The Anti-Inflammatory Capacity of Rapamycin in Aortic Vascular Smooth Muscle Cells from Marfan Syndrome Mice

Background: Vascular changes associated with the Marfan syndrome (MFS) are characterized by abnormally high matrix metalloproteinases (MMPs) expression in aortic smooth muscle cells, leading to the destruction of collagen fibers. MMPs are induced by cytokines, including tumor necrosis factor α (TNFα) and play a pivotal role in MFS and remodeling of the extracellular matrix. A hyperactive mechanistic target of rapamycin (mTOR) pathway leads to MMP2 and MMP9 overstimulation. Rapamycin is a potent and selective inhibitor of the mTOR protein kinase, regulating cell growth and metabolism. This study aims to characterize the effects of mTOR inhibition by rapamycin on MMPs and the pro-inflammatory cytokine TNFα.

Method: Cultured murine aortal smooth muscle cells (mAoSMC) from fibrillin-1-deficient Marfan mice (mgR/mgR) were used. Western blot analysis and immunocytochemical staining validated the inhibitory effect of 50 and 100 ng/mL rapamycin on mTORC1 and RPS6 protein abundance at the cellular level. The impact of these two rapamycin concentrations on MMP2, MMP9 and TNFα mRNA expression was analyzed by quantitative real-time PCR analysis. The protein abundance of MMPs was detected by Western blot analyses and TNFα by enzyme-linked immunosorbent assay (ELISA).

Results: The rapamycin concentrations used almost completely inhibited the protein abundance of phosphorylated mTOR and its downstream target ribosomal protein S6 kinase (RPS6) (p = 0.01). Treatment with rapamycin 50 or 100 ng/mL concentration dependently downregulated TNFα on both mRNA (70–52%, p > 0.05) and protein levels (63–47%, p > 0.05). MMP2 and MMP9 mRNA expression was also suppressed, where MMP2 inhibition was more pronounced by the rapamycin treatment. MMP2 mRNA expression was inhibited ~2-fold (p = 0.0001) through both concentrations. The MMP9 mRNA expression was significantly inhibited by 100 ng/mL rapamycin (p = 0.001), whereas the lower concentration had no effect. Only the MMP2 protein abundance was downregulated by 100 ng/mL rapamycin to 63% (p = 0.05) compared with the control.

Conclusion: In mAoSMCs isolated from MFS animals, the expression of mTOR, RPS6, TNFα, matrix metalloproteinase-2, and -9 were significantly suppressed by rapamycin, demonstrating its anti-inflammatory capacity.

Publikationsverlauf

Artikel online veröffentlicht:
28. Januar 2023