carbocyanines and Atherosclerosis

Atherosclerosis plaque is a major cause of cardiovascular diseases across the globe and a silent killer. There are no physical symptoms of the disease in its early stage and current diagnostic techniques cannot detect the small plaques effectively or safely. Plaques formed in blood vessels can cause serious clinical problems such as impaired blood flow or sudden death, regardless of their size. Thus, detecting early stage of plaques is especially more important to effectively reduce the risk of atherosclerosis. Nanoparticle based delivery systems are recognized as a promising option to fight against this disease, and various targeting ligands are typically used to improve their efficiency. So, the choice of appropriate targeting ligand is a crucial factor for optimal targeting efficiency. cRGD peptide and collagen IV targeting peptide, which binds with the α

Topics: Animals; Atherosclerosis; Carbocyanines; Collagen; Drug Carriers; Female; Ferric Compounds; Fluorescent Dyes; Magnetic Resonance Imaging; Mice; Mice, Knockout, ApoE; Nanoparticles; Optical Imaging; Peptides; RAW 264.7 Cells

The potential benefits of high-density lipoproteins (HDL) against atherosclerosis are attributed to its major protein component, apolipoprotein A-I (apoA-I). Most of the apoA-I in the vascular wall appears to be in its lipid-poor form. The latter, however, is subjected to degradation by proteases localized in atherosclerotic plaques, which, in turn, has been shown to negatively impact its atheroprotective functions. Here, we report the development and in vivo use of a bioactivatable near-infrared full-length apoA-I-Cy5.5 fluorescent probe for the assessment of apoA-I-degrading proteolytic activities.. Fluorescence quenching was obtained by saturation of Cy5.5 fluorophore molecules on apoA-I protein. ApoA-I cleavage led to near-infrared fluorescence enhancement. In vitro proteolysis of the apoA-I probe by a variety of proteases including serine, cysteine, and metalloproteases resulted in an up to 11-fold increase in fluorescence (n = 5, p ≤ 0.05).. We detected activation of the probe in atherosclerotic mice aorta sections using in situ zymography and showed that broad-spectrum protease inhibitors protected the probe from degradation, resulting in decreased fluorescence (-54%, n = 6 per group, p ≤ 0.0001). In vivo, the injected probe showed stronger fluorescence emission in the aorta of human apoB transgenic Ldlr. The use of this probe in different applications may help to assess new molecular mechanisms of atherosclerosis and may improve current HDL-based therapies by enhancing apoA-I functionality.

Topics: Animals; Aorta, Thoracic; Aortic Diseases; Apolipoprotein A-I; Apolipoprotein B-100; Atherosclerosis; Carbocyanines; Cathepsins; Cell Line; Chymases; Disease Models, Animal; Fluorescent Dyes; Humans; Kinetics; Macrophages; Matrix Metalloproteinase 12; Mice, Inbred C57BL; Mice, Knockout; Molecular Imaging; Peptide Hydrolases; Protein Stability; Proteolysis; Receptors, LDL; Spectrometry, Fluorescence; Spectroscopy, Near-Infrared; Trypsin

This study aims to explore non-invasive imaging of atherosclerotic plaque through magnetic resonance imaging (MRI) and near-infrared fluorescence (NIRF) by using profilin-1 targeted magnetic iron oxide nanoparticles (PF1-Cy5.5-DMSA-Fe3O4-NPs, denoted as PC-NPs) as multimodality molecular imaging probe in murine model of atherosclerosis.. PC-NPs were constructed by conjugating polyclonal profilin-1 antibody and NHS-Cy5.5 fluorescent dye to the surface of DMSA-Fe3O4-nanoparticles via condensation reaction. Murine atherosclerosis model was induced in apoE(-/-) mice by high fat and cholesterol diet (HFD) for 16 weeks. The plaque areas in aortic artery were detected with Oil Red O staining. Immunofluorescent staining and Western blot analysis were applied respectively to investigate profilin-1 expression. CCK-8 assay and transwell migration experiment were performed to detect vascular smooth muscle cells (VSMCs) proliferation. In vivo MRI and NIRF imaging of atherosclerotic plaque were carried out before and 36 h after intravenous injection of PC-NPs. Oil Red O staining showed that the plaque area was significantly increased in HFD group (p<0.05). Immunofluorescence staining revealed that profilin-1 protein was highly abundant within plaque in HFD group and co-localized with α-smooth muscle actin. Profilin-1 siRNA intervention could inhibit VSMCs proliferation and migration elicited by ox-LDL (p<0.05). In vivo MRI and NIRF imaging revealed that PC-NPs accumulated in atherosclerotic plaque of carotid artery. There was a good correlation between the signals of MRI and ex vivo fluorescence intensities of NIRF imaging in animals with PC-NPs injection.. PC-NPs is a promising dual modality imaging probe, which may improve molecular diagnosis of plaque characteristics and evaluation of pharmaceutical interventions for atherosclerosis.

Topics: Animals; Atherosclerosis; Carbocyanines; Carotid Arteries; Cell Proliferation; Ferric Compounds; Magnetic Resonance Imaging; Magnetite Nanoparticles; Male; Mice; Multimodal Imaging; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Optical Imaging; Profilins; Protein Binding; Succimer

The objective of this study is to develop a method for selective detection of the calcific (hydroxyapatite) component in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues ex vivo. This method uses a novel optical molecular imaging contrast dye, Cy-HABP-19, to target calcified cells and tissues.. A peptide that mimics the binding affinity of osteocalcin was used to label hydroxyapatite in vitro and ex vivo. Morphological changes in vascular smooth muscle cells were evaluated at an early stage of the mineralization process induced by extrinsic stimuli, osteogenic factors and a magnetic suspension cell culture. Hydroxyapatite components were detected in monolayers of these cells in the presence of osteogenic factors and a magnetic suspension environment.. Atherosclerotic plaque contains multiple components including lipidic, fibrotic, thrombotic, and calcific materials. Using optical imaging and the Cy-HABP-19 molecular imaging probe, we demonstrated that hydroxyapatite components could be selectively distinguished from various calcium salts in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues, carotid endarterectomy samples and aortic valves, ex vivo.. Hydroxyapatite deposits in cardiovascular tissues were selectively detected in the early stage of the calcification process using our Cy-HABP-19 probe. This new probe makes it possible to study the earliest events associated with vascular hydroxyapatite deposition at the cellular and molecular levels. This target-selective molecular imaging probe approach holds high potential for revealing early pathophysiological changes, leading to progression, regression, or stabilization of cardiovascular diseases.

Topics: Aorta; Aortic Valve; Atherosclerosis; Biomarkers; Calcinosis; Carbocyanines; Carotid Arteries; Cells, Cultured; Durapatite; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Heart Valve Diseases; Humans; Kinetics; Magnetic Fields; Microscopy, Fluorescence; Molecular Imaging; Molecular Probes; Muscle, Smooth, Vascular; Oligopeptides; Osteocalcin; Osteogenesis; Plaque, Atherosclerotic; Protein Binding; Time Factors; Vascular Calcification; X-Ray Microtomography

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.

Topics: Animals; Atherosclerosis; Carbocyanines; Ferritins; Humans; Macrophages; Magnetic Resonance Imaging; Magnetite Nanoparticles; Mice; Models, Theoretical; Plaque, Atherosclerotic

The aim of this study was to elucidate which of the following two factors plays a more important role in the localization of atherogenesis: the barrier function of the arterial endothelium modulated by wall shear stress or flow-dependent low-density lipoprotein (LDL) concentration at the blood/wall interface. To determine this, the rabbit aorto-iliac bifurcation was perfused with 3,3'-dioctadecylindocarbocyanine (DiI)-LDL solution under three different flow conditions: (i) forward flow (perfused in the in vivo flow direction); (ii) backward flow (perfused in a reversed flow direction); and (iii) static group (no flow). The results showed that there was a peak in the curve of DiI-LDL uptake distribution along the lateral wall of the bifurcation for all three groups, which was located in the branching areas where the endothelial cells were round and polygonal with no preferred orientation. Nevertheless, the peak of the forward flow group was much sharper than those of the other two groups. The overall DiI-LDL uptake was the highest for the static group. The present experimental study supports the concept that both the barrier function of the endothelium modulated by wall shear stress and the mass transport phenomenon of LDL concentration polarization are involved in the infiltration/accumulation of atherogenic lipids within the arterial wall. Nevertheless, the latter might play a larger role in the localization of atherogenesis.

Topics: Animals; Aorta; Aorta, Abdominal; Atherosclerosis; Biological Transport; Carbocyanines; Cholesterol, LDL; Endothelial Cells; Endothelium, Vascular; Lipoproteins, LDL; Male; Perfusion; Rabbits; Stress, Mechanical

To substantiate our hypothesis that concentration polarization of low-density lipoprotein (LDL) plays an important role in the localization of atherogenesis, we investigated the effects of wall shear stress and water filtration rate (or perfusion pressure) on the luminal surface LDL concentration (c(w)) and the LDL uptake by human vascular endothelial cells and smooth muscle cells co-cultured on a permeable membrane using a parallel-plate flow chamber technique and a flow cytometry method. The results indicated that the uptake of fluorescent labeled LDL (DiI-LDL) by the co-cultured cells was positively correlated with c(w) in a non-linear fashion. When c(w) was low, the uptake increased very sharply with increasing c(w). Then the increase became gradual and the uptake was seemingly leveled out when c(w) reached beyond 160 microg/ml. The present study therefore has provided further experimental evidence that concentration polarization may occur in the arterial system and have a positive correlation with the uptake of LDLs by the arterial wall, which gives support to our hypothesis regarding the localization of atherogenesis.

Topics: Atherosclerosis; Carbocyanines; Coculture Techniques; Endothelial Cells; Flow Cytometry; Fluorescent Dyes; Humans; Lipoproteins, LDL; Models, Cardiovascular; Myocytes, Smooth Muscle; Perfusion; Pressure; Rheology; Stress, Mechanical; Tunica Intima

Macrophages play an important role during the development and progression of atherosclerotic plaques. alphavbeta3 integrins are highly expressed by macrophages; thus, targeting alphavbeta3 may allow targeting of culprit macrophage-loaded atherosclerotic lesions in vivo.. An alphavbeta3-targeted Arg-Gly-Asp (RGD) peptide was labeled with the cyanine 5.5 (Cy 5.5) dye and applied to image atherosclerotic plaques in apolipoprotein E-deficient mice.. The peptide-dye conjugate binds to alphavbeta3 integrin-positive RAW264.7 macrophages with high affinity. Competition experiments confirmed binding specificity of the probe. A significant fluorochrome accumulation in atherosclerotic plaques was demonstrated 24 h after injection by fluorescence reflectance imaging, which was blocked with high efficiency by competition with the unlabeled peptide. Conversely, the nonconjugated dye revealed only a minor fluorescence signal in the plaques. Fluorescence microscopy revealed colocalization of the probe with macrophages in the plaque of a mouse model for accelerated atherosclerosis, which was corroborated in human carotid artery specimens. In addition to macrophage-associated signals, binding of the probe to the neointima or elastica of the arteries was observed.. RGD-Cy 5.5, combined with near-infrared optical imaging methods, allows the specific imaging of alphavbeta3-integrin expression on macrophages recruited to vascular lesions and may serve to estimate macrophage-bound inflammatory activity of atherosclerotic lesions.

Topics: Animals; Apolipoproteins E; Atherosclerosis; Carbocyanines; Cell Line; Flow Cytometry; Fluorescence; Fluorescent Dyes; Humans; Infrared Rays; Integrin alphaVbeta3; Macrophages; Mice; Mice, Knockout; Microscopy, Fluorescence; Peptides, Cyclic; Protein Binding; Substrate Specificity

Topics: Angiogenesis Inhibitors; Animals; Atherosclerosis; Atorvastatin; Carbocyanines; Cyclohexanes; Drug Carriers; Drug Synergism; Drug Therapy, Combination; Fatty Acids, Unsaturated; Heptanoic Acids; Heterocyclic Compounds, 1-Ring; Humans; Integrin alphaVbeta3; Magnetic Resonance Imaging; Nanoparticles; Neovascularization, Pathologic; Pyrroles; Sesquiterpenes; Time Factors

Studies were performed to develop a prolonged antiangiogenesis therapy regimen based on theranostic alpha(nu)beta(3)-targeted nanoparticles.. Antiangiogenesis therapy may normalize atherosclerotic plaque vasculature and promote plaque stabilization. alpha(nu)beta(3)-targeted paramagnetic nanoparticles can quantify atherosclerotic angiogenesis and incorporate fumagillin to elicit acute antiangiogenic effects.. In the first experiment, hyperlipidemic rabbits received alpha(nu)beta(3)-targeted fumagillin nanoparticles (0, 30, or 90 microg/kg) with either a continued high fat diet or conversion to standard chow. The antiangiogenic response was followed for 4 weeks by cardiac magnetic resonance (CMR) molecular imaging with alpha(nu)beta(3)-targeted paramagnetic nanoparticles. In a second 8-week study, atherosclerotic rabbits received atorvastatin (0 or 44 mg/kg diet) alone or with alpha(nu)beta(3)-targeted fumagillin nanoparticles (only week 0 vs. weeks 0 and 4), and angiogenesis was monitored with CMR molecular imaging. Histology was performed to determine the location of bound nanoparticles and to correlate the level of CMR enhancement with the density of angiogenic vessels.. The alpha(nu)beta(3)-targeted fumagillin nanoparticles reduced the neovascular signal by 50% to 75% at 1 week and maintained this effect for 3 weeks regardless of diet and drug dose. In the second study, atherosclerotic rabbits receiving statin alone had no antineovascular benefit over 8 weeks. The alpha(nu)beta(3)-targeted fumagillin nanoparticles decreased aortic angiogenesis for 3 weeks as in study 1, and readministration on week 4 reproduced the 3-week antineovascular response with no carry-over benefit. However, atorvastatin and 2 doses of alpha(nu)beta(3)-targeted fumagillin nanoparticles (0 and 4 weeks) achieved marked and sustainable antiangiogenesis. Microscopic studies corroborated the high correlation between CMR signal and neovessel counts and confirmed that the alpha(nu)beta(3)-targeted nanoparticles were constrained to the vasculature of the aortic adventia.. The CMR molecular imaging with alpha(nu)beta(3)-targeted paramagnetic nanoparticles demonstrated that the acute antiangiogenic effects of alpha(nu)beta(3)-targeted fumagillin nanoparticles could be prolonged when combined with atorvastatin, representing a potential strategy to evaluate antiangiogenic treatment and plaque stability.

Topics: Angiogenesis Inhibitors; Animals; Aorta, Thoracic; Atherosclerosis; Atorvastatin; Carbocyanines; Cyclohexanes; Disease Models, Animal; Drug Carriers; Drug Synergism; Drug Therapy, Combination; Fatty Acids, Unsaturated; Heptanoic Acids; Heterocyclic Compounds, 1-Ring; Integrin alphaVbeta3; Liver; Magnetic Resonance Imaging; Nanoparticles; Neovascularization, Pathologic; Pyrroles; Rabbits; Sesquiterpenes; Time Factors

We developed a new imaging probe for atherosclerotic lesion imaging by chemically conjugating an atherosclerotic plaque-homing peptide (termed the AP peptide) to hydrophobically modified glycol chitosan (HGC) nanoparticles. The AP peptide was previously discovered by using an in vivo phage display screening method. HGC nanoparticles were labeled with the near-infrared (NIR) fluorophore Cy5.5, yielding nanoparticles 314 nm in diameter. The binding characteristics of nanoparticles to cytokine (TNF-alpha)-activated bovine aortic endothelial cells (BAECs) were studied in vitro under static conditions and in a dynamic flow environment. AP-tagged HGC-Cy5.5 nanoparticles (100 microg/ml, 2 h incubation) bound more avidly to TNF-alpha-activated BAECs than to unactivated BAECs. Nanoparticles were mostly located in the membranes of BAECs, although some were taken up by the cells and were visible in the cytoplasm, suggesting that the AP peptides in HGC nanoparticles retained target selectivity for activated BAECs. Binding selectivity of AP-tagged HGC-Cy5.5 nanoparticles was also studied in vivo. NIR fluorescence imaging demonstrated that AP-tagged HGC-Cy5.5 nanoparticles bound better to atherosclerotic lesions in a low-density lipoprotein receptor-deficient (Ldlr(-/-)) atherosclerotic mouse than to such lesions in a normal mouse. These results suggest that the newly designed AP-tagged HGC-Cy5.5 nanoparticles may be useful for atherosclerotic lesion imaging, and may also be employed to elucidate pathophysiological changes, at the molecular level, on atherosclerotic endothelium.

Topics: Animals; Atherosclerosis; Binding Sites; Carbocyanines; Cattle; Cells, Cultured; Chitosan; Disease Models, Animal; Endothelial Cells; Fluorescent Dyes; Hydrophobic and Hydrophilic Interactions; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Confocal; Nanoparticles; Particle Size; Peptides; Protein Binding; Receptors, LDL; Spectroscopy, Near-Infrared; Staining and Labeling; Time Factors; Tumor Necrosis Factor-alpha

It has been shown that ED-B fibronectin (ED-B) is a potential target for plaque imaging. The aim of this study was to test a novel modified single chain anti-ED-B antibody (scFv) conjugated for near infrared fluorescence imaging (NIRF) with tetrasulfonated carbocyanine-maleimide (TSC-scFv) and to examine the association of ED-B with the presence of macrophages in a murine model of atherosclerosis. Expression of ED-B was observed in plaque areas in apolipoprotein E-deficient (apoE(-/-)) mice which increased with age and plaque load. Robust imaging was possible after explantation of the aorta and demonstrated a strong NIRF signal intensity in focal aortic and brachiocephalic plaque lesions, whereas no signals were found in undiseased areas. Plaque lesion ED-B was expressed by smooth muscle cell and was closely associated to macrophage infiltrates. Although not expressed by the same cell type, there was a significant correlation (p<0.01) between ED-B and macrophage immunoreactivity. In vitro human coronary and mouse smooth muscle cells significantly increased ED-B expression after angiotensin II and TNF-alpha treatment. This study demonstrates that plaque NIRF imaging is feasible with a novel single chain antibody and that ED-B expression is closely associated with inflammation in experimental atherosclerosis.

Topics: Animals; Antibodies; Aorta, Thoracic; Apolipoproteins E; Atherosclerosis; Carbocyanines; Cells, Cultured; Cholesterol, Dietary; Coronary Artery Disease; Disease Models, Animal; Feasibility Studies; Fibronectins; Fluorescent Antibody Technique, Direct; Fluorescent Dyes; Humans; Immunoglobulin Variable Region; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular

Whereas recent studies suggest that cholesterol plays important role in the regulation of membrane proteins, its effect on the interaction of the cell membrane with the underlying cytoskeleton is not well understood. Here, we investigated this by measuring the forces needed to extract nanotubes (tethers) from the plasma membrane, using atomic force microscopy. The magnitude of these forces provided a direct measure of cell stiffness, cell membrane effective surface viscosity and association with the underlying cytoskeleton. Furthermore, we measured the lateral diffusion constant of a lipid analog DiIC12, using fluorescence recovery after photobleaching, which offers additional information on the organization of the membrane. We found that cholesterol depletion significantly increased the adhesion energy between the membrane and the cytoskeleton and decreased the membrane diffusion constant. An increase in cellular cholesterol to a level higher than that in control cells led to a decrease in the adhesion energy and the membrane surface viscosity. Disassembly of the actin network abrogated all the observed effects, suggesting that cholesterol affects the mechanical properties of a cell through the underlying cytoskeleton. The results of these quantitative studies may help to better understand the biomechanical processes accompanying the development of atherosclerosis.

Topics: Animals; Atherosclerosis; Biomechanical Phenomena; Carbocyanines; Cattle; Cell Membrane; Cholesterol; Cytoskeleton; Endothelial Cells; Microscopy, Atomic Force; Microscopy, Fluorescence; Nanotubes; Photobleaching