The Effect of Maa Polymer-Stimulated Macrophages on Endothelial Cells

The
effect of MAA polymer-stimulated macrophages on endothelial cells.

Ilana
Talior-Volodarky¹_, David�Zhang¹,
Redouan Mahou¹and Michael Sefton¹

¹University
of Toronto, Institute of Biomaterials and Biomedical Engineering, Ontario,
Canada.

Polymers
based on methacrylic acid (MAA) were previously shown to have a therapeutic
effect on wound closure through the promotion of angiogenesis of surrounding
tissues. However, it is unclear how this polymer exerts these beneficial effects.

The
main goal of this current study was to understand and characterize the response
of endothelial cells and macrophages to MAA-co-IDA copolymer film (�MAA�, IDA=isodecyl
acrylate, in comparison to methyl methacrylate copolymer � MM-co-IDA, used as a
control) by identifying a critical signalling pathway, which is activated
during cell-material interaction.

MAA upregulated IGF-1 (Insulin like
Growth Factor 1), but not IGF-2 (Insulin like Growth Factor 2) in dTHP1cells and
mouse macrophages. It was hypothesized that IGF-1 in MAA-conditioned medium
stimulated HUVEC migration.

Scratch wound assay in HUVEC indicated
increased migration of the cells after their incubation with MAA-treated dTHP1
conditioned medium in comparison to MM-treated dTHP1 conditioned medium. The
enhanced effect was inhibited by IGF-1R inhibitor (AG1024).

Gene
expression analysis in dTHP1 and M0, M1 and M2 macrophages (BMDM � Bone Marrow-Derived
Macrophages) showed that MAA polymer increased the expression of IGFBP-3 (3.5-fold
in dTHP1; 3.8- fold in BMDM) relative to MM. IGFBP-3
is known to block IGF-1 access to the
IGF-1 receptor and thereafter deliver more IGF1 into the blood stream. Macrophages
also demonstrated decreased expression of IGF-R1 when treated with MAA
polymer. In contrast, HUVEC treated with MAA stimulated dTHP1 conditioned medium
showed increased expression of IGF-R1 (1.7-fold), but decreased expression of
IGFBP-3, which is opposite to dTHP1 cells treated with MAA polymer film.

Arg1 (M2 marker), was upregulated in both M0 and M1
macrophages treated with MAA polymer compared with control treatment, whereas,
the expression of M1 marker - iNOS � in M0 and M2 macrophages was unaffected in
MAA stimulated conditions. Exposure of MAA treated macrophages to AG1024 inhibitor
induced a reduction of Arg1 expression.

We related the in
vitro findings to the angiogenic response in vivo. All in vivo
results were consistent with in vitro studies: upregulation
of IGF-1 transcript level in MAA-treated animals was observed; Arginase-1
expression level was significantly increased in animals treated with MAA
polymer and iNOS expression was unaffected in
MAA stimulated conditions. Most importantly, AG1024 abrogated the increased
CD31+ vessels density seen with MAA implants, but had no effect on MM implants.

This data suggest
that MAA-co-IDA stimulates an IGF-1 mediated macrophage response to the biomaterial,
via macrophages polarization toward the M2 phenotype which in turn activates
endothelial cells required for the process of vascularisation.

Understanding the MAA stimulation mechanism can be used to
advance the knowledge of this biomaterial and cell or tissue interaction, thereby
facilitating the development of MAA polymer for new therapeutic applications,
such as revascularization and tissue regeneration.

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Key
words: Angiogenesis, IGF-1, macrophages and
endothelial cells.

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