Abstract 4366402: TGF-β1-Induced Phenotypic Switch in Endothelial and Mesenchymal Plasticity: The Role of CDK5

Background: The endothelial-to-mesenchymal transition (EndMT) is a phenotypic shift wherein endothelial cells acquire proliferative, mesenchymal-like properties, contributing to endothelial dysfunction, vascular remodeling, and the progression of diseases such as pulmonary arterial hypertension (PAH). While the upregulation of mesenchymal markers in TGF-β1-induced endothelial cells has been previously reported, the temporal dynamics, involvement of cell cycle pathways, and potential for phenotypic reversal remain poorly defined. Hypothesis: TGF-β1 drives EndMT in endothelial cells via activation of the CDK5 signaling pathway, and targeted inhibition of CDK5 can reverse this shift and restore endothelial identity. Methods: Mouse lung endothelial cells (mLECs) were isolated from Tg(TIE2GFP)287Sato/J mice using FACS and verified via cobblestone morphology and endothelial marker expression. Cells were treated with optimized TGF-β1 concentrations over time to induce controlled EndMT. CDK5’s role was evaluated using three inhibitors: PHA793887 (commercial), Compound-1 (kinases-promiscuous), and TK22 (novel, reversible). Various EndMT markers, cell proliferation, angiogenesis, and collagen gel contraction were assessed. Cells were also subjected to single-cell RNA sequencing (scRNA-seq) to analyze phenotypic transitions. Results: TGF-β1-treated mLECs exhibited elongated, fibroblast-like morphology with increased expression of αSMA, SM22α, and Calponin, indicating EndMT. CDK5 inhibition significantly reduced mesenchymal marker expression, with TK22 showing the strongest effect. Inhibition also reduced cell proliferation and collagen contraction. TK22 induction also restored angiogenic potential comparable to control cells. ScRNA-seq analysis showed phenotypic alterations in TGFB1 treatment with TK22 as a potential EndMT inhibitor. Conclusion: TGF-β1 induces EndMT in mLECs via the CDK5 pathway. Inhibition of CDK5, particularly with the novel compound TK22, effectively halts and reverses EndMT, offering a promising therapeutic avenue for treating PAH and related vascular pathologies.