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of PASMCs evoked by Hx and SU, reduced expression of Cyp1a1 and Sufu , which potentially arrested growth of PASMCs, and rescinded occlusion of PA in lungs of mice exposed to Hx+SU.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.