MMP-12 Deficiency Attenuates Angiotensin II-Induced Vascular Injury, M2 Macrophage Accumulation, and Skin and Heart Fibrosis

<div><p>MMP-12, a macrophage-secreted elastase, is elevated in fibrotic diseases, including systemic sclerosis (SSc) and correlates with vasculopathy and fibrosis. The goal of this study was to investigate the role of MMP-12 in cardiac and cutaneous fibrosis induced by angiotensin II infusion. Ang II-induced heart and skin fibrosis was accompanied by a marked increase of vascular injury markers, including vWF, Thrombospondin-1 (TSP-1) and MMP-12, as well as increased number of PDGFRβ⁺ cells. Furthermore Ang II infusion led to an accumulation of macrophages (Mac3⁺) in the skin and in the perivascular and interstitial fibrotic regions of the heart. However, alternatively activated (Arg 1⁺) macrophages were mainly present in the Ang II infused mice and were localized to the perivascular heart regions and to the skin, but were not detected in the interstitial heart regions. Elevated expression of MMP-12 was primarily found in macrophages and endothelial cells (CD31⁺) cells, but MMP-12 was not expressed in the collagen producing cells. MMP-12 deficient mice (MMP12KO) showed markedly reduced expression of vWF, TSP1, and PDGFRβ around vessels and attenuation of dermal fibrosis, as well as the perivascular fibrosis in the heart. However, MMP-12 deficiency did not affect interstitial heart fibrosis, suggesting a heterogeneous nature of the fibrotic response in the heart. Furthermore, MMP-12 deficiency almost completely prevented accumulation of Arg 1⁺ cells, whereas the number of Mac3⁺ cells was partially reduced. Moreover production of profibrotic mediators such as PDGFBB, TGFβ1 and pSMAD2 in the skin and perivascular regions of the heart was also inhibited. Together, the results of this study show a close correlation between vascular injury markers, Arg 1⁺ macrophage accumulation and fibrosis and suggest an important role of MMP-12 in regulating these processes.</p></div

MMP-12 deficiency inhibits production of PDGFBB, TGFβ1 and pSMAD2 in the Ang II treated skin and heart.

<p>A. IHC staining of PDGFBB, TGFβ1 and pSMAd2 was performed on paraffin sections from the skin. B. Immunofluorescence staining of TGFβ1 was performed on cryosections from the heart. Representative photographs are shown from four animals per group.</p

MMP12KO mice are partially protected from heart and skin fibrosis in the Ang II model.

<p>Picrosirius Red and Gomori's Trichrome staining was performed on paraffin sections respectively from the heart (<b>A</b>) and skin (<b>B</b>) of WT and MMP12KO mice infused with PBS or Ang II. Representative photographs are shown from five animals per group. <b>C</b>. Perivascular and interstitial collagen content in the heart was quantified using ImageJ software by measuring the ratio of the red area to the total area of the heart. <b>D</b>. Total hydroxyproline content in PBS- and Ang II-treated WT and MMP12KO mice. Values are the mean±SD of 6 mice in each group; *<i>p</i>≤0.05; **<i>p</i>≤0.01.</p

Ang II increases the number of PDGFRβ positive cells in mouse heart and skin.

<p>IHC staining of PDGFRβ was performed on paraffin sections from the heart (<b>A</b>) and skin (<b>B</b>) of PBS and Ang II treated WT mice. Representative photographs from five animals per group.</p

Ang II increases the number of TSP-1 positive cells in mouse heart and skin.

<p>IHC staining of TSP-1 was performed on paraffin sections from the heart (<b>A</b>) and skin (<b>B</b>) of PBS and Ang II treated WT mice. Representative photographs are shown from five animals per group. <b>C</b>. TSP-1 protein levels were increased in Ang II treated HDMECs in a dose dependent manner (*p≤0.05).</p

Ang II induces expression of vWF in mouse heart and skin.

<p>IHC staining of vWF was performed on paraffin sections from the heart (<b>A</b>) and skin (<b>B</b>) of PBS and Ang II treated WT mice. Representative photographs are shown from five animals per group. <b>C</b>. vWF protein levels were increased in Ang II treated HDMECs in a dose dependent manner (*p≤0.05).</p

Reduced levels of vascular and pericyte markers in MMP12KO mice.

<p><b>A</b>. IHC staining of vWF, TSP-1 and PDGFRβ was performed on paraffin sections from the heart of PBS and Ang II treated WT and MMP12KO mice. <b>B</b>. IHC staining of vWF, and PDGFRβ was performed on paraffin sections from the skin of PBS and Ang II treated WT and MMP12KO mice. Representative photographs are shown from three animals per group.</p

Ang II increases the number of MMP-12/CD31 positive cells in mouse heart and skin.

<p>Double IHC staining of MMP-12/CD31 was performed on paraffin sections from the heart (<b>A</b>) and skin (<b>B</b>) of PBS and Ang II treated WT mice. Representative photographs are shown from five animals per group. Arrows indicate double positive cells. <b>C</b>. MMP-12 protein levels were increased in Ang II treated HDMECs in a dose dependent manner (*p≤0.05).</p

Distribution of Mac3⁺ and Arg 1⁺ macrophages in the Ang II treated mouse skin.

<p>IHC staining of Mac3 (<b>A</b>) and Arg 1 (<b>B</b>) was performed on paraffin sections from skin of PBS and Ang II treated WT mice. Representative photographs are shown from four animals per group.</p

Ang II increases the number of MMP-12/Mac3 positive cells in mouse heart and skin.

<p>Double staining of MMP-12/Mac3 was performed on cryosections from the heart (<b>A</b>) and paraffin sections from the skin (<b>B</b>) of PBS and Ang II treated WT mice. Representative photographs are shown from five animals per group. Dotted arrows: Mac3 positive cells, arrowheads: MMP-12 positive cells, arrows: MMP-12/Mac3 double positive cells.</p