In recent years, it has become increasingly clear that the extra-renal effects of aldosterone play an important role in the pathogenesis of cardiovascular disease. Stroke is one of the leading causes of death in the Western world, and MR (mineralocorticoid receptor) antagonism is a potential preventative therapy for patients at risk of both ischaemic and haemorrhagic strokes. This protective effect of MR antagonism appears to occur at the level of the cerebral vasculature and may be related to the expression and activation of the EGFR (epidermal growth factor receptor) and the degree of vessel wall collagen deposition.
Stroke is a leading cause of morbidity and mortality in the Western world . The majority of strokes that occur in the population are ischaemic in nature; however, the therapeutic options for the treatment of these strokes are few . TPA (tissue plasminogen activator) is the only currently approved treatment for ischaemic stroke. Yet, only 3% of stroke patients receive this therapy , and 10% of these patients die before leaving hospital . Until our understanding of the factors affecting the outcome of stroke increases, the paucity of useful therapies to be administered post-stroke is unlikely to change. It would therefore seem prudent to also study the factors that increase an individual's risk of having a stroke with a view of developing preventative therapies. Recently, it has become increasingly clear that aldosterone has deleterious extra-renal effects that play an important role in the pathogenesis of cardiovascular disease and its associated end-organ damage. Several studies have suggested that MR (mineralocorticoid receptor) antagonism with eplerenone or spironolactone has beneficial effects on the cerebral vasculature that could function to reduce an individual's risk of having a stroke [5–8]. Our understanding of the molecular mechanisms underlying the effects of aldosterone in the vasculature is limited when compared with our knowledge of its effects in the kidney. However, one thing is clear: a nephrocentric view of aldosterone as a hormone that merely regulates salt and water balance is no longer appropriate. There are clear links between aldosterone and hypertension and between hypertension and stroke risk. Therefore this review will focus primarily on the potential effects of aldosterone in the cerebral vasculature and how that might affect the outcome of cerebral ischaemia. We will also discuss potential mechanisms for aldosterone-induced vascular changes and review the evidence that, in some models of hypertension, the MR is activated not by aldosterone but by glucocorticoids.
VASCULAR STRUCTURE AND STROKE
Hypertension is one of the primary risk factors for stroke  and, prior to discussing how aldosterone might affect the cerebral vasculature, it is necessary to consider how hypertension in general affects the cerebral vessels and the outcome of stroke. At the level of the vasculature, there are two ways that the outcome of ischaemia could be affected, a reduction in the number of collateral vessels would reduce blood flow and therefore increase infarct size. Similarly, remodelling of the vessels in a manner that would reduce the lumen diameter would also reduce flow and increase the damage post-stroke. As the number of collateral vessels present in the brain of SHRSP (stroke-prone spontaneously hypertensive rats) and WKY (Wistar–Kyoto) rats does not differ , a reduction in vessel lumen diameter appears to be the probable cause of the increased ischaemic injury in hypertensive rats. SHRSP, a model of essential hypertension and cerebrovascular disease, have been used extensively to investigate the factors affecting the outcome of both ischaemic and haemorrhagic strokes. SHRSP suffer spontaneous haemorrhagic strokes when fed a high-salt diet , and when cerebral ischaemia is induced experimentally by MCA (middle cerebral artery) occlusion SHRSP have significantly more neuronal damage than normotensive WKY rats .
Under non-ischaemic conditions, there is very little blood flow though the collateral vessels , but these vessels dilate in response to an ischaemic insult to increase perfusion, effectively ‘bypassing’ the blockage. The ability of these vessels to dilate is impaired in SHRSP and this may contribute to the larger infarct observed in these rats [14,15]. The impaired ability to dilate in response to ischaemia may be caused by vascular remodelling, which is a complex process and the subject of many excellent reviews [16,17]. Cerebral vessels from hypertensive rats undergo mostly inward eutrophic remodelling , which is characterized by a reduction in the vessel lumen and outer diameters without a significant change in wall thickness. Thus there is thought to be little cell proliferation, but that the existing cells rearrange themselves around a smaller lumen. This type of remodelling also occurs in patients with essential hypertension  and is thought to be particularly important in the cerebral vasculature because it impairs the vessel's ability to autoregulate  and dilate [14,15]. However, there is also a small amount of hypertrophy of the vessel walls of cerebral arterioles from SHRSP which have been shown to contain more VSMCs (vascular smooth muscle cells) and elastin than those from WKY rats . The hypertrophy appears to be pressure-dependent, whereas the rearrangement of the VSMC is pressure-independent . It should be noted that these studies assessed the lumen diameter of cerebral arterioles and not of the MCA. This also raises an important point: not all branches of the cerebrovascular tree respond to hypertension in the same way. For instance, our laboratory has recently shown that dietary potassium supplementation improves the compliance of the branches of the MCA from SHRSP, but has no effect on the MCA itself .
MINERALOCORTICOIDS AND STROKE
Various studies have suggested that elevated plasma aldosterone or MR activation increases both the risk of and severity of, cerebrovascular events. Studies of patients with PA (primary aldosteronism) performed by Conn et al. in the 1960s were some of the first to suggest a link between hypertension, stroke and elevated plasma aldosterone levels . Like PA patients, patients with increased plasma aldosterone caused by glucocorticoid-remediable hypertension have an increased frequency of stroke and hypertension [25,26]. Interestingly, when PA patients and patients with essential hypertension are compared, PA patients suffer from more strokes, despite having slightly lower BP (blood pressure) than patients with essential hypertension . This suggests that elevated plasma aldosterone may increase an individual's risk of having a stroke in a BP-independent manner. The link between aldosterone and stroke has been strengthened further by studies using inhibitors of the RAAS (renin–angiotensin–aldosterone system). RAAS inhibition confers a greater protection from stroke than can be accounted for by the reduction in BP alone [28,29], presenting the possibility that reduced aldosterone production in these patients is responsible for at least part of the beneficial effects. Importantly, it is now accepted that aldosterone is involved in 10–15% of the cases of hypertension classified previously as essential hypertension . If a causative link between aldosterone and stroke exists, then this finding significantly increases the percentage of the population at risk of a cerebrovascular event.
The link between mineralocorticoids and stroke has been established experimentally. Administration of MR antagonists (spironolactone or eplerenone) to SHRSP fed a high-salt or stroke-prone diet prevents spontaneous haemorrhagic strokes without lowering BP . Administration of captopril, an ACE (angiotensin-converting enzyme) inhibitor, to salt-loaded SHRSP has a similar effect. Interestingly, the beneficial effects of captopril are lost when a mineralocorticoid is administered with the ACE inhibitor [31,32], suggesting that some of the benefits of ACE inhibition result from a reduction in AngII (angiotensin II)-stimulated aldosterone release. Spironolactone treatment has extremely dramatic effects on the outcome of acute ischaemic strokes, in fact it reduces the damage caused by cerebral ischaemia by 50% in SHRSP . In this study , spironolactone had no effect on systolic BP, suggesting again that aldosterone has deleterious actions on the cerebral vasculature that occur independently of its ability to increase BP. The possibility that the effects of MR activation on the cerebral vasculature are BP-independent is corroborated further by studies showing that treatment of normotensive WKY rats with spironolactone also reduces the ischaemic cerebral infarct size .
MINERALOCORTICOIDS AND VESSEL STRUCTURE
As mentioned above, the structure of the cerebral vasculature is a major determinant of the outcome of cerebral ischaemia. The effect of spironolactone on MCA structure has been studied in SHRSP. Studies in which SHRSP were treated with spironolactone from 6–12 weeks of age showed that MR antagonism significantly increases the MCA lumen and outer diameters, but has no effect on vessel wall area. This is consistent with a prevention of the eutrophic remodelling during the development of hypertension . Interestingly, an increase in myogenic tone with spironolactone treatment was also observed in these rats; this may represent an improvement in the ability of these MCAs to autoregulate, which may, in turn, be the mechanism for the protective effects of spironolactone against haemorrhagic strokes. Although these studies suggest that spironolactone can prevent hypertension-driven vessel remodelling, the reversal of remodelling is more important clinically. Therefore the studies described above were repeated in adult SHRSP. SHRSP were treated with spironolatone from 12–18 weeks of age. Importantly, at 12 weeks of age MCA remodelling in SHRSP is marked and maintained, as there is no difference between the structure of the MCAs from the 12- and 18-week-old rats. Spironolactone treatment also increased the lumen and outer diameters of the MCAs from adult rats . What is of interest is that in the young rats (age 6–12 weeks) the prevention of the remodelling was complete, i.e. the vessels from spironolactone-treated SHRSP behaved for all intents and purposes like a vessel from a WKY rat. However, in the older rats (age 12–18 weeks), the lumen diameter of spironolactone-treated SHRSP was between that of control SHRSP and WKY rats; the lumen is significantly larger than the former and smaller than the latter. What prevents these older rats from ‘completely’ remodelling in response to spironolactone treatment is unclear; it could be an effect of the duration of the hypertension or it is possible that in the older rats the remodelling process is ‘set’, whereas in the young rats the process is still active and therefore more pliable.
Studies using ACE inhibitors and ARBs (angiotensin receptor blockers) also support a role for the RAAS in hypertensive remodelling of the cerebral vasculature. Both ACE inhibition and angiotensin receptor blockade increase cerebral arteriolar lumen diameter [17,32,35]. Whether this is an aldosterone-mediated effect or strictly mediated by AngII has not been investigated, but its appears a probable possibility in light of the studies of haemorrhagic stoke with ACE inhibition, which show that aldosterone administration rescues the SHRSP phenotype [31,32]. Interestingly, there appears to be some cross-talk between the MR and the AT1 receptor (AngII type 1 receptor), which is elevated in the cerebral microvessels from hypertensive rats . Jaffe and Mendelsohn  have shown that AngII regulates gene transcription in cultured VSMCs. Surprisingly, the effects of AngII could be inhibited by spironolactone, suggesting that the MR is involved in the AngII-dependent modulation of gene transcription and that AngII may transactivate the MR. Aldosterone synthase was not present in the VSMCs, ruling out the possibility that the actions of AngII were via local aldosterone production. If AngII transactivates the MR in the cerebral vasculature, this could potentially enhance the actions of aldosterone and increase the risk of stroke.
One caveat that must be included in the discussion of these studies is that they all took place in genetically hypertensive rats. This makes it difficult to distinguish the specific effects of MR activation compared with BP-dependent effects. One other concern is that spironolactone has several effects other than MR antagonism; of particular importance here is its antagonistic effects on the testosterone receptor. Short-term testosterone administration increases the lesion size after ischaemia/reperfusion injury in rats , thus it is possible that testosterone receptor antagonism could reduce infarct size. However, it should be noted that these studies were of transient focal ischaemia, the pathogenesis of which is different from that of permanent ischaemia. Additionally, there are also studies showing that, unlike spironolactone, testosterone has no effect on vessel structure . This, taken with the knowledge that eplerenone, a more specific MR antagonist with no antitestosterone actions, is as effective as spironolactone at reducing the number of haemorrhagic strokes in salt-loaded SHRSP  suggests that the effects of spironolactone on the vasculature are MR- and not testosterone-receptor-dependent.
To confirm further a role for MR activation in cerebral vessel remodelling, we have performed studies to directly activate the MR with DOCA (deoxycorticosterone acetate) . In these studies rats were treated with DOCA, but did not undergo uninephrectomy or receive salt water. This treatment causes a modest increase in systolic BP (157±5.9 in DOCA-treated rats compared with 124±3.1 mmHg in control rats) and an approx. 40% increase in the amount of damage caused by cerebral ischaemia. The effects on the MCA were also dramatic: DOCA treatment reduced the lumen and outer diameters. Surprisingly, DOCA treatment also caused a significant increase in vessel wall thickness, suggesting that direct MR activation perhaps has an effect on VSMC hypertrophy. These results challenge the accepted dogma that there is little proliferative remodelling in the cerebral vasculature, but the question of the BP-dependency of this effect remains.
A recent study from Watts et al.  may shed some light on this issue. In this study , aorta and vena cava from DOCA-salt hypertensive rats were compared. As expected, the aorta had marked remodelling, whereas none could be detected in the vena cava. Both vessel types were exposed to similar levels of circulating mineralocorticoids, but only the aorta was exposed to the increase in pressure, suggesting that an increase in BP may be required for MR-dependent remodelling to occur. However, it should be noted that remodelling is extremely difficult to assess in veins because they have so little smooth muscle mass and such a large amount of collagen. It is also possible that the vena cava and aorta have differential expression of the MR, but, to the best of our knowledge, there have been no studies comparing the expression of the MR between arteries and veins. It should also be noted that these studies were conducted in conduit vessels and that the response in resistance vessels may prove to be quite different.
Studies on the effects of aldosterone on vessel structure in humans are few. Recently, an elegant study compared the resistance vessels of patients with hypertension with PA patients . Interestingly, the pattern of remodelling in arteries from gluteal biopsies appears to be different between the two groups despite similar BP. Both the patients with hypertension and PA patients had increased media thickness and media/lumen ratio when compared with normotensive controls. However, only the PA patients had a significant reduction in lumen diameter, probably driven by an enhanced increase in the media thickness compared with the hypertensive patients . This suggests that, as indicated above, direct MR activation does stimulate a small amount of vascular hypertrophy.
The possibility that MR activation results in VSMC proliferation has been corroborated further by studies showing that aldosterone increases the levels of MDM2 (murine double minute type 2) protein in VSMCs . The MDM2 protein has been shown previously to be involved in cell cycle regulation . Interestingly, MDM2 levels were not only found to be increased in VSMCs treated with aldosterone, but also in resistance vessels from PA patients (Figure 1) .
Proposed mechanisms by which aldosterone alters vascular structure via genomic and non-genomic effects on the VSMC
It should also be pointed out that not all of the effects of aldosterone or spironolactone on the vasculature occur at the level of the smooth muscle. Recent studies have suggested that spironolactone has beneficial effects on endothelial cells. Several studies have shown that spironolactone treatment improves endothelium-dependent vasodilation in patients with congestive heart failure [45,46]. Studies using HUVECs (human umbilical vein endothelial cells) suggest that spironolactone prevents endothelial cell apoptosis induced by serum deprivation. Interestingly, however, this protective effect of spironolactone appears not to be mediated by its ability to antagonize the MR, but may instead be a function of its ability to activate the progesterone receptor . Other studies using HUVECs suggest that aldosterone can increase ROS (reactive oxygen species) generation from endothelial cells and increase the expression of the p47phox subunit of NADPH oxidase. The function of eNOS (endothelial nitric oxide synthase) is also reduced by aldosterone; this is caused by a reduction in the availability of the eNOS co-factor BH4 (5,6,7,8-tetrhydrobiopterin), activation of PP2A (protein phosphatase 2A)  and a reduction in eNOS phosphorylation . Although these studies may explain why spironolactone therapy increases endothelium-dependent vasodilation in heart failure patients and in PA patients [45,46], a note of caution is warranted: the concentration of aldosterone used in some of these studies was 100-fold higher than what would be considered to be physiological levels.
POTENTIAL MECHANISMS FOR THE EFFECTS OF MR ACTIVATION
As mentioned previously, our understanding of the molecular mechanisms underlying the effects of aldosterone in the vasculature are limited. Therefore we will limit our discussion to pathways which evidence suggests are important in cerebral vessel remodelling (Figure 1).
Enhanced EGFR [EGF (epidermal growth factor) receptor] expression
Studies on MCA structure after DOCA administration suggest there may be VSMC proliferation , and aldosterone has been shown to stimulate VSMC proliferation in culture . EGF and its receptor (EGFR) are possibly the best candidates for a signalling mechanism for this, and several studies have linked this pathway and aldosterone. EGF is a VSMC mitogen that has been implicated previously in the hypertensive remodelling process [51–53]. That said, when the effect of spironolactone on the MCA of SHRSP is considered , it is difficult to envision a role for cell proliferation in the remodelling process as spironolactone has no affect on the amount of vessel wall material. However, it should be noted that EGFR activation is not only involved in proliferation, but also in cell migration , as is required for the inward eutrophic remodelling process. Also, in our initial studies on the effects of spironolactone on the outcome of cerebral ischaemia , we observed an increase in the mRNA for EGF and EGFR in vessels from SHRSP compared with WKY rats. Importantly, spironolactone treatment reduced the expression of both the receptor and ligand . As discussed above, spironolactone prevents the hypertension-induced remodelling process , and the possibility that EGF is involved in this process cannot be ruled out.
Some of the first studies suggesting a link between aldosterone and the EGF signalling pathway were studies on vascular reactivity. Florian and Watts  showed that EGF causes a marked contraction of aorta from DOCA-salt hypertensive rats compared with control rats. Later studies showed that this was not just an effect of elevated BP . These studies utilized Wistar and Wistar–Furth rats: the former become hypertensive with uninephrectomy and administration of DOCA and salt, whereas the latter remain normotensive . Vessels from both Wistar and Wistar–Furth rats treated with DOCA responded to EGF by contracting more than their respective controls , this contraction appears to be dependent on activation of the PI3K (phosphoinositide 3-kinase) and ERK1/2 (extracellular-signal-regulated kinase 1/2) pathways . These studies also add weight to the argument that the effects of aldosterone on the vasculature occur independently of its effects on BP. Additional studies carried out to assess the effects of DOCA-salt administration on the expression of mRNA and protein for the EGFR showed that the mRNA levels were increased, but that the protein itself was not elevated . Proof that aldosterone increases the expression of EGFR protein in the vasculature remained elusive until recently. Grossmann et al.  have shown that aldosterone increases EGFR protein expression in aorta from adrenalectomized rats and in cultured human aortic VSMCs. These studies identified the area of the EGFR promoter with which the MR interacts. Importantly, the glucocorticoid receptor does not interact with this area, suggesting that the authors may have identified a novel mineralocorticoid-responsive element. The increase in expression of EGFR was confirmed to be due to increased EGFR production, as EGFR degradation was unaffected by aldosterone. The newly produced EGFR was also shown to be functional; EGF-induced ERK1/2 phosphorylation was also enhanced with aldosterone treatment.
Rapid EGFR and tyrosine kinase activation
The rapid non-genomic effects of aldosterone occur within minutes and are insensitive to protein synthesis inhibition or to the actions of classical MR antagonists . This suggests that aldosterone activates the EGFR and other tyrosine kinase pathways. The initial studies documenting the effects of aldosterone on the EGFR were carried out in CHO (Chinese-hamster ovary) cells  and in renal epithelial cells . In VSMCs, aldosterone and AngII act synergistically to increase EGFR activation. Low doses of aldosterone or AngII alone have no effect on proliferation, but when the two are combined they increase proliferation via an EGFR-dependent mechanism . In these studies, the increase in EGFR phosphorylation also resulted in an increase in the activation of ERK1/2. In other studies, the AngII-induced phosphorylation of ERK1/2 and JNK (c-Jun N-terminal kinase) has been shown to be enhanced by the presence of aldosterone. Interestingly, spironolactone prevented not only the response to AngII and aldosterone together, but also the increase in ROS production and EGFR activation caused by AngII alone . These studies suggest that AngII might stimulate the release of aldosterone from VSMCs and that this locally produced aldosterone is responsible for the effects that can be inhibited by spironolactone. The notion that the cardiovascular system can produce aldosterone has become increasingly controversial. Studies have suggested that VSMCs can produce significant amounts of aldosterone and that the enzymes responsible for aldosterone production are present in these cells [16,66–68]. However, more recent studies using state-of-the-art technology have failed to show any evidence of aldosterone production at least in the heart ; as yet the presence of aldosterone production in VSMCs has not been confirmed or refuted.
The non-genomic actions of aldosterone that could stimulate cell proliferation are not limited to the EGFR. Recently, BMK1 [big MAPK (mitogen-activated protein kinase) 1], a MAPK involved in cell proliferation, differentiation and survival, has been shown to be rapidly activated by aldosterone in VSMCs. This activation resulted in VSMC proliferation that could be inhibited by eplerenone . Aldosterone also activates p38 MAPK and NADPH oxidase; this effect appears to be dependent on c-Src activation as it can be inhibited by PP2, a Src-kinase inhibitor . In these studies, an increase in [3H]proline incorporation, a marker of collagen synthesis, was observed in response to aldosterone, and this will be discussed further below. These effects of aldosterone were enhanced in cells from hypertensive rats .
Care should be taken in the interpretation of these results; all of these studies of the rapid effects of aldosterone have been performed in cell culture, where the effects of flow and pressure are lost. How these rapid responses to aldosterone will affect homoeostasis is not clear. The question that remains is how physiologically relevant is an event that has only been shown to occur in cultured cells, which are of a proliferative and non-contractile phenotype?
Although most effects of aldosterone on tyrosine kinase activation have been described in VSMCs, there is some evidence that aldosterone activates tyrosine kinases in endothelial cells. Incubation of endothelial cells with aldosterone caused an increase in the mRNA for ACE; this was inhibited by genistein, a broad-spectrum tyrosine-kinase inhibitor, and by JAK2 (Janus kinase 2), Src and EGFR inhibitors. The phosphorylation of JAK2 was measured and, interestingly, it does not appear to follow the classical non-genomic timeline. The maximum activation of JAK2 appears to occur after approx. 2 h of incubation with aldosterone. It is also worth noting that the concentration of aldosterone used in these studies was extremely high (10−6 mol/l) .
The location of the receptor responsible for the rapid actions of aldosterone remains, in essence, a mystery. It has been proposed that it is a membrane-bound receptor that co-localizes with caveolin-1 . Conversely, others have suggested that an intracellular receptor is responsible for the rapid effects of aldosterone [65,74] and that the MR does not colocalize with α1-integrin, which is a membrane marker . However, care should be taken in the interpretation of this result; as these studies used a commercially available MR antibody directed to the classical MR, it is possible that the MR that causes the rapid non-genomic responses does not react with this antibody. Because so many of the rapid actions of aldosterone occur via membrane-associated proteins it appears more likely that the receptor responsible for this effect is membrane-bound itself.
Studies on the MCA show that when the MR is inhibited in hypertensive rats the vessels become less stiff or more compliant , and that the opposite is true when the MR is activated by DOCA . The suggestion that MR activation increases fibrosis and collagen deposition is not new and has been eloquently reviewed by other groups [75,76]. For instance, congestive heart failure increases aldosterone levels, which in turn appears to increase carotid artery stiffness and collagen deposition, that can be reduced by spironolactone . What is rather surprising is the apparent lack of understanding of how aldosterone modulates collagen deposition in the vasculature. In theory, collagen levels can increase in two ways, by an increase in collagen synthesis or by a reduction in collagen metabolism.
Some of the cell culture studies already discussed with reference to tyrosine kinase activation also reported an increase in [3H]proline incorporation into cells treated with aldosterone and that this response is enhanced in cells from hypertensive rats [70,71]. Although actual collagen release was not measured, these studies suggest an increase in collagen deposition in response to aldosterone that is exacerbated by hypertension. One well-designed study carried out using human aortic VSMCs suggests that aldosterone alone is not sufficient to increase collagen synthesis, but that aldosterone can potentiate H2O2-induced collagen accumulation . Interestingly, this effect of aldosterone was prevented by GM6001, a compound that inhibits the shedding of membrane-bound EGFR ligands and, therefore, reduces EGFR activation. An increase in EGFR activation by aldosterone in the presence of H2O2 was confirmed as was the ability of EGFR blockade to prevent the increase in collagen accumulation . Therefore these studies suggest that the interaction between EGFR and aldosterone may not only drive the remodelling process, but also the vessel stiffness that is observed with hypertension.
Studies on cardiac tissue have shed some light on the molecular mechanisms behind the increase in collagen deposition in the presence of aldosterone. Procollagens are processed into mature fibrillar collagen by procollagen C-proteinases. PCPE-1 (procollagen C-proteinase enhancer protein-1) increases the rate of activation of the procollagen C-proteinase to allow for increased mature collagen production . Collagen I and PCPE-1 expression are positively regulated in cultured cardiac myocytes by aldosterone . Recently, PCPE-1 has also been shown to be increased in the hearts of rats after myocardial infarction, a situation where aldosterone levels are markedly elevated. Importantly, the increase in PCPE-1 could be inhibited by spironolactone, suggesting an MR-dependent effect .
CTGF (connective tissue growth factor) is activated in situations where fibrosis occurs, promotes fibroblast proliferation and intercellular matrix deposition , and may also be regulated by aldosterone in the aorta. These studies showed that expression of this growth factor was higher in the vasculature of SHRs (sponteneously hypertensive rats) and that it could be lowered by a dose of eplerenone that had no effect on BP . CTGF does not cause cell proliferation, instead it appears to activate caspase 3 and induce apoptosis ; however, its role in the hypertensive remodelling process has not been completely investigated.
Surprisingly, not all studies report an increase in vessel collagen with aldosterone treatment. Aldosterone-salt treatment increases carotid artery stiffness, but this increase was related to an increase in fibronectin and not collagen or elastin . It is important to note that the rats used in this study were younger than those used by most groups to produce aldosterone- or DOCA-salt-dependent hypertension models, which may have affected the remodelling process.
Studies comparing PA patients with hypertensive subjects suggest that hypertension is not the sole regulator of collagen synthesis. An assessment of collagen levels in resistance vessels from gluteal biopsies showed that essential hypertension and PA increased the total collagen content in the vessel walls and the collagen III content, in particular. However, the magnitude of the increase was significantly greater in the PA patients compared with the essential hypertensive patients , indicating a role for aldosterone in the increased collagen deposition.
MMPs (matrix metaloproteinases) digest extracellular matrix components including collagen . The gelatinases (MMP-2 and -9) may play a role in the vascular remodelling caused by a combination of VSMC migration and extracellular matrix metabolism . One study showed that MMP-2 activity increases in the media of the coronary vessels from DOCA-salt-treated rats . As is so often the case with this model of hypertension, it is impossible to assess if this is a direct effect of aldosterone or due to the malignant hypertension that develops in these rats. However, a study using cultured cardiac myocytes has shown that aldosterone increases MMP-2 and -9 activity . Interestingly, the increase in activity appears to be caused by a rapid non-genomic activation of a PKC (protein kinase C) and ROS-dependent pathway that results in the activation of MEK (MAPK/ERK kinase)/ERK. Surprisingly, aldosterone has been shown to have no effect on the activity of the MMPs in cardiac fibroblasts , suggesting that the effects of aldosterone on MMP activity and expression are cell-type-dependent. Interestingly, MMPs are also thought to regulate VSMC proliferation by causing the release or activation of proteins with proliferative properties such as β-catenin .
MR SPECIFICITY FOR ALDOSTERONE
Antagonism of the MR is beneficial in several cardiovascular pathologies even when aldosterone levels are not elevated. In recent years, this has raised the question of whether a molecule other than aldosterone is responsible for increased MR activation in these conditions. Cortisol, the predominant glucocorticoid in humans, binds the MR with the same affinity as aldosterone, and circulates at much higher concentrations than aldosterone [92,93]. However, MR specificity for aldosterone is preserved by activity of the enzyme 11β-HSD2 (type 2 isoform of 11β-hydroxysteroid dehydrogenase) (Figure 2) [94,95]. 11β-HSD2 is expressed in aldosterone target tissues , including blood vessels, and converts cortisol into its inactive form cortisone (corticosterone to 11-dehydrocorticosterone in rodents), which does not have a high affinity for the MR. This process serves to protect the MR from cortisol occupancy. Aldosterone, on the other hand, is unaffected by 11β-HSD2. Owing to the higher levels of circulating glucocorticoids, inactivation of 11β-HSD2 allows increased cortisol occupancy of the MR, reducing its activation by aldosterone. The activity of 11β-HSD2 is impaired congenitally in a condition known as the syndrome of AME (apparent mineralocorticoid excess) or by excessive glycerrhetinic acid consumption, the active ingredient in liquorice [95,97,98]. Both conditions mimic mineralocorticoid excess as indicated by sodium retention, hypokalaemia and hypertension; however, it has been reported that, in patients with AME, plasma aldosterone and renin concentrations remain low while the ratio of cortisol to cortisone metabolites is elevated .
Proposed mechanism for the activation of the MR by glucocorticoids in VSMCs
Evidence suggests that, although cortisol binds the MR, binding alone may not be sufficient to activate the receptor. NAD+ is required for the conversion of cortisol into cortisone, and therefore activity of the enzyme 11β-HSD2 is dependent on the redox status of the cell. This concept has been reviewed in detail , and a study by Ward et al.  highlights the importance of increased ROS on the ability of cortisol to go beyond simply occupying the MR to activating it. In this study, endogenous cortisol did not activate MR in porcine coronary vessels until the redox state was altered as a result of angioplasty-induced damage. Additionally, a study by Young et al.  revealed that inhibition of 11β-HSD2 with carbenoxolone in combination with 0.9% NaCl mimicked the deleterious cardiac effects of deoxycorticosterone treatment, and these effects were reversed by the MR antagonist eplerenone. These studies demonstrate that, with increases in ROS, the redox state of the cell changes, and cortisol becomes an MR agonist, mimicking the effects of aldosterone. The mechanism by which this occurs, however, is not completely understood.
Although AME is quite rare, diseases in which oxidative stress is increased, such as obesity, diabetes and hypertension, may involve an impairment in the ability of 11β-HSD2 to protect the MR from cortisol binding due to alterations in the redox status. With this in mind, it seems possible that these disease states may be examples of disproportionate MR activation by glucocorticoids rather than aldosterone. This scenario may provide an explanation for the beneficial effects of MR antagonism observed in the RALES (Randomized Aldactone Evaluation Study) and EPHESUS (EPlerenone neuroHormonal Efficacy and SUrvival Study) trials in heart failure patients possessing normal plasma aldosterone levels, but probably having elevated oxidative stress [103,104]. A recent study also demonstrated the beneficial effects of MR antagonism with eplerenone in a low aldosterone model of heart failure in rats . Despite decreased renin activity and aldosterone concentrations, Dahl salt-sensitive rats with congestive heart failure had improvements in left ventricular hypertrophy, inflammation and oxidative stress with a non-antihypertensive dose of eplerenone. This study underscores further the notion that many cardiovascular pathologies may involve disproportionate MR activation via glucocorticoids, and patients with these conditions will probably benefit from treatment with an MR antagonist. As the incidences of diabetes and obesity increase in the population, so does the number of people suffering from oxidative stress. If the pathway described above is correct, this also dramatically increases the number of patients who will suffer from MR-dependent vascular diseases.
In this review, we have presented evidence that aldosterone or MR activation increases the risk of, and the damage caused by, a stroke via alterations in the structure of the cerebral blood vessels. It appears that the interaction between the MR and the EGFR, both at the genomic and non-genomic level, is a possible candidate for intervention in this pathway. Still several questions remain, not least of these is the BP-dependency of the effects of MR activation on the vasculature. Also, the physiological relevance of the rapid effects of aldosterone and the potential that multiple forms of the MR exist need to be evaluated, as does the notion that the MR can be activated in different ways depending on the oxidative state of the cell.
apparent mineralocorticoid excess
- AT1 receptor
AngII type 1 receptor
connective tissue growth factor
epidermal growth factor
endothelial nitric oxide synthase
human umbilical vein endothelial cell
type 2 isoform of 11β-hydroxysteroid dehydrogenase
Janus kinase 2
mitogen-activated protein kinase
middle cerebral artery
murine double minute type 2
procollagen C-proteinase enhancer protein-1
reactive oxygen species
stroke-prone spontaneously hypertensive rats
vascular smooth muscle cell
This work was supported by a grant from the National Institutes of Health (HL077385 to A. M. D.).