C046 - Turning off the Engine of Chronic Rejection: RhoA-ROCK Pathway Modulation of Macrophage Infiltration

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Author Block: A. Uosef, Surgery, Houston Methodist Hospital, Houston, TX
*Purpose: Chronic allograft rejection is driven by persistent macrophage infiltration, vascular remodeling, and progressive fibroinflammatory injury. Although macrophages are central mediators of chronic vasculopathy, the intracellular pathways guiding their recruitment, cytoskeletal behavior, and profibrotic activation remain incompletely defined. RhoA, a key regulator of actin dynamics, directs macrophage chemotaxis through CX3CR1 signaling, enabling migration toward graft-derived CX3CL1. Pharmacologic inhibition of this pathway has shown promise in mitigating chronic rejection, but the molecular reprogramming underlying these effects remains insufficiently characterized. This analysis integrates mechanistic studies of RhoA/CX3CR1 signaling with transcriptomic profiling of ROCK-inhibited macrophages to define a unified pathway regulating macrophage-driven chronic allograft injury.
*Methods: Mechanistic studies used macrophage-specific RhoA knockout mice in a chronic cardiac allograft model to evaluate graft infiltration, chemotaxis, cytoskeletal organization, and CX3CR1 trafficking. In vitro analyses assessed actin remodeling, polarization, and directed migration. Parallel experiments exposed mouse peritoneal macrophages and human monocyte-derived macrophages to Rezurock, fingolimod, or both. Bulk RNA sequencing, pathway enrichment, and protein expression assays were performed to characterize transcriptional and post-transcriptional effects on inflammatory and profibrotic programs.
*Results: RhoA deficiency markedly impaired CX3CR1-mediated macrophage recruitment by disrupting receptor recycling, cytoskeletal remodeling, and directional migration, resulting in reduced infiltration and attenuated chronic vasculopathy in vivo. Combined ROCK pathway inhibition with Rezurock and fingolimod reprogrammed macrophages at a global transcriptomic level, altering nearly 5,000 genes. This combination suppressed fibrosis-associated transcripts, while downregulating pathways linked to extracellular matrix organization, inflammatory activation, and cell-cycle progression. Corresponding protein levels of PTX3, CCL2, CCR2, TGF-β1, ROCK2, and Notch1 were reduced. Cytoskeletal and actin-assembly networks, key downstream targets of RhoA/ROCK signaling, were also significantly diminished.
*Conclusions: RhoA/CX3CR1 signaling and ROCK-dependent cytoskeletal pathways are essential regulators of macrophage recruitment and effector activity in chronic allograft injury. Genetic loss of RhoA restricts macrophage infiltration by impairing CX3CR1-dependent migration, while ROCK2 inhibition shifts macrophages toward a less inflammatory, less fibrotic phenotype. Together, these findings define a unified RhoA → ROCK → cytoskeletal axis that governs macrophage-driven chronic rejection and support Rezurock-based ROCK inhibition as a targeted therapeutic strategy to prevent long-term graft failure.