Key Words: atherosclerosis Ⅲ immunity Ⅲ tumor necrosis factor receptor superfamily 14 Ⅲ matrix metalloproteinases Ⅲ foam cells T umor necrosis factor (TNF)-␣ and CD40L play pivotal roles in the atherogenesis. TNF-␣ was found to be expressed in atherosclerotic plaques, 1,2 and TNF-␣ was also found to be colocalized with foam cells, smooth muscle cells (SMCs), 3,4 and mast cells. 5 CD40, a member of the TNF receptor superfamily (TNFRSF), is an integral membrane protein found on the surface of B lymphocytes, dendritic cells, hematopoietic progenitor cells, epithelial cells, and carcinomas. CD40 binds to a ligand (CD40L) which is a member of the TNF superfamily (TNFSF). 6 In atherosclerotic plaques, the expression of CD40L in T cells and the coexpression of CD40 and CD40L in vascular endothelial cells, SMCs, and macrophages were detected. 7 The interaction between CD40 and CD40L, similar to the interaction between TNF-␣ and its receptor, elicits diverse biological responses involved in atherosclerosis, such as the secretion of proinflammatory cytokines and matrix metalloproteinases (MMPs), and the expression of adhesion molecules and tissue factor. 8,9 These responses are known to make the plaque unstable. See page 1873Recently, the list of molecules belonging to TNFRSF has expanded significantly. TNFRSF14 (HVEM/HveA/ LIGHTR/TR2/ATAR) was initially identified as a cellular coreceptor for herpes simplex virus entry, hence, the name HVEM (herpes virus entry mediator, later named HveA [herpes virus entry protein A]). 10 TNFRSF14 has a wide tissue distribution and is prominently expressed by cells in lymphoid tissue, such as the spleen, and on peripheral blood leukocytes. TNFRSF14 mRNA was detected on resting and activated CD4ϩ and CD8ϩ T cells, on CD19ϩ B cells, and on monocytes. 14 We hypothesized that TNFRSF14, like the CD40/CD40L system, has a role in atherosclerosis. We analyzed the expression of TNFRSF14 in atherosclerotic plaques and the expression of proatherogenic cytokines and MMPs after stimulation of TNFRSF14 in THP-1 cells. Methods Histological AnalysisFor immunohistochemical analysis, carotid endarterectomy specimens were obtained from 13 patients, aged 63 to 81 years, who underwent the surgery at Samsung Seoul Hospital. The study was approved by an institutional review committee, and the subjects gave informed consent. Atherosclerotic plaque specimens were washed with saline and embedded in OCT (Miles Laboratories) to make frozen sections. Standard 5-m sections were stained by use of the LSAB kit (DAKO) according to the manual provided by the manufacturer. Double staining of CD68 and TNFRSF14 was performed by using an Animal Research Kit (DAKO) according to the manual provided by the manufacturer. Cell CultureHuman monocytic leukemia THP-1 cells 15 were obtained from the American Type Culture Collection. For the analysis of peripheral blood monocytes, whole blood was collected either in heparin Vacutainer or CTAD Diatubes (Becton Dickinson/Diagnostica Stago) containing dipyridamole and theophylline to pr...

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Inflammation-mediated endothelial cell (EC) dysfunction likely contributes to the pathogenesis of several vascular diseases including atherosclerosis. We found that stimulation of human umbilical vein ECs with lipopolysaccharide induced secretion of cyclophilin (CyPA) an intracellular protein belonging to the immunophilin family. We then found that when added exogenously CyPA has direct effects on ECs in vitro. At low concentrations (10 to 100 ng/ml) CyPA increased EC proliferation, migration, invasive capacity, and tubulogenesis. Gelatin zymography indicated increased secretion of active matrix metalloproteinase-2, a mediator of cell migration and angiogenesis. At high concentrations (eg, 2 g/ml) CyPA had opposite effects, decreasing EC migration and viability, possibly in relation to induction of Toll-like receptor-4 expression, detected by immunocytochemistry and flow cytometry. In vivo CyPA expression was not detectable in the luminal ECs of normal mouse carotid arteries but was rapidly induced after systemic lipopolysaccharide injection. In an experimental mouse model of atherosclerosis, CyPA expression was detected in the ECs of neocapillaries of carotid artery lesions, supporting its association with pathological angiogenesis suggested by our in vitro results. In conclusion, we found that CyPA has a biphasic activity on ECs in vitro and is up-regulated in vivo in ECs under pathological states. Our results suggest that CyPA is a novel paracrine and autocrine modulator of EC functions in immune-mediated vascular disease.

Objective Much of the genetic basis for Alzheimer disease (AD) is unexplained. We sought to identify novel AD loci using a unique family-based approach that can detect robust associations with infrequent variants (minor allele frequency <0.10). Methods We conducted a genome-wide association study in the Framingham Heart Study (FHS) (discovery) and NIA-LOAD (replication) family-based cohorts using an approach that accounts for family structure and calculates a risk score for AD as the outcome. Links between the most promising gene candidate and AD pathogenesis were explored in silico as well as experimentally in cell-based models and in human brain. Results Genome-wide significant association was identified with a PLXNA4 SNP (rs277470) located in a region encoding the semaphorin-3A (SEMA3A) binding domain (meta-analysis p value [meta-P]=4.1×10−8). A test for association with the entire region was also significant (meta-P=3.2×10−4). Transfection of SH-SY5Y cells or primary rat neurons with full-length PLXNA4 (TS1) increased tau phosphorylation when stimulated by SEMA3A. The opposite effect was observed when transfected with shorter isoforms (TS2 and TS3). However, transfection of any isoform into HEK293 cells stably expressing APP did not result in differential effects on APP processing or Aβ production. Late-stage AD cases (n=9) compared to controls (n=5) had 1.9-fold increased expression of TS1 in cortical brain tissue (P=1.6×10−4). Expression of TS1 was significantly correlated with the Clinical Dementia Rating score (ρ=0.75, P=2.2×10−4), plaque density (ρ=0.56, P=0.01) and Braak stage (ρ=0.54, P=0.02). Interpretation Our results indicate that PLXNA4 has a role in AD pathogenesis through isoform-specific effects on tau phosphorylation.

ipids are an essential component in the pathogenesis of atherosclerosis. As atherosclerosis advances, lipids play an important role in determining plaque vulnerability. [1][2][3] Vulnerable plaques are characterized by distinct morphological features: thin fibrous caps and enlarged lipid cores. 4 -6 The latter is composed of free and modified cholesterols, phospholipids, triacylglycerol, and fatty acids. 7 Cholesterols, which comprise the main component of these lipid cores, can exist in a soft gruel-like phase and in various crystallized forms such as plates, needles, and sometimes helices. 8,9 Recently, Virmani et al 6 reported that ruptured plaques contain greater amounts of cholesterol clefts or crystals in their necrotic cores than stable plaques, potentially indicating that these cholesterol clefts and crystals may be partly responsible for plaque vulnerability. However, little is known about the role of lipid crystals in the pathogenesis of atherosclerosis and plaque vulnerability, most likely because of the lack of currently available non-tissue-destructive imaging techniques and ex vivo chemical profiling tools.Nonlinear spectroscopic imaging modalities have the potential to visualize cellular organelles and tissue architecture with molecular specificity. Coherent anti-Stokes Raman scattering (CARS) microcopy has recently emerged as the most viable means for 3D chemical imaging of tissues. 10 -12 It works by probing intrinsic molecular vibrations, which obviates the need to label target molecules and fix specimens. 13 Original received September 8, 2009; revision received March 2, 2010; accepted March 9, 2010 CARS microscopy has been used in a full-scale biological study of lipid metabolism in a living organism after direct evidence of the undesirable bias associated with fluorescence labeling techniques was demonstrated. 14 Recently, a videorate CARS microscopy system has been developed for imaging skin tissue in vivo. 15,16 Because of the nonlinear nature of the CARS process, rapid scanning of the tight focal spot over the specimen permitted real-time acquisition of vibrational contrast images with 3D submicron resolution, 14 which is not possible with conventional Raman microscopes. CARS microscopy is particularly ideal for selective imaging of lipids because of the abundance of carbon-hydrogen (CH) bonds that exist in lipids as compared to the surrounding tissues. Lipids exhibit strong and distinct vibrational signatures in CARS spectra from 2700 to 3100 cm Ϫ1 . 15,16 Because of these unique characteristics, CARS microscopy is a suitable tool for atherosclerosis which is one of lipid-related diseases. Le et al applied CARS to image lipid-laden cells in atheroma, 17 and the morphological components of arterial walls were imaged as well 18 by integrating other nonlinear optics such as second harmonic generation and 2-photon auto-fluorescence (TPAF) into CARS. However, detailed chemical analysis of lipid composition beyond mere vibrational histology is still limited in the currently available CAR...

Abstract. The association between subclinical hypothyroidism and cardiovascular disease and the beneficial effect of levothyroxine replacement in subclinical hypothyroidism are still under debate. The present study was designed to determine whether subclinical hypothyroidism is associated with an increase in the intima-media thickness of the common carotid artery (C-IMT) and whether thyroid hormone replacement can reverse this change in the C-IMT. Patients with newly-diagnosed subclinical (n=36) and overt (n=40) hypothyroidism and healthy euthyroid individuals (n=32) participated in this study. All the patients were examined for clinical characteristics, and the serum lipid levels and the C-IMT were measured. Patients with subclinical hypothyroidism had a C-IMT measurement after 18 months of levothyroxine replacement. There were meaningful differences in total cholesterol and LDL-cholesterol levels between patients with subclinical hypothyroidism and euthyroidism. The subjects with subclinical and overt hypothyroidism had a greater C-IMT compared with euthyroid controls (0.66±0.10 and 0.70±0.11 vs. 0.57±0.08 mm, respectively; P < 0.05). After 12 months of euthyroidism, 28 of 36 patients with subclinical hypothyroidism completed the follow-up study. Thyroid hormone replacement significantly decreased the C-IMT (0.67±0.11 to 0.60±0.10 mm; P = 0.021) and improved the lipid profile. Based on multiple regression analysis, the decrement in LDL-cholesterol was independently associated with the regression of the C-IMT. Subclinical hypothyroidism was closely related to an increased C-IMT. Thyroid hormone replacement resulted in regression of the increased C-IMT, which was attributed to the improvement in the lipid profile. THYROID hormone has profound effects on the cardiovascular system [1]. In particular, overt hypothyroidism is known to be a risk factor for atherosclerosis and cardiovascular disease [2]. Subclinical hypothyroidism (i.e., increased serum thyrotropin concentration and normal serum thyroxine levels) is a common condition affecting 4% of the general population and 10% to 15% of the elderly [3]. Subclinical hypothyroidism, like over hypothyroidism, may be a risk factor for cardiovascular disease; however, it is controversial whether subclinical hypothyroidism results in an increased risk of atherosclerotic disease and whether the replacement of thyroid hormone reverses or prevents the risk of cardiovascular disease. Several studies have reported no association between cardiovascular risk and subclinical hypothyroidism [4,5]. Contrary to these results, a recent meta-analysis concluded that subclinical hypothyroidism was associated with atherosclerosis and coronary artery disease [6], and some placebo-controlled studies have shown the beneficial effect of levothyroxine replacement on cardiovascular risk and early atherosclerotic changes in patients with subclinical hypothyroidism [7,8].The intima-media thickness of the common carotid artery (C-IMT) is an established measure of subclinical atherosclerotic cha...

We describe the fabrication and characterization of poly(ethylene glycol) (PEG) hydrogel spheres containing the enzyme horseradish peroxidase (HRP) for application as optical nanosensors for hydrogen peroxide. HRP was encapsulated in PEG hydrogel spheres by reverse emulsion photopolymerization, yielding spheres with a size range from 250 to 350 nm. Encapsulated HRP activity and sensitivity to hydrogen peroxide were investigated by the Amplex Red assay based on the fluorescence response as a function of H2O2. These HRP-loaded spheres were then introduced to murine macrophages with Amplex Red in the culture media. After phagocytosis, the biocompatibility of spheres was determined by live cell staining using calcein AM (5 microM). The HRP-loaded PEG hydrogel spheres were activated (i.e., fluorescent oxidized Amplex Red produced within the spheres) by oxidative stresses such as exogenous H2O2 (100 microM) and lipopolysaccharide (1 microg/mL), which induced the production of endogenous peroxide inside macrophages. The results presented here indicate that after polymerization, the enzyme activity of HRP was still maintained and that using these HRP-containing nanospheres, peroxide production could be sensed locally within cells.

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