RESUMEN
BACKGROUND: Inflammation contributes to the pathogenesis of heart failure, but there is limited understanding of inflammation's potential benefits. Inflammatory cells secrete MYDGF (myeloid-derived growth factor) to promote tissue repair after acute myocardial infarction. We hypothesized that MYDGF has a role in cardiac adaptation to persistent pressure overload. METHODS: We defined the cellular sources and function of MYDGF in wild-type (WT), Mydgf-deficient (Mydgf-/-), and Mydgf bone marrow-chimeric or bone marrow-conditional transgenic mice with pressure overload-induced heart failure after transverse aortic constriction surgery. We measured MYDGF plasma concentrations by targeted liquid chromatography-mass spectrometry. We identified MYDGF signaling targets by phosphoproteomics and substrate-based kinase activity inference. We recorded Ca2+ transients and sarcomere contractions in isolated cardiomyocytes. Additionally, we explored the therapeutic potential of recombinant MYDGF. RESULTS: MYDGF protein abundance increased in the left ventricular myocardium and in blood plasma of pressure-overloaded mice. Patients with severe aortic stenosis also had elevated MYDGF plasma concentrations, which declined after transcatheter aortic valve implantation. Monocytes and macrophages emerged as the main MYDGF sources in the pressure-overloaded murine heart. While Mydgf-/- mice had no apparent phenotype at baseline, they developed more severe left ventricular hypertrophy and contractile dysfunction during pressure overload than WT mice. Conversely, conditional transgenic overexpression of MYDGF in bone marrow-derived inflammatory cells attenuated pressure overload-induced hypertrophy and dysfunction. Mechanistically, MYDGF inhibited G protein-coupled receptor agonist-induced hypertrophy and augmented SERCA2a (sarco/endoplasmic reticulum Ca2+-ATPase 2a) expression in cultured neonatal rat ventricular cardiomyocytes by enhancing PIM1 (Pim-1 proto-oncogene, serine/threonine kinase) expression and activity. Along this line, cardiomyocytes from pressure-overloaded Mydgf-/- mice displayed reduced PIM1 and SERCA2a expression, greater hypertrophy, and impaired Ca2+ cycling and sarcomere function compared with cardiomyocytes from pressure-overloaded WT mice. Transplanting Mydgf-/- mice with WT bone marrow cells augmented cardiac PIM1 and SERCA2a levels and ameliorated pressure overload-induced hypertrophy and dysfunction. Pressure-overloaded Mydgf-/- mice were similarly rescued by adenoviral Serca2a gene transfer. Treating pressure-overloaded WT mice subcutaneously with recombinant MYDGF enhanced SERCA2a expression, attenuated left ventricular hypertrophy and dysfunction, and improved survival. CONCLUSIONS: These findings establish a MYDGF-based adaptive crosstalk between inflammatory cells and cardiomyocytes that protects against pressure overload-induced heart failure.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Retículo Endoplásmico/fisiología , Insuficiencia Cardíaca/terapia , Interleucinas/uso terapéutico , Miocitos Cardíacos/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Interleucinas/farmacología , RatonesRESUMEN
Myeloid-derived growth factor (MYDGF in humans, Mydgf in mice) is a secreted protein with previously unknown biological functions. In a recent study, Mydgf was shown to mediate cardiac repair after acute myocardial infarction (MI) in mice. Lack of a sensitive assay to measure MYDGF in the circulation has hampered its further investigation. Here, we developed a liquid chromatography/multiple reaction monitoring-mass spectrometry MYDGF assay, employing SDS-PAGE-based protein fractionation to deplete high-abundant proteins and a stable isotope-labeled synthetic standard peptide for quantification. The assay had a lower limit of quantification of 0.8 ng/mL and a linear range up to 190 ng/mL. Within-run and total imprecision ranged from 8 to 17% and 11 to 20%, respectively. MYDGF plasma concentrations were not affected by either storage at room temperature for 4 h or up to three freeze-thaw cycles. Apparently healthy adults presented with a median (range) MYDGF concentration of 3.3 (1.3-6.7) ng/mL ( n = 120). MYDGF concentrations were elevated 2.7-fold ( P < 0.001) in patients with acute MI ( n = 101) and were associated with inflammatory biomarkers, renal dysfunction, and long-term cardiovascular mortality. The new assay and the favorable preanalytic characteristics of the analyte will facilitate studies into the pathophysiology of MYDGF and its potential use as a biomarker or protein therapeutic in patients with acute MI or other disease states.
Asunto(s)
Cromatografía Liquida/métodos , Interleucinas/sangre , Espectrometría de Masas/métodos , Infarto del Miocardio/sangre , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Interleucinas/química , Masculino , Persona de Mediana Edad , Proteolisis , Tripsina/química , Adulto JovenRESUMEN
BACKGROUND: Clinical trials of bone marrow cell-based therapies after acute myocardial infarction (MI) have produced mostly neutral results. Treatment with specific bone marrow cell-derived secreted proteins may provide an alternative biological approach to improving tissue repair and heart function after MI. We recently performed a bioinformatic secretome analysis in bone marrow cells from patients with acute MI and discovered a poorly characterized secreted protein, EMC10 (endoplasmic reticulum membrane protein complex subunit 10), showing activity in an angiogenic screen. METHODS: We investigated the angiogenic potential of EMC10 and its mouse homolog (Emc10) in cultured endothelial cells and infarcted heart explants. We defined the cellular sources and function of Emc10 after MI using wild-type, Emc10-deficient, and Emc10 bone marrow-chimeric mice subjected to transient coronary artery ligation. Furthermore, we explored the therapeutic potential of recombinant Emc10 delivered by osmotic minipumps after MI in heart failure-prone FVB/N mice. RESULTS: Emc10 signaled through small GTPases, p21-activated kinase, and the p38 mitogen-activated protein kinase (MAPK)-MAPK-activated protein kinase 2 (MK2) pathway to promote actin polymerization and endothelial cell migration. Confirming the importance of these signaling events in the context of acute MI, Emc10 stimulated endothelial cell outgrowth from infarcted mouse heart explants via p38 MAPK-MK2. Emc10 protein abundance was increased in the infarcted region of the left ventricle and in the circulation of wild-type mice after MI. Emc10 expression was also increased in left ventricular tissue samples from patients with acute MI. Bone marrow-derived monocytes and macrophages were the predominant sources of Emc10 in the infarcted murine heart. Emc10 KO mice showed no cardiovascular phenotype at baseline. After MI, however, capillarization of the infarct border zone was impaired in KO mice, and the animals developed larger infarct scars and more pronounced left ventricular remodeling compared with wild-type mice. Transplanting KO mice with wild-type bone marrow cells rescued the angiogenic defect and ameliorated left ventricular remodeling. Treating FVB/N mice with recombinant Emc10 enhanced infarct border-zone capillarization and exerted a sustained beneficial effect on left ventricular remodeling. CONCLUSIONS: We have identified Emc10 as a previously unknown angiogenic growth factor that is produced by bone marrow-derived monocytes and macrophages as part of an endogenous adaptive response that can be enhanced therapeutically to repair the heart after MI.
Asunto(s)
Proteínas Angiogénicas/metabolismo , Células de la Médula Ósea/metabolismo , Proteínas de la Membrana/metabolismo , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Neovascularización Fisiológica , Cicatrización de Heridas , Proteínas Angiogénicas/administración & dosificación , Proteínas Angiogénicas/deficiencia , Proteínas Angiogénicas/genética , Animales , Trasplante de Médula Ósea , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Genotipo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/metabolismo , Proteínas de la Membrana/administración & dosificación , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Miocardio/patología , Neovascularización Fisiológica/efectos de los fármacos , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Factores de Tiempo , Cicatrización de Heridas/efectos de los fármacos , Quinasas p21 Activadas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Large clostridial toxins mono-O-glucosylate small GTPases of the Rho and Ras subfamily. As a result of glucosylation, the GTPases are inhibited and thereby corresponding downstream signaling pathways are disturbed. Current methods for quantifying the extent of glucosylation include sequential [14 C]glucosylation, sequential [32 P]ADP-ribosylation, and Western Blot detection of nonglucosylated GTPases, with neither method allowing the quantification of the extent of glucosylation of an individual GTPase. Here, we describe a novel MS-based multiplexed MRM assay to specifically quantify the glucosylation degree of small GTPases. This targeted proteomics approach achieves a high selectivity and reproducibility, which allows determination of the in vivo substrate pattern of glucosylating toxins. As proof of principle, GTPase glucosylation was analyzed in CaCo-2 cells treated with TcdA, and glucosylation kinetics were determined for RhoA/B, RhoC, RhoG, Ral, Rap1, Rap2, (H/K/N)Ras, and R-Ras2.
Asunto(s)
Toxinas Bacterianas/análisis , Espectrometría de Masas/métodos , Proteínas de Unión al GTP Monoméricas/análisis , Proteómica/métodos , Células CACO-2 , Cromatografía Liquida , Glicosilación , Humanos , Transducción de SeñalRESUMEN
Tissue repair after myocardial infarction (MI) is guided by autocrine and paracrine-acting proteins. Deciphering these signals and their upstream triggers is essential when considering infarct healing as a therapeutic target. Here we perform a bioinformatic secretome analysis in mouse cardiac endothelial cells and identify cysteine-rich with EGF-like domains 2 (CRELD2), an endoplasmic reticulum stress-inducible protein with poorly characterized function. CRELD2 was abundantly expressed and secreted in the heart after MI in mice and patients. Creld2-deficient mice and wild-type mice treated with a CRELD2-neutralizing antibody showed impaired de novo microvessel formation in the infarct border zone and developed severe postinfarction heart failure. CRELD2 protein therapy, conversely, improved heart function after MI. Exposing human coronary artery endothelial cells to recombinant CRELD2 induced angiogenesis, associated with a distinct phosphoproteome signature. These findings identify CRELD2 as an angiogenic growth factor and unravel a link between endoplasmic reticulum stress and ischemic tissue repair.
Asunto(s)
Estrés del Retículo Endoplásmico , Células Endoteliales , Infarto del Miocardio , Neovascularización Fisiológica , Animales , Humanos , Masculino , Ratones , Inductores de la Angiogénesis/farmacología , Inductores de la Angiogénesis/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Neovascularización Fisiológica/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Effective tissue repair after myocardial infarction entails a vigorous angiogenic response, guided by incompletely defined immune cell-endothelial cell interactions. We identify the monocyte- and macrophage-derived cytokine METRNL (meteorin-like) as a driver of postinfarction angiogenesis and high-affinity ligand for the stem cell factor receptor KIT (KIT receptor tyrosine kinase). METRNL mediated angiogenic effects in cultured human endothelial cells through KIT-dependent signaling pathways. In a mouse model of myocardial infarction, METRNL promoted infarct repair by selectively expanding the KIT-expressing endothelial cell population in the infarct border zone. Metrnl-deficient mice failed to mount this KIT-dependent angiogenic response and developed severe postinfarction heart failure. Our data establish METRNL as a KIT receptor ligand in the context of ischemic tissue repair.
Asunto(s)
Adipoquinas , Citocinas , Infarto del Miocardio , Neovascularización Fisiológica , Factores de Crecimiento Nervioso , Proteínas Proto-Oncogénicas c-kit , Animales , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Células Endoteliales/metabolismo , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/genética , Ligandos , Macrófagos/metabolismo , Ratones , Ratones Mutantes , Infarto del Miocardio/complicaciones , Infarto del Miocardio/fisiopatología , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismoRESUMEN
Glycoprotein G (gG) from herpes simplex virus type 1 and 2 (HSV-1 and HSV-2, respectively) functions as a viral chemokine binding protein (vCKBP). Soluble recombinant forms of gG of HSV-1 and HSV-2 (SgG1 and SgG2, respectively) enhance chemokine-mediated leukocyte migration, in contrast to most known vCKBPs, including those from animal alpha-herpesviruses. Furthermore, both proteins bind to nerve growth factor (NGF), but only SgG2 enhances NGF-dependent neurite outgrowth. The basis and implications of this functional difference between the two proteins are still unknown. While gG1 and gG2 are positional homologues in the genome, they share very limited sequence homology. In fact, US4, the open reading frame encoding gG is the most divergent genetic locus between these viruses. Full-length gG1 and gG2 are type I transmembrane proteins located on the plasma membrane of infected cells and at the viral envelope. However, gG2 is larger than gG1 and is cleaved during protein maturation, secreting the N-terminal domain to the supernatant of infected cells, whereas gG1 is not. The enzyme involved in gG2 cleavage and the functional relevance of gG2 cleavage and secretion are unknown. We aim to identify the gG2 sequence required for cleavage to determine its functional role in future experiments. Our results prove the existence of at least two cleavage motifs in gG2 within the amino acid region 314-343. Transfer of this sequence to a fusion protein results in cleavage. Finally, we show that propeptide convertases like furin are responsible for gG2 cleavage.
Asunto(s)
Herpes Simple/virología , Herpesvirus Humano 2/fisiología , Dominios y Motivos de Interacción de Proteínas , Proteínas del Envoltorio Viral/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular , Cromatografía Liquida , Expresión Génica , Genes Reporteros , Humanos , Espectrometría de Masas , ProteolisisRESUMEN
Aiming at clinical translation, robust directed differentiation of human pluripotent stem cells (hPSCs), preferentially in chemically defined conditions, is a key requirement. Here, feasibility of suspension culture based hPSC-cardiomyocyte (hPSC-CM) production in low-cost, xeno-free media compatible with good manufacturing practice standards is shown. Applying stirred tank bioreactor systems at increasing dimensions, our advanced protocol enables routine production of about 1 million hPSC-CMs/mL, yielding â¼1.3 × 108 CM in 150 mL and â¼4.0 × 108 CMs in 350-500 mL process scale at >90% lineage purity. Process robustness and efficiency is ensured by uninterrupted chemical WNT pathway control at early stages of differentiation and results in the formation of almost exclusively ventricular-like CMs. Modulated WNT pathway regulation also revealed the previously unappreciated role of ROR1/CD13 as superior surrogate markers for predicting cardiac differentiation efficiency as soon as 72 h of differentiation. This monitoring strategy facilitates process upscaling and controlled mass production of hPSC derivatives.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Medios de Cultivo/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Reactores Biológicos , Antígenos CD13/genética , Antígenos CD13/metabolismo , Técnicas de Cultivo de Célula/métodos , Medios de Cultivo/química , Humanos , Mesodermo/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismoRESUMEN
Paracrine-acting proteins are emerging as a central mechanism by which bone marrow cell-based therapies improve tissue repair and heart function after myocardial infarction (MI). We carried out a bioinformatic secretome analysis in bone marrow cells from patients with acute MI to identify novel secreted proteins with therapeutic potential. Functional screens revealed a secreted protein encoded by an open reading frame on chromosome 19 (C19orf10) that promotes cardiac myocyte survival and angiogenesis. We show that bone marrow-derived monocytes and macrophages produce this protein endogenously to protect and repair the heart after MI, and we named it myeloid-derived growth factor (MYDGF). Whereas Mydgf-deficient mice develop larger infarct scars and more severe contractile dysfunction compared to wild-type mice, treatment with recombinant Mydgf reduces scar size and contractile dysfunction after MI. This study is the first to assign a biological function to MYDGF, and it may serve as a prototypical example for the development of protein-based therapies for ischemic tissue repair.