This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Devlin, A. M. Articles by Dominiczak, A. F. Search for Related Content PubMed PubMed Citation Articles by Devlin, A. M. Articles by Dominiczak, A. F. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information High Blood Pressure Related Collections Animal models of human disease Apoptosis Cell biology/structural biology Hypertension - basic studies Smooth muscle proliferation and differentiation

(Hypertension. 2000;36:110.)
© 2000 American Heart Association, Inc.

Scientific Contributions

DNA Synthesis and Apoptosis in Smooth Muscle Cells From a Model of Genetic Hypertension

Alison M. Devlin; James S. Clark; John L. Reid; Anna F. Dominiczak

From the Department of Medicine and Therapeutics, University of Glasgow, Western Infirmary, Glasgow, UK.

Correspondence to Dr Alison M Devlin, Laboratory of Cell Biology of Hypertension, Centre de Recherche-CHUM, 3850, rue Saint-Urbain, Montréal, Québec H2W 1T8, Canada. E-mail alisondevlin{at}hotmail.com

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract—The present study was designed to assess vascular smooth muscle cell (VSMC) proliferation and apoptosis in primary cultured VSMCs prepared from the aortic tunica media of adult (4 to 5 months old) age- and gender-matched groups of stroke-prone spontaneously hypertensive rats (SHRSP) and the normotensive reference strain, Wistar-Kyoto (WKY) rats. In the present study, VSMC proliferation was assessed with measurement of DNA synthesis in response to stimulation of G₀/G₁ arrested VSMCs with 10% serum, whereas apoptosis was measured in response to serum deprivation. Apoptosis in aortic VSMCs was assessed in vitro with the technique of Annexin V binding in combination with propidium iodide exclusion with bivariate flow cytometric analysis. The percentage of necrotic VSMCs in the cell populations was assessed simultaneously. The light-scattering properties of the cells were assessed to provide further information on cell shrinkage and chromatin condensation. Results of the present study have shown enhanced DNA synthesis in VSMCs from SHRSP (n=10; 5.2±0.9 cpmx10³/mg protein) compared with WKY (n=12; 2.4±0.7 cpmx10³ /mg protein; P<0.05, 95% CI, -5271 to -296). In addition, the results of the present study have demonstrated the role of serum in the survival of VSMCs in vitro, because SHRSP VSMCs underwent significantly more apoptosis in response to insult by serum deprivation (n=13; 10.21±1.8%) than WKY VSMCs (n=7; 3.44±1.4%; P<0.01, 95% CI, -11.5 to -2.0). Thus, it appears that both proliferation and apoptosis are enhanced in synthetic phenotype aortic medial VSMCs from the SHRSP in vitro.

Key Words: rats, inbred stroke-prone SHR • muscle, smooth, vascular • cells • apoptosis • annexin V

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Apoptosis, or programmed cell death, is a well-documented phenomenon in many cellular systems.¹ It plays a key role in tissue and organ development and has been identified in the physiological hypertrophy and remodeling of the vasculature that is found in hypertension, atherosclerosis, and restenosis after angioplasty.^{2 3} It has been postulated that apoptosis occurs as a consequence of a defective cell cycle, and it is known that damage to the cell cycle is an efficient trigger of apoptosis. The cell cycle regulatory molecules and signaling mechanisms involved in vascular smooth muscle cell (VSMC) proliferation and apoptosis are being elucidated in vitro,⁴ but the majority of these studies were not conducted with VSMCs from well-characterized models of genetic hypertension.

Previous in vitro studies have identified several factors that can modulate, in parallel or in opposition, VSMC proliferation and apoptosis. For example, DNA replication is inhibited and apoptosis is increased via the cAMP or nitric oxide pathway.⁵ In contrast, platelet-derived growth factor or insulin-like growth factor-1 inhibits apoptosis and promotes DNA replication. In addition, overexpression of the transcription factor c-myc or its adenoviral functional homolog, E1A, increases both proliferation and apoptosis in VSMCs.⁶ The effect of growth factors and cytokines on cell fate is also extremely tissue specific, as is illustrated in a recent study in which transforming growth factor-ß₁ was found to potentiate endothelial cell apoptosis yet inhibit VSMC apoptosis.⁷ In the context of hypertension, apoptosis has been shown to be enhanced in target organs of spontaneously hypertensive rats (SHR) in vivo, and it is also known that VSMCs from the SHR exhibit abnormal growth in vitro that is manifest as an accelerated entry into S phase of the cell cycle as well as a hyperresponsiveness to growth factors.^{2 8} The current status of the regulation of vascular cell apoptosis both in vivo and in vitro, including the gene products and the signaling pathways involved, was comprehensively reviewed by Orlov et al.⁹

One of the earliest features of apoptotic cells is the translocation of the negatively charged phospholipid species phosphatidylserine (PS) to the outer surface of the cell membrane. This feature was first reported to occur in cells of the immune system and was later demonstrated to unequivocally occur in apoptotic VSMCs.¹⁰ Furthermore, previous studies have shown that VSMCs undergo marked shrinkage and a reduced cell volume due to loss of intracellular water when undergoing death by apoptosis.¹¹ The differential staining of VSMCs with Annexin V protein and propidium iodide through dual color "bivariate" flow cytometry may be used to quantitatively assess apoptosis and necrosis in vitro.

Because the regulation of apoptosis in VSMCs derived from models of genetic hypertension remains poorly characterized, the aim of the present study was to assess VSMC proliferation as measured with [³H]thymidine incorporation into newly synthesized DNA and apoptosis, as measured with Annexin V binding, in vitro in primary cultured aortic VSMCs isolated from adult age- and gender-matched stroke-prone SHR (SHRSP) and Wistar-Kyoto (WKY) animals. This present work examines further the phenotypic differences in growth and susceptibility to apoptosis in VSMCs isolated from hypertensive compared with normotensive arteries with the use of one of the best characterized models of genetic hypertension, the SHRSP.^{12 13}

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Experimental Animals
Adult parental SHRSP and WKY animals were obtained from colonies established in the Department of Medicine and Therapeutics, University of Glasgow, which have been maintained since 1991 by brotherxsister mating as previously described.¹⁴ A total of 8 adult (4 to 5 months old) parental SHRSP (4 male, 4 female) and 6 adult (4 to 5 months old) parental WKY (3 male, 3 female) animals were used for the preparation of primary aortic medial VSMCs. Blood pressure measurements were made with tail-cuff plethysmography in conscious, restrained rats as previously described.^{12 13} Animals were sacrificed with halothane overdose, and tissue was removed as described later. These studies were approved by the Home Office according to regulations regarding experiments with animals in the United Kingdom. These regulations meet all the requirements of the American Physiological Society.

Preparation of Primary Aortic VSMCs From SHRSP and WKY
Animals were killed, and the full length of the thoracic aorta was carefully dissected. Primary VSMCs were isolated from the tunica media of thoracic aortas from SHRSP and WKY through a stepwise enzymatic digestion procedure as described previously.¹² VSMCs, which were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (vol/vol) fetal bovine serum, 10% (vol/vol) equine serum, L-glutamine (2 mmol/L), penicillin (100 U/mL), and streptomycin (100 µg/mL), had the usual growth characteristics and at confluence exhibited the typical "hill-and-valley" pattern and other VSMC characteristics as reported previously.¹²

Determination of VSMC DNA Synthesis In Vitro
VSMCs were used at passages 5 to 8 for all proliferation experiments. VSMCs were plated onto 24-well plates at the range of 3 to 4x10⁴ cells/mL, 1 mL/well, in complete DMEM growth medium. VSMCs were allowed to grow until they reached subconfluency (80% to 90% confluent), after which the growth medium was replaced with serum-free medium, to synchronize the VSMCs at G₀/G₁ phase of the cell cycle. After a 24-hour serum-deprivation period, VSMCs were treated with DMEM that contained 10% serum for 20 hours. At the end of this time period, fresh DMEM that contained 1 µCi/mL [³H]thymidine was added for an additional 6-hour incubation period. Labeled medium was then removed, and each well was washed twice with 1 mL PBS and then treated with 10% trichloroacetic acid. The precipitate was solubilized with 0.3N NaOH/0.1% SDS, and aliquots from 8 to 12 separate wells for each individual experimental treatment were counted in a liquid scintillation counter. Protein estimations were conducted according to the method of Lowry et al,¹⁵ and results are expressed as cpmx10³/mg protein.

Determination of VSMC Apoptosis In Vitro
VSMCs were used at passages 6 to 10 for all apoptosis experiments. VSMCs were plated onto 12-well plates at a density of 4.5x10⁴ cells/mL, 3 mL/well, in complete DMEM growth medium and allowed to grow for a period of 24 to 48 hours. DMEM was then removed, and wells were washed once with serum-free medium; then, VSMCs were treated with DMEM containing 10% serum or serum-free medium for 24 hours to induce death by apoptosis. VSMCs were then harvested and pooled with their respective supernatant, so as to include any detached apoptotic VSMCs. VSMCs were pelletted by centrifugation at 1200 rpm at 4°C and washed twice in chilled (4°C) PBS. Samples were resuspended in binding buffer (10 mmol/L HEPES/NaOH, pH 7.4, 140 mmol/L NaCl, and 2.5 mmol/L CaCl₂) at a concentration of 1x10⁶ cells/mL, to which was added sequentially 10 µL of 10 µg/mL fluorescein isothiocyanate–conjugated Annexin V protein (FITC Annexin V) followed by 10 µL of propidium iodide (50 µg/mL). There were 3 to 6 repeats for each experimental treatment conducted, and additional wells of VSMCs were harvested simultaneously for a series of controls to validate the correct electronic compensation of the fluorochromes.

After the addition of the fluorochromes and a 15-minute incubation period at 20°C to 25°C (room temperature) in the dark, binding was stopped by the addition of 400 µL of chilled binding buffer. Apoptotic cells were assessed by binding of Annexin V (FITC) protein to exposed PS residues along with propidium iodide exclusion to confirm the integrity of the cell membrane that remains intact during apoptosis. Characteristic changes in forward and side light-scattering properties,¹⁶ which are related to cell shrinkage and chromatin condensation, respectively, were used to confirm VSMC apoptosis in vitro. Furthermore, 3 traditional methods (microscopy, DNA flow cytometry, and DNA laddering electrophoresis) have previously been shown to correlate well with the Annexin V binding assay for the detection of apoptotic cells.¹⁷

Annexin V/Propidium Iodide Dual Color Flow Cytometry
Samples were analyzed (typically 10 000 to 20 000 cells per sample) with a FACScan benchtop analyzer (Becton-Dickinson UK Ltd.) with a 15-mW argon air-cooled laser with emission wavelength of 488 nm. Acquisition and analysis were performed with the CellQuest software package (Becton-Dickinson UK Ltd). The specificity of Annexin V (FITC) binding to VSMCs was checked in preliminary experiments in which binding was conducted in the presence or absence of 5 mmol/L EDTA. Almost 100% of VSMCs showed an increased fluorescence compared with the same cells stained in the presence of 5 mmol/L EDTA, which confirms the Ca²⁺ dependency of binding (data not shown). Representative compensated flow cytometric dot-plots are shown in Figure 1, in which viable cells that exclude both fluorochromes are Annexin V negative and propidium iodide negative, apoptotic cells are Annexin V positive and propidium iodide negative (R1), and necrotic cells are Annexin V positive and propidium iodide positive (R2). Figure 1A shows a representative flow cytometric dot-plot for VSMCs grown in the presence of 10% serum in which the apoptotic population (R1) equals 7.1% and the necrotic population (R2) equals 3.8%. Figure 1B shows a representative flow cytometric dot-plot for VSMCs after 24-hour serum deprivation in which the apoptotic population (R1) is increased to 13.4% and the necrotic population (R2) is increased to 9.6%.

View larger version (21K): [in this window] [in a new window]   Figure 1. Representative flow cytometric dot-plots of FITC-Annexin V/propidium iodide (PI) dual color flow cytometry for VSMCs grown in the presence of 10% serum (A) and after 24 hours of serum deprivation (B). Bottom left region of each quadrant shows the viable cells, which exclude PI and are negative for FITC-Annexin V binding. Bottom right region (R1) represents the apoptotic cells (FITC-Annexin V positive and PI negative), demonstrating cytoplasmic membrane integrity. Top right region (R2) contains the nonviable necrotic cells (positive for FITC-Annexin V binding and for PI uptake).

Statistical Analysis
The results are presented as mean±SEM. Statistical analysis was performed with Student’s unpaired t test. A value of P<0.05 was considered statistically significant.

Materials
DMEM, fetal bovine serum, equine serum, penicillin/streptomycin, and L-glutamine were from Gibco Life Technologies Ltd. Propidium iodide and other standard chemicals were purchased from Sigma Chemical Company Ltd. Annexin V (FITC) protein was from R&D Systems Europe Ltd. [methyl-³H]Thymidine (20 Ci/mmol) was from NEN/DuPont.

	Results

Top Abstract Introduction Methods Results Discussion References

 
A total of 8 separate preparations of primary VSMCs were established from 8 separate adult SHRSP animals, and 6 separate preparations of primary VSMCs were prepared from 6 separate adult WKY animals. Both groups of animals were age and gender matched, and the mean systolic blood pressures (SBPs) are given in Table 1. As expected, there was a highly significant difference in mean SBP between WKY (n=6; 128.7±1.5 mm Hg) and SHRSP (n=8; 170.1±5.2 mm Hg; P<0.0001, 95% CI, 28.9 to 54.0).

View this table: [in this window] [in a new window]   Table 1. Data on Blood Pressure, Age, and Gender of Rats

[³H]Thymidine Incorporation Into DNA in VSMCs From SHRSP and WKY
For [³H]thymidine incorporation experiments, aortic VSMCs from 5 (3 female, 2 male) SHRSP (SBP 163.2±2.9 mm Hg) and 4 (2 female, 2 male) WKY (SBP 129.75±1.5 mm Hg; P<0.001; 95% CI, 24.9 to 42.0) animals were used. Synchronized WKY and SHRSP VSMCs in vitro presented different responses in [³H]thymidine incorporation into new DNA in response to stimulation with 10% serum, which is the usual serum concentration used in cell growth studies. VSMCs from SHRSP (n=10; 5.2±0.9 cpmx10³/mg protein) incorporated significantly more [³H]thymidine into newly synthesized DNA than did control VSMCs from WKY (n=12; 2.4±0.7 cpmx10³/mg protein; P<0.05, 95% CI -5271 to -296) (Table 2).

View this table: [in this window] [in a new window]   Table 2. Growth of VSMCs in the Presence of 10% Serum as Determined with [3H]Thymidine Incorporation Into Newly Synthesized DNA

Apoptosis in VSMCs from SHRSP and WKY In Vitro
For in vitro apoptosis experiments with Annexin V (FITC) binding, primary aortic VSMCs from 5 (3 female, 2 male) SHRSP (SBP 172.2±8.3 mm Hg) and 4 (2 female, 2 male) WKY (SBP 127.75±1.7 mm Hg; P<0.01; 95% CI, 20.8 to 68.1) animals were used. A representative flow cytometric histogram for the binding of Annexin V (FITC) to VSMCs maintained in the presence of 10% serum or after treatment with serum deprivation for 24 hours is presented in Figure 2. The profile shows a shift in the FL-1 (FITC) Annexin V parameter, which corresponds to increased binding to PS residues exposed on the surface of VSMCs undergoing apoptosis in vitro.

View larger version (24K): [in this window] [in a new window]   Figure 2. Representative flow cytometric histogram for the binding of FITC-Annexin V to VSMCs maintained in the presence of 10% serum or VSMCs treated with serum deprivation for 24 hours. The profile shows increased fluorescence 1 (FL1) intensity, which corresponds to increased FITC-Annexin V binding to PS residues exposed on the surface of apoptotic VSMCs.

In response to serum deprivation, VSMCs from SHRSP underwent significantly more death by apoptosis as assessed with Annexin V binding compared with VSMCs from age- and gender-matched WKY animals (WKY: n=7, 3.44±1.4%; SHRSP: n=13, 10.21±1.8%; P<0.01; 95% CI, -11.5 to -2.0) (Figure 3). VSMCs from SHRSP also underwent more death by necrosis in response to serum deprivation compared with VSMCs from WKY (WKY: n=9, 3.02±0.6%; SHRSP: n=10, 5.16±0.83%; P=0.054; 95% CI, -4.32 to 0.05).

View larger version (18K): [in this window] [in a new window]   Figure 3. Apoptosis in primary aortic VSMCs in vitro under conditions of serum deprivation showing a comparison between VSMCs from the SHRSPGla (n=13) and the WKYGla (n=7). Cells were used at passages 6 to 10 for all experiments with 3 to 6 repeats for each experimental treatment conducted. **P<0.01 vs WKY.

Figure 4 displays representative forward light scatter (FSC), which is related to cell volume, versus side light scatter (SSC), which is related to granularity, cell frequency dot-plots for VSMCs in the presence of 10% serum (Figure 4A) compared with cells treated by serum deprivation for 24 hours (Figure 4B). A decrease in FSC and an increase in SSC is evident and corresponds to a decrease in VSMC size and an increase in cell granularity or density, which in turn corresponds to cell shrinkage and chromatin condensation.¹⁶ Therefore, the mean FSC parameter confirms VSMC volume decrease or shrinkage in response to serum deprivation treatment. In VSMCs from WKY (n=7), there is a reduction in mean FSC (cell volume) from 396.9±31 to 377.6±16 arbitrary units, whereas in VSMCs from SHRSP (n=15), there is a significant reduction in mean FSC from 384±17 to 337.9±9.9 arbitrary units (P<0.05; 95% CI, -86.7 to -5). However, the mean decrease in FSC in VSMCs from SHRSP compared with WKY in response to treatment with serum deprivation was nonsignificant. The results for cell volume in VSMCs from SHRSP and WKY before and after the induction of apoptosis are also summarized in Figure 4.

View larger version (34K): [in this window] [in a new window]   Figure 4. Representative FSC (cell volume) versus SSC (granularity) cell frequency dot-plots for VSMCs in the presence of 10% serum (A) and VSMCs treated for 24 hours with serum deprivation (B). Each plot represents a minimum of 10 000 cellular events, and the plots are representative of a typical experiment. A decrease in FSC and an increase in SSC is evident in accordance with decreased VSMC volume and increased VSMC granularity (or density). Bottom, Summary of data on cell volume changes in VSMCs from WKYGla and SHRSPGla. Data are expressed as mean±SEM. *P<0.05 for VSMCs in 0% serum compared with 10% serum.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This is the first study in which apoptosis has been assessed in conjunction with proliferation in primary cultured aortic VSMCs from SHRSP compared with WKY. Consistent with previous reports in the SHR model,^{2 8} we have documented enhanced growth that is accompanied by enhanced susceptibility to apoptosis in VSMCs in vitro from the SHRSP compared with the WKY. The results of the present in vitro study provide a further line of evidence to suggest that dysregulation of VSMC growth and apoptosis in hypertension is not merely secondary to increased blood pressure. Apoptosis was originally identified and reported on the basis of the unique cellular pathology and morphological changes that occur as revealed by electron microscopy; a technique that to this day remains a definitive means of identifying apoptotic cells.¹ However, as far as we are aware, this is one of the first reports of the suitable application of Annexin V/propidium iodide staining to quantify VSMC apoptosis in vitro, although other workers have previously applied the technique to quantify the incidence of B cell and endothelial cell apoptosis in vitro.¹⁸

In our previous in vivo pharmacological intervention studies in the SHRSP, we have shown that levels of angiotensin II (Ang II) contribute to the development of VSMC polyploidy and hypertrophy in the blood vessel wall via the Ang II type 1 (AT₁) receptor.^{12 13 19} In addition, previous studies in the SHR model have identified apoptosis as the cellular mechanism whereby vascular structure is normalized in response to AT₁ receptor antagonism in vivo.²⁰ Because polyploidy occurs when cells double their DNA content but fail to complete mitosis, it follows that the polyploid VSMCs in the blood vessel wall may represent a highly differentiated VSMC phenotype that is possibly more susceptible to death by apoptosis in vivo, although this has yet to be proved. However, the present study was conducted with primary aortic VSMCs in vitro, and it is well known that VSMCs dedifferentiate in vitro and adopt a fetal pattern of gene expression consistent with the "synthetic" phenotype. We have shown here that the abnormal proliferation of VSMCs from the model of genetic hypertension, the SHRSP, is associated with increased susceptibility to death by apoptosis once the serum mitogens and survival factors are completely withdrawn. A natural interpretation of this observation is that the mechanisms that control the cell cycle and apoptosis in VSMCs from the SHRSP are very closely related. Indeed, a recent report has documented a default molecule, which is expressed in the G₂M phase, that is responsible for the control of cell cycle progression or death by apoptosis.²¹ In this study, workers identified a protein that is an inhibitor of apoptosis known as survivin that may also preserve genetic fidelity, including the control of ploidy during cell division, although the study was not conducted in vascular cells.

The present study with the SHRSP model provides further insight to confirm that VSMC growth and apoptosis dysregulation occurs in genetic hypertension. However, it is pertinent to note that the primary VSMCs used in the present study were all prepared in an identical fashion from tightly age- and gender-matched animal groups because previous workers have reported that cultured VSMCs exhibit developmentally regulated growth phenotypes that mimic the in vivo pattern.²² For this reason, the phenotypic differences observed here in the rates of VSMC proliferation and apoptosis in the SHRSP relate to adult animals in which hypertension is well established.

However, in blood vessels of adult rodent models of hypertension, such as the SHR in vivo, the VSMC phenotype is highly differentiated, and recent studies with Ang II type 2 (AT₂) receptor knockout mouse strains have documented the important role of the AT₂ receptor subtype in the mediation of vasculogenesis and, more recently, in the mediation of VSMC differentiation.²³ Because this receptor subtype has recently been shown to mediate VSMC apoptosis in vitro²⁴ and has been previously shown to have different levels of expression in VSMCs from models of hypertension compared with respective controls,²⁵ it is also possible that the differences in growth and apoptosis in VSMCs isolated from the SHRSP compared with the WKY, as reported in the present study, may be related to altered AT₁/AT₂ receptor expression levels or function. Such cellular mechanisms are presently hypothetical because they were not assessed in this study. However, these hypotheses that remain to be fully tested in models of genetic hypertension in vivo and in vitro will be of major interest in future studies.

	Acknowledgments

 
This work was supported by the British Heart Foundation program and project grants RG 97009 and PG 96175 to Prof A.F. Dominiczak.

Received September 29, 1999; first decision October 28, 1999; accepted February 8, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Kerr JFR, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26:239–257.[Medline] [Order article via Infotrieve]

2. Hamet P, Richard L, Dam T-V, Teiger E, Orlov SN, Gaboury L, Gossard F, Tremblay J. Apoptosis in target organs of hypertension. Hypertension. 1995;26:642–648.[Abstract/Free Full Text]

3. Teiger E, Dam T-V, Richard L, Wisnewsky C, Tea B-S, Gaboury L, Tremblay J, Schwartz K, Hamet P. Apoptosis in pressure overload-induced heart hypertrophy in the rat. J Clin Invest. 1996;97:2891–2897.[Medline] [Order article via Infotrieve]

4. Macdonald K, Bennett MR. cdc25A is necessary but not sufficient for optimal c-myc–induced apoptosis and cell proliferation of vascular smooth muscle cells. Circ Res. 1999;84:820–830.[Abstract/Free Full Text]

5. Fukuo K, Hata S, Suhara T, Nakahashi T, Shinto Y, Tsujimoto Y, Morimoto S, Ogihara T. Nitric oxide induces upregulation of Fas and apoptosis in vascular smooth muscle. Hypertension. 1996;27:823–826.[Abstract/Free Full Text]

6. Bennett MR, Evan GI, Schwartz SM. Apoptosis of rat vascular smooth muscle cells is regulated by p53-dependent and -independent pathways. Circ Res. 1995;77:266–273.[Abstract/Free Full Text]

7. Pollman MJ, Naumovski L, Gibbons GH. Vascular cell apoptosis: cell type–specific modulation by transforming growth factor-ß₁ in endothelial cells versus smooth muscle cells. Circulation. 1999;99:2019–2026.[Abstract/Free Full Text]

8. Hadrava V, Tremblay J, Hamet P. Abnormalities in growth characteristics of aortic smooth muscle cells in spontaneously hypertensive rats. Hypertension. 1989;13:589–597.[Abstract/Free Full Text]

9. Orlov SN, deBlois D, Tremblay J, Hamet P. Apoptosis in hypertension: mechanisms and implications in vascular remodeling. Cardiovasc Risk Factors. 1999;9:67–79.

10. Bennett MR, Gibson DF, Schwartz SM, Tait JF. Binding and phagocytosis of apoptotic vascular smooth muscle cells is mediated in part by exposure of phosphatidylserine. Circ Res. 1995;77:1136–1142.[Abstract/Free Full Text]

11. Orlov SN, Dam T-V, Tremblay J, Hamet P. Apoptosis in vascular smooth muscle cells: role of cell shrinkage. Biochem Biophys Res Commun. 1996;221:708–715.[Medline] [Order article via Infotrieve]

12. Devlin AM, Gordon JF, Davidson AO, Clark JS, Hamilton CA, Morton JJ, Campbell AM, Reid JL, Dominiczak AF. The effects of perindopril on vascular smooth muscle polyploidy in stroke-prone spontaneously hypertensive rats. J Hypertens. 1995;13:211–218.[Medline] [Order article via Infotrieve]

13. Dominiczak AF, Devlin AM, Lee WK, Anderson NH, Bohr DF, Reid JL. Vascular smooth muscle polyploidy and cardiac hypertrophy in genetic hypertension. Hypertension. 1996;27:752–759.[Abstract/Free Full Text]

14. Davidson AO, Schork N, Jaques BC, Kelman AW, Sutcliffe RG, Reid JL, Dominiczak AF. Blood pressure in genetically hypertensive rats: influence of the Y chromosome. Hypertension. 1995;26:452–459.[Abstract/Free Full Text]

15. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin reagent. J Biol Chem. 1951;193:265–275.[Free Full Text]

16. Dive C, Gregory CD, Phipps DJ, Evans DL, Milner AE, Wyllie AH. Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry. Biochim Biophys Acta. 1992;1133:275–285.[Medline] [Order article via Infotrieve]

17. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for apoptosis: flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods. 1995;184:39–51.[Medline] [Order article via Infotrieve]

18. DeMeester SL, Qui Y, Buchman TG, Hotchkiss RS, Dunnigan K, Karl IE, Perren Cobb J. Nitric oxide inhibits stress-induced endothelial cell apoptosis. Crit Care Med. 1998;26:1500–1509.[Medline] [Order article via Infotrieve]

19. Devlin AM, Davidson AO, Gordon JF, Campbell AM, Morton JJ, Reid JL, Dominiczak AF. Vascular smooth muscle polyploidy in genetic hypertension: the role of angiotensin II. J Hum Hypertens. 1995;9:497–500.[Medline] [Order article via Infotrieve]

20. deBlois D, Tea B-S, Dam T-V, Tremblay J, Hamet P. Smooth muscle apoptosis during vascular regression in spontaneously hypertensive rats. Hypertension. 1997;29:340–349.[Abstract/Free Full Text]

21. Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC, Altieri DC. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature. 1998;396:580–584.[Medline] [Order article via Infotrieve]

22. Cook CL, Weiser MC, Schwartz PE, Jones CL, Majack RA. Developmentally timed expression of an embryonic growth phenotype in vascular smooth muscle cells. Circ Res. 1994;74:189–196.[Abstract/Free Full Text]

23. Yamada H, Akishita M, Ito M, Tamura K, Daviet L, Lehtonen JYA, Dzau VJ, Horiuchi M. AT₂ receptor and vascular smooth muscle cell differentiation in vascular development. Hypertension. 1999;33:1414–1419.[Abstract/Free Full Text]

24. Yamada T, Akishita M, Pollman MJ, Gibbons GH, Dzau VJ, Horiuchi M. Angiotensin II type 2 receptor mediates vascular smooth muscle cell apoptosis and antagonises angiotensin II type 1 receptor action: an in vitro gene transfer study. Life Sci. 1998;63:289–295.

25. Ichiki T, Kambayashi Y, Inagami T. Differential inducibility of angiotensin II AT₂ receptor between SHR and WKY vascular smooth muscle cells. Kidney Int. 1996;49:S14–S17.

This article has been cited by other articles:

				D. M. Fries, R. Lightfoot, M. Koval, and H. Ischiropoulos Autologous Apoptotic Cell Engulfment Stimulates Chemokine Secretion by Vascular Smooth Muscle Cells Am. J. Pathol., August 1, 2005; 167(2): 345 - 353. [Abstract] [Full Text] [PDF]

				J. Su, J. Li, W. Li, B. T. Altura, and B. M. Altura Cocaine Induces Apoptosis in Primary Cultured Rat Aortic Vascular Smooth Muscle Cells: Possible Relationship to Aortic Dissection, Atherosclerosis, and Hypertension International Journal of Toxicology, July 1, 2004; 23(4): 233 - 237. [Abstract] [Full Text] [PDF]

				A. Curcio, D. Torella, G. Cuda, C. Coppola, M. C. Faniello, F. Achille, V. G. Russo, M. Chiariello, and C. Indolfi Effect of stent coating alone on in vitro vascular smooth muscle cell proliferation and apoptosis Am J Physiol Heart Circ Physiol, March 1, 2004; 286(3): H902 - H908. [Abstract] [Full Text] [PDF]

				X.-L. Zheng, Y. Gui, G. Du, M. A. Frohman, and D.-Q. Peng Calphostin-C Induction of Vascular Smooth Muscle Cell Apoptosis Proceeds through Phospholipase D and Microtubule Inhibition J. Biol. Chem., February 20, 2004; 279(8): 7112 - 7118. [Abstract] [Full Text] [PDF]

				H. D. Intengan and E. L. Schiffrin Vascular Remodeling in Hypertension: Roles of Apoptosis, Inflammation, and Fibrosis Hypertension, September 1, 2001; 38(3): 581 - 587. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Devlin, A. M. Articles by Dominiczak, A. F. Search for Related Content PubMed PubMed Citation Articles by Devlin, A. M. Articles by Dominiczak, A. F. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information High Blood Pressure Related Collections Animal models of human disease Apoptosis Cell biology/structural biology Hypertension - basic studies Smooth muscle proliferation and differentiation