Hypertension, high blood pressure, is a common condition with more than three million cases in the United States per year. Pulmonary hypertension (PH), a chronic condition targeting the lungs, leading to vascular remodeling, right ventricle hypertrophy, and death in 50% of cases 5 years after diagnosis, is multi-factorial, not fully understood, and is without a permanent cure to this day. Utilizing multiple animal models involving xenobiotic injections, hypoxic conditions, growth factor receptor antagonists, and the combinations thereof have provided valuable insights into pathways and abnormalities associated with PH, however, the precise mechanisms remain unknown. In the last decade, it was discovered that a common feature of PH is the increase of oxidized lipids (especially the oxylipins of the lipoxygenase [LOX] pathway) in the plasma and lung tissues in not only human PAH patients but also in multiple animal models of PH. Animal studies demonstrated that apolipoprotein A-I (apoA-I) mimetic peptides ameliorated PH while lowering levels of oxylipins of the lipoxygenase pathway, including hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecadienoic acids (HODEs). Taking this one step further, myself and our collaborators have developed a new animal model of PH in which feeding wild type C57BL6/J mice 5μg per mouse per day of 15-HETE resulted in increases in right ventricle systolic pressure (RVSP), Fulton index, vascular remodeling, lung weight, and plasma levels of HETEs and HODEs. In this thesis, I set out to utilize our new animal model to explore the mechanisms by which oxylipins of the lipoxygenase pathway (focusing on 15-HETE) cause PH and apoA-I mimetic peptides (specifically Tg6F) prevent the development of PH. My hypothesis is that 15-HETE initiates, and Tg6F mitigates, intestinal inflammation by modulating pro-inflammatory lipids and immune responses, which result in PH. I utilized cell culture (rat intestinal epithelial cells; IEC-6 cells, mouse T-cells; TK-1 cells, and mouse pulmonary arterial endothelial cells, PAECs), and animal models (C57BL6/J and 12/15 lipoxygenase KO mice) to test my hypothesis. I determined that 15-HETE diet (i) induces the accumulation of oxylipins in the intestine and plasma, (ii) activates pulmonary arterial endothelial cells (PAEC), and (iii) increases CD8 cell mediated PAEC apoptosis. I demonstrated that Tg6F treatment prevented all the above changes and prevented PH. Further, analyses of RNA-seq data from lung samples of PAH patients, lung samples of 15-HETE fed mice, and intestinal samples of 15-HETE fed mice, identified interferon induced protein 44 (IFI44) as the only gene that was significantly increased between all three groups. IFI44 increases in the intestine as early as one week into the 3 week 15-HETE PH protocol, preceding the increase in IFI44 observed in the lungs, and when IFI44 is blocked via intratracheal instillation of siRNA, it prevented the onset of PH in mice on the 15-HETE diet when compared with those administered scrambled siRNA, establishing IFI44 as a novel target for preventing PH. Finally, I determined that anti-inflammatory short chain fatty acids (SCFAs) play a role in the development of PH and identified oral butyrate as a potential therapy for PH. Luminal microbiota analysis from mice fed a 15-HETE diet revealed that bacterial species that participate in SCFA production were significantly reduced. Tg6F fed mice, interestingly, had increased SCFA producing bacterial species and fecal pellet levels of the SCFAs butyrate, propionate, and acetate compared to 15-HETE fed mice. Furthermore, supplementing 15-HETE fed mice with sodium butyrate in their drinking water increased the abundance of SCFA producing bacteria, decreased intestine and lung expression of IFI44, prevented the onset of PH, and reduced RVSP of mice fed 15-HETE diet for 2 weeks before adding sodium butyrate to their diet in the third, suggesting that oral butyrate is a potential therapy for both prevention and progression of PH. Combined, these experiments not only establish the role of oxylipins of the LOX pathway in the onset of PH, but also reveal mechanisms by which it causes it and multiple targets for future therapeutic applications.
