Plaque Rupture Complications in Murine Atherosclerotic Vein Grafts Can Be Prevented by TIMP-1 Overexpression

<div><p>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, <em>in vivo</em> 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.</p> </div

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

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

Vein graft lesions show clear neovascularization.

<p><b>A</b>; Endothelial cell staining (CD31) of neovessels in a vein graft lesion. <b>B</b>; Erythrocytes are clearly visible in (*) and outside neovessels indicating that leaky vessels are present. <b>C</b>; VEGF staining of a vein graft with extravasated erythrocytes (*), a VEGF positive neovessel is depicted with #. <b>D</b>; Basement membrane staining with antibodies directed to laminin. Most neovessels in vein grafts stain positive. <b>E:</b> 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.</p

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

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

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

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

Vein graft lesion showing complex morphology including plaque rupture complications.

<p><b>A</b>; 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>B</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) <b>C</b>; Foamcell rich area including the start of a dissection. <b>D</b>; A small necrotic core with cholesterol clefts and foam cells can be seen with a SMC rich layer and endothelial cells on top. <b>E</b>; Calcification rich area (arrows) near the outer layer of the vessel wall. <b>F</b>; Area with extravasated erythrocytes (arrows) and neovascularization (#). <b>G</b>; Detailed photograph of dissection and extravasated erythrocytes 150 µm upstream in the lesion (arrows; erythrocytes stuck in the dissection).</p

TLR Accessory Molecule RP105 (CD180) Is Involved in Post-Interventional Vascular Remodeling and Soluble RP105 Modulates Neointima Formation

<div><p>Background</p><p>RP105 (CD180) is TLR4 homologue lacking the intracellular TLR4 signaling domain and acts a TLR accessory molecule and physiological inhibitor of TLR4-signaling. The role of RP105 in vascular remodeling, in particular post-interventional remodeling is unknown.</p><p>Methods and Results</p><p>TLR4 and RP105 are expressed on vascular smooth muscle cells (VSMC) as well as in the media of murine femoral artery segments as detected by qPCR and immunohistochemistry. Furthermore, the response to the TLR4 ligand LPS was stronger in VSMC from RP105^−/− mice resulting in a higher proliferation rate. In RP105^−/− mice femoral artery cuff placement resulted in an increase in neointima formation as compared to WT mice (4982±974 µm² vs.1947±278 µm²,p = 0.0014). Local LPS application augmented neointima formation in both groups, but in RP105^−/− mice this effect was more pronounced (10316±1243 µm² vs.4208±555 µm²,p = 0.0002), suggesting a functional role for RP105. For additional functional studies, the extracellular domain of murine RP105 was expressed with or without its adaptor protein MD1 and purified. SEC-MALSanalysis showed a functional 2∶2 homodimer formation of the RP105-MD1 complex. This protein complex was able to block the TLR4 response in whole blood ex-vivo. In vivo gene transfer of plasmid vectors encoding the extracellular part of RP105 and its adaptor protein MD1 were performed to initiate a stable endogenous soluble protein production. Expression of soluble RP105-MD1 resulted in a significant reduction in neointima formation in hypercholesterolemic mice (2500±573 vs.6581±1894 µm²,p<0.05), whereas expression of the single factors RP105 or MD1 had no effect.</p><p>Conclusion</p><p>RP105 is a potent inhibitor of post-interventional neointima formation.</p></div

Effect of garcinol treatment on inflammatory cell recruitment and CCL2 expression <i>in vivo</i>.

<p>(A) Quantification of CD45 positive cells (leukocytes) in the intima and media 3 days after cuff placement in ApoE*3-Leiden mice treated with garcinol (n = 6) or pluronic gel (n = 8). *<i>P</i><0.05, **<i>P</i><0.01. (B) Quantification of Mac3 positive cells (macrophages) in the intima and media 3 days after cuff placement in ApoE*3-Leiden mice treated with garcinol (n = 6) or pluronic gel (n = 6). **<i>P</i><0.01. (C) Quantification of CCL2 positive cells in the intima and media 3 days after cuff placement in ApoE*3-Leiden mice treated with garcinol (n = 6) or pluronic gel (n = 7). **<i>P</i><0.01, ***<i>P</i><0.001. Representative images of CD45, Mac3 and CCL2 staining of cuffed femoral arteries, scale bar = 20 μm. Results are mean±SEM.</p

Effect of PCAF deficiency on intimal hyperplasia and vascular smooth muscle cell content <i>in vivo</i>.

<p>Representative images of PCAF staining (A), scale bar = 100 μm. Quantification of intimal hyperplasia (B), intima/media ratio (C) and lumenstenosis (D) 21 days after cuff placement in WT (n = 7) and PCAF KO (n = 11) mice. ***<i>P</i><0.001. Representative images of elastin staining (E), scale bar = 50 μm. Quantification intimal (F) and medial (G) smooth muscle cell area (μm²) 21 days after cuff placement in WT (n = 7) and PCAF KO (n = 8) mice. **<i>P</i><0.001. (H) Representative images of smooth muscle actin (SMA) staining of cuffed femoral arteries, scale bar = 50 μm. Results are mean±SEM.</p

Neointima formation in WT and RP105^−/− mice.

<p>Neointima formation after femoral artery cuff placement in RP105^−/− and wild type mice. Areas of femoral arterial sections were quantified by using 6 sequential sections per segment and are expressed in micrometers squared (mean±SEM). Increased neointima formation in RP105^−/− mice compared to WT (wild type) controls (A). Increased intima/media ratio in RP105^−/− mice compared to WT controls (B). Representative pictures of Elastin von Giesson (C) HPS (D) and α-smooth muscle cell actin (F) of RP105^−/− and WT controls. * = P<0.05 Arrows indicate the Internal Elastic Lamina (IEL) and the External Elastic Lamina (EEL).</p