Page 86 - JCTR-11-5

Journal of Clinical and
Translational Research ROCK inhibition in chronic rejection

1. Introduction combined Rezurock/fingolimod treatment downregulates
genes associated with cell cycle progression, metabolic
Although short-term survival rates of transplanted and cytokine production pathways, and dysregulates
organs have reached satisfactory levels, long-term signaling pathways involved in macrophage recruitment.
survival remains a major post-transplantation challenge. Overall, our findings suggest that Rezurock/fingolimod,
According to the International Report on Organ Donation in combination, holds therapeutic potential in preventing
and Transplantation Activities from Global Observatory the chronic rejection-related functions of macrophages in
on Donation and Transplantation for 2022, and published organ transplantation.
1
data, approximately 157,000 solid organ transplants were
2
performed worldwide, including kidney (~102,090); liver 2. Materials and methods
(~37,436), heart (~8,988), lung (~6,784), pancreas (~2,026),
and small bowel (~170). However, around 70% percent 2.1. Mice handling
of transplant recipients experienced symptoms of organ All experiments were performed according to The
rejection within 10 years post-transplantation. Current Methodist Hospital Research Institute’s animal care and use
treatment approaches depend on organ type; however, the standards, based on the National Institute of Health (NIH)
most common treatments include immunosuppressants guidelines outlined in the Guide for the Care and Use of
and corticosteroids, which primarily target the immune Laboratory Animals (DHHS Publication No. [NIH] 85-23
response and reduce inflammation. The primary Revised 1985). The Institute also mandates compliance
3,4
histological features in biopsies of chronically rejected with the Public Health Service Policy on Humane Care and
organs are macrophage-driven narrowing or occlusion Use of Laboratory Animals and the NIH Principles for the
3,5
of the vessels and tissue fibrosis. Because fibrosis is an Utilization and Care of Vertebrate Animals Used in Testing,
important hallmark of chronic rejection, we hypothesize Research, and Training.
that targeting macrophages could become a new, efficient
approach in post-transplant treatment. In our quest for 2.2. Isolation and culture of peritoneal macrophages
clinically applicable anti-chronic rejection therapy, we Mouse peritoneal macrophages were isolated from the
showed that in a rodent cardiac transplantation model, peritoneal cavity of C57BL/6J mice (n = 25) obtained from
the pharmacologic inhibition of the Ras homolog Jackson Laboratory, Bar Harbor, USA and cultured as
family member A (RhoA)/Rho-associated coiled-coil previously described. 10
kinase (ROCK) pathway or macrophage-specific RhoA
knockout eliminates, through changes in the actin 2.3. RAW 264.7 macrophage culture
cytoskeleton, macrophage infiltration of the allograft RAW 264.7 cells (ATCC, Manassas, USA) were maintained
and inhibits chronic rejection. After testing several and cultured as previously described. 10
6,7
commercially available RhoA/ROCK inhibitors, we found
that Rezurock—Food and Drug Administration (FDA)- 2.4. Isolation and culture of peripheral blood
approved for the treatment of chronic graft-versus-host mononuclear cells from human blood and
disease—is superior in inhibiting fibrosis in the mouse differentiation of monocytes into macrophages
cardiac transplantation model. Molecularly, RhoA/ (human monocyte-derived macrophages)
8
ROCK signaling mediates cytoskeletal remodeling during Blood (500 mL) was purchased from the blood bank.
cell migration and polarization of macrophages and Fifty-milliliter stem cell tubes (SepMate™ Peripheral Blood
additionally affects the proliferation rates of immune Mononuclear Cell [PBMC] Isolation Tubes, STEMCELL
9
cells. Therefore, we believe that inhibitors of the RhoA/ Technologies, Vancouver, BC, Canada) were filled to the
ROCK pathway could represent a novel approach for 15 mL mark with Ficoll-Paque Plus (Cytiva, Marlborough,
post-transplant treatment. MA 01752, USA). After slowly adding blood to the 40 mL
Here, we studied the effects of Rezurock and fingolimod mark, the tubes were centrifuged at 700 × g for 15 min
combination on gene and protein expression in mouse and at room temperature (RT). The white PBMC layer was
human macrophages. Fingolimod, primarily recognized transferred to the 50 mL tubes, and after adding phosphate-
as a nonselective functional antagonist of sphingosine-1- buffered saline, centrifuged at 500 × g for 10 min at RT.
phosphate receptors, is FDA-approved for multiple sclerosis Cells were cultured in RPMI 1640 medium (11875-093,
treatment. However, our previous work demonstrated Gibco, Waltham, Massachusetts, USA. Supplemented
that fingolimod also inhibits the RhoA/ROCK pathway, with 10% fetal bovine serum, penicillin-streptomycin
although it is much less effective in inhibiting fibrosis (100 units/mL), and macrophage colony-stimulating
than Rezurock. Our transcriptomic analysis reveals that factor (Peprotech 300-25, Cranbury, New Jersey 08512,
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Volume 11 Issue 5 (2025) 80 doi: 10.36922/JCTR025270036