Abstract Body: Introduction: Our laboratory previously reported that the atypical cadherin FAT1 plays a pivotal role in mitigating arterial occlusion after injury by modulating the metabolism and proliferation of vascular smooth muscle cells (SMCs). Our findings demonstrated that FAT1 directly influences SMC behavior through cell-autonomous mechanisms involving mitochondrial respiration, but the precise molecular pathways underlying FAT1 function remain incompletely understood. A growing area of interest is the role of extracellular vesicles (EVs) in intercellular communication. EVs facilitate the transfer of molecular cargo among cells and tissues, thereby influencing various biological processes.
Hypothesis: In addition to its cell-autonomous functions, FAT1 exerts cell non-autonomous effects on neighboring cells via EVs.
Methods: EVs were isolated from supernatants of bovine, mouse, and human aortic SMCs by differential ultracentrifugation, and characterized by electron microscopy, dynamic light scattering, and Western blotting for common EV markers (CD63, CD9, Alix) as well as FAT1. To study how FAT1 affects EV function, we treated wild-type (WT) and FAT1-deficient (FAT1Δ) SMCs with EVs derived from either FAT1-deficient or WT SMCs. SMC proliferation was assessed using Alamar Blue and Ki67 immunofluorescence assays.
Results: EVs ranged in size from 20 to 120 nm. FAT1 was detected in EVs derived from all three species, including both full-length FAT1 (~510 kDa) and processed forms (~430, ~85, and ~65 kDa). Specificity of FAT1 detection was confirmed by ultrastructural immunogold labeling, siRNA-mediated knockdown, genetic inactivation (FAT1Δ), and use of multiple anti-FAT1 antibodies. Proliferation of FAT1Δ SMCs was approximately two-fold higher than WT SMCs. Notably, treatment with EVs derived from WT SMCs reduced the proliferation of FAT1Δ SMCs (p<0.01), bringing it back to levels comparable to control. Similarly, EVs isolated from bovine SMCs inhibited serum-stimulated proliferation of both bovine and human vascular SMCs. In contrast, EVs from FAT1Δ SMCs failed to exhibit anti-proliferative effects.
Conclusions: These findings suggest that FAT1-containing EVs from SMCs can inhibit SMC proliferation in a paracrine manner, contributing to the anti-proliferative properties of FAT1. This highlights the potential of FAT1-mediated EV signaling as a therapeutic target in vascular diseases associated with aberrant SMC proliferation, such as atherosclerosis and restenosis.