MMP-13 regulates growth of wound granulation tissue and modulates gene expression signatures involved in inflammation, proteolysis, and cell viability

Abstract

Proteinases play a pivotal role in wound healing by regulating cell-matrix interactions and availability of bioactive molecules. The role of matrix metalloproteinase-13 (MMP-13) in granulation tissue growth was studied in subcutaneously implanted viscose cellulose sponge in MMP-13 knockout (Mmp13(-/-)) and wild type (WT) mice. The tissue samples were harvested at time points day 7, 14 and 21 and subjected to histological analysis and gene expression profiling. Granulation tissue growth was significantly reduced (42%) at day 21 in Mmp13(-/-) mice. Granulation tissue in Mmp13(-/-) mice showed delayed organization of myofibroblasts, increased microvascular density at day 14, and virtual absence of large vessels at day 21. Gene expression profiling identified differentially expressed genes in Mmp13(-/-) mouse granulation tissue involved in biological functions including inflammatory response, angiogenesis, cellular movement, cellular growth and proliferation and proteolysis. Among genes linked to angiogenesis, Adamts4 and Npy were significantly upregulated in early granulation tissue in Mmp13(-/-) mice, and a set of genes involved in leukocyte motility including Il6 were systematically downregulated at day 14. The expression of Pdgfd was downregulated in Mmp13(-/-) granulation tissue in all time points. The expression of matrix metalloproteinases Mmp2, Mmp3, Mmp9 was also significantly downregulated in granulation tissue of Mmp13(-/-) mice compared to WT mice. Mmp13(-/-) mouse skin fibroblasts displayed altered cell morphology and impaired ability to contract collagen gel and decreased production of MMP-2. These results provide evidence for an important role for MMP-13 in wound healing by coordinating cellular activities important in the growth and maturation of granulation tissue, including myofibroblast function, inflammation, angiogenesis, and proteolysis.

Figure 1. Delayed growth of experimental granulation tissue in Mmp13^−/− mice.

Subcutaneous viscose cellulose sponges (VCS) implanted in wild type (WT) and MMP-13 knockout (Mmp13^−/−) mice were harvested at different time points, as indicated. (A) Hematoxylin-eosin staining of representative sections demonstrating reduced growth of granulation tissue in Mmp13^−/− mice at 21 d. The border of cellular granulation tissue is marked with dashed line. The area enclosed by a square is shown in (C) with higher magnification. (Scale bar = 1 mm). (B) The growth of granulation tissue inside VCS was quantified blinded by determining the portion of cellular tissue relative to the implant area in a tissue section. The border of granulation tissue was determined as exemplified with dashed lines in (A). (*P<0.05, Independent samples T-test, n = 5–6). (C) Higher resolution images from the tissue sections presented in (A) showing the border region at the endpoint of the granulation tissue (the area enclosed by a square in A). (s, implant surface; scale bar = 200 µm).

Figure 2. Delayed maturation of myofibroblasts in granulation tissue of Mmp13^−/− mice.

Sections of experimental granulation tissue of wild type (WT) and MMP-13 knockout (Mmp13^−/−) mice were stained with α-smooth muscle actin (α-SMA) antibody. (A) The panel shows three representative image pairs from comparable locations of WT and Mmp13^−/− granulation tissue at 7 d. α-SMA-positive myofibroblasts were detected close to implant surface (s). The staining pattern was denser and followed parallel orientation more strictly in WT mice compared to Mmp13^−/− granulation tissue. (B) (Upper panels) at 14 d, α-SMA-staining pattern was strong and comparable in WT and Mmp13^−/−. (Lower panels) representative image pair of WT and Mmp13^−/− granulation tissues at 21 d immunostained for α-SMA. The expression of α-SMA was evident in the inner parts of implants in WT mouse granulation tissue, whereas in the Mmp13^−/− granulation tissue α-SMA-positive cells were mainly abundant close to implant surface. (s, implant surface; scale bar = 100 µm).

Figure 3. Altered vascular pattern in granulation tissue of Mmp13^−/− mice.

(A) Sections of experimental granulation tissue of wild-type (WT) and MMP-13 knockout (Mmp13^−/−) mice harvested at indicated time points were immunostained for blood vessels using CD34 as a marker. The arrowheads indicate microvessels and medium sized vessels (diameter<40 µm) and arrows indicate large vessel structures (diameter>40 µm). (s, implant surface; scale bar = 200 µm. (B) The number and the diameter of CD34-positive blood vessels were determined in defined areas of cellular granulation tissues with digital image analysis. *Statistically significant difference in the density of microvessels (<10 µm) at 14 d and of the large vessels (>40 µm) at 21 d (P<0.05, MannWhitney U test, n = 5–6).

Figure 4. Comparison of gene expression profiles in granulation tissue of Mmp13^−/− and WT mice.

(A) Microarray data of MMP-13 knockout (Mmp13^−/−) and wild type (WT) mouse granulation tissue at 7, 14 and 21 d were analyzed for differential gene expression by comparing Mmp13^−/− granulation tissue samples to WT. The genes, which showed significant difference (P<0.05) and FC>0.75 in the expression are illustrated as heatmap. *Genes with FC>1 and P<0.001. (B) Differentially expressed genes at indicated time points were categorized based on molecular function according to Ingenuity Pathway Analysis® (IPA) software.

Figure 5. Molecular interactions of MMP-13 with differently regulated genes in Mmp13^−/− mouse granulation tissue compared to WT.

IPA software was employed to construct a molecular interaction network of MMP-13 with the genes that were differently expressed in MMP-13 knockout (Mmp13^−/−) granulation tissue compared to wild type (WT) in indicated time points. Interactions are based on the literature in Ingenuity Knowledge Base. The molecules with fold change (FC) >0.3 in one of the time points were included in the figure and the molecules with FC>0.5 and with P-value<0.05 in specific time point are highlighted with yellow color. Red color indicates upregulation and green color indicates downregulation in Mmp13^−/− mouse granulation tissue compared to WT. The intensity of the color implies the magnitude of FC. The arrows and lines indicate direct (solid line) and indirect (dashed line) functional and physical interactions. The arrows show the direction of the regulation.

Figure 6. Molecular interactions involved in biological functions cell movement of leukocytes and metabolism of protein at 14 d time point, and proliferation of connective tissue cells at 21 d.

The diagrams show the differentially regulated genes involved in biological functions and the molecular interactions based on the literature in Ingenuity Knowledge Base. The expression ratios in the MMP-13 knockout (Mmp13^−/−) granulation tissues compared to WT are visualized as heatmaps. Red color indicates upregulation and green color indicates downregulation in Mmp13^−/− mouse granulation tissues. The intensity of the color implies the magnitude of the FC. The arrows and lines indicate direct (solid line) and indirect (dashed line) functional and physical interactions. The arrows show the direction of regulation. (A) Functional analysis of differentially expressed genes (FC>0.5, P<0.05) in Mmp13^−/− granulation tissue compared to WT (14 d) revealed enrichment in the biological function cell movement of leukocytes (P<3.91E-10), which was predicted to be downregulated in Mmp13^−/− mice (regulation z-score -2.28). (B) Functional analysis was performed as in (A). Enrichment of differentially expressed genes was found in the biological function metabolism of protein (P<6.75E-05) in Mmp13^−/− granulation tissues, and the function was predicted to be downregulated (regulation z-score -2.40). (C) Functional analysis was performed as in (A). Enrichment of differentially expressed genes was found in the biological function proliferation of connective tissue cells at 21 d and the function was predicted to be upregulated in Mmp13^−/− granulation tissue compared to WT (regulation z-score 2.69).

Figure 7. The expression of Mmp2, Mmp3, Mmp9, Adamts4, and Npy mRNA in Mmp13^−/− and WT mouse granulation tissue.

(A) Microarray data of MMP-13 knockout (Mmp13^−/−) and wild type (WT) mouse granulation tissue at 7, 14 and 21 d were analyzed for MMP gene expression, and the signal intensities are illustrated as a heatmap. (B,C) Total RNA harvested from WT and Mmp13^−/− granulation tissues at the indicated time points was analyzed for Mmp2, Mmp3, Mmp9, Adamts4, and Npy mRNA levels by real-time qRT-PCR. A dot represents a mean of triplicate analysis of a sample with SD≤2% of the mean and the black horizontal bar represents the mean of the experimental replicates. The amplification result of a given mRNA was normalized for β-actin mRNA level in each sample. (*P<0.05, **P<0.001, ***P<0.0001, independent samples T-test, n = 4–6).

Figure 8. Reduced collagen gel contraction by Mmp13^−/− mouse skin fibroblasts.

(A) Skin fibroblasts (MSF) established from wild type (WT) and MMP-13 knockout (Mmp13^−/−) mice were cultured in mechanically unloaded (floating) 3D collagen gel at density 2×10⁵/ml for 24 h in the presence of 0.5% FCS, 10% FCS or 0.5% FCS+TGF-β (5 ng/ml), as indicated. The cells were fixed, stained with fluorescently labeled phalloidin and Hoechst, and photographed with 20× magnification to observe morphological appearance. In contrast to Mmp13^−/− MSF, WT fibroblasts displayed stellate morphology with numerous thick cell extensions in response to TGF-β or 10% FCS (Scale bar = 10 µm). (B) WT and Mmp13^−/− MSF were cultured in mechanically unloaded 3D collagen gel at density 5×10⁵/ml for 24 and 48 h in the presence of 10% FCS. Contraction of collagen gels was measured from digital images of the gels and is shown as relative to the original gel size. (*P<0.005 compared to control, Independent samples T-test, n = 4) (C) WT and Mmp13^−/− MSF were cultured in attached 3D collagen gel at density 5×10⁵/ml for 72 h in the presence of 10% FCS. Subsequently the gels were detached from the well walls and contraction was quantified after 24 h. (*P<0.005 compared to control. Independent samples T-test, n = 3). (D) MSF were cultured for 72 h in 3D collagen gel in the presence 10% FCS. Equal aliquots of conditioned media were analyzed in gelatinase zymography.