Differential regulation of endothelial and smooth muscle cell functions by miR-32-5p: Consequences for vascular remodeling in the context of cellular senescence

Background and purpose: Dysfunctional endothelial cells (EC) and smooth muscle cells (SMC) contribute to vascular remodeling and subsequent cardiovascular diseases. Specifically, senescent cells exacerbate vessel wall dysfunction by creating a pro-inflammatory and pro-thrombotic environment. Recent research has identified multiple microRNAs (miRNAs) as drivers of cellular senescence, emphasizing their importance in regulating SMC and EC function and suggesting them as potential targets for modulating vascular remodeling. This study examines the possible role of miR-32-5p in vascular cell function and its viability as a therapeutic target in vascular remodeling.

Material and methods: All experiments were conducted in (non-) senescent human coronary artery endothelial (HCAEC) and smooth muscle cells (HCASMC). First, miR-32-5p expression was screened in HCAECs and HCASMC in different cell culture models using qRT-PCR. The role of miR-32-5p in cellular functions such as migration, proliferation, and cell death were assessed via scratch wound and BrdU assays. Downstream targets were identified and their expression was evaluated on mRNA and protein levels. Immunofluorescence microscopy was used for analyzing cell morphology.

Results: MiR-32-5p expression was higher in HCASMC than in HCAEC and overall decreased in replicative senescent vascular cells (p<0.05). By stimulation with growth factors, HCASMC showed increased miR-32-5p expression (p<0.05). Pre-miR-32-5p transfection significantly enhanced HCASMC proliferation (p<0.05) but did not affect HCAEC. MiR-32-5p reduced migration in HCASMC (p<0.05) as well as in HCAEC and improved it after antagomiR interference. At the same time, in senescent cells, its knockdown resulted in reduced HCASMC migration and improved HCAEC migration (p<0.05). Transfection of miR-32-5p showed overall no apoptotic effects. In silico analysis identified several miR-32-5p targets in HCASMC and HCAEC. The miR-32-5p overexpression resulted in a stable downregulation on mRNA level, including KLF4 (p<0.05), CD69 (p<0.01), MAP2K4 (p<0.05), BCL211 (p<0.01), and PIK3R3 (p<0.05), where PIK3R3 protein levels were also significantly decreased (p<0.05). Further, CD69 and KLF4 expression was reduced in HCAECs (p<0.05). Senescence markers showed p21 upregulation and Lamin B1 downregulation in HCASMC (p<0.01), while p53 (p<0.01), p14ARF, p16INK4a, and p21 further increased in senescent cells (p<0.05). Cytokine analysis revealed IL8 decreases by pre-miR-transfection in both (non-) senescent HCASMC (p<0.01). Antagomir interference downregulated p53 in HCAEC (p<0.01), but other cytokines and senescence markers were unaffected. Immunofluorescence staining revealed miR-32-5p overexpression led to a rhomboid shape in HCASMC, without affecting HCAEC morphology.

Conclusion: These results indicate miR-32-5p as a substantial regulator of HCASMC function rather than HCAEC function, highlighting its potential as a therapeutic target. Especially the contrasting regulation in EC already suggests a pathway for more precise treatment possibilities in the future. To fully evaluate its role in the pathogenesis of atherosclerosis and therefore, its therapeutic potential in cardiovascular diseases, further investigations are needed. Additional experiments to examine downstream target interactions and senescence associated β-galactosidase staining will be performed.