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(Hypertension. 1997;29:776-780.)
© 1997 American Heart Association, Inc.

Articles

Is the Regulation of Apoptosis Altered in Smooth Muscle Cells of Adult Spontaneously Hypertensive Rats?

Javier Diez; Angel Panizo; Marta Hernandez; Javier Pardo

the Department of Vascular Pathophysiology, School of Medicine (J.D., M.H.), and Department of Pathology, University Clinic, School of Medicine (A.P., J.P.), University of Navarra, Pamplona, Spain.

Correspondence to Javier Diez, MD, PhD, Departamento de Fisiopatologia Vascular, Facultad de Medicina, C/ Irunlarrea s/n, 31080 Pamplona, Spain.

	Abstract

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Whereas the protein product of the Bcl-2 gene inhibits apoptosis, the protein product of the Bax gene acts as a promoter of apoptosis. To gain insight into the regulation of apoptosis in vascular smooth muscle cells in arterial hypertension, we investigated the expression of the proteins Bcl-2 and Bax in small intramyocardial arteries of 36-week-old normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). In addition, 16-week-old SHR were treated for 20 weeks with the angiotensin-converting enzyme inhibitor quinapril and killed at 36 weeks of age. We measured the percentages of smooth muscle cells expressing these proteins using monoclonal antibodies and the avidin-biotin immunoperoxidase method. Compared with WKY, untreated SHR exhibited increased (P<.001) Bcl-2 expression and similar Bax expression. Values of Bcl-2 measured in quinapril-treated SHR were significantly lower than values measured in untreated SHR and similar to values measured in WKY. Quinapril-treated SHR showed higher (P<.001) Bax expression than WKY and untreated SHR. Bcl-2 expression was directly correlated with systolic pressure. Inverse correlations were found between the expression of Bax and the activities of both cardiac and circulating angiotensin-converting enzyme. These findings suggest that smooth muscle cell apoptosis might be inhibited in small arteries of adult SHR as a consequence of an excess of the protein Bcl-2. In addition, our results suggest that chronic angiotensin-converting enzyme inhibition might restore the susceptibility to apoptosis in these cells through stimulation of the protein Bax.

Key Words: apoptosis • hypertension, arterial • muscle, smooth, vascular

	Introduction

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Apoptosis is a physiological mechanism of deleting cells that regulates cell mass and architecture in many tissues. Apoptosis is also called programmed cell death because a number of genes have been identified that regulate the apoptotic process, including genes that primarily suppress apoptosis, genes that act as effectors of apoptosis, and intermediate genes.¹ The Bcl-2 proto-oncogene family is critical for the regulation of apoptosis.² Bcl-2 family members come in two functional categories: those that inhibit apoptosis (ie, Bcl-2)³ and those that induce apoptosis (ie, Bax).⁴

Bcl-2 family members interact with each other. One aspect of the control of apoptosis occurs at the level of protein-protein interactions. Bcl-2 family members form both homodimers and heterodimers. For example, Bcl-2–related apoptosis inhibitors associate with Bcl-2–related apoptosis promoters, suggesting that the relationship between inhibitors and promoters is an antagonist one. The determining factor for cell viability can be the ratio of the level of the death-inhibiting Bcl-2 family member relative to that of the death-promoting Bcl-2 family member.⁵ Bcl-2, for example, interacts with Bax, and a high level of Bcl-2 relative to Bax promotes survival, whereas an excess of Bax relative to Bcl-2 promotes cell death.⁴

The finding that normal vascular SMCs, both animal and human, undergo apoptosis in culture^{6 7 8 9} indicates that normal SMCs possess the machinery to regulate apoptosis. For instance, the presence of the Bcl-2 protein is demonstrable in SMCs seen in the media of arteries^{10 11} and in nontransformed SMCs in culture.¹² In addition, the presence of the Bax protein has been localized in SMCs.¹¹ Thus, apoptosis may be one method of regulating SMC mass and architecture in the vessel wall.

A number of in vivo and in vitro studies have documented abnormal regulation of SMC growth in hypertension (see Reference 13 for review). Recently, Hamet¹⁴ proposed that alterations of apoptosis might be involved in abnormalities of SMC growth in hypertension; however, no data are yet available regarding in vivo regulation of SMC apoptosis in hypertension. We therefore analyzed the in vivo expression of the proteins Bcl-2 and Bax in SMCs from intramyocardial arteries of untreated adult SHR and adult SHR chronically treated with the ACEI quinapril.

	Methods

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Design
Sixteen-week-old male WKY (n=14) and SHR (n=15) were observed in a colony over 20 weeks and killed by decapitation for different studies. In addition, 16-week-old SHR (n=15) were treated with oral quinapril (10 mg/kg body wt per day) for 20 weeks and killed at 36 weeks of age (SHR-ACEI group) as previously described.¹⁵ All rats were provided by Iffa-Credo (L'Abresle, France).

Systolic pressure was measured every 2 weeks in all rats by the standard tail-cuff method¹⁶ with an LE 5007 Pressure Computer (Letica Scientific Instruments).

Before the rats were killed, they were anesthetized (50 mg/kg methohexital IP), and blood was obtained from the rat's eye by venipuncture. Once rats had been killed by decapitation, the heart was removed and the left ventricle dissected. The left ventricle was washed thoroughly with normal saline to remove contaminating blood and then immediately snap-frozen in liquid nitrogen and stored at -80°C for later biochemical and immunohistochemical studies.

All manipulations were carried out in accordance with institutional guidelines.

Measurement of ACE Activity
The measurement of ACE activity was based on the ability of the enzyme to cleave off a synthetic substrate, N-hippuryl-I-histidyl-I-leucine, into hippuric acid and the dipeptide histidyl-leucine (His-Leu).¹⁷ The left ventricle ACE activity was determined in homogenized tissue by measurement of the generation of His-Leu following the fluorometric assay previously described by Cushman and Cheung.¹⁸ Serum ACE activity was determined by measurement of liberated hippuric acid according to Hurst and Lovell-Smith.¹⁹

Immunohistochemical Study
Preparation of Samples
Serial coronal sections 5 µm thick from the equator of the left ventricle were fixed and embedded in paraffin as previously described.²⁰ The fixation and embedding procedures were performed simultaneously for WKY, SHR, and SHR-ACEI so as to avoid any differences in immunostaining caused by fixation time or embedding conditions. Similarly, all subsequent steps were performed on WKY, SHR, and SHR-ACEI tissues in parallel using exactly the same conditions and reagents.

All intramyocardial arteries with an external diameter ranging from 150 to 300 µm present in each section were analyzed with an automatic image analyzer (Microm IP 1.6). Arteries with these dimensions represented 25% to 30% of arterial vessels found in the section, the total number ranging from 5 to 10 per section.

Immunocytochemical Procedure
Two ventricular sections from each rat were independently processed for Bcl-2 and for Bax immunocytochemical detection following the avidin-biotin immunoperoxidase method of Gown and Vogel,²¹ as modified by Krajewski et al.¹¹ Antisera against Bcl-2 and Bax were kindly provided by Dr John C. Reed (La Jolla, Calif). Rabbit anti-mouse Bcl-2 and rabbit anti-mouse Bax antisera were raised against synthetic peptides corresponding to either amino acids 68 through 86 of the mouse Bcl-2²² protein or 43 through 61 of the mouse Bax protein.¹¹ After titration experiments performed in samples from WKY, the antibody against Bcl-2 was applied at a dilution of 1:8000, whereas the antibody against Bax was applied at a dilution of 1:2000. The same optimal dilutions were observed in titration experiments performed in samples from SHR and SHR-ACEI.

For all data presented, the specificity of the immunostaining results was confirmed by use of both preimmune serum, which produced entirely no background, and by preadsorption of anti–Bcl-2 and anti-Bax with competing peptides that abrogated the immunostaining (data not shown).

Immunostaining for the two above-mentioned antisera was assessed after exposure to diaminobenzidine, which acts as the substrate for the peroxidase enzyme, forming a brown-colored end product that precipitates at the site of the antigen, thereby enabling localization of that antigen within the tissue. Hematoxylin was then used in the preparations to counterstain nuclei a light blue color. In this manner, a greater contrast between positive nuclei and nuclei that are not positive was achieved. Cells with nuclei demonstrating intense brown staining were considered positive for either Bcl-2 or Bax in each case. Cells having very faint, granular brown staining were not regarded as positive.

Quantification of the Measurements
One section was analyzed per rat and per antibody. The nuclei of the SMCs were counted in 25 high-power fields of each artery present in the section. A high-power field is equal to an area of 0.142 mm² (x10 ocular, x40 objective). The high-power fields were chosen all along the circumference of the vessel wall. The total number of nuclei counted per high-power field ranged from 30 to 70. The percentage of SMCs with positive staining was determined in each high-power field, and the average percentage of SMCs with positive staining per high-power field was calculated in each section.

Assessment of the Reproducibility of the Measurements
Reproducibility of the method for immunocytochemical estimation of Bcl-2 and Bax expressions was assessed by analyzing interobserver and intraobserver variabilities. Interobserver variability was determined by having a second observer reanalyze staining from the original preparations analyzed by the first observer. Bcl-2 and Bax estimates measured by the two observers were strongly correlated (r=.97 and .98, respectively).

For determination of intraobserver variability, two more ventricular sections adjacent to the previous one were analyzed in each rat. The calculated coefficients of variation in two sections from the same vessel were less than 4% for Bcl-2 and less than 3% for Bax.

Statistical Analysis
All data are presented as mean±SE of the individual percentages recorded in rats from each group. Differences among groups were tested by one-way ANOVA. Subsequent analysis for significant differences was performed with a multiple comparison test (Scheffe method). Correlation coefficients were tested by multiple linear regression analysis. The significance level was assumed at a value of P<.05.

	Results

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Blood Pressure and ACE Activity
At the end of the experiment, systolic pressure was higher in SHR than WKY (Table). Systolic pressure decreased in SHR-ACEI compared with SHR. The systolic pressure values measured in SHR-ACEI were similar to those obtained in WKY (Table).

View this table: [in this window] [in a new window]   Table 1. Blood Pressure and Angiotensin-Converting Enzyme Activity in Normotensive and Hypertensive Rats

The Table shows that the values of both circulating and cardiac ACE activities were similar in SHR and WKY. Quinapril treatment inhibited the activity of the serum enzyme in SHR-ACEI compared with WKY and SHR (Table). The activity of the left ventricular enzyme was lesser in SHR-ACEI after treatment than in SHR (Table). Thus, the ability of quinapril to inhibit circulating ACE was higher than its ability to inhibit cardiac ACE. This is in agreement with recent data showing that in rats receiving quinapril, the circulating levels of angiotensin II decrease more than the cardiac levels of the peptide.²³

Expression of Proteins Bcl-2 and Bax
The photomicrographs in Fig 1 show Bcl-2 expression in small intramyocardial arteries representative of the three experimental rat groups. The percentage of SMCs expressing Bcl-2 was higher (P<.001) in SHR than WKY (26.48±0.27% versus 12.28±0.41%) (Fig 2). Compared with SHR, the percentage of SMCs expressing this protein in SHR-ACEI was decreased (P<.001) after treatment with quinapril (14.82±0.32%) (Fig 2). No differences were found in Bcl-2 expression between WKY and SHR-ACEI.

View larger version (402K): [in this window] [in a new window]   Figure 1. Photomicrographs from immunohistochemical staining with anti–Bcl-2 antiserum of small intramyocardial arteries from WKY (top), untreated SHR (middle), and quinapril-treated SHR (bottom). Bcl-2 protein expression is illustrated by the development of a brown-based chromogen apparent in the nuclei of SMCs located at the media of the vascular wall. (Original magnification x240.)

View larger version (28K): [in this window] [in a new window]   Figure 2. Percentage of SMCs expressing Bcl-2 protein in small intramyocardial arteries from normotensive WKY, untreated SHR, and quinapril-treated SHR (SHR-ACEI). *P<.001 compared with WKY and SHR-ACEI.

Photomicrographs in Fig 3 show Bax expression in arteries from the different rat groups. Fig 4 shows that the percentage of SMCs expressing this protein was similar in WKY (12.90±0.31%) and SHR (11.58±0.31%). However, SHR-ACEI exhibited an increased (P<.005) Bax expression (18.07±0.44%) compared with both WKY and SHR (Fig 4).

View larger version (386K): [in this window] [in a new window]   Figure 3. Photomicrographs from immunohistochemical staining with anti-Bax antiserum of small intramyocardial arteries from WKY (top), SHR (middle), and quinapril-treated SHR (bottom). Bax protein expression is illustrated by the development of a brown-based chromogen apparent in the nuclei of SMCs located at the media of the vascular wall. (Original magnification x240.)

View larger version (34K): [in this window] [in a new window]   Figure 4. Percentage of SMCs expressing Bax protein in small intramyocardial arteries from normotensive WKY, untreated SHR, and quinapril-treated SHR (SHR-ACEI). *P<.005 compared with WKY and SHR.

In order to elucidate whether changes in the branch level of the artery could influence the data, we ranked vessels in each preparation according to their external diameters (from 150 to 199 µm, from 200 to 249 µm, and from 250 to 300 µm) and calculated the average percentage of SMCs with positive staining per high-power field for each artery size. The comparison of Bcl-2 and Bax expressions present in each category of arteries among the three rat groups yielded the same differences previously noted above (data not shown).

The ratio of Bcl-2 to Bax (an inverse index of cell susceptibility to apoptosis) was increased (P<.01) in SHR compared with WKY (2.29±0.07 versus 0.99±0.04) (Fig 5). This parameter was normalized in SHR-ACEI after treatment with quinapril (0.82±0.04) (Fig 5).

View larger version (50K): [in this window] [in a new window]   Figure 5. Ratio of Bcl-2 to Bax (an inverse index of cell susceptibility to apoptosis) in SMCs of small intramyocardial arteries from normotensive WKY, untreated SHR, and quinapril-treated SHR (SHR-ACEI). *P<.01 compared with WKY and SHR-ACEI.

Bcl-2 was directly correlated with systolic pressure (r=.817, P<.001, y=-4.12+0.11x) in all rats. Bax was inversely correlated with left ventricular ACE activity (r=.773, P<.001, y=25.07-0.12x) and serum ACE activity (r=.779, P<.001, y=17.58-0.04x) in all rats. No correlation was found between Bcl-2 and Bax.

	Discussion

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One finding of the present study is that the expression of the oncoprotein Bcl-2 is abnormally increased in SMCs of small intramyocardial arteries of adult SHR and becomes normal in SHR treated with the ACEI quinapril.

It is unclear what mechanisms are responsible for these changes in Bcl-2 expression in the vessel wall of SHR. Although we found a direct association between Bcl-2 expression and blood pressure level, the effect of ACE inhibition on this oncoprotein suggests also a potential role for nonhemodynamic factors such as angiotensin II. It is known that both continuous mechanical strain²⁴ and angiotensin II^{25 26} may modulate in vitro the expression of a number of growth-related proto-oncogenes in SMCs. Thus, further studies are needed to elucidate whether the apoptosis-related Bcl-2 proto-oncogene is also modulated in vivo by the above factors in SMCs.

Some data suggest that Bcl-2 acts as an antiapoptotic protein in SMCs.²⁷ On the other hand, exposure of SMCs to an apoptotic stimulus (ie, calphostin C) leads to a decline in Bcl-2 expression, suggesting that this decline may be responsible for the apoptosis of SMCs.¹² Thus, Bcl-2 overexpression in SMCs of small arteries of SHR can be associated with a blunted response to apoptotic stimuli. This is further supported by the finding that the ratio of Bcl-2 to Bax is abnormally increased in these rats. Since Bcl-2 must dimerize with Bax to prevent apoptosis, a high ratio of Bcl-2 to Bax means more Bcl-2–Bax heterodimers, favoring survival, whereas a low ratio means more Bax homodimers, favoring death.^{4 5 28} This has been confirmed in experiments showing that apoptotic cells induced to die by overexpression of p53 protein did indeed reduce Bcl-2 expression and increase Bax expression.²⁹

Our proposal of diminished in vivo susceptibility to apoptosis in SMCs of SHR disagrees with recent data by Hamet et al³⁰ showing that cultured SMCs from the SHR aorta exhibited a higher response to apoptotic inducers than do SMCs from normotensive WKY. Besides methodological differences, intervascular differences may exist in the control of SMC apoptosis that account for the discrepancy. This is based on previous observations dealing with the multifactorial nature of SMC growth control and the possibility that SMCs are not homogeneous but composed of different SMC populations.³¹ This hypothesis may help to explain the origin of intervascular heterogeneity of SMC growth responses in SHR.^{32 33} On the other hand, no information is available on the evolution of apoptosis concomitant with hypertension. This is important because we studied adult 36-week-old SHR, whereas Hamet et al performed studies on SMCs from young 10- to 13-week-old SHR.

Another finding of the present study is that quinapril treatment stimulates expression of the oncoprotein Bax in SMCs of small intramyocardial arteries of adult SHR. Furthermore, an inverse association exists between Bax expression and ACE activity in SHR. Therefore, the possibility that the availability of angiotensin II and/or bradykinin participates in the in vivo regulation of Bax expression in SMCs requires further investigation.

As a consequence of the quinapril-induced stimulation of the oncoprotein Bax, the ratio of Bcl-2 to Bax was normalized in treated SHR. Thus, the decrease in angiotensin II and increase in bradykinin-related compounds (ie, nitric oxide) secondary to ACE inhibition could result in restoration of the susceptibility to apoptosis in SMCs of SHR. In this regard, it has been reported recently that angiotensin II inhibits apoptosis in SMCs via stimulation of the angiotensin II type 1 receptor³⁴ and that activation of nitric oxide synthesis leads to apoptosis in SMCs.^{35 36}

In conclusion, our findings suggest that apoptosis may be reversibly depressed in SMCs of small arteries of adult SHR. Since increased proliferation of SMCs has been described in SHR,¹³ our data add support to the hypothesis that an imbalance between proliferation and apoptosis alters SMC homeostasis in the arterial wall in genetic hypertension.¹⁴ However, we are aware that the findings of this study are descriptive in nature and that more data are required to provide direct evidence linking altered expression of Bcl-2 and Bax to depressed SMC apoptosis in SHR.

	Selected Abbreviations and Acronyms

 

ACE = angiotensin-converting enzyme ACEI = angiotensin-converting enzyme inhibitor SHR = spontaneously hypertensive rat(s) SMC = smooth muscle cell WKY = Wistar-Kyoto rat(s)

Received August 26, 1996; first decision September 25, 1996; accepted September 25, 1996.

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