Aldosterone Induced Galectin-3 Secretion In Vitro and In Vivo: From Cells to Humans

Context Patients with primary aldosteronism are associated with increased myocardial fibrosis. Galectin-3 is one of the most important mediators between macrophage activation and myocardial fibrosis. Objective To investigate whether aldosterone induces galectin-3 secretion in vitro and in vivo. Methods and Results We investigated the possible molecular mechanism of aldosterone-induced galectin-3 secretion in macrophage cell lines (THP-1 and RAW 264.7 cells). Aldosterone induced galectin-3 secretion through mineralocorticoid receptors via the PI3K/Akt and NF-κB transcription signaling pathways. In addition, aldosterone-induced galectin-3 expression enhanced fibrosis-related factor expression in fibroblasts. We observed that galectin-3 mRNA from peripheral blood mononuclear cells and serum galectin-3 levels were both significantly increased in mice implanted with aldosterone pellets on days 7 and 14. We then conducted a prospective preliminary clinical study to investigate the association between aldosterone and galectin-3. Patients with aldosterone-producing adenoma had a significantly higher plasma galectin-3 level than patients with essential hypertension. One year after adrenalectomy, the plasma galectin-3 level had decreased significantly in the patients with aldosterone-producing adenoma. Conclusion This study demonstrated that aldosterone could induce galectin-3 secretion in vitro and in vivo.


Introduction
An excess of aldosterone plays a pivotal role in the development and progression of cardiac diseases such as primary aldosteronism (PA) and heart failure (HF) [1,2]. PA affects 5-13% of patients with hypertension, and is characterized by an inappropriate production of aldosterone [1]. Increasing evidence indicates that PA is much more prevalent than previously believed, and that this disease is the most frequent cause of secondary hypertension [1]. Using aldosterone blockade with spironolactone has been shown to improve the outcome of HF patients [3], and is now the standard therapy for severe HF.
Aldosterone has been found to induce myocardial fibrosis in the cardiovascular system. In an animal study, aldosterone infusion with high salt intake induced cardiac hypertrophy and myocardial interstitial fibrosis and scarring in the right and left ventricles [4]. In clinical studies, patients with PA have been found to have more prominent left ventricular (LV) mass and cardiac fibrosis than patients with essential hypertension (EH), which may be due to the high plasma aldosterone levels in these patients [5,6]. Unilateral aldosterone-producing adenoma (APA) is the most common subtype of PA; it can be cured by adrenalectomy after which the increased LV mass and myocardial fibrosis regress [5,[7][8][9][10].
Despite the direct effect of aldosterone to induce fibrosis, aldosterone also induces macrophage activation and low grade inflammation, which may play an important role in cardiac fibrosis and myocardial dysfunction [2]. Deletion of mineralocorticoid receptors (MR) from macrophages has been shown to protect against deoxycorticosterone/salt-induced cardiac fibrosis, which implies that macrophages play an important role in aldosterone-induced cardiac fibrosis [11]. However, the mediators of aldosterone through inflammation to fibrosis are still unclear. Among a number of candidate mediators, galectin-3 was found to be one of the most likely candidates in several animal [12,13] and clinical studies [14,15]. Galectin-3 is a b-galactoside-binding animal lectin which is highly expressed and secreted by macrophages [16]. Galectin-3 has been shown to be involved in fibrosis in various organs, including the lungs [17], liver [18], kidneys [19], and heart [12]. In an animal model of renal fibrosis, knocking-out the galectin-3 gene was found to decrease renal fibrosis without changing macrophage recruitment or macrophage proinflammatory cytokine profiles [19]. In addition, the adoptive transfer of wild-type macrophages into galectin-3 knocked-out mice was found to restore the fibrotic phenotype in kidneys, suggesting that galectin-3 expression and secretion is a key mechanism linking macrophages to myofibroblast activation and tissue fibrosis [19]. A recent study further showed that aldosterone increased vascular galectin-3 expression, inflammation, and collagen formation in wild-type mice, whereas no changes occurred in galectin-3 knocked-out mice [20].
We hypothesized that aldosterone plays an important role in galectin-3 production in macrophages, which may further cause fibrosis in the cardiovascular system. Therefore, we conducted this study to evaluate galectin-3 secretion by aldosterone stimulation in vitro and vivo.

Materials and Methods
Cell cultures of THP-1, Raw 264.7, human fibroblast 293T and mouse fibroblast NIH/3T3 cell lines THP-1, RAW 264.7, 293T and NIH/3T3 cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). THP-1 and RAW 264.7 cell lines were maintained in RPMI 1640 medium supplemented with 2 mmol/L glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin to inhibit bacterial contamination, and 10% FCS for cell growth. The human fibroblast 293T and mouse fibroblast NIH/3T3 cell lines were cultured in DMEM containing 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin to inhibit bacterial contamination.

Detection of galectin-3 level by enzyme immunoassay
The level of galectin-3 in the cell culture supernatant was determined using a commercially available enzyme immunoassay (EIA) kit (R&D Systems) according to the manufacturer's instructions. The catalog numbers for human galectin-3 and mouse galectin-3 were DGAL30 and DY1197, respectively. Each measurement was performed in triplicate, and the average value was then recorded as ng/mL.

Preparation of conditioned medium
Cultures of the THP-1 and RAW 264.7 cells (2610 6 /10-mm dish) were rinsed twice with PBS and then cultured in 5 ml of serum-free RPMI 1640 with aldosterone (10 26 M) or vehicle treatment for 24 hours. The conditioned medium was collected and then clarified by centrifugation (4uC, 10,000 rpm, 5 minutes) to remove cell debris. A final solution of 25 mM HEPES buffer (pH 7.4), 1 mg ml 21 leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 0.02% NaN 3 , and 0.1% BSA (Sigma) was the added. The conditioned medium was then frozen and stored at 270uC until use.

RNA interference
Small interfering RNA (siRNA) duplexes were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The targeted siRNAs for human galectin-3, mouse galectin-3, and control siRNA were sc-155994, sc-35443 and sc-37007, respectively. The THP-1 and Raw 264.7 cells were transfected with siRNA at a concentration of 25 nM in serum-free Opti-MEM using the Oligofectamine method (Invitrogen, Carlsbad, CA, USA).

Cell growth determined by MTT assay
Human fibroblast 293T and mouse fibroblast NIH/3T3 cell lines were plated onto 96-well microplates at a density of 5610 3 cells/well with the conditioned media for cell growth assay. Briefly, the cells were cultured at 37uC for the indicated times, and 30 ml of MTT solution (5 mg/ml) was then added into each well followed by incubation for 4 hours in darkness. Formazan grains were dissolved in DMSO, and the absorbance at 570 nm was read using an ELISA plate reader.

Aldosterone release pellet implanted mouse model
Six-week-old ICR wild-type male mice were obtained from the Animal Center of the Medical College of National Taiwan University and kept in standard animal housing conditions under the guidelines for the care and use of experimental animals. The experiments were approved by the Animal Care and Use Committee of the Medical College of National Taiwan University. Mice with a similar body weight (25-30 g) were randomly divided into two treatment groups (vehicle and aldosterone; N = 5 per group). The mice were implanted subcutaneously with a 20-d continuous aldosterone release pellet (Innovative Research of America, Sarasota, FL). The vehicle control mice received a placebo pellet. Mice whole blood was collected on days 0, 7, and 14. The sera used for galectin-3 and total RNA were purified from peripheral blood mononuclear cells and used for galectin-3 mRNA analysis.

Patients
Seventeen patients with essential hypertension (EH) and 7 patients with APA who did not take spironolactone within the previous 6 months and who were prepared to receive adrenalectomy were enrolled. The patients were evaluated and registered in the Taiwan Primary Aldosteronism Investigation (TAIPAI) database. This database was established for quality assurance in one medical center (National Taiwan University Hospital, Taipei, Taiwan) and its three branch hospitals in different cities around Taiwan (National Taiwan University Hospital Yun-Lin branch, Yun-Lin, southern Taiwan; Far-Eastern Memorial Hospital, Taipei, northern Taiwan; and Tao-Yuan General Hospital, Tao-Yuan, central Taiwan) [24,25]. The medical history of each patient including demographic and medication data was carefully recorded. Serum biochemistry studies of these patients were performed at the initial evaluation at National Taiwan University Hospital. Plasma aldosterone concentration (PAC) was measured by radioimmunoassay using a commercial kit (Aldosterone Maia Kit; Adaltis Italia, Bologna, Italy), and plasma renin activity (PRA) was measured as the generation of angiotensin-I in vitro using a commercially available radioimmunoassay kit (Cisbio, Bedford, MA). This study was approved by the Institutional Review Board of National Taiwan University Hospital, and all subjects provided written informed consent, including for the storage of their information in the hospital database and usage for research.

Diagnosis of APA
The diagnosis of APA was established when all of the following ''modified 4 corners score'' criteria were met: (1) evidence of autonomous excess aldosterone production based on an aldosterone-renin ratio (ARR) .35 or urine $12 mg/24 hours, and a TAIPAI score of more than 60% [26] and post-saline loading PAC .10 ng/dl; (2) lateralization of aldosterone secretion in adrenal vein sampling or during dexamethasone suppression NP-59 SPECT/computed tomography [27]; (3) evidence of adenoma in computed tomography scans; and (4) pathologically proven adenoma after adrenalectomy if the patient received surgery, and cure of hypertension without anti-hypertensive agents or improved hypertension, potassium, PAC, and PRA as described.

Plasma galectin-3 analysis
Venous blood samples were collected in serum separation tubes after overnight fasting. After clotting and centrifugation, the serum was stored at 260 uC until analysis. Galectin-3 was measured by an ELISA kit (Bender Medsystems, Vienna, Austria) and measured on a Victor 2 plate reader (Perkin Elmer, Turku, Finland). Calibration of the assay was performed according to the manufacturer's protocol. Values were normalized to a standard curve. The intra-and inter-assay variances for galectin-3 were 5.6% and 8.6%, respectively.

Post-adrenalectomy follow-up
Clinical evaluation and plasma galectin-3 level measurements were performed 1 year after adrenalectomy. Hypertension was considered cured if the blood pressure decreased to 140/90 mm Hg or less after adrenalectomy, and anti-hypertensive medications were not required [28] one year post adrenalectomy [8]. The patients who were cured within one year but later developed hypertension were still classified as being cured.

Statistical analysis
In the cell study, each experiment was performed in triplicate and all experiments were repeated at least three times on different occasions. A t test was used to evaluate statistically significant differences between the aldosterone treatment and vehicle control groups in the specified tests. In the human study, data were expressed as mean 6SD. Comparisons for continuous data between the PA and EH patients were made using a t test. Differences between proportions were assessed by the chi-square or Fisher exact test. Comparisons between pre-operative and postoperative parameters were made using a paired t test. Pearson's correlation test was used to analyze the association between two parameters. Data of PAC, PRA, and ARR were log-transformed before the correlation study due to the non-normality which was determined by the Kolmogorov-Smirnov test. Before further analysis, the log-transformed data were tested again to assure the normality of distribution. A p value less than 0.05 was selected as the statistically significant cutoff value. A p value between 0.05 and 0.1 was considered to indicate borderline significance.

Results
Concentration and timing of aldosterone-induced galectin-3 secretion

Signal transduction pathway
The secretion of galectin-3 after aldosterone stimulation was inhibited by pretreated with 10 27 M spironolactone, but not by 10 27 M RU-486 (Figure 3, 4). This suggested that the secretion of galectin-3 was via mineralocorticoid receptors (MRs) rather than glucocorticoid receptors.
The induced secretion was blocked by LY294002, but not by PD98059, SB203580, Ro318220, or SP600125. This suggested that the induced secretion occurred via the PI3K/Akt pathway ( Figure 5A, 6A). To confirm that the PI3K/Akt pathway was involved in the aldosterone signaling, spironolactone was used to evaluate whether or not MRs were upstream of the aldosteroneinduced Akt phosphorylation. The result of p-Akt Western blotting revealed that the MRs were indeed upstream of the aldosteroneinduced Akt phosphorylation ( Figure 5B, 6B).
In transcriptional factor analysis, induction of galectin-3 secretion was blocked by the decoy ODN of NF-kB but not the decoy ODN of AP-1. This suggested that NF-kB is an important transcriptional factor in the secretion pathway ( Figure 5C, 6C). Spironolactone and LY294002 were used to evaluate the roles of MRs and PI3K/Akt in the aldosterone-induced NF-kB activation. The results of NF-kB nuclear and cytosol Western blotting showed that MRs and PI3K/Akt were upstream of the aldosteroneinduced NF-kB activation ( Figure 5D, 6D).

Aldosterone induced galectin-3 expression in vivo
To confirm the effect of aldosterone-induced galectin-3 expression in vivo, ICR wild-type male mice were implanted subcutaneously with a 20-d continuous aldosterone release pellet, and serum galectin-3 levels were measured. Galectin-3 mRNA was analyzed from total RNA purified from peripheral blood mononuclear cells. The results showed that both were significantly increased in the aldosterone pellet implanted mice on days 7 and 14 (Figure 8a, 8b).

Patient characteristics
Sixteen EH patients and 7 APA patients who did not take spironolactone and received adrenalectomy within 6 months were enrolled in this study. The clinical data are shown in Table 1. The APA patients had significantly higher PAC, ARR, log PAC, log ARR, and lower PRA than the patients with EH. Moreover, the APA patients had a significantly higher plasma galectin-3 level than the EH patients (2.060.9 vs. 1.360.7 ng/ml, p = 0.039). The plasma galectin-3 level was borderline significantly correlated with log PAC (r = 20.365, p = 0.087), but not log PRA, log ARR, systolic blood pressure, or diastolic blood pressure (all p.0.10).

Post-adrenalectomy follow-up
One year after adrenalectomy, 3 APA patients (38%) were cured of hypertension. In serological studies, log PAC significantly decreased and log PRA significantly increased (log PAC 1.860.3 to 1.560.1; log PRA 20.860.9 to 0.260.8, both p,0.05). The plasma galectin-3 level was also significantly decreased (from 2.160.9 to 1.260.7 ng/ml, p = 0.020).

Discussion
The results of this study demonstrate a novel signal transduction pathway of galectin-3 secretion by aldosterone in macrophage cell lines, and the impact of galectin-3 on collagen formation ( Figure  9). Furthermore, we found that aldosterone could induce galectin-3 secretion by macrophages in a mouse model, and an association between aldosterone and galectin-3 in a preliminary clinical study.
Aldosterone can induce myocardial and vascular fibrosis, and aldosterone infusion with high salt intake has been shown to induce cardiac hypertrophy, myocardial interstitial fibrosis and scarring in the right and left ventricles [4]. Furthermore, aldosterone infusion has also been shown to induce increased interstitial fibrosis in the atria and pulmonary arteries [29] which is not influenced by systemic blood pressure. In a rat model, infra-renal aorta banding was found to induce hypertension and cardiac hypertrophy, but not to increase myocardial fibrosis [30].
The proposed mechanism of aldosterone-induced myocardial fibrosis has changed in recent years. Aldosterone-induced macrophage activation and low grade inflammation have been proven to play important roles in cardiac fibrosis [2]. Furthermore, Rickard et al demonstrated that specific deletion of macrophage MRs prevented cardiac fibrosis induced by deoxycorticosterone in a mouse model [11].
Galectin-3 has also been reported to play an important role in failing hearts and HF-related cardiac fibrosis. Sharma et al reported that galectin-3 was one of the most robustly overexpressed mediators in failing hearts in microarray studies [12]. Ochieng et al reported that galectin-3 interacts with various ligands in the extracellular matrix (ECM), including laminin, synexin, integrins, and collagen [31], and Sharma et al suggested that activated cardiac macrophages produce galectin-3 and recognize the galectin-3 binding sites in cardiac fibroblasts and the ECM [12]. Sharma et al also showed that intrapericardial infusion of recombinant galectin-3 in healthy rats can result in a depressed LV ejection fraction and increased collagen volume fraction in the myocardium [12]. Further, they showed that cardiac macrophages were activated in failure-prone rat hearts, and that the expression of galectin-3 was increased and colocalized with activated myocardial macrophages [12]. Sharma et al also found galectin-3 binding sites in the ECM, and cardiac fibroblasts and galectin-3-induced cardiac fibroblast proliferation, collagen production, and cardiac dysfunction in rats. These findings support the relationship between inflammation and fibrosis, and provide a new insight into the mechanism in ECM turnover in HF. Further, the findings imply that galectin-3 may play an important role in the regulation of myocardial fibrosis and pathogenesis of HF. In our previous study on HF patients, serum galectin-3 was found to be significantly correlated with serum markers of the ECM, and that ECM turnover is an essential process in LV remodeling and interstitial fibrosis [15].
Galectin-3 is also a crucial factor in hyperaldosteronism in combination with hypertension-induced cardiac fibrosis [13] and vascular fibrosis [20]. A previous study reported that hyperaldo- steronism combined with hypertension stimulated macrophage infiltration in the heart, and enhanced the mRNA level of galectin-3 [13]. Furthermore, blocking the MRs with eplerenone could reverse the effect of aldosterone on cardiac fibrosis and cardiac galectin-3 mRNA production. These findings not only demonstrate the importance of aldosterone on cardiac fibrosis, but also suggest the possible effect of galectin-3 secretion by aldosterone. Our study provides further evidence of galectin-3 secretion by aldosterone stimulation and the possible signal transduction pathway of the process. In addition, this is the first study to provide human evidence of increased galectin-3 secretion in patients with hyperaldosteronism and the effect of adrenalectomy on galectin-3 level.
In the current study, aldosterone-induced galectin-3 secretion started from 10 27 M (1*10 27 M in the THP-1 cell study and 2.5*10 27 M in the RAW 264.7 cell study). A normal human PAC is around 0.2-1.0 *10 29 M, and the PAC in patients with PA is 2 to 10-fold higher than normal subjects. However, PA is a chronic disease. To investigate the long-term effects of aldosterone exposure we used a relatively high aldosterone concentration in the cell and animal studies.
Calvier et al detected intracellular galectin-3 mRNA and protein formation after aldosterone stimulation in vascular smooth muscle cells, and concluded that galectin-3 is essential in aldosterone-induced collagen production in vascular smooth muscle cells [32]. The aldosterone effect was found to be significant Conditional medium as described in (A) was used to treat 293T cells. After 24 hours, the expressions of fibronectin and procollagen (I) mRNA were determined by real-time quantitative RT-PCR. Data were compared between the indicated groups. A paired Student's t-test was used to evaluate statistically significant differences between the indicated groups. * P,0.05. (D) Effect of galectin-3 siRNA treatment on inhibiting aldosterone-induced galectin-3 expression. Raw 264.7 cells were pretreated with galectin-3 siRNA or control siRNA for 24 h, and then serum-starved for 24 h prior to 10 26 M aldosterone or vehicle treatment for 24 h. The conditional medium was collected, and the level of aldosterone was detected by enzyme immunoassay. (E) Effect of conditional medium from aldosterone-treated RAW 264.7 cells on fibroblast cell growth. The conditional medium was collected and cultured with the mouse fibroblast cell line NIH/3T3. The cell growth was determined by MTT assay. Data were recorded as relative cell growth in the indicated conditions with the vehicle-treated conditional medium in lane 1 defined as 100%. *, P,0.05 (n = 5). (F) Effect of conditional medium from aldosterone-treated RAW 264.7 cells on fibronectin and procollagen (I) mRNA expressions. Conditional medium as described in (E) was used to treat NIH/3T3 cells. After 24 h, the expressions of fibronectin and procollagen (I) mRNA were determined by real-time quantitative RT-PCR. Data were compared between the indicated groups. A paired Student's t-test was used to evaluate statistically significant differences between the indicated groups. *P,0.05. AD = aldosterone. doi:10.1371/journal.pone.0095254.g007 from 10 29 M in that study, which is lower than the dosage used in the current study. However, there are several differences between the two studies. First, Calvier et al detected intracellular galectin-3 mRNA and protein, and as a result, vascular smooth muscle cells acted as both producers and effectors of galectin-3. This result, however, fails to explain the findings of the study by Rickard et al [11], in which deletion of macrophage MRs was found to protect the process of cardiac fibrosis, implying that macrophage MRs are involved in the formation of cardiac fibrosis. Second, we detected the secretion of galectin-3 from macrophages in our study, whereas Calvier et al detected intracellular galectin-3 mRNA and protein, and it would be easier to detect changes in galectin-3 level intracellularly under the stimulation of a lower aldosterone concentration. Third, the tissue concentration of aldosterone is different from the plasma concentration. In the process of aldosterone-induced myocardial fibrosis, macrophages may be activated in cardiac tissue rather than in peripheral blood. In a previous study, the tissue aldosterone concentration was reported to be 6 pg/mg in atrial tissue [33]. However, the tissue concentration is measured from total tissue extract diluted in a fixed volume. Therefore, PAC and tissue aldosterone concentration cannot be compared directly.  Values are mean 6 SD or number (percentage). APA = aldosterone producing adenoma; EH = essential hypertension; SBP = systolic blood pressure; DBP = diastolic blood pressure; PAC = plasma aldosterone concentration; PRA = plasma renin activity; ARR = aldosterone-renin ratio. doi:10.1371/journal.pone.0095254.t001 There are some limitations to this study. First, this study focused on the process of aldosterone-induced galectin-3 secretion, and the relationship between aldosterone-induced galectin-3 secretion and fibrosis was only studied in cellular experiments. Further animal and human studies related to this topic are needed to obtain a full understanding of the mechanism. Second, the number of patients in the clinical study was small, and the clinical findings of this study should be confirmed by a larger clinical study. Further, the correlation between plasma aldosterone and galectin-3 concentration was borderline significant. This relationship should also be studied in a larger clinical study.

Conclusion
Aldosterone could induce galectin-3 secretion in macrophage cell lines and further enhance collagen formation by fibroblasts. The pathway was through mineralocorticoid receptors, and the PI3K/Akt and NF-kB transcription pathways. Galectin-3 secretion induced by aldosterone was further confirmed by an aldosterone-infusing mouse model. The association between aldosterone and galectin-3 was also found in a preliminary clinical study.