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1.
Circulation ; 146(14): 1033-1045, 2022 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-36004627

RESUMO

BACKGROUND: Cell-free DNA (cfDNA) is a noninvasive marker of cellular injury. Its significance in pulmonary arterial hypertension (PAH) is unknown. METHODS: Plasma cfDNA was measured in 2 PAH cohorts (A, n=48; B, n=161) and controls (n=48). Data were collected for REVEAL 2.0 (Registry to Evaluate Early and Long-Term PAH Disease Management) scores and outcome determinations. Patients were divided into the following REVEAL risk groups: low (≤6), medium (7-8), and high (≥9). Total cfDNA concentrations were compared among controls and PAH risk groups by 1-way analysis of variance. Log-rank tests compared survival between cfDNA tertiles and REVEAL risk groups. Areas under the receiver operating characteristic curve were estimated from logistic regression models. A sample subset from cohort B (n=96) and controls (n=16) underwent bisulfite sequencing followed by a deconvolution algorithm to map cell-specific cfDNA methylation patterns, with concentrations compared using t tests. RESULTS: In cohort A, median (interquartile range) age was 62 years (47-71), with 75% female, and median (interquartile range) REVEAL 2.0 was 6 (4-9). In cohort B, median (interquartile range) age was 59 years (49-71), with 69% female, and median (interquartile range) REVEAL 2.0 was 7 (6-9). In both cohorts, cfDNA concentrations differed among patients with PAH of varying REVEAL risk and controls (analysis of variance P≤0.002) and were greater in the high-risk compared with the low-risk category (P≤0.002). In cohort B, death or lung transplant occurred in 14 of 54, 23 of 53, and 35 of 54 patients in the lowest, middle, and highest cfDNA tertiles, respectively. cfDNA levels stratified as tertiles (log-rank: P=0.0001) and REVEAL risk groups (log-rank: P<0.0001) each predicted transplant-free survival. The addition of cfDNA to REVEAL improved discrimination (area under the receiver operating characteristic curve, 0.72-0.78; P=0.02). Compared with controls, methylation analysis in patients with PAH revealed increased cfDNA originating from erythrocyte progenitors, neutrophils, monocytes, adipocytes, natural killer cells, vascular endothelium, and cardiac myocytes (Bonferroni adjusted P<0.05). cfDNA concentrations derived from erythrocyte progenitor cells, cardiac myocytes, and vascular endothelium were greater in patients with PAH with high-risk versus low-risk REVEAL scores (P≤0.02). CONCLUSIONS: Circulating cfDNA is elevated in patients with PAH, correlates with disease severity, and predicts worse survival. Results from cfDNA methylation analyses in patients with PAH are consistent with prevailing paradigms of disease pathogenesis.


Assuntos
Ácidos Nucleicos Livres , Hipertensão Arterial Pulmonar , Idoso , Biomarcadores , Ácidos Nucleicos Livres/genética , Hipertensão Pulmonar Primária Familiar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Hipertensão Arterial Pulmonar/diagnóstico , Hipertensão Arterial Pulmonar/genética , Curva ROC
2.
Am J Physiol Lung Cell Mol Physiol ; 318(1): L98-L111, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31617731

RESUMO

Inflammatory cell infiltrates are a prominent feature of aberrant vascular remodeling in pulmonary arterial hypertension (PAH), suggesting that immune effector cells contribute to disease progression. Genome-wide blood expression profiling studies have attempted to better define this inflammatory component of PAH pathobiology but have been hampered by small sample sizes, methodological differences, and very little gene-level reproducibility. The current meta-analysis (seven studies; 156 PAH patients/110 healthy controls) was performed to assess the comparability of data across studies and to possibly derive a generalizable transcriptomic signature. Idiopathic (IPAH) compared with disease-associated PAH (APAH) displayed highly similar expression profiles with no differentially expressed genes, even after substantially relaxing selection stringency. In contrast, using a false discovery rate of ≤1% and I2 < 40% (low-to-moderate heterogeneity across studies) both IPAH and APAH differed markedly from healthy controls with the combined PAH cohort yielding 1,269 differentially expressed, unique gene transcripts. Bioinformatic analyses, including gene-set enrichment, which uses all available data independent of gene selection thresholds, identified interferon, mammalian target of rapamycin/p70S6K, stress kinase, and Toll-like receptor signaling as enriched mechanisms within the PAH gene signature. Enriched biological functions and diseases included tumorigenesis, autoimmunity, antiviral response, and cell death consistent with prevailing theories of PAH pathogenesis. Although otherwise indistinguishable, APAH (predominantly PAH due to systemic sclerosis) had a somewhat stronger interferon profile than IPAH. Meta-analysis defined a robust and generalizable transcriptomic signature in the blood of PAH patients that can help inform the identification of biomarkers and therapeutic targets.


Assuntos
Hipertensão Pulmonar Primária Familiar/genética , Hipertensão Arterial Pulmonar/genética , Transcriptoma/genética , Biomarcadores/metabolismo , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Reprodutibilidade dos Testes
3.
Pulm Circ ; 11(3): 20458940211022204, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34249330

RESUMO

Pulmonary arterial hypertension is characterized by endothelial dysfunction and microthrombi formation. The role of anticoagulation remains controversial, with studies demonstrating inconsistent effects on pulmonary arterial hypertension mortality. Clinical anticoagulation practices are currently heterogeneous, reflecting physician preference. This study uses thrombelastography and hematology markers to evaluate whether clot formation and fibrinolysis are abnormal in pulmonary arterial hypertension patients. Venous blood was collected from healthy volunteers (n = 20) and patients with pulmonary arterial hypertension (n = 20) on stable medical therapy for thrombelastography analysis. Individual thrombelastography parameters and a calculated coagulation index were used for comparison. In addition, hematologic markers, including fibrinogen, factor VIII activity, von Willebrand factor activity, von Willebrand factor antigen, and alpha2-antiplasmin, were measured in pulmonary arterial hypertension patients and compared to healthy volunteers. Between group differences were analyzed using t tests and linear mixed models, accounting for repeated measures when applicable. Although the degree of fibrinolysis (LY30) was significantly lower in pulmonary arterial hypertension patients compared to healthy volunteers (0.3% ± 0.6 versus 1.3% ± 1.1, p = 0.04), all values were within the normal reference range (0-8%). All other thrombelastography parameters were not significantly different between pulmonary arterial hypertension patients and healthy volunteers (p ≥ 0.15 for all). Similarly, alpha2-antiplasmin activity levels were higher in pulmonary arterial hypertension patients compared to healthy volunteers (103.7% ± 13.6 versus 82.6% ± 9.5, p < 0.0001), but all individual values were within the normal range (75-132%). There were no other significant differences in hematologic markers between pulmonary arterial hypertension patients and healthy volunteers (p ≥ 0.07 for all). Sub-group analysis comparing thrombelastography results in patients treated with or without prostacyclin pathway targeted therapies were also non-significant. In conclusion, treated pulmonary arterial hypertension patients do not demonstrate abnormal clotting kinetics or fibrinolysis by thrombelastography.

4.
J Heart Lung Transplant ; 36(9): 1004-1012, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28624139

RESUMO

BACKGROUND: Use of new genomic techniques in clinical settings requires that such methods are rigorous and reproducible. Previous studies have shown that quantitation of donor-derived cell-free DNA (%ddcfDNA) by unbiased shotgun sequencing is a sensitive, non-invasive marker of acute rejection after heart transplantation. The primary goal of this study was to assess the reproducibility of %ddcfDNA measurements across technical replicates, manual vs automated platforms, and rejection phenotypes in distinct patient cohorts. METHODS: After developing and validating the %ddcfDNA assay, we subjected the method to a rigorous test of its reproducibility. We measured %ddcfDNA in technical replicates performed by 2 independent laboratories and verified the reproducibility of %ddcfDNA patterns of 2 rejection phenotypes: acute cellular rejection and antibody-mediated rejection in distinct patient cohorts. RESULTS: We observed strong concordance of technical-replicate %ddcfDNA measurements across 2 independent laboratories (slope = 1.02, R2 > 0.99, p < 10-6), as well as across manual and automated platforms (slope = 0.80, R2 = 0.92, p < 0.001). The %ddcfDNA measurements in distinct heart transplant cohorts had similar baselines and error rates. The %ddcfDNA temporal patterns associated with rejection phenotypes were similar in both patient cohorts; however, the quantity of ddcfDNA was significantly higher in samples with severe vs mild histologic rejection grade (2.73% vs 0.14%, respectively; p < 0.001). CONCLUSIONS: The %ddcfDNA assay is precise and reproducible across laboratories and in samples from 2 distinct types of heart transplant rejection. These findings pave the way for larger studies to assess the clinical utility of %ddcfDNA as a marker of acute rejection after heart transplantation.


Assuntos
Ácidos Nucleicos Livres/análise , Rejeição de Enxerto/sangue , Transplante de Coração/efeitos adversos , Disfunção Primária do Enxerto/sangue , Doadores de Tecidos , Doença Aguda , Adulto , Biomarcadores/sangue , Feminino , Transplante de Coração/métodos , Humanos , Modelos Lineares , Masculino , Disfunção Primária do Enxerto/fisiopatologia , Reprodutibilidade dos Testes , Estatísticas não Paramétricas
5.
Thromb Haemost ; 112(4): 770-80, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25057108

RESUMO

Accurately detecting circulating endothelial cells (CECs) is important since their enumeration has been proposed as a biomarker to measure injury to the vascular endothelium. However, there is no single methodology for determining CECs in blood, making comparison across studies difficult. Many methods for detecting CECs rely on characteristic cell surface markers and cell viability indicators, but lack secondary validation. Here, a CEC population in healthy adult human subjects was identified by flow cytometry as CD45-, CD34dim that is comparable to a previously described CD45-, CD31bright population. In addition, nuclear staining with 7-aminoactinomycin D (7-AAD) was employed as a standard technique to exclude dead cells. Unexpectedly, the CD45-, CD34dim, 7-AAD- CECs lacked surface detectable CD146, a commonly used marker of CECs. Furthermore, light microscopy revealed this cell population to be composed primarily of large cells without a clearly defined nucleus. Nevertheless, immunostains still demonstrated the presence of the lectin Ulex europaeus and von Willebrand factor. Ultramicro analytical immunochemistry assays for the endothelial cell proteins CD31, CD34, CD62E, CD105, CD141, CD144 and vWF indicated these cells possess an endothelial phenotype. However, only a small amount of RNA, which was mostly degraded, could be isolated from these cells. Thus the majority of CECs in healthy individuals as defined by CD45-, CD34dim, and 7-AAD- have shed their CD146 surface marker and are senescent cells without an identifiable nucleus and lacking RNA of sufficient quantity and quality for transcriptomal analysis. This study highlights the importance of secondary validation of CEC identification.


Assuntos
Antígenos CD34/metabolismo , Separação Celular , Células Endoteliais/citologia , Antígenos Comuns de Leucócito/metabolismo , Adulto , Antígeno CD146/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular , Dactinomicina/análogos & derivados , Dactinomicina/sangue , Citometria de Fluxo , Células Endoteliais da Veia Umbilical Humana , Humanos , Indóis/química , Leucócitos Mononucleares/citologia , Microscopia , Microscopia de Fluorescência , Pessoa de Meia-Idade , Fenótipo , Projetos Piloto , Lectinas de Plantas/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , RNA/metabolismo , Fator de von Willebrand/metabolismo
6.
Trials ; 14: 91, 2013 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-23547564

RESUMO

BACKGROUND: Pulmonary arterial hypertension is a rare disorder associated with poor survival. Endothelial dysfunction plays a central role in the pathogenesis and progression of pulmonary arterial hypertension. Inflammation appears to drive this dysfunctional endothelial phenotype, propagating cycles of injury and repair in genetically susceptible patients with idiopathic and disease-associated pulmonary arterial hypertension. Therapy targeting pulmonary vascular inflammation to interrupt cycles of injury and repair and thereby delay or prevent right ventricular failure and death has not been tested. Spironolactone, a mineralocorticoid and androgen receptor antagonist, has been shown to improve endothelial function and reduce inflammation. Current management of patients with pulmonary arterial hypertension and symptoms of right heart failure includes use of mineralocorticoid receptor antagonists for their diuretic and natriuretic effects. We hypothesize that initiating spironolactone therapy at an earlier stage of disease in patients with pulmonary arterial hypertension could provide additional benefits through anti-inflammatory effects and improvements in pulmonary vascular function. METHODS/DESIGN: Seventy patients with pulmonary arterial hypertension without clinical evidence of right ventricular failure will be enrolled in a randomized, double-blinded, placebo-controlled trial to investigate the effect of early treatment with spironolactone on exercise capacity, clinical worsening and vascular inflammation in vivo. Our primary endpoint is change in placebo-corrected 6-minute walk distance at 24 weeks and the incidence of clinical worsening in the spironolactone group compared to placebo. At a two-sided alpha level of 0.05, we will have at least 84% power to detect an effect size (group mean difference divided by standard deviation) of 0.9 for the difference in the change of 6-minute walk distance from baseline between the two groups. Secondary endpoints include the effect of spironolactone on the change in placebo-corrected maximal oxygen consumption; plasma markers of vascular inflammation and peripheral blood mononuclear cell gene expression profiles; sympathetic nervous system activation, renin-angiotensin-aldosterone system activation and sex hormone metabolism; and right ventricular structure and function using echocardiography and novel high-resolution magnetic resonance imaging-based techniques. Safety and tolerability of spironolactone will be assessed with periodic monitoring for hyperkalemia and renal insufficiency as well as the incidence of drug discontinuation for untoward effects. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01712620.


Assuntos
Anti-Inflamatórios/uso terapêutico , Endotélio Vascular/efeitos dos fármacos , Tolerância ao Exercício/efeitos dos fármacos , Hipertensão Pulmonar/tratamento farmacológico , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Artéria Pulmonar/efeitos dos fármacos , Projetos de Pesquisa , Espironolactona/uso terapêutico , Anti-Inflamatórios/efeitos adversos , Biomarcadores/sangue , Células Cultivadas , Protocolos Clínicos , Diuréticos/uso terapêutico , Método Duplo-Cego , Endotélio Vascular/imunologia , Endotélio Vascular/fisiopatologia , Teste de Esforço , Hipertensão Pulmonar Primária Familiar , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Hipertensão Pulmonar/sangue , Hipertensão Pulmonar/diagnóstico , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/imunologia , Hipertensão Pulmonar/fisiopatologia , Mediadores da Inflamação/sangue , Maryland , Antagonistas de Receptores de Mineralocorticoides/efeitos adversos , Projetos Piloto , Artéria Pulmonar/imunologia , Artéria Pulmonar/fisiopatologia , Recuperação de Função Fisiológica , Espironolactona/efeitos adversos , Fatores de Tempo , Resultado do Tratamento , Caminhada
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