Therapeutic potential of the renin angiotensin system in ischaemic stroke

In the circulation, a drop in blood pressure (systemic hypotension) and/or blood volume results in juxtaglomerular cells within the kidneys to release renin (protease) whereas increased blood pressure (hypertension) inhibits renin release under normal circumstances. Circulating angiotensinogen is hydrolysed by renin to produce Angiotensin I (Ang I), which is then further converted by angiotensin converting enzyme (ACE) to generate biological active octapeptide, Ang II (Fig. 1). Ang II, a potent vasoconstrictor, acts by stimulating Ang II type 1 and type 2 receptors (AT₁R and AT₂R) [10]. Ang II exhibits a higher affinity to the widely expressed AT₁R whereby it exerts its main physiological effects by constricting blood vessels, increasing BP, and stimulating aldosterone release from adrenal glands, promoting water and salt reabsorption in the kidneys, thus, raising blood volume levels [10]. In the last decade, an ‘alternative axis’ has been identified involving the monocarboxypeptidase, ACE2, the biologically active peptide Ang-(1–7) and its G-protein coupled receptor, Mas (MasR). Ang-(1–7) is formed by the direct actions of ACE2 on Ang II or via ACE2 induced cleavage of Ang I, generating the nine amino acid peptide Ang-(1–9), which is further converted to Ang (1–7) by ACE or peptidases such as neprilysin (NEP) [8].

All the components of the ‘classical axis’ (angiotensinogen, renin, ACE, Ang II) have been identified within the brain parenchyma (see reviews [8, 11]). In addition, there is evidence that the ‘alternative axis’ is locally produced within the brain as ACE2 has been localised in neurons [12], astrocytes [13] and within the cerebrovasculature [14]. Similarly, the receptor subtypes responsible for mediating the functional effects of RAS peptides are expressed in neuronal and glial cells throughout the brain. For example, AT₁ and AT₂ receptors have been shown to be present in dopaminergic neurons, astrocytes and microglia from both human and primate brain tissue [15] and MasR shown to be expressed in neurons, astrocytes, microglia and cerebral endothelial cells in rodents [16, 17].

Over-activation of the ACE/Ang II/AT₁R axis is thought to contribute to the pathogenesis of AIS through its vasoconstrictor effects on cerebral vessels as well as its pro-inflammatory, pro-fibrotic and increased oxidative stress effects in the parenchyma [8]. For instance, in the brain, AT₁R knockout (KO) mice subjected to permanent middle cerebral artery occlusion (pMCAO) exhibit a larger metabolic penumbra volume (cerebral protein synthesis and ATP mismatch) and higher CBF within the core and penumbra when compared to wild type (WT) mice whereas mice over-expressing human renin and angiotensinogen genes have larger infarcts [18, 19]. Furthermore, Ang II has been shown to enhance the contractile response in isolated middle cerebral arteries (MCA) following MCAO via the AT₁R [20], therefore, worsening cerebral perfusion following ischaemia.

Interestingly, emerging evidence identifies the ‘alternative axis’ as an endogenous protective system, which acts to counteract the effects of the ‘classical axis’ by mediating vasodilation and anti-inflammatory, anti-oxidant and anti-apoptotic effects via MasR and AT₂R activation [21, 22]. Following focal cerebral ischaemia in the rat, AT₂Rs have been shown to be upregulated in the peri-infarct region of the cortex, and also within the selectively vulnerable regions of the cortex and hippocampus following global cerebral ischaemia [23, 24]. AT₂R KO mice exhibit less ischaemic damage compared to WT control animals following MCAO [25] suggesting a protective role for this receptor in the setting of cerebral ischaemia. In support of a role for this pathway following acute ischaemic stroke the ACE2/Ang-(1–7)/Mas axis has been shown to be upregulated. Brain expression (mRNA and protein levels) of both ACE2 and MasR are upregulated from as early as 6 h following permanent MCAO in the rat, peaking at 24 h post MCAO. This was associated with an concomitant increase in both circulating serum and cerebral Ang-(1–7) levels [16]. This increase occurs during the first critical hours after stroke when ischaemic damage and loss of potentially salvageable penumbra is taking place, suggesting a potential role for this pathway in the pathogenesis. Increasing exogenous Ang-(1–7) levels in the brain by central infusion prior to stroke has been shown to decrease infarct volume in rats [26], providing evidence of a protective role for this pathway post-stroke.

The current evidence suggests that there is an imbalance in the RAS following stroke with an enhanced activation of the ACE/Ang II/AT₁R pathway and that targeting the counter-regulatory ACE2/Ang-(1–7)/Mas axis may provide protection. This imbalance in the RAS may be exacerbated in the presence of known stroke risk factors such as gender and hypertension and therefore provide a potential therapeutic target in a subset of patients.

Men have a higher incidence of stroke, however, after the menopause the incidence of stroke in women rapidly increases and this is co-incident with the decrease in female sex hormones [27]. In addition, to sex differences in stroke incidence, it is also well established in experimental stroke studies that female animals have less ischaemic injury than their male counterparts and that this protection is lost after ovariectomy [28, 29]. Furthermore, cells exhibit sex specific differences in the specific mechanisms of cell death following cerebral ischaemia [30] therefore it is not unlikely that sex differences will exist in the RAS in the brain and elsewhere. It is widely accepted that the RAS is influenced by sex hormones with males and females exhibiting differential responses to stimulation and inhibition of the RAS [31]. Differences in vascular reactivity to Ang II and receptor expression have been shown to be influenced by sex hormones, with testosterone stimulating the ‘classical axis’ and oestrogen lowering the AT₁R:AT₂R ratio, thereby enhancing vasodilatation [32‐34]. Interestingly, in experimental animal models, female rats exhibit enhanced activity of the ACE2/Ang-(1–7)/Mas axis compared to males [31], showing increased renal Ang-(1–7) levels [35]. These findings may translate to humans, where normotensive healthy adult females have been shown to have higher plasma levels of Ang-(1–7) than their male counterparts [36]. These combined findings suggest that oestrogen may confer some degree of protection against stroke in premenopausal women by promoting increased activation of the ACE2/Ang-(1–7)/Mas axis and therefore, the loss of oestrogen associated with menopause may contribute to increased stroke risk through a loss of this enhanced protective pathway. In terms of brain expression, AT₁R expression has been shown to be lowered in rats treated with oestrogen plus ovariectomy compared to ovariectomy alone. In contrast, oestrogen treatment in ovariectomised rats resulted in an increased expression of the AT₂R compared to ovariectomised rats [37]. Similarly, mRNA expression of the AT₁R and ACE are increased in the brains of hypertensive ovariectomised rats compared to intact females [38].

Hypertension is the single most important modifiable risk factor for the development of stroke, clinical outcome is poorer in patients with hypertension during acute stroke and in experimental animal models of stroke, ischaemic damage is significantly increased in hypertensive animals [39, 40]. Dysregulation of the RAS has been implicated in the development of hypertension, where hyperactivity of Ang II and other RAS components lead to enhanced oxidative stress and inflammation. Preclinical models of genetic hypertension have demonstrated increased AT₁R expression in the vasculature of spontaneously hypertensive rats (SHR) compared to age-matched normotensive controls [41]. In addition to hyperactivity of the ‘classical axis’, dampening of the protective counter-regulatory axis is also evident, where hypertensive rat strains exhibit decreased ACE2 mRNA and protein expression compared to normotensive controls [42]. Chronic treatment of diabetic SHR rats with either an AT₁R blocker (olmesartan) or ACE inhibitor (enalapril) reverses the microcirculatory changes that occur in pial vessels (functional and structural rarefaction) of the brain resulting in an improved cerebral perfusion and reduced cerebral oxidative stress [43]. Candesartan treatment in salt loaded SHRSP rats was shown to increase endothelial cell progenitor (EPC) colony number and reduce oxidative stress levels in mononuclear cells. This study suggests that ARB treatment may also act to improve endothelial cell function and angiogenesis in the presence of hypertension [44]. These results demonstrate the influence of the ACE/Ang II/AT1R pathway on remodelling of the cerebral microvasculature and suggest that overactivation of this pathway may contribute to the pathology.

Therefore, manipulation of the RAS towards the protective ACE2/Ang-(1–7)/MasR pathway in the presence of co-morbidities may shift the balance to prevent the exacerbation of ischaemic damage following AIS.

In clinical trials, the effectiveness of ARB treatment in preventing vascular events and mortality following AIS is under debate. The LIFE trial compared losartan (AT₁R antagonist) and atenolol (selective β1 receptor blocker) treatment for preventing cerebral ischaemic events in patients with a clinical history of hypertension and left ventricular hypertrophy. After a follow up time of 4.8 years, the authors demonstrated that the use of losartan was associated with a decrease in the frequency of stroke [80]. Moreover the ACCESS trial was designed to assess the efficacy of a modest blood pressure reduction acutely after stroke (day 1 post stroke for 7 days) with patients receiving either candesartan cilexetil (AT₁R antagonist) or placebo treatment. Although the trial was stopped early it did demonstrate a reduced number of vascular events and decreased mortality in the candesartan treatment group [81]. In following years, the MOSES study investigated the effects of eprosartan or nitrendipine (calcium channel blocker) in hypertensive patients who had a cerebrovascular event within the last 24 months prior to recruitment. After 2.5 years follow up, it was identified that eprosartan group showed significantly less cases of cardiovascular and cerebrovascular events [82]. Furthermore, telmisartan, as an alternative therapy for cardiovascular disease patients who are intolerant to ACE inhibitors, was shown to induce a modest reduction in stroke incidence [83].

Despite the benefits of AT₁R antagonism observed, other studies showed contradictory effects. For instance, a study assessing whether blood pressure lowering with telmisartan treatment in ischaemic stroke patients affected the risk of recurrent cerebral events concluded after a 2.5 year follow up that telmisartan did not affect the risk of recurrent stroke nor other cardiovascular events [84]. More recently, the SCAST clinical trial investigated whether blood pressure lowering with candesartan in acute stroke patients is beneficial. After 6 months follow up, no beneficial effect of blood pressure lowering with candesartan was observed with no difference in the occurrence of vascular events. Plus, for functional outcome there was a less favourable modified Rankin score in the candesartan group albeit this was not statistically significant [85]. The results from clinical trials to date have primarily investigated the influence of modulation of the RAS in terms of stroke incidence or blood pressure lowering strategies following stroke however, the effect of RAS modulation acutely after ischaemic stroke in terms of outcome (i.e. penumbral salvage, lesion volume) has yet to be fully investigated.