Neutrophil production and traffic in the body compartments is finely controlled, and the strong evidences support the role of CXCL12/CXCR4 pathway on neutrophil trafficking to and from the bone marrow (BM). We recently showed that the glucocorticoid-regulated protein, Annexin A1 (AnxA1) modulates neutrophil homeostasis and here we address the effects of AnxA1 on steady-state neutrophil maturation and trafficking. For this purpose, AnxA1(-/-) and Balb/C wild-type mice (WT) were donors of BM granulocytes and mesenchymal stem cells and blood neutrophils. In vivo treatments with the pharmacological AnxA1 mimetic peptide (Ac2-26) or the formyl peptide receptor (FPR) antagonist (Boc-2) were used to elucidate the pathway of AnxA1 action, and with the cytosolic glucocorticoid antagonist receptor RU 38486. Accelerated maturation of BM granulocytes was detected in AnxA1(-/-) and Boc2-treated WT mice, and was reversed by treatment with Ac2-26 in AnxA1(-/-) mice. AnxA1 and FPR2 were constitutively expressed in bone marrow granulocytes, and their expressions were reduced by treatment with RU38486. Higher numbers of CXCR4(+) neutrophils were detected in the circulation of AnxA1(-/-) or Boc2-treated WT mice, and values were rescued in Ac2-26-treated AnxA1(-/-) mice. Although circulating neutrophils of AnxA1(-/-) animals were CXCR4(+) , they presented reduced CXCL12-induced chemotaxis. Moreover, levels of CXCL12 were reduced in the bone marrow perfusate and in the mesenchymal stem cell supernatant from AnxA1(-/-) mice, and in vivo and in vitro CXCL12 expression was re-established after Ac2-26 treatment. Collectively, these data highlight AnxA1 as a novel determinant of neutrophil maturation and the mechanisms behind blood neutrophil homing to BM via the CXCL12/CXCR4 pathway. J. Cell. Physiol. 231: 2418-2427, 2016. © 2016 Wiley Periodicals, Inc.

Atherosclerosis can be originated from the accumulation of modified cholesterol-rich lipoproteins in the arterial wall. The electronegative LDL, LDL(-), plays an important role in the pathogenesis of atherosclerosis once this cholesterol-rich lipoprotein can be internalized by macrophages, contributing to the formation of foam cells, and provoking an immune-inflammatory response. Herein, we engineered a nanoformulation containing highly pure surface-functionalized nanocapsules using a single-chain fragment variable (scFv) reactive to LDL(-) as a ligand and assessed whether it can affect the LDL(-) uptake by primary macrophages and the progression of atherosclerotic lesions in Ldlr−/− mice. The engineered and optimized scFv-anti-LDL(-)-MCMN-Zn nanoformulation is internalized by human and murine macrophages in vitro by different endocytosis mechanisms. Moreover, macrophages exhibited lower LDL(-) uptake and reduced mRNA and protein levels of IL1B and MCP1 induced by LDL(-) when treated with this new nanoformulation. In a mouse model of atherosclerosis employing Ldlr−/− mice, intravenous administration of scFv-anti-LDL(-)-MCMN-Zn nanoformulation inhibited atherosclerosis progression without affecting vascular permeability or inducing leukocytes-endothelium interactions. Together, these findings suggest that a scFv-anti-LDL(-)-MCMN-Zn nanoformulation holds promise to be used in future preventive and therapeutic strategies for atherosclerosis.

Agradeço a Deus pela vida. Aos meus pais, por permitirem que eu chagasse mais longe, meus desesperos e desafios foram superados por amor a vocês. Ao meu noivo, Ricardo Cestaro Chiqueto, pela paciência, amor e compreensão. O seu companheirismo, perdendo alguns finais de semana comigo para conclusão desse trabalho, o seu incentivo e as infinitas discussões científicas, faz deste trabalho, de algum modo, também ser seu. A minha orientadora, Sandra Helena Poliselli Farsky, pela dedicação e permitir fazer parte da sua equipe. Ao Prof. Dr. Raul Cavalcante Maranhão, coordenador do Laboratório de Lipídes do Instituto do Coração-InCor-e professor titular na Universidade de São Paulo, por fornecer as nanoemulsões. A todos que passaram pelo Laboratório de Toxicologia Experimental por compartilhar dos conhecimentos, das alegrias, das tristezas, das angústias, das frustrações e aprendizado, vocês fizeram parte desse processo de evolução. Natalia Rubio, amiga querida, tornou o caminho árduo mais suave. Muito obrigada por nunca se esquecer de mim. A todos os técnicos e funcionários do Departamento de Analises Clínicas e Toxicológicas, especialmente a Paula por facilitar a minha vida no laboratório, Samantha por estar atenta aos prazos e a D. Luzia, pelo café, por ensinar respeito e dedicação. A agencia de fomento CAPES, pela bolsa concedida. OBRIGADA!!!! Digo-vos, pois: Pedi e ser-vos-á dado; procurai e achareis; batei e abrir se-vos-á; porque todo aquele que pede, recebe; quem procura, encontra, e ao que bate, abrir-se-á. Qual o pai de entre vós que, se o filho lhe pedir pão, lhe dará uma pedra? Ou, se lhe pedir um peixe, lhe dará uma serpente? Ou, se lhe pedir um ovo, lhe dará um escorpião? Pois se vós, que sois maus, sabeis dar coisas boas aos vossos filhos, quanto mais o Pai do Céu dará o Espírito Santo àqueles que lho pedem!