Angiotensin II-induced TLR4 mediated abdominal aortic aneurysm in apolipoprotein E knockout mice is dependent on STAT3

Abdominal Aortic Aneurysm (AAA) is a major cause of mortality and morbidity in men over 65 years of age. Male apolipoprotein E knockout (ApoE^−/−) mice infused with angiotensin II (AngII) develop AAA. Although AngII stimulates both JAK/STAT and Toll-like receptor 4 (TLR4) signaling pathways, their involvement in AngII mediated AAA formation is unclear. Here we used the small molecule STAT3 inhibitor, S3I-201, the TLR4 inhibitor Eritoran and ApoE^−/−TLR4^−/− mice to evaluate the interaction between STAT3 and TLR4 signaling in AngII-induced AAA formation. ApoE^−/− mice infused for 28 days with AngII developed AAAs and increased STAT3 activation and TLR4 expression. Moreover, AngII increased macrophage infiltration and the ratio of M1 (pro-inflammatory)/M2 (healing) macrophages in aneurysmal tissue as early as 7–10 days after AngII infusion. STAT3 inhibition with S3I-201 decreased the incidence and severity of AngII-induced AAA formation and decreased MMP activity and the ratio of M1/M2 macrophages. Furthermore, AngII-mediated AAA formation, MMP secretion, STAT3 phosphorylation and the ratio of M1/M2 macrophages were markedly decreased in ApoE^−/−TLR4^−/− mice, and in Eritoran-treated ApoE^−/− mice. TLR4 and pSTAT3 levels were also increased in human aneurysmal tissue. These data support a role of pSTAT3 in TLR4 dependent AAA formation and possible therapeutic roles for TLR4 and/or STAT3 inhibition in AAA.     

Melatonin stabilizes rupture‐prone vulnerable plaques via regulating macrophage polarization in a nuclear circadian receptor RORα‐dependent manner

Rupture of vulnerable plaques is the main trigger of acute cardio‐cerebral vascular events, but mechanisms responsible for transforming a stable atherosclerotic into a vulnerable plaque remain largely unknown. Melatonin, an indoleamine hormone secreted by the pineal gland, plays pleiotropic roles in the cardiovascular system; however, the effect of melatonin on vulnerable plaque rupture and its underlying mechanisms remains unknown. Here, we generated a rupture‐prone vulnerable carotid plaque model induced by endogenous renovascular hypertension combined with low shear stress in hypercholesterolemic ApoE^?/? mice. Melatonin (10 mg/kg/d by oral administration for 9 weeks) significantly prevented vulnerable plaque rupture, with lower incidence of intraplaque hemorrhage (42.9% vs. 9.5%, P = 0.014) and of spontaneous plaque rupture with intraluminal thrombus formation (38.1% vs. 9.5%, P = 0.029). Mechanistic studies indicated that melatonin ameliorated intraplaque inflammation by suppressing the differentiation of intraplaque macrophages toward the proinflammatory M1 phenotype, and circadian nuclear receptor retinoid acid receptor‐related orphan receptor‐α (RORα) mediated melatonin‐exerted vasoprotection against vulnerable plaque instability and intraplaque macrophage polarization. Further analysis in human monocyte‐derived macrophages confirmed the role of melatonin in regulating macrophage polarization by regulating the AMPKα‐STATs pathway in a RORα‐dependent manner. In summary, our data provided the first evidence that melatonin‐RORα axis acts as a novel endogenous protective signaling pathway in the vasculature, regulates intraplaque inflammation, and stabilizes rupture‐prone vulnerable plaques.     

FNDC5 attenuates adipose tissue inflammation and insulin resistance via AMPK-mediated macrophage polarization in obesity

BackgroundObesity-induced chronic inflammation is critical in the pathogenesis of insulin resistance, and the recruitment and proinflammatory activation of adipose tissue macrophages (ATMs) is important for the development of this process. Here, we examined the effects of fibronectin type III domain-containing 5 (FNDC5) on inflammation and insulin resistance in high-fat diet-induced obese mice.Materials and MethodsMale wild-type (WT) and FNDC5^−/− mice were fed with standard chow (Ctrl) or high fat diet (HFD) for 20 weeks to induce obesity and insulin resistance. Firstly, effects of FNDC5 gene deletion on obesity, insulin resistance, macrophage accumulation and polarization and adipose tissue inflammation were determined in mice. Secondly, the macrophage polarity shift was further examined with flow cytometry in isolated stromal vascular fraction (SVF). Thirdly, the effects of exogenous FNDC5 on lipopolysaccharide (LPS)-induced macrophage polarization, inflammation and the underlying signaling mechanism were investigated in RAW264.7 macrophages and primary mouse peritoneal cavity macrophages (PMs). Finally, the therapeutic effects of FNDC5 overexpression were examined in HFD-induced obese WT and FNDC5^−/− mice.ResultsFNDC5 gene deletion aggravated obesity, insulin resistance, fat accumulation and inflammation accompanied with enhanced AMPK inhibition, macrophages recruitment and M1 polarization in mice fed with HFD. Exogenous FNDC5 inhibited LPS-induced M1 macrophage polarization and inflammatory cytokine production via AMPK phosphorylation in both RAW264.7 macrophages and PMs. FNDC5 overexpression attenuated insulin resistance, AMPK inhibition, M1 macrophage polarization and inflammatory cytokine production in adipose tissue of obese WT and FNDC5^−/− mice.ConclusionsFNDC5 attenuates adipose tissue inflammation and insulin resistance via AMPK-mediated macrophage polarization in HFD-induced obesity. FNDC5 plays several beneficial roles in obesity and may be used as a therapeutic regimen for preventing inflammation and insulin resistance in obesity and diabetes.     

Apolipoprotein E–deficient mice are resistant to the development of collagen‐induced arthritis

Objective

To determine whether elevated serum lipid levels resulting from feeding animals a high‐fat diet can affect the inflammatory process in C57BL/6 (B6) wild‐type (WT) and B6 ApoE^−/− mouse models of collagen‐induced arthritis (CIA).

Methods

Male B6 WT or ApoE^−/− mice were fed either a normal chow diet or a high‐fat diet. CIA was induced in mice at 12 weeks of age using type II chicken collagen, Freund's complete adjuvant, and, on occasion, a lipopolysaccharide boost. Expression levels of autoantibodies and cytokines were measured using enzyme‐linked immunosorbent assay and multiplex assay, respectively.

Results

Whereas B6 WT mice developed severe articular inflammation after collagen immunization, ApoE^−/− mice developed no clinical or histologic evidence of disease regardless of whether they had been fed a high‐fat diet or a normal chow diet. The fact that arthritis was not present in ApoE^−/− mice did not result from inadequate production of serum IgG2a collagen antibodies, since levels observed in ApoE^−/− mice were similar to those observed in arthritic B6 WT control mice. Critically, development of atherosclerosis in ApoE^−/− mice was not affected by the CIA protocol.

Conclusion

Our findings suggest that ApoE^−/− mice are resistant to the development of CIA. Intriguingly, induction of host autoimmunity in the absence of articular inflammation had no effect on atherosclerosis progression, suggesting that articular inflammatory load may be a critical risk factor in vascular pathology.

     

Functional blockage of EMMPRIN ameliorates atherosclerosis in apolipoprotein E-deficient mice

Background

Extracellular matrix metalloproteinase inducer (EMMPRIN), a 58-kDa cell surface glycoprotein, has been identified as a key receptor for transmitting cellular signals mediating metalloproteinase activities, as well as inflammation and oxidative stress. Clinical evidence has revealed that EMMPRIN is expressed in human atherosclerotic plaque; however, the relationship between EMMPRIN and atherosclerosis is unclear. To evaluate the functional role of EMMPRIN in atherosclerosis, we treated apolipoprotein E-deficient (ApoE^−/−) mice with an EMMPRIN function-blocking antibody.

Methods and results

EMMPRIN was found to be up-regulated in ApoE^−/− mice fed a 12-week high-fat diet in contrast to 12 weeks of normal diet. Administration of a function-blocking EMMPRIN antibody (100 μg, twice per week for 4 weeks) to ApoE^−/− mice, starting after 12 weeks of high-fat diet feeding caused attenuated and more stable atherosclerotic lesions, less reactive oxygen stress generation on plaque, as well as down-regulation of circulating interleukin-6 and monocyte chemotactic protein-1 in ApoE^−/− mice. The benefit of EMMPRIN functional blockage was associated with reduced metalloproteinases proteolytic activity, which delayed the circulating monocyte transmigrating into atherosclerotic lesions.

Conclusion

EMMPRIN antibody intervention ameliorated atherosclerosis in ApoE^−/− mice by the down-regulation of metalloproteinase activity, suggesting that EMMPRIN may be a viable therapeutic target in atherosclerosis.     

ClC-3 deficiency prevents atherosclerotic lesion development in ApoE−/− mice

BackgroundRecent evidence suggested that ClC-3, encoding Cl⁻ channel or Cl⁻/H⁺ antiporter, plays a critical role in regulation of a variety of physiological functions. However, remarkably little is known about whether ClC-3 is involved in atherosclerosis. This study aims to establish the involvement and direct role of ClC-3 in atherogenesis and underlying mechanisms by using ClC-3 and ApoE double null mice.Methods and resultsAfter a 16-week western-type high-fat diet, the ClC-3^+/+ApoE^−/− mice developed widespread atherosclerotic lesions in aorta. However, the lesion size was significantly reduced in aorta of ClC-3^−/−ApoE^−/− mice. Compared with the ClC-3^+/+ controls, there was significantly decreased ox-LDL binding and uptake in isolated peritoneal macrophages from ClC-3^−/− mice. Moreover, the expression of scavenger receptor SR-A, but not CD36, was significantly decreased in both ClC-3^−/− peritoneal macrophages and aortic lesions from ClC-3^−/−ApoE^−/− mice. These findings were further confirmed in ox-LDL-treated RAW264.7 macrophages, which showed that silence of ClC-3 inhibited SR-A expression, ox-LDL accumulation and foam cell formation, whereas overexpression of ClC-3 produced the opposite effects. In addition, ClC-3 siRNA significantly inhibited, whereas ClC-3 overexpression increased, the phosphorylation of JNK/p38 MAPK in ox-LDL-treated RAW264.7 foam cells. Pretreatment with JNK or p38 inhibitor abolished ClC-3-induced increase in SR-A expression and ox-LDL uptake. Finally, the increased JNK/p38 phosphorylation and SR-A expression induced by ClC-3 could be mimicked by reduction of [Cl⁻]_i by low Cl⁻ solution.ConclusionsOur findings demonstrated that ClC-3 deficiency inhibits atherosclerotic lesion development, possibly via suppression of JNK/p38 MAPK dependent SR-A expression and foam cell formation.     

Macrophage inflammasome mediates hyperhomocysteinemia-aggravated abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a serious vascular disease with high mortality. Our previous study suggested that hyperhomocysteinemia (HHcy) exaggerates the occurrence of AAA. Here, we investigated whether macrophage inflammasome is involved in HHcy-aggravated AAA formation. Two independent HHcy-aggravated AAA models, perivascular calcium phosphate–treated C57BL/6 mice and angiotensin II (Ang II)–infused apolipoprotein E-deficient (ApoE^−/−) mice were used. NLPR3, caspase 1, and interleukin-1β (IL-1β) levels were higher in aneurysmal lesions of both HHcy models compared to controls, preferentially in macrophages. Similarly, macrophage inflammasome activation was observed in vitro. Folic acid administration reversed the HHcy-accelerated AAA, with ameliorated activation of inflammasome in the tunica adventitia. Lentiviral silencing of NLRP3 significantly ameliorated HHcy-aggravated AAA formation. We observed increased mitochondrial production of reactive oxygen species (ROS) and energy switch from oxidative phosphorylation to glycolysis with excess Hcy in macrophages. Blocking mitochondrial ROS production in macrophages abolished inflammasome activation. Our study highlights the potential importance of macrophage inflammasome in the pathogenesis and development of HHcy-aggravated AAA.     

ApoE(-/-)/lysozyme M(EGFP/EGFP) mice as a versatile model to study monocyte and neutrophil trafficking in atherosclerosis

ObjectivesIntravital microscopy is a useful tool for studying leukocyte trafficking in atherosclerosis. However, distinction between various subclasses of leukocytes using this technology is lacking. Therefore, we generated ApoE^?/?/Lysozyme M^EGFP/EGFP mice and investigated whether targeted cell types could be visualized by in vivo microscopy and whether absence of lysozyme M will influence atherosclerosis.MethodsWe crossed male ApoE^?/? mice with mice homozygous for a knock-in mutation of enhanced green fluorescent protein (EGFP) in the lysozyme M locus (Lys^EGFP/EGFP) creating ApoE^?/?/Lys^EGFP/EGFP mice. Mice were sacrificed at the age of 26 weeks. Blood was collected for serum lipid analysis, differential white blood cell count and flow cytometry. Lesion area was determined on en face mounted aortas and sections from aortic roots were stained for immunohistochemistry. Atherosclerotic lesions were also studied by confocal- and intravital microscopy.ResultsBasic parameters, such as white blood cell count, cholesterol profile, lesion area and plaque composition was unaltered in ApoE^?/?/Lys^EGFP/EGFP mice compared to ApoE^?/? mice. Fluorescent neutrophils and monocytes were clearly visualized by intravital fluorescence and confocal microscopy. Fluorescent cells were distributed primarily in the periphery of atherosclerotic lesions indicating a preference for recruitment in these areas.ConclusionsApoE^?/?/Lys^EGFP/EGFP mice will serve as a useful model to study leukocyte trafficking in atherosclerosis and how different subsets of leukocytes influence atherogenesis.     

Interleukin-19 increases angiogenesis in ischemic hind limbs by direct effects on both endothelial cells and macrophage polarization

Hypoxia in ischemic limbs typically initiates angiogenic and inflammatory factors to promote angiogenesis in attempt to restore perfusion. There is a gap in our knowledge concerning the role of anti-inflammatory interleukins in angiogenesis, macrophage polarization, and endothelial cell activation. Interleukin-19 is a unique anti-inflammatory Th2 cytokine that promotes angiogenic effects in cultured endothelial cells (EC); the purpose of this study was to characterize a role for IL-19 in restoration of blood flow in hind-limb ischemia, and define potential mechanisms. Hind limb ischemia was induced by femoral artery ligation, and perfusion quantitated using Laser Doppler Perfusion Imaging (LDPI). Wild type mice which received i.p. injections of rIL-19 (10 ng/g/day) showed significantly increased levels of perfusion compared to PBS controls. LDPI values were significantly decreased in IL-19^−/− mice when compared to wild type mice. IL-19^−/− mice injected with rIL-19 had significantly increased LDPI compared with PBS control mice. Significantly increased capillary density was quantitated in rIL-19 treated mice, and significantly less capillary density in IL-19^−/− mice. Multiple cell types participate in IL-19 induced angiogenesis. IL-19 treatment of human microvascular EC induced expression of angiogenic cytokines. M2 macrophage marker and VEGF-A expression were significantly increased in macrophage and the spleen from rIL-19 injected mice, and M1 marker expression was significantly increased in the spleen from IL-19^−/− compared with controls. Plasma VEGF-A levels are higher in rIL-19 injected mice. IL-19 decreased the expression of anti-angiogenic IL-12 in the spleen and macrophage. This study is the first to implicate IL-19 as a novel pro-angiogenic interleukin and suggests therapeutic potential for this cytokine.     

Oscillatory shear stress upregulation of endothelial nitric oxide synthase requires intracellular hydrogen peroxide and CaMKII

We have previously shown that hydrogen peroxide (H(2)O(2)) upregulates endothelial nitric oxide synthase (eNOS) expression via a calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated mechanism whereas it also acutely activates eNOS enzyme. We hypothesized that oscillatory shear stress (OSS), which stimulates endogenous H(2)O(2), would have effects on eNOS expression and function similar to that of exogenous H(2)O(2). Exposure of bovine aortic endothelial cells to OSS (+/-15 dynes/cm(2)) increased eNOS mRNA expression by 3-fold. Pretreatment with either polyethylene glycol-catalase (PEG-CAT, a scavenger of H(2)O(2)) or KN93, an inhibitor of CaMKII, abolished this response. OSS activated CaMKII in an H(2)O(2)-dependent fashion whereas unidirectional laminar shear stress (LSS) inhibited CaMKII phosphorylation. Inhibition of c-Src (essential for LSS upregulation of eNOS) had no effect on OSS upregulation of eNOS. Additionally, OSS stimulated NO* production acutely. Scavenging of H(2)O(2) by PEG-CAT attenuated OSS stimulation of NO* by 50% whereas it had no effect on LSS regulation of NO* production. These data suggest that intracellular H(2)O(2) and CaMKII mediate OSS upregulation of eNOS. The acute activation of eNOS by OSS also partially requires H(2)O(2). As OSS has been shown previously to stimulate sustained production of superoxide (O(2)*-) which would inactivate NO*, these responses may represent attempted compensation to restore NO* bioavailability in areas exposed to OSS. Simultaneous stimulation of O(2)*- and NO* by this mechanism, however, could facilitate peroxynitrite formation and protein nitration, which may enhance atherosclerotic lesion formation. Both OSS and LSS upregulate eNOS expression but via different signaling mechanisms.     

Cellular repressor of E1A-stimulated genes inhibits inflammation to decrease atherosclerosis in ApoE−/− mice

AimsMacrophage inflammation response is important in the pathogenesis of atherosclerosis. We investigated the role and mechanism of cellular repressor of E1A-stimulated genes (CREG) in regulating TNF-α induced inflammation response in macrophages and explore whether CREG might be a therapeutic target for atherosclerosis.Method and resultsImmunostaining and western blotting showed that expression of CREG was reduced in human atherosclerotic coronary artery. In vivo experiments demonstrated that supplementation of recombinant CREG protein to ApoE^−/− mice fed with high fat diet alleviated aortic atherosclerosis development and inflammation. In vitro, macrophage from ApoE^−/− mice fed with high fat diet had lower level of CREG compared to control mice fed with normal diet. Immunohistochemical staining and western blotting further confirmed that CREG inhibited inflammatory response of macrophages induced by TNF-α. Supplementation of exogenous recombinant CREG protein or CREG gene silencing showed that CREG promoted autophagy in TNF-α treated macrophages. The use of autophagy inhibitors, 3-methyladenine and bafilomycin A, identified that CREG attenuated TNF-α induced inflammation by activate autophagy. In addition, supplementation of exogenous CREG protein stimulated expression and maturity of cathepsin B and cathepsin L and induced lysosome formation, whereas CREG deficiency reduced lysosomal formation.ConclusionCREG inhibits inflammation and promotes autophagy mediated by lysosome formation; it might be a potential therapeutic target in atherosclerosis.     

Social disruption stress increases IL-6 levels and accelerates atherosclerosis in ApoE-/- mice

IntroductionWe have previously shown that different forms of stress have distinctive effects on atherogenesis in mice. We showed that social stress increase atherosclerosis in ApoE^?/? mice, while more physical forms of stress do not. Here we evaluated the effect of social disruption (SDR) stress on atherogenesis and evaluated cytokine release after SDR-stress and five more physical stressors.MethodsMale ApoE^?/? mice were exposed to SDR-stress during 12 weeks, and atherosclerotic plaque area was assessed in aorta, aortic root and innominate artery. Further, male C57BL/6 mice were exposed to SDR-stress or five physical stressors, and cytokine and corticosterone levels were analyzed in plasma/serum samples immediately after stress.ResultsWe found a correlation between the level of SDR-stress and atherosclerotic plaque area in aorta and a numerical increased plaque area in aortic root. SDR stress did not affect histological features of plaque composition. However, SDR-stress increased levels of corticosterone, IL-6 and CXCL1. Plasma corticosterone increased for all five physical stressors, but IL-6 and CXCL1 only increased in the group exposed to restraint combined with rat odor.ConclusionsThese findings suggest that SDR-stress is indeed atherogenic, in contrast to our previous results using the physical stressors. A possible explanation to this difference is that SDR-stress, but not physical stressors, leads to release of the pro-inflammatory cytokines IL-6 and CXCL1.     

Loss of p27 phosphorylation at Ser10 accelerates early atherogenesis by promoting leukocyte recruitment via RhoA/ROCK

Reduced phosphorylation of the tumor suppressor p27^Kip1 (p27) at serine 10 (Ser10) is a hallmark of advanced human and mouse atherosclerosis. Apolipoprotein E-null mice defective for this posttranslational modification (apoE^−/−p27Ser10Ala) exhibited increased atherosclerosis burden at late disease states. Here, we investigated the regulation of p27 phosphorylation in Ser10 at the very initial stages of atherosclerosis and its impact on endothelial-leukocyte interaction and early plaque formation. Hypercholesterolemia in fat-fed apoE^−/− mice is associated with a rapid downregulation of p27-phospho-Ser10 in primary endothelial cells (ECs) and in aorta prior to the development of macroscopically-visible lesions. We find that lack of p27 phosphorylation at Ser10 enhances the expression of adhesion molecules in aorta of apoE^−/− mice and ECs, and augments endothelial-leukocyte interactions and leukocyte recruitment in vivo. These effects correlated with increased RhoA/Rho-associated coiled-coil containing protein kinase (ROCK) signaling in ECs, and inhibition of this pathway with fasudil reduced leukocyte-EC interactions to control levels in the microvasculature of p27Ser10Ala mice. Moreover, apoE^−/−p27Ser10Ala mice displayed increased leukocyte recruitment and homing to atherosusceptible arteries and augmented early plaque development, which could be blunted with fasudil. In conclusion, our studies demonstrate a very rapid reduction in p27-phospho-Ser10 levels at the onset of atherogenesis, which contributes to early plaque build-up through RhoA/ROCK-induced integrin expression in ECs and enhanced leukocyte recruitment.     

Impairing Eukaryotic Elongation Factor 2 Kinase Activity Decreases Atherosclerotic Plaque Formation

We tested whether loss of eukaryotic elongation factor 2 kinase (eEF2K) activity in macrophages suppresses development of atherosclerosis by transplanting bone marrow from mice with mutant eEF2K into ldlr^−/− mice. Sixteen weeks after high-fat diet feeding, mutant eEF2K hematopoietic chimeras had a dramatically reduced level of atherosclerotic plaque formation. M1-skewed macrophages from eEF2K knock-in mice have less tumour necrosis factor-α release and a lesser ability to induce expression of endothelial cell markers, providing a potential explanation for the role of eEF2K. Because eEF2K activity in cells of the hematopoietic compartment contributes to atherosclerosis development, drugs inhibiting eEF2K might have a beneficial effect in treatment of atherosclerosis.     

Class A scavenger receptor attenuates myocardial infarction-induced cardiomyocyte necrosis through suppressing M1 macrophage subset polarization

Classically (M1) and alternatively (M2) activated macrophage subsets play differential roles in left ventricular remodeling after myocardial infarction (MI). The precise mechanism underlying the regulation of M1/M2 polarization during MI is unknown. We hypothesized that class A scavenger receptor (SR-A), a key modulator of inflammation, may steer macrophage polarization, which in turn influences cardiomyocytes necrosis after MI. MI was induced in wild type (WT) and SR-A deficient (SR-A^−/−) mice by left anterior descending coronary artery ligation. Cardiac function deterioration, ventricular dilatation and fibrosis were all exacerbated in SR-A^−/− mice following MI compared to WT littermates. Meanwhile, enhanced M1 macrophage polarization was observed in SR-A^−/− mice, along with increased production of M1 signature cytokines including interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) as demonstrated by immunohistochemistry, flow cytometry, quantitative real-time PCR, and ELISA assays. Moreover, activation of the activated apoptosis signal regulating kinase 1 (ASK1)/p38 mitogen-activated protein kinase (MAPK)/nuclear factor-κB (NF-κB) signaling pathway was markedly elevated in SR-A^−/− animals post-MI. Most importantly, transplantation using bone marrow from SR-A^+/+ mice partially restored M1 macrophages and significantly augmented left ventricular fractional shortening in SR-A^−/− mice. SR-A attenuated MI-induced cardiac remodeling by suppressing macrophage polarization toward a skewed M1 phenotype, reducing secretion of IL-1β, IL-6, and TNF-α, and dampening the ASK1/p38/NF-κB signaling pathway. Therefore, SR-A may exert a protective effect against MI, which may represent a new interventional target for treatment of post-infarct remodeling and subsequent heart failure.     

Arthritis in mice that are deficient in interleukin‐1 receptor antagonist is dependent on genetic background

Objective

To determine the effect of deletion of interleukin‐1 receptor antagonist (IL‐1Ra) protein in an animal model of rheumatoid arthritis.

Methods

BALB/c mice deficient in IL‐1Ra (IL‐1Ra^−/−) were bred with collagen‐induced arthritis (CIA)–susceptible DBA/1 mice and B10 mice transgenic for HLA–DRB1*0101 (B10.DR1). After generation of IL‐1Ra^−/− mice on the DBA/1 and B10.DR1 backgrounds, the mice were observed for the development of spontaneous arthritis and immunized for induction of CIA.

Results

We found that although BALB/c mice deficient in IL‐1Ra (BALB/c^−/−) spontaneously developed chronic inflammatory arthritis, DBA/1 IL‐1Ra–deficient (DBA/1^−/−) and B10.DR1 IL‐1Ra–deficient (B10.DR1^−/−) mice did not. Splenocytes from BALB/c^−/− mice produced elevated levels of IL‐2, IL‐4, IL‐6, IL‐10, IL‐17, and granulocyte–macrophage colony‐stimulating factor in response to anti‐CD3 stimulation. After immunization with type II collagen (CII), DBA/1^−/− and B10.DR1^−/− mice had a significantly earlier onset of CIA, and with increased severity compared with IL‐1Ra^+/+ mice. Immunization of BALB/c^−/− mice with CII did not aggravate spontaneous arthritis. All of the immunized mice developed antibodies to CII that correlated with arthritis severity. Levels of antibody to CII in the BALB/c^−/− strain were relatively low.

Conclusion

These data indicate that the spontaneous arthritis of IL‐1Ra deficiency is highly dependent on non–major histocompatibility complex genes and that autoimmunity to CII is not the major disease‐inducing event. Class II immune response genes are more important for the regulation of CIA, and although these 2 models of arthritis share many pathogenic mechanisms, they also have significant differences.

     

Novel phytopeptide osmotin mimics preventive effects of adiponectin on vascular inflammation and atherosclerosis

IntroductionThe novel phytohormone, osmotin, has been reported to act like mammalian adiponectin through PHO36/AdipoR1 in various in vitro and in vivo models. However, there have been no reports regarding the precise effects of osmotin on atherosclerosis.MethodsWe assessed the atheroprotective effects of osmotin on inflammatory molecules in human umbilical vein endothelial cells (HUVECs), human leukemic monocyte (THP-1) adhesion, inflammatory responses, and foam cell formation in THP-1-derived macrophages, and the migration, proliferation, and extracellular matrix expression in human aortic smooth muscle cells (HASMCs). We examined whether 4-week infusion of osmotin could suppress the development of aortic atherosclerotic lesions in apolipoprotein E-deficient (ApoE^−/−) mice.ResultsAdipoR1 was abundantly expressed in HUVECs, HASMCs, THP-1, and derived macrophages. Osmotin suppressed lipopolysaccharide-induced upregulation of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin in HUVECs, and TNF-α-induced THP-1–HUVEC adhesion. In THP-1-derived macrophages, osmotin suppressed the inflammatory M1 phenotype, lipopolysaccharide-induced secretion of interleukin-6 and TNF-α, and oxidized low-density lipoprotein-induced foam cell formation associated with CD36 and acyl-CoA:cholesterol acyltransferase 1 downregulation and ATP-binding cassette transporter A1 upregulation. In HASMCs, osmotin suppressed angiotensin II-induced migration, proliferation, collagen-1 and fibronectin expression, and matrix metalloproteinase-2 activity without inducing apoptosis. Infusion of osmotin into ApoE^−/− mice prevented the development of aortic atherosclerotic lesions with reductions of intraplaque pentraxin-3 expression, fasting plasma glucose, and insulin resistance.ConclusionsThis study provided the first evidence that osmotin exerts preventive effects on vascular inflammation and atherosclerosis, which may facilitate the development of new therapeutic modalities for combating atherosclerosis and related diseases.     

Interleukin‐6 modulates production of T lymphocyte–derived cytokines in antigen‐induced arthritis and drives inflammation‐induced osteoclastogenesis

Objective

To determine the cellular mediators of antigen‐induced arthritis (AIA) and the relative contribution of members of the interleukin‐6 (IL‐6) family and tumor necrosis factor (TNF) in AIA.

Methods

AIA was induced in mice deficient in T and B lymphocytes, IL‐6 (IL‐6^−/−), TNF (TNF^−/−), IL‐11 receptor, and oncostatin M receptor, by immunization with methylated bovine serum albumin (mBSA) followed 7 days later by intraarticular injection of mBSA. Arthritis severity was assessed histologically, and T lymphocyte responses were assessed in vitro. Anti‐TNF neutralizing antibody was administered to wild‐type mice during AIA. Bone marrow osteoclasts were generated in vitro via culture with RANKL and macrophage colony‐stimulating factor.

Results

AIA was dependent on CD4+ T lymphocytes, but not CD8+ T lymphocytes or B cells. IL‐6^−/− mice had reduced AIA severity and fewer osteoclasts at sites of bone erosion. This protective effect was not seen with a deficiency of other IL‐6 family members and was similar to that in TNF^−/− mice or wild‐type mice receiving TNF blockade treatment. IL‐6^−/− CD4+ T lymphocytes from draining lymph nodes had reduced antigen‐induced proliferation and produced less IL‐17 and less RANKL, relative to osteoprotegerin, than cells from wild‐type mice. Bone marrow from IL‐6^−/− mice generated fewer osteoclasts in vitro than bone marrow from either wild‐type or TNF^−/− mice.

Conclusion

AIA is driven by CD4+ T lymphocytes. IL‐6 is an important mediator of bone destruction in AIA because it regulates T lymphocyte production of key osteoclastogenic cytokines and inflammation‐induced bone marrow osteoclast differentiation. These findings have implications for reducing bone and joint damage in rheumatoid arthritis.

     

Clopidogrel significantly lowers the development of atherosclerosis in ApoE-deficient mice in vivo

The anti-platelet drug clopidogrel has been shown to modulate adhesion molecule and cytokine expression, both playing an important role in the pathogenesis of atherosclerosis. The aim of this study was to investigate the impact of clopidogrel on the development and progression of atherosclerosis. ApoE^?/? mice fed an atherogenic diet (cholesterol: 1 %) for 6 months received a daily dose of clopidogrel (1 mg/kg) by i.p. injection. Anti-platelet treatment was started immediately in one experimental group, and in another group clopidogrel was started 2 month after beginning of the atherogenic diet. Blood was analysed at days 30, 60 and 120 to monitor the lipid profile. After 6 months the aortic arch and brachiocephalic artery were analysed by Sudan IV staining for plaque size and by morphometry for luminal occlusion. Serum levels of various adhesion molecules were investigated by ELISA and the cellular infiltrate was analysed by immunofluorescence. After daily treatment with 1 mg/kg clopidogrel mice showed a significant reduction of atherosclerotic lesions in the thoracic aorta and within cross sections of the aortic arch [plaque formation 55.2 % (clopidogrel/start) vs. 76.5 % (untreated control) n = 8, P < 0.05]. After treatment with clopidogrel P-/E-selectin levels and cytokine levels of MCP-1 and PDGFβ were significantly reduced as compared to controls. The cellular infiltrate showed significantly reduced macrophage and T-cell infiltration in clopidogrel-treated animals. These results show that clopidogrel can effectively delay the development and progression of ‘de-novo’ atherosclerosis. However, once atherosclerotic lesions were already present, anti-platelet treatment alone did not result in reverse remodelling of these lesions.     

Supplementation with carnosine decreases plasma triglycerides and modulates atherosclerotic plaque composition in diabetic apo E−/− mice

ObjectiveCarnosine has been shown to modulate triglyceride and glycation levels in cell and animal systems. In this study we investigated whether prolonged supplementation with carnosine inhibits atherosclerosis and markers of lesion stability in hyperglycaemic and hyperlipidaemic mice.MethodsStreptozotocin-induced diabetic apo E^−/− mice were maintained for 20 weeks, post-induction of diabetes. Half of the animals received carnosine (2 g/L) in their drinking water. Diabetes was confirmed by significant increases in blood glucose and glycated haemoglobin, plasma triglyceride and total cholesterol levels, brachiocephalic artery and aortic sinus plaque area; and lower body mass.ResultsProlonged carnosine supplementation resulted in a significant (∼20-fold) increase in plasma carnosine levels, and a significant (∼23%) lowering of triglyceride levels in the carnosine-supplemented groups regardless of glycaemic status. Supplementation did not affect glycaemic status, blood cholesterol levels or loss of body mass. In the diabetic mice, carnosine supplementation did not diminish measured plaque area, but reduced the area of plaque occupied by extracellular lipid (∼60%) and increased both macrophage numbers (∼70%) and plaque collagen content (∼50%). The area occupied by α-actin-positive smooth muscle cells was not significantly increased.ConclusionsThese data indicate that in a well-established model of diabetes-associated atherosclerosis, prolonged carnosine supplementation enhances plasma levels, and has novel and significant effects on atherosclerotic lesion lipid, collagen and macrophage levels. These data are consistent with greater lesion stability, a key goal in treatment of existing cardiovascular disease. Carnosine supplementation may therefore be of benefit in lowering triglyceride levels and suppressing plaque instability in diabetes-associated atherosclerosis.