The technique of Kern et al., (1983) [19] was adapted as follows: 1.5 x 10⁵ HBMEC were cultured in 24 multiwell plates filled with M131 containing 5% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin and 5% MVGS. After 18 hours at 37°C, the cells were carefully washed 2 times with RPMI and detached with a non-enzymatic solution containing accutase (PAA, France). The effect of aldosterone, aldactone and eplerenone on capillary formation was analyzed on low matrigel base in a medium devoid of growth factor. To this end, 96-well plates were pre-coated with matrigel (Becton-Dickinson, Le Pont de Claix, France) that was allowed to polymerize for 1-2 hours at 37° C. Thereafter, 100 μl HBMEC (3.5 x 10⁴ cells/ well) in M131, without FCS and MVGS, were seeded onto the matrigel-coated wells in presence of various concentrations and combinations of steroids. Following different incubation periods at 37° C, capillary tube formation was assessed using an inverted microscope fitted with a digital camera (Nikon-Diafot). The capillary network was quantitated by recording the number and length of connections between three or more capillary-like structures/field with the aid of the Archimed program, developed by Microvision [20].

6 x 10⁴ HBMEC were cultured in 24 well plates coated with 03% of gelatin in the presence of M131 containing 5% FCS and 5 % MVGS. After 18 h, the semi-confluent cells were dislodged by a cell scraper on a standardized surface. The effect of aldosterone (100 nM), eplerenone (400 nM), aldactone (400 nM), alone or in various combinations, on cell migration was quantitated by measuring the number of cells migrating to the wound edge after 6, 18, 27 and 48 h incubation in a medium containing only 2% FCS to avoid cell proliferation.

6 x 10⁴ HBMEC were cultured in 24 well plates in the presence of M 131 containing 10% FCS and aldosterone, eplerenone or aldactone in desired combinations. After 0, 18 and 36h, the cells were detached by trypsin and counted in the presence of Trypan blue.

The HBMEC cultures were exposed for 8 h to 100 nM aldosterone in M131, twenty five million cells were lysed in Trizol reagent and shipped to Memorec on dry ice. RNA was extracted and hybridized on PIQOR^tm microarrays, followed by linear amplification of RNA on oligo(dT)-T7 primed cDNA. Amplified RNA (aRNA) samples were quantitated by spectrophotometry and the sample quality was checked by gel electrophoresis (Bioanalyzer 2001, Agilent) as reported before [21]. The 1800 products in this microassay consisted of genes related to cytokines, monokines, growth factors, angiogenic effectors, cell metabolism, growth and malignant transformation. The experiment was repeated twice with four spots each time and is based on qPCR analysis.

The student’s t test was used to assess the significance between various groups; the p values are shown in figure legends.

Data in Fig. (1) show decreased number of HBMEC networks and capillary lengths as a result of exposure to either 100 nM aldosterone (b), 400 nM eplerenone (c) or a combination of the agonist plus the antagonist (d), 18 h after the cells were seeded on low Matrigel support; in contrast, aldosterone had no effect on high matrigel support [16].

Fig. (1) Inhibition of HBMEC capillary length and abundance by aldosterone and eplerenone. HBMEC (30,000 cells) were exposed to 100 nM aldosterone (b), 400 nM eplerenone (c), or the agonist plus the antagonist (d), for 18 h and thereafter layered on Matrigel for 18 h. Archimed program of Microvision was used to quantitate the capillary length (arrows in a) and the number of networks (*) compared to control (a) in Fig. (1).

Quantification by the Archimed program of Microvision revealed that the length of the capillaries decreased significantly from 17.000 mm in the control group (c) to 5.600 mm in the presence of 100 nM aldosterone, and to 11000 mm in the presence of eplerenone alone (Fig. 2A). The potent inhibitory effect of aldosterone was partially attenuated by eplerenone such that the capillary length increased from, 5600 mm in the presence of aldosterone alone, to 8200 mm when the antagonist competed with the agonist. This suggests that both the agonist and the antagonist compete for MCR occupancy albeit the underlying mechanisms remain to be elucidated. Similarly, the number of capillary networks diminished significantly from 23 in the control group (c) to 3 and 12.5, in the presence of aldosterone and eplerenone, respectively (Fig. 2B). Again, eplerenone partially reversed the effect of aldosterone to raise the number of networks from 3 in the presence of the agonist alone to 6 when the antagonist was combined with the agonist (Fig. 2B). Similar results were obtained with aldactone when used in place of eplerenone (not shown).

Fig. (2) Quantification of the number and the length of capillaries. The results, repeated three times, are expressed as the capillary length (Fig. 2A) and capillary number (Fig. 2B) as described in the Methods section. In Fig. (2A), compared to control all other columns show p<0.001; compared to eplerenone alone both aldosterone and aldosterone + eplerenone also show p<0.001. In Fig. (2B), compared to control, aldosterone alone, eplerenone alone or aldosterone + eplerenone = p<0.003; eplerenone vs aldosterone = p<0.003; eplerenone vs aldosterone + eplerenone = p<0.021.

Data in Fig. (3) show time dependent HBMEC migration to the wound edge on the gelatine support. The number of migrating cells diminished from 3120 in the control group (c) to 2810, 2175 and 2420 in the presence of aldosterone, eplerenone, and aldosterone plus eplerenone, respectively, 48 h after a wound had been excised on the cell layer. In this test, aldosterone neither inhibited cell migration nor modified the significant inhibition observed with eplerenone. It would therefore appear that eplerenone possesses innate capacity to inhibit cell migration which is not dependent upon the antagonism of aldosterone action. Further experiments are needed to elucidate the action of eplerenone which assumes importance in view of the clinical use of the steroid [22, 23]. Interestingly, aldactone was even more potent than eplerenone in this context (data not shown) which may stem from the fact that it activates receptors for several steroid hormones (24 in contrast to the specificity of eplerenone for the MCR [22, 23].

Fig. (3) The effect of aldosterone and eplerenone on HBMEC migration. HBMEC (200,000), cultivated in M131 + 10% fetal calf serum, were layered on 0.3% gelatin for 18 h to attain 90% confluence. A fissure was incised by the technique of the Wound-healing test (Mirshahi et al., 2006). The flasks were cultivated in the presence of M 131 + 2% fetal calf serum and the number of cells migrating to the wound edge was quantitated by Archimed program of Microvision 6h, 18h, 27h and 48h later. The experiment was repeated eight times using control cultures, 100 nM aldosterone, 400 nM eplerenone, agonist plus antagonist, as described in the Methods section. After 48 h at 37°C, compared to the control, eplerenone = p<0.003; aldosterone + eplerenone = p<0.003; aldosterone = not significant (NS); aldosterone vs eplerenone = NS; eplerenone vs aldosterone + eplerenone = NS.

Data in Fig. (4) show that HBMEC proliferation, over the 36 h period, decreased from 550,000 in the control group to 380,000, 455,000 and 420,000 living cells in the presence of aldosterone, eplerenone and aldosterone plus eplerenone, respectively. Eplerenone was significantly less effective than aldosterone in inhibiting HBMEC proliferation and the agonist completely reversed the statistically insignificant inhibition observed with the antagonist alone. Aldactone was again more potent than eplerenone (not shown) possibly due to its affinity for various classes of steroid receptors [24].

Fig. (4) Inhibition of HBMEC proliferation by aldosterone and eplerenone. HBMEC (60,000) were cultivated in M131 + 10% fetal calf serum, detached by acutase, exposed to trypan blue and the surviving cells were counted 0, 18 and 36 h later. The experiment was repeated five times with control cultures, 100 nM aldosterone, 400nM eplerenone, agonist plus antagonist, as detailed in the Methods section. After 36 h, compared to the control, aldosterone = p<0.001; eplerenone = p>0.05; aldosterone + eplerenone = p<0.001; aldosterone vs eplerenone = p<0.003.

Collectively, whereas eplerenone partially reversed both the angiogenic (Fig. 1) and growth (Fig. 4) inhibition by aldosterone the agonist did not influence cell migration at all (Fig. 3) which was effectively inhibited by eplerenone (Fig. 3). The reason(s) for these differences are not understood but could stem from the nature of gene(s) modified by MCR-mediated effect of aldosterone. In order to elucidate these relationships, a gene array analysis was performed to screen for those genes that could potentially respond to aldosterone. The 1800 products screened in this microassay represent genes that direct the synthesis of various cytokines, monokines, growth factors, angiogenic effectors, cell metabolism, cell growth and neoplasia.

Data in Fig. (5) shows that the 28S/18S ratios were 1.72 and 1.85, respectively, for the control and aldosterone treated groups. As the samples did not show any additional degradation peaks in the low molecular range they were suitable for gene array screening. Data in Table 1 show upregulation of 20 mRNAs, along with the concurrent downregulation of 10 mRNAs in HBMEC exposed for 8 h to 100 nM aldosterone. In particular, the increase in mRNA for VEGF, CXCR-4, CDKN1A, Egr-1, EPCR, BCL-2, CD-24 and P-21 along with the decrease in the mRNA for NOD2, L1CAM, CD-83, ITGB-7, Cyclin-D, Cyclin-A, Decorin, GADD-135 and MMP-12, is in keeping with the accepted up and down regulation of these various factors in capillary formation. The table also provides the various synonyms in contemporary use to denote these various gene products. Altogether, 36 genes were up-regulated and 29 genes were down-regulated (details not shown).

Fig. (5) Qualitative analysis of RNA used to assess gene modulation by aldosterone. Total RNA from HBMEC was electrophoresed on agarose (A) to verify the quality of mRNA. Electrophreograms (B) performed on the electrophoresis peaks permitted the calculation of 28S/18S ratio. A value greater than 1.5 indicated qualitative integrity of the RNAs. Here, the ratios were 1.72 for the control and 1.85 for the aldosterone treated group. Furthermore, the samples did not show any additional degradation peaks in the low molecular weight range. These samples were used for linear amplification of 1800 genes that code for cytokines, monokines, growth factors, angiogenic effectors and cell metabolism, normal and malignant growth, as detailed in the Methods section.

Table 1

Transactivation of Genes in HBMEC Exposed to 100 nM Aldosterone for 6 h, Assessed by Quantitative PCR