https://doi.org/10.1007/s00392-025-02625-4
1Universitätsklinikum Halle (Saale) Klinik und Poliklinik für Innere Medizin III Halle (Saale), Deutschland
MicroRNAs (miRs) have emerged as promising therapeutic targets in cardiovascular disease, given their capacity to regulate multiple signaling pathways and cellular functions. Neointima formation, which is a major complication associated with angioplasty, is primarily caused by changes in the behavior and function of vascular smooth muscle cells. Therefore, we suggest that targeting miR-31-5p could be an effective way to selectively modify smooth muscle cell functions and promote vascular healing and regeneration.
C57BL/6J mice (6 male, 3 months old) underwent wire-induced femoral artery injury. The femoral arteries were collected for miRNA analysis on days 7 and 21 post-injury. Quantitative real-time PCR (QRT-PCR) was used to confirm the expression of miR-31-5p in human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells (HCASMC). In vitro studies were conducted to investigate the effects of miR-31-5p on cellular functions, including migration (scratch assay), proliferation (BrdU assay), and apoptosis. Potential targets of miR-31-5p were identified through structured research on RNA binding prediction and RNA sequencing. The functional properties of the identified targets were further investigated in vascular cells. Immunofluorescence microscopy of murine aortic tissue was performed.
Expression analysis revealed a significant upregulation of miR-31-5p (p<0.0001) in murine femoral artery neointimal tissue at 7 and 21 days. In vitro, miR-31-5p was significantly upregulated in HCASMC (p<0.05) but not HCAEC following serum stimulation. On a functional level, miR-31-5p revealed dissenting effects on HCAEC and HCASMC. Whereas migration and proliferation are not altered in HCAECs, knockdown of miR-31-5p significantly reduced migration (p<0.01), proliferation (p<0.05), and increased apoptosis (p<0.05) in HCASMCs. Under these conditions, the release of cytokines like TNF-alpha and IL1-beta was also significantly reduced (p<0.05) in HCASMCs. An RNA sequencing approach identified STK40, LATS2, and GXYLT1 as targets of miR-31-5p regulation in HCASMC. Their regulation was confirmed on mRNA and protein levels. Transfection with anti-miR-31-5p resulted in a significant downregulation of STK40 and LATS2 (p<0.01 and p<0.05). Identified targets do not show regulations in HCAECs. Further investigations focused on the role of STK40, a kinase that does not affect cellular proliferation and migration in HCASMC. However, siRNA transfection for STK40 significantly reduces apoptosis (p<0.05). Additional experiments confirmed that STK40 regulation is important for anti-miR-31-5p-induced apoptosis in HCASMC (p<0.05). Optical analysis visualized the co-localization of smoothelin and STK40 in murine aortic cross sections.
In summary, our findings reveal a strong upregulation of miR-31-5p during neointima formation, highlighting its crucial role in promoting this process, specifically in HCASMC rather than HCAEC. Mechanistically, we identified STK40 as a direct target of miR-31-5p, which contributes to the observed apoptotic effects in HCASMC. These insights suggest that miR-31-5p is a promising target for selectively addressing HCASMC dysfunction following vascular intervention, offering the potential to reduce neointima formation in treated vessels.