β-Adrenergic Blocker, Carvedilol, Abolishes Ameliorating Actions of Adipose-Derived Stem Cell Sheets on Cardiac Dysfunction and Remodeling After Myocardial Infarction

Maya Adachi; Mai Watanabe; Yasutaka Kurata; Yumiko Inoue; Tomomi Notsu; Kenshiro Yamamoto; Hiromu Horie; Shogo Tanno; Maki Morita; Junichiro Miake; Toshihiro Hamada; Masanari Kuwabara; Naoe Nakasone; Haruaki Ninomiya; Motokazu Tsuneto; Yasuaki Shirayoshi; Akio Yoshida; Motonobu Nishimura; Kazuhiro Yamamoto; Ichiro Hisatome

doi:10.1253/circj.CJ-19-0261

Abstract

Background: Treatment of myocardial infarction (MI) includes inhibition of the sympathetic nervous system (SNS). Cell-based therapy using adipose-derived stem cells (ASCs) has emerged as a novel therapeutic approach to treat heart failure in MI. The purpose of this study was to determine whether a combination of ASC transplantation and SNS inhibition synergistically improves cardiac functions after MI.

Methods and Results: ASCs were isolated from fat tissues of Lewis rats. In in vitro studies using cultured ASC cells, mRNA levels of angiogenic factors under normoxia or hypoxia, and the effects of norepinephrine and a β-blocker, carvedilol, on the mRNA levels were determined. Hypoxia increased vascular endothelial growth factor (VEGF) mRNA in ASCs. Norepinephrine further increased VEGF mRNA; this effect was unaffected by carvedilol. VEGF promoted VEGF receptor phosphorylation and tube formation of human umbilical vein endothelial cells, which were inhibited by carvedilol. In in vivo studies using a rat MI model, transplanted ASC sheets improved contractile functions of MI hearts; they also facilitated neovascularization and suppressed fibrosis after MI. These beneficial effects of ASC sheets were abolished by carvedilol. The effects of ASC sheets and carvedilol on MI heart functions were confirmed by Langendorff perfusion experiments using isolated hearts.

Conclusions: ASC sheets prevented cardiac dysfunctions and remodeling after MI in a rat model via VEGF secretion. Inhibition of VEGF effects by carvedilol abolished their beneficial effects.

Developments of coronary angioplasty and bypass operation for patients with myocardial infarction (MI) have decreased their mortality and morbidity.¹ However, these treatments are still insufficient to prevent progression of cardiac remodeling and heart failure after MI. One of the reasons for this burden is activation of the sympathetic nervous system (SNS) after MI, which deteriorates cardiac functions.² Activation of β-adrenergic receptors (β-ARs) predisposes to apoptosis of cardiac myocytes,³^–⁶ suggesting the effectiveness of β-blockers. Clinical studies have shown that β-blockers could reduce incidence of cardiovascular events, as well as the mortality of patients with chronic heart failure.⁷

Carvedilol is a non-selective AR antagonist that blocks β1- and β2-ARs, as well as α1-AR.⁸ Carvedilol suppresses SNS activities, and decreases heart rate (HR) and contractility. This action is beneficial to patients with heart failure whose SNS is activated.⁹ Carvedilol also causes vasodilation and decreases peripheral vascular resistance without reflex tachycardia by concomitant blockade of α1- and β1-ARs.¹⁰

Cell-based regenerative therapy improved blood supply to the damaged heart, and minimized the area of infarction.¹¹^–¹³ These effects are mediated, in part, by cytokines such as hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) secreted by transplanted cells.¹⁴^–¹⁶ Direct injection of cells into the heart has been problematic because of a significant loss of live cells and pro-arrhythmic effects.¹⁷^,¹⁸ Tissue engineering using cell sheets has been developed to overcome these disadvantages; preparing cell sheets could improve viability of cells¹⁹^,²⁰ and prolong secretion of cytokines.²¹

Adipose-derived stem cells (ASCs), belonging to the family of mesenchymal stem cells (MSCs), secrete cytokines such as VEGF, HGF and fibroblast growth factor (FGF), and prevent cardiac dysfunction and remodeling after MI²²^,²³ by inducing neovascularization.²⁴^–²⁷ We have previously reported that ASC sheets decrease interstitial fibrosis, induce neovascularization, and prevent remodeling in rat MI hearts.

Catecholamines control mobilization and angiogenesis of bone marrow-derived endothelial progenitor cells.²⁸ Recently, norepinephrine has been reported to activate MSC chemotaxis by increasing stroma cell-derived factor-1 (SDF-1).²⁹ It has also been reported that activation of β-ARs is required to elevate α-AR expression and signaling and to regulate secretion of cytokines by MSCs.³⁰ Thus, carvedilol may influence the effects of ASC sheets on MI hearts via blocking actions of catecholamines and regulating secretion of cytokines such as VEGF.

In the present study, we examined whether administration of carvedilol before and after MI could influence ASC sheet effects on cardiac functions and remodeling after MI.

Methods

In Vitro Study

Engineering of ASC Sheets ASCs were isolated from the inguinal subcutaneous fat tissue of Lewis rats (200–250 g) and cultured as described previously.²⁴ To prepare cell sheets, ASCs (1×10⁶ cells; 3–4 passage) were cultured on 35-mm temperature-responsive culture dishes (UpCell, Cell Seed Inc., Tokyo, Japan) in an incubator at 37℃ for 48 h. They were then maintained at 20℃ for 1 h to be detached as an intact sheet.

Real-Time Reverse Transcriptase-Polymerase Chain Reaction Analysis ASCs were incubated at 37℃ under normal (21% O₂) or hypoxic (<2% O₂) conditions for 48 h using an AnaeroPack system (MITSUBISHI GAS CHEMICAL Inc., Tokyo, Japan). Total RNA was extracted from ASCs using a RNeasy Mini kit (QIAGEN inc., Valencia, CA, USA). Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of VEGF-A, HGF, FGF, hypoxia inducible factor-1α (HIF-1α), SDF-1, and β-actin was performed using the ABI 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Their mRNA levels were expressed as ratios to those of β-actin. mRNA levels of α1-, β1- and β2-ARs were determined by quantitative RT-PCR. The primers used are shown in Table.²⁴ The mRNA levels were determined by the comparative Ct method, and expressed as 2^−∆∆Ct.

Table. Polymerase Chain Reaction Primers

Gene name	Probe no.	Sequence
Rattus VEGF-A	#4	Forward 5’-TTAAACGAACGTACTTGCAGATG-3’
Rattus VEGF-A	#4	Reverse 5’-TCTAGTTCCCGAAACCCTGA-3’
Rattus HGF	#49	Forward 5’-GATTGGATCAGGACCTTGTGA-3’
Rattus HGF	#49	Reverse 5’-CCATTCTCATTTTGTGTTGTTCA-3’
Rattus bFGF	#7	Forward 5’-TCTTCCTGCGCATCCATC-3’
Rattus bFGF	#7	Reverse 5’-GCTTGGAGCTGTAGTTTGACG-3’
Rattus HIF-1α	#95	Forward 5’-AAGCACTAGACAAAGCTCACCTG-3’
Rattus HIF-1α	#95	Reverse 5’-TTGACCATATCGCTGTCCAC-3’
Rattus SDF-1	#21	Forward 5’-GCTCTGCATCAGTGACGGTA-3’
Rattus SDF-1	#21	Reverse 5’-GTTGAGGATTTTCAGATGTTTGAC-3’
Rattus β-actin	#115	Forward 5’-CTAAGGCCAACCGTGAAAAG-3’
Rattus β-actin	#115	Reverse 5’-GCCTGGATGGCTACGTACA-3’
Rattus α1-AR	–	Forward 5’-AGCCTCTGCACCATCTCTGT-3’
Rattus α1-AR	–	Reverse 5’-AAGGAGCACACGGAAGAGAA-3’
Rattus β1-AR	–	Forward 5’-GCTCTGGACTTCGGTAGACG-3’
Rattus β1-AR	–	Reverse 5’-ACTTGGGGTCGTTGTAGCAG-3’
Rattus β2-AR	–	Forward 5’-GAGCACAAAGCCCTCAAGAC-3’
Rattus β2-AR	–	Reverse 5’-TGGAAGGCAATCCTGAAATC-3’
Rattus β-actin	–	Forward 5’-CTAAGGCCAACCGTGAAAAG-3’
Rattus β-actin	–	Reverse 5’-CTCTCAGCTGTGGTGGTGAA-3’

AR, adrenergic receptor; bFGF, basic fibroblast growth factor; HGF, hepatocyte growth factor; HIF, hypoxia inducible factor; SDF, stroma cell-derived factor; VEGF, vascular endothelial growth factor.

Western Blotting ASCs were harvested and lysed by sonication in PBS supplemented with 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS and a protease mixture (Rosche Diagnostics, Tokyo, Japan). Proteins were separated on SDS-PAGE and electrotransferred to PVDF membranes. The membranes were immunoblotted with a rabbit polyclonal antibody against α1-AR or β1-AR (Abcam plc, Cambridge, UK).

Human umbilical vein endothelial cells (HUVECs; Takara Bio, Kusatsu, Japan) were harvested and lysed in PBS supplemented with 7 M urea, 2 M thiourea, 3% CHAPS and 1% TritonX-100. SDS-PAGE and membrane transfer were conducted as described above, and the membranes were proved by rabbit polyclonal antibodies against total VEGF receptor 2 (VEGFR2) and phospho-VEGFR2 (Tyr1175) (Cell Signaling Technology, Danvers, MA, USA). The blots were developed by using an ECL system (Amersham, Biosciences, Piscataway, NJ, USA).

Tube Formation Assay Matrigel matrix growth factor reduced (GFR; BD Biosciences Japan, Tokyo, Japan) was thawed, dispensed in 24-well plates, and allowed to solidify at 37℃ for 30 min. HUVECs were seeded at a density of 5×10⁴ cells per well, and incubated at 37℃ for 1 h. Then, a cell culture insert (0.8 µm pore; BD Biosciences Japan) was set on each well. HUVECs were resuspended in DMEM and the inserts were removed after 18 h. The network-like structures of HUVECs were examined under a microscope. Angiogenic activities were assessed by counting the branch point of tubes. We evaluated effects of VEGF and ASC-conditioned medium on tube formation in the absence or presence of carvedilol.

In Vivo Study

Applications of ASC Sheets and Carvedilol to a Rat MI Model Adult male syngeneic Lewis rats (200–250 g) obtained from Japan SLC Inc. (Hamamatsu, Japan) served as both cell donors and recipients. The experimental protocols were approved by the Institutional Animal Care and Use Committee, Faculty of Medicine, Tottori University. Rats were randomly assigned to 4 groups: (1) no treatment (Control MI group, n=7); (2) ASC sheet transplantation alone (ST group, n=9); (3) carvedilol treatment alone (Car group, n=12); and (4) both ASC sheet transplantation and carvedilol treatment (ST+Car group, n=10). For all the groups, acute MI was created by ligation of the left anterior descending artery, as described previously.³¹ Carvedilol at 5 or 20 mg·kg⁻¹·day⁻¹ was orally administered to the Car and ST+Car group rats for 6 weeks (from 7 days prior to the creation of MI to 5 weeks after MI). ASC sheet transplantation was performed for the ST and ST+Car group rats 7 days after the creation of MI. We confirmed that transplanted ASC sheets existed on the surface of hearts by luminescent signals of ASCs derived from transgenic rats harboring the luciferase gene (data not shown). Details of the experimental protocol are shown in Supplementary Figure 1.

Measurements of Blood Pressure and HR Systolic and diastolic blood pressure (SBP and DBP) and HR were measured by a tail-cuff system (BP-98A; Softron, Tokyo, Japan) a week before, just before, and 1, 3 and 5 weeks after MI (2 and 1 week before, just before, and 2 and 4 weeks after ASC sheet transplantation).

Echocardiography Cardiac function was evaluated by echocardiography just before and 1, 3 and 5 weeks after MI (7 days before, just before, and 2 and 4 weeks after ASC sheet transplantation) using a 12-MHz transducer (LOGIQ P5J and 12L; GE Healthcare, Fairfield, CT, USA). Short-axis two-dimensional images at the mid-papillary level of the left ventricle (LV) were stored. The end-systolic and end-diastolic cavity areas were determined by tracing the endocardial borders to calculate the fractional area shortening (FS). Left ventricular internal diameters at end-systole (LVESD) and end-diastole (LVEDD) were measured between the mitral valve and papillary muscles to calculate the ejection fraction (EF).

Measurement of Plasma Atrial Natriuretic Peptide (ANP) Levels Blood samples were obtained from the femoral vein just before and 5 weeks after MI (1 week before and 4 weeks after ASC sheet transplantation). ANP concentrations were measured by an ANP ELISA Kit (Assaypro, Saint Charles, MO, USA).

Histopathology Five weeks after MI (4 weeks after transplantation), hearts were dissected, fixed in 10% formalin, and embedded in paraffin. Sections were subjected to Masson’s trichrome staining to detect interstitial fibrosis. Transverse sections were obtained from the lateral wall, posterior wall, and septum. We determined the percentage of stained areas to the total sectional area by using a microscope (BZ9000; Keyence, Osaka, Japan). Each field was scanned, and digital images were analyzed by Keyence analysis software. The sections were also stained with an anti-von Willebrand factor (vWF) antibody (Abcam plc, Cambridge, UK) to detect capillaries in the border zone between the myocardium and scar. Capillaries were counted in 2 fields of transverse sections per animal. The number of capillaries in each field was averaged and expressed as capillary density.

Assessment of LV Functions in Isolated MI Hearts Whether carvedilol affects the action of ASC sheets on MI heart functions was also determined using the Langendorff heart isolated from male Lewis rats (300–350 g, 12 weeks old).³¹ Animals were handled in accordance with the Tottori University Guide for the Care and Use of Laboratory Animals. Isolation and coronary perfusion of hearts were executed with a Langendorff perfusion system (model 7523-40; Masterflex, Barrington, IL, USA), as described previously.³² Coronary arteries were perfused with a modified Tyrode’s solution [NaCl 144, KCl 5, CaCl₂ 1.5, MgCl₂ 0.9, HEPES 6, and glucose 5 (in mmol/L); pH 7.4 with NaOH] equilibrated with 100% oxygen.

Ventricular functions were assessed by measuring the left ventricular pressure (LVP) with a fluid-filled latex balloon inserted into the LV and inflated to give a LV end-diastolic pressure (LVEDP) of 5–15 mmHg. The transducer was connected to a PowerLab/8SP (AD Instruments, Castle Hill, NSW, Australia) to measure LVP and determine the maximum of LVP (LVP_max), LVEDP, LV developing pressure (LVDP), and maximum rates of LVP rise (dP/dt_max) and fall (dP/dt_min).

Data Analysis

All data are expressed as mean±SEM. A Student’s t-test and a Bonferroni multiple comparison test were used for comparisons of 2 groups and multiple (≥3) groups, respectively. A probability value of <0.05 was considered significant.

Results

Effects of Hypoxia on Expressions of Angiogenic Factor mRNAs in ASCs

We quantified mRNAs of angiogenic factors in ASCs after exposure to normoxia or hypoxia for 48 h (Figure 1). Levels of VEGF-A mRNA were significantly elevated under hypoxia. Levels of FGF mRNA tended to increase under hypoxia, whereas those of HGF, HIF-1α and SDF-1 mRNA significantly decreased.

Figure 1.

mRNA levels of vascular endothelial growth factor (VEGF)-A, hepatocyte growth factor (HGF), fibroblast growth factor (FGF), hypoxia inducible factor (HIF)-1α and stroma cell-derived factor (SDF)-1 in adipose-derived stem cells (ASCs) under normoxia or hypoxia. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed to determine mRNA expression levels. They were normalized to β-actin mRNA levels and expressed as ratios to the control values under normoxia (n=5). **P<0.01 (vs. Normoxia).

Expressions of α/β-AR mRNAs and Proteins in ASCs Under Hypoxia

We quantified mRNAs and proteins of α1-, β1- and β2-ARs expressed in ASCs under hypoxia. ASCs expressed mRNAs of α1- and β1-ARs, but not that of β2-AR (Supplementary Figure 2A). Expressions of α1-AR and β1-AR proteins in ASCs were confirmed by Western blotting (Supplementary Figure 2B).

Effects of Norepinephrine on Expressions of Angiogenic Factor mRNAs in ASCs

Treatment with norepinephrine (10 μmol/L) significantly increased the mRNA level of VEGF-A under hypoxia, but failed to change mRNA levels of HGF, FGF, HIF-1α and SDF-1 (Supplementary Figure 3). Even in the presence of carvedilol (10 μmol/L), norepinephrine significantly increased VEGF-A mRNA. Carvedilol did not significantly alter expressions of mRNAs of any cytokines in the presence or absence of norepinephrine.

Carvedilol Abolished the Beneficial Effects of ASC Sheets on Cardiac Functions in a Rat MI Model

We studied effects of ASC sheets and carvedilol on cardiac functions and remodeling after MI in vivo. EF was significantly lower in the MI group than in the sham-operated, non-MI group (data not shown). Four weeks after ASC sheet transplantation, EF and FS in the ST group, but not in the Car group or the ST+Car group, were significantly higher than those in the control MI group, without any significant difference in LVESD and LVEDD (Figure 2). There was no significant difference in SBP, DBP or HR among the 4 groups (Supplementary Figure 4).

Figure 2.

Effects of adipose-derived stem cell (ASC) sheet transplantation and/or carvedilol treatment on cardiac functions after myocardial infarction (MI). Ejection fraction (EF), fractional area shortening (FS), left ventricular end-systolic diameter (LVESD), and left ventricular end-diastolic diameter (LVEDD) were measured by echocardiography just before (0 weeks), 2 weeks after and 4 weeks after ASC sheet transplantation (1, 3 and 5 weeks after MI) for the control MI hearts (black crosses), and those treated with an ASC sheet alone (red circles), carvedilol alone (blue triangles) and both an ASC sheet and carvedilol (magenta squares). Carvedilol was orally administered to the carvedilol-treatment group rats at a dose of 5 mg·kg⁻¹·day⁻¹ for 6 weeks (Supplementary Figure 1). The numbers of experiments are given in parentheses (n=7–12). *P<0.05, **P<0.01 (vs. Control MI); ^##P<0.01 (vs. Carvedilol alone), ⁺⁺P<0.01 (vs. ASC sheet+Carvedilol).

The plasma level of ANP measured 4 weeks after transplantation was slightly lower in the ST group than in the control MI group, while the ANP level in the ST+Car group was comparable to that in the control MI group (Supplementary Figure 5).

Figure 3 shows LV functions of Langendorff-perfused hearts isolated from rats in the 4 groups. LVP_max, LVDP and dP/dt_min were significantly improved in the ST group in comparison to those in the control MI group, but were comparable in the control MI and ST+Car groups. Thus, the ASC-induced changes in LVP_max, LVDP and dP/dt_min were abolished by carvedilol treatment.

Figure 3.

Effects of adipose-derived stem cell (ASC) sheet transplantation and/or carvedilol treatment on systolic and diastolic functions of isolated myocardial infarction (MI) hearts. Maximum of LVP (LVP_max), left ventricular end-diastolic pressure (LVEDP), LV developing pressure (LVDP), and maximum rates of LVP rise (dP/dt_max) and fall (dP/dt_min) were determined using the Langendorff apparatus for MI hearts isolated 5 weeks after MI from rats in the control MI group without any treatments (black bar) and the groups with ASC sheet transplantation alone (red bar), carvedilol treatment alone (blue bar), and both ASC sheet transplantation and carvedilol treatment (magenta bar) (n=4 each). *P<0.05 (by Bonferroni test), ^#P<0.05 (by Student’s t-test).

Carvedilol Abolished the Beneficial Effects of ASC Sheets on Cardiac Fibrosis and Neovascularization After MI

Cardiac fibrosis and neovascularization were evaluated by the histological analyses 4 weeks after transplantation (5 weeks after MI). In the control MI group, extensive fibrosis occurred around the infarcted region. This fibrosis after MI was reduced in the ST group, but not in the ST+Car group (Figure 4A). Figure 4B shows the summary data obtained from 7 to 11 experiments. The extent of cardiac fibrosis was significantly lower in the ST group than in the control MI group. The reduction of fibrosis did not occur either in the Car or ST+Car group. The capillary density was remarkably increased in the ST group, as compared with those in the control MI and Car groups, although there was no difference between the control MI and ST+Car groups (Figure 5A). As summarized in Figure 5B, the capillary density was significantly higher in the ST group than in the control MI group, while comparable in the control MI and Car groups; it was significantly lower in the ST+Car group than in the ST group. Thus, carvedilol prevented the reduction of fibrosis and enhancement of neovascularization by ASC sheets without affecting them in itself.

Figure 4.

Effects of adipose-derived stem cell (ASC) sheet transplantation and/or carvedilol treatment on interstitial fibrosis in the remote zone of myocardial infarction (MI) hearts. (A) Representative pictures of interstitial fibrosis in MI hearts isolated from the Control MI, ASC sheet transplantation alone (ST), carvedilol treatment alone (Car) and ST+Car group rats 4 weeks after transplantation (5 weeks after MI). Picrosirius-red staining was performed to detect interstitial fibrosis. Scale bar=1 mm. Carvedilol was orally administered to the Car and ST+Car group rats at a dose of 5 mg·kg⁻¹·day⁻¹ for 6 weeks. (B) Summary of the prevalence of interstitial fibrosis in the remote zone of MI hearts from the 4 groups. The numbers of experiments are given in the parentheses for each group (n=7–11). *P<0.05, **P<0.01.

Figure 5.

Effects of adipose-derived stem cell (ASC) sheet transplantation and/or carvedilol treatment on neovascularization in the border zone of myocardial infarction (MI) hearts. (A) Representative images of capillary vessels stained with an antibody against von-Willebrand factor (vWF) in the border zone of MI hearts isolated from the Control MI, ASC sheet transplantation alone (ST), carvedilol treatment alone (Car) and ST+Car group rats 5 weeks after MI, that is, 4 weeks after ASC sheet transplantation (black arrows; scale bar=100 μm). The inset at the bottom left shows an expanded view of capillary vessels stained with the antibody against vWF (scale bar=100 μm). Carvedilol was administered, as described for Figure 4. (B) Summary of the numbers of vWF-positive vessels in the border zone of MI hearts from each group. The number of capillary vessels was determined for 2 selected fields in the border zone per animal and expressed as an averaged value. The numbers of experiments are given in parentheses (n=7–11). *P<0.05, **P<0.01.

Effects of Carvedilol on VEGF-Induced Phosphorylation of VEGF Receptors and Tube Formation in HUVECs

To clarify how carvedilol prevented the enhancement of neovascularization by ASC sheets, we examined effects of carvedilol on VEGF-induced phosphorylation of the VEGFR2 (Tyr1175) and tube formation of HUVECs. In the presence of VEGF, carvedilol (10μmol/L) significantly reduced the phospho-VEGFR2 (Tyr1175) without altering the total VEGFR2 protein level (Supplementary Figure 6). VEGF and ASC-conditioned medium enhanced tube formation by HUVECs, and these effects were abolished by 10 μmol/L carvedilol (Figure 6).

Figure 6.

Inhibitory effects of carvedilol on vascular endothelial growth factor (VEGF)- or adipose-derived stem cell (ASC) conditioned medium-induced enhancement of tube formation by human umbilical vein endothelial cells (HUVECs). (A) Effects on the VEGF-induced enhancement of tube formation. Representative images of tube formation (top) and averaged luminal areas of tubular structures (bottom) are shown. HUVECs were placed in Matrigel with or without VEGF (50 ng/mL) in the presence or absence of 10 μmol/L carvedilol (Car). After 8 h, tubular structures were imaged (magnification: ×100). Averaged lumen areas were determined using ImageJ software (n=6). **P<0.01. (B) Effects on ASC-conditioned medium-induced enhancement of tube formation. Representative images are shown. HUVECs were placed in Matrigel in the conditioned medium of ASCs (0.5×10⁵ cells) in the presence or absence of 10 μmol/L carvedilol. After 8-h incubation, tubular structures were imaged (magnification: ×100).

Discussion

In this study, we found that: (1) hypoxia increased VEGF-A mRNA in ASCs; (2) ASC sheet transplantation improved contractile functions of MI hearts, suppressed fibrosis, and enhanced neovascularization; (3) carvedilol treatment before and after MI abolished these beneficial actions of ASC sheets; and (4) carvedilol abolished promoting effects of VEGF on VEGFR2 (Tyr1175) phosphorylation and tube formation of HUVECs.

ASCs have been reported to improve cardiac functions after MI by inducing neovascularization and suppressing fibrosis. These actions of ASCs may be attributable to the release of cytokines and/or their trans-differentiation into cardiomyocytes.³¹ We reported that ASC sheets survived on infarcted areas as layers for at least 2 weeks after transplantation, and that vWF-positive capillaries were created under the sheet.³¹ ASCs secrete VEGF, FGF and HGF in vitro and in vivo, which may contribute to the increment of capillaries.³² In this study, the mRNA level of VEGF-A was significantly increased in ASCs under hypoxia. ASCs expressed mRNAs of α1- and β1-ARs, and norepinephrine further increased VEGF-A mRNA in ASCs under hypoxia. It has been reported that increased expression of α-ARs and enhancement of their downstream signaling play an important role in the secretion of cytokines by MSCs.³⁰ Thus, ASCs may exert beneficial effects on MI hearts via the norepinephrine-induced enhancement of VEGF secretion. In this study, the expression of HIF-1α mRNA was significantly suppressed under hypoxia, which was very similar to our previous data.³² There was a difference in mRNA level of HIF-1α between our study and a previous study,³³ which may be attributable to the difference in the timing of measurements of HIF-1α mRNA.

Carvedilol is a third-generation vasodilating β-blocker that has been shown to reduce morbidity and mortality of patients with heart failure. Inhibition of β-ARs on cardiomyocytes prevents their responses to the SNS, leading to decreased HR and contractility. This action of the agent is beneficial for patients with heart failure whose SNS is activated.⁹ Carvedilol has a relatively weak inhibitory action on α1-ARs, which leads to decreased peripheral vascular resistance and an antihypertensive effect; there happens no reflex tachycardia due to simultaneous blockade of β1-ARs in the heart.¹⁰ In this study, carvedilol did not affect norepinephrine-induced elevation of VEGF-A mRNA in ASCs, suggesting that the norepinephrine effect was not mediated by β- or α1-ARs. Further experiments are necessary to identify the signaling pathway responsible for the norepinephrine-induced elevation of VEGF-A mRNA.

The most prominent finding in the present study was that carvedilol abolished beneficial effects of ASC sheets on contractile functions, neovasculization and fibrosis of MI hearts. Although several reports indicate that administration of carvedilol after MI exerts cardioprotective actions,³⁴ there have been no reports to clarify whether treatment with carvedilol prior to MI could improve contractile functions of MI heats. Following our previous report showing the effects of pretreatment with an angiotensin II receptor blocker, irbesartan, on cardiac functions,³² we examined the effects of oral administration of carvedilol at 5 and 20 mg·kg⁻¹·day⁻¹ before and after MI on the contractile function of MI hearts. As shown in Figure 2 and Supplementary Figure 7, carvedilol at 5 mg·kg⁻¹·day⁻¹ did not influence contractile functions of MI hearts, whereas carvedilol at the higher dose of 20 mg·kg⁻¹·day⁻¹ significantly improved the MI heart function 1 week after MI (just before ASC sheet transplantation). In contrast, treatment with carvedilol at 5 mg·kg⁻¹·day⁻¹ abolished the beneficial effects of ASC sheets on contractile functions, neovasculization and fibrosis of MI hearts. To confirm the present study results from the in vivo measurement of LV functions, we performed the Langendorff experiment to measure LV functions of isolated MI hearts 5 weeks after MI (4 weeks after transplantation). Although ASC sheets improved systolic and diastolic functions of isolated MI hearts, carvedilol abolished the beneficial effects of ASC sheets (Figure 3), which is consistent with the results from the in vivo experiment (Figure 2).

Carvedilol did not reduce the mRNA level of VEGF-A in the absence or presence of norepinephrine, indicating that preventive effects of carvedilol on ASC sheet-induced cardioprotection are not ascribable to its inhibition of VEGF-A transcription or reductions in the norepinephrine action. VEGF promoted phosphorylation of VEGFR2 (Tyr1175) and tube formation of HUVECs, which were abolished by carvedilol. Consistent with our finding, β-blockers such as carvedilol and labetalol have been reported to exert anti-angiogenic effects on neuroblastoma cells; this action was potentiated by propranolol, nebivolol, atenolol or metoprolol.³⁵ Besides, carvedilol inhibited angiogenesis through the VEGF-Src-ERK signaling pathway in cirrhosis livers.³⁶ Ding et al³⁶ demonstrated that carvedilol inhibited tube formation of HUVECs induced by VEGF-A through inhibition of phosphorylation of VEGFR2, ERK and Src. This finding was partly confirmed by the fact that carvedilol inhibited the VEGF- or ASC-conditioned medium-induced enhancement of the tube formation by HUVECs (Figure 6). This pathway may be involved in carvedilol inhibition of the ASC sheet effects observed in the present study.

Although the precise mechanism that underlies the carvedilol effects on ASC sheet-induced cardioprotection is unknown, a clinical implication of this study may be clear. ASC sheets would be applicable to drug-refractory patients who wait for heart transplantation. Given the observed carvedilol effects, we have to pay attention to the period and timing of carvedilol administration before and after ASC sheet transplantation. We cannot apply the present study results to clinical settings directly, because effects of carvedilol on MI hearts and ASC sheet therapy may depend on the method of its administration; β-blockers, including carvedilol, improved prognosis of MI patients when administered with a time lag of more than 24 h after MI, but failed to do so when administered immediately after MI.³¹ These results suggest that carvedilol should not be administered to patients before or immediately after MI. We need further investigations on whether carvedilol administered 24 h or later after MI (post-treatment with the agent) inhibits cardioprotective actions of ASC sheets. Carvedilol at 25 or 50 mg/day have been reported to be prescribed to patients with chronic heart failure,³⁷^,³⁸ and Watanabe et al³⁹ reported that carvedilol doses of 25 and 50 mg for humans correspond to those of 2 and 5 mg/kg, respectively, for rats, when corrected by body surface areas. Therefore, our experimental condition of the carvedilol dose (5 mg·kg⁻¹·day⁻¹) may be applicable to clinical settings. Nevertheless, differences in carvedilol effects between the groups, with its treatment only after MI and both before and after MI, and the effects of carvedilol at a broader range of doses should be determined in future studies.

There is a limitation in the present study. MI damage just before ASC sheet transplantation (1 week after MI) should be the same in the 4 groups to assess the ameliorating effects of ASC sheets on dysfunctions and remodeling of MI hearts properly. In the present study, however, it was impossible to confirm that the MI damage was comparable among the 4 groups because we aimed to evaluate the effects of oral administration of carvedilol before MI (before ASC sheet transplantation); pretreatment with carvedilol may affect myocardial damage caused by MI.

Funding

None.

Conflicts of Interest

Dr. Hisatome reported receiving lecturer’s fee from Teijin Pharma Co. Ltd, Sanwa Kagaku Kenkyusho Co. Ltd, Feizer Co. Ltd. and Fuji Yakuhin Co. Ltd., and research grants from Daiichi Sankyo Co., Bayer Yakuhin, Ltd., Teijin Pharma Co. Ltd, Fuji Yakuhin Co. Ltd and Sanwa Kagaku Kenkyusho Co. Ltd.

Supplementary Files

Please find supplementary file(s);

http://dx.doi.org/10.1253/circj.CJ-19-0261

References

1. Pavillard LE, Marín-Aguilar F, Bullon P, Cordero MD. Cardiovascular diseases, NLRP3 inflammasome, and western dietary patterns. Pharmacol Res 2018; 131: 44–50.
2. Dzau VJ, Antman EM, Black HR, Hayes DL, Manson JE, Plutzky J, et al. The cardiovascular disease continuum validated: Clinical evidence of improved patient outcomes: Part I: Pathophysiology and clinical trial evidence (risk factors through stable coronary artery disease). Circulation 2006; 114: 2850–2870.
3. Singh K, Communal C, Sawyer DB, Colucci WS. Adrenergic regulation of myocardial apoptosis. Cardiovasc Res 2000; 45: 713–719.
4. Amin P, Singh M, Singh K. β-adrenergic receptor-stimulated cardiac myocyte apoptosis: Role of β1 integrins. J Signal Transduct 2011; 2011: Article ID 179057.
5. Goldspink DF, Burniston JG, Ellison GM, Clark WA, Tan LB. Catecholamine-induced apoptosis and necrosis in cardiac and skeletal myocytes of the rat in vivo: The same or separate death pathways? Exp Physiol 2004; 89: 407–416.
6. Krishnamurthy P, Subramanian V, Singh M, Singh K. Beta1 integrins modulate beta-adrenergic receptor-stimulated cardiac myocyte apoptosis and myocardial remodeling. Hypertension 2007; 49: 865–872.
7. Chia N, Fulcher J, Keech A. Beta-blocker, angiotensin-converting enzyme inhibitor/angiotensin receptor blocker, nitrate-hydralazine, diuretics, aldosterone antagonist, ivabradine, devices and digoxin (BANDAID²): An evidence-based mnemonic for the treatment of systolic heart failure. Intern Med J 2016; 46: 653–662.
8. Feuerstein GZ, Bril A, Ruffolo RR. Protective effects of carvedilol in the myocardium. Am J Cardiol 1997; 80: 41L–45L.
9. Manurung D, Trisnohadi HB. Beta blockers for congestive heart failure. Acta Med Indones 2007; 39: 44–48.
10. Ruffolo RR, Gellai M, Hieble JP, Willette RN, Nichols AJ. The pharmacology of carvedilol. Eur J Clin Pharmacol 1990; 38(Suppl 2): S82–S88.
11. Kawamura M, Miyagawa S, Miki K, Saito A, Fukushima S, Higuchi T, et al. Feasibility, safety, and therapeutic efficacy of human induced pluripotent stem cell-derived cardiomyocyte sheets in a porcine ischemic cardiomyopathy model. Circulation 2012; 126: S29–S37.
12. Menasché P, Alfieri O, Janssens S, McKenna W, Reichenspurner H, Trinquart L, et al. The Myoblast Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) trial: First randomized placebo-controlled study of myoblast transplantation. Circulation 2008; 117: 1189–1200.
13. Bolli R, Tang XL, Sanganalmath SK, Rimoldi O, Mosna F, Abdel-Latif A, et al. Intracoronary delivery of autologous cardiac stem cells improves cardiac function in a porcine model of chronic ischemic cardiomyopathy. Circulation 2013; 128: 122–131.
14. Zhao T, Zhao W, Meng W, Liu C, Chen Y, Gerling IC, et al. VEGF-C/VEGFR-3 pathway promotes myocyte hypertrophy and survival in the infarcted myocardium. Am J Transl Res 2015; 7: 697–709.
15. Rong SL, Wang XL, Zhang CY, Song ZH, Cui LH, He XF, et al. Transplantation of HGF gene-engineered skeletal myoblasts improve infarction recovery in a rat myocardial ischemia model. PLoS One 2017; 12: e0175807.
16. Liu J, Wu P, Wang Y, Du Y, A N, Liu S, et al. Ad-HGF improves the cardiac remodeling of rat following myocardial infarction by upregulating autophagy and necroptosis and inhibiting apoptosis. Am J Transl Res 2016; 8: 4605–4627.
17. Raedschelders K, Ansley DM, Chen DD. The cellular and molecular origin of reactive oxygen species generation during myocardial ischemia and reperfusion. Pharmacol Ther 2012; 133: 230–255.
18. Song H, Cha MJ, Song BW, Kim IK, Chang W, Lim S, et al. Reactive oxygen species inhibit adhesion of mesenchymal stem cells implanted into ischemic myocardium via interference of focal adhesion complex. Stem Cells 2010; 28: 555–563.
19. Hamdi H, Planat-Benard V, Bel A, Puymirat E, Geha R, Pidial L, et al. Epicardial adipose stem cell sheets results in greater post-infarction survival than intramyocardial injections. Cardiovasc Res 2011; 91: 483–491.
20. Patila T, Miyagawa S, Imanishi Y, Fukushima S, Siltanen A, Mervaala E, et al. Comparison of arrhythmogenicity and proinflammatory activity induced by intramyocardial or epicardial myoblast sheet delivery in a rat model of ischemic heart failure. PLoS One 2015; 10: e0123963.
21. Memon IA, Sawa Y, Fukushima N, Matsumiya G, Miyagawa S, Taketani S, et al. Repair of impaired myocardium by means of implantation of engineered autologous myoblast sheets. J Thorac Cardiovasc Surg 2005; 130: 1333–1341.
22. Sekiya N, Matsumiya G, Miyagawa S, Saito A, Shimizu T, Okano T, et al. Layered implantation of myoblast sheets attenuates adverse cardiac remodeling of the infarcted heart. J Thorac Cardiovasc Surg 2009; 138: 985–993.
23. Gnecchi M, Zhang Z, Ni A, Dzau VJ. Paracrine mechanisms in adult stem cell signaling and therapy. Circ Res 2008; 103: 1204–1219.
24. Harada Y, Yamamoto Y, Tsujimoto S, Matsugami H, Yoshida A, Hisatome I. Transplantation of freshly isolated adipose tissue-derived regenerative cells enhances angiogenesis in a murine model of hind limb ischemia. Biomed Res 2013; 34: 23–29.
25. Miranville A, Heeschen C, Sengenès C, Curat CA, Busse R, Bouloumié A. Improvement of postnatal neovascularization by human adipose tissue-derived stem cells. Circulation 2004; 110: 349–355.
26. Nakagami H, Maeda K, Morishita R, Iguchi S, Nishikawa T, Takami Y, et al. Novel autologous cell therapy in ischemic limb disease through growth factor secretion by cultured adipose tissue-derived stromal cells. Arterioscler Thromb Vasc Biol 2005; 25: 2542–2547.
27. Rehman J, Traktuev D, Li J, Merfeld-Clauss S, Temm-Grove CJ, Bovenkerk JE, et al. Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation 2004; 109: 1292–1298.
28. Jiang Q, Ding S, Wu J, Liu X, Wu Z. Norepinephrine stimulates mobilization of endothelial progenitor cells after limb ischemia. PLoS One 2014; 9: e101774.
29. Wu B, Wang L, Yang X, Mao M, Ye C, Liu P, et al. Norepinephrine inhibits mesenchymal stem cell chemotaxis migration by increasing stromal cell-derived factor-1 secretion by vascular endothelial cells via NE/abrd3/JNK pathway. Exp Cell Res 2016; 349: 214–220.
30. Tyurin-Kuzmin PA, Fadeeva JI, Kanareikina MA, Kalinina NI, Sysoeva VY, Dyikanov DT, et al. Activation of β-adrenergic receptors is required for elevated α1A-adrenoreceptors expression and signaling in mesenchymal stromal cells. Sci Rep 2016; 6: 32835.
31. Otsuki Y, Nakamura Y, Harada S, Yamamoto Y, Ogino K, Morikawa K, et al. Adipose stem cell sheets improved cardiac function in the rat myocardial infarction, but did not alter cardiac contractile responses to beta-adrenergic stimulation. Biomed Res 2015; 36: 11–19.
32. Yamamoto K, Kurata Y, Inoue Y, Adachi M, Tsuneto M, Miake J, et al. Pretreatment with an angiotensin II receptor blocker abolished ameliorating actions of adipose-derived stem cell sheets on cardiac dysfunction and remodeling after myocardial infarction. Regen Ther 2018; 9: 79–88.
33. Basic J, Stojkovic S, Assadian A, Rauscher S, Duschek N, Kaun C, et al. The relevance of vascular endothelial growth factor, hypoxia inducible factor-1 alpha, and clusterin in carotid plaque instability. J Stroke Cerebrovasc Dis 2019; 28: 1540–1545.
34. Sia YT, Parker TG, Tsoporis JN, Liu P, Adam A, Rouleau JL. Long-term effects of carvedilol on left ventricular function, remodeling, and expression of cardiac cytokines after large myocardial infarction in the rat. J Cardiovasc Pharmacol 2002; 39: 73–87.
35. Pasquier E, Street J, Pouchy C, Carre M, Gifford AJ, Murray J, et al. β-blockers increase response to chemotherapy via direct antitumour and anti-angiogenic mechanisms in neuroblastoma. Br J Cancer 2013; 108: 2485–2494.
36. Ding Q, Tian XG, Li Y, Wang QZ, Zhang CQ. Carvedilol may attenuate liver cirrhosis by inhibiting angiogenesis through the VEGF-Src-ERK signaling pathway. World J Gastroenterol 2015; 21: 9566–9576.
37. Anderson JL, Lutz JR, Gilbert EM, Sorensen SG, Yanowitz FG, Menlove RL, et al. A randomized trial of low-dose beta-blockade therapy for idiopathic dilated cardiomyopathy. Am J Cardiol 1985; 55: 471–475.
38. Bristow MR, Gilbert EM, Abraham WT, Adams KF, Fowler MB, Hershberger RE, et al. Carvedilol produces dose-related improvements in left ventricular function and survival in subjects with chronic heart failure. MOCHA Investigators. Circulation 1996; 94: 2807–2816.
39. Watanabe K, Ohta Y, Nakazawa M, Higuchi H, Hasegawa G, Naito M, et al. Low dose carvedilol inhibits progression of heart failure in rats with dilated cardiomyopathy. Br J Pharmacol 2000; 130: 1489–1495.

Corresponding author

Correction information

Register with J-STAGE for free!