Abstract 14665: Cybrid Analysis Delineates Nuclear and Mitochondrial Contributions to Oxidative Stress in Pulmonary Arterial Hypertension

Rationale : Heritable pulmonary arterial hypertension (HPAH) is a serious vasculopathy caused by remodeling of pulmonary arterioles. It is inherited in an autosomal dominant pattern with reduced penetrance, suggesting additional triggers are needed for disease development. Past study shows increased DNA damage and total reactive oxygen species (ROS) in lymphoblastoid cells (LCL) from PAH patients and unaffected relatives, indicating a potential genetic component.

Hypothesis: Increased oxidative stress is an independent genetically-determined modifier of PAH susceptibility.

Methods: LCL from patients, relatives and unrelated controls were analyzed for total and mitochondrial (mt) ROS and mt function (Seahorse). Untargeted metabolomics were performed by Metabolon. Nuclear vs mt gene contribution was studied using cybrids created by transferring the mitochondria from high (HC) and low ROS LCL (LC) to mt negative 143B Rho0 cells.

Results: Metabolomics revealed significant differences between PAH and control LCL in the TCA cycle and glutathione metabolism relating to oxidative stress. HPAH LCL showed significant upregulation of both ROS species compared to controls. LCL from relatives clustered into two groups, one with increased mtROS, the other comparable to controls . Like PAH lung vascular cells, LCL with increased mtROS had reduced spare respiratory capacity (SRC) suggesting dysfunctional mitochondria, but no glycolytic switch. Cybrid analysis revealed that HCs had significantly higher basal respiration (p=0.02), with SRC trending higher compared to LCs, indicating a functional electron transport chain. These cells also had elevated mtROS (p=0.04), suggesting reduced antioxidants.

Conclusions: HPAH is a complex disease, and mutation status alone doesn’t determine its susceptibility. Patient LCL recapitulate some of the metabolomic pathways dysregulated in lung vascular cells. Oxidative stress in LCL extends to some unaffected relatives, suggesting this is an independent genetic trait that modifies PAH risk. Our cybrid analysis, novel in PAH highlights the importance of nuclear-mt crosstalk. WGS is underway to identify nuclear variants that could affect the mt, and act as potential modifiers and second hits in PAH pathogenesis.