Serotonin transporter, sex, and hypoxia: microarray analysis in the pulmonary arteries of mice identifies genes with relevance to human PAH

Abstract

Pulmonary arterial hypertension (PAH) is up to threefold more prevalent in women than men. Female mice overexpressing the serotonin transporter (SERT; SERT+ mice) exhibit PAH and exaggerated hypoxia-induced PAH, whereas male SERT+ mice remain unaffected. To further investigate these sex differences, microarray analysis was performed in the pulmonary arteries of normoxic and chronically hypoxic female and male SERT+ mice. Quantitative RT-PCR analysis was employed for validation of the microarray data. In relevant groups, immunoblotting was performed for genes of interest (CEBPβ, CYP1B1, and FOS). To translate clinical relevance to our findings, CEBPβ, CYP1B1, and FOS mRNA and protein expression was assessed in pulmonary artery smooth muscle cells (PASMCs) derived from idiopathic PAH (IPAH) patients and controls. In female SERT+ mice, multiple pathways with relevance to PAH were altered. This was also observed in chronically hypoxic female SERT+ mice. We selected 10 genes of interest for qRT-PCR analysis (FOS, CEBPβ, CYP1B1, MYL3, HAMP2, LTF, PLN, NPPA, UCP1, and C1S), and 100% concordance was reported. Protein expression of three selected genes, CEBPβ, CYP1B1, FOS, was also upregulated in female SERT+ mice. Serotonin and 17β-estradiol increased CEBPβ, CYP1B1, and FOS protein expression in PASMCs. In addition, CEBPβ, CYP1B1, and FOS mRNA and protein expression was also increased in PASMCs derived from IPAH patients. Here, we have identified a number of genes that may predispose female SERT+ mice to PAH, and these findings may also be relevant to human PAH.

A Sex-Specific MicroRNA-96/5-Hydroxytryptamine 1B Axis Influences Development of Pulmonary Hypertension

“…Total RNA extraction and Taqman quantitative RT-PCR were conducted as described previously (16,20) and in the online supplement.…”

“…Remodeled pulmonary arteries were investigated by elastin-picrosirius red staining as described previously (16,20) and in the online supplement.…”

Activity of the Estrogen-Metabolizing Enzyme Cytochrome P450 1B1 Influences the Development of Pulmonary Arterial Hypertension

“…Specifically, multiple lines of evidence converge to implicate the estrogen pathway in PAH pathogenesis. 10 -12 First, physiological concentrations (Ϸ1 nmol/L) of 17␤ estradiol (the predominant circulating estrogen in premenopausal women) are sufficient to increase expression of the estrogen-metabolizing enzyme cytochrome P450 1B1 (CYP1B1) 13 and to stimulate the proliferation of human pulmonary artery smooth muscle cells (hPASMCs). 14,15 Second, the aberrant expression of CYP1B1 is reported in bone morphogenetic protein type-II (BMPR-II)-affected female HPAH patients 16,17 and hPASMCs iso-lated from IPAH patients.…”

“…14,15 Second, the aberrant expression of CYP1B1 is reported in bone morphogenetic protein type-II (BMPR-II)-affected female HPAH patients 16,17 and hPASMCs iso-lated from IPAH patients. 13 Third, female susceptibility is observed in mice expressing the human serotonin transporter gene construct (SERT; SERT ϩ mice), and this is dependent on the presence of circulating 17␤ estradiol. 14 Intriguingly, CYP1B1 is also increased in the pulmonary arteries of these mice.…”

Endothelial Apoptosis in Pulmonary Hypertension Is Controlled by a microRNA/Programmed Cell Death 4/Caspase-3 Axis

“…27,29 Briefly, protein was extracted from PAECs and lung tissues by sonication, ice solubilization, and cellular fractionation before quantification (Protein Assay Kit, BioRad) and electrophoresis separation. For protein immunodetection, anti-PDCD4 (1:1000, Abcam), anti-caspase-3 (pro-and cleaved-; 1:1000, Cell Signal), and anti-GAPDH (1:5000, Abcam) were used.…”