Everolimus-eluting stents stabilize plaque inflammation in vivo: assessment by intravascular fluorescence molecular imaging.

AIMS: Inflammation drives atherosclerosis complications and is a promising therapeutic target for plaque stabilization. At present, it is unknown whether local stenting approaches can stabilize plaque inflammation in vivo. Here, we investigate whether everolimus-eluting stents (EES) can locally suppress plaque inflammatory protease activity in vivo using intravascular near-infrared fluorescence (NIRF) molecular imaging. METHODS AND RESULTS: Balloon-injured, hyperlipidaemic rabbits with atherosclerosis received non-overlapping EES and bare metal stents (BMS) placement into the infrarenal aorta (n = 7 EES, n = 7 BMS, 3.5 mm diameter x 12 mm length). Four weeks later, rabbits received an injection of the cysteine protease-activatable NIRF imaging agent Prosense VM110. Twenty-four hours later, co-registered intravascular 2D NIRF, X-ray angiography and intravascular ultrasound imaging were performed. In vivo EES-stented plaques contained substantially reduced NIRF inflammatory protease activity compared with untreated plaques and BMS-stented plaques (P = 0.006). Ex vivo macroscopic NIRF imaging of plaque protease activity corroborated the in vivo results (P = 0.003). Histopathology analyses revealed that EES-treated plaques showed reduced neointimal and medial arterial macrophage and cathepsin B expression compared with unstented and BMS-treated plaques. CONCLUSIONS: EES-stenting stabilizes plaque inflammation as assessed by translational intravascular NIRF molecular imaging in vivo. These data further support that EES may provide a local approach for stabilizing inflamed plaques.

Improving quantification of intravascular fluorescence imaging using structural information.

Intravascular near-infrared fluorescence (iNIRF) imaging can enable the in vivo visualization of biomarkers of vascular pathology, including high-risk plaques. The technique resolves the bio-distribution of systemically administered fluorescent probes with molecular specificity in the vessel wall. However, the geometrical variations that may occur in the distance between fibre-tip and vessel wall can lead to signal intensity variations and challenge quantification. Herein we examined whether the use of anatomical information of the cross-section vessel morphology, obtained from co-registered intravascular ultrasound (IVUS), can lead to quantification improvements when fibre-tip and vessel wall distance variations are present. The algorithm developed employs a photon propagation model derived from phantom experiments that is used to calculate the relative attenuation of fluorescence signals as they are collected over 360° along the vessel wall, and utilizes it to restore accurate fluorescence readings. The findings herein point to quantification improvements when employing hybrid iNIRF, with possible implications to the clinical detection of high-risk plaques or blood vessel theranostics.

In vivo near infrared fluorescence (NIRF) intravascular molecular imaging of inflammatory plaque, a multimodal approach to imaging of atherosclerosis.

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.(1) Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.(4) While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.(2) The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis. Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.(2,5) Inflammatory cysteine proteases have been well studied using activatable NIRF probes(10), and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis(8). In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.(3,6,7) In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology(6), in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.(10) Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque. In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,(6) is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.(11,12) The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

Modular fluorescent-labeled siderophore analogues.

Biomimetic analogues 1 of the microbial siderophore (iron carrier) ferrichrome were labeled via piperazine with various fluorescent markers at a site not interfering with iron binding or receptor recognition (compounds 10-12). These iron carriers were built from a tetrahedral carbon symmetrically extended with three strands, each containing an amino acid (G = glycyl, A = alanyl, L = leucyl and P = phenylalanyl) and terminated by a hydroxamic acid, which together define an octahedral iron-binding domain. A fourth exogenous strand provided the site for connecting various fluorescent markers via a short bifunctional linker. Iron(III) titrations, along with fluorescence spectroscopy, generated quenching of fluorescence emission of some of the probes used. The quenching process fits the Perrin model which reinforces the intramolecular quenching process, postulated previously.1 All tested compounds, regardless of their probe size, polarity, or the linker binding them to the siderophore analogue, promote growth of Pseudomonas putida with the same efficacy as the nonlabeled analogues 1, with the added benefit of signaling microbial activity by fluorescence emission. All G derivatives of compounds 10-12 were found to parallel the behavior of natural ferrichrome, whereas A derivatives mediated only a modest iron(III) uptake by P. putida. Incubation of various Pseudomonas strains with iron(III)-loaded G derivatives resulted in the build-up of the labels' fluorescence in the culture medium to a much larger extent than from the corresponding A derivatives. The fluorescence buildup corresponds to iron utilization by the cells and the release of the fluorescent labeled desferrisiderophore from the cell to the media. The fact that the microbial activity of these compounds is not altered by attachment of various fluorescent markers via a bifunctional linker proposes their application as diagnostic tools for detecting and identifying pathogenic microorganisms.

Iron uptake in Ustilago maydis: tracking the iron path.

In this study, we monitored and compared the uptake of iron in the fungus Ustilago maydis by using biomimetic siderophore analogs of ferrichrome, the fungal native siderophore, and ferrioxamine B (FOB), a xenosiderophore. Ferrichrome-iron was taken up at a higher rate than FOB-iron. Unlike ferrichrome-mediated uptake, FOB-mediated iron transport involved an extracellular reduction mechanism. By using fluorescently labeled siderophore analogs, we monitored the time course, as well as the localization, of iron uptake processes within the fungal cells. A fluorescently labeled ferrichrome analog, B9-lissamine rhodamine B, which does not exhibit fluorescence quenching upon iron binding, was used to monitor the entry of the compounds into the fungal cells. The fluorescence was found intracellularly 4 h after the application and later was found concentrated in two to three vesicles within each cell. The fluorescence of the fluorescently labeled FOB analog CAT18, which is quenched by iron, was visualized around the cell membrane after 4 h of incubation with the ferrated (nonfluorescent) compounds. This fluorescence intensity increased with time, demonstrating fungal iron uptake from the siderophores, which remained extracellular. We here introduce the use of fluorescent biomimetic siderophores as tools to directly track and discriminate between different pathways of iron uptake in cells.

Simulation of fractograms of fat emulsions in power-programmed sedimentation field-flow fractionation (SdFFF).

A quasi-empirical approach to the simulation of fractograms was examined to verify that the elution behavior of emulsions in power-based field programmed sedimentation field-flow fractionation (SdFFF) is consistent and predictable. The approach was applied to Intralipid, a commercial soybean emulsion and to an investigational medium chain triglyceride emulsion. The simulations predicted the fractograms that were obtained under various conditions of field strength, field decay and velocity of the suspending fluid, using distribution parameters obtained from one preliminary measurement of size distribution profile. Predicted fractograms were compared to experimental ones, under various fractionating powers. Good agreement was observed in most cases, in which interference of the secondary relaxation effects was not effective. The agreement confirmed the applicability of the approach to emulsions and that the simulations can be used instead of actual experiments for the optimization of their characterization by power-programmed SdFFF.

In vivo and in vitro elimination of aliphatic polyanhydrides.

The hydrolysis and elimination processes of biopolyanhydrides has been studied using a polymer series of linear aliphatic diacids. This polymer series degrade into their monomer counterparts at about the same rate but differ in the water solubility of their degradation products. Polymers based on natural diacids of the general structure -[OOC-(CH2)x-CO]n- where x is between 4 and 12 were implanted subcutaneously in rats and the elimination of the polymers from the implantation site was observed. The in vitro hydrolysis of this polymer series was studied by monitoring the weight loss, release of monomer degradation products and the content of anhydride bonds in the polymer as a function of time. A dependence was found between the monomer solubility and the rate of polymer elimination in both in vivo and in vitro. The elimination time for polymers based on soluble monomers (x = 4-8) was 7-14 days, while the polymers based on low monomer solubility (x = 10-12) were eliminated only after 8 weeks. The in vitro degradation of polyanhydrides in the presence of several common hydrolytic enzymes found around implants did not affect polymer degradation. All polymers were biocompatible and found to be useful as carriers for drug delivery.

Chemical interactions between drugs containing reactive amines with hydrolyzable insoluble biopolymers in aqueous solutions.

Chemical reactions between drugs containing reactive amines with hydrolyzable polymers in buffer solutions were investigated. Phenylalkylamines with increasing nucleophilic reactivity were used as model drugs. Solutions of phenylalkylamines were reacted heterogeneously with representative biodegradable polyanhydride and polyester powders in various pH solutions, and the recovery of the amines from the solutions was determined. Poly(sebacic acid), a reactive polyanhydride, reacted by amide formation with the tested amines and their respective HCl salts when exposed to physiologic pH (pH 7.4). However, at pH 5.0 no interaction occurred. The aromatic polyanhydride, PCPP, and the polyesters based on lactic acid and caprolactone did react with the amine derivatives at pH 7.4, but at a slower rate. The reaction can be avoided with appropriate salt derivatives of the amines.

Arteriovenous malformations of the superior cerebellar artery: excision via an occipital transtentorial approach.

Arteriovenous malformations (AVMs) of the superior cerebellar artery (SCA) are unusual and difficult lesions to treat, representing less than half of all AVMs located in the posterior fossa. Traditional approaches for surgical extirpation include the subtemporal transtentorial and suboccipital supracerebellar routes. On the basis of our recent experience with three SCA-supplied AVMs, we advocate an occipital transtentorial approach similar to that used for neoplasms of the pineal gland. Exposure of the AVM from above and in the midline provides superior visualization of the deep veins, the SCA arborization in the retrocollicular space, and the rostral cerebellum, without exposing the temporal lobe and the 4th nerve to surgical trauma in a narrow, confined space. Superior cerebellar AVMs that arise from the caudal branch of the SCA on the superolateral aspect of the hemisphere are more easily handled by standard suboccipital methods.

Parenteral aluminum administration in the dog: II. Induction of osteomalacia and effect on vitamin D metabolism.

There is an association between bone aluminum (Al) accumulation and dialysis-associated osteomalacia (OM). To study whether Al is pathogenic in OM, quantitative bone histomorphometry was done in six dogs before (Bx 1) and after (Bx 2) 3 to 5 weeks of intravenous Al administration (1 mg Al /kg/day). Bone Al was determined by histochemical and chemical methods. The percent osteoid rose from 2.8 +/- 0.8 to 7.0 +/- 4.3% (mean +/- SD), P less than 0.05, and osteoid width increased from 5.7 +/- 0.6 to 8.0 +/- 1.2 mu, P less than 0.01, after Al. Bone Al rose from 1.3 +/- 1.6 to 94.0 +/- 19.0 mg/kg after Al, and the severity of OM, expressed as either percent forming surface or percent osteoid, correlated with bone Al measured histochemically and expressed as either percent surface or percent area of trabecular bone staining for Al (r = 0.85 - 0.90, P less than 0.01). Poor tetracycline uptake (six dogs), which indicates impaired mineralization, and little or no separation of tetracycline labels (four dogs) were noted at Bx 2; thus, bone apposition and formation rates were below the limits of detection. Resorptive surface did not change but trabecular volume, expressed as percent of tissue volume, fell from 22.1 +/- 3.0 to 17.1 +/- 1.4%, P less than 0.05. Serum levels of 1,25(OH)2D fell from 26.8 +/- 9.1 to 4.5 +/- 5.5 pg/ml after 17 days of Al; serum 25(OH)D levels were unchanged. These data indicate that Al can cause OM and that its severity correlates with the bone Al content.2 +

Parenteral aluminum administration in the dog: I. Plasma kinetics, tissue levels, calcium metabolism, and parathyroid hormone.

Aluminum (Al) may cause both osteomalacia and encephalopathy in dialysis patients. Little is known about the biology of Al. This study examined the initial distribution kinetics of Al and its biological effects after injections of 1 mg/kg/day into dogs for 3 to 5 weeks. Following one intravenous dose, the plasma half-life (x +/- SE) was 276 +/- 51.8 min, with an apparent volume of distribution of 1.30 +/- 0.17 liters or 5.90 +/- 0.30% body wt; 10 to 21% of administered Al was excreted in the urine over 150 min, and the renal contribution to plasma clearance of Al correlated with GFR (r = 0.77, P less than 0.05). The total plasma clearance of Al (4.43 +/- 2.83 ml/min) exceeded the renal contribution to plasma clearance (1.94 +/- 0.36 ml/min) in each dog, and in only two instances did the renal contribution reach 50% of total plasma clearance. Serum calcium rose from 9.4 +/- 0.2 to 11.1 +/- 0.3 mg/dl and immunoreactive parathyroid hormone (iPTH) fell by 27 +/- 4% following one Al injection. With repeated Al injections, serum calcium increased from baseline levels of 10.2 +/- 0.07 mg/dl to 11.1 +/- 0.22 and 11.3 +/- 0.46 mg/dl after 1 and 2 weeks, respectively. Renal function declined in all dogs, and serum creatinine exceeded 3.5 mg/dl in four; over the 5 weeks of study, serum calcium correlated with serum creatinine (r = 0.91, P less than 0.001). Liver, kidney, and spleen showed the highest tissue content of Al, and there was substantial uptake by bone; the parathyroid content of Al was modest.(ABSTRACT TRUNCATED AT 250 WORDS)

C-reactive protein in pediatrics.

CRP levels have been found to be helpful in differential diagnosis and in following the clinical course and response to treatment of various disorders. Of particular interest is their value in the diagnosis of serious bacterial infections in neonates, such as meningitis, septicemia, and osteomyelitis. While no individual has been reported to be deficient in CRP, it is possible that abnormalities of CRP synthesis and function will be found. As increasing knowledge of the role of CRP accumulates, it is conceivable that CRP may acquire some therapeutic value. Increasing availability of rapid quantitative methods for CRP is likely to result in greater clinical usefulness.