RESUMEN
Resident macrophages densely populate the normal arterial wall, yet their origins and the mechanisms that sustain them are poorly understood. Here we use gene-expression profiling to show that arterial macrophages constitute a distinct population among macrophages. Using multiple fate-mapping approaches, we show that arterial macrophages arise embryonically from CX3CR1(+) precursors and postnatally from bone marrow-derived monocytes that colonize the tissue immediately after birth. In adulthood, proliferation (rather than monocyte recruitment) sustains arterial macrophages in the steady state and after severe depletion following sepsis. After infection, arterial macrophages return rapidly to functional homeostasis. Finally, survival of resident arterial macrophages depends on a CX3CR1-CX3CL1 axis within the vascular niche.
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Autorrenovación de las Células , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Monocitos/citología , Monocitos/metabolismo , Receptores de Quimiocina/metabolismo , Animales , Receptor 1 de Quimiocinas CX3C , Supervivencia Celular , Quimiocina CX3CL1/metabolismo , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Inmunofenotipificación , Macrófagos/inmunología , Macrófagos/microbiología , Masculino , Ratones , Ratones Transgénicos , Fenotipo , Unión Proteica , Nicho de Células Madre , TranscriptomaRESUMEN
Adverse cardiac outcomes in COVID-19 patients, particularly those with preexisting cardiac disease, motivate the development of human cell-based organ-on-a-chip models to recapitulate cardiac injury and dysfunction and for screening of cardioprotective therapeutics. Here, we developed a heart-on-a-chip model to study the pathogenesis of SARS-CoV-2 in healthy myocardium established from human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and a cardiac dysfunction model, mimicking aspects of preexisting hypertensive disease induced by angiotensin II (Ang II). We recapitulated cytopathic features of SARS-CoV-2-induced cardiac damage, including progressively impaired contractile function and calcium handling, apoptosis, and sarcomere disarray. SARS-CoV-2 presence in Ang II-treated hearts-on-a-chip decreased contractile force with earlier onset of contractile dysfunction and profoundly enhanced inflammatory cytokines compared to SARS-CoV-2 alone. Toward the development of potential therapeutics, we evaluated the cardioprotective effects of extracellular vesicles (EVs) from human iPSC which alleviated the impairment of contractile force, decreased apoptosis, reduced the disruption of sarcomeric proteins, and enhanced beta-oxidation gene expression. Viral load was not affected by either Ang II or EV treatment. We identified MicroRNAs miR-20a-5p and miR-19a-3p as potential mediators of cardioprotective effects of these EVs.
Asunto(s)
Angiotensina II , COVID-19 , Células Madre Pluripotentes Inducidas , Dispositivos Laboratorio en un Chip , Miocitos Cardíacos , Humanos , Angiotensina II/farmacología , Apoptosis/efectos de los fármacos , COVID-19/virología , COVID-19/metabolismo , Citocinas/metabolismo , Vesículas Extracelulares/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , MicroARNs/metabolismo , MicroARNs/genética , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/virología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , SARS-CoV-2/fisiologíaRESUMEN
The prognosis and treatment outcomes of heart failure (HF) patients rely heavily on disease etiology, yet the majority of underlying signaling mechanisms are complex and not fully elucidated. Phosphorylation is a major point of protein regulation with rapid and profound effects on the function and activity of protein networks. Currently, there is a lack of comprehensive proteomic and phosphoproteomic studies examining cardiac tissue from HF patients with either dilated dilated cardiomyopathy (DCM) or ischemic cardiomyopathy (ICM). Here, we used a combined proteomic and phosphoproteomic approach to identify and quantify more than 5,000 total proteins with greater than 13,000 corresponding phosphorylation sites across explanted left ventricle (LV) tissue samples, including HF patients with DCM vs. nonfailing controls (NFC), and left ventricular infarct vs. noninfarct, and periinfarct vs. noninfarct regions of HF patients with ICM. Each pair-wise comparison revealed unique global proteomic and phosphoproteomic profiles with both shared and etiology-specific perturbations. With this approach, we identified a DCM-associated hyperphosphorylation cluster in the cardiomyocyte intercalated disc (ICD) protein, αT-catenin (CTNNA3). We demonstrate using both ex vivo isolated cardiomyocytes and in vivo using an AAV9-mediated overexpression mouse model, that CTNNA3 phosphorylation at these residues plays a key role in maintaining protein localization at the cardiomyocyte ICD to regulate conductance and cell-cell adhesion. Collectively, this integrative proteomic/phosphoproteomic approach identifies region- and etiology-associated signaling pathways in human HF and describes a role for CTNNA3 phosphorylation in the pathophysiology of DCM.
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Cardiomiopatía Dilatada , Insuficiencia Cardíaca , Animales , Ratones , Humanos , Cardiomiopatía Dilatada/metabolismo , Ventrículos Cardíacos/metabolismo , Fosforilación , Proteómica , Miocardio/metabolismo , Insuficiencia Cardíaca/metabolismo , alfa Catenina/metabolismoRESUMEN
Heart disease remains a leading cause of death in North America and worldwide. Despite advances in therapies, the chronic nature of cardiovascular diseases ultimately results in frequent hospitalizations and steady rates of mortality. Systems biology approaches have provided a new frontier toward unraveling the underlying mechanisms of cell, tissue, and organ dysfunction in disease. Mapping the complex networks of molecular functions across the genome, transcriptome, proteome, and metabolome has enormous potential to advance our understanding of cardiovascular disease, discover new disease biomarkers, and develop novel therapies. Computational workflows to interpret these data-intensive analyses as well as integration between different levels of interrogation remain important challenges in the advancement and application of systems biology-based analyses in cardiovascular research. This review will focus on summarizing the recent developments in network biology-level profiling in the heart, with particular emphasis on modeling of human heart failure. We will provide new perspectives on integration between different levels of large "omics" datasets, including integration of gene regulatory networks, protein-protein interactions, signaling networks, and metabolic networks in the heart.
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Enfermedades Cardiovasculares , Humanos , Enfermedades Cardiovasculares/genética , Multiómica , Biología de Sistemas , Genoma , Metaboloma , Biología Computacional/métodosRESUMEN
Heart failure (HF) is associated with pathological remodeling of the myocardium, including the initiation of fibrosis and scar formation by activated cardiac fibroblasts (CFs). Although early CF-dependent scar formation helps prevent cardiac rupture by maintaining the heart's structural integrity, ongoing deposition of the extracellular matrix in the remote and infarct regions can reduce tissue compliance, impair cardiac function, and accelerate progression to HF. In our study, we conducted mass spectrometry (MS) analysis to identify differentially altered proteins and signaling pathways between CFs isolated from 7 day sham and infarcted murine hearts. Surprisingly, CFs from both the remote and infarct regions of injured hearts had a wide number of similarly altered proteins and signaling pathways that were consistent with fibrosis and activation into pathological myofibroblasts. Specifically, proteins enriched in CFs isolated from MI hearts were involved in pathways pertaining to cell-cell and cell-matrix adhesion, chaperone-mediated protein folding, and collagen fibril organization. These results, together with principal component analyses, provided evidence of global CF activation postinjury. Interestingly, however, direct comparisons between CFs from the remote and infarct regions of injured hearts identified 15 differentially expressed proteins between MI remote and MI infarct CFs. Eleven of these proteins (Gpc1, Cthrc1, Vmac, Nexn, Znf185, Sprr1a, Specc1, Emb, Limd2, Pawr, and Mcam) were higher in MI infarct CFs, whereas four proteins (Gstt1, Gstm1, Tceal3, and Inmt) were higher in MI remote CFs. Collectively, our study shows that MI injury induced global changes to the CF proteome, with the magnitude of change reflecting their relative proximity to the site of injury.
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Infarto del Miocardio , Remodelación Ventricular , Animales , Modelos Animales de Enfermedad , Fibroblastos/patología , Fibrosis , Proteínas con Dominio LIM , Ratones , Proteínas de Microfilamentos , Infarto del Miocardio/genética , Miocardio/patología , Miofibroblastos/patologíaRESUMEN
Cardiovascular diseases remain the most rapidly rising contributing factor of all-cause mortality and the leading cause of inpatient hospitalization worldwide, with costs exceeding $30 billion annually in North America. Cell surface and membrane-associated proteins play an important role in cardiomyocyte biology and are involved in the pathogenesis of many human heart diseases. In cardiomyocytes, membrane proteins serve as critical signaling receptors, Ca2+ cycling regulators, and electrical propagation regulators, all functioning in concert to maintain spontaneous and synchronous contractions of cardiomyocytes. Membrane proteins are excellent pharmaceutical targets due to their uniquely exposed position within the cell. Perturbations in cardiac membrane protein localization and function have been implicated in the progression and pathogenesis of many heart diseases. However, previous attempts at profiling the cardiac membrane proteome have yielded limited results due to poor technological developments for isolating hydrophobic, low-abundance membrane proteins. Comprehensive mapping and characterization of the cardiac membrane proteome thereby remains incomplete. This review will focus on recent advances in mapping the cardiac membrane proteome and the role of novel cardiac membrane proteins in the healthy and the diseased heart.
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Membrana Celular/metabolismo , Cardiopatías/metabolismo , Proteínas de la Membrana/metabolismo , Miocitos Cardíacos/metabolismo , Proteómica , Animales , Difusión de Innovaciones , Predicción , Cardiopatías/patología , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Miocitos Cardíacos/patología , Proteómica/historia , Proteómica/tendenciasRESUMEN
Phospholamban (PLN) plays a central role in Ca2+ homeostasis in cardiac myocytes through regulation of the sarco(endo)plasmic reticulum Ca2+-ATPase 2A (SERCA2A) Ca2+ pump. An inherited mutation converting arginine residue 9 in PLN to cysteine (R9C) results in dilated cardiomyopathy (DCM) in humans and transgenic mice, but the downstream signaling defects leading to decompensation and heart failure are poorly understood. Here we used precision mass spectrometry to study the global phosphorylation dynamics of 1,887 cardiac phosphoproteins in early affected heart tissue in a transgenic R9C mouse model of DCM compared with wild-type littermates. Dysregulated phosphorylation sites were quantified after affinity capture and identification of 3,908 phosphopeptides from fractionated whole-heart homogenates. Global statistical enrichment analysis of the differential phosphoprotein patterns revealed selective perturbation of signaling pathways regulating cardiovascular activity in early stages of DCM. Strikingly, dysregulated signaling through the Notch-1 receptor, recently linked to cardiomyogenesis and embryonic cardiac stem cell development and differentiation but never directly implicated in DCM before, was a prominently perturbed pathway. We verified alterations in Notch-1 downstream components in early symptomatic R9C transgenic mouse cardiomyocytes compared with wild type by immunoblot analysis and confocal immunofluorescence microscopy. These data reveal unexpected connections between stress-regulated cell signaling networks, specific protein kinases, and downstream effectors essential for proper cardiac function.
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Cardiomiopatía Dilatada/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Transducción de Señal , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Cardiomiopatía Dilatada/genética , Modelos Animales de Enfermedad , Humanos , Ratones Transgénicos , Mutación , Miocardio/metabolismo , Miocardio/patología , Fosfoproteínas/genética , Fosforilación , Proteoma/genéticaRESUMEN
The mammalian nucleus has invaginations from the cytoplasm, termed nucleoplasmic reticulum (NR). With increased resolution of cellular imaging, progress has been made in understanding the formation and function of NR. In fact, nucleoplasmic Ca2+ homeostasis has been implicated in the regulation of gene expression, DNA repair, and cell death. However, the majority of studies focus on cross-sectional or single-plane analyses of NR invaginations, providing an incomplete assessment of its distribution and content. Here, we provided advanced imaging and three-dimensional reconstructive analyses characterizing the molecular constituents of nuclear invaginations in the nucleoplasm in HEK293 cells, murine C2C12 muscle cells, and cardiac myocytes. We demonstrated the presence of critical Ca2+ regulatory channels, including sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a), stromal interaction molecule 1 (STIM1), and Ca2+ release-activated Ca2+ channel protein 1 (ORAI1), in the nucleoplasm in isolated primary mouse cardiomyocytes. We have shown for the first time the presence of STIM1 and ORAI1 in the nucleoplasm, suggesting the presence of store-operated calcium entry (SOCE) mechanism in nucleoplasmic Ca2+ regulation. These results show that nucleoplasmic invaginations contain continuous endoplasmic reticulum components, mitochondria, and intact nuclear membranes, highlighting the extremely detailed and complex nature of this organellar structure.
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Retículo Endoplásmico/ultraestructura , Imagenología Tridimensional/métodos , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Mitocondrias Cardíacas/ultraestructura , Mioblastos/ultraestructura , Miocitos Cardíacos/ultraestructura , Membrana Nuclear/ultraestructura , Retículo Sarcoplasmático/ultraestructura , Animales , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Señalización del Calcio , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Masculino , Ratones , Mitocondrias Cardíacas/metabolismo , Mioblastos/metabolismo , Miocitos Cardíacos/metabolismo , Membrana Nuclear/metabolismo , Proteína ORAI1/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Molécula de Interacción Estromal 1/metabolismo , TransfecciónRESUMEN
Phospholamban (PLN) is an effective inhibitor of the sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA). Here, we examined PLN stability and degradation in primary cultured mouse neonatal cardiomyocytes (CMNCs) and mouse hearts using immunoblotting, molecular imaging, and [(35)S]methionine pulse-chase experiments, together with lysosome (chloroquine and bafilomycin A1) and autophagic (3-methyladenine and Atg5 siRNA) antagonists. Inhibiting lysosomal and autophagic activities promoted endogenous PLN accumulation, whereas accelerating autophagy with metformin enhanced PLN degradation in CMNCs. This reduction in PLN levels was functionally correlated with an increased rate of SERCA2a activity, accounting for an inotropic effect of metformin. Metabolic labeling reaffirmed that metformin promoted wild-type and R9C PLN degradation. Immunofluorescence showed that PLN and the autophagy marker, microtubule light chain 3, became increasingly colocalized in response to chloroquine and bafilomycin treatments. Mechanistically, pentameric PLN was polyubiquitinylated at the K3 residue and this modification was required for p62-mediated selective autophagy trafficking. Consistently, attenuated autophagic flux in HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1-null mouse hearts was associated with increased PLN levels determined by immunoblots and immunofluorescence. Our study identifies a biological mechanism that traffics PLN to the lysosomes for degradation in mouse hearts.
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Autofagia , Proteínas de Unión al Calcio/metabolismo , Metformina/farmacología , Miocitos Cardíacos/efectos de los fármacos , Animales , Células HEK293 , Humanos , Lisosomas/metabolismo , Ratones , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Proteolisis , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/fisiología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/fisiología , UbiquitinaciónRESUMEN
Cardiac fibroblasts (CFs) represent a major subpopulation of cells in the developing and adult heart. Cardiomyocyte (CM) and CF intercellular communication occurs through paracrine interactions and modulate myocyte development and stress response. Detailed proteomic analysis of the CF secretome in normal and stressed conditions may offer insights into the role of CF in heart development and disease. Primary neonatal mouse CFs were isolated and cultured for 24 h in 21% (normoxic) or 2% (hypoxic) O2. Conditioned medium was separated to obtain exosomes (EXO) and EXO-depleted secretome fractions. Multidimensional protein identification technology was performed on secreted fractions. Whole cell lysate data were also generated to provide subcellular context to the secretome. Proteomic analysis identified 6163 unique proteins in total. Statistical (QSpec) analysis identified 494 proteins differentially expressed between fractions and oxygen conditions. Gene Ontology enrichment analysis revealed hypoxic conditions selectively increase expression of proteins with extracellular matrix and signaling annotations. Finally, we subjected CM pretreated with CF secreted factors to hypoxia/reoxygenation. Viability assays suggested altered viability due to CF-derived factors. CF secretome proteomics revealed differential expression based on mode of secretion and oxygen levels in vitro.
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Fibroblastos/metabolismo , Hipoxia/patología , Miocardio/citología , Animales , Animales Recién Nacidos , Células Cultivadas , Exosomas/metabolismo , Fibroblastos/patología , Ratones , Miocardio/química , Oxígeno/farmacología , Proteínas/análisis , Proteínas/efectos de los fármacos , Proteoma/efectos de los fármacos , Proteoma/metabolismoRESUMEN
In this study we carried out a mass spectrometry-based proteome analysis of human fetal atria and ventricles. Heart protein lysates were analyzed on the Q-Exactive mass spectrometer in biological triplicates. Protein identification using MaxQuant yielded a total of 2754 atrial protein groups (91%) and 2825 ventricular protein groups (83%) in at least 2 of the 3 runs with ≥ 2 unique peptides. Statistical analyses using fold-enrichment (>2) and p-values (≤ 0.05) selected chamber-enriched atrial (134) and ventricular (81) protein groups. Several previously characterized cardiac chamber-enriched proteins were identified in this study including atrial isoform of myosin light chain 2 (MYL7), atrial natriuretic peptide (NPPA), connexin 40 (GJA5), and peptidylglycine alpha-amidating monooxygenase (PAM) for atria, and ventricular isoforms of myosin light chains (MYL2 and MYL3), myosin heavy chain 7 (MYH7), and connexin 43 (GJA1) for ventricle. Our data was compared to in-house generated and publicly available human microarrays, several human cardiac proteomes, and phenotype ontology databases.
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Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/metabolismo , Proteínas Musculares/metabolismo , Proteoma/metabolismo , Feto/metabolismo , Expresión Génica , Humanos , Proteínas Musculares/genética , Especificidad de Órganos , Proteoma/genética , ProteómicaRESUMEN
Calnexin (CNX) is an endoplasmic reticulum (ER) quality control chaperone that has been implicated in ER stress. ER stress is a prominent pathological feature of various pathologic conditions, including cardiovascular diseases. However, the role of CNX and ER stress has not been studied in the heart. In the present study, we aimed to characterize the role of CNX in cardiomyocyte physiology with respect to ER stress, apoptosis, and cardiomyocyte Ca(2+) cycling. We demonstrated significantly decreased CNX mRNA and protein levels by LentiVector mediated transduction of targeting shRNAs. CNX silenced cardiomyocytes exhibited ER stress as evidenced by increased GRP78 and ATF6 protein levels, increased levels of spliced XBP1 mRNA, ASK-1, ERO1a, and CHOP mRNA levels. CNX silencing also led to significant activation of caspases-3 and -9. This activation of caspases was associated with hallmark morphological features of apoptosis including loss of sarcomeric organization and nuclear integrity. Ca(2+) imaging in live cells showed that CNX silencing resulted in Ca(2+) transients with significantly larger amplitudes but decreased frequency and Ca(2+) uptake rates in the basal state. Interestingly, 5 mM caffeine stimulated Ca(2+) transients were similar between control and CNX silenced cardiomyocytes. Finally, we demonstrated that CNX silencing induced the expression of the L-type voltage dependent calcium channel (CAV1.2) but reduced the expression of the sarcoplasmic reticulum ATPase (SERCA2a). In conclusion, this is the first study to demonstrate CNX has a specific role in cardiomyocyte viability and Ca(2+) cycling through its effects on ER stress, apoptosis and Ca(2+) channel expression.
Asunto(s)
Apoptosis , Señalización del Calcio , Calnexina/metabolismo , Estrés del Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Miocitos Cardíacos/metabolismo , Interferencia de ARN , Factor de Transcripción Activador 6/genética , Factor de Transcripción Activador 6/metabolismo , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Cafeína/farmacología , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio/efectos de los fármacos , Calnexina/genética , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Supervivencia Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/patología , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Vectores Genéticos , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Lentivirus/genética , MAP Quinasa Quinasa Quinasa 5/genética , MAP Quinasa Quinasa Quinasa 5/metabolismo , Ratones , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Oxidorreductasas , ARN Mensajero/metabolismo , Factores de Transcripción del Factor Regulador X , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Factores de Tiempo , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transducción Genética , Transfección , Proteína 1 de Unión a la X-BoxRESUMEN
Current protocols for the screening of prostate cancer cannot accurately discriminate clinically indolent tumors from more aggressive ones. One reliable indicator of outcome has been the determination of organ-confined versus nonorgan-confined disease but even this determination is often only made following prostatectomy. This underscores the need to explore alternate avenues to enhance outcome prediction of prostate cancer patients. Fluids that are proximal to the prostate, such as expressed prostatic secretions (EPS), are attractive sources of potential prostate cancer biomarkers as these fluids likely bathe the tumor. Direct-EPS samples from 16 individuals with extracapsular (n = 8) or organ-confined (n = 8) prostate cancer were used as a discovery cohort, and were analyzed in duplicate by a nine-step MudPIT on a LTQ-Orbitrap XL mass spectrometer. A total of 624 unique proteins were identified by at least two unique peptides with a 0.2% false discovery rate. A semiquantitative spectral counting algorithm identified 133 significantly differentially expressed proteins in the discovery cohort. Integrative data mining prioritized 14 candidates, including two known prostate cancer biomarkers: prostate-specific antigen and prostatic acid phosphatase, which were significantly elevated in the direct-EPS from the organ-confined cancer group. These and five other candidates (SFN, MME, PARK7, TIMP1, and TGM4) were verified by Western blotting in an independent set of direct-EPS from patients with biochemically recurrent disease (n = 5) versus patients with no evidence of recurrence upon follow-up (n = 10). Lastly, we performed proof-of-concept SRM-MS-based relative quantification of the five candidates using unpurified heavy isotope-labeled synthetic peptides spiked into pools of EPS-urines from men with extracapsular and organ-confined prostate tumors. This study represents the first efforts to define the direct-EPS proteome from two major subclasses of prostate cancer using shotgun proteomics and verification in EPS-urine by SRM-MS.
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Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas de Secreción Prostática/análisis , Proteínas de Secreción Prostática/orina , Proteínas 14-3-3/análisis , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Exonucleasas/análisis , Exorribonucleasas , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/análisis , Marcaje Isotópico , Masculino , Proteínas Oncogénicas/análisis , Antígeno Prostático Específico/metabolismo , Análisis por Matrices de Proteínas , Proteína Desglicasa DJ-1 , Proteoma/análisis , Inhibidor Tisular de Metaloproteinasa-1/análisis , Transglutaminasas/análisisRESUMEN
The ex vivo endothelialization of small diameter vascular prostheses can prolong their patency. Here, we demonstrate that heterotypic interactions between human adipose tissue-derived endothelial cells and perivascular cells can be exploited to accelerate the endothelialization of an electrospun ionomeric polyurethane scaffold. The scaffold was used to physically separate endothelial cells from perivascular cells to prevent their diffuse neo-intimal hyperplasia and spontaneous tubulogenesis, yet enable their paracrine cross-talk to accelerate the integration of the endothelial cells into a temporally stable endothelial lining of a continuous, elongated, and aligned morphology. Perivascular cells stimulated endothelial basement membrane protein production and suppressed their angiogenic and inflammatory activation to accelerate this biomimetic morphogenesis of the endothelium. These findings demonstrate the feasibility and underscore the value of exploiting heterotypic interactions between endothelial cells and perivascular cells for the fabrication of an endothelial lining intended for small diameter arterial reconstruction. STATEMENT OF SIGNIFICANCE: Adipose tissue is an abundant, accessible, and uniquely dispensable source of endothelial cells and perivascular cells for vascular tissue engineering. While their spontaneous self-assembly into microvascular networks is routinely exploited for the vascularization of engineered tissues, it threatens the temporal stability of an endothelial lining intended for small diameter arterial reconstruction. Here, we demonstrate that an electrospun polyurethane scaffold can be used to physically separate endothelial cells from perivascular cells to prevent their spontaneous capillary morphogenesis, yet enable their cross-talk to promote the formation of a stable endothelium. Our findings demonstrate the feasibility of engineering an endothelial lining from human adipose tissue, poising it for the rapid ex vivo endothelialization of small diameter vascular prostheses in an autologous, patient-specific manner.
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Células Endoteliales , Poliuretanos , Humanos , Poliuretanos/metabolismo , Endotelio , Tejido Adiposo/metabolismo , Ingeniería de Tejidos , Prótesis VascularRESUMEN
Cell-based models that mimic in vivo heart physiology are poised to make significant advances in cardiac disease modeling and drug discovery. In these systems, cardiomyocyte (CM) contractility is an important functional metric, but current measurement methods are inaccurate and low-throughput or require complex setups. To address this need, we developed a standalone noninvasive, label-free ultrasound technique operating at 40-200 MHz to measure the contractile kinetics of cardiac models, ranging from single adult CMs to 3D microtissue constructs in standard cell culture formats. The high temporal resolution of 1000 fps resolved the beat profile of single mouse CMs paced at up to 9 Hz, revealing limitations of lower speed optical based measurements to resolve beat kinetics or characterize aberrant beats. Coupling of ultrasound with traction force microscopy enabled the measurement of the CM longitudinal modulus and facile estimation of adult mouse CM contractile forces of 2.34 ± 1.40 µN, comparable to more complex measurement techniques. Similarly, the beat rate, rhythm, and drug responses of CM spheroid and microtissue models were measured, including in configurations without optical access. In conclusion, ultrasound can be used for the rapid characterization of CM contractile function in a wide range of commonly studied configurations ranging from single cells to 3D tissue constructs using standard well plates and custom microdevices, with applications in cardiac drug discovery and cardiotoxicity evaluation.
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Células Madre Pluripotentes Inducidas , Ratones , Animales , Miocitos Cardíacos , Células Cultivadas , Descubrimiento de Drogas , Dispositivos Laboratorio en un ChipRESUMEN
The exosome is a secreted microvesicle that has been shown to contain genetic material and proteins and is involved in multiple levels of cellular communication. The cardiovascular exosome proteome is a promising subproteome that warrants investigation since a detailed understanding of its role in the heart should improve our comprehension of intercellular communication in the heart, and may even assist in biomarker discovery. Indeed, uncovering the role of the exosome in cardiovascular physiology could be accomplished with the application of scientific approaches and insights gained from studies of exosomes in other fields, such as cancer biology and immunology, where much of the established knowledge of the exosome has been generated. In the present review, we discuss the relevant literature and examine areas of investigation that would bring the cardiovascular exosome to the forefront of intercellular communication in the heart.
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Enfermedades Cardiovasculares/metabolismo , Exosomas/metabolismo , Proteoma/metabolismo , Animales , Enfermedades Cardiovasculares/tratamiento farmacológico , Comunicación Celular , Portadores de Fármacos/metabolismo , Humanos , Miocardio/metabolismo , Proteoma/aislamiento & purificaciónRESUMEN
In-depth proteomic analyses offer a systematic way to investigate protein alterations in disease and, as such, can be a powerful tool for the identification of novel biomarkers. Here, we analyzed proteomic data from a transgenic mouse model with cardiac-specific overexpression of activated calcineurin (CnA), which results in severe cardiac hypertrophy. We applied statistically filtering and false discovery rate correction methods to identify 52 proteins that were significantly different in the CnA hearts compared to controls. Subsequent informatic analysis consisted of comparison of these 52 CnA proteins to another proteomic dataset of heart failure, three available independent microarray datasets, and correlation of their expression with the human plasma and urine proteome. Following this filtering strategy, four proteins passed these selection criteria, including myosin heavy chain 7, insulin-like growth factor-binding protein 7, annexin A2, and desmin. We assessed expression levels of these proteins in mouse plasma by immunoblotting, and observed significantly different levels of expression between healthy and failing mice for all four proteins. We verified antibody cross-reactivity by examining human cardiac explant tissue by immunoblotting. Finally, we assessed protein levels in plasma samples obtained from four unaffected and four heart failure patients and demonstrated that all four proteins increased between twofold and 150-fold in heart failure. We conclude that MYH7, IGFBP7, ANXA2, and DESM are all excellent candidate plasma biomarkers of heart failure in mouse and human.
Asunto(s)
Anexina A2/sangre , Desmina/sangre , Insuficiencia Cardíaca/sangre , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Cadenas Pesadas de Miosina/sangre , Animales , Biomarcadores/sangre , Calcineurina/genética , Calcineurina/metabolismo , Análisis por Conglomerados , Bases de Datos Factuales , Modelos Animales de Enfermedad , Ventrículos Cardíacos/química , Humanos , Ratones , Ratones Transgénicos , Miocardio/química , Neoplasias/metabolismo , Proyectos Piloto , ProteómicaRESUMEN
Preeclampsia (PE) adversely impacts ~5% of pregnancies. Despite extensive research, no consistent biomarkers or cures have emerged, suggesting that different molecular mechanisms may cause clinically similar disease. To address this, we undertook a proteomics study with three main goals: (1) to identify a panel of cell surface markers that distinguish the trophoblast and endothelial cells of the placenta in the mouse; (2) to translate this marker set to human via the Human Protein Atlas database; and (3) to utilize the validated human trophoblast markers to identify subgroups of human preeclampsia. To achieve these goals, plasma membrane proteins at the blood tissue interfaces were extracted from placentas using intravascular silica-bead perfusion, and then identified using shotgun proteomics. We identified 1181 plasma membrane proteins, of which 171 were enriched at the maternal blood-trophoblast interface and 192 at the fetal endothelial interface with a 70% conservation of expression in humans. Three distinct molecular subgroups of human preeclampsia were identified in existing human microarray data by using expression patterns of trophoblast-enriched proteins. Analysis of all misexpressed genes revealed divergent dysfunctions including angiogenesis (subgroup 1), MAPK signaling (subgroup 2), and hormone biosynthesis and metabolism (subgroup 3). Subgroup 2 lacked expected changes in known preeclampsia markers (sFLT1, sENG) and uniquely overexpressed GNA12. In an independent set of 40 banked placental specimens, GNA12 was overexpressed during preeclampsia when co-incident with chronic hypertension. In the current study we used a novel translational analysis to integrate mouse and human trophoblast protein expression with human microarray data. This strategy identified distinct molecular pathologies in human preeclampsia. We conclude that clinically similar preeclampsia patients exhibit divergent placental gene expression profiles thus implicating divergent molecular mechanisms in the origins of this disease.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Proteínas de la Membrana/metabolismo , Placenta/metabolismo , Preeclampsia/metabolismo , ARN Mensajero/metabolismo , Algoritmos , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Inteligencia Artificial , Teorema de Bayes , Biomarcadores/metabolismo , Endoglina , Endotelio/metabolismo , Femenino , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Expresión Génica , Células Gigantes/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Placenta/patología , Preeclampsia/diagnóstico , Preeclampsia/genética , Embarazo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Programas Informáticos , Investigación Biomédica Traslacional , Trofoblastos/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Cardiac-specific overexpression of a constitutively active form of calcineurin A (CNA) leads directly to cardiac hypertrophy in the CNA mouse model. Because cardiac hypertrophy is a prominent characteristic of many cardiomyopathies, we deduced that delineating the proteomic profile of ventricular tissue from this model might identify novel, widely applicable therapeutic targets. Proteomic analysis was carried out by subjecting fractionated cardiac samples from CNA mice and their WT littermates to gel-free liquid chromatography linked to shotgun tandem mass spectrometry. We identified 1,918 proteins with high confidence, of which 290 were differentially expressed. Microarray analysis of the same tissue provided us with alterations in the ventricular transcriptome. Because bioinformatic analyses of both the proteome and transcriptome demonstrated the up-regulation of endoplasmic reticulum stress, we validated its occurrence in adult CNA hearts through a series of immunoblots and RT-PCR analyses. Endoplasmic reticulum stress often leads to increased apoptosis, but apoptosis was minimal in CNA hearts, suggesting that activated calcineurin might protect against apoptosis. Indeed, the viability of cultured neonatal mouse cardiomyocytes (NCMs) from CNA mice was higher than WT after serum starvation, an apoptotic trigger. Proteomic data identified α-crystallin B (Cryab) as a potential mediator of this protective effect and we showed that silencing of Cryab via lentivector-mediated transduction of shRNAs in NCMs led to a significant reduction in NCM viability and loss of protection against apoptosis. The identification of Cryab as a downstream effector of calcineurin-induced protection against apoptosis will permit elucidation of its role in cardiac apoptosis and its potential as a therapeutic target.
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Calcineurina/metabolismo , Retículo Endoplásmico/metabolismo , Miocardio/metabolismo , Cadena B de alfa-Cristalina/metabolismo , Animales , Apoptosis/fisiología , Calcineurina/genética , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patología , Expresión Génica , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ratones , Ratones Transgénicos , Miocardio/citología , Análisis por Matrices de Proteínas , Proteómica , ARN Interferente Pequeño/genética , Estrés Fisiológico , Cadena B de alfa-Cristalina/antagonistas & inhibidores , Cadena B de alfa-Cristalina/genéticaRESUMEN
Here, we describe a protocol for purifying functional clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) from Staphylococcus aureus within 24 h and over 90% purity. SaCas9 purification begins with immobilized metal affinity chromatography, followed by cation exchange chromatography, and ended with centrifugal concentrators. The simplicity, cost-effectiveness, and reproducibility of such protocols will enable general labs to produce a sizable amount of Cas9 proteins, further accelerating CRISPR research.