Abstract
Primary aldosteronism has been associated with myocardial fibrosis, and is the most common cause of secondary hypertension. We previously showed that aldosterone can induce the secretion of galectin-3. The aim of this study was to investigate the association between myocardial fibrosis and plasma galectin-3 level in patients with primary aldosteronism. We prospectively analyzed 11 patients with aldosterone-producing adenoma (APA) who received adrenalectomy from December 2006 to October 2008, and 17 patients with essential hypertension as controls. Levels of plasma galectin-3 were determined in both groups, and both groups underwent echocardiography with cyclic variations of integrated backscatter (CVIBS) to characterize tissue initially and 1 year after surgery in the APA group. Diastolic blood pressure, concentration of plasma aldosterone and aldosterone-renin ratio were significantly higher, and serum potassium level and plasma renin activity significantly lower in the APA group compared to the controls. In addition, left ventricular mass index was significantly higher and CVIBS significantly lower in the APA group (7.3±2.0 vs 9.2±1.7 dB, p=0.015). Furthermore, the concentration of plasma galectin-3 was significantly higher in the APA group (2.1±0.9 vs 1.1±0.6 ng/mL, p=0.005) compared to the controls. CVIBS was correlated to plasma galectin-3 level. In the APA group, CVIBS increased significantly (7.3±2.0 to 9.2±2.4 dB, p=0.032) and plasma galectin-3 decreased (2.1±0.9 to 1.2±0.6, p=0.049) 1 year postadrenalectomy. The patients with APA had increased myocardial fibrosis, and this was associated with a higher plasma galectin-3 level. Both increased myocardial fibrosis and plasma galectin-3 level recovered at least partially after adrenalectomy.
Trial registration number 200611031R; Results.
Significance of this study
What is already known about this subject?
Patients with primary aldosteronism have higher left ventricular mass and degree of cardiac fibrosis compared to those with essential hypertension, possibly due to increases in plasma aldosterone.
Aldosterone-induced increment in left ventricular mass, and cardiac fibrosis can be reversed in patients with unilateral aldosterone-producing adenomas by performing adrenalectomy.
Aldosterone induces macrophages to secrete galectin-3, which is a possible mediator linking macrophages to myocardial fibrosis.
What are the new findings?
Patients with unilateral aldosterone-producing adenoma had increased myocardial fibrosis, and this was associated with a higher plasma galectin-3 level.
The increased myocardial fibrosis was associated with a higher plasma galectin-3 level.
Both increased myocardial fibrosis and plasma galectin-3 level recovered at least partially after adrenalectomy.
How might these results change the focus of research or clinical practice?
These results suggest that of galectin-3 may play a role in aldosterone-induced myocardial fibrosis, which we hope will encourage further basic or large prospective clinical studies on this topic.
Galetcin-3 may be a target of treatment for aldosterone-induced myocardial fibrosis.
Primary aldosteronism (PA) is characterized by excess aldosterone production, and reportedly affects 5–13% of patients with hypertension.1 Patients with PA have higher rates of left ventricular hypertrophy (LVH) and more severe cardiac and vascular fibrosis than those with essential hypertension (EH), which are hemodynamically independent factors.2–5 Unilateral aldosterone-producing adenoma (APA) is a common type of PA which can be cured with adrenalectomy.2 It has also been reported that increases in left ventricular (LV) mass and cardiac fibrosis can be reversed after adrenalectomy.2 ,6 ,7
Characterization of tissue using ultrasound with cyclic variation of integrated backscatter (CVIBS) has been shown to be a useful non-invasive tool to assess collagen content in the myocardium in hypertensive patients8 and patients with PA.3 ,9 In previous studies, we described increased myocardial fibrosis (evaluated by CVIBS or CVIBS combined with serological fibrotic markers) in patients with APA, and improvements in patients receiving adrenalectomy.6 ,7 These studies highlight the significant influence of aldosterone on myocardial fibrosis.
However, the mechanism by which aldosterone induces cardiac fibrosis has yet to be elucidated unclear. Recent studies have shown that aldosterone-induced macrophage activation and low grade inflammation play important roles in cardiac fibrosis and myocardial dysfunction induced by aldosterone.10 ,11 We previously reported that aldosterone induces macrophages to secrete galectin-3, which is a possible mediator linking macrophages to myocardial fibrosis.12 However, the relationships among aldosterone, galectin-3 and myocardial fibrosis have yet to be clarified in humans. Therefore, the aim of this study was to investigate these possible relationships.
Materials and methods
Patients
We enrolled 17 patients with EH as the control group and 11 with APA who had not taken spironolactone within the past 6 months and who agreed to undergo adrenalectomy. EH was diagnosed by excluding clinical and biochemical factors associated with secondary hypertension. The patients were evaluated and registered in the Taiwan Primary Aldosteronism Investigation (TAIPAI) database.13–15 Among the participants, seven patients with APA and 15 patients with EH were included in our previous study on whether the secretion of galectin-3 is induced by aldosterone.12 In addition, six patients with APA and 10 patients with EH had also been enrolled in another of our previous studies to evaluate whether CVIBS can be used to examine increases in myocardial fibrosis.4
The medical history and use of medications were carefully recorded in each patient. Serum biochemistry studies were performed at the initial evaluation. Plasma aldosterone concentration (PAC) and plasma renin activity (PRA), defined as the level of angiotensin-I in vitro, using radioimmunoassay kits (Aldosterone Maia Kit; Adaltis Italia, Bologna, Italy, and Cisbio, Bedford, Massachusetts, USA, respectively). The patients underwent echocardiography 3 months presurgery and then 1 year postsurgery, with levels of plasma galectin-3 being measured at the same time. This study was approved by the Institutional Review Board of National Taiwan University Hospital, and all participants provided written informed consent.
Diagnosis criteria for APA
The confirmation of PA and the differentiation of subtypes according to the TAIPAI group have been described in previous studies.15–17 Briefly, a diagnosis of APA was made according to these factors: (1) autonomous excess aldosterone production defined as an aldosterone-to-renin activity ratio (ARR) of more than 35, a TAIPAI score higher than 60%,18 and post-saline loading PAC of more than 10 ng/dL; (2) adenoma as seen on preoperative CT; (3) lateralization of aldosterone secretion in adrenal venous samples or during dexamethasone suppression NP-59 SPECT/CT;19 and (4) for the patients receiving surgery, pathologically proven adenoma after adrenalectomy, with the subsequent development of a cure defined as either hypertension without the need for antihypertensive medications or improvements in hypertension, potassium level, PAC and PRA.15 ,16
Echocardiography
In this study, echocardiography was performed using a Hewlett-Packard Sonos 5500 ultrasound system (Palo Alto, California, USA) equipped with a S3 transducer. The measurements included two-dimensional, M-mode, Doppler ultrasound and CVIBS. The dimensions of the left ventricle, wall thickness and left ventricular ejection fraction (M-mode) were measured via a parasternal long-axis view according to the recommendations of the American Society of Echocardiography. We used Devereux and Reichek20 method to calculate the left ventricular mass index (LVMI). E wave, E wave deceleration time, A wave, and transmitral flow velocity were measured in apical four-chamber view using Doppler ultrasound.
Cyclic variation of integrated backscatter
CVIBS data were analyzed using Acoustic Densitometry software (Hewlett-Packard, Andover, Massachusetts, USA).21 The software displayed grayscale images that were created in proportion to the amplitude of the integrated backscatter. The images were displayed at 30 frames/second, with a dynamic integrated backscatter signal range of ∼ 60 dB. After scan conversion, 60 frames from consecutive cardiac cycles were displayed. All raw data were stored in a computer for subsequent analysis. The integrated backscatter data were processed using linear compression.
Mid-posterior segments of the LV myocardium in the parasternal long-axis view were selected as regions of interest. Integrated backscatter data were quantified by placing a 21×21 pixel region of interest on the myocardium on a frozen image. The regions of interest were designed to fit within the boundary of the subendocardial myocardium in each frame throughout the cardiac cycle. Changes in integrated backscatter amplitude over time were measured in each frame of the whole cardiac cycle, and then plotted as a curve of integrated backscatter versus time. The amplitude of CVIBS was calculated in dB as the difference between maximum and minimum values in a cardiac cycle, and the average of two consecutive beats was used for analysis.
Postadrenalectomy follow-up
Follow-up biochemical tests for PAC, PRA and galectin-3 and echocardiography were performed 1 year postadrenalectomy. Hypertension was considered to have been cured if the patients had a blood pressure of 140/90 mm Hg or lower and did not require antihypertensive medications,22 all of which had to be achieved within 1 year postadrenalectomy.23 If these patients subsequently developed hypertension, they were still defined as having been cured.
Plasma galectin-3
Venous blood samples were taken after an overnight fast. After clotting and centrifugation, the plasma was stored at −60°C until analysis. Levels of galectin-3 were measured using an ELISA kit (Bender Medsystems, Vienna, Austria) and a Victor 2 plate reader (Perkin Elmer, Turku, Finland) according to the manufacturers' protocols. Values were normalized to a standard curve. The intra-assay and interassay variations were 5.6% and 8.6%, respectively.12
Statistical analysis
Data were expressed as mean±SD. T tests were used to compare continuous variables between groups, and the χ2 test or Fisher's exact test to assess differences, as appropriate. Paired t tests were used to compare preoperative and postoperative variables, with Pearson's correlation to analyze associations between two variables. PAC, PRA, and ARR values were log-transformed before analysis due to non-normality as shown by the Kolmogorov-Smirnov test. The log-transformed data were then tested again to ensure that they were normally distributed before subsequent analysis. SPSS V.18.0 (SPSS Inc, Chicago, Illinois, USA) was used for all statistical analyses, and a p value of < 0.05 was considered to indicate statistical significance.
Results
Patients
We enrolled 11 patients with APA who underwent adrenalectomy and 17 patients with EH as the control group (table 1). The APA group had a significantly higher diastolic blood pressure, PAC and log ARR, and a significantly lower serum potassium level and PRA than the controls. In addition, a higher proportion of the APA group were prescribed with α-blockers.
Echocardiography
The patients with APA had a thicker interventricular septum, LV posterior wall and LVMI than the controls (table 2). In addition, CVIBS was significantly lower in the APA group compared to the controls (7.3±2.0 vs 9.2±1.7 dB, p=0.015).
Plasma galectin-3
The APA group had a significantly higher level of plasma galectin-3 (2.1±0.9 vs 1.1±0.6 ng/mL, p=0.005) than the controls. CVIBS was significantly correlated with plasma galectin-3 level (r=−0.409, p=0.047).
Postadrenalectomy
One-year postadrenalectomy, there were significant decreases in the use of antihypertensive medications and log ARR, and significant increases in log PRA and serum potassium level (table 3). Five of the 11 patients were cured of hypertension. In addition, CVIBS increased significantly (7.3±2.0 to 9.2±2.4 dB, p=0.032), and plasma galectin-3 level decreased (2.1±0.9 to 1.2±0.6, p=0.049; figure 1).
Discussion
In this study, we demonstrated that: (1) the patients with PA had lower CVIBS and higher plasma galectin-3 levels than the patients with EH; (2) CVIBS was significantly correlated with plasma galectin-3 level; and (3) adrenalectomy resulted in improvements in the lower CVIBS and higher levels of plasma glaectin-3. A reduction in myocardial fibrosis after adrenalectomy has previously been reported.6 ,7 However, to the best of our knowledge, this is the first study to report the association between myocardial fibrosis and level of plasma galectin-3 as evaluated by CVIBS.
A lower CVIBS implies a higher degree of myocardial fibrosis in patients with APA, and the postoperative increase in CVIBS implies that adrenalectomy reversed myocardial fibrosis in these patients. In addition, the association between plasma galectin-3 and CVIBS suggests that galectin-3 may also be associated with myocardial fibrosis. This study provides clinical evidence of the role of galectin-3 in aldosterone-induced myocardial fibrosis, which we previously demonstrated in a basic study.12 To the best of our knowledge, this is the first study to report a relationship between galectin-3 and myocardial fibrosis in patients with PA.
PA is characterized by elevated aldosterone levels and lower renin levels, and prolonged exposure to high levels of aldosterone has been shown to cause damage to the cardiovascular system independently of blood pressure.24 ,25 In addition, high levels of aldosterone have been reported to lead to progressive changes in the dimensions of the left ventricle and higher levels of collagen deposition independently of a hemodynamic effect.2 ,26 ,27 Therefore, patients with PA are at a higher risk of LV hypertrophy, myocardial fibrosis, and diastolic dysfunction than patients with EH.2 ,28 ,29
In previous animal studies, a combination of aldosterone infusion and high salt intake have resulted in increased collagen deposition in bilateral ventricles,30 bilateral atria and adventitia of the pulmonary artery.31 This reported increase in fibrosis suggests that the fibrosis induced by aldosterone is hemodynamically independent, via a mechanism recently postulated by Rickard et al.11 In addition to the association with myocardial fibrosis, aldosterone-induced macrophage activation leading to low-grade inflammation has also been reported, and this in turn has been reported to play a key role in cardiac fibrosis and myocardial dysfunction.10 The deletion of macrophage mineralocorticoid receptors has been reported to protect against salt-induced and deoxycorticosterone-induced cardiac fibrosis, and this implies that macrophages play a pivotal role in cardiac fibrosis induced by aldosterone.11 The mediators of aldosterone through macrophages to fibrosis have yet to be elucidated, however galectin-3 is likely to be one of the most important.32 ,33 In an animal model, galectin-3 was found to play an important role in hyperaldosteronism combined with high-blood pressure-induced cardiac fibrosis.34 In that model, hyperaldosteronism combined with high-blood pressure was found to stimulate macrophage infiltration into the heart and enhance the transcription of galectin-3 mRNA.34 The results of the current study demonstrate the importance of aldosterone in cardiac fibrosis and the possible effect of aldosterone in inducing galectin-3. In our previous study, we found evidence that aldosterone could induce the secretion of galectin-3 and proposed a possible signal transduction pathway,12 and also that aldosterone-induced galectin-3 expression enhanced fibrosis related to factor expression in fibroblasts. Recent studies have reported that pharmacological inhibition or genetic disruption of galectin-3 rescues aldosterone-induced cardiac fibrosis and dysfunction.35 ,36 These findings suggest the important role that galectin-3 plays in aldosterone-induced myocardial fibrosis, and the possibility that galectin-3 could be a target of treatment in aldosterone-induced myocardial fibrosis. The current study provides clinical evidence of the relationships among aldosterone, galectin-3, and myocardial fibrosis. However, although significant, the correlation between galectin-3 and CVIBS was moderate (r=−0.409), which implies that galectin-3 may not be the sole reason for fibrosis. Further large scale studies are needed to elucidate this issue.
In clinical studies regarding myocardial fibrosis, a myocardial biopsy is still the gold standard to assess collagen content, although its use is limited by the invasive nature. Myocardial extracellular matrix is an important source of myocardial integrated backscatter,37 and high levels of myocardial extracellular collagen deposition may alter the myocardium, myofibril orientation and water content.38 Therefore, integrated backscatter studies provide an opportunity to investigate the content of myocardial collagen in patients with hypertension21 ,39 and PA.2 ,3 Since patients with PA tend to have a greater degree of myocardial fibrosis, they also have lower CVIBS than patient with EH,3 as demonstrated in the current study.
There are several limitations to this study. First, the measurement of CVIBS depends on the angle between the ultrasound beam and fiber orientation.38 To minimize bias in these measurements, we used the mid-posterior segment of the left ventricle where the myocardial fibers were perpendicular to the ultrasound beam. In some patients, individual variations in the position of the heart resulted in perfect alignment with the segments perpendicular to the incident ultrasound beam. Second, it would not have been ethical to routinely perform endomyocardial biopsies in our patients, and thus we were unable to use biopsy findings to assess the severity of LV fibrosis. Third, the patients with EH had higher diastolic blood pressure than those with APA, which may have led to bias in the results. Fourth, we did not obtain serial plasma samples after adrenalectomy. Therefore, we do not know how rapidly the galectin-3 levels declined. Fifth, we did not check other key inflammatory markers such as interleukin-6 or monocyte chemoattractant protein-1, which would have allowed us to further investigate the potential mechanisms. Sixth, there is no standard assay for galectin-3, and different galectin-3 assays make it difficult to compare results between studies.
In conclusion, the patients with APA had increased myocardial fibrosis which was associated with increased plasma galectin-3 levels. Both increased myocardial fibrosis and plasma galectin-3 levels recovered at least partially after adrenalectomy.
Acknowledgments
The authors thank Ms Fen-Fun Hsien for medical technological support and the staff of the Second Core Lab of Department of Medical Research in National Taiwan University Hospital for technical assistance.
Footnotes
Collaborators The members of the Taiwan Primary Aldosteronism Investigation (TAIPAI) Study Group are Vin-Cent Wu, MD, PhD, Yen-Hung Lin, MD, PhD, Yi-Luwn Ho, MD, PhD, Hung-Wei Chang, MD, PhD, Lian-Yu Lin, MD, PhD, Fu-Chang Hu, MS, ScD, Kao-Lang Liu, MD, Shuo-Meng Wang, MD, Kuo-How Huang, MD, PhD, Yung-Ming Chen, MD, Chin-Chi Kuo, MD, Shih-Chieh Chueh, MD, PhD, Ching-Chu Lu, MD, Fang-Chi Chang, MD, Shih-Cheng Liao, MD, Ruoh-Fang Yen, MD, PhD, Wei-Chou Lin, MD, PhD, Bor-Sen Hsieh, MD, PhD and Kwan-Dun Wu, MD, PhD.
Contributors C-WL, K-DW, and YLH. conceived and designed the experiments. V-CW, Y-TL, X-MW, and C-SH collected and analyzed the data. Y-H.L and C-WL wrote the paper.
Funding This study was supported by the National Taiwan University Hospital, NTUH 102-S2096, NTUH 103-M254, NTUH 103-S2447, NTU, Taiwan National Science Council, NSC 102-2314-B-002-056, NSC 102-2314-B-002-078-MY3.
Competing interests None declared.
Patient consent Obtained.
Ethics approval Institutional Review Board of National Taiwan University Hospital.
Provenance and peer review Not commissioned; externally peer reviewed.