EXPLORING THE ROLE OF THE INTEGRATED STRESS RESPONSE IN PULMONARY VASCULAR DISEASE

Pulmonary hypertension (PH) is a family of diseases that consist of high blood pressure in the pulmonary vasculature. Pulmonary veno-occlusive disease (PVOD) is a rare form of PH characterized by progressive obstruction of the smallest branches of the pulmonary veins with patchy capillary proliferation, leading to right heart failure and eventual death. Heritable PVOD is known to be caused by a biallelic mutation in the EIF2AK4 gene, specifically, the GCN2 gene. This gene is part of the integrated stress response (ISR), which is regulated by four kinases, including GCN2. Our primary research goal was to determine why PVOD is caused by GCN2 mutation, because there are no known cells in the pulmonary vasculature that activate the ISR through GCN2. We also wanted to investigate non-canonical stimuli for GCN2 activation, and determine the best way to visualize ISR activation. 

To explore these research goals, we developed methods for sectioning and staining murine lungs via immunofluorescence, staining for two endothelial cell markers. We also activated the ISR in vitro and probed for downstream products of ISR induction, phospho-eIF2A and ATF4. The development of this assay showed that ISR can be induced via GCN2 with arsenic, serum starvation, and doxycycline. Additionally, we utilized Gibson cloning to clone SPOTlight, a reporter that measures ISR state-dependent protein synthesis, into 293T cells in order to visualize ISR in specific cells. Future directions with this research will utilize the methods that I developed to further investigate the connection between PVOD and GCN2 mutation. 

For more information: ljchudac@eckerd.edu

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