Early Stages Of Atherogenesis Research Articles

Single-cell transcriptomics reveals subtype-specific molecular profiles in Nrf2-deficient macrophages from murine atherosclerotic aortas.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional regulator of antioxidant and anti-inflammatory response in all cell types. It also activates the transcription of genes important for macrophage function. Nrf2 activity declines with age and has been closely linked to atherosclerosis, but its specific role in this vascular pathology is not clear. Atherosclerotic plaques contain several macrophage subsets with distinct, yet not completely understood, functions in the lesion development. The aim of this study was to analyze the transcriptome of diverse Nrf2-deficient macrophage subpopulations from murine atherosclerotic aortas. Mice with transcriptionally inactive Nrf2 in Cdh5-expressing cells (Nrf2 Cdh5tKO) were used in the experiments. These mice lack transcriptional Nrf2 activity in endothelial cells, but also in a proportion of leukocytes. We confirmed that the bone marrow-derived and tissue-resident macrophages isolated from Nrf2 Cdh5tKO mice exhibit a significant decline in Nrf2 activity. Atherosclerosis was induced in Nrf2 Cdh5tKO and appropriate control mice via adeno-associated viral vector (AAV)-mediated overexpression of murine proprotein convertase subtilisin/kexin type 9 (Pcsk9) in the liver and high-fat diet feeding. After 21 weeks, live aortic cells were sorted on FACS and single-cell RNA sequencing (scRNA-seq) was performed. Unsupervised clustering singled out 13 distinct aortic cell types. Among macrophages, 9 subclusters were identified. Differential gene expression analysis revealed cell subtype-specific expression patterns. A subset of inflammatory macrophages from atherosclerotic Nrf2 Cdh5tKO mice demonstrated downregulation of DNA replication genes (e.g. Mcm7, Lig1, Pola1) concomitant with upregulation of DNA damage sensor Atr gene. Atherosclerotic Nrf2 Cdh5tKO Lyve1+ resident macrophages showed strong upregulation of IFN-stimulated genes, as well as changes in the expression of death pathways-associated genes (Slc40a1, Bcl2a1). Furthermore, we observed subtype-specific expression of core ferroptosis genes (e.g. Cp, Hells, Slc40a1) in inflammatory versus tissue resident macrophages. This observation suggested a link between ferroptosis and inflammatory microenvironment appearing at a very early stage of atherogenesis. Our findings indicate that Nrf2 deficiency in aortic macrophages leads to subtype-specific transcriptomic changes associated with inflammation, iron homeostasis, cell injury or death pathways. This may help understanding the role of aging-associated decline of Nrf2 activity and the function of specific macrophage subtypes in atherosclerotic lesion development.

Abstract 462: Transcriptomic Analysis Reveals That Intimal Monocytes Drive Inflammation In Early Atherogenesis

Hypercholesterolemia (HC) is a key risk factor for atherosclerosis, a chronic inflammatory disease that causes myocardial infarction and stroke. Although all regions of arteries are exposed to HC, atherosclerosis develops in discrete regions, such as the lesser curvature (LC) of the mouse aortic arch. In Ldlr -/- mice HC induces intimal myeloid cells to develop into the foam cells of nascent lesions. The purpose of this study is to characterize the transcriptomic changes occurring in the aortic intimal myeloid cells during the earliest stages of atherogenesis, and correlate them to cellular responses. Intimal cells were isolated from the LC of the ascending aortic arch of Ldlr -/- mice at 0, 5, 14, and 56 days of CRD feeding using en face enzymatic digestion and cell microisolation. Bulk RNA-seq and RT-qPCR revealed that genes associated with lipid-loaded macrophages (e.g., Abcg1 , Lgals3 ) were progressively elevated over time. Atherogenesis-associated inflammatory transcripts and pathways, such as leukocyte transendothelial migration, cell adhesion, and cytokine/chemokine signalling were significantly elevated after 14d of CRD, but not at 5d. Single cell transcriptomic analysis of the mouse aortic arch intima of Ldlr -/- mice revealed multiple sub-populations of myeloid cells. With our intima specific approach, we have uncovered a diverse landscape of intimal myeloid cells; including monocytes, macrophages, foam cells, DCs, and neutrophils. Atherogenesis-associated inflammatory transcripts were scarcely detected in macrophage and foam cell clusters, but were prominent in non-macrophage cell types. Ccr2 + intimal monocytes exhibited a hypercholesterolemia induced increase in inflammatory pathways within 5 days of CRD feeding. Furthermore, analysis of monocyte and macrophage clusters suggests that this inflammatory signature dissipates as recently recruited monocytes transition into a macrophage/foam cell fate. We are using Ccr2 + fate mapping models, to interrogate the hypercholesterolemia-induced transition of inflammatory monocytes within a growing lesion. We suggest that the atherogenesis-associated inflammation may be initiated by recently recruited monocytes, and may not originate directly from lipid loaded macrophages.

Effect of Bariatric Surgery on Intima Media Thickness: A Systematic Review and Meta-Analysis

Background: Obesity, and in particular extreme obesity, as a global health problem is an important risk factor for many diseases, including atherosclerotic cardiovascular disease (ACVD). Bariatric surgery might stop or slow atherogenesis by decreasing excessive weight in the early stages of atherogenesis, by suppressing low-grade systemic inflammation as well as by inhibiting oxidative stress and endothelial dysfunction. The aim of this meta-analysis was to provide an answer to whether bariatric surgery has a significant effect on intima-media thickness (IMT) which is a surrogate marker of early atherosclerosis and has a good correlation with atherosclerotic coronary heart disease. Methods: A systematic literature search in PubMed, Scopus, Embase, and Web of Science as well as grey literature was performed from inception to 1 July 2022. The meta-analysis was performed using Comprehensive Meta-Analysis (CMA) V3 software. Overall, the estimate of effect size was measured by a random effects meta-analysis. To account for the heterogeneity of studies regarding study design, characteristics of the populations, and treatment duration, a random-effects model (using the DerSimonian–Laird method) and the generic inverse variance weighting approach were used. To assess the existence of publication bias in the meta-analysis, the funnel plot, Begg’s rank correlation, and Egger’s weighted regression tests were used. Results: The meta-analysis of 30 trials, including 1488 subjects, demonstrated a significant decrease in IMT after bariatric surgery. The reduction in IMT was also robust in the leave-one-out sensitivity analysis. It must be stressed that the results of the random-effects meta-regression did not suggest any relationship between the changes in IMT and delta body mass index (BMI) or duration of follow-up after the bariatric surgery. However, the subgroup analyses showed a better IMT reduction after laparoscopic sleeve gastrectomy (LSG) when compared to Roux-en-Y gastric bypass (RYGB). Within a year, the IMT follow-up values showed a further improvement. Conclusions: Bariatric surgery significantly reduced IMT. Significant associations were found between the surgery type and IMT changes, as well as a significant effect of follow-up duration on the changes of IMT after bariatric surgery.

Giant multinucleated endothelial cells are associated with local accumulation of LDL and CD45+ lymphocytes in the intima of human aorta

Background and Aims : At the early stages of atherogenesis, low-density lipoprotein (LDL) particles and immune cells penetrate into the intimal layer of the arterial wall through the endothelium. In adult humans, the luminal surface of the arterial wall is a heterogeneous monolayer of cells with varying morphology, including typical endothelial cells (ECs) and multinucleated variant endothelial cells (MVECs). We hypothesized that distribution of MVECs in the endothelial monolayer can explain the distribution pattern of early atherosclerotic lesions.

Abstract 412: Transcriptomic Analysis Reveals That Lipid Loading Of Distinct Intimal Myeloid Cell Subpopulations Precedes Inflammation In Early Atherogenesis

Hypercholesterolemia (HC) is a key risk factor of atherosclerosis, a chronic inflammatory disease that causes myocardial infarction and stroke. Although all regions of arteries are exposed to HC, atherosclerosis develops in discrete regions, such as the lesser curvature (LC) of the mouse aortic arch. Dietary HC in Ldlr -/- mice induces intimal myeloid cells to develop into foam cells, constituting nascent lesions. The purpose of this study is to characterize the transcriptomic changes occurring in the aortic intima during the earliest stages of atherogenesis, and correlate them to cellular responses. Using en face enzymatic digestion and intimal cell microisolation, transcriptomic analysis was performed on the LC. Intimal cells were isolated from the LC of the ascending aortic arch of Ldlr -/- mice at 0, 5, and 14 days of CRD feeding. Bulk RNA-seq and RT-qPCR found that genes associated with lipid-loaded macrophages (e.g., Abcg1, Lgals3 ) were progressively elevated at 5 and 14 days of CRD. Atherogenesis-associated inflammatory transcripts and pathways, such as leukocyte transendothelial migration, cell adhesion, and cytokine/chemokine signalling were significantly elevated after 14d of CRD, but not detected at 5d. Single cell transcriptomic analysis of the mouse aortic arch intima of Ldlr -/- mice revealed multiple sub-populations of myeloid, endothelial, and other cell types. A shift in the C1qb + , Mmp12 + myeloid subpopulations was observed as the duration of cholesterol feeding increased - with expansion of a macrophage population rich in lipid-responsive transcripts. This finding is consistent with an increase in macrophage number and the presence of Nile Red + CD68 + foam cells observed by immunoconfocal microscopy at 5 days of CRD. However, a robust inflammatory signature was not detected in this macrophage cluster. Interestingly, an inflammatory signature similar to that reported in early atherogenesis was identified in a distinct Ccr2 + myeloid population with no signature of lipid loading. This suggests that the inflammation associated with atherogenesis may be a result of intercellular crosstalk between distinct myeloid subpopulations within the intima and may not originate directly from lipid loaded macrophages.

Local Accumulation of Lymphocytes in the Intima of Human Aorta Is Associated with Giant Multinucleated Endothelial Cells: Possible Explanation for Mosaicism of Atherosclerosis.

Distribution of different types of atherosclerotic lesions in the arterial wall is not diffuse, but is characterized by mosaicism. The causes of such distribution remain to be established. At the early stages of atherogenesis, low-density lipoprotein (LDL) particles and immune cells penetrate into the intimal layer of the arterial wall through the endothelium. In adult humans, the luminal surface of the arterial wall is a heterogeneous monolayer of cells with varying morphology including typical endothelial cells (ECs) and multinucleated variant endothelial cells (MVECs). We hypothesized that distribution of MVECs in the endothelial monolayer can be related to the distribution pattern of early atherosclerotic lesions. We obtained en face preparations of intact adult (22–59 years old) aortic wall sections that allowed us to study the endothelial monolayer and the subendothelial layer. We compared the distribution of MVECs in the endothelial monolayer with the localization of early atherosclerotic lesions in the subendothelial layer, which were characterized by lipid accumulation and immune cell recruitment. In primary culture, MVECs demonstrated increased phagocytic activity compared to mononuclear ECs. Moreover, we have shown that unaffected aortic intima contained associates formed as a result of aggregation and/or fusion of LDL particles that are non-randomly distributed. This indicated that MVECs may be involved in the accumulation of LDL in the subendothelial layer through increased transcytosis. Interaction of LDL with subendothelial cells of human aorta in primary culture increased their adhesive properties toward circulating immune cells. Study of unaffected aortic intima revealed non-random distribution of leukocytes in the subendothelial layer and increased localization of CD45+ leukocytes in the subendothelial layer adjacent to MVECs. Together, our observations indicate that MVECs may be responsible for the distribution of atherosclerotic lesions in the arterial wall by participating in LDL internalization and immune cell recruitment.

Local accumulation of LDL and CD45+ lymphocytes in the intima of human aorta is associated with giant multinucleated endothelial cells

Background and Aims: At the early stages of atherogenesis, low-density lipoprotein (LDL) particles and immune cells penetrate into the intimal layer of the arterial wall through the endothelium. In adult humans, the luminal surface of the arterial wall is a heterogeneous monolayer of cells with varying morphology, including typical endothelial cells (ECs) and multinucleated variant endothelial cells (MVECs). We hypothesized that distribution of MVECs in the endothelial monolayer can explain the distribution pattern of early atherosclerotic lesions.

Lower extremity arterial calcifications assessed by multislice CT as a correlate to coronary artery disease

BackgroundPatients with peripheral artery disease (PAD) frequently have concomitant coronary artery disease (CAD) and display a higher risk for myocardial infarction, stroke, and death due to cardiovascular events. In order to prevent cardiovascular events, there is an increasing interest in new markers of atherosclerosis. Vascular calcifications (VC) are often present in the early stages of atherogenesis and could be considered an early marker. The aim of this study is to correlate the extent of lower limbs, aortic, and coronary arterial calcification diagnosed by non-contrast multislice CT with the severity of coronary artery disease diagnosed by conventional coronary angiography.ResultsThere is borderline significant association between CAD (Gensini score) and each of total lower limb and aorto-iliac calcifications. There is significant association between the number of diseased coronaries and lower limb calcifications. Also, there is significant association between the coronary artery calcifications and lower limb arterial calcifications (total and segmental).Diabetes mellitus and hypertension are significantly associated with lower limb calcification (total, aorto-iliac, and infra-popliteal). Moreover, lower limb arterial calcifications (total and segmental) are positively correlated with increasing age.ConclusionLower limb arterial calcifications, as diagnosed by non-contrast MSCT, is a noninvasive measure for evaluation of the atherosclerotic burden that correlate to the CAD; it can aid to refine risk stratification and the need for more aggressive preventive strategies.

Nutraceuticals for prevention of atherosclerosis: Targeting monocyte infiltration to the vascular endothelium.

Cardiovascular disease (CVD) is the leading cause of death, globally, and is a serious problem in developing countries. Preventing atherosclerosis is key to reducing the risk of developing CVD. Similar to carcinogenesis, atherogenesis can be divided into four stages: initiation, promotion, progression, and acute events. The current study focuses on the promotion stage, which is characterized by circular monocyte penetration into vascular endothelial cells, monocyte differentiation into macrophages, and the formation of foam cells. This early stage of atherogenesis is a major target for nutraceuticals. We discuss nutraceuticals that can potentially inhibit monocyte adhesion to the vascular endothelium, thereby preventing the promotional stage of atherosclerosis. The mechanisms through which these nutraceuticals prevent monocyte adhesion are classified according to the following targets: NF-κB, ROS, MAPKs, and AP-1. Additionally, we discuss promising targets for nutraceuticals that can regulate monocyte adhesion to the endothelium. PRACTICAL APPLICATIONS: Introduction of atherogenesis with initiation, promotion, progression, and acute events provide specific information and factors for each step in the development of atherosclerosis. Functional food or pharmaceutical researchers can set target stages and use them to develop materials that control atherosclerosis. In particular, because it focuses on vascular inflammation via interaction between monocytes and vascular endothelial cells, it provides specific information to researchers developing functional foods that regulate this process. Therefore, this manuscript, unlike previous papers, will provide material information and potential mechanisms of action to researchers who want to develop functional foods that control vascular inflammation rather than vascular lipids.

4-Phenylbutyrate protects against atherosclerotic lesion growth by increasing the expression of HSP25 in macrophages and in the circulation of Apoe-/- mice.

Endoplasmic reticulum stress plays an important role in cardiovascular disease (CVD) and atherosclerosis. We aimed to assess the ability of 4-phenylbutyrate (4-PBA), a small chemical chaperone administered via drinking water, to reduce atherosclerotic lesion size in chow-fed apolipoprotein (Apo) e-/- mice and to identify mechanisms that contribute to its antiatherogenic effect. Chow-fed 17-wk-old female Apoe-/- mice treated with 4-PBA-supplemented drinking water for 5 wk exhibited smaller lesions as well as increased plasma levels of heat shock protein (HSP) 25, the mouse homolog of human HSP27, compared with controls. In addition, 4-PBA inhibited cell death and increased HSP27 expression as measured by real-time PCR and immunoblotting, as well as induced nuclear localization of its transcription factor, heat shock factor 1, in human monocyte/macrophage (THP-1) cells. Furthermore, HSP27 small interfering RNA diminished the protective effect of 4-PBA on THP-1 macrophage attachment and differentiation. In summary, drinking water containing 4-PBA attenuated early lesion growth in Apoe-/- mice fed a chow diet and increased expression of HSP25 and HSP27 in macrophages and HSP25 in the circulation of Apoe-/- mice. Given that increased expression of HSP27 is inversely correlated with CVD risk, our findings suggest that 4-PBA protects against the early stages of atherogenesis in part by enhancing HSP27 levels, leading to inhibition of both macrophage cell death and monocyte-macrophage differentiation.-Lynn, E. G., Lhoták, Š., Lebeau, P., Byun, J. H., Chen, J., Platko, K., Shi, C., O'Brien, E. R., Austin, R. C. 4-Phenylbutyrate protects against atherosclerotic lesion growth by increasing the expression of HSP25 in macrophages and in the circulation of Apoe-/- mice.

Glucose-6-phosphate dehydrogenase deficiency and risk of cardiovascular disease: A propensity score-matched study

Background and aimsCardiovascular disease (CVD) is associated with high morbidity and mortality. Studies in animal models and humans suggested that glucose‒6‒phosphate dehydrogenase (G6PD) deficiency, a genetically inherited condition causing haemolytic anemia, may be a risk factor for CVD. This hypothesis was tested in a large cohort from Northern Sardinia, where the population prevalence of G6PD deficiency is the highest in the Mediterranean area. MethodsA retrospective observational case‒control study was performed using clinical records of 9604 patients undergoing digestive endoscopy between 2002 and 2017, with a known G6PD status and a complete clinical history including CVD and leading CVD risk factors. To circumvent covariates imbalance between cases and controls, a 1:2 propensity score‒matched analysis was performed. ResultsMajor predictors of CVD, as expected, were age (OR 1.07; 95%CI 1.06–1.08), male sex (1.63; 95%CI 1.29–2.06), high blood pressure (OR 1.46; 95%CI 1.16–1.84), smoking (OR 3.03; 95%CI 2.42–3.79), diabetes (OR 1.65; 95%CI 1.23–2.21) and hypercholesterolemia (OR 2.20; 95%CI 1.71–2.84). The propensity score matching procedure resulted in 1123 G6PD deficient patients and 2246 patients with normal enzyme activity. When G6PD status was regressed on the CVD, including propensity score as a continuous covariate, an OR of 1.71 (95%CI 1.17–2.49; p = 0.006) was obtained. ConclusionsG6PD deficiency is significantly associated with increased risk of CVD, although the underlying mechanisms are still poorly understood. The loss of important protective pathways against oxidative stress, especially in the early stages of atherogenesis, might play a crucial role.

C-Reactive Protein in Atherothrombosis and Angiogenesis.

C-reactive protein (CRP) is a short pentraxin mainly found as a pentamer in the circulation, or as non-soluble monomers CRP (mCRP) in tissues, exerting different functions. This review is focused on discussing the role of CRP in cardiovascular disease, including recent advances on the implication of CRP and its forms specifically on the pathogenesis of atherothrombosis and angiogenesis. Besides its role in the humoral innate immune response, CRP contributes to cardiovascular disease progression by recognizing and binding multiple intrinsic ligands. mCRP is not present in the healthy vessel wall but it becomes detectable in the early stages of atherogenesis and accumulates during the progression of atherosclerosis. CRP inhibits endothelial nitric oxide production and contributes to plaque instability by increasing endothelial cell adhesion molecules expression, by promoting monocyte recruitment into the atheromatous plaque and by enzymatically binding to modified low-density lipoprotein. CRP also contributes to thrombosis, but depending on its form it elicits different actions. Pentameric CRP has no involvement in thrombogenesis, whereas mCRP induces platelet activation and thrombus growth. In addition, mCRP has apparently contradictory pro-angiogenic and anti-angiogenic effects determining tissue remodeling in the atherosclerotic plaque and in infarcted tissues. Overall, CRP contributes to cardiovascular disease by several mechanisms that deserve an in-depth analysis.

Enzymatically modified LDL, atherosclerosis and beyond: paving the way to acceptance.

The eLDL (enzymatically modified LDL) hypothesis proposes that modification of LDL during atherogenesis occurs through the action of ubiquitous hydrolytic enzymes. eLDL is recognized by multiple macrophage receptors. Following cellular uptake, eLDL triggers atherosclerotic lesion initiation with reversion or progression depending on the balance between cholesterol insudation and depletion. The effects of eLDL on cellular constituents of the atherosclerotic lesion comprise both pro- and anti-inflammatory mechanisms. eLDL triggered complement activation is centrally involved in atherosclerosis with the first CRP (C-reactive protein)-dependent activation step to prevail at the early stages of atherogenesis (lesion initiation with reversion), and the second situation to gain dominance as local concentrations of eLDL surpass a critical threshold (lesion initiation with progression). Thus, CRP-mediated lipoprotein removal likely underlies the regression of early lesions, which occurs continuously through life. Perhaps CRP should be considered as an antiatherogenic agent and the question whether it is an innocent bystander or proatherogenic culprit is not really to the point. The observed association between CRP and atherosclerosis might simply be reverse causation: atherosclerotic disease progression induces CRP.

Erythrocyte Efferocytosis by the Arterial Wall Promotes Oxidation in Early-Stage Atheroma in Humans.

Since red blood cells (RBCs) are the predominant cellular blood component interacting with the arterial wall, we explored the role of RBCs efferocytosis by vascular smooth muscle cells (vSMCs) in the initiation of human atheroma. The comparison of human healthy aortas with aortic fatty streaks or fibroatheromas revealed that RBC angiophagy is implicated from the earliest stages of atherogenesis, as documented by the concomitant detection of redox-active iron, hemoglobin, glycophorin A, and ceroids. RBCs infiltration in the arterial wall was associated with local lipid and protein oxidation, as well as vascular response (expression of heme oxygenase-1 and of genes related to iron metabolism as well as those encoding for phagocytosis). These effects were recapitulated in vitro when vSMCs were co-cultured with phosphatidyl-exposing senescent (s) RBCs but not with fresh RBCs. VSMCs engulfing sRBC increased their intracellular iron content, accumulated hemoglobin, lipids, and activated their phagolysosomes. Strikingly, injections of sRBCs into rats promoted iron accumulation in the aortic wall. In rabbits, hypercholesterolemia increased circulating senescent RBCs and induced the subendothelial accumulation of iron-rich phagocytic foam cells. RBCs bring cholesterol and iron/heme into the vascular wall and interact with vSMCs that phagocytize them. This study presents a previously unforeseen mechanism of plaque formation that implicates intimal RBC infiltration as one of the initial triggers for foam cell formation and intimal oxidation. Pathogenic effects exerted by several metabolic and hemodynamic factors may rely on their effect on RBC biology, thereby impacting how RBCs interact with the vascular wall.

Histamine promotes the differentiation of macrophages from CD11b+ myeloid cells and formation of foam cells through a Stat6-dependent pathway

Background and aimsThe enzyme histidine decarboxylase (Hdc), which generates histamine, is highly expressed in CD11b+Gr-1+ myeloid cells that play a critical role in infection, inflammation and tumorigenesis. The aim of this study was to explore the role of Hdc-expressing CD11b+ myeloid cells or histamine in atherogenesis. MethodsHdc-EGFP bacterial artificial chromosome (BAC) transgenic reporter mice (Hdc-EGFP) were used to track Hdc expression during the development of atherosclerosis. The expression of EGFP fluorescence was examined by immunofluorescence staining in a variety of adult tissues. Wild-type (WT), Apoe knockout (Apoe−/−), Hdc knockout (Hdc−/−), and Stat6 knockout (Stat6−/−) mice were used. Serum concentration of histamine was determined with ELISA. Changes in subsets of immune cells were evaluated by flow cytometry (FACS). Non-invasive tracking of the expression of CD11b+ myeloid cells was tested using 125I-anti-CD11b SPECT/CT imaging in the early stages of atherogenesis. Microarray analysis and RT-PCR were applied to detect gene expressions while Western blot was used to assess protein levels. ResultsUsing Hdc-EGFP transgenic mice, we demonstrated that Hdc+CD11b+ myeloid cells increase in the circulation in response to hypercholesterolemia and contribute to foam cell formation in atherosclerosis. Histamine deficiency in Hdc knockout (Hdc−/−) mice repressed the differentiation of CD11b+Ly6Chigh classically activated M1-type macrophages and CD11b+CD11c+ dendritic cells (DCs), which was associated with downregulation of signal transducer and activator of transcription 6 (Stat6) expression. Furthermore, the results of in vivo and in vitro studies demonstrated that histamine could promote macrophage differentiation and foam cell formation via the Stat6 signal. ConclusionsModulation of histamine and Stat6-signaling may represent an attractive therapeutic strategy for the prevention or treatment of atherosclerosis.

Circulating Mitochondrial (mt) DNA Fragments as Precursors of Inflammation in Early Stages of Atherogenesis

Atherosclerosis and its vascular complications are the leading cause of morbidity and mortality among Western populations. It is widely accepted that inflammation plays a major role in all stages of atherogenesis, including its initial phases. However, the mechanisms of initiation of inflammatory response predisposing to the development of atherosclerosis are not well understood. Mitochondrial dysfunction underlies many key processes in the development of atherosclerosis, including generation of reactive oxygen species, apoptosis, and inflammation. There is growing evidence that oxidative mtDNA damage can directly promote atherosclerosis. The mechanism linking mitochondrial damage to atherogenesis remains unclear, but may include initiation of inflammation by circulating mtDNA fragments known as DAMPs (Damage Associated Molecular Patterns). Previously, we have demonstrated that mice deficient in DNA repair glycosylase Ogg1 and fed a high fat diet developed larger fatty streaks in the aortic valve as a sign of early atherogenesis that was correlated with higher mtDNA oxidative damage in cardiac tissue and elevated content of plasma mtDNA fragments in comparison to wild type animals. In the present study we tested the hypothesis that development of atherosclerosis in animal model is associated with increased abundance of circulating pro‐inflammatory mtDNA fragments. ApoE‐null and wild type mice of increasing age were analyzed for atherosclerotic burden within the aortic valve and for abundance of mtDNA fragments in plasma. ApoE‐null mice develop atherosclerotic plaque in the aortic valve either spontaneously with age or after feeding a high fat diet. Early lesions were detected in ApoE‐null mice by 3 months of age whereas in 5‐month old animals they became prominent. Atherosclerotic burden reached 30% of valve area in 10‐month old animals and remained at that level in 12‐month old mice. Wild type animals did not develop lesions within the aortic valve at any age. The abundance of circulating mtDNA fragments was higher in ApoE‐null mice in all age groups when compared to wild type animals. Importantly, the largest relative increase in mtDNA fragments (5 to 6.5 fold vs. age‐matched controls) was observed in ApoE‐null mice at 3 and 5 months. These results indicate that circulating mtDNA fragments could be precursors of inflammation development in early stages of atherogenesis and suggest that efficient maintenance of mtDNA integrity could be an important target for atheroprotection.Support or Funding InformationNIH R01 HL113614

Antioxidant Properties of Polysaccharide Extract from Juice and Pomace of Psidium guajava (Pink Guava) and its Mitigating Effect in Early Stage of Atherogenesis

A number of significant herbal drug interactions have their origins in the alteration of cytochrome P450 (CYP) activity by various phytochemicals. In the present study, inhibition potential of Terminalia arjuna extracts and its constituent gallic acid (GA) and ellagic acid (EA) to cause herb-drug interactions through rat liver cytochrome enzymes, CYP3A4, and CYP2D6 was evaluated, a rapid RP-HPLC method was developed for quantitative estimation of GA and EA in the extract. In vitro safety of the extract and active components were evaluated through CYP450 inhibition method using pooled rat microsomes and high throughput fluorometric assay with CYP3A4 and CYP2D6. The binding mode and the molecular interaction of GA and EA within the CYP3A4 and CYP2D6 active site were demonstrated using an in-silico docking studies. From CYP450-CO Complex assay, the inhibitory potential of T. arjuna standardized extract (31.02 ± 2.24%), was found to be less than positive control. In high throughput i¬‚uorometric assay, T. arjuna extracts exhibited higher IC50 values (48.06 ± 1.14-57.89 ± 2.15 μg/mL) compared to positive inhibitors and lower than GA (66.54 ± 1.04-83.84 ± 1.06 μg/mL), EA (69.47 ± 1.18-102.69 ± 2.87 μg/mL) on CYP3A4 and CYP2D6. Based on the inhibitory potential of test samples, it can be concluded that T. arjuna and its standardized bioactive molecules could produce weak interaction potential when co-administered with conventional medicines.

Different effects of anthocyanins and phenolic acids from wild blueberry (Vaccinium angustifolium) on monocytes adhesion to endothelial cells in a TNF-α stimulated proinflammatory environment.

Monocyte adhesion to the vascular endothelium is a crucial step in the early stages of atherogenesis. This study aims to investigate the capacity of an anthocyanin (ACN) and phenolic acid (PA) rich fraction (RF) of a wild blueberry, single ACNs (cyanidin, malvidin, delphinidin) and related metabolites (protocatechuic, syringic, and gallic acid) to counteract monocytes (THP-1) adhesion to human umbilical vein endothelial cells (HUVECs) in a tumor necrosis factor α (TNF-α) mediated proinflammatory environment. HUVECs were incubated with different concentrations (from 0.01 to 10 μg/mL) of the compounds for 24 h. Labeled monocytic THP-1 cells were added to HUVECs and their adhesion was induced by TNF-α (100 ng/mL). ACN-RF reduced THP-1 adhesion to HUVECs with a maximum effect at 10 μg/mL (-33%). PA-RF counteracted THP-1 adhesion at 0.01, 0.1, and 1 μg/mL (-45, -48.7, and -27.6%, respectively), but not at maximum concentration. Supplementation with gallic acid reduced THP-1 adhesion to HUVECs with a maximum effect at 1 μg/mL (-29.9%), while malvidin-3-glucoside and syringic acid increased the adhesion. No effect was observed for the other compounds. These results suggest that ACNs/PA-RF may prevent atherogenesis while the effects of the single ACNs and metabolites are controversial and merit further exploration.

Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice.

Exercise is an effective approach for primary and secondary prevention of cardiovascular diseases (CVD) and loss of muscular mass and function. Its benefits are widely documented but incompletely characterized. It has been reported that exercise can induce changes in the expression of antioxidant enzymes including Sod2, Trx1, Prdx3 and Gpx1 and limits the rise in oxidative stress commonly associated with CVD. These enzymes can be subjected to epigenetic regulation, such as DNA methylation, in response to environmental cues. The aim of our study was to determine whether in the early stages of atherogenesis, in young severely dyslipidemic mice lacking LDL receptors and overexpressing human ApoB100 (LDLR-/-; hApoB+/+), exercise regulates differentially the expression of antioxidant enzymes by DNA methylation in the skeletal muscles that consume high levels of oxygen and thus generate high levels of reactive oxygen species. Expression of Sod2, Txr1, Prdx3 and Gpx1 was altered by 3 months of exercise and/or severe dyslipidemia in 6-mo dyslipidemic mice. Of these genes, only Gpx1 exhibited changes in DNA methylation associated with dyslipidemia and exercise: we observed both increased DNA methylation with dyslipidemia and a transient decrease in DNA methylation with exercise. These epigenetic alterations are found in the second exon of the Gpx1 gene and occur alongside with inverse changes in mRNA expression. Inhibition of expression by methylation of this specific locus was confirmed in vitro. In conclusion, Gpx1 expression in the mouse skeletal muscle can be altered by both exercise and dyslipidemia through changes in DNA methylation, leading to a fine regulation of free radical metabolism.

Characterizing the hematopoietic stem/progenitor cells in the placenta and umbilical cord blood from normal and preeclamptic subjects

Homocysteine lowering with folic acid has been shown to be ineffective in reducing vascular events in patients with established cardiovascular disease. However, it may be beneficial in the early stages of atherosclerosis particularly for the population at risk. We have investigated the effect of folic acid administration on homocysteine levels, endothelial function, and leukocyte adhesion molecules in atorvastatin-treated patients. Thirty-nine patients were randomized to folic acid 5 mg daily or no additional treatment. Plasma total homocysteine levels were determined by high-performance liquid chromatography. Biochemical and hematologic tests were performed using routine automated analyzer methods. We examined the expression of l-selectin (CD62L); α-subunits of integrins LFA-1 (CD11a), Mac-1 (CD11b), and VLA-4 (CD49d); and β-subunit of β-2 integrins (CD18) by an immunofluorescence method using single-step staining of whole blood with monoclonal antibodies. The fluorescence was quantified by flow cytometry. Reactivity of the forearm skin microvascular bed to thermal and ischemic stimuli was measured using laser Doppler flowmetry. Postocclusive and thermal reactive hyperemia tests were performed. Statistical analysis used 2-sample t test and paired t test. Homocysteine levels decreased significantly in the folic acid–treated group (11.78 ± 2.7 vs 9.75 ± 1.89 μmol/L, P < .0001) but did not change in the control group (11.17 ± 2.09 vs 11.88 ± 2.88 μmol/L). There was no effect of folic acid treatment on leukocyte expression of cell adhesion molecules or microvascular reactivity. The lack of effect of folic acid on microvascular reactivity and leukocyte adhesion molecules supports the evidence against the beneficial effects of folic acid administration in the early stages of atherogenesis.