RESUMEN
Preterm birth affects ~10% of pregnancies in the US. Despite familial associations, identifying at-risk genetic loci has been challenging. We built deep learning and graphical models to score mutational effects at base resolution via integrating the pregnant myometrial epigenome and large-scale patient genomes with spontaneous preterm birth (sPTB) from European and African American cohorts. We uncovered previously unidentified sPTB genes that are involved in myometrial muscle relaxation and inflammatory responses and that are regulated by the progesterone receptor near labor onset. We studied genomic variants in these genes in our recruited pregnant women administered progestin prophylaxis. We observed that mutation burden in these genes was predictive of responses to progestin treatment for preterm birth. To advance therapeutic development, we screened ~4000 compounds, identified candidate molecules that affect our identified genes, and experimentally validated their therapeutic effects on regulating labor. Together, our integrative approach revealed the druggable genome in preterm birth and provided a generalizable framework for studying complex diseases.
Asunto(s)
Nacimiento Prematuro , Recién Nacido , Femenino , Humanos , Embarazo , Nacimiento Prematuro/genética , Progestinas , Sitios Genéticos , MutaciónRESUMEN
Worldwide, the incidence of both preterm births and chronic lung disease of infancy, or bronchopulmonary dysplasia, remains high. Infants with bronchopulmonary dysplasia have larger and fewer alveoli, a lung pathology that can persist into adulthood. Although recent data point to a role for hypoxia-inducible factor-1α (HIF-1α) in mediating pulmonary angiogenesis and alveolarization, the cell-specific role of HIF-1α remains incompletely understood. Thus, we hypothesized that HIF-1α, in a distinct subset of mesenchymal cells, mediates postnatal alveolarization. To test the hypothesis, we generated mice with a cell-specific deletion of HIF-1α by crossing SM22α promoter-driven Cre mice with HIF-1αflox/flox mice (SM22α-HIF-1α-/-), determined SM-22α-expressing cell identity using single-cell RNA sequencing, and interrogated samples from preterm infants. Deletion of HIF-1α in SM22α-expressing cells had no effect on lung structure at day 3 of life. However, at 8 days, there were fewer and larger alveoli, a difference that persisted into adulthood. Microvascular density, elastin organization, and peripheral branching of the lung vasculature were decreased in SM22α-HIF-1α-/- mice, compared with control mice. Single-cell RNA sequencing demonstrated that three mesenchymal cell subtypes express SM22α: myofibroblasts, airway smooth muscle cells, and vascular smooth muscle cells. Pulmonary vascular smooth muscle cells from SM22α-HIF-1α-/- mice had decreased angiopoietin-2 expression and, in coculture experiments, a diminished capacity to promote angiogenesis that was rescued by angiopoietin-2. Angiopoietin-2 expression in tracheal aspirates of preterm infants was inversely correlated with overall mechanical ventilation time, a marker of disease severity. We conclude that SM22α-specific HIF-1α expression drives peripheral angiogenesis and alveolarization in the lung, perhaps by promoting angiopoietin-2 expression.
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Angiopoyetina 2 , Displasia Broncopulmonar , Subunidad alfa del Factor 1 Inducible por Hipoxia , Animales , Humanos , Recién Nacido , Ratones , Angiopoyetina 2/metabolismo , Displasia Broncopulmonar/genética , Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Recien Nacido Prematuro , Pulmón/patologíaRESUMEN
Pulmonary angiogenesis is essential for alveolarization, the final stage of lung development that markedly increases gas exchange surface area. We recently demonstrated that activation of the nuclear factor kappa-B (NFκB) pathway promotes pulmonary angiogenesis during alveolarization. However, the mechanisms activating NFκB in the pulmonary endothelium, and its downstream targets are not known. In this study, we sought to delineate the specific roles for the NFκB activating kinases, IKKα and IKKß, in promoting developmental pulmonary angiogenesis. Microarray analysis of primary pulmonary endothelial cells (PECs) after silencing IKKα or IKKß demonstrated that the 2 kinases regulate unique panels of genes, with few shared targets. Although silencing IKKα induced mild impairments in angiogenic function, silencing IKKß induced more severe angiogenic defects and decreased vascular cell adhesion molecule expression, an IKKß regulated target essential for both PEC adhesion and migration. Taken together, these data show that IKKα and IKKß regulate unique genes in PEC, resulting in differential effects on angiogenesis upon inhibition, and identify IKKß as the predominant regulator of pulmonary angiogenesis during alveolarization. These data suggest that therapeutic strategies to specifically enhance IKKß activity in the pulmonary endothelium may hold promise to enhance lung growth in diseases marked by altered alveolarization.
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Células Endoteliales/enzimología , Regulación del Desarrollo de la Expresión Génica , Quinasa I-kappa B/genética , Pulmón/enzimología , Neovascularización Fisiológica/genética , Animales , Animales Recién Nacidos , Apoptosis/genética , Adhesión Celular , Movimiento Celular , Proliferación Celular , Células Endoteliales/citología , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/metabolismo , Pulmón/citología , Pulmón/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/genética , FN-kappa B/metabolismo , Organogénesis/genética , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Although the accumulation of amyloidogenic proteins in neuroinflammatory conditions is generally considered pathologic, in a murine model of multiple sclerosis, amyloid-forming fibrils, comprised of hexapeptides, are anti-inflammatory. Whether these molecules modulate systemic inflammatory conditions remains unknown. We hypothesized that an amylin hexapeptide that forms fibrils can attenuate the systemic inflammatory response in a murine model of sepsis. To test this hypothesis, mice were pre-treated with either vehicle or amylin hexapeptide (20 µg) at 12 hours and 6 hours prior to intraperitoneal (i.p.) lipopolysaccharide (LPS, 20 mg/kg) administration. Illness severity and survival were monitored every 6 hours for 3 days. Levels of pro- (IL-6, TNF-α, IFN-γ) and anti-inflammatory (IL-10) cytokines were measured via ELISA at 1, 3, 6, 12, and 24 hours after LPS (i.p.). As a metric of lung injury, pulmonary artery endothelial cell (PAEC) barrier function was tested 24 hours after LPS administration by comparing lung wet-to-dry ratios, Evan's blue dye (EBD) extravasation, lung histology and caspase-3 activity. Compared to controls, pretreatment with amylin hexapeptide significantly reduced mortality (p<0.05 at 72 h), illness severity (p<0.05), and pro-inflammatory cytokine levels, while IL-10 levels were elevated (p<0.05). Amylin pretreatment attenuated LPS-induced lung injury, as demonstrated by decreased lung water and caspase-3 activity (p<0.05, versus PBS). Hence, in a murine model of systemic inflammation, pretreatment with amylin hexapeptide reduced mortality, disease severity, and preserved lung barrier function. Amylin hexapeptide may represent a novel therapeutic tool to mitigate sepsis severity and lung injury.
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Lesión Pulmonar Aguda/tratamiento farmacológico , Proteínas Amiloidogénicas/farmacología , Polipéptido Amiloide de los Islotes Pancreáticos/química , Pulmón/efectos de los fármacos , Oligopéptidos/farmacología , Sepsis/tratamiento farmacológico , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/mortalidad , Proteínas Amiloidogénicas/síntesis química , Animales , Caspasa 3/genética , Caspasa 3/inmunología , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Células Endoteliales/patología , Femenino , Regulación de la Expresión Génica , Inflamación , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Lipopolisacáridos/administración & dosificación , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Oligopéptidos/síntesis química , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/inmunología , Arteria Pulmonar/patología , Sepsis/inducido químicamente , Sepsis/inmunología , Sepsis/mortalidad , Análisis de Supervivencia , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Pulmonary artery smooth muscle cells (PASMCs) express endothelin (ET-1), which modulates the pulmonary vascular response to hypoxia. Although cross-talk between hypoxia-inducible factor-1α (HIF-1α), an O2-sensitive transcription factor, and ET-1 is established, the cell-specific relationship between HIF-1α and ET-1 expression remains incompletely understood. We tested the hypotheses that in PASMCs 1) HIF-1α expression constrains ET-1 expression, and 2) a specific microRNA (miRNA) links HIF-1α and ET-1 expression. In human (h)PASMCs, depletion of HIF-1α with siRNA increased ET-1 expression at both the mRNA and protein levels ( P < 0.01). In HIF-1α-/- murine PASMCs, ET-1 gene and protein expression was increased ( P < 0.0001) compared with HIF-1α+/+ cells. miRNA profiles were screened in hPASMCs transfected with siRNA-HIF-1α, and RNA hybridization was performed on the Agilent (Santa Clara, CA) human miRNA microarray. With HIF-1α depletion, miRNA-543 increased 2.4-fold ( P < 0.01). In hPASMCs, miRNA-543 overexpression increased ET-1 gene ( P < 0.01) and protein ( P < 0.01) expression, decreased TWIST gene expression ( P < 0.05), and increased ET-1 gene and protein expression, compared with nontargeting controls ( P < 0.01). Moreover, we evaluated low passage hPASMCs from control and patients with idiopathic pulmonary arterial hypertension (IPAH). Compared with controls, protein expression of HIF-1α and Twist-related protein-1 (TWIST1) was decreased ( P < 0.05), and miRNA-543 and ET-1 expression increased ( P < 0.001) in hPASMCs from patients with IPAH. Thus, in PASMCs, loss of HIF-1α increases miRNA-543, which decreases Twist expression, leading to an increase in PASMC ET-1 expression. This previously undescribed link between HIF-1α and ET-1 via miRNA-543 mediated Twist suppression represents another layer of molecular regulation that might determine pulmonary vascular tone.
Asunto(s)
Endotelina-1/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , MicroARNs/biosíntesis , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/metabolismo , Animales , Células Cultivadas , Endotelina-1/genética , Regulación de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Ratones Noqueados , MicroARNs/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
Compromised pulmonary endothelial cell (PEC) barrier function characterizes acute respiratory distress syndrome (ARDS), a cause of substantial morbidity and mortality. Survival from ARDS is greater in children compared with adults. Whether developmental differences intrinsic to PEC barrier function contribute to this survival advantage remains unknown. To test the hypothesis that PEC barrier function is more well-preserved in neonatal lungs compared with adult lungs in response to inflammation, we induced lung injury in neonatal and adult mice with systemic lipopolysaccharide (LPS). We assessed PEC barrier function in vivo and in vitro, evaluated changes in the expression of focal adhesion kinase 1 (FAK1) and phosphorylation in response to LPS, and determined the effect of FAK silencing and overexpression on PEC barrier function. We found that LPS induced a greater increase in lung permeability and PEC barrier disruption in the adult mice, despite similar degrees of inflammation and apoptosis. Although baseline expression was similar, LPS increased FAK1 expression in neonatal PEC but increased FAK1 phosphorylation and decreased FAK1 expression in adult PEC. Pharmacologic inhibition of FAK1 accentuated LPS-induced barrier disruption most in adult PEC. Finally, in response to LPS, FAK silencing markedly impaired neonatal PEC barrier function, whereas FAK overexpression preserved adult PEC barrier function. Thus, developmental differences in FAK expression during inflammatory injury serve to preserve neonatal pulmonary endothelial barrier function compared with that of adults and suggest that intrinsic differences in the immature versus pulmonary endothelium, especially relative to FAK1 phosphorylation, may contribute to the improved outcomes of children with ARDS.
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Apoptosis , Barrera Alveolocapilar/enzimología , Células Endoteliales/enzimología , Endotelio/enzimología , Quinasa 1 de Adhesión Focal/metabolismo , Transducción de Señal , Animales , Barrera Alveolocapilar/crecimiento & desarrollo , Barrera Alveolocapilar/patología , Células Endoteliales/patología , Endotelio/patología , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Inflamación/inducido químicamente , Inflamación/enzimología , Inflamación/patología , Lipopolisacáridos/toxicidad , RatonesRESUMEN
The importance of gaining insight into the mechanisms underlying uterine quiescence and contractility is highlighted by the absence of an effective strategy to prevent or treat preterm labor, the greatest cause of perinatal mortality and morbidity worldwide. Although current evidence suggests that in myometrial smooth muscle cells (mSMCs) calcium homeostasis is modulated near term to promote uterine contractility, the efficacy of blocking voltage-operated calcium channels is limited by dose-related cardiovascular side effects. Thus, we considered whether uterine contractility might be modulated by calcium entry via transient receptor potential vanilloid 4 (TRPV4) channels. In mSMC, TRPV4 gene and protein expression increased with gestation, and TRPV4-mediated Ca(2+) entry and contractility were increased in mSMC from pregnant compared to nonpregnant rats. Cell membrane TRPV4 expression was specifically increased, whereas the expression of ß-arrestin-1 and ß-arrestin-2, molecules that can sequester TRPV4 in the cytoplasm, decreased. Physical interaction of ß-arrestin-2 and TRPV4 was apparent in nonpregnant, but absent in pregnant, mouse uterus. Moreover, direct pharmacologic activation of TRPV4 increased uterine contraction, but oxytocin-induced myometrial contraction was blocked by pharmacologic inhibition of TRPV4 and decreased in mice with global deletion of TRPV4. Finally, TRPV4 channel blockade prolonged pregnancy in two distinct in vivo murine models of preterm labor, whereas the absence of either ß-arrestin-1 or ß-arrestin-2 increased susceptibility to preterm labor. These data suggest that TRPV4 channel activity modulates uterine contractility and might represent a therapeutic target to address preterm labor.
Asunto(s)
Canales Catiónicos TRPV/metabolismo , Útero/fisiología , Animales , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Colágeno/farmacología , Citosol/efectos de los fármacos , Citosol/metabolismo , Femenino , Geles , Eliminación de Gen , Humanos , Activación del Canal Iónico/efectos de los fármacos , Lipopolisacáridos , Ratones Endogámicos C57BL , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miometrio/citología , Trabajo de Parto Prematuro , Oxitocina/farmacología , Embarazo , Unión Proteica/efectos de los fármacos , Ratas Sprague-Dawley , Contracción Uterina/efectos de los fármacos , Útero/efectos de los fármacos , beta-Arrestinas/metabolismoRESUMEN
Endothelin-1 (ET-1) increases pulmonary vascular tone through direct effects on pulmonary artery smooth muscle cells (PASMC) via membrane-bound ET-1 receptors. Circulating ET-1 contributes to vascular remodeling by promoting SMC proliferation and migration and inhibiting SMC apoptosis. Although endothelial cells (EC) are the primary source of ET-1, whether ET-1 produced by SMC modulates pulmonary vascular tone is unknown. Using transgenic mice created by crossbreeding SM22α-Cre mice with ET-1(flox/flox) mice to selectively delete ET-1 in SMC, we tested the hypothesis that PASMC ET-1 gene expression modulates the pulmonary vascular response to hypoxia. ET-1 gene deletion and selective activity of SM22α promoter-driven Cre recombinase were confirmed. Functional assays were performed under normoxic (21% O2) or hypoxic (5% O2) conditions using murine PASMC obtained from ET-1(+/+) and ET-1(-/-) mic and in human PASMC (hPASMC) after silencing of ET-1 using siRNA. Under baseline conditions, there was no difference in right ventricular systolic pressure (RVSP) between SM22α-ET-1(-/-) and SM22α-ET-1(+/+) (control) littermates. After exposure to hypoxia (10% O2, 21-24 days), RVSP was and vascular remodeling were less in SM22α-ET-1(-/-) mice compared with control littermates (P < 0.01). Loss of ET-1 decreased PASMC proliferation and migration and increased apoptosis under normoxic and hypoxic conditions. Exposure to selective ET-1 receptor antagonists had no effect on either the hypoxia-induced hPASMC proliferative or migratory response. SMC-specific ET-1 deletion attenuates hypoxia-induced increases in pulmonary vascular tone and structural remodeling. The observation that loss of ET-1 inhibited SMC proliferation, survival, and migration represents evidence that ET-1 derived from SMC plays a previously undescribed role in modulating the response of the pulmonary circulation to hypoxia. Thus PASMC ET-1 may modulate vascular tone independently of ET-1 produced by EC.
Asunto(s)
Endotelina-1/biosíntesis , Regulación de la Expresión Génica , Hipoxia/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/metabolismo , Animales , Movimiento Celular/genética , Proliferación Celular/genética , Supervivencia Celular/genética , Células Cultivadas , Enfermedad Crónica , Células Endoteliales/metabolismo , Células Endoteliales/patología , Endotelina-1/genética , Silenciador del Gen , Humanos , Hipoxia/genética , Hipoxia/patología , Hipoxia/fisiopatología , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/patología , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Remodelación Vascular/genéticaRESUMEN
To test, whether 10 genes, diagnostic of renal allograft rejection in blood, are able to diagnose and predict cardiac allograft rejection, we analyzed 250 blood samples from heart transplant recipients with and without acute rejection (AR) and with cytomegalovirus (CMV) infection by QPCR. A QPCR-based logistic regression model was built on 5 of these 10 genes (AR threshold composite score >37% â=âAR) and tested for AR prediction in an independent set of 109 samples, where it correctly diagnosed AR with 89% accuracy, with no misclassifications for AR ISHLT grade 1b. CMV infection did not confound the AR score. The genes correctly diagnosed AR in a blood sample within 6 months prior to biopsy diagnosis with 80% sensitivity and untreated grade 1b AR episodes had persistently elevated scores until 6 months after biopsy diagnosis. The gene score was also correlated with presence or absence of cardiac allograft vasculopathy (CAV) irrespective of rejection grade. In conclusion, there is a common transcriptional axis of immunological trafficking in peripheral blood in both renal and cardiac organ transplant rejection, across a diverse recipient age range. A common gene signature, initially identified in the setting of renal transplant rejection, can be utilized serially after cardiac transplantation, to diagnose and predict biopsy confirmed acute heart transplant rejection.
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Infecciones por Citomegalovirus/complicaciones , Expresión Génica , Rechazo de Injerto/diagnóstico , Trasplante de Corazón , Trasplante de Riñón , Adulto , Anciano , Aloinjertos , Biomarcadores/sangre , Infecciones por Citomegalovirus/sangre , Femenino , Rechazo de Injerto/sangre , Rechazo de Injerto/complicaciones , Humanos , Masculino , Persona de Mediana EdadRESUMEN
RATIONALE: Hypoxia-inducible factor-1α (HIF-1α), an oxygen (O2)-sensitive transcription factor, mediates transcriptional responses to low-O2 tension states. Although acute hypoxia causes pulmonary vasoconstriction and chronic hypoxia can cause vascular remodeling and pulmonary hypertension, conflicting data exist on the role of HIF-1α in modulating pulmonary vascular tone. OBJECTIVE: To investigate the role of smooth muscle cell (SMC)-specific HIF-1α in regulating pulmonary vascular tone. METHODS AND RESULTS: Mice with an SMC-specific deletion of HIF-1α (SM22α-HIF-1α(-/-)) were created to test the hypothesis that pulmonary artery SMC (PASMC) HIF-1α modulates pulmonary vascular tone and the response to hypoxia. SM22α-HIF-1α(-/-) mice exhibited significantly higher right ventricular systolic pressure compared with wild-type littermates under normoxia and with exposure to either acute or chronic hypoxia in the absence of histological evidence of accentuated vascular remodeling. Moreover, myosin light chain phosphorylation, a determinant of SMC tone, was higher in PASMCs isolated from SM22α-HIF-1α(-/-) mice compared with wild-type PASMCs, during both normoxia and after acute hypoxia. Further, overexpression of HIF-1α decreased myosin light chain phosphorylation in HIF-1α-null SMCs. CONCLUSIONS: In both normoxia and hypoxia, PASMC HIF-1α maintains low pulmonary vascular tone by decreasing myosin light chain phosphorylation. Compromised PASMC HIF-1α expression may contribute to the heightened vasoconstriction that characterizes pulmonary hypertension.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Músculo Liso Vascular/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Arteria Pulmonar/metabolismo , Vasoconstricción , Enfermedad Aguda , Animales , Células Cultivadas , Enfermedad Crónica , Modelos Animales de Enfermedad , Genotipo , Humanos , Hipoxia/genética , Hipoxia/fisiopatología , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Persona de Mediana Edad , Proteínas Musculares/genética , Fenotipo , Fosforilación , Regiones Promotoras Genéticas , Interferencia de ARN , Factores de Tiempo , TransfecciónRESUMEN
Increased pulmonary artery endothelial cell (PAEC) endothelium-dependent nitric oxide synthase (eNOS) activity mediates perinatal pulmonary vasodilation. Compromised eNOS activity is central to the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN). Voltage-derived anion channel (VDAC)-1 was recently demonstrated to bind eNOS in the systemic circulation. We hypothesized that VDAC isoforms modulate eNOS activity in the pulmonary circulation, and that decreased VDAC expression contributes to PPHN. In PAECs derived from an ovine model of PPHN: (1) there is eNOS activity, but not expression; and (2) VDAC1 and -2 proteins are decreased. Immunocytochemistry, coimmunoprecipitation, and in situ proximity ligation assays in human PAECs (hPAECs) demonstrate binding between eNOS and both VDAC1 and -2, which increased upon stimulation with NO agonists. The ability of agonists to increase the eNOS/VDAC interaction was significantly blunted in hypertensive, compared with normotensive, ovine PAECs. Depletion of VDAC2, but not VDAC1, blocked the agonist-induced increase in eNOS activity in hPAECs. Overexpression of VDAC2 in hypertensive PAECs increased eNOS activity. Binding of VDAC2 enhances eNOS activity in the pulmonary circulation, and diminished VDAC2 constrains eNOS in PAECs derived from fetal lambs with chronic intrauterine pulmonary hypertension. We speculate that decreases in VDAC2 may contribute to the limited eNOS activity that characterizes pulmonary hypertension.
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Células Endoteliales/enzimología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Arteria Pulmonar/patología , Canal Aniónico 2 Dependiente del Voltaje/metabolismo , Animales , Calcimicina/farmacología , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/patología , Endotelio Vascular/patología , Expresión Génica , Histamina/fisiología , Humanos , Recién Nacido , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/genética , Síndrome de Circulación Fetal Persistente/enzimología , Síndrome de Circulación Fetal Persistente/metabolismo , Síndrome de Circulación Fetal Persistente/patología , Unión Proteica , Mapeo de Interacción de Proteínas , Ovinos , Canal Aniónico 1 Dependiente del Voltaje/genética , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Canal Aniónico 2 Dependiente del Voltaje/genéticaRESUMEN
Noninvasive methods to diagnose rejection of renal allografts are unavailable. Mass spectrometry followed by multiple-reaction monitoring provides a unique approach to identify disease-specific urine peptide biomarkers. Here, we performed urine peptidomic analysis of 70 unique samples from 50 renal transplant patients and 20 controls (n = 20), identifying a specific panel of 40 peptides for acute rejection (AR). Peptide sequencing revealed suggestive mechanisms of graft injury with roles for proteolytic degradation of uromodulin (UMOD) and several collagens, including COL1A2 and COL3A1. The 40-peptide panel discriminated AR in training (n = 46) and test (n = 24) sets (area under ROC curve >0.96). Integrative analysis of transcriptional signals from paired renal transplant biopsies, matched with the urine samples, revealed coordinated transcriptional changes for the corresponding genes in addition to dysregulation of extracellular matrix proteins in AR (MMP-7, SERPING1, and TIMP1). Quantitative PCR on an independent set of 34 transplant biopsies with and without AR validated coordinated changes in expression for the corresponding genes in rejection tissue. A six-gene biomarker panel (COL1A2, COL3A1, UMOD, MMP-7, SERPING1, TIMP1) classified AR with high specificity and sensitivity (area under ROC curve = 0.98). These data suggest that changes in collagen remodeling characterize AR and that detection of the corresponding proteolytic degradation products in urine provides a noninvasive diagnostic approach.
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Rechazo de Injerto/orina , Trasplante de Riñón , Péptidos/orina , Enfermedad Aguda , Adolescente , Biomarcadores/orina , Niño , Preescolar , Femenino , Humanos , Masculino , Adulto JovenRESUMEN
The molecular and cellular mechanisms underlying nephropathic cystinosis, which exhibits generalized proximal tubular dysfunction and progressive renal failure, remain largely unknown. Renal biopsies from patients with this disorder can reveal abnormally large mitochondria, but the relevance of this and other ultrastructural abnormalities is unclear. We studied the ultrastructure of fibroblasts and renal proximal tubular epithelial cells from patients with three clinical variants of cystinosis: Nephropathic, intermediate, and ocular. Electron microscopy revealed the presence of morphologically abnormal mitochondria and abnormal patterns of mitochondrial autophagy (mitophagy) with a high number of autophagic vacuoles and fewer mitochondria (P < 0.02) in nephropathic cystinosis. In addition, we observed increased apoptosis in renal proximal tubular epithelial cells, greater expression of LC3-II/LC3-I (microtubule-associated protein 1 light chain 3), and significantly more autophagosomes in the nephropathic variant. The autophagy inhibitor 3-methyl adenine rescued cell death in cystinotic cells. Cystinotic cells had increased levels of beclin-1 and aberrant mitochondrial function with a significant decrease in ATP generation and an increase in reactive oxygen species. This study provides ultrastructural and functional evidence of abnormal mitophagy in nephropathic cystinosis, which may contribute to the renal Fanconi syndrome and progressive renal injury.
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Apoptosis/fisiología , Autofagia/fisiología , Cistinosis/fisiopatología , Fibroblastos/patología , Mitocondrias/patología , Adenosina Trifosfato/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Beclina-1 , Células Cultivadas , Células Epiteliales/patología , Células Epiteliales/ultraestructura , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Humanos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/ultraestructura , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/ultraestructuraRESUMEN
A disparity remains between graft survival of renal allografts from deceased donors and from living donors. A better understanding of the molecular mechanisms that underlie this disparity may allow the development of targeted therapies to enhance graft survival. Here, we used microarrays to examine whole genome expression profiles using tissue from 53 human renal allograft protocol biopsies obtained both at implantation and after transplantation. The gene expression profiles of living-donor kidneys and pristine deceased-donor kidneys (normal histology, young age) were significantly different before reperfusion at implantation. Deceased-donor kidneys exhibited a significant increase in renal expression of complement genes; posttransplantation biopsies from well-functioning, nonrejecting kidneys, regardless of donor source, also demonstrated a significant increase in complement expression. Peritransplantation phenomena, such as donor death and possibly cold ischemia time, contributed to differences in complement pathway gene expression. In addition, complement gene expression at the time of implantation was associated with both early and late graft function. These data suggest that complement-modulating therapy may improve graft outcomes in renal transplantation.
Asunto(s)
Proteínas del Sistema Complemento/metabolismo , Riñón/metabolismo , Donadores Vivos , Trasplantes , Adolescente , Adulto , Factores de Edad , Biopsia , Muerte Encefálica , Niño , Preescolar , Isquemia Fría , Femenino , Perfilación de la Expresión Génica , Supervivencia de Injerto , Humanos , Lactante , Riñón/patología , Trasplante de Riñón , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Trasplante Homólogo , Adulto JovenRESUMEN
Previously, we related fibronectin (Fn1) mRNA translation to an interaction between an AU-rich element in the Fn1 3' UTR and light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. Since human fibrosarcoma (HT1080) cells produce little fibronectin and LC3, we used these cells to investigate how LC3-mediated Fn1 mRNA translation might alter tumor growth. Transfection of HT1080 cells with LC3 enhanced fibronectin mRNA translation. Using polysome analysis and RNA-binding assays, we show that elevated levels of translation depend on an interaction between a triple arginine motif in LC3 and the AU-rich element in Fn1 mRNA. Wild-type but not mutant LC3 accelerated HT1080 cell growth in culture and when implanted in SCID mice. Comparison of WT LC3 with vector-transfected HT1080 cells revealed increased fibronectin-dependent proliferation, adhesion and invasion. Microarray analysis of genes differentially expressed in WT and vector-transfected control cells indicated enhanced expression of connective tissue growth factor (CTGF). Using siRNA, we show that enhanced expression of CTGF is fibronectin dependent and that LC3-mediated adhesion, invasion and proliferation are CTGF dependent. Expression profiling of soft tissue tumors revealed increased expression of both LC3 and CTGF in some locally invasive tumor types.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Fibronectinas , Fibrosarcoma , Proteínas Asociadas a Microtúbulos/metabolismo , Biosíntesis de Proteínas , Animales , Adhesión Celular , Línea Celular , Proliferación Celular , Factor de Crecimiento del Tejido Conjuntivo/genética , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Perfilación de la Expresión Génica , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/genética , Invasividad Neoplásica , Análisis de Secuencia por Matrices de Oligonucleótidos , Polirribosomas/metabolismo , RatasRESUMEN
Microarray technologies have both fascinated and frustrated the transplant community since their introduction roughly a decade ago. Fascination arose from the possibility offered by the technology to gain a profound insight into the cellular response to immunogenic injury and the potential that this genomic signature would be indicative of the biological mechanism by which that stress was induced. Frustrations have arisen primarily from technical factors such as data variance, the requirement for the application of advanced statistical and mathematical analyses, and difficulties associated with actually recognizing signature gene-expression patterns and discerning mechanisms. To aid the understanding of this powerful tool, its versatility, and how it is dramatically changing the molecular approach to biomedical and clinical research, this teaching review describes the technology and its applications, as well as the limitations and evolution of microarrays, in the field of organ transplantation. Finally, it calls upon the attention of the transplant community to integrate into multidisciplinary teams, to take advantage of this technology and its expanding applications in unraveling the complex injury circuits that currently limit transplant survival.
Asunto(s)
Investigación Biomédica/métodos , ADN/genética , Expresión Génica , Rechazo de Injerto/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Trasplante de Órganos/métodos , Rechazo de Injerto/fisiopatología , Humanos , Monitoreo Fisiológico/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/tendenciasRESUMEN
Microarray technology is a powerful tool in the discovery of new biomarkers for disease. After solid organ transplantation, where the detection of rejection is usually made on invasive biopsies, it could be hypothesized that noninvasive transcriptional profiling of peripheral blood will reveal rejection-specific expression patterns from circulating immune cells. However, in kidney transplant rejection, the analysis of gene expression data in whole blood has proven difficult for detecting significant genes specific for acute graft rejection. Previous studies have demonstrated that the abundance of globin genes in whole blood may mask the underlying biological differences between whole blood samples. In the present study, we compared the gene expression profiles of peripheral blood of nine stable renal allograft recipients with seven matched patients having an ongoing acute renal transplant rejection, using four different protocols of preparation, amplification, and synthesis of cRNA or cDNA and hybridization on the Affymetrix platform. We demonstrated that the globin reduction method is not sufficient to unmask clinically relevant rejection-specific transcriptome profiles in whole blood. Applying an additional mathematical depletion of the globin genes improves the efficacy of globin reduction but cannot remove the confounding influence of globin gene hybridization. Sampling of peripheral blood leukocytes alone, without the confounding influence of globin mRNA, provides sensitive and specific peripheral signatures for graft rejection, with many of these signals overlapping with rejection-driven tissue (kidney)-specific signatures from matched biopsies. Similar applications may exist for array-based biomarker discovery for other diseases associated with changes in leukocyte trafficking, activation, or function.
Asunto(s)
Globinas/genética , Rechazo de Injerto/genética , Trasplante de Riñón/métodos , Adolescente , Biomarcadores/sangre , Niño , Preescolar , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Rechazo de Injerto/sangre , Humanos , Lactante , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN/sangre , ARN/genética , Trasplante HomólogoRESUMEN
Murine light chain 3 (LC3) exists as two isoforms, LC3alpha and beta: LC3beta is an RNA-binding protein that enhances fibronectin (FN) mRNA translation, and is also a marker of autophagy. We report embryonic expression patterns for LC3alpha and LC3beta, with some overlap but notable differences in the brain, and in tissues of non-neuronal origin. LC3beta knockout (-/-) mice develop normally without a compensatory increase in LC3alpha. LC3beta-/- embryonic fibroblasts (MEFs) exhibit reduced FN synthesis but maintain wild type (WT) levels of FN protein. No significant changes in proteins associated with FN turnover, i.e., caveolin-1, LRP-1, or matrix metalloproteinases were identified. Autophagosomes form in amino acid-starved LC3beta-/-MEFs, and Caesarean-delivered pups survive as long as WT pups without an increase in LC3-related proteins linked to autophagy. These results suggest novel compensatory mechanisms for loss of LC3beta, ensuring proper FN accumulation and autophagy during fetal and neonatal life.