The effect of cold exposure on serum cholesterol is dependent upon ApoE.

BACKGROUND: Several lines of evidence indicate that cold stimulation may not only activate brown adipose tissue (BAT) and the white adipose tissue (WAT), but also regulate the lipid metabolism and influence the development of atherosclerosis. However, the study of cold exposure affecting cholesterol metabolism have opposite results in different experiments, and Apolipoprotein E (ApoE) may play an important role. There is still a lack of complete research to illustrate this problem. METHODS: In this study, we first analyzed and discussed the activation of interscapular brown adipose tissue (iBAT), inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) under cold exposure (4 °C) in male wild-type C57BL/6 J (WT) and ApoE-deficient mice (ApoE-/-) fed high-fat diet (HFD) for 4 weeks. Subsequently, we investigated the effect of cold exposure on blood lipid profiles in both models. We further explored whether cold exposure can reduce serum cholesterol. RESULTS: In both WT and ApoE-/- mice, cold exposure activates iBAT and iWAT, as well as hardly affects eWAT. In WT mice,4 weeks cold exposure (4 °C) reduces serum triglyceride by 28%, cholesterol by 30% and LDL-cholesterol by 63%. In ApoE-/- mice, cold stimulation decreases serum triglyceride by 59%, but increases cholesterol by 20% and LDL-cholesterol by 25%. CONCLUSIONS: Based on these findings, we conclude that cold exposure decreases serum cholesterol is dependent upon the existence of ApoE.

Dietary Sterols and Sterol Oxidation Products on Atherosclerosis: An Insight Provided by Liver Proteomic and Lipidomic.

SCOPE: The development of atherosclerosis is closely associated with disorder of lipid metabolism. Dietary sterols and their oxidation products play a role in the pathogenesis of atherosclerosis. However, their effects on liver lipid metabolism during the atherosclerosis remain unknown. METHODS AND RESULTS: Here, we apply lipidomic and proteomic analysis on liver of ApoE-/- mice feed with phytosterols, cholesterol oxidation products (COPs), or phytosterol oxidation products (POPs) to profile lipid species and reveal the underlying mechanism. Dietary exposure of phytosterols, COPs, and POPs all reduce the accumulation of liver triglyceride (TG), but COPs and POPs accelerate the fibrosis of liver. Lipidomic analysis reveals that phytosterols mainly decrease the levels of phosphatidylinositol (PI), while COPs and POPs both increase the level of digalactosyldiacylglycerol (DGDG) and reduce TG with long-chain polyunsaturated fatty acids. Besides, COPs up-regulated levels of lipids associate with atherosclerosis risk, such as phosphatidylcholines (PC), phosphatidylethanolamine (PE) and ceramides (Cer). POPs down-regulate the level of acyl carnitine (AcCa). Furthermore, proteomic analysis shows that COPs promote oxidative phosphorylation and POPs inhibit the beta oxidation of fatty acids. CONCLUSIONS: This study reveals that phytosterols, COPs, and POPs differently change the composition and metabolism of glycerophospholipids, sphingolipids, and glycerolipids in liver of ApoE-/- mice.

IL-10-Dependent Amelioration of Chronic Inflammatory Disease by Microdose Subcutaneous Delivery of a Prototypic Immunoregulatory Small Molecule.

One of the interventional strategies to reestablish the immune effector/regulatory balance, that is typically altered in chronic inflammatory diseases (CID), is the reinforcement of endogenous immunomodulatory pathways as the one triggered by interleukin (IL)-10. In a recent work, we demonstrated that the subcutaneous (sc) administration of an IL-10/Treg-inducing small molecule-based formulation, using a repetitive microdose (REMID) treatment strategy to preferentially direct the effects to the regional immune system, delays the progression of atherosclerosis. Here we investigated whether the same approach using other IL-10-inducing small molecule, such as the safe, inexpensive, and widely available polyphenol curcumin, could induce a similar protective effect in two different CID models. We found that, in apolipoprotein E deficient mice, sc treatment with curcumin following the REMID strategy induced atheroprotection that was not consequence of its direct systemic lipid-modifying or antioxidant activity, but instead paralleled immunomodulatory effects, such as reduced proatherogenic IFNγ/TNFα-producing cells and increased atheroprotective FOXP3+ Tregs and IL-10-producing dendritic and B cells. Remarkably, when a similar strategy was used in the neuroinflammatory model of experimental autoimmune encephalomyelitis (EAE), significant clinical and histopathological protective effects were evidenced, and these were related to an improved effector/regulatory cytokine balance in restimulated splenocytes. The essential role of curcumin-induced IL-10 for neuroprotection was confirmed by the complete abrogation of the clinical effects in IL-10-deficient mice. Finally, the translational therapeutic prospection of this strategy was evidenced by the neuroprotection observed in mice starting the treatment one week after disease triggering. Collectively, results demonstrate the power of a simple natural IL-10-inducing small molecule to tackle chronic inflammation, when its classical systemic and direct pharmacological view is shifted towards the targeting of regional immune cells, in order to rationally harness its immunopharmacological potential. This shift implies that many well-known IL-10-inducing small molecules could be easily reformulated and repurposed to develop safe, innovative, and accessible immune-based interventions for CID.

Activity of Selected Group of Monoterpenes in Alzheimer's Disease Symptoms in Experimental Model Studies-A Non-Systematic Review.

Alzheimer's disease (AD) is the leading cause of dementia and cognitive function impairment. The multi-faced character of AD requires new drug solutions based on substances that incorporate a wide range of activities. Antioxidants, AChE/BChE inhibitors, BACE1, or anti-amyloid platelet aggregation substances are most desirable because they improve cognition with minimal side effects. Plant secondary metabolites, used in traditional medicine and pharmacy, are promising. Among these are the monoterpenes-low-molecular compounds with anti-inflammatory, antioxidant, enzyme inhibitory, analgesic, sedative, as well as other biological properties. The presented review focuses on the pathophysiology of AD and a selected group of anti-neurodegenerative monoterpenes and monoterpenoids for which possible mechanisms of action have been explained. The main body of the article focuses on monoterpenes that have shown improved memory and learning, anxiolytic and sleep-regulating effects as determined by in vitro and in silico tests-followed by validation in in vivo models.

Accelerated atherosclerosis caused by serum amyloid A response in lungs of ApoE-/- mice.

Airway exposure to eg particulate matter is associated with cardiovascular disease including atherosclerosis. Acute phase genes, especially Serum Amyloid A3 (Saa3), are highly expressed in the lung following pulmonary exposure to particles. We aimed to investigate whether the human acute phase protein SAA (a homolog to mouse SAA3) accelerated atherosclerotic plaque progression in Apolipoprotein E knockout (ApoE-/- ) mice. Mice were intratracheally (i.t.) instilled with vehicle (phosphate buffered saline) or 2 µg human SAA once a week for 10 weeks. Plaque progression was assessed in the aorta using noninvasive ultrasound imaging of the aorta arch as well as by en face analysis. Additionally, lipid peroxidation, SAA3, and cholesterol were measured in plasma, inflammation was determined in lung, and mRNA levels of the acute phase genes Saa1 and Saa3 were measured in the liver and lung, respectively. Repeated i.t. instillation with SAA caused a significant progression in the atherosclerotic plaques in the aorta (1.5-fold). Concomitantly, SAA caused a statistically significant increase in neutrophils in bronchoalveolar lavage fluid (625-fold), in pulmonary Saa3 (196-fold), in systemic SAA3 (1.8-fold) and malondialdehyde levels (1.14-fold), indicating acute phase response (APR), inflammation and oxidative stress. Finally, pulmonary exposure to SAA significantly decreased the plasma levels of very low-density lipoproteins - low-density lipoproteins and total cholesterol, possibly due to lipids being sequestered in macrophages or foam cells in the arterial wall. Combined these results indicate the importance of the pulmonary APR and SAA3 for plaque progression.

Thrombin-Par1 signaling axis disrupts COP9 signalosome subunit 3-mediated ABCA1 stabilization in inducing foam cell formation and atherogenesis.

ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1) play a vital role in promoting cholesterol efflux. Although, the dysregulation of these transporters was attributed as one of the mechanisms of atherogenesis, what renders their dysfunction is not well explored. Previously, we have reported that thrombin without having any effect on ABCG1 levels depletes ABCA1 levels affecting cholesterol efflux. In this study, we explored the mechanisms underlying thrombin-induced depletion of ABCA1 levels both in macrophages and smooth muscle cells. Under normal physiological conditions, COP9 signalosome subunit 3 (CSN3) was found to exist in complex with ABCA1 and in the presence of proatherogenic stimulants such as thrombin, ABCA1 was phosphorylated and dissociated from CSN3, leading to its degradation. Forced expression of CSN3 inhibited thrombin-induced ABCA1 ubiquitination and degradation, restored cholesterol efflux and suppressed foam cell formation. In Western diet (WD)-fed ApoE-/- mice, CSN3 was also disassociated from ABCA1 otherwise remained as a complex in Chow diet (CD)-fed ApoE-/- mice. Interestingly, depletion of CSN3 levels in WD-fed ApoE-/- mice significantly lowered ABCA1 levels, inhibited cholesterol efflux and intensified foam cell formation exacerbating the lipid laden atherosclerotic plaque formation. Mechanistic studies have revealed the involvement of Par1-Gα12-Pyk2-Gab1-PKCθ signaling in triggering phosphorylation of ABCA1 and its disassociation from CSN3 curtailing cholesterol efflux and amplifying foam cell formation. In addition, although both CSN3 and ABCA1 were found to be colocalized in human non-lesion coronary arteries, their levels were decreased as well as dissociated from each other in advanced atherosclerotic lesions. Together, these observations reveal for the first time an anti-atherogenic role of CSN3 and hence, designing therapeutic drugs protecting its interactions with ABCA1 could be beneficial against atherosclerosis.

Apolipoprotein E isoforms differentially regulate matrix metallopeptidase 9 function in Alzheimer's disease.

Apolipoprotein E (APOE) has been shown to influence amyloid-ß (Aß) clearance from the brain in an isoform-specific manner. Our prior work showed that Aß transit across the blood-brain-barrier was reduced by apoE4, compared to other apoE isoforms, due to elevated lipoprotein receptor shedding in brain endothelia. Recently, we demonstrated that matrix metallopeptidase 9 (MMP-9) induces lipoprotein receptor proteolysis in an apoE isoform-dependent manner, which impacts Aß elimination from the brain. The current studies interrogated the relationship between apoE and MMP-9 and found that apoE impacted proMMP-9 cellular secretion from brain endothelia (apoE2 < apoE3 = apoE4). In a cell-free assay, apoE dose-dependently reduced MMP-9 activity, with apoE4 showing a significantly weaker ability to inhibit MMP-9 function than apoE2 or apoE3. Finally, we observed elevated MMP-9 expression and activity in the cerebrovasculature of both human and animal AD brain specimens with an APOE4 genotype. Collectively, these findings suggest a role for apoE in regulating MMP-9 disposition and may describe the effect of apoE4 on Aß pathology in the AD brain.

The SGLT2 inhibitor canagliflozin suppresses lipid synthesis and interleukin-1 beta in ApoE deficient mice.

Sodium-glucose cotransporter 2 inhibitors such as canagliflozin lower blood glucose and reduce cardiovascular events in people with type 2 diabetes through mechanisms that are not fully understood. Canagliflozin has been shown to increase the activity of the AMP-activated protein kinase (AMPK), a metabolic energy sensor important for increasing fatty acid oxidation and energy expenditure and suppressing lipogenesis and inflammation, but whether AMPK activation is important for mediating some of the beneficial metabolic effects of canagliflozin has not been determined. We, therefore, evaluated the effects of canagliflozin in female ApoE-/- and ApoE-/-AMPK ß1-/- mice fed a western diet. Canagliflozin increased fatty acid oxidation and energy expenditure and lowered adiposity, blood glucose and the respiratory exchange ratio independently of AMPK ß1. Canagliflozin also suppressed liver lipid synthesis and the expression of ATP-citrate lyase, acetyl-CoA carboxylase and sterol response element-binding protein 1c independently of AMPK ß1. Canagliflozin lowered circulating IL-1ß and studies in bone marrow-derived macrophages indicated that in contrast with the metabolic adaptations, this effect required AMPK ß1. Canagliflozin had no effect on the size of atherosclerotic plaques in either ApoE-/- and ApoE-/-AMPK ß1-/- mice. Future studies investigating whether reductions in liver lipid synthesis and macrophage IL-1ß are important for the cardioprotective effects of canagliflozin warrant further investigation.

The Important Interface Between Apolipoprotein E and Neuroinflammation in Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent form of neurodegenerative disease, currently affecting over 5 million Americans with projections expected to rise as the population ages. The hallmark pathologies of AD are Aß plaques composed of aggregated beta-amyloid (Aß), and tau tangles composed of hyperphosphorylated, aggregated tau. These pathologies are typically accompanied by an increase in neuroinflammation as an attempt to ameliorate the pathology. This idea has pushed the field toward focusing on mechanisms and the influence neuroinflammation has on disease progression. The vast majority of AD cases are sporadic and therefore, researchers investigate genetic risk factors that could lead to AD. Apolipoprotein E (ApoE) is the largest genetic risk factor for developing AD. ApoE has 3 isoforms-ApoE2, ApoE3, and ApoE4. ApoE4 constitutes an increased risk of AD, with one copy increasing the risk about 4-fold and two copies increasing the risk about 15-fold compared to those with the ApoE3 allele. ApoE4 has been shown to play a role in Aß deposition, tau tangle formation, neuroinflammation and many subsequent pathways. However, while we know that ApoE4 plays a role in these pathways and virtually all aspects of AD, the exact mechanism of how ApoE4 impacts AD progression is murky at best and therefore the role ApoE4 plays in these pathways needs to be elucidated. This review aims to discuss the current literature regarding the pathways and mechanisms of ApoE4 in AD progression with a focus on its role in neuroinflammation.

sLRP1 (Soluble Low-Density Lipoprotein Receptor-Related Protein 1): A Novel Biomarker for P2Y12 (P2Y Purinoceptor 12) Receptor Expression in Atherosclerotic Plaques.

OBJECTIVE: Recent studies suggest that the P2Y12 (P2Y purinoceptor 12) receptor of vascular smooth muscle cells in atherosclerotic plaques aggravates atherosclerosis, and P2Y12 receptor inhibitors such as CDL (clopidogrel) may effectively treat atherosclerosis. It is imperative to identify an effective biomarker for reflecting the P2Y12 receptor expression on vascular smooth muscle cells in plaques. Approach and Results: We found that there was a positive correlation between the level of circulating sLRP1 (soluble low-density lipoprotein receptor-related protein 1) and the number of LRP1+ α-SMA+ (α-smooth muscle actin), P2Y12+, or P2Y12+ LRP1+ cells in plaques from apoE-/- mice fed a high-fat diet. Furthermore, activation of the P2Y12 receptor increased the expression and shedding of LRP1 in vascular smooth muscle cells by inhibiting cAMP (3'-5'-cyclic adenosine monophosphate)/PKA (protein kinase A)/SREBP-2 (sterol regulatory element binding transcription factor 2). Conversely, genetic knockdown or pharmacological inhibition of the P2Y12 receptor had the opposite effects. Additionally, CDL decreased the number of lesional LRP1+ α-SMA+ cells and the levels of circulating sLRP1 by activating cAMP/PKA/SREBP-2 in apoE-/- mice fed a high-fat diet. CONCLUSIONS: Our study suggests that sLRP1 may be a biomarker that reflects the P2Y12 receptor level in plaques and has the potential to be an indicator for administering P2Y12 receptor inhibitors for patients with atherosclerosis.

Effect of ApoE isoforms on mitochondria in Alzheimer disease.

OBJECTIVE: To test the hypothesis that ApoE isoforms affect mitochondrial structure and function that are related to cognitive impairment in Alzheimer disease (AD), we systematically investigated the effects of ApoE isoforms on mitochondrial biogenesis and dynamics, oxidative stress, synapses, and cognitive performance in AD. METHODS: We obtained postmortem human brain tissues and measured proteins that are responsible for mitochondrial biogenesis (peroxisome proliferator-activated receptor-gamma coactivator-1α [PGC-1α] and sirtuin 3 [SIRT3]), for mitochondrial dynamics (mitofusin 1 [MFN1], mitofusin 2 [MFN2], and dynamin-like protein 1 [DLP1]), for oxidative stress (superoxide dismutase 2 [SOD2] and forkhead-box protein O3a [Foxo3a]), and for synapses (postsynaptic density protein 95 [PSD95] and synapsin1 [Syn1]). A total of 46 cases were enrolled, including ApoE-ɛ4 carriers (n = 21) and noncarriers (n = 25). RESULTS: Levels of these proteins were compared between ApoE-ɛ4 carriers and noncarriers. ApoE-ɛ4 was associated with impaired mitochondrial structure and function, oxidative stress, and synaptic integrity in the human brain. Correlation analysis revealed that mitochondrial proteins and the synaptic protein were strongly associated with cognitive performance. CONCLUSION: ApoE isoforms influence mitochondrial structure and function, which likely leads to alteration in oxidative stress, synapses, and cognitive function. These mitochondria-related proteins may be a harbinger of cognitive decline in ApoE-ɛ4 carriers and provide novel therapeutic targets for prevention and treatment of AD.

Altered Tregs Differentiation and Impaired Autophagy Correlate to Atherosclerotic Disease.

Atherosclerosis is a progressive vascular disease representing the primary cause of morbidity and mortality in developed countries. Formerly, atherosclerosis was considered as a mere passive accumulation of lipids in blood vessels. However, it is now clear that atherosclerosis is a complex and multifactorial disease, in which the involvement of immune cells and inflammation play a key role. A variety of studies have shown that autophagy-a cellular catalytic mechanism able to remove injured cytoplasmic components in response to cellular stress-may be proatherogenic. So far, in this context, its role has been investigated in smooth muscle cells, macrophages, and endothelial cells, while the function of this catabolic protective process in lymphocyte functionality has been overlooked. The few studies carried out so far, however, suggested that autophagy modulation in lymphocyte subsets may be functionally related to plaque formation and development. Therefore, in this research, we aimed at better clarifying the role of lymphocyte subsets, mainly regulatory T cells (Tregs), in human atherosclerotic plaques and in animal models of atherosclerosis investigating the contribution of autophagy on immune cell homeostasis. Here, we investigate basal autophagy in a mouse model of atherosclerosis, apolipoprotein E (ApoE)-knockout (KO) mice, and we analyze the role of autophagy in driving Tregs polarization. We observed defective maturation of Tregs from ApoE-KO mice in response to tumor growth factor-ß (TGFß). TGFß is a well-known autophagy inducer, and Tregs maturation defects in ApoE-KO mice seem to be related to autophagy impairment. In this work, we propose that autophagy underlies Tregs maturation, advocating that the study of this process in atherosclerosis may open new therapeutic strategies.

Protocatechuic Acid Inhibits Vulnerable Atherosclerotic Lesion Progression in Older Apoe-/- Mice.

BACKGROUND: Normalization of arterial inflammation inhibits atherosclerosis. The preventive role for protocatechuic acid (PCA) in early-stage atherosclerosis is well recognized; however, its therapeutic role in late-stage atherosclerosis remains unexplored. OBJECTIVE: We investigated whether PCA inhibits vulnerable atherosclerosis progression by normalizing arterial inflammation. METHODS: Thirty-wk-old male apolipoprotein E-deficient (Apoe-/-) mice with vulnerable atherosclerotic lesions in the brachiocephalic artery were fed the AIN-93G diet alone (control) or supplemented with 0.003% PCA (wt:wt) for 20 wk. Lesion size and composition, IL-1ß, and NF-κB in the brachiocephalic arteries, and serum lipid profiles, oxidative status, and proinflammatory cytokines (e.g., IL-1ß, monocyte chemoattractant protein-1, and serum amyloid A) were measured. Moreover, the effect of PCA on the inflammation response was evaluated in efferocytic macrophages from C57BL/6J mice. RESULTS: Compared with the control treatment, dietary PCA supplementation significantly reduced lesion size (27.5%; P < 0.05) and also improved lesion stability (P < 0.05) as evidenced by increased thin fibrous cap thickness (31.7%) and collagen accumulation (58.3%), reduced necrotic core size (37.6%) and cellular apoptosis (73.9%), reduced macrophage accumulation (45.1%), and increased vascular smooth muscle cell accumulation (51.5%). Moreover, PCA supplementation inhibited IL-1ß expression (53.7%) and NF-κB activation (64.4%) in lesions. However, PCA supplementation did not change serum lipid profiles, total antioxidant capacity, and inflammatory cytokines. In efferocytic macrophages, PCA at 0.5 and 1 µmol/L inhibited Il1b/IL-1ß mRNA (27.2-46.5%) and protein (29.2-49.6%) expression and NF-κB activation (67.0-80.3%) by upregulation of MER proto-oncogene tyrosine kinase (MERTK) and inhibition of mitogen-activated protein kinase 3/1 (MAPK3/1). Strikingly, the similar pattern of the MERTK and MAPK3/1 changes in lesional macrophages of mice after PCA intervention in vivo was recapitulated. CONCLUSION: PCA inhibits vulnerable lesion progression in mice, which might partially be caused by normalization of arterial inflammation by upregulation of MERTK and inhibition of MAPK3/1 in lesional macrophages.

Transglutaminase 2 limits the extravasation and the resultant myocardial fibrosis associated with factor XIII-A deficiency.

BACKGROUND AND AIMS: Transglutaminase (TG) 2 and Factor (F) XIII-A have both been implicated in cardiovascular protection and repair. This study was designed to differentiate between two competing hypotheses: that TG2 and FXIII-A mediate these functions in mice by fulfilling separate roles, or that they act redundantly in this respect. METHODS: Atherosclerosis was assessed in brachiocephalic artery plaques of fat-fed mixed strain apolipoprotein (Apo)e deficient mice that lacked either or both transglutaminases. Cardiac fibrosis was assessed both in the mixed strain mice and also in C57BL/6J Apoe expressing mice lacking either or both transglutaminases. RESULTS: No difference was found in the density of buried fibrous caps within brachiocephalic plaques from mice expressing or lacking these transglutaminases. Cardiac fibrosis developed in both Apoe/F13a1 double knockout and F13a1 single knockout mice, but not in Tgm2 knockout mice. However, concomitant Tgm2 knockout markedly increased fibrosis, as apparent in both Apoe/Tgm2/F13a1 knockout and Tgm2/F13a1 knockout mice. Amongst F13a1 knockout and Tgm2/F13a1 knockout mice, the extent of fibrosis correlated with hemosiderin deposition, suggesting that TG2 limits the extravasation of blood in the myocardium, which in turn reduces the pro-fibrotic stimulus. The resulting fibrosis was interstitial in nature and caused only minor changes in cardiac function. CONCLUSIONS: These studies confirm that FXIII-A and TG2 fulfil different roles in the mouse myocardium. FXIII-A protects against vascular leakage while TG2 contributes to the stability or repair of the vasculature. The protective function of TG2 must be considered when designing clinical anti-fibrotic therapies based upon FXIII-A or TG2 inhibition.

Physical exercise inhibits atherosclerosis development by regulating the expression of neuropeptide Y in apolipoprotein E-deficient mice.

AIMS: Population-based studies have shown that exercise has anti-atherosclerotic effects, but the mechanisms underlying this cardiac protection are poorly understood. The aim of this study was to investigate if the anti-atherosclerotic effects of exercise are associated with changes in neuropeptide Y (NPY) expression in apolipoprotein E-deficient (ApoE-/-) mice. MAIN METHODS: Thirty-one male ApoE-/- mice were randomly divided into regular exercise (5 days/week), occasional exercise (1-2 days/week), and sedentary groups. After 8 weeks, atherosclerotic burden and plaque stability were measured by histological and morphological analysis. Quantitative real-time PCR and immunohistochemistry were used to measure the expression of NPY and its receptors in the aorta. KEY FINDINGS: Eight weeks of occasional exercise was equally effective as regular exercise at preventing atherosclerotic plaque formation and enhancing atherosclerotic plaque stability. This was shown by increased plaque collagen and smooth muscle cell content and decreased plaque lipid and macrophage content. The expression of NPY and its receptors in the vasculature was decreased in the regular exercise and occasional exercise groups, and this expression was significantly correlated with the progress of atherosclerosis. Moreover, exercise may reduce the activity of macrophages by down-regulating the expression of NPY Y1 receptors, thereby reducing the release of inflammatory cytokines. SIGNIFICANCE: These results suggest that exercise training can attenuate plaque burden and enhance atherosclerotic plaque stability. The anti-atherosclerotic effect of exercise appears to be, at least in part, dependent on down-regulation of the expression of NPY and its receptors (especially Y1 receptors) in the aorta.

Artemisinin attenuates the development of atherosclerotic lesions by the regulation of vascular smooth muscle cell phenotype switching.

AIMS: The purpose of this study was to investigate the therapeutic effect of artemisinin (ART) on atherosclerosis and explore the molecular mechanisms involved by RNA sequencing (RNA-Seq). MAIN METHODS: Eight-week-old male ApoE-/- mice were treated with ART for eight weeks. Atherosclerotic lesion sizes were determined by Oil Red O staining, and RNA-Seq was used to detect the profile of differentially expressed genes following the administration of ART. The expressions of contractile phenotypic markers were detected by western blot and qRT-PCR, and the ability of the MOVAS cells to migrate and proliferate were assessed using the wound healing and CCK8 assays. KEY FINDINGS: Artemisinin treatment significantly reduced plaque area in the ApoE-/- mice and increased the expression of contractile phenotypic markers. RNA-Seq of aorta tissue revealed a distinct change in gene expression patterns after the mice were treated with ART. Our bioinformatics analysis demonstrated that the most prominently enriched pathway was a set of genes involved in vascular smooth muscle contractile function. Using an in vitro cell model, we demonstrated that ART could effectively reverse PDGF-activated MOVAS migration and proliferation, and elevate the level of proteins involved in the contractile phenotype. SIGNIFICANCE: We provide in vivo and in vitro evidence supporting a role for ART in the suppression of atherosclerosis, partly through the inhibition of vascular smooth muscle cell phenotype switching to a de-differentiated phenotype. These data further advances our understanding for a potential role for ART and suggests that ART is an excellent candidate for the treatment of atherosclerosis.

APOE modifies the interaction of entorhinal cerebral blood flow and cortical thickness on memory function in cognitively normal older adults.

OBJECTIVE: The ε4 allele of the apolipoprotein E (APOE) gene increases risk for cognitive decline in normal and pathologic aging. However, precisely how APOE ε4 exerts its negative impact on cognition is poorly understood. The present study aimed to determine whether APOE genotype (ε4+ vs. ε4-) modifies the interaction of medial temporal lobe (MTL) resting cerebral blood flow (CBF) and brain structure (cortical thickness [CT], volume [Vo]) on verbal memory performance. METHODS: Multiple linear regression models were employed to investigate relationships between APOE genotype, arterial spin labeling MRI-measured CBF and FreeSurfer-based CT and Vo in four MTL regions of interest (left and right entorhinal cortex and hippocampus), and verbal memory performance among a sample of 117 cognitively normal older adults (41 ε4+, 76 ε4-) between the ages of 64 and 89 (mean age â€‹= â€‹73). RESULTS: Results indicated that APOE genotype modified the interaction of CBF and CT on memory in the left entorhinal cortex, such that the relationship between entorhinal CBF and memory was negative (lower CBF was associated with better memory) in non-carriers with higher entorhinal CT, positive (higher CBF was associated with better memory) in non-carriers with lower entorhinal CT, and negative (higher CBF was associated with worse memory) in ε4 carriers with lower entorhinal CT. CONCLUSIONS: Findings suggest that older adult APOE ε4 carriers may experience vascular dysregulation and concomitant morphological alterations in the MTL that interact to negatively affect memory even in the absence overt clinical symptoms, providing potential insight into the mechanistic link between APOE ε4 and detriments in cognition. Moreover, findings suggest a distinct multimodal neural signature in ε4 carriers (higher CBF and lower CT in the entorhinal cortex) that could aid in the identification of candidates for future clinical trials aimed at preventing or slowing cognitive decline. Differential findings with respect to ε4 carriers and non-carriers are discussed in the context of neurovascular compensation.

Caspase-1 Activation Is Related With HIV-Associated Atherosclerosis in an HIV Transgenic Mouse Model and HIV Patient Cohort.

OBJECTIVE: Atherosclerotic cardiovascular disease (ASCVD) is an increasing cause of morbidity and mortality in people with HIV since the introduction of combination antiretroviral therapy. Despite recent advances in our understanding of HIV ASCVD, controversy still exists on whether this increased risk of ASCVD is due to chronic HIV infection or other risk factors. Mounting biomarker studies indicate a role of monocyte/macrophage activation in HIV ASCVD; however, little is known about the mechanisms through which HIV infection mediates monocyte/macrophage activation in such a way as to engender accelerated atherogenesis. Here, we experimentally investigated whether HIV expression is sufficient to accelerate atherosclerosis and evaluated the role of caspase-1 activation in monocytes/macrophages in HIV ASCVD. Approach and Results: We crossed a well-characterized HIV mouse model, Tg26 mice, which transgenically expresses HIV-1, with ApoE-/- mice to promote atherogenic conditions (Tg26+/-/ApoE-/-). Tg26+/-/ApoE-/- have accelerated atherosclerosis with increased caspase-1 pathway activation in inflammatory monocytes and atherosclerotic vasculature compared with ApoE-/-. Using a well-characterized cohort of people with HIV and tissue-banked aortic plaques, we documented that serum IL (interleukin)-18 was higher in people with HIV compared with non-HIV-infected controls, and in patients with plaques, IL-18 levels correlated with monocyte/macrophage activation markers and noncalcified inflammatory plaques. In autopsy-derived aortic plaques, caspase-1+ cells and CD (clusters of differentiation) 163+ macrophages correlated. CONCLUSIONS: These data demonstrate that expression of HIV is sufficient to accelerate atherogenesis. Further, it highlights the importance of caspase-1 and monocyte/macrophage activation in HIV atherogenesis and the potential of Tg26+/-/ApoE-/- as a tool for mechanistic studies of HIV ASCVD.

SGLT-2 (Sodium-Glucose Cotransporter 2) Inhibition Reduces Ang II (Angiotensin II)-Induced Dissecting Abdominal Aortic Aneurysm in ApoE (Apolipoprotein E) Knockout Mice.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a pathological condition of permanent vessel dilatation that predisposes to the potentially fatal consequence of aortic rupture. SGLT-2 (sodium-glucose cotransporter 2) inhibitors have emerged as powerful pharmacological tools for type 2 diabetes mellitus treatment. Beyond their glucose-lowering effects, recent studies have shown that SGLT-2 inhibitors reduce cardiovascular events and have beneficial effects on several vascular diseases such as atherosclerosis; however, the potential effects of SGLT-2 inhibition on AAA remain unknown. This study evaluates the effect of oral chronic treatment with empagliflozin-an SGLT-2 inhibitor-on dissecting AAA induced by Ang II (angiotensin II) infusion in apoE (apolipoprotein E)-/- mice. Approach and Results: Empagliflozin treatment significantly reduced the Ang II-induced increase in maximal suprarenal aortic diameter in apoE-/- mice independently of blood pressure effects. Immunohistochemistry analysis revealed that empagliflozin diminished Ang II-induced elastin degradation, neovessel formation, and macrophage infiltration at the AAA lesion. Furthermore, Ang II infusion resulted in a marked increase in the expression of chemokines (CCL-2 [chemokine (C-C motif) ligand 2] and CCL-5 [chemokine (C-C motif) ligand 5]), VEGF (vascular endothelial growth factor), and MMP (matrix metalloproteinase)-2 and MMP-9 in suprarenal aortic walls of apoE-/- mice, and all were reduced by empagliflozin cotreatment. Western blot analysis revealed that p38 MAPK (p38 mitogen-activated protein kinase) and NF-κB (nuclear factor-κB) activation was also reduced in the suprarenal aortas of apoE-/- mice cotreated with empagliflozin. Finally, in vitro studies in human aortic endothelial cells and macrophages showed that empagliflozin inhibited leukocyte-endothelial cell interactions and release of proinflammatory chemokines. CONCLUSIONS: Pharmacological inhibition of SGLT-2 by empagliflozin inhibits AAA formation. SGLT-2 inhibition might represent a novel promising therapeutic strategy to prevent AAA progression.

Chronic iron overload intensifies atherosclerosis in apolipoprotein E deficient mice: Role of oxidative stress and endothelial dysfunction.

AIMS: We previously demonstrated that iron overload induces endothelial dysfunction and oxidative stress, which could increase the risk for atherosclerosis. However, the iron-related harmfulness under a genetic predisposition to atherosclerosis is still unclear. Here, we have tested the hypothesis that chronic iron overload may change vascular reactivity associated with worsening of the atherosclerotic process in apolipoprotein E knockout (apoE(-/-)) mice. MAIN METHODS: Serum and aortas of wild-type (WT) and apoE(-/-) mice injected with iron-dextran (IO, 10 mg/mouse/day, ip) or saline 5 times a week for 4 weeks, were used. KEY FINDINGS: Iron overload increased serum levels of iron and biomarkers of liver injury and oxidative stress, and iron deposition in the aorta in both lines, but only apoE(-/-) IO mice had intensified hypercholesterolemia and atherosclerosis. By scanning electron microscopy, the small endothelial structural damage caused by iron in WT was worsened in the apoE(-/-) group. However, endothelial dysfunction was found only in the apoE(-/-) IO group, identified by impaired relaxation to acetylcholine and hyperreactivity to phenylephrine associated with reduced nitric oxide modulation. Moreover, tiron and indomethacin attenuated reactivity to phenylephrine with greater magnitude in aortas of the apoE(-/-) IO group. Confirming, there were changes in the antioxidant (superoxide dismutase and catalase) activity, increased expression of cyclooxygenase-2 in the aorta and elevated levels of thromboxane A2 and prostacyclin metabolites in the urine of apoE(-/-) IO. SIGNIFICANCE: Our results showed that chronic iron overload intensifies the atherosclerotic process and induces endothelial dysfunction in atherosclerotic mice, probably due to the oxidative stress and the imbalance between the relaxing and contractile factors synthesized by the damaged endothelium.