The outcome of the deployment of cardiovascular stents in coronary arteries compromised by atherosclerosis may be affected by the deposition of a myofibroblast-driven neointimal tissue and consequent re-blockage of the vessel lumen known as instent restenosis (ISR). Its incidence is particularly high in people with diabetes unless drug-eluting stents (DES) are implanted as alternatives to the traditional bare metal stents (BMS) made of stainless steel (ST). However, the long-term outcome of the use of DES is under debate due to evidence of late thrombosis. Monocytes/macrophages (MM) play a key role in ISR participating in the different phases of the host response to the implant. This in-vitro study investigates differences in the distribution and response of MM that may be significant for a better understanding of the causes of the increased ISR occurrence among diabetic patients, particularly focusing on the trans-differentiation potential of MM into myofibroblast-like cells when in contact with ST. The overnight incubation of MM freshly isolated from blood from healthy donors (n=6) in high glucose (4S00mglL) and high free fatty acid (sodium palmitate 0.4mM) concentrations, alone or in combination, aimed at creating a diabetes-mimicking experimental model. MM response to ST seemed exacerbated by the syoergistic effect of the palmitate and glucose as shown by the occurrence of features characteristic of activation as well as by their trend to fuse into the formation of giant cells as shown by scanning electron microscopy. Similarly results from RT-PCR (n=6) suggested an increase in the expression of the mRNA for HAS3, the enzyme responsible for the synthesis of low molecular weight hyaluronic acid characteristic of the inflammatory process. Furthermore, the presence of MM positive for a-actin, as shown by immunocytochemistry, seemed to confirm the occurrence of early changes in their phenotype. This was accompanied by a significant (p<O.OS or p<O.OOS after paired Hest) increase in the release of PDGF-BB, growth factor characterizing the post-inflammatory condition, as well as in the release of 1NF-a confirming the short-term metal-induced inflammatory reaction from MM incubated on ST in comparison to control surfaces as shown by ELISA tests. Flow cytometry on buffy coats collected from three cohorts of donors (control, type 1 and type 2 diabetes) (n=7) suggested that variations among donors with diabetes in the number of circulating activated macrophages and haematopoietic progenitor cells may, once compared to clinical data, help to identify patients at high risk of developing ISR. The hypothesis of the contribution of MM in the deposition of restenotic neointimal tissue through the transformation into myofibroblasts was reinforced by results from flow cytometry that identified, both in freshly isolated buffy coats and after contact with surfaces, subpopulations co-expressing a-actin and markers for MM (CDI4 or CD68). This was also supported by the imununostaining of the cells after overnight incubation on the surfaces where a-actin-positive cells were seen. Furthermore, ELISA test on the supernatants (n=10) suggested that ST triggered a significant increase in the release of PDGF-BB and 1NF-a when compared to control surfaces. This study reinforced the hypothesis that MM can contribute to ISR through phenotypical changes and that the acute synergistic effect of glucose and lipids can affect their level of activation.

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