Pharmacological inhibition of COP9 signalosome deneddylation activity by CSN5i-3 limits neointimal hyperplasia without affecting vascular smooth muscle cell dedifferentiation markers

Background: Vascular smooth muscle cell (VSMC) phenotypic remodeling is pivotal to neointimal hyperplasia (NH), a common pathological response to vascular injury and a known complication of vascular procedures. CSN5/JAB1 is an essential catalytic component of the COP9 signalosome (CSN) responsible for deneddylation of cullin-RING ligases, a key regulatory mechanism maintaining cellular protein homeostasis. CSN5i-3, a novel chemical inhibitor of CSN deneddylase has previously demonstrated potent antiproliferative effects in cancer, yet its relevance to vascular pathology remains unexplored. Here, we investigated whether targeted pharmacological inhibition of CSN deneddylase function using CSN5i-3 could influence NH following arterial injury. Methods and Results: Wild-type mice were treated with CSN5i-3 (20 mg/kg/day, i.p.) or vehicle and subjected to left common carotid artery (LCCA) ligation. Ligated and control (right common carotid artery, RCCA) arteries were excised on the 7th day of treatment and assessed for pharmacodynamic markers, the effect on VSMC phenotypic modulation, and neointima formation. CSN5i-3 treatment regime did not affect mouse body weight but, consistent with selective deneddylase inhibition, CSN5i-3 treated arteries exhibited increased neddylated Cullin1 and decreased Skp2 protein levels. Interestingly, the treatment did not alter the ligation-induced changes in the mRNA levels of MYOCD, KLF4 or SMC differentiation markers in vessel wall, suggesting that VSMC dedifferentiation into a synthetic state proceeds independently of CSN deneddylase activity. However, CSN5i-3 markedly attenuated the ligation-induced increases in COL1A1, FN1, CCND1, and CCND2, genes associated with extracellular matrix deposition and cell-cycle progression. Moreover, CSN5i-3 significantly repressed the proliferative response to vascular injury. Ligation-induced PCNA upregulation was significantly suppressed in CSN5i-3 treated mice; and dual Ki67/SM22α immunostaining demonstrated a pronounced reduction in VSMC proliferation accompanied by markedly attenuated NH in CSN5i-3 treated mice, indicating a protective effect of CSN deneddylase inhibition on arterial remodeling following injury. Conclusion: Collectively, these findings demonstrate that CSN deneddylase activity is not required for mature VSMC dedifferentiation into synthetic VSMCs but promotes the proliferation of dedifferentiated VSMCs, which suggests the dedifferentiation and proliferation of VSMCs in response to injury are separatable processes. Pharmacological targeting of CSN deneddylase activity with CSN5i-3 effectively suppresses NH, highlighting the CSN as a promising therapeutic target for limiting restenosis and other occlusive vascular diseases. Funding Source: NIH grants HL085629, HL072166, and HL153614 (to X.W.). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.