Variable cartilage degradation in mice with diet-induced metabolic dysfunction: food for thought.

OBJECTIVE: Human cohort studies have demonstrated a role for systemic metabolic dysfunction in osteoarthritis (OA) pathogenesis in obese patients. To explore the mechanisms underlying this metabolic phenotype of OA, we examined cartilage degradation in the knees of mice from different genetic backgrounds in which a metabolic phenotype was established by various dietary approaches. DESIGN: Wild-type C57BL/6J mice and genetically modified mice (hCRP, LDLr-/-. Leiden and ApoE*3Leiden.CETP mice) based on C57BL/6J background were used to investigate the contribution of inflammation and altered lipoprotein handling on diet-induced cartilage degradation. High-caloric diets of different macronutrient composition (i.e., high-carbohydrate or high-fat) were given in regimens of varying duration to induce a metabolic phenotype with aggravated cartilage degradation relative to controls. RESULTS: Metabolic phenotypes were confirmed in all studies as mice developed obesity, hypercholesteremia, glucose intolerance and/or insulin resistance. Aggravated cartilage degradation was only observed in two out of the twelve experimental setups, specifically in long-term studies in male hCRP and female ApoE*3Leiden.CETP mice. C57BL/6J and LDLr-/-. Leiden mice did not develop HFD-induced OA under the conditions studied. Osteophyte formation and synovitis scores showed variable results between studies, but also between strains and gender. CONCLUSIONS: Long-term feeding of high-caloric diets consistently induced a metabolic phenotype in various C57BL/6J (-based) mouse strains. In contrast, the induction of articular cartilage degradation proved variable, which suggests that an additional trigger might be necessary to accelerate diet-induced OA progression. Gender and genetic modifications that result in a humanized pro-inflammatory state (human CRP) or lipoprotein metabolism (human-E3L.CETP) were identified as important contributing factors.

The AT04A vaccine against proprotein convertase subtilisin/kexin type 9 reduces total cholesterol, vascular inflammation, and atherosclerosis in APOE*3Leiden.CETP mice.

AIMS: Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a promising therapeutic target for the treatment of hypercholesterolaemia and atherosclerosis. PCSK9 binds to the low density lipoprotein receptor and enhances its degradation, which leads to the reduced clearance of low density lipoprotein cholesterol (LDLc) and a higher risk of atherosclerosis. In this study, the AT04A anti-PCSK9 vaccine was evaluated for its therapeutic potential in ameliorating or even preventing coronary heart disease in the atherogenic APOE*3Leiden.CETP mouse model. METHODS AND RESULTS: Control and AT04A vaccine-treated mice were fed western-type diet for 18 weeks. Antibody titres, plasma lipids, and inflammatory markers were monitored by ELISA, FPLC, and multiplexed immunoassay, respectively. The progression of atherosclerosis was evaluated by histological analysis of serial cross-sections from the aortic sinus. The AT04A vaccine induced high and persistent antibody levels against PCSK9, causing a significant reduction in plasma total cholesterol (-53%, P < 0.001) and LDLc compared with controls. Plasma inflammatory markers such as serum amyloid A (SAA), macrophage inflammatory protein-1ß (MIP-1ß/CCL4), macrophage-derived chemokine (MDC/CCL22), cytokine stem cell factor (SCF), and vascular endothelial growth factor A (VEGF-A) were significantly diminished in AT04A-treated mice. As a consequence, treatment with the AT04A vaccine resulted in a decrease in atherosclerotic lesion area (-64%, P = 0.004) and aortic inflammation as well as in more lesion-free aortic segments (+119%, P = 0.026), compared with control. CONCLUSIONS: AT04A vaccine induces an effective immune response against PCSK9 in APOE*3Leiden.CETP mice, leading to a significant reduction of plasma lipids, systemic and vascular inflammation, and atherosclerotic lesions in the aorta.

Molecular mechanisms responsible for the differential effects of apoE3 and apoE4 on plasma lipoprotein-cholesterol levels.

OBJECTIVE: The goal of this study was to understand the molecular basis of how the amino acid substitution C112R that distinguishes human apolipoprotein (apo) E4 from apoE3 causes the more proatherogenic plasma lipoprotein-cholesterol distribution that is known to be associated with the expression of apoE4. APPROACH AND RESULTS: Adeno-associated viruses, serotype 8 (AAV8), were used to express different levels of human apoE3, apoE4, and several C-terminal truncation and internal deletion variants in C57BL/6 apoE-null mice, which exhibit marked dysbetalipoproteinemia. Plasma obtained from these mice 2 weeks after the AAV8 treatment was analyzed for cholesterol and triglyceride levels, as well as for the distribution of cholesterol between the lipoprotein fractions. Hepatic expression of apoE3 and apoE4 induced similar dose-dependent decreases in plasma cholesterol and triglyceride to the levels seen in control C57BL/6 mice. Importantly, at the same reduction in plasma total cholesterol, expression of apoE4 gave rise to higher very low-density lipoprotein-cholesterol (VLDL-C) and lower high-density lipoprotein-cholesterol levels relative to the apoE3 situation. The C-terminal domain and residues 261 to 272 in particular play a critical role, because deleting them markedly affected the performance of both isoforms. CONCLUSIONS: ApoE4 possesses enhanced lipid and VLDL-binding ability relative to apoE3, which gives rise to impaired lipolytic processing of VLDL in apoE4-expressing mice. These effects reduce VLDL remnant clearance from the plasma compartment and decrease the amount of VLDL surface components available for incorporation into the high-density lipoprotein pool, accounting for the more proatherogenic lipoprotein profile (higher VLDL-C/high-density lipoprotein-cholesterol ratio) occurring in apoE4-expressing animals compared with their apoE3 counterparts.

Apolipoprotein E controls cerebrovascular integrity via cyclophilin A.

Human apolipoprotein E has three isoforms: APOE2, APOE3 and APOE4. APOE4 is a major genetic risk factor for Alzheimer's disease and is associated with Down's syndrome dementia and poor neurological outcome after traumatic brain injury and haemorrhage. Neurovascular dysfunction is present in normal APOE4 carriers and individuals with APOE4-associated disorders. In mice, lack of Apoe leads to blood-brain barrier (BBB) breakdown, whereas APOE4 increases BBB susceptibility to injury. How APOE genotype affects brain microcirculation remains elusive. Using different APOE transgenic mice, including mice with ablation and/or inhibition of cyclophilin A (CypA), here we show that expression of APOE4 and lack of murine Apoe, but not APOE2 and APOE3, leads to BBB breakdown by activating a proinflammatory CypA-nuclear factor-κB-matrix-metalloproteinase-9 pathway in pericytes. This, in turn, leads to neuronal uptake of multiple blood-derived neurotoxic proteins, and microvascular and cerebral blood flow reductions. We show that the vascular defects in Apoe-deficient and APOE4-expressing mice precede neuronal dysfunction and can initiate neurodegenerative changes. Astrocyte-secreted APOE3, but not APOE4, suppressed the CypA-nuclear factor-κB-matrix-metalloproteinase-9 pathway in pericytes through a lipoprotein receptor. Our data suggest that CypA is a key target for treating APOE4-mediated neurovascular injury and the resulting neuronal dysfunction and degeneration.

Reducing human apolipoprotein E levels attenuates age-dependent Aß accumulation in mutant human amyloid precursor protein transgenic mice.

Apolipoprotein E4 (apoE4) plays a major role in the pathogenesis of Alzheimer's disease. Brain amyloid-ß (Aß) accumulation depends on age and apoE isoforms (apoE4 > apoE3) both in humans and in transgenic mouse models. Brain apoE levels are also isoform dependent, but in the opposite direction (apoE4 < apoE3). Thus, one prevailing hypothesis is to increase brain apoE expression to reduce Aß levels. To test this hypothesis, we generated mutant human amyloid precursor protein transgenic mice expressing one or two copies of the human APOE3 or APOE4 gene that was knocked in and flanked by LoxP sites. We report that reducing apoE3 or apoE4 expression by 50% in 6-month-old mice results in efficient Aß clearance and does not increase Aß accumulation. However, 12-month-old mice with one copy of the human APOE gene had significantly reduced Aß levels and plaque loads compared with mice with two copies, regardless of which human apoE isoform was expressed, suggesting a gene dose-dependent effect of apoE on Aß accumulation in aged mice. Additionally, 12-month-old mice expressing one or two copies of the human APOE4 gene had significantly higher levels of Aß accumulation and plaque loads than age-matched mice expressing one or two copies of the human APOE3 gene, suggesting an isoform-dependent effect of apoE on Aß accumulation in aged mice. Moreover, Cre-mediated APOE4 gene excision in hippocampal astrocytes significantly reduced insoluble Aß in adult mice. Thus, reducing, rather than increasing, apoE expression is an attractive approach to lowering brain Aß levels.

Apolipoprotein e3 as a risk factor for Alzheimer's disease under conditions of nutritional imbalance.

The presence of one or more copies of the E4 allele of apolipoprotein E (ApoE) is strongly associated with of Alzheimer's disease (AD). The impact of E4 on neurodegeneration is potentiated by dietary oxidative challenge. Our prior studies in transgenic mice demonstrate that, in the face of dietary oxidative challenge, E3 does not provide any further protection than E4 or lack of murine ApoE for aggression, oxidative damage, presenilin-1 expression, and gamma-secretase activity, and provides only partial reduction in phospho-tau levels. Extrapolation of these findings to the human condition leads us to hypothesize that the E3 allele may not provide sufficient neuroprotection under conditions of dietary compromise and/or oxidative challenge. Epidemiological evidence is consistent with this possibility. The E3 allele is approximately half as effective compared to E2 at buffering the impact of a single E4 allele. In addition, the risk of AD increases linearly for the genotypes E2/2, E2/3, and E3/3. It has been proposed that that clinical manifestation of AD may in some cases require the convergence of 2 or more risk factors. We hypothesize that the combined impact of dietary oxidative stress and either the ApoE3 or E4 genotype represents one such condition.

Observation of the density and size of cells in hippocampus and vascular lesion in thalamus of GFAP-apoE transgenic mice.

OBJECTIVE: Apolipoprotein E (apoE) is associated with increased risk of age-related diseases, such as Alzheimer's disease (AD) and cerebrovascular disease (CVD). The present study aims to investigate the age-related general morphological changes of the brain in GFAP-apoE transgenic mice, especially the alterations in number and size of hippocampal pyramidal cells and the microvascular lesions in the thalamus. METHODS: Nine female apoE4/4 mice were divided into 3 groups (n=3 in each group): 3-4 months (young group), 9-10 months (middle-aged group) and 20-21 months (old group). Age-matched apoE3/3 mice were employed as control group (n=3 in each group). The paraffin sections of brain tissue were stained by 2 conventional staining methods, thionin staining and hematoxylin-esion(HE) staining, the former of which was to observe the hippocampal cells, while the latter was used to examine the brain microvasculature. RESULTS: There was no apparent difference in the cortical layer between apoE3/3 and apoE4/4 mice, neither any significant difference in the number of cells in hippocampal CA1-CA3 subfields between apoE3/3 and apoE4/4 mice at various age points (P>0.05). However, the mean size of pyramidal cells in CA1 subfield in apoE3/3 and apoE4/4 mice decreased as mice were getting older (P<0.001). At the age of 20-21 months, this cellular atrophy in apoE4/4 mice was more severe than that in old apoE3/3 mice (P<0.05). Furthermore, microvascular lesion in the thalamus was detected in all the 3 old apoE4/4 mice, at varying degrees (5.24%, 1.41% and 3.97%, respectively), while only one apoE3/3 mouse exhibited microvascular lesion in the thalamus, at a low level (0.85%). CONCLUSION: The current study suggests that the cell size in hippocampal CA1 subfield decreases with aging, irrespective of apoE genotype. Cellular atrophy in CA1 subfield and the microvascular lesion in the thalamus are both more severe in old apoE4/4 mice as compared with those in age-matched apoE3/3 mice. Doubts still exist on whether the decreased cell size in hippocampal CA1 subfield in old apoE4/4 mice is associated with dysfunction in learning and memory and whether the microvascular lesions indicate a higher risk of stroke in human apoE4 allele mice. To clarify these issues, further investigations are needed.

Role of apolipoprotein E in anxiety.

Anxiety is most common among Alzheimer's disease (AD) patients with an age at onset under age 65. Apolipoprotein E4 (apoE4) is a risk factor for developing AD at an earlier age and might contribute to this effect. In mice, apoE plays a role in the regulation of anxiety, which might involve histamine receptor-mediated signaling and steroidogenesis in the adrenal gland. In addition, human apoE isoforms have differential effects on anxiety in adult mice lacking apoE and probable AD patients. Compared to wild-type mice, mice lacking apoE and apoE4 mice showed pathological alterations in the central nucleus of the amygdala, which is involved in regulation of anxiety. ApoE4, but not mice lacking apoE, or apoE3 mice showed impaired dexamethasone suppression of plasma corticosterone. Understanding how apoE modulates measures of anxiety might help the developments of therapeutic targets to reduce or even prevent measures of anxiety in health and in dementing illnesses.