Additional file 1: Figure S1. of Endothelium-derived microparticles from chronically thromboembolic pulmonary hypertensive patients facilitate endothelial angiogenesis

Endothelial tube formation induced by CTEPH microparticles. HPAECs were cultured on Matrigel in serum-reduced (0.5 % serum) medium with different numbers of CTEPH microparticles, as indicated. Tube formation was scored after 18 h of incubation. **P < 0.01, comparison with untreated controls. Figure S2. Tube formation in HPAECs incubated with TGF-β and MPs. Representative images of tube formation in HPAECs incubated with MP fractions (full and endoglin−) from healthy and CTEPH plasma, with or without TGF-β (10 ng/mL;18 h). Bar = 50 μm. Figure S3. Endoglin levels in full MP fractions (containing endoglin+ MPs) and endoglin-depleted (endoglin−) MP fractions. Endoglin+ MPs were removed by Dynabeads-mediated immunoprecipitation and endoglin levels were measured with Human Endoglin/CD105 Quantikine ELISA Kit.***P < 0.001, Student t-test, n = 6. Figure S4. Pro-angiogenic factors in CTEPH EMP fraction. (a) Proteome Profiler™ Human Angiogenesis Array membrane was incubated with CTEPH EMPs (image representative of n = 2). (R) shows reference spots, (1) corresponds to Serpin E1 and (2) corresponds to uPA, as indicated. Full information about the microarray layout can be found on https://resources.rndsystems.com/pdfs/datasheets/ary007.pdf . A schematic diagram of microarray is shown in (b). (DOCX 479 kb

Hepatic glucose production in mice fed a high fat diet.

<p>mRNA levels of PEPCK and G6Pase (A) and triglyceride (TG) content (B) in the liver of male wild type and endoglin heterozygous mice fed a high fat diet for 16 weeks. 18S was used as an internal control. n = 6–8. **p<0.01.</p

Levels of plasma metabolites and hormones in mice fed a high fat diet.

<p>Serum levels of insulin (A), glucose (B), TG (C), NEFAS (D), and cholesterol (E) in male wild type and endoglin heterozygous mice fed a high fat diet for 16 weeks. n = 6–8. **p<0.01.</p

Body weight, body composition, food intake, and metabolic parameters in mice fed a high fat diet.

<p>Body weight (A), fat mass (B), non-fat mass (C), food intake (D), total energy expenditure (E), energy expenditure corrected by non-fat mass (F), total locomotor activity (G), locomotor activity corrected by non-fat mass (H), respiratory quotient during light phase (I), respiratory quotient during dark phase (J), and 48 h profile of RQ (K) in male wild type and endoglin heterozygous mice fed a high fat diet for 16 weeks. Measurements were done during 48 h. n = 6–8.</p

Glucose homeostasis and insulin sensitivity in mice fed a standard diet.

<p>Basal glucose levels (A), glucose tolerance test (B), respective area under the curve (C), insulin tolerance test (% of glucose levels represented against t0) (D), and respective area under the curve (E) in 8-week male wild type (WT) and endoglin heterozygous (HZ) mice fed a standard diet. n = 6–8. *p<0.05.</p

Glucose homeostasis and insulin sensitivity in mice fed a high fat diet.

<p>Basal glucose levels (A), glucose tolerance test (B), respective area under the curve (C), insulin tolerance test (% of glucose levels represented against t0) (D), and respective area under the curve (E) in male wild type (WT) and endoglin heterozygous (HZ) mice fed a high fat diet for 16 weeks. n = 6–8. *p<0.05.</p

Insulin signaling and glucose uptake in mice fed a high fat diet.

<p>Protein levels of NFkB, pAKT, AKT, and PTEN in the liver (A), and muscle (B) of male wild type and endoglin heterozygous mice fed a high fat diet for 16 weeks. Protein levels of NFkB, pAKT (Ser473), AKT, PTEN, and Glut4 in the white adipose tissue (C) of male wild type (+/+) and endoglin heterozygous (+/−) mice fed a high fat diet for 16 weeks. All the samples (+/+ and +/−) for each protein were analyzed within the same gel, and the lines represent splicings of the gels. n = 6–8. *p<0.05.</p

Schematic domain organization of human endoglin and ALK1 and western blots of endoglin domains.

<p>Bar diagram of (A) endoglin and (B) ALK1 with the domains indicated and highlighted in different styles. TM, transmembrane region, ZP, zona pellucida. The putative Asn glycosylation sites Asn88, Asn102, Asn121, Asn134 and Asn306 of endoglin and Asn98 of ALK1 are labelled within green ovals. The constructs used in this study and the domains encompassed by these are shown below the bar diagram of the respective full length protein. (C) Western blots of endoglin constructs. Endo₃₃₈, Endo₃₆₂ and LG-Endo_EC were analyzed by 10% SDS polyacrylamide electrophoresis gel followed by western blotting with an anti-His₆ antibody. Samples reduced with dithithreitol (DTT) were incubated for 1 h with 10 mM of this reagent at 65°C. All samples (0.5 µg of protein) were then denaturated by boiling at 95°C for 5 minutes prior to charging onto the gel. The molecular weight markers (M) are indicated at the left of the samples. For both Endo₃₆₂ and LG-Endo_EC, dimeric species are visible, while Endo₃₃₈ was only observed in monomeric form.</p

Functional interactions between endoglin, ALK1 and BMP-9.

<p>The interactions of (A) Endo_EC, (B) ALK1_EC, (E) Endo₃₃₈ and (F) Endo₃₆₂ with BMP-9 were investigated by SPR. While HG-Endo_EC and Endo₃₆₂ dissociated slowly, Endo₃₃₈ dissociated much faster, indicating a rigid body type of binding as opposed to an induced fit mechanism. For affinity measurements, the indicated recombinant proteins were injected at six concentrations ranging from 12.5 to 400 nM over BMP-9 (which was immobilized on a CM5 sensor chip by amine coupling) to generate sensorgrams (colored curves). When testing competition between HG-Endo_EC and HG-ALK1_EC (C and D) the chip was first pre-equilibrated with 750 mM of either HG-Endo_EC (C, left) or HG-ALK1_EC (D, left) before injecting the various concentration of the second ligand, showing the curve for the highest concentrations of the 2^nd ligand. Both HG-ALK1_EC (C, right) and HG-Endo_EC (D, right) yielded, after subtracting the background, similar results to those in runs in which no first ligand was preequilibrated before injecting the second ligand (D right vs. E; C right vs. B). This leads to the conclusion that endoglin and ALK1 bind independently to different sites on BMP-9. The kinetic parameters for the interaction were determined by global fitting (curves in black) of the 1∶1 Langmuir binding model to these data, providing values for the association (k_a) and dissociation (k_d) rate constants and the dissociation affinity constant (K_D).</p

Analysis of ligand binding to BMP-9 assessed by Surface Plamson Resonance.

<p>Kinetic analysis of endoglin and ALK1 binding to BMP-9 was performed in triplicates on a Biacore T100 at 25°C as described in the experimental procedures. Data were globally fit to a 1∶1 binding model using the Biacore T100 evaluation software.</p