Plasma Cell-Free DNA Predicts Survival and Maps Specific Sources of Injury in Pulmonary Arterial Hypertension.

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.

Pulmonary arterial hypertension patients display normal kinetics of clot formation using thrombelastography.

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.

Isolation of a circulating CD45-, CD34dim cell population and validation of their endothelial phenotype.

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.