METHODS. On line data supplement
Subjects
All subjects gave written informed consent to participate in the study, and the local committee on human research approved the study protocol. The study conformed with the principles of the Declaration of Helsinki. The study population consisted of 28 Caucasian patients (17 men and 11 women; mean age 60 years; range 37 to 75) with repeated documented elevated systolic blood pressure (SBP) of >139 mm Hg and diastolic blood pressure (DBP) of >89 mm Hg who were referred to our clinic for evaluation and treatment of arterial hypertension. No patient had received previous treatment with angiotensin converting enzyme inhibitors, AT1 receptor antagonists or calcium channel blockers. All patients had appropriate clinical and laboratory evaluation to exclude secondary hypertension and coronary artery disease as previously described.1 The ejection fraction was normal in all patients.
No wash-out phase was performed to assure continuous antihypertensive treatment (required by the ethics committee). Patients were randomized according to their previous antihypertensive treatment with diuretic agents, antiadrenergic drugs, and combined treatment. After randomization, 14 patients were assigned to losartan (losartan group) and 14 patients to amlodipine (amlodipine group) treatment. The dosages of losartan and amlodipine were titrated to achieve the therapeutic goal of SBP and DBP <140 and 90 mm Hg, respectively. After titration all patients in the losartan group and in the amlodipine group were receiving daily dosages of 50 mg and 10 mg, respectively, during 12 months.
A group of 8 hearts collected from a total of 100 autopsies performed at the University Clinic of Navarra during 1998 and 1999 served as control for cardiac apoptosis after cardiovascular disease was excluded.
Assessment of Left Ventricular Mass
Two-dimensional, targeted M-mode ultrasound recordings were obtained in each patient as previously described.13 Left ventricular mass and interventricular septal thickness (IVST) were measured, and left ventricular mass index (LVMI) was calculated by dividing left ventricular mass by body surface area. The presence of left ventricular hypertrophy (LVH) was defined as LVMI > 104 g/m2 in women and > 116 g/m2 in men, and/or IVST >11 mm.2
Collection of Tissue Samples
Transvenous endomyocardial biopsies were taken from the mid area of the interventricular septum with a bioptome Cordis 96 cm (7F) under fluoroscopic guidance after angiographic examination, at baseline and after 1 year of treatment in hypertensive patients. The biopsy procedure was well tolerated in all cases and no complications were recorded. In control hearts, septal specimens were also taken to assess apoptosis. Myocardial samples were immediately fixed in 10% buffered formalin, embebbed in paraffin, and serially sectioned in 4-mm-thick sections. Histological evaluation was performed without knowledge of from which patient the tissue section had been obtained.
In Situ Detection of Apoptosis
The TUNEL methodology used for in-situ labelling of DNA fragments was the same as recently described,3,4 with small modifications. Tissue sections were permeabilized with 0.01% saponine (Sigma). TdT reaction buffer consisted on: sodium cacodolyte 140 mmol/L, Tris 30 mmol/L, cobalt chloride 1.5 mmol/L, biotin-15-deoxyuridin triphosphate 0.5 mmol/L (Boehringer Mannheim) and TdT 0.125 U/ml (Boehringer Mannheim). The reaction was stopped with 50g/L of dry-skim milk diluted in 4xSSC, and incorporated biotined nucleotides were visualized with 4 mg/mL of fluorescein-isothiocyanate (FITC) conjugated with extravidin (Sigma) diluted in the stop buffer with 0.1% Triton-X-100. After mounting with Vectashield mounting medium containing 1.5mg/mL of 4’,6’-diamino-2-phenylindole (DAPI) (Vector) the analysis was performed with an epifluorescence microscope equipped with two filters that allowed detection of DAPI and FITC stained, respectively. Pre-treatment with DNase and addition of TdT reaction buffer without enzyme were used as positive and negative controls, respectively. The discrimination of the TUNEL positive nuclei between cardiomyocytes and non-cardiomyocytes was performed according to the cytological characteristics of the different cellular types.5 The determination of the total number of cardiomyocytes and non-cardiomyocytes was performed on a serial section from the one employed for the TUNEL, stained with Masson trichrome, and was based on the cytological characteristics of the different cellular types and the differential staining.5 Eighty one percent of the non cardiomyocyte cell population was estimated to be fibroblasts. The presence of apoptotic nuclei was expressed by means of apoptotic index that was the number of apoptotic nuclei per 106 total nuclei. Three apoptotic indexes were evaluated: a global index for total cells, an index for cardiomyocytes and an index for non-cardiomyocyte cells.
For immunohistochemistry of caspase-3 the slides were treated 3 times for 5 minutes with citric acid 10 mmol/L in the microwave and tissues were blocked with 4% normal goat serum. The primary antibody (DAKO) was incubated at 4ºC overnight, at a 1:100 dilution in PBS. The secondary antibody (Santa Cruz) was incubated at a 1:100 dilution in PBS and the ABC kit (Vectastain) was applied at a dilution 1:100 in distilled water. Positive staining was visualized with DAB plus (Boehringer Mannheim) and tissues were counterstained with Harris hematoxylin (Sigma).
Western Blot Analysis
Immunoblot assay of Bax-a and Bcl-2 was performed as recently described,11 with small modifications. Aliquots containing 20 mg of total protein were diluted in 4X sample buffer (40% b-mercaptoethanol, 8% SDS, 40% glycerol, 0.025 % bromophenol blue, and 0.25 mmol/L Tris, pH 6.4). Once obtained, the filters were blocked for 30 minutes at room temperature with 0.05% Tween and 10% dry skim milk in TBS (150 mmol/L NaCl, 10 mmol/L Tris, pH 8). Specific rabbit polyclonal antibodies anti-Bcl-2 and anti-Bax-a (Santa Cruz) were incubated at 1:400 and 1:200 in 0.05% Tween and 2% dry skim milk in TBS, respectively, at 4ºC overnight. Bands were detected by peroxidase-conjugated anti-rabbit IgG (Amersham) at 1:5000 in TBS and visualized with the ECL-Plus chemiluminiscence system (Amersham). The specificity of the bands was checked by preadsorbing anti-Bcl-2 and anti-Bax-a antibodies with the corresponding synthetic peptides. Autoradiograms were analysed using an automatic densitometer (Bio-Rad).The ratio Bax-a:Bcl-2 was calculated as an index of susceptibility to apoptosis. 6
Statistical Analysis
Differences in baseline parameters between hypertensives and normotensives or between hypertensives treated with losartan and hypertensives treated with amlodipine, were tested by a Student’s t test for unpaired data once normality was demonstrated (Shapiro-Wilks and Kolmogorov-Smirnov tests); otherwise, a nonparametric test (Mann Whitney U test) was used. Differences between hypertensives before and after treatment were tested by a Student’s t test for paired data once normality was demonstrated (Shapiro-Wilks and Kolmogorov-Smirnov tests); otherwise, a nonparametric test (Wilcoxon test) was used. Categorical variables were analyzed by the c2 Fisher exact test when necessary. Values are expressed as mean ± standard error. A value of P<0.05 was considered statistically significant.
References
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2. Díez J, Laviades C, Mayor G, Gil MJ, Monreal I. Increased serum concentrations of procollagen peptides in essential hypertension: relation to cardiac alterations. Circulation. 1995;91:1450-1456.
3. Fortuño MA, Ravassa S, Etayo JC, Díez J. Overexpression of Bax-a protein and enhanced apoptosis in the left ventricle of spontaneously hypertensive rats: effects of AT1 blockade with losartan. Hypertension. 1998;32:280-286.
4. Ravassa S, Fortuño MA, González A, López B, Zalba G, Fortuño A, Díez J. Mechanisms of increased susceptibility to angiotensin II-induced apoptosis in ventricular cardiomyocytes of spontaneously hypertensive rats. Hypertension 2000;36:1065-1071.
5. Burkitt HG, Young B, Heath JW. Wheater’s functional histology. Churchill Livingstone Ed. 1996.
6. Kroemer G. The protooncogene Bcl-2 and its role in regulating apoptosis. Nature Med. 1997;3:614-620.
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