Fig 2: SCUBE1 Is Enriched in Pulmonary ECs and Down-Regulated by Triggering Factors Driving PAH(A) Abundance of secreted SCUBE1 protein in conditioned media from human pulmonary arterial endothelial cells (PAECs) and pulmonary arterial smooth muscle cells (PASMCs) was determined by immunoblotting (n = 3 samples per group). (B,C) PAECs were treated with control small, interfering ribonucleic acid (si-NC) or small, interfering ribonucleic acid specific to BMPR2 (si-BMPR2) for 72 h after which (B) SCUBE1 messenger ribonucleic acid (mRNA) expression was measured by reverse transcription, quantitative polymerase chain reaction of cellular homogenates (n = 3 samples per group) and SCUBE1 protein abundance was quantified by (C) enzyme-linked immunosorbent assay of conditioned media (n = 4 samples per group). (D,E) Similarly, PAECs were stimulated with hypoxia or interleukin (IL)-1β for 48 h, after which SCUBE1 (D) mRNA (n = 3 samples per group) and (E) secreted protein (n = 3 samples per group), were quantified by reverse transcription, quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. (F) Immunoblot of PAEC homogenates (n = 3 samples per group) after the above-mentioned treatments. Immunoblots are representative of 3 independent experiments with band intensities quantified by densitometry. Values are presented as mean ± SD. The p values were calculated by Student's t-test, 1-way analysis of variance with post hoc Bonferroni test. The comparisons with p > 0.05 were not explicitly stated in the panels. AU = arbitrary units; PAH = pulmonary arterial hypertension.

Fig 3: Plasma SCUBE1 Levels Are Inversely Correlated With Disease Severity in Patients with WSPH Group 1 PAHPlasma SCUBE1 concentration was compared across WSPH group 1 PAH patients with overall mean pulmonary artery pressure (mPAP) and calculated pulmonary vascular resistance (PVR) with Spearman correlation (A,C) or trend of change analysis in quartiles (binned based on minimum, 25th percentile, median, 75th percentile, and maximum) of mPAP (first quartile: 15 to 35 mm Hg; second quartile: 36 to 44 mm Hg; third quartile: 45 to 51 mm Hg; fourth quartile: 52 to 86 mm Hg) (B), or PVR (first quartile: 1.30 to 4.06 Wood units [WU]; second quartile: 4.07 to 5.31 WU; third quartile: 5.32 to 8.42 WU; fourth quartile: 8.42 to 20.0 WU) (D). (E to G) Transthoracic echocardiography images from the patient cohort managed at University of Pittsburgh Medical Center with WSPH group 1 PAH (n = 49 patients) were reviewed, and right ventricular (RV) dimensions and tricuspid annular plane systolic excursion (TAPSE) were directly measured from original transthoracic echocardiography images. Plasma SCUBE1 levels were compared within the population based on the degree of RV hypertrophy (E), RV dilation (F), or TAPSE tertile (binned based on minimum, 33th percentile, 66th percentile, and maximum. First quartile: 2.3 to 2.8 cm; second quartile: 1.7 to 2.3 cm; third quartile: 1.2 to 1.6 cm) (G). Grouped data are presented as median with interquartile range. The p values were calculated by Mann-Whitney U tests for pairwise comparisons, Kruskal-Wallis test for the change across quartiles and tertiles, and Spearman correlation analysis (rho: correlation coefficient). Abbreviations as in Figures 1 and 6.

Fig 4: HIF1A Expression Is Induced by Both Hypoxia and Il-1β and Mediates SCUBE1 Down-Regulation(A) PAECs were treated with si-NC, si-BMPR2, hypoxia, and IL-1β for 48 h, and HIF1A protein expression was determined by immunoblotting (n = 3 samples per group). (B) PAECs were treated with either si-NC or small, interfering ribonucleic acid to HIF1A and exposed to normoxia, hypoxia, or IL-1β, followed by immunoblotting for SCUBE1 (n = 3 samples per group). (C) In addition to the above-mentioned conditions, PAECs were also treated with si-BMPR2, and mRNA expression was determined by reverse transcription, quantitative polymerase chain reaction (n = 3 to 6 samples per group). (D) BMPR2 mRNA expression was quantified in PAECs treated with either si-NC or si-HIF1A (n = 3 samples per group). Immunoblots are representative of 3 independent experiments with band intensities quantified by densitometry. Values are presented as mean ± SD. The p values were calculated by Student's t-test, 1-way analysis of variance with post hoc Bonferroni test. The comparisons with p > 0.05 were not explicitly stated in the panels. Abbreviations as in Figure 1.

Fig 5: Plasma SCUBE1 Levels Are Decreased in Patients With WSPH Group 1 PAH(A) Plasma was collected from the pulmonary arteries of patients with World Symposium on Pulmonary Hypertension (WSPH) group 1 (n = 62) at the time of right heart catheterization. Peripheral plasma samples were collected from patients without PH (non-PH) (n = 56), patients with chronic obstructive pulmonary disease (COPD) (n = 39), and acute lung injury (ALI) (n = 39). SCUBE1 protein was quantified and compared across cohorts. (B) SCUBE1 protein was quantified in lung tissue obtained from rapid autopsy or lung transplant of individuals with WSPH group 1 PAH (n = 8), non-PH (n = 11), or COPD (n = 20). (C) Plasma was collected from group 2 PH (n = 16), and SCUBE1 protein levels were compared with group 1 PAH plasma samples. (D) SCUBE1 protein was quantified in serum samples collected from patients with coronary angiogram–confirmed coronary artery disease (CAD) (n = 22) and non-CAD control subjects (n = 21). (E) SCUBE1 protein was quantified in myocardium tissue homogenates obtained from rapid autopsy or heart transplant of nondiseased individuals (Ctl) (n = 12), patients with nonischemic cardiomyopathy (NICM) (n = 12) and ischemic cardiomyopathy (ICM) (n = 12). (F) Receiver-operating characteristic (ROC) curve for sensitivity and specificity analysis between PAH and a combined non-PAH cohort composed of control, COPD, and ALI patients (Clopper-Pearson method). Grouped data are presented as medians with interquartile range. The p values were calculated by Mann-Whitney U tests for pairwise comparisons, and Kruskal-Wallis test with post hoc Dunn Multiple Comparison test. The comparisons with p > 0.05 were not explicitly stated in the panels. AUC = area under the curve; other abbreviations as in Figures 1 and 5.