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1.
Am J Physiol Renal Physiol ; 326(3): F511-F533, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38234298

RESUMO

Antibodies are one of the most used reagents in scientific laboratories and are critical components for a multitude of experiments in physiology research. Over the past decade, concerns about many biological methods, including those that use antibodies, have arisen as several laboratories were unable to reproduce the scientific data obtained in other laboratories. The lack of reproducibility could be largely attributed to inadequate reporting of detailed methods, no or limited verification by authors, and the production and use of unvalidated antibodies. The goal of this guideline article is to review best practices concerning commonly used techniques involving antibodies, including immunoblotting, immunohistochemistry, and flow cytometry. Awareness and integration of best practices will increase the rigor and reproducibility of these techniques and elevate the quality of physiology research.


Assuntos
Anticorpos , Reprodutibilidade dos Testes , Imuno-Histoquímica , Citometria de Fluxo , Especificidade de Anticorpos
2.
PLoS Pathog ; 18(2): e1010342, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35192678

RESUMO

Viral infection of the heart is a common but underappreciated cause of heart failure. Viruses can cause direct cardiac damage by lysing infected cardiomyocytes. Inflammatory immune responses that limit viral replication can also indirectly cause damage during infection, making regulatory factors that fine-tune these responses particularly important. Identifying and understanding these factors that regulate cardiac immune responses during infection will be essential for developing targeted treatments for virus-associated heart failure. Our laboratory has discovered Brain Expressed X-linked protein 1 (BEX1) as a novel stress-regulated pro-inflammatory factor in the heart. Here we report that BEX1 plays a cardioprotective role in the heart during viral infection. Specifically, we adopted genetic gain- and loss-of-function strategies to modulate BEX1 expression in the heart in the context of coxsackievirus B3 (CVB3)-induced cardiomyopathy and found that BEX1 limits viral replication in cardiomyocytes. Interestingly, despite the greater viral load observed in mice lacking BEX1, inflammatory immune cell recruitment in the mouse heart was profoundly impaired in the absence of BEX1. Overall, the absence of BEX1 accelerated CVB3-driven heart failure and pathologic heart remodeling. This result suggests that limiting inflammatory cell recruitment has detrimental consequences for the heart during viral infections. Conversely, transgenic mice overexpressing BEX1 in cardiomyocytes revealed the efficacy of BEX1 for counteracting viral replication in the heart in vivo. We also found that BEX1 retains its antiviral role in isolated cells. Indeed, BEX1 was necessary and sufficient to counteract viral replication in both isolated primary cardiomyocytes and mouse embryonic fibroblasts suggesting a broader applicability of BEX1 as antiviral agent that extended to viruses other than CVB3, including Influenza A and Sendai virus. Mechanistically, BEX1 regulated interferon beta (IFN-ß) expression in infected cells. Overall, our study suggests a multifaceted role of BEX1 in the cardiac antiviral immune response.


Assuntos
Infecções por Coxsackievirus , Insuficiência Cardíaca , Miocardite , Viroses , Animais , Antivirais/farmacologia , Enterovirus Humano B , Fibroblastos , Camundongos , Miócitos Cardíacos , Viroses/genética , Replicação Viral
3.
Am J Physiol Cell Physiol ; 322(3): C354-C369, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35044859

RESUMO

Suppressing mineralocorticoid receptor (MR) activity with MR antagonists is therapeutic for chronic skeletal muscle pathology in Duchenne muscular dystrophy (DMD) mouse models. Although mechanisms underlying clinical MR antagonist efficacy for DMD cardiomyopathy and other cardiac diseases are defined, mechanisms in skeletal muscles are not fully elucidated. Myofiber MR knockout improves skeletal muscle force and a subset of dystrophic pathology. However, MR signaling in myeloid cells is known to be a major contributor to cardiac efficacy. To define contributions of myeloid MR in skeletal muscle function and disease, we performed parallel assessments of muscle pathology, cytokine levels, and myeloid cell populations resulting from myeloid MR genetic knockout in muscular dystrophy and acute muscle injury. Myeloid MR knockout led to lower levels of C-C motif chemokine receptor 2 (CCR2)-expressing macrophages, resulting in sustained myofiber damage after acute injury of normal muscle. In acute injury, myeloid MR knockout also led to increased local muscle levels of the enzyme that produces the endogenous MR agonist aldosterone, further supporting important contributions of MR signaling in normal muscle repair. In muscular dystrophy, myeloid MR knockout altered cytokine levels differentially between quadriceps and diaphragm muscles, which contain different myeloid populations. Myeloid MR knockout led to higher levels of fibrosis in dystrophic diaphragm. These results support important contributions of myeloid MR signaling to skeletal muscle repair in acute and chronic injuries and highlight the useful information gained from cell-specific genetic knockouts to delineate mechanisms of pharmacological efficacy.


Assuntos
Diafragma/metabolismo , Macrófagos/metabolismo , Doenças Musculares/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Músculo Quadríceps/metabolismo , Receptores de Mineralocorticoides/metabolismo , Aldosterona/metabolismo , Animais , Compostos de Bário , Cloretos , Citocinas/genética , Citocinas/metabolismo , Diafragma/imunologia , Diafragma/patologia , Modelos Animais de Doenças , Feminino , Fibrose , Macrófagos/imunologia , Masculino , Camundongos Endogâmicos mdx , Camundongos Knockout , Doenças Musculares/induzido quimicamente , Doenças Musculares/imunologia , Doenças Musculares/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/imunologia , Distrofia Muscular de Duchenne/patologia , Músculo Quadríceps/imunologia , Músculo Quadríceps/patologia , Receptores CCR2/genética , Receptores CCR2/metabolismo , Receptores de Mineralocorticoides/genética , Transdução de Sinais
4.
Am J Pathol ; 191(4): 730-747, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33497702

RESUMO

Duchenne muscular dystrophy (DMD) is a genetic, degenerative, striated muscle disease exacerbated by chronic inflammation. Mdx mice in the genotypic DMD model poorly represent immune-mediated pathology observed in patients. Improved understanding of innate immunity in dystrophic muscles is required to develop specific anti-inflammatory treatments. Here, inflammation in mdx mice and the more fibrotic utrn+/-;mdx Het model was comprehensively investigated. Unbiased analysis showed that mdx and Het mice contain increased levels of numerous chemokines and cytokines, with further increased in Het mice. Chemokine and chemokine receptor gene expression levels were dramatically increased in 4-week-old dystrophic quadriceps muscles, and to a lesser extent in diaphragm during the early injury phase, and had a small but consistent increase at 8 and 20 weeks. An optimized direct immune cell isolation method prevented loss of up to 90% of macrophages with density-dependent centrifugation previously used for mdx flow cytometry. Het quadriceps contain higher proportions of neutrophils and infiltrating monocytes than mdx, and higher percentages of F4/80Hi, but lower percentages of F4/80Lo cells and patrolling monocytes compared with Het diaphragms. These differences may restrict regenerative potential of dystrophic diaphragms, increasing pathologic severity. Fibrotic and inflammatory gene expression levels are higher in myeloid cells isolated from Het compared with mdx quadriceps, supporting Het mice may represent an improved model for testing therapeutic manipulation of inflammation in DMD.


Assuntos
Distrofina/metabolismo , Inflamação/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/patologia , Animais , Inflamação/patologia , Macrófagos/metabolismo , Camundongos Transgênicos , Monócitos/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Músculos Respiratórios/metabolismo , Músculos Respiratórios/patologia
5.
Pharmacol Res ; 170: 105606, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34119620

RESUMO

Gender bias in cardiovascular disease has been extensively documented in epidemiological and clinical studies. Despite this, the precise molecular mechanisms underlying these disparities between men and women are poorly understood. It is clear that physiological concentrations of estradiol, such as those present in pre-menopausal women, exert cardioprotective effects that are absent in men or in post-menopausal women. These cardioprotective effects, in part, are due to the estrogen receptor-mediated modulation of the immune system including T-cells. Estrogen receptors (ERs) are widely expressed in different T-cell subsets which are known to play an indispensable role in the progression of cardiovascular disease. Because T-cells can be polarized into several distinct subsets depending on the activation milieu, they can have many different, potentially opposing functions, and it is unclear what roles estrogen receptor signaling may play in mediating these functions. This is further complicated by the discrete and often antagonistic actions of different ERs on T-cell biology which dictate the balance between numerous ER-dependent signaling pathways. While myriad effects of estrogen in T-cells are relevant for many cardiovascular diseases, their widespread effects on several other (patho)physiological systems introduce several obstacles to understanding ER signaling and its precise effects on the immune system. This review aims to provide a more comprehensive summary of the mechanisms of estrogen receptor-mediated modulation of T-cell function, polarization, and cytokine production in the context of cardiovascular disease.


Assuntos
Doenças Cardiovasculares/metabolismo , Sistema Cardiovascular/metabolismo , Estradiol/metabolismo , Receptores de Estrogênio/metabolismo , Linfócitos T/metabolismo , Animais , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/fisiopatologia , Sistema Cardiovascular/imunologia , Sistema Cardiovascular/fisiopatologia , Citocinas/metabolismo , Feminino , Humanos , Ligantes , Ativação Linfocitária , Masculino , Fenótipo , Caracteres Sexuais , Transdução de Sinais , Linfócitos T/imunologia
6.
Circulation ; 139(2): 206-221, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30586716

RESUMO

BACKGROUND: Heart failure (HF) is a state of inappropriately sustained inflammation, suggesting the loss of normal immunosuppressive mechanisms. Regulatory T-lymphocytes (Tregs) are considered key suppressors of immune responses; however, their role in HF is unknown. We hypothesized that Tregs are dysfunctional in ischemic cardiomyopathy and HF, and they promote immune activation and left ventricular (LV) remodeling. METHODS: Adult male wild-type C57BL/6 mice, Foxp3-diphtheria toxin receptor transgenic mice, and tumor necrosis factor (TNF) α receptor-1 (TNFR1)-/- mice underwent nonreperfused myocardial infarction to induce HF or sham operation. LV remodeling was assessed by echocardiography as well as histological and molecular phenotyping. Alterations in Treg profile and function were examined by flow cytometry, immunostaining, and in vitro cell assays. RESULTS: Compared with wild-type sham mice, CD4+Foxp3+ Tregs in wild-type HF mice robustly expanded in the heart, circulation, spleen, and lymph nodes in a phasic manner after myocardial infarction, beyond the early phase of wound healing, and exhibited proinflammatory T helper 1-type features with interferon-γ, TNFα, and TNFR1 expression, loss of immunomodulatory capacity, heightened proliferation, and potentiated antiangiogenic and profibrotic properties. Selective Treg ablation in Foxp3-diphtheria toxin receptor mice with ischemic cardiomyopathy reversed LV remodeling and dysfunction, alleviating hypertrophy and fibrosis, while suppressing circulating CD4+ T cells and systemic inflammation and enhancing tissue neovascularization. Tregs reconstituted after ablation exhibited restoration of immunosuppressive capacity and normalized TNFR1 expression. Treg dysfunction was also tightly coupled to Treg-endothelial cell contact- and TNFR1-dependent inhibition of angiogenesis and the mobilization and tissue infiltration of CD34+Flk1+ circulating angiogenic cells in a C-C chemokine ligand 5/C-C chemokine receptor 5-dependent manner. Anti-CD25-mediated Treg depletion in wild-type mice imparted similar benefits on LV remodeling, circulating angiogenic cells, and tissue neovascularization. CONCLUSIONS: Proinflammatory and antiangiogenic Tregs play an essential pathogenetic role in chronic ischemic HF to promote immune activation and pathological LV remodeling. The restoration of normal Treg function may be a viable approach to therapeutic immunomodulation in this disease.


Assuntos
Cardiomiopatias/imunologia , Mediadores da Inflamação/imunologia , Infarto do Miocárdio/imunologia , Linfócitos T Reguladores/imunologia , Função Ventricular Esquerda , Remodelação Ventricular , Proteínas Angiogênicas/metabolismo , Animais , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Fibrose , Mediadores da Inflamação/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/imunologia , Miocárdio/metabolismo , Miocárdio/patologia , Neovascularização Fisiológica , Fenótipo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/metabolismo
7.
Int J Mol Sci ; 21(10)2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32429244

RESUMO

Excessive inflammation after myocardial infarction (MI) can promote infarct expansion and adverse left ventricular (LV) remodeling. L-4F, a mimetic peptide of apolipoprotein A-I (apoA-I), exhibits anti-inflammatory and anti-atherogenic properties; however, whether L-4F imparts beneficial effects after myocardial infarction (MI) is unknown. Here we demonstrate that L-4F suppresses the expansion of blood, splenic, and myocardial pro-inflammatory monocytes and macrophages in a mouse model of reperfused MI. Changes in immune cell profiles were accompanied by alleviation of post-MI LV remodeling and dysfunction. In vitro, L-4F also inhibited pro-inflammatory and glycolytic gene expression in macrophages. In summary, L-4F treatment prevents prolonged and excessive inflammation after MI, in part through modulation of pro-inflammatory monocytes and macrophages, and improves post-MI LV remodeling. These data suggest that L-4F could be a used as a therapeutic adjunct in humans with MI to limit inflammation and alleviate the progression to heart failure.


Assuntos
Apolipoproteína A-I/metabolismo , Monócitos/patologia , Infarto do Miocárdio/fisiopatologia , Peptídeos/farmacologia , Remodelação Ventricular/efeitos dos fármacos , Animais , Plasticidade Celular/efeitos dos fármacos , Inflamação/patologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Células RAW 264.7 , Sístole/efeitos dos fármacos , Disfunção Ventricular Esquerda/fisiopatologia
8.
J Mol Cell Cardiol ; 129: 247-256, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30880251

RESUMO

Inflammation has recently gained tremendous attention as a key contributor in several chronic diseases. While physiological inflammation is essential to counter a wide variety of damaging stimuli and to improve wound healing, dysregulated inflammation such as in the myocardium and vasculature can promote cardiovascular diseases. Given the high severity, prevalence, and economic burden of these diseases, understanding the factors involved in the regulation of physiological inflammation is essential. Like other complex biological phenomena, RNA-based processes are emerging as major regulators of inflammatory responses. Among such processes are cis-regulatory elements in the mRNA of inflammatory genes, noncoding RNAs directing the production or localization of inflammatory cytokines/chemokines, or pathogenic RNA driving inflammatory responses. In this review, we describe several specific RNA-based molecular mechanisms by which physiological inflammation pertaining to cardiovascular diseases is regulated. These include the role of AU-rich element-containing mRNAs, long non-coding RNAs, microRNAs, and viral RNAs.


Assuntos
Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/patologia , Inflamação/genética , Inflamação/patologia , RNA/metabolismo , Elementos Ricos em Adenilato e Uridilato/genética , Animais , Humanos , Modelos Biológicos , RNA/genética , RNA Viral/genética , RNA Viral/metabolismo
9.
Am J Physiol Heart Circ Physiol ; 317(3): H658-H666, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31373510

RESUMO

Immune activation post-myocardial infarction is an orchestrated sequence of cellular responses to effect tissue repair and healing. However, excessive and dysregulated inflammation can result in left ventricular remodeling and pathological alterations in the structural and mechanical attributes of the heart. Identification of key pathways and critical cellular mediators of inflammation is thus essential to design immunomodulatory therapies for myocardial infarction and ischemic heart failure. Despite this, the experimental approaches to isolate mononuclear cells from the heart are diverse, and detailed protocols to enable maximum yield of live cells in the shortest time possible are not readily available. Here, we describe optimized protocols for the isolation, fixation, and flow cytometric characterization of cardiac CD45+ leukocytes. These protocols circumvent time-consuming coronary perfusion and density-mediated cell-separation steps, resulting in high cellular yields from cardiac digests devoid of contaminating intravascular cells. Moreover, in contrast to methanol and acetone, we show that cell fixation using 1% paraformaldehyde is most optimal as it does not affect antibody binding or cellular morphology, thereby providing a considerable advantage to study activation/infiltration-associated changes in cellular granularity and size. These are highly versatile methods that can easily be streamlined for studies requiring simultaneous isolation of immune cells from different tissues or deployment in studies containing a large cohort of samples with time-sensitive constraints.NEW & NOTEWORTHY In this article, we describe optimized protocols for the isolation, fixation, and flow cytometric analysis of immune cells from the ischemic/nonischemic hearts. These protocols are optimized to process several samples/tissues, simultaneously enabling maximal yield of immune cells in the shortest time possible. We show that the low-speed centrifugation can be used as an effective alternative to lengthy coronary perfusion to remove intravascular cells, and sieving through 40-µm filter can replace density-mediated mononuclear cell separation which usually results in 50-70% cell loss in the sedimented pellets. We also show that cell fixation using 1% paraformaldehyde is better than the organic solvents such as methanol and acetone for flow cytometric analysis.


Assuntos
Separação Celular/métodos , Fixadores/química , Citometria de Fluxo/métodos , Leucócitos/imunologia , Infarto do Miocárdio/imunologia , Miocárdio/imunologia , Fixação de Tecidos/métodos , Animais , Biomarcadores/análise , Centrifugação com Gradiente de Concentração , Modelos Animais de Doenças , Imunofenotipagem , Antígenos Comuns de Leucócito/análise , Leucócitos/patologia , Masculino , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Miocárdio/patologia
10.
Basic Res Cardiol ; 112(2): 19, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28238121

RESUMO

In the failing heart, iNOS is expressed by both macrophages and cardiomyocytes. We hypothesized that inflammatory cell-localized iNOS exacerbates left ventricular (LV) remodeling. Wild-type (WT) C57BL/6 mice underwent total body irradiation and reconstitution with bone marrow from iNOS-/- mice (iNOS-/-c) or WT mice (WTc). Chimeric mice underwent coronary ligation to induce large infarction and ischemic heart failure (HF), or sham surgery. After 28 days, as compared with WTc sham mice, WTc HF mice exhibited significant (p < 0.05) mortality, LV dysfunction, hypertrophy, fibrosis, oxidative/nitrative stress, inflammatory activation, and iNOS upregulation. These mice also exhibited a ~twofold increase in circulating Ly6Chi pro-inflammatory monocytes, and ~sevenfold higher cardiac M1 macrophages, which were primarily CCR2- cells. In contrast, as compared with WTc HF mice, iNOS-/-c HF mice exhibited significantly improved survival, LV function, hypertrophy, fibrosis, oxidative/nitrative stress, and inflammatory activation, without differences in overall cardiac iNOS expression. Moreover, iNOS-/-c HF mice exhibited lower circulating Ly6Chi monocytes, and augmented cardiac M2 macrophages, but with greater infiltrating monocyte-derived CCR2+ macrophages vs. WTc HF mice. Lastly, upon cell-to-cell contact with naïve cardiomyocytes, peritoneal macrophages from WT HF mice depressed contraction, and augmented cardiomyocyte oxygen free radicals and peroxynitrite. These effects were not observed upon contact with macrophages from iNOS-/- HF mice. We conclude that leukocyte iNOS is obligatory for local and systemic inflammatory activation and cardiac remodeling in ischemic HF. Activated macrophages in HF may directly induce cardiomyocyte contractile dysfunction and oxidant stress upon cell-to-cell contact; this juxtacrine response requires macrophage-localized iNOS.


Assuntos
Insuficiência Cardíaca/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Remodelação Ventricular/fisiologia , Animais , Western Blotting , Ecocardiografia , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Imuno-Histoquímica , Isquemia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
11.
Circ Res ; 114(2): 266-82, 2014 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-24186967

RESUMO

RATIONALE: The role of mononuclear phagocytes in chronic heart failure (HF) is unknown. OBJECTIVE: Our aim was to delineate monocyte, macrophage, and dendritic cell trafficking in HF and define the contribution of the spleen to cardiac remodeling. METHODS AND RESULTS: We evaluated C57Bl/6 mice with chronic HF 8 weeks after coronary ligation. As compared with sham-operated controls, HF mice exhibited: (1) increased proinflammatory CD11b+ F4/80+ CD206- macrophages and CD11b+ F4/80+ Gr-1(hi) monocytes in the heart and peripheral blood, respectively, and reduced CD11b+ F4/80+ Gr-1(hi) monocytes in the spleen; (2) significantly increased CD11c+ B220- classical dendritic cells and CD11c+ low)B220+ plasmacytoid dendritic cells in both the heart and spleen, and increased classic dendritic cells and plasmacytoid dendritic cells in peripheral blood and bone marrow, respectively; (3) increased CD4+ helper and CD8+ cytotoxic T-cells in the spleen; and (4) profound splenic remodeling with abundant white pulp follicles, markedly increased size of the marginal zone and germinal centers, and increased expression of alarmins. Splenectomy in mice with established HF reversed pathological cardiac remodeling and inflammation. Splenocytes adoptively transferred from mice with HF, but not from sham-operated mice, homed to the heart and induced long-term left ventricular dilatation, dysfunction, and fibrosis in naive recipients. Recipient mice also exhibited monocyte activation and splenic remodeling similar to HF mice. CONCLUSIONS: Activation of mononuclear phagocytes is central to the progression of cardiac remodeling in HF, and heightened antigen processing in the spleen plays a critical role in this process. Splenocytes (presumably splenic monocytes and dendritic cells) promote immune-mediated injurious responses in the failing heart and retain this memory on adoptive transfer.


Assuntos
Insuficiência Cardíaca/imunologia , Inflamação/imunologia , Miocárdio/imunologia , Fagócitos/imunologia , Baço/imunologia , Remodelação Ventricular , Transferência Adotiva , Animais , Apresentação de Antígeno , Biomarcadores/metabolismo , Células Cultivadas , Quimiotaxia , Doença Crônica , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Ativação de Macrófagos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Monócitos/metabolismo , Miocárdio/metabolismo , Fagócitos/metabolismo , Fagócitos/transplante , Transdução de Sinais , Baço/metabolismo , Esplenectomia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Tempo
13.
Anal Bioanal Chem ; 405(5): 1547-57, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23090650

RESUMO

Novel drug delivery systems capable of continuous sustained release of therapeutics have been studied extensively for use in the prevention and management of chronic diseases. The use of these systems holds promise as a means to achieve higher patient compliance while improving therapeutic index and reducing systemic toxicity. In this work, an implantable nanochannel drug delivery system (nDS) is characterized and evaluated for the long-term sustained release of atorvastatin (ATS) and trans-resveratrol (t-RES), compounds with a proven role in managing atherogenic dyslipidemia and promoting cardioprotection. The primary mediators of drug release in the nDS are nanofluidic membranes with hundreds of thousands of nanochannels (up to 100,000/mm(2)) that attain zero-order release kinetics by exploiting nanoconfinement and molecule-to-surface interactions that dominate diffusive transport at the nanoscale. These membranes were characterized using gas flow analysis, acetone diffusion, and scanning and transmission electron microscopy (SEM, TEM). The surface properties of the dielectric materials lining the nanochannels, SiO(2) and low-stress silicon nitride, were further investigated using surface charge analysis. Continuous, sustained in vitro release for both ATS and t-RES was established for durations exceeding 1 month. Finally, the influence of the membranes on cell viability was assessed using human microvascular endothelial cells. Morphology changes and adhesion to the surface were analyzed using SEM, while an MTT proliferation assay was used to determine the cell viability. The nanochannel delivery approach, here demonstrated in vitro, not only possesses all requirements for large-scale high-yield industrial fabrication, but also presents the key components for a rapid clinical translation as an implantable delivery system for the sustained administration of cardioprotectants.


Assuntos
Anticolesterolemiantes/administração & dosagem , Sistemas de Liberação de Medicamentos/instrumentação , Implantes de Medicamento/química , Ácidos Heptanoicos/administração & dosagem , Membranas Artificiais , Pirróis/administração & dosagem , Estilbenos/administração & dosagem , Vasodilatadores/administração & dosagem , Atorvastatina , Linhagem Celular , Sobrevivência Celular , Difusão , Desenho de Equipamento , Humanos , Nanoestruturas/química , Resveratrol
14.
Carcinogenesis ; 33(8): 1608-15, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22696595

RESUMO

Many chemopreventive agents have encountered bioavailability issues in pre-clinical/clinical studies despite high oral doses. We report here a new concept utilizing polycaprolactone implants embedded with test compounds to obtain controlled systemic delivery, circumventing oral bioavailability issues and reducing the total administered dose. Compounds were released from the implants in vitro dose dependently and for long durations (months), which correlated with in vivo release. Polymeric implants of curcumin significantly inhibited tissue DNA adducts following the treatment of rats with benzo[a]pyrene, with the total administered dose being substantially lower than typical oral doses. A comparison of bioavailability of curcumin given by implants showed significantly higher levels of curcumin in the plasma, liver and brain 30 days after treatment compared with the dietary route. Withaferin A implants resulted in a nearly 60% inhibition of lung cancer A549 cell xenografts, but no inhibition occurred when the same total dose was administered intraperitoneally. More than 15 phytochemicals have been tested successfully by this formulation. Together, our data indicate that this novel implant-delivery system circumvents oral bioavailability issues, provides continuous delivery for long durations and lowers the total administered dose, eliciting both chemopreventive/chemotherapeutic activities. This would also allow the assessment of activity of minor constituents and synthetic metabolites, which otherwise remain uninvestigated in vivo.


Assuntos
Anticarcinógenos/administração & dosagem , Neoplasias Pulmonares/prevenção & controle , Animais , Anticarcinógenos/farmacocinética , Disponibilidade Biológica , Preparações de Ação Retardada , Implantes de Medicamento , Feminino , Humanos , Camundongos , Camundongos Nus , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual
15.
Front Cardiovasc Med ; 9: 992653, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36093172

RESUMO

CD4+ T-cells facilitate wound healing post-myocardial infarction (MI) but promote left-ventricular (LV) remodeling during ischemic heart failure (HF; 8 weeks post-MI). Therefore, it is critical to understand if sustained CD4+ T-cell activation leads to this pathological response, or if phenotypically different T-cells are activated during MI vs. HF. Using flow cytometry, we found that cardiac CD4+ T-cells exhibit two distinct patterns of transmigration. First pattern consisted of a rapid CD4+ T-cell response with maximal levels seen at 3 days post-MI which return to baseline by 14 days. However, during HF we observed a 2nd phase of activation and CD4+ T-cells were ∼20-fold higher in HF as compared to sham-operated mice. Importantly, these biphasic kinetics were observed with all major T-cell subsets such as Th1, Th2, Th17, and regulatory T-cells suggesting a global change. To determine the role of this 2nd peak of T-cell activation, CD4-iDTR mice were generated and treated with DT every 10 from 28 days post-MI to deplete CD4+ T-cells during chronic HF. While littermate control mice showed increased end-systolic and end-diastolic volumes (ESV and EDV) and decreased ejection fraction (EF) from 4 to 8 weeks post-MI, depletion of CD4+ T-cells in Cre + mice significantly blunted LV remodeling and inhibited progressive increases in the EDV and ESV, and reduction in EF. This suggests that CD4+ T-cell responses occurring during HF are different than those occurring during MI and promote LV remodeling and progressive cardiac dysfunction. Temporal immunomodulation of CD4+ T-cells could be a translatable modality for ischemic HF.

16.
JACC Basic Transl Sci ; 7(10): 1038-1049, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36337927

RESUMO

CD4+ T cells turn pathological during heart failure (HF). We show that the expression of tumor necrosis factor (TNF)-α and tumor necrosis factor receptor (TNFR1) increases in HF-activated CD4+ T cells. However, the role of the TNF-α/TNFR1 axis in T-cell activation/proliferation is unknown. We show that TNFR1 neutralization during T-cell activation (ex vivo) or the loss of TNFR1 in adoptively transferred HF-activated CD4+ T cells (in vivo) augments their prosurvival and proliferative signaling. Importantly, TNFR1 neutralization does not affect CD69 expression or the pathological activity of HF-activated TNFR1-/- CD4+ T cells. These results show that during HF TNFR1 plays an important role in quelling prosurvival and proliferative signals in CD4+ T cells without altering their pathological activity.

17.
JCI Insight ; 7(19)2022 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-36040807

RESUMO

Mineralocorticoid receptor antagonists (MRAs) slow cardiomyopathy in patients with Duchenne muscular dystrophy (DMD) and improve skeletal muscle pathology and function in dystrophic mice. However, glucocorticoids, known antiinflammatory drugs, remain a standard of care for DMD, despite substantial side effects. Exact mechanisms underlying mineralocorticoid receptor (MR) signaling contribution to dystrophy are unknown. Whether MRAs affect inflammation in dystrophic muscles and how they compare with glucocorticoids is unclear. The MRA spironolactone and glucocorticoid prednisolone were each administered for 1 week to dystrophic mdx mice during peak skeletal muscle necrosis to compare effects on inflammation. Both drugs reduced cytokine levels in mdx quadriceps, but prednisolone elevated diaphragm cytokines. Spironolactone did not alter myeloid populations in mdx quadriceps or diaphragms, but prednisolone increased F4/80hi macrophages. Both spironolactone and prednisolone reduced inflammatory gene expression in myeloid cells sorted from mdx quadriceps, while prednisolone additionally perturbed cell cycle genes. Spironolactone also repressed myeloid expression of the gene encoding fibronectin, while prednisolone increased its expression. Overall, spironolactone exhibits antiinflammatory properties without altering leukocyte distribution within skeletal muscles, while prednisolone suppresses quadriceps cytokines but increases diaphragm cytokines and pathology. Antiinflammatory properties of MRAs and different limb and respiratory muscle responses to glucocorticoids should be considered when optimizing treatments for patients with DMD.


Assuntos
Distrofia Muscular de Duchenne , Miosite , Animais , Citocinas/metabolismo , Fibronectinas/metabolismo , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos mdx , Antagonistas de Receptores de Mineralocorticoides/metabolismo , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/complicações , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Prednisolona/metabolismo , Prednisolona/farmacologia , Prednisolona/uso terapêutico , Receptores de Mineralocorticoides/metabolismo , Receptores de Mineralocorticoides/uso terapêutico , Espironolactona/metabolismo , Espironolactona/farmacologia , Espironolactona/uso terapêutico
18.
Circ Heart Fail ; 15(7): e008997, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35730443

RESUMO

BACKGROUND: CD4+ T cells temporally transition from protective to pathological during ischemic heart failure (HF; 8 weeks postmyocardial infarction). Cellular mechanisms mediating this shift are unknown. METHODS: RNA-sequencing of cardiac CD4+ T cells and flow cytometric analysis of immune cells was conducted. RESULTS: RNA-sequencing of CD4+ T cells from the failing hearts of male mice indicated activation of ER (estrogen receptor)-α signaling. Flow cytometric analysis showed that ERα in CD4+ T cells decreases significantly at 3-day postmyocardial infarction but increases during HF. To antagonize ERα, we tested a novel ERß agonist (OSU-ERb-012) to inhibit T cells and blunt left ventricular remodeling. Proliferation assays showed that OSU-ERb-012 dose-dependently inhibited proliferation and proinflammatory cytokine expression in anti-CD3/CD28 stimulated splenic T cells isolated from both the sexes. For in vivo efficacy, 10- to 12-week-old male and ovariectomized female mice were randomized at 4 weeks postmyocardial infarction and treated with either vehicle or drug (60 mg/kg per day; oral). While vehicle-treated HF mice displayed progressive left ventricular dilatation with significantly increased end-systolic and end-diastolic volumes from 4 to 8 weeks postmyocardial infarction, treatment with OSU-ERb-012 significantly blunted these changes and stopped left ventricular remodeling in both the sexes. Reduction in tibia-normalized heart and left ventricular weights, cardiomyocyte hypertrophy and interstitial fibrosis further supported these results. Additionally, OSU-ERb-012 treatment selectively inhibited cardiac, splenic, and circulating CD4+ T cells without affecting other myeloid and lymphoid cells in the HF mice. CONCLUSIONS: Our studies indicate that ERß agonists and OSU-ERb-012, in particular, could be used as selective immunomodulatory drugs to inhibit CD4+ T cells during chronic HF.


Assuntos
Insuficiência Cardíaca , Infarto do Miocárdio , Animais , Doença Crônica , Receptor alfa de Estrogênio , Receptor beta de Estrogênio/fisiologia , Receptor beta de Estrogênio/uso terapêutico , Estrogênios/uso terapêutico , Feminino , Ativação Linfocitária , Masculino , Camundongos , Infarto do Miocárdio/metabolismo , RNA/uso terapêutico , Receptores de Estrogênio/uso terapêutico , Remodelação Ventricular/fisiologia
19.
Sci Adv ; 8(42): eabo1244, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36269835

RESUMO

Mitochondrial-associated membranes (MAMs) are known to modulate organellar and cellular functions and can subsequently affect pathophysiology including myocardial ischemia-reperfusion (IR) injury. Thus, identifying molecular targets in MAMs that regulate the outcome of IR injury will hold a key to efficient therapeutics. Here, we found chloride intracellular channel protein (CLIC4) presence in MAMs of cardiomyocytes and demonstrate its role in modulating ER and mitochondrial calcium homeostasis under physiological and pathological conditions. In a murine model, loss of CLIC4 increased myocardial infarction and substantially reduced cardiac function after IR injury. CLIC4 null cardiomyocytes showed increased apoptosis and mitochondrial dysfunction upon hypoxia-reoxygenation injury in comparison to wild-type cardiomyocytes. Overall, our results indicate that MAM-CLIC4 is a key mediator of cellular response to IR injury and therefore may have a potential implication on other pathophysiological processes.

20.
Pharm Res ; 28(5): 1121-30, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21311958

RESUMO

PURPOSE: The introduction of curcumin into clinics is hindered by its low water solubility and poor bioavailability. To overcome these limitations, we developed curcumin implants using poly (ε-caprolactone) as the polymeric matrix. METHODS: Implants were prepared by melt-extrusion method; in vitro drug release was optimized for effects of polymer composition, drug load, surface area and water-soluble additives. Implants were also tested under in vivo conditions for cumulative curcumin release, and liver concentration was correlated with its efficacy to modulate selected xenobiotic-metabolizing enzymes (CYP1A1 and GSTM). RESULTS: Drug release from implants followed biphasic release pattern with Higuchi kinetics and was proportional to the surface area of implants. Drug release increased proportionately from 2 to 10% (w/w) drug load, and incorporation of 10% (w/w) of water-soluble additives (F-68, PEG 8000 and cyclodextrin) did not significantly alter the drug release. In vivo drug release was found to be ~1.8 times higher than in vitro release. Curcumin was detected at 60 ± 20 ng/g in the liver after four days of implantation and was almost constant (8-15 ng/g) for up to 35 days. This time-dependent drop in curcumin level was found to be due to induction of CYP1A1 and GSTM (µ) enzymes which led to increased metabolism of curcumin. CONCLUSION: Our data showed that these implants were able to release curcumin for long duration and to modulate liver phase I and phase II enzymes, demonstrating curcumin's biological efficacy delivered via this delivery system.


Assuntos
Curcumina/administração & dosagem , Curcumina/farmacocinética , Preparações de Ação Retardada/química , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacocinética , Animais , Curcumina/metabolismo , Curcumina/farmacologia , Citocromo P-450 CYP1A1/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Fígado/efeitos dos fármacos , Fígado/enzimologia , Polímeros/química , Ratos , Ratos Sprague-Dawley
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