Results
G6PD inhibition decreased chronic Hx- and Hx+SU-induced PH in mice: PH was induced by exposing C57BL/6J mice to Hx and Hx+SU (Figure 1A). C57BL/6J mice exposed to Hx and Hx+SU had higher RVSP, RVEDP, and arterial elastance (Ea) than Nx mice (Figure 1B and Table 1). In Hx+SU group, RVSP, RVEDP, Ea, and LV stiffness were higher than those of Hx group (Figure 1C and D, and Table 1). Furthermore, cardiac index (CI) was decreased in Hx and Hx+SU, and more so in Hx+SU mice (Table 1).
In Hx mice, we first established the maximum tolerated dose (MTD) of G6PD inhibitor (4091). The MTD in Hx mice was 15 mg.kg-1.day-1 beyond which PDD4091 depressed LV function. More importantly, PDD4091 had a reasonably wide therapeutic window (0.01 to 15 mg.kg-1.day-1) with an EC50 of 0.26±0.10 and 0.58±0.36 mg.kg-1.day-1 reduced both RVSP and RVEDP. G6PD inhibitor, PDD4091, treatment to Hx mice decreased the elevated RVSP and RVEDP in a dose-dependent manner (Figure 2C and D top panel). Moreover, PDD4091 (1.5 mg.kg-1.day-1) treatment to both Hx and Hx+SU mice efficaciously and comparably reduced (by 50%) the elevated RVSP and RVEDP (Figure 2C and D bottom panel) and Ea (Table 1). Fulton’s index was increased in Hx and Hx+SU groups compared with Nx group. G6PD inhibitor reduced elevated Fulton’s index in Hx and Hx+SU groups (Table 1), and increased CI in both groups (Table 1).
G6PD inhibitor did not cause toxicity in mice: To examine whether G6PD inhibitor caused toxicity in mice, we measured hematocrit and performed blood chemistry analysis to assess organ (such as; liver, pancreas, and kidney) damage/function. As expected, mice exposed to Hx and Hx+SU had higher hematocrit than their respective controls, Nx and Nx+SU (Table 1). Treating Hx and Hx+SU mice with PDD4091 (1.5 mg.kg-1.day-1) for 3 weeks reduced the elevated hematocrit to the Nx levels in Hx, but not in Hx+SU, mice (Table 1). The blood chemistry revealed that PDD4091 treatment normalized electrolyte levels and did not cause toxicity in mice (Table 2).
Loss-of-function Mediterranean G6pd variant normalized RV pressures and decreased RV hypertrophy elevated by SU/Hx/Nx exposure in rats: Although Hx+SU mouse model is used to study pathogenesis of PH, it is not a perfect experimental model of human PH (Vitali et al., 2014). Therefore, we also tested our hypothesis in a SU/Hx/Nx rat model. PH was induced by exposing rats to SU/Hx/Nx, as shown in figure 2A, in Wild-type SD rats and in the G6PDS188F rat model mimicking human condition (Kitagawa et al., 2020; in press). These G6PDS188F rats have less G6PD activity in the heart and lungs as compared to wild-type SD rats (Figure 2B). SU/Hx/Nx increased RVSP (by 69.8% ; Figure 2C) and Fulton’s index (by 32.2%; Figure 2E), compared to Nx wild-type SD rats. Intriguingly, RVSP (Figure 2C), RVDP (Figure 2D), and RV hypertrophy (Figure 2E), were reduced significantly in SU/Hx/Nx G6PDS188F rats as compared to SU/Hx/Nx wild-type SD rats.
G6PD inhibition relaxed pre-contracted PA, decreased PASMC growth, and reduced PA remodeling in Hx+SU mice: PA remodeling is the hallmark of severe PH. Hyperplasic and apoptosis-resistant PA endothelial cells and PASMCs contribute to hypertensive remodeling (Morrell et al., 2009). Previously, we and others proposed that SMCs switch from a differentiated to a dedifferentiated phenotype in PA of hypertensive patients and animals and contribute to PA remodeling (Chettimada, Gupte, Rawat, Gebb, McMurtry & Gupte, 2015; Sahoo et al., 2016; Zhou, Negash, Liu & Raj, 2009). Dedifferentiated SMCs are hyper-proliferative, migratory, and secretory (Frismantiene, Philippova, Erne & Resink, 2018). Previous studies show that the Hx+SU mouse model of PH has more severe PA remodeling than Hx mice (Vitali et al., 2014). Therefore, we determined whether G6PD inhibition relaxes PA in ex vivo studies, stunts the growth of PASMCs exposed to Hx and SU in cell culture, and reduces remodeling of PA in Hx+SU mice. Our results demonstrated PDD4091 dose-dependently relaxed PA pre-contracted with KCl (Figure 3A). Application of PDD4091 (1 µmol/L) for 48 hours to PASMCs cultured in normoxia decreased cell numbers (Figure 3B) and in addition attenuated the cell growth evoked by Hx and Hx+SU (Figure 3C). Treatment of Hx+SU mice with PDD4091 (1.5 mg.kg-1.day-1) for 3 weeks abrogated the occlusive pulmonary vascular remodeling (Figure 3D).
Gene expression is altered in lungs of Hx and Hx+SU mice: To discover the genetic and epigenetic determinants of PASMC growth in the PA wall and remodeling of PA in Hx and Hx+SU, we first performed RNA-seq analysis in lungs of mice exposed to Nx, Hx, and Hx+SU. The results revealed that out of 159 and 97 genes upregulated in lungs of Hx vs Nx and Hx+SU vs Nx mice, respectively, only 3 genes were commonly upregulated in both groups (Figure 4A). Whereas 1511 and 1523 genes were downregulated in lungs of Hx vs Nx and Hx+SU vs Nx mice, respectively, 1085 genes were commonly downregulated in both groups (Figure 4A). Transcription factor binding site enrichment analysis using oPOSSUM (Kwon, Arenillas, Worsley Hunt & Wasserman, 2012) disclosed TCFCP2l1, KLF4, and E2F1 as the most enriched TFBS in genes upregulated in the Hx group and HIF1A::ARNT, KLF4, and SP1 as the most enriched TFBS in genes upregulated in the Hx+SU group (Figure 4B top panels). HOXA5, PDX1, and PRRX2 were the most enriched TFBS in genes downregulated in the Hx and Hx+SU groups (Figure 2B bottom panels). Suppressor of fused (Sufu ) homolog and Cytochrome P450 1A1 (Cyp1a1 ) genes were, respectively, upregulated >100- and >15-fold in lungs of mice exposed to Hx+SU, but not to Hx, while all genes downregulated >20-fold were common in lungs of mice exposed to Hx+SU and Hx (Figure 4C).
Methylation of DNA is decreased in lungs of Hx and Hx+SU mice:Epigenetic modifications, including methylation of DNA, are incriminated in the pathogenesis of PH (Cheng, Wang & Du, 2019; Joshi et al., 2020). Recently, we reported that downregulation of ten-eleven translocation 2 (Tet2 ) DNA demethylase in lungs of Hx Sv129J mice lackingCyp2c44 gene contributes to the genesis of PH, and also demonstrated that inhibition of G6PD was ineffective in reducing PH in hypoxic Tet2-/- mice (Joshi et al., 2020). Therefore, we postulated that downregulation of Tet2 by Hx may augment DNA methylation in C57BL/J mice and mediate maladaptive gene expression. Unexpectedly, we found that expression of Tet1 but not of Tet2 or Tet3 was reduced in lungs of C57BL/J mice exposed to Hx (Figure 5A and Table 3). In addition, expression ofDnmt3b but not of Dnmt3a or Dnmt1a was decreased (Figure 5A and Table 3). Concomitantly, we found that 45,321 CpG regions genome-wide were differentially methylated, and out of which 46.53% regions were hypermethylated and 53.47% were hypomethylated in lungs of mice exposed to Hx as compared to Nx (Figure 5B). While 46,286 CpG regions genome-wide were differentially methylated, and out of which 46.15% regions were hypermethylated and 53.85% were hypomethylated in lungs of mice exposed to Hx+SU as compared to Nx (Figure 5C). Therefore, there were 0.38% more hypomethylated and less hypermethylated CpG regions in lungs of C57BL/J mice exposed to Hx+SU than Hx. Furthermore, two genes, Cyp1a1 and Kcng3 , out of 12 differentially regulated genes by Hx and Hx+SU (Figure 4C), were hypomethylated in lungs of both Hx and Hx+SU mice (Figure 5D). It is noteworthy that CpG regions 2890 bp from the transcription start site of Cyp1a1 gene were hypomethylated by Hx and Hx+SU (Table 4).
G6PD inhibition decreased expression of Cyp1a1 andSufu genes in lungs of mice and in human PASMCs exposed to Hx+SU : Next, we determined whether inhibition of G6PD activity, which hypermethylated Cyp1a1 gene in lungs of Hx mice (Table 4), decreased expression of Cyp1a1 and Sufu in lungs of Hx and Hx+SU mice and in human PASMCs exposed to Hx+SU. Treatment of PDD4091 (1.5 mg.kg-1.day-1) for 3 weeks to Hx mice and application of PDD4091 (1 µM) to human PASMCs for 48 hr rescinded the Hx+SU-induced Cyp1a1 and Sufu expression in lungs (Figure 6A, B) and in human PASMCs (Figure 6C).