Page 90 - JCTR-11-5

Journal of Clinical and
Translational Research ROCK inhibition in chronic rejection

whereas 310 genes were downregulated exclusively in some of the genes downregulated by Rezurock and the
Rezurock-treated macrophages (Figure 3A and B). The Rezurock/fingolimod combination, but not by fingolimod
genes downregulated after combination treatment were alone. We previously published the expression data of
related to extracellular matrix organization, blood vessel these fibrosis-related proteins (some selected from Table 1
development, neutrophil degranulation, cell–cell adhesion, and others from independent fibrosis studies) following
and cytokine production (Figure 3C; Table S5). The treatment with Rezurock or fingolimod separately. Here,
10
downregulated genes specific to Rezurock treatment were we compared the expression of fibrotic pathway-related
involved in lipid metabolism, atherosclerosis, hemostasis, proteins in control (DMSO-treated), Rezurock-only,
cytokine signaling, neutrophil signaling, lipid localization, fingolimod-only, and Rezurock/fingolimod combination-
and regulation of the actin cytoskeleton (Figure 3D; treated treatment.
Table S6). The genes downregulated in both Rezurock We evaluated the expression levels of ROCK1, ROCK2,
and combination treatments were related to the cell Notch1, PTX3, collagen Type I, CCL2, CCR2, and TGF-
cycle, translation, chromosome organization, regulation β1 in RAW 264.7 mouse macrophages (Figure 6). We
of cellular stress, adaptive immune response, and found that the expression of ROCK1, ROCK2, Notch1,
chromosome segregation (Figure 3E; Table S7). Among the and collagen Type I was similar across Rezurock
upregulated genes, 357 were unique to Rezurock treatment
(Figure 4A and B), 821 were uniquely upregulated after alone, fingolimod alone, and the Rezurock/fingolimod
Rezurock/fingolimod treatment, and 1,554 genes were combination treatments. However, for PTX3, CCL2,
shared between the two treatments. The genes upregulated CCR2, and TGF-β1, the combination treatment had a
only after combination treatment were related to Golgi much stronger inhibitory effect compared to either drug
vesicle transport, histone modification, the Rho GTPase alone (Figure 6). We also analyzed the protein expression
pathway, protein modification, DNA methylation, and of ROCK1, ROCK2, and Notch1 following Rezurock,
the mitogen-activated protein kinase signaling pathway fingolimod, and combination treatment in HMDMs
(Figure 4C; Table S8). The genes shared between Rezurock (Figure 7). All three treatments reduced the levels of these
and combination treatments were associated with protein proteins. However, the Rezurock/fingolimod combination
processing in the endoplasmic reticulum (ER), metabolic was significantly more effective at reducing Notch1 and
processes, autophagy, GTPase activity, and membrane ROCK2 expression in HMDMs than either individual
trafficking (Figure 4D; Table S9). The genes upregulated treatment.
only in Rezurock-treated macrophages were related to In addition, we performed GO enrichment analysis
ER-to-Golgi transport, lysozyme pathway, fatty acyl- of Stat3/Stat5-related pathways among DEGs in
coenzyme A synthesis, membrane lipid metabolism, macrophages treated with Rezurock, fingolimod, or their
protein localization to the ER, neutrophil degranulation, combination (Figure 8; Tables S13-S15). Macrophages
and apoptotic signaling (Figure 4E; Table S10). treated with either drug, or the combination, exhibited
In summary, treatment with Rezurock/fingolimod differential modulation of immune-related pathways.
had a more pronounced effect on the mouse macrophage GO enrichment analysis revealed significant changes in
transcriptome than individual treatment with either cytokine signaling and Stat3/Stat5-associated processes,
Rezurock or fingolimod. The shared 87 downregulated with the strongest enrichment observed following
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genes were mostly related to antigen processing, cytokine combination treatment (Figure 8A-C). Heatmap analysis
production, chemokine signaling, regulation of leukocyte showed more pronounced transcriptional changes
differentiation, cell activation, macrophage markers, G alpha following Rezurock treatment compared to fingolimod
signaling, and interleukin (IL)-2 production (Figure 5A-C; alone, while the combination treatment induced the most
Table S11). Among the upregulated genes, 35 were shared extensive modulation, suggesting enhanced macrophage
by all three treatment groups (Figure 5D and E). These 35 reprogramming (Figure 8D-F).
genes were associated with protein folding, adipogenesis,
transcription, immunoglobulin production, and cell–cell 4. Discussion
adhesion (Figure 5F; Table S12). Chronic rejection of transplanted organs remains
incurable, and long-term organ survival rates continue to
3.3. Effect of Rezurock/fingolimod combination on be unsatisfactory. Existing therapies primarily focus on
3,4
the fibrosis pathway-related proteins inhibiting T-cell proliferation and activation to prevent
3,4
Rezurock prevents fibrosis more effectively than other graft rejection. However, we and others hypothesize that
RhoA/ROCK inhibitors. It downregulates genes involved targeting macrophages could offer additional therapeutic
in fibrosis and collagen deposition pathways. Table 1 lists benefits, as they are well-established contributors to

Volume 11 Issue 5 (2025) 84 doi: 10.36922/JCTR025270036