Y-Chromosome Gene, Uty, Protects Against Pulmonary Hypertension by Reducing Proinflammatory Chemokines

Abstract

Rationale: Idiopathic pulmonary arterial hypertension (PAH) is a terminal pulmonary vascular disease characterized by increased pressure, right ventricular failure, and death. PAH exhibits a striking sex bias and is up to four times more prevalent in females. Understanding the molecular basis behind sex differences could help uncover novel therapies. Objectives: We previously discovered that the Y chromosome is protective against hypoxia-induced experimental pulmonary hypertension (PH), which may contribute to sex differences in PAH. Here, we identify the gene responsible for Y-chromosome protection, investigate key downstream autosomal genes, and demonstrate a novel preclinical therapy. Methods: To test the effect of Y-chromosome genes on PH development, we knocked down each Y-chromosome gene expressed in the lung by means of intratracheal instillation of siRNA in gonadectomized male mice exposed to hypoxia and monitored changes in right ventricular and pulmonary artery hemodynamics. We compared the lung transcriptome of Uty knockdown mouse lungs to those of male and female PAH patient lungs to identify common downstream pathogenic chemokines and tested the effects of these chemokines on human pulmonary artery endothelial cells. We further inhibited the activity of these chemokines in two preclinical pulmonary hypertension models to test the therapeutic efficacy. Measurements and Main Results: Knockdown of the Y-chromosome gene Uty resulted in more severe PH measured by increased right ventricular pressure and decreased pulmonary artery acceleration time. RNA sequencing revealed an increase in proinflammatory chemokines Cxcl9 and Cxcl10 as a result of Uty knockdown. We found CXCL9 and CXCL10 significantly upregulated in human PAH lungs, with more robust upregulation in females with PAH. Treatment of human pulmonary artery endothelial cells with CXCL9 and CXCL10 triggered apoptosis. Inhibition of Cxcl9 and Cxcl10 expression in male Uty knockout mice and CXCL9 and CXCL10 activity in female rats significantly reduced PH severity. Conclusions:Uty is protective against PH. Reduction of Uty expression results in increased expression of proinflammatory chemokines Cxcl9 and Cxcl10, which trigger endothelial cell death and PH. Inhibition of CLXC9 and CXLC10 rescues PH development in multiple experimental models.

Keywords: CXCL10; CXCL9; endothelial cells; pulmonary arterial hypertension; sex differences.

Figure 1.

Lung-specific knockdown of Uty, but not other Y-chromosome (ChrY) genes expressed in the lung, eliminates ChrY protection against hypoxia (Hx)-induced pulmonary hypertension. (A) Experimental protocol: 30 days after gonadectomy (GDX), male mice receive scramble siRNA or siRNA targeting ChrY genes every 5 days and are placed in Hx for 3 weeks. (B–E) Right ventricular systolic pressure (RVSP) measured by direct RV catheterization for (B) Kdm5d, (C) Ddx3y, (D) Eif2s3y, and (E) Uty. (F) Pulmonary artery acceleration time (PAAT) measured by echocardiography. (G) Fulton index (RV/LV + IVS). *P < 0.05. IVS = intraventricular septum; LV = left ventricle; ns = not significant.

Figure 2.

Proinflammatory chemokines CXCL9 and CXCL10 are autosomal downstream deterrents of Uty protection and are upregulated in PAH patient lungs in a sex-specific manner. (A) Comparison of RNA-sequencing (RNAseq) data from Si-Scrm and Si-Uty Hx mouse lungs revealed 523 differently expressed genes (DEGs) as represented by vertical columns on a heatmap depicting log fold change in expression. (B) Pathways enriched with DEGs include inflammation, cell signaling, extracellular matrix (ECM), transcription, and translation. (C) Integration of RNAseq data with an online microarray dataset of male and female human PAH lung samples revealed Cxcl9/CXCL9 and Cxcl10/CXCL10 as upregulated in PAH females (versus PAH males) and Si-Uty mice (versus Si-Scrm) as depicted in a heatmap representing individual patients’ relative gene expression. (D) Relative expression of CXCL9 and CXCL10 in human healthy and PAH lungs as measured by real-time quantitative PCR (qPCR). (E–G) Representative immunofluorescence staining of CXCL9 (E) and CXCL10 (G) in male and female lung tissue from healthy and diseased (PAH) patients. (F) Relative expression of CXCL9 and CXCL10 in human male and female PAH lungs as measured by real-time qPCR. Scale bars, 100 μm. *P < 0.05, **P < 0.01, and ***P < 0.001. Hx = hypoxia; KD = knockdown; WT = wild-type.

Figure 3.

Global Uty-KO promotes more severe PH, which is prevented by KD of Cxcl9 and Cxcl10. (A) Experimental protocol: 30 days after GDX, age-matched global Uty-KO or WT male mice receive scramble siRNA or siRNA targeting Cxcl9 and Cxcl10 genes every 3–5 days and are placed in Hx for 3 weeks. (B) RVSP measured by direct RV catheterization. (C) PAAT measured by echocardiography. (D) Fulton index (RV/LV + IVS). (E) Quantification of Cxcl9 and Cxcl10 transcripts in whole lung tissue by means of real-time qPCR (normalized to Si-Scrm). *P < 0.05 and ***P < 0.001. GDX = gonadectomy; HX = hypoxia; IVS = intraventricular septum; KD = knockdown; KO = knockout; LV = left ventricle; ns = not significant; PAAT = pulmonary artery acceleration time; PH = pulmonary hypertension; RV = right ventricle; RVSP = right ventricular systolic pressure; WT = wild type.

Figure 4.

Uty, Cxcl9, and Cxcl10 are colocalized in macrophages, and Uty expression is inversely related to Cxcl9 and Cxcl10 expression. (A–D) Representative images depicting colocalization of in situ probes labeling Cd68/CD68 (red), Uty/UTY (green), and Cxcl9/CXCL9 (white) in mouse (A) and human lung sections (C). Representative images depicting colocalization of in situ probes labeling Cd68/CD68 (red), Uty/UTY (green), and Cxcl10/CXCL10 (white) in mouse (B) and human lung sections (D). (E) Schematic of bone marrow–derived macrophage (BMDM) in vitro experiments. (F and G) Relative expression of Cxcl9 (F) and Cxcl10 (G), as measured by real-time qPCR in BMDMs isolated from WT and Uty-KO mice. *P < 0.05 and **P < 0.01. Scale bars, 50 μm. KO = knockout; WT = wild type.

Figure 5.

CXCL9 and CXCL10 recombinant protein triggers pulmonary artery endothelial cell (PAEC) dysfunction. (A) Schematic of human PAEC (hPAEC) experiments. (B and C) Quantification of percent apoptosis measured in PAECs treated with CXCL9 (B) or CXCL0 (C), as measured by cleaved caspase-3 immunofluorescence. (D) Representative images of cleaved caspase-3 (CC-3; red) immunofluorescence in vehicle, CXCL9-treated, and CXCL10-treated cells. *P < 0.05. Scale bar, 50 μm.

Figure 6.

Blocking the activity of CXCL9 and CXCL10 is sufficient to rescue PH in two preclinical rat models. Schematic of in vivo experiments in female rats with (A) monocrotaline (MCT)-induced PH and (B) Sugen 5416-Hx (SuHx)–induced PH. All MCT model measurements are listed in the left column (A, C, E, G, I, and K), and SuHx measurements are listed in the right column (B, D, F, H, J, and L). (C and D) RVSP, (E and F) PAAT, and (G and H) Fulton index (RV/LV + IVS) measured in AMG487 (AMG)-treated PH rats compared with vehicle-treated PH controls. (I and J) Quantification and (K and L) representative images of apoptotic EC cells in CD31 (yellow) and cleaved caspase-3 (CC-3; pink) labeled sections from vehicle (VEH) and AMG-treated lungs. *P < 0.05 and **P < 0.01. Scale bars, 50 μm. CTRL = control; EC = endothelial control; INH = inhibition; IVS = intraventricular septum; LV = left ventricle; ns = not significant; PAAT = pulmonary artery acceleration time; RV = right ventricle; RVSP = right ventricular systolic pressure.

Figure 7.

Proposed mechanism of Uty/Cxcl9/Cxcl10 axis in PH pathogenesis. Uty absence (in females) or reduced expression (in males with PAH or Uty-KD or in KO mice) results in an upregulation of proinflammatory chemokines Cxcl9 and Cxcl10 in the lung. CXCL9 and CXCL10 trigger vascular EC dysfunction, resulting in increased PH severity. Blocking CXCL9 and CXCL10 activity by pharmacologically inhibiting their shared receptor, CXCR3 is a novel treatment strategy to rescue PH development. EC = endothelial control; KD = knockdown; KO = knockout; PAH = pulmonary arterial hypertension; PH = pulmonary hypertension.