Comparative transcriptomic analysis of macrophages treated with a combination of ROCK pathway inhibitors

Background: At present, there is no therapy for the long-term (chronic) rejection of transplanted organs. This condition leads to tissue fibrosis and occlusion of the blood vessels. Aim: The overall goal of the current research is to identify a clinically applicable therapy for chronic rejection in transplanted organs. Our previous study showed that inhibitors of the RhoA/Rock pathway, such as Rezurock and fingolimod, prevent chronic rejection in rodent transplantation models, with Rezurock being superior in reducing fibrosis. Materials and methods: In this study, we analyzed the effect of a Rezurock and fingolimod combination on the transcriptome of mouse peritoneal macrophages and protein expression in both mouse and human macrophages. Results: The Rezurock/fingolimod combination resulted in the differential expression of 4,855 genes (2,477 downregulated and 2378 upregulated). Downregulated genes were related to fibrotic pathways, extracellular matrix, blood vessel development, cell adhesion, and cytokine production. Protein expression analysis showed that Rezurock/fingolimod treatment had a significantly stronger effect on the expression of pentraxin 3, chemokine (C-C motif) ligand 2, C-C motif chemokine receptor 2, and transforming growth factor beta 1 in mouse macrophages, and was much more effective in reducing the expression of Notch1 and Rho-associated coiled-coil kinase 2 in human macrophages compared to individual treatments. Conclusion: Rezurock/fingolimod treatment not only affects fibrotic pathways but also downregulates genes related to cell cycle progression and cytokine production and disrupts macrophage recruitment signaling. These findings indicate that Rezurock, alone or in combination with other immunomodulators, may be a promising candidate for clinical therapy targeting chronic rejection.