123 4 Proteomic signature of macrophages cultured on collagen type 1 mice [15]. In our study, the higher levels of glycolytic enzymes in macrophages grown on collagen did not functionally translate into higher glycolytic activity, based on the extracellular acidification rate analysis. This may have been the result of a compensatory lower activity of those enzymes, as there appeared to be a negative correlation between enzyme expression and activity. However, for proper correlation analysis, both analysis of enzyme concentration and activity would need to be performed on the same sample, which was not the case in our study. Explanations of why more protein expression did not translate into more enzymatic activity could be found in changes in protein folding, post-translational changes, regulatory changes [16], or allosteric regulation. For instance, phosphofructokinase has been shown to be allosterically regulated by ATP, AMP, fructose 1,6-bisphosphate, fructose 2,6-biphosphate, and citrate [17]. Another possible explanation could be the contributions of different enzyme isoforms. All enzymes of interest have more than one isoform, while the enzymatic activity tests we performed are not isoform-specific. The proteome analysis, however, assessed each specific isoform. Therefore, any difference in activity may have been obscured by contributions of other isoforms. Importantly, our results highlight that using only one analytical approach could lead to biased results when studying metabolic behavior of macrophages. Multiple techniques should be used when possible to gain complementary information [18]. Even though pathway analysis of differentially expressed proteins in macrophages on fibrous versus globular collagen did not highlight any specific metabolic pathways, four of those proteins are part of the mitochondrial respiratory chain. These were cytochrome b-c1, part of the ubiquinol-cytochrome c complex, elongation factorlike GTPase 1, NADH dehydrogenase (ubiquinone), and V-type proton ATPase. The mitochondrial respiratory chain is one of the main energy sources used by the cells and is composed of five different multi-subunit respiratory complexes. Increased oxidative phosphorylation and fatty acid oxidation are usually associated with an alternatively activated, anti-inflammatory phenotype [19]. This is in line with our previous finding demonstrating that Ym1 is increased in macrophages cultured on fibrous collagen [11] and would further explain why we do not see any differences in the glycolytic function. The most affected protein by collagen and its morphology was the anti-angiogenic glycoprotein pigment epithelium-derived factor (PEDF), encoded by the Serpinf1 gene. PEDF can be secreted by macrophages and is known to bind to collagen type I in a microstructure-dependent manner. Therefore, it is suggested to be involved in collagen fibril assembly [20]. Furthermore, treatment of macrophages with PEDF has been shown to increase macrophage migration [21], which could explain the
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