Discussion
The results of this study provided evidence that downregulation of the
epigenetic modifiers Tet1 and Dnmt3b and hypomethylation
of DNA altered gene expression in lungs of Hx- and Hx+SU-induced PH
mice. Furthermore, pharmacologic inhibition of G6PD activity relaxed
pre-contracted PA, decreased growth of PASMCs evoked by Hx and SU,
reduced expression of Cyp1a1 and Sufu , which potentially
arrested growth of PASMCs, and rescinded occlusion of PA in lungs of
mice exposed to Hx+SU. Additionally, we demonstrated attenuation of
SU/Hx/Nx-induced PH in a loss-of-function Mediterranean G6pdvariant rat model. These results suggest G6PD is a common factor for the
genesis of PH in Hx and Hx+SU mice and rats. Since a selective inhibitor
of G6PD activity decreased occlusive remodeling of PA and alleviated PH
induced by Hx and Hx+SU in mice without causing toxicity, we propose
that G6PD might be a safe pharmacotherapeutic target to reduce PH in
humans.
Hx and Hx+SU rat and mouse models are routinely used to study the
pathology of PH (Stenmark, Meyrick, Galie, Mooi & McMurtry, 2009). We
observed in this study that mice exposed to Hx for 6 weeks and to Hx+SU
for 3 weeks developed PH, which was more severe in Hx+SU than Hx group.
In chronically Hx (3 weeks) mice, vasoconstriction and muscularization
of small arteries, but not obliterative remodeling of PA, contribute to
increased pulmonary arterial pressure and RV pressure overload
(Stenmark, Meyrick, Galie, Mooi & McMurtry, 2009). The more severe PH
in Hx+SU mice is attributed to the formation of angio-obliterative
lesions in addition to vasoconstriction and muscularization (Vitali et
al., 2014). To support our findings of pharmacologic G6PD inhibition in
the Hx+SU mice, we also used the SU/Hx/Nx rat model of PH and found that
the hypertension was reduced in rats expressing
G6PDS188F, a Mediterranean G6pd variant that
has 80% less activity than wild-type G6PD. Thus, our results indicate
that inhibition of G6PD activity by either pharmacologic or genetic
interventions reduces remodeling of PA and elevated RV pressure/overload
in PH mice and rats.
The above observations raise the question of whether the underlying
genetic determinants of PH in mice exposed to Hx and Hx+SU are same or
different? To seek answers, we performed RNA-seq analysis in lungs which
revealed that >1000 downregulated genes and only 3
upregulated genes, driven by different transcription factors, were
common between the two models. Most striking difference was noticed in
>15-fold increase of Sufu and Cyp1a1 genes in
lungs of mice exposed to Hx+SU but not to Hx. Furthermore, exposure to
SU increased expression of both SUFU and CYP1A1 genes in
Hx but not in Nx human PASMCs. While these results are consistent with a
recent study that indicates HIF::ART-driven Cyp1a1 gene is
upregulated in lungs of rats exposed to SU/Hx/Nx and in human PASMCs by
SU (Dean et al., 2018), an increase of Sufu in lungs of PH mice
and human PASMCs has not been reported. CYP1A1 is an
estrogen-metabolizing enzyme that produces mitogenic metabolites of
estrogen in human PASMCs (Dean et al., 2018) and SUFU is a negative
regulator of hedgehog signaling, which controls cell proliferation
during development in invertebrates and vertebrates (Briscoe & Therond,
2013; Liu, 2019). Increased CYP1A1 contributes to the pathogenesis of PH
in SU/Hx/Nx rats (Dean et al., 2018). Our results suggest that increased
CYP1A1 and SUFU signaling may have a potential role in the genesis of
occlusive lesion formation in Hx+SU mice. Since transcription ofCYP1A1 was arrested and that of SUFU was partially
decreased in mice lungs and in human PASMCs by G6PD inhibition,
transcription of CYP1A1 and SUFU genes in lungs and PASMCs
exposed to Hx+SU is potentially controlled by G6PD. Therefore, we
propose inhibition of G6PD activity could be useful in reversing the
elevated expression of the pathogenic CYP1A1 and SUFUgenes in PH.
We and others have recently proposed that DNA methylation and other
epigenetic modifications potentially promote aberrant/maladaptive gene
expression, a determinant of inflammatory and hyperproliferative cell
phenotype, in remodeled PA (Hu, Zhang, Laux, Pullamsetti & Stenmark,
2019; Joshi et al., 2020). Furthermore, we recently showed that
expression of Tet2 , a DNA demethylase considered as a master
regulator of differentiated fate of SMC phenotype (Liu et al., 2013),
was downregulated in lungs of Sv129J mice with a Cyp2c44 gene
knockout (Joshi et al., 2020). Therefore, we assumed that a loss of TET2
modifies DNA methylation and initiates maladaptive gene expression in
lungs of mice exposed to Hx and Hx+SU. Unexpectedly, expression ofTet1 , but not of Tet2, and Dnmt3b was downregulated
in lungs of C57BL/J mice exposed to Hx and Hx+SU. We propose genetic
variations and differences in gene regulation between Sv129J and C57BL/J
mice (Hashimoto et al., 2020) may be the cause of Tet1 andDnmt3b downregulation, but not of other isoforms of DNA
demethylases and methyltransferases, in response to stress observed in
C57BL/J mice. Since G6PD inhibition prevented downregulation ofTet1 and Dnmt3b in lungs of Hx mice, it appears that G6PD,
directly or indirectly, suppressed transcription of Tet1 andDnmt3b in lungs of Hx and Hx+SU C57BL/J mice. TET proteins are
involved in the regulation of hematopoietic stem cell homeostasis, and
hematological malignancies and diseases (Nakajima & Kunimoto, 2014).
Although loss of single TET protein is not sufficient to promote
malignancies (An et al., 2015), TET1 and TET2 have been shown to,
respectively, repress and promote osteogenesis and adipogenesis
(Cakouros et al., 2019). Furthermore, inhibition of TET1 blocks
expression of large-conductance Ca2+-activated
K+ channel β1 subunit in uterine arteries of pregnant
rats (Hu et al., 2017). Expression of this channel is a marker of
differentiated SMCs. Therefore, downregulation of Tet1 could
imply that: 1) SMCs are dedifferentiated and 2) decreased
Ca2+-activated K+ channels
contribute to constrict PAs and increase pressure in lungs of Hx and
Hx+SU mice. Therefore, altogether these results suggest that DNA
methylation modulated by G6PD is functionally important in gene
regulation and substantiate our previous finding that G6PD is a
regulator of DNA methyltransferases and demethylase, which plays a
crucial role in remodeling of PA (Joshi et al., 2020).
Transcription of the many genes, including the Cyp1a1 gene that
promotes PASMC proliferation (Dean et al., 2018), was repressed through
hypermethylation of the DNA evoked by G6PD inhibition. In contrast,
transcription of Sufu in mouse lungs evoked by Hx+SU was not
regulated by the methylation of DNA. These results suggest G6PD
inhibition activated other mechanisms of gene expression in addition to
differential methylation of the DNA, and these mechanisms worked
independently but synergistically to regulate gene expression in lungs
of Hx and Hx+SU mice.
In addition to arresting maladaptive gene expression in vascular cells
of the PA wall and reducing cell growth in occlusive pulmonary arterial
disease, G6PD inhibitor, PDD4091, dose-dependently relaxed
pre-contracted PAs. By using 17-ketosteroids (dehydroepiandrosterone
(DHEA) and epiandrosterone – a DHEA metabolite), which inhibit G6PD
activity, and siRNA-mediated knockdown of G6pd , we have
previously shown that reduced G6PD elicits relaxation of pre-contracted
pulmonary artery (Gupte, Li, Okada, Sato & Oka, 2002) and reduces RV
pressures in Hx and SU/Hx/Nx rats (Chettimada, Gupte, Rawat, Gebb,
McMurtry & Gupte, 2015; Chettimada et al., 2012). Recently, we found
that arteries of G6PDS188F rats as compared to
wild-type SD rats constrict less in response to nitric oxide synthase
inhibitor and L-type Ca2+ channel opener (Kitagawa,
2020). Therefore, these studies and our current findings collectively
suggest that G6PD inhibition reduces the elevated RV pressures in Hx-
and Hx+SU-induced PH by dilating PAs and reducing PA remodeling.
In conclusion, our results collectively demonstrate that G6PD activity
is an important contributor to differential DNA methylation,
aberrant/maladaptive gene expression, and remodeling of PA in Hx and
Hx+SU mice. The inhibition of G6PD activity abrogated pulmonary vascular
cell remodeling in vivo . As a consequence, the inhibition of G6PD
activity by pharmacologic and genetic manipulations improved the
hemodynamics in mouse and rat models of PH. Therefore, G6PD inhibitor,
N-[(3β,5α)-17-Oxoandrostan-3-yl]sulfamide (PDD4091), might be
employed in the future as a pharmacotherapeutic agent to treat different
forms of PH.