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. Author manuscript; available in PMC: 2020 Oct 22.
Published in final edited form as: Circulation. 2019 Aug 29;140(17):1409–1425. doi: 10.1161/CIRCULATIONAHA.119.040629

Figure 4. BMPR2 deficiency coupled with increased 5-LO-mediated immunity induces a proliferative and inflammatory endothelial phenotype.

Figure 4.

To mimic BMPR2 signaling loss in vitro, PAECs from control donor were infected with lentiviral shBmpr2 or control lentivirus (shCtr) and were compared with cells collected from hPAH patient bearing Bmpr2 mutations. PAECs were subsequently cultured in 400nM LTB4 or vehicle in the serum free medium for 7 days to assess the effects of increased 5-LO-mediated inflammation. (A and B) Representative flow cytometry contour plots and quantification of cleaved caspase 3. n=6. (C and D) Representative flow cytometry contour plots and quantification of Ki67. n=6. (E and F) Representative PAEC phase-contrast light microscopy images and quantification. n=6; scale bar, 100μm. (G and H) Representative confocal images and quantification of cells stained with tight junction marker, VE-cadherin (green). DAPI (blue) identifies nuclei. Red arrow heads point to the discontinuous cellular junctions in G. n=6. Scale bar, 100μm. (I and J) Representative confocal staining and quantification of 5-LO (green). DAPI (blue) identifies nuclei. White arrows point to endogenous 5-LO expression in the cytoplasm or in the nucleus in I; white arrow heads indicate the nuclear envelope and ER and Golgi membrane localized 5-LO in I. n=6. Scale bar, 100μm. (K) Measurements of leukotriene B4 (LTB4) in PAEC culture medium. n=6. (L) Quantification of Illrl (interleukin 1 receptor 1), Il6r (IL6 receptor), Tlr2 (Toll-like receptor 2) and Tlr4. (M) Model of PAEC transformation: enhanced 5-LO/LTB4 inflammatory responses cause a step-wise transformation of PAECs with compromised BMPR2 signaling. 1) LTB4 induces PAEC apoptosis within 24hrs of culture. 2) The surviving, apoptosis-resistant cells become hyperproliferative on day 3, and then 3) transform into a mesenchymal-cell like phenotype on day 5. These cells acquire nuclear envelope-localized 5-LO to produce endogenous LTB4 and express molecular signature of IL-1, IL-6 and TLR inflammatory pathways. BMPR2-conserved PAECs with LTB4 culture do not under undergo the inflammatory transformation. All data are presented as means and SEM; Kruskal-Wallis test; ns, not significant, *p< 0.05, **p < 0.01.