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Am J Physiol Heart Circ Physiol 293: H1590-H1595, 2007. First published June 1, 2007; doi:10.1152/ajpheart.00431.2007
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Factors released from embryonic stem cells inhibit apoptosis of H9c2 cells

Dinender K. Singla and Debbie E. McDonald

Department of Medicine, Division of Cardiology, Cardiovascular Research Institute, University of Vermont, College of Medicine, Colchester, Vermont

Submitted 7 April 2007 ; accepted in final form 26 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our recent study (Singla DK, Hacker TA, Ma L, Douglas PS, Sullivan R, Lyons GE, Kamp TJ, J Mol Cell Cardiol 40: 195–200, 2006) suggests that transplanted embryonic stem (ES) cells subsequent to myocardial infarction differentiate into the major cell types in the heart and improve cardiac function. However, the extent of regeneration is relatively meager compared with the observed functional improvement. The mechanisms underlying their improved function are completely unknown. In this report, we provide evidence using a cell culture model system for novel mechanisms that involve the release of cytoprotective, anti-apoptotic factor(s) from ES cells and inhibit H2O2-induced apoptosis in the rat cardiomyocyte-derived cell line H9c2. Conditioned medium (CM) from growing mouse ES cells treated with and without H2O2 was generated. Apoptosis was induced after exposure to H2O2 in H9c2 cells for 2 h followed by replacement with fresh cell culture or ES cell-CM. After 24 h, H9c2 cells treated with both ES cell-CMs demonstrated significantly decreased apoptosis, as determined by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining, apoptotic ELISA, caspase-3 activity, and DNA ladder. Next, using Luminex technology, we examined the presence of antiapoptotic proteins cystatin c, osteopontin, and clusterin and anti-fibrotic, tissue inhibitor of metalloproteinase-1 (TIMP-1) in both ES cell-CMs. The levels of released factors were 2- to 170-fold higher than those in H9c2 cell-CM. Antiapoptotic effects of ES cell-CM were significantly inhibited with TIMP-1 antibody, suggesting that TIMP-1 is an important factor to inhibit apoptosis. Furthermore, we used CM from an TIMP-1-overexpressing cell line and demonstrated that H2O2-induced apoptosis in the H9c2 cells was significantly inhibited. These observations demonstrate that factors released from ES cells contain antiapoptotic factors and that the effects are mediated by TIMP-1. Moreover, these findings suggest that released factors might be useful for therapeutic applications in ischemic heart disease as well as for many other diseases.

hydrogen peroxide; tissue inhibitor of metalloproteinase-1

MYOCARDIAL INFARCTION (MI) is a major cause of heart failure and death. Death of myocytes within the infarct and peri-infarct regions occurs as a result of both necrosis and apoptosis (1–4, 13). This cell loss may be partially offset by natural regeneration of cardiac myocytes in adult hearts (6). Cell transplantation to treat heart disease has been studied in various animal models (9, 14, 16, 19, 25). Cell types used include ventricular cardiomyocytes, skeletal myoblasts, smooth muscle cells, fetal and embryonic cardiomyocytes, bone marrow stromal, hematopoietic stem cells, and mouse embryonic stem (ES) cells (9, 14, 16, 19, 25).

Pluripotent ES cells hold significant appeal for cell therapy because they have the ability to differentiate into any cell type in the body, including the various cell types found in the heart (26). Prior studies have suggested that mouse ES cells transplanted after MI in the rat heart can differentiate into cardiac myocytes (5), and, as our group (25) recently showed, they can differentiate into vascular smooth muscle and endothelial cells as well. We and others have also shown that transplantation of mouse ES cells improves cardiac function despite low amounts of regeneration that are likely to be insufficient to compensate for the cell loss due to apoptosis and/or necrosis (5, 20, 25). Notably, ~80–90% of the transplanted cells die (7, 24). This might alter the cardiac microenvironment by accumulation of factors released from both dying and surviving cells.

Accordingly, we hypothesized that substances released from ES cells contain cytoprotective factors that inhibit stress-induced apoptosis. To test this hypothesis, we prepared conditioned medium (CM) from cultured ES cells and from ES cells in which apoptosis was induced by H2O2, which simulates the oxidative stress typical of the post-MI heart. We delineated the factors released from ES cells and their effects on H2O2-induced apoptosis in the cardiomyocyte H9c2 cell line.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell lines and CM.

Mouse ES cells (line CGR8) were passaged and maintained in DMEM (Invitrogen) supplemented with leukemia inhibitory factor (LIF), sodium pyruvate, glutamine, penicillin-streptomycin, beta-mercaptoethanol, nonessential amino acids, and 15% FBS (Invitrogen) as reported previously (27). The rat cardiomyocyte-derived cell line H9c2 was obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in DMEM containing 10% FBS, 2 mM L-glutamine, and 25 µg/ml gentamycin at 37°C in a humidified atmosphere of 5% CO2.

Mouse ES cells at 9,000 ES cells/cm2 were grown in Petri dishes containing cell culture medium with LIF for 24 h. Cells were treated with and without H2O2 (400 µM) for 2 h, which was then replaced with fresh cell culture medium without LIF for 48 h. After 48 h, supernatants (CM) from both groups were collected and labeled as ES cell-CM or ES cell-H2O2-CM. To prepare H9c2 cell-CM, 500 cells/cm2 were grown, and supernatant (CM) was collected after 48 h. Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1)-CM was prepared as reported (23) and was kindly provided to us by Dr. David Denhardt (Piscataway, NJ). In brief, human TIMP-1 was subcloned into the pIZ/V5-His plasmid in a frame with the COOH-terminal histidine tag. BTI-TN-5B1-4 insect cells were then transfected with the pIZ/hTIMP-1V5-His plasmid. The CM from cell culture-maintained transfected cells was collected for up to 2 mo.

CM from groups was filtered (0.22-µm cellulose syringe filter) and tested for the presence of cytokine and growth factors and used to assess effects on H2O2-induced apoptosis in H9c2 cells.

For blocking experiments, ES cell-CM was incubated with anti-TIMP-1 antibody or control non-TIMP-1 IgG antibody (Santa Cruz Biotechnology) for 1 h at 4°C. Immunoprecipitation was performed by further incubation with 20 µl of protein A-agarose on a rotating platform at 4°C for overnight. Supernatant was collected by centrifugation at 3,000 rpm for 5–10 min and used to examine its effect on apoptosis.

Preparation of apoptotic cell culture model. H9c2 cells were cultured (500 cells/cm2) for 24 h. We assessed the optimal dose of H2O2 (Sigma) by exposing the cells to concentrations ranging from 50 to 400 µM for 2 h followed by replacement with fresh cell culture medium. Cells were cultured for an additional 24 h and then counted in each of 24 wells based on morphology. Rod-shaped cells were considered to be alive, whereas round cells were considered to be dead. The 400 µM H2O2 concentration was found to produce the greatest amount of apoptosis and was used in subsequent experiments in which H9c2 cells were exposed to H2O2 (400 µM) for 2 h and then replaced with fresh cell culture medium, ES cell-CM, ES cell-H2O2-CM, H9c2 cell-CM, or TIMP-1-CM. Apoptosis was examined by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) staining, apoptotic ELISA kit, caspase-3 activity, and DNA laddering.

Detection of apoptosis by TUNEL staining. Cells were fixed in 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate followed by proteinase K (25 µg/ml in 100 mM Tris·HCl). An in situ apoptotic cell death detection kit (TMR red, Roche Applied Bio Sciences) based on TUNEL assay was used as per manufacturer's instruction to detect apoptotic cells. Negative controls were included in each case by omitting TUNEL enzyme terminal deoxynucleotidyl transferase (TdT) reaction mixture and incubating the cells with label solution. Sections were mounted with Antifade Vectashield mounting medium containing 4'-6-diamidino-2-phenylindole (DAPI; Vector Laboratories) to stain nuclei and examined with a Zeiss Axiovert 200 microscope. The percentage of apoptotic nuclei was calculated by counting the total number of TUNEL-stained nuclei divided by total DAPI-positive nuclei.

Cell death detection ELISA. The cell death detection ELISA plus kit (Roche Applied Sciences) was used to measure histone-bound DNA fragments in an ELISA format. Medium was used as reported in a previous study (18) to examine apoptosis collected at 24 h from different groups.

Caspase-3 activity assay. H9c2 cells (1 x 106) were cultured in 150-mm2 petri dishes for 2–3 days. Cells were treated with and without H2O2 and then replaced with cell culture medium or CM from the different groups. Caspase-3 activity was measured by a caspase-3 colorimetric activity assay kit from BioVision. Cells were washed with PBS and enzymatically dissociated with trypsin-EDTA (Invitrogen) and then collected in the cell lysis buffer provided in the kit. The cell lysate was centrifuged for 1–2 min at 10,000 g. Protein concentrations were measured in the supernatant by a Bio-Rad assay. Caspase-3 activity was performed as per the instructions provided in the kit and measured at 405 nm in a microtiter plate reader.

Agarose gel electrophoresis for DNA fragmentation. To characterize the extent of DNA fragmentation by agarose gel electrophoresis after treatment with H2O2 and CM, we used the apoptotic DNA ladder kit (Roche Applied Diagnostics). H9c2 cells (1 x 106), treated or untreated, were suspended in lysis buffer, and DNA was extracted according to the manufacturer's instructions. DNA concentrations were measured with a spectrophotometer. In brief, 5 µl of DNA were diluted in 995 µl of water, and optical density was read at 260 nm with the use of water as a blank. DNA concentration (µg/µl) was calculated as optical density at 260 nm x 9.88 (9.88 is a multiplication factor that accounts for the 1:200 dilution and the optical density of DNA in water). DNA sample (3 µg) was used to analyze DNA fragmentation on 1% agarose gels stained with ethidium bromide. The gels were visualized under UV light, and photographs were taken.

Cytokine and growth factor analysis. ES cell-CM, H9c2 cell-CM, or control cell culture medium was prepared and mailed to Rule Based Medicine (Austin, TX) where cytokine or growth factor analysis using Luminex technology was performed. In brief, using automated pipetting, an aliquot of each sample is introduced into one of the capture microsphere multiplexes of the rodent antigen mitogen-activated protein. These mixtures of sample and capture microspheres are thoroughly mixed and incubated at room temperature for 1 h. Multiplexed cocktails of biotinylated, and reporter antibodies for each multiplex are then added robotically and, after thorough mixing, incubated for an additional 1 h at room temperature. Multiplexes are developed using an excess of streptavidin-phycoerythrin solution, which is thoroughly mixed into each multiplex and incubated for 1 h at room temperature. The volume of each multiplexed reaction is reduced by vacuum filtration, and the volume is increased by dilution into matrix buffer for analysis. Analysis was performed in a Luminex 100 instrument, and the resulting data stream was interpreted by use of proprietary data analysis software developed at Rule Based Medicine. This technology detected a total of 69 factors present in the CMs.

TIMP-1 ELISA assay. The detection of TIMP-1 in CMs was performed with the mouse TIMP-1 ELISA sandwich kit (RayBiotech) in accordance with the kit instructions. In brief, 100 µl of CMs (1:200 dilution) and standards (recombinant mouse TIMP-1 provided with the kit) were added to a 96-well ELISA plate. After washings, plates were incubated with 100 µl of biotinylated anti-mouse TIMP-1 antibody for 1 h at room temperature followed by horseradish peroxidase-conjugated streptavidin. The reaction was detected with tetramethylbenzidine one-step substrate reagent followed by stop solution. The 96-well ELISA plate was read at 450 nm in a microtiter plate reader. TIMP-1 concentration in the samples was analyzed from the standard curve performed according to the kit directions.

Data analysis. Significance of differences between values was assessed with the Excel program t-test. All values are means ± SE. Statistical significance was assigned at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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We first determined the optimal concentration of H2O2 for induction of apoptosis. H9c2 cells treated with 50–400 µM of H2O2 demonstrated morphological changes as shown in Fig. 1, A and B, and had decreased cell survival at tested concentrations (Fig. 1C). The maximum decrease in cell survival (58%) occurred at 400 µM H2O2 (P < 0.05).


Figure 1
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  		Fig. 1. Effects on H9c2 cell viability after exposure to H2O2. A: representative photomicrograph of untreated H9c2 cells grown in control medium for 24 h. B: H9c2 cells exposed to 50–400 µM H2O2 for 2 h and then placed in normal cell culture control medium for 24 h. C: histogram showing number of H9c2 cells exposed to different concentrations of H2O2.

 
Using TUNEL staining, we determined the effect of ES cell-CM and ES cell-H2O2-CM on H2O2-induced apoptosis in H9c2 cells. Figure 2, A–C, demonstrates the presence of total nuclei stained with DAPI, and Fig. 2, D–F, shows TUNEL-stained nuclei. Merged nuclei are shown in Fig. 2, G–H. The percentage of TUNEL-stained nuclei was 28% in H2O2-treated H9c2 cells compared with <1% in the control group (P < 0.05, Fig. 3A). The percentage of apoptotic nuclei induced by H2O2 was significantly reduced (P < 0.05) after treatment with both ES cell-CMs (Fig. 3A). Quantitative apoptosis measured by the ELISA assay was significantly reduced (Fig. 3B; P < 0.05) with both ES cell-CMs. However, apoptosis was not inhibited with H9c2 cell-CM used as control (data not shown). With the use of gel electrophoresis, DNA fragmentation was observed in the H2O2-treated H9c2 cells, whereas no DNA fragmentation was seen in the untreated control group (Fig. 3C). Importantly, DNA fragmentation was inhibited after treatment with both ES cell-CMs (Fig. 3C).


Figure 2
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  		Fig. 2. Effects of embryonic stem (ES) cell-conditioned medium (CM) on H9c2 cell apoptosis after exposure to H2O2. Representative photomicrographs show total nuclei stained with 4'-6-diamidino-2-phenylindole (DAPI) in blue (A–C), terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL)-stained nuclei in red (D–F), and merged images (G–I). +ve, Positive. Magnification = x10.

 

Figure 3
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  		Fig. 3. Effects of both of the ES cell-CMs on H9c2 cell-induced apoptosis, as determined by TUNEL staining (A), apoptotic ELISA (B), DNA fragmentation (C), and caspase-3 activity (D) after exposure to H2O2. A: histogram shows quantitative apoptotic nuclei (n = 4–6 fields/well in each condition). Data are from set of 3 or 4 independent experiments. B: histogram shows quantitative apoptotic ELISA (see MATERIALS AND METHODS). Data are from set of 8–10 independent experiments. C: representative photomicrographs of DNA fragmentation isolated from different preparations of H9c2 cells performed on 1% agarose gel electrophoresis (A, 1-kb molecular marker; B, H9c2 cells grown in control cell culture medium shows no DNA fragmentation; C, H9c2 cells exposed to H2O2 demonstrate clear DNA ladder and DNA ladder is inhibited when cells are grown in the presence of CMs; D, ES cell-CM; E, ES cell-H2O2-CM). Data are from set of 2–4 independent experiments. D: histogram shows quantitative caspase-3 activity examined by ELISA kit. Data are from set of 8–10 independent experiments. In A, B, and D, C represents control. *P < 0.05 vs. H2O2 group.

 
It has been reported that caspase-3 activity plays a major role in stress-induced apoptosis in H9c2 cells. We examined whether ES cell-CMs had an effect on caspase-3 activity in H9c2 cells induced to undergo apoptosis. Our data demonstrate that caspase-3 activity was significantly higher (P < 0.05) in H2O2-induced apoptosis in H9c2 cells. This increase was inhibited by both ES cell-CMs (P < 0.05) (Fig. 3D).

Next, we determined the presence of cytokine or growth factors in the CM that provided protection from H2O2-induced apoptosis. The 69 cytokine or growth factors analyzed in the present study are listed in Table 1 in the supplemental data (supplemental data for this article is available online at the Am J Physiol Heart Circ Physiol website). Proteins released in the CM by ES cells, in ES cells exposed to H2O2, and from the H9c2 cells that were present at levels greater than those found in the control cell culture medium (containing serum background levels) are listed in Table 2 in the supplemental data (supplemental data for this article is available online at the Am J Physiol Heart Circ Physiol website). Four major antiapoptotic proteins were released in the ES cell-CM or ES cell-H2O2-CM at these levels, specifically, cystatin-c (2- to 4-fold), clusterin (3- to 5-fold), osteopontin (16- to 31-fold), and TIMP-1 (107- to 171-fold), which were significantly different from their levels in H9c2 cell-CM (Table 1).


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  		Table 1. List of anti-apoptotic factors released in the ES cell- and H9c2 cell-derived CM

 
TIMP-1 is a potent inhibitor of matrix metalloproteinases (MMPs) and also plays a major role in adverse cardiac remodeling in the heart. Accordingly, we determined whether TIMP-1 plays a role in ES cell-CM inhibition of apoptosis in H9c2 cells. TIMP-1 ELISA data were in concordance with the Luminex data, demonstrating significant amounts (P < 0.05) of TIMP-1 in both ES cell-CMs and 10% TIMP-1-CM used in the study (Fig. 4). We then blocked TIMP-1 containing ES cell-CM with an anti-TIMP-1 antibody, using a non-TIMP-1 IgG antibody as a control. Figure 5 demonstrates that the TIMP-1 antibody abolished the protective antiapoptotic effects of ES cell-CM. Non-TIMP-1 IgG antibody did not inhibit the antiapoptotic effects of ES cell-CM (data not shown). These data suggest that TIMP-1 is the major factor present in the ES cell-CM that inhibits apoptosis.


Figure 4
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  		Fig. 4. Mouse tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) ELISA kit was used to measure the total amount of TIMP-1 protein present in the ES cell-CM, ES cell-H2O2-CM, and 10% TIMP-1-CM. Note that TIMP-1 was undetectable in the control cell culture medium. Data are from set of 4–6 independent experiments. *P < 0.05 vs. control (C).

 

Figure 5
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  		Fig. 5. Histogram shows that TIMP-1 antibody blocks the antiapoptotic effects of ES cell-CM on H9c2 cells exposed to H2O2. ES cell-CM was blocked by incubation with anti-TIMP-1 antibody at different concentrations, as explained in MATERIALS AND METHODS. ES cell-CM containing TIMP-1 shows no decrease in apoptosis compared with H2O2 group. ES cell-CM without TIMP-1 antibody inhibits apoptosis, as assessed by the apoptotic ELISA kit. Data are from set of 4–8 independent experiments. *P < 0.05 vs. H2O2 group, #P = not significant vs. H2O2 group.

 
Next, we determined the direct role of TIMP-1 in H2O2-induced apoptosis in H9c2 cells. TIMP-1-CM obtained from TIMP-1-overexpressing cells demonstrated significant (P < 0.05) inhibition of H2O2-induced apoptosis in H9c2 cells. This was confirmed by TUNEL staining (Fig. 6 A), ELISA (Fig. 6B), and caspase-3 activity (Fig. 6C). Importantly, the amount of apoptosis inhibited with ES cell-CM determined with apoptotic ELISA kit was significantly lower (P < 0.05) than with TIMP-1-CM (Fig. 6B). Apoptotic ELISA data suggest that other factors present in the ES cell-CM may also contribute to inhibition of H2O2-induced apoptosis in H9c2 cells.


Figure 6
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  		Fig. 6. Effects of TIMP-1-CM on H9c2 cell apoptosis after exposure to H2O2. A: histogram shows quantitative apoptotic nuclei per well (n = 4–6 fields/well in each condition and data are from set of 3 or 4 independent experiments). B: histogram shows quantitative apoptotic ELISA (see MATERIALS AND METHODS). Data are from set of 8–10 independent experiments. *P < 0.05 vs. H2O2 group; #P < 0.05 vs. H2O2 group and ES cell-CM. C: histogram shows quantitative caspase-3 activity examined by ELISA kit. Data are from set of 4–6 independent experiments. *P < 0.05 vs. H2O2 group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major new findings of the present study are as follows: 1) H2O2-induced cell death in H9c2 cells is apoptotic in nature; 2) CM from ES cells or ES cells treated with H2O2 inhibits H2O2-induced apoptosis in H9c2 cells; 3) both of the ES cell-CMs contained antiapoptotic (osteopontin, clusterin, cystatin) and antifibrotic (TIMP-1) factors; 4) TIMP-1 present in the ES cell-CM inhibited H2O2-induced apoptosis in H9c2 cells; and 5) TIMP-1-CM obtained from TIMP-1-overexpressing cells also inhibited H2O2-induced apoptosis.

Apoptosis has been shown to be an important factor in the development and progression of MI, cardiac remodeling, and eventually chronic heart failure (1–4, 13). It is well established that H2O2 induces apoptosis in isolated adult cardiomyocytes and in H9c2 cells (10, 22). In the present study, we showed that treatment of H9c2 cells with H2O2 induces dose-dependent decreases in cell survival. Apoptotic detection methods, including TUNEL staining, ELISA, DNA ladder, and caspase-3 activity, indicate that the cell death observed in the H9c2 cells is apoptotic in nature. We also demonstrated for the first time that cytoprotective factors were released in both of the ES cell-CMs and that these inhibit H2O2-induced apoptosis in H9c2 cells. Notably, our data suggest that exposure of ES cells to H2O2 further increases the amount of antiapoptotic factors (Table 1) present in the CM. These findings suggest that factors released from surviving and from dying cells after exposure to H2O2 protect against stress-induced apoptosis. Our data are consistent with recent findings suggesting that factors released from hypoxia-induced Akt-mesenchymal stem cells subjected to hypoxia inhibit apoptosis in isolated cardiomyocytes (8). In the present study, we demonstrated effects of released factors from ES cells. The recent reports suggest that progenitor cell-specific markers such as Flk-1 (endothelial cells) and Nkx2.5 (cardiac myocyte) could be used for the preselection of endothelial or cardiomyocyte cell-specific lineage from mouse ES cells (11, 29). Importantly, both of the cell-specific, marker-isolated preselected cells could be significantly different in characteristics vs. ES cells used in the present study. However, future studies are warranted to isolate CM from preselected ES cells to determine the released factors and their protective effects on apoptosis.

Next, we used cell culture model system to establish that released factors from ES cells inhibit H9c2 cell-induced apoptosis. The present study opens new avenues to investigate whether protective effects of released factors can also be seen in other major cell types present in the heart, such as endothelial or vascular smooth muscle cells.

We identified four major antiapoptotic factors, osteopontin, clusterin, cystatin-c and TIMP-1, in the ES cell-CM with and without exposure to H2O2. These factors have been shown to be antiapoptotic in cancer cells (15, 17), neuroblastoma cells (30), PC-12 cells (21), and endothelial cells (12) but not in isolated cardiomyocytes or in the rat cardiomyocyte-derived cell line H9c2. TIMP-1 is antifibrotic in the heart because of inhibition of MMPs (28). In the present study, the antibody-blocking experiments suggest that TIMP-1 is the key factor present in the ES cell-CMs that inhibit apoptosis. Furthermore, we also demonstrated for the first time that TIMP-1-CM inhibits apoptosis in H9c2 cells. However, further mechanisms of inhibited H9c2 cell apoptosis are required. Moreover, our data are consistent with the antiapoptotic properties of TIMP-1 observed in cancer and endothelial cells (15, 17). Further studies are required to delineate whether the antiapoptotic effects of TIMP-1 on cardiac cell types are independent of its ability to inhibit MMP activity. Importantly, we have previously shown that transplanted, undifferentiated ES cells can be successfully engrafted and differentiated and that they are associated with significant improvement in cardiac function; however, the extent of regeneration itself does not appear sufficient to account for these observations (25). Thus our data generated in the stress-induced cell culture model system suggest inhibition of apoptosis typical of the oxidative stress-induced post-MI heart following ES cell transplantation. We hypothesize that improved function in our previous study (25) could be caused by factors released from transplanted ES cells that inhibit cardiomyocyte apoptosis.

In conclusion, we report that ES cells treated with and without H2O2 release factors that provide protection against stress-induced apoptosis in a cell culture model system. We also demonstrate that TIMP-1 is a key factor present in ES cell-CM or ES cell-H2O2-CM responsible for inhibition of apoptosis. However, further transplantation studies with ES cells and ES cell-derived CM are required to identify the released factors in vivo, levels of TIMP-1, and the potential mechanisms of action of the released factors, which may have important implications for treatment of various heart diseases.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
We acknowledge partial support provided by American Heart Association Scientist Development Grant 0430227N (to D. K. Singla).


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. K. Singla, Biomolecular Science Center, University of Central Florida, 4000 Central Florida Blvd., BMS224, Orlando, FL, 32817

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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