Early Atherosclerotic Lesion Formation Research Articles

Alternate day fasting aggravates atherosclerosis through the suppression of hepatic ATF3 in Apoe−/− mice

Abstract Atherosclerosis is the major contributor to cardiovascular mortality worldwide. Alternate day fasting (ADF) has gained growing attention due to its metabolic benefits. However, the effects of ADF on atherosclerotic plaque formation remain inconsistent and controversial in atherosclerotic animal models. The present study was designed to investigate the effects of ADF on atherosclerosis in apolipoprotein E-deficient (Apoe−/−) mice. Eleven-week-old male Apoe−/− mice fed with Western diet (WD) were randomly grouped into ad libitum (AL) group and ADF group, and ADF aggravated both the early and advanced atherosclerotic lesion formation, which might be due to the disturbed cholesterol profiles caused by ADF intervention. ADF significantly altered cholesterol metabolism pathways and down-regulated integrated stress response (ISR) in the liver. The hepatic expression of activating transcription factor 3 (ATF3) was suppressed in mice treated with ADF and hepatocyte-specific overexpression of Aft3 attenuated the effects of ADF on atherosclerotic plaque formation in Apoe−/− mice. Moreover, the expression of ATF3 could be regulated by Krüppel-like factor 6 (KLF6) and both the expressions of ATF3 and KLF6 were regulated by hepatic cellular ISR pathway. In conclusion, ADF aggravates atherosclerosis progression in Apoe−/− mice fed on WD. ADF inhibits the hepatic ISR signaling pathway and decreases the expression of KLF6, subsequently inhibiting ATF3 expression. The suppressed ATF3 expression in the liver mediates the deteriorated effects of ADF on atherosclerosis in Apoe−/− mice. The findings suggest the potentially harmful effects when ADF intervention is applied to the population at high risk of atherosclerosis.

Interleukin-23 receptor expressing γδ T cells locally promote early atherosclerotic lesion formation and plaque necrosis in mice.

Atherosclerosis is a chronic inflammatory disease of the vessel wall controlled by local and systemic immune responses. The role of interleukin-23 receptor (IL-23R), expressed in adaptive immune cells (mainly T-helper 17 cells) and γδ T cells, in atherosclerosis is only incompletely understood. Here, we investigated the vascular cell types expressing IL-23R and addressed the function of IL-23R and γδ T cells in atherosclerosis. IL-23R+ cells were frequently found in the aortic root in contrast to the aorta in low-density lipoprotein receptor deficient IL-23R reporter mice (Ldlr-/-Il23rgfp/+), and mostly identified as γδ T cells that express IL-17 and GM-CSF. scRNA-seq confirmed γδ T cells as the main cell type expressing Il23r and Il17a in the aorta. Ldlr-/-Il23rgfp/gfp mice deficient in IL-23R showed a loss of IL-23R+ cells in the vasculature, and had reduced atherosclerotic lesion formation in the aortic root compared to Ldlr-/- controls after 6 weeks of high-fat diet feeding. In contrast, Ldlr-/-Tcrδ-/- mice lacking all γδ T cells displayed unaltered early atherosclerotic lesion formation compared to Ldlr-/- mice. In both HFD-fed Ldlr-/-Il23rgfp/gfp and Ldlr-/-Tcrδ-/- mice a reduction in the plaque necrotic core area was noted as well as an expansion of splenic regulatory T cells. In vitro, exposure of bone marrow-derived macrophages to both IL-17A and GM-CSF induced cell necrosis, and necroptotic RIP3K and MLKL expression, as well as inflammatory mediators. IL-23R+ γδ T cells are predominantly found in the aortic root rather than the aorta and promote early atherosclerotic lesion formation, plaque necrosis, and inflammation at this site. Targeting IL-23R may thus be explored as a therapeutic approach to mitigate atherosclerotic lesion development.

Zerumbone ameliorates the induced atherosclerosis-initiated inflammatory conditions through suppression of proinflammatory mediators and inflammatory cytokines

Background: Zerumbone (ZER) is a naturally occurring monosesquiterpine and dietary compound from Zingiber zerumbet Smith, an edible ginger. It has been shown to possess anti-inflammatory activities. Inflammation plays an important role in all stages of atherosclerosis. However, little is known about ZER's anti-inflammatory role in atherosclerosis. Objectives: This study investigated the anti-inflammatory effect of ZER in mitigating the formation and development of early atherosclerotic lesions in rabbits fed with high-cholesterol diet. Materials and Methods: A total of 72 New Zealand White rabbits were used in two experimental studies carried out at different times. The first experiment was carried out to investigate the prophylactic effects of ZER in preventing premature atheromas plaque formation. In contrast, the second experiment was carried out to assess the therapeutic efficacy of ZER in reducing atheromas lesion progression and dissemination. Results: ZER significantly (P

P7142-AG impacts on endothelial cell activation and endothelial cell viability in vitro and impairs endothelial repair in vivo

Abstract Background The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) is a known modulator of inflammation and few studies have addressed its influence on myeloid cells in the context of atherogenesis. However, the impact of 2-AG on endothelial cell function has not been studied before. Methods Endothelial repair was studied in two treatment groups of wildtype mice following electrical denudation of the common carotid artery at a length of 3000 μm. One group received the monoacylglycerol lipase (MAGL)-inhibitor JZL184 [5 mg/kg i.p.], which impairs 2-AG degradation and thus causes elevated 2-AG levels, the other group received vehicle. The residual endothelial gap at five days in either group was visualized by Evan's blue staining. In vitro, the effect of 2-AG on human coronary artery endothelial cell (HCAEC) viability was assessed by an XTT-based assay. Endothelial activation was studied by an adhesion assay of THP-1 monocytes to 2-AG-preconditioned HCAEC. HCAEC migration, ROS-production, expression of NADPH oxidases, and secretion of inflammatory cytokines were assessed by Boyden chamber, qPCR, and colorimetric assays. Results Treatment with JZL184 produced a significant increase in 2-AG levels and impaired reendothelialisation in wildtype mice following electrical injury of the common carotid artery. The residual denudation at 5 days yielded 2291±286 μm in JZL184-treated animals vs. 1505±223 μm in vehicle treated controls (n=18–19; p<0.05). In vitro, JZL184 significantly reduced viability of HCAEC at 24 hours (0.31±0.10 vs. 1.00±0.08; n=3; p<0.01). Finally, 2-AG promoted HCAEC activation resulting in a significant increase in THP-1 monocyte adhesion to HCAEC following pre-treatment of HCAEC with 2-AG (0.17±0.03 THP-1 cells per HCAEC vs. 0.07±0.01 THP-1 cells per HCAEC; n=3; p<0.05). Besides, HCAEC migration, ROS-production, expression of NADPH oxidases and secretion of inflammatory cytokines were unaffected by 2-AG. Conclusion Elevated 2-AG levels appear to hamper endothelial repair and to promote HCAEC activation and cell death. Our data suggest that besides its influence on myeloid cells, 2-AG is also adverse to endothelial integrity which might promote early atherosclerotic lesion formation. Thus, decreasing vascular 2-AG levels might represent a promising therapeutic strategy for the prevention of atherosclerosis and coronary heart disease.

Proatherogenic effects of 4-hydroxynonenal

Among the various mechanisms involved in atherogenesis, the oxidative theory of atherosclerosis relies on the oxidation of low density lipoprotein (LDL) in the vascular wall and their implication in the formation of early atherosclerotic lesions. Reactive oxygen species (ROS) and oxidants generated by activated endothelium, initiate LDL oxidation in the intima, which generates a huge variety of lipid peroxidation products (LPPs), exhibiting atherogenic, pro-inflammatory and pro-apoptotic properties. Reactive carbonyl compounds (RCCs), are a family of highly reactive agents generated during polyunsaturated fatty acid (PUFA) peroxidation. RCCs covalently bind to nucleophilic group of proteins, peptides, phospholipids and nucleic acids, thereby generating a “carbonyl stress”. Among RCCs, 4-hydroxy-2-nonenal (HNE) exerts its atherogenic effects through several mechanisms, by targeting lipoproteins or cellular components. HNE generated during LDL oxidation is able to form HNE-apoB adducts, which are recognized by scavenger-receptors of macrophagic cells, thereby leading to foam cell formation. HNE can be released during the degradation of oxLDL, or generated through oxidative stress and PUFA peroxydation in cell membranes. The biological effects of HNE on vascular cells depend on its local concentration and on the expression of detoxifying systems, such as glutathione S-transferase, aldose reductase, and aldehyde dehydrogenase (ALDH), which rapidly neutralize and remove HNE from cells. Physiological concentrations (0.1-1 µmol/L) of HNE induce hormetic and adaptive responses, and transcription factors (Nrf2) that increase cell resistance to oxidative attack and other stresses, while moderate HNE concentrations, (1 to 10 µmol/L), trigger the accumulation of HNE-adducts and a variety of biological responses, such as inflammation and cell proliferation. Higher HNE concentrations, (above 10-20 µmol/L), induce cell dysfunction and apoptosis. However, important variations are observed in atherosclerotic lesions, from the lipid core to the periphery of the plaque, with very different local outcomes. HNE can modify signaling proteins involved in atherosclerotic plaque remodeling, particularly growth factor receptors (PDGFR, EGFR), cell cycle proteins, mitochondrial and endoplasmic reticulum components or extracellular matrix proteins, which progressively alters smooth muscle cell proliferation, angiogenesis and induces apoptosis. HNE-adducts accumulate in the lipidic necrotic core of advanced atherosclerotic lesions and may locally contribute to macrophage and smooth muscle cell apoptosis, which contributes to plaque destabilization and rupture, thereby increasing the risk of athero-thrombotic events.

Corrigendum to "Inhibitory Effect of a French Maritime Pine Bark Extract-Based Nutritional Supplement on TNF-α-Induced Inflammation and Oxidative Stress in Human Coronary Artery Endothelial Cells".

[This corrects the article DOI: 10.1155/2015/260530.].

Impact of the Consumption of Tea Polyphenols on Early Atherosclerotic Lesion Formation and Intestinal Bifidobacteria in High-Fat-Fed ApoE-/- Mice.

There is an increasing interest in the effect of dietary polyphenols on the intestinal microbiota and the possible associations between this effect and the development of some cardiovascular diseases, such as atherosclerosis (AS). However, limited information is available on how these polyphenols affect the gut microbiota and AS development. This study was designed to evaluate the modulation of dietary tea polyphenols (TPs) on intestinal Bifidobacteria (IB) and its correlation with AS development in apolipoprotein E-deficient (ApoE−/−) mice. Fifty C57BL/6 ApoE−/− mice were randomized into one of the five treatment groups (n = 10/group): control group fed normal diet (CK); a group fed a high-fat diet (HFD); and the other three groups fed the same HFD supplemented with TPs in drinking water for 16 weeks. The total cholesterol and low-density lipoprotein cholesterol (LDL-C) were decreased significantly (P < 0.05) after TP interference. In addition, the TP diet also decreased the plaque area/lumen area (PA/LA) ratios (P < 0.01) in the TP diet group. Interestingly, copies of IB in the gut of ApoE−/− mice were notably increased with TP interference. This increase was dose dependent (P < 0.01) and negatively correlated with the PA/LA ratio (P < 0.05). We conclude that TPs could promote the proliferation of the IB, which is partially responsible for the reduction of AS plaque induced by HFD.

Atherogenesis in human - clinical aspects of circulating immune complexes

It has been suggested that circulating immune complexes containing low density lipoproteins (LDL-CIC) play a role in atherogenesis and are involved in the formation of early atherosclerotic lesions. The complexes, as well as anti-LDL antibody were found in the blood of patients with atherosclerotic process in various cardiovascular diseases, well as in the blood of animals with experimentally modulated atherosclerosis. One can assume that the presence anti-LDL antibodies in blood is a result of an immune response that is induced by modification of lipoproteins. LDL-CIC differ from native LDL in many aspects. They have much lower levels of sialic acid, a smaller diameter and a higher density electronegativity than native LDL. The fraction of the LDL-CIC in serum is an important manifestation of the atherosclerotic process. LDL-CIC, unlike the native LDL is able to induce intracellular accumulation of neutral lipids, especially esterified cholesterol in cell cultures obtained from healthy human aortic intima and macrophages in culture. After removal of the LDL-CIC, the serum of CHD-patients loses its atherogenic properties. The titer of the LDL-CIC in the blood serum significantly correlate with the progression of atherosclerosis and in vivo has the highest diagnostic yield of measured among other lipid parameters. Increasing CIC- cholesterol could also increase the risk of coronary artery atherosclerosis.

Abstract 189: Increased Atherosclerosis And Plasma Lipids In Apoe-pon1 Dko Mice As Compared To Ldlr-pon1 Dko Mice.

Background: Atherosclerosis is a multi-step process that includes early atherosclerotic macrophage foam cell lesion formation, proliferation and migration of smooth muscle cells into the intima, and the formation of complex necrotic and calcified lesions. In this study, we determined the role of dyslipidemia and carotid/innominate atherosclerosis versus the lack of ApoE and the enzyme PON 1 in atherosclerosis. Hypothesis: ApoE-PON1 double knockout (DKO) mice, by virtue of age-dependent and profound atherosclerosis in brachiocephalic trunk (innominate artery) even on a normal diet, might be more susceptible to atherosclerosis followed by stroke as compared to age-matched wild type mice. Methods and results: APOE-PON1 DKO and LDLR-PON1 DKO mice were developed using ApoE-/-, PON1-/-, and LDLR-/- mice of C57BL/6 background and were maintained until 18months of age on normal chow. Serum lipids and atherosclerotic lesions were quantified. A significant decrease in levels of TRG, TC, LDL-C and VLDL-C was observed in LDLR-PON1 DKO as compared to ApoE-PON1 DKO group animals, whereas, an increase in HDL levels was observed in LDLR-PON1 DKO mice. No significant change in glucose levels was observed. Increased lesions were observed in ApoE-PON1 DKO mice as compared to the LDLR-PON1 DKO mice. Conclusion: Our studies suggest the lack of ApoE might be the reason behind increased cholesterol levels and greater atherosclerosis. This is the first study to demonstrate the importance of PON1, in addition to ApoE, and also provide valuable insights as to where therapy should be targeted.

Early atherosclerosis development in female apolipoprotein E-deficient mice is associated with an increased vascular oxidative stress

Objective: The mechanisms of atherosclerosis development in the apolipoprotein E-deficient (apoE−/−) mice have been clearly described. In this genetically modified strain, oxidative stress contributes with atherosclerotic lesion progression. However, there are controversial criteria on the vulnerability of female apoE−/− mouse to oxidative stress and atherosclerosis during the fertile period. Thus, the aim of the present work was to examine the implication of vascular oxidative stress during early atherosclerosis development in young apoE−/− female mice. Methods: AQ1 Please provide a brief methodology Results: Hematoxylin/eosin staining demonstrated the presence of early atherosclerotic lesions in the apoE−/− mice, which progressed with age. Dihydroethidium staining of aortic sections revealed a significant increase of superoxide anion generation in apoE−/− compared with the wild type mice. The increment of this reactive oxygen species was associated, at least, with the overexpression of Nox2 mRNA levels. In addition, the 8-week-old apoE−/− mice had a significant increment of lipid and protein damage, as well as a disruption of superoxide dismutase and catalase activity, together with a deprivation of reduced glutathione and an impairment of nitric oxide availability. Conclusion: The present study shows that early atherosclerotic lesion formation in young apoE−/− female mice is associated with an increment of Nox2-mediated reactive oxygen species generation with the subsequent redox disruption, suggesting a key role of oxidative stress during atherogenesis in fertile apoE−/− female mice.

Phage-display-guided nanocarrier targeting to atheroprone vasculature.

In regions of the circulation where vessels are straight and unbranched, blood flow is laminar and unidirectional. In contrast, at sites of curvature, branch points, and regions distal to stenoses, blood flow becomes disturbed. Atherosclerosis preferentially develops in these regions of disturbed blood flow. Current therapies for atherosclerosis are systemic and may not sufficiently target these atheroprone regions. In this study, we sought to leverage the alterations on the luminal surface of endothelial cells caused by this atheroprone flow for nanocarrier targeting. In vivo phage display was used to discover unique peptides that selectively bind to atheroprone regions in the mouse partial carotid artery ligation model. The peptide GSPREYTSYMPH (PREY) was found to bind 4.5-fold more avidly to the region of disturbed flow and was used to form targeted liposomes. When administered intravenously, PREY-targeted liposomes preferentially accumulated in endothelial cells in the partially occluded carotid artery and other areas of disturbed flow. Proteomic analysis and immunoblotting indicated that fibronectin and Filamin-A were preferentially bound by PREY nanocarriers in vessels with disturbed flow. In additional experiments, PREY nanocarriers were used therapeutically to deliver the nitric oxide synthase cofactor tetrahydrobiopterin (BH4), which we have previously shown to be deficient in regions of disturbed flow. This intervention increased vascular BH4 and reduced vascular superoxide in the partially ligated artery in wild-type mice and reduced plaque burden in the partially ligated left carotid artery of fat fed atheroprone mice (ApoE(-/-)). Targeting atheroprone sites of the circulation with functionalized nanocarriers provides a promising approach for prevention of early atherosclerotic lesion formation.

ACE2 and Ang-(1-7) protect endothelial cell function and prevent early atherosclerosis by inhibiting inflammatory response.

Angiotensin-converting enzyme 2 (ACE2) is a counter-regulator against ACE by converting angiotensin II (Ang-II) to Ang-(1-7), but the effect of ACE2 and Ang-(1-7) on endothelial cell function and atherosclerotic evolution is unknown. We hypothesized that ACE2 overexpression and Ang-(1-7) may protect endothelial cell function by counterregulation of angiotensin II signaling and inhibition of inflammatory response. We used a recombinant adenovirus vector to locally overexpress ACE2 gene (Ad-ACE2) in human endothelial cells in vitro and in apoE-deficient mice in vivo. The Ang II-induced MCP-1, VCAM-1 and E-selectin expression, endothelial cell migration and adhesion of human monocytic cells (U-937) to HUVECs by ACE2 gene transfer were evaluated in vitro. Accelerated atherosclerosis was studied in vivo, and atherosclerosis was induced in apoE-deficient mice which were divided randomly into four groups that received respectively a ACE2 gene transfer, Ad-ACE2, Ad-EGFP, Ad-ACE2 + A779, an Ang-(1-7) receptor antagonist, control group. After a gene transfer for 4 weeks, atherosclerotic pathology was evaluated. ACE2 gene transfer not only promoted HUVECs migration, inhibited adhesion of monocyte to HUVECs and decreased Ang II-induced MCP-1, VCAM-1 and E-selectin protein production in vitro, but also decreased the level of MCP-1, VCAM-1 and interleukin 6 and inhibit atherosclerotic plaque evolution in vivo. Further, administration of A779 increased the level of MCP-1, VCAM-1 and interleukin 6 in vivo and led to further advancements in atherosclerotic extent. ACE2 and Ang-(1-7) significantly inhibit early atherosclerotic lesion formation via protection of endothelial function and inhibition of inflammatory response.

Inhibitory Effect of a French Maritime Pine Bark Extract-Based Nutritional Supplement on TNF-α-Induced Inflammation and Oxidative Stress in Human Coronary Artery Endothelial Cells.

Oxidative stress and inflammation, leading to endothelial dysfunction, contribute to the pathogenesis of atherosclerosis. The popularity of natural product supplements has increased in recent years, especially those with purported anti-inflammatory and/or antioxidant effects. The efficacy and mechanism of many of these products are not yet well understood. In this study, we tested the antioxidant and anti-inflammatory effects of a supplement, HIPER Health Supplement (HIPER), on cytokine-induced inflammation and oxidative stress in human coronary artery endothelial cells (HCAECs). HIPER is a mixture of French maritime pine bark extract (PBE), honey, aloe vera, and papaya extract. Treatment for 24 hours with HIPER reduced TNF-α-induced reactive oxygen species (ROS) generation that was associated with decreased NADPH oxidase 4 and increased superoxide dismutase-1 expression. HIPER inhibited TNF-α induced monocyte adhesion to HCAECs that was in keeping with decreased expression of vascular cell adhesion molecule-1 and intercellular cell adhesion molecule-1 and decreased nuclear factor-kappa B (NF-κB) activation. Further investigation of mechanism showed HIPER reduced TNF-α induced IκBα and p38 and MEK1/2 MAP kinases phosphorylation. Our findings show that HIPER has potent inhibitory effects on HCAECs inflammatory and oxidative stress responses that may protect against endothelial dysfunction that underlies early atherosclerotic lesion formation.

Role of platelet CD40 ligand for endothelial cell-monocyte interaction in the presence of flow

CD40 ligand (CD154)-induced ultra-large von Willebrand factor (vWF) multimer-mediated endothelial cell-platelet-monocyte interaction may play an important role in adaptive and maladaptive vascular remodeling processes. Here we analyzed the impact of and conditions favouring the deposition of these multimers on the endothelial cell (EC) surface by way of CD40-CD154 co-stimulation in settings mimicking different forms of blood flow. Upon exposure to low oscillatory shear stress and sCD154, a release of vWF multimers comparable to histamine stimulation was monitored on the EC surface in a string-like fashion. Moreover, ex vivo perfused carotid arteries of wild type mice at low laminar shear stress rates showed a luminal release of vWF as ultra-large vWF multimers (ULVWF) upon stimulation with sCD154 which was absent in blood vessels of CD40 knockout mice. The observed CD40- and flow-dependent vWF release from intact endothelial cells and subsequent vWF multimer formation may facilitate adhesion and subsequent activation of circulating platelets at atherosclerotic predilection sites, which are characterized by disturbed flow patterns. This in turn may amplify endothelial cell-monocyte interaction, thus possibly initiating or promoting early atherosclerotic lesion formation.

Abstract 315: Caspase-1 Activation is Critical for Endothelial Cell Activation, Monocyte Recruitment, and Early Atherogenesis

Activation of caspase-1(Casp-1), an inflammatory protease in a protein complex termed inflammasome, has been demonstrated to play an essential role in sensing environmental and metabolic danger signals and initiating inflammation. Our goal of this study is to examine a novel hypothesis that Casp-1 activation in endothelial cells (ECs) plays a critical role in promoting EC activation and accelerating early atherogenesis. We first found that hyperlipidemia induced casp-1 expression and activation in mouse aorta along with atherogenesis in apolipoprotein E (ApoE)-/- mice with high fat (HF) diet feeding. By generating the ApoE-/-/Casp-1-/- mice, we then found that the deficiency of Casp-1 in ApoE-/- background significantly attenuated early atherosclerotic lesion formation in mouse aortic sinus (42% reduction comparing to that ApoE-/- mice (n=9)) when fed with a HF diet for 3 weeks. Mechanistically, we found that the ApoE-/-/Casp-1-/- mice had significantly less CD11b+/F4/80- monocytes (0.2% vs 0.4% in ApoE-/- mice(n=10)) and CD11b+/F4/80+ macrophages (0.9% vs 1.8% in ApoE-/- mice) infiltration into mouse aorta after 3-week HF diet. Moreover, we found that there were less cytokine and chemokine expressions in the aorta of ApoE-/-/Casp-1-/- mice in comparison to that of ApoE-/- mice. Furthermore, the deficiency of Casp-1 attenuated hyperlipidemia-induced EC activation by inhibiting adhesion molecules including intercellular adhesion molecule 1, vascular cell adhesion molecule 1 and E-selectin up-regulation both in mouse aorta and primary mouse aortic ECs. In in vitro experiments, we found that oxidized low density lipoprotein (ox-LDL) and two oxLDL active components, lysophosphatidylcholine and lysophosphatidic acid, induced Casp-1 activation through Reactive Oxygen Species pathway in human aortic ECs (HAECs). Finally, Casp-1 inhibitors attenuated oxLDL-induced monocyte adhesion to HAECs. Collectively, our data have demonstrated that Casp-1/inflammasome in ECs can sense hyperlipidemia and become activated, which drive EC activation and monocyte recruitment into aorta and promote early atherogenesis.

Activation of inflammatory cells and cytokines by peptide epitopes in vitro: a simple in-vitro screening assay for prioritizing them for in-vivo studies

Antigen-specific immune modulation is an attractive approach to atherosclerosis treatment. The aim of this study was to develop an in-vitro assay to screen peptide molecules for their inflammatory propensity. Human dendritic cells derived from CD14(+) monocytes were activated using peptides derived from apolipoprotein B100 (ApoB), heat shock protein 60 (HSP60) and complement cascade (peptide A) in vitro, and used for priming autologous T cells. Proliferation of T cells, their differentiation to regulatory cells (Treg) and their cytokine profile were studied. The efficacy of the peptides in preventing atherosclerosis was studied in ApoB(tm2Sgy)/Ldlr(tm1Her/J) knockout mice. ApoB and HSP60 peptides induced T-cell proliferation and expansion of regulatory T cells with interleukin-10 and transforming growth factor-β secretion. In comparison, peptide A was a poor stimulator of T cells and was found to induce tumor necrosis factor-α secretion by activated T cells. ApoB and HSP60 peptides were found to reduce early atherosclerotic lesion formation in mice by 32.1 and 33.5%, respectively, while the reduction with peptide A was 5.7%. Thus the in-vitro assay shows an apparent correlation with in-vivo activity and can be developed as a screening assay to prioritize the candidate molecules for animal efficacy testing.

Adventitial fibroblasts from apoE(−/−) mice exhibit the characteristics of transdifferentiation into myofibroblasts

Adventitial fibroblasts (AFs) are the main cell type in the adventitia, however, their role in atherosclerosis remains unclear. We have investigate the role of AFs in atherosclerotic lesion formation by comparing the characteristics of AFs from apoE(-/-) to C57BL/6 mice. A minority of AFs from apoE(-/-) mice expressed α-SM-actin, but no α-SM-actin-positive cells were found in AFs from C57BL/6 mice. The content of total collagens, and the mRNA levels of collagen I and collagen III in AFs of apoE(-/-) mice, were higher than in C57BL/6 mice. AFs from apoE(-/-) mice proliferate and migrate faster, and synthesized more TGF-β(1) , MCP-1, and PDGF-b AFs from apoE(-/-) mice have the characteristics of transdifferentiation into myofibroblasts, including enhanced proliferation and migration, along with synthesis of collagens and cytokines compared to AFs from C57BL/6 mice. The histological and functional characteristics of AFs may contribute to early atherosclerotic lesion formation.

Chlamydia pneumoniae infection acts as an endothelial stressor with the potential to initiate the earliest heat shock protein 60-dependent inflammatory stage of atherosclerosis

We identified increased expression and redistribution of the intracellular protein 60-kDa human heat shock protein (hHSP60) (HSPD1) to the cell surface in human endothelial cells subjected to classical atherosclerosis risk factors and subsequent immunologic cross-reactivity against this highly conserved molecule, as key events occurring early in the process of atherosclerosis. The present study aimed at investigating the role of infectious pathogens as stress factors for vascular endothelial cells and, as such, contributors to early atherosclerotic lesion formation. Using primary donor-matched arterial and venous human endothelial cells, we show that infection with Chlamydia pneumoniae leads to marked upregulation and surface expression of hHSP60 and adhesion molecules. Moreover, we provide evidence for an increased susceptibility of arterial endothelial cells for redistribution of hHSP60 to the cellular membrane in response to C. pneumoniae infection as compared to autologous venous endothelial cells. We also show that oxidative stress has a central role to play in endothelial cell activation in response to chlamydial infection. These data provide evidence for a role of C. pneumoniae as a potent primary endothelial stressor for arterial endothelial cells leading to enrichment of hHSP60 on the cellular membrane and, as such, a potential initiator of atherosclerosis.

Emerging Engineered Magnetic Nanoparticulate Probes for Molecular MRI of Atherosclerosis: How Far Have We Come?

Atherosclerosis is a chronic, progressive, immunoinflammatory disease of the large and medium-sized arteries, and a major cause of cardiovascular diseases. Atherosclerosis often progresses silently for decades until the occurrence of a major catastrophic clinical event such as myocardial infarction, cardiac arrest and stroke. The main challenge in the diagnosis and management of atherosclerosis is to develop a safe, noninvasive technique that is accurate and reproducible, which can detect the biologically active high-risk vulnerable plaques (with ongoing active inflammation, angiogenesis and apoptosis) before the occurrence of an acute clinical event. This article reviews the events involved in the pathogenesis of atherosclerosis in light of recently advanced understanding of the molecular pathogenesis of the disease. Next, we elaborate on the interesting developments in molecular MRI, by describing the recently engineered magnetic nanoparticulate probes targeting clinically promising molecular and cellular players/processes, involved in early atherosclerotic lesion formation to plaque rupture and erosion.

The double‐edged sword of fibronectin in atherosclerosis

Atherosclerosis, the leading cause of death in Westernized countries, derives from a non‐resolving inflammation of the large arteries. A critical step in the initiation of an atherosclerotic plaque is the expansion of intimal space (the area between the endothelial cell (EC) lining and the medial smooth muscle cell layer) in susceptible regions of arteries (Tabas et al, 2007). The disease‐prone locations are largely determined by arterial blood flow characteristics, with branch points and the lesser curvatures of arteries, where the flow is turbulent, being particularly disposed to plaque formation. At these sites, the normally thin intimal space expands with the deposition of extracellular matrix (ECM), which then serves as a sticky surface to trap LDL particles that are constantly crossing the endothelial layer to enter the artery. One reason for this retention is that the apoB100 molecule on the LDL particle has a specific domain that binds tightly to the ECM, and genetic causation studies in a mouse model of atherosclerosis have shown that when this domain is deleted, plaque burden is significantly reduced despite equivalent hyperlipidemia (Skalen et al, 2002). As implied by the ‘Response to Retention’ hypothesis (Tabas et al, 2007), the retained lipoprotein particles set in motion the invasion of the arterial wall by monocytes that quickly become macrophages that engorge themselves on cholesterol and the other lipids to form foam cells. Foam cells not only physically expand the plaque, but by secreting a variety of chemokines and cytokines, they also create and amplify an inflammatory state (Fig 1). Figure 1. Plasma derived FN promotes atherogenesis in mouse models of atherosclerosis. pFN, synthesized in the liver, is deposited in susceptible regions of the arteries and contributes to early atherosclerotic lesion formation and the recruitment of smooth muscle cells that shape the fibrous cap of advanced lesions (upper panel). …