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Keywords:

  • Angiotensin;
  • Bone marrow stromal cells;
  • Transdifferentiation;
  • Stem cell transplantation

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

To improve the modest efficacy of mesenchymal stem cell (MSC) transplantation, the treatment of human MSCs with angiotensin receptor blockers (ARBs) was investigated. MSCs were cultured with or without the medium containing 3 μmol/l of ARBs before cardiomyogenic induction. After cardiomyogenic induction in vitro, cardiomyogenic transdifferentiation efficiency (CTE) was calculated by immunocytochemistry using anticardiac troponin-I antibody. In the nude rat chronic myocardial infarction model, we injected MSCs pretreated with candesartan (A-BM; n = 18) or injected MSCs without pretreatment of candesartan (BM; n = 25), each having survived for 2 weeks. The left ventricular function, as measured by echocardiogram, was compared with cardiomyogenic transdifferentiation in vivo, as determined by immunohistochemistry. Pretreatment with ARBs significantly increased the CTE in vitro (10.1 ± 0.8 n = 12 vs. 4.6 ± 0.3% n = 25, p < .05). Transplantation of candesartan-pretreated MSCs significantly improved the change in left ventricular ejection fraction (BM; −7.2 ± 2.0 vs. A-BM; 3.3 ± 2.3%). Immunohistochemistry revealed significant improvement of cardiomyogenic transdifferentiation in A-BM in vivo (BM; 0 ± 0 vs. A-BM; 0.014 ± 0.006%). Transplantation of ARB-pretreated MSCs significantly improved cardiac function and can be a promising cardiac stem cell source from which to expect cardiomyogenesis. STEM CELLS 2011;29:1405–1414


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Regeneration therapies have attracted a great deal of medical attention. Various cellular resources such as embryonic stem cells [1], mesenchymal stem cells (MSCs) [2], mononuclear cells [3, 4], and endothelial progenitor cells (EPCs) [5] have been candidates for the regeneration therapies. The majority of cells derived from bone marrow (BM) consist of blood cells in various stages of differentiation; however, BM also contains, hematopoietic stem cells, EPCs, and MSCs. MSCs have characteristics of replication competence and multipotency [2, 6–8], as reported in numerous studies of MSCs.

Mesenchymal cells are classified as somatic stem cells and exist in BM stroma, dermis, skeletal muscle, uterine endometrial gland [9], umbilical cord blood [7, 10], placenta [11], amniotic membrane [6], etc. They are known to be capable of transdifferentiating into bone, cartilage, skeletal muscles, fats, ligaments, vascular endothelium, smooth muscle, and cardiomyocytes. Among the various mesenchymal cell sources, BM-derived MSCs (BM-MSCs) can be used in an autologous manner; therefore, there are no immunological problems in transplantations. However, in terms of cardiomyogenic transdifferentiation, the efficiency of human BM-MSCs is extremely low [8] in vitro, and efficiency of human BM-MSC transplantation is modest in in vivo [12, 13] and in clinical trials [14, 15]. The limited effect in clinical trials may be due to low angiogenic and paracrine effect of human BM-MSCs, low cardioprotective effect on host myocardium, and partially due to low cardiomyogenic transdifferentiation efficiency (CTE) [8]. We have previously shown that human mesenchymal cells derived from younger populations, that is, endometrial gland [9], umbilical cord blood [10], placenta [11], and amniotic membrane [6] have a high CTE and a beneficial effect on cardiac function. Therefore, we hypothesized that mesenchymal cells obtained from younger populations might have a better effect on regeneration therapies. As angiotensin receptor blocker (ARB) was known to have the potential to play a role in the anti-aging effect, we postulated that ARB might improve the efficacy of BM-MSCs on cardiac stem cell therapy.

Stimulation of angiotensin receptors is known to be related to adipogenic transdifferentiation of human BM-MSCs [16]. In the brain ischemic reperfusion model, BM-MSC transplantation significantly reduced the brain infarction area via improvement of brain blood flow and reduction of oxidative stress [17]. The effect of BM-MSC transplantation was abolished by knocking out the angiotensin-II (AT) receptor type-II (AT2R). On the other hand, this effect was restored by pretreatment with ARB for BM-MSCs in the culture. These facts suggest that ARB and stimulation of AT receptor may play a significant role in causing the angiogenesic effect of BM-MSC transplantation. Therefore, in this study, we investigated the effect of ARB on CTE of human BM-MSCs in vitro and in vivo, and efficacy of BM-MSC transplantation on cardiac function in the myocardial infarction (MI) model in vivo.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

BM-Derived MSCs

Yub623 (RIKEN Cell bank, Cell No. HMS0017, Tokyo, Japan) cells were used as BM-MSCs in this study. Yub623 is a fibroblast-like shaped human MSC (hMSC) derived from neonatal human BM from a finger of patients with polydactyly. Cells were cultured in high-glucose supplemented Dulbecco's modified Eagle's medium containing 10% human serum.

Cardiomyogenic Induction and Chemical Agents

The method of cardiomyogenic induction in vitro was described previously (Supporting Information Material and Method-1) [6, 8–11]. In short, enhanced green fluorescent protein (EGFP) labeled BM-MSCs were cocultured with murine cardiomyocytes. In this system, the incidence of cell fusion was approximately 0.3% and the evidence of cell fusion-independent cardiomyogenesis was extensively shown in the previous studies [6, 8–11, 18, 19]. BM-MSCs were preincubated with chemical agent-containing medium for 2 weeks before coculture and/or cultured with chemical agent-containing medium after coculture. In this study, we used 3 μmol/l of telmisartan (tel), candesartan (cnd), losartan (los), olmesartan (olm), and valsartan (val) as an AT receptor blocker (ARB), 3 μmol/l of PD123319 (pd) as a specific AT type-I blocker; enalaprilat (ena) and captopril (cap) as an angiotensin converting enzyme (ACE) inhibitor; 3 μmol/l of aliskiren (ali) as a direct rennin inhibitor; 1 μmol/l of AT; and 10 μmol/l of GW9662 (gw) as a peroxisome proliferators-activated receptor-γ (PPAR-γ) blocker. Evaluation of efficiency of cardiomyogenic transdifferentiation was described previously [6, 10, 11]. In short, cocultivated BM-MSCs were enzymatically isolated, a smear sample was made, and then immunocytochemistry using mouse monoclonal antibody against anticardiac troponin-I (Trop-I, #4T21 Hytest, Euro, Finland) antibody was performed (described later). Isolated cells (spherical shape), in which Trop-I colocalized with EGFP at the cytoplasm were considered as Trop-I/EGFP double positive cells. The CTE was defined as the incidence of Trop-I/EGFP double positive cells in EGFP-positive BM-MSCs. The incidence of cell fusion was not affected by ARB treatment (0.30% to 0.39%) in this study.

Immunocytochemistry and Immunohistochemistry

A laser confocal microscope (FV1000, Olympus, Tokyo, Japan) was used. As described previously [6, 8–11, 18, 19], samples were stained with Trop-I with mouse monoclonal antibody (sigma) and rabbit polyclonal anti-connexin 43 antibody (sigma) diluted 1:300 overnight at 4°C, then stained with TRITC-conjugated anti-mouse IgG antibody (Sigma) and Cy5-conjugated anti-rabbit IgG antibody (Chemicon) diluted 1:100, containing 4′-6-diamidino-2-phenylindole (Wako) at 1:300 for 30 minutes at 25–28°C.

Enzyme-Linked Immunosorbent Assay

Angiogenic humoral factors (angiogenin, angiotensin-2, epidermal growth factor [EGF], basic fibroblast growth factor, heparin-binding EGF-like growth factor, hepatocyte growth factor, phosphatidylinositol-glycan biosynthesis class F protein, and vascular endothelial growth factor) in culture medium supernatant (cultured with 10% serum-containing medium for 7 days) were measured by enzyme-linked immunosorbent assay [19]. The assay was performed with Quantibody Human Angiogenesis Array I kit (RayBiotec, Inc. GA) and was conducted according to manufacturer recommended protocol.

Gene Chip Analysis

Human genome-wide gene expression was examined with the Human Genome U133A Probe array (Affymetrix), which contains the oligonucleotide probe set for approximately 23,000 full-length genes and expressed sequence tags as described previously [11, 20].

Transplantation of ARB-Pretreated BM-MSCs in MI Model In Vivo

MI was induced in the open chests of anesthetized female F344 nude rats (Clea Japan, Inc., 6 weeks of age) as described previously [6, 9, 19]. Two weeks after MI, 1–2 × 106 of EGFP-labeled BM-MSCs were injected into the myocardium at the border zone of the MI. Two weeks after the first operation, rats with MI were randomized in a blind study of the following groups: the sham operated group (Sham), the (CNT), the CNT with plain BM-MSC transplanted group (BM), and the MI+candesartan-pretreated BM-MSC transplanted group (A-BM). After cellular transplantation, TCV-116 (stable form of candesartan; 0.5 mg/kg/day) was orally administered in some of the experiments (+A). Randomization occurred immediately before echocardiogram. Immediately before cell transplantation, two-dimensional and M-mode echocardiographic (8.5 MHz linear transducer; EnVisor C, Phillips Medical System, Andover, MA) images were obtained to assess left ventricular (LV) end-diastolic dimension and LV end-systolic dimension (LVESD) at the mid-papillary muscle level by a single blinded observer. Two weeks after the transplantation, a similar echocardiogram was performed again. LV percentage fractional shortening, thickness of anterior wall (AW), and thickness of posterior wall were calculated from five to six traces and averaged. LV pressure, brain natriuretic peptide (BNP), body weight, and heart weight (wet) were measured as described previously. Tissue samples were obtained by slicing along the short axis of the left ventricle, for every 1 mm of depth. After masson trichrom staining, the area of fibrosis was digitized from each slice, and then the percentage fibrosis volume in the LV myocardium was calculated as described previously [6, 19]. Immunohistochemical analysis was performed to observe CTE in vivo as described previously (Supporting Information Material and Method-2). Immunohistochemical analysis was performed using anti-rat CD34 antibody (1:200 R&D Systems; AF4117) to evaluate vascular density. Then, biotinylated goat immunoglobulins (Dako; E0466) were used as a second antibody, next, strept avidin biotin complex (ABC) complex/horseradish peroxidase (HRP) (Dako; K0377), and, finally, 3,3′-Diaminobenzidine substrate (Wako; K3183500) were used. The images were digitized and the percentage brown pixel area of the capillary vessels was counted in the peri-infarct normal zone (NZ) and the center of the MI zone (MI) using a light microscope at 10× magnification. The areas in five high-power fields were calculated and averaged.

Statistical Analysis

All data are shown as mean value ± SE. The difference between mean values was determined with one-way analysis of variance (ANOVA) test or one-way repeated measures ANOVA test and Bonferroni post hoc test. Statistical significance was set at p < .05.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Pretreatment with ARB Increased Efficiency of Cardiomyogenic Transdifferentiation Via AT2R

Administration of 3 μmol/l of popular ARBs (tel, can, los, olm, and val) did not cause any significant change in morphology of BM-MSCs (Supporting Information Fig. 1A, 1B), while improved CTE in vitro was observed (Fig. 1A and Supporting Information Fig. 1C–1P). In our pilot study, we tested dose-response effect of ARBs and confirmed that this effect was saturated at the concentration of 3 μmol/l (CTE at control, 0.03, 0.3, 3, and 30 μmol/l of cnd were 3.0 ± 0.3, 3.5 ± 0.2, 4.8 ± 0.3, 8.9 ± 0.4, and 8.1 ± 0.5%, respectively). Therefore, in this study, we selected 3 μmol/l as a default concentration of ARBs. To clarify the target of the ARBs, val was administrated only before the coculture or only after the coculture (Fig. 1B). Administration of val after the start of coculture (±) caused modest improvement of CTE; on the other hand, administration of val before the start of coculture (±) significantly increased CTE, suggesting that val modified the character of the BM-MSCs so as to be able to cause higher CTE. To determine whether the effect of the ARBs was mediated by AT receptor type-I (AT1R) or AT2R, we used val as AT1R specific blocker and pd as AT2R specific blocker (Fig. 1C). Administration of pd did not affect CTE, while val increased CTE significantly. Furthermore, CTE with both val and pd administered did not show an additional increase (rather, tended to show a statistically nonsignificant decrease). These data suggest that blockade of AT1R plays a pivotal role in ARB-dependent CTE increase. We have previously reported that PPAR-γ activator has an ability to increase CTE of BM-MSCs [19], and some of the ARBs, that is, tel, have a potential to activate the PPAR-γ. To clarify that the mechanism of ARB-induced CTE increase was mediated via PPAR-γ activation effect, we used gw as a specific blocker for PPAR-γ (Fig. 1D). The gw partially blocked tel-induced CTE increase; on the other hand, it did not block cnd-induced CTE increase. These data suggest that the effect of cnd on CTE was independent from PPAR-γ activation. In our previous study, the effect of pio was completely blocked by gw [19]; therefore, the gw-insensitive tel-induced CTE increase was caused by a PPAR-γ-independent mechanism. On the other hand, administration of AT did not affect CTE in the absence of ARB, while administration of AT significantly increased CTE in the presence of ARB (Fig. 1E). These data suggest both blockade of AT1R and stimulation of AT2R increase CTE. The increase in CTE was also observed by administration of ACE inhibitors ena or cap (Fig. 1F), suggesting the source of AT in this system is autocrine of angiotensin-I from BM-MSCs and local ACE activity. Furthermore, the effect was not blocked by the specific renin blocker, ali (Fig. 1G); therefore, angiotensinogen does not play a role as an AT source in this system, but a local angiotensin-generating system may play a role in this phenomenon.

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Figure 1. Improvement of cardiomyogenic transdifferentiation efficiency (CTE) of bone marrow-derived mesenchymal stem cells (BM-MSC) by blockade of renin-angiotensin system in vitro. The calculated rate of cardiac troponin-I positive cells in enhanced green fluorescent protein-positive cells are averaged and shown as CTE. (A): The effect of pretreatment with telmisartan (tel), candesartan (cnd), losartan (los), olmesartan (olm), and valsartan (val) on CTE of human BM-MSCs are shown. CNT denoted CTE of control MSCs. These ARBs increase CTE significantly. (B): Condition of pretreatment of val (before slash) and val treatment after induction (after slash) are shown in the bottom. Pretreatment of val significantly increased CTE and was essential for val-induced CTE increase. Val treatment after induction moderately increased CTE. (C): The effect of combination of val as a specific angiotensin-II (AT) receptor type-I (AT1R) blocker and PD123319 (pd) as a specific AT2R blocker to CTE is shown. The pd did not affect CTE. (D): The effect of GW9662 (gw) as a specific peroxisome proliferators-activated receptor-γ (PPAR-γ) blocker on tel-induced CTE increase and cnd-induced CTE increase are shown. The blockade of PPAR-γ partially blocked the tel-induced CTE increase and did not affect cnd-induced CTE increase. (E): The effect of additional application AT in the presence or in the absence of cnd is shown. AT alone did not affect CTE; however, AT significantly increased CTE in the presence of cnd. (F): Dose-response effect of pretreatment with enalaprilat (ena) and captoril (cap) as angiotensin converting enzyme inhibitors (ACEI). ACEI significantly improves CTE in a dose-dependent manner. (G): The effect of aliskiren (ali) as a renin inhibitor on CTE is shown. Ali did not affect CTE. *p<0.05. Abbreviations: ali, aliskiren; AT, angiotensin-II; cap, captoril; cnd, candesartan; CNT, control; EGFP, enhanced green fluorescent protein; ena, enalaprilat; gw, GW9662; los, losartan; olm, olmesartan; pd, PD123319; Tel, telmisartan; Trop-I, troponin-I; val, valsartan.

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The Effect of ARB-Treated BM-MSC Transplantation on Cardiac Function In Vivo

The BM-MSCs were transplanted into the hearts of nude rats with chronic MI, in vivo, and the effect on cardiac function was examined. Representative M-mode echocardiographic images at 2 weeks after transplantation are shown (Fig. 2A). In the CNT group, akinesis and thinning of AW are observed. There were no marked changes in the BM group, while in A-BM group, the motion of AW markedly improved. The same trend was also observed in the ARB orally administered group (+A group). The changes in echocardiographic parameters between the immediately before the transplantation group (post MI 2 weeks) and the 2 weeks after transplantation group (post MI 4 weeks) are compared (Fig. 3). Changes in LV ejection fraction (ΔLVEF) were decreased as a function of time, even 2 weeks after the MI, which may be due to LV remodeling. The transplantation of plain BM-MSCs (BM) did not have an effect on ΔLVEF; on the other hand, candesartan-pretreated BM-MSCs (A-BM) significantly improved ΔLVEF. The degree of improvement was marked when candesartan was orally administered (A-BM-A). Change in end-diastolic diameter of LV (ΔLVEDD) did not differ among the groups; on the other hand, change in LVESD (ΔLVESD) was significantly improved in A-BM group (vs. BM group) and A-BM+A group (vs. BM+A group), suggesting transplantation of candesartan-pretreated BM-MSCs significantly improved systolic function. Other echocardiographic parameter did not differ among the groups. There was no difference in the changes in body weight, serum BNP concentration, heart weight, LV systolic pressure, or LV end-diastolic pressure among the groups (Fig. 4). LV dP/dt was significantly improved by candesartan-pretreatment (A-BM vs. BM) with BM-MSCs; however, there was no additional effect of candesartan-pretreatment in the group of candesartan oral administration group (N.S. CNT-A vs. A-BM+A).

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Figure 2. Confocal laser microscopic images of the immunocytochemical analysis of transdifferentiated cardiomyocytes. Confocal microscopic images of immunocytochemistry after cardiomyogenic induction using anti-cardiac troponin-I (red: Trop-I) revealed significant augmentation of enhanced green fluorescent protein (EGFP) (green)/Trop-I double positive cardiomyocytes (white arrow) by candesartan (cnd) (D–F) pretreatment, while EGFP/Trop-I double positive cells were rare in CNT (A–C). Area within the dotted yellow box is expanded and shown in (G, H). Clear striation staining pattern of Trop-I was observed in every EGFP-positive cell. The striating pattern of EGFP and Trop-I appeared in alternation, suggesting that the Trop-I was expressed in the EGFP-positive cells. Scale bar = 20 μm. Abbreviations: cnd, candesartan; CNT, control; DAPI, 4′-6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; Trop-I, troponin-I.

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Figure 3. Effect of candesartan-pretreated bone marrow-derived mesenchymal stem cell (BM-MSC) transplantation and/or oral administration of candesartan on echocardiographic parameters in vivo. (A): Representative trace of M-mode echocardiogram from Sham-operated nude rats, control myocardial infarction (MI) (CNT), MI with BM-MSCs transplantation (BM), candesartan-pretreated BM (A-BM), and oral administration of candesartan after the transplantation (CNT+A, BM+A, A-BM+A) is shown. Changes in left ventricular ejection fraction (LVEF) from 2 to 4 weeks (B; ΔLVEF), LV end-diastolic dimension (C; ΔLVEDD), and LV end-systolic dimension (D; ΔLVESD) are averaged and shown. (E): Calculated LVEF from each group at 2 weeks after first operation are shown. There was no statistical significance; however, the degree of percentage EF tends to be worse in the oral administration series (right columns separated by dotted bar). Candesartan-pretreated BM significantly improved LVESD, consequently improved LVEF. *p<0.05. Abbreviations: BM, bone marrow; CNT, control; LVEDD, left ventricular end-diastolic dimension; LVEF, left ventricular ejection fraction; LVESD, left ventricular end-systolic dimension.

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Figure 4. Effect of candesartan-pretreated bone marrow-derived mesenchymal stem cell (BM-MSC) transplantation and/or oral administration of candesartan on body weight, serum BNP concentration, and hemodynamic parameters. There was no difference in (A) changes in body weight, (B) heart weight, (C) BNP concentration, (D) left ventricular (LV) end-systolic pressure, or (E) end-diastolic pressure. (D): Effect of BM-MSCs on LV positive dP/dt is significantly improved by pretreatment with pioglitazone. (F): The LV dP/dt was significantly improved by transplantation of candesartan-pretreated BM-MSC (A-BM). *p<0.05. Abbreviations: BM, bone marrow; BNP, brain natriuretic peptide; CNT, control MI; LV, left ventricle; LVEDP, left ventricular end- pressure; LVSP, left ventricular systolic pressure.

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In this study, the beneficial effect was observed even in the ARB-pretreated BM-MSC transplantation group. The effect of ARB is known to cause an irreversible biological change in the cell, the “so-called” memory effect; therefore, such memory effect might affect cardiac function in vivo. To check this possibility, we cultured three groups of BM-MSCs: cells with candesartan for 2 weeks (ARB), cells without candesartan (CNT), and cells with candesartan for 1 week followed by 1 week without candesartan (1 week-ARB: wash-out for 1 week). The GeneChip analysis was performed among them, then the hierarchical clustering was used using the average distance method [20]. The gene expression pattern of 1 week-ARB was similar to CNT; therefore, the effect of ARB on BM-MSCs was reversible from the aspect of genechip analysis.

Incidence of Myocardial Transdifferentiation of ARB-Pretreated BM-MSCs In Vivo

To evaluate myocardial transdifferentiation of BM-MSCs in vivo, immunohistochemical analysis was performed. Antibodies against cardiac troponin-I (Trop-I) and connexin 43 were used. Confocal laser microscopic images could not detect EGFP-positive cardiomyocytes having clear striation staining pattern of Trop-I in the BM group. Sometimes enucleated EGFP-positive fragments of the cell at the center of the MI zone were observed, but taking the number of the injected EGFP-positive cells into account, the incidence seemed to be rare, as was reported previously [6, 19]. On the other hand, EGFP-positive and Trop-I double positive cells with clear striation staining pattern were observed at the marginal zone of the MI area in the candesartan-pretreated BM-MSC transplanted group (A-BM, Fig. 5F–5I). The oral administration of candesartan increased the incidence of survival of the EGFP/Trop-I double positive cells in vivo (A-BM+A, Fig. 5A–5E, 5J).

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Figure 5. Both pretreatment and oral administration of candesartan significantly improved the incidence of survival of bone marrow-derived mesenchymal stem cell (BM-MSC)-derived cardiomyocytes in vivo. Confocal laser microscopic image of immunohistochemistry using anti-cardiac troponin-I antibody (red; Trop-I) is shown. (A–C): Lower magnification view for enhanced green fluorescent protein (EGFP) (green; A), Trop-I (B), and 4′-6-diamidino-2-phenylindole (Blue; E) is shown. After transplantation of candesartan-pretreated BM-MSCs in the presence of oral administration of candesartan (A-BM+A), EGFP-positive cells can be observed at the margin of the myocardial infarction (MI), but there were many EGFP/Trop-I double positive cardiomyocytes survived at the peri-MI zone (A). (D): Higher magnification view of merged image is shown. (E): The Trop-I positive cells are surrounded by dot-like staining of connexin 43 (white; Cx43). (F): Higher magnification view clearly shows striation staining pattern of Trop-I in the EGFP-positive cells. (G): At the center of MI zone (A-BM group), many EGFP-positive cells were enucleated and were negative for Trop-I. (H, I): However, there were some EGFP, Trop-I double positive rod-shaped cells at the center of MI zone. (J): The percentage of EGFP/Trop-I double positive cells in the injected EGFP-positive cells was averaged and is shown. By pretreatement with candesartan, the rate was significantly improved (A-BM vs. BM), and oral administration of candesartan additionally improved the incidence of EGFP/Trop-I double positive cells in vivo. Scale bars = 50 μm (A–C, E, F), = 100 μm (D), = 200 μm (G), and = 25 μm (H, I), respectively. *p<0.05. Abbreviations: BM, bone marrow; DAPI, 4′-6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; Trop-I, troponin-I.

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Genesis of Angiogenic Humoral Factors Derived from BM-MSCs by ARB

Angiogenic humoral factors were detected in the supernatant of the culture medium of BM-MSCs, suggesting that they are secreted from BM-MSCs, as reported previously [19]. However, the administration of 3 μmol/l of candesartan did not significantly affect the concentration of these angiogenic factors (Fig. 6). On the other hand, the angiogenic effect of candesartan-pretreated BM-MSCs was observed in vivo (Fig. 7A, 7B). In the peri-MI NZ, a CD34 positive area was not different among CNT, BM, and A-BM groups (without oral administration of candesartan). On the other hand, in the MI area, a CD34 positive area was significantly higher in A-BM group (vs. BM group). Oral administration of candesartan, significantly increased the CD34 area (CNT+A vs. CNT) in the peri-MI normal area and significantly increased it in the MI area. Masson trichrome staining and calculated MI volume at 2 weeks after transplantation (Fig. 7C, 7D) showed significant reduction of MI volume by pretreatment with candesartan of engrafted BM-MSCs (BM vs. A-BM) and the effect of pretreatment was not significantly augmented by the oral administration of candesartan.

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Figure 6. Secretion of angiogenic humoral factors from bone marrow-derived mesenchymal stem cells (BM-MSCs) into the culture medium supernatant and the effect of candesartan in vitro. Concentration of angiogenic humoral factors in (A) angiogenin, (B) angiotensin-2 (ANG-2), (C) epidermal growth factor (EGF), (D) basic fibroblast growth factor, (E) heparin-binding EGF-like growth factor, (F) hepatocyte growth factor, (G) phosphatidylinositol-glycan biosynthesis class F protein, and (H) vascular endothelial growth factor in culture medium was measured by enzyme-linked immunosorbent assay and averaged. Candesartan (cnd) treatment did not cause any significant change in angiogenic humoral factors secretion from BM-MSCs into the culture medium. Abbreviations: ANG-2, angiotensin-2; bFGF, basic fibroblast growth factor; cnd, candesartan; EGF, epidermal growth factor; HB-EGF, heparin-binding EGF-like growth factor; HGF, hepatocyte growth factor; hMSC, human mesenchymal stem cell; PIGF, phosphatidylinositol-glycan biosynthesis class F protein; VEGF, vascular endothelial growth factor.

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Figure 7. Effect of bone marrow-derived mesenchymal stem cell (BM-MSC) transplantation and/or treatment with candesartan on vessel density and infarction size in the heart in vivo. (A): The percentage of CD34 positive area in control myocardial infarction (MI) (CNT), MI with bone candesartan-pretreated BM-MSCs transplantation (BM), candesartan-pretreated BM (A-BM), and additional oral administration of candesartan after the transplantation (CNT+A, BM+A, A-BM+A) are calculated and averaged. (B): Representative microscopic image of immunohistochemistry using anti-CD34 antibody to detect vessels at center of MI zone and peri-MI normal zone (non-MI) are shown. Scale bar = 20 μm. Pretreatment with candesartan significantly increased vessel density at MI zone; on the other hand, oral administration of candesartan significantly increased vessel density at non-MI zone. (C): Representative masson-trichrom staining of the heart at the tendinous cord level of CNT, BM, and A-BM are shown. The digitized data were measured and calculated in (D). By the candesartan-pretreatment, BM-MSC transplantation significantly decreased in percentage fibrosis volume. Scale bar = 5 mm. *p<0.05. Abbreviations: BM, bone marrow; CNT, control; MI, myocardial infarction.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

The Effect of Pretreatment with ARB in Human Neonatal BM-MSCs

The ARB did not affect the morphology of BM-MSCs and did not increase secretion of angiogenic humoral factors from BM-MSCs. The pretreatment with ARB significantly increased the CTE in vitro and in vivo. As pretreatment with ARB was essential for the effect on CTE, we concluded that the effect of ARB is not mediated by murine cultured myocardium, but directly affects BM-MSCs themselves, modifying the character of BM-MSCs. As the effect was not mediated by PD123319 as a selective AT2R blocker, the effect of ARB was mediated by the blockade of AT1R. In our previous article [19], activation of PPAR-γ significantly increased the CTE in BM-MSCs and the effect was completely blocked by GW9662, as a specific blocker of PPAR-γ receptor. The effect of telmisartan, which is known to have the strongest PPAR-γ activation activity among the ARBs, on CTE was partially blocked by GW9662, suggesting that the effect of ARBs is not mediated by PPAR-γ receptor activation activity. The molecular mechanism of the effect of ARBs on CTE is still unclear. Further experiments should be done.

In the absence of valsartan as an AT1R selective blocker, administration of AT did not affect CTE; however, in the presence of valsartan, AT significantly increased CTE, suggesting that the relative stimulation of AT2R increased CTE. Furthermore, AT in culture medium seems to be generated by ACE activity in BM-MSCs, as the administration of ACE inhibitor to the BM-MSCs in culture significantly increased CTE in vitro. Furthermore, aliskiren did not affect the CTE; therefore, rennin and angiotensinogen did not play a role, but the angiotensin-I in the culture medium or autocrine from BM-MSCs must be a major source for AT.

Mechanism of Improving Systolic Function with ARB

Although EGFP-positive cardiomyocytes were observed in the candesartan-treated BM-MSC transplanted group, the number of them seems to be low for causing improvement in systolic function in vivo, as was seen in this study.

Concordant with the previous in vivo study [8] and clinical study [14], in the absence of BM-MSC transplantation, oral administration of candesartan suppressed the post-MI LV remodeling and progressive worsening of LVEF (CNT vs. CNT+A) at 2 weeks after MI. Furthermore, in this study, even in the absence of oral administration, the beneficial effect was observed in the candesartan-pretreated BM-MSC transplantation group. In this study, the effect of default BM-MSC transplantation was modest and there was no statistical significance from the control MI group. These data suggest that the ARBs modify the biology of BM-MSC, which play an important role in suppressing post-MI LV remodeling. This trend was observed in hemodynamic parameters and histological data. Pretreatment with candesartan significantly improved the efficacy of BM-MSC transplantation in augmentation of LV dP/dt and reduction in MI volume. Such cardioprotective effect of ARB-pretreated BM-MSCs may be due to augmentation of angiogenic effect and/or anti-apoptotic paracrine effect of BM-MSCs by pretreatment with ARB. The beneficial effect of ARB-pretreated BM-MSCs was also reported in the ischemia-reperfusion brain injury model [17], in which it was pointed out that both the stimulation of AT2R and blockade of AT1R have a significant effect on reducing brain damage in vivo and this data well correlated with our CTE data in vitro. In this study, the effect can be observed even by BM-MSC transplantation at 2 weeks after MI; therefore, the BM-MSC-induced angiogenesis might have suppressed ongoing post-MI LV remodeling. In this study, there was discrepancy between the angiogenic effect of ARB-pretreatment in BM-MSCs in vitro and in vivo. We speculated that additional angiogenic effect of BM-MSC transplantation by ARB-pretreatment might require graft-host interaction, that is, immunological reaction or inflammation in the host myocardium.

Cell Fusion-Independent Cardiomyogenic Transdifferentiation

Extensive evidence of cell fusion-independent cardiomyogenic transdifferentiation of human MSCs was presented in our previous study [6, 9–11, 19]. In this study, the incidence of cell fusion was approximately 1% and it was not affected by ARB pretreatment; therefore, the increase in EGFP-positive cardiomyocytes by ARB treatment was due to an increase in efficiency of cardiomyogenic transdifferentiation in vitro. Furthermore, there were no EGFP/Trop-I double positive rod shaped cardiomyocytes in the default BM-MSC transplanted group; on the other hand, the appearance of significant numbers of EGFP/Trop-I double positive cardiomyocytes was observed in ARB-pretreated BM-MSC transplanted group. This suggests an improvement of CTE of BM-MSCs in vivo by ARB pretreatment. Taking into account our previous study and our present in vitro experiment, we concluded that our observed EGFP/Trop-I double positive cells in vivo are caused by cardiomyogenic transdifferentiation.

Clinical Application

The efficacy of human BM-MSC transplantation had been modest [14, 15], and a new method for BM-MSC transplantation that will gain dramatic improvement in efficacy is expected. Genetic modification, that is, over-expression of the AKT-gene was reported to improve efficacy of BM-MSC transplantation in vivo [21]; however, use of such genetically modified cells raises a safety concern, that is, tumorgenecity. In comparison with the genetic modification, modification of BM-MSCs by ARBs, which are commonly used for heart failure patients, is a method that is ready to use for clinical patients.

In addition to the beneficial efficacy for cardiac function, this experimental model may also give us a clue to improving CTE in vivo, which is very essential for cardiac regenerative therapy. The precise mechanism for cardiomyogenic transdifferentiation of human BM-MSCs has been unclear. As the incidence of cardiomyogenic transdifferentiation of human BM-MSCs is extremely rare, it has been impossible to statistically analyze the effect on CTE of various drugs or interventions in vivo. Therefore, there has been no systematic strategy for improvement of CTE of BM-MSCs until our previous article [6, 9–11, 19]. Our in vivo model of ARB-treated BM-MSCs is able to statistically analyze the effects of drugs on CTE, which is important for further improvement of CTE. In vitro, the pioglitazone's effect on CTE was independent from the effect of ARB; therefore, the additional administration of pioglitazone, as a PPAR-γ activator may be expected to improve CTE further. Further experiments should be done.

Study Limitation

In our previous study, we have used BM-MSCs obtained from a 41-year-old and a 90-year-old men. The CTE results were 1% and 0.3% in vitro [19], respectively. In this study, the CTE of default BM-MSCs from neonates was approximately 3%–5%. This data implies BM-MSCs obtained from younger generations that may have higher cardiomyogenic transdifferentiation ability. As ARB is known to have a potential for an anti-aging effect, the effect of ARB on BM-MSCs might increase the CTE by ARB's anti-aging effect on BM-MSCs. Further experiments should be done on this issue.

In vivo MI model was performed by two series (Sham, CNT, BM, A-BM series and CNT-A, BM-A, A-BM-A series) at different periods. As it was difficult to control the size of the MI at the coronary ligation, the size of the MI of later series are slightly larger (N.S.) than the former series. Therefore, we did not perform statistical analysis on some parameters between the series (separated by dotted line in the figures). The serum BNP level and the size of percentage MI volume are slightly larger in the later series. In this study, intra-individual difference values were compared with the values of the two series.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Pretreatment with angiotensin receptor blockers (ARBs) in culture activate human marrow-derived mesenchymal stem cells by angiotensin-II receptor type 1 blockade. ARBs-pretreated human marrow-derived msenchymal stem cells was significantly improved cardiomyogenic transdifferentiation efficiency in vitro and in vivo, and transplantation of the ARBs-pretreated cells significantly improved cardiac function and can be a promising cardiac stem cell source from which to expect cardiomyogenesis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

The research was partially supported by a grant from the Ministry of Education, Science and Culture, Japan. A part of this work was undertaken at the Keio Integrated Medical Research Center.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
STEM_691_sm_SuppInfo.doc30KSupporting Information
STEM_691_sm_SuppFig1.tif3461KSupporting Information Figure 1. Laser confocal microscopic view of immunocytochemistry of enzymatically isolated transdifferentiated marrow-derived mesenchymal stem cells to calculate cardiomyogenic transdifferentiation efficiency. (A,B) Phase contrast image of candesartan-administered (B; cnd) marrow-derived mesenchymal stem cells (MSCs), was not changed significantly compared to the default state (A; CNT). (C,D) Representative high magnification view of confocal laser microscopic image of immunocytochemistry is shown. After cardiomyogenic induction (co-culture with murine cardiomyocyte) cells were enzymatically isolated and a smear sample was made. Cells were stained by anti-cardiac troponin-I (Red; Trop-I, C). Trop-I was localized at the cytoplasm. (D) Merged image is shown. EGFP (Green)/Trop-I double positive cells were observed as orange, and calculated. Representative laser confocal microscopic view of telmisartan-pretreated cells (E,F, tel-), candesartan-pretreated cells (G,H, cnd-), losartan-pretreated cells (I,J, los-), olmesartan-pretreated cells (K,L, olm-), valsartan-pretreated cells (M,N, val), and default MSC (O,P, CNT) are shown. Allows denoted EGFP/Trop-I double positive cells.

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