Estrogen deficiency worsens steatohepatitis in mice fed high-fat and high-cholesterol diet

Recent studies indicate an accelerated progression of nonalcoholic steatohepatitis (NASH) in postmenopausal women. Hypercholesterolemia, an important risk factor for NASH progression, is often observed after menopause. This study examined the effects of estrogen on NASH in ovariectomized (OVX) mice fed a high-fat and high-cholesterol (HFHC) diet. To investigate the effects of estrogen deficiency, OVX mice and sham-operated (SO) mice were fed normal chow or HFHC diet for 6 wk. Next, to investigate the effects of exogenous estrogen replenishment, OVX mice fed with HFHC diet were treated with implanted hormone release pellets (containing 17β-estradiol or placebo vehicle) for 6 wk. OVX mice on the HFHC diet showed enhanced liver injury with increased liver macrophage infiltration and elevated serum cholesterol levels compared with SO-HFHC mice. Hepatocyte monocyte chemoattractant protein-1 (MCP1) protein expression in OVX-HFHC mice was also enhanced compared with SO-HFHC mice. In addition, hepatic inflammatory gene expressions, including monocytes chemokine (C-C motif) receptor 2 (CCR2), were significantly elevated in OVX-HFHC mice. Estrogen treatment improved serum cholesterol levels, liver injury, macrophage infiltration, and inflammatory gene expressions in OVX-HFHC mice. Moreover, the elevated expression of liver CCR2 and MCP1 were decreased by estrogen treatment in OVX-HFHC mice, whereas low-density lipoprotein dose dependently enhanced CCR2 expression in THP1 monocytes. Our study demonstrated that estrogen deficiency accelerated NASH progression in OVX mice fed HFHC diet and that this effect was improved by estrogen therapy. Hypercholesterolemia in postmenopausal women would be a potential risk factor for NASH progression.     

Chemokine receptor CCR1 disruption in bone marrow cells enhances atherosclerotic lesion development and inflammation in mice

Several chemokines or chemokine receptors are involved in atherogenesis. CCR1 is expressed by macrophages and lymphocytes, two major cell types involved in the progression of atherosclerosis, and binds to lesion-expressed ligands. We examined the direct role of the blood-borne chemokine receptor CCR1 in atherosclerosis by transplanting bone marrow cells from either CCR1+/+ or CCR1-/- mice into low-density lipoprotein-receptor (LDLr)-deficient mice. After exposure to an atherogenic diet for 8 weeks, no differences in fatty streak size or composition were detected between the 2 groups. After 12 weeks of atherogenic diet, however, an unexpected 70% increase in atherosclerotic lesion size in the thoracic aorta was detected in the CCR1-/- mice, accompanied by a 37% increase in the aortic sinus lesion area. CCR1-/- mice showed enhanced basal and concanavalin A-stimulated IFN-gamma production by spleen T cells and enhanced plaque inflammation. In conclusion, blood-borne CCR1 alters the immuno-inflammatory response in atherosclerosis and prevents excessive plaque growth and inflammation.     

A critical function of Th17 proinflammatory cells in the development of atherosclerotic plaque in mice

Considerable evidence supports that the CD4(+) T cell-mediated immune response contributes to the development of atherosclerotic plaque. However, the effects of Th17 cells on atherosclerosis are not thoroughly understood. In this study, we evaluated the production and function of Th17 and Th1 cells in atherosclerotic-susceptible ApoE(-/-) mice. We observed that the proportion of Th17 cells, as well as Th1, increased in atherosclerotic ApoE(-/-) mice compared with nonatherosclerotic wild-type littermates. In ApoE(-/-) mice with atherosclerosis, the expression of IL-17 and retinoic acid-related orphan receptor γt was substantially higher in the arterial wall with plaque than in the arterial wall without plaque. Increased Th17 cells were associated with the magnitude of atherosclerotic plaque in ApoE(-/-) mice. Importantly, treatment of ApoE(-/-) mice with neutralizing anti-IL-17 Ab dramatically inhibited the development of atherosclerotic plaque, whereas rIL-17 application significantly promoted the formation of atherosclerotic plaque. These data demonstrate that Th17 cells play a critical role in atherosclerotic plaque formation in mice, which may have implications in patients with atherosclerosis.     

Angiotensin-converting Enzyme Inhibition Down-regulates the Pro-atherogenic Chemokine Receptor 9 (CCR9)-Chemokine Ligand 25 (CCL25) Axis

Many experimental and clinical studies suggest a relationship between enhanced angiotensin II release by the angiotensin-converting enzyme (ACE) and the pathophysiology of atherosclerosis. The atherosclerosis-enhancing effects of angiotensin II are complex and incompletely understood. To identify anti-atherogenic target genes, we performed microarray gene expression profiling of the aorta during atherosclerosis prevention with the ACE inhibitor, captopril. Atherosclerosis-prone apolipoprotein E (apoE)-deficient mice were used as a model to decipher susceptible genes regulated during atherosclerosis prevention with captopril. Microarray gene expression profiling and immunohistology revealed that captopril treatment for 7 months strongly decreased the recruitment of pro-atherogenic immune cells into the aorta. Captopril-mediated inhibition of plaque-infiltrating immune cells involved down-regulation of the C-C chemokine receptor 9 (CCR9). Reduced cell migration correlated with decreased numbers of aorta-resident cells expressing the CCR9-specific chemoattractant factor, chemokine ligand 25 (CCL25). The CCL25-CCR9 axis was pro-atherogenic, because inhibition of CCR9 by RNA interference in hematopoietic progenitors of apoE-deficient mice significantly retarded the development of atherosclerosis. Analysis of coronary artery biopsy specimens of patients with coronary artery atherosclerosis undergoing bypass surgery also showed strong infiltrates of CCR9-positive cells in atherosclerotic lesions. Thus, the C-C chemokine receptor, CCR9, exerts a significant role in atherosclerosis.     

TLR/MyD88 and liver X receptor alpha signaling pathways reciprocally control Chlamydia pneumoniae-induced acceleration of atherosclerosis

Experimental and clinical studies link Chlamydia pneumoniae infection to atherogenesis and atherothrombotic events, but the underlying mechanisms are unclear. We tested the hypothesis that C. pneumoniae-induced acceleration of atherosclerosis in apolipoprotein E (ApoE)(-/-) mice is reciprocally modulated by activation of TLR-mediated innate immune and liver X receptor alpha (LXRalpha) signaling pathways. We infected ApoE(-/-) mice and ApoE(-/-) mice that also lacked TLR2, TLR4, MyD88, or LXRalpha intranasally with C. pneumoniae followed by feeding of a high fat diet for 4 mo. Mock-infected littermates served as controls. Atherosclerosis was assessed in aortic sinuses and in en face preparation of whole aorta. The numbers of activated dendritic cells (DCs) within plaques and the serum levels of cholesterol and proinflammatory cytokines were also measured. C. pneumoniae infection markedly accelerated atherosclerosis in ApoE-deficient mice that was associated with increased numbers of activated DCs in aortic sinus plaques and higher circulating levels of MCP-1, IL-12p40, IL-6, and TNF-alpha. In contrast, C. pneumoniae infection had only a minimal effect on atherosclerosis, accumulation of activated DCs in the sinus plaques, or circulating cytokine increases in ApoE(-/-) mice that were also deficient in TLR2, TLR4, or MyD88. However, C. pneumoniae-induced acceleration of atherosclerosis in ApoE(-/-) mice was further enhanced in ApoE(-/-)LXRalpha(-/-) double knockout mice and was accompanied by higher serum levels of IL-6 and TNF-alpha. We conclude that C. pneumoniae infection accelerates atherosclerosis in hypercholesterolemic mice predominantly through a TLR/MyD88-dependent mechanism and that LXRalpha appears to reciprocally modulate and reduce the proatherogenic effects of C. pneumoniae infection.     

Expression of the Lyst(beige) mutation is atheroprotective in chow-fed apolipoprotein E-deficient mice

Lyst(beige) mice crossed with hyperlipidemic low density lipoprotein receptor-deficient mice (BgLDLr(-/-)) display increased lesion area and a more stable lesion morphology. To verify that the beige phenotype is not unique to LDLr(-/-) mice, we examined atherosclerosis in beige, apolipoprotein E-deficient mutant mice (BgApoE(-/-)). Severe diet-induced hyperlipidemia in BgApoE(-/-) mice resulted in increased aortic sinus lesion areas compared with controls. Minimal aortic lesions were observed in both genotypes on a chow diet. Nevertheless, BgApoE(-/-) mice displayed drastically reduced aortic sinus lesion growth. Reconstitution with bone marrow (BM) from green fluorescent protein mice created chimeric animals that allowed for the identification of donor-derived cells within lesions. Expressing the beige mutation exclusively in BM-derived cells had no impact on plaque development, yet the beige mutation in all cells except the BM-derived cells led to significantly larger aortic sinus lesion areas. Both mRNA and secreted protein levels of monocyte chemoattractant protein 1 were altered in quiescent and phorbol ester-stimulated cultured macrophages, vascular smooth muscle cells, and aortic endothelial cells isolated from BgApoE(-/-) mice. Thus, expression of the beige mutation in all cell types involved in lesion development contributed to atheroprotection in chow-fed ApoE(-/-) mice.     

Differential role of CCR2 in islet and heart allograft rejection: tissue specificity of chemokine/chemokine receptor function in vivo

Chemokines have a pivotal role in the mobilization and activation of specific leukocyte subsets in acute allograft rejection. However, the role of specific chemokines and chemokine receptors in islet allograft rejection has not been fully elucidated. We now show that islet allograft rejection is associated with a steady increase in intragraft expression of the chemokines CCL8 (monocyte chemoattractant protein-2), CCL9 (monocyte chemoattractant protein-5), CCL5 (RANTES), CXCL-10 (IFN-gamma-inducible protein-10), and CXCL9 (monokine induced by IFN-gamma) and their corresponding chemokine receptors CCR2, CCR5, CCR1, and CXCR3. Because CCR2 was found to be highly induced, we tested the specific role of CCR2 in islet allograft rejection by transplanting fully MHC mismatched islets from BALB/c mice into C57BL/6 wild-type (WT) and CCR2-deficient mice (CCR2-/-). A significant prolongation of islet allograft survival was noted in CCR2-/- recipients, with median survival time of 24 and 12 days for CCR2-/- and WT recipients, respectively (p < 0.0001). This was associated with reduction in the generation of CD8+, but not CD4+ effector alloreactive T cells (CD62L(low)CD44(high)) in CCR2-/- compared with WT recipients. In addition, CCR2-/- recipients had a reduced Th1 and increased Th2 alloresponse in the periphery (by ELISPOT analysis) as well as in the grafts (by RT-PCR). However, these changes were only transient in CCR2-/- recipients that ultimately rejected their grafts. Furthermore, in contrast to the islet transplants, CCR2 deficiency offered only marginal prolongation of heart allograft survival. This study demonstrates the important role for CCR2 in early islet allograft rejection and highlights the tissue specificity of the chemokine/chemokine receptor system in vivo in regulating allograft rejection.     

The chemokine receptor CCR5 is not a necessary inflammatory mediator in kainic acid-induced hippocampal injury: evidence for a compensatory effect by increased CCR2 and CCR3

Chemokines and their receptors have been strongly implicated in the inflammatory process. However, their roles in excitotoxic brain injury are largely unknown. In this study we used C-C chemokine receptor 5 (CCR5) knockout (KO) mice to investigate the role of CCR5 in neurodegeneration induced by intranasal administration of the excitotoxin kainic acid (KA). Although KA treatment resulted in an increased CCR5 mRNA level in the hippocampi of wild-type mice, a CCR5 deficiency in KO mice did not affect either the clinical and pathological changes in vivo or the neuronal susceptibilities to KA insult in vitro. KA treatment stimulated mRNA expression of the monocyte chemoattractant protein-2 (MCP-2) in both the wild-type and KO mice. KA treatment did not affect mRNA levels for the macrophage inflammatory protein-1alpha (MIP-1alpha) or the regulated upon activation normal T cells expressed and secreted protein (RANTES) in either wild-type or CCR5 KO mice. CCR2 mRNA expression was undetectable in the hippocampi of wild-type mice regardless of KA treatment. In contrast, CCR5 KO mice showed CCR2 mRNA expression that was remarkably increased after KA treatment. KA treatment did not affect CCR3 mRNA expression in the wild-type mice, whereas KO mice showed both a higher basal level of CCR3 mRNA expression as well as a strong upregulation following KA treatment. These results indicate that CCR5 is not a necessary inflammatory mediator in KA induced neurodegeneration. The roles of CCR5 in excitotoxic injury in CCR5 deficient mice are compensated by increased CCR2 and CCR3 expression, which share the common MCP-2 ligand with CCR5.     

Chemokine Receptor CCR8 Is Required for Lipopolysaccharide-Triggered Cytokine Production in Mouse Peritoneal Macrophages

Chemokine (C-C motif) receptor 8 (CCR8), the chemokine receptor for chemokine (C-C motif) ligand 1 (CCL1), is expressed in T-helper type-2 lymphocytes and peritoneal macrophages (PMφ) and is involved in various pathological conditions, including peritoneal adhesions. However, the role of CCR8 in inflammatory responses is not fully elucidated. To investigate the function of CCR8 in macrophages, we compared cytokine secretion from mouse PMφ or bone marrow-derived macrophages (BMMφ) stimulated with various Toll-like receptor (TLR) ligands in CCR8 deficient (CCR8^{- /-}) and wild-type (WT) mice. We found that CCR8^-/- PMφ demonstrated attenuated secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 when stimulated with lipopolysaccharide (LPS). In particular, LPS-induced IL-10 production absolutely required CCR8. CCR8-dependent cytokine secretion was characteristic of PMφ but not BMMφ. To further investigate this result, we selected the small molecule compound R243 from a library of compounds with CCR8-antagonistic effects on CCL1-induced Ca²⁺ flux and CCL1-driven PMφ aggregation. Similar to CCR8^-/- PMφ, R243 attenuated secretion of TNF-α, IL-6, and most strikingly IL-10 from WT PMφ, but not BMMφ. CCR8^-/- PMφ and R243-treated WT PMφ both showed suppressed c-jun N-terminal kinase activity and nuclear factor-κB signaling after LPS treatment when compared with WT PMφ. A c-Jun signaling pathway inhibitor also produced an inhibitory effect on LPS-induced cytokine secretion that was similar to that of CCR8 deficiency or R243 treatment. As seen in CCR8^-/- mice, administration of R243 attenuated peritoneal adhesions in vivo. R243 also prevented hapten-induced colitis. These results are indicative of cross talk between signaling pathways downstream of CCR8 and TLR-4 that induces cytokine production by PMφ. Through use of CCR8^-/- mice and the new CCR8 inhibitor, R243, we identified a novel macrophage innate immune response pathway that involves a chemokine receptor.     

Urine proteome analysis reflects atherosclerotic disease in an ApoE-/- mouse model and allows the discovery of new candidate biomarkers in mouse and human atherosclerosis

Noninvasive diagnosis of atherosclerosis via single biomarkers has been attempted but remains elusive. However, a previous polymarker or pattern approach of urine polypeptides in humans reflected coronary artery disease with high accuracy. The aim of the current study is to use urine proteomics in ApoE(-/-) mice to discover proteins with pathophysiological roles in atherogenesis and to identify urinary polypeptide patterns reflecting early stages of atherosclerosis. Urine of ApoE(-/-) mice either on high fat diet (HFD) or chow diet was collected over 12 weeks; urine of wild type mice on HFD was used to exclude diet-related proteome changes. Capillary electrophoresis coupled to mass spectrometry (CE-MS) of samples identified 16 polypeptides specific for ApoE(-/-) mice on HFD. In a blinded test set, these polypeptides allowed identification of atherosclerosis at a sensitivity of 90% and specificity of 100%, as well as monitoring of disease progression. Sequencing of the discovered polypeptides identified fragments of α(1)-antitrypsin, epidermal growth factor (EGF), kidney androgen-regulated protein, and collagen. Using immunohistochemistry, α(1)-antitrypsin, EGF, and collagen type I were shown to be highly expressed in atherosclerotic plaques of ApoE(-/-) mice on HFD. Urinary excretion levels of collagen and α(1)-antitrypsin fragments also significantly correlated with intraplaque collagen and α(1)-antitrypsin content, mirroring plaque protein expression in the urine proteome. To provide further confirmation that the newly identified proteins are relevant in humans, the presence of collagen type I, α(1)-antitrypsin, and EGF was also confirmed in human atherosclerotic disease. Urine proteome analysis in mice exemplifies the potential of a novel multimarker approach for the noninvasive detection of atherosclerosis and monitoring of disease progression. Furthermore, this approach represents a novel discovery tool for the identification of proteins relevant in murine and human atherosclerosis and thus also defines potential novel therapeutic targets.     

Experimental autoimmune encephalomyelitis on the SJL mouse: effect of gamma delta T cell depletion on chemokine and chemokine receptor expression in the central nervous system

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system (CNS) that is a model for multiple sclerosis. Previously, we showed that depletion of gamma delta T cells significantly reduced clinical and pathological signs of disease, which was associated with reduced expression of IL-1 beta, IL-6, TNF-alpha, and lymphotoxin at disease onset and a more persistent reduction in IFN-gamma. In this study, we analyzed the effect of gamma delta T cell depletion on chemokine and chemokine receptor expression. In the CNS of control EAE mice, mRNAs for RANTES, eotaxin, macrophage-inflammatory protein (MIP)-1 alpha, MIP-1 beta, MIP-2, inducible protein-10, and monocyte chemoattractant protein-1 were detected at disease onset, increased as disease progressed, and fell as clinical signs improved. In gamma delta T cell-depleted animals, all of the chemokine mRNAs were reduced at disease onset; but at the height of disease, expression was variable and showed no differences from control animals. mRNA levels then fell in parallel with control EAE mice. ELISA data confirmed reduced expression of MIP-1 alpha and monocyte chemoattractant protein-1 at disease onset in gamma delta T cell-depleted mice, and total T cell numbers were also reduced. In normal CNS mRNAs for CCR1, CCR3, and CCR5 were observed, and these were elevated in EAE animals. mRNAs for CCR2 were also detected in the CNS of affected mice. Depletion of gamma delta T cells reduced expression of CCR1 and CCR5 at disease onset only. We conclude that gamma delta T cells contribute to the development of EAE by promoting an inflammatory environment that serves to accelerate the inflammatory process in the CNS.     

Circulating adhesion molecules in apoE-deficient mouse strains with different atherosclerosis susceptibility

Recruitment of inflammatory cells in the arterial wall by vascular adhesion molecules plays a key role in development of atherosclerosis. Apolipoprotein E-deficient (apoE(-/-)) mice have spontaneous hyperlipidemia and develop all phases of atherosclerotic lesions. We sought to examine plasma levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) and sP-selectin in two apoE(-/-) strains C57BL/6 (B6) and BALB/c with early or advanced lesions. Mice were fed chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. On either diet, BALB/c.apoE(-/-) mice developed much smaller atherosclerotic lesions and displayed significantly lower levels of sVCAM-1 and sP-selectin than B6.apoE(-/-) mice. The Western diet significantly elevated sVCAM-1 levels in both strains and sP-selectin levels in B6.apoE(-/-) mice. BALB/c.apoE(-/-) mice exhibited 2-fold higher HDL cholesterol levels on the chow diet and 15-fold higher HDL levels on the Western diet than B6.apoE(-/-) mice, although the two strains had comparable levels of total cholesterol and triglyceride. Thus, increased atherosclerosis is accompanied by increases in circulating VCAM-1 and P-selectin levels in the two apoE(-/-) mouse strains, and the high HDL level may protect against atherosclerosis by inhibiting the expression of adhesion molecules in BALB/c.apoE(-/-) mice.     

Mast cell deficiency attenuates progression of atherosclerosis and hepatic steatosis in apolipoprotein E-null mice

Mast cells are important cells of the immune system and are recognized as participants in the pathogenesis of atherosclerosis. In this study, we evaluated the role of mast cells on the progression of atherosclerosis and hepatic steatosis using the apolipoprotein E-deficient (ApoE(-/-)) and ApoE(-/-)/mast cell-deficient (Kit(W-sh/W-sh)) mouse models maintained on a high-fat diet. The en face analyses of aortas showed a marked reduction in plaque coverage in ApoE(-/-)/Kit(W-sh/W-sh) compared with ApoE(-/-) after a 6-mo regimen with no significant change noted after 3 mo. Quantification of intima/media thickness on hematoxylin and eosin-stained histological cross sections of the aortic arch revealed no significant difference between ApoE(-/-) and ApoE(-/-)/Kit(W-sh/W-sh) mice. The high-fat regimen did not induce atherosclerosis in either Kit(W-sh/W-sh) or wild-type mice. Mast cells with indications of degranulation were seen only in the aortic walls and heart of ApoE(-/-) mice. Compared with ApoE(-/-) mice, the serum levels of total cholesterol, low-density lipoprotein and high-density lipoprotein were decreased by 50% in ApoE(-/-)/Kit(W-sh/W-sh) mice, whereas no appreciable differences were noted in serum levels of triglycerides or very low density lipoprotein. ApoE(-/-)/Kit(W-sh/W-sh) mice developed significantly less hepatic steatosis than ApoE(-/-) mice after the 3-mo regimen. The analysis of Th1/Th2/Th17 cytokine profile in the sera revealed significant reduction of interleukin (IL)-6 and IL-10 in ApoE(-/-)/Kit(W-sh/W-sh) mice compared with ApoE(-/-) mice. The assessment of systemic generation of thromboxane A(2) (TXA(2)) and prostaglandin I(2) (PGI(2)) revealed significant decrease in the production of PGI(2) in ApoE(-/-)/Kit(W-sh/W-sh) mice with no change in TXA(2). The decrease in PGI(2) production was found to be associated with reduced levels of cyclooxygenase-2 mRNA in the aortic tissues. A significant reduction in T-lymphocytes and macrophages was noted in the atheromas of the ApoE(-/-)/Kit(W-sh/W-sh) mice. These results demonstrate the direct involvement of mast cells in the progression of atherosclerosis and hepatic steatosis.     

CCR5 deficiency aggravates crescentic glomerulonephritis in mice

The chemokine receptor CCR5 is predominantly expressed on monocytes and Th1-polarized T cells, and plays an important role in T cell and monocyte recruitment in inflammatory diseases. To investigate the functional role of CCR5 in renal inflammation, we induced a T cell-dependent model of glomerulonephritis (nephrotoxic serum nephritis) in CCR5(-/-) mice. Induction of nephritis in wild-type mice resulted in up-regulation of renal mRNA expression of the three CCR5 chemokine ligands, CCL5 (15-fold), CCL3 (4.9-fold), and CCL4 (3.4-fold), in the autologous phase of the disease at day 10. The up-regulated chemokine expression was paralleled by infiltration of monocytes and T cells, followed by renal tissue injury, albuminuria, and loss of renal function. Nephritic CCR5(-/-) mice showed a 3- to 4-fold increased renal expression of CCL5 (61.6-fold vs controls) and CCL3 (14.1-fold vs controls), but not of CCL4, in comparison with nephritic wild-type mice, which was accompanied by augmented renal T cell and monocyte recruitment and increased lethality due to uremia. Furthermore, CCR5(-/-) mice showed an increased renal Th1 response, whereas their systemic humoral and cellular immune responses were unaltered. Because the CCR5 ligands CCL5 and CCL3 also act via CCR1, we investigated the effects of the pharmacological CCR1 antagonist BX471. CCR1 blockade in CCR5(-/-) mice significantly reduced renal chemokine expression, T cell infiltration, and glomerular crescent formation, indicating that increased renal leukocyte recruitment and consecutive tissue damage in nephritic CCR5(-/-) mice depended on functional CCR1. In conclusion, this study shows that CCR5 deficiency aggravates glomerulonephritis via enhanced CCL3/CCL5-CCR1-driven renal T cell recruitment.     

Atorvastatin Improves Plaque Stability in ApoE-Knockout Mice by Regulating Chemokines and Chemokine Receptors

It is well documented that statins protect atherosclerotic patients from inflammatory changes and plaque instability in coronary arteries. However, the underlying mechanisms are not fully understood. Using a previously established mouse model for vulnerable atherosclerotic plaque, we investigated the effect of atorvastatin (10 mg/kg/day) on plaque morphology. Atorvastatin did not lower plasma total cholesterol levels or affect plaque progression at this dosage; however, vulnerable plaque numbers were significantly reduced in the atorvastatin-treated group compared to control. Detailed examinations revealed that atorvastatin significantly decreased macrophage infiltration and subendothelial lipid deposition, reduced intimal collagen content, and elevated collagenase activity and expression of matrix metalloproteinases (MMPs). Because vascular inflammation is largely driven by changes in monocyte/macrophage numbers in the vessel wall, we speculated that the anti-inflammatory effect of atorvastatin may partially result from decreased monocyte recruitment to the endothelium. Further experiments showed that atorvastatin downregulated expression of the chemokines monocyte chemoattractant protein (MCP)-1, chemokine (C-X3-C motif) ligand 1 (CX3CL1) and their receptors CCR2 and, CX3CR1, which are mainly responsible for monocyte recruitment. In addition, levels of the plasma inflammatory markers C-reactive protein (CRP) and tumor necrosis factor (TNF)-α were also significantly decrease in atorvastatin-treated mice. Collectively, our results demonstrate that atorvastatin can improve plaque stability in mice independent of plasma cholesterol levels. Given the profound inhibition of macrophage infiltration into atherosclerotic plaques, we propose that statins may partly exert protective effects by modulating levels of chemokines and their receptors. These findings elucidate yet another atheroprotective mechanism of statins.     

Immunoglobulin activation of T cell chemoattractant expression in fibroblasts from patients with Graves' disease is mediated through the insulin-like growth factor I receptor pathway

Graves' disease (GD) is associated with T cell infiltration, but the mechanism for lymphocyte trafficking has remained uncertain. We reported previously that fibroblasts from patients with GD express IL-16, a CD4-specific chemoattractant, and RANTES, a C-C chemokine, in response to GD-specific IgG (GD-IgG). We unexpectedly found that these responses result from a functional interaction between GD-IgG and the insulin-like growth factor (IGF)-I receptor (IGF-IR). IGF-I and the IGF-IR-specific IGF-I analog, des(1-3), mimic the effects of GD-IgG. Neither GD-IgG nor IGF-I activates chemoattractant expression in control fibroblasts from donors without GD. Interrupting IGF-IR function with specific receptor-blocking Abs or by transiently transfecting fibroblasts with a dominant negative mutant IGF-IR completely attenuates signaling provoked by GD-IgG. Moreover, GD-IgG displaces specific (125)I-labeled IGF-I binding to fibroblasts and attenuates IGF-IR detection by flow cytometry. These findings identify a novel disease mechanism involving a functional GD-IgG/IGF-IR bridge, which potentially explains T cell infiltration in GD. Interrupting this pathway may constitute a specific therapeutic strategy.     

Monitoring therapeutical intervention with ezetimibe using targeted near-infrared fluorescence imaging in experimental atherosclerosis

Ezetimibe (EZE), an inhibitor of cholesterol absorption, reduces atherosclerosis in apolipoprotein E-deficient (apoE(-/-)) mice. The matrix protein ED-B fibronectin (ED-B) is upregulated in atherosclerotic lesions. Using a novel conjugate for near-infrared fluorescence (NIRF) imaging targeting ED-B, we studied the effect of EZE on plaque lesion formation in apoE(-/-) mice. ApoE(-/-) mice received EZE (5 mug/kg/d) or chow up to the age of 4, 6, and 8 months. NIRF imaging of aortic lesions was performed 24 hours after intravenous application ex vivo and in vivo. Plaque lesion formation was analyzed by histology and immunohistochemistry. Aortic lesion formation detected by Sudan staining and NIRF imaging was significantly reduced at 6 and 8 months (p < .001). Plaque areas determined by NIRF imaging significantly correlated with Sudan staining (p < .001). EZE treatment resulted in a significant reduction in plaque macrophage and ED-B immunoreactivity (both p < .05) in brachiocephalic lesions. There was a significant reduction in plaque size in brachiocephalic arteries in 8-month-old mice treated with EZE compared with mice during short-term treatment (p < .05), indicating EZE plaque regression. Targeted NIRF imaging showed a correlation to histologic lesion extension during therapeutical intervention in experimental atherosclerosis.     

Targeting CCL2-CCR4 axis suppress cell migration of head and neck squamous cell carcinoma

For head and neck squamous cell carcinoma (HNSCC), the local invasion and distant metastasis represent the predominant causes of mortality. Targeted inhibition of chemokines and their receptors is an ongoing antitumor strategy established on the crucial roles of chemokines in cancer invasion and metastasis. Herein, we showed that C-C motif chemokine ligand 2 (CCL2)- C-C motif chemokine receptor 4 (CCR4) signaling, but not the CCL2- C-C motif chemokine receptor 2 (CCR2) axis, induces the formation of the vav guanine nucleotide exchange factor 2 (Vav2)- Rac family small GTPase 1 (Rac1) complex to activate the phosphorylation of myosin light chain (MLC), which is involved in the regulation of cell motility and cancer metastasis. We identified that targeting CCR4 could effectively interrupt the activation of HNSCC invasion and metastasis induced by CCL2 without the promoting cancer relapse observed during the subsequent withdrawal period. All current findings suggested that CCL2-CCR4-Vav2-Rac1-p-MLC signaling plays an essential role in cell migration and cancer metastasis of HNSCC, and CCR4 may serve as a new potential molecular target for HNSCC therapy.Subject terms: Head and neck cancer, Cell migration, Cancer therapy     

Npp1 promotes atherosclerosis in ApoE knockout mice

Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) generates inorganic pyrophosphate (PP(i)), a physiologic inhibitor of hydroxyapatite deposition. In a previous study, we found NPP1 expression to be inversely correlated with the degree of atherosclerotic plaque calcification. Moreover, function-impairing mutations of ENPP1, the gene encoding for NPP1, are associated with severe, artery tunica media calcification and myointimal hyperplasia with infantile onset in human beings. NPP1 and PP(i) have the potential to modulate atherogenesis by regulating arterial smooth muscle cell (SMC) differentiation and function, including increase of pro-atherogenic osteopontin (OPN) expression. Hence, this study tested the hypothesis that NPP1 deficiency modulates both atherogenesis and atherosclerotic intimal plaque calcification. Npp1/ApoE double deficient mice were generated by crossing mice bearing the ttw allele of Enpp1 (that encodes a truncation mutation) with ApoE null mice and fed with high-fat/high-cholesterol atherogenic diet. Atherosclerotic lesion area and calcification were examined at 13, 18, 23 and 28 weeks of age. The aortic SMCs isolated from both ttw/ttw ApoE(-/-) and ttw/+ ApoE(-/-) mice demonstrated decreased Opn expression. The 28-week-old ttw/ttw ApoE(-/-) and ttw/+ ApoE(-/-) had significantly smaller atherosclerotic lesions compared with wild-type congenic ApoE(-/-) mice. Only ttw/ttw but not ttw/+ mice developed artery media calcification. Furthermore in ttw/+ mice, there was a tendency towards increased plaque calcification compared to ApoE(-/-) mice without Npp1 deficiency. We conclude that Npp1 promotes atherosclerosis, potentially mediated by Opn expression in ApoE knockout mice.