Transglutaminase 2 moderates the expansion of mouse abdominal aortic aneurysms.

OBJECTIVE: Previously published work has indicated that transcripts encoding transglutaminase 2 (TG2) increase markedly in a rat model of abdominal aortic aneurysm. This study determines whether TG2 and the related TG, factor XIII-A (FXIII-A), protect against aortic aneurysm development in mice. METHODS: C57BL/6J wild-type, Tgm2 -/- knockout, F13a1 -/- knockout, and Tgm2 -/- /F13a1 -/- double knockout mice were subjected to laparotomy and periaortic application of CaCl2. RESULTS: Tgm2 -/- mice showed slightly greater aortic dilatation at 6 weeks after treatment when compared with wild type. However, vessels from Tgm2 -/- mice, but not wild-type mice, continued to dilate up to 6 months after injury and by 24 weeks, a greater number of Tgm2 -/- mice had developed aneurysms (16/17 vs 10/19; P = .008). Laparotomy resulted in a high death rate in F13a1 -/- knockout mice, more frequently from cardiac complications than from hemorrhage, but among F13a1 -/- mice that survived for 6 weeks after CaCl2 treatment, abdominal aortic aneurysm diameter was unaltered relative to wild-type mice. Laparotomy resulted in a higher death rate among Tgm2 -/- /F13a1 -/- double knockout mice, owing to an increased frequency of delayed bleeding. Surprisingly, Tgm2 -/- /F13a1 -/- double knockout mice showed a trend toward decreased dilatation of the aorta 6 weeks after injury, and this finding was replicated in Tgm2 -/- /F13a1 -/- mice subjected to carotid artery injury. Levels of transcripts encoding TG2 were not increased in the aortas of injured wild-type or F13a1 -/- knockout mice relative to uninjured mice, although changes in the levels of other transcripts accorded with previous descriptions of the CaCl2 aneurysm model in mice. CONCLUSIONS: Knockout of Tgm2, but not F13a1 exacerbates aortic dilatation, suggesting that TG2 confers protection. However, levels of TG2 messenger RNA are not acutely elevated after injury. FXIII-A plays a role in preventing postoperative damage after laparotomy, confirming previous reports that it prevents distal organ damage after trauma. TG2 promotes wound healing after surgery and, in its absence, the bleeding diathesis associated with FXIII-A deficiency is further exposed.

Galectin-3 Identifies a Subset of Macrophages With a Potential Beneficial Role in Atherosclerosis.

OBJECTIVE: Galectin-3 (formerly known as Mac-2), encoded by the LGALS3 gene, is proposed to regulate macrophage adhesion, chemotaxis, and apoptosis. We investigated the role of galectin-3 in determining the inflammatory profile of macrophages and composition of atherosclerotic plaques. Approach and Results: We observed increased accumulation of galectin-3-negative macrophages within advanced human, rabbit, and mouse plaques compared with early lesions. Interestingly, statin treatment reduced galectin-3-negative macrophage accrual in advanced plaques within hypercholesterolemic (apolipoprotein E deficient) Apoe-/- mice. Accordingly, compared with Lgals3+/+:Apoe-/- mice, Lgals3-/-:Apoe-/- mice displayed altered plaque composition through increased macrophage:smooth muscle cell ratio, reduced collagen content, and increased necrotic core area, characteristics of advanced plaques in humans. Additionally, macrophages from Lgals3-/- mice exhibited increased invasive capacity in vitro and in vivo. Furthermore, loss of galectin-3 in vitro and in vivo was associated with increased expression of proinflammatory genes including MMP (matrix metalloproteinase)-12, CCL2 (chemokine [C-C motif] ligand 2), PTGS2 (prostaglandin-endoperoxide synthase 2), and IL (interleukin)-6, alongside reduced TGF (transforming growth factor)-ß1 expression and consequent SMAD signaling. Moreover, we found that MMP12 cleaves macrophage cell-surface galectin-3 resulting in the appearance of a 22-kDa fragment, whereas plasma levels of galectin-3 were reduced in Mmp12-/-:Apoe-/- mice, highlighting a novel mechanism where MMP12-dependent cleavage of galectin-3 promotes proinflammatory macrophage polarization. Moreover, galectin-3-positive macrophages were more abundant within plaques of Mmp12-/-:Apoe-/- mice compared with Mmp12+/+:Apoe-/- animals. CONCLUSIONS: This study reveals a prominent protective role for galectin-3 in regulating macrophage polarization and invasive capacity and, therefore, delaying plaque progression.

Transglutaminase 2 limits the extravasation and the resultant myocardial fibrosis associated with factor XIII-A deficiency.

BACKGROUND AND AIMS: Transglutaminase (TG) 2 and Factor (F) XIII-A have both been implicated in cardiovascular protection and repair. This study was designed to differentiate between two competing hypotheses: that TG2 and FXIII-A mediate these functions in mice by fulfilling separate roles, or that they act redundantly in this respect. METHODS: Atherosclerosis was assessed in brachiocephalic artery plaques of fat-fed mixed strain apolipoprotein (Apo)e deficient mice that lacked either or both transglutaminases. Cardiac fibrosis was assessed both in the mixed strain mice and also in C57BL/6J Apoe expressing mice lacking either or both transglutaminases. RESULTS: No difference was found in the density of buried fibrous caps within brachiocephalic plaques from mice expressing or lacking these transglutaminases. Cardiac fibrosis developed in both Apoe/F13a1 double knockout and F13a1 single knockout mice, but not in Tgm2 knockout mice. However, concomitant Tgm2 knockout markedly increased fibrosis, as apparent in both Apoe/Tgm2/F13a1 knockout and Tgm2/F13a1 knockout mice. Amongst F13a1 knockout and Tgm2/F13a1 knockout mice, the extent of fibrosis correlated with hemosiderin deposition, suggesting that TG2 limits the extravasation of blood in the myocardium, which in turn reduces the pro-fibrotic stimulus. The resulting fibrosis was interstitial in nature and caused only minor changes in cardiac function. CONCLUSIONS: These studies confirm that FXIII-A and TG2 fulfil different roles in the mouse myocardium. FXIII-A protects against vascular leakage while TG2 contributes to the stability or repair of the vasculature. The protective function of TG2 must be considered when designing clinical anti-fibrotic therapies based upon FXIII-A or TG2 inhibition.

Neointima development in externally stented saphenous vein grafts.

INTRODUCTION: The main limitation of coronary artery bypass grafting (CABG) is rapid neointimal hyperplasia leading to graft failure. AIM: To assess plaque formation in saphenous vein grafts (SVG) covered by an external Dacron stent in comparison with the classical technique. MATERIAL AND METHODS: In the study group vein grafts covered by external stent mesh made of Dacron were implanted. An intravascular ultrasonography (IVUS) study was performed in 35 aorto-coronary SVG covered by an external Dacron stent and in 64 normal SVG during the first year after CABG. In each SVG 25 mm of good quality IVUS image, volumes of lumen, plaque (neointima), outer border of the vein graft (external SVG) and adventitia were calculated in three time periods: 0-130 days, 130-260 days and 260-390 days. RESULTS: Between the first and second time period, lumen volume (mm3) was reduced from 10.33 ±4.4, to 6.80 ±2.23 in the second period and 5.69 ±1.26 in the third one. This effect was much less marked in normal grafts. The corresponding lumen volume (mm3) was: 10.90 ±3.9, 9.15 ±2.94 and 8.92 ±2.93 in consecutive time periods. Plaque volume (mm3) did not change in control grafts during the course of the study, but it increased very significantly in stented grafts from 0.86 ±1.24 in the first period to 2.70 ±1.58 in the second and 3.29 ±2.66 in the third one. CONCLUSIONS: The experimental technique of implanting SVG covered with an external elastic Dacron stent seems to be inferior to traditional ones. This is probably due to the more complicated process of vein implantation and higher micro-injury occurrence during the surgery.

Differential effects of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 on atherosclerosis and monocyte/macrophage invasion.

AIMS: MMPs contribute to atherosclerotic plaque progression and instability, but the relative potency of their endogenous tissue inhibitors of metalloproteinases (TIMPs) as protective factors has not been defined. We therefore investigated the impact of TIMP-1 and TIMP-2 knockout on atherosclerotic plaque burden and composition in apolipoprotein E-knockout (Apoe(-/-)) mice and studied the underlying effects on monocyte/macrophage behaviour. METHODS AND RESULTS: Analysis of brachiocephalic artery plaques revealed comparable atherosclerotic lesion areas between TIMP-1(-/-) Apoe(-/-) or TIMP-2(-/-) Apoe(-/-) double deficient mice and relevant age-matched, strain-matched Apoe(-/-) controls after 8 weeks of high-fat feeding. However, lesions from TIMP-2(-/-) Apoe(-/-) mice had higher levels of markers associated with plaque vulnerability, including increased macrophage: vascular smooth muscle cell ratios, larger necrotic core areas, reduced collagen contents, increased macrophage proliferation, and apoptosis frequencies, compared with TIMP-1(-/-)Apoe(-/-) and controls. In contrast, TIMP-1(-/-) Apoe(-/-) animals only had a significant reduction in vascular smooth muscle cell content compared with Apoe(-/-) controls. In vitro and in vivo findings implicated heightened monocyte/macrophage invasion in the detrimental effects observed on atherosclerotic plaque composition in TIMP-2(-/-) Apoe(-/-) mice. Moreover, TIMP-2 specifically decreased MMP-14-dependent monocyte/macrophage infiltration into sites of experimentally induced inflammation and established atherosclerotic lesions. CONCLUSION: Our data demonstrate that TIMP-2 plays a greater protective role than TIMP-1 during the pathogenesis of atherosclerosis, in part by suppressing MMP-14-dependent monocyte/macrophage accumulation into plaques.

Normal Bone Deposition Occurs in Mice Deficient in Factor XIII-A and Transglutaminase 2.

Transglutaminase activity has been widely implicated in bone deposition. A predominant role has been proposed for factor (F)XIII-A and a subsidiary role suggested for the homologous protein, transglutaminase 2. Full-length FXIII-A is an 83kDa protransglutaminase that is present both in plasma and also in haematopoietic and connective tissue lineages. Several studies have reported expression in murine cells, including osteocytes, of a 37 kDa protein that reacts with the monoclonal anti-FXIII-A antibody AC-1A1. This protein was presumed to be a catalytically active fragment of FXIII-A-83 and to play a major role in bone deposition. We detected a 37 kDa AC-1A1 reactive protein in FXIII-A mRNA negative cell lines and in tissues from FXIII-A(-/-) mice. By mass spectrometric sequencing of AC-1A1 immunoprecipitates, we identified this protein as transaldolase-1, and confirmed that recombinant transaldolase-1 is recognised by AC-1A1. We have also shown that bone deposition is normal in FXIII-A(-/-).TG2(-/-) double knockout mice, casting doubt on the role of transglutaminases in bone mineralisation. Various studies have used antibody AC-1A1 for immunohistochemistry or immunofluorescence. We observe strong FXIII-A dependent staining in paraffin embedded mouse heart sections, with relatively low background in non-expressing mouse cells. In contrast, FXIII-A independent staining predominates in cultured human cells using a standard immunofluorescence procedure. Immunofluorescence is present in membrane compartments that are expected to lack transaldolase, indicating that other off-target antigens are recognised by AC-1A1. This has significant implications for studies that have used this approach to define the subcellular trafficking of FXIII-A in osteocytes.

Cartilage oligomeric matrix protein (COMP) in murine brachiocephalic and carotid atherosclerotic lesions.

OBJECTIVE: To investigate the hypothesis that COMP can influence the morphology, stability and size of murine atherosclerotic lesions. METHODS: ApoE- and ApoE/COMP-knockout mice were fed a high-fat diet to develop atherosclerotic plaques at lesion sites of three different types; inflammatory and fibrous plaques induced in the carotid artery by low or oscillatory shear stress, respectively, and spontaneously developing plaques in the brachiocephalic artery. The localization of COMP in the plaques and the effect of COMP deficiency on plaque development were evaluated. RESULTS: COMP immunoreactivity was observed in about half of the investigated plaques from the ApoE null mice, mainly located along the intima-medial border. There were no significant differences in the size of inflammatory and fibrous carotid plaques between the genotypes. Plaques in the brachiocephalic artery from ApoE mice lacking COMP were increased in size with 54%. In these plaques the collagen content was also increased by 48%. There were no differences in relative collagen content in inflammatory and fibrous carotid plaques between genotypes. Polarized light microscopy showed that the increase in total collagen in brachiocephalic plaques was more than proportionally accounted for by an increase in thicker collagen fibrils. CONCLUSION: We have shown that COMP deficiency has a significant impact on atherosclerotic plaque morphology and size. Our data also suggest that an altered collagen metabolism may be an important mechanism in this finding.

Hearts from mice fed a non-obesogenic high-fat diet exhibit changes in their oxidative state, calcium and mitochondria in parallel with increased susceptibility to reperfusion injury.

RATIONALE: High-fat diet with obesity-associated co-morbidities triggers cardiac remodeling and renders the heart more vulnerable to ischemia/reperfusion injury. However, the effect of high-fat diet without obesity and associated co-morbidities is presently unknown. OBJECTIVES: To characterize a non-obese mouse model of high-fat diet, assess the vulnerability of hearts to reperfusion injury and to investigate cardiac cellular remodeling in relation to the mechanism(s) underlying reperfusion injury. METHODS AND RESULTS: Feeding C57BL/6J male mice high-fat diet for 20 weeks did not induce obesity, diabetes, cardiac hypertrophy, cardiac dysfunction, atherosclerosis or cardiac apoptosis. However, isolated perfused hearts from mice fed high-fat diet were more vulnerable to reperfusion injury than those from mice fed normal diet. In isolated cardiomyocytes, high-fat diet was associated with higher diastolic intracellular Ca2+ concentration and greater damage to isolated cardiomyocytes following simulated ischemia/reperfusion. High-fat diet was also associated with changes in mitochondrial morphology and expression of some related proteins but not mitochondrial respiration or reactive oxygen species turnover rates. Proteomics, western blot and high-performance liquid chromatography techniques revealed that high-fat diet led to less cardiac oxidative stress, higher catalase expression and significant changes in expression of putative components of the mitochondrial permeability transition pore (mPTP). Inhibition of the mPTP conferred relatively more cardio-protection in the high-fat fed mice compared to normal diet. CONCLUSIONS: This study shows for the first time that high-fat diet, independent of obesity-induced co-morbidities, triggers changes in cardiac oxidative state, calcium handling and mitochondria which are likely to be responsible for increased vulnerability to cardiac insults.

Endothelium-specific insulin resistance leads to accelerated atherosclerosis in areas with disturbed flow patterns: a role for reactive oxygen species.

OBJECTIVE: Systemic insulin resistance is associated with a portfolio of risk factors for atherosclerosis development. We sought to determine whether insulin resistance specifically at the level of the endothelium promotes atherosclerosis and to examine the potential involvement of reactive oxygen species. METHODS: We cross-bred mice expressing a dominant negative mutant human insulin receptor specifically in the endothelium (ESMIRO) with ApoE(-/-) mice to examine the effect of endothelium-specific insulin resistance on atherosclerosis. RESULTS: ApoE(-/-)/ESMIRO mice had similar blood pressure, plasma lipids and whole-body glucose tolerance, but blunted endothelial insulin signalling, in comparison to ApoE(-/-) mice. Atherosclerosis was significantly increased in ApoE(-/-)/ESMIRO mice at the aortic sinus (226 ± 16 versus 149 ± 24 × 10(3) µm(2), P = 0.01) and lesser curvature of the aortic arch (12.4 ± 1.2% versus 9.4 ± 0.9%, P = 0.035). Relaxation to acetylcholine was blunted in aorta from ApoE(-/-)/ESMIRO mice (Emax 65 ± 41% versus 103 ± 6%, P = 0.02) and was restored by the superoxide dismutase mimetic MnTMPyP (Emax 112 ± 15% versus 65 ± 41%, P = 0.048). Basal generation of superoxide was increased 1.55 fold (P = 0.01) in endothelial cells from ApoE(-/-)/ESMIRO mice and was inhibited by the NADPH oxidase inhibitor gp91ds-tat (-12 ± 0.04%, P = 0.04), the NO synthase inhibitor L-NMMA (-8 ± 0.02%, P = 0.001) and the mitochondrial specific inhibitor rotenone (-23 ± 0.04%, P = 0.006). CONCLUSIONS: Insulin resistance specifically at the level of the endothelium leads to acceleration of atherosclerosis in areas with disturbed flow patterns such as the aortic sinus and the lesser curvature of the aorta. We have identified a potential role for increased generation of reactive oxygen species from multiple enzymatic sources in promoting atherosclerosis in this setting.

Low carbohydrate, high protein diet promotes atherosclerosis in apolipoprotein E/low-density lipoprotein receptor double knockout mice (apoE/LDLR(-/-)).

Although in apoE/LDLR(-/-) mice atherosclerotic plaques develop spontaneously, various atherogenic diets (e.g. Western diet) are frequently used to accelerate the disease in this model. The objective of this study was to compare the effects on atherosclerosis of Western diet and other types of high-fat, high cholesterol, hypertriglyceridemic diets with the effects of the low carbohydrate, high protein (LCHP) diet. 16-18 week old mice with pre-established atherosclerosis were assigned to experimental groups and fed for the next 10 weeks with control diet, margarine diet (margarine 7%), hypertrigliceridemic diet (fructose 62%), high-fat diet (Western diet), high cholesterol diet (egg yolk diet) or with LCHP diet. No differences in body weight were observed among experimental groups. Plasma cholesterol concentration was significantly increased in egg yolk diet- and LCHP diet-fed apoE/LDLR(-/-) mice as compared to other types of diets. Plasma concentration of triacylglycerols was significantly elevated in egg yolk diet- and LCHP diet-fed apoE/LDLR(-/-) mice. The area of atherosclerotic plaques in the aortic root was substantially increased in LCHP diet-fed mice as compared to other types of diets. Furthermore, in brachiocephalic arteries of LCHP diet-fed mice there was evidence of plaque rupture. In conclusion, the LCHP diet promoted atherosclerosis in apoE/LDLR(-/-) mice more intensively than classical Western diet and favored the development of unstable lesions.

[Periprocedural and late complications after percutaneous closure of patent foramen ovale: a single centre experience].

BACKGROUND: Patent foramen ovale (PFO) is a potential risk factor for ischaemic stroke in young individuals. An interventional method of secondary stroke prevention in PFO patients is its percutaneous closure. AIM: To assess safety and effectiveness (i.e. lack of residual shunt) of percutaneous PFO closure in patients with history of cryptogenic cerebrovascular event. METHODS: 149 patients (56 men/93 women), aged 39 ± 12 years, underwent percutaneous PFO closure. The implantation was performed under local anaesthesia, guided by trans-oesophageal echocardiography (TEE) and fluoroscopy. Follow-up trans-thoracic echocardiography (TTE) was performed at 1 month and follow-up TEE at 6-months. In cases of residual shunt, additional TEE was performed after ensuing 6 months. RESULTS: Effective PFO closure (no residual shunt) was achieved in 91.3% patients at 6 months and 95.3% patients at 12 months. In 2 patients transient atrial fibrillation was observed during the procedure. In 2 patients, a puncture site haematoma developed and in 1 patient superficial thrombophlebitis was noted. In 1 patient a small pericardial effusion was observed, which resolved at day 3 post-procedurally, after administration of non-steroidal anti-inflammatory drugs. CONCLUSIONS: Percutaneous PFO closure seems to be a safe procedure when performed in a centre with adequate expertise with regard to these procedures.

Genetic inactivation of IL-1 signaling enhances atherosclerotic plaque instability and reduces outward vessel remodeling in advanced atherosclerosis in mice.

Clinical complications of atherosclerosis arise primarily as a result of luminal obstruction due to atherosclerotic plaque growth, with inadequate outward vessel remodeling and plaque destabilization leading to rupture. IL-1 is a proinflammatory cytokine that promotes atherogenesis in animal models, but its role in plaque destabilization and outward vessel remodeling is unclear. The studies presented herein show that advanced atherosclerotic plaques in mice lacking both IL-1 receptor type I and apolipoprotein E (Il1r1⁻/⁻Apoe⁻/⁻ mice) unexpectedly exhibited multiple features of plaque instability as compared with those of Il1r1⁺/⁺Apoe⁻/⁻ mice. These features included reduced plaque SMC content and coverage, reduced plaque collagen content, and increased intraplaque hemorrhage. In addition, the brachiocephalic arteries of Il1r1⁻/⁻Apoe⁻/⁻ mice exhibited no difference in plaque size, but reduced vessel area and lumen size relative to controls, demonstrating a reduction in outward vessel remodeling. Interestingly, expression of MMP3 was dramatically reduced within the plaque and vessel wall of Il1r1⁻/⁻Apoe⁻/⁻ mice, and Mmp3⁻/⁻Apoe⁻/⁻ mice showed defective outward vessel remodeling compared with controls. In addition, MMP3 was required for IL-1-induced SMC invasion of Matrigel in vitro. Taken together, these results show that IL-1 signaling plays a surprising dual protective role in advanced atherosclerosis by promoting outward vessel remodeling and enhancing features of plaque stability, at least in part through MMP3-dependent mechanisms.

CD55 deficiency protects against atherosclerosis in ApoE-deficient mice via C3a modulation of lipid metabolism.

Atherosclerosis, the leading cause of death in the Western world, is driven by chronic inflammation within the artery wall. Elements of the complement cascade are implicated in the pathogenesis, because complement proteins and their activation products are found in the atherosclerotic plaque. We examined the role of CD55, a membrane inhibitor of the complement component 3 (C3) convertase, which converts C3 into C3a and C3b, in atherosclerosis. CD55-deficient (CD55(-/-)) mice were crossed onto the atherosclerosis-prone apolipoprotein E (apoE)-deficient (apoE(-/-)) background. High fat-fed male apoE(-/-)/CD55(-/-) mice were strongly protected from developing atherosclerosis compared with apoE(-/-) controls. Lipid profiling showed significantly lower levels of triglycerides, nonesterified fatty acids, and cholesterol in apoE(-/-)/CD55(-/-) mice than that in controls after high-fat feeding, whereas body fat in apoE(-/-)/CD55(-/-) mice content was increased. Plasma levels of C3 fell, whereas concentrations of C3adesArg (alias acylation stimulating protein; ASP), produced by serum carboxypeptidase N-mediated desargination of C3a, increased in nonfasted high fat-fed apoE(-/-)/CD55(-/-) mice, indicating complement activation. Thus, complement dysregulation in the absence of CD55 provoked increased C3adesArg production that, in turn, caused altered lipid handling, resulting in atheroprotection and increased adiposity. Interventions that target complement activation in adipose tissue should be explored as lipid-decreasing strategies.

Inhibition of rupture of established atherosclerotic plaques by treatment with apolipoprotein A-I.

AIMS: Plasma concentrations of high-density lipoprotein (HDL)-cholesterol correlate inversely with the incidence of myocardial infarction in humans. We investigated the effect of treatment with human apolipoprotein A-I (apoA-I), the principal protein of HDL, on plaque disruption in an animal model. METHODS AND RESULTS: Seventy apolipoprotein E knockout mice were induced to develop atherosclerotic lesions in the brachiocephalic artery by feeding a high-fat diet for 9 weeks. Mice then received twice-weekly treatment with human apoA-I (8 mg/kg) or vehicle, for 2 weeks. The incidence of acute plaque disruption was reduced by 65% in mice receiving apoA-I (P < 0.01). Plaques in treated mice had a more stable phenotype, with an increase in smooth muscle cell (SMC): macrophage ratio (P = 0.05), principally the consequence of an increase in the number of SMC in plaques. In the fibrous cap, there were reductions in matrix metalloproteinase-13 (-69%, P < 0.0001) and S100A4, a marker of SMC de-differentiation (-60%, P < 0.0001). These results indicate that 2 weeks of treatment with small amounts of human apoA-I produces more stable plaques in a mouse model. CONCLUSION: Treatment with apoA-I has the potential to stabilize plaques and prevent plaque rupture in humans.

A selective matrix metalloproteinase-12 inhibitor retards atherosclerotic plaque development in apolipoprotein E-knockout mice.

OBJECTIVE: Matrix metalloproteinase (MMP)-12 has been implicated in plaque progression and instability and is also amenable to selective inhibition. In this study, we investigated the influence of a greater than 10-fold selective synthetic MMP-12 inhibitor on plaque progression in the apolipoprotein E knockout mouse model of atherosclerosis. METHODS AND RESULTS: A phosphinic peptide (RXP470.1) that is a potent, selective murine MMP-12 inhibitor significantly reduced atherosclerotic plaque cross-sectional area by approximately 50% at 4 different vascular sites in male and female apolipoprotein E knockout mice fed a Western diet. Furthermore, RXP470.1 treatment resulted in less complex plaques with increased smooth muscle cell:macrophage ratio, less macrophage apoptosis, increased cap thickness, smaller necrotic cores, and decreased incidence of calcification. Additional in vitro and in vivo findings indicate that attenuated monocyte/macrophage invasion and reduced macrophage apoptosis probably underlie the beneficial effects observed on atherosclerotic plaque progression with MMP-12 inhibitor treatment. CONCLUSIONS: Our data demonstrate that a selective MMP-12 inhibitor retards atherosclerosis development and results in a more fibrous plaque phenotype in mice. Our study provides proof of principle to motivate translational work on MMP-12 inhibitor therapy in humans.

The fat-fed apolipoprotein E knockout mouse brachiocephalic artery in the study of atherosclerotic plaque rupture.

Atherosclerosis has been studied in animals for almost a century, yet the events leading up to the rupture of an atherosclerotic plaque (the underlying cause of the majority of fatal thrombosis formation) have only been studied in the past decade, due in part to the development of a mouse model of spontaneous plaque rupture. Apolipoprotein E knockout mice, when fed a high-fat diet, consistently develop lesions in the brachiocephalic artery that rupture at a known time point. It is therefore now possible to observe the development of lesions to elucidate the mechanisms behind the rupture of plaques. Critics argue that the model does not replicate the appearance of human atherosclerotic plaque ruptures. The purpose of this review is to highlight the reasons why we should be looking to the apolipoprotein E knockout mouse to further our understanding of plaque rupture.

Adeno-associated virus serotypes 7 and 8 outperform serotype 9 in expressing atheroprotective human apoE3 from mouse skeletal muscle.

Intramuscular injection of adeno-associated viral (AAV) vectors is potentially a safe, minimally invasive procedure for the long-term gene expression of circulating antiatherogenic proteins. Here, we compare secretion and atheroprotective effects of human apoE3 after injection of 3 pseudotyped AAV vectors (AAV2/7, AAV2/8, or AAV2/9), driven by the CMV enhancer/chicken ß-actin (CAG) promoter, into skeletal muscle of hyperlipidemic apolipoprotein E-deficient (apoE⁻/⁻) mice. Vector viabilities were verified by transducing cultured C2C12 mouse myotubes and assessing secretion of human apoE3 protein. Both hind limb tibialis anterior muscles of female C57BL/6 apoE⁻/⁻ mice, 2 months old and fed a high-fat diet, were each injected with 1 x 10¹° vector genomes of AAV vector. Identical noninjected mice served as controls; and blood was collected at weeks 0, 1, 2, 4, and 13. At termination (13 weeks), the brachiocephalic artery was excised; and after staining sections, plaque morphometry and fractional lipid content were quantified by computerized image analysis. Intramuscular injection of AAV2/7 and AAV2/8 vectors produced up to 2 µg human apoE3 per milliliter plasma, just below the threshold to reverse dyslipoproteinemia. AAV2/9 was notably less effective, mice having a 3-fold lower level of plasma apoE3 at 13 weeks and a 50% greater burden of atherosclerotic plaque lipid in their brachiocephalic arteries. We conclude that although vector refinement is needed to exploit fully apoE3 atheroprotective functions, AAV2/7 and AAV2/8 are promising gene transfer vectors for muscle-based expression of antiatherogenic circulating proteins.

MMP-7 mediates cleavage of N-cadherin and promotes smooth muscle cell apoptosis.

AIMS: Vascular smooth muscle cell (VSMC) apoptosis can lead to thinning of the fibrous cap and plaque instability. We previously showed that cell-cell contacts mediated by N-cadherin reduce VSMC apoptosis. This study aimed to determine whether matrix-degrading metalloproteinase (MMP)-dependent N-cadherin cleavage causes VSMC apoptosis. METHODS AND RESULTS: Induction of human VSMC apoptosis using different approaches, including 200 ng/mL Fas ligand (Fas-L) and culture in suspension, caused N-cadherin cleavage and resulted in the appearance of a C-terminal fragment of N-cadherin (approximately 35 kDa). Appearance of this fragment during apoptosis was inhibited by 47% with the broad-spectrum MMP inhibitor BB-94. We observed retarded cleavage of N-cadherin after treatment with Fas-L in aortic mouse VSMCs lacking MMP-7. Furthermore, VSMC apoptosis, measured by quantification of cleaved caspase-3, was 43% lower in MMP-7 knockout mouse VSMCs compared with wild-type VSMCs following treatment with Fas-L. Addition of recombinant active MMP-7 increased the amount of N-cadherin fragment by 82% and augmented apoptosis by 53%. The involvement of MMP-7 was corroborated using human cells, where a MMP-7 selective inhibitor reduced the amount of fragment formed by 51%. Importantly, we observed that treatment with Fas-L increased levels of active MMP-7 by 80%. Finally, we observed significantly increased cleavage of N-cadherin, MMP-7 activity, and apoptosis in human atherosclerotic plaques compared with control arteries, and a significant reduction in apoptosis in atherosclerotic plaques from MMP-7 knockout mice. CONCLUSION: This study demonstrates that MMP-7 is involved in the cleavage of N-cadherin and modulates VSMC apoptosis, and may therefore contribute to plaque development and rupture.