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1.
Cells ; 11(3)2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35159221

RESUMO

Background: Atherosclerosis is a chronic inflammatory disease where macrophages participate in the progression of the disease. However, the role of resident-like macrophages (res-like) in the atherosclerotic aorta is not completely understood. Methods: A single-cell RNA sequencing analysis of CD45+ leukocytes in the atherosclerotic aorta of apolipoprotein E-deficient (Apoe-/-) mice on a normal cholesterol diet (NCD) or a high cholesterol diet (HCD), respecting the side-to-specific predisposition to atherosclerosis, was performed. A population of res-like macrophages expressing hyaluronan receptor LYVE-1 was investigated via flow cytometry, co-culture experiments, and immunofluorescence in human atherosclerotic plaques from carotid artery disease patients (CAD). Results: We identified 12 principal leukocyte clusters with distinct atherosclerosis disease-relevant gene expression signatures. LYVE-1+ res-like macrophages, expressing a high level of CC motif chemokine ligand 24 (CCL24, eotaxin-2), expanded under hypercholesteremia in Apoe-/- mice and promoted VSMC phenotypic modulation to osteoblast/chondrocyte-like cells, ex vivo, in a CCL24-dependent manner. Moreover, the abundance of LYVE-1+CCL24+ macrophages and elevated systemic levels of CCL24 were associated with vascular calcification and CAD events. Conclusions: LYVE-1 res-like macrophages, via the secretion of CCL24, promote the transdifferentiation of VSMC to osteogenic-like cells with a possible role in vascular calcification and likely a detrimental role in atherosclerotic plaque destabilization.


Assuntos
Aterosclerose , Doenças das Artérias Carótidas , Receptores de Hialuronatos , Hipercolesterolemia , Placa Aterosclerótica , Calcificação Vascular , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Doenças das Artérias Carótidas/metabolismo , Quimiocina CCL24 , Colesterol/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Hipercolesterolemia/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Músculo Liso Vascular/metabolismo , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo , RNA-Seq , Calcificação Vascular/metabolismo , Proteínas de Transporte Vesicular/metabolismo
2.
Int J Mol Sci ; 23(1)2021 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-35008765

RESUMO

(1) Background: Monocytes and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome orchestrate lipid-driven amplification of vascular inflammation promoting the disruption of the fibrous cap. The components of the NLRP3 inflammasome are expressed in macrophages and foam cells within human carotid atherosclerotic plaques and VSMCs in hypertension. Whether monocytes and NLRP3 inflammasome activation are direct triggers of VSMC phenotypic switch and plaque disruption need to be investigated. (2) Methods: The direct effect of oxLDL-activated monocytes in VSMCs co-cultured system was demonstrated via flow cytometry, qPCR, ELISA, caspase 1, and pyroptosis assay. Aortic roots of VSMCs lineage tracing mice fed normal or high cholesterol diet and human atherosclerotic plaques were used for immunofluorescence quantification of NLRP3 inflammasome activation/VSMCs phenotypic switch. (3) Results: OxLDL-activated monocytes reduced α-SMA, SM22α, Oct-4, and upregulation of KLF-4 and macrophage markers MAC2, F4/80 and CD68 expression as well as caspase 1 activation, IL-1ß secretion, and pyroptosis in VSMCs. Increased caspase 1 and IL-1ß in phenotypically modified VSMCs was detected in the aortic roots of VSMCs lineage tracing mice fed high cholesterol diet and in human atherosclerotic plaques from carotid artery disease patients who experienced a stroke. (4) Conclusions: Taken together, these results provide evidence that monocyte promote VSMC phenotypic switch through VSMC NLRP3 inflammasome activation with a likely detrimental role in atherosclerotic plaque stability in human atherosclerosis.


Assuntos
Aterosclerose/metabolismo , Aterosclerose/patologia , Inflamassomos/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Aterosclerose/complicações , Aterosclerose/genética , Doenças das Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/patologia , Transdiferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipercolesterolemia/complicações , Hipercolesterolemia/patologia , Interleucina-1beta/metabolismo , Lipoproteínas LDL/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
3.
Stroke ; 50(8): 2207-2215, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31272316

RESUMO

Background and Purpose- After ischemic injury, microglia and infiltrated macrophages may acquire different polarization phenotypes promoting inflammation and injury (M1) or repair and protection (M2). There is evidence that immunomodulation, via type 2 helper T-cells (Th2) cytokines, exerts neuroprotection after ischemia. We investigated the consequences of simultaneous genetic deletion of Th2 cytokines (IL [interleukin]-4, IL-5, IL-9, IL-13) on the histopathologic outcome, microglia and infiltrated macrophages markers, and ischemic microenvironment at different time points after ischemic injury in mice subjected to permanent occlusion of the middle cerebral artery. Methods- Wild-type and Th2 cytokine-deficient mice (4KO) were subjected to permanent occlusion of the middle cerebral artery by electrocoagulation and followed up to 5 weeks after permanent occlusion of the middle cerebral artery. Neuropathologic outcome was assessed at 24 hours (n=6), 7 days (n=6), and 5 weeks (n=6-7) by examination of the ischemic lesion, neuronal count, microglia and infiltrated macrophages markers, brain atrophy, collagen deposition, and GFAP (glial fibrillary acidic protein) immunohistochemistry. Selected gene expression was investigated at 7 days (n=6). Results- 4KO mice showed no difference in lesion and neuronal count 7 days and up to 5 weeks after permanent occlusion of the middle cerebral artery compared with wild type. Ischemic 4KO mice had lower CD16/32 expression at 24 hours, lower CD11b and CD16/32 expression at 7 days than wild type. They had higher CD206 expression at 24 hours, higher CD206 and arginase1 at 7 days, and increased mRNA for CXCL9 (chemokine [C-X-C motif] ligand 9) compared with wild type. Additional histopathologic analysis, including brain atrophy, gliotic scar, and collagenous scar confirmed no difference between genotypes at 5 weeks. Conclusions- This study casts light on the proposed neuroprotective function of Th2 cytokines, showing that combined IL-4, IL-5, IL-9, IL-13 deletion does not affect the neuropathologic response to ischemic stroke in the subacute and chronic phases. Our findings indicate that Th2 cytokines are not an essential neuroimmunological cue able to drive the brain's ischemic outcome.


Assuntos
Isquemia Encefálica/genética , Encéfalo/patologia , Interleucinas/genética , Acidente Vascular Cerebral/genética , Animais , Encéfalo/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Interleucinas/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Knockout , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia
4.
ESC Heart Fail ; 5(5): 818-829, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30099854

RESUMO

AIMS: Myocarditis is often associated with parvovirus B19 (B19V) persistence, which can induce vascular damage. Based on the antiviral and anti-inflammatory properties of telbivudine, we aimed to evaluate its efficacy to protect B19V-infected endothelial cells in vitro and to treat chronic lymphocytic myocarditis patients with B19V transcriptional activity. METHODS AND RESULTS: We evaluated the endothelial-protective potential of telbivudine in human microvascular endothelial cells-1, which were infected with B19V. Treatment with 10 ng/mL of telbivudine decreased the B19V-induced endothelial cell apoptosis and endothelial-to-mesenchymal transition. Along with this finding, telbivudine reduced the expression of transforming growth factor-ß1 and of tenascin-C. The endothelial-protective properties of telbivudine were also found in tumour necrosis factor-α-stressed human microvascular endothelial cells-1. In addition, oxidative stress in angiotensin II-stressed and transforming growth factor-ß1-stressed HL-1 cardiomyocytes and fibroblasts, respectively, was reduced upon telbivudine treatment, illustrating that telbivudine exerts multimodal protective effects. Based on these in vitro findings, four patients severely suffering from an endomyocardial biopsy-proven myocarditis associated with B19V transcriptional activity (VP1/VP2-mRNA positive) were treated with telbivudine (600 mg/dL) for 6 months in a single-patient-use approach. Follow-up biopsies 6 months after treatment showed that VP1/VP2-mRNA levels and CD3 cells decreased in all patients and were associated with an improvement in ejection fraction and New York Heart Association class. These findings were paralleled by a drop in tenascin-C expression as shown via matrix-assisted laser desorption ionization-imaging mass spectrometry. CONCLUSIONS: Telbivudine exerts endothelial-protective effects in B19V-infected endothelial cells and improves chronic myocarditis associated with B19V transcriptional activity. These findings will be further evaluated in the clinical exploratory trial: the PreTopic study.


Assuntos
DNA Viral/análise , Ventrículos do Coração/diagnóstico por imagem , Linfócitos/patologia , Miocardite/tratamento farmacológico , Miocárdio/patologia , Parvovirus B19 Humano/genética , Telbivudina/uso terapêutico , Antivirais/uso terapêutico , Apoptose , Linhagem Celular , Doença Crônica , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Endotélio Vascular/virologia , Feminino , Citometria de Fluxo , Humanos , Linfócitos/virologia , Masculino , Pessoa de Meia-Idade , Miocardite/patologia , Miocardite/virologia , Miocárdio/metabolismo
5.
Sci Rep ; 8(1): 2820, 2018 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-29434214

RESUMO

Inflammation in myocarditis induces cardiac injury and triggers disease progression to heart failure. NLRP3 inflammasome activation is a newly identified amplifying step in the pathogenesis of myocarditis. We previously have demonstrated that mesenchymal stromal cells (MSC) are cardioprotective in Coxsackievirus B3 (CVB3)-induced myocarditis. In this study, MSC markedly inhibited left ventricular (LV) NOD2, NLRP3, ASC, caspase-1, IL-1ß, and IL-18 mRNA expression in CVB3-infected mice. ASC protein expression, essential for NLRP3 inflammasome assembly, increased upon CVB3 infection and was abrogated in MSC-treated mice. Concomitantly, CVB3 infection in vitro induced NOD2 expression, NLRP3 inflammasome activation and IL-1ß secretion in HL-1 cells, which was abolished after MSC supplementation. The inhibitory effect of MSC on NLRP3 inflammasome activity in HL-1 cells was partly mediated via secretion of the anti-oxidative protein stanniocalcin-1. Furthermore, MSC application in CVB3-infected mice reduced the percentage of NOD2-, ASC-, p10- and/or IL-1ß-positive splenic macrophages, natural killer cells, and dendritic cells. The suppressive effect of MSC on inflammasome activation was associated with normalized expression of prominent regulators of myocardial contractility and fibrosis to levels comparable to control mice. In conclusion, MSC treatment in myocarditis could be a promising strategy limiting the adverse consequences of cardiac and systemic NLRP3 inflammasome activation.


Assuntos
Inflamassomos/metabolismo , Células-Tronco Mesenquimais/fisiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Cardiomiopatias/metabolismo , Caspase 1/metabolismo , Infecções por Coxsackievirus/virologia , Coração/fisiologia , Humanos , Inflamação/patologia , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miocardite/virologia , Miocárdio/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia
6.
Circ Heart Fail ; 10(11)2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29158436

RESUMO

BACKGROUND: The alarmins S100A8 and S100A9 are damage-associated molecular patterns, which play a pivotal role in cardiovascular diseases, inflammation, and viral infections. We aimed to investigate their role in Coxsackievirus B3 (CVB3)-induced myocarditis. METHODS AND RESULTS: S100A8 and S100A9 mRNA expression was 13.0-fold (P=0.012) and 5.1-fold (P=0.038) higher in endomyocardial biopsies from patients with CVB3-positive myocarditis compared with controls, respectively. Elimination of CVB3 led to a downregulation of these alarmins. CVB3-infected mice developed an impaired left ventricular function and displayed an increased left ventricular S100A8 and S100A9 protein expression versus controls. In contrast, CVB3-infected S100A9 knockout mice, which are also a complete knockout for S100A8 on protein level, showed an improved left ventricular function, which was associated with a reduced cardiac inflammatory and oxidative response, and lower CVB3 copy number compared with wild-type CVB3 mice. Exogenous application of S100A8 to S100A9 knockout CVB3 mice induced a severe myocarditis similar to wild-type CVB3 mice. In CVB3-infected HL-1 cells, S100A8 and S100A9 enhanced oxidative stress and CVB3 copy number compared with unstimulated infected cells. In CVB3-infected RAW macrophages, both alarmins increased MIP-2 (macrophage inflammatory protein-2) chemokine expression, which was reduced in CVB3 S100A8 knockdown versus scrambled siRNA CVB3 cells. CONCLUSIONS: S100A8 and S100A9 aggravate CVB3-induced myocarditis and might serve as therapeutic targets in inflammatory cardiomyopathies.


Assuntos
Calgranulina A/metabolismo , Calgranulina B/metabolismo , Infecções por Coxsackievirus/metabolismo , Enterovirus Humano B/patogenicidade , Miocardite/metabolismo , Miócitos Cardíacos/metabolismo , Adulto , Animais , Calgranulina A/deficiência , Calgranulina A/genética , Calgranulina B/genética , Estudos de Casos e Controles , Quimiocina CXCL2/metabolismo , Infecções por Coxsackievirus/diagnóstico , Infecções por Coxsackievirus/genética , Infecções por Coxsackievirus/virologia , Modelos Animais de Doenças , Enterovirus Humano B/genética , Feminino , Fibrose , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/metabolismo , Macrófagos/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Miocardite/diagnóstico , Miocardite/genética , Miocardite/virologia , Miócitos Cardíacos/patologia , Miócitos Cardíacos/virologia , Infiltração de Neutrófilos , Estresse Oxidativo , Células RAW 264.7 , Interferência de RNA , RNA Mensageiro/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Transdução de Sinais , Transfecção , Função Ventricular Esquerda
7.
Stem Cells Transl Med ; 6(12): 2135-2145, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29024485

RESUMO

Left ventricular (LV) diastolic dysfunction is among others attributed to cardiomyocyte stiffness. Mesenchymal stromal cells (MSC) have cardiac-protective properties. We explored whether intravenous (i.v.) application of PLacenta-eXpanded (PLX) MSC-like cells (PLX) improves LV diastolic relaxation in streptozotocin (STZ)-induced diabetic mice and investigated underlying mechanisms. Diabetes mellitus was induced by STZ application (50 mg/kg body weight) during five subsequent days. One week after the first STZ injection, PLX or saline were i.v. applied. Two weeks later, mice were hemodynamically characterized and sacrificed. At this early stage of diabetic cardiomyopathy with low-grade inflammation and no cardiac fibrosis, PLX reduced LV vascular cell adhesion molecule-1, transforming growth factor-ß1, and interferon-γ mRNA expression, induced the percentage of circulating regulatory T cells, and decreased the splenic pro-fibrotic potential in STZ mice. STZ + PLX mice exhibited higher LV vascular endothelial growth factor mRNA expression and arteriole density versus STZ mice. In vitro, hyperglycemic PLX conditioned medium restored the hyperglycemia-impaired tube formation and adhesion capacity of human umbelical vein endothelial cells (HUVEC) via increasing nitric oxide (NO) bioavailability. PLX further induced the diabetes-downregulated activity of the NO downstream protein kinase G, as well as of protein kinase A, in STZ mice, which was associated with a raise in phosphorylation of the titin isoforms N2BA and N2B. Concomitantly, the passive force was lower in single isolated cardiomyocytes from STZ + PLX versus from STZ mice, which led to an improvement of LV diastolic relaxation. We conclude that i.v. PLX injection improves diabetes mellitus-associated diastolic performance via decreasing cardiomyocyte stiffness. Stem Cells Translational Medicine 2017;6:2135-2145.


Assuntos
Diabetes Mellitus Experimental/terapia , Cardiomiopatias Diabéticas/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Função Ventricular , Animais , Células Cultivadas , Diástole , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Placenta/citologia , Gravidez
8.
Circ Heart Fail ; 10(9)2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28912259

RESUMO

BACKGROUND: The cytoplasmatic pattern recognition receptor, NOD2 (nucleotide-binding oligomerization domain 2), belongs to the innate immune system and is among others responsible for the recognition of single-stranded RNA. With Coxsackievirus B3 (CVB3) being a single-stranded RNA virus, and the recent evidence that the NOD2 target, NLRP3 (NOD-like receptor family, pyrin domain containing 3) is of importance in the pathogenesis of CVB3-induced myocarditis, we aimed to unravel the role of NOD2 in CVB3-induced myocarditis. METHODS AND RESULTS: Endomyocardial biopsy NOD2 mRNA expression was higher in CVB3-positive patients compared with patients with myocarditis but without evidence of persistent CVB3 infection. Left ventricular NOD2 mRNA expression was also induced in CVB3-induced myocarditis versus healthy control mice. NOD2 knockdown(-/-) mice were rescued from the detrimental CVB3-mediated effects as shown by a reduced cardiac inflammation (less cardiac infiltrates and suppression of proinflammatory cytokines), cardiac fibrosis, apoptosis, lower CAR (Coxsackievirus and adenovirus receptor) expression and CVB3 copy number, and an improved left ventricular function in NOD2-/- CVB3 mice compared with wild-type CVB3 mice. In agreement, NOD2-/- decreased the CVB3-induced inflammatory response, CVB3 copy number, and apoptosis in vitro. NOD2-/- was further associated with a reduction in CVB3-induced NLRP3 expression and activity as evidenced by lower ASC (apoptosis-associated speck-like protein containing a CARD) expression, caspase 1 activity, or IL-1ß (interleukin-1ß) protein expression under in vivo and in vitro CVB3 conditions. CONCLUSIONS: NOD2 is an important mediator in the viral uptake and inflammatory response during the pathogenesis of CVB3 myocarditis.


Assuntos
Infecções por Coxsackievirus/metabolismo , Enterovirus Humano B/metabolismo , Miocardite/metabolismo , Miocárdio/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Adaptadoras de Sinalização CARD , Estudos de Casos e Controles , Caspase 1/metabolismo , Linhagem Celular , Infecções por Coxsackievirus/imunologia , Infecções por Coxsackievirus/prevenção & controle , Infecções por Coxsackievirus/virologia , Modelos Animais de Doenças , Enterovirus Humano B/genética , Enterovirus Humano B/imunologia , Predisposição Genética para Doença , Interações Hospedeiro-Patógeno , Humanos , Interleucina-1beta/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocardite/imunologia , Miocardite/prevenção & controle , Miocardite/virologia , Miocárdio/imunologia , Miocárdio/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína Adaptadora de Sinalização NOD2/deficiência , Proteína Adaptadora de Sinalização NOD2/genética , Fenótipo , Interferência de RNA , Transdução de Sinais , Transfecção , Regulação para Cima
9.
Stem Cells Transl Med ; 6(4): 1249-1261, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28186704

RESUMO

Mesenchymal stromal cell (MSC) application in Coxsackievirus B3 (CVB3)-induced myocarditis reduces myocardial inflammation and fibrosis, exerts prominent extra-cardiac immunomodulation, and improves heart function. Although the abovementioned findings demonstrate the benefit of MSC application, the mechanism of the MSC immunomodulatory effects leading to a final cardioprotective outcome in viral myocarditis remains poorly understood. Monocytes are known to be a trigger of myocardial tissue inflammation. The present study aims at investigating the direct effect of MSC on the mobilization and trafficking of monocytes to the heart in CVB3-induced myocarditis. One day post CVB3 infection, C57BL/6 mice were intravenously injected with 1 x 106 MSC and sacrificed 6 days later for molecular biology and flow cytometry analysis. MSC application reduced the severity of myocarditis, and heart and blood pro-inflammatory Ly6Chigh and Ly6Cmiddle monocytes, while those were retained in the spleen. Anti-inflammatory Ly6Clow monocytes increased in the blood, heart, and spleen of MSC-treated CVB3 mice. CVB3 infection induced splenic myelopoiesis, while MSC application slightly diminished the spleen myelopoietic activity in CVB3 mice. Left ventricular (LV) mRNA expression of the chemokines monocyte chemotactic protein-1 (MCP)-1, MCP-3, CCL5, the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, the pro-inflammatory cytokines interleukin-6, interleukin-12, tumor necrosis factor-α, the pro-fibrotic transforming growth factorß1, and circulating MCP-1 and MCP-3 levels decreased in CVB3 MSC mice, while LV stromal cell-derived factor-1α RNA expression and systemic levels of fractalkine were increased in CVB3 MSC mice. MSC application in CVB3-induced myocarditis modulates monocytes trafficking to the heart and could be a promising strategy for the resolution of cardiac inflammation and prevention of the disease progression. Stem Cells Translational Medicine 2017;6:1249-1261.


Assuntos
Infecções por Coxsackievirus/complicações , Células-Tronco Mesenquimais/fisiologia , Miocardite/etiologia , Miocardite/terapia , Miocárdio/citologia , Animais , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiocina CCL7/metabolismo , Humanos , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Fator de Necrose Tumoral alfa/metabolismo
10.
J Inflamm (Lond) ; 13: 25, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27486384

RESUMO

BACKGROUND: Obesity is associated with vascular inflammation, fibrosis and reduced high-density lipoproteins (HDL)-cholesterol. We aimed to investigate whether adenoviral gene transfer with human apolipoprotein (apo) A-I (Ad.A-I), the main apo of HDL, could exert immunomodulatory effects and counteract vascular inflammation and fibrosis in ob/ob mice. METHODS: Ad.A-I transfer was performed in 8 weeks (w) old ob/ob mice, which were sacrificed 7 w later. The aorta was excised for mRNA analysis and the spleen for splenocyte isolation for subsequent flow cytometry and co-culture with murine fibroblasts. HDL was added to mononuclear cells (MNC) and fibroblasts to assess their impact on adhesion capacity and collagen deposition, respectively. RESULTS: Ad.A-I led to a 1.8-fold (p < 0.05) increase in HDL-cholesterol versus control ob/ob mice at the day of sacrifice, which was paralleled by a decrease in aortic TNF-α and VCAM-1 mRNA expression. Pre-culture of MNC with HDL decreased their adhesion to TNF-α-activated HAEC. Ad.A-I exerted immunomodulatory effects as evidenced by a downregulation of aortic NOD2 and NLRP3 mRNA expression and by a 12 %, 6.9 %, and 15 % decrease of the induced proliferation/activity of total splenic MNC, CD4+, and CD8+ cells in ob/ob Ad.A-I versus control ob/ob mice, respectively (p < 0.05). Ad.A-I further reduced aortic collagen I and III mRNA expression by 62 % and 66 %, respectively (p < 0.0005), and abrogated the potential of ob/ob splenocytes to induce the collagen content in murine fibroblasts upon co-culture. Finally, HDL decreased the TGF-ß1-induced collagen deposition of murine fibroblasts in vitro. CONCLUSIONS: Apo A-I transfer counteracts vascular inflammation and fibrosis in ob/ob mice.

11.
Stem Cells Transl Med ; 5(12): 1707-1718, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27460853

RESUMO

: Cardiac-derived adherent proliferating cells (CardAPs) are cells derived from human endomyocardial biopsy specimens; they share several properties with mesenchymal stromal cells. The aims of this study were to evaluate whether intramyocardial injection of CardAPs modulates cardiac fibrosis and hypertrophy in a mouse model of angiotensin II (Ang II)-induced systolic heart failure and to analyze underlying mechanisms. Intramyocardial application of 200,000 CardAPs improved left ventricular function. This was paralleled by a decline in left ventricular remodeling, as indicated by a reduction in cardiac fibrosis and hypertrophy. CardAPs reduced the ratio of the left ventricle to body weight and cardiac myosin expression (heavy chain), and decreased the Ang II-induced phosphorylation state of the cardiomyocyte hypertrophy mediators Akt, extracellular-signal regulated kinase (ERK) 1, and ERK2. In accordance with the antifibrotic and antihypertrophic effects of CardAPs shown in vivo, CardAP supplementation with cardiac fibroblasts decreased the Ang II-induced reactive oxygen species production, α-SMA expression, fibroblast proliferation, and collagen production. Coculture of CardAPs with HL-1 cardiomyocytes downregulated the Ang II-induced expression of myosin in HL-1. All antifibrotic and antihypertrophic features of CardAPs were mediated in a nitric oxide- and interleukin (IL)-10-dependent manner. Moreover, CardAPs induced a systemic immunomodulation, as indicated by a decrease in the activity of splenic mononuclear cells and an increase in splenic CD4CD25FoxP3, CD4-IL-10, and CD8-IL-10 T-regulatory cells in Ang II mice. Concomitantly, splenocytes from Ang II CardAPs mice induced less collagen in fibroblasts compared with splenocytes from Ang II mice. We conclude that CardAPs improve Ang II-induced cardiac remodeling involving antifibrotic and antihypertrophic effects via paracrine actions and immunomodulatory properties. SIGNIFICANCE: Despite effective pharmacological treatment with angiotensin II type I receptor antagonists or angiotensin II-converting enzyme inhibitors, morbidity and mortality associated with heart failure are still substantial, prompting the search of novel therapeutic strategies. There is accumulating evidence supporting the use of cell therapy for cardiac repair. This study demonstrates that cells derived from human endomyocardial biopsies, cardiac-derived adherent proliferating cells (CardAPs), have the potential to reduce angiotensin II-induced cardiac remodeling and improve left ventricular function in angiotensin II mice. The mechanism involves antifibrotic and antihypertrophic effects via paracrine actions and immunomodulatory properties. These findings support the potential of CardAPs for the treatment of heart failure.


Assuntos
Angiotensina II/farmacologia , Miocárdio/patologia , Remodelação Vascular/efeitos dos fármacos , Adulto , Animais , Biópsia , Cardiomegalia/patologia , Proliferação de Células/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Colágeno/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Humanos , Imunomodulação/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Estresse Oxidativo/efeitos dos fármacos , Células Estromais/citologia , Função Ventricular Esquerda/efeitos dos fármacos
12.
Mediators Inflamm ; 2015: 308185, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25814787

RESUMO

Introduction. Mesenchymal stromal cells (MSC) have immunomodulatory features. The aim of this study was to investigate the migration and homing potential of endogenous circulating MSC in virus negative inflammatory cardiomyopathy (CMi). Methods. In 29 patients with (n = 23) or without (n = 6) CMi undergoing endomyocardial biopsies (EMB), transcardiac gradients (TCGs) of circulating MSC were measured by flow cytometry from blood simultaneously sampled from aorta and coronary sinus. The presence of MSC in EMB, cardiac inflammation, and SDF-1α mRNA expression were detected via immunohistochemistry and real-time PCR. Results. MSC defined as CD45(-)CD34(-)CD11b(-)CD73(+)CD90(+) cells accounted for 0.010 [0.0025-0.048]%/peripheral mononuclear cell (PMNC) and as CD45(-)CD34(-)CD11b(-)CD73(+)CD105(+) cells for 0.019 [0.0026-0.067]%/PMNC, both with similar counts in patients with or without cardiac inflammation. There was a 29.9% (P < 0.01) transcardiac reduction of circulating MSC in patients with CMi, correlating with the extent of cardiac inflammation (P < 0.05, multivariate analysis). A strong correlation was found between the TCG of circulating MSC and numbers of MSC (CD45(-)CD34(-)CD90(+)CD105(+)) in EMB (r = -0.73, P < 0.005). SDF-1α was the strongest predictor for increased MSC in EMB (P < 0.005, multivariate analysis). Conclusions. Endogenous MSC continuously migrate to the heart in patients with CMi triggered by cardiac inflammation.


Assuntos
Cardiomiopatias/patologia , Inflamação/patologia , Células-Tronco Mesenquimais/fisiologia , Miocárdio/patologia , Adolescente , Adulto , Idoso , Biópsia , Movimento Celular , Quimiocina CCL2/fisiologia , Quimiocina CXCL12/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
13.
Atherosclerosis ; 232(1): 1-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24401210

RESUMO

Stimulation of the liver X receptor (LXR) is associated with anti-inflammatory and vascular-protective effects under hyperlipemic conditions. We examined whether LXR stimulation influences TNF-α-induced endothelial dysfunction under normolipemic conditions. Endothelium-dependent vasorelaxation of aortic rings was determined in an organ water bath. Human umbilical vein endothelial cells (HUVEC) were exposed to TNF-α (10 ng/ml) in the presence or absence of 5 µM of the LXR agonist T0901317 or GW3965 and changes in TNF-α-induced endothelial cell apoptosis, inflammation, oxidative stress, and NO metabolism were analyzed. T0901317 improved TNF-α-impaired endothelium-dependent relaxation of aortic rings in response to acetylcholine. T0901317 decreased the TNF-α-induced apoptosis and inflammation as indicated by a decrease in caspase 3/7 activity, VCAM-1 mRNA expression and subsequent mononuclear cell adhesion. Furthermore, T0901317 reduced the expression of the oxidative stress markers: AT1R, NOX4, and p22phox and normalized the TNF-α-induced NOX activity to basal levels. In line with the reduced AT1R expression, T0901317 impaired the Ang II responsiveness. T0901317 influenced NO metabolism as indicated by a decrease in TNF-α-upregulated arginase activity, a reversal of TNF-α-induced downregulation of argininosuccinate synthase mRNA expression and eNOS expression to basal levels and a raise in NO production. Furthermore, T0901317 decreased the TNF-α-induced superoxide and nitrotyrosine production, but did not upregulate the TNF-α-downregulated eNOS dimer/monomer ratio. Silencing of LXRß, but not of LXRα, abrogated the anti-apoptotic effects of T0901317. We conclude that LXR agonism improves TNF-α-impaired endothelial function via its anti-apoptotic, anti-inflammatory, and anti-oxidative properties and its capacity to restore TNF-α-impaired NO bioavailability independent of its cholesterol-modulating effects.


Assuntos
Colesterol/metabolismo , Hidrocarbonetos Fluorados/farmacologia , Receptores Nucleares Órfãos/agonistas , Sulfonamidas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Antioxidantes/metabolismo , Aorta/metabolismo , Benzoatos/farmacologia , Benzilaminas/farmacologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Endotélio/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Receptores X do Fígado , Masculino , NADPH Oxidases/metabolismo , Óxido Nítrico/metabolismo , Receptores Nucleares Órfãos/metabolismo , Estresse Oxidativo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio
14.
PLoS One ; 7(7): e41047, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22815907

RESUMO

Systemic application of mesenchymal stromal cells (MSCs) in inflammatory cardiomyopathy exerts cardiobeneficial effects. The mode of action is unclear since a sufficient and long-acting cardiac homing of MSCs is unlikely. We therefore investigated the regulation of the immune response in coxsackievirus B3 (CVB3)-induced acute myocarditis after intravenous application of MSCs. Wildtype mice were infected with CVB3 and treated with either PBS, human MSCs or human cardiac fibroblasts intravenously 1 day after infection. Seven days after infection, MSCs could be detected in the spleen, heart, pancreas, liver, lung and kidney, whereby the highest presence was observed in the lung. MSCs increased significantly the myocardial expression of HGF and decreased the expression of the proinflammatory cytokines TNFα, IL1ß and IL6 as well as the severity of myocarditis and ameliorated the left ventricular dysfunction measured by conductance catheter. MSCs upregulated the production of IFNγ in CD4+ and CD8+ cells, the number of IL10-producing regulatory T cells and the apoptosis rate of T cells in the spleen. An increased number of CD4+CD25+FoxP3 could be found in the spleen as well as in the circulation. In contrast, application of human cardiac fibroblasts had no effect on the severity of myocarditis and the systemic immune response observed after MSCs-administration. In conclusion, modulation of the immune response in extracardiac organs is associated with cardiobeneficial effects in experimental inflammatory cardiomyopathy after systemic application of MSCs.


Assuntos
Fibroblastos/citologia , Fatores de Transcrição Forkhead/metabolismo , Células-Tronco Mesenquimais/citologia , Miocardite/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Cardiomiopatias/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Citometria de Fluxo/métodos , Hemodinâmica , Humanos , Sistema Imunitário , Hibridização In Situ , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miocardite/imunologia , Fator de Necrose Tumoral alfa/metabolismo
15.
Graefes Arch Clin Exp Ophthalmol ; 250(8): 1221-9, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22222923

RESUMO

BACKGROUND: Supplementation of staurosporine is the method of choice for differentiating the solely existing retinal ganglion cell (RGC)-5 cell line. This differentiation was initially claimed to be in the absence of apoptosis, but some publications supposed the induction of apoptosis during staurosporine induced RGC-5 differentiation. In respect to these inconsistencies in the literature, we investigated in detail whether RGC-5 cell differentiation by staurosporine induces apoptosis or not. METHODS: Amounts of 50 nM, 200 nM, 300 nM, and 600 nM of staurosporine were supplemented on RGC-5 cells for 24 h. Cell morphology and cell death, via propidium iodide staining, were evaluated with phase contrast and fluorescence microscopy, respectively. Cell amount, cell proliferation, and cell viability were analyzed by crystal violet staining, CFSE flow cytometry, and MTS assay, respectively. Apoptosis was determined by analyzing caspase 3/7 activity, Annexin-V+/ 7AAD- cells and the quotient of Bax to Bcl-2 mRNA expression via caspase 3/7 activity assay, flow cytometry, and real-time PCR, respectively. RESULTS: RGC-5 cells started to change their morphology and their expression of neuronal markers at 50 nM of staurosporine. This was associated with apoptosis and cell death, as indicated by a 2.1-fold (p < 0.0005) increase in caspase 3/7 activity, a 1.2-fold (p < 0.05) induction of Annexin-V+/ 7AAD- cells, and a 12-fold (p < 0.0005) increase in propidium iodide positive cells, respectively. Furthermore, staurosporine led to a dose-dependent increase in apoptosis and reduction in cell viability, cell density, and cell proliferation. CONCLUSIONS: The lowest staurosporine concentration inducing RGC-5 cell differentiation is accompanied by apoptosis and cell death.


Assuntos
Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células Ganglionares da Retina/patologia , Estaurosporina/farmacologia , Anexina A5/metabolismo , Caspase 3/metabolismo , Caspase 7/metabolismo , Morte Celular , Proliferação de Células , Sobrevivência Celular , Dactinomicina/análogos & derivados , Dactinomicina/metabolismo , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/metabolismo , Células Ganglionares da Retina/metabolismo , Proteína X Associada a bcl-2/genética
16.
PLoS One ; 6(12): e28513, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22174827

RESUMO

BACKGROUND: Under conventional heart failure therapy, inflammatory cardiomyopathy typically has a progressive course, indicating a need for alternative therapeutic strategies to improve long-term outcomes. We recently isolated and identified novel cardiac-derived cells from human cardiac biopsies: cardiac-derived adherent proliferating cells (CAPs). They have similarities with mesenchymal stromal cells, which are known for their anti-apoptotic and immunomodulatory properties. We explored whether CAPs application could be a novel strategy to improve acute Coxsackievirus B3 (CVB3)-induced myocarditis. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the safety of our approach, we first analyzed the expression of the coxsackie- and adenovirus receptor (CAR) and the co-receptor CD55 on CAPs, which are both required for effective CVB3 infectivity. We could demonstrate that CAPs only minimally express both receptors, which translates to minimal CVB3 copy numbers, and without viral particle release after CVB3 infection. Co-culture of CAPs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis and viral progeny release. In addition, CAPs reduced CD4 and CD8 T cell proliferation. All CAPs-mediated protective effects were nitric oxide- and interleukin-10-dependent and required interferon-γ. In an acute murine model of CVB3-induced myocarditis, application of CAPs led to a decrease of cardiac apoptosis, cardiac CVB3 viral load and improved left ventricular contractility parameters. This was associated with a decline in cardiac mononuclear cell activity, an increase in T regulatory cells and T cell apoptosis, and an increase in left ventricular interleukin-10 and interferon-γ mRNA expression. CONCLUSIONS: We conclude that CAPs are a unique type of cardiac-derived cells and promising tools to improve acute CVB3-induced myocarditis.


Assuntos
Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/virologia , Enterovirus/fisiologia , Miocardite/patologia , Miocardite/virologia , Miocárdio/patologia , Animais , Apoptose , Antígenos CD55/metabolismo , Adesão Celular , Proliferação de Células , Sobrevivência Celular , Transplante de Células , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Infecções por Coxsackievirus/complicações , Infecções por Coxsackievirus/fisiopatologia , Humanos , Imunomodulação , Injeções Intravenosas , Interferon gama/metabolismo , Interleucina-10/metabolismo , Camundongos , Contração Miocárdica/fisiologia , Miocardite/complicações , Miocardite/fisiopatologia , Óxido Nítrico/metabolismo , Receptores Virais/metabolismo , Linfócitos T Reguladores/imunologia
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