Lipoprotein Receptor Double-knockout Mice Research Articles

Enhanced Muscle Strength in Dyslipidemic Mice and Its Relation to Increased Capacity for Fatty Acid Oxidation.

Dyslipidemia is commonly linked to skeletal muscle dysfunction, accumulation of intramyocellular lipids, and insulin resistance. However, our previous research indicated that dyslipidemia in apolipoprotein E and low-density lipoprotein receptor double knock-out mice (ApoE/LDLR -/-) leads to improvement of exercise capacity. This study aimed to investigate in detail skeletal muscle function and metabolism in these dyslipidemic mice. We found that ApoE/LDLR -/- mice showed an increased grip strength as well as increased troponins, and Mhc2 levels in skeletal muscle. It was accompanied by the increased skeletal muscle mitochondria numbers (judged by increased citrate synthase activity) and elevated total adenine nucleotides pool. We noted increased triglycerides contents in skeletal muscles and increased serum free fatty acids (FFA) levels in ApoE/LDLR -/- mice. Importantly, Ranolazine mediated inhibition of FFA oxidation in ApoE/LDLR -/- mice led to the reduction of exercise capacity and total adenine nucleotides pool. Thus, this study demonstrated that increased capacity for fatty acid oxidation, an adaptive response to dyslipidemia leads to improved cellular energetics that translates to increased skeletal muscle strength and contributes to increased exercise capacity in ApoE/LDLR -/- mice.

Alcohol consumption combined with dietary low-carbohydrate/high-protein intake increased the left ventricular systolic dysfunction risk and lethal ventricular arrhythmia susceptibility in apolipoprotein E/low-density lipoprotein receptor double-knockout mice.

Alcohol abuse is positively associated with cardiovascular disease. Dietary low-carbohydrate/high-protein (LCHP) intake confers a greater mortality risk. Here, the impact of ethanol consumption in combination with dietary LCHP intake on left ventricular (LV) systolic function and lethal ventricular arrhythmia susceptibility were investigated in apolipoprotein E/low-density lipoprotein receptor double-knockout (AL) mice. The underlying mechanisms, cardiac sympathovagal balance, beta-adrenergic receptor (ADRB) levels, and gap junction channel protein connexin 43 (Cx43) expression, were examined. Male AL mice fed an LCHP diet with or without ethanol were bred for 16 weeks. Age-matched male AL and wild-type mice received standard chow diet and served as controls. The following were used to assess LV systolic function, lethal ventricular arrhythmia susceptibility, cardiac sympathovagal balance, Cx43 expression, and ADRB levels: The results demonstrated that ethanol consumption in combination with dietary LCHP intake worsened LCHP-induced LV systolic dysfunction in AL mice and enhanced their susceptibility in the ventricular arrhythmia-evoked test. There were concomitant increases in LV weight, LF/HF ratio shown by HRV, TH, ADRB1, ADRB2, and Cx43 expressions by LV fluorescence immunohistochemistry, and LV Cx43 messenger ribonucleic acid expression by PCR. In AL mice, alcohol consumption combined with dietary LCHP intake may thus promote a shift in cardiac sympathovagal balance toward sympathetic predominance, the increases in beta-adrenergic receptors (ADRB1 and ADRB2), and then affect the gap junction channel protein Cx43, which in turn could contribute to increased risks of LV systolic dysfunction and susceptibility to lethal ventricular arrhythmia.

Western diet in ApoE-LDLR double-deficient mouse model of atherosclerosis leads to hepatic steatosis, fibrosis, and tumorigenesis

Nonalcoholic fatty liver disease has been linked to cardiovascular diseases and atherosclerosis. The aim of the current study was to characterize the hepatic pathology leading to fibrosis and tumors in a murine model of atherosclerosis. Male apolipoprotein E/low-density lipoprotein receptor double-knockout mice (AL) mice were fed with a high fat and high cholesterol western diet for 35 weeks (AL mice on WD). Protein and mRNA analysis as well as micro-computed tomography (micro-CT) were performed to assess oxidative stress, liver damage, inflammation, fibrosis, signaling pathways, vascularization, and tumorigenesis. Controls were chosen to distinguish between genetically and dietary effects in steatohepatitis and associated tumorigenesis. Hepatic inflammation and dyslipidemia were increased in AL mice on WD compared with wild-type mice on WD. Uniquely, AL mice on WD showed a spontaneous development of tumors (30% of cases) and thickening of intrahepatic vessel walls. Functionally relevant underlying signaling pathways such as NF-κB, Stat3, JNK, and AKT were differentially regulated between AL and wild-type mice on WD. Micro-CT was capable of visualizing and quantitatively distinguishing tumor neovascularization from vascularization in non-neoplastic liver tissue. AL mice on WD diet represent a novel model combining atherosclerosis and nonalcoholic fatty liver disease. Signaling pathways of liver cell damage and compensatory liver regeneration in combination with enhanced inflammation appear to be crucial for the spontaneous development of tumors in AL mice on WD. Micro-CT represents a new and powerful technique for the ultrastructural and three-dimensional assessment of the vascular architecture of liver tumors.

The effects of diet on occlusive coronary artery atherosclerosis and myocardial infarction in scavenger receptor class B, type 1/low-density lipoprotein receptor double knockout mice.

Deficiency of the high-density lipoprotein receptor, scavenger receptor class B, type I (SR-BI), in apolipoprotein E knockout or hypomorphic mice, respectively, results in spontaneous or diet-inducible occlusive coronary artery (CA) atherosclerosis, myocardial infarction, and early death. Here, we examine effects of SR-BI deficiency on cardiovascular phenotypes in low-density lipoprotein receptor (LDLR) knockout mice fed different atherogenic diets. SR-BI/LDLR double knockout and control LDLR knockout mice were fed atherogenic diets containing different amounts of fat, cholesterol, and sodium cholate. Double knockout mice fed atherogenic diets high in cholesterol exhibited significantly reduced survival compared with LDLR knockout mice fed the same diets. In addition to increased diet-accelerated aortic sinus atherosclerosis, we observed significant diet-induced CA atherosclerosis in double knockout mice and diet-dependent accumulation of platelets in CA atherosclerotic plaques. This was accompanied by substantial myocardial fibrosis in double knockout mice fed high cholesterol diets. Atherogenic diet fed double knockout mice also exhibited higher circulating cytokine levels, monocytosis with increased proportions of Ly6C(hi) and Ly6C(int) monocytes, and higher adhesion molecule expression in CA endothelial cells compared with control LDLR knockout mice. Diet-accelerated atherosclerosis and occlusive, platelet-rich CA disease in SR-BI/LDLR double knockout mice is affected by amounts of cholesterol and cholate in atherogenic diets and is accompanied by increased expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in CAs and increased Ly6C(hi) and Ly6C(int) monocytes in circulation. The increased vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in CA endothelial cells in SR-BI-deficient mice likely explains their increased susceptibility to atherosclerosis in CAs.

Impaired endothelial function may be due to decreased aortic tetrahydrobiopterin, assessed by a new flow-mediated vasodilation in vivo in hypercholesterolemic/atherogenic mice

Tetrahydrobiopterin (BH4) is an important cofactor for endothelial nitric oxide synthase activity. The relationship between endothelial function in vivo and aortic BH4 level is not fully understood, however. In the present study, we aimed to clarify whether reduction of aortic BH4 levels contributes to endothelial dysfunction in vivo using spontaneously hyperlipidemic mice. To estimate endothelial function in vivo and in real-time state, we developed a flow-mediated vasodilation (FMV) method in mice, which measured changes in the diameter of the femoral artery in response to increased blood flow. C57BL/6 mice and apoE/low-density lipoprotein receptor double knock-out mice were fed a low-fat diet (LFD) or a high-fat diet (HFD) for 12 weeks from 6 weeks of age. HFD feeding impaired FMV in double knock-out mice, but not in C57BL/6 mice. Furthermore, HFD feeding reduced plasma NOx concentration and aortic BH4 level in double knock-out mice. Conversely, exogenous injection of BH4 (2 mg/kg) markedly increased aortic BH4 levels and restored endothelial function. In conclusion, we demonstrated that HFD feeding impaired nitric oxide-mediated endothelial function and reduced BH4 level in vivo, and that acute augmentation of aortic BH4 levels improved endothelial function. These findings indicate that BH4 is a critical determinant of nitric oxide-mediated endothelial function in hypercholesterolemia.

The putative role of leukotrienes in experimental atherogenesis

Since inflammation plays an important role in atherogenesis, during recent years it has become apparent that the 5-lipoxygenase (5-LO) pathway may take a significant part in the pathogenesis of atherosclerosis. These data raised the possibility that antileukotriene drugs may be an effective treatment regimen in atherosclerosis. This review describes the research performed on the apolipoprotein E/low-density lipoprotein receptor-double knockout mice as a model of atherosclerosis. The study has shown that 5-LO activating protein inhibitors and leukotriene receptor blockers decrease atherosclerosis in atherosclerotic mice. The article also discusses the importance of these findings for the future use in the clinic.

Defining and Defending Murine Models of Plaque Rupture

There is general agreement that atherosclerotic plaque rupture is a major clinical problem and that finding ways to prevent it would be greatly facilitated by an animal model of plaque rupture. The article by Schwartz et al1 seeks to clarify and define the terms used in descriptions of disrupted atherosclerotic plaques, and then to apply these to murine models in order to assess their similarities to and differences from human ruptured lesions. On the face of it this seems a reasonable thing to do, but it has some serious conceptual problems. The first is the issue of whether it is useful at all to provide definitions of disrupted plaques, especially when these are based on histopathology. The second concerns the transferability of such definitions from human to mouse. I would like also to deal with issues relating to reevaluation of existing mouse models of plaque rupture. What do we gain from framing scientific definitions of real-world phenomena? In the context of the ruptured plaque, Schwartz et al1 argue that this would provide an agreed upon and consistent framework that would make it easier for different labs to compare and contrast their data. There is obviously virtue in this as a general principle in science, but we have to consider the possibility that in the developing field of plaque rupture it may be premature. We do not know why plaques rupture, we do not know whether “plaque erosions” are simply the adjacent manifestations of ruptures that are out of the plane of the section, and we do not know whether “plaque fissures” arise through a different set of processes to plaque ruptures. In other words, this means that we do not know whether ruptures, erosions and fissures are ontogenically similar or different. We are therefore moving beyond the boundaries …

A model of neointima formation in the atherosclerotic carotid artery of mice

To establish a simple, reproducible model of neointima formation in mice with atherosclerotic vessels. Apolipoprotein E/low density lipoprotein receptor double knockout mice were fed an atherogenic diet. Carotid artery injury was induced by separate ligation of the external and internal carotid artery immediately distal to the bifurcation. Mice with normal vessels were used for comparison. Monocytes and macrophages were detected with immunohistochemistry using CD68 antibodies. The transcription factor nuclear factor kappa B was also detected by immunohistochemistry. Expression of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) was evaluated by real time polymerase chain reaction. Neointima and media areas were digitally measured and analyzed. Four weeks later carotid artery ligation had induced neointima formation proximal to the ligation site, apparent as a smooth muscle cell alpha-actin positive layer intimal to the lamina elastica interna. The shape and size of the lesions were reproducible. Nuclear localization of nuclear factor kappa B was found, and the expression of TNF-alpha and IL-1beta increased after injury. CD68 positive cells were detected in the lumen, in the media and in the neointima. We have established in atherosclerotic mice a reproducible model of arterial injury with inflammation and neointima formation.

Does Homocysteine Promote Atherosclerosis?

The hypothesis that homocysteine is atherogenic was proposed more than 30 years ago by Kilmer McCully,1 who observed vascular lesions in children with inherited disorders of methionine metabolism. Since McCully’s pioneering observations in 1969, a large number of epidemiological studies have confirmed that an elevation of total plasma homocysteine (tHcy) is prevalent in patients with stroke, myocardial infarction, peripheral vascular disease, and venous thrombosis.2 A significant association between hyperhomocysteinemia and clinical cardiovascular events has been observed in several large prospective studies, although a few prospective studies have failed to demonstrate this association.2 Nevertheless, hyperhomocysteinemia is now considered by many an independent risk factor for atherosclerotic vascular disease.3 See p 1470 Homocysteine is a thiol amino acid, but only a small fraction ( 15 μmol/L and may be caused by genetic defects, renal insufficiency, certain drugs, or nutritional deficiencies of folate, vitamin B6, or vitamin B12.5 Even a mild elevation of plasma tHcy to levels within the high-to-normal range (10 to 15 μmol/L) may increase cardiovascular risk.2 Because plasma tHcy can often be lowered by oral administration of folic acid or combinations of B vitamins, there is growing enthusiasm for treatment of hyperhomocysteinemia as a strategy for prevention of cardiovascular disease and …

Direct effect of an acyl-CoA:cholesterol acyltransferase inhibitor, F-1394, on atherosclerosis in apolipoprotein E and low density lipoprotein receptor double knockout mice.

The acyl-CoA:cholesterol acyltransferase (ACAT) enzyme is thought to be responsible for foam cell formation and the subsequent progression of atherosclerosis. The apolipoprotein E and low density lipoprotein receptor double knockout (apoE/LDLr-DKO) mouse is an animal model that develops severe hyperlipidaemia and atherosclerosis. Here we have examined the effect of oral administration of an ACAT inhibitor, F-1394, on atherosclerosis in apoE/LDLr-DKO mice fed a regular chow diet. In en face analysis, a dose of 10, 30, or 100 mg kg(-1) day(-1) F-1394 for 10 weeks reduced the extent of lesions visible in the aorta by 24, 28 and 38%, respectively, as detected by staining with oil red O, without affecting serum cholesterol level in these mice. At the highest dose 100 mg kg(-1) day(-1) of F-1394, the reduction was statistically significant. For quantitative analysis of the cellular and non-cellular components comprising the lesions at the aortic sinus, the effects of an oral dose of 100 mg kg(-1) day(-1) F-1394 for 15 weeks were studied. There was a significant reduction (31.9%) in the oil-red O-stained area in cross-sections of the aortic sinus. In addition, the neointimal area, as well as levels of ACAT-1 protein tended to be decreased (15.2 and 25.8%, respectively, not significant). However, the areas containing macrophages, smooth muscle cells, and collagen were not affected by F-1394. In vitro, F-1394 attenuated foam cell formation in mouse peritoneal macrophages. These results indicate that ACAT may be primarily responsible for lipid accumulation in atherosclerotic lesions, and that its inhibition diminishes the lipid deposition via a direct effect on macrophages in the arterial wall.