Plaque rupture complications in murine atherosclerotic vein grafts can be prevented by TIMP-1 overexpression

Abstract

The current study describes the incidence and phenotype of plaque rupture complications in murine vein grafts. Since matrix metalloproteinases (MMPs) are highly involved in atherosclerotic plaque vulnerability and plaque rupture, we hypothesized that this model can be validated by overexpression of the MMP inhibitor TIMP-1. First we studied 47 vein grafts in hypercholesterolemic ApoE3*Leiden mice for the incidence of plaque complications. In 79% of these grafts, extensive lesions with plaque rupture complications like dissections, intraplaque hemorrhages or erosions with intramural thrombi were found. Next, in vivo Near-InfraRed-Fluorescence imaging demonstrated that electroporation mediated TIMP-1-overexpression reduced local MMP activity in vein grafts by 73% (p<0.01). This led to a 40% reduction in lesion-size after 28d (p = 0.01) and a more stable lesion phenotype with significant more smooth muscle cells (135%), collagen (47%) and significant less macrophages (44%) and fibrin (55%) than controls. More importantly, lesions in the TIMP-1 group showed a 90% reduction of plaque complications (10/18 of control mice showed plaque complications versus 1/18 in TIMP-1 treated mice). Murine vein grafts are a relevant spontaneous model to study plaque stability and subsequent hemorrhagic complications, resulting in plaque instability. Moreover, inhibition of MMPs by TIMP-1-overexpression resulted in decreased plaque progression, increased stabilization and decreased plaque rupture complications in murine vein grafts.

Figure 1. Vein graft lesion showing complex morphology including plaque rupture complications.

A; Cross-section stained with a Masson trichrome staining of a vein graft in an ApoE3Leiden mouse 28 days after surgery. Coloured squares depict location of photographs B–G. L represents lumen and A; adventitia B; Part of the lesion showing foamcells (white arrows) and SMC’s, with a smooth muscle cell rich cap underneath an intact endothelial layer (black arrows; endothelial cells) C; Foamcell rich area including the start of a dissection. D; A small necrotic core with cholesterol clefts and foam cells can be seen with a SMC rich layer and endothelial cells on top. E; Calcification rich area (arrows) near the outer layer of the vessel wall. F; Area with extravasated erythrocytes (arrows) and neovascularization (#). G; Detailed photograph of dissection and extravasated erythrocytes 150 µm upstream in the lesion (arrows; erythrocytes stuck in the dissection).

Figure 2. Vein graft lesions show clear neovascularization.

A; Endothelial cell staining (CD31) of neovessels in a vein graft lesion. B; Erythrocytes are clearly visible in (*) and outside neovessels indicating that leaky vessels are present. C; VEGF staining of a vein graft with extravasated erythrocytes (*), a VEGF positive neovessel is depicted with #. D; Basement membrane staining with antibodies directed to laminin. Most neovessels in vein grafts stain positive. E: SMC staining of a vein graft. ^# marks SMC positive neovessels and * depicts neovessels lacking pericytes. These neovessels without pericytes are frequently found in regions with extravasated erythrocytes. Insets are 10x magnifications of vein grafts.

Figure 3. Divers forms of plaque rupture complications can be seen in vein graft lesions.

A; Vein graft with a dissection (arrow) starting at the lumen of the vessel wall with small fibrin depositions (*) and erythrocytes (#) in the clearly visible gap. B; Vein graft lesion with leaky vessels and extravasated erythrocytes (#) C; Vein graft showing massive intramural thrombi consisting of layers of fibrin (*) and diffuse erythrocytes (#) with erosion extending to the outer part of the vessel wall D; Vein graft showing combined leaky vessels with extravasated erythrocytes (#) and dissection in the outer part of the vessel wall (black arrow). E; Vein graft lesion with leaky vessels showing extravasation of erythrocytes (#) and erosion with small mural thrombi (*).

Figure 4. Quantification of vein grafts without complications (Control), and vein grafts with complications, namely plaque hemorrhage, dissections or erosions (n = 10/group).

A; Vessel wall area measurements B; Quantification of lumen area C; Total vessel area (combined lumen and vessel wall area, as a measure for outward remodeling) D; Correlation between the vessel wall area and the length of the plaque rupture complications.

Figure 5. In vivo MMP activity in mice A; Representative Near-InfraRed-Fluorescence images of enzymatic MMP activity in mice.

No signal was detected in mice without MMPSense. B; Quantification of the MMP activity in the vein graft region (n = 5/group).

Figure 6. Quantitative measurements on vein graft lesions in mice that overexpress Luciferase, TIMP-1 or TIMP-3.

A; Representative cross-sections of vein grafts in mice 28 days after surgery (Hematoxilin-Phloxine-Saffron staining). B; Quantitative measurements of total vessel area, luminal area and lesion area C; Graph showing the length of the plaque rupture complications. D; Quantitative measurement of percentage collagen in vein grafts in Luciferase, TIMP-1 or TIMP-3 overexpressing mice. E–G; Quantitative measurement of percentage SMC actin, macrophages and fibrin H–K; Typical examples for all (immuno)histochemical stainings.