Gene Therapy in a Humanized Mouse Model of Familial Hypercholesterolemia Leads to Marked Regression of Atherosclerosis

Familial hypercholesterolemia (FH) is an autosomal codominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Homozygous FH patients (hoFH) have severe hypercholesterolemia leading to life threatening atherosclerosis in childhood and adolescence. Mice with germ line interruptions in the Ldlr and Apobec1 genes (Ldlr(-/-)Apobec1(-/-)) simulate metabolic and clinical aspects of hoFH, including atherogenesis on a chow diet.In this study, vectors based on adeno-associated virus 8 (AAV8) were used to deliver the gene for mouse Ldlr (mLDLR) to the livers of Ldlr(-/-)Apobec1(-/-) mice. A single intravenous injection of AAV8.mLDLR was found to significantly reduce plasma cholesterol and non-HDL cholesterol levels in chow-fed animals at doses as low as 3×10(9) genome copies/mouse. Whereas Ldlr(-/-)Apobec1(-/-) mice fed a western-type diet and injected with a control AAV8.null vector experienced a further 65% progression in atherosclerosis over 2 months compared with baseline mice, Ldlr(-/-)Apobec1(-/-) mice treated with AAV8.mLDLR realized an 87% regression of atherosclerotic lesions after 3 months compared to baseline mice. Immunohistochemical analyses revealed a substantial remodeling of atherosclerotic lesions.Collectively, the results presented herein suggest that AAV8-based gene therapy for FH may be feasible and support further development of this approach. The pre-clinical data from these studies will enable for the effective translation of gene therapy into the clinic for treatment of FH

Disruption of Nrf2, a Key Inducer of Antioxidant Defenses, Attenuates ApoE-Mediated Atherosclerosis in Mice

Background: Oxidative stress and inflammation are two critical factors that drive the formation of plaques in atherosclerosis. Nrf2 is a redox-sensitive transcription factor that upregulates a battery of antioxidative genes and cytoprotective enzymes that constitute the cellular response to oxidative stress. Our previous studies have shown that disruption of Nrf2 in mice (Nrf2-/-) causes increased susceptibility to pulmonary emphysema, asthma and sepsis due to increased oxidative stress and inflammation. Here we have tested the hypothesis that disruption of Nrf2 in mice causes increased atherosclerosis. Principal Findings: To investigate the role of Nrf2 in the development of atherosclerosis, we crossed Nrf2-/- mice with apoliporotein E-deficient (ApoE-/- mice. ApoE-/- and ApoE-/- Nrf2-/- mice were fed an atherogenic diet for 20 weeks, and plaque area was assessed in the aortas. Surprisingly, ApoE-/- Nrf2-/- mice exhibited significantly smaller plaque area than ApoE-/- controls (11.5% vs 29.5%). This decrease in plaque area observed in ApoE-/- Nrf2-/- mice was associated with a significant decrease in uptake of modified low density lipoproteins (AcLDL) by isolated macrophages from ApoE-/- Nrf2-/- mice. Furthermore, atherosclerotic plaques and isolated macrophages from ApoE-/- Nrf2-/- mice exhibited decreased expression of the scavenger receptor CD36. Conclusions: Nrf2 is pro-atherogenic in mice, despite its antioxidative function. The net pro-atherogenic effect of Nrf2 may be mediated via positive regulation of CD36. Our data demonstrates that the potential effects of Nrf2-targeted therapies on cardiovascular disease need to be investigated.9 page(s

Regression and stabilization of advanced murine atherosclerotic lesions: a comparison of LDL lowering and HDL raising gene transfer strategies

Both reductions in atherogenic lipoproteins and increases in high-density lipoprotein (HDL) levels may affect atherosclerosis regression. Here, the relative potential of low-density lipoprotein (LDL) lowering and HDL raising gene transfer strategies to induce regression of complex murine atherosclerotic lesions was directly compared. Male C57BL/6 LDL receptor (LDLr)−/− mice were fed an atherogenic diet (1.25% cholesterol and 10% coconut oil) to induce advanced atherosclerotic lesions. A baseline group was killed after 6 months and remaining mice were randomized into a control progression (Adnull or saline), an apolipoprotein (apo) A-I (AdA-I), an LDLr (AdLDLr), or a combined apo A-I/LDLr (AdA-I/AdLDLr) adenoviral gene transfer group and followed-up for another 12 weeks with continuation of the atherogenic diet. Gene transfer with AdLDLr decreased non-HDL cholesterol levels persistently by 95% (p < 0.001) compared with baseline. This drastic reduction of non-HDL cholesterol levels induced lesion regression by 28% (p < 0.001) in the aortic root and by 25% (p < 0.05) in the brachiocephalic artery at 12 weeks after transfer. Change in lesion size was accompanied by enhanced plaque stability, as evidenced by increased collagen content, reduced lesional macrophage content, a drastic reduction of necrotic core area, and decreased expression of inflammatory genes. Elevated HDL cholesterol following AdA-I transfer increased collagen content in lesions, but did not induce regression. Apo A-I gene transfer on top of AdLDLr transfer resulted in additive effects, particularly on inflammatory gene expression. In conclusion, drastic lipid lowering induced by a powerful gene transfer strategy leads to pronounced regression and stabilization of advanced murine atherosclerosis

Hyperlipidemia and Atherosclerotic Lesion Development in Ldlr-Deficient Mice on a Long-Term High-Fat Diet

BACKGROUND: Mice deficient in the LDL receptor (Ldlr(-/-) mice) have been widely used as a model to mimic human atherosclerosis. However, the time-course of atherosclerotic lesion development and distribution of lesions at specific time-points are yet to be established. The current study sought to determine the progression and distribution of lesions in Ldlr(-/-) mice. METHODOLOGY/PRINCIPAL FINDINGS: Ldlr-deficient mice fed regular chow or a high-fat (HF) diet for 0.5 to 12 months were analyzed for atherosclerotic lesions with en face and cross-sectional imaging. Mice displayed significant individual differences in lesion development when fed a chow diet, whereas those on a HF diet developed lesions in a time-dependent and site-selective manner. Specifically, mice subjected to the HF diet showed slight atherosclerotic lesions distributed exclusively in the aortic roots or innominate artery before 3 months. Lesions extended to the thoracic aorta at 6 months and abdominal aorta at 9 months. Cross-sectional analysis revealed the presence of advanced lesions in the aortic sinus after 3 months in the group on the HF diet and in the innominate artery at 6 to 9 months. The HF diet additionally resulted in increased total cholesterol, LDL, glucose, and HBA1c levels, along with the complication of obesity. CONCLUSIONS/SIGNIFICANCE: Ldlr-deficient mice on the HF diet tend to develop site-selective and size-specific atherosclerotic lesions over time. The current study should provide information on diet induction or drug intervention times and facilitate estimation of the appropriate locations of atherosclerotic lesions in Ldlr(-/-) mice

Phagocytosis of Cholesteryl Ester Is Amplified in Diabetic Mouse Macrophages and Is Largely Mediated by CD36 and SR-A

Type 2 diabetes (T2D) is associated with accelerated atherosclerosis, which accounts for approximately 75% of all diabetes-related deaths. Here we investigate the link between diabetes and macrophage cholesteryl ester accumulation. When diabetic (db/db) mice are given cholesteryl ester intraperitoneally (IP), peritoneal macrophages (PerMΦs) recovered from these animals showed a 58% increase in intracellular cholesteryl ester accumulation over PerMΦs from heterozygote control (db/+) mice. Notably, PerMΦ fluid-phase endocytosis and large particle phagocytosis was equivalent in db/+and db/db mice. However, IP administration of CD36 and SR-A blocking antibodies led to 37% and 25% reductions in cholesteryl ester accumulation in PerMΦ. Finally, in order to determine if these scavenger receptors (SRs) were part of the mechanism responsible for the increased accumulation of cholesteryl esters observed in the diabetic mouse macrophages, receptor expression was quantified by flow cytometry. Importantly, db/db PerMΦs showed a 43% increase in CD36 expression and an 80% increase in SR-A expression. Taken together, these data indicate that direct cholesteryl ester accumulation in mouse macrophages is mediated by CD36 and SR-A, and the magnitude of accumulation is increased in db/db macrophages due to increased scavenger receptor expression

LXR Deficiency Confers Increased Protection against Visceral Leishmania Infection in Mice

Leishmania spp. are protozoan single-cell parasites that are transmitted to humans by the bite of an infected sand fly, and can cause a wide spectrum of disease, ranging from self-healing skin lesions to potentially fatal systemic infections. Certain species of Leishmania that cause visceral (systemic) disease are a source of significant mortality worldwide. Here, we use a mouse model of visceral Leishmania infection to investigate the effect of a host gene called LXR. The LXRs have demonstrated important functions in both cholesterol regulation and inflammation. These processes, in turn, are closely related to lipid metabolism and the development of atherosclerosis. LXRs have also previously been shown to be involved in protection against other intracellular pathogens that infect macrophages, including certain bacteria. We demonstrate here that LXR is involved in susceptibility to Leishmania, as animals deficient in the LXR gene are much more resistant to infection with the parasite. We also demonstrate that macrophages lacking LXR kill parasites more readily, and make higher levels of nitric oxide (an antimicrobial mediator) and IL-1β (an inflammatory cytokine) in response to Leishmania infection. These results could have important implications in designing therapeutics against this deadly pathogen, as well as other intracellular microbial pathogens

Tumor Necrosis Factor Receptor Associated Factor 6 Is Not Required for Atherogenesis in Mice and Does Not Associate with Atherosclerosis in Humans

BACKGROUND: Tumor necrosis factor receptor-associated factors (TRAFs) are important signaling molecules for a variety of pro-atherogenic cytokines including CD40L, TNF alpha, and IL1beta. Several lines of evidence identified TRAF6 as a pro-inflammatory signaling molecule in vitro and we previously demonstrated overexpression of TRAF6 in human and Murine atherosclerotic plaques. This study investigated the role of TRAF6-deficiency in mice developing atherosclerosis, a chronic inflammatory disease. METHODOLOGY/PRINCIPAL FINDINGS: Lethally irradiated low density lipoprotein receptor-deficient mice (TRAF6(+/+)/LDLR(-/-)) were reconstituted with TRAF6-deficient fetal liver cells (FLC) and consumed high cholesterol diet for 18 weeks to assess the relevance of TRAF6 in hematopoietic cells for atherogenesis. Additionally, TRAF6(+/-)/LDLR(-/-) mice received TRAF6-deficient FLC to gain insight into the role of TRAF6 deficiency in resident cells. Surprisingly, atherosclerotic lesion size did not differ between the three groups in both aortic roots and abdominal aortas. Similarly, no significant differences in plaque composition could be observed as assessed by immunohistochemistry for macrophages, lipids, smooth muscle cells, T-cells, and collagen. In accord, in a small clinical study TRAF6/GAPDH total blood RNA ratios did not differ between groups of patients with stable coronary heart disease (0.034+/-0.0021, N = 178), acute coronary heart disease (0.029+/-0.0027, N = 70), and those without coronary heart disease (0.032+/-0.0016, N = 77) as assessed by angiography. CONCLUSION: Our study demonstrates that TRAF6 is not required for atherogenesis in mice and does not associate with clinical disease in humans. These data suggest that pro- and anti-inflammatory features of TRAF6 signaling outweigh each other in the context of atherosclerosis

Cholesterol Crystals Activate the NLRP3 Inflammasome in Human Macrophages: A Novel Link between Cholesterol Metabolism and Inflammation

Chronic inflammation of the arterial wall is a key element in the pathogenesis of atherosclerosis, yet the factors that trigger and sustain the inflammation remain elusive. Inflammasomes are cytoplasmic caspase-1-activating protein complexes that promote maturation and secretion of the proinflammatory cytokines interleukin(IL)-1beta and IL-18. The most intensively studied inflammasome, NLRP3 inflammasome, is activated by diverse substances, including crystalline and particulate materials. As cholesterol crystals are abundant in atherosclerotic lesions, and IL-1beta has been linked to atherogenesis, we explored the possibility that cholesterol crystals promote inflammation by activating the inflammasome pathway.Here we show that human macrophages avidly phagocytose cholesterol crystals and store the ingested cholesterol as cholesteryl esters. Importantly, cholesterol crystals induced dose-dependent secretion of mature IL-1beta from human monocytes and macrophages. The cholesterol crystal-induced secretion of IL-1beta was caspase-1-dependent, suggesting the involvement of an inflammasome-mediated pathway. Silencing of the NLRP3 receptor, the crucial component in NLRP3 inflammasome, completely abolished crystal-induced IL-1beta secretion, thus identifying NLRP3 inflammasome as the cholesterol crystal-responsive element in macrophages. The crystals were shown to induce leakage of the lysosomal protease cathepsin B into the cytoplasm and inhibition of this enzyme reduced cholesterol crystal-induced IL-1beta secretion, suggesting that NLRP3 inflammasome activation occurred via lysosomal destabilization.The cholesterol crystal-induced inflammasome activation in macrophages may represent an important link between cholesterol metabolism and inflammation in atherosclerotic lesions

Trypanosoma cruzi Epimastigotes Are Able to Store and Mobilize High Amounts of Cholesterol in Reservosome Lipid Inclusions

Reservosomes are lysosome-related organelles found in Trypanosoma cruzi epimastigotes. They represent the last step in epimastigote endocytic route, accumulating a set of proteins and enzymes related to protein digestion and lipid metabolism. The reservosome matrix contains planar membranes, vesicles and lipid inclusions. Some of the latter may assume rectangular or sword-shaped crystalloid forms surrounded by a phospholipid monolayer, resembling the cholesterol crystals in foam cells.Using Nile Red fluorimetry and fluorescence microscopy, as well as electron microscopy, we have established a direct correlation between serum concentration in culture medium and the presence of crystalloid lipid inclusions. Starting from a reservosome purified fraction, we have developed a fractionation protocol to isolate lipid inclusions. Gas-chromatography mass-spectrometry (GC-MS) analysis revealed that lipid inclusions are composed mainly by cholesterol and cholesterol esters. Moreover, when the parasites with crystalloid lipid-loaded reservosomes were maintained in serum free medium for 48 hours the inclusions disappeared almost completely, including the sword shaped ones.Taken together, our results suggest that epimastigote forms of T. cruzi store high amounts of neutral lipids from extracellular medium, mostly cholesterol or cholesterol esters inside reservosomes. Interestingly, the parasites are able to disassemble the reservosome cholesterol crystalloid inclusions when submitted to serum starvation

Assessing the Effects of Responsible Leadership and Ethical Conflict on Behavioral Intention

[[abstract]]This study develops a research model that elaborates how responsible leadership and ethical conflict influence employees from the perspectives of role theory and attachment theory. Its empirical results reveal that turnover intention indirectly relates to ethical conflict and responsible leadership via the mediating mechanisms of organizational identification and organizational uncertainty. At the same time, helping intention indirectly relates to ethical conflict and responsible leadership only through organizational identification. Finally, the managerial implications for international business and research limitations based on the empirical results are discussed.[[notice]]補正完