Endothelial Pannexin 1 Channels Control Inflammation by Regulating Intracellular Calcium.

The proinflammatory cytokine IL-1ß is a significant risk factor in cardiovascular disease that can be targeted to reduce major cardiovascular events. IL-1ß expression and release are tightly controlled by changes in intracellular Ca2+ ([Ca2+]i), which has been associated with ATP release and purinergic signaling. Despite this, the mechanisms that regulate these changes have not been identified. The pannexin 1 (Panx1) channels have canonically been implicated in ATP release, especially during inflammation. We examined Panx1 in human umbilical vein endothelial cells following treatment with the proinflammatory cytokine TNF-α. Analysis by whole transcriptome sequencing and immunoblot identified a dramatic increase in Panx1 mRNA and protein expression that is regulated in an NF-κB-dependent manner. Furthermore, genetic inhibition of Panx1 reduced the expression and release of IL-1ß. We initially hypothesized that increased Panx1-mediated ATP release acted in a paracrine fashion to control cytokine expression. However, our data demonstrate that IL-1ß expression was not altered after direct ATP stimulation in human umbilical vein endothelial cells. Because Panx1 forms a large pore channel, we hypothesized it may permit Ca2+ diffusion into the cell to regulate IL-1ß. High-throughput flow cytometric analysis demonstrated that TNF-α treatments lead to elevated [Ca2+]i, corresponding with Panx1 membrane localization. Genetic or pharmacological inhibition of Panx1 reduced TNF-α-associated increases in [Ca2+]i, blocked phosphorylation of the NF-κB-p65 protein, and reduced IL-1ß transcription. Taken together, the data in our study provide the first evidence, to our knowledge, that [Ca2+]i regulation via the Panx1 channel induces a feed-forward effect on NF-κB to regulate IL-1ß synthesis and release in endothelium during inflammation.

Magnetic resonance enterography for predicting the clinical course of Crohn's disease strictures.

BACKGROUND AND AIMS: Strictures are the most common Crohn's disease complication, but their natural history is unknown. This study aimed to characterize inflammation, predict prognosis, and understand the impact of drug therapy using magnetic resonance enterography (MRE). METHODS: Patients with a stricture diagnosed on MRE over a 5-year period were reviewed for MRE disease extent and inflammation, clinical course, C-reactive protein, response to anti-TNF therapy, endoscopic dilatation, hospitalization, and surgery. RESULTS: 136 patients had 235 strictures (77, one and 59, ≥ 2 strictures). TREATMENT: 46% of patients underwent surgery after a median 6 months; median follow-up for those not requiring surgery was 41 months. Predictors of surgery: Hospitalization because of obstruction predicted subsequent surgery (OR 2.50; 95% CI 1.06-5.90) while anti-TNF therapy commenced at stricture diagnosis was associated with a reduced risk (OR 0.23; 95% CI 0.05-0.99). MRE characteristics associated with surgery were proximal bowel dilatation ≥ 30-mm diameter (OR 2.98; 95% CI 1.36-6.55), stricture bowel wall thickness ≥ 10-mm (OR 2.42; 95% CI 1.11-5.27), and stricture length > 5-cm (OR 2.56; 95% CI 1.21-5.43). 81% of patients with these three adverse MRE features required surgery versus 17% if none were present (P < 0.001). Accuracy for these three MRE variables predicting surgery was high (AUC 0.76). CONCLUSION: Magnetic resonance enterography findings in Crohn's disease strictures are highly predictive of the disease course and the need for future surgery. MRE may also identify who would benefit from treatment intensification. Anti-TNF therapy is associated with reduced risk of surgery and appears to alter the natural history of this complication.

The role of microRNA-155/liver X receptor pathway in experimental and idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is progressive and rapidly fatal. Improved understanding of pathogenesis is required to prosper novel therapeutics. Epigenetic changes contribute to IPF; therefore, microRNAs may reveal novel pathogenic pathways. OBJECTIVES: We sought to determine the regulatory role of microRNA (miR)-155 in the profibrotic function of murine lung macrophages and fibroblasts, IPF lung fibroblasts, and its contribution to experimental pulmonary fibrosis. METHODS: Bleomycin-induced lung fibrosis in wild-type and miR-155-/- mice was analyzed by histology, collagen, and profibrotic gene expression. Mechanisms were identified by in silico and molecular approaches and validated in mouse lung fibroblasts and macrophages, and in IPF lung fibroblasts, using loss-and-gain of function assays, and in vivo using specific inhibitors. RESULTS: miR-155-/- mice developed exacerbated lung fibrosis, increased collagen deposition, collagen 1 and 3 mRNA expression, TGF-ß production, and activation of alternatively activated macrophages, contributed by deregulation of the miR-155 target gene the liver X receptor (LXR)α in lung fibroblasts and macrophages. Inhibition of LXRα in experimental lung fibrosis and in IPF lung fibroblasts reduced the exacerbated fibrotic response. Similarly, enforced expression of miR-155 reduced the profibrotic phenotype of IPF and miR-155-/- fibroblasts. CONCLUSIONS: We describe herein a molecular pathway comprising miR-155 and its epigenetic LXRα target that when deregulated enables pathogenic pulmonary fibrosis. Manipulation of the miR-155/LXR pathway may have therapeutic potential for IPF.

Distinct Phosphorylation Clusters Determine the Signaling Outcome of Free Fatty Acid Receptor 4/G Protein-Coupled Receptor 120.

It is established that long-chain free fatty acids includingω-3 fatty acids mediate an array of biologic responses through members of the free fatty acid (FFA) receptor family, which includes FFA4. However, the signaling mechanisms and modes of regulation of this receptor class remain unclear. Here, we employed mass spectrometry to determine that phosphorylation of mouse (m)FFAR4 occurs at five serine and threonine residues clustered in two separable regions of the C-terminal tail, designated cluster 1 (Thr(347), Thr(349), and Ser(350)) and cluster 2 (Ser(357)and Ser(361)). Mutation of these phosphoacceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation. Rather, an inhibitor of Gq/11proteins completely prevented receptor signaling to ERK1/2. By contrast, the recruitment of arrestin 3, receptor internalization, and activation of Akt were regulated by mFFA4 phosphorylation. The analysis of mFFA4 phosphorylation-dependent signaling was extended further by selective mutations of the phosphoacceptor sites. Mutations within cluster 2 did not affect agonist activation of Akt but instead significantly compromised receptor internalization and arrestin 3 recruitment. Distinctly, mutation of the phosphoacceptor sites within cluster 1 had no effect on receptor internalization and had a less extensive effect on arrestin 3 recruitment but significantly uncoupled the receptor from Akt activation. These unique observations define differential effects on signaling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signaling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode) at the C terminus of the receptor.

Soluble ST2 is regulated by p75 neurotrophin receptor and predicts mortality in diabetic patients with critical limb ischemia.

OBJECTIVE: The p75 neurotrophin receptor (p75(NTR)) contributes to diabetes mellitus-induced defective postischemic neovascularization. The interleukin-33 receptor ST2 is expressed as transmembrane (ST2L) and soluble (sST2) isoforms. Here, we studied the following: (1) the impact of p75(NTR) in the healing of ischemic and diabetic calf wounds; (2) the link between p75(NTR) and ST2; and (3) circulating sST2 levels in critical limb ischemia (CLI) patients. METHODS AND RESULTS: Diabetes mellitus was induced in p75(NTR) knockout (p75KO) mice and wild-type (WT) littermates by streptozotocin. Diabetic and nondiabetic p75KO and WT mice received left limb ischemia induction and a full-thickness wound on the ipsilateral calf. Diabetes mellitus impaired wound closure and angiogenesis and increased ST2 expression in WT, but not in p75KO wounds. In cultured endothelial cells, p75(NTR) promoted ST2 (both isoforms) expression through p38(MAPK)/activating transcription factor 2 pathway activation. Next, sST2 was measured in the serum of patients with CLI undergoing either revascularization or limb amputation and in the 2 nondiabetic groups (with CLI or nonischemic individuals). Serum sST2 increased in diabetic patients with CLI and was directly associated with higher mortality at 1 year from revascularization. CONCLUSIONS: p75(NTR) inhibits the healing of ischemic lower limb wounds in diabetes mellitus and promotes ST2 expression. Circulating sST2 predicts mortality in diabetic CLI patients.

Impaired renal function impacts negatively on vascular stiffness in patients with coronary artery disease.

BACKGROUND: Chronic kidney disease (CKD) and coronary artery disease (CAD) are independently associated with increased vascular stiffness. We examined whether renal function contributes to vascular stiffness independently of CAD status. METHODS: We studied 160 patients with CAD and 169 subjects without CAD. The 4-variable MDRD formula was used to estimate glomerular filtration rate (eGFR); impaired renal function was defined as eGFR <60 mL/min. Carotid-femoral pulse wave velocity (PWV) was measured with the SphygmoCor® device. Circulating biomarkers were assessed in plasma using xMAP® multiplexing technology. RESULTS: Patients with CAD and impaired renal function had greater PWV compared to those with CAD and normal renal function (10.2 [9.1;11.2] vs 7.3 [6.9;7.7] m/s; P < 0.001). In all patients, PWV was a function of eGFR (ß = -0.293; P < 0.001) even after adjustment for age, sex, systolic blood pressure, body mass index and presence or absence of CAD. Patients with CAD and impaired renal function had higher levels of adhesion and inflammatory molecules including E-selectin and osteopontin (all P < 0.05) compared to those with CAD alone, but had similar levels of markers of oxidative stress. CONCLUSIONS: Renal function is a determinant of vascular stiffness even in patients with severe atherosclerotic disease. This was paralleled by differences in markers of cell adhesion and inflammation. Increased vascular stiffness may therefore be linked to inflammatory remodeling of the vasculature in people with impaired renal function, irrespective of concomitant atherosclerotic disease.

COVID-19 booster vaccination during pregnancy enhances maternal binding and neutralizing antibody responses and transplacental antibody transfer to the newborn.

The immune response to COVID-19 booster vaccinations during pregnancy for mothers and their newborns and the functional response of vaccine-induced antibodies against Omicron variants are not well characterized. We conducted a prospective, multicenter cohort study of participants vaccinated during pregnancy with primary or booster mRNA COVID-19 vaccines from July 2021 to January 2022 at 9 academic sites. We determined SARS-CoV-2 binding and live virus and pseudovirus neutralizing antibody (nAb) titers pre- and post-vaccination, and at delivery for both maternal and infant participants. Immune responses to ancestral and Omicron BA.1 SARS-CoV-2 strains were compared between primary and booster vaccine recipients in maternal sera at delivery and in cord blood, after adjusting for days since last vaccination. A total of 240 participants received either Pfizer or Moderna mRNA vaccine during pregnancy (primary 2-dose series: 167; booster dose: 73). Booster vaccination resulted in significantly higher binding and nAb titers, including to the Omicron BA.1 variant, in maternal serum at delivery and in cord blood compared to a primary 2-dose series (range 0.44-0.88 log10 higher, p < 0.0001 for all comparisons). Live virus nAb to Omicron BA.1 were present at delivery in 9 % (GMT ID50 12.7) of Pfizer and 22 % (GMT ID50 14.7) of Moderna primary series recipients, and in 73 % (GMT ID50 60.2) of mRNA boosted participants (p < 0.0001), although titers were significantly lower than to the D614G strain. Transplacental antibody transfer was efficient for all regimens with median transfer ratio range: 1.55-1.77 for IgG, 1.00-1.78 for live virus nAb and 1.79-2.36 for pseudovirus nAb. COVID-19 mRNA vaccination during pregnancy elicited robust immune responses in mothers and efficient transplacental antibody transfer to the newborn. A booster dose during pregnancy significantly increased maternal and cord blood binding and neutralizing antibody levels, including against Omicron BA.1. Findings support the use of a booster dose of COVID-19 vaccine during pregnancy.

IL-33 induces skin inflammation with mast cell and neutrophil activation.

Psoriasis is a common chronic autoimmune condition of the skin characterized by hyperplasia of epidermal keratinocytes associated with pro-inflammatory cytokines. IL-33 is a new member of the IL-1 superfamily that signals through the ST2 receptor and was originally defined as an inducer of T helper 2 (Th2) cytokines. Recently, broader immune activatory potential has been defined for IL-33 particularly via mast cell activation and neutrophil migration. Here, we show that ST2(-/-) mice exhibit reduced cutaneous inflammatory responses compared with WT mice in a phorbol ester-induced model of skin inflammation. Furthermore, injections of IL-33 into the ears of mice induce an inflammatory skin lesion. This inflammatory response was partially dependent on mast cells as mast cell-deficient mice (Kit(W-sh/W-sh) ) showed delayed responses to IL-33. IL-33 also recruited neutrophils to the ear, an effect mediated in part by increased production of the chemokine KC (CXCL1). Finally, we show that IL-33 expression is up-regulated in the epidermis of clinical psoriatic lesions, compared with healthy skin. These results therefore demonstrate that IL-33 may play a role in psoriasis-like plaque inflammation. IL-33 targeting may provide a new treatment strategy for psoriasis.

Biodistribution and retargeting of FX-binding ablated adenovirus serotype 5 vectors.

A major limitation for adenoviral transduction in vivo is the profound liver tropism of adenovirus type 5 (Ad5). Recently, we demonstrated that coagulation factor X (FX) binds to Ad5-hexon protein at high affinity to mediate hepatocyte transduction after intravascular delivery. We developed novel genetically FX-binding ablated Ad5 vectors with lower liver transduction. Here, we demonstrate that FX-binding ablated Ad5 predominantly localize to the liver and spleen 1 hour after injection; however, they had highly reduced liver transduction in both control and macrophage-depleted mice compared with Ad5. At high doses in macrophage-depleted mice, FX-binding ablated vectors transduced the spleen more efficiently than Ad5. Immunohistochemical studies demonstrated transgene colocalization with CD11c(+), ER-TR7(+), and MAdCAM-1(+) cells in the splenic marginal zone. Systemic inflammatory profiles were broadly similar between FX-binding ablated Ad5 and Ad5 at low and intermediate doses, although higher levels of several inflammatory proteins were observed at the highest dose of FX-binding ablated Ad5. Subsequently, we generated a FX-binding ablated virus containing a high affinity Ad35 fiber that mediated a significant improvement in lung/liver ratio in macrophage-depleted CD46(+) mice compared with controls. Therefore, this study documents the biodistribution and reports the retargeting capacity of FX binding-ablated Ad5 vectors in vitro and in vivo.

Interleukin-21--a biomarker of importance in predicting myocardial function following acute infarction?

INTRODUCTION: Following acute myocardial infarction (AMI), the acute inflammatory response contributes to wound healing but also to progressive myocardial injury. Interleukin-21 (IL-21) plays a key role in immunoregulation; whether IL-21 is associated with left ventricular (LV) remodelling after AMI is unknown. METHODS: Plasma IL-21 concentrations were measured in 100 patients (age 58.9 ± 12.0 years, 77% male) admitted with AMI and LV dysfunction, at baseline (mean 46 h) and again at 24 weeks; cardiac magnetic resonance and measurement of B-type natriuretic peptide, monocyte chemoattractant protein-1, matrix metalloproteinase (MMP)-2, -3, -9, and tissue inhibitor of metalloproteinase (TIMP)-1, -2, -4 occurred at both time-points. Remodelling was defined as change in LV end-systolic volume index (ΔLVESVI). RESULTS: Plasma IL-21 concentration was unchanged over time (48.1 [SD 35.4]pg/mL at baseline vs. 48.8 [61.3]pg/mL at 24 weeks, p=0.92). Baseline IL-21 correlated significantly with ΔLVESVI (r=0.30, p=0.005) and change in LV end-diastolic volume index (r=0.33, p=0.003). On multivariate analysis, plasma IL-21 was an independent predictor of remodelling. IL-21 was also significantly associated with higher TIMP-4 concentrations and lower MMP-9 concentrations at baseline. CONCLUSIONS: IL-21 predicts adverse remodelling following AMI in patients with LV dysfunction. Whether it plays a direct pathophysiological role in remodelling merits further study.

Interleukin-33 induces protective effects in adipose tissue inflammation during obesity in mice.

RATIONALE: Chronic low-grade inflammation involving adipose tissue likely contributes to the metabolic consequences of obesity. The cytokine interleukin (IL)-33 and its receptor ST2 are expressed in adipose tissue, but their role in adipose tissue inflammation during obesity is unclear. OBJECTIVE: To examine the functional role of IL-33 in adipose tissues and investigate the effects on adipose tissue inflammation and obesity in vivo. METHODS AND RESULTS: We demonstrate that treatment of adipose tissue cultures in vitro with IL-33 induced production of Th2 cytokines (IL-5, IL-13, IL-10) and reduced expression of adipogenic and metabolic genes. Administration of recombinant IL-33 to genetically obese diabetic (ob/ob) mice led to reduced adiposity, reduced fasting glucose and improved glucose and insulin tolerance. IL-33 also induced accumulation of Th2 cells in adipose tissue and polarization of adipose tissue macrophages toward an M2 alternatively activated phenotype (CD206(+)), a lineage associated with protection against obesity-related metabolic events. Furthermore, mice lacking endogenous ST2 fed high-fat diet had increased body weight and fat mass and impaired insulin secretion and glucose regulation compared to WT controls fed high-fat diet. CONCLUSIONS: In conclusion, IL-33 may play a protective role in the development of adipose tissue inflammation during obesity.

Mast cells express IL-17A in rheumatoid arthritis synovium.

The proinflammatory cytokine IL-17A is considered a crucial player in rheumatoid arthritis (RA) pathogenesis. In experimental models of autoimmune arthritis, it has been suggested that the cellular source of IL-17A is CD4(+) T cells (Th17 cells). However, little is known about the source of IL-17 in human inflamed RA tissue. We explored the cellular sources of IL-17A in human RA synovium. Surprisingly, only a small proportion of IL-17-expressing cells were T cells, and these were CCR6 negative. Unexpectedly, the majority of IL-17A expression colocalized within mast cells. Furthermore, we demonstrated in vitro that mast cells produced RORC-dependent IL-17A upon stimulation with TNF-alpha, IgG complexes, C5a, and LPS. These data are consistent with a crucial role for IL-17A in RA pathogenesis but suggest that in addition to T cells innate immune pathways particularly mediated via mast cells may be an important component of the effector IL-17A response.

IL-33 reduces macrophage foam cell formation.

The development of atherosclerosis, a chronic inflammatory disease characterized by the formation of arterial fibrotic plaques, has been shown to be reduced by IL-33 in vivo. However, whether IL-33 can directly affect macrophage foam cell formation, a key feature of atherosclerotic plaques, has not been determined. In this study, we investigated whether IL-33 reduces macrophage foam cell accumulation in vivo and if IL-33 reduces their formation in vitro using THP-1 and primary human monocyte-derived macrophages. In Apolipoprotein E(-/-) mice fed on a high fat diet, IL-33 treatment significantly reduced the accumulation of macrophage-derived foam cells in atherosclerotic plaques. IL-33 also reduced macrophage foam cell formation in vitro by decreasing acetylated and oxidized low-density lipoprotein uptake, reducing intracellular total and esterified cholesterol content and enhancing cholesterol efflux. These changes were associated with IL-33-mediated reduction in the expression of genes involved in modified low-density lipoprotein uptake, such as CD36, and simultaneous increase in genes involved in cholesterol efflux, including Apolipoprotein E, thereby providing a mechanism for such an action for this cytokine. IL-33 also decreased the expression of key genes implicated in cholesterol esterification and triglyceride storage, including Acyl-CoA:cholesterol acyltransferase 1 and Adipocyte differentiation-related protein. Furthermore, using bone marrow-derived macrophages from ST2(-/-) mice, we demonstrate that the IL-33 receptor, ST2, is integral to the action of IL-33 on macrophage foam cell formation. In conclusion, IL-33 has a protective role in atherosclerosis by reducing macrophage foam cell formation suggesting that IL-33 maybe a potential therapeutic agent against atherosclerosis.

Intensive drug therapy versus standard drug therapy for symptomatic intestinal Crohn's disease strictures (STRIDENT): an open-label, single-centre, randomised controlled trial.

BACKGROUND: Strictures are the most common structural complication of Crohn's disease. Surgery and endoscopic balloon dilation are the main treatments; drug therapy has been considered contraindicated. Given that most strictures have an inflammatory component, we aimed to find out whether strictures are responsive to drug treatment and whether intensive drug therapy is more effective than standard drug therapy. METHODS: This open-label, single-centre, randomised controlled trial was performed in one specialist inflammatory bowel disease centre in Australia. Patients aged 18 years or older with Crohn's disease were included. Eligible patients had a de novo or postoperative anastomotic intestinal stricture on MRI or ileocolonoscopy, symptoms consistent with chronic or subacute intestinal obstruction (postprandial abdominal pain in the presence of a confirmed stricture), and evidence of active intestinal inflammation. Patients were randomly assigned (2:1) to receive intensive high-dose adalimumab (160 mg adalimumab once per week for 4 weeks followed by 40 mg every 2 weeks, with escalation of dose at 4 months and 8 months if assessment of disease activity indicated active inflammation) plus thiopurine (initial dose of azathioprine 2·5 mg/kg or mercaptopurine 1·5 mg/kg, with dose adjustment based on thiopurine metabolite testing) or standard adalimumab monotherapy (160 mg at week 0, 80 mg at week 2, then 40 mg every 2 weeks) using stratified fixed block randomisation. Stratification factors were stricture dilation at study baseline colonoscopy and current biologic drug use. The primary endpoint was improvement (decrease) in the 14-day obstructive symptom score at 12 months by one or more points compared with baseline. This trial is registered with ClinicalTrials.gov, NCT03220841, and is completed. FINDINGS: Between Sept 10, 2017, and Sept 6, 2019, 123 patients were screened and 77 randomly assigned to intensive adalimumab plus thiopurine treatment (n=52) or standard adalimumab treatment (n=25). At 12 months, improvement in obstructive symptom score was noted in 41 (79%) of 52 patients in the intensive treatment group and 16 (64%) of 25 in the standard treatment group (odds ratio [OR] 2·10 [95% CI 0·73-6·01]; p=0·17). Treatment failure occurred in five (10%) patients in the intensive treatment group versus seven (28%) in the standard treatment group (OR 0·27 [95% CI 0·08-0·97]; p=0·045); four patients in each group required stricture surgery (0·44 [0·10-1·92]; p=0·27). Crohn's Disease Activity Index was less than 150 in 36 (69%) patients in the intensive treatment group versus 15 (60%) in the standard treatment group (1·50 [0·56-4·05]; p=0·42). MRI at 12 months showed improvement using the stricture MaRIA score (≥25%) in 31 (61%) of 51 versus seven (28%) of 25 patients (3·99 [1·41-11·26]; p=0·0091). MRI complete stricture resolution was seen in ten (20%) versus four (16%) patients (1·28 [0·36 to 4·57]; p=0·70). Intestinal ultrasound at 12 months showed improvement (>25%) in bowel wall thickness in 22 (51%) of 43 versus seven (33%) of 21 patients (2·10 [0·71 to 6·21]; p=0·18). Faecal calprotectin normalised in 32 (62%) versus 11 (44%) patients (2·04 [0·77-5·36]; p=0·15). Normalisation of CRP was seen in 32 (62%) versus 11 (44%) patients (2·04 [0·77-5·36]; p=0·15). Eight (15%) patients in the intensive treatment group and four (16%) in the standard treatment group reported serious adverse events. No deaths occurred during the study. INTERPRETATION: Crohn's disease strictures are responsive to drug treatment. Most patients had improved symptoms and stricture morphology. Treat-to-target therapy intensification resulted in less treatment failure, a reduction in stricture-associated inflammation, and greater improvement in stricture morphology, although these differences were not significantly different from standard therapy. FUNDING: Australian National Health and Medical Research Council, Gastroenterological Society of Australia Ferring IBD Clinician Establishment Award, Australasian Gastro Intestinal Research Foundation, AbbVie, and the Spotlight Foundation.

Simultaneous activation of the liver X receptors (LXRα and LXRß) drives murine collagen-induced arthritis disease pathology.

BACKGROUND: It has previously been shown that dual activation of the Liver X Receptors (LXRα and LXRß) by the agonist, GW3965, enhances pathology in a murine model of collagen-induced arthritis. OBJECTIVE: To determine whether LXRα or LXRß have discrete roles in driving articular inflammation. METHODS: Arthritis was induced in male C57BL/6 wild-type (WT), LXRα-/-, LXRß-/- and LXRα/ß double KO mice by injection with type II collagen and treated with 30 mg/kg of the LXR agonist GW3965 or vehicle control. The mice were monitored for articular inflammation and cartilage degradation by scoring for clinical signs of arthritis and by histological examination of the joints. RESULTS: Administration of 30 mg/kg GW3965 significantly increases the severity of arthritis in WT but not LXRα-/-, LXRß-/- or LXRα/ß KO mice as assessed by an increase in the clinical score, paw thickness and articular histological analysis. CONCLUSION: The proinflammatory effects associated with the administration of GW3965 are mediated specifically through LXRs. The absence of increased disease severity in the LXRα-/- and LXRß-/- GW3965-treated groups shows for the first time that agonism of both LXRα and LXRß is required to drive proinflammatory pathways in vivo.

Animal models of rheumatoid arthritis.

Animal models have been used extensively in studies of rheumatoid arthritis pathogenesis. Despite the inherent limitations of all animal models, several rodent models have significantly progressed our understanding of the fundamental mechanisms underpinning rheumatoid arthritis and contributed to several current major advances in treatment. These models include the induced arthritis models such as collagen-induced arthritis, collagen-antibody-induced arthritis, zymosan-induced arthritis, and the methylated BSA model, and the genetically manipulated or spontaneous arthritis models such as the TNF-alpha-transgenic mouse, K/BxN mouse, and the Skg mouse. Here, we describe these animal models and discuss their advantages and limitations.

miR-34a(-/-) mice are susceptible to diet-induced obesity.

OBJECTIVE: MicroRNA (miR)-34a regulates inflammatory pathways, and increased transcripts have been observed in serum and subcutaneous adipose of subjects who have obesity and type 2 diabetes. Therefore, the role of miR-34a in adipose tissue inflammation and lipid metabolism in murine diet-induced obesity was investigated. METHODS: Wild-type (WT) and miR-34a(-/-) mice were fed chow or high-fat diet (HFD) for 24 weeks. WT and miR-34a(-/-) bone marrow-derived macrophages were cultured in vitro with macrophage colony-stimulating factor (M-CSF). Brown and white preadipocytes were cultured from the stromal vascular fraction (SVF) of intrascapular brown and epididymal white adipose tissue (eWAT), with rosiglitazone. RESULTS: HFD-fed miR-34a(-/-) mice were significantly heavier with a greater increase in eWAT weight than WT. miR-34a(-/-) eWAT had a smaller adipocyte area, which significantly increased with HFD. miR-34a(-/-) eWAT showed basal increases in Cd36, Hmgcr, Lxrα, Pgc1α, and Fasn. miR-34a(-/-) intrascapular brown adipose tissue had basal reductions in c/ebpα and c/ebpß, with in vitro miR-34a(-/-) white adipocytes showing increased lipid content. An F4/80(high) macrophage population was present in HFD miR-34a(-/-) eWAT, with increased IL-10 transcripts and serum IL-5 protein. Finally, miR-34a(-/-) bone marrow-derived macrophages showed an ablated CXCL1 response to tumor necrosis factor-α. CONCLUSIONS: These findings suggest a multifactorial role of miR-34a in controlling susceptibility to obesity, by regulating inflammatory and metabolic pathways.

TNFalpha increases the inflammatory response to vascular balloon injury without accelerating neointimal formation.

There is now clear evidence for a contributory role of inflammatory processes to restenosis following vascular balloon injury and stent implantation. The aim of the present study was to study the effects of TNFalpha, administered locally in vivo immediately following balloon angioplasty, on the leukocyte adhesive response and extent of neointimal formation in a rabbit model of subclavian artery injury. Initial in vitro studies were performed with normal isolated artery rings to assess the vascular adhesive response to TNFalpha or IL-1beta. Pre-incubation with either cytokine prior to addition of (51)Cr-labelled leukocytes enhanced the adhesion of leukocytes to the artery in both a time- and concentration-dependent manner. Although both cytokines induced an increase in the expression of the adhesion molecules ICAM-1 and VCAM-1, only antibodies to ICAM-1 blocked the enhanced adhesion induced by the cytokines. In artery segments retrieved from rabbits that had previously undergone subclavian artery angioplasty either 24 h or 8 days previously, there was an injury-induced increase in adhesion of leukocytes assessed ex vivo. In segments obtained from rabbits that received a 15 min local infusion of TNFalpha (2 ng/min) to the injured artery immediately after the angioplasty procedure, leukocyte adhesion assessed ex vivo was further significantly enhanced. The pro-adhesive effect of TNFalpha was associated with an increased expression of both ICAM-1 and VCAM-1. However, TNFalpha administration did not alter the extent of neointimal formation observed 8 days after injury. These findings suggest that while TNFalpha may play a role following vascular injury, it does not act alone to induce neointimal formation. Thus anti-inflammatory strategies targeted at multiple cytokines may be more appropriate than targeting a single cytokine to reduce the response to vascular injury.

Reducing In-Stent Restenosis: Therapeutic Manipulation of miRNA in Vascular Remodeling and Inflammation.

BACKGROUND: Drug-eluting stents reduce the incidence of in-stent restenosis, but they result in delayed arterial healing and are associated with a chronic inflammatory response and hypersensitivity reactions. Identifying novel interventions to enhance wound healing and reduce the inflammatory response may improve long-term clinical outcomes. Micro-ribonucleic acids (miRNAs) are noncoding small ribonucleic acids that play a prominent role in the initiation and resolution of inflammation after vascular injury. OBJECTIVES: This study sought to identify miRNA regulation and function after implantation of bare-metal and drug-eluting stents. METHODS: Pig, mouse, and in vitro models were used to investigate the role of miRNA in in-stent restenosis. RESULTS: We documented a subset of inflammatory miRNAs activated after stenting in pigs, including the miR-21 stem loop miRNAs. Genetic ablation of the miR-21 stem loop attenuated neointimal formation in mice post-stenting. This occurred via enhanced levels of anti-inflammatory M2 macrophages coupled with an impaired sensitivity of smooth muscle cells to respond to vascular activation. CONCLUSIONS: MiR-21 plays a prominent role in promoting vascular inflammation and remodeling after stent injury. MiRNA-mediated modulation of the inflammatory response post-stenting may have therapeutic potential to accelerate wound healing and enhance the clinical efficacy of stenting.

NCX4016 (NO-aspirin) reduces infarct size and suppresses arrhythmias following myocardial ischaemia/reperfusion in pigs.

1. The effect of the nitro-derivative of aspirin, NCX4016, was assessed on ischaemic ventricular arrhythmias and myocardial infarct size in anaesthetized pigs in comparison to native aspirin. 2. Pigs were given aspirin (10 mg kg(-1); n=6), low dose NCX4016 (18.4 mg kg(-1); n=6) or high dose NCX4016 (60 mg kg(-1); n=7) orally for 5 days prior to coronary occlusion and reperfusion. None of the interventions had any effect on baseline haemodynamics prior to coronary occlusion in comparison to control pigs (n=9). Aspirin and high dose NCX4016 both prevented the generation of thromboxane A(2) from platelets activated ex vivo with A23187 (30 microM), whereas all three interventions markedly attenuated platelet aggregation in response to collagen in whole blood in comparison to controls. 3. None of the drug interventions had any effect on the incidence of ventricular fibrillation (VF) during myocardial ischaemia (100% in all groups). However, 60 mg kg(-1) NCX4016 significantly attenuated the total number of premature ventricular beats (PVB's) (62+/-16 vs 273+/-40 in control pigs; P<0.05) during the first 30 min of occlusion. The higher dose of NCX4016 also significantly reduced myocardial infarct size (22.6+/-3.7% of area at risk vs 53.0+/-2.8% of area at risk in control pigs; P<0.05). 4. These results suggest that the nitro-derivative of aspirin, NCX4016, is an effective antiplatelet agent, which unlike aspirin also reduces the extent of myocardial injury following ischaemia and reperfusion.