The effect of low-dose proteasome inhibition on pre-existing atherosclerosis in LDL receptor-deficient mice

Dysfunction of the ubiquitin-proteasome system (UPS) has been implicated in atherosclerosis development. However, the nature of UPS dysfunction has been proposed to be specific to certain stages of atherosclerosis development, which has implications for proteasome inhibition as a potential treatment option. Recently, low-dose proteasome inhibition with bortezomib has been shown to attenuate early atherosclerosis in low-density lipoprotein receptor-deficient (LDLR(-/-)) mice. The present study investigates the effect of low-dose proteasome inhibition with bortezomib on pre-existing advanced atherosclerosis in LDLR(-/-) mice. We found that bortezomib treatment of LDLR(-/-) mice with pre-existing atherosclerosis does not alter lesion burden. Additionally, macrophage infiltration of aortic root plaques, total plasma cholesterol levels, and pro-inflammatory serum markers were not influenced by bortezomib. However, plaques of bortezomib-treated mice exhibited larger necrotic core areas and a significant thinning of the fibrous cap, indicating a more unstable plaque phenotype. Taking recent studies on favorable effects of proteasome inhibition in early atherogenesis into consideration, our data support the hypothesis of stage-dependent effects of proteasome inhibition in atherosclerosis

Computer-assisted ex vivo, normothermic small bowel perfusion

Background: In the present study, a technique for computer-assisted, normothermic, oxygenated, ex vivo, recirculating small bowel perfusion was established as a tool to investigate organ pretreatment protocols and ischemia/reperfusion phenomena. A prerequisite for the desired setup was an organ chamber for ex vivo perfusion and the use of syngeneic whole blood as perfusate. Methods: The entire small bowel was harvested from Lewis rats and perfused in an organ chamber ex vivo for at least 2 h. The temperature was kept at 37 degrees C in a water bath. Three experimental groups were explored, characterized by different perfusion solutions. The basic perfusate consisted of syngeneic whole blood diluted with either NaCl, Krebs' solution or Krebs' solution and norepinephrine to a hematocrit of 30%. In addition, in each group l-glutamine was administered intraluminally. The desired perfusion pressure was 100 mm Hg which was kept constant with a computer-assisted data acquisition software, which measured an-line pressure, oxygenation, flow, temperature and pH and adjusted the pressure by changing the flow via a peristaltic pump. The viability of the preparation was tested by measuring oxygen consumption and maltose absorption, which requires intact enzymes of the mucosal brush border to break down maltose into glucose. Results: Organ perfusion in group 1 (dilution with NaCl) revealed problems such as hypersecretion into the bowel lumen, low vascular resistance and no maltose uptake. In contrast a viable organ could be demonstrated using Krebs' solution as dilution solution. The addition of norepinephrine led to an improved perfusion over the entire perfusion period. Maltose absorption was comparable to tests conducted with native small bower. Oxygen consumption was stable during the 2-hour perfusion period. Conclusions: The ex vivo perfusion system established enables small bowel perfusion for at least 2 h. The viability of the graft could be demonstrated. The perfusion time achieved is sufficient to study leukocyte/lymphocyte interaction with the endothelium of the graft vessels. In addition, a viable small bowel, after 2 h of ex vivo perfusion, facilitates testing of pretreatment protocols for the reduction of the immunogenicity of small bowel allografts. Copyright (C) 2000 S. Karger AG, Basel

Microsurgical Technique of Simultaneous Pancreas/Kidney Transplantation in the Rat: Clinical Experience and Review of the Literature

Background: For experimental basic research, standardized transplantation models reflecting technical and immunologic aspects are necessary. This article describes an experimental model of combined pancreas/kidney transplantation (PKTx) in detail. Materials and Methods: Donor rats underwent en bloc pancreatectomy and nephrectomy. Revascularization was performed using the aorta with the superior mesenteric artery and the inferior vena cava with the portal vein. Exocrine drainage of the pancreas took place over a segment of the duodenum which was transplanted side-to-side to the jejunum. The kidney vessels were transplanted end-to-side. The ureter was anastomosed by patch technique. Postoperatively, serum parameters were monitored daily. Biopsies for histopathology were taken on days 5, 8 and 12. Results: All 12 recipients survived the combined PKTx without serious surgical complications. One thrombosis of the portal vein led to organ failure. Blood glucose levels were normal by the 3rd postoperative day. The transplanted duodenal segment showed slight villous atrophy, and the kidneys were well perfused without vascular complications. The anastomosis between ureter and bladder was leakproof. Conclusions: Excellent graft function and survival rates can be achieved due to simplified operation technique and short operation time. It may thus have high clinical relevance to immunologic issues within the scope of basic research. Copyright (C) 2009 S. Karger AG, Base

Initial interaction of citrate-coated iron oxide nanoparticles with the glycocalyx of THP-1 monocytes assessed by real-time magnetic particle spectroscopy and electron microscopy

Interaction with biological material can alter physicochemical parameters of magnetic nanoparticles and might thereby change their magnetic behavior with potentially important implications for various nanoparticle applications. Little is known about changes of the magnetic behavior that occur during the initial phase of cell binding and uptake. We investigate the magnetic behavior of very small superparamagnetic iron-oxide nanoparticles (VSOP) during initial contact with THP-1 monocytes. We combine real-time magnetic particle spectroscopy (MPS), a fast and sensitive method for specific detection of magnetic nanoparticles in biological specimen with high-pressure-freezing/freeze-substitution transmission electron microscopy (HPF/FS-TEM), enabling us to generate snapshots of the interaction of VSOP with the cellular glycocalyx. MPS reveals significant changes of the dynamic magnetic behavior within seconds after VSOP injection into monocyte suspensions that correlate with the formation of nanoparticle clusters in the glycocalyx. The combination of real-time MPS and HPF/FS-TEM provides an ideal platform to analyze magnetic behaviors of nanoparticles upon interaction with cells and tissues

Macrophage uptake switches on OCT contrast of superparamagnetic nanoparticles for imaging of atherosclerotic plaques

Background: Optical coherence tomography (OCT) is an intravascular, high-resolution imaging technique that is used to characterize atherosclerotic plaques. However, the identification of macrophages as important markers of inflammation and plaque vulnerability remains difficult. Here, we investigate whether the uptake of very small iron oxide particles (VSOP) in macrophages, that cluster in phagolysosomes and allow high-quality magnetic resonance imaging (MRI) of atherosclerotic plaques, and uptake of ferumoxytol nanoparticles enhance detection of macrophages by OCT. Materials and methods: RAW 264.7 macrophage cells were incubated with VSOP (1 and 2 mM Fe) that have been clinically tested and ferumoxytol (8.9 mM Fe) that is approved for iron deficiency treatment and currently investigated as an MRI contrast agent. The light scattering of control macrophages, nanoparticle-labeled macrophages (2,000,000 in 500 mu L) and nanoparticle suspensions was measured in synchronous wavelength scan mode using a fluorescence spectrophotometer. For OCT analyses, pellets of 8,000,000 non-labeled, VSOP-labeled and ferumoxytol-labeled RAW 264.7 macrophages were imaged and analyzed on an OPTIS (TM) OCT imaging system. Results: Incubation with 1 and 2 mM VSOP resulted in uptake of 7.1 +/- 1.5 and 12 +/- 1.5 pg Fe per cell, which increased the backscattering of the macrophages in spectrophotometry 2.5- and 3.6-fold, whereas incubation with 8.9 mM Fe ferumoxytol resulted in uptake of 6.6 +/- 2 pg Fe per cell, which increased the backscattering 1.5-fold at 700 nm. In contrast, backscattering of non-clustered nanoparticles in suspension was negligible. Accordingly, OCT imaging could visualize significantly increased backscattering and signal attenuation of nanoparticle-labeled macrophages in comparison with controls. Conclusion: We conclude that VSOP and, to a lesser extent, ferumoxytol increase light scattering and attenuation when taken up by macrophages and can serve as a multimodal imaging probe for MRI and OCT to improve macrophage detection in atherosclerotic plaques by OCT in the future