Effects of PBM Treatments on Fibroblast MMP-2, MMP-9, TIMP-1 and TIMP-2 Expression and on MMPs' Functionality  
Further experiments were aimed at the evaluation of the expression and functionality of the enzymes involved in the digestion of components of the ECM such as collagen, namely MMP-2 and MMP-9 gelatinases and of their specific inhibitors TIMP-1 and TIMP-2. Indeed, ECM/collagen homeostasis and remodeling are dependent on the balanced coordination between MMPs and TIMPs [36]. As assessed by confocal immunofluorescence and western blotting analyses, NIH/3T3 fibroblasts subjected to red PBM up-regulated either MMP-2 or MMP-9 expression (Figure 9A,B,E,FQ and Figure 10A,B) and concomitantly down-regulated TIMP-1 and TIMP-2 expression as compared to control cells (Figure 9I,J,M,N,R and Figure 10C). By contrast, NIR and violet-blue PBM reduced MMP-2 and MMP-9 production (Figure 9C,D,G,H,Q and Figure 10A,B) while they enhanced the expression of TIMP-1 and TIMP-2 (Figure 9K,L,O,P,R and Figure 10C). Figure 8: Confocal immunofluorescence analysis of \(\alpha\)-sma expression in NIH/3T3 fibroblasts performed 24 h after cell exposure to red (635 nm), NIR (808 nm) and violet-blue (405 nm) PBM. (**A–D**) Representative confocal fluorescence micrographs of fixed cells immunost to detect \(\alpha\)-sma (green). Nuclei are marked in red with PI. Scale bar: 25 μm. Figure 9: Confocal immunofluorescence analysis of the expression of MMP-2 and MMP-9 and of their inhibitors, TIMP-1 and TIMP-2, in NIH/3T3 fibroblasts 24 h after cell exposure to red (635 nm), NIR (808 nm) and violet-blue (405 nm) PBM. Cells not subjected to PBM treatments served as control. **(A–P)** Representative confocal fluorescence micrographs of fixed cells immunostained for detection of **(A–D)** MMP-2, **(E–H)** MMP-9, **(I–L)** TIMP-1 and **(M–P)** TIMP-2 (green). Nuclei are labeled in red with PI. Scale bar: 50 μm. **(Q,R)** Histograms showing the densitometric analysis of **(Q)** MMP-2 and MMP-9 and of **(R)** TIMP-1 and TIMP-2 fluorescence intensity performed on digitized images. Significance of difference in Q: * \(p<0.01\) vs. relative control; \(\oslash\)\(p<0.01\) vs. 635 nm; \(p=0.067\) MMP-2, 405 nm vs. 808 nm; \(p=0.06\), MMP-9 405 nm vs. 808 nm. Significance of difference in R: * \(p<0.01\) vs. relative control; \(\oslash\)\(p<0.01\) vs. relative control; \(\oslash\)\(p<0.01\) vs. relative 635 nm, \(\#\)\(p<0.01\) vs. relative 808 nm. In particular, MMP-2 resulted mainly distributed in the cytoplasm with a spotted staining (Figure 9A-D) whereas MMP-9 was localized along the cytoskeletal filaments (Figure 9E-H). Both TIMP-1 and TIMP-2 appeared evenly distributed within the cytoplasm (Figure 9I-P). Likewise, murine fibroblasts, HDF-\(\alpha\), 24 h after NIR PBM treatment, down-regulated the expression of MMP-2 (Figure 7D-F) and MMP-9 (Figure 7G-I). These data well correlated with the increased and reduced gelatinase activity observed in fibroblasts after exposure to red PBM and after NIR or violet-blue PBM respectively (Figure 10D). In fact, utilizing a fluorescent gelatin degradation test, it was found that the intensity of the fluorescent signal augmented in the red PBM samples indicating that these treated fibroblasts were able to synthesize functional MMPs, whereas it decreased in NIR or violet-blue PBM ones (Figure 10D). Figure 10: Analysis of the expression and activity of MMP-2 and MMP-9 and of the expression of their inhibitors, TIMP-1 and TIMP-2, in NIH/3T3 fibroblasts 24 h after cell exposure to red (635 nm), NIR (808 nm) and violet-blue (405 nm) PBM. Cells not subjected to PBM treatments served as control. **(A-C)** Representative blots of western blotting analysis of **(A)** MMP-2 (72 kDa), **(B)** MMP-9 (82 kDa), **(C)** TIMP-1 (26 kDa) and TIMP-2 (21 kDa). The densitometric analysis of the bands normalized to \(\beta\)-actin (42 kDa) are reported in the histograms. **(D)** Gelatinase assay. NIH/3T3 fibroblasts were seeded on a fluorescein-labeled gelatin substrate (DQ\({}^{\text{TM}}\) gelatin).