Vascular Smooth Muscle-Secreted Exosomal X26nt Impedes Atherosclerosis Progression via the c-FOS/XBP1/SOD1 Axis.

Background: Atherosclerosis is a chronic immune-inflammatory disorder in which vascular smooth muscle cell (VSMC) phenotypic modulation plays a critical role in plaque development and instability. Endoplasmic reticulum (ER) stress and its downstream effector, XBP1s, have been shown to influence VSMC behavior. During XBP1 mRNA splicing, a 26-nucleotide RNA fragment (X26nt) is excised, yet its biological significance remains poorly understood. Exosomes derived from VSMCs have been implicated in mediating intercellular signaling under inflammatory and stress conditions. However, the potential role of X26nt in vascular regulation, particularly via exosomal pathways, has not been investigated.

Methods: Atherosclerosis was induced in ApoE-/- mice using a high-fat diet. Ox-LDL-treated VSMCs were used for in vitro studies. Histology, qPCR, and Western blot were conducted. Exosomes from IRE1α- or XBP1-knockdown VSMCs were isolated and used to treat Ox-LDL-exposed VSMCs to assess X26nt effects. Luciferase assays and ChIP were used to explore mechanisms. AAV2-SM22a-ZsGreen-26nt vectors were constructed to evaluate X26nt effects in vivo.

Results: X26nt levels in exosomes increased with arterial medial thickening in atherosclerosis. In vitro, exosomal X26nt decreased ER stress, suppressed mitophagy, and upregulated SOD1 in VSMCs. Exosomes from IRE1α- or XBP1-knockdown VSMCs reversed the protective phenotype. Mechanistically, X26nt bound the 3'UTR of XBP1 and c-Fos, reducing their expression. ChIP confirmed c-Fos directly activated XBP1 transcription. In vivo, AAV2-X26nt delivery elevated SOD1, reduced mitophagy, and attenuated vascular remodeling.

Conclusion: This study identified exosomal X26nt as a novel regulator of VSMC phenotypic switching and oxidative stress through the c-Fos/XBP1/SOD1 axis. These findings highlight the functional relevance of ER stress-derived noncoding RNAs in vascular remodeling and suggest that targeting exosomal RNAs, such as X26nt, may represent a promising therapeutic strategy for atherosclerosis and related cardiovascular diseases.