| pA2 online © Copyright 2004 The British Pharmacological Society |
096P
GKT, University of London Winter Meeting December 2003 |
| Nifedipine
induces apoptosis in the A7r5 vascular smooth muscle cell line KE Kerry, RD Jones, KS Channer & TH Jones. Hormone and Vascular Biology Group, Academic Unit of Endocrinology, Division of Genomic Medicine, University of Sheffield & Department of Cardiology, Royal Hallamshire Hospital, Sheffield, UK. |
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Print abstractVascular remodelling is an important process in the pathogenesis of hypertension (Heagerty et al., 1993), in which alterations in the distribution of vascular smooth muscle cells (VSMCs) plays an important role. Apoptosis of these cells has been suggested to be a key contributor towards this process (Bennet et al., 1999), and reports indicate that certain drugs currently used in anti-hypertensive therapy may bring about their effects by promoting VSMC apoptosis. Apoptosis involves a number of complex molecular events, and alterations in intracellular calcium levels have been demonstrated to act as a trigger for apoptosis in VSMCs. In this study we have utilised the A7r5 rat thoracic aorta VSMC line to investigate the effects of the L-type calcium channel antagonist nifedipine on VSMC apoptosis.
A7r5 cells were plated at 150000cells/well in 6-well tissue culture plates in 10% foetal bovine serum supplemented DMEM, and left to adhere over night. Media was then replaced with phenol red free DMEM supplemented with 2% foetal bovine serum, containing nifedipine (1, 10 or 100µM) or DMSO (0.1%) control for either 24 or 48 hours at 37°C. Adherent cells were removed with 0.25% tyrpsin-EDTA and combined with floating cells via centrifugation at 1000rpm for 5minutes, washed in PBS, and resuspended in annexin-binding buffer. Cells were then incubated with 5µl of FITC-conjugated annexin V for 15- minutes before the addition of 10µl of 100µg/ml propidium iodide. Apoptotic cells were then identified via fluorescence-activated cell sorting.
Nifedipine induced significant levels of apoptosis in A7r5 cells in both a time and concentration dependent manner (Table 1).
| Stage of cell death |
DMSO
Ctrl
|
Nifedipine
1µM
|
Nifedipine10µM
|
Nifedipine100µM
|
| Viable cells (%) | ||||
| 24hr |
85.1
(0.67)
|
85.0
(1.06)
|
83.2
(0.55)
|
65.2
(2.21)*
|
| 48hr |
82.6
(0.87)
|
83.6
(0.88)
|
68.7
(2.81)*
|
17.6
(3.62)*
|
| Early apoptotic cells (%) | ||||
| 24hr |
4.2
(0.25)
|
4.8
(0.41)
|
5.6
(0.34)
|
7.6
(0.60)*
|
| 48hr |
3.6
(0.32)
|
5.0
(0.25)
|
6.3
(0.91)
|
33.1
(2.40)*
|
| Mid apoptotic cells (%) | ||||
| 24hr |
3.6
(0.38)
|
3.9
(0.42)
|
4.7
(0.42)
|
10.8
(0.74)*
|
| 48hr |
3.8
(0.25)
|
4.5
(0.27)
|
7.4
(0.74)
|
27.5
(3.41)*
|
| Late apoptotic/necrotic cells (%) | ||||
| 24hr |
3.9
(0.24)
|
4.3
(0.30)
|
4.5
(0.24)
|
11.9
(1.75)*
|
| 48hr |
5.5
(0.57)
|
4.2
(0.33)
|
10.0
(0.63)*
|
17.7
(1.08)*
|
Table1. Effect of nifedipine on A7r5 VSMC apoptosis. * = p<0.05 compared to DMSO control via 1-way ANOVA followed by Dunnett's test. Results as mean % cells counted (S.E.M), n=8.
In conclusion, this apoptotic action of nifedipine may be important in explaining how nifedipine, and certain other anti-hypertensive drugs, contribute towards vascular remodelling in the treatment of hypertensive heart disease.
Bennet MR et al.
(1999) Cardiovasc. Res., 41: 361-368.
Heagerty AM et al. (1993) Hypertension, 21: 391-397.