Inhibition of Nuclear Factor of Activated T-Cells (NFAT) Suppresses Accelerated Atherosclerosis in Diabetic Mice

OBJECTIVE OF THE STUDY: Diabetic patients have a much more widespread and aggressive form of atherosclerosis and therefore, higher risk for myocardial infarction, peripheral vascular disease and stroke, but the molecular mechanisms leading to accelerated damage are still unclear. Recently, we showed that hyperglycemia activates the transcription factor NFAT in the arterial wall, inducing the expression of the pro-atherosclerotic protein osteopontin. Here we investigate whether NFAT activation may be a link between diabetes and atherogenesis. METHODOLOGY AND PRINCIPAL FINDINGS: Streptozotocin (STZ)-induced diabetes in apolipoprotein E(-/-) mice resulted in 2.2 fold increased aortic atherosclerosis and enhanced pro-inflammatory burden, as evidenced by elevated blood monocytes, endothelial activation- and inflammatory markers in aorta, and pro-inflammatory cytokines in plasma. In vivo treatment with the NFAT blocker A-285222 for 4 weeks completely inhibited the diabetes-induced aggravation of atherosclerosis, having no effect in non-diabetic mice. STZ-treated mice exhibited hyperglycemia and higher plasma cholesterol and triglycerides, but these were unaffected by A-285222. NFAT-dependent transcriptional activity was examined in aorta, spleen, thymus, brain, heart, liver and kidney, but only augmented in the aorta of diabetic mice. A-285222 completely blocked this diabetes-driven NFAT activation, but had no impact on the other organs or on splenocyte proliferation or cytokine secretion, ruling out systemic immunosuppression as the mechanism behind reduced atherosclerosis. Instead, NFAT inhibition effectively reduced IL-6, osteopontin, monocyte chemotactic protein 1, intercellular adhesion molecule 1, CD68 and tissue factor expression in the arterial wall and lowered plasma IL-6 in diabetic mice. CONCLUSIONS: Targeting NFAT signaling may be a novel and attractive approach for the treatment of diabetic macrovascular complications

Fidelity and moderating factors in complex interventions: a case study of a continuum of care program for frail elderly people in health and social care

<p>Abstract</p> <p>Background</p> <p>Prior studies measuring fidelity of complex interventions have mainly evaluated adherence, and not taken factors affecting adherence into consideration. A need for studies that clarify the concept of fidelity and the function of factors moderating fidelity has been emphasized. The aim of the study was to systematically evaluate implementation fidelity and possible factors influencing fidelity of a complex care continuum intervention for frail elderly people.</p> <p>Methods</p> <p>The intervention was a systematization of the collaboration between a nurse with geriatric expertise situated at the emergency department, the hospital ward staff, and a multi-professional team with a case manager in the municipal care services for older people. Implementation was evaluated between September 2008 and May 2010 with observations of work practices, stakeholder interviews, and document analysis according to a modified version of The Conceptual Framework for Implementation Fidelity.</p> <p>Results</p> <p>A total of 16 of the 18 intervention components were to a great extent delivered as planned, while some new components were added to the model. No changes in the frequency or duration of the 18 components were observed, but the dose of the added components varied over time. Changes in fidelity were caused in a complex, interrelated fashion by all the moderating factors in the framework, i.e., context, staff and participant responsiveness, facilitation, recruitment, and complexity.</p> <p>Discussion</p> <p>The Conceptual Framework for Implementation Fidelity was empirically useful and included comprehensive measures of factors affecting fidelity. Future studies should focus on developing the framework with regard to how to investigate relationships between the moderating factors and fidelity over time.</p> <p>Trial registration</p> <p>ClinicalTrials.gov, <a href="http://www.clinicaltrials.gov/ct2/show/NCT01260493">NCT01260493</a>.</p

A Genome-Wide Association Study of Diabetic Kidney Disease in Subjects With Type 2 Diabetes

dentification of sequence variants robustly associated with predisposition to diabetic kidney disease (DKD) has the potential to provide insights into the pathophysiological mechanisms responsible. We conducted a genome-wide association study (GWAS) of DKD in type 2 diabetes (T2D) using eight complementary dichotomous and quantitative DKD phenotypes: the principal dichotomous analysis involved 5,717 T2D subjects, 3,345 with DKD. Promising association signals were evaluated in up to 26,827 subjects with T2D (12,710 with DKD). A combined T1D+T2D GWAS was performed using complementary data available for subjects with T1D, which, with replication samples, involved up to 40,340 subjects with diabetes (18,582 with DKD). Analysis of specific DKD phenotypes identified a novel signal near GABRR1 (rs9942471, P = 4.5 x 10(-8)) associated with microalbuminuria in European T2D case subjects. However, no replication of this signal was observed in Asian subjects with T2D or in the equivalent T1D analysis. There was only limited support, in this substantially enlarged analysis, for association at previously reported DKD signals, except for those at UMOD and PRKAG2, both associated with estimated glomerular filtration rate. We conclude that, despite challenges in addressing phenotypic heterogeneity, access to increased sample sizes will continue to provide more robust inference regarding risk variant discovery for DKD.Peer reviewe

Autoantibodies against aldehyde-modified collagen type IV are associated with risk of development of myocardial infarction

Background: Oxidation of LDL particles entrapped in the extracellular matrix of the arterial wall is a key factor in the development of atherosclerosis. Lipid oxidation products, such as malondialdehyde (MDA), react with surrounding extracellular matrix proteins and cause modifications that are recognized by the immune system. MDA modification of collagen type IV is increased in carotid lesions from symptomatic patients and correlates with autoantibodies against MDA-modified collagen type IV in plasma. Objective: The aim of this study was to determine whether autoantibodies against MDA-modified collagen type IV predict risk of development of myocardial infarction (MI). Methods: Plasma levels of MDA-modified collagen type IV IgM and IgG antibodies were analysed by enzyme-linked immunosorbent assay in 385 subjects with incident MI during 13 years of follow-up and 410 age- and sex-matched controls in the Malmö Diet and Cancer study. Results: MDA-modified collagen type IV IgG levels were higher in cases with incident MI than in controls. Subjects in the highest tertile of MDA-modified collagen type IV IgG had an increased risk of MI (hazard ratio 1.56, 95% confidence interval 1.22-2.00, P for trend 0.0004). This association remained significant after adjusting for factors included in the Framingham risk score and diabetes. High levels of MDA-collagen type IV IgG were associated with increased carotid intima-media thickness and elevated plasma levels of matrix metalloproteinase 10 and 12. Conclusions: Immune responses against MDA-modified collagen type IV are associated with more severe carotid disease and increased risk of MI. These immune responses may reflect LDL oxidation in the artery wall, but could also affect the atherosclerotic disease process

Activation of immune responses against the basement membrane component collagen type IV does not affect the development of atherosclerosis in ApoE-deficient mice

Oxidation of low-density lipoprotein (LDL) in the arterial extracellular matrix results in malondialdehyde (MDA)-modifications of surrounding matrix proteins. We have recently demonstrated an association between high levels of autoantibodies against MDA-modified collagen type IV and risk for development of myocardial infarction. Collagen type IV is an important component of the endothelial basement membrane and influences smooth muscle cell function. We hypothesized that immune responses against collagen type IV could contribute to vascular injury affecting the development of atherosclerosis. To investigate this possibility, we induced an antibody-response against collagen type IV in apolipoprotein E (Apo E)-deficient mice. Female ApoE −/− mice on C57BL/6 background were immunized with α1α2 type IV collagen chain peptides linked to the immune-enhancer PADRE, PADRE alone or PBS at 12 weeks of age with three subsequent booster injections before the mice were killed at 23 weeks of age. Immunization of PADRE alone induced autoantibodies against PADRE, increased IL-4 secretion from splenocytes and reduced SMC content in the subvalvular plaques. Immunization with peptides of α1α2 type IV collagen chains induced a strong IgG1antibody response against collagen type IV peptides without affecting the distribution of T cell populations, plasma cytokine or lipid levels. There were no differences in atherosclerotic plaque development between collagen α1α2(IV)-PADRE immunized mice and control mice. Our findings demonstrate that the presence of antibodies against the basement membrane component collagen type IV does not affect atherosclerosis development in ApoE −/− mice. This suggests that the association between autoantibodies against collagen type IV and risk for myocardial infarction found in humans does not reflect a pathogenic role of these autoantibodies

Cloning, structural characterization and functional expression of a zebrafish bradykinin B2-related receptor.

The actions of bradykinin (BK) in mammals are mediated through the activation of the B1 and B2 BK receptors. The only BK receptor that has been cloned from a non-mammalian species is a B2-like receptor from the chicken (termed the ornithokinin receptor). Pharmacological studies have demonstrated the presence of BK receptors in tissues of teleost fishes, such as trout and cod, but the ligand-binding properties of these receptors differ appreciably from those of the mammalian and chicken receptors. We report here the cloning of a B2-like receptor in zebrafish that shares 35% identity with human B2 and 30% identity with human B1. Phylogenetic analyses confirm a closer relationship with B2 than B1. The receptor gene was mapped to linkage group 17, which is syntenic to the human B2-B1 gene region. After functional expression of the zebrafish B2 receptor in mammalian cells, nanomolar concentrations of trout BK ([Arg0,Trp5,Leu8]-BK) and the derivative [des-Arg0,Trp5,Leu8]-BK (where 'des' indicates a missing amino acid) induced a significant transient rise in intracellular free Ca2+. The B1-selective analogue [Arg0,Trp5,Leu8,des-Arg9]-BK was inactive at nanomolar concentrations. Taken together, these results strongly support the gene's identity as a piscine orthologue of the mammalian B2 receptor

An osteopontin-derived peptide inhibits human hair growth at least in part by decreasing fibroblast growth factor-7 production in outer root sheath keratinocytes

Background: Given that unwanted hair growth (hirsutism, hypertrichosis) can cause major psychological distress, new pharmacological treatment strategies with safe and effective hair growth inhibitors that do not destroy the hair follicle (HF) and its stem cells need to be developed. Objectives: To establish if osteopontin-derived fragments may modulate human hair growth given that human HFs express the multifunctional, immunomodulatory glycoprotein, osteopontin. Methods: Our hypothesis was tested ex vivo and in vivo by using a newly generated, toxicologically well-characterized, modified osteopontin-derived peptide (FOL-005), which binds to the HF. Results: In organ-cultured human HFs and scalp skin, and in human scalp skin xenotransplants onto SCID mice, FOL-005 treatment (60 nmol L−1 to 3 μmol L−1) significantly promoted premature catagen development without reducing the number of keratin 15-positive HF stem cells or showing signs of drug toxicity. Genome-wide DNA microarray, quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry revealed decreased expression of the hair growth promoter, fibroblast growth factor-7 (FGF7) by FOL-005, while cotreatment of HFs with recombinant FGF7 partially abrogated FOL-005-induced catagen promotion. Conclusions: With caveats in mind, our study identifies this osteopontin-derived peptide as an effective, novel inhibitory principle for human hair growth ex vivo and in vivo, which deserves systematic clinical testing in hirsutism and hypertrichosis. What's already known about this topic?. The treatment of unwanted hair growth (hypertrichosis, hirsutism) lacks pharmacological intervention, with only few and often unsatisfactory treatments available. Osteopontin is prominently expressed in human HFs and has been reported to be elevated during catagen in the murine hair cycle. What does this study add?. We tested the effects on hair growth of a novel, osteopontin-derived fragment (FOL-005) ex vivo and in vivo. In human hair follicles, high-dose FOL-005 significantly reduces hair growth both ex vivo and in vivo. What is the translational message?. High-dose FOL-005 may provide a new therapeutic opportunity as a treatment for unwanted hair growth

Cartilage oligomeric matrix protein associates with a vulnerable plaque phenotype in human atherosclerotic plaques

Background and Purpose- Extracellular matrix proteins are important in atherosclerotic disease by influencing plaque stability and cellular behavior but also by regulating inflammation. COMP (cartilage oligomeric matrix protein) is present in healthy human arteries and expressed by smooth muscle cells. A recent study showed that transplantation of COMP-deficient bone marrow to apoE-/- mice increased atherosclerotic plaque formation, indicating a role for COMP also in bone marrow-derived cells. Despite the evidence of a role for COMP in murine atherosclerosis, knowledge is lacking about the role of COMP in human atherosclerotic disease. Methods- In the present study, we investigated if COMP was associated with a stable or a vulnerable human atherosclerotic plaque phenotype by analyzing 211 carotid plaques for COMP expression using immunohistochemistry. Results- Plaque area that stained positive for COMP was significantly larger in atherosclerotic plaques associated with symptoms (n=110) compared with asymptomatic plaques (n=101; 9.7% [4.7-14.3] versus 5.6% [2.8-9.8]; P=0.0002). COMP was positively associated with plaque lipids (r=0.32; P=0.000002) and CD68 cells (r=0.15; P=0.036) but was negatively associated with collagen (r=-0.16; P=0.024), elastin (r=-0.14; P=0.041), and smooth muscle cells (r=-0.25; P=0.0002). COMP was positively associated with CD163 (r=0.37; P=0.00000006), a scavenger receptor for hemoglobin/haptoglobin and a marker of Mhem macrophages, and with intraplaque hemorrhage, measured as glycophorin A staining (r=0.28; P=0.00006). Conclusions- The present study shows that COMP is associated to symptomatic carotid atherosclerosis, CD163-expressing cells, and a vulnerable atherosclerotic plaque phenotype in humans