Somatic ablation of IKKß in liver and leukocytes is not tolerated in obese mice but hepatic IKKß deletion improves fatty liver and insulin sensitivity.

The kinase IKKß controls pro-inflammatory gene expression, and its activity in the liver and leukocytes was shown to drive metabolic inflammation and insulin resistance in obesity. However, it was also proposed that liver IKKß signaling protects obese mice from insulin resistance and endoplasmic reticulum (ER) stress by increasing XBP1s protein stability. Furthermore, mice lacking IKKß in leukocytes display increased lethality to lipopolysaccharides. This study aims at improving our understanding of the role of IKKß signaling in obesity. We induced IKKß deletion in hematopoietic cells and liver of obese mice by Cre-LoxP recombination, using an INF-inducible system, or a liver-specific IKKß deletion in obese mice by adenovirus delivery of the Cre recombinase. The histopathological, immune, and metabolic phenotype of the mice was characterized. IKKß deletion in the liver and hematopoietic cells was not tolerated in mice with established obesity exposed to the TLR3 agonist poly(I:C) and exacerbated liver damage and ER-stress despite elevated XBP1s. By contrast, liver-specific ablation of IKKß in obese mice reduced steatosis and improved insulin sensitivity in association with increased XBP1s protein abundance and reduced expression of de-novo lipogenesis genes. We conclude that IKKß blockage in liver and leukocytes is not tolerated in obese mice exposed to TLR3 agonists. However, selective hepatic IKKß ablation improves fatty liver and insulin sensitivity in association with increased XBP1s protein abundance and reduced expression of lipogenic genes.

The selective alpha7 nicotinic acetylcholine receptor agonist AR-R17779 does not affect ischemia-reperfusion brain injury in mice.

Inflammation plays a central role in stroke-induced brain injury. The alpha7 nicotinic acetylcholine receptor (α7nAChR) can modulate immune responses in both the periphery and the brain. The aims of the present study were to investigate α7nAChR expression in different brain regions and evaluate the potential effect of the selective α7nAChR agonist AR-R17779 on ischemia-reperfusion brain injury in mice. Droplet digital PCR (ddPCR) was used to evaluate the absolute expression of the gene encoding α7nAChR (Chrna7) in hippocampus, striatum, thalamus and cortex in adult, naïve mice. Mice subjected to transient middle cerebral artery occlusion (tMCAO) or sham surgery were treated with α7nAChR agonist AR-R17779 (12 mg/kg) or saline once daily for 5 days. Infarct size and microglial activation 7 days after tMCAO were analyzed using immunohistochemistry. Chrna7 expression was found in all analyzed brain regions in naïve mice with the highest expression in cortex and hippocampus. At sacrifice, white blood cell count was significantly decreased in AR-R17779 treated mice compared with saline controls in the sham groups, although, no effect was seen in the tMCAO groups. Brain injury and microglial activation were evident 7 days after tMCAO. However, no difference was found between mice treated with saline or AR-R17779. In conclusion, α7nAChR expression varies in different brain regions and, despite a decrease in white blood cells in sham mice receiving AR-R17779, this compound does not affect stroke-induced brain injury.

α1-Microglobulin (A1M) Protects Human Proximal Tubule Epithelial Cells from Heme-Induced Damage In Vitro.

Oxidative stress is associated with many renal disorders, both acute and chronic, and has also been described to contribute to the disease progression. Therefore, oxidative stress is a potential therapeutic target. The human antioxidant α1-microglobulin (A1M) is a plasma and tissue protein with heme-binding, radical-scavenging and reductase activities. A1M can be internalized by cells, localized to the mitochondria and protect mitochondrial function. Due to its small size, A1M is filtered from the blood into the glomeruli, and taken up by the renal tubular epithelial cells. A1M has previously been described to reduce renal damage in animal models of preeclampsia, radiotherapy and rhabdomyolysis, and is proposed as a pharmacological agent for the treatment of kidney damage. In this paper, we examined the in vitro protective effects of recombinant human A1M (rA1M) in human proximal tubule epithelial cells. Moreover, rA1M was found to protect against heme-induced cell-death both in primary cells (RPTEC) and in a cell-line (HK-2). Expression of stress-related genes was upregulated in both cell cultures in response to heme exposure, as measured by qPCR and confirmed with in situ hybridization in HK-2 cells, whereas co-treatment with rA1M counteracted the upregulation. Mitochondrial respiration, analyzed with the Seahorse extracellular flux analyzer, was compromised following exposure to heme, but preserved by co-treatment with rA1M. Finally, heme addition to RPTE cells induced an upregulation of the endogenous cellular expression of A1M, via activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-pathway. Overall, data suggest that A1M/rA1M protects against stress-induced damage to tubule epithelial cells that, at least partly, can be attributed to maintaining mitochondrial function.

Bis(maltolato)oxovanadium(IV) Induces Angiogenesis via Phosphorylation of VEGFR2.

VEGFR2 and VEGF-A play a pivotal role in the process of angiogenesis. VEGFR2 activation is regulated by protein tyrosine phosphatases (PTPs), enzymes that dephosphorylate the receptor and reduce angiogenesis. We aim to study the effect of PTPs blockade using bis(maltolato)oxovanadium(IV) (BMOV) on in vivo wound healing and in vitro angiogenesis. BMOV significantly improves in vivo wound closure by 45% in C57BL/6JRj mice. We found that upon VEGFR2 phosphorylation induced by endogenously produced VEGF-A, the addition of BMOV results in increased cell migration (45%), proliferation (40%) and tube formation (27%) in HUVECs compared to control. In a mouse ex vivo, aortic ring assay BMOV increased the number of sprouts by 3 folds when compared to control. However, BMOV coadministered with exogenous VEGF-A increased ECs migration, proliferation and tube formation by only 41%, 18% and 12% respectively and aortic ring sprouting by only 1-fold. We also found that BMOV enhances VEGFR2 Y951 and p38MAPK phosphorylation, but not ERK1/2. The level of phosphorylation of these residues was the same in the groups treated with BMOV supplemented with exogenous VEGF-A and exogenous VEGF-A only. Our study demonstrates that BMOV is able to enhance wound closure in vivo. Moreover, in the presence of endogenous VEGF-A, BMOV is able to stimulate in vitro angiogenesis by increasing the phosphorylation of VEGFR2 and its downstream proangiogenic enzymes. Importantly, BMOV had a stronger proangiogenic effect compared to its effect in coadministration with exogenous VEGF-A.

Dietary Polyunsaturated Fatty Acids Promote Neutrophil Accumulation in the Spleen by Altering Chemotaxis and Delaying Cell Death.

Neutrophils are the most abundant circulating leukocytes in humans and are essential for the defense against invading pathogens. Like many other cells of an organism, neutrophils can be highly influenced by the diet. We have previously described that mice fed a high-fat diet rich in polyunsaturated fatty acids (HFD-P) present a higher frequency of neutrophils in bone marrow than mice fed a high-fat diet rich in saturated fatty acids (HFD-S). Interestingly, such an increase correlated with improved survival against bacterium-induced sepsis. In this study, we aimed to investigate the effects of dietary polyunsaturated and saturated fatty acids on neutrophil homeostasis. We found that HFD-P specifically induced the accumulation of neutrophils in the marginal pools of the spleen and liver. The accumulation of neutrophils in the spleen was a result of a dual effect of polyunsaturated fatty acids on neutrophil homeostasis. First, polyunsaturated fatty acids enhanced the recruitment of neutrophils from the circulation into the spleen via chemokine secretion. Second, they delayed neutrophil cell death in the spleen. Interestingly, these effects were not observed in mice fed a diet rich in saturated fatty acids, suggesting that the type of fat rather than the amount of fat mediates the alterations in neutrophil homeostasis. In conclusion, our results show that dietary polyunsaturated fatty acids have a strong modulatory effect on neutrophil homeostasis that may have future clinical applications.

Testosterone is an endogenous regulator of BAFF and splenic B cell number.

Testosterone deficiency in men is associated with increased risk for autoimmunity and increased B cell numbers through unknown mechanisms. Here we show that testosterone regulates the cytokine BAFF, an essential survival factor for B cells. Male mice lacking the androgen receptor have increased splenic B cell numbers, serum BAFF levels and splenic Baff mRNA. Testosterone deficiency by castration causes expansion of BAFF-producing fibroblastic reticular cells (FRCs) in spleen, which may be coupled to lower splenic noradrenaline levels in castrated males, as an α-adrenergic agonist decreases splenic FRC number in vitro. Antibody-mediated blockade of the BAFF receptor or treatment with the neurotoxin 6-hydroxydopamine revert the increased splenic B cell numbers induced by castration. Among healthy men, serum BAFF levels are higher in men with low testosterone. Our study uncovers a previously unrecognized regulation of BAFF by testosterone and raises important questions about BAFF in testosterone-mediated protection against autoimmunity.

Diabetes negatively affects cortical and striatal GABAergic neurons: an effect that is partially counteracted by exendin-4.

Type 2 diabetic (T2D) patients often develop early cognitive and sensorimotor impairments. The pathophysiological mechanisms behind these problems are largely unknown. Recent studies demonstrate that dysfunctional γ-aminobutyric acid (GABAergic) neurons are involved in age-related cognitive decline. We hypothesized that similar, but earlier dysfunction is taking place under T2D in the neocortex and striatum (two brain areas important for cognition and sensorimotor functions). We also hypothesized that the T2D-induced effects are pharmacologically reversible by anti-diabetic drugs targeting the glucagon-like peptide-1 receptor (GLP-1R). We determined the effect of T2D on cortical and striatal GABAergic neurons positive for glutamic acid decarboxylase-67 (GAD67), calbindin (CB), parvalbumin (PV) and calretinin (CR) by using immunohistochemistry and quantitative microscopy. Young and middle-aged T2D Goto-Kakizaki (GK) (a model of spontaneous T2D) and Wistar rats were used. Furthermore, we determined the therapeutic potential of the GLP1-R agonist exendin-4 (Ex-4) by treating middle-aged GK rats for 6 weeks with 0.1 µg/kg Ex-4 twice daily. We show that T2D reduced the density of GAD67-positive neurons in the striatum and of CB-positive neurons in both striatum and neocortex. T2D also increased the average volume of PV-positive interneurons in the striatum. Ex-4 treatment increased the density of CB-positive neurons in the striatum of GK rats. Our data demonstrate that T2D negatively affects GAD67 and CB-positive GABAergic neurons in the brain during aging, potentially identifying some of the pathophysiological mechanisms to explain the increased prevalence of neurological complications in T2D. We also show a specific, positive effect of Ex-4 on striatal CB-positive neurons, which could be exploited in therapeutic perspective.

Six Tissue Transcriptomics Reveals Specific Immune Suppression in Spleen by Dietary Polyunsaturated Fatty Acids.

Dietary polyunsaturated fatty acids (PUFA) are suggested to modulate immune function, but the effects of dietary fatty acids composition on gene expression patterns in immune organs have not been fully characterized. In the current study we investigated how dietary fatty acids composition affects the total transcriptome profile, and especially, immune related genes in two immune organs, spleen (SPL) and bone marrow cells (BMC). Four tissues with metabolic function, skeletal muscle (SKM), white adipose tissue (WAT), brown adipose tissue (BAT), and liver (LIV), were investigated as a comparison. Following 8 weeks on low fat diet (LFD), high fat diet (HFD) rich in saturated fatty acids (HFD-S), or HFD rich in PUFA (HFD-P), tissue transcriptomics were analyzed by microarray and metabolic health assessed by fasting blood glucose level, HOMA-IR index, oral glucose tolerance test as well as quantification of crown-like structures in WAT. HFD-P corrected the metabolic phenotype induced by HFD-S. Interestingly, SKM and BMC were relatively inert to the diets, whereas the two adipose tissues (WAT and BAT) were mainly affected by HFD per se (both HFD-S and HFD-P). In particular, WAT gene expression was driven closer to that of the immune organs SPL and BMC by HFDs. The LIV exhibited different responses to both of the HFDs. Surprisingly, the spleen showed a major response to HFD-P (82 genes differed from LFD, mostly immune genes), while it was not affected at all by HFD-S (0 genes differed from LFD). In conclusion, the quantity and composition of dietary fatty acids affected the transcriptome in distinct manners in different organs. Remarkably, dietary PUFA, but not saturated fat, prompted a specific regulation of immune related genes in the spleen, opening the possibility that PUFA can regulate immune function by influencing gene expression in this organ.

Dietary polyunsaturated fatty acids increase survival and decrease bacterial load during septic Staphylococcus aureus infection and improve neutrophil function in mice.

Severe infection, including sepsis, is an increasing clinical problem that causes prolonged morbidity and substantial mortality. At present, antibiotics are essentially the only pharmacological treatment for sepsis. The incidence of resistance to antibiotics is increasing; therefore, it is critical to find new therapies for sepsis. Staphylococcus aureus is a major cause of septic mortality. Neutrophils play an important role in the defense against bacterial infections. We have shown that a diet with high levels of dietary saturated fatty acids decreases survival in septic mice, but the mechanisms behind this remain elusive. The aim of the present study was to investigate how the differences in dietary fat composition affect survival and bacterial load after experimental septic infection and neutrophil function in uninfected mice. We found that, after S. aureus infection, mice fed a polyunsaturated high-fat diet (HFD-P) for 8 weeks had increased survival and decreased bacterial load during sepsis compared with mice fed a saturated high-fat diet (HFD-S), similar to mice fed a low-fat diet (LFD). Uninfected mice fed HFD-P had a higher frequency of neutrophils in bone marrow than mice fed HFD-S. In addition, mice fed HFD-P had a higher frequency of neutrophils recruited to the site of inflammation in response to peritoneal injection of thioglycolate than mice fed HFD-S. Differences between the proportion of dietary protein and carbohydrate did not affect septic survival at all. In conclusion, polyunsaturated dietary fat increased both survival and efficiency of bacterial clearance during septic S. aureus infection. Moreover, this diet increased the frequency and chemotaxis of neutrophils, key components of the immune response to S. aureus infections.

α7 Nicotinic acetylcholine receptor is expressed in human atherosclerosis and inhibits disease in mice--brief report.

OBJECTIVE: Cholinergic pathways of the autonomic nervous system are known to modulate inflammation. Because atherosclerosis is a chronic inflammatory condition, we tested whether cholinergic signaling operates in this disease. We have analyzed the expression of the α7 nicotinic acetylcholine receptor (α7nAChR) in human atherosclerotic plaques and studied its effects on the development of atherosclerosis in the hypercholesterolemic Ldlr(-/-) mouse model. APPROACH AND RESULTS: α7nAChR protein was detected on T cells and macrophages in surgical specimens of human atherosclerotic plaques. To study the role of α7nAChR signaling in atherosclerosis, male Ldlr(-/-) mice were lethally irradiated and reconstituted with bone marrow from wild-type or α7nAChR-deficient animals. Ablation of hematopoietic cell α7nAChR increased aortic atherosclerosis by 72%. This was accompanied by increased aortic interferon-γ mRNA, implying increased Th1 activity in the absence of α7nAChR signaling. CONCLUSIONS: The present study shows that signaling through hematopoietic α7nAChR inhibits atherosclerosis and suggests that it operates by modulating immune inflammation. Given the observation that α7nAChR is expressed by T cells and macrophages in human plaques, our findings support the notion that cholinergic regulation may act to inhibit disease development also in man.

Exendin-4 reduces ischemic brain injury in normal and aged type 2 diabetic mice and promotes microglial M2 polarization.

Exendin-4 is a glucagon-like receptor 1 agonist clinically used against type 2 diabetes that has also shown neuroprotective effects in experimental stroke models. However, while the neuroprotective efficacy of Exendin-4 has been thoroughly investigated if the pharmacological treatment starts before stroke, the therapeutic potential of the Exendin-4 if the treatment starts acutely after stroke has not been clearly determined. Further, a comparison of the neuroprotective efficacy in normal and aged diabetic mice has not been performed. Finally, the cellular mechanisms behind the efficacy of Exendin-4 have been only partially studied. The main objective of this study was to determine the neuroprotective efficacy of Exendin-4 in normal and aged type 2 diabetic mice if the treatment started after stroke in a clinically relevant setting. Furthermore we characterized the Exendin-4 effects on stroke-induced neuroinflammation. Two-month-old healthy and 14-month-old type 2 diabetic/obese mice were subjected to middle cerebral artery occlusion. 5 or 50 µg/kg Exendin-4 was administered intraperitoneally at 1.5, 3 or 4.5 hours thereafter. The treatment was continued (0.2 µg/kg/day) for 1 week. The neuroprotective efficacy was assessed by stroke volume measurement and stereological counting of NeuN-positive neurons. Neuroinflammation was determined by gene expression analysis of M1/M2 microglia subtypes and pro-inflammatory cytokines. We show neuroprotective efficacy of 50 µg/kg Exendin-4 at 1.5 and 3 hours after stroke in both young healthy and aged diabetic/obese mice. The 5 µg/kg dose was neuroprotective at 1.5 hour only. Proinflammatory markers and M1 phenotype were not impacted by Exendin-4 treatment while M2 markers were significantly up regulated. Our results support the use of Exendin-4 to reduce stroke-damage in the prehospital/early hospitalization setting irrespectively of age/diabetes. The results indicate the polarization of microglia/macrophages towards the M2 reparative phenotype as a potential mechanism of neuroprotection.

Metoprolol reduces proinflammatory cytokines and atherosclerosis in ApoE-/- mice.

A few studies in animals and humans suggest that metoprolol (ß1-selective adrenoceptor antagonist) may have a direct antiatherosclerotic effect. However, the mechanism behind this protective effect has not been established. The aim of the present study was to evaluate the effect of metoprolol on development of atherosclerosis in ApoE(-/-) mice and investigate its effect on the release of proinflammatory cytokines. Male ApoE(-/-) mice were treated with metoprolol (2.5 mg/kg/h) or saline for 11 weeks via osmotic minipumps. Atherosclerosis was assessed in thoracic aorta and aortic root. Total cholesterol levels and Th1/Th2 cytokines were analyzed in serum and macrophage content in lesions by immunohistochemistry. Metoprolol significantly reduced atherosclerotic plaque area in thoracic aorta (P < 0.05 versus Control). Further, metoprolol reduced serum TNFα and the chemokine CXCL1 (P < 0.01 versus Control for both) as well as decreasing the macrophage content in the plaques (P < 0.01 versus Control). Total cholesterol levels were not affected. In this study we found that a moderate dose of metoprolol significantly reduced atherosclerotic plaque area in thoracic aorta of ApoE(-/-) mice. Metoprolol also decreased serum levels of proinflammatory cytokines TNFα and CXCL1 and macrophage content in the plaques, showing that metoprolol has an anti-inflammatory effect.

Toll-like receptor-3 activation increases the vulnerability of the neonatal brain to hypoxia-ischemia.

Susceptibility and progression of brain injury in the newborn is closely associated with an exacerbated innate immune response, but the underlying mechanisms are often unclear. Toll-like receptors (TLRs) are important innate immune sensors that may influence the vulnerability of the developing brain. In the current study, we provide novel data to show that activation of the viral innate immune receptor TLR-3 sensitizes the neonatal brain to subsequent hypoxic-ischemic (HI) damage. Poly inosinic:poly cytidylic acid (Poly I:C), a synthetic ligand for TLR-3, was administered to neonatal mice 14 h before cerebral HI. Activation of TLR-3 before HI increased infarct volume from 3.0 ± 0.5 to 15.4 ± 2.1 mm³ and augmented loss of myelin basic protein from 13.4 ± 6.0 to 70.6 ± 5.3%. The sensitizing effect of Poly I:C was specific for the TLR-3 pathway because mice deficient in the TLR-3 adaptor protein Toll/IL-1R domain-containing adaptor molecule-1 (TRIF) did not develop larger brain damage. The increased vulnerability was associated with a TRIF-dependent heightened inflammatory response, including proinflammatory cytokines, chemokines, and the apoptosis-associated mediator Fas, whereas there was a decrease in reparative M2-like CD11b⁺ microglia and phosphorylation of Akt. Because TLR-3 is activated via double-stranded RNA during most viral infections, the present study provides evidence that viral infections during pregnancy or in the neonate could have great impact on subsequent HI brain injury.

Toll-like receptor 3 and 4 signalling through the TRIF and TRAM adaptors in haematopoietic cells promotes atherosclerosis.

AIMS: Members of the Toll-like receptor (TLR) family initiate innate immune responses and were recently shown to play a role in atherosclerosis. However, the mechanisms that link TLR ligation to vascular inflammation and atherogenesis remain unclear. To identify which signalling pathways downstream of TLRs in immune cells are pro-atherogenic, we analysed the role of the TLR-specific adaptors MyD88 adaptor-like (MAL), TRIF-related adaptor molecule (TRAM), and TIR-domain-containing adaptor-inducing interferon-ß (TRIF) in atherosclerosis. METHODS AND RESULTS: Using a bone-marrow transplantation strategy into low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice, we could specifically study the absence of the TLR adaptors in immune cells. We showed that haematopoietic deficiency of TRAM and TRIF, but not MAL, reduces atherosclerosis without affecting cholesterol metabolism. This was mediated by decreased aortic inflammation, indicated by lower aortic levels of pro-inflammatory mediators, and reduced influx of macrophages and T cells. Furthermore, by studying Tlr3(-/-) chimeric Ldlr(-/-) mice, we found that deleting TLR3 in immune cells significantly reduced both aortic inflammation and atherosclerotic burden. CONCLUSIONS: By studying hypercholesterolaemic mice with defects in TLR-signalling adaptors, we demonstrated that deleting either TRAM or TRIF in immune cells is sufficient to attenuate vessel inflammation and protect against atherosclerosis. In addition, these adaptors elicit partly different sets of inflammatory mediators and can independently inhibit the disease process. Furthermore, we identify TLR3 as a pro-atherogenic receptor in haematopoietic immune cells. The identification of these pro-atherogenic pathways downstream of TLR3 and TLR4 contributes to a better understanding of TLRs and their signalling pathways in the pathogenesis of atherosclerosis.

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

INTRODUCTION: We 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. METHODS: Male 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. RESULTS: We 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. CONCLUSIONS: These 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.

Identification of a danger-associated peptide from apolipoprotein B100 (ApoBDS-1) that triggers innate proatherogenic responses.

BACKGROUND: Subendothelial deposited low-density lipoprotein particles are a known inflammatory factor in atherosclerosis. However, the causal components derived from low-density lipoprotein are still poorly defined. Apolipoprotein B100 (ApoB100) is the unexchangeable protein component of low-density lipoprotein, and the progression of atherosclerosis is associated with immune responses to ApoB100-derived peptides. In this study, we analyzed the proinflammatory activity of ApoB100 peptides in atherosclerosis. METHODS AND RESULTS: By screening a peptide library of ApoB100, we identified a distinct native peptide referred to as ApoB100 danger-associated signal 1 (ApoBDS-1), which shows sequence-specific bioactivity in stimulation of interleukin-8, CCL2, and interleukin-6. ApoBDS-1 activates mitogen-activated protein kinase and calcium signaling, thereby effecting the expression of interleukin-8 in innate immune cells. Ex vivo stimulation of carotid plaques with ApoBDS-1 enhances interleukin-8 and prostaglandin E2 release. Furthermore, we demonstrated that ApoBDS-1-positive peptide fragments are present in atherosclerotic lesions using immunoassays and that low-molecular-weight fractions isolated from plaque show ApoBDS-1 activity inducing interleukin-8 production. CONCLUSIONS: Our data show that ApoBDS-1 is a previously unrecognized peptide with robust proinflammatory activity, contributing to the disease-promoting effects of low-density lipoprotein in the pathogenesis of atherosclerosis.

Rip2 deficiency leads to increased atherosclerosis despite decreased inflammation.

RATIONALE: The innate immune system and in particular the pattern-recognition receptors Toll-like receptors have recently been linked to atherosclerosis. Consequently, inhibition of various signaling molecules downstream of the Toll-like receptors has been tested as a strategy to prevent progression of atherosclerosis. Receptor-interacting protein 2 (Rip2) is a serine/threonine kinase that is involved in multiple nuclear factor-κB (NFκB) activation pathways, including Toll-like receptors, and is therefore an interesting potential target for pharmaceutical intervention. OBJECTIVE: We hypothesized that inhibition of Rip2 would protect against development of atherosclerosis. METHODS AND RESULTS: Surprisingly, and contrary to our hypothesis, we found that mice transplanted with Rip2(-/-) bone marrow displayed markedly increased atherosclerotic lesions despite impaired local and systemic inflammation. Moreover, lipid uptake was increased whereas immune signaling was reduced in Rip2(-/-) macrophages. Further analysis in Rip2(-/-) macrophages showed that the lipid accumulation was scavenger-receptor independent and mediated by Toll-like receptor 4 (TLR4)-dependent lipid uptake. CONCLUSIONS: Our data show that lipid accumulation and inflammation are dissociated in the vessel wall in mice with Rip2(-/-) macrophages. These results for the first time identify Rip2 as a key regulator of cellular lipid metabolism and cardiovascular disease.

Nuclear factor {kappa}B-mediated transactivation of telomerase prevents intimal smooth muscle cell from replicative senescence during vascular repair.

OBJECTIVE: To gain insights into mechanisms by which intimal hyperplasia interferes with the repair process by investigating expression and function of the catalytic telomerase reverse transcriptase (TERT) subunit after vascular injury. METHODS AND RESULTS: Functional telomerase is essential to the replicative longevity of vascular cells. We found that TERT was de novo activated in the intima of injured arteries, involving activation of the nuclear factor κB pathway. Stimulation of the isolated intimal smooth muscle cell (SMC) by basic fibroblast growth factor or tumor necrosis factor α resulted in increased TERT activity. This depends on the activation of c-Myc signaling because mutation of the E-box in the promoter or overexpression of mitotic arrest deficient 1 (MAD1), a c-Myc competitor, abrogated the transcriptional activity. Inhibition of nuclear factor κB in both intimal SMCs and the injured artery attenuated TERT transcriptional activity through reduction of c-Myc expression. Pharmacological blockade of TERT led to SMC senescence. Finally, depletion of telomerase function in mice resulted in severe intimal SMC senescence after vascular injury. CONCLUSIONS: These results support a model in which vascular injury induces de novo expression of TERT in intimal SMCs via activation of nuclear factor κB and upregulation of c-Myc. The resumed TERT activity is critical for intimal hyperplasia.

T cell-mediated inflammation in adipose tissue does not cause insulin resistance in hyperlipidemic mice.

Obesity is associated with chronic inflammation in adipose tissue. Proinflammatory cytokines including tumor necrosis factor-alpha and interleukin-6 secreted by adipose tissue during the metabolic syndrome are proposed to cause local and general insulin resistance and promote development of type 2 diabetes. We have used a compound mutant mouse, Apoe(-/-)xCD4dnTGFbR, with dysregulation of T-cell activation, excessive production of proinflammatory cytokines, hyperlipidemia, and atherosclerosis, to dissect the role of inflammation in adipose tissue metabolism. These mice are lean, which avoids confounding effects of concomitant obesity. Expression and secretion of a set of proinflammatory factors including tumor necrosis factor-alpha, interferon-gamma, and monocyte chemoattractant protein-1 was increased in adipose tissue of Apoe(-/-)xCD4dnTGFbR mice, as was the enzyme 11beta-hydroxysteroid dehydrogenase type 1, which converts cortisone to bioactive cortisol. Interleukin-6, which has an inhibitory glucocorticoid response element in its promoter, was not upregulated. In spite of intense local inflammation, insulin sensitivity was not impaired in adipose tissue of Apoe(-/-)xCD4dnTGFbR mice unless exogenous interleukin-6 was administered. In conclusion, T-cell activation causes inflammation in adipose tissue but does not lead to insulin resistance in this tissue in the absence of interleukin-6.

Blood pressure is the major driving force for plaque formation in aortic-constricted ApoE-/- mice.

OBJECTIVE: Using an aortic constriction model in mice, we studied whether the increase in pressure or the activation of the renin-angiotensin system (RAS) and its main receptors is the main driving force for plaque progression. METHODS: Male ApoE mice underwent sham surgery or placement of a suprarenal silver clip around the aorta (AoC). Half the group was treated with the selective AT1 receptor antagonist losartan (30 mg/kg per day) for 4 weeks. RESULTS: Anesthetized mean arterial pressure (MAP) was increased in AoC mice compared to sham (106 +/- 3 versus 90 +/- 1 mmHg, P < 0.001). Losartan reduced MAP in sham mice (78 +/- 2 mmHg, P < 0.01) but not in AoC (AoC losartan 104 +/- 2 mmHg). Plasma renin concentration (PRC) was increased in AoC mice compared to sham [1.6 +/- 0.3 versus 0.8 +/- 0.2 milliGoldblatt units (mGU)/ml, P < 0.001]. Losartan treatment augmented this difference (18.7 +/- 3.7 versus 4.6 +/- 1.7 mGU/ml, P < 0.01). AT2 receptor mRNA expression was increased 5.8-fold by aortic constriction in thoracic aorta (P < 0.05) and the major site for expression of the AT2 receptor protein was within the plaques. The plaque area was increased in AoC mice compared to sham (0.61 +/- 0.09 versus 0.07 +/- 0.01%, P < 0.001); however, losartan did not alter plaque area. CONCLUSIONS: Our data do not support a role for the AT1 receptor in the progression of atherosclerosis in this model, since blockade with losartan did not alter plaque distribution. Furthermore, we found no support for the counteraction of atherogenesis by increased activity of the RAS acting on the AT2 receptor. Our data suggest that increased pressure is the main driving force for atherosclerosis in this model.