M. C. Zuniga1,2, G. Raghuraman1,2, W. Zhou1,2 1VA Palo Alto Healthcare Systems,Vascular Surgery,Palo Alto, CA, USA 2Stanford University,Vascular Surgery,Palo Alto, CA, USA
Introduction: Resistin, an adipokine with inflammatory properties, has been associated with atherosclerosis and cardiovascular disease. However, its specific mechanisms of action in key cells involved in atherosclerotic plaque formation are not well defined. We have shown that resistin promotes vascular smooth muscle cell (VSMC) dysfunction at a pathological concentration. The purpose of this study was to examine the direct effect of resistin on macrophage-related inflammation and its indirect effects on VSMCs in the presence of macrophages.
Methods: Human monocytes were isolated from healthy controls and differentiated into macrophages (MΦ). MΦ were treated with resistin at a “high” physiological level (10 ng/ml) for 18 h, with or without selective PKC-ε inhibitor, εV1-2, at 1 µM. MΦ supernatants (conditioned media) were analyzed with ELISA for pro-inflammatory cytokines. MΦ were assessed for gene and protein expression of inflammatory markers using RT-PCR, immunocytochemistry (ICC) and Western blot, respectively. Conditioned media (CM) generated from MΦ cultures were used to treat human coronary artery smooth muscle cells (HCASMCs) for 24 h. HCASMCs were evaluated for changes in proliferation using the MTT assay and flow cytometry, as well as gene expression changes of nuclear factor-kappa-B (NF-kB) subunits and of proliferation-associated genes.
Results: Physiological levels of resistin triggered increased gene expression and protein production of the inflammatory cytokines TNF-α, CD40L, IL-6, and resistin in MΦ (Figure 1 A-D). MΦ showed significantly upregulated expression of membrane receptor CD40 and downregulated CD206 (mannose receptor) after resistin treatment. Inhibition of PKC-ε reduced gene expression of inflammatory cytokines and protein expression of CD40. HCASMCs exposed to CM from resistin-treated MΦ showed significantly increased proliferation, while blocking PKC-ε in MΦ during resistin treatment reduced CM-induced proliferation in HCASMCs (Figure 1 E). Furthermore, HCASMCs upregulated gene expression of NF-kB1 (p50), NF-kB2 (p52), cyclin-D1 and proliferating cell nuclear antigen, whereas this effect was not observed in HCASMCs treated with CM from control MΦ.
Conclusion: Resistin at a high physiologic concentration promotes pro-inflammatory MΦ transformation via PKC-ε, and it induces VSMC dysfunction indirectly through MΦ. Targeting resistin may represent a therapeutic strategy in atherosclerosis-associated complications. Further in vivo investigations are warranted.
