Elsevier

Neuropharmacology

Volume 71, August 2013, Pages 154-163
Neuropharmacology

Anti-inflammatory effects of angiotensin-(1-7) in ischemic stroke

https://doi.org/10.1016/j.neuropharm.2013.03.025Get rights and content

Highlights

  • Ang-(1-7) reduces infarct & improves function 24 h after ischemic stroke in rat model.

  • Ang-(1-7) blunts stroke-induced increases in iNOS, cytokines, and CD11b/microglia.

  • Ang-(1-7) blunts LPS-induced increases in NO production in vitro, using cultured glia.

  • Ang-(1-7) receptor, Mas, is present on cortical microglia and neurons in vivo.

  • Suggests an anti-inflammatory role for Ang-(1-7)'s therapeutic effect in stroke.

Abstract

Previously we demonstrated that central administration of angiotensin-(1-7) [Ang-(1-7)] into rats elicits significant cerebroprotection against ischemic stroke elicited by endothelin-1 induced middle cerebral artery occlusion. Ang-(1-7), acting via its receptor Mas, reduced cerebral infarct size, and rats exhibited improved performance on neurological exams. These beneficial actions of Ang-(1-7) were not due to inhibition of the effects of endothelin-1 on cerebral vasoconstriction or effects on cerebral blood flow, and so we considered other potential mechanisms. Here we investigated the possibility that the Ang-(1-7)-induced cerebroprotection involves an anti-inflammatory effect, since stroke-induced cerebral damage includes an excessive intracerebral inflammatory response. Our quantitative RT-PCR analyses revealed that central Ang-(1-7) treatment attenuates the increased expression of mRNAs for inducible nitric oxide synthase (iNOS), several pro-inflammatory cytokines and cluster of differentiation molecule 11b (microglial marker) within the cerebral cortex following endothelin-1 induced stroke. Western blotting confirmed similar changes in iNOS protein expression in the cerebral cortex. In support of these observations, immunostaining revealed the presence of immunoreactive Mas on activated microglia within the cerebral cortical infarct zone, and in vitro experiments demonstrated that lipopolysaccharide-induced increases in nitric oxide production in glial cultures are attenuated by Ang-(1-7) acting via Mas. Collectively these findings demonstrate an anti-inflammatory action of Ang-(1-7) in the brain, and suggest that the cerebroprotective action of this peptide in ischemic stroke may involve effects on nitric oxide generation by microglia.

Introduction

Stroke is the fourth leading cause of death in the United States and a major cause of serious, long-term disability (Roger et al., 2012). While there have been many efforts to develop therapeutic approaches for stroke, very little progress has been made to counteract stroke damage and limit long-term disability. Mounting evidence indicates that the renin-angiotensin system (RAS) is a potential therapeutic target for ischemic stroke, as over activation of the angiotensin converting enzyme/angiotensin II/angiotensin II type 1 receptor (ACE/Ang II/AT1R) arm of the RAS is highly involved in the processes that induce cerebral damage following ischemia. Specifically, numerous studies in animal models of experimental stroke have shown that ACE inhibitors and AT1R blockers (ARBs) decrease cortical/subcortical infarct size and the ensuing neurological deficits in animal models of stroke (Groth et al., 2003; Thone-Reineke et al., 2006). Importantly, a number of human clinical trials have also indicated that ACE inhibitors and ARBs can reduce cardiovascular risk and prevent stroke (Dahlöf et al., 2002; Papademetriou et al., 2004; Reboldi et al., 2008).

While Ang II acting via AT1R activation is well known to exert deleterious actions in stroke and other cardiovascular diseases, there is accumulating evidence that the more recently discovered angiotensin converting enzyme 2/angiotensin-(1-7)/Mas (ACE2/Ang-(1-7)/Mas) axis of the RAS exerts beneficial actions in several cardiovascular diseases (Santos et al., 2008; Ferreira et al., 2010). Activating this protective arm of the RAS appears to have potential for treating hypertension, hypertension related pathology, pulmonary hypertension, myocardial infarction, and heart failure based on its ability to counteract the ACE/Ang II/AT1R axis (Castro-Chaves et al., 2010). In the brain, Ang-(1-7) is primarily generated by the action of ACE2 on Ang II, and its effects are mediated by its receptor, Mas (Santos et al., 2003). In recent studies, we demonstrated that the intracerebral damage and neurological deficits elicited by endothelin-1 (ET-1)-induced middle cerebral artery occlusion (MCAO), a model of ischemic stroke, are significantly reduced by intracerebroventricular (ICV) administration of either exogenous Ang-(1-7) or an activator of ACE2, prior to and during the stroke period (Mecca et al., 2011). These beneficial actions of Ang-(1-7) were not due to inhibition of the effects of ET-1 on cerebral vasoconstriction or effects on cerebral blood flow.

The aim of the present study was to investigate the mechanism of this Ang-(1-7) induced cerebroprotection, as understanding these processes would further support the rationale for activating the ACE2/Ang-(1-7)/Mas axis as a potential stroke therapy. Since stroke-induced cerebral damage includes an excessive intracerebral pro-inflammatory response leading to neuronal death (Jin et al., 2010; Iadecola and Anrather, 2011; Lambertsen et al., 2012), in the current study we investigated whether the cerebroprotective actions of Ang-(1-7) in ischemic stroke are associated with anti-inflammatory actions of this peptide.

Section snippets

Animals and ethical approval

For the experiments described here, we used adult male Sprague Dawley (SD) rats (250–275 g) or FVB mice (25–30 g) purchased from Charles River Farms (Wilmington, MA, USA). In addition, Sprague Dawley pups (derived from in-house breeding colony) were used to generate the cell cultures. Brains from FVB/N-Mas-deficient (Mas−/−) mice were obtained from Dr. Michael Bader (Max Delbrück Center for Molecular Medicine, Berlin, Germany) and Dr. Robson Santos (Federal University of Minas Gerais, Belo

Ang-(1-7) reduces intracerebral infarct size 24 h after ET-1-induced MCAO

Previously we demonstrated that ICV treatment with Ang-(1-7) prior to and during induction of ischemic stroke via ET-1-induced MCAO (Mecca et al., 2011) decreased the size of the intracerebral infarct and improved performance on several neurological exams, when measured 72 h after MCAO. Furthermore, in the same study we demonstrated that Ang-(1-7) did not alter blood pressure, percent change in MCA branch vessel diameter or percent change in cerebral blood flow (Mecca et al., 2011). Using the

Discussion

In this study we investigated potential mechanisms that underlie the cerebroprotective action of Ang-(1-7) in ischemic stroke. The major novel findings are: (i) Ang-(1-7) blunts the increases in the levels of iNOS, pro-inflammatory cytokines and CD11b (a marker of macrophage/microglial activation) that occur in the ipsilateral hemisphere following MCAO-induced ischemic stroke; (ii) Ang-(1-7) blunts LPS-induced increases in NO production in cultured glia (mixed culture of microglia and

Acknowledgments

This work was supported by grants from the American Heart Association Greater Southeast Affiliate (09GRNT2060421), the American Medical Association, and from the University of Florida Clinical and Translational Science Institute. Robert Regenhardt received predoctoral fellowship support from the University of Florida Multidisciplinary Training Program in Hypertension (T32 HL-083810). Adam Mecca is a NIH/NINDS, NRSA predoctoral fellow (F30 NS-060335). Support from University of Florida HHMI

References (38)

  • L.K. Becker et al.

    Immunofluorescence localization of the receptor Mas in cardiovascular-related areas of the rat brain

    Am. J. Physiol. Heart Circ. Physiol.

    (2007)
  • P. Castro-Chaves et al.

    New pathways of the renin-angiotensin system: the role of ACE2 in cardiovascular pathophysiology and therapy

    Expert Opin. Ther. Targets

    (2010)
  • J. El Khoury et al.

    Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like disease

    Nat. Med.

    (2007)
  • A.J. Ferreira et al.

    Therapeutic implications of the vasoprotective axis of the renin-angiotensin system in cardiovascular diseases

    Hypertension

    (2010)
  • P.E. Gallagher et al.

    Distinct roles for ANG II and ANG-(1-7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes

    Am. J. Physiol. Cell. Physiol.

    (2006)
  • J.F. Giani et al.

    Angiotensin-(1-7) improves cardiac remodeling and inhibits growth-promoting pathways in the heart of fructose-fed rats

    Am. J. Physiol. Heart Circ. Physiol.

    (2010)
  • J.F. Giani et al.

    Angiotensin-(1-7) reduces proteinuria and diminishes structural damage in renal tissue of stroke-prone spontaneously hypertensive rats

    Am. J. Physiol. Renal Physiol.

    (2011)
  • W. Groth et al.

    Chronic pretreatment with candesartan improves recovery from focal cerebral ischaemia in rats

    J. Hypertens.

    (2003)
  • F. Guo et al.

    Astroglia are a possible cellular substrate of angiotensin(1-7) effects in the rostral ventrolateral medulla

    Cardiovasc. Res.

    (2010)
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