A miRNA-based approach for prevention of SMC apoptosis in unstable plaques

Jan Köster (Halle (Saale))1, K. Kalies (Halle (Saale))1, S. Böttcher (Halle (Saale))1, S. Gürlach (Halle (Saale))1, F. Daniel (Halle (Saale))1, L. Hehl (Halle (Saale))1, K. Knöpp (Halle (Saale))1, J. Dutzmann (Halle (Saale))1, D. G. Sedding (Halle (Saale))1

1Universitätsklinikum Halle (Saale) Klinik und Poliklinik für Innere Medizin III Halle (Saale), Deutschland


Apoptosis of smooth muscle cells (SMCs) drives the destabilization of atherosclerotic lesions.
This process contributes to plaque rupture through various mechanisms, including Phosphatidylserine exposure and plaque microcalcification. Aversion of SMC apoptosis in late-stage plaques would allow for the targeted therapy of patients at risk through causal prevention of plaque rupture.

BIM inhibits anti-apoptotic pathways and directly exerts pro-apoptotic signaling, thus contributing to apoptosis in atherosclerosis.

Previously, we showed, miR-92b-3p to be centrally involved in SMC functional capacity, altering both migration and proliferation and further regulating pivotal SMC target genes, both on mRNA and protein levels.

Here, we investigate the role of miR-92b-3p in the regulation of intrinsic SMC apoptosis as a possible therapeutic axis, targeting plaque stability.

Working within a model of primary human vascular cells, we used a variety of in vitro methods, including PCR, Western Blot, Live cell imaging and ELISA. We further used lipotransfection for transient miR-92b-3p knockdown or elevation and an siRNA transfection model.

In a murine model of late-stage atherosclerosis, miR-92b-3p expression was elevated (p<0.001). Notably, both in vivo and in vitro, miR-92b-3p exhibited stronger expression in SMCs compared to endothelial cells (ECs) (p<0.01, p<0.01). Mir-92b-3p demonstrated upregulation in proliferating SMCs, relative to those cultured in basal medium (p<0.05). Correspondingly, a predicted target of miR-92b-3p, BIM, exhibited downregulation under growth medium conditions compared to basal medium treatment (p<0.001). Altering miR-92b-3p levels resulted in changed Bim expression, showing upregulation post-knockdown of miR-92b-3p and downregulation following pre-miR-92b-3p introduction into SMCs, both at the mRNA level and protein levels after transfection (p<0.01, p<0.05). Yet, we could also show miR-92b-3p to regulate PTEN levels, which is another potent regulator of apoptosis in SMCs. (p<0,05)

Consequently, after miR-92b-3p knockdown apoptosis was elevated (p<0.05), and cell loss was shown in live cell imaging (p<0,05) in proliferative SMCs, but not ECs. Interestingly, senescent SMCs exhibited BIM regulation at the mRNA level but did not undergo apoptosis after anti-miR-92b-3p transfection (p<0.01). Neither did basal medium-treated SMCs (p<0,05).

The effect of miR-92b-3p on apoptosis proved to be contingent on BIM, as siRNA-mediated knockdown of BIM in SMCs resulted in the absence of apoptosis following miR-92b-3p knockdown. Additionally, upregulation of miR-92b-3p in SMCs undergoing apoptosis exhibited a protective effect against apoptosis (p<0.05).
miR-92b-3p alteration also steered interleukin production of SMCs, influencing TNF-alpha and IL-6 production, which are involved in apoptotic chain reactions. (p<0,05)

In conclusion, miR-92b-3p emerges as a robust controller of intrinsic apoptosis specific in SMCs by targeting BIM. Notably, these regulatory impacts were confined to proliferative SMCs, which hold a central position in the biology of atherosclerotic lesions. Considering these findings, the prospect of elevating miR-92b-3p levels, perhaps through drug-miRNA interaction, surfaces as a promising strategy in patients at risk. This intervention could be a cost-efficient and approachable therapeutic strategy as drugs, exerting such influence on miR-92b-3p levels, are already commercially available and in clinical use.

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