Chemokines are critically involved in the development of chronic inflammatory-associated diseases such as atherosclerosis. We hypothesised that targeted delivery of compounds to the surface of activated endothelial cells (EC) interferes with chemokine/receptor interaction and thereby efficiently blocks inflammation. We developed PEGylated target-sensitive liposomes (TSL) encapsulating a CCR2 antagonist (Teijin compound 1) coupled with a specific peptide recognized by endothelial VCAM-1 (Vp-TSL-Tj). TSL were characterized for size (by dynamic light scattering), the amount of peptide coupled at the surface of liposomes and Teijin release (by HPLC). We report that Vp-TSL-Tj binds specifically to activated EC in vitro and in vivo, release the entrapped Teijin and prevent the transmigration of monocytes through activated EC. This is the first evidence that nanocarriers transporting and releasing chemokine inhibitors at specific pathological sites reduce the chemokine-dependent inflammatory process. Abstract:Chemokines are critically involved in the development of chronic inflammatory-associated diseases such as atherosclerosis. We hypothesised that targeted delivery of compounds to the surface of activated endothelial cells (EC) interferes with chemokine/receptor interaction and thereby efficiently blocks inflammation. We developed PEGylated target-sensitive liposomes (TSL) encapsulating a CCR2 antagonist (Teijin compound 1) coupled with a specific peptide recognized by endothelial VCAM-1 (Vp-TSL-Tj). TSL were characterized for size (by dynamic light scattering), the amount of peptide coupled at the surface of liposomes and Teijin release (by HPLC). We report that Vp-TSL-Tj binds specifically to activated EC in vitro and in vivo, release the entrapped Teijin and prevent the transmigration of monocytes through activated EC. This is the first evidence that nanocarriers transporting and releasing chemokine inhibitors at specific pathological sites reduce the chemokine-dependent inflammatory process.

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In the atherosclerotic plaque, smooth muscle cells (SMC) acquire an inflammatory phenotype. Resistin and fractalkine (CX3CL1) are found in human atheroma and not in normal arteries. CX3CL1 and CX3CR1 are predominately associated with SMC. We have questioned whether resistin has a role in the expression of CX3CL1 and CX3CR1 in SMC thus contributing to the pro-inflammatory status of these cells. Cultured human aortic SMC were stimulated with 100 ng/ml resistin for 4, 6, 12, and 24 h, and then CX3CL1 and CX3CR1 expression was assessed by quantitative reverse transcription with the polymerase chain reaction and Western blot. We found that resistin up-regulated CX3CL1 and CX3CR1 in SMC and induced the phosphorylation of p38MAPK and STAT3. Inhibitors of p38MAPK, JAK-STAT, NF-kB, and AP-1 significantly reduced CX3CL1 and CX3CR1 expression. Knockdown of STAT1 and STAT3 with decoy oligodeoxinucleotides and the silencing of p65 and cjun with short interfering RNA decreased CX3CL1 and CX3CR1 expression. Anti-TLR4 antibody and pertussis toxin also reduced CX3CL1 and CX3CR1 protein expression. xCELLigence experiments revealed that resistin probably uses Gi-proteins for its effect on SMC. The CX3CL1 induced by resistin exhibited a chemotactic effect on monocyte transmigration. Thus, (1) resistin contributes to the pro-inflammatory state of SMC by the up-regulation of CX3CL1 and CX3CR1 expression via a mechanism involving NF-kB, AP-1, and STAT1/3 transcription factors, (2) resistin employs TLR4 and Gi-protein signaling for its effect on SMC, (3) CX3CL1 induced by resistin is functional in monocyte chemotaxis. The data reveal new mechanisms by which resistin promotes the inflammatory phenotype of SMC.

The cross-talk between SMC and monocytes augments the inflammatory response in both cell types as revealed by the increased expression of TNFα, IL-1β, IL-6, CX3CR1 and MMPs. Up-regulation of TNFα, CX3CR1 and MMP-9 is further increased upon interaction of SMC with activated monocytes and is dependent on fractalkine/CXRCR1 pair. These data imply that the fractalkine/CX3RCR1 axis may represent a therapeutic target to impede the inflammatory process associated with atherosclerosis.

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