Lipid-rich Atherosclerotic Lesions Research Articles

Serum amyloid A and interleukin -1β facilitate LDL transcytosis across endothelial cells and atherosclerosis via NF-κB/caveolin-1/cavin-1 pathway

Inflammatory molecules play important roles in atherosclerosis. We aimed to illustrate the roles of serum amyloid A (SAA), and interleukin (IL)-1β in low density lipoproteins (LDL) transcytosis and atherosclerosis. Effects of SAA and IL-1β on transcytosis of LDL were measured by an in vitro LDL transcytosis model. NF-κB/caveolin-1/cavin-1 pathway activation was investigated by Western blots and ELISA. Effects of SAA and IL-1β on the retention of LDL in aorta of C57BL/6J mice were detected by IVIS spectrum. Effects of SAA and IL-1β on atherosclerosis in Apoe-/- mice were examined by Oil Red O staining. SAA and IL-1β stimulated LDL transcytosis across endothelial cells (ECs), which was accompanied by an increase in LDL uptake by ECs. SAA and IL-1β enhanced the activity of nuclear factor (NF)-κB, consequently facilitating an up-regulation of proteins involved in caveolae formation, including caveolin-1 and cavin-1, along with an assembly of NLRP3 inflammasome. Furthermore, SAA- and IL-1β-induced effects were blocked by NF-κB subunit p65 siRNA. Meanwhile, SAA- and IL-1β-induced LDL transcytosis were effectively blocked by caveolin-1 siRNA or cavin-1 siRNA. Interestingly, SAA and IL-1β facilitated LDL entering into the aorta of C57BL/6J mice. In Apoe-/- mice, SAA and IL-1β increased the areas of lipid-rich atherosclerotic lesions in the both ascending and root of aorta. Furthermore, a significant increase in the NLRP3 inflammasome, accompanied by accumulation of cavin-1 and caveolin-1, was observed in the aortic endothelium of Apoe-/- mice. SAA and IL-1β accelerated LDL transcytosis via the NF-κB/caveolin-1/cavin-1 axis.

Photoacoustic imaging of carotid artery atherosclerosis.

We introduce a method for photoacoustic imaging of the carotid artery, tailored toward detection of lipid-rich atherosclerotic lesions. A common human carotid artery was obtained at autopsy, embedded in a neck mimicking phantom and imaged with a multimodality imaging system using interstitial illumination. Light was delivered through a 1.25-mm-diameter optical probe that can be placed in the pharynx, allowing the carotid artery to be illuminated from within the body. Ultrasound imaging and photoacoustic signal detection is achieved by an external 8-MHz linear array coupled to an ultrasound imaging system. Spectroscopic analysis of photoacoustic images obtained in the wavelength range from 1130 to 1250 nm revealed plaque-specific lipid accumulation in the collagen structure of the artery wall. These spectroscopic findings were confirmed by histology.

Stretchable electrochemical impedance sensors for intravascular detection of lipid-rich lesions in New Zealand White rabbits

Flexible electronics have enabled catheter-based intravascular sensing. However, real-time interrogation of unstable plaque remains an unmet clinical challenge. Here, we demonstrate the feasibility of stretchable electrochemical impedance spectroscopy (EIS) sensors for endoluminal investigations in New Zealand White (NZW) rabbits on diet-induced hyperlipidemia. A parylene C (PAC)-based EIS sensor mounted on the surface of an inflatable silicone balloon affixed to the tip of an interrogating catheter was deployed (1) on the explants of NZW rabbit aorta for detection of lipid-rich atherosclerotic lesions, and (2) on live animals for demonstration of balloon inflation and EIS measurements. An input peak-to-peak AC voltage of 10 mV and sweeping-frequency from 300 kHz to 100 Hz were delivered to the endoluminal sites. Balloon inflation allowed EIS sensors to be in contact with endoluminal surface. In the oxidized low-density-lipoprotein (oxLDL)-rich lesions from explants of fat-fed rabbits, impedance magnitude increased significantly by 1.5-fold across the entire frequency band, and phase shifted ~5° at frequencies below 10 kHz. In the lesion-free sites of the normal diet-fed rabbits, impedance magnitude increased by 1.2-fold and phase shifted ~5° at frequencies above 30 kHz. Thus, we demonstrate the feasibility of stretchable intravascular EIS sensors for identification of lipid rich lesions, with a translational implication for detecting unstable lesions.

Noninvasive Detection of Lipids in Atherosclerotic Plaque Using Ultrasound Thermal Strain Imaging: In Vivo Animal Study

This study sought to examine the feasibility of in vivo detection of lipids in atherosclerotic plaque (AP) by ultrasound (US) thermal (or temporal) strain imaging (TSI). Intraplaque lipid content is thought to contribute to plaque stability. Lipid exhibits a distinctive physical characteristic of temperature-dependent US speed compared with water-bearing tissues. As tissue temperature changes, US radiofrequency (RF) echoes shift in time of flight, which produces an apparent strain (thermal or temporal strain [TS]). US heating-imaging pulse sequences and transducers were designed and integrated into commercial US scanners for US-TSI of arterial segments. US-RF data were collected while gradually increasing tissue temperature. Phase-sensitive speckle tracking was applied to reconstruct TS maps coregistered to B-scans. Segments from injured atherosclerotic and uninjured nonatherosclerotic common femoral arteries (CFA) in cholesterol-fed New Zealand rabbits, and segments from control normal diet-fed rabbits (N =14) were scanned in vivo at different time points up to 12 weeks. Lipid-rich atherosclerotic lesions exhibited distinct positive TS (+0.19 ± 0.08%) compared with that in nonatherosclerotic (-0.10 ± 0.13%) and control (-0.09 ± 0.09%) segments (p < 0.001). US-TSI enabled serial monitoring of lipids during atherosclerosis development. The coregistered set of morphological and compositional information of US-TSI showed good agreement with histology. US-TSI successfully detected and longitudinally monitored lipid progression in atherosclerotic CFA. US-TSI of relatively superficial arteries may be a modality that could be integrated into a commercial US system for noninvasive lipid detection in AP.

Abstract 553: Expression of TNFalfa and CCL18 is Increased as a Result of Cholesterol Accumulation in Macrophages

Innate immunity plays important role in atherogenesis. Immune reaction in artery wall involves both pro-and anti-inflammatory cytokines. We investigated cytokines’ mRNA expression in uninvolved and atherosclerotic human aortic intima. TNF-α and CCL18 were localized in autopsy aortic samples using immunohistochemistry. Expression of cytokine mRNA was measured by PCR or qPCR in the same samples. Primary culture of blood-derived monocytes/macrophages from healthy donors was used to study TNF-α and CCL18 mRNA expression induced by IFN-gamma and IL-4, respectively. Intracellular lipid accumulation was induced by incubation of cultured cells with atherogenic modified LDL isolated from blood of patients with documented coronary atherosclerosis. Immunohistochemistry demonstrated that the number of cytokine-positive cells (both TNF-α and CCL18) were increased in atherosclerotic lesions as compared with unaffected aortic intima. Cytokines were associated with different cell types including macrophages and non-macrophage cells. Both TNF-α and CCL18 were manly occurred in macrophages. Rice of both cytokines’ mRNA expression measured by PCR was observed in atherosclerotic lesion. In lipid-rich atherosclerotic lesion cytokine expression was the most considerable. Correlation between lipid content in aortic intima and cytokine expression was found. To elucidate the mechanism of elevation of lipid-associated cytokine expression, cell culture study was performed. Cultured monocytes/macrophages were incubated with atherogenic modified LDL of atherosclerotic patients and non-atherogenic LDL from healthy donors. In contrast to non-atherogenic LDL patients’ LDL induced intracellular cholesterol accumulation and increased mRNA expression of both TNF-α and CCL18 in cell culture. Thus, i ntracellular lipid accumulation caused by atherogenic modified LDL from atherosclerotic patients induces overexpression of TNFa and CCL18. This finding can explain the elevation of TNF-α and CCL18 expression in lipid-rich atherosclerotic lesions of human aorta.

Elevated electrochemical impedance in the endoluminal regions with high shear stress: Implication for assessing lipid-rich atherosclerotic lesions

Identifying metabolically active atherosclerotic lesions remains an unmet clinical challenge during coronary intervention. Electrochemical impedance (EIS) increased in response to oxidized low density lipoprotein (oxLDL)-laden lesions. We hereby assessed whether integrating EIS with intravascular ultrasound (IVUS) and shear stress (ISS) provided a new strategy to assess oxLDL-laden lesions in the fat-fed New Zealand White (NZW) rabbits. A micro-heat transfer sensor was deployed to acquire the ISS profiles at baseline and post high-fat diet (HD) in the NZW rabbits (n=8). After 9 weeks of HD, serum oxLDL levels (mg/dL) increased by 140 fold, accompanied by a 1.5-fold increase in kinematic viscosity (cP) in the HD group. Time-averaged ISS (ISSave) in the thoracic aorta also increased in the HD group (baseline: 17.61±0.24 vs. 9 weeks: 25.22±0.95dyne/cm(2), n=4), but remained unchanged in the normal diet group (baseline: 22.85±0.53dyn/cm(2) vs. 9 weeks: 22.37±0.57dyne/cm(2), n=4). High-frequency intravascular ultrasound (IVUS) revealed atherosclerotic lesions in the regions with augmented ISSave, and concentric bipolar microelectrodes demonstrated elevated EIS signals, which were correlated with prominent anti-oxLDL immuno-staining (oxLDL-free regions: 497±55Ω, n=8 vs. oxLDL-rich lesions: 679±125Ω, n=12, P<0.05). The equivalent circuit model for tissue resistance between the lesion-free and ox-LDL-rich lesions further validated the experimental EIS signals. By applying electrochemical impedance in conjunction with shear stress and high-frequency ultrasound sensors, we provided a new strategy to identify oxLDL-laden lesions. The study demonstrated the feasibility of integrating EIS, ISS, and IVUS for a catheter-based approach to assess mechanically unstable plaque.

Transforming growth factor-β and atherosclerosis: interwoven atherogenic and atheroprotective aspects.

Age-related progression of cardiovascular disease is by far the largest health problem in the US and involves vascular damage, progressive vascular fibrosis and the accumulation of lipid-rich atherosclerotic lesions. Advanced lesions can restrict flow to key organs and can trigger occlusive thrombosis resulting in a stroke or myocardial infarction. Transforming growth factor-beta (TGF-β) is a major orchestrator of the fibroproliferative response to tissue damage. In the early stages of repair, TGF-β is released from platelets and activated from matrix reservoirs; it then stimulates the chemotaxis of repair cells, modulates immunity and inflammation and induces matrix production. At later stages, it negatively regulates fibrosis through its strong antiproliferative and apoptotic effects on fibrotic cells. In advanced lesions, TGF-β might be important in arterial calcification, commonly referred to as "hardening of the arteries". Because TGF-β can signal through multiple pathways, namely the SMADs, a MAPK pathway and the Rho/ROCK pathways, selective defects in TGF-β signaling can disrupt otherwise coordinated pathways of tissue regeneration. TGF-β is known to control cell proliferation, cell migration, matrix synthesis, wound contraction, calcification and the immune response, all being major components of the atherosclerotic process. However, many of the effects of TGF-β are essential to normal tissue repair and thus, TGF-β is often thought to be "atheroprotective". The present review attempts to parse systematically the known effects of TGF-β on both the major risk factors for atherosclerosis and to isolate the role of TGF-β in the many component pathways involved in atherogenesis.

Increased Neovascularization in Advanced Lipid-Rich Atherosclerotic Lesions Detected by Gadofluorine-M-Enhanced MRI

Background— Inflammation and neovascularization may play a significant role in atherosclerotic plaque progression and rupture. We evaluated gadofluorine-M-enhanced MRI for detection of plaque inflammation and neovascularization in an animal model of atherosclerosis. Methods and Results— Sixteen rabbits with aortic plaque and 6 normal control rabbits underwent gadofluorine-M-enhanced MRI. Eight rabbits had advanced atherosclerotic lesions, whereas the remaining 8 had early lesions. Magnetic resonance atherosclerotic plaque enhancement was meticulously compared with plaque inflammation and neovessel density as assessed by histopathology. Advanced plaques and early atheroma were enhanced after gadofluorine-M injection. Control animals displayed no enhancement. After accounting for the within-animal correlation of observations, mean contrast-to-noise ratio was significantly higher in advanced plaques than compared with early atheroma (4.29�0.21 versus 3.00�0.32; P =0.004). Macrophage density was higher in advanced plaques in comparison to early atheroma (geometric mean=0.50 [95% CI, 0.19 to 1.03] versus 0.25 [0.07 to 0.42]; P =0.05). Furthermore, higher neovessel density was observed in advanced plaques (1.83 [95% CI, 1.51 to 2.21] versus 1.29 [0.99 to 1.69]; P =0.05). The plaque accumulation of gadofluorine-M correlated with increased neovessel density as shown by linear regression analysis ( r =0.67; P &lt;0.001). Confocal and fluorescence microscopy revealed colocalization of gadofluorine-M with plaque areas containing a high density of neovessels. Conclusion— Gadofluorine-M-enhanced MRI is effective for in vivo detection of atherosclerotic plaque inflammation and neovascularization in an animal model of atherosclerosis. These findings suggest that gadofluorine-M enhancement reflects the presence of high-risk plaque features believed to be associated with plaque rupture. Gadofluorine-M plaque enhancement may therefore provide functional assessment of atherosclerotic plaque in vivo.

The immunoadhesin glycoprotein VI-Fc regulates arterial remodelling after mechanical injury in ApoE−/− mice

Rupture of advanced atherosclerotic plaques initiates platelet activation and aggregation as subendothelial collagen is exposed. Platelet collagen receptor glycoprotein VI (GPVI) was found to bind preferentially to the core region of human plaques. Consequently, platelets contribute to inflammatory processes and trigger atherosclerotic lesion progression. In this study, we examined binding of soluble platelet collagen receptor GPVI-Fc to atherosclerotic lesions and its effect on platelet-triggered athero-progression and neointima formation after wire-induced carotid injury. For binding studies after ligation-induced arterial injury, the left common carotid artery of C57BL/6J mice was ligated. For binding studies at spontaneously formed atherosclerotic lesion sites, Apolipoprotein E-deficient (ApoE(-/-)) mice were fed a 0.25% cholesterol diet over 16 weeks. Binding of [(124)I]GPVI-Fc was monitored by autoradiography 48 h after intravenous injection and by immunostaining. To study the effect of GPVI-Fc on neointima formation vs. control-Fc, a wire-induced injury of the left A. carotis communis of ApoE(-/-)-mice was performed. Mice were treated intraperitoneally with GPVI-Fc for 8 days and neointima formation was assessed 4 weeks after intervention. [(124)I]GPVI-Fc preferentially bound to injury sites after carotid ligation in C57BL/6J mice and to lipid-rich atherosclerotic lesions of the carotid artery and aortic arch in uninjured ApoE(-/-)-mice. Histological examinations of wire-injured carotid arteries showed that neointima formation was significantly reduced in GPVI-Fc-treated ApoE(-/-) mice compared to ApoE(-/-) mice receiving control-Fc (P < 0.05). GPVI-Fc preferentially bound to sites of vascular injury and was able to inhibit neointima formation after wire-induced vascular injury in ApoE(-/-) mice. Thus, soluble GPVI-Fc might be also a promising compound to attenuate lesion progression after plaque rupture.

Characterisation of atherosclerotic lesions with scanning electron microscopy (SEM) of wet tissue

Atherosclerotic cardiovascular disease (CVD) is the universal leading cause of mortality and a major cause of morbidity. Additionally, the global epidemic of diabetes is associated with considerable cardiovascular mortality risk due to accelerated premature atherosclerosis. Development of effective therapies for atherosclerosis is dependent upon improved tools to assess atherosclerotic lesion progression in animal models. We present a novel technique that utilises scanning electron microscopy (SEM) for imaging wet biological specimens, thus enabling rapid and high-resolution imaging of atherosclerotic lesions. This wet SEM technique was used in an apoE-deficient mice model for morphological characterisation of early and advanced atherosclerotic lesions. Further demonstration of lipid-rich atherosclerotic lesions was carried out with osmium tetroxide staining for cholesterol. Gold immunolabelling of specific epitopes was applied in identification of the cellular and molecular components within the atherosclerotic lesions, namely foam cells, smooth muscle cells and collagen. The wet SEM technique demonstrates an accurate and detailed structural evaluation of the pathological process of atherosclerosis. Understanding the mechanisms that precipitate the atherosclerotic process, utilising this novel technique, may assist in the development of innovative therapeutic interventions for CVD management and prevention in the general population and in those with diabetes.

Reduced In Vivo Aortic Uptake of Radiolabeled Oxidation-Specific Antibodies Reflects Changes in Plaque Composition Consistent With Plaque Stabilization

Labeled oxidation-specific antibodies (Ox-AB) detect, quantify, and noninvasively image lipid-rich atherosclerotic lesions. However, it is unknown whether Ox-AB detect plaque stabilization. The aortic uptake of intravenously injected 125I-MDA2 (Ox-AB to malondialdehyde [MDA]-low-density lipoprotein [LDL]) was quantitated in: (1) LDL receptor-/- mice with established atherosclerosis continued on Western diet (Progression) or switched to chow (Regression) or chow+vitamins E and C (Regression-VIT) for 6 months; and (2) Watanabe rabbits (3- to 57-months old) with naturally evolved atherosclerotic lesions. In mice, the Progression group had more extensive atherosclerosis, higher 125I-MDA2 uptake, high concordance of Sudan (lipid)-staining and 125I-MDA2 uptake, and stronger oxidized LDL (OxLDL) and macrophage immunostaining than both Regression groups. In contrast, the Regression groups showed Sudan-positive lesions with focally diminished 125I-MDA2 uptake, which coincided with reduced OxLDL and macrophages but more smooth muscle cells (SMCs) and collagen. In rabbits, areas of increased 125I-MDA2 uptake were associated with high Sudan concordance and strong immunostaining for OxLDL and macrophages. Interestingly, advanced lesions with focally diminished 125I-MDA2 uptake showed stronger immunostaining for SMCs and collagen, particularly at the fibrous cap. Ox-AB uptake is focally diminished in plaques displaying accepted features of plaque stability. Imaging techniques to detect the presence and depletion of OxLDL may be useful in assessing plaque stabilization.

Clinical characteristics of coronary artery calcification in diabetic patients

Coronary artery calcification is frequently seen in cardiovascular disease in diabetic patients, and usually indicates the severity of coronary stenosis. They occur in the intima of the lipid-rich atherosclerotic coronary lesions. The mechanism of the coronary calcification is still not known, though several proteins such as AGE are thought to induce the calcium accumulation in the atheroma.

Noninvasive imaging of oxidized Low-Density Lipoprotein in atherosclerotic plaques with tagged oxidation-specific antibodies

The lipid component of vulnerable atherosclerotic lesions strongly determines the propensity for plaque rupture. Oxidized low-density lipoprotein (OxLDL) is a major component of the plaque lipid pool and contributes significantly to the inflammatory milieu. Because oxidation-specific antibodies (Ox-AB) have been shown to selectively immunostain human atherosclerotic lesions, we hypothesized that tagged Ox-AB may be useful for noninvasive imaging of atherosclerosis. Indeed, intravenously injected (125)I-MDA2, a prototype murine monoclonal Ox-AB, has strong selectivity for and accurately identifies lipid-rich atherosclerotic lesions in their entirety. Plaque uptake of (125)I-MDA2 strongly correlates with extent of atherosclerosis, measured by percentage of surface area and aortic weight, and thus provides an accurate quantitative measure of atherosclerotic burden. Dietary regression studies in mice have shown that (125)I-MDA2 uptake is more sensitive to "regression," not measured by physical dimensions but by depletion of OxLDL, suggesting a novel mechanism of plaque stabilization. In vivo gamma-camera scintigraphy with (99m)Tc-MDA2 revealed that imaging of lipid and oxidation-rich atherosclerotic lesions is feasible with this technique. We have developed IK17, the first human Ox-AB, which recognizes a unique oxidation-specific epitope, and may have significant advantages over murine antibodies. Ox-AB may be tagged with appropriate labels for use in nuclear, magnetic resonance, or ultrasound imaging. Potential applications include early diagnosis of lipid-rich atherosclerotic lesions, screening, and serial follow-up of high-risk individuals, identifying vulnerable plaques, and evaluating novel drug therapies on progression and regression of atherosclerosis.

Non-invasive imaging of vulnerable plaques by molecular targeting of oxidized LDL with tagged oxidation-specific antibodies.

The concept of the "vulnerable" plaque has recently emerged to explain how quiescent atherosclerotic lesions evolve to cause clinical events. Vulnerable plaques are generally non-obstructive, asymptomatic lesions that may abruptly rupture and induce thrombotic occlusion leading to tissue ischemia and its attendant sequelae. They are responsible for over 50% of cases of sudden death and acute myocardial infarction. The lipid component of vulnerable plaques, which is abundant and highly enriched in oxidized low-density lipoprotein (OxLDL), strongly contributes to their propensity to rupture through physical, inflammatory, and thrombogenic properties. We hypothesized that OxLDL would serve as an ideal target to detect vulnerable plaques. In a series of experimental studies, we have shown that oxidation-specific antibodies (Ox-AB) specifically accumulate in vivo within lipid-rich atherosclerotic lesions but not normal arteries, provide a quantitative measure of the content of OxLDL, allow detection of atherosclerosis progression and regression in the context of enhanced or reduced OxLDL content and non-invasively image atherosclerotic lesions. Ox-AB may be tagged with appropriate labels for use in nuclear scintigraphy, magnetic resonance, or ultrasound imaging. Potential research and clinical applications include studying the natural history of atherosclerosis in animal models and humans, evaluating novel drug or genetic therapies on progression and regression of atherosclerosis, evaluating plaque stability, screening and serial follow-up of high-risk individuals, non-invasive imaging of vulnerable plaques, and assessing the clinical efficacy of new treatments of atherosclerosis.

Toll-like receptor-4 is expressed by macrophages in murine and human lipid-rich atherosclerotic plaques and upregulated by oxidized LDL.

Inflammation is implicated in atherogenesis and plaque disruption. Toll-like receptor 2 (TLR-2) and TLR-4, a human homologue of drosophila Toll, play an important role in the innate and inflammatory signaling responses to microbial agents. To investigate a potential role of these receptors in atherosclerosis, we assessed the expression of TLR-2 and TLR-4 in murine and human atherosclerotic plaques. Aortic root lesions of high-fat diet-fed apoE-deficient mice (n=5) and human coronary atherosclerotic plaques (n=9) obtained at autopsy were examined for TLR-4 and TLR-2 expression by immunohistochemistry. Aortic atherosclerotic lesions in all apoE-deficient mice expressed TLR-4, whereas aortic tissue obtained from control C57BL/6J mice showed no TLR-4 expression. All 5 lipid-rich human plaques expressed TRL-4, whereas the 4 fibrous plaques and 4 normal human arteries showed no or minimal expression. Serial sections and double immunostaining showed TLR-4 colocalizing with macrophages both in murine atherosclerotic lesions and at the shoulder region of human coronary artery plaques. In contrast to TLR-4, none of the plaques expressed TLR-2. Furthermore, basal TLR-4 mRNA expression by human monocyte-derived macrophages was upregulated by ox-LDL in vitro. Our study demonstrates that TLR-4 is preferentially expressed by macrophages in murine and human lipid-rich atherosclerotic lesions, where it may play a role to enhance and sustain the innate immune and inflammatory responses. Moreover, upregulation of TLR-4 in macrophages by oxidized LDL suggests that TLR-4 may provide a potential pathophysiological link between lipids and infection/inflammation and atherosclerosis.

Adenovirus-mediated overexpression of tissue inhibitor of metalloproteinase-1 reduces atherosclerotic lesions in apolipoprotein E-deficient mice.

To define the role of metalloproteinases (MMPs) in the development of lipid-rich atherosclerotic lesions in relation to the balance between proteolytic and antiproteolytic activities, we investigated the impact of adenovirus-mediated elevation in the circulating levels of human tissue inhibitor of MMP (TIMP-1) in atherosclerosis-susceptible apolipoprotein E-deficient (apoE(-/-)) mice. Infusion of apoE(-/-) mice fed a lipid-rich diet with rAd.RSV.TIMP-1 (1x10(11) viral particles) resulted in high hepatic expression of TIMP-1. At 2 weeks after injection, plasma TIMP-1 levels ranged from 7 to 24 micrograms/mL (mean 14.8+/-6.8). Marked overexpression of TIMP-1 was transient, with levels of TIMP-1 decreasing to 2.5 to 8 micrograms/mL (mean 4.3+/-2.1) at 4 weeks. Plasma lipid and lipoprotein levels in mice treated with rAd.RSV.TIMP-1 were similar to those treated with rAd.RSV.betaGal. However, rAd.RSV.TIMP-1-infused mice displayed a marked reduction (approximately 32%; P<0.05) in mean lesion area per section (512+/-121 micrometers(2)x10(3); n=12 sections from 4 animals) as compared with rAd.RSV.betaGal-infused mice (750+/-182 micrometers(2)x10(3); n=12 sections from 4 animals). Similarly, marked reduction in macrophage deposition as well as MMP-2, MMP-3, and MMP-13 antigens was observed. Histological and immunohistologic analyses of atherosclerotic lesions revealed increases in collagen, elastin, and smooth muscle alpha-actin content in mice treated with rAd.RSV.TIMP-1. These qualitative and quantitative features were the consequence of TIMP-1 infiltration from plasma to arterial intima, as immunohistochemical analyses revealed an abundance of TIMP-1 specifically in lesions of rAd.RSV. TIMP-1-treated mice.

Distinct patterns of transforming growth factor-beta isoform and receptor expression in human atherosclerotic lesions. Colocalization implicates TGF-beta in fibrofatty lesion development.

Some animal studies suggest that transforming growth factor-beta (TGF-beta) protects vessels from atherosclerosis by preventing intima formation, but others indicate a role in vessel proteoglycan accumulation and lipoprotein retention. To distinguish between these possibilities in humans, immunohistochemical studies were performed examining the coexpression of TGF-beta isoforms and the TGF-beta receptors ALK-5 and TbetaR-II in aorta during the various stages of atherosclerotic lesion development. The spatial relationships between TGF-beta1, TGF-beta3, ALK-5, and TbetaR-II expression were compared in aortic segments from 21 subjects. Nonatherosclerotic intima contained predominantly TGF-beta1, low concentrations of TbetaR-II, and barely detectable amounts of ALK-5. In contrast, fatty streaks/fibrofatty lesions contained high concentrations of both TGF-beta isoforms. Smooth muscle cells (SMCs), macrophages, and foam cells of macrophage and SMC origin contributed to these high levels. These lesions also contained high, colocalized concentrations of ALK-5 and TbetaR-II. Despite fibrous plaques containing TGF-beta1, its receptors were at detection limits. We found no evidence for truncated TbetaR-II expression in either normal intima or the various atherosclerotic lesions. TGF-beta appears to be most active in lipid-rich aortic intimal lesions. The findings support the hypothesis that TGF-beta contributes primarily to the pathogenesis of lipid-rich atherosclerotic lesions by stimulating the production of lipoprotein-trapping proteoglycans, inhibiting smooth muscle proliferation, and activating proteolytic mechanisms in macrophages.

Tissue factor modulates the thrombogenicity of human atherosclerotic plaques.

The thrombogenicity of a disrupted atherosclerotic lesion is dependent on the nature and extent of the plaque components exposed to flowing blood together with local rheology and a variety of systemic factors. We previously reported on the different thrombogenicity of the various types of human atherosclerotic lesions when exposed to flowing blood in a well-characterized perfusion system. This study examines the role of tissue factor in the thrombogenicity of different types of atherosclerotic plaques and their components. Fifty human arterial segments (5 foam cell-rich, 9 collagen-rich, and 10 lipid-rich atherosclerotic lesions and 26 normal, nonatherosclerotic segments) were exposed to heparinized blood at high shear rate conditions in the Badimon perfusion chamber. The thrombogenicity of the arterial specimens was assessed by 111In-labeled platelets. After perfusion, specimens were stained for tissue factor by use of an in situ binding assay for factor VIIa. Tissue factor in specimens was semiquantitatively assessed on a scale of 0 to 3. Platelet deposition on the lipid-rich atheromatous core was significantly higher than on all other substrates (P = .0002). The lipid-rich core also exhibited the most intense tissue factor staining (3 +/- 0.1 arbitrary units) compared with other arterial components. Comparison of all specimens showed a positive correlation between quantitative platelet deposition and tissue factor staining score (r = .35, P < .01). Our results show that tissue factor is present in lipid-rich human atherosclerotic plaques and suggest that it is an important determinant of the thrombogenicity of human atherosclerotic lesions after spontaneous or mechanical plaque disruption.

Influence of lipoprotein lipids, dietary fat and smoking on macrophage degradation of native and oxidized low density lipoprotein

Relatively little is known about the biological mechanisms by which lipoproteins promote atherogenesis. It has, however, been shown that structural modification of low density lipoprotein (LDL), such as by oxidation, results in their uptake and degradation by intimal macrophages and consequently leads to formation of lipid-rich atherosclerotic lesions. The aim of the present investigation was to study the influence of dietary intake of fat, lipoprotein lipid composition, smoking and gender on macrophage degradation of LDL before and after oxidation. The study group consisted of 48 males and 56 females with hyperlipidemia taking part in the open prerandomization phase of the Probucol Quantitative Regression Swedish Trial (PQRST). Analysis including lipoprotein determinations, dietary and smoking habit interviews, LDL degradation by macrophages, LDL receptor binding and LDL thiobarbituric acid reactive substance (TBARS) levels before and after copper ion-induced oxidation was done during the pre-andomization phase of the study. Increased plasma and very low density lipoprotein (VLDL) triglyceride levels were associated with an increased macrophage degradation of native LDL, whereas no such association was found after oxidation of LDL. The dietary intake of polyunsaturated fatty acids (PUFA) was also inversely related to the degradation of native LDL by macrophages, but increased the rate at which oxidized LDL was degraded. Smoking and gender did not influence the rate of macrophage degradation of native or oxidized LDL. It is concluded that hypertriglyceridemia is associated with an increased macrophage degradation of LDL. This may represent a mechanism by which hypertriglyceridemia promotes atherosclerosis.

Human macrophage scavenger receptors: primary structure, expression, and localization in atherosclerotic lesions.

Two types of cDNAs for human macrophage scavenger receptors were cloned from a cDNA library derived from the phorbol ester-treated human monocytic cell line THP-1. The type I and type II human scavenger receptors encoded by these cDNAs are homologous (73% and 71% amino acid identity) to their previously characterized bovine counterparts and consist of six domains: cytoplasmic (I), membrane-spanning (II), spacer (III), alpha-helical coiled-coil (IV), collagen-like (V), and a type-specific C-terminal (VI). The receptor gene is located on human chromosome 8. The human receptors expressed in CHO-K1 cells mediated endocytosis of modified low density lipoproteins. Two mRNAs, 4.0 and 3.2 kilobases, have been detected in human liver, placenta, and brain. Immunohistochemical studies using an anti-peptide antibody which recognizes human scavenger receptors indicated the presence of the scavenger receptors in the macrophages of lipid-rich atherosclerotic lesions, suggesting the involvement of scavenger receptors in atherogenesis.