Apolipoprotein E-knockout Mice Research Articles

FC 120: Lipid Changes as Early Indicator for Diabetes Induced Renal Pathology

Abstract BACKGROUND AND AIMS Diabetic nephropathy (DN), chronic loss of kidney function due to diabetes mellitus, is a common complication in diabetes which currently lacks effective long-term treatment. Microalbuminuria is now the earliest marker for DN, thereby disregarding renal changes that occur before this first indication of decreased kidney function. For better diagnosis and treatment strategies, it is important to study diabetes induced early renal changes. Renal-specific changes of the lipid metabolism could serve as an indicator for early kidney damage. Mass spectrometry imaging (MSI) allows to study the lipid metabolism in the context of tissue histology without the need for labeling (i.e. antibody or radioactive). Existing literature applying this technique focuses on later stages of diabetic kidney disease, thereby overlooking its potential to serve in an earlier stage of diagnosis. The aim of this project is to identify renal cell-specific lipid changes using MSI which could serve as damage markers for early diabetes induced renal pathology. METHOD To study diabetic renal changes, apolipoprotein E-knockout mice were treated with streptozotocin (STZ) and put on an enriched cholesterol diet to induce diabetes. After 12 weeks, both control (n = 4) and diabetic (n = 4) mice were sacrificed and kidneys were harvested for immunohistochemical and matrix assisted laser desorption/ionization (MALDI) MSI. Post-MSI immunofluorescent staining in combination with tissue morphology was used to identify different renal cell types. Analysis of MALDI-MSI data allows us to spatially segment the results per cell type for further statistical analysis of the metabolic MSI data (Figure 1A). RESULTS Two weeks after STZ induction, blood glucose levels of the diabetic mice were significantly elevated compared to control. Spatial segmentation analysis of the MSI data revealed that in both groups different renal cell types could clearly be distinguished based on their metabolic profile. Using the immunofluorescence-based cell type annotation, we zoomed in on various morphological regions of the kidney to specifically test for changes in lipid profiles. Here, the proximal tubular cells in the cortex and the outer stripe of outer medulla had the highest number of significantly changed lipids (Figure 1B). Focusing the analysis on these two groups of cells revealed specific molecular signatures to be discriminative between diabetes and control (Figure 1C). CONCLUSION Using MSI, we were able to identify renal cell type-specific differentially expressed lipids in diabetic mice compared to the healthy control group. The proximal tubular cells in the cortex and outer stripe of the outer medulla had the most altered lipid composition. These molecular signatures might serve as an early indicator of diabetes induced renal changes, thereby opening up a potential window for treatment before the kidney is damaged beyond repair.

Abstract 314: The Alteration In Cell Mechanics In Atherosclerosis

Atherosclerosis remains a major cause of cardiovascular disease. It is well known that the cholesterol accumulated in the macrophage-derived foam cells is the major component of atherosclerotic plaque. However, the growing evidence suggests that about 40% of the total foam cells in the atherosclerotic plaque were vascular smooth muscle cells (VSMCs)-derived. VSMCs are the major cellular components of arterial wall and undergo phenotypic switching from a functional contractile phenotype to a synthetic proliferative phenotype during the development of atherosclerosis. In addition, the surrounding environment of VSMCs within vascular wall changes with alterations in extracellular matrix (ECM) stiffness and composition. The main goal of this study is to test hypothesis that VSMC mechanics undergoes significant changes in response to these surrounding microenvironment alteration in the progression of atherosclerosis. We employed an innovative approach integrating a series of novel experimental approaches and data-driven mathematical models to monitor the real time mechanics of VSMCs isolated from western diet fed apolipoprotein-E knockout (ApoE -/- ) and wild type (WT) mice. The results demonstrated significant differences in the actin cytoskeletal organization and cell mechanics between ApoE -/- VSMCs and WT cells, which may trigger the VSMC migration and contribute to the formation of the arterial plaque. These interesting finding will lead to a novel therapeutic strategy for the pharmaceutical treatment of atherosclerosis by interfering the alteration in VSMC mechanics.

Chitin oligosaccharides alleviate atherosclerosis progress in ApoE-/- mice by regulating lipid metabolism and inhibiting inflammation

Atherosclerosis is driven both by hyperlipidemia and inflammation. Chitin oligosaccharides (NACOS) have shown pharmacological effects on multiple diseases via hypolipidemic and/or anti-inflammatory activities. The present study aims to evaluate whether NACOS treatment can prevent atherosclerosis induced by a high-fat-diet (HFD) in Apolipoprotein E-knockout (ApoE-/-) mice. Results showed that 300 and 900mg/kg b.w./day NACOS supplementation for 14 weeks significantly decreased atherosclerotic lesions up to 45% and 67% in compared with the HFD (P < 0.05), as measured in the valve area of the aortic root. Further, NACOS supplementation significantly reduced the serum hyperlipidemia and circulating proinflammatory cytokines including interleukin-1β, interleukin-6, monocyte chemoattractant protein-1 and tumor necrosis factor-α. NACOS decreased the hepatic Hmgcr to reduce cholesterol synthesis, activated the genes involved in reverse cholesterol transport to enhance cholesterol efflux and excretion, and reduced the intestinal Npc1L1 to lower cholesterol absorption. Additionally, NACOS enhanced cecum short chain fatty acids production and intestinal integrity. Thus, NACOS supplementation ameliorated atherosclerosis via altering lipid metabolism and reducing inflammation. These findings indicate that NACOS may be a potential functional food material for attenuating atherosclerosis development.

Adoptive transfer of metabolically reprogrammed macrophages for atherosclerosis treatment in diabetic ApoE−/- mice

Atherosclerosis is characterized by inflammation in the arterial wall, which is known to be exacerbated by diabetes. Therapeutic repression of inflammation is a promising strategy for treating atherosclerosis. In this study, we showed that diabetes aggravated atherosclerosis in apolipoproteinE knockout (ApoE−/-) mice, in which increased expression of long-chain acyl-CoA synthetase 1 (Acsl1) in macrophages played an important role. Knockdown of Acsl1 in macrophages (MφshAcsl1) reprogrammed macrophages to an anti-inflammatory phenotype, especially under hyperglycemic conditions. Injection of MφshAcsl1 reprogrammed macrophages into streptozotocin (STZ)-induced diabetic ApoE−/- mice (ApoE−/-+ STZ) alleviated inflammation locally in the plaque, liver and spleen. Consistent with the reduction in inflammation, plaques became smaller and more stable after the adoptive transfer of reprogrammed macrophages. Taken together, our findings indicate that increased Acsl1 expression in macrophages play a key role in aggravated atherosclerosis of diabetic mice, possibly by promoting inflammation. Adoptive transfer of Acsl1 silenced macrophages may serve as a potential therapeutic strategy for atherosclerosis.

Expression and Significance of Programmed Death-1 and Its Ligands in the Accelerated Formation of Atherosclerosis in an Induced Murine Lupus Model.

Atherosclerosis (AS) is a chronic inflammatory disease that occurs in artery walls, which seriously affects the survival and prognosis of patients with systemic lupus erythematosus (SLE). Immune and inflammatory responses have notable effects on all stages of AS. In this study, we modeled SLE combined with AS in vivo via intraperitoneal injection of pristane (2,6,10,14-tetramethylpentadecane) into apolipoprotein E-knockout (ApoE-/- ) mice that had accelerated atherosclerotic lesions compared with wild-type (WT) ApoE-/- mice. In pristane-induced ApoE-/- mice, expression of programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) in peripheral blood and on the surfaces of atherosclerotic lesions significantly increased, and levels of proinflammatory cytokines, namely, interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in peripheral blood were elevated. We did not detect expression of programmed death-ligand 2 (PD-L2) in the arterial plaques of either pristane-induced or WT ApoE-/- mice, nor did we observe any significant difference in PD-L2 expression in peripheral blood between the two groups. Taken together, these results suggested that PD-1/PD-L1 signaling pathway might play an important regulatory role in the progression of AS in an induced murine lupus model which implies a potential target for treatment of AS in SLE.

Inhaled silica nanoparticles exacerbate atherosclerosis through skewing macrophage polarization towards M1 phenotype

Background and aimsExposure to environmental nanoparticles is related to the adverse impact on health, including cardiovascular system. Various forms of nanoparticles have been reported to interact with endothelium and induce inflammation. However, the potential role of nanoparticles in the pathogenesis of atherosclerosis and their mechanisms of action are still unclear. The aim of this study was to investigate the effect of two broadly used nanomaterials, which also occur in natural environment - silicon oxide (SiO2) and ferric oxide (Fe2O3) in the form of nanoparticles (NPs) - on the development of atherosclerosis. MethodsWe used apolipoprotein E-knockout mice exposed to silica and ferric oxide nanoparticles in a whole body inhalation chamber. ResultsInhaled silica nanoparticles augmented the atherosclerotic lesions and increased the percentage of pro-inflammatory M1 macrophages in both the plaque and the peritoneum in apoE-/- mice. Exposure to ferric oxide nanoparticles did not enhance atherogenesis process, however, it caused significant changes in the atherosclerotic plaque composition (elevated content of CD68-positive macrophages and enlarged necrotic core accompanied by the decreased level of M1 macrophages). Both silica and ferric oxide NPs altered the phenotype of T lymphocytes in the spleen by promoting polarization towards Th17 cells. ConclusionsExposure to silica and ferric oxide nanoparticles exerts impact on atherosclerosis development and plaque composition. Pro-atherogenic abilities of silica nanoparticles are associated with activation of pro-inflammatory macrophages.

Natural Killer T Cells Are Involved in Atherosclerotic Plaque Instability in Apolipoprotein-E Knockout Mice.

The infiltration and activation of macrophages as well as lymphocytes within atherosclerotic lesion contribute to the pathogenesis of plaque rupture. We have demonstrated that invariant natural killer T (iNKT) cells, a unique subset of T lymphocytes that recognize glycolipid antigens, play a crucial role in atherogenesis. However, it remained unclear whether iNKT cells are also involved in plaque instability. Apolipoprotein E (apoE) knockout mice were fed a standard diet (SD) or a high-fat diet (HFD) for 8 weeks. Moreover, the SD- and the HFD-fed mice were divided into two groups according to the intraperitoneal injection of α-galactosylceramide (αGC) that specifically activates iNKT cells or phosphate-buffered saline alone (PBS). ApoE/Jα18 double knockout mice, which lack iNKT cells, were also fed an SD or HFD. Plaque instability was assessed at the brachiocephalic artery by the histological analysis. In the HFD group, αGC significantly enhanced iNKT cell infiltration and exacerbated atherosclerotic plaque instability, whereas the depletion of iNKT cells attenuated plaque instability compared to PBS-treated mice. Real-time PCR analyses in the aortic tissues showed that αGC administration significantly increased expressional levels of inflammatory genes such as IFN-γ and MMP-2, while the depletion of iNKT cells attenuated these expression levels compared to those in the PBS-treated mice. Our findings suggested that iNKT cells are involved in the exacerbation of plaque instability via the activation of inflammatory cells and upregulation of MMP-2 in the vascular tissues.

Abstract 13322: Development of the Preclinical Science Integration and Translation (PRESCIANT) Method and Application to the Apolipoprotein E Knockout Mouse Atherosclerosis Model

Introduction: Animal models of human diseases are used extensively to interrogate molecular mechanisms in a reductionist fashion. We tested whether aggregation and integration of preclinical data can identify new causative pathways that faithfully model human disease mechanisms. We have termed this novel technique, the Preclinical Science Integration and Translation (PRESCIANT) method. Methods and Results: Data were extracted from 716 manuscripts in the journal Arteriosclerosis, Thrombosis, and Vascular Biology from 1995-2019 using the apolipoprotein E knockout mouse (ApoE-KO) to study atherosclerosis. We identified 360 unique studies in which genes were experimentally perturbed in ApoE-KO mice to impact atherosclerotic plaque size and/or composition. Impacts of the interventions on plaque size, inflammation, and lipid content were analyzed using Ingenuity Pathway Analysis. The top upstream regulatory network (sc-58125, a COX2 inhibitor) linked 37.2% (134) of the genes implicated in atherosclerosis into a single network. Further, Ingenuity Pathway Analysis identified TREM1 signaling, LXR/RXR activation, and renin-angiotensin signaling as the top 3 pathways associated with changes in atherosclerosis parameters. Specifically, early atherogenesis genes were enriched with pathways associated with inflammatory cell migration and infiltration (including TREM1) whereas late atherosclerosis genes were associated with cell metabolism and survival (including LXR/RXR activation). These two pathways were interrogated in a clinical cohort of 88,660 patients by testing for association between genetically predicted expression of the human homologs of mouse pathway genes and a composite phenotype composed of 27 human atherosclerosis diagnoses. There was a significant enrichment (p&lt;0.01 for both pathways) in the number of human homologs associated with atherosclerosis, including 12 (57.2%) genes in the TREM1 pathway and 15 (53.6%) in the LXR/RXR pathway. Conclusion: PRESCIANT can successfully leverage decades of animal investigations to translate results from large-volume singular preclinical studies to make novel causal inferences into human disease.

Abstract 9079: Omentin Reduces Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in Apolipoprotein E Knockout Mice

Background: Abdominal aortic aneurysm (AAA) is a life-threatening disease worldwide. Obesity is associated with an increased risk of AAA. Omentin is an adipokine, which is downregulated by obesity. Here we investigated the effect of omentin on angiotensin (Ang) II-induced AAA formation in apolipoprotein-E knockout (apoE-KO) mice. Methods and Results: ApoE-KO mice were crossed with transgenic mice expressing the human omentin in fat tissue (OMT-Tg mice). ApoE-KO/OMT-Tg and apoE-KO mice were subjected to continuous Ang II infusion. ApoE-KO/OMT-Tg mice exhibited a reduced maximal diameter of AAA compared with apo-E KO mice evaluated by macroscopic and ultrasound analyses. ApoE-KO/OMT-Tg mice showed a reduction of elastic fiber degradation in aorta compared with apoE-KO mice. ApoE-KO/OMT-Tg mice also displayed reduced expression levels of matrix metalloproteinase (MMP) 9, MMP2, pro-inflammatory mediators and macrophage surface marker, F4/80, in aortic walls compared with apo-E-KO mice. Conversely, intravenous administration of adenoviral vectors expressing omentin (Ad-OMT) significantly reduced maximal diameter and elastic fiber degradation of abdominal aorta after Ang II infusion in apoE-KO mice compared with control Ad-β-gal treatment. Treatment of human monocyte-derived macrophages with omentin protein attenuated activity and expression of MMP9 and NF-κB phosphorylation after stimulation with lipopolysaccharide. Treatment of human aortic smooth muscle cells with omentin protein attenuated activity and expression of MMP2 after stimulation with tumor necrosis factor α. The suppressive effects of omentin on MMP9 and MMP2 expression in macrophages and vascular smooth muscle cells were reversed by inhibition of PI3-kinase/Akt signaling or blockade of integrin αVβ3. Finally, continuous administration of PI3-kinase inhibitor LY294002 reversed the suppressive effects of Ad-OMT on maximal diameter and elastic fiber degradation of abdominal aorta in apoE-KO mice after Ang II infusion. Conclusion: Our data indicate that omentin acts as an adipokine that can attenuate Ang II-induced development of AAA by reducing MMP9 and MMP2 expression through the integrin αVβ3/PI3-kinase/Akt signaling in the vascular wall.

Cell proliferation detected using [18F]FLT PET/CT as an early marker of abdominal aortic aneurysm

BackgroundAbdominal aortic aneurysm (AAA) is a focal aortic dilatation progressing towards rupture. Non-invasive AAA-associated cell proliferation biomarkers are not yet established. We investigated the feasibility of the cell proliferation radiotracer, fluorine-18-fluorothymidine ([18F]FLT) with positron emission tomography/computed tomography (PET/CT) in a progressive pre-clinical AAA model (angiotensin II, AngII infusion). Methods and ResultsFourteen-week-old apolipoprotein E-knockout (ApoE−/−) mice received saline or AngII via osmotic mini-pumps for 14 (n = 7 and 5, respectively) or 28 (n = 3 and 4, respectively) days and underwent 90-minute dynamic [18F]FLT PET/CT. Organs were harvested from independent cohorts for gamma counting, ultrasound scanning, and western blotting. [18F]FLT uptake was significantly greater in 14- (n = 5) and 28-day (n = 3) AAA than in saline control aortae (n = 5) (P < 0.001), which reduced between days 14 and 28. Whole-organ gamma counting confirmed greater [18F]FLT uptake in 14-day AAA (n = 9) compared to saline-infused aortae (n = 4) (P < 0.05), correlating positively with aortic volume (r = 0.71, P < 0.01). Fourteen-day AAA tissue showed increased expression of thymidine kinase-1, equilibrative nucleoside transporter (ENT)-1, ENT-2, concentrative nucleoside transporter (CNT)-1, and CNT-3 than 28-day AAA and saline control tissues (n = 3 each) (all P < 0.001). Conclusions[18F]FLT uptake is increased during the active growth phase of the AAA model compared to saline control mice and late-stage AAA.

Abstract P435: Probiotic Supplements Could Prevent Diet-induced Mitochondrial Dysfunction In The Liver Hepatocytes Of Apolipoprotein-e Knockout Mice

A poor diet can modify the intestinal microbiota, known as gut dysbiosis, which causes hepatocyte mitochondrial dysfunction through the release of endotoxins in the portal vein. Mitochondrial dysfunction in the liver leads to non-alcoholic fatty liver disease (NAFLD). Epidemiological evidence shows that most deaths from NAFLD are related to cardiovascular diseases (CVD) demonstrated by impaired vasodilatation as well as increased arterial stiffness, calcification, inflammation, and intimal-medial thickness. Therefore, the objective of our study was to investigate the potential of probiotic supplements to prevent a diet-induced mitochondrial dysfunction in hepatocytes. Apolipoprotein-E knockout mice—genetically modified animals that develop dyslipidemia—were placed in 3 diet groups including a control, western style (high in fats and carbohydrates), as well as a low-carbohydrate high-protein diet. Each group was then further divided in 3 subgroups based on the dose of probiotics administered, including a control, low (0.5 B/dose) and high (5 B/dose) dose of probiotics, for a total of 9 groups (n= 8-12 animals/group). The probiotic supplements contained a 1:1 ratio of Lactobacillus Helveticus and Bifidobacterium Bifidum . The bodyweight and water intake were monitored weekly. After 6 weeks, the liver mitochondria were analyzed by high resolution respirometry using a sequential substrate addition protocol (malate, octanoylcarnitine, ADP, glutamate, pyruvate, cytochrome C, succinate and FCCP) to investigate mitochondrial maximal respiration, leak, membrane damage and uncoupling. Our preliminary results show that the groups that received a high dose of probiotics gained less weight, had a higher respiration following the addition of ADP (a biomarker of complex V efficiency) and a lower respiration following FCCP (a biomarker of mitochondrial uncoupling), as compared to control. Supplements of probiotics could promote weight loss and prevent a diet-induced mitochondrial dysfunction in liver hepatocytes, an important risk factor for NAFLD and therefore, CVD. This project could translate into future research directions in preventative medicine, for example by investigating the potential of probiotics in the prevention of mitochondrial dysfunction in other organs, including the heart and the vascular system.

Aggregate Trends of Apolipoprotein E on Cognition in Transgenic Alzheimer's Disease Mice.

Background:Apolipoprotein E (APOE) genotypes typically increase risk of amyloid-β deposition and onset of clinical Alzheimer’s disease (AD). However, cognitive assessments in APOE transgenic AD mice have resulted in discord.Objective:Analysis of 31 peer-reviewed AD APOE mouse publications (n = 3,045 mice) uncovered aggregate trends between age, APOE genotype, gender, modulatory treatments, and cognition.Methods:T-tests with Bonferroni correction (significance = p < 0.002) compared age-normalized Morris water maze (MWM) escape latencies in wild type (WT), APOE2 knock-in (KI2), APOE3 knock-in (KI3), APOE4 knock-in (KI4), and APOE knock-out (KO) mice. Positive treatments (t+) to favorably modulate APOE to improve cognition, negative treatments (t–) to perturb etiology and diminish cognition, and untreated (t0) mice were compared. Machine learning with random forest modeling predicted MWM escape latency performance based on 12 features: mouse genotype (WT, KI2, KI3, KI4, KO), modulatory treatment (t+, t–, t0), mouse age, and mouse gender (male = g_m; female = g_f, mixed gender = g_mi).Results:KI3 mice performed significantly better in MWM, but KI4 and KO performed significantly worse than WT. KI2 performed similarly to WT. KI4 performed significantly worse compared to every other genotype. Positive treatments significantly improved cognition in WT, KI4, and KO compared to untreated. Interestingly, negative treatments in KI4 also significantly improved mean MWM escape latency. Random forest modeling resulted in the following feature importance for predicting superior MWM performance: [KI3, age, g_m, KI4, t0, t+, KO, WT, g_mi, t–, g_f, KI2] = [0.270, 0.094, 0.092, 0.088, 0.077, 0.074, 0.069, 0.061, 0.058, 0.054, 0.038, 0.023].Conclusion:APOE3, age, and male gender was most important for predicting superior mouse cognitive performance.

Regulation of NADPH Oxidase-Mediated Superoxide Production by Acetylation and Deacetylation.

Oral treatment of apolipoprotein E-knockout (ApoE-KO) mice with the putative sirtuin 1 (SIRT1) activator resveratrol led to a reduction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in the heart. In contrast, the SIRT1 inhibitor EX527 enhanced the superoxide production in isolated human polymorphonuclear granulocytes. In human monocytic THP-1 cells, phorbol ester-stimulated superoxide production was enhanced by inhibitors of histone deacetylases (HDACs; including quisinostat, trichostatin A (TSA), PCI34051, and tubastatin A) and decreased by inhibitors of histone acetyltransferases [such as garcinol, curcumin, and histone acetyltransferase (HAT) Inhibitor II]. These results indicate that protein acetylation and deacetylation may represent crucial mechanisms regulating NADPH oxidase-mediated superoxide production. In cell-free systems, incubation of recombinant Rac1 with SIRT1 resulted in decreased Rac1 acetylation. Mass spectrometry analyses identified lysine 166 (K166) in Rac1 as a residue targeted by SIRT1. Deacetylation of Rac1 by SIRT1 markedly reduced the interaction of Rac1 with p67phox in in vitro assays. Computational modeling analyses revealed that K166 deacetylation of Rac1 led to a 5-fold reduction in its binding affinity to guanosine-5'-triphosphate, and a 21-fold decrease in its binding potential to p67phox. The latter is crucial for Rac1-mediated recruitment of p67phox to the membrane and for p67phox activation. In conclusion, both SIRT1 and non-sirtuin deacetylases play a role in regulating NADPH oxidase activity. Rac1 can be directly deacetylated by SIRT1 in a cell-free system, leading to an inhibition of Rac1-p67phox interaction. The downstream targets of non-sirtuin deacetylases are still unknown. The in vivo significance of these findings needs to be investigated in future studies.

Atherosclerosis Impairs Naive CD4 T-Cell Responses via Disruption of Glycolysis.

CD4 T cells are important regulators of atherosclerotic progression. The metabolic profile of CD4 T cells controls their signaling and function, but how atherosclerosis affects T-cell metabolism is unknown. Here, we sought to determine the impact of atherosclerosis on CD4 T-cell metabolism and the contribution of such metabolic alterations to atheroprogression. Using PCR arrays, we profiled the expression of metabolism genes in CD4 T cells from atherosclerotic apolipoprotein-E knockout mice fed a Western diet. These cells exhibited dysregulated expression of genes critically involved in glycolysis and fatty acid degradation, compared with those from animals fed a standard laboratory diet. We examined how T-cell metabolism was changed in either Western diet–fed apolipoprotein-E knockout mice or samples from patients with cardiovascular disease by measuring glucose uptake, activation, and proliferation in CD4 T cells. We found that naive CD4 T cells from Western diet–fed apolipoprotein-E knockout mice failed to uptake glucose and displayed impaired proliferation and activation, compared with CD4 T cells from standard laboratory diet–fed animals. Similarly, we observed that naive CD4 T-cell frequencies were reduced in the circulation of human subjects with high cardiovascular disease compared with low cardiovascular disease. Naive T cells from high cardiovascular disease subjects also showed reduced proliferative capacity. These results highlight the dysfunction that occurs in CD4 T-cell metabolism and immune responses during atherosclerosis. Targeting metabolic pathways within naive CD4 T cells could thus yield novel therapeutic approaches for improving CD4 T-cell responses against atheroprogression.

Antioxidant and Anti-atherogenic Properties of Prosopis strombulifera and Tessaria absinthioides Aqueous Extracts: Modulation of NADPH Oxidase-Derived Reactive Oxygen Species.

Despite popular usage of medicinal plants, their effects as cardiovascular protective agents have not been totally elucidated. We hypothesized that treatment with aqueous extract from Prosopis strombulifera (AEPs) and Tessaria absinthioides (AETa), Argentinian native plants, produces antioxidant effects on vascular smooth muscle cells (VSMCs) and attenuates atherogenesis on apolipoprotein E-knockout (ApoE-KO) mice. In VSMCs, both extracts (5–40 μg/ml) inhibited 10% fetal calf serum-induced cell proliferation, arrested cell in G2/M phase, reduced angiotensin II-induced reactive oxygen species (ROS) generation, and decreased NADPH oxidase subunit expression. In ApoE-KO mice, extracts significantly reduced triglycerides and lipid peroxidation [plasma thiobarbituric acid reactive substances (TBARS)], increased plasma total antioxidant status (TAS), and improved glutathione peroxidase activity in the liver. Under high-fat diet (HFD), both extracts were able to inhibit O2– generation in the aortic tissue and caused a significant regression of atheroma plaques (21.4 ± 1.6% HFD group vs. 10.2 ± 1.2%∗ AEPs group and 14.3 ± 1.0%∗ AETa group; ∗p < 0.01). Consumption of AEPs and AETa produces antioxidant/antimitogenic/anti-atherosclerotic effects, and their use may be beneficial as a complementary strategy regarding cardiovascular disease therapies.

Aneurysm severity is suppressed by deletion of CCN4

Patients with abdominal aortic aneurysms are frequently treated with high-risk surgery. A pharmaceutical treatment to reverse aneurysm progression could prevent the need for surgery and save both lives and healthcare resources. Since CCN4 regulates cell migration, proliferation and apoptosis, processes involved in aneurysm progression, it is a potential regulator of aneurysm progression. We investigated the role of CCN4 in a mouse aneurysm model, using apolipoprotein-E knockout (ApoE−/−) mice fed high fat diet and infused with Angiotensin II (AngII). Blood pressure was similarly elevated in CCN4−/−ApoE−/− mice and CCN4+/+ApoE−/− mice (controls) in response to AngII infusion. Deletion of CCN4 significantly reduced the number of ruptured aortae, both thoracic and abdominal aortic area, and aneurysm grade score, compared to controls. Additionally, the frequency of vessel wall remodelling and the number of elastic lamina breaks was significantly suppressed in CCN4−/−ApoE−/− mice compared to controls. Immunohistochemistry revealed a significantly lower proportion of macrophages, while the proportion of smooth muscle cells was not affected by the deletion of CCN4. There was also a reduction in both proliferation and apoptosis in CCN4−/−ApoE−/− mice compared to controls. In vitro studies showed that CCN4 significantly increased monocyte adhesion beyond that seen with TNFα and stimulated macrophage migration by more than threefold. In summary, absence of CCN4 reduced aneurysm severity and improved aortic integrity, which may be the result of reduced macrophage infiltration and cell apoptosis. Inhibition of CCN4 could offer a potential therapeutic approach for the treatment of aneurysms.

Protective Role of Dietary Capsanthin in a Mouse Model of Nonalcoholic Fatty Liver Disease.

Capsanthin is the main carotenoid compound in red paprika (Capsicum annuum L.). However, little is known about the beneficial effects of capsanthin in nonalcoholic fatty liver disease (NAFLD). In this study, the hepatoprotective activity of capsanthin was investigated in a mouse model of NAFLD. Apolipoprotein-E knockout mice were fed with normal diet, Western-type diet (WD, NAFLD model), WD with capsanthin (0.5 mg/kg of body weight/day, CAP), WD with capsanthin-rich extract (25 mg/kg of body weight/day; CRE), or WD with red paprika powder (25 mg/kg of body weight/day, RPP) for 12 weeks. The carotenoid content in CRE or RPP was analyzed using ultraperformance liquid chromatography. The capsanthin concentration in CRE was 2067 mg/100 g of dry weight, which was 63% of total carotenoids. The oral administration of CRE or capsanthin significantly reduced the WD-induced increase in body weight and lipid accumulation in the liver (vs. the RPP group). In addition, CRE or capsanthin significantly inhibited the WD-induced increase in cholesterol and low-density lipoprotein levels. Furthermore, CRE or capsanthin showed reduced levels of plasma alanine and aspartate aminotransferase (ALT and AST, respectively), suggesting a steatohepatitis protective effect. Capsanthin regulated mRNA levels of peroxisome proliferator-activated receptor alpha (Pparα), carnitine palmitoyltransferase 1A (Cpt1a), acyl-CoA oxidase 1 (Acox1), and sterol regulatory element binding protein-1c (Srebp1c), which are associated with hepatic fatty acid metabolism. Overall, our results suggest that the capsanthin of red paprika plays a protective role against hepatic steatosis/steatohepatitis in NAFLD.

Omentin attenuates angiotensin II-induced abdominal aortic aneurysm formation in apolipoprotein E-knockout mice.

Abdominal aortic aneurysm (AAA) is an increasing and life-threatening disease. Obesity contributes to an increased risk of AAA. Omentin is a circulating adipokine, which is downregulated in obese complications. Here, we examined whether omentin could modulate angiotensin (Ang) II-induced AAA formation in apolipoprotein E-knockout (apoE-KO) mice. apoE-KO mice were crossed with transgenic mice expressing the human omentin gene in fat tissue (OMT-Tg mice) to generate apoE-KO/OMT-Tg mice. apoE-KO/OMT-Tg and apoE-KO mice were subjected to continuous Ang II infusion by using osmotic mini pumps. apoE-KO/OMT-Tg mice exhibited a lower incidence of AAA formation and a reduced maximal diameter of AAA compared with apoE-KO mice. apoE-KO/OMT-Tg mice showed attenuated disruption of medial elastic fibres in response to Ang II compared with apoE-KO mice. apoE-KO/OMT-Tg mice also displayed reduced expression levels of matrix metalloproteinase (MMP) 9, MMP2, and pro-inflammatory genes in aortic walls compared with apoE-KO mice. Furthermore, systemic administration of omentin also attenuated AAA formation and disruption of medial elastic fibres in response to Ang II in apoE-KO mice. Treatment of human monocyte-derived macrophages with omentin protein attenuated expression of MMP9 and pro-inflammatory mediators, and MMP9 activation after stimulation with lipopolysaccharide. Treatment of human vascular smooth muscle cells (VSMCs) with omentin protein reduced expression and activation of MMP2 after stimulation with tumour necrosis factor α. Omentin treatment increased phosphorylation levels of Akt in human macrophages and VSMCs. The suppressive effects of omentin on MMP9 and MMP2 expression were reversed by inhibition of integrin-αVβ3/PI3-kinase/Akt signalling in macrophages and VSMCs, respectively. These data suggest that omentin acts as an adipokine that can attenuate Ang II-induced development of AAA through suppression of MMP9 and MMP2 expression and inflammatory response in the vascular wall.

Pyk2/MCU Pathway as a New Target for Reversing Atherosclerosis.

Objective: Multiple mechanisms including vascular endothelial cell damage have a critical role in the formation and development of atherosclerosis (AS), but the specific molecular mechanisms are not exactly clarified. This study aims to determine the possible roles of proline-rich tyrosine kinase 2 (Pyk2)/mitochondrial calcium uniporter (MCU) pathway in AS mouse model and H2O2-induced endothelial cell damage model and explore its possible mechanisms.Approach and Results: The AS mouse model was established using apolipoprotein E-knockout (ApoE–/–) mice that were fed with a high-fat diet. It was very interesting to find that Pyk2/MCU expression was significantly increased in the artery wall of atherosclerotic mice and human umbilical vein endothelial cells (HUVECs) attacked by hydrogen peroxide (H2O2). In addition, down-regulation of Pyk2 by short hairpin RNA (shRNA) protected HUVECs from H2O2 insult. Furthermore, treatment with rosuvastatin on AS mouse model and H2O2-induced HUVEC injury model showed a protective effect against AS by inhibiting the Pyk2/MCU pathway, which maintained calcium balance, prevented the mitochondrial damage and reactive oxygen species production, and eventually inhibited cell apoptosis.Conclusion: Our results provide important insight into the initiation of the Pyk2/MCU pathway involved in AS-related endothelial cell damage, which may be a new promising target for atherosclerosis intervention.

HDL Improves Cholesterol and Glucose Homeostasis and Reduces Atherosclerosis in Diabetes-Associated Atherosclerosis.

Background and Aims Apolipoprotein A-I (ApoA-I), the main component of high-density lipoprotein (HDL), not only promotes reverse cholesterol transport (RCT) in atherosclerosis but also increases insulin secretion in pancreatic β-cells, suggesting that interventions which raise HDL levels may be beneficial in diabetes-associated cardiovascular disease (CVD). Previously, we showed that TNF-related apoptosis-inducing ligand (TRAIL) deletion in Apolipoprotein Eknockout (Apoe−/−) mice results in diabetes-accelerated atherosclerosis in response to a “Western” diet. Here, we sought to identify whether reconstituted HDL (rHDL) could improve features of diabetes-associated CVD in Trail−/−Apoe−/− mice. Methods and Results Trail−/−Apoe−/− and Apoe−/− mice on a “Western” diet for 12 weeks received 3 weekly infusions of either PBS (vehicle) or rHDL (containing ApoA-I (20 mg/kg) and 1-palmitoyl-2-linoleoyl phosphatidylcholine). Administration of rHDL reduced total plasma cholesterol, triglyceride, and glucose levels in Trail−/−Apoe−/− but not in Apoe−/− mice, with no change in weight gain observed. rHDL treatment also improved glucose clearance in response to insulin and glucose tolerance tests. Immunohistological analysis of pancreata revealed increased insulin expression/production and a reduction in macrophage infiltration in mice with TRAIL deletion. Furthermore, atherosclerotic plaque size in Trail−/−Apoe−/− mice was significantly reduced associating with increased expression of the M2 macrophage marker CD206, suggesting HDL's involvement in the polarization of macrophages. rHDL also increased vascular mRNA expression of RCT transporters, ABCA1 and ABCG1, in Trail−/−Apoe−/− but not in Apoe−/− mice. Conclusions. rHDL improves features of diabetes-associated atherosclerosis in mice. These findings support the therapeutic potential of rHDL in the treatment of atherosclerosis and associated diabetic complications. More studies are warranted to understand rHDL's mechanism of action.