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Keywords:

  • Adipose-derived stem cells;
  • Myocardial infarction;
  • Echocardiography;
  • Angiogenesis;
  • Paracrine factors

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 Conflict of Interest
  10. REFERENCES
  11. Supporting Information

The rationale of this article is enhancing the therapeutic potential of stem cells in ischemic microenvironments by novel preconditioning strategies is critical for improving cellular therapy. We tested the hypothesis that inhibition of phosphodiesterase-5 (PDE-5) with sildenafil (Viagra) or knockdown with a silencing vector in adipose-derived stem cells (ASCs) would improve their survival and enhance cardiac function following myocardial implantation in vivo. ASCs were treated with sildenafil or PDE-5 silencing vector short hairpin RNA (shRNAPDE-5) and subjected to simulated ischemia/reoxygenation in vitro. Both sildenafil and shRNAPDE-5 significantly improved viability, decreased necrosis, apoptosis, and enhanced the release of growth factors, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF), and insulin-like growth factor. Inhibition of protein kinase G reversed these effects. To show the beneficial effect of preconditioned ASCs in vivo, adult male CD-1 mice underwent myocardial infarction. Preconditioned ASCs (4 × 105) were directly injected intramyocardially. Preconditioned ASC-treated hearts showed consistently superior cardiac function when compared with nonpreconditioned ASCs after 4 weeks of treatment. This was associated with significantly reduced fibrosis, increased vascular density, and decreased resident myocyte apoptosis when compared with mice receiving nonpreconditioned ASCs. VEGF, b-FGF, and Angiopoietin-1 were also significantly elevated 4 weeks after cell therapy with preconditioned ASCs. We conclude that preconditioning by inhibition of PDE-5 can be a powerful novel approach to improve stem cell therapy following myocardial infarction. STEM CELLS 2012; 30:326–335.


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 Conflict of Interest
  10. REFERENCES
  11. Supporting Information

Acute myocardial infarction (MI) is a major cause of morbidity and mortality worldwide. Despite advances in the treatment of MI, congestive heart failure secondary to infarction continues to be a major complication. The main factor leading to the progression of heart failure is the irreversible loss of cardiomyocytes due to necrosis and apoptosis following ischemic injury. To overcome myocyte loss and the heart's limited self-regeneration capacity, recent research has focused on transplantation of stem cells to differentiate and replenish the loss of myocytes. Various animal studies have shown the potential to regenerate myocardium, improve perfusion to the infarct area, and improve cardiac function [1–4]. Although cardiac performance by cell-based therapy has improved, unsatisfactory cell retention and transplant survival still plague this technique. The current transplantation strategies achieve modest engraftment of donor stem cells in the infarcted myocardium, primarily due to the rapid and massive loss of donor stem cells [5, 6]. Several factors influence the accelerated cell death in the infarcted myocardium. These include the ischemic and cytokine-rich microenvironment, mechanical injury, maladaptation, and origin and quality of the donor cell preparation [7]. Therefore, enhancing stem cell survival in the ischemic microenvironment is of paramount importance in improving cardiac regeneration. Previous studies have shown that treatment of bone marrow stem cells with hypoxia improved survival postengraftment in the infarcted heart [8], increased proliferation rate and differentiation in addition to modulation of their paracrine activity [9]. In addition, pharmacological preconditioning agents including diazoxide, an opener of mitochondrial KATP channel [10], vascular endothelial growth factor 2 (VEGF)-2 [11], and insulin-like growth factor-1 (IGF-1) [12] have been shown to promote myogenic response of stem cells following transplantation in the myocardium. Nevertheless, newer and safer strategies to improve the long-lasting regenerative potential of stem cells are critical for their clinical utility.

Recent studies have suggested the role cyclic guanosine monophosphate (cGMP)-mediated nitric oxide (NO) signaling plays in the differentiation of embroyonic stem cells into myocardial cells [13, 14]. To this context, the potential role of phosphodiesterase-5 (PDE-5) inhibitors including sildenafil in preconditioning of stem cells appears promising. These drugs inhibit PDE-5, the predominant enzyme in the corpus cavernosum, which plays an essential role in vascular smooth muscle contraction through specific regulation of cGMP [15]. Several studies from our laboratory have demonstrated that PDE-5 inhibitors induce a powerful protective effect against ischemia/reperfusion injury [16–20], doxorubicin-induced cardiomyopathy [21, 22], and MI-induced heart failure in mice [23]. The cardioprotective effect is attributed to limiting apoptosis and necrosis through several mechanisms. These include enhanced expression of endothelial nitric oxide synthase (NOS) and inducible NOS, activation of protein kinase C and protein kinase G (PKG), phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), PKG-dependent phosphorylation of glycogen synthase kinase 3β (GSK-3β), upregulation of Bcl-2/Bax, and opening of the mitochondrial KATP channels [16–20, 23–25]. In addition, it has been shown that the long-acting PDE-5 inhibitor, tadalafil, provides longer and sustained protection of mesenchymal stem cells and promotes their survival and proliferation in the infarcted heart [26].

In this study, we tested the hypothesis whether PDE-5 inhibition could improve the survival of adipose-derived stem cells (ASCs), which may lead to enhanced cardiac function following MI in mice. Specifically, we addressed the following questions: (a) Does PDE-5 inhibition by sildenafil or genetic knockdown with a silencing vector improves survival following simulated ischemia/reoxygenation (SI/RO) injury in vitro? (b) What is the role of cGMP-dependent PKG signaling pathway in protection of ASCs? (c) Can in vivo transplantation of ASCs after PDE-5 inhibition ex vivo improve left ventricle (LV) function following MI? (d) What is the role of paracrine mechanisms in enhancing cytoprotective effects of PDE-5 inhibition? Our results show that PDE-5 inhibition is a powerful new preconditioning strategy to increase viability of ASCs in vitro and enhancing their therapeutic potential as shown by reduced fibrosis, cardiomyocyte apoptosis, improved vascular density, and cardiac function in mice following MI.

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 Conflict of Interest
  10. REFERENCES
  11. Supporting Information

Animals

Adult male outbred CD-1 mice (∼30 g) were supplied by Harlan Sprague Dawley (Indianapolis, IN). All animal experiments were conducted under the guidelines on humane use and care of laboratory animals for biomedical research published by National Institutes of Health (No. 85-23, revised 1996).

Isolation of ASCs

Epicardial adipose tissue harvested from voluntary patients undergoing heart transplant was used for isolation of ASCs. The tissue was minced and digested with Collagenase type VIII in phosphate-buffered saline (PBS) for 90 minutes at 37°C with slight shaking. Collagenase mixture was filtered through a 100-μm nylon mesh filter. Filtrate was centrifuged at 800g for 10 minutes. The supernatant was discarded, and the pellet was washed with Hank's balanced salt solution. Centrifugation and wash steps were repeated twice. Freshly isolated ASCs were plated with α minimum essential medium with 20% fetal bovine serum and 1% Pen/Strep. Culture medium was changed every 3–4 days, and nonadherent hematopoietic cells were removed during this process. Subsequent passages were performed with a 0.25% trypsin solution containing 0.01% EDTA for 6 minutes at 37°C.

Immunocytochemistry

ASCs were cultured on sterile glass coverslips and fixed by incubation in 4% paraformaldehyde/PBS for 20 minutes and permeabilized with 1.0% Triton X-100 in PBS for 10 minutes. Intracellular staining patterns and distribution of PDE-5 protein were analyzed by immunostaining with incubation of respective antibody at 4°C overnight (1:500 dilution) followed by incubation of Alexa Fluor 594 conjugated secondary antibody at 37°C for 1 hour (1:1,000 dilution). Staining of 4′,6-diamino-2-phenylindole (Sigma Aldrich, St. Louis, MO, www.sigmaaldrich.com) was used to visualize all nuclei.

Western Blot Analysis

Total soluble protein was extracted from the cells with reporter lysis buffer (Promega, Madison, WI, www.promega.com). Homogenate was centrifuged at 14,000g for 10 minutes at 4°C, and the supernatant was recovered as the total cellular protein. Total protein (50 μg) from each sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and then blocked with 5% nonfat dry milk in Tris-buffered saline. The membrane was subsequently incubated with a primary antibody at a dilution of 1:500 for each of the respective proteins, that is, PDE-5A (rabbit polyconal), PKG, and β-actin (goat polyclonal, Santa Cruz, Santa Cruz, CA, www.scbt.com). The membrane was washed and incubated with horseradish peroxidase-conjugated secondary antibody (1:2,000 dilution, 1 hour at room temperature). The membranes were developed using enhanced chemiluminescence system (ECL Plus; GE Life Sciences, Pittsburgh, PA, www.gelifesciences.com) and exposed to x-ray film.

Preparation of Short Hairpin RNAs (shRNAs)

PDE-5 gene silencing shRNA (inserted into microRNA-155 cassette) and gfp-PDE-5 fusion protein both coupled to a cytomegalovirus (CMV) promoter and incorporated into adenoviral vectors were generated by Zhang et al. [27]. The mouse PKGI, shRNA expression vector for PKG, was constructed using the pSilencer adeno1.0-CMV system from Ambion (Adenoviral shRNA expression Vector System) as described previously [20].

SI/RO Protocol

ASCs were incubated at 37°C and 5% CO2 for 2 hours, with or without 10 μM sildenafil. This dose was selected based on its protective effect against SI/RO injury in adult cardiomyocytes [24]. A subset of ASCs were treated with PKG inhibitor KT 5823 (2 μM) with or without sildenafil for 2 hours. Another subset of ASCs were transduced with an adenoviral vector containing scrambled control shRNA (shRNACon ASC), PDE-5 shRNA (shRNAPDE-5 ASC), or PKG shRNA (shRNAPKG ASC) in serum-free growth medium for 24 hours (Supporting Information Fig. S1A). ASCs were then subjected to SI for 15 hours by replacing the cell medium with an “ischemia buffer” that contained 118 mM NaCl, 24 mM NaHCO3, 1.0 mM NaH2PO4, 2.5 mM CaCl2·2H2O, 1.2 mM MgCl2, 20 mM sodium lactate, 16 mM KCl, 10 mM 2-deoxyglucose (pH adjusted to 6.2) as reported previously [24]. Cells were incubated in an anoxic chamber at 37°C during the entire SI period. RO was accomplished by replacing ischemic buffer with normal cell medium under normoxic conditions. Cell necrosis and apoptosis were assessed after 1 hour or 18 hours of RO, respectively.

Evaluation of Cell Viability and Apoptosis

Trypan blue exclusion assay was used to assess cell necrosis. Floating and attached cells were collected by centrifugation, and cell pellets were resuspended and mixed with 20 μl of 0.4% trypan blue dye. After 5 minutes, dead cells, stained by trypan blue, were counted using a hemocytometer. The number of dead cells was counted from five randomly chosen fields and expressed as a percentage of the total number of cells. We also measured lactate dehydrogenase (LDH) release into the cellular medium spectrophotometrically using an LDH assay kit (Sigma Aldrich). Cell viability was measured with CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit (Promega) according to the manufacturer's instructions. Apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining using a commercial kit (Millipore, Billerica, MA, www.millipore.com) that detects nuclear DNA fragmentation via a peroxidase assay as reported previously [1]. In brief, after SI and 18 hours of RO, cells were fixed by 4% formaldehyde/PBS at 4°C for 25 minutes and then subjected to TUNEL assay according to the manufacturer's protocol. The slides were developed with Nova Red peroxidase substrate (Vector Labs, Burlingame, CA, www.vectorlabs.com), counterstained with hemotoxylin and mounted using Permount solution (Fisher Scientific, Waltham, MA, www.fishersci.com). Stained cells were examined under a Nikon Eclipse TE 800 microscope.

Measurement of cGMP, Cyclic Adenosine Monophosphate, and PKG Activity

cGMP activity assay was performed using cGMP Direct Immunoassay Kit (Biovision), which provided a direct competitive immunoassay for sensitive and quantitative determination of cGMP as per manufacturer's instructions. Cellular levels of cyclic adenosine monophosphate (cAMP) were measured using bioluminescent assay, cAMP-Glo (Promega) as per manufacturer's instructions. Cardiac PKG activity was examined using a commercially available PKG activity kit (Cyclex, Nagano, Japan, www.cyclex.co.jp/home_etop.html) in whole cell lysates. The activity was measured according to the manufacturer's instructions. Spectrophotometric absorbance was measured at 450 nm. Results were normalized as per mg of protein.

MI Protocol

Adult CD-1 mice underwent permanent occlusion of the left anterior descending (LAD) coronary artery as described previously [2]. In brief, the animals were anesthetized with pentobarbital (70 mg/kg i.p.), intubated orotracheally, and ventilated on a positive-pressure ventilator. The tidal volume was set at 0.2 ml, and the respiratory rate was adjusted to 133 cycles per minute. All surgical procedures were carried out under sterile conditions. A left thoracotomy was performed at the 4th intercostal space, and the heart was exposed by stripping the pericardium. The LAD was identified and ligated with 7-0 silk suture mounted on a tapered needle (BV-1; Ethicon). After occlusion, the air was expelled from the chest. The animals were extubated and then received intramuscular doses of analgesia (buprenex; 0.02 mg/kg) and antibiotic (Gentamicin; 0.7 mg/kg; for 3 days).

Transplantation of ASCs

Immediately after ligation, 4 × 105 ASCs (total of 30 μl) were injected at three injection sites into anterior and lateral wall of the LV bordering the infarction. The control group was injected with identical volume of PBS at similar sites (Supporting Information Fig. S1B). The chests were sutured, and animals were allowed to recover. The hearts were harvested for histological studies 4 weeks postcellular transplantation.

Echocardiographic Studies

Echocardiograms were obtained using the Vevo770 imaging system (VisualSonics, Toronto, Canada, www.visualsonics.com) prior to surgery (baseline) and 4 weeks after surgery prior to sacrificing the animal. The mice were anesthetized with pentobarbital (30 mg/kg i.p.). The chest was carefully shaved and ultrasound gel was used on the thorax to optimize visibility during the exam. A 30-MHz probe was used to obtain two-dimensional, M-mode and Doppler imaging from parasternal short-axis view at the level of the papillary muscles and the apical four-chamber view. The M-mode images of the LV were obtained, and systolic and diastolic wall thickness (anterior and posterior) and LV end-systolic and end-diastolic diameters (LVESD and LVEDD, respectively) were measured. LV fractional shortening (FS) was calculated as (LVEDD − LVESD)/LVEDD × 100. Ejection fraction (EF) was calculated using the Teichholz formula.

Histology

Tranverse sections of the median third of the LV were flash frozen in liquid nitrogen and processed for cryosectioning. Apoptosis was examined using TUNEL assay according to manufacturer's instructions. Apoptotic rate within the peri-infarct regions was calculated at ×40 magnification under light microscopy.

Myocardial fibrosis was examined to address prevalence of scar formation within the LV. Heart sections (5 μm) were stained with Masson's trichrome (Sigma Aldrich). Fibrosis and the total LV area of each image were measured using computer morphometry (Bioquant) and expressed as a percentage of LV. The assignment of animals to treatment was random although fibrosis was not measured in a blinded fashion.

Determination of Vascular Density

Tissue sections were stained with CD-31 antibody (Millipore) for evaluation of vascular density. For quantification of positively stained vessels, five sections within the peri-infarcted area of each animal were analyzed in a nonblinded way as described previously [28]. Blood vessels were detected at low magnification, ×200.

Measurement of VEGF, Basic Fibroblast Growth Factor, Angiopoietin-1, and IGF by ELISA

For in vitro studies involving SI/RO in ASCs, the levels of VEGF, basic fibroblast growth factor (b-FGF), and IGF released from ASCs into culture medium were measured by ELISA kit according to manufacturer's instructions (R&D Systems, Minneapolis, MN, www.rndsystems.com). Basal medium was used as a control. The absorbance was measured at 450 and 570 nm. In the in vivo mouse studies, whole blood was drawn in sodium citrate tubes at sacrifice and immediately centrifuged at 1,000g at 4°C for 10 minutes. The supernatant was collected and used in ELISA-based Quantikine assay (R&D Systems) for human VEGF, b-FGF, and angiopoietin-1 (Ang-1) as per manufacturer's instructions.

Data Analysis and Statistics

Data are presented as mean ± SE. The differences between groups were analyzed with one-way analysis of variance followed by Student–Newman–Keuls post hoc test for pairwise comparison. p < .05 was considered to be statistically significant.

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 Conflict of Interest
  10. REFERENCES
  11. Supporting Information

Cytoprotection by PDE-5 Inhibition

Immunostaining showed that ASCs express PDE-5 which was localized within the cytoplasm (Fig. 1A). Moreover, adenoviral infection was confirmed by the presence of green fluorescent protein (GFP) expression in shRNACON and shRNAPDE-5. PDE-5 expression was also confirmed by Western blot and real-time polymerase chain reaction. shRNAPDE-5 efficiently silenced PDE-5 in ASCs at gene and protein expression levels (Fig. 1B, 1C). Adenovirally transduced or normal cultured ASCs were treated with or without 10 μM sildenafil for 2 hours. The percentage of trypan blue-positive (necrotic) cells increased to 24.2 ± 3.7 as compared to non-SI/RO controls (0.6 ± 0.1) following SI (15 hours)/RO (1 hour) (n = 8, p < .01). Sildenafil treatment reduced cell death as measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium–based cell incorporation (Supporting Information Fig. S2A) and necrosis as shown by decrease in trypan blue-positive cells to 5.7 ± 1.6% (n = 8, p < .01; Fig. 2A). PDE-5 knockdown by shRNAPDE-5 conferred a similar protective effect when compared with the scrambled shRNACON ASCs (7.1 ± 0.9% vs. 29.3 ± 1.3%, p < .01, n = 8; Fig. 1C). Also, combination of shRNACON with sildenafil increased cell viability and reduced necrosis (Supporting Information Fig. S2A, Fig. 2A). Similar results were obtained when LDH release in the medium was used as a marker of necrosis in ASCs (Supporting Information Fig. S2B).

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Figure 1. PDE-5 expression in ASCs. (A): Immunohistochemical staining of PDE-5 (red), GFP (green), DAPI (blue), and overlay in ASCs (×40 magnification). (B): Western blot analysis showing knockdown of PDE-5 with shRNAPDE-5 (representative lanes from continuous blot). (C): Real-time PCR showing reduced PDE-5 expression. Abbreviations: ASC, adipose-derived stem cell; DAPI, 4′,6-diamino-2-phenylindole; GFP, green fluorescent protein; PCR, polymerase chain reaction; PDE-5, phosphodiesterase-5; shRNA, short hairpin RNA; shRNACON ASC, ASCs transduced with an adenoviral vector containing scrambled control shRNA; shRNAPDE-5 ASC, ASCs transduced with an adenoviral vector containing PDE-5 shRNA.

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Figure 2. Effect of PDE-5 inhibition on protection of ASCs. Quantitative data showing the effect of Sil or shRNAPDE-5 on necrosis and apoptosis following SI-RO as determined by trypan blue staining (A) and TUNEL assay (B) (*, p < .01 vs. SI-RO, shRNACON ASC; n = 8). Abbreviations: ASC, adipose-derived stem cell; PDE-5, phosphodiesterase-5; shRNA, short hairpin RNA; SI–RO, simulated ischemia-reoxygenation; Sil, sildenafil; shRNACON ASC, ASCs transduced with an adenoviral vector containing scrambled control shRNA; shRNAPDE-5 ASC, ASCs transduced with an adenoviral vector containing PDE-5 shRNA; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

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After 15 hours of SI and 18 hours of RO, apoptotic nuclei (TUNEL-positive cells) increased from 2.0 ± 0.5% (in nonischemic control group) to 18.3 ± 2.5% of total cells (p < .01, n = 8). PDE-5 inhibition resulted in reduction of TUNEL-positive cells to 5.7 ± 2.1% in sildenafil-treated ASCs and 6.4 ± 0.9% in shRNAPDE-5 ASCs (p < .01 vs. SI-RO, n = 8; Fig. 2B, Supporting Information Fig. S2C).

Effect of PDE-5 Inhibition on cGMP and PKG Activity

PDE-5 inhibition by sildenafil or shRNAPDE-5 resulted in nearly identical increase in cGMP levels (0.9 ± 0.01 and 0.9 ± 0.02 pmol/mg of protein, respectively) as compared to nontreated ASCs (0.7 ± 0.03) and shRNACON ASCs (0.7 ± 0.03; p < .05, n = 4). shRNACON had no effect on cGMP formation with sildenafil treatment (Fig. 3A). Both sildenafil and shRNAPDE-5 had no effect on cAMP levels (Fig. 3B). Also, sildenafil and shRNAPDE-5 increased PKG enzymatic activity (A450 per mg protein) when compared with control ASCs (Fig. 3D).

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Figure 3. PDE-5 inhibition increases cGMP and PKG activity. cGMP (A) and cAMP (B) concentrations in ASCs following preconditioning with Sil and shRNAPDE-5. (C): Western blot analysis showing knockdown of PKG expression. (D): PKG activity in ASCs (*p < .05 vs. other groups; n = 4). Abbreviations: ASC, adipose-derived stem cell; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; PDE-5, phosphodiesterase-5; PKG, protein kinase G; shRNA, short hairpin RNA; shRNACON ASC, ASCs transduced with an adenoviral vector containing scrambled control shRNA; shRNAPDE-5 ASC, ASCs transduced with an adenoviral vector containing PDE-5 shRNA; shRNAPKG ASC, ASCs transduced with an adenoviral vector containing PKG shRNA; Sil, sildenafil.

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To determine the cause and effect relationship of PKG in sildenafil-induced survival of ASCs following SI/RO, we used shRNA knockdown of PKG and pharmacological inhibition approach as reported previously [25]. ASCs infected with shRNAPKG caused 60% knockdown of PKG (Fig. 3C). Moreover, sildenafil-induced PKG activity was inhibited in shRNAPKG ASCs or by cotreatment with PKG inhibitor, KT 5823 (Fig. 3D). The protective effect of sildenafil against necrosis and apoptosis (Fig. 2) was attenuated by KT 5823 and in shRNAPKG ASCs (Fig. 4, Supporting Information Fig. S3).

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Figure 4. Sil protects ASCs against ischemia/reoxygenation injury through PKG. (A): Quantitative data showing cell necrosis assessed by trypan blue exclusion assay. (B): Apoptosis assessed by TUNEL staining (*, p < .01 vs. all groups; n = 8). Abbreviations: ASC, adipose-derived stem cell; PKG, protein kinase G; SI–RO, simulated ischemia–reoxygenation; shRNAPKG ASC, ASCs transduced with an adenoviral vector containing PKG shRNA; Sil, sildenafil; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

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PDE-5 Inhibition Enhances Release of Growth Factors

We examined the effect of PDE-5 inhibition in ASCs on release of growth factors, VEGF, b-FGF, and IGF-1 in vitro. No differences in their secretion were observed between sildenafil-treated, shRNAPDE-5 and nontreated ASCs under normal conditions. Following SI/RO, both sildenafil and knockdown with shRNAPDE-5 increased the release of b-FGF (1.7-fold), IGF-1(1.5-fold), and VEGF (1.4-fold) when compared with SI/RO control. Inhibition of PKG blocked the enhanced secretion of the growth factors (Fig. 5).

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Figure 5. PDE-5 inhibition increases the release of growth factors in ASCs. Sil and PDE-5 knockdown with shRNAPDE-5 augmented the release of (A) b-FGF, (B) IGF, and (C) VEGF after simulated ischemia/reoxygenation which is blocked by PKG inhibitor KT 5823 (KT) and shRNAPKG (*, p < .001 vs. all groups; n = 4). Abbreviations: ASC, adipose-derived stem cell; b-FGF, basic fibroblast growth factor; IGF, insulin-like growth factor; PKG, protein kinase G; PDE-5, phosphodiesterase-5; shRNA, short hairpin RNA; shRNAPKG ASC, ASCs transduced with an adenoviral vector containing PKG shRNA; shRNACON ASC, ASCs transduced with an adenoviral vector containing scrambled control shRNA; shRNAPDE-5 ASC, ASCs transduced with an adenoviral vector containing PDE-5 shRNA; Sil, sildenafil; VEGF, vascular endothelial growth factor.

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Preconditioned ASCs Improve Cardiac Function, Reduce Fibrosis and Apoptosis

Supporting Information Figure S4A shows representative M-mode images 4 weeks after MI. Significant functional loss continued over the following 28 days in saline-treated hearts. Echocardiography recordings show that values for all measures of LV function decreased significantly in hearts treated with PBS post-MI when compared with sham. Conversely, hearts injected with ASCs demonstrated a trend toward an increase in function. However, treatment with preconditioned ASCs demonstrated significant improvement in function when compared with nonpreconditioned cells or PBS. Specifically, LVEDD, LVESD, EF, and FS were improved significantly in mice receiving sildenafil-treated ASCs when compared with mice receiving nontreated ASCs at 4 weeks after MI (Fig. 6A–6C, Supporting Information Fig. S4B). Administration of ASCs preconditioned with shRNAPDE-5 also caused similar enhanced preservation of cardiac function and attenuation of cardiac remodeling when compared with ASCs treated with control vector. There were no significant differences in the echo parameters or heart rate in mice receiving shRNACON ASCs and nontreated ASC (Fig. 6A–6C, Supporting Information Fig. S4C).

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Figure 6. Transplantation of preconditioned ASCs improves cardiac function and remodeling and reduces myocardial fibrosis, apoptosis and increases vascular density following myocardial infarction. Bar diagram showing quantitative data of hearts treated with preconditioned ASCs as compared with nontreated ASCs control following MI. (A): Left ventricle end-diastolic diameter; (B) end-systolic diameter; (C) ejection fraction (*, p < .05 vs. MI and MI + ASC; n ≥ 5). (D): Fibrosis measured by Masson's trichrome staining of tissue sections from the various groups. (E): Bar diagram showing quantitative data of TUNEL positive cells. (F): Vascular density measured by immunostaining of sections with CD31 (*, p < .05 vs. MI and MI + ASC; n = 4). (G): Representative images of Masson's trichrome staining of tissue sections from the various groups. Abbreviations: ASC, adipose-derived stem cell; MI, myocardial infarction; PDE-5, phosphodiesterase-5; shRNA, short hairpin RNA; shRNACON ASC, ASCs transduced with an adenoviral vector containing scrambled control shRNA; shRNAPDE-5 ASC, ASCs transduced with an adenoviral vector containing PDE-5 shRNA; Sil, sildenafil; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

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The fibrosis was 55%–60% lower in hearts receiving preconditioned ASCs when compared with nontreated ASCs (7.1 ± 0.7% with sildenafil-treated ASCs; 6.8 ± 1.7% with shRNAPDE-5 ASCs vs. 14.9 ± 3.8% with nontreated ASCs, n = 5, p < .05). All cell treatment groups showed significant reduction in fibrosis compared with PBS controls (31.3 ± 3.8%; Fig. 6D, 6G). Also, the reduction in fibrosis seen in sildenafil- and shRNAPDE-5-treated ASCs correlated with reduction of cardiomyocyte apoptosis. PBS-treated mice 4 weeks post-MI had a resident myocyte apoptotic rate of 3.3 ± 0.3%, which was reduced to 1.9 ± 0.2% in nontreated ASC group. Mice injected with sildenafil or shRNAPDE-5 ASCs showed even further reduction in apoptosis (0.9 ± 0.14% with sildenafil and 0.8 ± 0.12% with shRNAPDE-5 ASCs, p < .05 vs. PBS control, n = 5; Fig. 6E).

Preconditioned ASCs Increase Vascular Density and Secretion of Growth Factors

Rapid restoration of blood supply to the ischemic region is critical for stabilizing the border region of the infarct and supporting viable and regenerating myocardium. We used CD-31 positive staining to determine the vascular density in the border zone. Transplantation of ASCs significantly increased vascular density when compared with PBS-treated hearts (Fig. 6F). The vascular density in sildenafil- and shRNAPDE-5 ASC-treated mice was enhanced when compared with nontreated ASC mice (8.3 ± 1.3 in sildenafil-treated ASCs; 7.8 ± 0.5 shRNAPDE-5 ASCs vs. 4.5 ± 0.6 vessels/high-powered field, p < .01, n = 4; Fig. 6F). Moreover, the plasma levels of b-FGF, Ang-1, and VEGF were increased in mice receiving sildenafil or shRNAPDE-5 preconditioned ASCs when compared with nontreated ASCs (p < .001, n = 5; Fig. 7A–7C).

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Figure 7. Transplantation of preconditioned ASCs enhances the release of growth factors following MI. Plasma levels of (A) b-FGF, (B) Ang-1, and (C) VEGF were increased in mice receiving sil or shRNAPDE-5 preconditioned ASCs as compared to nontreated ASCs (*, p < .001 vs. MI and MI + ASC, n = 5). Abbreviations: ASC, adipose-derived stem cell; Ang-1, angiopoietin-1; b-FGF, basic fibroblast growth factor; MI, myocardial infarction; PDE-5, phosphodiesterase-5; shRNA, short hairpin RNA; shRNAPDE-5 ASC, ASCs transduced with an adenoviral vector containing PDE-5 shRNA; Sil, sildenafil; VEGF, vascular endothelial growth factor.

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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 Conflict of Interest
  10. REFERENCES
  11. Supporting Information

Cardiac repair via cellular transplantation has generated considerable enthusiasm in recent years although the optimal cells for cardiac repair remain to be identified. In this study, we chose adipose tissue which is an excellent source for stem cell-based treatment of injured myocardium because it is relatively easy to harvest, available in sufficient quantities, and yields a significantly higher number of uncommitted stem cells compared with other sources such as bone marrow. Moreover, the use of adipose tissue as the source of these cells obviates the need for prior cell expansion in vitro, thus potentially allowing for immediate autologous cell transplantation for acute infarction. Prior studies have shown that ASCs have the ability to differentiate into multiple mesenchymal cell types including endothelial cells [29, 30] and cardiomyocytes [31, 32]. Human ASCs have been shown to preserve heart function following MI [33]. In this study, we investigated the feasibility of PDE-5 inhibition as a strategy to precondition human ASCs for improving their efficacy in vivo after cardiac transplantation. The rationale for this approach was the established powerful preconditioning-like effect of PDE-5 inhibitors in cardiomyocytes [24, 25] and against ischemia/reperfusion injury in heart [16, 17, 19, 34] established by us. Our results show that preconditioned ASCs by PDE-5 inhibition significantly improved their ability to survive SI/RO injury in vitro. Moreover, we observed significant release of proangiogenic/prosurvival growth factors including VEGF, b-FGF, IGF, and Ang-1. The intramyocardial injection of preconditioned ASCs into the border zone induced angiogenesis, suppressed fibrosis, decreased apoptosis, and significantly improved function when compared with nonpreconditioned ASCs. More importantly, we provide the first evidence for robust expression of PDE-5 in the isolated ASCs. PDE-5 knockdown with a silencing vector significantly reproduced the effect of sildenafil in survival against cell death as well as release of growth factors. These data not only rule out the potential off target effects of sildenafil in ASCs but also provide us a genetic approach to precondition the stem cells possibly for improving survival after transplantation. To our knowledge, this is the first study showing PDE-5 as a target gene/enzyme to improve survival of ASCs under ischemic conditions in vitro and in vivo. These are clinically significant observations because improving stem cell survival by exploiting novel therapeutic targets with clinically approved drugs would directly impact the prognostic outcome of stem cell therapy in the heart.

We have previously shown that sildenafil induces a cytoprotective effect in cardiomyocytes through NO- and cGMP-dependent activation of PKG, which results in opening of mitochondrial KATP channels [24, 25, 35]. While the comprehensive cytoprotective signaling pathways following PDE-5 inhibition in ASCs remains to be investigated, it is clear that cGMP (but not cAMP) was elevated and PKG was involved in protecting ASCs against ischemic injury (Figs. 3, 4). PKG is a serine/threonine protein kinase that has two isozymes (type I and type II; i.e., PKGI and PKGII). PKGIα is mainly found in lung, heart, platelets, and cerebellum, whereas PKGIβ is highly expressed in smooth muscles of uterus, vessels, intestine, and trachea [36]. We had shown that sildenafil activated PKG-dependent signaling cascade that involved phosphorylation of ERK and inhibition of GSK-3β thus leading to cytoprotection [25]. Moreover, gene transfer of PKGIα in cardiomyocytes in the absence of sildenafil or other Pathophysiological stimuli (such as ischemic preconditioning) resulted in a cytoprotective phenotype that was associated with the phosphorylation of Akt, ERK, and c-Jun N-terminal kinase and increased Bcl-2 expression [37]. It is quite likely that a similar cascade of signaling events leads to survival of ASCs after SI.

A significant improvement in cardiac function was observed 4 weeks after transplantation of preconditioned ASCs. Moreover, the superior functional improvement compared with nonpreconditioned ASC-injected group was associated with enhanced vascular density (Fig. 6F), decrease in fibrosis and apoptosis (Fig. 6D, 6E). The significantly improved vascular density observed could be due to the release of growth factors with angiogenic potential, including b-FGF, Ang-1, IGF-1, and VEGF, which were significantly higher in the sildenafil- and shRNAPDE-5-treated ASCs. Moreover, the release of growth factors was blunted under conditions where PKG signaling was disrupted (Fig. 5) suggesting a critical role of cGMP–PKG pathway in their secretion. These results are supported by other studies on downregulation of VEGF expression with the inhibition of the downstream kinase of PKG, GSK3β [38], which are also activated by sildenafil treatment in cardiomyocytes and exert protective effect against SI/RO injury [25]. ASCs have been shown to secrete VEGF and HGF, which possess both angiogenic and antiapoptotic effects on both myocardial and endothelial cells [39]. Moreover, PDE-5 inhibition induces expression of VEGF and Ang-1, which correlated with significant reduction in infarct size, cardiomyocyte and endothelial apoptosis while promoting angiogenesis following ischemia/reperfusion [40]. Similarly, overexpression of Ang-1, VEGF, or b-FGF improves cell survival, neovascularization, and cardiac function by limiting the remodeling process in the scar while decreasing apoptosis of myocytes in the peri-infarct region [41–43]. Transplantation of ASCs along with their secretion of VEGF, bFGF, and IGF has been shown to upregulate Bcl-2, which results in the decrease in myocyte apoptosis in vitro and in vivo [42]. Our results demonstrate that transplantation of preconditioned ASCs abrogated resident cardiomyocyte apoptosis seen in nontreated ischemic hearts suggesting that the protective effect is attributed to increased duration and release of paracrine factors in the ischemic myocardium.

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 Conflict of Interest
  10. REFERENCES
  11. Supporting Information

In conclusion, we have shown that in vitro preconditioning of ASCs by PDE-5 inhibition improves survival under conditions of ischemia/RO. The preconditioned ASCs ameliorate LV remodeling, preserve LV function, and reduced cardiomyocyte apoptosis and fibrosis possibly by improving stem cell survival and paracrine effects. We propose that in vitro preconditioning of ASCs by inhibition of PDE-5 with small molecule drugs or gene silencing vectors can be a powerful new approach to improve stem cell therapy following MI in patients. Particularly, the easy availability of ASCs from humans combined with the preconditioning by inhibition of PDE-5 may hold great promise for initiation of clinical trials in patients with heart failure.

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 Conflict of Interest
  10. REFERENCES
  11. Supporting Information

This study was supported by grants from National Institutes of Health (HL51045, HL79424, and HL93685) to Rakesh C. Kukreja, National Scientist Development grant from the American Heart Association (10SDG3770011) to Fadi N. Salloum, and Predoctoral Fellowship from the American Heart Association (09PRE2250905) to Nicholas N. Hoke.

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  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. Disclosure of Potential Conflict of Interest
  10. REFERENCES
  11. Supporting Information
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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 Conflict of Interest
  10. REFERENCES
  11. Supporting Information

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

FilenameFormatSizeDescription
STEM_789_sm_suppFigure1.pdf1082KFigure S1. Experimental Protocol. (A). In vitro protocol, arrows indicate time points for treatment, performance of simulated ischemia/reoxygenation, and measurement of various parameters for each experimental group. (B). In vivo protocol. Arrowheads indicate sites of injection of adipose-derived stem cells in the border zone of the infarcted heart.
STEM_789_sm_suppFigure2.pdf957KFigure S2. Effect of PDE-5 inhibition on protection of ASCs. Quantitative data showing the effect of sildenafil (SIL) or shRNAPDE-5 on necrosis following SI-RO as determined by MTS cell viability assay (A) and LDH release (B). (C) Representative images of TUNEL assay (10 x magnification). (* indicates p<0.01 versus SI-RO, shCON ASC; n=8).
STEM_789_sm_suppFigure3.pdf955KFigure S3. Sildenafil protects ASCs against ischemia/reoxygenation injury through PKG. Quantitative data showing the effect of KT 5823 (KT) and shRNAPKG on necrosis following SIRO as determined by MTS cell viability assay (A) and LDH release (B). (C) Representative images of TUNEL assay (10 x magnification). (* indicates p<0.01 versus SI-RO, shCON ASC; n=8).
STEM_789_sm_suppFigure4.pdf1183KFigure S4. Transplantation of preconditioned ASCs improves cardiac function and remodeling following myocardial infarction. (A) Representative M-mode images showing preservation of LV contractility of hearts treated with preconditioned ASCs as compared with non-treated ASCs control following myocardial infarction (MI). (B). Bar diagram showing quantitative data of LV fractional shortening.

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