Objective—: It has been shown that CD40-TRAF6 axis in leukocytes plays a significant role in neointimal formation after carotid ligation. Because CD40 and TRAF6 are expressed not only in leukocytes but also in vascular cells, we examined the role of CD40 contributed by vascular wall cells in neointimal formation after carotid ligation in an atherogenic environment. Methods and Results—: Both CD40 and TRAF6 in medial smooth muscle cells (SMCs) was upregulated significantly at 3 days and more prominently at 7 days after injury in wildtype mice, but the TRAF6 upregulation was abolished in CD40 ^−/− mice. In vitro, TRAF6 expression was induced by cytokines (tumor necrosis factor -α, interleukin-1β) via a NF-κB–dependent manner in wildtype SMCs, but this induction was blocked in CD40-deficient SMCs. Bone marrow chimeras revealed a comparable reduction in neointimal formation and lumen stenosis in mice lacking either vascular wall- or bone marrow-associated CD40. Lacking vascular wall-associated CD40 resulted in a significant reduction in monocyte/macrophage accumulation, NF-κB activation, and multiple proinflammatory mediators (ICAM-1, VCAM-1, MCP-1, MMP-9, tissue factor). In vitro data confirmed that CD40 deficiency or TRAF6 knockdown suppressed CD40L-induced proinflammatory phenotype of SMCs by inhibition of NF-κB activation. Moreover, both in vivo and in vitro data showed that CD40 deficiency prevented injury-induced SMC apoptosis but did not affect SMC proliferation and migration. Conclusion—: CD40 signaling through TRAF6 in vascular SMCs seems to be centrally involved in neointimal formation in a NF-κB–dependent manner. Modulating CD40 signaling on local vascular wall may become a new therapeutic target against vascular restenosis.

The threat to public health posed by rapidly increasing levels of cadmium (Cd) in the environment is receiving worldwide attention. Although, Cd is known to be absorbed into the body and causes non-negligible damage to the liver, the detailed mechanisms underlying its hepatoxicity are incompletely understood. In the present study, investigated the effect of TNFAIP3 and α-ketoglutarate (AKG) on Cd-induced liver inflammation and hepatocyte death. Male C57BL/6 mice were exposed to cadmium chloride (1.0 mg/kg) while being fed a diet with 2 % AKG for two weeks. We found that Cd induced hepatocyte injury and inflammatory infiltration. In addition, TNFAIP3 expression was inhibited in the liver tissues and cells of CdCl₂-treated mice. Mouse hepatocyte-specific TNFAIP3 overexpression by tail vein injection of an adeno-associated virus (AAV) vector effectively alleviated Cd-induced hepatic necrosis and inflammation, which was mediated by the NF-κB signaling pathway. Notably, this inhibitory effect of TNFAIP3 on Cd-induced liver injury was dependent on AKG. Exogenous addition of AKG prevented Cd exposure-induced increases in serum ALT, AST and LDH levels, production of pro-inflammatory cytokines, activation of the NF-κB signaling pathway, and even significantly reduced Cd-induced oxidative stress and hepatocyte death. Mechanistically, AKG exerted its anti-inflammatory effect by promoting the hydroxylation and degradation of HIF1A to reduce its Cd-induced overexpression in vivo and in vitro, avoiding the inhibition of the TNFAIP3 promoter by HIF1A. Moreover, the protective effect of AKG was significantly weaker in Cd-treated primary hepatocytes transfected with HIF1A pcDNA. Overall, our results reveal a novel mechanism of Cd-induced hepatotoxicity.

Transplant arteriosclerosis is a leading cause of late allograft loss. Medial smooth muscle cell (SMC) apoptosis is considered to be an important event in transplant arteriosclerosis. However, the precise contribution of medial SMC apoptosis to transplant arteriosclerosis and the underlying mechanisms remain unclear. We transferred wild‐type p53 to induce apoptosis of cultured SMCs. We found that apoptosis induces the production of SDF‐1α from apoptotic and neighboring viable cells, resulting in increased SDF‐1α in the culture media. Conditioned media from Ltv‐p53‐transferred SMCs activated PI3K/Akt/mTOR and MAPK/Erk signaling in a SDF‐1α‐dependent manner and thereby promoted mesenchymal stem cell (MSC) migration and proliferation. In a rat aorta transplantation model, lentivirus‐mediated BclxL transfer selectively inhibits medial SMC apoptosis in aortic allografts, resulting in a remarkable decrease of SDF‐1α both in allograft media and in blood plasma, associated with diminished recruitment of CD90⁺CD105⁺ double‐positive cells and impaired neointimal formation. Systemic administration of rapamycin or PD98059 also attenuated MSC recruitment and neointimal formation in the aortic allografts. These results suggest that medial SMC apoptosis is critical for the development of transplant arteriosclerosis through inducing SDF‐1α production and that MSC recruitment represents a major component of vascular remodeling, constituting a relevant target and mechanism for therapeutic interventions.

Transplant arteriosclerosis (TA) remains the major cause of chronic graft failure in solid organ transplantation. The phenotypic modulation of vascular smooth muscle cells (VSMCs) is a key event for the initiation and progression of neointimal formation and TA. This study aims to explore the role and underlying mechanism of phosphoinositide 3-kinases γ (PI3Kγ) in VSMC phenotypic modulation and TA. The rat model of aortic transplantation was established to detect PI3Kγ expression and its role in neointimal formation and vascular remodeling in vivo. PI3Kγ shRNA transfection was employed to knockdown PI3Kγ gene. Aortic VSMCs was cultured and treated with TNF-α to explore the role and molecular mechanism of PI3Kγ in VSMC phenotypic modulation. Activated PI3Kγ/p-Akt signaling was observed in aortic allografts and in TNF-α-treated VSMCs. Lentivirus-mediated shRNA transfection effectively inhibited PI3Kγ expression in medial VSMCs while restoring the expression of VSMC contractile genes, associated with impaired neointimal formation in aortic allografts. In cultured VSMCs, PI3Kγ blockade with pharmacological inhibitor or genetic knockdown markedly abrogated TNF-α-induced downregulation of VSMC contractile genes and increase in cellular proliferation and migration. Moreover, SOX9 located in nucleus competitively inhibited the interaction of Myocardin and SRF, while PI3Kγ inhibition robustly reduced SOX9 expression and its nuclear translocation and repaired the Myocardin/SRF association. These results suggest that PI3Kγ plays a critical role in VSMC phenotypic modulation via a SOX9-dependent mechanism. Therefore, PI3Kγ in VSMCs may represent a promising therapeutic target for the treatment of TA. FUND: National Natural Science Foundation of China.

As a gene therapy strategy, RNA interference (RNAi) offers tremendous tumor therapy potential. However, its therapeutic efficacy is restricted by its inferior ability for targeted delivery and cellular uptake of small interfering RNA (siRNA). This study sought to develop a dual-ligand nanoparticle (NP) system loaded with siRNA to promote targeted delivery and therapeutic efficacy. We synthesized a dual receptor-targeted chitosan nanosystem (GCGA), whose target function was controlled by the ligands of galactose of lactobionic acid (LA) and glycyrrhetinic acid (GA). By loading siPAK1, an siRNA targeting P21-activated kinase 1 (PAK1), a molecular-targeted therapeutic dual-ligand NP (GCGA–siPAK1) was established. We investigated the synergistic effect of these two targeting units in hepatocellular carcinoma (HCC). In particular, GCGA–siPAK1 enhanced the NP targeting ability and promoted siPAK1 cell uptake. Subsequently, dramatic decreases in cell proliferation, invasion, and migration, with an apparent increase in cell apoptosis, were observed in treated cells. Furthermore, this dual-ligand NP gene delivery system demonstrated significant anti-tumor effects in tumor-bearing mice. Finally, we illuminated the molecular mechanism, whereby GCGA–siPAK1 promotes endogenous cell apoptosis through the PAK1/MEK/ERK pathway. Thus, the dual-target property effectively promotes the HCC therapeutic effect and provides a promising gene therapy strategy for clinical applications.