LDLR-null Mice Research Articles

Abstract 2139: Galectin-3 Plays A Critical Intracellular Role In Macrophages By Promoting The Repair, Removal, And Replacement Of Damaged Lysosomes And Protecting Against Atherosclerosis

Galectin-3 (Gal3) is a β-galactoside-binding lectin predominantly known as a secreted inflammatory biomarker in cardiovascular disease, with significantly elevated circulating levels in patients with atherosclerosis and myocardial infarction. However, the molecular mechanisms of Galectin-3 action and whether its intracellular role contributes to atherogenesis remain unclear. Using several databases of bulk and single cell RNAseq, we first show that Gal3 transcripts are upregulated during plaque progression with particular abundance in the myeloid/macrophage lineage and foamy macrophages. Furthermore, Gal3 transcripts correlate significantly with increases in a network of lysosomal genes, suggesting a possible link between macrophage Gal3 and lysosomal function. Indeed, instigation of lysosome membrane damage in primary macrophages via cholesterol crystals and LLOMe triggers robust recruitment of Gal3 to lysosomes by sensing exposed carbohydrate moieties of proteins including Lamp1. Gal3 recruitment initiates a two-pronged lysosomal recovery program composed of either lysosomal repair involving the ESCRT complex or lysosomal removal and replacement involving autophagy/lysophagy and TFEB-mediated lysosomal biogenesis. Gal3-KO macrophages corroborate these findings displaying blunted ESCRT recruitment, diminished autophagy and TFEB activation, and resultant accumulation of damaged lysosomes. Gal3-deficiency also results in enhanced apoptosis and inflammasome/IL-1β activation upon triggering of lysosomal stress in macrophages. These findings are recapitulated in vivo, where Gal3-null bone marrow transplanted in atherogenic LDLR-null mice yields increased lesion size as well as altered plaque composition with macrophage accumulation, apoptosis, and necrotic core formation, characteristic features of the advanced plaque. Taken together, our data implicate unique and previously unrecognized protective function for intracellular Galectin-3 in macrophages and atherosclerosis which are distinct from its traditional role as an inflammatory biomarker in cardiovascular disease.

Abstract 522: Macrophage Galectin 3 Protects Against Atherosclerosis By Promoting Repair, Removal And Replacement Of Damaged Lysosomes

Understanding the derangements in cellular processes that underlie macrophage dysfunction is critical to the development of future therapeutics. The autophagy-lysosome system in macrophages plays critical role in the degradation and recycling of long-lived/damaged intracellular material. Atherosclerotic plaque macrophages develop an impairment in autophagy and lysosomes which is a contributing factor to plaque progression. Thus, one can surmise that harnessing programs of lysosomal repair and lysosomal biogenesis in macrophages can have atheroprotective effects. A class of important regulators of the autophagy-lysosomal system are cytosolic lectins. When the lysosome membrane becomes damaged, Galectin-3 (Gal3) recognizes the damage and recruits autophagic receptors and delivers the cargo to autophagosomes. Additionally, Gal3 may recruit the ESCRT complex and facilitate the repair and restoration of lysosomes. We hypothesized that Gal3 provides a protective role in atherogenic macrophages, by repairing or removing damaged lysosomes, thus preventing the relevant deleterious signaling pathways such as inflammasome activation and apoptosis. We utilized Gal3-KO macrophages and mice to study the role of Gal3 in atherosclerosis. First, we show that Gal3-KO macrophages are highly sensitive to lysosome membrane damaging agents including atherogenic oxLDL and cholesterol crystals, exhibiting decreased autophagy and protein aggregate clearance compared to the WT controls. Furthermore, the recruitment of lysosome repair mechanisms involving the ESCRT complex are blunted. Such blunting of critical repair mechanisms results in increased macrophage apoptosis and inflammasome/IL-1β activation. These findings are also recapitulated in vivo, where bone marrow transplants of atherogenic LDLR-null mice with Gal3-deficient bone marrow results in increased plaque size and altered plaque composition with significant macrophage accumulation, apoptosis, and necrotic core formation, characteristic features of advanced plaques. Overall, our data demonstrate important roles for Gal3 in macrophage lysosome repair mechanisms which are a critical response against atherosclerosis.

Lack of Mitogen-activated Kinase Phosphatase-5 in Macrophages Protects Ldlr-null Mice against Atherogenesis

Lack of Mitogen-activated Kinase Phosphatase-5 in Macrophages Protects Ldlr-null Mice against Atherogenesis

Cardiovascular protection in females linked to estrogen-dependent inhibition of arterial stiffening and macrophage MMP12.

Arterial stiffening is a consequence of aging and a cholesterol-independent risk factor for cardiovascular disease (CVD). Arterial stiffening and CVD show a sex bias, with men more susceptible than premenopausal women. How arterial stiffness and sex interact at a molecular level to confer risk of CVD is not well understood. Here, we used the sexual dimorphism in LDLR-null mice to show that the protective effect of female sex on atherosclerosis is linked to reduced aortic stiffness and reduced expression of matrix metalloproteinase-12 (MMP12) by lesional macrophages. Deletion of MMP12 in LDLR-null mice attenuated the male sex bias for both arterial stiffness and atherosclerosis, and these effects occurred despite high serum cholesterol. Mechanistically, we found that oxidized LDL stimulates secretion of MMP12 in human as well as mouse macrophages. Estrogen antagonizes this effect by downregulating MMP12 expression. Our data support cholesterol-independent causal relationships between estrogen, oxidized LDL-induced secretion of macrophage MMP12, and arterial stiffness that protect against atherosclerosis in females and emphasize that reduced MMP12 functionality can confer atheroprotection to males.

Inflammasome Activation Aggravates Cutaneous Xanthomatosis and Atherosclerosis in ACAT1 (Acyl-CoA Cholesterol Acyltransferase 1) Deficiency in Bone Marrow

Objective- ACAT1 (Acyl-CoA cholesterol acyltransferase 1) esterifies cellular free cholesterol, thereby converting macrophages to cholesteryl ester-laden foam cells in atherosclerotic lesions and cutaneous xanthoma. Paradoxically, however, loss of ACAT1 in bone marrow causes the aggravation of atherosclerosis and the development of severe cutaneous xanthoma in hyperlipidemic mice. Recently, it has been reported that cholesterol crystals activate NLRP3 (NACHT, LRR [leucine-rich repeats], and PYD [pyrin domain] domain-containing protein 3) inflammasomes, thereby contributing to the development of atherosclerosis. The present study aimed to clarify the role of NLRP3 inflammasomes in the worsening of atherosclerosis and cutaneous xanthoma induced by ACAT1 deficiency. Approach and Results- Ldlr-null mice were transplanted with bone marrow from WT (wild type) mice and mice lacking ACAT1, NLRP3, or both. After the 4 types of mice were fed high-cholesterol diets, we compared their atherosclerosis and skin lesions. The mice transplanted with Acat1-null bone marrow developed severe cutaneous xanthoma, which was filled with numerous macrophages and cholesterol clefts and had markedly increased expression of inflammatory cytokines, and increased atherosclerosis. Loss of NLRP3 completely reversed the cutaneous xanthoma, whereas it improved the atherosclerosis only partially. Acat1-null peritoneal macrophages showed enhanced expression of CHOP (C/EBP [CCAAT/enhancer binding protein] homologous protein) and TNF-α (tumor necrosis factor-α) but no evidence of inflammasome activation, after treatment with acetylated LDL (low-density lipoprotein). Conclusions- Elimination of ACAT1 in bone marrow-derived cells aggravates cutaneous xanthoma and atherosclerosis. The development of cutaneous xanthoma is induced mainly via the NLRP3 inflammasome activation.

Role of enterocyte stearoyl-Co-A desaturase-1 in LDLR-null mice

After crossing floxed stearoyl-CoA desaturase-1 (Scd1fl/fl) mice with LDL receptor-null (ldlr-/-) mice, and then Villin Cre (VilCre) mice, enterocyte Scd1 expression in Scd1fl/fl/ldlr-/-/VilCre mice was reduced 70%. On Western diet (WD), Scd1fl/fl/ldlr-/- mice gained more weight than Scd1fl/fl/ldlr-/-/VilCre mice (P < 0.0023). On WD, jejunum levels of lysophosphatidylcholine (LysoPC) 18:1 and lysophosphatidic acid (LPA) 18:1 were significantly less in Scd1fl/fl/ldlr-/-/VilCre compared with Scd1fl/fl/ldlr-/- mice (P < 0.0004 and P < 0.026, respectively). On WD, Scd1fl/fl/ldlr-/-/VilCre mice compared with Scd1fl/fl/ldlr-/- mice had lower protein levels of lipopolysaccharide-binding protein (LBP), cluster of differentiation 14 (CD14), toll-like receptor 4 (TLR4), and myeloid differentiation factor-88 (MyD88) in enterocytes and plasma, and less dyslipidemia and systemic inflammation. Adding a concentrate of tomatoes transgenic for the apoA-I mimetic peptide 6F (Tg6F) to WD resulted in reduced enterocyte protein levels of LBP, CD14, TLR4, and MyD88 in Scd1fl/fl/ldlr-/- mice similar to that seen in Scd1fl/fl/ldlr-/-/VilCre mice. Adding LysoPC 18:1 to WD did not reverse the effects of enterocyte Scd1 knockdown. Adding LysoPC 18:1 (but not LysoPC 18:0) to chow induced jejunum Scd1 expression and increased dyslipidemia and plasma serum amyloid A and interleukin 6 levels in Scd1fl/fl/ldlr-/- mice, but not in Scd1fl/fl/ldlr-/-/VilCre mice. We conclude that enterocyte Scd1 is partially responsible for LysoPC 18:1- and WD-induced dyslipidemia and inflammation in ldlr-/- mice.

Loss of Rictor in Monocyte/Macrophages Suppresses Their Proliferation and Viability Reducing Atherosclerosis in LDLR Null Mice.

Rictor is an essential component of mammalian target of rapamycin (mTOR) complex 2 (mTORC2), a conserved serine/threonine kinase that may play a role in cell proliferation, survival and innate or adaptive immune responses. Genetic loss of Rictor inactivates mTORC2, which directly activates Akt S473 phosphorylation and promotes pro-survival cell signaling and proliferation. To study the role of mTORC2 signaling in monocytes and macrophages, we generated mice with myeloid lineage-specific Rictor deletion (MRictor-/-). These MRictor-/- mice exhibited dramatic reductions of white blood cells, B-cells, T-cells, and monocytes but had similar levels of neutrophils compared to control Rictor flox-flox (Rictorfl/fl) mice. MRictor-/- bone marrow monocytes and peritoneal macrophages expressed reduced levels of mTORC2 signaling and decreased Akt S473 phosphorylation, and they displayed significantly less proliferation than control Rictorfl/fl cells. In addition, blood monocytes and peritoneal macrophages isolated from MRictor-/- mice were significantly more sensitive to pro-apoptotic stimuli. In response to LPS, MRictor-/- macrophages exhibited the M1 phenotype with higher levels of pro-inflammatory gene expression and lower levels of Il10 gene expression than control Rictorfl/fl cells. Further suppression of LPS-stimulated Akt signaling with a low dose of an Akt inhibitor, increased inflammatory gene expression in macrophages, but genetic inactivation of Raptor reversed this rise, indicating that mTORC1 mediates this increase of inflammatory gene expression. Next, to elucidate whether mTORC2 has an impact on atherosclerosis in vivo, female and male Ldlr null mice were reconstituted with bone marrow from MRictor-/- or Rictorfl/fl mice. After 10 weeks of the Western diet, there were no differences between the recipients of the same gender in body weight, blood glucose or plasma lipid levels. However, both female and male MRictor-/- → Ldlr-/- mice developed smaller atherosclerotic lesions in the distal and proximal aorta. These lesions contained less macrophage area and more apoptosis than lesions of control Rictorfl/fl → Ldlr-/- mice. Thus, loss of Rictor and, consequently, mTORC2 significantly compromised monocyte/macrophage survival, and this markedly diminished early atherosclerosis in Ldlr-/- mice. Our results demonstrate that mTORC2 is a key signaling regulator of macrophage survival and its depletion suppresses early atherosclerosis.

Dyslipidemia, steatohepatitis and atherogenesis in lipodystrophic apoE deficient mice with Seipin deletion

SEIPIN is an integral membrane protein located in the endoplasmic reticulum, regulating adipocytes differentiation and lipolysis. Deficiency of Seipin in mice causes severe general lipodystrophy, accompanied by insulin resistance, postprandial hypertriglyceridemia and steatohepatitis. In atherosclerosis-prone Ldlr null (Ldlr−/−) mice, lipodystrophy caused by Seipin deletion even led to severe hypercholesteremia and accelerated atherogenesis, when challenged with an atherogenic diet. However, whether the phenotypes observed in Seipin−/−Ldlr−/− mice were a common consequence due to lipodystrophy, rather than genetic background restricted or diet dependent, was unknown. Herein we explored the lipodystrophy-related dyslipidemia, steatohepatitis and atherogenesis in another atherosclerosis-prone murine model, apolipoprotein E null (apoE−/−) mice. Besides, we also compared phenotypes between sexes in apoE−/− mice with Seipin deletion (Seipin−/−apoE−/−). We found that compared with apoE−/− controls, Seipin−/−apoE−/− mice also developed severe general lipodystrophy with hyperlipidemia, steatohepatitis and increased atherogenesis. Although the severity of adipose loss in female and male Seipin−/−apoE−/− mice were similar, hyperlipidemia, steatohepatitis and atherosclerosis were less severe in females than in males. Therefore, we demonstrated that lipodystrophy-related metabolic disorders, caused by Seipin deletion, were independent of genetic background and experimental diet, as seen in Ldlr−/− and apoE−/− mice. However, gender factor affected the disease progression, with females more resistant to developing lipodystrophy-related metabolic consequences.

Lack of Lamin A/C in immune cells accelerates atherosclerosis development in Ldlr-null mice

Aim: Nuclear A-type lamins (Lamin A/C) play structural and functional roles in many cell types. We have recently shown that lamin A/C promote the activation of naïve T-cells. Since both innate and acquired immune responses regulate atherosclerosis, here we investigated whether lamin A/C expression in immune cells affects disease development.

Sequential colonization of periodontal pathogens in induction of periodontal disease and atherosclerosis in LDLRnull mice.

Periodontal disease (PD) and atherosclerotic vascular disease (ASVD) are both chronic inflammatory diseases with a polymicrobial etiology and have been epidemiologically associated. The purpose is to examine whether periodontal bacteria that infect the periodontium can also infect vascular tissues and enhance pre-existing early aortic atherosclerotic lesions in LDLRnull mice. Mice were orally infected with intermediate bacterial colonizer Fusobacterium nucleatum for the first 12 weeks followed by late bacterial colonizers (Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia) for the remaining 12 weeks mimicking the human oral microbiota ecological colonization. Genomic DNA from all four bacterial was detected in gingival plaque by PCR, consistently demonstrating infection of mouse gingival surfaces. Infected mice had significant levels of IgG and IgM antibodies, alveolar bone resorption, and showed apical migration of junctional epithelium revealing the induction of PD. These results support the ability of oral bacteria to cause PD in mice. Detection of bacterial genomic DNA in systemic organs indicates hematogenous dissemination from the gingival pockets. Bacterial infection did not alter serum lipid fractions or serum amyloid A levels and did not induce aortic atherosclerotic plaque. This is the first study examining the causal role of periodontal bacteria in induction of ASVD in LDLRnull mice.

Tg6F ameliorates the increase in oxidized phospholipids in the jejunum of mice fed unsaturated LysoPC or WD

Mouse chow supplemented with lysophosphatidylcholine with oleic acid at sn-1 and a hydroxyl group at sn-2 (LysoPC 18:1) increased LysoPC 18:1 in tissue of the jejunum of LDL receptor (LDLR)-null mice by 8.9 ± 1.7-fold compared with chow alone. Western diet (WD) contained dramatically less phosphatidylcholine 18:1 or LysoPC 18:1 compared with chow, but feeding WD increased LysoPC 18:1 in the jejunum by 7.5 ± 1.4-fold compared with chow. Feeding LysoPC 18:1 or feeding WD increased oxidized phospholipids in the jejunum by 5.2 ± 3.0-fold or 8.6 ± 2.2-fold, respectively, in LDLR-null mice (P < 0.0004), and 2.6 ± 1.5-fold or 2.4 ± 0.92-fold, respectively, in WT C57BL/6J mice (P < 0.0001). Adding 0.06% by weight of a concentrate of transgenic tomatoes expressing the 6F peptide (Tg6F) decreased LysoPC 18:1 in the jejunum of LDLR-null mice on both diets (P < 0.0001), and prevented the increase in oxidized phospholipids in the jejunum in LDLR-null and WT mice on both diets (P < 0.008). Tg6F decreased inflammatory cells in the villi of the jejunum, decreased dyslipidemia, and decreased systemic inflammation in LDLR-null and WT mice on both diets. We conclude that Tg6F reduces diet-induced inflammation by reducing the content of unsaturated LysoPC and oxidized phospholipids in the jejunum of mice.

Abstract 2: Loss of Rictor in Macrophages Suppresses Their Viability and Reduces Atherosclerosis in LDLR Null Mice

The mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that plays a central role in the regulation of cell viability, growth and metabolism. mTOR complex 2 (mTORC2) directly activates phosphorylation of Akt at S 473 , promoting pro-survival signaling. Rictor is an essential component of mTORC2, and genetic loss of Rictor inactivates the complex. To examine whether macrophage mTORC2 signaling has an impact on atherosclerosis, we transplanted male Ldlr null mice with bone marrow isolated from male mice with myeloid-specific Rictor deletion ( Rictor -/- , n=9) and control marrow from Rictor flox-flox mice ( Rictor flox/flox ; n=10). Compared to control mice reconstituted with Rictor flox/flox cells, the recipients of Rictor -/- bone marrow cells exhibited dramatic changes in blood cells including lower levels of white blood cells, B-cells, T-cells and monocytes but had similar levels of neutrophils. After 8 weeks of the Western diet, both groups of recipients had similar levels of body weight, blood glucose, plasma total cholesterol and triglycerides. However, Rictor -/- → Ldlr -/- mice developed smaller atherosclerotic lesions in the proximal and distal aorta (46 and 40% reduction, respectively). These lesions contained less macrophage area and more apoptotic macrophages than lesions of control Rictor flox/flox → Ldlr -/- mice. Importantly, blood monocytes and peritoneal macrophages isolated from Rictor -/- → Ldlr -/- mice were more sensitive to apoptotic stimuli compared to control Rictor flox/flox cells. In response to LPS, Rictor -/- macrophages exhibited the M1 phenotype with high levels of pro-inflammatory gene expression. Both Rictor -/- blood monocytes and macrophages had lower levels of Il10 gene expression than Rictor flox/flox cells. Thus, loss of Rictor and, consequently, mTORC2 in monocyte/macrophages significantly compromises their survival, and this markedly diminishes early atherosclerosis in Ldlr -/- mice. Our results indicate that mTORC2 is a key signaling regulator of macrophage survival and inflammatory responses and promote atherosclerosis.

Abstract 11679: Similarities Between the Villi of the Jejunum and the Human Artery Wall May Contribute to Diet-Induced Dyslipidemia and Systemic Inflammation in LDLR Null Mice

In early atherosclerosis, LDL in the human artery wall is seeded with reactive oxygen species resulting in the formation of oxidized phospholipids that promote infiltration of monocytes, which convert into macrophages. We recently reported that supplementing standard mouse chow with lysophosphatidylcholine (LysoPC) 18:1 caused dyslipidemia and systemic inflammation in LDLR null mice. We now report that feeding chow supplemented with LysoPC 18:1 (1 mg LysoPC 18:1 per gram chow) or feeding a Western diet (WD) to LDLR null mice results in an increase in oxidized phospholipids in the villi of the jejunum as detected with the E06 antibody. On a diet of standard mouse chow, 2.2 ± 0.2% of the area of the villi of the jejunum stained positively for oxidized phospholipids. After two weeks of feeding standard mouse chow supplemented with LysoPC 18:1 or feeding WD, the area staining positively for oxidized phospholipids increased to 4.0 ± 0.5%, and 7.3 ± 0.8%, respectively (p = 0.0043, and p &lt; 0.0001, respectively). If 0.06% by weight of a concentrate of transgenic tomato expressing an apoA-I mimetic peptide (Tg6F) was added to the chow supplemented with LysoPC 18:1 or was added to WD, the area staining positively for oxidized phospholipids decreased to 2.1 ± 0.3%, and 2.3 ± 0.4%, respectively (p = 0.0029, and p &lt;0.0001, respectively). Concomitant with the increase in oxidized phospholipids there was a significant increase in macrophages as detected by F4/80 staining. On chow 0.8 ± 0.2% of the area of the villi of the jejunum stained positively for macrophages. After two weeks of feeding chow supplemented with LysoPC 18:1 or feeding WD, the area staining positively for macrophages increased to 1.6 ± 0.2%, and 4.4 ± 0.4%, respectively (p = 0.0095, and p &lt; 0.0001, respectively). If 0.06% by weight of Tg6F was added to the chow supplemented with LysoPC 18:1 or was added to WD, the area staining positively for macrophages decreased to 0.9 ± 0.2%, and 3.0 ± 0.3%, respectively (p = 0.0169, and p &lt; 0.0001, respectively). These data support the hypothesis that LysoPC 18:1 and WD lead to a sequence of events in the villi of jejunum that is very similar to that seen in the human artery wall, and may contribute to diet-induced dyslipidemia and systemic inflammation in LDLR null mice.

89 - Hematopoietic Glutaredoxin 1 Deficiency Promotes Tissue Inflammation and Accelerates Diet-Induced Obesity and Atherosclerosis in LDLR Null Mice

Glutaredoxins (Grxs) and thioredoxins (Trxs) play important roles in maintaining intracellular thiol-redox homeostasis by scavenging reactive oxygen species. However, few Grxs and Trxs have been functionally characterized in Apis cerana cerana. In this study, we identified three genes, AccGrx1, AccGrx2, and AccTrx1, and investigated their connection to antioxidant defense. AccGrx1 and AccGrx2 were mainly detected in dark-eyed pupae, whereas AccTrx1 was highly concentrated in 15-day postemergence adults. The expression levels of AccGrx1 and AccTrx1 were the highest in fat body and epidermis, respectively. However, the expression level of AccGrx2 was the highest in muscle, followed by the epidermis. AccGrx1, AccGrx2, and AccTrx1 were induced by 4, 16, and 42 °C; H2O2; and pesticide (acaricide, paraquat, cyhalothrin, and phoxime) treatments and repressed by UV light. AccGrx1 and AccGrx2 were upregulated by HgCl2 treatment, whereas AccTrx1 was downregulated. We investigated the knockdown of AccGrx1, AccGrx2, AccTpx-3, and AccTrx1 in A. cerana cerana and surprisingly found that knockdown of the these four genes enhanced the enzymatic activities of CAT and POD; the metabolite contents of hydrogen peroxide, carbonyls, and ascorbate; and the ratios of GSH/GSSG and NADP+/NADPH. In addition, we also analyzed the transcripts of other antioxidant genes and found that some were upregulated and others were downregulated, revealing that the upregulated genes may be involved in compensating for the knockdown of AccGrx1, AccGrx2, AccTpx-3, and AccTrx1. Taken together, these results suggest that AccGrx1, AccGrx2, AccTpx-3, and AccTrx1 may play critical roles in antioxidant defense.

Cannabinoid Receptor Type 2 (CB2) Dependent and Independent Effects of WIN55,212-2 on Atherosclerosis in Ldlr-null Mice.

WIN55,212-2, a potent synthetic agonist of cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2), reduces atherosclerosis in apolipoprotein E (ApoE) null mice. Although pharmacologic evidence suggests the anti-atherosclerotic effects of WIN55,212-2 are mediated via CB2, this remains to be confirmed by genetic studies. Therefore, in this study, we investigated the effects of WIN55,212-2 on development of atherosclerosis in low-density lipoprotein receptor (Ldlr) null mice with and without homozygous deletion of the CB2 gene. After 6 weeks on an atherogenic diet, groups of CB2+/+ and CB2-/- Ldlr-null mice received a daily intraperitoneal injection of WIN55,212-2 or vehicle. After two weeks, plasma lipid levels and atherosclerosis in the aortic root were quantified. Plasma cholesterol and triglyceride levels did not differ between CB2+/+ and CB2-/- mice and WIN55,212-2 had no effect on total cholesterol levels in either genotype. However, triglyceride levels in both CB2+/+ and CB2-/- mice were significantly lowered by WIN55,212-2. The size of aortic root lesions did not differ significantly between CB2+/+ and CB2-/- mice with or without WIN55,212-2 treatment. However, WIN55,212-2 treatment significantly lowered lesional macrophage accumulation in CB2+/+ mice, and lesional smooth muscle content in both CB2+/+ and CB2-/- mice. Lesional apoptosis was also greater in CB2+/+ mice compared to CB2-/-mice, and only reduced by WIN55,212-2 in CB2+/+ mice. Collagen content and elastin fiber fragmentation were unaffected by genotype or WIN55,212-2. WIN55,212-2 treatment does not alter lesion size in Ldlr null-mice, but does modify lesion cellularity via CB2-dependent and CB2-independent mechanisms.

Cellular cholesterol modulates the differentiation of satellite cells to brown fat in lcat-deficient ldlr-null mice

Cellular cholesterol modulates the differentiation of satellite cells to brown fat in lcat-deficient ldlr-null mice

Abstract 3: Cholesterol Crystals Produced by Endothelial Cells During Hyperlipidemia Initiate Atherogenesis

Atherosclerosis is caused by prolonged dyslipidemia and chronic inflammation. Although much is known about advanced stages of atherosclerosis, events leading to its initiation are poorly understood. Cholesterol crystals (CC) have been associated with advanced plaque, but their origin and potential role in the initiation of atherosclerosis and early plaque development are unknown. Using polarized light, scanning and transmission electron microscopy we show for the first time that CC are present in the very early plaque induced after only 1 week of high fat diet in ldlr-null mice. We determined that the source of CC is the endothelial monolayer by treatment of human aortic endothelial cells with LDL for 5 days. Other cell types like monocytes, fibroblasts and human umbilical vein endothelial cells failed to form CC upon LDL, AcLDL or oxLDL treatment. To identify in vitro formed CC we used polarized light, transmission electron and confocal microscopy. Furthermore, we identified that CC are generated within autophagolysosomes of the endothelium. The formation of CC by endothelial cells is accompanied with changes in intracellular cAMP levels. The consequences of early subendothelial CC formation are incomplete junction formation with subsequent impaired endothelial barrier integrity, processes known to be involved in atherosclerosis initiation and progression. Interestingly, increase of intracellular cAMP inhibited endothelial CC production in vitro and in vivo. By performing western Blot, flow cytometry and various microscopy techniques we could show that the inhibition of CC-formation as the results of cAMP increase is accompanied by a decrease autophagolysosomes as the CC producing organelles. We could furthermore demonstrate that this protective effect of cAMP could be inhibited by additional application of an eNOS inhibitor. In summary, we could demonstrate that CC are present in very early stages of atherosclerosis. These early subendothelial CC depositions are produced by autophagolysosomes within the endothelial monolayer in a cAMP/eNOS dependent manner. With these findings we might have found the so far missing link to initiation of plaque development in atherosclerosis.

Abstract 549: Loss of Macrophage Akt2/Akt3 or Akt1/Akt2 Decreases Cell Survival and Suppresses Early Atherosclerosis in Ldlr Null Mice

Akt is a serine/threonine-specific protein kinase that plays a key role in multiple cellular processes including glucose metabolism and cell survival. Macrophages express three Akt isoforms, Akt1, Akt2 and Akt3 which have been ascribed a number of isoform specific effects but may also be functionally redundant in terms of Akt signaling. Here we hypothesize that dramatic reduction of Akt content with loss of two isoforms will suppress Akt signaling in macrophages, reducing their survival and modulating the development of atherosclerosis. To test this hypothesis, we generated mice with combined deficiency Akt2/Akt3 in hematopoietic cells. Peritoneal macrophages isolated from these mice preserved a similar level of Akt phosphorylation when treated with insulin but exhibited a dramatic increase in apoptosis in response to several different factors that induce ER stress compared to WT or single Akt2 or Akt3 knockout macrophages. Male LDLR null mice reconstituted with double Akt2/Akt3 knockout hematopoietic cells and fed the Western diet for 8 weeks developed smaller (57.6 % reduction) atherosclerotic lesions with more apoptotic macrophages in the proximal aorta than control WT→LDLR -/- mice, in the absence of changes in plasma lipid levels. Thus, loss of Akt2/Akt3 in macrophages increases their sensitivity to apoptosis and decreases atherosclerosis. Next, we generated mice with combined deficiency of Akt1/Akt2 in hematopoietic cells. Again, loss of a single Akt1 or Akt2 isoform in macrophages had no impact on cell apoptosis, whereas combined Akt1/Akt2 deficiency did not change Akt phosphorylation but markedly compromised their sensitivity to different apoptotic stimuli. Furthermore, male and female LDLR -/- mice transplanted with Akt1/Akt2 knockout hematopoietic cells had significantly smaller atherosclerotic lesions with increased macrophage apoptosis than control WT→LDLR -/- mice. Taken together these data indicate that a dramatic reduction of Akt protein in macrophages with loss of either of the Akt1/Akt2 or Akt2/Akt3 pairs of isoforms significantly compromises cell survival and this diminishes early atherosclerosis.

Renoprotective Effects of Vitex megapotamica (Spreng.) Moldenke in C57BL/6 LDLr-Null Mice Undergoing High Fat Diet.

Although Vitex megapotamica (Spreng.) Moldenke is used in Brazilian folk medicine as hypolipidemic drug no study has been conducted to evaluate the effects of this species in an experimental model of atherosclerosis. So, the aim of this study was to evaluate the possible renoprotective activity of methanolic extract obtained from Vitex megapotamica (MEVM) using C57BL/6 LDLr-null mice submitted to high fat diet (HFD). MEVM was orally administered at doses of 30, 100, and 300 mg/kg, for three weeks, starting from the 2nd week of HFD. Systolic blood pressure (SBP) and diuretic activity were measured weekly. At the end of experiments the serum lipids, atherogenic index serum (AIS), oxidative stress, and markers of renal function were determined. HFD induced a significant increase in the systolic blood pressure, dyslipidemia, increase in AIS, and lipid peroxidation accompanied by an important reduction in renal function. Treatment with MEVM was able to prevent increase in SBP, total cholesterol, triglycerides, AIS, urea, and creatinine levels in LDLr-null mice. These effects were accompanied by a significant reduction in oxidative stress and renal injury. The data reported here support the potential of Vitex megapotamica as candidate to be an herbal medicine used in cardiovascular or renal diseases.

The apolipoprotein-AI mimetic peptide L4F at a modest dose does not attenuate weight gain, inflammation, or atherosclerosis in LDLR-null mice.

ObjectiveHigh density lipoprotein (HDL) cholesterol levels are inversely related to cardiovascular disease risk and associated with a reduced risk of type 2 diabetes. Apolipoprotein A-I (apoA-I; major HDL protein) mimetics have been reported to reduce atherosclerosis and decrease adiposity. This study investigated the effect of L4F mimetic peptide and apoA-I overexpression on weight gain, insulin resistance, and atherosclerosis in an LDL receptor deficient (Ldlr-/-) model fed a high fat high sucrose with cholesterol (HFHSC) diet.MethodsStudies in differentiated 3T3-L1 adipocytes tested whether L4F could inhibit palmitate-induced adipocyte inflammation. In vivo studies used male Ldlr-/- mice fed a HFHSC diet for 12 weeks and were injected daily with L4F (100 µg/mouse) subcutaneously during the last 8 weeks. Wild-type and apoA-I overexpressing Ldlr-/- mice were fed HFHSC diet for 16 weeks.ResultsNeither L4F administration nor apoA-I overexpression affected weight gain, total plasma cholesterol or triglycerides in our studies. While pre-treatment of 3T3-L1 adipocytes with either L4F or HDL abolished palmitate-induced cytokine expression in vitro, L4F treatment did not affect circulating or adipose tissue inflammatory markers in vivo. Neither L4F administration nor apoA-I overexpression affected glucose tolerance. ApoA-I overexpression significantly reduced atherosclerotic lesion size, yet L4F treatment did not affect atherosclerosis.ConclusionOur results suggest that neither L4F (100 µg/day/mouse) nor apoA-I overexpression affects adiposity or insulin resistance in this model. We also were unable to confirm a reduction in atherosclerosis with L4F in our particular model. Further studies on the effect of apoA-I mimetics on atherosclerosis and insulin resistance in a variety of dietary contexts are warranted.