Clinical and Translational Medicine · 16 authors, 2 centres
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This study demonstrates that Rnd3 protects against atherosclerosis by inhibiting endothelial cell pyroptosis. Mechanistically, Rnd3 directly interacts with TRAF6 to promote its K48-linked ubiquitination and degradation while suppressing K63-linked ubiquitination, thereby inhibiting NF-κB/NLRP3 signaling. These findings identify Rnd3 as a potential therapeutic target for atherosclerosis.
**Background:** Atherosclerosis is a chronic inflammatory disease driven by endothelial cell (EC) injury and pyroptosis. Rnd3 (RhoE) is a small GTPase with protective roles in cardiovascular diseases, but its function in atherosclerosis was unknown. TRAF6 is an E3 ubiquitin ligase that activates NF-κB via K63-linked ubiquitination and is degraded via K48-linked ubiquitination. This study investigated whether Rnd3 regulates EC pyroptosis through TRAF6 ubiquitination.
**Methods:** Apoe^KO^ mice were fed a high-fat diet (HFD) for 8 weeks to induce atherosclerosis. Endothelial-specific Rnd3 transgenic (Rnd3^ECTG^) and knockout (Rnd3^ECKO^) mice were generated and crossed with Apoe^KO^ mice. Primary mouse aortic ECs were isolated and treated with oxLDL (25 μg/mL for 24 h) to induce pyroptosis. Protein interactions were assessed by co-immunoprecipitation (Co-IP), LC-MS/MS, and molecular docking. Ubiquitination was analyzed by IP with ubiquitin-specific antibodies. Atherosclerotic lesions were quantified by Oil Red O staining. Macrophage migration and phagocytosis were evaluated using transwell and oxLDL uptake assays.
**Key Results:** In Apoe^KO^ mice, ECs showed increased pyroptosis markers (GSDMD-N, NLRP3, Caspase1) and decreased Rnd3 expression compared to WT. Overexpression of Rnd3 in ECs (Apoe^KO^Rnd3^ECTG^) reduced aortic plaque area from 36.3±1.8% to 18.8±1.4% (p<0.05) and aortic root plaque area from 17.9±1.3×10^5 μm² to 9.6±1.2×10^5 μm² (p<0.05). Rnd3 overexpression also decreased EC pyroptosis markers and reduced macrophage migration and oxLDL uptake. Conversely, EC-specific Rnd3 knockout (Apoe^KO^Rnd3^ECKO^) increased plaque area to 39.4±2.3% vs 31.3±1.8% in Apoe^KO^WT (p<0.05) and elevated pyroptosis markers. Mechanistically, Rnd3 directly interacted with the ring finger domain (amino acids 65-88) of TRAF6. Rnd3 overexpression decreased K63-linked ubiquitination of TRAF6 and increased K48-linked ubiquitination, leading to TRAF6 degradation and reduced NF-κB activation (P-P65). Rnd3 knockdown had opposite effects. TRAF6 knockdown via AAV9-shTRAF6 reduced plaque area by 52.6±3.8% in Apoe^KO^Rnd3^ECKO^ mice and by 31.8±2.9% in Apoe^KO^WT mice, and offset the increase in GSDMD-N and P-P65 caused by Rnd3 knockout.
**Clinical Implications:** This study identifies Rnd3 as a critical suppressor of EC pyroptosis in atherosclerosis through regulation of TRAF6 ubiquitination. By promoting K48-linked degradation and inhibiting K63-linked activation of TRAF6, Rnd3 attenuates NF-κB/NLRP3 signaling and subsequent pyroptosis. These findings suggest that enhancing Rnd3 expression or activity could be a novel therapeutic strategy for atherosclerosis. The study also highlights TRAF6 as a potential downstream target, as its knockdown partially reversed the pro-atherosclerotic effects of Rnd3 deficiency. Limitations include the use of murine models and the need for validation in human cells and other atherosclerosis models.