Detection of macrophage activity in atherosclerosis in vivo using multichannel, high-resolution laser scanning fluorescence microscopy

Molecular and cellular mechanisms of atherogenesis and its treatment are largely being unraveled by in vitro techniques. We describe methodology to directly image macrophage cell activity in vivo in a murine model of atherosclerosis using laser scanning fluorescence microscopy (LSFM) and a macrophage-targeted, near-infrared fluorescent (NIRF) magnetofluorescent nanoparticle (MFNP). Atherosclerotic apolipoprotein E deficient (apoE -/-) mice (n=10) are injected with MFNP or 0.9% saline, and wild-type mice (n=4) are injected with MFNP as additional controls. After 24 h, common carotid arteries are surgically exposed and prepared for LSFM. Multichannel LSFM of MFNP-enhanced carotid atheroma (5x5-microm in-plane resolution) shows a strong focal NIRF signal, with a plaque target-to-background ratio of 3.9+/-1.8. Minimal NIRF signal is observed in control mice. Spectrally resolved indocyanine green (ICG) fluorescence angiograms confirm the intravascular location of atheroma. On ex vivo fluorescence reflectance imaging, greater NIRF plaque signal is seen in apoE -/- MFNP mice compared to controls (p<0.01). The NIRF signal correlates well with immunostained macrophages, both by stained surface area (r=0.77) and macrophage number (r=0.86). The validated experimental methodology thus establishes a platform for investigating macrophage activity in atherosclerosis in vivo, and has implications for the detection of clinical vulnerable plaques.     

Human ferritin cages for imaging vascular macrophages

Atherosclerosis is a leading cause of death worldwide. Macrophages are key components of vascular inflammation, which contributes to the development and complications of atherosclerosis. Ferritin, an iron storage and transport protein, has been found to accumulate in macrophages in human atherosclerotic plaques. We hypothesized that ferritin could serve as an intrinsic nano-platform to target delivery of imaging agents to vascular macrophages to detect high-risk atherosclerotic plaques. Here we show that engineered human ferritin protein cages, either conjugated to the fluorescent Cy5.5 molecule or encapsulating a magnetite nanoparticle, are taken up in vivo by macrophages in murine atherosclerotic carotid arteries and can be imaged by fluorescence and magnetic resonance imaging. These results indicate that human ferritin can serve as a nanoparticle platform to image vascular inflammation in vivo.     

In vivo near-infrared dual-axis confocal microendoscopy in the human lower gastrointestinal tract

Near-infrared confocal microendoscopy is a promising technique for deep in vivo imaging of tissues and can generate high-resolution cross-sectional images at the micron-scale. We demonstrate the use of a dual-axis confocal (DAC) near-infrared fluorescence microendoscope with a 5.5-mm outer diameter for obtaining clinical images of human colorectal mucosa. High-speed two-dimensional en face scanning was achieved through a microelectromechanical systems (MEMS) scanner while a micromotor was used for adjusting the axial focus. In vivo images of human patients are collected at 5 frames/sec with a field of view of 362×212 μm(2) and a maximum imaging depth of 140 μm. During routine endoscopy, indocyanine green (ICG) was topically applied a nonspecific optical contrasting agent to regions of the human colon. The DAC microendoscope was then used to obtain microanatomic images of the mucosa by detecting near-infrared fluorescence from ICG. These results suggest that DAC microendoscopy may have utility for visualizing the anatomical and, perhaps, functional changes associated with colorectal pathology for the early detection of colorectal cancer.     

Synthesis, characterization, and in vivo diagnostic applications of hyaluronic acid immobilized gold nanoprobes

Herein we describe a new class of multifunctional gold nanoprobes for ultra-sensitive optical detection of reactive oxygen species (ROS) and hyaluronidase (HAdase). The nanoprobes were fabricated by end-immobilizing near-infrared fluorescence (NIRF) dye labeled hyaluronic acid (HA) onto the surface of gold nanoparticles (AuNPs). The nanoprobes effectively induced nanoparticle surface energy transfer (NSET) between NIRF dyes and AuNPs. When the surface immobilized HA was cleaved by ROS and HAdase, strong fluorescence recovery signals were attained with extreme sensitivity. In live animal models of rheumatoid arthritis (RA) and metastatic tumor, local arthritic inflammation and tumor sites were clearly identified upon systemic injection of the nanoprobes. These results suggest that the gold nanoprobes can be exploited not only as in vitro molecular and cellular imaging sensors for ROS and HAdase, but also as in vivo optical imaging agents for detection of local HA degrading diseases such as RA and tumor.     

Coronary hemodynamics and atherosclerotic wall stiffness: a vicious cycle

Local hemodynamic environment, including low shear stress and increased tensile stress, determines the localization, growth and progression of coronary atherosclerosis. As atherosclerotic lesions evolve, the diseased coronary arteries undergo local quantitative and qualitative changes in their wall, and progressively become stiff. Arterial stiffening amplifies the atherogenic local hemodynamic environment, initiating a self-perpetuating vicious cycle, which drives the progression of atherosclerosis and the formation of atherosclerotic plaque. In vivo evidence indicates that endothelial dysfunction is associated with arterial stiffness, an association that creates a challenging perspective of utilizing stiffness as an early marker of endothelial dysfunction and future atherosclerosis. Coronary stiffening is also associated with vascular remodeling, which is a major determinant of the natural history of atherosclerotic plaques. Thus, arterial stiffness may constitute a useful marker for the identification of the remodeling pattern, in particular expansive remodeling, which is closely associated with high-risk plaques. The early identification of endothelial dysfunction, or a high-risk plaque may enable the early adoption of preventive measures to improve endothelial function, or justify pre-emptive local interventions in high-risk regions to prevent future acute coronary syndromes. Further experimental and perspective clinical studies are needed for the investigation of these perspectives, whereas the development of new modalities for non-invasive and reliable assessment of coronary stiffness is anticipated to serve these studies.     

Alterations of endothelin-converting enzyme expression in early and advanced stages of human coronary atherosclerosis

Endothelin-1 is a potent vasoconstrictor and exhibits a mitogenic activity on vascular smooth muscle cells (SMCs). Endothelin-converting enzyme (ECE) is the final key enzyme of endothelin-1 processing. We studied the immunolocalization of ECE in human coronary atherosclerotic lesions with different disease stages. Frozen sections of normal coronary arteries with diffuse intimal thickening (n=13) and those of coronary arteries with early (n=10) or advanced atherosclerotic plaques (n=13) were studied. Monoclonal antibodies used were directed against SMCs, macrophages, endothelial cells, and ECE. For the identification of cell types that express ECE, double immunostaining analysis was also used. In normal coronary arteries, ECE immunoreactivity was observed in luminal endothelial cells and medial SMCs. Early atherosclerotic plaques, which consisted predominantly of SMCs, showed enhanced ECE expression in luminal endothelial cells and intimal SMCs. In advanced atherosclerotic plaques, distinct ECE expression was found in accumulated macrophages and in endothelial cells of intraplaque microvessels, while luminal endothelial cells showed relatively weak immunoreactivity for ECE. In conclusion, the present study demonstrates that the major cell types expressing ECE within the plaques are different between early and advanced stages of human coronary atherosclerosis. Enhanced ECE expression and possible endothelin-1 generation may contribute to SMC proliferation and vasoconstriction in early atherosclerotic stages, and may promote plaque destabilization in advanced atherosclerotic stages.     

Near-infrared fluorescence heptamethine carbocyanine dyes mediate imaging and targeted drug delivery for human brain tumor

Brain tumors and brain metastases are among the deadliest malignancies of all human cancers, largely due to the cellular blood–brain and blood–tumor barriers that limit the delivery of imaging and therapeutic agents from the systemic circulation to tumors. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. Here we identified and synthesized a group of near-infrared fluorescence (NIRF) heptamethine carbocyanine dyes and derivative NIRF dye-drug conjugates for effective imaging and therapeutic targeting of brain tumors of either primary or metastatic origin in mice, which is mechanistically mediated by tumor hypoxia and organic anion-transporting polypeptide genes. We also demonstrate that these dyes, when conjugated to chemotherapeutic agents such as gemcitabine, significantly restricted the growth of both intracranial glioma xenografts and prostate tumor brain metastases and prolonged survival in mice. These results show promise in the application of NIRF dyes as novel theranostic agents for the detection and treatment of brain tumors.     

Receptor-targeted iron oxide nanoparticles for molecular MR imaging of inflamed atherosclerotic plaques

In a number of literature reports iron oxide nanoparticles have been investigated for use in imaging atherosclerotic plaques and found to accumulate in plaques via uptake by macrophages, which are critical in the process of atheroma initiation, propagation, and rupture. However, the uptake of these agents is non-specific; thus the labeling efficiency for plaques in vivo is not ideal. We have developed targeted agents to improve the efficiency for labeling macrophage-laden plaques. These probes are based on iron oxide nanoparticles coated with dextran sulfate, a ligand of macrophage scavenger receptor type A (SR-A). We have sulfated dextran-coated iron oxide nanoparticles (DIO) with sulfur trioxide, thereby targeting our nanoparticle imaging agents to SR-A. The sulfated DIO (SDIO) remained mono-dispersed and had an average hydrodynamic diameter of 62 nm, an r(1) relaxivity of 18.1 mM(-1) s(-1), and an r(2) relaxivity of 95.8 mM(-1) s(-1) (37 °C, 1.4 T). Cell studies confirmed that these nanoparticles were nontoxic and specifically targeted to macrophages. In vivo MRI after intravenous injection of the contrast agent into an atherosclerotic mouse injury model showed substantial signal loss on the injured carotid at 4 and 24 h post-injection of SDIO. No discernable signal decrease was seen at the control carotid and only mild signal loss was observed for the injured carotid post-injection of non-sulfated DIO, indicating preferential uptake of the SDIO particles at the site of atherosclerotic plaque. These results indicate that SDIO can facilitate MRI detection and diagnosis of vulnerable plaques in atherosclerosis.     

Lipopolysaccharide-Induced Proliferation of the Vasa Vasorum in a Rabbit Model of Atherosclerosis as Evaluated by Contrast-Enhanced Ultrasound Imaging and Histology

Whether lipopolysaccharide (LPS) can promote vasa vasorum (VV) proliferation for atherosclerosis in vivo is unclear. Eighteen rabbits with atherosclerosis were randomly assigned into one of three groups of six. Group A received biweekly injections of 10 mL saline after 2 weeks of balloon injury. Groups B and C received biweekly intravenous injections of 3.0 μg LPS in 10 mL saline at weeks 10 and 4, respectively, until study termination. LPS significantly increased the levels of triglycerides and C-reactive protein and decreased the level of high-density lipoprotein cholesterol. Group C had significant larger plaques and more macrophages than group A (p = 0.01 and p < 0.001, respectively). Contrast enhancement ultrasound imaging and histological detection demonstrated that plaques in group C had a significantly higher VV density than that in group A (p = 0.009 and p = 0.002, respectively). In summary, VV proliferation for plaque destabilization can be accelerated by LPS-induced systemic inflammation and changes in lipid profiles.     

兔不稳定斑块的18F-FDG PET/CT显像及其病理学检测

 目的 探讨氟18标记的脱氧葡萄糖 (18F-FDG) PET/CT无创检测不稳定斑块的可行性。方法 用新西兰雄性大白兔建立动脉粥样硬化模型,注射18F-FDG后行腹主动脉PET/CT成像,离体动脉数码照相,测定动脉片段的放射强度,选取不稳定和稳定斑块各30块,比较其放射强度,采用免疫组化方法观察斑块中的巨噬细胞和平滑肌细胞并计数。结果 PET/CT活体成像可见实验组沿腹主动脉走行的不均匀的放射性分布,与离体动脉的斑块分布基本一致;不稳定斑块组的靶-非靶比值和巨噬细胞数明显高于稳定斑块组(P＜0.01),而平滑肌细胞数明显降低(P＜0.01);靶-非靶比值与斑块中的巨噬细胞数呈正相关(r=0.815,P＜0.01),而与平滑肌细胞数呈负相关(r=-0.684,P＜0.01)。结论18F-FDG PET/CT无创检测实验性不稳定斑块具有一定的可行性。     

Pathology of myocardial infarction,coronary artery disease,plaque disruption,and the vulnerable atherosclerotic plaque

Despite scientific advances, cardiovascular disease remains the leading cause of death in developed countries. The pathologic process responsible for the majority of this mortality is atherosclerosis. Human atherosclerosis is characterized by the transition of arteries through distinct pathologic stages. Initially, there is vascular wall activation, characterized by the formation of a smooth muscle cell rich intimal hyperplasia/thickening. The thickened intima promotes the lipid and macrophage accumulation characteristic of atherosclerosis. In some patients, the atherosclerotic plaque becomes disrupted stimulating formation of luminal thrombus and acute clinical events. Understanding the pathology of such vulnerable plaques has been a challenging and controversial area of investigation. Recent prospective longitudinal imaging studies of human coronary arteries have confirmed earlier pathologic observations reporting pathologic features that predispose to acute events in some patients include plaques with a thin fibrous cap overlying a large lipid-rich necrotic core as well as plaques with severe stenosis.     

Convergence of Nanotechnology and Cardiovascular Medicine

Advances in the emergence of biological probes, materials, and analytical tools limited to the nanoscale size range, collectively referred to as 'nanotechnology', are increasingly being applied to the understanding and treatment of the major pathophysiological problems in cardiovascular medicine. Analytical techniques based on high-resolution microscopy and molecular-level fluorescence excitation processes capable of detecting nanoscale interactions have been used to elucidate cardiovascular pathology. Nanotechnology has also significantly impacted diagnostic intervention in cardiology, with the use of nanoparticles as contrast agents, for targeted biomedical imaging of vulnerable plaques, for detection of specific pathologic targets signaling the onset of atherosclerosis, and for tracking inflammatory events. Real-time nanoscale biosensors can be used to measure cardiovascular biomarkers, and nanopore sequencing has the potential to speed up the analysis of gene expression in cardiovascular disease. Potential therapeutic applications include the use of nanomaterials in cardiovascular devices, for delivery of drugs and bioactive molecules, or in novel technologies for reducing cholesterol accumulation and for dissolving clots.     

Apolipoprotein E localization in human coronary atherosclerotic plaques by in situ hybridization and immunohistochemistry and comparison with lipoprotein lipase.

Apolipoprotein E (apo E) mediates both lipid accumulation by and removal from cells and may be secreted by both macrophages and smooth muscle cells in vitro, but its cellular source in atherosclerotic plaques is not known. Lipoprotein lipase (LPL) also enhances cell lipid accumulation and is synthesized by macrophage foam cells in atherosclerotic plaques. To determine the cellular source of apo E in human coronary atherosclerotic lesions and its relationship to LPL synthesis, in situ hybridization and immunohistochemistry were performed on 12 atherosclerotic plaques and six nondiseased coronary artery segments from 10 cardiac transplant recipients. Apo E messenger RNA was localized to both non-foam cell and foam cell macrophages in plaques, but not to other cell types, and was not detected in nonatherosclerotic arteries. Half of the regions with non-foam cell macrophages expressed neither apo E nor LPL messenger RNA, whereas 86% of macrophage foam cell-containing regions contained both messenger RNAs. Polyclonal antisera raised against human apo E localized apo E protein to the surface of macrophages and surrounding matrix in plaques but not in control coronary segments. An LPL-specific monoclonal antibody demonstrated that, similar to apo E, LPL protein on foam cell and non-foam cell macrophages was detected in atherosclerotic lesions, but LPL was also localized to intimal muscle smooth muscle cells and was not distributed as widely in association with matrix as was apo E. The expression of both apo E and LPL in atherosclerotic lesions but not in normal intima suggest that these molecules play a role in lipid metabolism in atherosclerosis.     

Tracer-cocktail injections for combined pre- and intraoperative multimodal imaging of lymph nodes in a spontaneous mouse prostate tumor model

To improve surgical guidance toward prostate draining lymph nodes, we investigate the potential of intraoperative fluorescence imaging and combined pre- and intraoperative multimodality imaging approaches. Transgenic adenocarcinoma mouse prostate mice with spontaneous prostate tumors are injected intratumorally with: 1. a cocktail of patent blue (Pb) and indocyanine green (ICG); 2. a cocktail of albumin radiocolloids (99mTc-NanoColl), Pb, and ICG; or 3. a cocktail of radiolabeled albumin (99mTc-Vasculosis), Pb, and ICG. The distribution of these imaging agents over the lymph nodes (LNs) are studied at different time points after injection. We find that at 60-min postinjection, ICG significantly improves the detection of the LNs compared to Pb, 53 versus 7%, respectively. Moreover, a cocktail of ICG and 99mTc-NanoColl improves the fluorescent detection rate to 86%, equalling that of the clinically applied 99mTc-NanoColl. A similar overlap is observed in our initial clinical pilot data. Fluorescent detection of the LNs using a ICG with 99mTc-Vasculosis gives similar results as "free" ICG (58%; 60 min). A 99mTc-NanoColl, Pb, and cocktail ICG enriches the standard 99mTc-NanoColl approach by adding optical detection of the sentinel lymph nodes. Furthermore, this approach improves fluorescent-based guidance and enables both accurate surgical planning and intraoperative detection, based on a single injection.     

Macrophage myeloperoxidase regulation by granulocyte macrophage colony-stimulating factor in human atherosclerosis and implications in acute coronary syndromes

Inflammation and oxidative stress contribute to the pathogenesis of many human diseases including atherosclerosis. Advanced human atheroma contains high levels of the enzyme myeloperoxidase that produces the pro-oxidant species, hypochlorous acid (HOCl). This study documents increased numbers of myeloperoxidase-expressing macrophages in eroded or ruptured plaques causing acute coronary syndromes. In contrast, macrophages in human fatty streaks contain little or no myeloperoxidase. Granulocyte macrophage colony-stimulating factor, but not macrophage colony-stimulating factor, selectively regulates the ability of macrophages to express myeloperoxidase and produce HOCl in vitro. Moreover, myeloperoxidase-positive macrophages in plaques co-localized with granulocyte macrophage colony-stimulating factor. Pro-inflammatory stimuli known to be present in human atherosclerotic plaque, including CD40 ligand, lysophosphatidylcholine, or cholesterol crystals, could induce release of myeloperoxidase from HOCl production by macrophages in vitro. HOCl-modified proteins accumulated at ruptured or eroded sites of human coronary atheroma. These results identify granulocyte macrophage colony-stimulating factor as an endogenous regulator of macrophage myeloperoxidase expression in human atherosclerosis and support a particular role for the myeloperoxidase-expressing macrophages in atheroma complication and the acute coronary syndromes.     

Type I collagen gene expression in human atherosclerosis. Localization to specific plaque regions.

Because collagen is a major component of the human atherosclerotic plaque, factors controlling collagen synthesis may have a profound influence on the volume growth of these intimal lesions. In human arteries, we compared normal vs atherosclerotic media vs intimas for type I collagen gene expression using immunocytochemistry and in situ messenger RNA hybridization with subsequent correlations with plaque topographical features. We also determined the associations of such collagen gene expression with proximity to monocyte/macrophages and T lymphocytes. Type I collagen synthesis appears to be upregulated in atherosclerotic plaques compared with their underlying medias and normal internal mammary arteries and coronary diffuse intimal thickenings. At least in established and advanced coronary and carotid plaques, type I collagen gene expression is focal and especially prevalent in fibrous cap and vascularized regions. Although macrophages and type I procollagen messenger RNA and protein are both found in atherosclerotic plaques, no apparent spatial correlation between macrophage presence and type I procollagen presence was found within these atherosclerotic intimas. Type I procollagen presence appears to be negatively associated with the spatial presence of T cells. Thus, human atherosclerotic plaques exhibit nonuniform patterns of type I collagen gene expression. Although the biochemical determinants of this focal gene expression have yet to be determined, it is conceivable that stimulatory/inhibitory cytokines and other factors (eg hemodynamics) play important roles in determining the focal nature of collagen synthesis in atherosclerosis.     

ASSOCIATION OF CORONARY PLAQUE RUPTURE AND ATHEROSCLEROTIC INFLAMMATION

Coronary plaque inflammation may promote plaque rupture and thrombosis. To test this hypothesis, 351 coronary plaques from 83 patients were formalin-fixed and stained with haematoxylin and eosin. There were six groups: (1) ruptured plaques; (2) intact plaques from recently infarcted hearts; (3) plaques from hearts with severe coronary atherosclerosis without identifiable thrombosis; (4) native explanted hearts with severe coronary atherosclerosis; (5) cardiac transplant atherosclerosis; and (6) fatalities unrelated to coronary atherosclerosis. Selected arteries were immunostained for leukocyte markers and serially sectioned to identify plaque rupture. There were infiltrates of CD68-positive macrophages and CD3- and CD8-positive T cells adjacent to all plaque ruptures. Labelling with HLA-DR and CD30 indicated inflammatory cell activation. Plaque rupture was strongly statistically associated with the severity and frequency of superficial plaque inflammation but not that of deep plaque inflammation. Although atherosclerotic inflammation has been identified adjacent to rupture, this is its first comparison with control plaques. These results support the concept that inflammation in the fibrous cap is particularly associated with plaque rupture. © 1997 by John Wiley & Sons, Ltd.     

Photosensitizer delivery to vulnerable atherosclerotic plaque: comparison of macrophage-targeted conjugate versus free chlorin(e6)

We have previously shown that a conjugate (MA-ce6) between maleylated serum albumin and the photosensitizer chlorin(e6) (ce6) is targeted in vitro to macrophages via class A scavenger receptors. We now report on the ability of this conjugate to localize in macrophage-rich atherosclerotic plaques in vivo. Both the conjugate and the free photosensitizer ce6 are studied after injection into New Zealand White rabbits that are rendered atherosclerotic by a combination of aortic endothelial injury and cholesterol feeding into normal rabbits. Rabbits are sacrificed at 6 and 24 h after injection and intravascular fluorescence spectroscopy is carried out by fiber-based fluorimetry in intact blood-filled arteries. Surface spectrofluorimetry of numbered excised aortic segments together with injured and normal iliac arteries is carried out, and quantified ce6 content by subsequent extraction and quantitative fluorescence determination of the arterial segments and also of nontarget organs. There is good agreement between the various techniques for quantifying ce6 localization, and high contrast between arteries from atherosclerotic and normal rabbits is obtained. Fluorescence correlates with the highest burden of plaque in the aorta and the injured iliac artery. The highest accumulation in plaques is obtained using MA-ce6 at 24 h. Free ce6 gives better accumulation at 6 h compared to 24 h. The liver, spleen, lung, and gall bladder have the highest uptake in nontarget organs. Macrophage-targeted photosensitizer conjugates may have applications in both detecting and treating inflamed vulnerable plaque.     

Experimental Induction of Atheroarteriosclerosis by the Synergy of Allergic injury to Arteries and Lipid-Rich Diet: II. Effect of Repeatedly Injected Foreign Protein in Rabbits Fed a Lipid-Rich, Cholesterol-Poor Diet

Rabbits fed a lipid-rich, cholesterol-poor diet and given concomitant injections of foreign protein, over a period as long as 17 months, developed in their coronary arteries both a) proliferative fibromuscular intimal thickening closely resembling the diffuse intimal thickening that commonly occurs in coronary arteries of man, and b) fatty-proliferative fibromuscular intimal thickening that closely resembles coronary atherosclerosis in man. In contrast, rabbits of another group that were concurrently fed the same diet for as long as 22 months without injections of foreign protein developed changes in arteries of their hearts that resemble neither coronary atherosclerosis nor diffuse intimal thickening in man. Fatty-proliferative changes in aortas of the first group of rabbits are strikingly greater and more closely resemble human aortic atherosclerosis than those in the latter group. In the course of the experiments, the average serum cholesterol was not significantly different in the two groups of rabbits. It was approximately 200 to 250 mg%, which is the average serum cholesterol in adult humans in the United States. These experiments support the hypothesis that the synergy of arterial injury, in particular immunologic injury, and a diet rich in lipid can lead to atherosclerosis in man.     

Collagen fiber pathology in atherosclerotic plaques of the coronary arteries in ischemic heart disease

Structure-metabolic changes of collagen fibers (CF) in atherosclerosis plaques of the coronary arteries in the conditions of ischemic heart disease (IHD) have been studied. Segments of the coronary arteries were received from 68 men after a coronary artery bypass grafting. CF was study with using of the Van Gieson's and the Masson's methods. Histologic slices were studied by polarization microscopy. The atherosclerosis plaques with IHD were notable for lipidosis of CF. We've suspected lipidosis of CF is a crucial factor for the development of atherosclerosis plaques instability. Evident lipidosis of CF was attended with destructive changes probably resulted in accumulation of atheromatous mass in atherosclerosis plaques.