Derivation and characterisation of endothelial cells from patients with chronic thromboembolic pulmonary hypertension

Figure 1. PEA samples. (a, b) Specimens collected during PEA from CTEPH patients were stained by hematoxylin and eosin and stained for elastin by orcein stain. (c) PEA samples presented an intact endothelium as shown by endothelial markers CD31 and vWF, an enlarged α-SMA negative neointima and an α-SMA positive organized media. (d) PEA samples showed the presence of mature microvessels. (E–f) Coagulant material showed a fibrous structure lacking cells.

Figure 2. CTEPH-EC characterization. (a) Images showing expansion of CTEPH-EC isolated from PEA specimen, (i) minced PEA material, (ii) endothelial colony at day 10, (iii) day 14 and (iv) confluent CTEPH-EC. (b) Representative flow cytometry histograms of HPAE, CTEPH-EC and smooth muscle cells (SMC) labelled with antibodies against endothelial surface makers (CD31, UEA-1, vWF, VE-CAD, eNOS) and muscular markers (α-SMA and calponin) (n = 10). (c) Representative images of HPAE, CTEPH-EC, and SMC were immune-labelled with antibodies against endothelial makers (CD31, CAV1, vWF, VE-CAD, eNOS) and muscular markers (α-SMA and calponin) (n = 10). Nuclei were counterstained using DAPI (blue) × 40 magnification.

Figure 3. Expression of cell-specific markers in CTEPH-EC. (a) CTEPH-EC presented endothelial markers at the mRNA level and were negative for muscular markers (myocardin). Values expressed as mean ± SD, n = 5 HPAE, n = 11 CTEPH-EC and n = 8 SMC independent experiments performed in triplicate, p < 0.05*, p < 0.01**, p < 0.0001****, Mann–Whitney U test comparing two groups and Kruskal–Wallis test followed by Dunn’s multiple comparisons test for three groups. (b) CD31, eNOS, vWF and Cav-1 protein levels in CTEPH-EC compared to HPAE. Values expressed as mean ± SD, n = 3 HPAE, n = 7 CTEPH-EC independent experiments, p < 0.05*, Mann–Whitney U test, (full-length gels are presented in supplementary material Fig. 7). The intensity of the individual bands was quantified using freely available Image Lab software (Biorad laboratories), version 6.1.0 build7, http://www.biorad.com. (c) CTEPH-EC and HPAE expression levels of HOXD3, -8 and -9 compared to HMVEC-L. Values expressed as mean ± SD, n = 5 HPAE, n = 9 CTEPH-EC and n = 5 HMVEC-L independent experiments performed in triplicate, p < 0.05*, p < 0.01**, Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Statistical analysis was…

Figure 4. Proliferation capacities of CTEPH-EC. (a) Proliferative capacity of CTEPH-EC as population doubling time. n = 3 HPAE, CTEPH-EC, n = 5 independent experiments performed in triplicate (b) Viability of CTEPH-EC compared to HPAE evaluated by MTT assay. Values expressed as mean ± SD, n = 5 HPAE, n = 8 CTEPH-EC independent experiments performed in triplicate, p < 0.05*, p < 0.01**, Mann–Whitney U test. (c) Quantification and stain of proliferative marker Ki-67 in CTEPH-EC and HPAE at different passages. Values expressed as mean ± SD, n = 5 HPAE, n = 8 CTEPH-EC; independent experiments p < 0.05*, p < 0.01**, Mann–Whitney U test, (d) Clonogenic potential of CTEPH-EC expressed in percentage. Values expressed as mean ± SD, n = 6 HPAE, n = 6 CTEPH-EC; independent experiments, p < 0.05*, Mann–Whitney U test. (E) Cellular adhesion of CTEPH-EC compared to HPAE. n = 3 HPAE, n = 6 CTEPH-EC. Statistical analysis was performed with GraphPad Prism 7 software, version 7.0e, serial number:GP7-0633739-R###-#####, https://www.graphpad.com.

Figure 5. Apoptosis in CTEPH-EC. (a) mRNA expression profile of apoptotic markers caspase-3, -8 and -9 in CTEPH-EC compared to HPAE. Values expressed as mean ± SD, n = 5 HPAE, n = 11 CTEPH-EC independent experiments performed in triplicate, p < 0.05*, Mann–Whitney U test. (b) Cellular perimeter, area, and diameter of CTEPH-EC and HPAE measured at different passages. Values expressed as mean ± SD, n = 8 HPAE, n = 8 CTEPH-EC independent experiments performed in triplicate. p < 0.05*, p < 0.0001****, two-way ANOVA followed by Tukey´s multiple comparisons test. (c) Representative images of CTEPH-EC and HPAE cells at different cell passages. Cellular circumference, area and diameter were measured in triplicate fields of 5–10 cells/picture (20 × magnification) using freely available imaging processing ImageJ software, version:2.1.0/1.53c, http://imagej.net/contributors. Statistical analysis was performed with GraphPad Prism 7 software, version 7.0e, serial number:GP7-0633739-R###-#####, https://www.graphpad.com.

Figure 6. Functional characterization of CTEPH-EC. (a) In vitro angiogenic potential of CTEPH-EC quantified by the number of branching points, tube length, cell covered area and number of loops. Representative pictures of spontaneous tube formation in HPAE and CTEPH-EC, n = 9 HPAE, n = 10 CTEPH-EC independent experiments performed in triplicate, p < 0.05*, p < 0.01**, Mann–Whitney U test, values expressed as mean ± SD. (b) Quantification of cell connections and geometrical features measured at 24, 72 and 120 h and representative pictures of HPAE and CTEPH-EC. p < 0.05*, p < 0.05*, Mann–Whitney U test values expressed as mean ± SD. (c) Migration capacity of CTEPH-EC and HPAE in the wound healing assay. Percentage of wound closure is measured at 8, 24, 32 and 48 h and plotted as the percentage of closure over the average area of width. Representative pictures of wound closure at 0 h and 24 h in HPAE and CTEPH-EC, n = 6 HPAE, n = 7 CTEPH-EC, p < 0.01**, Mann–Whitney U test at 8 h, 24 h, 32 h and 48 h, values expressed as mean ± SD. (d) In vivo angiogenic potential of CTEPH-EC- and HPAE-embedded sponge pellets quantified by the number of vessels in the sponge sections. Representative pictures…

Figure 7. Mitochondria in CTEPH-EC. (a) Visualization of the mitochondrial structure of cultured CTEPH-EC and HPAE by electron microscopy. (b) Oxygen consumption was measured in CTEPH-EC and HPAE by high-resolution respirometry (Oroboros Instruments). Contribution of the individual complexes of the mitochondrial respiratory chain to total cellular respiration was determined by the use of substrates or inhibitors of specific mitochondrial respiratory chain complexes. At last, proton leak was measured in CTEPH-EC and HPAE. Values expressed as mean ± SD, n = 5 HPAE, n = 5 CTEPH-EC independent experiments, p < 0.05*, p < 0.01**, Mann–Whitney U test. (c, d) mRNA expression and protein expression of fusion (MFN1, MFN2, and OPA1) and fission (DRP1) related modulators in CTEPH-EC and HPAE. n = 5 HPAE, n = 11 CTEPH-EC independent experiments performed in triplicate for mRNA expression for MFN1, MFN2, DRP1; and n = 4 HPAE, n = 11 CTEPH-EC independent experiments performed in triplicate for mRNA expression for OPA1. For western blot experiments, values expressed as mean ± SD, n = 3 HPAE, n = 7 CTEPH-EC, p < 0.05*, Mann–Whitney U test, (full-length gels are presented in supplementary material Fig. 8).…

Figure 8. Oxidative stress in CTEPH-EC. (a) ROS levels in CTEPH-EC and HPAE were visualized by fluorogenic dye MitoSOX (red) n = 5 HPAE, n = 7 CTEPH-EC independent experiments, p < 0.01**, Mann–Whitney U test, values expressed as mean ± SD and CellROX (green) n = 6 HPAE, n = 6 CTEPH-EC, p > 0.05. (b) Oxidation status of proteins in CTEPH-EC, HPAE, patient serum and serum from healthy volunteers. n = 3 HPAE, n = 7 CTEPH-EC; n = 3 HPAE, n = 6 HV, p < 0.05*, Mann–Whitney U test, values expressed as mean ± SD. The intensity of the bands was quantified using Image Lab software (Biorad laboratories), version 6.1.0 build7, http://www.biorad.com. (c) mRNA expression of SOD1 and SOD2 in CTEPH-EC and HPAE. Values expressed as mean ± SD, n = 6 HPAE, n = 10 CTEPH-EC independent experiments performed in triplicate, p < 0.05*, Mann–Whitney U test. (d) Staining of DNA damage induced by oxidative stress in PEA specimen using 8-hydroxy-2′-deoxyguanosine (8-OHdG). Statistical analysis was performed with GraphPad Prism 7 software, version 7.0e, serial number:GP7-0633739-R###-#####, https://www.graphpad.com.