D28-04 Interleukin-1 Signaling Mediates Endothelial Reprogramming in Pulmonary Hypertension

Rationale

Severe pulmonary arterial hypertension (PAH) involves endothelial cell (EC) barrier dysfunction, abnormal EC proliferation, and migration. ECs undergo endothelial-to-mesenchymal transition (EndMT), affecting vascular complications in PAH. The molecular mechanisms of EndMT in PAH are not fully understood. Inflammatory signals interact with vascular cells, with elevated interleukin-1 (IL-1) levels found in severe PAH patients. Animal studies indicate that blocking IL-1 receptor (IL-1R1) protects against PAH, but the downstream signaling pathway of IL-1R1 remains largely unexplored.

Hypothesis

IL-1R1 contributes to EC-to-EndMT, and therefore, targeting the downstream activation of IL-1R1 either genetically or pharmacologically reduces pulmonary vascular remodeling and right ventricular failure (RV) in PAH patients.

Methods

Single-cell and bulk RNA sequencing were conducted on lungs and RV of PAH patients and mouse models with pulmonary hypertension (PH). Validation involved immunohistochemistry, immunoblotting, and qRT-PCR in both humans and mice. Cell culture experiments included deleting IL-1R1, IRAK1, and IRAK4 and assessing EndMT gene expression and function.

Results

Human pulmonary EC (HPEC) clusters from PAH patients showed significant upregulation of EndMT genes compared with HPECs from failed donors (FD). This was linked to higher levels of IL-1 in PAH EC clusters than in FD controls. Interestingly, in the PAH clusters, levels of IL-1R1 and its downstream kinase interleukin-1 activated kinase 1 (IRAK1) were markedly higher than in the FD controls. In contrast, IRAK4 levels were not significantly altered. Hypoxia is a major driver of EndMT activation in PAH models, and in our culture studies, exposing HPECs to hypoxia (1% v/v) significantly increased IL-1R1 protein and mRNA levels after 24-48 hours. After 24 hours of hypoxia, control cells showed a significant increase in mRNA and protein levels of EndMT markers, including Fibronectin, TWIST1, TGFβ2, and SNAIL2. Knockdown of IRAK1 or IL-1R1 reduced this induction. IRAK1 knockdown also resulted in a significant decrease in EC migration and Ki67-positive cells after 24 hours of hypoxia compared with controls. Immunohistochemical analysis revealed a significant increase in IL-1R1/IRAK1 and SNAIL1 levels in ECs of pulmonary arteries and the right ventricles in PAH patients, compared with healthy controls.

Conclusions

Silencing IL-1R1/IRAK1 reduced EndMT formation under hypoxic conditions and led to significant functional changes. The increased expression of IL-1R1/IRAK1 in ECs from patients with PAH suggests a new role for IRAK1 in EndMT and PAH progression, making IRAK1 a potential therapeutic target.

This abstract is funded by: None