Journal: Pulmonary Circulation

Article Title: Transmembrane Protein 100 Expression on Endothelial Cells Vascularizing Thrombi in Chronic Thromboembolic Pulmonary Hypertension Modulates TGFβ1−ALK1 Signaling During Angiogenesis

doi: 10.1002/pul2.70253

Figure Lengend Snippet: (A) Schematic drawing depicting the analysis of CTEPH‐ECs, HPAECs, and HSaVECs. The panel was created with BioRender.com. (B) TMEM100 mRNA expression in CTEPH‐ECs ( n = 20 biological replicates; ECs isolated from central PEA samples marked with “full” and distal PEA samples with “open” circles) compared to HPAECs ( n = 3 biological replicates examined in 3–6 independent experiments) and HSaVECs ( n = 1 biological replicate examined in four independent experiments). (C and D) TMEM100 protein expression in CTEPH‐ECs ( n = 3 biological replicates examined in 3 independent experiments), HPAECs ( n = 2 biological replicates examined in 2 – 3 independent experiments), and HSaVECs ( n = 1 biological replicate examined in 5 independent experiments). Quantitative analysis (C). Representative images (D). Expression of CD31/PECAM1 is shown to demonstrate EC marker expression. (E and F) Immunohistochemical detection of TMEM100 on cross‐sections through PEA specimens from patients with CTEPH ( n = 6 biological replicates) or PE ( n = 3 biological replicates examined in 2 independent experiments). Representative images (E). Quantitative analysis (F). Findings after omission of the first antibody (negative control) are also shown. ALK1 (G) and ALK5 (H) mRNA expression in CTEPH‐ECs ( n = 12 and n = 8 biological replicates, respectively) versus HPAECs ( n = 3 biological replicates examined in 2–3 independent experiments) and HSaVECs ( n = 1 biological replicate examined in 5 independent experiments). (I) TMEM100 mRNA expression in HPAECs ( n = 2 biological replicates examined in 2 independent experiments), control or after TGFβ1 stimulation (10 ng/mL for 24 h), alone or in the presence of the ALK1 inhibitor K02288 (10 µM) or the ALK5 inhibitor SB431542 (10 µM). ALK1 (J) and ALK5 (K) mRNA expression in HPAECs ( n = 2 biological replicates examined in 3 independent experiments), control or after TGFβ1 stimulation, and TMEM100 siRNA transfection. ALK1 (L) and ALK5 (M) mRNA expression in HPAECs ( n = 2 biological replicates examined in 2–3 independent experiments), alone or in the presence of an ALK5 (L) or ALK1 (M) inhibitor. Representative images (N) and quantitative analysis of the cumulative tube length (O) of CTEPH‐ECs ( n = 1 biological replicate examined in 4 independent experiments) cultivated on Matrigel for 5 h and the effects of TGFβ1 stimulation (10 ng/mL for 24 h), control or TMEM100 siRNA transfection, ALK1, or ALK5 inhibition. Exact p values were determined using unpaired Student's t ‐test (F, L, and M), one‐way ANOVA, Sidak's multiple comparisons test (G, H, I, and O), or Kruskal–Wallis, Dunn's multiple comparisons test (B, C, J, and K), based on the Shapiro–Wilk test for normal distribution. Quantitative data are shown as mean ± SEM or median with interquartile range, depending on the presence of normal distribution or not. Scale bars in (D), (E), and (N) denote 100 µm. (P) Visual summary of the findings of the study. The panel was created with Inkscape v.1.4.2.

Article Snippet: CTEPH‐ECs (Passage 1; Ethics Approval AZ 199/15) were isolated from CTEPH PEA specimens, as described in Bochenek et al. [ ], and compared to human pulmonary arterial ECs (HPAECs; PromoCell, C‐12241, Lot Numbers 458Z016.13 and 482Z007.13; ATCC, PCS‐100‐022, Lot Number 59880292) and ECs from the human saphenous vein (HSaVECs; PromoCell, C‐12231, Lot Number 455Z015.1), a frequent origin of blood clots embolizing into the lungs.

Techniques: Expressing, Isolation, Marker, Immunohistochemical staining, Negative Control, Control, Transfection, Inhibition