6 Accentuation of the transcription factor Smad3 or CTGF in the arterial wall has been proven to stimulate intimal hyperplasia, one of the main conversions observed during arterial remodeling in chronic rejection. In detail, transforming growth factor-␤ (TGF-␤) has been reported to activate the transcription factor Smad3 in medial smooth muscle cells, causing them to secrete CTGF, which in turn stimulates adventitial fibroblasts to migrate, proliferate, produce collagen, and transform into myofibroblasts.6 In addition, CTGF expression has been shown to be induced in experimental models of chronic cardiac allograft rejection.7–9 A polymorphism located in the promoter region of the gene (rs6918698), 945 bp upstream from the ATG start codon, has been reported to induce its transcriptional activity possibly due to a shift of the binding transcription factors.10 In detail, the presence of cytosine at the corresponding position increases the affinity of the region for the specificity protein (Sp)1 transcription factor and enhances the transcriptional activity of the gene.10 In Brazilian and Chinese fishermen infected with Schistosomes, rs6918698CC has been associated with hepatic fibrosis, but this association could not be replicated for 2 other groups of patients, leading to the assumption that the rs6918698 polymorphism may be in linkage disequilibrium with some other causal single nucleotide polymorphism (SNP).11 Our aim was to explore the contribution of CTGF to the development of CAV, one of the main processes that characterize the progression of chronic rejection to patients who had undergone cardiac allograft transplantation. In this direction we explored for a possible association between the rs6918698 polymorphism and the development of CAV. Furthermore, we attempted to assess whether the presence of the studied SNP and/or the onset of CAV could consequently influence CTGF expression. Quantification of CTGF expression has been attempted through the detection of serum protein levels along with the relative quantification of myocardial transcribed CTGF messenger RNA (mRNA) levels.

Materials and methods The Ethics Committee of the Onassis Cardiac Surgery Center approved this study.

Patients and samples This prospective study included 72 adults who received heart allografts from 1996 to 2009 at the Onassis Cardiac Surgery Center. As part of our standard protocol, all heart transplant patients received anti-thymocyte globulin as induction therapy and statins at moderate doses, with routine evaluation for myopathic complications. Extraction of donor DNA was performed from whole blood samples at the time of transplantation and stored for subsequent analyses. For the assessment of CAV, patients underwent coronary angiography and intravascular ultrasound (IVUS)

imaging during their routine hospital visits. The surveillance CAV protocol briefly included coronary angiography and IVUS imaging at 1 month, 1 year, and yearly thereafter, or sooner if clinically indicated in case of cardiac events or abnormal echocardiographic indices. Patients were included in the CAV group if newly detected (not previously observed) maximal intimal thickness in the proximal left anterior descending artery exceeded 0.5 mm and/or if angiographic findings substantiated the development of CAV. At that point, patients were switched to an immunosuppression regimen that comprised everolimus in addition to a reduced dose of calcineurin inhibitor according to the therapeutic protocol followed in our hospital. Samples of whole blood and serum, along with routine transplant surveillance biopsy specimens were collected during routine periodic examination of the recipients at least 1 year after transplantation. The median time points (months after transplantation) of sampling between patients with and without CAV (No-CAV) were comparable, [61 (range, 13–167) vs 64 (range, 13–165) for serum samples and 65 (range, 12–167) vs 59 (range, 23–165) for biopsy samples]. Assessment of the “everolimus” effect on serum CTGF levels was performed by comparing CTGF levels in serum samples from 10 patients immediately after diagnosis of CAV and before everolimus administration with CTGF levels in samples obtained from the same patients at a median of 2 months (range, 1– 6 months) after the addition of everolimus in the immunosuppression scheme.

CTGF polymorphism genotyping A polymorphism corresponding to the position ⫺945 from the ATG start codon of the gene (rs6918698) was analyzed. The analysis encompassed all recipients and all but 3 donors whose DNA had not been extracted and stored during transplantation. Genotyping was performed with the use of a sequence-specific primer-polymerase chain reaction (PCR) assay, as described by Fonseca et al.10 The DNA used as template was extracted from whole blood samples with the QIAamp DNA Blood mini kit (Qiagen, Valencia, CA).

Measurement of CTGF protein Plasma levels of CTGF protein for the study patients were measured using a commercially available sandwich enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (USCN Life Science Inc, Wuhan, China). In 10 patients, CTGF levels were determined twice — before and after the switch of their immunosuppression regimen. The lower limit of detection of the method was 8.2 pg/ml, and all samples were analyzed simultaneously. Seven samples that displayed CTGF serum levels above the upper limit of detection (2,098.4 pg/ml), even after their dilution by 10 times, were assigned the maximum value of 20,984 pg/ml.

RNA extraction and real-time PCR analysis Total RNA was isolated from transplant biopsies using the RNeasy Fibrous Tissue Mini kit (Qiagen). The RNA samples were separated in 1% agarose gels containing ethidium bromide and their quality was determined by the visibility of 18S and 28S RNA bands. Total RNA of high quality was processed to

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complimentary DNA (cDNA) with the use of an oligo-dT primer provided in the Transcription First Strand cDNA synthesis kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. The primer pair used for amplification of the human CTGF gene was 5=-CGACTGGAAGACACGTTTGG-3= and 5=-AGGCTTGGAGATTTTGGGAG3=. As an internal standard, a fragment of ␤-actin was amplified with the use of the primer pair 5=-GATCATTGCTCCTCCTGAGC-3= and 5=-ACTCCTGCTTGCTGATCCAC-3=. The primers were designed to overlap exon junctions to avoid the amplification of DNA. Amplification reactions were performed in a 20-␮l volume of the LightCycler FastStart DNA Master SYBR Green I mixture (Roche Diagnostics) with the addition of primers and MgCl2 at a final concentration of 0.5 ␮mol/liter and 2 mmol/ liter, respectively. All reactions were performed in triplicate in a LightCycler 2.0 with the following thermal cycling conditions: 95°C for 10 minutes, 45 cycles of 95°C for 10 seconds, 58°C for 10 seconds, and 72°C for 20 seconds. To confirm the specificity of amplification PCR products were subjected to a melting curve analysis and electrophoresis in a 4% agarose gel.

Statistical analyses Values are expressed as the mean ⫾ standard deviation or median with interquartile range. Statistical analyses were performed using SPSS 17.0 software (SPSS Inc, Chicago, IL). All tests were 2-tailed, and differences at values of p ⬍ 0.05 were considered significant. The Fisher’s exact test was used to estimate the association of the genotyped SNP with the development of CAV. The difference of the plasma CTGF concentration among different groups was tested by the Kruskal-Wallis or Mann-Whitney test as appropriate. The significance of the different serum CTGF serum protein levels before and after the addition of everolimus to the immunosuppression regimen of the patients that developed CAV was estimated by the Wilcoxon rank sum test. Correlation of serum CTGF concentration and CTGF mRNA expression in the myocardial biopsy specimens was tested by the Kendall’s ␶. The difference of CTGF mRNA expression between recipients with and without CAV was assessed by the Mann-Whitney test.

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Results Clinical characteristics of recipients The study included 72 recipient-donor pairs (recipients: 58 males, 14 females) with a mean age at transplantation of 38.28 ⫾ 13.66 years (range, 14 – 63 years). Follow-up had an average duration of 71.9 ⫾ 45.4 months. Transplantation was performed on a background of dilated (48 patients), ischemic (16 patients), hypertrophic (4 patients), restrictive (2 patients), or valvular (1 patient) cardiomyopathy. One recipient was originally diagnosed with amyloidosis. Dyslipidemia was present in 24 patients, diabetes in 10, and mild hypertension in 4 at the time of transplantation. There was no significant difference between the distribution of the patients with dyslipidemia, diabetes, and hypertension to the CAV and No-CAV groups. Proximal left anterior descending artery intimal thickness was significantly different between CAV (30 patients) and No-CAV (42 patients) groups (0.9 ⫾ 0.4 vs. 0.3 ⫾ 0.1 respectively; p ⬍ 0.001). Everolimus was added to the CAV group at a median of 29.5 months (range, 16.5–54.5 months) after transplantation. Two recipients died, 1 patient underwent retransplantation after loss of graft function due to development of CAV, and the recipient originally diagnosed with amyloidosis subsequently required kidney transplantation.

Association of CTGF polymorphism with the development of CAV In our analysis, a polymorphism corresponding to the promoter region of the CTGF gene (rs6918698) was studied. The genotype distribution of CTGF was not in accordance with Hardy-Weinberg equilibrium (p ⬍ 0.001). The association of the genotyped SNP (CTGF ⫺945C/G) with the detection of CAV is presented in Table 1. Recipient genotype was associated with the development of CAV (p ⫽ 0.014), and recipient genotypes, including the C allele (CC

Table 1 Genotype Distributions in Donors and Recipients According to the Occurrence of Cardiac Allograft Vasculopathy During Recipient Follow-up Genotype

rs6918698

CAV n (%)

No CAV n (%)

Recipient

GG CG CC CC⫹CG vs GG Total GG CG CC CC⫹CG vs GG Total

6 17 7 24 30 10 9 11 20 30

19 10 13 23 42 12 13 14 27 39

Donor

(20) (57) (23) (80) (100) (33) (30) (37) (67) (100)

CAV, cardiac allograft vasculopathy; CI, confidence interval; OR, odds ratio. a Assessed by Fisher’s exact test.

(45) (24) (31) (55) (100) (31) (33) (36) (69) (100)

OR (95% CI)

p-valuea 0.014

3.3 (1.1–9.7)

0.044 ⬎0.999

0.9 (0.3–2.5)

⬎0.999

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and CG genotype), were high-risk genotypes for CAV development (odds ratio [OR], 3.30, 95% confidence interval [CI], 1.12–9.74; p ⫽ 0.044). These promising data, along with the observation that the deposition of ECM is mainly mediated by donor fibroblasts and cardiomyocytes,12 led us to extend our study to include donor genotypes. However, our findings did not support any association between the C allele in the donor genotype and the development of CAV (p ⬎ 0.999).

Serum CTGF concentration in relation to the development of CAV Serum CTGF concentration was measured in all study patients because the detected C allele of the rs6918698 polymorphism has been shown in vitro and in vivo to change the balance of the basic transcriptional activity of the CTGF gene toward enhanced transcription.10 Nevertheless, no association could be established between the presence of the C allele in the recipient and/or donor genotype and serum CTGF protein levels (data not shown). On the contrary and despite the association of the C allele with the development of CAV, patients who had developed CAV displayed significantly lower serum CTGF protein levels than patients who did not exhibit CAV (306.31 [interquartile range (IQR), 158.51–1,262.63] pg/ml vs 747.06 [IQR, 344.70 – 2,662.13] pg/ml, p ⫽ 0.038; Figure 1). We hypothesized that this controversy could be partially attributed to the switch of the pharmacologic regimen in all patients who developed CAV (ie, the addition of everolimus to their immunosuppression therapy). To investigate this hypothesis, we examined serum samples from 10 patients before and after the addition of everolimus to their immunosuppression regimen. CTGF serum protein levels were

Figure 2 Serum levels of connective tissue growth factor (CTGF) measured by enzyme-linked immunosorbent assay in 10 cardiac allograft recipients were significantly reduced after the addition of everolimus to the immunosuppression regimen. Statistical significance (p ⫽ 0.009) was estimated by the Wilcoxon rank sum test.

significantly higher before the administration of everolimus (471.90 [IQR, 268.15–1,005.44] pg/ml vs 195.77 [IQR, 141.99 –514.94] pg/ml, p ⫽ 0.009), suggesting an active role of everolimus to the regulation of the CTGF expression (Figure 2). Consequently, no correlation could be established between the extent of CAV defined by IVUS imaging and the serum CTGF concentration (data not shown).

Elevated intragraft CTGF expression is associated with CAV

Figure 1 Box and whisker plot displays the serum concentrations of connective tissue growth factor (CTGF) protein according to the occurrence of cardiac allograft vasculopathy (CAV). The horizontal line in the middle of each box indicates the median, the top and bottom borders of the box mark the 75th and 25th percentiles, respectively, the whiskers mark the 10th and 90th percentiles, and the 5th and 95th percentiles are marked as circled dots. The difference of the concentration in each group was tested by the Mann-Whitney test.

To study the expression pattern of the CTGF gene in myocardial biopsy specimens, levels of CTGF mRNA were quantified by real-time PCR in 14 patients who had not developed CAV and 9 patients who had developed CAV. Expression levels were calculated as a ratio between CTGF and the reference gene ␤-actin to correct for the variation in the amount of RNA. Relative expression levels were determined after the normalization of results against the median CTGF expression level in biopsy specimens from patients who had not developed CAV. CTGF mRNA expression was estimated to be approximately twice as much in the CAV patients than in the No-CAV patients (2.18 [IQR, 1.22– 5.44] vs 1 [IQR, 0.57–1.55], p ⫽ 0.013; Figure 3), suggesting an important role of this protein during the development of this fibrotic disorder.

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Connective Tissue Growth Factor and CAV

Figure 3 Box and whisker plot displays connective tissue growth factor (CTGF) expression according to the occurrence of cardiac allograft vasculopathy (CAV). The horizontal line in the middle of each box indicates the median, the top and bottom borders of the box mark the 75th and 25th percentiles, respectively, and the whiskers mark the 10th and 90th percentiles. The difference of CTGF mRNA expression in each group was tested by the Mann-Whitney test.

No correlation could be established between serum CTGF protein levels and CTGF mRNA transcription in the myocardial biopsy specimens, undermining the merit of serum CTGF as a potential biomarker for the development of chronic rejection. Similarly, CTGF mRNA levels did not correlate with the severity of CAV as quantified by the maximum intimal thickness (data not shown).

Discussion CAV remains of primary concern in heart transplant recipients, having distinct features from atherosclerosis,13 and although studies have focused on the driving mechanisms of CAV development, these mechanisms are poorly understood and probably involve immunologic and non-immunologic triggers.14 We investigated for a possible link between the development of CAV, one of the main processes that characterize the phenotype of chronic rejection in patients who had undergone cardiac allograft transplantation, and CTGF, a major pro-fibrotic factor that frequently acts downstream of TGF-␤–mediated fibrogenic pathways. We focused on a polymorphism (rs6918698) participating in a Sp-factor binding site located in the promoter region of CTGF. Our data support an association between the rs6918698 polymorphism in the genotype of heart allograft recipients and the development of CAV (p ⫽ 0.014), whereas recipients carrying the C allele, which has been associated with enhanced transcriptional activity, were high-risk genotypes for development of CAV. These findings come in agreement with data from experimental models of chronic cardiac allograft rejection that support an increased CTGF expression during the development of this pathologic condition.7–9

885 Cardiac fibroblasts are one of the most important cell types that produce and maintain myocardial ECM balance,12 and therefore, the excessive deposition of ECM in the setting of cardiac allograft chronic rejection might be donor derived. Our study was extended to include donor genotypes, but no association could be established between the polymorphism and the development of CAV. In this regard, CTGF protein produced by recipient infiltrating cells could induce, in a “feed forward” process, the expression of CTGF by donor cardiomyocytes as has been observed in vitro.15 In support of this hypothesis, it has been found in sex-mismatched transplantations that 3% to 15% of the neointimal smooth muscle cells are recipient derived.16 Furthermore and in addition to cardiac fibroblasts and cardiac myocytes that have been reported to produce CTGF, ␥␦-T cells also express CTGF mRNA and produce CTGF protein.17,18 Data from the heart and kidney of experimental models showed that CTGF expression colocalizes very closely with infiltrated leucocytes.7,19 Serum CTGF has been proposed as a potential biomarker of cardiac dysfunction and its concentration was positively correlated with New York Heart Association heart failure functional class.20,21 CTGF serum levels were also linked to the degree of liver fibrosis of patients infected with hepatitis C virus, even though no association could be established between the genotypes and the alleles for 6 polymorphisms (including rs6918698) and the severity of hepatic fibrosis.22 In our cohort, serum CTGF levels were not associated to the recipient or donor genotype, despite data supporting the linkage of the C allele with enhanced transcriptional activity of the gene.10 Furthermore and surprisingly, patients who had developed CAV displayed significantly lower serum CTGF protein levels than patients who did not have CAV. Because the expression of CTGF has been reported to be affected by various pharmacologic agents like 3-hydroxy3 methyl-glutaryl-coenzyme A reductase inhibitors (statins),23 angiotensin II type 1 receptor antagonists (candesartan).24 and mammalian target of rapamycin (mTOR) inhibitors (rapamycin),25 our hypothesis was that the addition of everolimus to the immunosuppression regimen of patients who presented CAV might account for the puzzling results. In defense of this hypothesis, serum samples taken from 10 patients displayed significantly lower protein CTGF levels after the addition of everolimus (Figure 2). No correlation could be detected between serum CTGF protein levels and myocardial CTGF mRNA levels. Besides, CTGF is a matricellular protein that exerts its action at a local level, and therefore, it is conceivable that its presence in the serum does not quantitatively correlate with its presence at different tissues. Furthermore, the exact effect of the administration of everolimus seems to be context and tissue dependent because CTGF mRNA expression has been reported to be induced by the administration of everolimus in the heart and kidney of transgenic rats harboring human renin and angiotensinogen genes,19 whereas the administration of sirolimus, another mTOR inhibitor, in renal transplant recipients with biopsy-proven chronic allo-

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graft nephropathy revealed that sirolimus attenuates renal CTGF expression.26 Regardless of everolimus, several groups have reported data from experimental models indicating that CTGF is upregulated and functions as a downstream mediator of fibrosis in chronic cardiac allograft rejection.7–9 In the human setting of our study, the relative CTGF mRNA expression was estimated to be approximately twice as much in the CAV patients than in the No-CAV patients (Figure 3), although CTGF mRNA transcription did not correlate with the severity of CAV as quantified by the maximum intimal thickness (data not shown). Because all of the CAV patients in our study were receiving everolimus at the time of biopsy, it is difficult to discern the relative contribution of everolimus and CAV on the mRNA expression that was observed. In this regard there are 3 distinct possibilities: 1. Everolimus, similarly to what was observed in serum, reduces CTGF myocardial expression. In that case, the observed elevated levels of CTGF mRNA in the CAV group would have been more intense had not the patients received everolimus. 2. Everolimus induces myocardial CTGF expression, similarly to what was observed by Finckenberg et al19 in their transgenic rats harboring human renin and angiotensinogen genes. In that case the observed elevated levels of CTGF mRNA in the CAV patients would be partially everolimus driven. 3. Everolimus does not affect myocardial CTGF expression, and the observed levels can be attributed to the development of CAV alone. Our data do not permit us to distinguish between these possibilities because the Hospital Ethics Committee limited the number of myocardial biopsy specimens we could obtain to those that were part of the routine protocol and prevented us from acquiring additional specimens from patients at the onset of CAV and before the administration of everolimus. Connective tissue growth factor has been proposed to be a useful prognostic marker because its levels in various biologic fluids, such as blood plasma, urine, and vitreous, were found to correlate with the presence or the progression of various fibrotic diseases.27 In the setting of cardiac allograft transplantation, serum protein levels of CTGF were reduced by the administration of everolimus and therefore could not be associated with the development of CAV. Nevertheless, myocardial CTGF mRNA levels were elevated in patients who developed CAV, pointing to an active role of this protein during the progression of chronic allograft rejection in accordance with extensive data from animal models7–9 that support the contribution of CTGF to the development of cardiac fibrosis. In the human setting, however, the pathogenesis of CAV is still obscure and complex; therefore, it remains possible that the CTGF concentration differences observed in our study constitute an epiphenomenon.

The role of CTGF was further supported by the association of the rs6918698 polymorphism with the development of CAV, a polymorphism that has been reported to enhance the transcriptional activity of the CTGF gene. This association awaits confirmation by a study of significantly larger number of patients. The exact mechanism for this association remains to be resolved as the presence of the mutated allele could either constitute a causal parameter or alternatively could represent an accelerator of the progression of this pathologic condition.

Disclosure statement This work was supported by a research grant from the Central Hellenic Health Council (KESY), Ministry of Health. None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to disclose.

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