RESUMO
Chronic proteolytic disruption of elastic fibres within the abdominal aortic wall results in wall vessel expansion to form rupture-prone abdominal aortic aneurysms (AAA). Arresting AAA growth is not possible as adult vascular smooth muscle cells (SMCs) poorly auto-regenerate and repair elastic fibres. Thus, there is a need to identify alternate cell sources capable of robust elastic matrix assembly to overcome elastolysis in the AAA wall. Previously, we demonstrated the superior elastogenic properties of rat bone marrow mesenchymal stem cell (BM-MSC)-derived SMCs (BM-SMCs) relative to aneurysmal and healthy rat aortic SMCs. In the present study, we investigate how phenotypic coordinates of the derived BM-SMCs, in turn dependent on conditions of BM-MSC differentiation, impact their elastic matrix synthesis abilities. More specifically, we investigated how glucose content, serum levels and the presence of transforming growth factor (TGF)-ß1 supplements alone or together with platelet-derived growth factor (PDGF-BB) in the differentiation medium influence phenotype of, and elastogenesis by derived rat BM-SMCs. BM-SMCs generated in low-glucose and 10% v/v serum conditions in the presence of TGF-ß1 with or without PDGF-BB exhibited a mature phenotype characterized by contractility and migrative tendencies similar to healthy rat aortic SMCs, and yet capable of robust tropoelastin (precursor) synthesis and assembly of a fibrous, highly crosslinked elastic matrix. Thus, we have identified metrics and conditions for selecting BM-SMCs with superior elastogenesis for in situ elastic matrix regeneration. Future studies will focus on characterizing these specific BM-SMC subtypes for their pro-elastogenic and anti-proteolytic effects on aneurysmal SMCs to confirm their preferred use for therapy aimed at AAA tissue regenerative repair. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Aneurisma da Aorta Abdominal/terapia , Células da Medula Óssea/citologia , Elasticidade , Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/citologia , Miócitos de Músculo Liso/citologia , Regeneração , Animais , Aneurisma da Aorta Abdominal/patologia , Becaplermina/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Glucose/análise , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fenótipo , Ratos Sprague-Dawley , Regeneração/efeitos dos fármacos , Soro/metabolismoRESUMO
Aortic valve disease (AVD) is a common condition with a progressive natural history, and presently, there are no pharmacologic treatment strategies. Elastic fiber fragmentation (EFF) is a hallmark of AVD, and increasing evidence implicates developmental elastic fiber assembly defects. Emilin1 is a glycoprotein necessary for elastic fiber assembly that is present in both developing and mature human and mouse aortic valves. The Emilin1-deficient mouse (Emilin1-/- ) is a model of latent AVD, characterized by activated TGFß/MEK/p-Erk signaling and upregulated elastase activity. Emilin1-/- aortic valves demonstrate early EFF and aberrant angiogenesis followed by late neovascularization and fibrosis. The objective of this study was to test the effectiveness of three different targeted therapies. Aged (12-14 months) Emilin1-/- mice were treated with refametinib (RDEA-119, MEK1/2 inhibitor), doxycycline (elastase inhibitor), or G6-31 (anti-VEGF-A mouse antibody) for 4 weeks. Refametinib- and doxycycline-treated Emilin1-/- mice markedly reduced MEK/p-Erk activation in valve tissue. Furthermore, both refametinib and doxycycline attenuated elastolytic cathepsin K, L, MMP-2, and MMP-9 activation, and abrogated macrophage and neutrophil infiltration in Emilin1-/- aortic valves. RNAseq analysis was performed in aortic valve tissue from adult (4 months) and aged (14 months) Emilin1-/- and age-matched wild-type control mice, and demonstrated upregulation of genes associated with MAPK/MEK/p-Erk signaling and elastases at the adult stage and inflammatory pathways at the aged stage controlling for age. These results suggest that Erk1/2 signaling is an important modulator of early elastase activation, and pharmacological inhibition using refametinib may be a promising treatment to halt AVD progression.
Assuntos
Anticorpos/uso terapêutico , Valva Aórtica/efeitos dos fármacos , Difenilamina/análogos & derivados , Doxiciclina/uso terapêutico , Cardiopatias Congênitas/tratamento farmacológico , Doenças das Valvas Cardíacas/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Sulfonamidas/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/imunologia , Animais , Anticorpos/farmacologia , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Doença da Válvula Aórtica Bicúspide , Difenilamina/farmacologia , Difenilamina/uso terapêutico , Modelos Animais de Doenças , Progressão da Doença , Doxiciclina/farmacologia , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/patologia , Doenças das Valvas Cardíacas/metabolismo , Doenças das Valvas Cardíacas/patologia , Camundongos , Camundongos Knockout , Elastase Pancreática/metabolismo , Sulfonamidas/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Abdominal aortic aneurysms (AAAs) involve slow proteolysis and loss of structural matrix components (collagen and elastin), which lead to wall thinning, weakening and ultimate rupture. At this time, no established non-surgical therapy is available to slow or arrest AAA growth. Inhibiting matrix metalloproteases (MMPs; e.g. MMP2 and -9) overexpressed within AAAs is insufficient to arrest AAA growth, since resident smooth muscle cells (SMCs) are poorly elastogenic and cannot overcome elastolysis to reinstate a healthy elastic matrix. Towards overcoming this limitation, this first study sought to determine the utility of rat bone marrow mesenchymal stem cell (BM-MSC)-derived SMCs to stimulate elastin and elastic matrix synthesis and assembly by aneurysmal SMCs (EaRASMCs). BM-MSCs were successfully differentiated into cells of an SMC lineage (SMLCs). Our study indicates that BM-MSC-derived SMLCs secrete trophic factors, contained in conditioned medium (CM) from their cultures, that, when exposed to EaRASMC cultures in real time, stimulate elastin precursor and matrix deposition and crosslinking by these elastogenically deficient cells, with added benefits in terms of attenuating MMPs, specifically MMP9. The results thus lend support to a proposed cell therapy for AAAs, based on the use of BM-MSC-derived SMLCs. Although we observed no particular improvement in elastic fibre formation, no attenuation of MMP2 activity and increase in amounts of active MMP2 enzyme, we believe that this study justifies follow-up studies to improve upon these outcomes. Future studies will explore the effects of concentrated CM collected from long-term SMLC cultures on EaRASMCs and also investigate the elastogenic output of SMLCs themselves. Copyright © 2014 John Wiley & Sons, Ltd.
Assuntos
Aneurisma da Aorta Abdominal/patologia , Células da Medula Óssea/citologia , Elastina/metabolismo , Células-Tronco Mesenquimais/citologia , Miócitos de Músculo Liso/patologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem da Célula/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Meios de Cultivo Condicionados/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Citometria de Fluxo , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/ultraestrutura , Comunicação Parácrina/efeitos dos fármacos , Ratos Sprague-DawleyRESUMO
Arresting or regressing growth of abdominal aortic aneurysms (AAAs), localized expansions of the abdominal aorta are contingent on inhibiting chronically overexpressed matrix metalloproteases (MMPs)-2 and -9 that disrupt elastic matrix within the aortic wall, concurrent with providing a stimulus to augmenting inherently poor auto-regeneration of these matrix structures. In a recent study we demonstrated that localized, controlled and sustained delivery of doxycycline (DOX; a tetracycline-based antibiotic) from poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs), enhances elastic matrix deposition and MMP-inhibition at a fraction of the therapeutically effective oral dose. The surface functionalization of these NPs with cationic amphiphiles, which enhances their arterial uptake, was also shown to have pro-matrix regenerative and anti-MMP effects independent of the DOX. Based on the hypothesis that the incorporation of superparamagnetic iron oxide NPs (SPIONs) within these PLGA NPs would enhance their targetability to the AAA site under an applied external magnetic field, we sought to evaluate the functional effects of NPs co-encapsulating DOX and SPIONs (DOX-SPION NPs) on elastic matrix regeneration and MMP synthesis/activity in vitro within aneurysmal smooth muscle cell (EaRASMC) cultures. The DOX-SPION NPs were mobile under an applied external magnetic field, while enhancing elastic matrix deposition 1.5-2-fold and significantly inhibiting MMP-2 synthesis and MMP-2 and -9 activities, compared to NP-untreated control cultures. These results illustrate that the multifunctional benefits of NPs are maintained following SPION co-incorporation. Additionally, preliminary studies carried out demonstrated enhanced targetability of SPION-loaded NPs within proteolytically-disrupted porcine carotid arteries ex vivo, under the influence of an applied external magnetic field. Thus, this dual-agent loaded NP system proffers a potential non-surgical option for treating small growing AAAs, via controlled and sustained drug release from multifunctional, targetable nanocarriers. STATEMENT OF SIGNIFICANCE: Proactive screening of high risk elderly patients now enables early detection of abdominal aortic aneurysms (AAAs). There are no established drug-based therapeutic alternatives to surgery for AAAs, which is unsuitable for many elderly patients, and none which can achieve restore disrupted and lost elastic matrix in the AAA wall, which is essential to achieve growth arrest or regression. We have developed a first generation design of polymer nanoparticles (NPs) for AAA tissue localized delivery of doxycycline, a modified tetracycline drug at low micromolar doses at which it provides both pro-elastogenic and anti-proteolytic benefits that can augment elastic matrix regenerative repair. The nanocarriers themselves are also uniquely chemically functionalized on their surface to also provide them pro-elastin-regenerative & anti-matrix degradative properties. To provide an active driving force for efficient uptake of intra-lumenally infused NPs to the AAA wall, in this work, we have rendered our polymer NPs mobile in an applied magnetic field via co-incorporation of super-paramagnetic iron oxide NPs. We demonstrate that such modifications significantly improve wall uptake of the NPs with no significant changes to their physical properties and regenerative benefits. Such NPs can potentially stimulate structural repair in the AAA wall following one time infusion to delay or prevent AAA growth to rupture. The therapy can provide a non-surgical treatment option for high risk AAA patients.
Assuntos
Preparações de Ação Retardada/administração & dosagem , Dextranos/administração & dosagem , Doxiciclina/administração & dosagem , Nanopartículas de Magnetita/administração & dosagem , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos da radiação , Regeneração/efeitos dos fármacos , Animais , Células Cultivadas , Preparações de Ação Retardada/química , Preparações de Ação Retardada/efeitos da radiação , Dextranos/efeitos da radiação , Doxiciclina/química , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/efeitos da radiação , Campos Magnéticos , Nanopartículas de Magnetita/efeitos da radiação , Masculino , Nanocápsulas/administração & dosagem , Nanocápsulas/química , Nanocápsulas/efeitos da radiação , Doses de Radiação , Ratos , Ratos Sprague-Dawley , Regeneração/efeitos da radiaçãoRESUMO
The pathogenesis of pulmonary fibrosis involves lung epithelial injury and aberrant proliferation of fibroblasts, and results in progressive pulmonary scarring and declining lung function. In vitro, fibroblast growth factor (FGF) 2 promotes myofibroblast differentiation and proliferation in cooperation with the profibrotic growth factor, transforming growth factor-ß1, but the in vivo requirement for FGF2 in the development of pulmonary fibrosis is not known. The bleomycin model of lung injury and pulmonary fibrosis was applied to Fgf2 knockout (Fgf2(-/-)) and littermate control mice. Weight loss, mortality, pulmonary fibrosis, and histology were analyzed after a single intranasal dose of bleomycin. Inflammation was evaluated in bronchoalveolar lavage (BAL) fluid, and epithelial barrier integrity was assessed by measuring BAL protein and Evans Blue dye permeability. Fgf2 is expressed in mouse and human lung epithelial and inflammatory cells, and, in response to bleomycin, Fgf2(-/-) mice have significantly increased mortality and weight loss. Analysis of BAL fluid and histology show that pulmonary fibrosis is unaltered, but Fgf2(-/-) mice fail to efficiently resolve inflammation, have increased BAL cellularity, and, importantly, deficient recovery of epithelial integrity. Fgf2(-/-) mice similarly have deficient recovery of club cell secretory protein(+) bronchial epithelium in response to naphthalene. We conclude that FGF2 is not required for bleomycin-induced pulmonary fibrosis, but rather is essential for epithelial repair and maintaining epithelial integrity after bleomycin-induced lung injury in mice. These data identify that FGF2 acts as a protective growth factor after lung epithelial injury, and call into question the role of FGF2 as a profibrotic growth factor in vivo.