Association of apoptosis inhibitor of macrophage (AIM) expression with urinary protein and kidney dysfunction.

BACKGROUND: Apoptosis inhibitor of macrophage (AIM) expressed on macrophages prolongs inflammation by protecting macrophages from apoptosis. Most circulating AIM co-exists with immunoglobulin M (IgM). AIM's pathophysiological role in relation to IgM remains unclear. Here we evaluated the glomerular expression/deposition of AIM and IgM in the kidney using immunohistochemistry and its associations with clinical manifestations in 43 patients with biopsy-confirmed kidney diseases. METHODS: Kidney biopsy tissue from all patients was immunostained for AIM and IgM. Staining patterns and percent stained areas within the glomeruli were determined. Cells expressing AIM were identified by co-staining with macrophage and endothelial cell surface markers. Correlations between staining results and clinical parameters were evaluated using univariate and multivariate analyses. RESULTS: AIM was deposited in various areas, such as mesangial and capillary area. A part of AIM expression was localized to CD68-positive macrophages in the glomerulus. Amount of glomerular expression was positively correlated with urinary protein in patients with severe proteinuria (urinary protein ≥0.5 g/day) and kidney dysfunction [estimated glomerular filtration ratio (eGFR) <60 ml/min/1.73 m2]. Urinary protein was higher in patients exhibiting overlapping glomerular expression of AIM and IgM. Annual eGFR decline rate negatively correlated with AIM-positive area. AIM-positive area and initial serum creatinine were independently associated with decreased kidney function. CONCLUSION: AIM expression in the kidney was associated with urinary protein and decline in kidney function. Co-expression with IgM appeared to exacerbate AIM's deleterious effects on kidney function. Combined glomerular AIM and IgM expression is a candidate prognostic index for kidney disease.

Increased expression of lysosome membrane protein 2 in glomeruli of patients with idiopathic membranous nephropathy.

Urinary microvesicles constitute a rich source of membrane-bound and intracellular proteins that may provide important clues of pathophysiological mechanisms in renal disease. In the current study, we analyzed and compared the proteome of urinary microvesicles from patients with idiopathic membranous nephropathy (iMN), idiopathic focal segmental glomerulosclerosis (iFSGS), and normal controls using an approach that combined both proteomics and pathology analysis. Lysosome membrane protein-2 (LIMP-2) was increased greater than twofold in urinary microvesicles obtained from patients with iMN compared to microvesicles of patients with iFSGS and normal controls. Immunofluorescence analysis of renal biopsies confirmed our proteomics findings that LIMP-2 was upregulated in glomeruli from patients with iMN but not in glomeruli of diseased patients (iFSGS, minimal change nephropathy, IgA nephropathy, membranoproliferative glomerulonephritis) and normal controls. Confocal laser microscopy showed co-localization of LIMP-2 with IgG along the glomerular basement membrane. Serum antibodies against LIMP-2 could not be detected. In conclusion, our data show the value of urinary microvesicles in biomarker discovery and provide evidence for de novo expression of LIMP-2 in glomeruli of patients with iMN.

Keratin pearl degradation in oral squamous cell carcinoma: reciprocal roles of neutrophils and macrophages.

BACKGROUND: We have reported that neutrophilic infiltration was associated with round-shaped dyskeratosis foci, a kind of keratin pearl, of oral carcinoma in situ and that those inflammatory cells are recruited from intra-epithelially entrapped blood vessels. Based on these lines of evidence, we have formulated a hypothesis that keratin pearls are terminally degraded by neutrophils. To confirm this hypothesis, we investigated immunohistochemically stepwise degradation of keratin pearls in oral squamous cell carcinoma (SCC) to clarify any other type scavenger cells in addition to neutrophils are involved in this particular degradation process. METHODS: Neutrophils (neutrophil elastase) and macrophage subpopulations (CD68, CD163 and CD204) were immunohistochemically localized in 30 cases of oral SCC with typical round-shaped keratin pearls. SCC cells were revealed by immunohistochemistry for keratin (K) 17, and blood vessels were demonstrated by CD31. RESULTS: Keratin pearl degradation process was divided into four steps: (i) intact stage: no macrophage infiltration but minimal neutrophils were found in keratin pearls; (ii) neutrophil recruit stage: no macrophage infiltration but focal neutrophilic infiltration within the pearls; (iii) neutrophil predominant stage: dense neutrophil infiltration with minimal macrophages and segregated keratinized cancer cells strongly positive for K17; and (iv) macrophage predominant stage: dense infiltration of CD68-, CD163 (mononuclear)- and CD204 (multinucleated)-positive macrophages engulfing detached keratinized SCC cells. CONCLUSION: Keratin pearl degradation in oral SCC is strictly regulated by two types of scavenger cells: neutrophils, which perform initial tasks, and macrophages, which reciprocally take over from neutrophils the role to finalize the degradation processes.

Expression of CD163, interleukin-10, and interferon-gamma in oral squamous cell carcinoma: mutual relationships and prognostic implications.

Tumor-associated macrophages (TAMs) and their associated inflammatory cytokines represent the major inflammatory component of the stroma of many tumors and can affect prognosis in the case of neoplasms. The objective of this study was to determine the prognostic significance of CD163(+) cells, interleukin-10 (IL-10), and interferon-gamma (IFN-γ) in oral lesions associated with oral squamous cell carcinoma (OSCC). The levels of CD163, IFN-γ, and IL-10 in the tissue samples of 240 patients with OSCC and 58 patients with other oral lesions were assessed by immunohistochemistry. Individuals with low IFN-γ levels, high IL-10 levels, and low CD163 levels were of special concern with respect to OSCC progression. We found that high levels of CD163, or a combination of low IFN-γ levels, high IL-10 levels, and low CD163 levels, were associated with poorer overall survival (OS). CD163(+) cells provide better predictive power for OS in comparison with traditional markers, such as clinical stage and lymph node metastasis. Therefore, CD163(+) cells may be effective prognostic predictors of OSCC. IL-10 may also indicate poor outcomes when IFN-γ secretion is low and the cells are CD163(-) .

Effect of bacterial peptidoglycan on erythrocyte death and adhesion to endothelial cells.

Peptidoglycans, bacterial wall components, have previously been shown to trigger eryptosis, the suicidal erythrocyte death, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Phosphatidylserine exposing erythrocytes adhere to the vascular wall at least partially by interaction of erythrocytic phosphatidylserine with endothelial CXC chemokine ligand 16 (CXCL16). The present study explored whether peptidoglycan exposure fosters the adhesion of erythrocytes to human umbilical vein endothelial cells (HUVEC). To this end, HUVEC were treated for 48 h with peptidoglycan (10 µg/ml) and CXCL16 abundance determined by confocal microscopy and FACS analysis. Moreover, human erythrocytes were exposed for 48 h to peptidoglycan (10 µg/ml) and phosphatidylserine exposure estimated from binding of fluorescent annexin-V, cell volume from forward scatter in FACS analysis and erythrocyte adhesion to human umbilical vein endothelial cells (HUVEC) from trapping of labeled erythrocytes in a flow chamber. As a result, bacterial peptidoglycan exposure was followed by increased CXCL16 expression in HUVEC as well as erythrocyte shrinkage, phosphatidylserine exposure and adhesion to HUVEC under flow conditions at arterial shear rates. The adhesion was significantly attenuated but not abrogated in the presence of either, erythrocyte phosphatidylserine-coating annexin-V (5 µl/ml) or CXCL16 neutralizing antibody directed against endothelial CXCL16 (4 µg/ml). In conclusion, exposure to peptidoglycan increases endothelial CXCL16 expression and leads to eryptosis followed by phosphatidylserine- and CXCL16-mediated adhesion of eryptotic erythrocytes to vascular endothelial cells.

An electrochemiluminescence assay for gp340 (DMBT1).

Gp340 is a member of the scavenger receptor cysteine-rich family of innate immune molecules and also functions as a tumor suppressor. This study describes a picogram-level assay using electrochemiluminescence technology on the MesoScale Discovery platform. Antibodies were evaluated and the best pair was used to assay whole-mouth stimulated saliva and cervical/vaginal lavage. The assay was tested using specimens obtained from healthy volunteers to determine if gp340 concentration in saliva correlates with levels in vaginal lavage fluid. Interestingly, no correlation was determined between gp340 content in these two fluids.

Cancer-associated fibroblasts and CD163-positive macrophages in oral squamous cell carcinoma: their clinicopathological and prognostic significance.

BACKGROUND: Stromal cells are believed to affect cancer invasion and metastasis. The purpose of this study was to evaluate the distribution of cancer-associated fibroblasts (CAFs) and the incidence of tumor-associated macrophages (TAMs) in oral squamous cell carcinoma (OSCC), focusing on clinicopathological factors and patient prognosis, as well as cancer invasion. METHODS: The study included 108 patients with OSCC. Anti-α-smooth muscle actin, CD68, and CD163 antibodies were used to identify CAFs and TAMs. CAFs were divided into 4 grades on the basis of staining intensity: negative (0), scanty (1), focal (2), and abundant (3). The most intensive areas of macrophage concentration in each tumor invasive stroma were also evaluated. RESULTS: The cancer specimens were divided into Grade 0/1, Grade 2, and Grade 3 on the basis of CAF grade. In addition, they were divided into low- and high-grade groups on the basis of the number of CD68-positive and CD163-positive macrophages. The latter were significantly increased in the Grade 2 CAF group compared to the Grade 0/1 group (P = 0.009). Kaplan-Meier and multivariate survival analyses revealed that Grade 2 CAFs (P = 0.003) and high CD163-positive macrophage levels (P = 0.007) significantly correlated with a poor outcome in patients with OSCC, and that a high CD163-positive macrophage level was a significant and an independent prognostic factor (P = 0.045). CONCLUSIONS: Cancer-associated fibroblasts and CD163-positive macrophages may be potential prognostic predictors of OSCC.

Repurposing an atherosclerosis targeting peptide for tumor imaging.

Cancer and atherosclerosis are chronic diseases that share common characteristics at both early and advanced stages and can arise from multiple factors. Both diseases are characterized by uncontrolled cell proliferation, inflammation, angiogenesis and apoptosis. Herein we investigated the ability of a peptide (CTHRSSVVC), that was previously reported to bind atherosclerotic lesions to home in the tumor microenvironment. The CTHRSSVVC peptide was synthesized on solid phase and N-terminally labeled with a sulfo-Cy5 dye. The specific binding to macrophage was evaluated in vitro with flow cytometry and immunofluorescence and in vivo for tumor targeting in BALB/c mice bearing a 4T1 tumor using optical imaging. The sulfo-Cy5-CTHRSSVVC peptide was synthesized in greater than 99% purity. No selective binding of the sulfo-Cy5-CTHRSSVVC peptide to macrophages in vitro was observed, however in vivo the sulfo-Cy5-CTHRSSVVC peptide accumulated in the 4T1 tumor, with a tumor-to-normal tissue ratio of 7.21 ± 1.44 at 2 h post injection. Ex vivo analysis of tumor tissue by confocal microscopy suggested that the sulfo-Cy5-CTHRSSVVC peptide had accumulated in the stroma of the tumor specifically, in regions of spindle shaped cells. In conclusion, although the target for the sulfo-Cy5-CTHRSSVVC peptide remains to be identified, the Cy5-CTHRSSVVC peptide warrants further investigation as a tumor imaging agent.

PbSR is synthesized in macrogametocytes and involved in formation of the malaria crystalloids.

Crystalloids are transient organelles that form in developing malaria ookinetes and disappear after ookinete-to-oocyst transition. Their origins and functions remain poorly understood. The Plasmodium berghei scavenger receptor-like protein PbSR is essential for mosquito-to-host transmission of the parasite: PbSR knockout parasites produce normal numbers of oocysts that fail to form sporozoites, pointing to a role for PbSR in the oocyst during sporogony. Here, using fluorescent protein tagging and targeted gene disruption, we show that PbSR is synthesized in macrogametocytes, gets targeted to the crystalloids of developing ookinetes and is involved in crystalloid formation. While oocyst sporulation rates of PbSR knockout parasites are highly reduced in parasite-infected mosquitoes, sporulation rates in vitro are not adversely affected, supporting the view that mosquito factors could be involved in the PbSR loss-of-function phenotype. These findings are the first to identify a parasite protein involved with the crystalloid organelle, and suggest a novel protein-trafficking mechanism to deliver PbSR to the oocysts.

Endocytosis of advanced glycation end-products in bovine choriocapillaris endothelial cells.

OBJECTIVES: Age-related changes in Bruch's membrane (BM), situated between the retina and the choroid, include the accumulation of advanced glycation end-products (AGEs) and other modified macromolecules. An important clearance mechanism for such molecules is endocytosis via macrophages and specialized endothelial cells. This study examined the endocytic clearance of AGEs in choriocapillaris endothelial (CCE) cells, which are strategically located beneath the BM. MATERIALS AND METHODS: Bovine CCE cultures were incubated with radiolabeled or fluorescently labeled AGE-modified bovine serum albumin (AGE-BSA). Cells and tissues were examined for the expression of two AGE-binding scavenger receptors, stabilin-1 and -2, by immunohistochemistry and Western blotting, and with antibody inhibition studies. RESULTS: CCE cells effectively endocytosed AGE-BSA via a scavenger receptor-mediated pathway. A polyclonal antibody against stabilin-2 significantly inhibited endocytosis (40%). Tissue sections stained positive for stabilin-2 and -1 in the choriocapillaris layer, which was confirmed by immunoblots of cell extracts. Colocalization of stabilin-1 and -2 and internalized AGE-BSA was seen in early endosomes. CONCLUSIONS: CCEs actively endocytose AGE-BSA and express the scavenger receptors, stabilin-1 and -2, of which at least stabilin-2 is involved in AGE-BSA uptake. Impairment of this stabilin-mediated clearance function may contribute to depositions of waste macromolecules in BM and subsequent retinopathy.

Regulation of CXCL16 expression and secretion by myeloid cells is not altered in rheumatoid arthritis.

OBJECTIVE: Chemokine (C-X-C motif) ligand 16 (CXCL16) is secreted by macrophages and dendritic cells (DCs) to attract memory type T cells. CXCL16 expression is increased in arthritic joints of patients with rheumatoid arthritis (RA) and a role for CXCL16 has been suggested in the pathogenesis of RA. To date, little is known about the regulation of CXCL16 on monocytes/macrophages and DCs. The aim of this study was to elucidate how CXCL16 expression is regulated in healthy donors and patients with RA. METHODS: CD14+cells were isolated from the peripheral blood or synovial fluid of patients with RA and healthy controls, differentiated into different types of dendritic cells or macrophages and stimulated with various cytokines or lipopolysaccharide (LPS). Cell surface proteins, including surface CXCL16, were measured by flow cytometry and soluble CXCL16 was measured by ELISA. RESULTS: Distinct types of dendritic cells constitutively express and secrete CXCL16, which is not affected by maturation. Monocytes rapidly upregulate membrane-bound CXCL16 expression and release soluble CXCL16 upon culture. CXCL16 expression by monocytes is transiently inhibited by the Toll-like receptor (TLR)4 ligand LPS. Th2 type cytokines inhibit soluble CXCL16, whereas T helper (Th)1 cell stimulus enhances its release. In RA monocytes/macrophages, neither CXCL16 expression, nor CXCL16 regulation is different from healthy controls. CONCLUSIONS: Culture of monocytes is the main trigger for CXCL16 surface expression in vitro, which is not altered in RA. Together our data suggest that the increased CXCL16 expression in patients with RA is likely to be caused by increased influx of monocytes rather than intrinsic differences in CXCL16 regulation.

Systemic deficiency of the MAP kinase-activated protein kinase 2 reduces atherosclerosis in hypercholesterolemic mice.

Atherosclerosis is a chronic inflammatory disease and represents the major cause of cardiovascular morbidity and mortality. A critical regulator of inflammatory processes represents the mitogen-activated protein kinase-activated protein kinase-2 (MK2). Therefore, we investigated the functional role of MK2 in atherogenesis in hypercholesterolemic mice as well as potentially underlying mechanisms in vivo and in vitro. Activation of MK2 (phospho-MK2) was predominantly detected in the endothelium and macrophage-rich plaque areas within aortas of hypercholesterolemic LDL receptor-deficient mice (ldlr(-/-)). Systemic MK2 deficiency of hypercholesterolemic ldlr(-/-) mice (ldlr(-/-)/mk2(-/-)) significantly decreased the accumulation of lipids and macrophages in the aorta after feeding an atherogenic diet for 8 and 16 weeks despite a significant increase in proatherogenic plasma lipoproteins compared with ldlr(-/-) mice. Deficiency of MK2 significantly decreased oxLDL-induced foam cell formation in vitro, diet-induced foam cell formation in vivo, and expression of scavenger receptor A in primary macrophages. In addition, systemic MK2 deficiency of hypercholesterolemic ldlr(-/-) mice significantly decreased the aortic expression of the adhesion molecule VCAM-1 and the chemokine MCP-1, key mediators of macrophage recruitment into the vessel wall. Furthermore, silencing of MK2 in endothelial cells by siRNA reduced the IL-1beta-induced expression of VCAM-1 and MCP-1. MK2 critically promotes atherogenesis by fostering foam cell formation and recruitment of monocytes/macrophages into the vessel wall. Therefore, MK2 might represent an attractive novel target for the treatment of atherosclerotic cardiovascular disease.

Expression and tissue localization of collectin placenta 1 (CL-P1, SRCL) in human tissues.

Collectin placenta-1 (CL-P1), also known as scavenger receptor with C-type lectin (SRCL), is a type II membrane glycoprotein that shares structural features with both collectins and type A scavenger receptors. CL-P1 was originally cloned from the placenta and found to be associated with endothelial cells. It binds via its lectin domain to desialyated Lewis X containing glycoproteins and it is able to facilitate internalization of bound ligands. Via positively charged residues in the collagen-like region it binds to negatively charged components of microbial membranes. It has previously been proposed that CL-P1 plays a role in the host defense system and in the clearance of glycoproteins from the blood. With the aims of determining the detailed tissue expression of human CL-P1 we expressed CL-P1 recombinantly in both E. coli and CHO cells, and raised monoclonal antibodies against human CL-P1. Three monoclonal antibodies were characterized and used in immunohistochemical analyses of a panel of cryo- and formalin-fixed sections. We find that CL-P1 mainly associates with cytotrophoblasts and syncytiotrophoblasts of the placenta, alveolar macrophages and to a less degree with macrophage-like and stromal cells of the tonsils. By real-time RT-PCR we verified that the placenta is also the main organ of CL-P1 synthesis. The only source of endothelial cells whereto CL-P1 associates are umbilical cord vein endothelial cells (human umbilical vein endothelial cells, HUVEC). In vitro cultured HUVECs express both the CL-P1 mRNA and show anti-CL-P1 immunoreactivity but CL-P1 locates mainly to the cytosol and not to the membrane of these cells. We conclude that CL-P1 is not a common membrane protein on endothelial cells found in normal tissues under steady state conditions.

CXCR6 is expressed in human prostate cancer in vivo and is involved in the in vitro invasion of PC3 and LNCap cells.

In spite of the clinical importance of prostate cancer (PCa) bone metastasis, the precise mechanisms for the directed migration of malignant cells remain unclear. In the present study, the expression of CXCR6 in human PCa and benign prostatic hyperplasia samples, and the expression of CXCL16 in human osseous tissues were determined by immunohistochemistry. It was found that the level of CXCR6 protein expression was elevated in human malignant prostate tumors, and CXCL16 was expressed positively by human osteocytes in vivo. The in vitro experiments further confirmed that the PCa cell lines PC3 and LNCap expressed CXCR6 at both the mRNA and protein levels, and exogenous CXCL16 has the potential to stimulate the invasion of PC3 and LNCap. To further elucidate the role of the CXCL16-CXCR6 axis in PCa progression, we compared the expression of CXCR6 and CXCR4 in human PCa tissues and the effects of CXCL16 and CXCL12 on the in vitro invasion of PC3 and LNCap cells. It was shown that CXCR6 and CXCR4 proteins were coexpressed and elevated in human PCa samples, and CXCL16 and CXCL12 promoted the invasion of PC3 and LNCap via their respective receptors. Furthermore, in contrast to CXCL12, which enhanced the activity of matrix metalloproteinase (MMP) 9 and MMP2 in PC3 and LNCap, CXCL16 ligation resulted in stronger MMP9 and MMP2 activity in LNCap but not in PC3. Our results suggest that besides CXCL12/CXCR4, CXCL16/CXCR6 might be another important factor involved in PCa bone metastasis.

CXCR6-CXCL16 interaction in the pathogenesis of Juvenile Idiopathic Arthritis.

In order to evaluate the role of CXCR6/CXCL16 in driving lymphocyte migration into inflamed joints of children with oligoarticular Juvenile Idiopathic Arthritis (JIA) we analysed CXCR6 expression and functional capability in lymphocytes from synovial fluid (SF) by flow cytometry, by real-time polymerase chain reaction (RT-PCR) and migration assays. Furthermore, CXCR6 and CXCL16 expression in synovial tissue (ST) was analysed by immunohistochemistry. T cells isolated from SF of patients with JIA expressed CXCR6 which was functionally active as shown by chemotactic assays. The same cells expressed CXCR3 and it exerted a migratory activity in response to CXCL10. CXCL16 and CXCR6 were intensively expressed on the synovium cells, respectively on macrophages, synoviocytes and endothelial cells and on lymphocytes, synoviocytes and endothelial cells. Taken together, these data suggest that CXCR6 and CXCR3 act coordinately with respective ligands and are involved in the pathophysiology of JIA-associated inflammatory processes.

Mobilization of endothelial progenitor cells in fracture healing and distraction osteogenesis.

INTRODUCTION: Fracture healing and distraction osteogenesis (DO) are unique postnatal bone formation processes, and neovascularization is critically required for successful bone regeneration. We investigated endothelial progenitor cell (EPC) mobilization during bone regeneration, and the possible contribution of EPCs to increased vascularization and new bone formation, especially in DO. METHODS: Mouse tibia fracture and rat tibia DO models were used in this study. The proportion of EPCs among the peripheral and splenic mononuclear cells (MNCs) was determined by examining the endothelial lineage staining characteristics and EPC cell surface markers. Messenger RNA expression of molecules related to EPC mobilization and homing at the fracture site were analyzed by ribonuclease protection assay and reverse-transcription polymerase chain reaction. In the rat tibia DO model, we measured blood flow during DO, and determined the distribution of ex vivo-expanded and intravenously-infused EPCs. RESULTS: The proportion of EPCs among the peripheral and splenic MNCs increased after fracture, peaked on post-fracture day 3, and returned to basal levels during the healing period. Messenger RNA expression of EPC mobilizing cytokines such as vascular endothelial growth factor (VEGF), stem cell factor, monocyte chemoattractant protein-1, and stromal cell-derived factor-1, were upregulated at the fracture callus. The plasma VEGF levels peaked prior to the increase in the EPC proportion. Adhesion molecules involved in EPC homing were expressed at the fracture callus. In the DO model, the temporal pattern of the increase in the EPC proportion was similar to that in the fracture healing model, but the EPC proportion increased again during the distraction and consolidation phases. The distraction gap was relatively ischemic during the distraction phase and blood flow increased profusely later in the consolidation phase. The number of EPCs homing to the bone regeneration site in the DO model correlated with the number of transplanted EPCs in a dose-dependent manner. CONCLUSIONS: These findings suggest that signals from the bone regeneration site mobilize EPCs from the bone marrow into the peripheral circulation. Increased EPC mobilization and homing may contribute to neovascularization and thus to new bone formation in fracture healing and DO.

Molecular Chaperone Receptors.

Extracellular heat shock proteins (HSP) play important roles in cell signaling and immunity. Many of these effects are mediated by surface receptors expressed on a wide range of cell types. We have investigated the nature of such proteins by cloning candidate receptors into cells (CHO-K1) with the rare property of being null for HSP binding. Using this approach we have discovered that Hsp70 binds avidly to at least two classes of receptors including: (1) c-type lectin receptors (CLR) and (2) scavenger receptors (SR). However, the structural nature of the receptor-ligand interactions is not clear at this time. Hsp70 can bind to LOX-1 (a member of both the CLR and SR), with the c-type lectin binding domain (CTLD) as well as the SR family members SREC-I and FEEL-1/CLEVER-1/STABILIN-1, which by contrast have arrays of EGF-like repeats in their extracellular domains. In this chapter we will discuss: (1) methods for discovery of HSP receptors, (2) approaches to the study of individual receptors in cells that contain multiple such receptors, and (3) methods for investigating HSP receptor function in vivo.

Synthetic retinoid Am80 reduces scavenger receptor expression and atherosclerosis in mice by inhibiting IL-6.

BACKGROUND: Macrophage scavenger receptors facilitate the uptake of modified low-density lipoprotein (LDL), formation of foam cells, and development of atherosclerosis. Given that proinflammatory cytokines, including IL-6, can modulate the macrophage foaming process, the aim of the present study was to determine whether the synthetic retinoic acid receptor-alpha/beta-specific agonist Am80, which is also an IL-6 inhibitor, can modulate macrophage lipid accumulation and foam cell formation. METHODS AND RESULTS: Am80 suppressed IL-6 production induced by 12-myristate 13-acetate (PMA) or angiotensin II in mouse Raw264 macrophages. It also suppressed expression of the 2 major scavenger receptors (scavenger receptor-A [SR-A] and CD36), in part by inhibiting IL-6, and inhibited macrophage foam cell formation. Systemic administration of Am80 led to reductions in the areas of atherosclerotic lesions and foam cell accumulation in the aortas of apolipoprotein E (apoE)-deficient mice and reduced serum concentrations of IL-6 and IL-1beta without affecting body weights, serum lipid profiles or IL-10 levels. CONCLUSIONS: Am80 suppresses scavenger receptor expression and macrophage foam cell formation in vitro and prevents atherogenesis in apoE-deficient mice in vivo. This suggests Am80 is a novel candidate agent that could be highly useful in the prevention and treatment of atherosclerosis.

Effects of soy pinitol on the pro-inflammatory cytokines and scavenger receptors in oxidized low-density lipoprotein-treated THP-1 macrophages.

Pinitol, a methylated form of D-chiro-inositol, acts as a insulin mediator. We investigated the effects of soy pinitol on the factors involved in foam cell formation using differentiated THP-1 macrophages. Pinitol slightly inhibited the lipid-laden foam cell formation by oxidized low-density lipoprotein (oxLDL) in a dose-dependent manner. Tumor necrosis factor-alpha and monocyte chemoattractant protein-1 releases were significantly reduced by pinitol treatment (0.05-0.5 mM), whereas interleukin-1beta and interleukin-8 secretions were significantly reduced in low-dose pinitol (0.05 or 0.1 mM) and 0.5 mM pinitol-treated cells, respectively, compared to no pinitol-treated cells. Gene expressions of CD36 and CD68 were significantly down-regulated by 0.05-0.5 mM pinitol compared to the oxLDL-treated control cells. Matrix metalloproteinase-9 gene expression was significantly decreased in 0.05-0.5 mM pinitol-treated cells compared to the no pinitol-treated macrophages. We conclude that pinitol has some inhibitory effects on foam cell formation by reducing lipid accumulation, secretion, and expression of some cytokines and macrophage scavenger receptor expression via its insulin-like action.

Gliclazide inhibits differentiation-associated biologic events in human monocyte-derived macrophages.

We investigated the in vitro effect of gliclazide on human monocyte-derived macrophage scavenger receptor expression and activity, foam cell formation, and lipopolysaccharide-induced cytokine production. Differentiation of human monocytes into macrophages in the presence of gliclazide (1-10 microg/mL) decreased CD36 expression by 20% to 50%, with maximal effect occurring at 2.5 microg/mL (P<.05). This effect was mimicked by vitamin E (50 micromol/L) and N-acetyl-L-cysteine (10 mmol/L). Incubation of the cells with gliclazide and N-acetyl-L-cysteine also reduced CD36 activity by 30% (P<.02). Despite these effects, neither gliclazide nor vitamin E did affect foam cell formation. In contrast, gliclazide significantly reduced lipopolysaccharide-stimulated macrophage tumor necrosis factor alpha and interleukin 6 secretion (P<.05). Overall, these data indicate that gliclazide, at concentrations in the therapeutic range, may regulate some key biologic events associated with the process of monocyte differentiation into macrophages.