Hypercholesterolemic Low-density Lipoprotein Receptor Research Articles

High-density lipoprotein regulates angiogenesis by affecting autophagy via miRNA-181a-5p.

We previously demonstrated that normal high-density lipoprotein (nHDL) can promote angiogenesis, whereas HDL from patients with coronary artery disease (dHDL) is dysfunctional and impairs angiogenesis. Autophagy plays a critical role in angiogenesis, and HDL regulates autophagy. However, it is unclear whether nHDL and dHDL regulate angiogenesis by affecting autophagy. Endothelial cells (ECs) were treated with nHDL and dHDL with or without an autophagy inhibitor. Autophagy, endothelial nitric oxide synthase (eNOS) expression, miRNA expression, nitric oxide (NO) production, superoxide anion (O2•-) generation, EC migration, and tube formation were evaluated. nHDL suppressed the expression of miR-181a-5p, which promotes autophagy and the expression of eNOS, resulting in NO production and the inhibition of O2•- generation, and ultimately increasing in EC migration and tube formation. dHDL showed opposite effects compared to nHDL and ultimately inhibited EC migration and tube formation. We found that autophagy-related protein 5 (ATG5) was a direct target of miR-181a-5p. ATG5 silencing or miR-181a-5p mimic inhibited nHDL-induced autophagy, eNOS expression, NO production, EC migration, tube formation, and enhanced O2•- generation, whereas overexpression of ATG5 or miR-181a-5p inhibitor reversed the above effects of dHDL. ATG5 expression and angiogenesis were decreased in the ischemic lower limbs of hypercholesterolemic low-density lipoprotein receptor null (LDLr-/-) mice when compared to C57BL/6 mice. ATG5 overexpression improved angiogenesis in ischemic hypercholesterolemic LDLr-/- mice. Taken together, nHDL was able to stimulate autophagy by suppressing miR-181a-5p, subsequently increasing eNOS expression, which generated NO and promoted angiogenesis. In contrast, dHDL inhibited angiogenesis, at least partially, by increasing miR-181a-5p expression, which decreased autophagy and eNOS expression, resulting in a decrease in NO production and an increase in O2•- generation. Our findings reveal a novel mechanism by which HDL affects angiogenesis by regulating autophagy and provide a therapeutic target for dHDL-impaired angiogenesis.

LAG3 Regulates T Cell Activation and Plaque Infiltration in Atherosclerotic Mice

BackgroundThe immune checkpoint receptor lymphocyte-activation gene 3 (LAG3) is a new target for immune checkpoint blockade (ICB), but the effects of LAG3 on atherosclerosis are not known. ObjectivesThe aim of the study was to evaluate the role of LAG3 on plaque inflammation using murine hypercholesterolemic models of atherosclerosis. MethodsTo study the role of LAG3 in atherosclerosis, we investigated both bone marrow chimeras lacking LAG3 in hematopoietic cells as well as global Lag3-/- knockout mice. Effects of anti-LAG3 monoclonal antibody monotherapy and combination therapy with anti-programmed cell death protein 1 (PD-1) were tested in hypercholesterolemic low-density lipoprotein receptor knockout (Ldlr-/-) mice and evaluated by histology and flow cytometry. ResultsLAG3-deficiency or treatment with blocking anti-LAG3 monoclonal antibodies led to increased levels of both interferon gamma–producing T helper 1 cells and effector/memory T cells, balanced by increased levels of regulatory T cells. Plaque size was affected by neither LAG3 deficiency nor LAG3 blockade, although density of T cells in plaques was 2-fold increased by loss of LAG3. Combination therapy of anti-PD-1 and anti-LAG3 had an additive effect on T cell activation and cytokine production and promoted plaque infiltration of T cells. ConclusionsLoss of LAG3 function promoted T cell activation and accumulation in plaques while not affecting plaque burden. Our report supports further clinical studies investigating cardiovascular risk in patients treated with anti-LAG3 ICB.

Bone Marrow Ts65Dn Trisomy-Induced Changes in Platelet Functionality and Lymphocytopenia Do Not Impact Atherosclerosis Susceptibility in Mice.

The genetic disorder Down syndrome is associated with a decreased susceptibility for atherosclerotic cardiovascular disease. Hematological and immune abnormalities occur frequently in Down syndrome patients. We evaluated, in a preclinical setting, the impact of a Down syndrome-like hematological/immune phenotype on atherosclerosis susceptibility. Hereto, hypercholesterolemic low-density lipoprotein receptor knockout mice were transplanted with bone marrow from either a trisomic Ts65Dn mouse or euploid wild-type control and subsequently fed a Western-type diet to induce the development of atherosclerotic lesions. T and B cell concentrations were markedly reduced in blood of Ts65Dn bone marrow recipients (p < 0.001). Expression levels of the pro-atherogenic scavenger receptor CD36 were respectively 37% and 59% lower (p < 0.001) in trisomic monocytes and macrophages. However, these combined effects did not translate into an altered atherosclerosis susceptibility. Notably, blood platelet numbers were elevated in Ts65Dn bone marrow recipients (+57%; p < 0.001), which was paralleled by higher platelet GPVI protein expression (+35%; p < 0.001) and an enhanced collagen-induced platelet activation (p < 0.001). In conclusion, we have shown that providing mice with a Down syndrome-like hematological profile does not change the susceptibility to atherosclerosis. Furthermore, our studies have uncovered a novel effect of the trisomy on platelet functionality that may be relevant in human clinical settings.

Angiogenic and Antiangiogenic mechanisms of high density lipoprotein from healthy subjects and coronary artery diseases patients

Normal high-density lipoprotein (nHDL) in normal, healthy subjects is able to promote angiogenesis, but the mechanism remains incompletely understood. HDL from patients with coronary artery disease may undergo a variety of oxidative modifications, rendering it dysfunctional; whether the angiogenic effect is mitigated by such dysfunctional HDL (dHDL) is unknown. We hypothesized that dHDL compromises angiogenesis. The angiogenic effects of nHDL and dHDL were assessed using endothelial cell culture, endothelial sprouts from cardiac tissue from C57BL/6 mice, zebrafish model for vascular growth and a model of impaired vascular growth in hypercholesterolemic low-density lipoprotein receptor null(LDLr-/-)mice. MiRNA microarray and proteomic analyses were used to determine the mechanisms. Lipid hydroperoxides were greater in dHDL than in nHDL. While nHDL stimulated angiogenesis, dHDL attenuated these responses. Protein and miRNA profiles in endothelial cells differed between nHDL and dHDL treatments. Moreover, nHDL suppressed miR-24-3p expression to increase vinculin expression resulting in nitric oxide (NO) production, whereas dHDL delivered miR-24-3p to inhibit vinculin expression leading to superoxide anion (O2•-) generation via scavenger receptor class B type 1. Vinculin was required for endothelial nitric oxide synthase (eNOS) expression and activation and modulated the PI3K/AKT/eNOS and ERK1/2 signaling pathways to regulate nHDL- and VEGF-induced angiogenesis. Vinculin overexpression or miR-24-3p inhibition reversed dHDL-impaired angiogenesis. The expressions of vinculin and eNOS and angiogenesis were decreased, but the expression of miR-24-3p and lipid hydroperoxides in HDL were increased in the ischemic lower limbs of hypercholesterolemic LDLr-/- mice. Overexpression of vinculin or miR-24-3p antagomir restored the impaired-angiogenesis in ischemic hypercholesterolemic LDLr-/- mice. Collectively, nHDL stimulated vinculin and eNOS expression to increase NO production by suppressing miR-24-3p to induce angiogenesis, whereas dHDL inhibited vinculin and eNOS expression to enhance O2•- generation by delivering miR-24-3p to impair angiogenesis, and that vinculin and miR-24-3p may be therapeutic targets for dHDL-impaired angiogenesis.

Hematopoietic Stabilin-1 deficiency does not influence atherosclerosis susceptibility in LDL receptor knockout mice

Background and aimsStabilin-1 (STAB1) is a scavenger receptor expressed on alternatively activated macrophages and sinusoidal endothelial cells. Its ligands include oxidized low-density lipoprotein (LDL) and the extracellular matrix glycoprotein SPARC and it is present in both human and murine atherosclerotic lesions. We aimed to investigate the effect of specific deletion of STAB1 in bone marrow-derived cells, including macrophages on atherosclerotic lesion formation in mice. MethodsLethally irradiated hypercholesterolemic LDL receptor knockout mice received either wildtype (WT) or STAB1 knockout (STAB1 KO) bone marrow. Bone marrow transplanted mice were fed a Western-type diet for 9 weeks to induce atherosclerotic lesion formation. ResultsInterestingly, LDL receptor knockout mice reconstituted with STAB1 KO bone marrow showed increased body weight gain (two-way ANOVA: p < 0.001) and larger white adipocyte cell sizes (43% increase in cell area; p < 0.05) as compared to WT bone marrow transplanted mice, which correlated positively (r = 0.82; p < 0.001). This was paralleled by a significant increase in white adipose tissue relative mRNA expression levels of the adipokine leptin (+94% p < 0.05). Despite these changes, no differences in serum lipid levels, the extent of in vivo macrophage foam cell formation or circulating leukocyte concentrations were observed. Moreover, the size and composition of atherosclerotic lesions was not different between the two experimental groups. ConclusionsBone marrow-specific Stabilin-1 deletion does not affect the susceptibility for atherosclerosis in mice. However, the increased body weight gain and adipocyte cell size highlight a potential role for leukocyte STAB1 in the development of metabolic disorders.

Fibroblast Growth Factor-2 Is Required for Vasa Vasorum Plexus Stability in Hypercholesterolemic Mice

Vasa vasorum are angiogenic in advanced stages of human atherosclerosis and hypercholesterolemic mouse models. Fibroblast growth factor-2 (FGF-2) is the predominant angiogenic growth factor in the adventitia and plaque of hypercholesterolemic low-density lipoprotein receptor-deficient/apolipoprotein B(100/100) mice (DKO). FGF-2 seems to play a role in the formation of a distinct vasa vasorum network. This study examined the vasa vasorum structure and its relationship to FGF-2. DKO mice treated with saline, antiangiogenic recombinant plasminogen activator inhibitor-1(23) (rPAI-1(23)), or soluble FGF receptor 1 were perfused with fluorescein-labeled Lycopersicon esculentum lectin. Confocal images of FGF-2-probed descending aorta adventitia show that angiogenic vasa vasorum form a plexus-like network in saline-treated DKO similar to the FGF-2 pattern of distribution. Mice treated with rPAI-1(23) and soluble FGF receptor 1 lack a plexus; FGF-2 and vasa vasorum density and area are significantly reduced. A perlecan/FGF-2 complex is critical for plexus stability. Excess plasmin produced in rPAI-1(23)-treated DKO mice degrades perlecan and destabilizes the plexus. Plasmin activity and plaque size measured in DKO and DKO/plasminogen activator inhibitor-1(-)(/-) mice demonstrate that elevated plasmin activity contributes to reduced plaque size. An FGF-2/perlecan complex is required for vasa vasorum plexus stability. Elevated plasmin activity plays a significant inhibitory role in vasa vasorum plexus and plaque development.

Anti-Atherogenic Effects of a Phenol-Rich Fraction from Brazilian Red Wine (Vitis labrusca L.) in Hypercholesterolemic Low-Density Lipoprotein Receptor Knockout Mice

Moderate wine intake (i.e., 1-2 glasses of wine a day) is associated with a reduced risk of morbidity and mortality from cardiovascular disease. The aim of this study was to evaluate the anti-atherosclerotic effects of a nonalcoholic ethyl acetate fraction (EAF) from a South Brazilian red wine obtained from Vitis labrusca grapes. Experiments were carried out on low-density lipoprotein (LDL) receptor knockout (LDLr⁻/⁻) mice, which were subjected to a hypercholesterolemic diet and treated with doses of EAF (3, 10, and 30 mg/kg) for 12 weeks. At the end of the treatment, the level of plasma lipids, the vascular reactivity, and the atherosclerotic lesions were evaluated. Our results demonstrated that the treatment with EAF at 3 mg/kg significantly decreased total cholesterol, triglycerides, and LDL plus very low-density lipoprotein levels compared with control hypercholesterolemic mice. The treatment of mice with EAF at 3 mg/kg also preserved the vasodilatation induced by acetylcholine on isolated thoracic aorta from hypercholesterolemic LDLr⁻/⁻ mice. This result is in agreement with the degree of lipid deposit on arteries. Taken together, the results show for the first time that the lowest concentration of an EAF obtained from a red wine produced in southern Brazil significantly reduced the progression of atherosclerosis in mice.

Positive correlation between elevated plasma cholesterol levels and cognitive impairments in LDL receptor knockout mice: relevance of cortico-cerebral mitochondrial dysfunction and oxidative stress

Convergent epidemiological, clinical, and experimental findings indicate that hypercholesterolemia contributes to the onset of Alzheimer's disease (AD)-like dementia, but the exact underlying mechanisms remains unknown. In this study, we evaluated the cognitive performance of mice submitted to a model of hypercholesterolemia, as well as its relationship with mitochondrial dysfunction and oxidative stress, two key events involved in AD pathogenesis. Wild-type C57bl/6 or low density lipoprotein receptor (LDLr)-deficient mice were fed with either standard or cholesterol-enriched diet for a 4-week period and tested for spatial learning and memory in the object location task. LDLr⁻/⁻ mice displayed spatial learning and memory impairments regardless of diet. Moreover, LDLr⁻/⁻ mice fed cholesterol-enriched diet presented a significant decrease in the mitochondrial complexes I and II activities in the cerebral cortex, which were negatively correlated with respective blood cholesterol levels. Additionally, hypercholesterolemic LDLr⁻/⁻ mice presented a significant decrease in glutathione levels, about 40% increase in the thiobarbituric acid-reactive substances levels, as well as an imbalance between the peroxide-removing-related enzymes glutathione peroxidase/glutathione reductase activities in the cerebral cortex. These findings indicate a significant relationship between hypercholesterolemia, cognitive impairment, and cortico-cerebral mitochondrial dysfunctional/oxidative stress. Because of the involvement of such alterations in AD patients, our data render this mouse model of hypercholesterolemia a useful approach to comprehend the molecular events mediating AD pathogenesis.

Antiangiogenic Activity of rPAI-1 23 Promotes Vasa Vasorum Regression in Hypercholesterolemic Mice Through a Plasmin-Dependent Mechanism

The antiangiogenic activity of rPAI-1(23), a truncated plasminogen activator inhibitor-1 (PAI-1) protein, induces vasa vasorum collapse and significantly reduces plaque area and plaque cholesterol in hypercholesterolemic low-density lipoprotein receptor-deficient/apolipoprotein B48-deficient mice. The objective of this study was to examine rPAI-1(23)-stimulated mechanisms that cause vasa vasorum collapse. The rPAI-1(23) protein opposed PAI-1 antiproteolytic function by stimulating a 1.6-fold increase in plasmin activity compared with the saline-treated counterpart. The increased proteolytic activity corresponded to increased activity of matrix metalloproteinase-3 and degradation of fibrin(ogen), nidogen, and perlecan in the adventitia of descending aortas. PAI-1 activity was reduced by 48% in response to rPAI-1(23); however, PAI-1 protein expression levels were similar in the rPAI-1(23)- and saline-treated hypercholesterolemic mice. Coimmunoprecipitation assays demonstrated a novel PAI-1-plasminogen complex in protein from the descending aorta of rPAI-1(23)- and saline-treated mice, but complexed PAI-1 was 1.6-fold greater in rPAI-1(23)-treated mice. Biochemical analyses demonstrated that rPAI-1(23) and PAI-1 binding interactions with plasminogen increased plasmin activity and reduced PAI-1 antiproteolytic activity. We conclude that rPAI-1(23) causes regression or collapse of adventitial vasa vasorum in hypercholesterolemic mice by stimulating an increase in plasmin activity. The rPAI-1(23)-enhanced plasmin activity was achieved through a novel mechanism by which rPAI-1(23) and PAI-1 bound plasminogen in a cooperative manner to increase plasmin activity and reduce PAI-1 activity.

Lower expression of PKAα impairs insulin secretion in islets isolated from low-density lipoprotein receptor (LDLR−/−) knockout mice

Hypercholesterolemic low-density lipoprotein receptor knockout mice (LDLR−/−) show normal whole-body insulin sensitivity, but impaired glucose tolerance due to a reduced insulin secretion in response to glucose. Here, we investigate the possible mechanisms involved in such a defect in isolated LDLR−/− mice islets. Low-fat chow–fed female and male mice aged 20 weeks, LDLR−/− mice, and wild-type (WT) mice were used in this study. Static insulin secretion, cytoplasmatic Ca2+ analysis, and protein expression were measured in islets isolated from LDLR−/− and WT mice. At basal (2.8 mmol/L) and stimulatory (11.1 mmol/L) glucose concentrations, the insulin secretion rates induced by depolarizing agents such as KCl, l-arginine, and tolbutamide were significantly reduced in LDLR−/− when compared with control (WT) islets. In addition, KCl-induced Ca2+ influx at 2.8 mmol/L glucose was lower in LDLR−/− islets, suggesting a defect downstream of the substrate metabolism step of the insulin secretion pathway. Insulin secretion induced by the protein kinase A (PKA) activators forskolin and 3-isobutyl-1-methyl-xanthine, in the presence of 11.1 mmol/L glucose, was lower in LDLR−/− islets and was normalized in the presence of the protein kinase C pathway activators carbachol and phorbol 12-myristate 13-acetate. Western blotting analysis showed that phospholipase Cβ2 expression was increased and PKAα was decreased in LDLR−/− compared with WT islets. Results indicate that the lower insulin secretion observed in islets from LDLR−/− mice at postprandial levels of glucose can be explained, at least in part, by the reduced expression of PKAα in these islets.

Deletion of EP4 on Bone Marrow–Derived Cells Enhances Inflammation and Angiotensin II–Induced Abdominal Aortic Aneurysm Formation

To examine whether a lack of prostaglandin E receptor 4 (EP4) on bone marrow-derived cells would increase local inflammation and enhance the formation of abdominal aortic aneurysm (AAA) in vivo. Prostaglandin E(2) (PGE(2)) through activation of EP4, can mute inflammation. Hypercholesterolemic low-density lipoprotein receptor knockout (LDLR(-/-)) mice transplanted with either EP4(+/+) (EP4(+/+)/LDLR(-/-)) or EP4(-/-) (EP4(-/-)/LDLR(-/-)) bone marrow received infusions of angiotensin II to induce AAA. Deficiency of EP4 on bone marrow-derived cells increased the incidence (50% of male EP4(+/+)/LDLR(-/-) mice versus 88.9% of male EP4(-/-)/LDLR(-/-) mice developed AAA; and 22% of female EP4(+/+)/LDLR(-/-) mice versus 83.3% of female EP4(-/-)/LDLR(-/-) mice developed AAA) and severity of AAA, increased monocyte chemoattractant protein-1 (2.72-fold in males and 1.64-fold in females), and enhanced infiltration of macrophages (3.8-fold in males and 2.44-fold in females) and T cells (1.88-fold in males and 1.66-fold in females) into AAA lesions. Lack of EP4 on bone marrow-derived cells augmented elastin fragmentation, increased apoptotic markers, and decreased smooth muscle cell accumulation within AAA lesions. Deficiency of EP4 on bone marrow-derived cells boosted inflammation and AAA formation induced by angiotensin II in hyperlipidemic mice. This study affirms the pathophysiologic importance of PGE(2) signaling through EP4 as an endogenous anti-inflammatory pathway involved in experimental aneurysm formation.

High cholesterol‐induced neuroinflammation and amyloid precursor protein processing correlate with loss of working memory in mice

Recent findings suggest that hypercholesterolemia may contribute to the onset of Alzheimer's disease-like dementia but the underlying mechanisms remain unknown. In this study, we evaluated the cognitive performance in rodent models of hypercholesterolemia in relation to neuroinflammatory changes and amyloid precursor protein (APP) processing, the two key parameters of Alzheimer's disease pathogenesis. Groups of normal C57BL/6 and low density lipoprotein receptor (LDLR)-deficient mice were fed a high fat/cholesterol diet for an 8-week period and tested for memory in a radial arm maze. It was found that the C57BL/6 mice receiving a high fat diet were deficient in handling an increasing working memory load compared with counterparts receiving a control diet while the hypercholesterolemic LDLR-/- mice showed impaired working memory regardless of diet. Immunohistochemical analysis revealed the presence of activated microglia and astrocytes in the hippocampi from high fat-fed C57BL/6 mice and LDLR-/- mice. Consistent with a neuroinflammatory response, the hyperlipidemic mice showed increased expression of cytokines/mediators including tumor necrosis factor-alpha, interleukin-1beta and -6, nitric oxide synthase 2, and cycloxygenase 2. There was also an induced expression of the key APP processing enzyme i.e. beta-site APP cleaving enzyme 1 in both high fat/cholesterol-fed C57BL/6 and LDLR-/- mice accompanied by an increased generation of C-terminal fragments of APP. Although ELISA for beta-amyloid failed to record significant changes in the non-transgenic mice, a threefold increase in beta-amyloid 40 accumulation was apparent in a strain of transgenic mice expressing wild-type human APP on high fat/cholesterol diet. The findings link hypercholesterolemia with cognitive dysfunction potentially mediated by increased neuroinflammation and APP processing in a non-transgenic mouse model.

Mangifera indica L. extract (Vimang ®) and its main polyphenol mangiferin prevent mitochondrial oxidative stress in atherosclerosis-prone hypercholesterolemic mouse

Atherosclerosis is linked to a number of oxidative events ranging from low-density lipoprotein (LDL) oxidation to the increased production of intracellular reactive oxygen species (ROS). We have recently demonstrated that liver mitochondria isolated from the atherosclerosis-prone hypercholesterolemic LDL receptor knockout (LDLr −/−) mice have lower content of NADP(H)-linked substrates than the controls and, as consequence, higher sensitivity to oxidative stress and mitochondrial membrane permeability transition (MPT). In the present work, we show that oral supplementation with the antioxidants Mangifera indica L. extract (Vimang ®) or its main polyphenol mangiferin shifted the sensitivity of LDLr −/− liver mitochondria to MPT to control levels. These in vivo treatments with Vimang ® and mangiferin also significantly reduced ROS generation by both isolated LDLr −/− liver mitochondria and spleen lymphocytes. In addition, these antioxidant treatments prevented mitochondrial NAD(P)H-linked substrates depletion and NADPH spontaneous oxidation. In summary, Vimang ® and mangiferin spared the endogenous reducing equivalents (NADPH) in LDLr −/− mice mitochondria correcting their lower antioxidant capacity and restoring the organelle redox homeostasis. The effective bioavailability of these compounds makes them suitable antioxidants with potential use in atherosclerosis susceptible conditions.

Oxidative stress in hypercholesterolemic LDL (low-density lipoprotein) receptor knockout mice is associated with low content of mitochondrial NADP-linked substrates and is partially reversed by citrate replacement

We have previously proposed that hypercholesterolemic LDL receptor knockout (k/o) mice mitochondria possess a lower antioxidant capacity due to a large consumption of reducing equivalents from NADPH to sustain high rates of lipogenesis. In this work, we tested the hypothesis that this k/o mice mitochondrial oxidative stress results from the depletion of NADPH-linked substrates. In addition, the oxidative stress was further characterized by showing a lower mitochondrial GSH/GSSG ratio and a higher liver content of protein carbonyls as compared to controls. The activity of the antioxidant enzyme system glutathione reductase/peroxidase did not differ in k/o and control mitochondria. The faster spontaneous oxidation of endogenous NADPH in the k/o mitochondria was prevented by the addition of exogenous catalase, indicating that this oxidation is mediated by mitochondrially generated H 2O 2. The higher rate of H 2O 2 production was also prevented by the addition of exogenous isocitrate that maintains NADP fully reduced. The hypothesis that high rates of lipogenesis in the k/o cells decrease mitochondrial NADPH/NADP + ratio due to consumption of NADPH-linked substrates was supported by two findings: (i) oxygen consumption supported by endogenous NAD(P)H-linked substrates was slower in k/o than in control mitochondria, but was similar in the presence of exogenous isocitrate; (ii) in vivo treatment of k/o mice with sodium citrate/citric acid drinking solution for 2 weeks partially restored both the rate of oxygen consumption supported by NAD(P)H-linked substrates and the mitochondrial capacity to sustain reduced NADPH. In conclusion, the data demonstrate that the mitochondrial oxidative stress in hypercholesterolemic LDL receptor knockout mice is the result of a low content of mitochondrial NADPH-linked substrates in the intact animal that can be, at least in part, replenished by oral administration of citrate.

Granulocyte Macrophage Colony-Stimulating Factor Regulates Dendritic Cell Content of Atherosclerotic Lesions

Recent evidence suggests that dendritic cells may play an important role in atherosclerosis. Based primarily on previous in vitro studies, we hypothesized that granulocyte macrophage colony-stimulating factor (GM-CSF)-deficient mice would have decreased dendritic cells in lesions. To test this, we characterized gene targeted GM-CSF(-/-) mice crossed to hypercholesterolemic low-density lipoprotein receptor null mice. Our results provide conclusive evidence that GM-CSF is a major regulator of dendritic cell formation in vivo. Aortic lesion sections in GM-CSF(-/-) low-density lipoprotein receptor null animals showed a dramatic 60% decrease in the content of dendritic cells as judged by CD11c staining but no change in the overall content of monocyte-derived cells. The GM-CSF-deficient mice exhibited a significant 20% to 50% decrease in the size of aortic lesions, depending on the location of the lesions. Other prominent changes in GM-CSF(-/-) mice were decreased lesional T cell content, decreased autoantibodies to oxidized lipids, and striking disruptions of the elastin fibers adjacent to the lesion. Given that GM-CSF is dramatically induced by oxidized lipids in endothelial cells, our data suggest that GM-CSF serves to regulate dendritic cell formation in lesions and that this, in turn, influences inflammation, plaque growth and possibly plaque stability.

Delayed Arteriogenesis in Hypercholesterolemic Mice

Hypercholesterolemia has been reported to inhibit ischemia-induced angiogenesis. To address its effects on arteriogenesis, we investigated arterial growth in hypercholesterolemic low-density lipoprotein receptor(-/-)/ApoB-48(-/-) (HCE) mice. The extent and the time course of arteriogenesis after femoral artery ligation was evaluated in HCE and strain-matched control mice. Distal limb perfusion was measured by laser Doppler imaging, whereas MRI was used to visualize arterial flow and micro-computed tomography to assess vascular growth. After femoral artery ligation, serial laser Doppler imaging demonstrated significantly delayed restoration of perfusion in untreated HCE compared with control mice (day 3, 0.09 versus 0.19, P<0.05). Treatment with Ad-PR39 in control mice led to a significant restoration of arterial blood flow and tissue perfusion at day 3, whereas in HCE mice, hindlimb perfusion began increasing only by day 7. Micro-CT analysis confirmed increased growth of smaller arterioles (16 to 63 microm in diameter) in the Ad-PR39-treated control compared with HCE mice. The delay in arteriogenesis in HCE mice correlated with delayed tissue appearance of F4/80+ cells. Analysis of gene expression after Ad-PR39 treatment demonstrated that HCE mice had significantly reduced expression of FGF receptor 1, hypoxia-inducible factor-1alpha, vascular cell adhesion molecule-1, macrophage scavenger receptor-1, and cyclophilin A compared with controls 3 days after arterial ligation that equalized by day 7, mimicking relative changes in arteriogenesis and tissue perfusion. Hypercholesterolemia results in delayed native arteriogenesis because of reduced early monocyte/macrophage influx and delayed and impaired arterial growth response to growth factor therapy.

Beneficial effects of antioxidants and L-arginine on oxidation-sensitive gene expression and endothelial NO synthase activity at sites of disturbed shear stress.

Atherogenesis is enhanced in arterial segments exposed to disturbed blood flow, indicating the active participation of the hemodynamic environment in lesion formation. Turbulent shear stress selectively regulates responsive genes in the endothelium and increases the damage induced by free radicals. The purpose of the present study was to evaluate the effects of intervention with antioxidants and l-arginine on endothelial NO synthase (eNOS) and oxidation-sensitive gene perturbation induced by disturbed flow in vitro and in vivo. Both human endothelial cells exposed to shear stress and high atherosclerosis-prone areas of hypercholesterolemic low-density lipoprotein receptor knockout (LDLR(-/-)) mice showed increased activities of redox-transcription factors (ELK-1, p-Jun, and p-CREB) and decreased expression of eNOS. Intervention with antioxidants and l-arginine reduced the activation of redox-transcription factors and increased eNOS expression in cells and in vivo. These results demonstrate that atherogenic effects induced by turbulent shear stress can be prevented by cotreatment with antioxidants and l-arginine. The therapeutic possibility to modulate shear stress-response genes may have important implications for the prevention of atherosclerosis and its clinical manifestations.

Apolipoprotein E knock-out mice are highly susceptible to endotoxemia and Klebsiella pneumoniae infection

Lipoproteins are able to neutralize bacterial lipopolysaccharide (LPS) and thereby inhibit the proinflammatory cytokine response. In a previous study, we demonstrated that hypercholesterolemic low density lipoprotein receptor knock-out (LDLr–/–) mice are protected against lethal endotoxemia and gram-negative infection. In the present study we investigated the susceptibility of apolipoprotein E knock-out mice (apoE–/–) to LPS and to Klebsiella pneumoniae. These mice have increased plasma lipoprotein concentrations in the very low density lipoprotein (VLDL)-sized fraction. Despite 8-fold higher plasma cholesterol levels compared to controls, and in contrast to LDLr–/– mice, apoE–/– mice were significantly more susceptible to endotoxemia and to K. pneumoniae infection. Circulating TNFα concentrations after intravenously injected LPS were 4- to 5-fold higher in apoE–/– mice, whereas IL-1α, IL-1β, and IL-6 did not differ. This TNF response was not due to an increased cytokine production capacity of cells from apoE–/– mice, as ex vivo cytokine production in response to LPS did not differ between apoE–/– and control mice. The LPS-neutralizing capacity of apoE–/– plasma was significantly less than that of controls. Most likely, the absence of apoE itself in the knock-out mice explains the failure to neutralize LPS, despite the very high cholesterol concentrations.—de Bont, N., M. G. Netea, P. N. M. Demacker, I. Verschueren, B. J. Kullberg, K. W. van Dijk, J. W. M. van der Meer, and A. F. H. Stalenhoef. Apolipoprotein E knockout-mice are highly susceptible to endotoxemia and Klebsiella pneumoniae infection. J. Lipid Res. 1999. 40: 680–685.