NYMC Student Theses and Dissertations

Date of Award

5-28-2025

Document Type

Doctoral Dissertation - Open Access

Degree Name

Doctor of Philosophy

Department

Pharmacology

First Advisor

Sachin Gupte

Abstract

Pulmonary hypertension is an under-recognized global health epidemic which is estimated to affect 1% of the population. It is associated with sustained mean pulmonary artery pressure greater than 20 mmHg, pulmonary artery remodeling, smooth muscle and endothelial cell proliferation and subsequent increased right ventricle hypertrophy due to increased afterload. Glucose-6-phosphate dehydrogenase (G6PD) is one of the key enzymes in the pentose phosphate pathway and has been previously linked to the development of pulmonary hypertension. G6PD deficiency is the most common human enzymopathy which affects approximately 400 million people worldwide and is a result of agricultural and epidemiological evolution in different parts of the world. G6PD, a X-linked gene, is highly polymorphic and more than 200 single nucleotide polymorphisms are found within the coding region. One G6PD variant, Mediterranean Ser188Phe, G6PDS188F, results in 80-90% decrease in G6PD activity and is known to be protective against vascular disease. Another G6PD variant, African Asn126Asp, G6PDN126D, results in 10-20% decrease in G6PD activity and increases susceptibility to vascular disease. The mechanism associated with the development of pulmonary hypertension is still unclear. The literature suggests that many variables contribute to the development of pulmonary hypertension such as metabolic reprogramming, epigenetics (DNA and histone methylation, histone acetylation), and altered gene expression. Consequently, we postulate that African variant G6PDN126D but not Mediterranean variant G6PDS188F, increases pulmonary artery remodeling and the risk of developing pulmonary hypertension, by modifying 3D genomic organization and DNA methylation that increases maladaptive signaling molecules.

Sprague-Dawley rats underwent CRISPR-Cas9 genome editing in which there was a substitution of either N126D or S188F in the G6PD coding region, and these animals and their age-matched wild-type littermates were randomly injected with Sugen-5416 (SU; 20mg/kg S.C. suspended in DMSO) and kept in ambient condition (normoxia; Nx). After 8 weeks, hemodynamic measurements revealed that G6PDN126D + SU/Nx rats had increased right ventricle systolic pressure, while G6PDS188F + SU/Nx did not, indicating that only G6PDN126D + SU/Nx rats developed pulmonary hypertension. G6PDN126D + SU/Nx rat lungs were formalin-fixed, and then paraffin embedded, and further wall thickness assessments displayed pulmonary artery remodeling. Interestingly, metabolomic analysis displayed that G6PDN126D + SU/Nx rat lungs had metabolic reprogramming resulting in increases glutathione disulfide and 2-hydroxyglutarate, which are indicative of increased oxidoreductive stress. The TCA cycle was upregulated in G6PDN126D + SU/Nx rat lungs, causing increased energy to shunt through the purine salvage pathway to break down purines into uric acid, which is known to occur in pulmonary hypertension. Vasoactive mediators were altered in G6PDN126D + SU/Nx but not G6PDS188F + SU/Nx rat lungs, leading to increased inositol triphosphate and calcium signaling, thrombin-antithrombin complex, and decreased nitric oxide. Serpine1, a gene that encodes plasminogen activator inhibitor-1 (PAI-1), was upregulated in G6PDN126D + SU/Nx rat lungs only, and resulted in binding to NOS3, which accounted for the decreased nitric oxide, and induced senescence and migration of smooth muscle cells.

Unbiased bulk RNASeq revealed that genes encoding chromatin related proteins, oncoproteins, plasminogen activator inhibitor-1, cytokines, and chemokines, were upregulated in G6PDN126D + SU/Nx compared with G6PDS188F + SU/Nx rat lungs. Further single nucleus RNA sequencing depicted that more cell types of G6PDN126D + SU/Nx rat lungs had upregulated Serpine1 and inflammatory cytokines. Upon more specific clustering of the cell types, it was shown that basophils and endothelial progenitor cells were present in the lungs of G6PDN126D + SU/Nx rats only. Next, we assessed how epigenetics was altered in these G6PD variants with whole genome methylation studies and found that Tet1, Tet2 and Tet3 were increased in G6PDS188F rat lungs and there was a 3.4-fold difference in 5-hmC to 5-mC in G6PDS188F + SU/Nx rat lungs, indicating that these lungs favor demethylation and transcriptional activation. Upon investigation of the 3D genome via Hi-C sequencing, we determined that there are changes in transcription activation via the presence of topologically associated domains and correspond loops in the chromatin at the gene loci of Tet2 on chromosome 2 for G6PDS188F rat lungs. These data suggest that perhaps epigenetics and the organization of the 3D-genome are interrelated in maintaining Tet2 driven transcriptional activation in G6PDS188F rat lungs, and this maintenance protects G6PDS188F rats, but not G6PDN126D rats from the maladaptive signaling molecules that occur during the development of Sugen-5416 induced pulmonary hypertension.

Understanding how gene expression is altered at the physiological, epigenetics, and 3D-genomic organization gives further insight into the role of G6PD variants in vascular disease. Elucidating the role of ethnic G6PD polymorphisms in tyrosine kinase inhibitor (TKI)-induced experimental PH will give critical insight into translational medicine. Furthermore, these findings suggest that carefully screening patients who are given TKIs for diseases such as cancer is necessary.

Keywords

G6PD, pulmonary hypertension, vascular disease, epigenetics, 3D genomics, thrombosis, oxidative stress

Disciplines

Animal Experimentation and Research | Cellular and Molecular Physiology | Genomics | Medicine and Health Sciences | Pharmacology | Respiratory System

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