Transplanted Human Amniotic Epithelial Cells Secrete Paracrine Proangiogenic Cytokines in Rat Model of Myocardial Infarctio

Abstract

Human amniotic epithelial cells (h-AECs) have been shown to differentiate into cardiomyocyte-like cells in vivo that can regenerate myocardial tissue and improve cardiac function in a rat model of myocardial infarction (MI). In this study, we investigated the paracrine factors released from h-AECs under hypoxic conditions to elucidate the possible mechanisms underlying this previously reported phenomenon of h-AEC-mediated cardiac repair. We used hypoxic cell culture conditions to simulate myocardial infarction in vitro. In comparison to normal conditions, we found that h-AECs secreted higher levels of several cytokines, including angiogenin (ANG), epidermal growth factor (EGF), interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1. To determine whether transplanted h-AECs express these proangiogenic cytokines in vivo, we ligated the coronary artery of rats to cause MI and injected either h-AECs or saline into the infarcted area. We found that the infarct and border zones of rat myocardium treated with h-AECs had higher expression levels of the human-origin cytokines ANG, EGF, IL-6, and MCP-1 compared to the tissues of saline-treated rats. In conclusion, h-AECs secreted proangiogenic cytokines in a rat model of MI, which may suggest that the paracrine effect by h-AECs could regenerate myocardial tissue and improve cardiac function.

Keywords

Amniotic epithelial cells Myocardial infarction (MI)Paracrine Cytokine

Introduction

Myocardial infarction (MI) is a central cause of heart failure and a global health concern (24). Among the treatments for MI, repair of damaged myocardium is a potential treatment of the underlying pathology. To this end, cell-based therapy has been investigated to regenerate damaged myocardium after MI using a wide range of cell types (18,21).

Human amniotic epithelial cells (h-AECs) are characterized by low immunogenicity and anti-inflammatory properties. They are easily isolated from human placenta with few ethical issues (20,25,28) and have been found to differentiate into cardiomyocytes in a rat model of MI (17). These characteristics make h-AECs good candidates for cell therapy. This research group recently demonstrated that h-AECs differentiated into cardiomyocyte-like cells in vivo and regenerated myocardial tissue in a rat model of MI, improving cardiac function (7). However, the mechanisms underlying these effects remain to be elucidated.

Previous studies have reported that adult stem cells repair the heart after MI through paracrine mechanisms. For example, it has been reported that bone marrow mononuclear cells regenerate infarcted myocardium through paracrine mechanisms (23). In addition, Perez-Ilzarbe et al. demonstrated the paracrine effects of transplanted human skeletal myoblasts in a rat model of MI (19). Another recent report has shown that bone marrow stem cells improve cardiac function by paracrine factors (26).

Therefore, we hypothesized that the improvements in myocardial function and regeneration of the myocardium in MI after h-AEC therapy could be associated with paracrine effects. This study was aimed first at analyzing the in vitro cytokine expression profile in hypoxic h-AECs in comparison with normoxic h-AECs, second at determining whether these cytokines were released in a rat model of MI, and finally at confirming the origin of released cytokines by h-AECs in vivo.

Materials and Methods

Isolation and Culture of h-AECs

Following informed consent, cryopreserved h-AECs (K-STEMCELL, Seoul, South Korea) provided as an anonymized sample from the stem cell bank and derived from one donor were used for the study. In brief, the amnion layer was mechanically peeled from human placenta and digested with trypsin-EDTA (Gibco, Grand Island, NY, USA). After centrifugation, the cells were cultured overnight at 37°C in amniotic epithelial cell media (K-STEMCELL) supplemented with 10% fetal bovine serum (Gibco). The next day, nonadherent cells were removed by washing with phosphate-buffered saline (Gibco), and adherent cells were kept in culture until the cells reached 80–90% confluence, which was defined as passage 0. The h-AECs had been cryopreserved after 9 days of passage 0 culture, which is formed by seeding and plating cells obtained directly from primary amnion tissue. Expanded h-AECs were aliquoted and stored in liquid nitrogen until further use. The procedure was approved by the Hanyang University Institutional Review Board (HYI-13-012-4).

Hypoxic Treatment

To simulate MI and stimulate cytokine secretion, h-AECs were cultured under hypoxic conditions using an MIC-101 modular incubator chamber (Billups-Rothenberg, Del Mar, CA, USA), which was flushed with 95% N₂ and 5% CO₂, sealed, and placed at 37°C for up to 24 h as indicated. Control cells were cultured in a standard incubator under normal conditions (21% O₂ and 5% CO₂) (9).

Preparation of h-AEC-Conditioned Medium

To obtain conditioned media, h-AECs (1 × 10⁶ cells/ dish) were grown in 100-mm Petri dishes (eight dishes; BD Falcon, San Jose, CA, USA) to approximately 90% confluence in three to four different experiments. h-AECs (approximately 5 × 10⁶ cells/dish) were rinsed with serum-free medium and cultured for 24 h in serum-free amniotic epithelial cell media. The 4 ml of conditioned media was centrifuged and stored frozen at 75°C until use, and 2 ml each of conditioned media was used for an antibody-based protein array and ELISA analysis.

Antibody-Based Protein Array

Conditioned media from h-AECs cultured under normal and hypoxic conditions were analyzed using an antibody-based protein array (10). The protein array kit RayBio® Human Cytokine Antibody Array 6 was performed according to the manufacturer's instruction (AAH-CYT-6; RayBiotech, Norcross, GA, USA).

ELISA Analysis

Cytokine concentrations were measured using ELISA kits [eBioscience, San Diego, CA, USA for monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-6; RayBiotech, for angiogenin (ANG) and epidermal growth factor (EGF)]. ELISA cytokine analysis was performed according to the manufacturer's instructions (2). All assays were performed in duplicate.

Western Blot Analysis

All cells were lysed with RIPA buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Next, protein extracts were loaded onto SDS-PAGE gels (8% acrylam-ide; GenDEPOT, Barker, TX, USA) and transferred to 0.45-μm immobilon polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked and then incubated with primary antibodies against hypoxia-inducible factor (HIF)-1α (1:500; Cell Signaling Technology, Beverly, MA, USA), and then incubated with HRP-conjugated anti-rabbit antibody (1:2,000; Jackson ImmunoResearch, West Grove, PA, USA).

Animals

Male athymic nude rats (NIH-rnu; Taconic, Germantown, NY, USA), 6 weeks of age and weighing 200–250 g, were used in this experiment. All experiments were performed in compliance with the ARRIVE guidelines for research (13), and the Hanyang University Institutional Animal Care and Use Committee approved all protocols.

Myocardial Infarction and Cell Implantation

We created a rat model of MI by ligation of the left anterior descending (LAD) coronary artery, as described previously (15,16). The rats were anesthetized with a mixture of ketamine hydrochloride (100 mg/kg; Yuhan Corp., Seoul, South Korea) and xylazine hydrochloride (10 mg/kg; Rompun; Bayer, Puteaux, France). The heart was exposed using a left anterior thoracotomy, and the mid portion of the LAD was ligated with 6-0 polypropylene (Prolene®; Ethicon, Hamburg, Germany) at the level of the left atrial appendage. Ten days after LAD ligation, the rats were randomly divided into two groups of eight rats each. One group was injected with h-AECs and the other with saline. To trace the fate of the transplanted cells, cultured h-AECs were labeled with chloromethyl-benzamidodialkylcarbocyanine (Cell Tracker”: CM-DiI; Molecular Probes, Eugene, OR, USA) according to the manufacturer's protocol (27). Using a 30-gauge needle (Profil needle; Shinchang Medical Co., Seoul, South Korea), we injected each heart with either 100 μl of the saline containing cultured h-AECs (1 × 10⁶ cells) or saline at three different sites 2 mm apart from the border toward the center of the infarct and from the lateral left ventricle to the septum. One day or 1 week after treatment with h-AECs or saline, myocardial tissues from the infarct zone (IZ), border zone (BZ), and remote zone (RZ) were snap frozen in liquid nitrogen and used for reverse transcription-polymerase chain reaction (RT-PCR) analysis. The experimental design is described in Figure 1.

Figure 1.

Experimental scheme. LAD, left anterior descending; AEC, amniotic epithelial cell; RT-PCR, reverse-transcription polymerase chain reaction; MT, Masson's trichrome.

Immunofluorescence Analysis

To investigate the fate of the h-AECs labeled with CM-DiI in the cardiac tissue after transplantation, immunofluorescence analysis was performed as previously described (12). Briefly, myocardial tissue harvested 1 week after treatment with h-AECs or saline was snap frozen and sliced into 4-μm sections with a microtome (CM1510S-3; Leica, Wetzlar, Germany). The sections were incubated with antibodies against myosin heavy chain (MHC; 1:100; Novus Biologicals, Littleton, CO, USA) at 4°C overnight, and then the sections were incubated with the fluorescein isothiocyanate-conjugated anti-goat IgG secondary antibody (Alexa 488; 1:250; Life Technologies, Carlsbad, CA, USA).

Masson's Trichrome Staining and TUNEL Assay

One week after the LAD ligation, heart tissues were fixed in 10% neutral-buffered formalin (BBC Biochemical, Mount Vernon, WA, USA) and embedded in paraplast (McCormick Scientific, St. Louis, MO, USA). The extent of myocardial fibrosis was determined by visualizing fibrotic tissue using Masson's trichrome (MT) staining. After deparaffinization, slides were dipped into Bouin's solution (Sigma-Aldrich, St. Louis, MO, USA) and kept for 1 h at 60°C. The slides were then washed in water and dipped into Biebrich scarlet-acid fuchsin solution (Sigma-Aldrich), phosphomolybdic–phosphotungstic acid (JUNSEI, Tokyo, Japan), and aniline blue (JUNSEI). Apoptotic myocytes of the infracted myocardium were evaluated using the TUNEL assay in paraffin sections with an In Situ Cell Death Detection kit (Roche, Mannheim, Germany). The stained sections were photographed using a light microscope (Leica DM 4000B).

RT-PCR

For RT-PCR, 1 μg of RNA samples was reverse transcribed with Moloney murine leukemia virus reverse tran-scriptase (Invitrogen, Carlsbad, CA, USA). PCR mixtures contained 1 μl cDNA, 2 μl 10× PCR buffer, 2 μl dNTP, 0.2 μl Taq DNA polymerase (iNtRON, Seongnam, South Korea), and 0.4 μl of 25 pmol/μl of sense and antisense primers (Bioneer, Daejeon, South Korea). The human- and rat-specific primers for the target genes ANG, EGF, IL-6, MCP-1, and GAPDH are listed in Table 1. We performed PCR (C1000^TM Thermal Cycler; Bio-Rad Laboratories, Richmond, CA, USA) under the following conditions: initial denaturation at 95°C for 5 min, 35 amplification cycles (denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min), and a final extension at 72°C for 5 min. The products were visualized on 2% agarose gels (Invitrogen) stained with ethidium bromide (Invitrogen) using Gel-Doc 2000 (Bio-Rad Laboratories). The band intensity was determined with Quantity One software (Bio-Rad Laboratories) (22).

Table 1.

Primer Sequences

Primer	Sequences	Size (bp)
hANG	F: 5′-CGG-GGC-CTG-ACC-TCA-CCC-TGC-AAA-3′	207
	R: 5′-TCT-GAA-CCC-CGC-TGT-GGC-TCG-GTA-3′
rANG	F: 5′- CTG-CAA-AGA-GGT-CAA-CAC-CTT-TAT-C-3′	307
	R: 5′- GGT-GAG-GGG-AGA-TAA-AAA-GAG-AAC-A-3′
hEGF	F: 5′-GAT-GGA-ACA-GAC-TAT-GGA-ACT-CTG-C-3′	351
	R: 5′-AGT-CCA-GAA-TAA-TCT-CTT-GGC-CAT-T-3′
rEGF	F: 5′-AAT-AAG-GGT-GAA-CAA-GAG-GAC-TGG-T-3′	318
	R: 5′-TCT-GGT-ACA-TAA-GCC-TTA-GGG-GAG-T-3′
hIL-6	F: 5′-GGC-ACC-TCA-GAT-TGT-TGT-TGT-TAA-T-3′	318
	R: 5′-TGA-GGT-AAG-CCT-ACA-CTT-TCC-AAG-A-3′
rIL-6	F: 5′-CAA-GAG-ACT-TCC-AGC-CAG-TTG-3′	612
	R: 5′-GCC-GAG-TAG-ACC-TCA-TAG-TGA-C-3′
hMCP-1	F: 5′-TGC-TGC-TAT-AAC-TTC-ACC-AAT-A-3′	259
	R: 5′-TGG-GGA-AAG-CTA-GGG-GAA-AAT-A-3′
rMCP-1	F: 5′-ATG-CAG-GTC-TCT-GTC-ACG-CTT-CTG-GGC-3′	447
	R: 5′-CTA-GTT-CTC-TGT-CAT-ACT-GGT-CAC-3′
rGAPDH	F: 5′-GAC-AAC-TTT-GGC-ATC-GTG-GA-3′	133
	R: 5′-ATG-CAG-GGA-TGA-TGT-TCT-GG-3′

ANG, angiogenin; EGF, epidermal growth factor; IL, interleukin; MCP, monocyte chemoattractant protein.

Statistical Analysis

All data are presented as mean ± standard deviation (SD). All data were analyzed with SPSS software version 17 (IBM, Armonk, NY, USA). Data were analyzed using a Student's t-test (for single comparisons) or a one-way ANOVA (for multiple comparisons), and post hoc multiple comparisons were made with Tukey's test (equal variances assumed). Values of p < 0.05 were considered statistically significant.

Results

Western Blot Analysis of HIF-1a Protein in h-AECs Under Hypoxic Conditions

We cultured h-AECs under hypoxic conditions (95% N₂ and 5% CO₂) for 1, 2, 4, 8, and 24 h. Western blot analysis revealed higher HIF-1α expression levels in cells exposed to hypoxic conditions compared to those under normal conditions (Fig. 2).

Figure 2.

Western blotting of hypoxia-inducible factor (HIF)-1α protein under hypoxic conditions. h-AECs were exposed to hypoxic conditions for 1, 2, 4, 8, and 24 h. HIF-1α protein was detected under hypoxic conditions but was not detected under normoxic conditions.

In Vitro Cytokine Release by h-AECs Under Hypoxic Conditions

To measure the cytokine levels released by h-AECs, we performed antibody-based protein array analysis of h-AEC-conditioned medium for 24 h under hypoxic or normal conditions using 60 human cytokines. Representative blots from these media showed the normal and hypoxic medium density ratios for each factor. The cytokines with at least a 1.5-fold increase in concentration in hypoxic-conditioned medium were ANG, EGF, IL-6, and MCP-1. Compared to normal conditions, hypoxic treatment significantly increased the release of cytokines ANG (100.00 ± 0.23% vs. 112.91 ± 2.54%, p < 0.01), EGF (136.80 ± 5.43%, p < 0.01), IL-6 (110.64 ± 2.60%, p < 0.01), and MCP-1 (200.00 ± 3.58%, p < 0.01). Similar expression patterns of all tested cytokines were found at the mRNA level (Fig. 3). The protein levels of ANG, EGF, IL-6, and MCP-1 in h-AEC-conditioned medium were further quantified by ELISA, and the results were consistent with those from the antibody-based protein array (Fig. 4). The respective amounts of each protein from normal medium versus hypoxic media were as follows: ANG (1.27 ± 0.03 pg/μg vs. 15.45 ± 0.74 pg/μg, p < 0.01) EGF (0.87 ± 0.96 pg/μg vs. 71.22 ± 1.94 pg/μg, p < 0.01), IL-6 (0.87 ± 0.21 pg/μg vs. 5.99 ± 0.81 pg/μg, p < 0.01), and MCP-1 (7.33 ± 2.06 pg/ μg vs. 48.57 ± 1.44 pg/μg, p < 0.01). These data indicate that the protein levels of these cytokines were significantly higher in h-AECs cultured in hypoxic conditions compared to those under normal conditions.

Figure 3.

Antibody-based protein array of h-AEC media under normoxic and hypoxic conditions. (A) In representative arrays of h-AEC media under normoxic and hypoxic conditions, a number of cytokines were released, including ANG, EGF, IL-6, and MCP-1. (B) Protein signals were quantified, normalized to the normoxic conditions, and compared with the negative control of the array. Levels of ANG, EGF, IL-6, and MCP-1 were significantly higher after 24 h of hypoxia compared to levels under normoxic conditions. All data are expressed as mean ± SD of three to four different experiments. *p < 0.01 versus negative control (N). ANG, angiogenin; EGF, epidermal growth factor; IL, interleukin; MCP, monocyte chemoattractant protein.

Figure 4.

Cytokine secretion from human amniotic epithelial cells cultured under normoxic and hypoxic conditions. Protein concentrations of (A) ANG, (B) EGF, (C) IL-6, and (D) MCP-1 in conditioned media were determined by ELISA. The protein levels of ANG, EGF, IL-6, and MCP-1 were significantly higher after 24 h of hypoxia compared to levels under normoxic conditions. All data are expressed as mean ± SD of three to four different conditioned media. *p < 0.01 versus normoxic conditions.

Rat Model of MI

MI was confirmed in the rats using MT staining and TUNEL assay. After 1 week, fibrosis was evident in the left ventricular wall of the infarcted myocardium by MT staining (Fig. 5A, B). The amount of apoptotic cardiomyocytes in the infarcted myocardium was determined by TUNEL assay (Fig. 5C).

Figure 5.

Fibrosis of myocardium and apoptosis of cardiomyocytes at 1 week after MI. (A, B) Representative images of MT staining of whole heart tissue. (C) Representative photomicrographs showing TUNEL assay in the peri-infarct region. (B, C) Higher magnification views of the boxed regions in (A). Scale bars: 200 μm.

Detection of h-AECs in Rat Myocardium

The labeling efficiency of CM-DiI is greater than 95% in vitro prior to cell transplantation. One week after cell transplantation, immunofluorescence analysis detected CM-DiI-positive cells in the infarcted regions of the rat myocardium (Fig. 6).

Figure 6.

One week after cell transplantation, fluorescence microscopy detected h-AECs within the infarcted region of the rat myocardium. Frozen tissue sections were stained with antibodies against myosin heavy chain (MHC); h-AECs (CM-DiI-positive cells) in myocardium (arrowhead); DAPI nuclear stain, blue in (A); CM-DiI, red in (B); MHC, green in (C). (D) is a merged image of (A, B, and C). Scale bars: 100 μm. CM-DiI, Cell tracker”; MHC, myosin heavy chain.

In Vivo Cytokine Release by h-AECs

To examine whether the same cytokines released by h-AECs under hypoxic in vitro conditions were present in vivo, we performed RT-PCR of cytokine mRNA in the infarcted myocardium using human- and rat-specific primers. After transplantation of h-AECs into the rat model of MI, rat mRNA expression levels of ANG, EGF, IL-6, and MCP-1 were not significantly different between groups. However, 1 day after h-AEC injection, human mRNA expression of ANG, EGF, IL-6, and MCP-1 was detected in the IZ and BZ. These cytokines were not detected in the saline-injected group. Furthermore, 1 week after cell transplantation, ANG and MCP-1 human mRNAs were still detected in the IZ and BZ of h-AEC-treated hearts, but EGF and IL-6 mRNAs were no longer detected (Fig. 7).

Figure 7.

hAECs released cytokines in vivo in a rat model of MI. Animals were sacrificed 1 day or 1 week after h-AEC or saline injection. Regional tissue samples were taken from the IZ, BZ, and RZ for RT-PCR analysis. In infarcted h-AEC-injected hearts, h-AECs released human cytokines including hANG, hEGF, hIL-6, and hMCP-1, which were detected by RT-PCR. In contrast, levels of rat cytokines were similar in the h-AEC-treated and saline-treated groups. Cytokines were detected only in the IZ and BZ of the infarcted h-AEC-injected hearts. One week after treatment, bands for hANG and hMCP-1 were detected; bands for hEGF and hIL-6 were no longer detected.

Discussion

Our previous study showed that myocardial injections of h-AECs regenerated myocardium and improved cardiac function in a rat model of MI (7). The present study aimed to elucidate the possible mechanisms underlying these effects. Data showed that h-AECs secreted ANG, EGF, IL-6, and MCP-1 under hypoxic conditions in vitro. We also detected these cytokines released by h-AECs in our in vivo rat model of MI. These proangiogenic cytokines may be associated with the structural and functional improvements observed after h-AEC treatment of MI.

In the previous report, only 3% of transplanted h-AECs were differentiated into cardiomyocyte-like cells in vivo (7). Given that only a few transdifferentiated h-AECs were found in the IZ, it is unlikely that they increased myocardial gene and protein expression; it is more likely that paracrine effects resulted in the observed increases (7). Similarly, Feygin et al. reported that bioenergetic improvements in MI after mesenchymal stem cell transplantation are most likely due to trophic effects of the transplanted cells, in light of the low fraction of transplanted cell engraftment (8). Other studies have also confirmed a paracrine origin of the cytoprotective effects of cytokines produced by bone marrow stem cells on infarcted hearts (23,26). In addition, human skeletal myoblasts have been shown to contribute to cardiac repair through paracrine mechanisms (19).

We demonstrated that h-AECs released cytokines ANG, EGF, IL-6, and MCP-1 when exposed to hypoxic conditions in vitro (Figs. 3 and 4). We also found increased levels of these cytokines in rats with MI after h-AEC injection (Fig. 7). The roles of ANG, EGF, and IL-6 in angiogenesis are mediated by induction of VEGF, a potent angiogenic agent (3,5,14). In addition, MCP-1 is a major chemoattractant for monocytes/macrophages and plays a key role in macrophage infiltration into the perivascular compartment, where they orchestrate processes that ultimately lead to collateral vessel growth (1). Macrophage infiltration plays a pivotal role in tissue repair (6), and myocardial regeneration after MI is associated with increased angiogenesis. Given these findings, our results suggest that the proangiogenic cytokines released by h-AECs may enhance neovascularization and the proliferation of endothelial cells in damaged myocardium. We hypothesize that the angiogenic effects of these cytokines released from transplanted h-AECs are mediated through multiple pathways, including paracrine effects on local vascular cells and chemoattractant-mediated effects. This may lead to the mobilization, proliferation, and/or homing of circulating stem cells and/or endogenous progenitor cells at the sites of injury (11).

In this study, we tested whether h-AECs release cytokines in a rat model of MI. One day and 1 week after h-AEC injection, proangiogenic cytokines including ANG, EGF, IL-6, and MCP-1 were detected in the myocardial IZ and BZ by RT-PCR. Although we did not measure cytokine levels at 4 weeks after h-AEC injection, which is when our previous report found regenerated myocardium and improved cardiac function, our findings provide new insights into the paracrine mechanisms of transplanted h-AECs in a rat model of MI.

The origin of the increased cytokine release in infarcted myocardium was determined by our xeno-h-AEC transplantation study. In this study, mRNA expression of the human-origin proangiogenic cytokines ANG, EGF, IL-6, and MCP-1 increased significantly after h-AEC injection (Fig. 7). A previous study using a mouse model of MI reported that transplanted human cardiosphere-derived cells secreted human-origin cytokines and resulted in endogenous regeneration (4). In addition, cytokines secreted by transplanted human skeletal myoblast cells may contribute to cardiac repair (19). Similarly, our results suggest that h-AECs injected into infarcted rat myocardium release proangiogenic cytokines that aid in myocardial regeneration and improved cardiac function.

This study has several limitations. First, the number of subjects was small. Second, the secretion and activity of the cytokines released by h-AECs were not measured, and cytokine quantification results were not confirmed in vivo. Third, measuring cytokines at 4 weeks after h-AEC transplantation is needed. Finally, the functional roles of the measured cytokines were not determined. Further studies are needed to determine the role of each cytokine and its molecular mechanisms in myocardial repair.

In conclusion, our data showed that h-AECs secrete proangiogenic cytokines including ANG, EGF, IL-6, and MCP-1 in vitro, and that h-AECs secreted these cytokines in a rat model of MI. Our data support the hypothesis that myocardial repair and functional improvement previously reported in a rat model of MI are associated with the paracrine effects of human cytokines derived from h-AECs such as ANG, EGF, IL-6, and MCP-1. Further research into the mechanisms of action of the secreted factors has important implications on the development of novel molecular therapies for the treatment of MI.

Footnotes

Acknowledgments

This work was supported by the research fund of Hanyang University HY-2013. The authors declare no conflicts of interest.

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