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).