Abstract Rationale Pulmonary arterial hypertension (PAH) is characterized by progressive pulmonary vascular adverse remodeling, which over time progresses to right ventricular (RV) failure. Pulmonary arterial endothelial cell (PAEC) dysfunction and smooth muscle cell (PASMC) proliferation are key mechanisms in PH pathophysiology. In line with recent research highlighting the gut-lung axis, our lab was the first to develop a novel model of pulmonary hypertension by feeding wild-type mice a 15-hydroxyeicosatetraenoic acid (15-HETE)-enriched diet for three weeks. However, the molecular mechanisms by which PH is induced by 15-HETE remain unclear. Methods Three week 15-HETE diet regimen (5ug/day) was used to induce PH in male wildtype C57BL6/J mice. Weekly echocardiography was performed to assess pulmonary artery acceleration time (PAAT), and direct catheterization at the end of the feeding protocol measured right ventricular systolic pressure (RVSP), confirming the PH development. Mice jejunal enterocytes were FACS-isolated and used for RNA-sequencing. Differentially regulated lipid metabolism genes were selected and validated experimentally by RT-qPCR. Mouse plasma oxidized lipids were measured by Liquid Chromatography-Mass Spectrometry (LC-MS/MS). In-vitro human healthy male PAEC and PASMC were incubated with oleate, and inflammatory cytokines and proliferation were measured. IEC-6 (a rat intestinal epithelial cell line) cells were transfected with either control or Scd1 siRNA, treated with 15-HETE, and their conditioned medium was transferred to PAECs to assess endothelial dysfunction. Results Mice on 15-HETE diet had significantly lower PAAT and higher RVSP compared to control mice on chow diet confirming PH development. RNA-seq of enterocytes isolated from 15HETE mice revealed upregulation of Stearoyl-CoA Desaturase-1 (Scd-1), which is an enzyme responsible for producing monounsaturated fatty acids (MUFAs). LC-MS/MS revealed a higher level of MUFAs including palmitoleate and oleate in plasma of mice on a 15-HETE diet compared to chow diet. Incubation of PAEC with oleate increased IL-1β expression. The conditioned medium of oleate-treated PAEC was able to increase PASMC proliferation. In addition, conditioned medium from 15-HETE treated IEC-6 cells that were preincubated with Scd1 siRNA showed reduced expression of IL-1β and increased expression of anti-apoptotic marker MCL1 in PAECs, when compared to PAEC treated with conditioned medium from 15-HETE-treated IEC-6 cells that were preincubated with a control siRNA. Conclusions Together, our data suggest that a 15-HETE-enriched diet upregulates Scd-1 expression in enterocytes, leading to an increase in oleate in the circulation. Oleate induces PAEC dysfunction and through paracrine signaling promote PASMC proliferation. Silencing enterocyte SCD-1 prevented 15-HETE induced PAEC dysfunction in vitro. This abstract is funded by: This work was supported by the National Institutes of Health R01HL162124 (M.E., S.T.R.), R01HL147586 (M.E.), 1R01HL174472 (M.E.), and R01HL159865 (M.E.).
Hatamnejad et al. (Fri,) studied this question.