Low-density Lipoprotein Receptor Knockout Mice Research Articles

Apolipoprotein J ablation triggers atherosclerosis in a chow diet-fed LDLR-KO mice

Background and Aims: Apolipoprotein J (apoJ) is an extracellular chaperone involved in the clearance of misfolded proteins, which is partly transported in blood bound to lipoproteins and is highly expressed in atherosclerotic arteries. However, the role of apoJ in atherogenesis is unclear. This study analyzes the effect of the genetic ablation of apoJ in LDL receptor knockout (LDLR-KO) mice.

Low-sodium diet increases insulin resistance and favors fat accumulation in the liver of LDL receptor knockout mice

Low-sodium diet increases insulin resistance and favors fat accumulation in the liver of LDL receptor knockout mice

High-phosphorus diets reduce aortic lesions and cardiomyocyte size and modify lipid metabolism in Ldl receptor knockout mice

The consumption of phosphorus in Western populations largely exceeds the recommended intake, while vitamin D supply is often insufficient. Both situations are linked to an increased cardiovascular risk. A 17-week two-factorial study with Ldl receptor-/- mice was conducted to investigate the cardiovascular impact of dietary phosphorus [adequate (0.3%; P0.3) vs. high (1.5%; P1.5)] in combination with a low (50 IU/kg; D50) or adequate vitamin D diet (1000 IU/kg; D1000). The data demonstrate that mice fed the P1.5 vs. P0.3 diets developed smaller vascular lesions (p = 0.013) and cardiac hypotrophy (p = 0.011), which were accompanied by diminished IGF1 and insulin signalling activity in their hearts. Vitamin D showed no independent effect on atherogenesis and heart morphology. Feeding P1.5 vs. P0.3 diets resulted in markedly reduced serum triacylglycerols (p < 0.0001) and cholesterol (p < 0.0001), higher faecal lipid excretion (p < 0.0001) and a reduced mRNA abundance of hepatic sterol exporters and lipoprotein receptors. Minor hypocholesterolaemic and hypotriglyceridaemic effects were also found in mice fed the D1000 vs. D50 diets (p = 0.048, p = 0.026). To conclude, a high phosphorus intake strongly affected the formation of vascular lesions, cardiac morphology, and lipid metabolism, although these changes are not indicative of an increased cardiovascular risk.

Effects of Oat Fiber Intervention on Cognitive Behavior in LDLR-/- Mice Modeling Atherosclerosis by Targeting the Microbiome-Gut-Brain Axis.

It is known that cardiovascular disease can result in cognitive impairment. However, whether oat fiber improves cognitive behavior through a cardiovascular-related mechanism remains unclear. The present work was aimed to elucidate the potential of oat fiber on cognitive behavior by targeting the neuroinflammation signal and microbiome-gut-brain axis in a mouse model of atherosclerosis. Male low-density lipoprotein receptor knock-out (LDLR-/-) mice were treated with a high fat/cholesterol diet without or with 0.8% oat fiber for 14 weeks. Behavioral tests indicated that LDLR-/- mice exhibited a significant cognitive impairment; however, oat fiber can improve cognitive behavior by reducing latency to the platform and increasing the number of crossing and swimming distance in the target quadrant. Oat fiber can inhibit Aβ plaque processing in both the cortex and hippocampus via decreasing the relative protein expression of GFAP and IBα1. Notably, oat fiber inhibited the nod-like receptor family pyrin domain-containing 3 inflammasome activation and blocked the toll-like receptor 4 signal pathway in both the cortex and hippocampus, accompanied by a reduction of circulating serum lipopolysaccharide. In addition, oat fiber raised the expressions of short-chain fatty acid (SCFA) receptors and tight junction proteins (zonula occludens-1 and occludin) and improved intestinal microbiota diversity via increasing the contents of gut metabolites SCFAs. In summary, the present study provided experimental evidence that dietary oat fiber retarded the progression of cognitive impairment in a mouse model of atherosclerosis. Mechanistically, the neuroprotective potential was related to oat fiber and its metabolites SCFAs on the diversity and abundance of gut microbiota that produced anti-inflammatory metabolites, leading to repressed neuroinflammation and reduced gut permeability through the microbiome-gut-brain axis.

Therapeutic effects of herbal formula Huangqisan on metabolic disorders via SREBF1, SCD1 and AMPK signaling pathway

ObjectiveMetabolic syndrome is a complex medical condition that has become an alarming epidemic, but an effective therapy for this disease is still lacking. The use of the herbal formula Huangqisan (HQS) to treat diabetes is documented in the Chinese medical literature as early as 1117 A.D.; however, its therapeutic effects and underlying mechanisms remain elusive. MethodsTo investigate the beneficial effects of HQS on metabolic disorders, high-fat diet-induced obesity (DIO), leptin receptor dysfunction (db/db) and low-density lipoprotein receptor-knockout (LDLR−/−) mice were used. Obese mice were treated with either HQS or vehicle. Blood, liver tissue, white fat tissue and brown adipose tissue were harvested at the end of the treatment. Metabolic disease-related parameters were evaluated to test effects of HQS against diabetes, obesity and hyperlipidemia. Aortic arches from LDLR−/− mice were analyzed to investigate the effects of HQS on atherosclerosis. RNA-sequence, quantitative real-time polymerase chain reaction and Western blot were performed to investigate the mechanisms of HQS against metabolic disorder. ResultsHQS lowered body weight, fasting blood glucose and serum lipid levels and improved glucose tolerance and insulin sensitivity in DIO mice and db/db mice (P < 0.05). HQS also blocked atherosclerotic plaque formation in LDLR−/− mice. HQS suppressed de novo lipid synthesis by reducing the expression of messenger RNA for sterol regulatory element-binding factor 1, stearyl coenzyme A desaturase 1 and fatty acid synthase, and enhancing adenosine 5′-monophosphate-activated protein kinase signaling in both in vivo and in vitro experiments, indicating potential mechanisms for HQS’s activity against diabetes. ConclusionHQS is effective for reversing metabolic disorder and has the potential to be used as therapy for metabolic syndrome.

High Cholesterol Diet Exacerbates Blood-Brain Barrier Disruption in LDLr-/- Mice: Impact on Cognitive Function.

Background:Evidence has revealed an association between familial hypercholesterolemia and cognitive impairment. In this regard, a connection between cognitive deficits and hippocampal blood-brain barrier (BBB) breakdown was found in low-density lipoprotein receptor knockout mice (LDLr–/–), a mouse model of familial hypercholesterolemia.Objective:Herein we investigated the impact of a hypercholesterolemic diet on cognition and BBB function in C57BL/6 wild-type and LDLr–/–mice.Methods:Animals were fed with normal or high cholesterol diets for 30 days. Thus, wild-type and LDLr–/–mice were submitted to memory paradigms. Additionally, BBB integrity was evaluated in the mice’s prefrontal cortices and hippocampi.Results:A tenfold elevation in plasma cholesterol levels of LDLr–/–mice was observed after a hypercholesterolemic diet, while in wild-type mice, the hypercholesterolemic diet exposure increased plasma cholesterol levels only moderately and did not induce cognitive impairment. LDLr–/–mice presented memory impairment regardless of the diet. We observed BBB disruption as an increased permeability to sodium fluorescein in the prefrontal cortices and hippocampi and a decrease on hippocampal claudin-5 and occludin mRNA levels in both wild-type and LDLr–/–mice treated with a hypercholesterolemic diet. The LDLr–/–mice fed with a regular diet already presented BBB dysfunction. The BBB-increased leakage in the hippocampi of LDLr–/–mice was related to high microvessel content and intense astrogliosis, which did not occur in the control mice.Conclusion:Therefore, LDLr–/–mice seem to be more susceptible to cognitive impairments and BBB damage induced by exposure to a high cholesterol diet. Finally, BBB disruption appears to be a relevant event in hypercholesterolemia-induced brain alterations.

Anti-atherogenic properties of Kgengwe (Citrullus lanatus) seed powder in low-density lipoprotein receptor knockout mice are mediated through beneficial alterations in inflammatory pathways.

Kgengwe fruits are commonly consumed in sub-Saharan countries. Recent reports indicated low coronary artery disease rates in those regions. To investigate anti-atherogenic properties and potential mechanisms of action of Kgengwe seed powder (KSP), male low-density lipoprotein receptor knockout (LDL-r-KO) mice were fed with an atherogenic diet supplemented with (treated, n = 10) or without (controls, n = 10) 10% (w/w) KSP for 20weeks. Proximate analysis revealed that KSP contained 38% fibre and 15% lipids. KSP supplementation was not associated with significant changes in body weight gain rate, food intake, and plasma lipid levels. However, the average atherosclerotic lesion size in the aortic roots in the KSP-treated group was 58% smaller than that in the control group (0.26 vs 0.11 mm2, p< 0.05). This strong anti-atherogenic effect was associated with significant increases in the average plasma levels of certain cytokines such as IL-10 (6 vs 13 pg/mL, p< 0.05), GM-CSF (0.1 vs 0.2 pg/mL, p< 0.05), and EPO (7 vs 16 pg/mL, p< 0.05) along with reductions in the average levels of plasma MCP-1 (19 vs 14 pg/mL, p< 0.05) and MIP-2 (28 vs 13 pg/mL, p< 0.05). Except for relatively high levels of saturated fatty acids, KSP possesses balanced nutrient compositions with strong anti-atherogenic properties, which may be mediated through alterations in inflammatory pathways. Additional studies warrant confirmation and mechanism(s) of action of such effects. Novelty: Kgengwe seeds prevent atherogenesis in LDL-r-KO mice. Kgengwe seeds increase circulating levels of IL-10 and EPO. No reduction in plasma total cholesterol levels.

Ablation of Galectin-12 Inhibits Atherosclerosis through Enhancement of M2 Macrophage Polarization

The formation of foam cells, which are macrophages that have engulfed oxidized low-density lipoprotein (OxLDL), constitutes the first stage in the development of atherosclerosis. Previously, we found that knocking down galectin-12, a negative regulator of lipolysis, leads to reduced secretion of monocyte chemoattractant protein-1 (MCP-1), a chemokine that plays an important role in atherosclerosis. This prompted us to study the role of galectin-12 in atherosclerosis. With that aim, we examined foam cell formation in Gal12‒/‒ murine macrophages exposed to OxLDL and acetylated LDL (AcLDL). Then, we generated an LDL receptor and galectin-12 double knockout (DKO) mice and studied the effect of galectin-12 on macrophage function and atherosclerosis. Lastly, we evaluated the role of galectin-12 in human THP-1 macrophages using a doxycycline-inducible conditional knockdown system. Galectin-12 knockout significantly inhibited foam cell formation in murine macrophages through the downregulation of cluster of differentiation 36 (CD36), and the upregulation of ATP Binding Cassette Subfamily A Member 1 (ABCA1), ATP Binding Cassette Subfamily G Member 1 (ABCG1), and scavenger receptor class B type 1 (SRB1). Consistent with this, galectin-12 knockdown inhibited foam cell formation in human macrophages. In addition, the ablation of galectin-12 promoted M2 macrophage polarization in human and murine macrophages as evidenced by the upregulation of the M2 marker genes, CD206 and CD163, and downregulation of the M1 cytokines, tumor necrosis factor α (TNF- α), interleukin-6 (IL-6), and MCP-1. Moreover, the ablation of galectin-12 decreased atherosclerosis formation in DKO mice. Based on these results, we propose galectin-12 as a potential therapeutic target for atherosclerosis.

North American Wild Rice-Attenuated Hyperglycemia in High-Fat-Induced Obese Mice: Involvement of AMP-Activated Protein Kinase.

Previous studies indicated that North American wild rice (WIR) reduced atherosclerosis and vascular inflammation in low-density lipoprotein receptor knockout mice. The effects of WIR on hyperglycemia in diabetic animal models have not been documented. The present study aims to determine the impact of WIR on glucose metabolism in high-fat (HF)-induced diabetic mice and a key modulator. Male C57 BL/J6 mice were treated with a control diet and a HF diet supplemented with 26% (weight/weight, a substitute for carbohydrates in the diet) of WIR or white rice (WHR) (n = 8/group) for 11 weeks. HF + WHR diet significantly increased fasting plasma glucose, cholesterol, triglycerides, insulin, insulin resistance, monocyte adhesion, and the levels of relevant inflammatory mediators (tumor necrotic factor-α, plasminogen activator inhibitor-1, and monocyte chemotactic protein-1) in mice compared to the control diet (p < 0.01). HF + WIR significantly reduced HF diet-induced metabolic and inflammatory changes compared to the HF + WHR diet (p < 0.01). Metabolomics analysis indicated that an array of metabolites related to glucose metabolism was significantly more abundant in WIR than in WHR, including adenosine 5'-monophosphate (AMP), a potent agonist for AMP-activated protein kinase or AMPK. WIR normalized HF diet-induced reduction in the abundance of phospho-AMPKα in skeletal muscle, liver, and adipose tissue from the mice. The findings for the first time demonstrated that WIR decreased HF diet-induced hyperglycemia in mice compared to WHR. The metabolic benefits of WIR may result, at least in part, from the activation of AMPKα in insulin-sensitive tissue in the mice.

A chalcone derivative, 1m-6, exhibits atheroprotective effects by increasing cholesterol efflux and reducing inflammation-induced endothelial dysfunction.

Atherosclerosis, resulting from lipid dysregulation and vascular inflammation, causes atherosclerotic cardiovascular disease (ASCVD), which contributes to morbidity and mortality worldwide. Chalcone and its derivatives possess beneficial properties, including anti-inflammatory, antioxidant and antitumour activity with unknown cardioprotective effects. We aimed to develop an effective chalcone derivative with antiatherogenic potential. Human THP-1 cells and HUVECs were used as in vitro models. Western blots and real-time PCRs were performed to quantify protein, mRNA and miRNA expressions. The cholesterol efflux capacity was assayed by 3 H labelling of cholesterol. LDL receptor knockout (Ldlr-/- ) mice fed a high-fat diet were used as an in vivo atherogenesis model. Haematoxylin and eosin and oil red O staining were used to analyse plaque formation. Using ATP-binding cassette transporter A1 (ABCA1) expression we identified the chalcone derivative, 1m-6, which enhances ABCA1 expression and promotes cholesterol efflux in THP-1 macrophages. Moreover, 1m-6 stabilizes ABCA1 mRNA and suppresses the expression of potential ABCA1-regulating miRNAs through nuclear factor erythroid 2-related factor 2 (Nrf2)/haem oxygenase-1 (HO-1) signalling. Additionally, 1m-6 significantly inhibits TNF-α-induced expression of adhesion molecules, vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), plus production of proinflammatory cytokines via inhibition of JAK/STAT3 activation and the modulation of Nrf2/HO-1 signalling in HUVECs. In atherosclerosis-prone mice, 1m-6 significantly reduces lipid accumulation and atherosclerotic plaque formation. Our study demonstrates that 1m-6 produces promising atheroprotective effects by enhancing cholesterol efflux and suppressing inflammation-induced endothelial dysfunction, which opens a new avenue for treating ASCVD. This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.23/issuetoc.

The Impact of Dietary Supplementation of Whole Foods and Polyphenols on Atherosclerosis.

The purpose of this review is to highlight current research on the benefits of supplementation with foods with a diverse polyphenol composition, including fruits, vegetables, nuts, grains, oils, spices, and teas in blunting atherosclerosis. We searched PubMed for publications utilizing whole food or polyphenols prepared from whole foods in Apolipoprotein E (ApoE) or Low-Density Lipoprotein Receptor (LDLR) knockout mice, and identified 73 studies in which plaque was measured. The majority of the studies reported a reduction in plaque. Nine interventions showed no effect, while three using Agaricus blazei mushroom, HYJA-ri-4 rice variety, and safrole-2’, 3’-oxide (SFO) increased plaque. The mechanisms by which atherosclerosis was reduced include improved lipid profile, antioxidant status, and cholesterol clearance, and reduced inflammation. Importantly, not all dietary interventions that reduce plaque showed an improvement in lipid profile. Additionally, we found that, out of 73 studies, only 9 used female mice and only 6 compared both sexes. Only one study compared the two models (LDLR vs. ApoE), showing that the treatment worked in one but not the other. Not all supplementations work in both male and female animals, suggesting that increasing the variety of foods with different polyphenol compositions may be more effective in mitigating atherosclerosis.

Effects of moderate aerobic exercise on thoracic aortic remodeling of female LDL-receptor knockout ovariectomized mice

Menopause is a major factor involved in dyslipidemia increasing the risk of atherosclerosis which may be reversed by a routine of aerobic physical activity. Thus, this study aimed to analyze the effects of aerobic training on the thoracic aorta of female LDL-receptor knockout mice submitted to estrogen deprivation. Fifteen genetically modified female mice, knockout for the low-density lipoprotein receptor (LDL-Knockout group) were used as experimental groups and fifteen wild female mice (C57BL/6 J) were used as control groups. Animals were divided as (n = 5/per group): sedentary control (SC); sedentary control ovariectomized (SCO); trained control ovariectomized (TCO); LDL-Knockout sedentary (KS); LDL-Knockout sedentary ovariectomized (KOS); and LDL-Knockout trained ovariectomized (KOT). Immunohistochemical techniques for TIMP-1 and metalloproteinases 2 and 9 were used to evaluate thoracic aorta remodeling. Picrosirius stain was used to highlight the collagen fibers. Verhoff-Van Gienson was used for the quantitative analyses of elastic lamellae. Our results demonstrate a positive remodeling promoted by physical exercise in ovariectomized and dyslipidemic animals. However, further studies are needed including the evaluation of inflammatory markers present in dyslipidemia.

Subcutaneous Administration of Apolipoprotein J-Derived Mimetic Peptide d-[113-122]apoJ Improves LDL and HDL Function and Prevents Atherosclerosis in LDLR-KO Mice.

Mimetic peptides are potential therapeutic agents for atherosclerosis. d-[113–122]apolipoprotein (apo) J (d-[113–122]apoJ) is a 10-residue peptide that is predicted to form a class G* amphipathic helix 6 from apoJ; it shows anti-inflammatory and anti-atherogenic properties. In the present study, we analyzed the effect of d-[113–122]apoJ in low-density lipoprotein receptor knockout mice(LDLR-KO) on the development of atherosclerosis and lipoprotein function. Fifteen-week-old female LDLR-KO mice fed an atherogenic Western-type diet were treated for eight weeks with d-[113–122]apoJ peptide, a scrambled peptide, or vehicle. Peptides were administered subcutaneously three days per week (200 µg in 100 µL of saline). After euthanasia, blood and hearts were collected and the aortic arch was analyzed for the presence of atherosclerotic lesions. Lipoproteins were isolated and their composition and functionality were studied. The extent of atherosclerotic lesions was 43% lower with d-[113–122]apoJ treatment than with the vehicle or scramble. The lipid profile was similar between groups, but the high-density lipoprotein (HDL) of d-[113–122]apoJ-treated mice had a higher antioxidant capacity and increased ability to promote cholesterol efflux than the control group. In addition, low-density lipoprotein (LDL) from d-[113–122]apoJ-treated mice was more resistant to induced aggregation and presented lower electronegativity than in mice treated with d-[113–122]apoJ. Our results demonstrate that the d-[113–122]apoJ peptide prevents the extent of atherosclerotic lesions, which could be partially explained by the improvement of lipoprotein functionality.

Deletion of fibroblast activation protein provides atheroprotection.

Fibroblast activation protein (FAP) is upregulated at sites of tissue remodelling including chronic arthritis, solid tumours, and fibrotic hearts. It has also been associated with human coronary atherosclerotic plaques. Yet, the causal role of FAP in atherosclerosis remains unknown. To investigate the cause-effect relationship of endogenous FAP in atherogenesis, we assessed the effects of constitutive Fap deletion on plaque formation in atherosclerosis-prone apolipoprotein E (Apoe) or low-density lipoprotein receptor (Ldlr) knockout mice. Using en face analyses of thoraco-abdominal aortae and aortic sinus cross-sections, we demonstrate that Fap deficiency decreased plaque formation in two atherosclerotic mouse models (-46% in Apoe and -34% in Ldlr knockout mice). As a surrogate of plaque vulnerability fibrous cap thickness was used; it was increased in Fap-deficient mice, whereas Sirius red staining demonstrated that total collagen content remained unchanged. Using polarized light, atherosclerotic lesions from Fap-deficient mice displayed increased FAP targets in terms of enhanced collagen birefringence in plaques and increased pre-COL3A1 expression in aortic lysates. Analyses of the Stockholm Atherosclerosis Gene Expression data revealed that FAP expression was increased in human atherosclerotic compared to non-atherosclerotic arteries. Our data provide causal evidence that constitutive Fap deletion decreases progression of experimental atherosclerosis and increases features of plaque stability with decreased collagen breakdown. Thus, inhibition of FAP expression or activity may not only represent a promising therapeutic target in atherosclerosis but appears safe at the experimental level for FAP-targeted cancer therapies.

Modulation of SIRT1-mediated signaling cascades in the liver contributes to the amelioration of nonalcoholic steatohepatitis in high fat fed middle-aged LDL receptor knockout mice by dihydromyricetin

Dihydromyricetin (DMY) is the most abundant flavonoid in Ampelopsis grossedentata possessing many pharmacological activities. But less is known about its protective effect against nonalcoholic steatohepatitis (NASH) in the context of metabolic syndrome. The present study is aimed to evaluate the pharmacological effects of DMY on NASH induced by feeding a high fat diet to 12-mo-old male LDLr−/− mice for 12 weeks and its molecular mode of action. At the end of the experiment, the blood samples and liver tissues of mice were collected for analysis. The results showed that DMY treatment improved the steatosis, inflammation and fibrosis which are three main aspects of NASH and some of the metabolic basal characteristics. The underlying mechanisms include regulating key regulators of lipid metabolism, oxidative stress, inflammation and fibrosis. Notably, DMY treatment increased hepatic sirtuin 1 (SIRT1) activity and protein expression. DMY also enhanced deacetylation of liver kinase B1 (LKB1) and nuclear transcription factor kappa B (NF-kB). Furthermore, in cultured hepatocyte cells, the benefits of DMY on lipid accumulation, oxidative stress and inflammation as well as the above related genes were abrogated in hepatocytes transfected with SIRT1 siRNA. These results suggest that modulation of SIRT1-mediated signaling cascades contributes to the amelioration of NASH by DMY and DMY may serve as a potentialtherapeuticcandidate for human NASH.

In Search for Genes Related to Atherosclerosis and Dyslipidemia Using Animal Models.

Atherosclerosis is a multifactorial chronic disease that affects large arteries and may lead to fatal consequences. According to current understanding, inflammation and lipid accumulation are the two key mechanisms of atherosclerosis development. Animal models based on genetically modified mice have been developed to investigate these aspects. One such model is low-density lipoprotein (LDL) receptor knockout (KO) mice (ldlr−/−), which are characterized by a moderate increase of plasma LDL cholesterol levels. Another widely used genetically modified mouse strain is apolipoprotein-E KO mice (apoE−/−) that lacks the primary lipoprotein required for the uptake of lipoproteins through the hepatic receptors, leading to even greater plasma cholesterol increase than in ldlr−/− mice. These and other animal models allowed for conducting genetic studies, such as genome-wide association studies, microarrays, and genotyping methods, which helped identifying more than 100 mutations that contribute to atherosclerosis development. However, translation of the results obtained in animal models for human situations was slow and challenging. At the same time, genetic studies conducted in humans were limited by low sample sizes and high heterogeneity in predictive subclinical phenotypes. In this review, we summarize the current knowledge on the use of KO mice for identification of genes implicated in atherosclerosis and provide a list of genes involved in atherosclerosis-associated inflammatory pathways and their brief characteristics. Moreover, we discuss the approaches for candidate gene search in animals and humans and discuss the progress made in the field of epigenetic studies that appear to be promising for identification of novel biomarkers and therapeutic targets.

Characterization of a mouse model to study the relationship between apical periodontitis and atherosclerosis.

First, to determine the feasibility of using the low-density lipoprotein receptor knockout (LDLR KO) mouse model to study apical periodontitis (AP). Secondly, to investigate the causal relationship between AP and atherosclerosis. It was hypothesized that it would be feasible to induce AP and atherosclerosis in LDLR KO mice and find a difference in atherosclerosis between AP and Sham groups. Using a published methodology, AP was induced in LDLR KO mice by exposing the dental pulp of the four first molars (Tx). Shams received only anaesthesia. Mice were fed a high fat, Western-type diet (WTD), to induce atherosclerosis. At 16weeks, mice were euthanized and aortas collected to measure atherosclerosis lesion burden (oil red O staining). Periapical lesions were validated using micro-CT and histology. Systemic inflammation was measured using a cytokine array. Both groups developed a similar degree of atherosclerosis (mean lesion area 7.46±0.44% in the Tx group compared with 7.65±0.46%, in the Sham group, P=0.77), and a similar degree of inflammation. Periapical lesions (PALs) in all four molars were only identified in a small subset of Tx mice. A novel mouse model, which combines AP and CVD, was created. This model allows investigation of the relationship between the two diseases, whilst avoiding other potential common confounders. Although no difference in the degree of atherosclerosis was found between the groups, more studies in which the number of periapical lesions, changes in systemic inflammation and the degree of atherosclerosis are correlated are necessary to ultimately determine the impact of AP on CVD.

Development of Fractalkine-Targeted Nanofibers that Localize to Sites of Arterial Injury.

Atherosclerosis is the leading cause of death and disability around the world, with current treatments limited by neointimal hyperplasia. Our goal was to synthesize, characterize, and evaluate an injectable, targeted nanomaterial that will specifically bind to the site of arterial injury. Our target protein is fractalkine, a chemokine involved in both neointimal hyperplasia and atherosclerosis. We showed increased fractalkine staining in rat carotid arteries 24 h following arterial injury and in the aorta of low-density lipoprotein receptor knockout (LDLR-/-) mice fed a high-fat diet for 16 weeks. Three peptide amphiphiles (PAs) were synthesized: fractalkine-targeted, scrambled, and a backbone PA. PAs were ≥90% pure on liquid chromatography/mass spectrometry (LCMS) and showed nanofiber formation on transmission electron microscopy (TEM). Rats systemically injected with fractalkine-targeted nanofibers 24 h after carotid artery balloon injury exhibited a 4.2-fold increase in fluorescence in the injured artery compared to the scrambled nanofiber (p < 0.001). No localization was observed in the non-injured artery or with the backbone nanofiber. Fluorescence of the fractalkine-targeted nanofiber increased in a dose dependent manner and was observed for up to 48 h. These data demonstrate the presence of fractalkine after arterial injury and the localization of our fractalkine-targeted nanofiber to the site of injury and serve as the foundation to develop this technology further.

Cow's milk polar lipids reduce atherogenic lipoprotein cholesterol, modulate gut microbiota and attenuate atherosclerosis development in LDL-receptor knockout mice fed a Western-type diet

Milk sphingomyelin (SM), a polar lipid (PL) component of milk fat globule membranes, is protective against dyslipidemia. However, it is unclear whether ingestion of milk PLs protect against atherosclerosis. To determine this, male LDLr−/− mice (age 6 weeks) were fed ad libitum either a high-fat, added-cholesterol diet (CTL; 45% kcal from fat, 0.2% cholesterol by weight; n=15) or the same diet supplemented with 1% milk PL (1% MPL; n=15) or 2% milk PL (2% MPL; n=15) added by weight from butter serum. After 14 weeks on diets, mice fed 2% MPL had significantly lower serum cholesterol (−51%) compared to CTL (P<.01), with dose-dependent effects in lowering VLDL- and LDL-cholesterol. Mice fed 2% MPL displayed lower inflammatory markers in the serum, liver, adipose and aorta. Notably, milk PLs reduced atherosclerosis development in both the thoracic aorta and the aortic root, with 2% MPL-fed mice having significantly lower neutral lipid plaque size by 59% (P<.01) and 71% (P<.02) compared to CTL, respectively. Additionally, the 2% MPL-fed mice had greater relative abundance of Bacteroidetes, Actinobacteria and Bifidobacterium, and lower Firmicutes in cecal feces compared to CTL. Milk PL feeding resulted in significantly different microbial communities as demonstrated by altered beta diversity indices. In summary, 2% MPL strongly reduced atherogenic lipoprotein cholesterol, modulated gut microbiota, lowered inflammation and attenuated atherosclerosis development. Thus, milk PL content may be important to consider when choosing dairy products as foods for cardiovascular disease prevention.

5-Aminolevulinic Acid Attenuates Atherosclerotic Plaque Progression in Low-Density Lipoprotein Receptor-Deficient Mice by Heme Oxygenase-1 Induction.

Background: Recently, 5-aminolevulinic acid (ALA) has been reported to modulate inflammatory development via an antioxidant effect. Hence, the aim of this study was to determine the anti-atherosclerotic effect of ALA.Methods and Results: Low-density lipoprotein (LDL) receptor knockout mice were fed the following diets for 24 weeks: normal diet (n=6); 1.25% cholesterol diet (high-cholesterol diet, HCD; n=7); HCD+ALA (46 mg/kg/day; n=10); and HCD+ezetimibe (5 mg/kg/day; n=10). At 40 weeks, HCD+ALA had reduced LDL cholesterol (320±68 vs. 379±49 mg/dL), triglyceride (141±44 vs. 195±49 mg/dL) and oxidized LDL (380±40 vs. 422±64 pg/mL) compared with HCD only. En face lesion area for the entire aortic surface was significantly smaller in mice that received HCD+ALA than in mice that received only HCD (32±5% vs. 39±4%, P<0.05). ALA intake exogenously increased tissue heme oxygenase-1 (HO-1) level in plaque composite tissue of the carotid arterial wall compared with HCD only (18±8 vs. 12±3 pg/μL, P<0.05), and HO-1-positive plaque showed modest NADPH oxidase 4 expression.Conclusions: ALA intake induces exogenous production of HO-1 at plaque sites, and improves lipid profiles and attenuation of atherosclerotic plaque progression in vivo.