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1.
Cardiovasc Diabetol ; 22(1): 214, 2023 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-37592236

RESUMO

BACKGROUND: Cardiac steatosis is an early yet overlooked feature of diabetic cardiomyopathy. There is no available therapy to treat this condition. Tyrosine kinase inhibitors (TKIs) are used as first or second-line therapy in different types of cancer. In cancer patients with diabetes mellitus, TKIs reportedly improved glycemic control, allowing insulin discontinuation. They also reduced liver steatosis in a murine model of non-alcoholic fatty liver disease. The present study aimed to determine the therapeutic effect of the second-generation TKI Dasatinib on lipid accumulation and cardiac function in obese, type 2 diabetic mice. We also assessed if the drug impacts extra-cardiac fat tissue depots. METHODS: Two studies on 21-week-old male obese leptin receptor mutant BKS.Cg-+Leprdb/+Leprdb/OlaHsd (db/db) mice compared the effect of Dasatinib (5 mg/kg) and vehicle (10% DMSO + 90% PEG-300) given via gavage once every three days for a week or once every week for four weeks. Functional and volumetric indices were studied using echocardiography. Post-mortem analyses included the assessment of fat deposits and fibrosis using histology, and senescence using immunohistochemistry and flow cytometry. The anti-adipogenic action of Dasatinib was investigated on human bone marrow (BM)-derived mesenchymal stem cells (MSCs). Unpaired parametric or non-parametric tests were used to compare two and multiple groups as appropriate. RESULTS: Dasatinib reduced steatosis and fibrosis in the heart of diabetic mice. The drug also reduced BM adiposity but did not affect other fat depots. These structural changes were associated with improved diastolic indexes, specifically the E/A ratio and non-flow time. Moreover, Dasatinib-treated mice had lower levels of p16 in the heart compared with vehicle-treated controls, suggesting an inhibitory impact of the drug on the senescence signalling pathway. In vitro, Dasatinib inhibited human BM-MSC viability and adipogenesis commitment. CONCLUSIONS: Our findings suggest that Dasatinib opposes heart and BM adiposity and cardiac fibrosis. In the heart, this was associated with favourable functional consequences, namely improvement in an index of diastolic function. Repurposing TKI for cardiac benefit could address the unmet need of diabetic cardiac steatosis.


Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Hepatopatia Gordurosa não Alcoólica , Humanos , Masculino , Animais , Camundongos , Dasatinibe/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Fibrose , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico
2.
Arterioscler Thromb Vasc Biol ; 39(6): 1113-1124, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31018661

RESUMO

Objective- To determine the role of the oncofetal protein TPBG (trophoblast glycoprotein) in normal vascular function and reparative vascularization. Approach and Results- Immunohistochemistry of human veins was used to show TPBG expression in vascular smooth muscle cells and adventitial pericyte-like cells (APCs). ELISA, Western blot, immunocytochemistry, and proximity ligation assays evidenced a hypoxia-dependent upregulation of TPBG in APCs not found in vascular smooth muscle cells or endothelial cells. This involves the transcriptional modulator CITED2 (Atypical chemokine receptor 3 CBP/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich tail) and downstream activation of CXCL12 (chemokine [C-X-C motif] ligand-12) signaling through the CXCR7 (C-X-C chemokine receptor type 7) receptor and ERK1/2 (extracellular signal-regulated kinases 1/2). TPBG silencing by siRNA transfection downregulated CXCL12, CXCR7, and pERK (phospho Thr202/Tyr204 ERK1/2) and reduced the APC migratory and proangiogenic capacities. TPBG forced expression induced opposite effects, which were associated with the formation of CXCR7/CXCR4 (C-X-C chemokine receptor type 4) heterodimers and could be contrasted by CXCL12 and CXCR7 neutralization. In vivo Matrigel plug assays using APCs with or without TPBG silencing evidenced TPBG is essential for angiogenesis. Finally, in immunosuppressed mice with limb ischemia, intramuscular injection of TPBG-overexpressing APCs surpassed naïve APCs in enhancing perfusion recovery and reducing the rate of toe necrosis. Conclusions- TPBG orchestrates the migratory and angiogenic activities of pericytes through the activation of the CXCL12/CXCR7/pERK axis. This novel mechanism could be a relevant target for therapeutic improvement of reparative angiogenesis.


Assuntos
Movimento Celular , Glicoproteínas de Membrana/metabolismo , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Pericitos/metabolismo , Veia Safena/metabolismo , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Membro Posterior , Humanos , Isquemia/genética , Isquemia/metabolismo , Isquemia/fisiopatologia , Isquemia/cirurgia , Masculino , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Nus , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Pericitos/transplante , Fosforilação , Receptores CXCR/genética , Receptores CXCR/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo
3.
Mol Ther ; 26(12): 2823-2837, 2018 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-30274787

RESUMO

MicroRNAs regulate endothelial function and angiogenesis, but their implication in pericyte biology remains undetermined. A PCR array, covering a panel of 379 human microRNAs, showed microRNA-532-5p to be one of the most differentially modulated by hypoxia, which was confirmed by qPCR in both skeletal muscle and adventitial pericytes. Furthermore, microRNA-532-5p was upregulated in murine muscular pericytes early after experimentally induced ischemia, decreasing below baseline after reperfusion. Transfection of human pericytes with anti-microRNA, microRNA-mimic, or controls indicates microRNA-532-5p modulates pro-angiogenic activity via transcriptional regulation of angiopoietin-1. Tie-2 blockade abrogated the ability of microRNA-532-5p-overexpressing pericytes to promote endothelial network formation in vitro. However, angiopoietin-1 is not a direct target of microRNA-532-5p. In silico analysis of microRNA-532-5p inhibitory targets associated with angiopoietin-1 transcription indicated three potential candidates, BACH1, HIF1AN, and EGLN1. Binding of microRNA-532-5p to the BACH1 3' UTR was confirmed by luciferase assay. MicroRNA-532-5p silencing increased BACH1, while a microRNA-532-5p mimic decreased expression. Silencing of BACH1 modulated angiopoietin-1 gene and protein expression. ChIP confirmed BACH1 transcriptional regulation of angiopoietin-1 promoter. Finally, microRNA-532-5p overexpression increased pericyte coverage in an in vivo Matrigel assay, suggesting its role in vascular maturation. This study provides a new mechanistic understanding of the transcriptional program orchestrating angiopoietin-1/Tie-2 signaling in human pericytes.


Assuntos
Angiopoietina-1/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Pericitos/metabolismo , Interferência de RNA , Comunicação Autócrina , Biomarcadores , Perfilação da Expressão Gênica , Genes Reporter , Humanos , Hipóxia , Comunicação Parácrina , Fenótipo , Transcriptoma
4.
Circ Res ; 116(10): e81-94, 2015 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-25801898

RESUMO

RATIONALE: Optimization of cell therapy for cardiac repair may require the association of different cell populations with complementary activities. OBJECTIVE: Compare the reparative potential of saphenous vein-derived pericytes (SVPs) with that of cardiac stem cells (CSCs) in a model of myocardial infarction, and investigate whether combined cell transplantation provides further improvements. METHODS AND RESULTS: SVPs and CSCs were isolated from vein leftovers of coronary artery bypass graft surgery and discarded atrial specimens of transplanted hearts, respectively. Single or dual cell therapy (300 000 cells of each type per heart) was tested in infarcted SCID (severe combined immunodeficiency)-Beige mice. SVPs and CSCs alone improved cardiac contractility as assessed by echocardiography at 14 days post myocardial infarction. The effect was maintained, although attenuated at 42 days. At histological level, SVPs and CSCs similarly inhibited infarct size and interstitial fibrosis, SVPs were superior in inducing angiogenesis and CSCs in promoting cardiomyocyte proliferation and recruitment of endogenous stem cells. The combination of cells additively reduced the infarct size and promoted vascular proliferation and arteriogenesis, but did not surpass single therapies with regard to contractility indexes. SVPs and CSCs secrete similar amounts of hepatocyte growth factor, vascular endothelial growth factor, fibroblast growth factor, stem cell factor, and stromal cell-derived factor-1, whereas SVPs release higher quantities of angiopoietins and microRNA-132. Coculture of the 2 cell populations results in competitive as well as enhancing paracrine activities. In particular, the release of stromal cell-derived factor-1 was synergistically augmented along with downregulation of stromal cell-derived factor-1-degrading enzyme dipeptidyl peptidase 4. CONCLUSIONS: Combinatory therapy with SVPs and CSCs may complementarily help the repair of infarcted hearts.


Assuntos
Infarto do Miocárdio/cirurgia , Miocárdio/patologia , Miócitos Cardíacos/transplante , Neovascularização Fisiológica , Pericitos/transplante , Regeneração , Transplante de Células-Tronco , Proteínas Angiogênicas/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Fibrose , Hemodinâmica , Humanos , Camundongos SCID , Contração Miocárdica , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Comunicação Parácrina , Pericitos/metabolismo , Fenótipo , Recuperação de Função Fisiológica , Veia Safena/citologia , Fatores de Tempo , Remodelação Ventricular
5.
JACC Basic Transl Sci ; 7(3): 207-219, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35411313

RESUMO

Surgical treatment of congenital heart defects affecting the right ventricular outflow tract often requires complex reconstruction and multiple reoperations. With a randomized controlled trial, we compared a novel tissue-engineered small intestine submucosa-based graft for pulmonary artery reconstruction (seeded with mesenchymal stem cells derived from Wharton's Jelly) with conventional small intestine submucosa in growing piglets. Six months after implantation, seeded grafts showed integration with host tissues at cellular level and exhibited growth potential on transthoracic echocardiography and cardiovascular magnetic resonance. Our seeded graft is a promising biomaterial for pulmonary artery reconstruction in pediatric patients with right ventricular outflow tract abnormalities.

6.
Antioxid Redox Signal ; 34(15): 1151-1164, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-33226850

RESUMO

Aims: To ascertain if human pericytes produce SPARC (acronym for Secreted Protein Acidic and Cysteine Rich), a matricellular protein implicated in the regulation of cell proliferation, migration, and cell-matrix interactions; clarify if SPARC expression in cardiac pericytes is modulated by hypoxia; and determine the functional consequences of SPARC silencing. Results: Starting from the recognition that the conditioned media (CM) of human pericytes promote proliferation and migration of cardiac stromal cells, we screened candidate mediators by mass-spectrometry analysis. Of the 14 high-confidence proteins (<1% FDR) identified in the bioactive fractions of the pericyte CM, SPARC emerged as the top-scored matricellular protein. SPARC expression was validated using ELISA and found to be upregulated by hypoxia/starvation in pericytes that express platelet-derived growth factor receptor α (PDGFRα). This subfraction is acknowledged to play a key role in extracellular matrix remodeling. Studies in patients with acute myocardial infarction showed that peripheral blood SPARC correlates with the levels of creatine kinase Mb, a marker of cardiac damage. Immunohistochemistry analyses of infarcted hearts revealed that SPARC is expressed in vascular and interstitial cells. Silencing of SPARC reduced the pericyte ability to secrete collagen1a1, without inhibiting the effects of CM on cardiac and endothelial cells. These data indicate that SPARC is enriched in the bioactive fraction of the pericyte CM, is induced by hypoxia and ischemia, and is essential for pericyte ability to produce collagen. Innovation: This study newly indicates that pericytes are a source of the matricellular protein SPARC. Conclusion: Modulation of SPARC production by pericytes may have potential implications for postinfarct healing.


Assuntos
Cadeia alfa 1 do Colágeno Tipo I/genética , Infarto do Miocárdio/genética , Miócitos Cardíacos/metabolismo , Osteonectina/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Hipóxia Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , Creatina Quinase Forma MB/genética , Células Endoteliais/metabolismo , Matriz Extracelular/genética , Regulação da Expressão Gênica/genética , Humanos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Miócitos Cardíacos/patologia , Pericitos/metabolismo , Secretoma/metabolismo
7.
Pharmacol Ther ; 171: 83-92, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27889329

RESUMO

The recent development of tissue engineering provides exciting new perspectives for the replacement of failing organs and the repair of damaged tissues. Perivascular cells, including vascular smooth muscle cells, pericytes and other tissue specific populations residing around blood vessels, have been isolated from many organs and are known to participate to the in situ repair process and angiogenesis. Their potential has been harnessed for cell therapy of numerous pathologies; however, in this Review we will discuss the potential of perivascular cells in the development of tissue engineering solutions for healthcare. We will examine their application in the engineering of vascular grafts, cardiac patches and bone substitutes as well as other tissue engineering applications and we will focus on their extensive use in the vascularization of engineered constructs. Additionally, we will discuss the emerging potential of human pericytes for the development of efficient, vascularized and non-immunogenic engineered constructs.


Assuntos
Vasos Sanguíneos/citologia , Pericitos/citologia , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/metabolismo , Terapia Baseada em Transplante de Células e Tecidos/métodos , Humanos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Neovascularização Fisiológica/fisiologia
8.
J Am Heart Assoc ; 4(6): e002043, 2015 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-26080813

RESUMO

BACKGROUND: Living grafts produced by combining autologous heart-resident stem/progenitor cells and tissue engineering could provide a new therapeutic option for definitive correction of congenital heart disease. The aim of the study was to investigate the antigenic profile, expansion/differentiation capacity, paracrine activity, and pro-angiogenic potential of cardiac pericytes and to assess their engrafting capacity in clinically certified prosthetic grafts. METHODS AND RESULTS: CD34(pos) cells, negative for the endothelial markers CD31 and CD146, were identified by immunohistochemistry in cardiac leftovers from infants and children undergoing palliative repair of congenital cardiac defects. Following isolation by immunomagnetic bead-sorting and culture on plastic in EGM-2 medium supplemented with growth factors and serum, CD34(pos)/CD31(neg) cells gave rise to a clonogenic, highly proliferative (>20 million at P5), spindle-shape cell population. The following populations were shown to expresses pericyte/mesenchymal and stemness markers. After exposure to differentiation media, the expanded cardiac pericytes acquired markers of vascular smooth muscle cells, but failed to differentiate into endothelial cells or cardiomyocytes. However, in Matrigel, cardiac pericytes form networks and enhance the network capacity of endothelial cells. Moreover, they produce collagen-1 and release chemo-attractants that stimulate the migration of c-Kit(pos) cardiac stem cells. Cardiac pericytes were then seeded onto clinically approved xenograft scaffolds and cultured in a bioreactor. After 3 weeks, fluorescent microscopy showed that cardiac pericytes had penetrated into and colonized the graft. CONCLUSIONS: These findings open new avenues for cellular functionalization of prosthetic grafts to be applied in reconstructive surgery of congenital heart disease.


Assuntos
Cardiopatias Congênitas/cirurgia , Pericitos/citologia , Engenharia Tecidual/métodos , Meios de Cultura , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Pericitos/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Células-Tronco/citologia , Células-Tronco/fisiologia , Transplante de Tecidos/métodos
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