The renin-angiotensin system (RAS) peptides play an important role in inflammation. Resolution of inflammation contributes to restore tissue homeostasis, and it is characterized by neutrophil apoptosis and their subsequent removal by macrophages, which are remarkable plastic cells involved in the pathophysiology of diverse inflammatory diseases. However, the effects of RAS peptides on different macrophage phenotypes are still emerging. Here, we evaluated the effects of angiotensin-(1-7) (Ang-(1-7)) and the most novel RAS peptide, alamandine, on resting (M0), proinflammatory M(LPS+IFN-γ), and anti-inflammatory M(IL-4) macrophage phenotypes in vitro, as well as on specific immune cell populations and macrophage subsets into the pleural cavity of LPS-induced pleurisy in mice. Our results showed that Ang-(1-7) and alamandine, through Mas and MrgD receptors, respectively, do not affect M0 macrophages but reduce the proinflammatory TNF-α, CCL2, and IL-1β transcript expression levels in LPS+IFN-γ-stimulated macrophages. Therapeutic administration of these peptides in LPS-induced inflammation in mice decreased the number of neutrophils and M1 (F4/80lowGr1+CD11bmed) macrophage frequency without affecting the other investigated macrophage subsets. Our data suggested that both Ang-(1-7) and alamandine, through their respective receptors Mas and MrgD, promote an anti-inflammatory reprogramming of M(LPS+IFN-γ)/M1 macrophages under inflammatory circumstances and potentiate the reprogramming induced by IL-4. In conclusion, our work sheds light on the emerging proresolving properties of Ang-(1-7) and alamandine, opening new avenues for the treatment of inflammatory diseases.

Aims: The renin–angiotensin system (RAS) plays an important role in the pathophysiology of vascular diseases, especially as a mediator of inflammation and tissue remodelling. Alamandine (Ala1-angiotensin-(1-7)) is a new biologically active peptide from the RAS, interacting with Mas-related G-protein-coupled receptor member D. Although a growing number of studies reveal the cardioprotective effects of alamandine, there is a paucity of data on its participation in vascular remodelling associated events. In the present study, we investigated the effects of alamandine on ascending aorta remodelling after transverse aortic constriction (TAC) in mice. Methods and results: C57BL/6J male mice were divided into the following groups: Sham (sham-operated), TAC (operated) and TAC+ALA (operated and treated with alamandine-HPβCD (2-Hydroxypropyl-β-cyclodextrin), 30 μg/kg/day, by gavage). Oral administration of alamandine for 14 days attenuated arterial remodelling by decreasing ascending aorta media layer thickness and the cells density in the adventitia induced by TAC. Alamandine administration attenuated ascending aorta fibrosis induced by TAC, through a reduction in the following parameters; total collagen deposition, expression collagen III and transforming growth factor-β (TGF-β) transcripts, matrix metalloproteinases (MMPs) activity and vascular expression of MMP-2. Importantly, alamandine decreased vascular expression of proinflammatory genes as CCL2, tumour necrosis factor α (TNF-α) and interleukin-1β (IL-1β), and was able to increase expression of MRC1 and FIZZ1, pro-resolution markers, after TAC surgery. Conclusion: Alamandine treatment attenuates vascular remodelling after TAC, at least in part, through anti-fibrotic and anti-inflammatory effects. Hence, this work opens new avenues for the use of this heptapeptide also as a therapeutic target for vascular disease.

Objective: Alamandine is the newest identified peptide of the renin-angiotensin system (RAS) and has protective effects in the cardiovascular system. While it is well known the involvement of classical RAS components in the genesis and progression of cardiac remodelling, less is known about the effects of alamandine. Therefore, in the present study, we investigated the effects of alamandine on cardiac remodelling induced by transverse aortic constriction (TAC) in mice. Methods and results: Male mice (C57BL/6), 10-12 weeks age, were divided in three groups: SHAM, TAC and TAC+ALA (30 µg/kg/day alamandine, for 14 days). The TAC surgery was performed under ketamine and xylazine anesthesia. At the end of treatment, the animals were submitted to echocardiographic examination and subsequently euthanized for tissue collection. TAC induced myocyte hypertrophy, collagen deposition and, the expression of MMP-2 and TGF-b in the left ventricle. These markers of cardiac remodelling were reduced by oral treatment with alamandine. Western blotting analysis showed that alamandine prevents the increase in ERK1/2 phosphorylation, and reverts the decrease in AMPKa phosphorylation induced by TAC. While both TAC and TAC+ALA increased SERCA2 expression, the phosphorylation of phospholambam in Thr17 residue was increased solely in alamandine treated group. The echocardiographic data showed that there are no functional or morphological alterations after two weeks of TAC. Conclusions: Alamandine treatment prevents myocyte hypertrophy and cardiac fibrosis induced by TAC. Our results reinforce the cardioprotective role of alamandine and highlight its therapeutic potential for treating heart diseases related to pressure overload conditions.

Subcutaneous implantation of synthetic materials and biomedical devices often induces abnormal tissue healing - the foreign body reaction-which impairs their function. In particular, Interferon-γ (IFN-γ) is a critical endogenous mediator of inflammation and plays a key role in a wide variety of biological responses including tissue healing. However, the contribution of endogenous IFN-γ on different features of the foreign body response induced by synthetic implants regarding neovascularization, inflammation, and fibrogenesis is not well known. Here, we evaluated inflammatory angiogenesis and fibrogenesis induced by implantation of polyether-polyurethane sponges in mice targeted disrupted of the interferon-γ gene (IFN-γ ) and wild-type (WT). The hemoglobin content, the number of vessels, and blood flow (evaluated by LDPI-laser Doppler perfusion imaging) were decreased in the implants from IFN-γ as compared to WT mice. Likewise, neutrophils and macrophages accumulation (MPO and NAG activities, respectively) was decreased in IFN-γ implants. Interestingly, while the local content of VEGF, TNF-α, CXCL-1/KC, as measured by ELISA, and iNOS expression, as measured by qPCR, were significantly reduced, the content of IL-10 was greatly increased in the implants from IFN-γ mice as compared to WT mice. No alterations were observed in CCL-2/MCP-1 levels. Lastly, the collagen deposition, assessed by Picro-Sirius red-stained histological sections, was also reduced in IFN-γ implants. Altogether, these data suggest that IFN-γ activity contributes to inflammatory angiogenesis and fibrogenesis in synthetic implants and that lack of IFN-γ expression attenuates foreign body reaction to implants in mice. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2243-2250, 2018.