ABSTRACT

Placenta growth factor (PlGF) is known to inhibit apoptosis in cells such as trophoblast or endothelial cells. Although PlGF is expressed in heart tissue in vivo and in vitro, little is known about its anti-apoptotic effects in cardiomyocytes. H9c2 cells (a rat cardiomyoblast cell line) were used to determine if PlGF can protect these cells from oxidative-induced apoptosis.

Cells were cultured and then treated with hydrogen peroxide (H2O2) to induce apoptosis. An H2O2 dose response curve was performed and showed increased concentrations of H2O2 were associated with increased percent apoptosis: 25uM H2O2 = 36.77

± 0.09%; 50uM H2O2 = 47.53

± 0.05%; and 100uM H2O2 = 61.36

± 0.05% apoptosis. Some cultures were then pre-treated with either 10ng/mL or 50ng/mL of exogenous recombinant mouse PlGF for 24 hours and then incubated with 50uM H2O2 for 2 hours. Cells were stained with 4',6-diamidino-2 phenylindole (DAPI) , a fluorescent cell stain that binds strongly to DNA and then fixed with ice cold acetone. Apoptotic cells were recognized by the condensed, fragmented, and degraded nuclei with fluorescence microscopy. Random fields of cells from each treatment group were chosen to count the apoptotic cells and percent of apoptosis was calculated for each field.

Control cultures showed 12.41

± 0.01% apoptosis, H2O2+0ng/mL PlGF = 46.49

± 0.02% apoptosis, H2O2+10ng/mL PlGF = 30.48

± 0.06% apoptosis, and H2O2+50ng/mL PlGF = 33.70% ± 0.06% apoptosis. In conclusion, we have developed a consistent method to induce apoptosis in H9c2 cells and used this to show that although exogenous PlGF tended to protect H9c2 cells from oxidative-induced apoptosis, this difference did not attain statistical significance.

BACKGROUND INFLUENCE OF EXOGENOUS PlGF ON APOPTOSIS IN THE H9c2 CARDIOMYOBLAST CELL LINE

Although the placental growth factor (PlGF) is a vital player in pregnancy little is known about its protective effects in cardiac tissue, specifically in preventing apoptosis. Apoptosis is the cell’s suicide regulation in which a dying cell will start to degrade itself and prepare for recycling (Crow et al., Circ Res., 2004). Apoptosis is a pressing issue because it has been shown that about 5-30% of cells in an ischemic area tend to be induced (Olivettii et al., J Mol Cell Cardiol., 1996). Factors capable of inducing apoptosis in cardiomyoblasts include a lack of nutrients, reactive oxygen species, drugs, and hypoxia (Mani et al., J Am Coll Cardiol., 2003). The importance of preventing cardiac necrosis provides insurmountable due to the numerous other side effects associated cardiac dysfunction. Cardiac necrosis produces a loss of cells which in turn means fewer cells are able to contract and gives such side effects as ischemia around the body. Ischemia is known to decrease vital organ or tissue function and could eventually lead to death. Past studies show PlGF’s synergistic effect with vascular endothelial growth factors promote angiogenesis and inhibit the detrimental effects of apoptosis in endothelial cells (Semenza et al., J Clin. Invest., 2001). Hydrogen peroxide is able to induce apoptosis in these cardiomyoblasts because as a toxic intermediate of cellular respiration H2O2 will peroxidize the mitochondrial membrane releasing cyctochrome c, a pro-apoptotic molecule (Elsevier. Kumar et al: Robbins Basic Pathology 8e). PlGF has been proven to protect cells such as trophoblast or endothelial cells from induction of apoptosis (Torry DS et al. J Reprod Immunol., 2003). However, it is not known if PlGF will rescue H9c2 cells from H2O2-induced apoptosis.

College of Pharmacy and Health Sciences, Drake University, Des Moines, Iowa.

METHODS Cell Culture:

H9c2 cardiomyoblasts were purchased from American Culture Collection (ATCC). Cells were cultured and grown in DMEM, sodium pyruvate (100mM) , and 10% FCS. They were passaged at less than 90% confluency using Trypsin EDTA.

Apoptotis Induction by Hydrogen Perioxide (H202):

Cells at 70% confluency were incubated with various concentrations of H2O2 as described (Chen et al., Archives of Biochem and Biophys, 2000) for 2 hours. A dose response curve was performed using concentrations of 25, 50, and 100uM H2O2. Results from those studies determined an optimal concentration of H2O2 to use in the PlGF studies described below.

Exogenous PlGF Administration:

Recombinant mouse PlGF (R+D Systems, MN) was added to the treatment groups at concentrations of 0ng/mL, 10ng/mL, and 50 ng/mL 24 hours before addition of H2O2. A control group was also used where no peroxide treatment was present to compare effects of PlGF. A (+) control was run using 50uM staurosporine, a known inducer of apoptosis as well as a (-) control in the absence of H2O2 and PlGF to determine the baseline rate of apoptosis in our culture conditions. All PlGF treatments were carried out using 50 uM H2O2 optimum dose.

Apoptosis Assessment:

After treatments the H2O2 was removed and the cells were rinsed quickly with PBS. 4',6-diamidino-2-phenylindole (DAPI 400ug/mL) and propidium iodide (2.5mg/mL) were then used to measure apoptosis and necrosis respectively. DAPI is known to integrate into all DNA and therefore allowed the identification and counting of condensed chromatin. Cells were incubated with the dyes for 15 minutes, rinsed with PBS, fixed with ice cold acetone, coverslipped, and observed under fluorescence microscopy. Representative photomicrographs were obtained with Zeiss Axioshop software coupled to a Zeiss MRc5 digital camera. Random fields were chosen to determine the number of cells exhibiting condensed chromatin as a percent of the total number of cells assessed. A minimum of 200 nuclei/treatment condition were counted. CaspACE-FITC-VAD-FMK marker purchased from Promega was added to measure capase activity. After activation, the marker would bind to the active site and fluoresce green.

Effects of Exogenous PlGF Control

Timothy Alberts, Sivakanthan Kasinathan, Kaitlin Theis, Ronald Torry (Mentor).

50 uM H2O2

Recombinant mouse PlGF from R+D Systems (Minneapolis, MN). Cells were incubated with PlGF for 24 hrs. prior to 50 uM H2O2 apoptotic induction. PlGF pretreatment produced a decrease in percent apoptosis, however the difference was not statistically significant.

RESULTS

DAPI

CaspACE In Situ Marker

Merged FITC-Caspase CaspACE FITC-VAD-FMK in situ marker binding to activated caspase and fluorescing green (top, right). DAPI was used to show nuclei of all cells (top, left). The images were merged (bottom, center). This pictures affirms the assay to measure apoptosis through observing caspase activity.

Apoptotic Pathway H2O2 Dose Response

Increasing concentrations of H2O2 showed increase in the percentage of apoptosis. Results coincided with the positive control, Staurosporine

Nuclear Diameter

Microphotograph taken of random fields and Axiovision software used to measure nuclear diameter. Diameters treated with 50uM H2O2 were statistically smaller. A smaller nuclear diameter is associated with apoptosis.

SUMMARY AND CONCLUSION

A dose response curve shows increasing H2O2 concentration is associated with an increased percent of apoptosis. Nuclear diameters were significantly smaller in H2O2-treated cells. PlGF was added to cells treated with the optimum dose of 50uM H2O2 and tended to show inhibition of apoptosis.

Conclusion: We have shown that hypoxia increases PlGF expression in cardiomyocytes and this growth factor can inhibit apoptosis in other cells. However, it is not known if PlGF can limit apoptosis in cardiac myocytes. We have developed a consistent method to induce apoptosis in H9c2 cells and used this to show that although exogenous PlGF up to 50ng/ml tended to protect H9c2 cells from oxidative-induced apoptosis, this difference did not attain statistical significance.

References:

Crow et al. “The Mitochondrial Death Pathway and Cardiac Myocyte Apoptosis.” Circulation Research 95 (2004) 957-970 Olivettii et al. “Acute Myocardial Infarction in Humans is Associated with Activation of Programmed Myocyte Cell Death in the Surviving Portion of the Heart.” J. Mol. Cell Cardiol 28:9 (1996) 2005-20016 Mani et al. “Myocyte Apoptosis: Programming Ventricular Remodeling” Journal of the American College Cardiology 41:5 (2003) 753-764 Semenza “Regulation of hypoxia-induced angiogenesis: a chaperone escorts VEGF to the dance” Journal of Clinical Investigation 108 (2001) 39-40 “Cellular Adaptations, Cell Injury, and Cell Death”. In: Pathologic Basis of Disease (8e). Kumar et al, Ed. Elsevier, Philadelphia. (2005) pg. 16 Torry DS, et al., ”Expression and function of placenta growth factor: implications for abnormal placentation..” J. Soc.Gynecol Invest 10(4):178-188, 2003 Chen et al. “Hydrogen Peroxide Dose Dependent Induction of Cell Death of Hypertrophy in Cardiomyocytes.” Arch Biochem Biophys 373 (2002) 242-248