Institute of Molecular Medicine, Department of Cardiovascular and Renal Physiology, University of Southern Denmark, Odense, Denmark
Correspondence: Ulrike M Steckelings, IMM–Department of Cardiovascular and Renal Physiology, University of Southern Denmark, JB Winsløws Vej 21–3, 5000 Odense C, Denmark. Email: email@example.com
In recent years it has been firmly established that apart from the classic renin–angiotensin system (RAS) comprising angiotensin (Ang) II, angiotensin converting enzyme (ACE; responsible for AngII generation) and the angiotensin AT1 receptor (AT1R), there also exist protective arms of the RAS that comprise the angiotensin AT2 receptor (AT2R), Ang-(1–7), ACE2 (mainly responsible for Ang-(1–7) synthesis) and Mas, the receptor for Ang-(1–7).
Stimulation of AT2R promotes neuronal differentiation, neurite outgrowth and axonal regeneration, which results in an acceleration of repair and improved functional outcome after injury of peripheral nerves or the spinal cord.
Stimulation of AT2R and the receptor Mas has been shown to reduce infarct size and ameliorate neurological deficits in various animal models of stroke. The underlying mechanisms of action are comprised of activation of direct neurotrophic, anti-inflammatory and anti-oxidant pathways, as well as the augmentation of cerebral blood flow.
Cognitive function is improved by AT2R stimulation due, at least in part, to increased cerebral blood flow. There is indirect evidence that Ang-(1–7) could also play a role in protection against cognitive decline, but studies confirming this have not yet been published.
In view of the data reviewed in this article, it can be assumed that the protective arms of the RAS are putative targets in the treatment of neurological diseases, which involve tissue damage or cognitive impairment.
Tropomyosin-related kinase (Trk) family of receptor tyrosine kinases A and B
The renin–angiotensin system (RAS) is critical for the maintenance of normal extracellular fluid volume and cardiovascular homeostasis. These important functions of the RAS include effects at multiple peripheral organs and actions within the areas of the brain that are involved in cardiovascular control and fluid balance. With very good reason, the RAS, when over activated, is considered to be a disease-promoting and -sustaining system within humans. Enhanced signalling of angiotensin (Ang) II via its AT1 receptor (AT1R) elicits well-established detrimental effects in cardiovascular and kidney disease and in neural pathologies such as stroke, Alzheimer's disease, Parkinson's disease, depression and bipolar disorder.[3-8] This had led to the dogma that the RAS, aside from its major physiological roles in cardiovascular regulation and fluid balance, is a harmful system. However, it is apparent that more than one of the other components of the RAS, namely angiotensin AT2 receptors (AT2R), angiotensin-(1–7) and its receptor Mas, exert significant beneficial actions in mammalian diseases, effects that are often in opposition to the harmful effects of AngII via activation of AT1R and also in opposition to the effects of cytokines or growth factors. This prompted the idea that protective arms or axes of the RAS exist, but they are still considered secondary in nature to the harmful RAS. In a way this view of the AT2R–Ang-(1–7)–Mas axes being secondary is justified, because AT1R-mediated actions seem always dominant. However, it has been shown in a multitude of animal studies that the protective RAS axes are able to ameliorate AT1R-mediated disease-promoting actions; the course of several cardiovascular, renal or neurological diseases is more severe if one or the other protective arms of the RAS axis is absent. These observations have led to the conclusion that stimulation of the protective RAS axes, and thus reinforcement of their natural ability to ameliorate the progress of various diseases, may be an interesting, novel therapeutic concept. Consequently, a series of drugs are currently in preclinical or clinical development that are aimed at increasing the synthesis of Ang-(1–7) or stimulating the AT2R or the receptor Mas. It must be pointed out that targeting the protective RAS in a pharmacological way would allow for much stronger effects than physiological stimulation of the protective RAS axes, because drugs can be dosed much higher than an endogenous substance would be increased in plasma or tissue, at least as long as side-effects or loss of specificity and selectivity of the drugs are avoided.
“AT2R and receptor Mas are neuroprotective”
In the present article we make the argument that, at least if regarded from a pharmacological point of view, the protective components of the RAS are more than secondary arms or axes of this important system. Our focus is on the nervous system, because there are many examples of how AngII–AT2R and Ang-(1–7)]–Mas can exert beneficial actions in neurological diseases.
In the following sections we review state-of-the art knowledge about the physiological role and the therapeutic potential of the protective RAS axes in various pathological neurological conditions, such as stroke, cognitive decline, neuronal injury and neurodegenerative disease.
Protective Arms of the RAS in Traumatic Neuronal Injury
One of the first descriptions of a functional effect of the AT2R was the observation that cells of neuronal origin (PC12W or NG-108 cells) treated in vitro with the AT2R agonist CGP42112A or AngII would react with differentiation resulting in the elongation of neurites.[13, 14] Subsequently, it was found that AT2R-induced neuronal differentiation and/or neurite outgrowth is mediated by complexes of AT2R-interacting protein (ATIP) with SH2 domain-containing phosphatase 1 (SHP-1), nitric oxide (NO), activation of mitogen-activated protein kinases (MAPK) and the methyl methanesulphonate sensitive 2 (MMS2) gene.[15-17] These entirely unexpected findings, which were mainly made by the Unger, Horiuchi and Gallo-Payet groups, pointed to, but did not prove, neuroregenerative properties of the AT2R. In subsequent experiments it was demonstrated that AT2R stimulation promoted an acceleration of axonal regeneration in injured peripheral and central nerves. Indeed, it was found that axonal regeneration was accelerated in the optic nerve after nerve crush. Nerve fibres stained for Growth associated protein -43, which is part of the neuronal growth cone, regularly crossed the lesion site in animals treated locally with an AngII-soaked foam, whereas in vehicle-treated animals, although an increased number of GAP-43 fibres was also detected, they rarely crossed the lesion. This effect of AngII was mediated by the AT2R because it was completely blocked by the AT2R antagonist PD123177. In a follow-up study in rats undergoing sciatic nerve injury, promotion of axonal regeneration and also myelination in response to AT2R stimulation was confirmed. Moreover, in that study it was shown, for the first time, that axonal regeneration resulted in functional improvement. The spirit of these studies from the 1980s and 1990s was picked up on very recently in a study demonstrating that pharmacological stimulation of the AT2R is able to improve neurological outcome and initiate neuroregenerative effects in one of the most devastating kinds of traumatic nerve injury, namely injury of the spinal cord. Using the non-peptide, orally active AT2R agonist compound 21 (C21; Vicore Pharma, Gothenburg, Sweden), which is a long awaited tool for AT2R research and one of the drugs in development targeting the protective RAS, the Steckelings and Unger groups demonstrated that, in mice, AT2R-induced axonal regeneration resulting in improved neurological outcome was due to an increased synthesis of brain-derived neurotrophic factor (BDNF) and of the neurotrophin receptors TrkA and TrkB.
“the AT2R promotes nerve regeneration”
To the best of our knowledge, drugs targeting the Ang-(1–7)–Mas axis have not yet been tested in models of traumatic nerve injury.
Interestingly, the initial observation that AT2R stimulation leads to the outgrowth of neurites has led to the concept that AT2R blockade may be able to prevent or reverse pathological outgrowth of neurites in dorsal root ganglia, which is part of the pathology underlying neuropathic pain. Consequently, the AT2R-antagonist PD123319, which is a standard tool in AT2R research, and some analogues of this molecule have been further developed into orally active drugs. The lead compound of this family of molecules, EMA 401, has been tested in a phase II clinical study for the treatment of postherpetic neuropathic pain and is currently being tested in a Phase II study for the treatment of chemotherapy induced neuropathic pain. PD123319 is a standard tool in AT2R research and some analogues of this molecule have been further developed into orally active drugs, the lead compound of which is EMA 401. Two very recent publications provide experimental evidence that EMA 401 and analogues indeed reduce neurite density of human dorsal root ganglia and neurite length in AngII-treated rat dorsal root ganglia and that they exert antinociceptive actions.[22, 23] However, neither of these two studies provided conclusive proof that the compounds tested are AT2R antagonists.
Until now there has been no evidence indicating that AT2R agonists or AT1R blockers (by indirect AT2R stimulation) would induce pain.
Protective Effects of the AT2R in Ischaemic Stroke
The detrimental impact of AT1R signalling in the setting of stroke has been clearly illustrated in various rodent models of hypoxia, and has been further confirmed in human clinical trials in which AT1R blockade improved outcomes. In experimental settings, AT1R blockade may exert beneficial effects during ischaemic insult due to the ‘unmasking’ of the neuroprotective AT2R.[25, 26] Indeed, the fact that infarct volume following stroke is exacerbated in AT2R-knockout mice points (indirectly) to a protective role of the AT2R in stroke. In this context, a range of experiments in which AT2R have been directly stimulated in the brain have recently been performed. In an ischaemic model of stroke in conscious spontaneously hypertensive rats (SHR), using endothelin (ET)-1 to locally occlude the middle cerebral artery (MCA), McCarthy et al. found that direct AT2R stimulation prior to stroke with the classic AT2R agonist CGP42112 administered intracerebroventricularly (i.c.v.) dose-dependently reduced the severity of neuronal injury. This protection was evident not only in the penumbral region of the stroke, but also in the core of the infarct, which has previously been resistant to other treatments. Furthermore, when treatment was delayed until 6 h after stroke induction, the same degree of neuroprotection was afforded by i.c.v. administration of CGP42112, indicating that protective pathways associated with the AT2R are still effective at reducing damage after ischaemia has already occurred. Moreover, in both the pretreatment and delayed treatment protocols, the benefit evoked by AT2R stimulation was independent of any changes in blood pressure and was evident in the absence of AT1R blockade. Thus, the AT2R is a clinically relevant therapeutic target. When considering the cellular targets contributing to the AT2R-mediated protection, CGP42112 was found to markedly increase microglial activation and reduce oxidative stress, both of which are likely to augment neuronal survival.[27, 28] Furthermore, CGP42112 reduced infarct volume and enhanced cerebral blood flow (CBF) in anaesthetized mice when administered peripherally during reperfusion, indicating that there may also be a vascular component to the treatment effect. Similarly, although not directly assessing brain injury, Gelosa et al. found that oral administration of C21 delayed spontaneous brain abnormalities and extended life expectancy in stroke-prone SHR fed a high-salt diet. Although previous studies have indicated that C21 is unable to gain access to the brain via the blood–brain barrier, it is well established that during stroke there is a disruption of the blood–brain barrier allowing usually impermeable compounds to diffuse into the central nervous system (CNS), which would account for the preservation of neuronal integrity following the systemic administration of C21. Although the exact mechanisms by which the AT2R is protecting the brain from damage remain to be fully elucidated, it is likely that a number of signalling cascades contribute to the treatment effect, including activation of direct neurotrophic, anti-inflammatory and anti-oxidant pathways, as well as the augmentation of CBF.
“stroke outcome is improved by AT2R agonists”
The influence of the peripheral immune system in centrally mediated events following stroke has recently been linked with the AT2R. Exogenously administered haematopoietic stem cells have been shown to protect against ischaemic damage during the acute phase of stroke and to restore injured neurons in the later phase of stroke recovery. However, these protective effects were absent when the stem cells originated from a mouse lacking the AT2R, indicating that this receptor is essential for the prevention of neuronal injury. Similarly, deletion of the AT2R from bone marrow stromal cells severely blunted the protection elicited by the cells during occlusion of the MCA. Specifically, wild-type bone marrow stromal cells significantly improved neurological function, reduced infarct volume and attenuated the inflammatory response as a result of hypoxia, but these effects were completely absent when the bone marrow stromal cells were AT2R deficient.
Apart from the effects in acute stroke described above, the AT2R seems to be fundamentally involved in the protective effects of preconditioning against ischaemic brain injury in newborn rats, which involves vascular endothelial growth factor-driven maintenance of microvessels.
As reviewed above, there are now several lines of evidence that emphasise the importance of both the central and systemic AT2R population in improving stroke outcome, highlighting the AT2R as an exciting prospect for future research, either alone or in combination with other stroke therapies.
Protective Effects of the Angiotensin-Converting Enzyme 2–Ang-(1–7)–Mas Axis in Ischaemic Stroke
It is now established that that Ang-(1–7) acting via its receptor Mas exerts beneficial actions in various cardiovascular, renal and metabolic diseases,[10, 11, 37] effects that are often in opposition to the actions of AngII acting via the AT1R. In the brain, Ang-(1–7) is primarily formed via the degradation of AngII by angiotensin-converting enzyme (ACE) 2, and immunostaining has revealed that Mas are widely distributed in the cerebrum, including the cerebral cortex and basal ganglia. Taking this into account, the possibility that Ang-(1–7) may have a beneficial effect against the cerebral damage and deficits produced by cerebral ischaemic stroke was investigated. This idea was strengthened by the demonstration that ACE inhibitors or angiotensin receptor blockers (ARB), drugs that are effective against experimental ischaemic stroke and that have beneficial effects in stroke patients, increase ACE2 and Ang-(1–7) levels in plasma and various tissues.[40, 41]
The Sumners group demonstrated recently that i.c.v. infusion of Ang-(1–7) prior to and during ischaemic stroke elicited by ET-1-induced MCA occlusion (MCAO) produced a significant reduction in the resulting intracerebral (cortical and striatal) infarct. This decrease in intracerebral infarct size was associated with increased neuron survival in the cortex and striatum, as well as decreases in the behavioural deficits caused by MCAO, as evidenced by a battery of neurological tests. Furthermore, i.c.v. administration of the novel ACE2 activator diminazine aceturate under similar conditions as those used for Ang-(1–7) effectively decreased the intracerebral infarct and behavioural deficits resulting from ET-1-induced MCAO. These protective actions of Ang-(1–7) and diminazine aceturate were abolished by coadministration of the receptor Mas blocker A-779.
An obvious candidate for the mechanism underlying the cerebroprotective action of Ang-(1–7) is a direct vascular action, because acute peripheral infusion of low doses of this peptide increases CBF and decreases vascular resistance in the brain. Furthermore, Ang-(1–7) can elicit relaxation of canine MCA. However, the observed beneficial actions of Ang-(1–7) and diminazine aceturate during ischaemic stroke were due neither to inhibition of the effects of ET-1 on MCA vasoconstriction nor effects on CBF, and so anti-inflammatory mechanisms were considered.
The expression of many genes, including those for proinflammatory cytokines (PIC), chemokines and some NO synthase (NOS) isozymes, is increased in the cerebral cortical infarct zone following ischaemic stroke and contributes to neurotoxicity. Clues concerning the mechanism underlying the protective action of Ang-(1–7) in ischaemic stroke have come from quantitative real-time polymerase chain reaction analysis of the expression of inflammatory genes within the cortex and striatum after MCAO. These analyses revealed that the marked increase in inducible NOS (NOS2) gene expression that occurs within the cerebrum ipsilateral to the stroke is greatly reduced by Ang-(1–7). Furthermore, results also indicate that the significant increases in the expression of mRNAs for interleukin (IL)-1β (IL1B) and IL-6 (IL6), both of which are PIC, and for CD11b (ITGAM), a marker of microglial activation, that occur in the cerebrum after stroke are all significantly attenuated by Ang-(1–7). These data suggest that an anti-inflammatory action of Ang-(1–7) may underlie its protective effect in ischaemic stroke. This view is reinforced by the finding that microglia within the cerebrum express immunoreactive Mas and by a recent study indicating that the neuroprotective action of Ang-(1–7) involves Mas-mediated suppression of the nuclear factor (NF)-κB pathway.
“ACE2/Ang-(1-7)/Mas protects against stroke”
In summary, the data so far demonstrate, in principle, that the ACE2–Ang-(1–7)/Mas axis can exert cerebroprotective actions in ischaemic stroke. It is now essential to demonstrate that Ang-(1–7) is as effective in reducing cerebral damage and behavioural deficits when administered after an ischaemic insult.
Role of AT2R Stimulation in Cognitive Function
In the brain, the AT2R is reported to be expressed in areas related to learning and control of motor activity, as well as in the vasculature.[9, 48, 49] Previous reports suggest that AT2R stimulation is involved in axonal regeneration and memory and behaviour.[50, 51] As mentioned above, it was demonstrated that stimulation of the AT2R may promote cell differentiation and regeneration in neuronal tissue and that AT2R stimulation supports neuronal survival and neurite outgrowth in response to ischaemia-induced neuronal injury. Moreover, the Gallo-Payet group reported that AngII induces neural differentiation and neurite outgrowth via mitogen-activated protein kinase or NO through AT2R activation and that the AT2R is involved in cerebellar development.[16, 17, 52] The Horiuchi group further demonstrated that AT2R (AGTR2) mRNA expression was significantly increased in the ischaemic side of the brain after MCAO and that the passive avoidance rate as an indicator of cognitive function was significantly impaired in AT2R-null compared with wild-type mice. Worse performance of AT2R-deficient mice in a spatial memory task and in a one-way active avoidance task was also reported by Maul et al. Treatment with the ARB valsartan prevented cognitive decline in wild-type mice, but this effect was weaker in AT2R-null mice, suggesting that AT2R stimulation during ARB treatment is important. Similar results were found in a model of scopolamine-induced memory impairment, in which the beneficial effect of the ARB candesartan was blunted by pharmacological blockade of the AT2R with PD123319. Moreover, it was observed that the ischaemic area was significantly larger in AT2R-deficient mice after MCAO, with a decrease in CBF and an increase in superoxide production. The ARB valsartan, at a non-hypotensive dose, significantly reduced the ischaemic area, neurological deficit and decrease in CBF, as well as superoxide production and NADPH oxidase activity, in wild-type mice with MCAO. In comparison, these inhibitory actions of the ARB were weaker in AT2R-deficient mice, pointing to a significant contribution of ‘indirect’ AT2R stimulation by AngII, levels of which had been elevated in response to ARB treatment. Li et al. and Faure et al. made similar observations in rats, in which a protective effect of pretreatment with candesartan or irbesartan on stroke outcome was lost when the AT2R and/or AT4 receptor was blocked.
“AT2R stimulation improves cognitive function”
Horiuchi and colleagues observed that direct stimulation of the AT2R by C21 enhanced cognitive function with an increase in CBF and hippocampal field excitatory post-synaptic potential, and that C21 promoted neurite outgrowth of cultured mouse hippocampal neurons. The pathological relevance of direct AT2R stimulation with C21 in spatial learning using an Alzheimer's disease mouse model with i.c.v. injection of β-amyloid was also investigated, with findings showing that C21 treatment prevented cognitive decline in this model.
Peroxisome proliferator-activated receptor (PPAR) γ activation in the brain has been reported to prevent brain damage via anti-inflammatory effects in cells such as neurons, endothelial cells, astrocytes and microglia, as well as having anti-oxidative actions and improving endothelial function.[61, 62] Moreover, β-amyloid protein clearance and neural stem cell proliferation have been reported to be enhanced by PPARγ activation.[61, 63] It has been reported that AngII induces PPARγ activation in PC12W cells via AT2R activation, suggesting that possible cross-talk between AT2R activation and PPARγ stimulation in the brain could contribute to more protective effects against ischaemic brain damage and cognitive impairment. It was recently demonstrated that direct AT2R stimulation by C21 accompanied by PPARγ activation ameliorated insulin resistance in Type 2 diabetic mice due, at least in part, to improvement of adipocyte dysfunction and protection of pancreatic β-cells. However, the possible beneficial roles of AT2R activation with PPARγ stimulation in improving cognitive function have to be investigated and clarified in more detail.
Possible Roles of Ang-(1–7) in Cognitive Function
Angiotensin-(1–7) has been shown to occur endogenously within the brain, and studies have suggested that the ACE2–Ang-(1–7)–Mas axis plays a role in the neural control of blood pressure and in regulating CBF.[37, 43] Furthermore, other studies have indicated that Ang-(1–7) increases bradykinin levels, NO release and endothelial NOS expression in the brain, the latter findings suggesting a further mechanism for the protective effects of this peptide against cerebrovascular disease.[37, 43, 66, 67]
Aside from these vascular actions, it has been reported that Ang-(1–7) enhances long-term potentiation in the CA1 region of the hippocampus in mice, implying its distinct function in mechanisms of learning and memory. The hippocampus is one of the regions that exhibits high levels of receptor Mas expression and Lazaroni et al. suggested that the functionality of the Ang-(1–7)–Mas axis is essential for normal object recognition memory processing. It is also possible that, due to the above noted cerebrovascular effects, the ACE2–Ang-(1–7)–Mas axis may protect against cognitive decline, but this is an area that remains to be investigated.
Protective Axes of the RAS in CNS Inflammation
There is evidence that the ‘classical’ ACE–AngII–AT1R axis is involved in the pathogenesis of inflammatory diseases of the CNS, such as multiple sclerosis (MS). In the September 2009 issue of the Proceedings of the National Academy of Sciences USA, Platten et al. and Stegbauer et al.[70, 71] reported in two independent studies that the RAS is activated in lesions of the brain and spinal cord in animals with experimental autoimmune encephalomyelitis, a model of MS. Moreover, they both showed that inhibition of the ACE–AngII–AT1R axis by renin inhibitors, ACE inhibitors or AT1R antagonists ameliorated neurological deficits, and even attenuated the autoimmune response.
Measurement of components of the protective arms of the RAS in the cerebrospinal fluid of patients with MS revealed that the imbalance of the RAS in this disease seems not only to involve the classical, but also the protective RAS because levels of ACE were found to be increased, whereas levels of ACE2 were decreased.
For several reasons it can be hypothesized that stimulation of the protective RAS may have beneficial therapeutic effects in MS: (i) stimulation of the protective RAS would counterbalance the disturbance of ‘RAS homeostasis’ in MS by opposing the overactivated classical RAS and by activating the downregulated protective RAS; (ii) the anti-inflammatory features of the protective arms of the RAS could diminish the inflammatory response, including T cell and microglial activation; and (iii) because data indicate that the AT2R can accelerate remyelination, this may also contribute to an improvement of neuronal signal transmission and function.
“the protective RAS attenuates CNS inflammation”
The therapeutic potential of the protective arms of the RAS has not been conclusively tested in models of MS, but preliminary data from the Steckelings and Unger groups suggest that stimulation of the AT2R has a positive influence on disease progression.
In general, several studies have shown that both the AT2R and receptor Mas mediate anti-inflammatory effects in the CNS, which may influence the course of various diseases in a beneficial way. For example, as reviewed above, stimulation of both the AT2R and receptor Mas has been shown to attenuate inflammation related to ischaemic tissue injury in stroke.[27, 39] Furthermore, reduced central inflammation may contribute to brain modulation of blood pressure, as has been suggested in a study in which overexpression of ACE2 in the hypothalamic paraventricular nucleus lowered AngII-induced hypertension, coinciding with a reduction in local expression of proinflammatory cytokines.
Important anti-inflammatory mechanisms of AT2R and receptor Mas stimulation seem to be inhibition of NF-kB activity[47, 75] and a reduction in oxidative stress.[27, 76] Whether these mechanisms play a role in further diseases or conditions involving CNS inflammation remains to be explored in future studies.
The studies reviewed in this article that tested the effects of stimulation of the AT2R or the receptor Mas in models of neurological disease revealed that both therapeutic approaches have an ameliorating effect on pathological conditions of the CNS. Stimulation of either receptor seems to act partly by direct, neuroprotective effects, partly by anti-inflammatory effects and partly by improving the cerebral circulation (for a summary, see Fig. 1). A further possibility that should be considered is that activation of AT2R or Mas positively influences the neurogenic processes that occur following CNS injury, such as neural progenitor cell proliferation and migration. However, at this time there is no evidence that Mas or AT2R function in this regard. It is clear that whenever a receptor Mas agonist and an AT2R agonist have been tested in the same disease model, the similarity of functional and molecular effects is striking. Therefore, it is tempting to speculate that both receptors may share downstream signalling cascades, possibly as a result of dimerization. This hypothesis is currently being investigated.
Encouraging data from receptor Mas or AT2R stimulation in animal models of neurological and other diseases has promoted the development of drugs that directly or indirectly stimulate AT2R or Mas. Such developments comprise Ang-(1–7) analogues or formulations that increase the metabolic stability of Ang-(1–7), receptor Mas agonists that are structurally unrelated to Ang-(1–7), activators of ACE2 and peptide or non-peptide AT2R agonists.
Although the translation of treatment strategies that were successful in preclinical models of CNS diseases into the clinical situation has turned out to be difficult, and in most cases unsuccessful, it is encouraging that both Mas and AT2R are able to positively influence the course of neurological (and other) diseases not by a single mechanism, but rather by a combination of mechanisms. Nonetheless, only clinical studies will ultimately indicate whether novel drugs targeting the protective arms of the RAS will be effective therapeutics in neurological diseases. Because some of these drugs have already successfully passed Phase II clinical studies (although not for neurological indications), an answer to the question of effectiveness likely will not be long in coming.
“the protective RAS: a drug target in neurology?”
The authors' work reported herein was partially supported by grants from the Ministry of Education, Science, Sports and Culture of Japan to MH, the American Heart Association Greater South-east Grant (09GRNT2060421) to CS and the National Health and Medical Research Council of Australia (APP1007986) to REW.
U.M.Steckelings has received modest research support from Vicore Pharma.