Cathepsin K Deficiency Prevents the Aggravated Vascular Remodeling Response to Flow Cessation in ApoE-/- Mice

Abstract

Cathepsin K (catK) is a potent lysosomal cysteine protease involved in extracellular matrix (ECM) degradation and inflammatory remodeling responses. Here we have investigated the contribution of catK deficiency on carotid arterial remodeling in response to flow cessation in apoE-/- and wild type (wt) background. Ligation-induced hyperplasia is considerably aggravated in apoE-/- versus wt mice. CatK protein expression was significantly increased in neointimal lesions of apoE-/- compared with wt mice, suggesting a role for catK in intimal hyperplasia under hyperlipidemic conditions. Surprisingly, CatK deficiency completely blunted the augmented hyperplastic response to flow cessation in apoE-/-, whereas vascular remodeling in wt mice was unaffected. As catK deficiency did neither alter lesion collagen content and elastic laminae fragmentation in vivo, we focused on effects of catK on (systemic) inflammatory responses. CatK deficiency significantly reduced circulating CD3 T-cell numbers, but increased the regulatory T cell subset in apoE-/- but not wt mice. Moreover, catK deficiency changed CD11b+Ly6G-Ly6C high monocyte distribution in apoE-/- but not wt mice and tended to favour macrophage M2a polarization. In conclusion, catK deficiency almost completely blunted the increased vascular remodeling response of apoE-/- mice to flow cessation, possibly by correcting hyperlipidemia-associated pro-inflammatory effects on the peripheral immune response.

Fig 1. CatK expression in macrophage-rich (apoE^-/-) and macrophage-poor (wt) flow cessation-induced intimal lesions.

A. Representative pictures of catK staining in wt and apoE^-/- mice. B. Relative catK-positivity was calculated by dividing the anti-catK Ab stained area by the total intima area (P = 0.027; n = 5 for wt, n = 12 for apoE^-/-). C-D. Representative examples of CatK (red) double staining with Mac2+ macrophages (left panel, green) or α-actin+ SMCs (right panel, green). DAPI (Blue) was used to stain cell nuclei. Arrows indicate double-positive cells.

Fig 2. Effect of catK deficiency on intimal hyperplasia.

A. Representative micrographs showing HE staining of intima lesions in wt, catK^-/-, apoE^-/- and catK^-/-//apoE^-/- mice. B. CatK deficiency did not affect lumen, intima and total vessel area in wt mice. CatK deficiency significantly reduced intima (P = 0.019) and total vessel area (P = 0.024) in apoE^-/- mice. Values represent the mean ± SEM. (n = 6–8 for wt and catK^-/- mice, n = 14–15 for apoE^-/- and catK^-/-//apoE^-/- mice).

Fig 3. CatK deficiency did not affect neointimal lesion composition.

(A) Macrophage, CD3 and SMC content in the macrophage-rich lesions of apoE^-/- mice or in macrophage-poor lesions of wt mice was not altered by CatK deficiency. The number of MAC3-positive, CD3-positive and α-SMA-positive cells in the intima area were corrected for the total number of cells in the entire intima area. CatK deficiency did not change collagen content in the intima area in apoE^-/- and wt mice (B). CatK deficiency did not significantly alter cell density in the intima area in apoE^-/- and wt mice (C). Total cell number in neointima lesion was corrected for the corresponding lesion area. Values represent the mean ± SEM. (n = 6–8 for wt and catK^-/- mice, n = 14–15 for apoE^-/- and catK^-/-//apoE^-/- mice). D: CatK inhibition did not affect in vitro primary mouse SMC proliferation. E: CatK deficiency did not affect medial elastin break frequency in apoE^-/- and in WT mice. Frequency is expressed as average number of breaks per cross-section.

Fig 4. Flow-cytometric analysis of the effect of catK deficiency on CD3⁺, B, NK, CD4+CD25+Foxp3 regulatory T cells and CD11b+Ly6G-Ly6C^high/- monocytes in spleen and blood.

Percentage of CD3⁺ T cells in spleen (P = 0.025) and NK cells in blood (P = 0.025) were significantly reduced in catK^-/-//apoE^-/- mice compared with apoE^-/- (A). The regulatory T cell population defined as CD4+CD25+Foxp3+ cells, was significantly increased in catK^-/-//apoE^-/- mice in blood (B, P = 0.025). CD11b+Ly6G-Ly6C^high monocytes were significantly increased (P = 0.035) and CD11b+Ly6G-Ly6C^- monocytes significantly reduced (P = 0.047) in blood of catK^-/-//apoE^-/- compared with apoE^-/- mice (C). CatK deficiency in the wt mice did not affect peripheral immune activity (D, E and F). Values represent the mean ± SEM. (n = 6 for each group).

Fig 5. Effect of catK deficiency on M1 and M2 macrophage marker expression of non-stimulated BMM.

BMM from wt, catK^-/-, apoE^-/- and catK^-/-//apoE^-/- mice were isolated and BMM cell lysates were analyzed for M1 and M2 marker expression. CatK^-/-//apoE^-/- -derived BMM increased arginase-1 expression (C, P = 0.004) and significantly decreased IL-10 expression (E, P = 0.004) compared with apoE^-/- -derived-BMM. While IL-18 (A) and Mannose Receptor (D) were not affected, catK deficiency tended to decrease Nos2 expression in ApoE^-/- BMM (B). Values represent the mean ± SEM. (n = 6 for each group).

Fig 6. CatK defiency increases expression of the M2a marker Mac-2/Galectin-3 in ApoE^-/- mice.

(A) Representative pictures of Mac-2 (upper panels) and Arg-1 (lower panels) staining in apoE^-/- and catK^-/-//apoE^-/- mice. (B) While Arg-1 positive cells were scarce and not different between groups, CatK deficiency increased Mac-2 positive cells in the intima of ApoE^-/- mice. The number of Mac-2 and Arg-1 positive cells in the neointima were corrected for corresponding lesion area or total cells, respectively. Values represent the mean ± SEM. (n = 4–6 for each group).