Figure 7.

Sex-specific increased expression of cyclin A (ccna1) promotes cell cycle progression and hyperproliferation of female (F) PAEC_PAH. A: The RNA sequencing of RNA isolated from female and male (M), PAEC_PAH, and lung tissue of EH_ITSN-KO^ITSN+/– mice shows log₂ fold change in cyclin A1 and cyclin D2 levels. Human female and male PAEC_ND-Ctrl and wild-type (wt) mice served as controls. The cyclin D2 expression does not show significant changes in EH_ITSN-KO^ITSN+/– mouse lung; false-discovery rate–adjusted P value. B: The murine and human cyclin A1 promoters, indicating the Elk1 binding motif (bold), conserved in mouse and human. Elk1 binds to sequences centered on the GGA trinucleotide motif (http://www.ensembl.org/index.html, last accessed January 19, 2020). C: Expression of cyclin A1 in synchronized male and female PAEC_ND-Ctrl and PAEC_PAH. Cells were synchronized using 2 μg/mL aphidicolin (G₁/S phase) and 0.1 μg/mL nocodazole (G₂/M phase). The dotted vertical lines separate the ccna1 and actin immunoreactivities in the Input (In; not synchronized cells), S, and G₂/M phases of the cell cycle. D: The histogram shows the ratio cyclin A1/actin, as determined by densitometry of three representative X-ray films, demonstrating the sex-specific increase in cyclin A1 expression in the S and G₂/M phases of the synchronized PAEC_PAH. E and F: Real-time quantitative PCR of mRNA isolated from EH_ITSN-KO^ITSN+/– mice (E) and Sugen5416 inhibitor/hypoxia (Su/Hx) rats (F). The wt animals served as controls. Data are shown as means ± SD and are representative for 3 independent experiments performed in triplicate (D–F). n = 5 mice per experimental condition (E); n = 3 rats per experimental condition (F). ∗P < 0.05, ∗∗∗∗P < 0.0001. DU, densitometric units; Elk1, ETS domain containing protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ND-Ctrl, nondisease controls; PAEC_PAH, pulmonary artery endothelial cells of pulmonary arterial hypertension patients.