Bart J. M. van Vlijmen

Induction of atherosclerotic plaque rupture in apolipoprotein E-/- mice after adenovirus-mediated transfer of p53

Background—The presence of the tumor-suppressor gene p53 in advanced atherosclerotic plaques and the sensitivity to p53-induced cell death of smooth muscle cells isolated from these plaques have fueled speculation about the role of p53 in lesion destabilization and plaque rupture. In this study, we describe a strategy to promote (thrombotic) rupture of preexisting atherosclerotic lesions using p53-induced lesion remodeling. Methods and Results—Carotid atherogenesis was initiated in apolipoprotein E knockout mice by placement of a perivascular silastic collar. The resulting plaques were incubated transluminally with recombinant adenovirus carrying either a p53 or &bgr;-galactosidase (lacZ) transgene. p53 transfection was restricted to the smooth muscle cell-rich cap of the plaque and led to an increase in cap cell apoptosis 1 day after transfer. p53 overexpression resulted in a marked decrease in the cellular and extracellular content of the cap, reflected by a markedly reduced cap/intima ratio (0.21±0.04 versus 0.46±0.03, P <0.001). The latter is a characteristic feature of plaque vulnerability to rupture, and whereas spontaneous rupture of p53-treated lesions was rare, it was found in 40% of cases after treatment with the vasopressor compound phenylephrine (P =0.003). Conclusions—We have demonstrated a potential role of p53-induced remodeling in atherosclerotic plaque destabilization. Being the first example of inducible rupture at a predefined location, this model offers a unique opportunity to delineate the processes that precede rupture and to evaluate plaque-stabilizing therapies.

Transgenic mouse models to study the role of APOE in hyperlipidemia and atherosclerosis

Transgenic technologies have provided a series of very useful mouse models to study hyperlipidemia and atherosclerosis. Normally, mice carry cholesterol mainly in the high density lipoprotein (HDL) sized lipoproteins, and have low density lipoprotein (LDL) and very low density lipoprotein (VLDL) cholesterol levels. These low LDL and VLDL levels are due to the very rapid metabolism of remnant clearance in mice, which hamper metabolic studies. In addition, due to the lack of atherogenic lipoproteins, mice will not readily develop atherosclerosis. This situation has changed completely, because to date, most known genes in lipoprotein metabolism have been used in transgenesis to obtain mice in which genes have been silenced or overexpressed. These experiments have yielded many mouse strains with high plasma lipid levels and a greater susceptibility for developing atherosclerosis. One of the most widely used strains are knock-out mice deficient for apoE, which is one of the central players in VLDL metabolism. Subsequently, a wide variety of other transgenic studies involving APOE have been performed elucidating the role of apoE and apoE mutants in lipolysis, remnant clearance, cellular cholesterol efflux and atherogenesis. In addition, the APOE mouse models are excellent tools for the development of gene therapy for hyperlipidemias.

The B domain of coagulation factor VIII interacts with the asialoglycoprotein receptor

Summary.  Background: Coagulation factor VIII (FVIII) is a heavily glycosylated heterodimeric plasma protein that consists of a heavy (domains A1‐A2‐B) and light chain (domains A3‐C1‐C2). It has been well established that the clearance of FVIII from the circulation involves mechanisms that are sensitive to the low‐density lipoprotein receptor (LDLR) family antagonist receptor‐associated protein (RAP), including LDLR‐related protein. Because FVIII clearance in the presence of a bolus injection of RAP still occurs fairly efficient, also RAP‐independent mechanisms are likely to be involved. Objectives: In the present study, we investigated the interaction of FVIII with the endocytic lectin asialoglycoprotein receptor (ASGPR) and the physiological relevance thereof. Methods and results: Surface plasmon resonance studies demonstrated that FVIII dose‐dependently bound to ASGPR with high affinity (Kd ≈ 2 nm). FVIII subunits were different in that only the heavy chain displayed high‐affinity binding to ASGPR. Studies employing a FVIII variant that lacks the B domain revealed that FVIII‐ASGPR complex assembly is driven by structure elements within the B domain of the heavy chain. The FVIII heavy chain‐ASGPR interaction required calcium ions and was inhibited by soluble d‐galactose. Furthermore, deglycosylation of the FVIII heavy chain by endoglycosidase F completely abrogated the interaction with ASGPR. In clearance experiments in mice, the FVIII mean residence time was prolonged by the ASGPR‐antagonist asialo‐orosomucoid (ASOR). Conclusions: We conclude that asparagine‐linked oligosaccharide structures of the FVIII B domain recognize the carbohydrate recognition domains of ASGPR and that an ASOR‐sensitive mechanism, most likely ASGPR, contributes to the catabolism of coagulation FVIII in vivo.

Macrophage LRP1 contributes to the clearance of von Willebrand factor.

The relationship between low-density lipoprotein receptor-related protein-1 (LRP1) and von Willebrand factor (VWF) has remained elusive for years. Indeed, despite a reported absence of interaction between both proteins, liver-specific deletion of LRP1 results in increased VWF levels. To investigate this discrepancy, we used mice with a macrophage-specific deficiency of LRP1 (macLRP1(-)) because we previously found that macrophages dominate VWF clearance. Basal VWF levels were increased in macLRP1(-) mice compared with control mice (1.6 ± 0.4 vs 1.0 ± 0.4 U/mL). Clearance experiments revealed that half-life of human VWF was significantly increased in macLRP1(-) mice. Ubiquitous blocking of LRP1 or additional lipoprotein receptors by overexpressing receptor-associated protein in macLRP1(-) mice did not result in further rise of VWF levels (0.1 ± 0.2 U/mL), in contrast to macLRP1(+) mice (rise in VWF, 0.8 ± 0.4 U/mL). This points to macLRP1 being the only lipoprotein receptor regulating VWF levels. When testing the mechanism(s) involved, we observed that VWF-coated beads adhered efficiently to LRP1 but only when exposed to shear forces exceeding 2.5 dyne/cm(2), implying the existence of shear stress-dependent interactions. Furthermore, a mechanism involving β2-integrins that binds both VWF and LRP1 also is implicated because inhibition of β2-integrins led to increased VWF levels in control (rise, 0.19 ± 0.16 U/mL) but not in macLRP1(-) mice (0.08 ± 0.15 U/mL).

Genetic inflammatory factors predict restenosis after percutaneous coronary interventions

Background— Restenosis is a negative effect of percutaneous coronary intervention (PCI). No clinical factors are available that allow good risk stratification. However, evidence exists that genetic factors are important in the restenotic process as well as in the process of inflammation, a pivotal factor in restenosis. Association studies have identified genes that may predispose to restenosis, but confirmation by large prospective studies is lacking. Our aim was to identify polymorphisms and haplotypes in genes involved in inflammatory pathways that predispose to restenosis. Methods and Results— The GENetic DEterminants of Restenosis (GENDER) project is a multicenter prospective study, including 3104 consecutive patients after successful PCI. Forty-eight polymorphisms in 34 genes in pathways possibly involved in the inflammatory process were analyzed. The 16Gly variant of the β2-adrenergic receptor gave an increased risk of target vessel revascularization (TVR). The rare alleles of the CD14 gene (−260T/T), colony-stimulating factor 2 gene (117Thr/Thr), and eotaxin gene (−1328A/A) were associated with decreased risk of TVR. However, through the use of multiple testing corrections with permutation analysis, the probability of finding 4 significant markers by chance was 12%. Conclusions— Polymorphisms in 4 genes considered involved in the inflammatory reaction showed an association with TVR after PCI. Our results may contribute to the unraveling of the restenotic process. Given the explorative nature of this analysis, our results need to be replicated in other studies.

Key role of platelet procoagulant activity in tissue factor-and collagen-dependent thrombus formation in arterioles and venules in vivo differential sensitivity to thrombin inhibition.

Objective: Blood coagulation and platelet activation are mutually dependent processes, but contribute differently to venous and arterial thrombosis. We investigated the interplay of these processes in vivo in a mouse model of arteriolar and venular thrombus formation.

Dual PPARα/γ Agonist Tesaglitazar Reduces Atherosclerosis in Insulin-Resistant and Hypercholesterolemic ApoE*3Leiden Mice

Objective—We investigated whether the dual PPAR&agr;/&ggr; agonist tesaglitazar has anti-atherogenic effects in ApoE*3Leiden mice with reduced insulin sensitivity. Methods and Results—ApoE*3Leiden transgenic mice were fed a high-fat (HF) insulin-resistance–inducing diet. One group received a high-cholesterol (HC) supplement (1% wt/wt; HC group). A second group received the same HC supplement along with tesaglitazar (T) 0.5 &mgr;mol/kg diet (T group). A third (control) group received a low-cholesterol (LC) supplement (0.1% wt/wt; LC group). Tesaglitazar decreased plasma cholesterol by 20% compared with the HC group; cholesterol levels were similar in the T and LC groups. Compared with the HC group, tesaglitazar caused a 92% reduction in atherosclerosis, whereas a 56% reduction was seen in the cholesterol-matched LC group. Furthermore, tesaglitazar treatment significantly reduced lesion number beyond that expected from cholesterol lowering and induced a shift to less severe lesions. Concomitantly, tesaglitazar reduced macrophage-rich and collagen areas. In addition, tesaglitazar reduced inflammatory markers, including plasma SAA levels, the number of adhering monocytes, and nuclear factor &kgr;B-activity in the vessel wall. Conclusions—Tesaglitazar has anti-atherosclerotic effects in the mouse model that go beyond plasma cholesterol lowering, possibly caused by a combination of altered lipoprotein profiles and anti-inflammatory vascular effects.

Macrophage p53 controls macrophage death in atherosclerotic lesions of apolipoprotein E deficient mice

The cellular composition of atherosclerotic lesions is determined by many factors including cell infiltration, proliferation and cell death. Tumor suppressor gene p53 has been shown to regulate both cell proliferation and cell death in many cell types. In the present study, we investigated the role of macrophage p53 in the pathogenesis of early and advanced atherosclerosis. Using the Cre-loxP system we found that absence of macrophage p53 (p53(del)) strongly reduces apoptosis of macrophages both in early and advanced atherosclerotic lesions (-59% and -37%, respectively). Consequently, in advanced atherosclerosis, reduced apoptosis upon absence of macrophage p53, coincided with increased acellular necrotic core formation (+96%), increased macrophage content (+24%), and reduced cholesterol cleft accumulation (-41%). Proliferation was not affected by the absence of macrophage p53 in both early and advanced atherosclerosis. However, these significant changes in lesional cell death did not affect total lesion area in both early and advanced atherosclerosis, neither in the aortic root nor in the aortic arch and thoracic aorta in ApoE-deficient mice. Our data demonstrate that macrophage p53 is an important regulator of macrophage apoptosis, thereby preventing necrotic death of lesional macrophages. The regulation of this cell death balance directly affects lesion composition.

Hepatic lipid accumulation, altered very low density lipoprotein formation and apolipoprotein E deposition in apolipoprotein E3-Leiden transgenic mice

BACKGROUND/AIM Apolipoprotein (apo) E-deficiency leads to hepatic steatosis and impaired Very Low Density Lipoprotein (VLDL)-triglyceride production rates in mice. A mutant apoE isoform, apoE3-Leiden, is associated with a dominantly inherited form of dysbetalipoproteinemia in humans. The aim of this study was to evaluate the effects of APOE*3-Leiden expression on hepatic lipid content, VLDL formation and liver morphology in mice. METHODS Comparison of lipid parameters and liver morphology in mouse strains with different expression of the APOE*3-Leiden transgene with and without co-expression of human APOCI. RESULTS Hepatic triglyceride content was increased to maximally 233% of control values, depending on hepatic APOE*3-Leiden expression. Hepatic secretion of VLDL-associated triglycerides was impaired (-20%) in high-expressing transgenics, with a concomitant increase from 1.6 to 8.1 of the apoB48/ apoB100 ratio in newly-formed VLDL. Hepatocytes of the transgenic mice contained characteristic inclusions, up to 20 microm in diameter, in numbers dependent on APOE*3-Leiden expression and independent of APOCI expression. These inclusions contained material positively reacting with antihuman apoE antibodies. Immunogold-labeling confirmed the presence of apoE3-Leiden within these inclusions and also revealed the presence of the mutant protein on sinusoidal membranes, in multivesicular bodies and in peroxisomes, i.e., a distribution pattern similar to that of endogenous apoE in rodents. Nascent VLDL particles associated with the Golgi apparatus were also labeled. CONCLUSION This study has demonstrated that introduction of human apoE3-Leiden in mice, in addition to its reported effects on lipolysis and lipoprotein clearance, leads to hepatic deposition of the mutant apolipoprotein, development of fatty liver and to altered hepatic VLDL secretion. The latter findings are consistent with a role of apoE in the regulation of intrahepatic lipid metabolism.

Macrophage retinoblastoma deficiency leads to enhanced atherosclerosis development in ApoE-deficient mice

The cellular composition of an atherosclerotic lesion is determined by cell infiltration, proliferation, and apoptosis. The tumor suppressor gene retinoblastoma (Rb) has been shown to regulate both cell proliferation and cell death in many cell types. To study the role of macrophage Rb in the development of atherosclerosis, we used apoE‐deficient mice with a macrophage‐restricted deletion of Rb (Rbdel mice) and control littermates (Rbfl mice). After 12 wk feeding a cholesterol‐rich diet, the Rbdel mice showed a 51% increase in atherosclerotic lesion area with a 39% increase in the relative number of advanced lesions. Atherosclerotic lesions showed a 13% decrease in relative macrophage area and a 46% increase in relative smooth muscle cell area, reflecting the more advanced state of the lesions. The increase in atherosclerosis was independent of in vitro macrophage modified lipoprotein uptake or cytokine production. Whereas macrophage‐restricted Rb deletion did not affect lesional macrophage apoptosis, a clear 2.6‐fold increase in lesional macrophage proliferation was observed. These studies demonstrate that macrophage Rb is a suppressing factor in the progression of atherosclerosis by reducing macrophage proliferation.—Boesten, L. S. M., Zadelaar, A. S. M., van Nieuwkoop, A., Hu, L., Jonkers, J., van de Water, B., Gijbels, M. J. J. van der Made, I., de Winther, M. P. J., Havekes, L. M., van Vlijmen, B. J. M. Macrophage retinoblastoma deficiency leads to enhanced atherosclerosis development in ApoE‐deficient mice. FASEB J. 20, E18–E26 (2006)