Progression Of Human Atherosclerosis Research Articles

Abstract 2082: Neutrophil Programming In Atherosclerosis

Emerging evidence suggest the involvement of neutrophils during the progression of human atherosclerosis through secreting inflammatory mediators and causing damages to endothelial vasculature. However, compared to other well-recognized immune cells such as monocytes/macrophages, mechanisms underlying the regulation of neutrophils during atherosclerosis are relatively less studied and understood. This presents a major impediment in our thorough understanding of the pathogenesis of atherosclerosis. In this study, we reported that both murine and human neutrophil population possesses a minor homeostatic resolving subset characterized by reduced expression of inflammatory mediators (LTB4, elastase), elevated expression of anti-inflammatory resolving mediators (RvD1, CD200R), correlated with reduced expression of inflammatory signaling adaptor TRAM. We observed that TRAM deficient neutrophils are effectively reprogrammed into the resolving state, and can potently improve vascular integrity and suppress atherosclerosis pathogenesis when adoptively transfused into recipient atherosclerotic animals. Mechanistically, TRAM deficiency reduces the expression of LOX5AP, dislodges nuclear localization of LOX5, and favors the generation of RvD1 instead of LTB4. We also observed that human TRAM-low neutrophils are less inflammatory, as reflected in reduced swarming and reduced induction of LTB4/elastase. TRAM serves as a stress sensor of oxLDL and/or free cholesterol, triggers inflammatory signaling processes that facilitate elastase release. Our data defines a novel resolving neutrophil subset due to reduced TRAM expression which may hold therapeutic potential for reducing inflammation and treating atherosclerosis.

Plaque‐Specific Adhesive Balloons Coated with Calcium Phosphate Nanoparticles Loaded with Rapamycin for Atherosclerosis Therapy

AbstractDrug‐coated balloons (DCBs), serving as a superior alternative to stents, are at the cutting edge of clinical innovations in atherosclerosis management. Nevertheless, the prevalent high flow rates in arterial vascular result in short drug residence times, significantly compromising DCBs’ therapeutic effectiveness. Drawing inspiration from the mechanisms of vascular calcification, a rapamycin‐loaded calcium phosphate nanoparticle (CaP@Rapa) coating for balloons is pioneered. This coating functions as a potent drug anchor in arteries, facilitating the adhesion of rapamycin to plaques and thereby enhancing the local concentration of the rapamycin. To evaluate its therapeutic efficacy in vivo, the rabbit iliac artery atherosclerosis model is innovatively developed, which authentically mirrors the pathological progression of atherosclerosis in humans. The enhanced localized rapamycin retention, along with its pH‐sensitive sustained release characteristics, culminate in substantial reductions in plaque volume, a decrease in intimal hyperplasia, and mitigation of local inflammation in model rabbits. To further substantiate the translational potential of CaP@Rapa balloons, the research on the pig coronary atherosclerosis model is expanded, which yielded results solidifying the clinical promise of CaP@Rapa balloons. In summary, this research not only offers a more effective and reliable alternative to traditional DCB but also paves the way for further clinical trials.

RNA-binding protein HuR controls vascular endothelial cell inflammatory responses to tumor necrosis factor-A and is associated with atherosclerosis progression in humans

RNA-binding protein HuR controls vascular endothelial cell inflammatory responses to tumor necrosis factor-A and is associated with atherosclerosis progression in humans

Ambient Air Pollution and Atherosclerosis: Recent Updates.

Purpose of ReviewDuring the past century, exposure to particulate matter (PM) air pollution < 2.5 μm in diameter (PM2.5) has emerged as an all-pervading element of modern-day society. This increased exposure has come at the cost of heightened risk for cardiovascular (CV) morbidity and mortality. Not only can short-term PM2.5 exposure trigger acute CV events in susceptible individuals, but longer-term exposure over years augments CV risk to a greater extent in comparison with short-term exposure. The purpose of this review is to examine the available evidence for how ambient air pollution exposure may precipitate events at various time frames.Recent FindingsRecent epidemiological studies have demonstrated an association between ambient PM2.5 exposure and the presence and progression of atherosclerosis in humans. Multiple animal exposure experiments over two decades have provided strong corroborative evidence that chronic exposure in fact does enhance the progression and perhaps vulnerability characteristics of atherosclerotic lesions.SummaryEvidence from epidemiological studies including surrogates of atherosclerosis, human translational studies, and mechanistic investigations utilizing animal studies have improved our understanding of how ambient air pollution may potentiate atherosclerosis and precipitate cardiovascular events. Even so, future research is needed to fully understand the contribution of different constituents in ambient air pollution–mediated atherosclerosis as well as how other systems may modulate the impact of exposure including adaptive immunity and the gut microbiome. Nevertheless, due to the billions of people continually exposed to PM2.5, the long-term pro-atherosclerotic effects of this ubiquitous air pollutant are likely to be of enormous and growing global public health importance.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11883-021-00958-9.

Semaphorin 7A Promotes VEGFA/VEGFR2-Mediated Angiogenesis and Intraplaque Neovascularization in ApoE-/- Mice.

Excessive neovascularization of atherosclerotic lesions increases plaque vulnerability and the susceptibility to rupture. Semaphorin 7A (Sema7A), a semaphorin family member, was recently reported to promote atherosclerotic plaque formation by mediating d-flow-induced endothelial phenotypic change and leukocyte adhesion. To extend our understanding of the proatherogenic role of Sema7A, we investigated the role of endothelial Sema7A in angiogenesis and atherosclerotic neovascularization. Sema7A overexpression in human umbilical vein endothelial cells (HUVECs) significantly upregulated VEGFA/VEGFR2 and promoted cell migration and angiogenesis. This enhancing effect was eliminated by the blockage of Sema7A receptor, β1 integrin. Inhibition of FAK or ERK1/2 downstream of β1 integrin signaling significantly inhibited cell migration and angiogenesis via ROCK (Rho-associated coiled forming protein kinase) and MYPT (myosin phosphatase targeting subunit), which are responsible for actin polymerization. Consistently, in vivo studies showed a remarkable reduction in VEGFA/VEGFR2 expression and neovascularization in the atherosclerotic plaques of Sema7A-/-ApoE-/- mice compared with Sema7A+/+ApoE-/- littermates. Supportively, Sema7A deficiency reduced the accumulation of T cells, macrophages, and dendritic cells, and enhanced plaque stability in ApoE-/- mice. Together, our findings show that Sema7A promotes VEGFA/VEGFR2-mediated neovascularization in a β1 integrin-dependent manner, supporting a crucial role of Sema7A in the progression of human atherosclerosis.

Loss of Sphingosine-1-Phoshate Receptor 3 Expression is Associated with Atherosclerosis in Humans

Introduction and Objectives: A role for Sphingosine-1-phosphate receptors (S1PR) in atherosclerosis has been suggested from animal experiments but no data from humans are available. This study was designed to measure the expression of S1PR in various human vascular beds and to analyze potential associations between receptor expression and plaque burden. Methods: Tissue samples from 6 arteries (coronary a., CO, carotid a., CA, ascending aorta , AS, descending aorta, DE, abdominal aorta, AB, and iliac a., IL) were collected from randomly selected people (N=163) during autopsies. For each subject, a comprehensive plaque score (CPS) was generated based on plaque in the arteries harvested. Expression of S1PR1-3 was measured by qPCR and data were expressed as log2. Statistical analyses were performed by appropriate non-parametric tests. Results: Among the 6 arteries, expression of each of the three S1PR measured differs significantly. The highest expression of all S1PR was measured in CO and the lowest in AS and DE. In all 6 arteries, the highest positive correlation was found between S1PR1 and S1PR2 expression with Spearman coefficients (r) between 0.59 (P=2xE-17) in IL and 0.74 (P=1xE-29) in AS. When the expression of S1PR was compared between the different arteries, the most significant associations were always found for S1PR3. Most strikingly, subjects with increasing CPS show a considerable decrease in S1PR3 expression in all arteries, which is independent of age. Interestingly, S1PR3 expression also decreases with increasing CPS even in arteries that themselves were scored healthy. Conclusion: Loss of S1PR3 expression is associated with increasing plaque burden. This effect appears to be of systemic nature as also arteries are concerned that themselves are free of plaque. This finding underlines the systemic nature of atherosclerosis and identifies the S1P/S1PR3-axis as a potential target to slow progression of atherosclerosis in humans.

Dicer in Macrophages Prevents Atherosclerosis by Promoting Mitochondrial Oxidative Metabolism.

Alternative macrophage activation, which relies on mitochondrial oxidative metabolism, plays a central role in the resolution of inflammation and prevents atherosclerosis. Moreover, macrophages handle large amounts of cholesterol and triglycerides derived from the engulfed modified lipoproteins during atherosclerosis. Although several microRNAs regulate macrophage polarization, the role of the microRNA-generating enzyme Dicer in macrophage activation during atherosclerosis is unknown. To evaluate the role of Dicer in atherosclerosis, Apoe-/- mice with or without macrophage-specific Dicer deletion were fed a high-fat diet for 12 weeks. Anti-argonaute 2 RNA immunoprecipitation chip and RNA deep sequencing combined with microRNA functional screening were performed in the Dicer wild-type and knockout bone marrow-derived macrophages to identify the individual microRNAs and the mRNA targets mediating the phenotypic effects of Dicer. The role of the identified individual microRNA and its target in atherosclerosis was determined by tail vein injection of the target site blockers in atherosclerotic Apoe-/- mice. We show that Dicer deletion in macrophages accelerated atherosclerosis in mice, along with enhanced inflammatory response and increased lipid accumulation in lesional macrophages. In vitro, alternative activation was limited whereas lipid-filled foam cell formation was exacerbated in Dicer-deficient macrophages as a result of impaired mitochondrial fatty acid oxidative metabolism. Rescue of microRNA (miR)-10a, let-7b, and miR-195a expression restored the oxidative metabolism in alternatively activated Dicer-deficient macrophages. Suppression of ligand-dependent nuclear receptor corepressor by miR-10a promoted fatty acid oxidation, which mediated the lipolytic and anti-inflammatory effect of Dicer. miR-10a expression was negatively correlated to the progression of atherosclerosis in humans. Blocking the interaction between ligand-dependent nuclear receptor corepressor and miR-10a by target site blockers aggravated atherosclerosis development in mice. Dicer plays an atheroprotective role by coordinately regulating the inflammatory response and lipid metabolism in macrophages through enhancing fatty acid-fueled mitochondrial respiration, suggesting that promoting Dicer/miR-10a-dependent metabolic reprogramming in macrophages has potential therapeutic implications to prevent atherosclerosis.

Amlodipine Inhibits Vascular Cell Senescence and Protects Against Atherogenesis Through the Mechanism Independent of Calcium Channel Blockade.

Vascular cells have a finite lifespan and eventually enter irreversible growth arrest called cellular senescence. We have previously suggested that vascular cell senescence contributes to the pathogenesis of human atherosclerosis. Amlodipine is a mixture of two enantiomers, one of which (S- enantiomer) has L-type channel blocking activity, while the other (R+ enantiomer) shows ~1000-fold weaker channel blocking activity than S- enantiomer and has other unknown effects. It has been reported that amlodipine inhibits the progression of atherosclerosis in humans, but the molecular mechanism of this beneficial effect remains unknown. Apolipoprotein E-deficient mice on a high-fat diet were treated with amlodipine, its R+ enantiomer or vehicle for eight weeks. Compared with vehicle treatment, both amlodipine and the R+ enantiomer significantly reduced the number of senescent vascular cells and inhibited plaque formation to a similar extent. Expression of the pro-inflammatory molecule interleukin-1β was markedly upregulated in vehicle-treated mice, but was inhibited to a similar extent by treatment with amlodipine or the R+ enantiomer. Likewise, activation of p53 (a critical inducer of senescence) was markedly suppressed by treatment with amlodipine or the R+ enantiomer. These results suggest that amlodipine inhibits vascular cell senescence and protects against atherogenesis at least partly by a mechanism that is independent of calcium channel blockade.

The role of CD36 receptor in the pathogenesis of atherosclerosis.

Atherosclerosis is a progressive, chronic inflammation in artery walls. Oxidized low density lipoproteins (ox-LDL) play an important role in atherosclerotic plaque formation. ox-LDL are taken up by macrophages mainly through scavenger receptors, among which CD36 is considered to be the most important. Animal studies have shown that crossing atherogenic mice with a strain lacking the expression of CD36 prevented the development of atherosclerosis despite a diet rich in saturated LCFA. In humans, autopsy studies performed in obese patients have demonstrated increased expression of CD36 receptor on macrophages, comprised within atherosclerotic plaques. Until recently it had been believed that CD36 is a major player in atherosclerosis progression in humans. However, recent studies challenge this conviction, showing increased incidence of coronary heart disease in the subgroup of patients with decreased expression of CD36. This article reviews the role of CD36 receptor in the development of atherosclerosis. The authors also discuss current possibilities to interfere with CD36, their potential benefits and hazards.

Protective effects of bezafibrate against elaidic acid-induced accumulation of lipid droplets in monocytic cells

Some factors related to diet, such as trans fatty acids (TFA), are known to be involved in the progression of atherosclerosis in humans. Thus, the aim of our study was (i) to evaluate the effects of three dietary free fatty acids (FFA) (elaidic (EA), oleic (OA) and palmitic acid (PA)) on U937 human monocytes, and (ii) to study the eventual benefits of bezafibrate (BZF), a pan-agonist for PPAR isoforms (α, γ and δ) in U937 cells treated with FFA. Morphologic and functional changes were investigated by microscopic and flow cytometric methods. Cellular lipid content, lipid droplets and FA composition were identified and studied. All analyses were also realized in association with or without BZF. Contrary to OA and PA, EA slightly induced both propidium iodide-positive cells and mitochondrial depolarization. In addition, in contrast to OA and PA, EA induced only a slight increase in superoxide anion production. However, EA and OA promoted cytoplasmic lipid droplets accumulation. Only EA and OA significantly increased CD36 expression. It is noteworthy that BZF had a more or less pronounced protective effect against EA-, OA- and PA-induced side effects: BZF attenuated the impaired cell viability and inflammatory response, decreased superoxide anion production and prevented the accumulation of neutral and polar lipids. The effects were less pronounced with OA and PA than with EA. Altogether, our data revealed a benefit of BZF on the side effects induced especially with EA. It may thus be of interest in preventing the early stages of atherosclerotic plaque formation.

Atheroprotective remodelling of vascular dermatan sulphate proteoglycans in response to hypercholesterolaemia in a rat model

Proteoglycan accumulation within the arterial intima has been implicated in atherosclerosis progression in humans. Nevertheless, hypercholesterolaemia is unable to induce intimal thickening and atheroma plaque development in rats. The study was performed to analyse proteoglycans modifications in rats fed with a high-cholesterol diet to understand whether vascular wall remodelling protects against lesions. Sections obtained from rat aortas showed normal features, in intimal-to-media ratio and lipid accumulation. However, focal endothelial hyperplasia and neo-intima rearrangement were observed in high-cholesterol animals. Besides, hypercholesterolaemia induced an inflammatory microenviroment. We determined the expression of different proteoglycans from aortic cells by Western blot and observed a diminished production of decorin and biglycan in high-cholesterol animals compared with control (P<0.01 and P<0.05, respectively). Versican was increased in high-cholesterol animals (P<0.05), whereas perlecan production showed no differences. No modification of the total content of glycosaminoglycans (GAGs) was found between the two experimental groups. In contrast, the chondroitin sulphate/dermatan sulphate ratio was increased in the high-cholesterol group as compared to the control (0.56 and 0.34, respectively). Structural alterations in the disaccharide composition of galactosaminoglycans were also detected by HPLC, as the ratio of 6-sulphate to 4-sulphate disaccharides was increased in high-cholesterol animals (P<0.05). Our results suggest that attenuation of decorin and biglycan expression might be an effective strategy to inhibit the first step in atherogenesis, although specific GAG structural modification associated with the development of vascular disease took place. Results emphasize the potential application of therapies based on vascular matrix remodelling to treat atherosclerosis.

Macrophage Mitochondrial Oxidative Stress Promotes Atherosclerosis and Nuclear Factor-κB–Mediated Inflammation in Macrophages

Mitochondrial oxidative stress (mitoOS) has been shown to correlate with the progression of human atherosclerosis. However, definitive cell type-specific causation studies in vivo are lacking, and the molecular mechanisms of potential proatherogenic effects remain to be determined. Our aims were to assess the importance of macrophage mitoOS in atherogenesis and to explore the underlying molecular mechanisms. We first validated Western diet-fed Ldlr(-/-) mice as a model of human mitoOS-atherosclerosis association by showing that non-nuclear oxidative DNA damage, a marker of mitoOS in lesional macrophages, correlates with aortic root lesion development. To investigate the importance of macrophage mitoOS, we used a genetic engineering strategy in which the OS suppressor catalase was ectopically expressed in mitochondria (mCAT) in macrophages. MitoOS in lesional macrophages was successfully suppressed in these mice, and this led to a significant reduction in aortic root lesional area. The mCAT lesions had less monocyte-derived cells, less Ly6c(hi) monocyte infiltration into lesions, and lower levels of monocyte chemotactic protein-1. The decrease in lesional monocyte chemotactic protein-1 was associated with the suppression of other markers of inflammation and with decreased phosphorylation of RelA (NF-κB p65), indicating decreased activation of the proinflammatory NF-κB pathway. Using models of mitoOS in cultured macrophages, we showed that mCAT suppressed monocyte chemotactic protein-1 expression by decreasing the activation of the IκB-kinase β-RelA NF-κB pathway. MitoOS in lesional macrophages amplifies atherosclerotic lesion development by promoting NF-κB-mediated entry of monocytes and other inflammatory processes. In view of the mitoOS-atherosclerosis link in human atheromata, these findings reveal a potentially new therapeutic target to prevent the progression of atherosclerosis.

Low Levels of Antibodies Against Phosphorylcholine Predict Development of Stroke in a Population-Based Study From Northern Sweden

Natural immunoglobulin M antibodies specific for phosphorylcholine (anti-PC) have been implicated in atherosclerosis. We have shown previously that high levels of anti-PC predict a slower progression of atherosclerosis in humans and that low levels of anti-PC are associated with higher risk for cardiovascular disease. Here we determine the association between anti-PC and the incidence of stroke. Using a nested case control study design, we examined 227 incident cases (125 men and 102 women) of first-time stroke and 455 age- and sex-matched controls identified during a 13-year time period (1985 to 1999) within the population-based cohorts of the Västerbotten Intervention Project (VIP) and the World Health Organization Monitoring Trends and Determinants in Cardiovascular Disease (WHO MONICA) project in Northern Sweden. Odds ratios of stroke with 95% CIs with adjustments for age, gender, smoking, serum cholesterol, diabetes, body mass index, and blood pressure were determined. Anti-PC levels were measured using ELISA. A significant association between low levels of anti-PC at baseline and incident stroke was seen for the whole group of anti-PC levels below the 30th percentile (multivariately adjusted odds ratio, 1.62; CI, 1.11 to 2.35). Analyses of gender-specific associations indicated fairly strong associations for females, especially at the lowest 30th percentile (multivariately adjusted odds ratio, 2.65; CI, 1.41 to 4.95). No associations were noted for men. Low anti-PC is a novel independent risk marker for development of stroke. Measurements of anti-PC could be used to identify immunodeficient subjects at an increased risk for stroke. The possibility that such subjects might be targets for novel modes of treatment such as immunotherapies deserves further investigation.

The thyroid hormone mimetic compound KB2115 lowers plasma LDL cholesterol and stimulates bile acid synthesis without cardiac effects in humans

Atherosclerotic cardiovascular disease is a major problem despite the availability of drugs that influence major risk factors. New treatments are needed, and there is growing interest in therapies that may have multiple actions. Thyroid hormone modulates several cardiovascular risk factors and delays atherosclerosis progression in humans. However, use of thyroid hormone is limited by side effects, especially in the heart. To overcome this limitation, pharmacologically selective thyromimetics that mimic metabolic effects of thyroid hormone and bypass side effects are under development. In animal models, such thyromimetics have been shown to stimulate cholesterol elimination through LDL and HDL pathways and decrease body weight without eliciting side effects. We report here studies on a selective thyromimetic [KB2115; (3-[[3,5-dibromo-4-[4-hydroxy-3-(1-methylethyl)-phenoxy]-phenyl]-amino]-3-oxopropanoic acid)] in humans. In moderately overweight and hypercholesterolemic subjects KB2115 was found to be safe and well tolerated and elicited up to a 40% lowering of total and LDL cholesterol after 14 days of treatment. Bile acid synthesis was stimulated without evidence of increased cholesterol production, indicating that KB2115 induced net cholesterol excretion. KB2115 did not provoke detectable effects on the heart, suggesting that the pharmacological selectivity observed in animal models translates to humans. Thus, selective thyromimetics deserve further study as agents to treat dyslipidemia and other risk factors for atherosclerosis.

C1-Esterase Inhibitor Protects Against Neointima Formation After Arterial Injury in Atherosclerosis-Prone Mice

Although activation of the complement system has been implicated in the progression of human atherosclerosis, its function during arterial remodeling after injury has not been investigated. Here, we examined the contribution of the complement cascade to neointima formation in apolipoprotein E-deficient mice using a C1-esterase inhibitor (C1-inhibitor). Apolipoprotein E-deficient mice fed an atherogenic diet were subjected to wire-induced endothelial denudation of the carotid artery and treated with C1-inhibitor (Berinert; 15 IU i.v.) or vehicle perioperatively and subsequently every 2 days. The effectiveness of C1-inhibitor treatment was confirmed by measurement of plasma C1-inhibitor activity. A significant reduction in serum triglyceride levels was observed in C1-inhibitor-treated mice, whereas cholesterol levels did not differ. After 3 weeks, neointimal area was significantly reduced in C1-inhibitor-treated mice versus controls, whereas medial area was unaltered. This was associated with a significant decrease in neointimal and medial macrophage and CD3+ T-cell content. Expression of C3 mRNA was significantly reduced in plaques of C1-inhibitor-treated mice 10 days after injury, as assessed by reverse-transcription polymerase chain reaction. The peak in serum C3 levels after injury was markedly downregulated by C1-inhibitor, as evidenced by ELISA. Immunohistochemistry revealed strong expression of C3 and C3c, which colocalized to plaque macrophages and was reduced in C1-inhibitor-treated mice. C1-inhibitor impaired monocyte arrest on activated endothelium and platelets under flow conditions in vitro and leukocyte recruitment to carotid arteries 1 day after injury in vivo. C1-inhibitor limits neointimal plaque formation and inflammation. This may involve blockade of complement activation, inhibition of leukocyte recruitment, and reduced triglyceride levels, thus providing a multimodal approach to treat arterial disease.

Management of Atherosclerosis With Antiallergic Medicine: A Lesson From the Mouse Model

Evaluation of: Sun J, Sukhova GK, Wolters PJ et al.: Mast cells promote atherosclerosis by releasing pro-inflammatory cytokines. Nat. Med. 13, 719-724 (2007). Mast cells are important components in human allergic response and innate immunity. These cells have been implicated in the pathogenesis of atherosclerosis since the 1950s, and a series of studies have proposed their roles in the pathologic events critical to atherogenesis. Despite these studies and hypotheses, there is no evidence to suggest a direct participation of these allergic cells in atherosclerosis. Using mast cell-deficient mice and intravenous mast cell reconstitution technology with a conventional mouse atherosclerosis model, Dr Sun et al. revealed that mast cells contribute to atherogenesis by releasing proinflammatory cytokines, which are utilized to stimulate vascular cell-protease expression and further tissue remodeling. Mice that lack these cells are resistant to diet-induced atherosclerosis. These data suggest that stabilization of mast cells with antiallergic medicine may be utilized in controlling or preventing the initiation and progression of atherosclerosis in humans.

Heat Shock Proteins and Autoimmunity in Patients with Carotid Atherosclerosis

Studies aimed at elucidating the pathogenetic mechanisms underlying the initiation and progression of human atherosclerosis have emphasized the central role of inflammatory and immune cells. Atherosclerotic plaques are infiltrated by activated macrophages, T and B lymphocytes, plasma cells, and mast cells, releasing inflammatory molecules, which amplify the severity of the disease. Endothelial cells subjected to various stress conditions express increased amounts of heat shock proteins (HSPs), some of the most successfully conserved proteins throughout evolution. Many experimental observations reviewed in this article draw attention to several HSPs targeted by a specific cellular and humoral immune response in patients with atherosclerotic disease. The review also reports preliminary data obtained by our group on the possible role of HSP90 as a candidate autoantigen in carotid atherosclerosis. Our study deals with the presence of specific antibodies and T cells directed against HSP90 in patients with carotid atherosclerotic plaques. In 60% of these subjects' sera but in none of the sera from healthy controls immunoblotting (IB) detected the presence of specific antibodies. Moreover, 20% of peripheral blood mononuclear cells (PBMC) samples from patients but none from healthy subjects proliferated in response to human purified HSP90. In vitro experiments showed an upregulation of HSP90 expression in endothelial cells exposed to oxidative stress by treatment with H(2)O(2) and greater release of soluble HSP90 in culture supernatants from H(2)O(2)-treated cells than from untreated cells.

Plasminogen Activator Inhibitor 1 Deficiency Reduces the Progression of Atherosclerosis in a Murine Model of Human Familial Hypercholesterolemia.

Atherosclerosis has been described as a “self-perpetuating” inflammatory disease which progresses in discrete stages and involves a number of cell types and effector molecules. Lipid metabolic disorders are heavily involved in the genesis and progression of atherosclerosis in humans, and thrombotic complications substantially contribute to the coronary artery disease that frequently accompanies atherosclerosis. A relevant mouse model of human atherosclerosis with elevated LDL-cholesterol (C) is one in which both the Ldlr and Apobec1 genes are deleted (Ldlr−/−/Apobec1−/−), since these mice lack the ability to convert apoB-100 to apoB-48 in liver and also are defective in LDL clearance. We have generated Ldlr−/−/Apobec1−/−/PAI-1−/−and characterized atherosclerosis progression in these mice. VLDL, LDL, HDL, and total C levels were identical between Ldlr−/−/Apobec1−/−and Ldlr−/−/Apobec1−/−/PAI-1−/−mice (VLDL-C; 30.3±1.8 vs 26.0±1.7 (mg/dl), LDL-C; 268.7±6.3 vs 274.1±13.1 (mg/dl), HDL-C; Total-C; 353.8±8.1 vs 356.8±14.5 (mg/dl), respectively). However, both the plaque size in the aortic sinus and the extent of plaque area in aorta were significantly smaller in Ldlr−/−/Apobec1−/−/PAI-1−/−mice relative to Ldlr−/−/Apobec1−/−mice (plaque in aortic sinus; 158792.9±11958.2 vs 227243.5±15291.1 (μm2), plaque area in aorta; 3.90±0.34 vs 5.10±0.27 (%), respectively). To elucidate the cellular functions, especially macrophages, in the plaque, bone marrow-derived macrophages (BMMs) were isolated and utilized for Dil-oxidized (Ox) LDL uptake assays and migration assays using 8 μm pore culture inserts. There were no significant differences in Dil-OxLDL uptake by BMMs between the two genotypes. The kinetics of migration of BMMs was more rapid in Ldlr−/−/Apobec1−/−/PAI-1−/−cells than Ldlr−/−/Apobec1−/−cells. Furthermore, thioglycollate-induced intraperitoneal migration of macrophages was increased in Ldlr−/−/Apobec1−/−/PAI-1−/−mice vs Ldlr−/−/Apobec1−/−mice. Results from these studies indicate that cell migratory differences may contribute to attenuating atheroma formation in a PAI-1 deficient state.

Regulation of Macrophage Foam Cell Formation by αVβ3 Integrin: Potential Role in Human Atherosclerosis

The accumulation of macrophage foam cells in atherosclerotic lesions is associated with both initiation and progression of this disease. Scavenger receptors CD36 and SRA are the primary receptors responsible for conversion of macrophages into foam cells. Integrin alphaVbeta3 plays a role in the differentiation of several cell types, but its involvement in the transition of macrophages into foam cells and the potential role of this receptor in atherosclerosis have not been examined. Using an in vitro model of single surface receptor activation by binding with an immobilized monoclonal antibody specific to alphaVbeta3 integrin we show that ligation of alphaVbeta3 integrin prevents differentiation of blood monocytes and macrophages into the foam cell phenotype via coordinate down-regulation of CD36 and SRA. This effect of alphaVbeta3 integrin ligation can be reproduced by contact with endothelial cells, whereas the inhibition of alphaVbeta3 receptor ligation restores the uptake of oxidized low-density lipoprotein. Moreover, we found that alphaVbeta3 integrin is readily detected in situ on macrophages in early and advanced atherosclerotic lesions and that in vitro exposure to oxidized low-density lipoprotein up-regulates alphaVbeta3 integrin expression. We hypothesize that alphaVbeta3 integrin regulates macrophage functional maturation into foam cells in a persistent manner, and therefore, by targeting alphaVbeta3 receptor it could potentially be possible to regulate progression of atherosclerosis in humans.

Multifunctional roles of macrophages in the development and progression of atherosclerosis in humans and experimental animals.

In atherosclerosis, macrophages are important for intracellular lipid accumulation and foam cell formation. Monocytes respond to chemotactic factors, cytokines, and macrophage growth factors produced by vascular endothelial cells, smooth muscle cells, and infiltrated cells, by migrating from peripheral blood into the arterial intima and differentiating into macrophages in atherosclerotic lesions. Although various chemotactic factors are known to induce monocyte migration, monocyte chemoattractant protein-1 is the most important and powerful inducer of migration into atherosclerotic lesions. Macrophage colony-stimulating factor is crucial for monocyte/macrophage differentiation and proliferation, and for the survival of macrophages in these lesions. A minor population of macrophages can proliferate in the atherosclerotic lesions themselves, particularly in the early stage. The macrophages express a variety of receptors, particularly scavenger receptors, and take up modified lipoproteins, including oxidized low-density lipoprotein, beta-very-low-density lipoprotein, and/or enzymatically degraded low-density lipoprotein. These cells accumulate cholesterol esters in the cytoplasm, which leads to foam cell formation in lesion development. Among various scavenger receptors, class A type I and type II macrophage scavenger receptors (MSR-A I,II) play the most important role in the uptake of oxidized low-density lipoprotein by macrophages. In addition, macrophages and macrophage-derived foam cells produce ceroid and advanced glycation end-products (AGEs) and accumulate these substances in their cytoplasm. Extracellularly generated AGEs are taken up by macrophages via receptors for AGEs, including MSR-AI,II. Most foam cells die in loco because of apoptosis, and some foam cells escape from the lesions into peripheral blood. Macrophages also play multifaceted roles in inducing plaque rupture, blood coagulation, and fibrinolysis via the production of various enzymes, activators, inhibitors, and bioactive mediators. During the development of atherosclerosis, macrophages interact with vascular endothelial cells, medial smooth muscle cells, and infiltrated inflammatory cells, particularly T cells and dendritic cells. This review, based on data accumulated in studies of atherosclerosis in humans and experimental animals, focuses on the multifunctional roles of macrophages in the pathogenesis and progression of atherosclerosis.