INFLUENCE OF EXOGENOUS PlGF ON APOPTOSIS IN THE H9c2 CARDIOMYOBLAST CELL LINE
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-2phenylindole (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
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.
Timothy Alberts, Sivakanthan Kasinathan, Kaitlin Theis, Ronald Torry (Mentor).
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.
Control
RESULTS
CaspACE In Situ Marker
Effects of Exogenous PlGF
FITC-Caspase
DAPI
Merged
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.
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.
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.
Apoptotic Pathway
H2O2 Dose Response
Nuclear Diameter
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
Increasing concentrations of H2O2 showed increase in the
percentage of apoptosis. Results coincided with the
positive control, Staurosporine
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.
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