Effects of Spironolactone on Cerebral Vessel Structure in Rats With Sustained Hypertension

Abstract

Background

Spironolactone prevents eutrophic middle cerebral artery (MCA) remodeling in young stroke-prone spontaneously hypertensive rats (SHRSP). Clinically, it is more relevant to identify treatments that improve vessel structure after hypertension and remodeling has developed. We hypothesized that spironolactone would increase the MCA lumen diameter and reduce the wall/lumen ratio in SHRSP treated from 12 to 18 weeks of age.

Methods

Twelve-week-old male SHRSP were treated with spironolactone (SHRSP + spir: 25mg/kg/day) for 6 weeks and were compared at 18 weeks to age matched untreated SHRSP and Wistar Kyoto (WKY) rats. MCA structure was assessed by pressure myography. The WKY rats were included to provide an indication of the magnitude of the hypertensive MCA remodeling.

Results

Spironolactone had no effect on blood pressure as measured by telemetry. MCA myogenic tone was enhanced in the SHRSP + spir. Spironolactone increased the MCA lumen diameter (SHRSP: 223.3 ± 9.7µm, SHRSP + spir: 283.7 ± 10.1µm, WKY: 319.5 ± 8.8µm, analysis of variance (ANOVA) P < 0.05) and reduced the wall/lumen ratio (SHRSP: 0.107 ± 0.007, SHRSP + spir: 0.078 ± 0.006, WKY: 0.047 ± 0.002, ANOVA P < 0.05). Vessel wall stiffness was unchanged by spironolactone. Collagen 1 and 4 mRNA expression was increased in cerebral vessels from SHRSP compared to WKY rats; collagen 1 was reduced by spironolactone. Western blot analysis showed that active matrix metalloproteinase (MMP)-13 expression was increased by spironolactone treatment. The expression of intercellular adhesion molecule 1 (ICAM-1), a marker of inflammation, was increased in SHRSP and reduced by spironolactone.

Conclusions

These studies provide evidence that chronic mineralocorticoid receptor (MR) antagonism improves cerebral vessel structure after remodeling has developed in a model of human essential hypertension.

American Journal of Hypertension, advance online publication 24 February 2011; doi:10.1038/ajh.2011.20

Ischemic strokes are a leading cause of morbidity and mortality in the United States,¹ and the treatment options available are extremely limited. Hypertension is a primary risk factor for stroke, and elevated blood pressure alters the cerebral vasculature in a manner that increases the risk of an ischemic stroke occurring² and impairs the ability of the vessels to dilate in response to an ischemic insult,³ thus increasing the damage caused by a stroke. Stroke-prone spontaneously hypertensive rats (SHRSP) have been used extensively to study the effects of vascular structure on the outcome of cerebral ischemia; SHRSP suffer hemorrhagic strokes when fed a high-salt diet,⁴ and when ischemia is induced by middle cerebral artery (MCA) occlusion they have significantly larger infarcts than normotensive Wistar Kyoto (WKY) rats.⁵ Cerebral arterioles from hypertensive rats undergo an inward eutrophic remodeling characterized by a reduction in the lumen and outer diameters without a change in wall area.⁶ In the MCA we have also found a reduction in the lumen and outer diameter in SHRSP compared to WKY rats.⁷ The presence of a smaller lumen is particularly important in cerebral vessels because it impairs the vessel's ability to autoregulate⁸ and dilate.³ This reduction in lumen diameter may contribute to the large cerebral infarcts observed with MCA occlusion in SHRSP.⁹ Therefore, treatments that increase the lumen diameter could be effective primary prevention strategies to reduce stroke risk.

There has been a meteoric rise in interest in the cardiovascular effects of aldosterone or activation of its receptor, the mineralocorticoid receptor (MR).¹⁰ The MR is present on vascular smooth muscle and endothelial cells,^11,12 and we have previously shown robust MR expression in the cerebral vasculature.¹³ There are clear links between aldosterone and stroke.¹⁴ Thirteen percent of patients with primary aldosteronism have a history of stroke compared to only three percent of patients with essential hypertension matched for age, gender, and blood pressure.¹⁵ Similarly, patients with increased plasma aldosterone, caused by glucocorticoid-remediable hypertension, have an increased frequency of stroke.¹⁶ Importantly, aldosterone is involved in 10–15% of the cases of hypertension previously classified as essential hypertension.¹⁷ Thus, a causative link between aldosterone and stroke would significantly increase the population considered at risk for a cerebrovascular event.

The link between mineralocorticoids and stroke has also been established in rodents. MR antagonists (spironolactone and eplerenone) prevent spontaneous hemorrhagic strokes without lowering blood pressure in SHRSP that fed a high-salt diet.^18,19 We have shown that spironolactone treatment dramatically reduces the damage caused by cerebral ischemia in SHRSP⁵ and prevents eutrophic MCA remodeling.⁷ The converse is also true; direct MR activation with deoxycorticosterone acetate in Wistar rats causes an increase in the damage resulting from cerebral ischemia.²⁰ These studies were conducted using young rats and were designed to test the involvement of the MR in the development of cerebral vessel remodeling. It is unclear whether MR activation is involved in the maintenance of remodeling and whether MR antagonism has beneficial effects on the cerebral vasculature after hypertensive remodeling has occurred. This is clinically relevant because blood pressure is only controlled in 25–40% of patients.²¹

Previous studies suggest that interruption of the renin–angiotensin–aldosterone system improves vessel structure in situations where the vessels are markedly remodeled. This has been shown in the cerebral vessels in SHRSP treated with the angiotensin-converting enzyme inhibitors, cilazapril²² and perindopril,²³ and in SHR treated with the angiotensin II receptor blocker, telmisartan.²⁴ In all these studies blood pressure was also reduced by the treatment. Angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers inhibit the actions of angiotensin II on the vasculature, but also reduce aldosterone levels, making it impossible to separate the effects mediated by the angiotensin II receptors and the MR. Therefore, we hypothesized that chronic spironolactone treatment would reverse MCA remodeling in male SHRSP treated from 12 to 18 weeks of age. This will be evidenced by an increase in the MCA lumen diameter and a reduction in the wall/lumen ratio. WKY rats were included in this study to provide an indicator of the magnitude of remodeling in the SHRSP.

Methods

Animals and treatments. Male SHRSP were obtained from the breeding colonies housed at the Medical College of Georgia and Michigan State University. Twelve-week-old male SHRSPs were treated with spironolactone (SHRSP + spir, 25mg/kg/day) in the food for 6 weeks, control SHRSPs were untreated. Age matched WKY rats were purchased from Harlan (Indianapolis, IN). Animals were maintained in a temperature-controlled environment on a 12-h light/dark cycle and were provided regular chow (Harlan Teklad, diet #8656), and tap water ad libitum. The Institutional Animal Care and Use Committees at the Medical College of Georgia and Michigan State University approved the experimental protocols, following protocols outlined by the American Physiological Society.

Blood pressure measurement. Blood pressure was continuously monitored using telemetry (Data Sciences, St Paul, MN) with the transmitter placed in the abdominal aorta, as previously described.²⁵

Artery preparation. MCA structure and function was assessed by pressure myography as described previously.^7,13 Briefly, MCAs were isolated and placed in cold physiological salt solution (in mmol/l: 141.9 NaCl, 4.7 KCl, 1.7 MgSO₄, 0.5 EDTA, 2.8 CaCl₂, 10.0 HEPES, 1.2 KH₂PO₄, and 5.0 glucose). The first branch-free segment of the MCA most proximal to the Circle of Willis was mounted on two glass micropipettes in a small vessel arteriograph (Living Systems Instrumentation, Burlington, VT). Vessels were bathed with warm oxygenated physiological salt solution and the intralumenal pressure was set at 75mm Hg, vessels were allowed to equilibrate for 30min. Vessels that did not hold pressure were discarded. Myogenic tone, expressed as a percentage of the passive lumen diameter, was calculated from the lumen diameters at 75 and 125mm Hg intralumenal pressure under active and passive conditions using the following formula: % Myogenic Tone = (1−(ID_A/ID_P)) × 100, where ID_A is inner diameter under active conditions and ID_P is inner diameter under passive conditions.^7,26 For the analysis of vessel structure, the vessels were bathed in calcium-free physiological salt solution containing 2mmol/l ethylene glycol tetraacetic acid and the intralumenal pressure was increased from 0 to 180mm Hg in 20mm Hg increments. Video microscopy was used to measure lumen diameter, external diameter, and wall thickness at each pressure after a 5-min equilibration. The wall/lumen ratio, circumferential wall stress, and wall strain were calculated using the method of Baumbach and Hadju.²⁷ The elastic modulus (β-coefficient) was calculated from the stress/strain curves for the individual vessels, these curves were fitted to an exponential model (y = ae^βx) where β is the slope of the curve: the higher the β-coefficient, the stiffer the vessel.

Slot and western blotting. Cerebral vessels (MCA, anterior and posterior communicating and ophthalmic arteries) were collected for analysis of collagen 1 and 4 expression by slot blot and matrix metalloproteinase (MMP)-13 expression by western blot using previously described techniques.²⁸

Reverse transcriptase-PCR. RNA was extracted from cerebral vessels using TRIZOL reagent (Invitrogen, Carlsbad, CA) following the manufacture's protocol. 1µg of RNA was reverse transcribed using Superscript II (Invitrogen). Real-time PCR was performed using TAQMAN primers (Applied Biosystems, Foster City, CA). mRNA expression was measured for MMP-2, MMP-9, MMP-13, tissue inhibitor of MMP-2, collagen 1, and collagen 4. Expression of MKI67, a marker of cell proliferation, Bcl-2, an antiapoptotic marker, and BAD, a proapoptotic marker were also measured. The following inflammatory markers were studied, intercellular adhesion molecule 1 (ICAM-1), osteopontin, and tumor necrosis factor-α. MR expression was also assessed. RPL32 was used for normalization. Fold changes, from WKY expression, were calculated using the 2^−ΔΔCt method.²⁹

Statistics. Data were analyzed by two-way repeated measures analysis of variance (ANOVA), with a Bonferroni multiple comparison post-test for all dose and pressure responses. All other data that passed the equal variance test were analyzed by one-way ANOVA with a Bonferroni post-test. ANOVA on the ranks followed by a Tukey's post-test was used when the equal variance test failed. A P value of 0.05 or below was considered significant. All values presented are mean ± s.e.m.; n represents the number of animals.

Results

Physiological parameters

Body weight was significantly increased in the SHRSP + spir (336.2 ± 4.0g) and in the WKY rats (322.1 ± 4.0g) compared to SHRSP (305.8 ± 8.8g; P < 0.05). There was no difference in body weight between the SHRSP + spir and WKY rats. Spironolactone treatment had no effect on plasma potassium levels in the SHRSP (4.5 ± 0.1 vs. 4.7 ± 0.1mmol/l, SHRSP + spir vs. SHRSP; P > 0.05). WKY rats had the lowest plasma potassium level (4.2 ± 0.1mmol/l). Spironolactone treatment did not alter mean arterial pressure in SHRSP over the course of treatment. Mean arterial pressure was increased in both groups of SHRSP compared to WKY rats (Figure 1).

Figure 1. Spironolactone has no effect on blood pressure in male stroke-prone spontaneously hypertensive rats (SHRSP). Male SHRSPs were treated with spironolactone (25mg/kg/day) for 6 weeks from 12 weeks of age. Blood pressure was measured continuously using telemetry with the telemeter placed in the abdominal aorta. The average of the daytime blood pressure (12h) is shown for each day of treatment. The results are expressed as the mean ± s.e.m. Wistar Kyoto (WKY) rats had lower blood pressure than both SHRSP groups, spironolactone did not affect blood pressure (*P ≤ 0.05).

Myogenic tone in the MCA

The ability of the MCA to generate tone was assessed at intralumenal pressures of 75 and 125mm Hg. Tone generation, expressed as a percentage of the passive lumen diameter, was increased in the MCA of SHRSP ± spir compared to SHRSP and WKY rats at 75mm Hg (P ≤ 0.05). There was no significant difference in the tone at 125mm Hg (P = 0.12), although there was a trend toward an increase in the MCAs from SHRSP + spir (Figure 2).

Figure 2. Spironolactone increases middle cerebral artery tone. Stroke-prone spontaneously hypertensive rats (SHRSP) were treated with spironolactone (SHRSP + spir; 25mg/kg/day; n = 6) for 6 weeks from 12 weeks of age. Myogenic tone, expressed as a percentage of the passive diameter, was measured at 75 and 125mm Hg using the following formula: % Myogenic Tone = (1–(ID_A/ID_P)) × 100 where ID_A is inner diameter under active conditions and ID_P is inner diameter under passive conditions. *indicates significant differences from SHRSP (n = 9) and Wistar Kyoto (WKY) rats (n = 9), results are shown as the mean ± s.e.m.

MCA passive structure

Before investigating whether spironolactone improves MCA structure after hypertension has developed, it was necessary to show that the remodeling we have previously observed in 12-week-old SHRSP is present and unchanged in 18-week-old SHRSP. We have shown that MCAs from 12-week-old male SHRSP exhibit eutrophic remodeling, indicated by smaller outer and lumen diameters, with no difference in wall thickness when compared to WKY rats at an intralumenal pressure of 80mm Hg (Table 1). The data in Table 1 for the 12-week-old SHRSP and WKY rats has been published previously and is included for comparison.⁷ There were no significant differences in MCA structure between 12- and 18-week-old control SHRSP over the range of intralumenal pressures studied.

Table 1.

Comparison of middle cerebral artery structure between 12 and 18-week-old male control SHRSP to establish pre-existing vascular remodeling in the 12-week old SHRSP

Spironolactone increased the MCA lumen (Figure 3a) and outer (Figure 3b) diameters compared to control SHRSP. The lumen diameter was increased by ~26% in SHRSP + spir but the lumen diameter did not reach the level of a WKY rat. Spironolactone increased the outer diameter of the MCA to a level such that it was not different from the outer diameter of the WKY MCAs (Figure 3b). The wall/lumen ratio was decreased in MCAs from SHRSP + spir compared to SHRSP, but remained greater than WKY rats (Figure 3c). Spironolactone treatment did not alter MCA wall thickness (Figure 3d). The β-coefficient, calculated from the individual stress/strain curves, was used as a measure of vessel stiffness. There was no significant difference in the β-coefficient between groups (SHRSP + spir: 4.32 ± 0.27, SHRSP: 5.08 ± 0.28, WKY: 5.00 ± 0.48), although there was a trend toward a decreased β-coefficient in the MCA of the spironolactone-treated SHRSP.

Figure 3. The effects of spironolactone on cerebral vessel structure. Male stroke-prone spontaneously hypertensive rats (SHRSP) were treated with spironolactone (25mg/kg/day) for 6 weeks from 12 weeks of age, vessels were collected from 18-week-old rats. Measurements of (a) passive middle cerebral artery (MCA) lumen and (b) outer diameters were obtained over a range of intralumenal pressures on a pressurized arteriograph in calcium-free physiological salt solution. (c) MCA wall to lumen ratios were calculated and (d) wall thickness was measured. *P < 0.05, brackets indicate group differences, analysis of variance. WKY, Wistar Kyoto rats.

Collagen and matrix MMP expression

Reverse transcriptase-PCR analysis showed increased expression of the mRNA for collagen 1 and 4 in SHRSP rats. Spironolactone reduced collagen 1 mRNA expression to a level between the SHRSP and WKY rats such that the expression in SHRSP + spir was not different from either group (Table 2). However, slot blot analysis of collagen expression did not show significant differences between the three groups of rats for either collagen 1 or 4 (Figure 4a,b). No differences in the mRNA expression of MMP-2 or MMP-9 were observed between the groups. There was a trend toward an increase in the mRNA expression of MMP-13 in the SHRSP and the SHRSP + spir but this did not reach statistical significance (Table 2). Western blot analysis of MMP-13 protein expression showed no difference in the expression of pro-MMP-13 but a significant increase in the level of active MMP-13 in the SHRSP + spir (Figure 4c). The mRNA expression of tissue inhibitor of MMP-2 was also unchanged between the groups (Table 2).

Table 2.

Fold change in mRNA expression in 18-week-old WKY, SHRSP, and SHRSP ± spir cerebral vessels

Figure 4. Effects of spironolactone (spir) on (a) collagen 1, (b) collagen 4, and (c) matrix metalloproteinase (MMP)-13 expression. Male stroke-prone spontaneously hypertensive rats (SHRSP) were treated with spironolactone (25mg/kg/day) from 12 to 18 weeks of age, vessels were collected from 18-week-old rats. * indicates a significant difference from SHRSP (n = 6 in each group).

Inflammation, proliferation, apoptosis, and MR expression

The mRNA expression of ICAM-1 was increased in SHRSP compared to WKY rats, ICAM-1 expression was reduced by spironolactone treatment and was not different between the SHRSP + spir and WKY rats (Table 2). Osteopontin levels were increased in both groups of SHRSP compared to WKY rats. Tumor necrosis factor-α mRNA expression was unchanged between the groups. There was a trend toward an increase in MKI67 expression in both SHRSP groups, but this did not reach statistical significance. There were no differences in the expression of BAD and Bcl-2 between the groups. The expression of the MR was not different between the groups (Table 2).

Discussion

The studies presented here suggest that MR antagonism changes the cerebral vessels of SHRSP to make their lumen and outer diameters more similar to those of WKY rats. Previously, we have shown that spironolactone treatment prevents eutrophic MCA remodeling in young rats without lowering blood pressure.⁷ The current study was designed to test the possibility that spironolactone would have beneficial effects in vessels that have already undergone marked remodeling and therefore could be potentially beneficial in patients with chronic hypertension. Therefore, we began spironolactone treatment once there was established MCA remodeling.^7,30 The effect of spironolactone on the MCA suggests that either aldosterone or MR activation acts to maintain the structural changes seen in the adult SHRSP compared to WKY rats. In the presence of a MR antagonist the maintenance signal for vessel remodeling is switched off and the structure of the MCA changes.

One caveat to the current study is that we used spironolactone to block the MR, spironolactone has antiandrogenic and progestogenic effects. The binding affinity of spironolactone at the androgen and progesterone receptor is considerably lower than the affinity for the MR.³¹ However, we cannot be certain that the effects observed are solely due to MR blockade. We felt its use was warranted here to allow for the comparison between the development of remodeling in our previous study⁷ and the maintenance of remodeling studied here. We have conducted studies using the more specific MR antagonist, eplerenone, in younger SHRSP and we found that the results obtained were similar to the results obtained with spironolactone; this lead us to believe that the effects observed are MR dependent. Moreover, we have conducted studies to activate the MR using deoxycorticosterone acetate²⁰ and we observed the effects that are essentially opposite to those observed with MR antagonists. Importantly, studies using SHRSP fed the stroke-prone high-salt diet show that spironolactone and eplerenone have extremely similar effects on the cerebral vasculature under these conditions.^18,19 Combined, these studies make us feel confident that the effects of spironolactone observed here occur at the level of the MR. It is also important to consider that spironolactone is still being actively prescribed and studies are advocating its use in patients with resistant hypertension.³² Therefore, studying the effects of spironolactone remains clinically relevant.

To the best of our knowledge, this is the first study showing that MR antagonism promotes beneficial structural changes in the vasculature of rats with sustained hypertension. Studies in normotensive Sprague–Dawley rats suggest that MR antagonists can reverse the age dependent increase in collagen in carotid arteries without changing the wall thickness.³³ Other studies using inhibitors of other components of the renin–angiotensin–aldosterone system demonstrate beneficial effects on vascular structure in humans and animals with hypertension.³⁴ As mentioned previously, angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers improve cerebral vessel structure in hypertensive rats.^22–24 While blood pressure was reduced in these studies, it seems that pressure lowering alone is not the mechanism for the beneficial effects observed because blood pressure lowering agents that do not affect the renin–angiotensin–aldosterone system do not change vessel structure.^23,35 In the current study, spironolactone had no effect on blood pressure as assessed by telemetry. This adds strength to the argument that interruption of the renin–angiotensin–aldosterone system affects the cerebral vasculature in a blood pressure–independent manner. Interestingly, blood pressure is still increasing slowly in the SHRSP between 12 and 18 weeks of age. However, our studies strongly suggest that vascular structure did not change during this time in SHRSP (Table 1). This presents the interesting possibility that MCA structure only responds to increases in blood pressure to a certain level, and any further change in pressure has no effect. This again suggests that blood pressure and vascular remodeling are at least in part independent from each other.

We observed an increase in myogenic tone in the MCA from spironolactone-treated SHRSP. Myogenic tone plays an important role in the maintenance of cerebral vessel autoregulation²⁶ and is influenced by various factors including metabolic and neural inputs.³⁶ Cerebral vessel autoregulation maintains appropriate brain perfusion and protects the microvasculature during fluctuations in blood pressure. The effect of blood pressure on tone generation seems to be rather controversial; increased tone,³⁷ unchanged tone,³⁸ and reduced tone³⁹ have all been reported in hypertensive rats. Importantly, the pattern of changes in tone observed in the current study are very similar to those previously observed by our group in younger SHRSP.⁷ The increase in the myogenic tone observed in MCAs from spironolactone-treated SHRSP at 75mm Hg may be an indication that the autoregulatory behavior of the MCA improved with spironolactone treatment. The improved ability of the vessel to generate tone will allow for better protection of the downstream arterioles from blood pressure fluctuations. Previous studies have shown that spironolactone protects SHRSP from spontaneous hemorrhagic strokes;¹⁸ enhanced myogenic tone may be responsible for this. The mechanism by which spironolactone increases tone remains unclear. Spironolactone does not increase vessel wall material, therefore the enhanced tone is not merely a function of increased muscle mass. This suggests that the change in regulation of tone may occur at a molecular level. Rho kinase is intrinsically involved in the control of MCA tone.⁴⁰ However, it is unlikely that Rho kinase is involved in the increased tone observed with spironolactone treatment because MR activation, not antagonism, increases Rho kinase activation.⁴¹

The MCA remodeling in SHRSP is classified as true remodeling because the lumen diameters at 0mm Hg intralumenal pressure were different from the WKY lumen diameters.⁶ Interestingly, spironolactone completely corrected the outer diameter of the MCA but only partially corrected the lumen diameter and wall to lumen ratio. This change appears to have necessitated a small increase in growth in the spironolactone-treated SHRSP. Both groups of SHRSP exhibited a trend toward increased expression of MKI67, a marker of cell proliferation.⁴² The pro- and antiapoptotic markers were unchanged between the groups. This suggests that there may be more smooth muscle cell proliferation in the SHRSP + spir, this may be advantageous to compensate for the increased vessel size without a change in blood pressure. Despite the potential increase in growth, the wall area of the MCAs from SHRSP + spir was not increased. This is consistent with previous studies showing that spironolactone prevented eutrophic remodeling in the MCAs from SHRSP.⁷ It is interesting to note that we observed a difference in the wall thickness of the vessel between the 18-week-old SHRSP and WKY rats that was not as evident when we studied younger rats.⁷ This finding is not without precedence, Izzard et al. showed that there was no difference between SHRSP and WKY MCA wall thickness in 5–6-week-old rats, but in 20–24-week-old rats the wall was thicker in the SHRSPs.⁴³

MCA stiffness was not altered by spironolactone treatment. Changes in collagen expression can result in changes in vessel wall stiffness. Interestingly, expression of the mRNA for collagen 1 and 4 was increased in SHRSP. In the presence of spironolactone collagen 1 expression was not significantly different from either the control SHRSP or the WKY rats, suggesting that spironolactone had partially reduced collagen 1 mRNA expression to bring it to a level midway between the SHRSP and WKY rats. Spironolactone had no effect on collagen 4 mRNA expression. However, we could not detect a difference in the expression of either collagen by slot blot despite an increase in the expression of active MMP-13 protein expression in the SHRSP + spir. MMP-13 is a collagenase that degrades fibrillar collagen; therefore, one would have expected a reduction in collagen expression in the SHRSP + spir. The disparate results for collagen protein and mRNA expression may reflect a change in the turnover or degradation of the mRNA between the groups. It may also be a function of the methodology used to measure collagen expression. For both collagen 1 and 4 protein expression there was a trend toward the expression levels being higher in the SHRSP than in the SHRSP + spir or the WKY rats, it is possible that the use of immunostaining to detect and quantify collagen expression would have yielded a significantly different result. Surprisingly little is known about the regulation of collagen, particularly collagen 4 in the cerebral vasculature. The lack of a spironolactone-mediated reduction in collagen levels in the SHRSP is somewhat surprising and at odds with many studies in the peripheral vasculature. For example, Bentos et al. showed that spironolactone treatment of young SHR rats prevented aortic collagen deposition.⁴⁴ As mentioned previously, spironolactone reduces aging-associated collagen deposition in carotid arteries.³³ Both the carotid arteries and the aorta experience higher blood pressures than the MCA, and one can see why structural proteins such as collagen would be more highly regulated in these vessels than in the smaller vessels that experience lower pressures.

We recognize that a caveat to this study is the lack of a direct measure of MMP activity. MMP-13 activity is difficult to measure in the small samples available from the cerebral vasculature. Therefore, western blots to assess this expression of pro- and active MMP-13 were used as a surrogate for activity. We also recognized that MMP-2 and -9 are important factors in vessel remodeling. Their mRNA expression was unchanged in any of the groups studied. We have also failed to observe any MMP-9 activity in naive cerebral vessels. Therefore, we chose to focus our studies on MMP-13. It should be noted that we do not believe that MMP-13 is responsible for the difference between WKY and SHRSP, but MMP-13 may be responsible for the difference between SHRSP and SHRSP + spir.

Chronic inflammation plays a key role in the pathogenesis of cardiovascular disease.^45,46 Recently several studies have linked aldosterone and inflammation in the cardiovascular system.⁴⁷ Notably, aldosterone increases ICAM-1 expression in human umbilical vein, aortic and coronary artery endothelial cells.^7,12,48 In the current study, we assessed the mRNA expression of ICAM-1, osteopontin, and tumor necrosis factor-α to provide an indication of vascular inflammation. Osteopontin and ICAM-1 were increased in the vessels from SHRSP and ICAM-1 expression was reduced by spironolactone, suggesting that the MR regulates ICAM-1 expression in the cerebral vasculature. Surprisingly, the expression of tumor necrosis factor-α was unaffected in the 18-week-old SHRSP. It should be noted that this is just a snapshot of the inflammatory profile of the vessels and it is possible that the temporal regulation of vascular inflammation looks quite different.

In conclusion, we have demonstrated that spironolactone reverses eutrophic MCA remodeling in SHRSP in a blood pressure–independent manner. Vessel remodeling increases the risk of and the damage caused by cerebral ischemia and studies suggest that small vessel remodeling is present before the detection of hypertension.⁴⁹ Therefore, it is prudent to identify mechanisms that could ameliorate remodeling in patients with hypertension to reduce the risk of stroke and other hypertension-related pathologies. The notion that MR blockade could improve the vascular changes and prevent the end organ damage seen in hypertension is appealing. MR antagonists are currently in use clinically and in general, well-tolerated. Therefore, the possibility that MR blockade could be used as an add-on therapy to reduce the risk of cerebrovascular disease warrants further investigation.

Disclosure:

The authors declared no conflict of interest.