Temporal regulation of expression of immediate early and second phase transcripts by endothelin-1 in cardiomyocytes

Background: Endothelin-1 stimulates Gq protein-coupled receptors to promote proliferation in dividing cells or hypertrophy in terminally differentiated cardiomyocytes. In cardiomyocytes, endothelin-1 rapidly (within minutes) stimulates protein kinase signaling, including extracellular-signal regulated kinases 1/2 (ERK1/2; though not ERK5), with phenotypic/physiological changes developing from approximately 12 h. Hypertrophy is associated with changes in mRNA/protein expression, presumably consequent to protein kinase signaling, but the connections between early, transient signaling events and developed hypertrophy are unknown. Results: Using microarrays, we defined the early transcriptional responses of neonatal rat cardiomyocytes to endothelin-1 over 4 h, differentiating between immediate early gene (IEG) and second phase RNAs with cycloheximide. IEGs exhibited differential temporal and transient regulation, with expression of second phase RNAs within 1 h. Of transcripts upregulated at 30 minutes encoding established proteins, 28 were inhibited >50% by U0126 (which inhibits ERK1/2/5 signaling), with 9 inhibited 25-50%. Expression of only four transcripts was not inhibited. At 1 h, most RNAs (approximately 67%) were equally changed in total and polysomal RNA with approximately 17% of transcripts increased to a greater extent in polysomes. Thus, changes in expression of most protein-coding RNAs should be reflected in protein synthesis. However, approximately 16% of transcripts were essentially excluded from the polysomes, including some protein-coding mRNAs, presumably inefficiently translated. Conclusion: The phasic, temporal regulation of early transcriptional responses induced by endothelin-1 in cardiomyocytes indicates that, even in terminally differentiated cells, signals are propagated beyond the primary signaling pathways through transcriptional networks leading to phenotypic changes (that is, hypertrophy). Furthermore, ERK1/2 signaling plays a major role in this response

Myocardial depressant effects of interleukin 6 in meningococcal sepsis are regulated by p38 mitogen-activated protein kinase

Our findings demonstrate an integral role of the p38 mitogen-activated protein kinase pathway in interleukin 6-mediated cardiac contractile dysfunction and inotrope insensitivity. Dysregulation of the p38 mitogen-activated protein kinase pathway in meningococcal septicemia suggests that this pathway may be an important target for novel therapies to reverse myocardial dysfunction in patients with meningococcal septic shock who are not responsive to inotropic support

Development of a peer support intervention to encourage dietary behaviour change towards a Mediterranean diet in adults at high cardiovascular risk.

BACKGROUND: Mediterranean diet (MD) interventions are demonstrated to significantly reduce cardiovascular disease (CVD) risk but are typically resource intensive and delivered by health professionals. There is considerable interest to develop interventions that target sustained dietary behaviour change and that are feasible to scale-up for wider public health benefit. The aim of this paper is to describe the process used to develop a peer support intervention to encourage dietary behaviour change towards a MD in non-Mediterranean adults at high CVD risk. METHODS: The Medical Research Council (MRC) and Behaviour Change Wheel (BCW) frameworks and the COM-B (Capability, Opportunity, Motivation, Behaviour) theoretical model were used to guide the intervention development process. We used a combination of evidence synthesis and qualitative research with the target population, health professionals, and community health personnel to develop the intervention over three main stages: (1) we identified the evidence base and selected dietary behaviours that needed to change, (2) we developed a theoretical basis for how the intervention might encourage behaviour change towards a MD and selected intervention functions that could drive the desired MD behaviour change, and (3) we defined the intervention content and modelled outcomes. RESULTS: A theory-based, culturally tailored, peer support intervention was developed to specifically target behaviour change towards a MD in the target population. The intervention was a group-based program delivered by trained peer volunteers over 12-months, and incorporated strategies to enhance social support, self-efficacy, problem-solving, knowledge, and attitudes to address identified barriers to adopting a MD from the COM-B analysis. CONCLUSIONS: The MRC and BCW frameworks provided a systematic and complementary process for development of a theory-based peer support intervention to encourage dietary behaviour change towards a MD in non-Mediterranean adults at high CVD risk. The next step is to evaluate feasibility, acceptability, and diet behaviour change outcomes in response to the peer support intervention (change towards a MD and nutrient biomarkers) using a randomized controlled trial design

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

A qualitative analysis exploring preferred methods of peer support to encourage adherence to a Mediterranean diet in a Northern European population at high risk of cardiovascular disease.

BACKGROUND: Epidemiological and randomised controlled trial evidence demonstrates that adherence to a Mediterranean diet (MD) can reduce cardiovascular disease (CVD) risk. However, methods used to support dietary change have been intensive and expensive. Peer support has been suggested as a possible cost-effective method to encourage adherence to a MD in at risk populations, although development of such a programme has not been explored. The purpose of this study was to use mixed-methods to determine the preferred peer support approach to encourage adherence to a MD. METHODS: Qualitative (focus groups) and quantitative methods (questionnaire and preference scoring sheet) were used to determine preferred methods of peer support. Sixty-seven high CVD risk participants took part in 12 focus groups (60% female, mean age 64 years) and completed a questionnaire and preference scoring sheet. Focus group data were transcribed and thematically analysed. RESULTS: The mean preference score (1 being most preferred and 5 being least preferred) for group support was 1.5, compared to 3.4 for peer mentorship, 4.0 for telephone peer support and 4.0 for internet peer support. Three key themes were identified from the transcripts: 1. Components of an effective peer support group: discussions around group peer support were predominantly positive. It was suggested that an effective group develops from people who consider themselves similar to each other meeting face-to-face, leading to the development of a group identity that embraces trust and honesty. 2. Catalysing Motivation: participants discussed that a group peer support model could facilitate interpersonal motivations including encouragement, competitiveness and accountability. 3. Stepping Stones of Change: participants conceptualised change as a process, and discussed that, throughout the process, different models of peer support might be more or less useful. CONCLUSION: A group-based approach was the preferred method of peer support to encourage a population at high risk of CVD to adhere to a MD. This finding should be recognised in the development of interventions to encourage adoption of a MD in a Northern European population

Protease Activated Receptor Signaling Is Required for African Trypanosome Traversal of Human Brain Microvascular Endothelial Cells

Human African trypanosomiasis, or sleeping sickness, occurs when single-cell trypanosome protozoan parasites spread from the blood to brain over the blood-brain barrier (BBB). This barrier is composed of brain microvascular endothelial cells (BMECs) especially designed to keep pathogens out. Safe drugs for treating sleeping sickness are lacking and alternative treatments are urgently required. Using our human BMEC BBB model, we previously found that a parasite protease, brucipain, induced calcium activation signals that allowed this barrier to open up to parasite crossing. Because human BMECs express protease-activated receptors (PARs) that trigger calcium signals in BMECs, we hypothesized a functional link between parasite brucipain and BMEC PARs. Utilizing RNA interference to block the production of one type of PAR called PAR-2, we hindered the ability of trypanosomes to both open up and cross human BMECs. Using gene-profiling methods to interrogate candidate BMEC pathways specifically triggered by brucipain, several pathways that potentially link brain inflammatory processes were identified, a finding congruent with the known role of PAR-2 as a mediator of inflammation. Overall, our data support a role for brucipain and BMEC PARs in trypanosome BBB transmigration, and as potential triggers for brain inflammation associated with the disease

Beyond the Global Brain Differences:Intraindividual Variability Differences in 1q21.1 Distal and 15q11.2 BP1-BP2 Deletion Carriers

BACKGROUND: Carriers of the 1q21.1 distal and 15q11.2 BP1-BP2 copy number variants exhibit regional and globalbrain differences compared with noncarriers. However, interpreting regional differences is challenging if a globaldifference drives the regional brain differences. Intraindividual variability measures can be used to test for regionaldifferences beyond global differences in brain structure.METHODS: Magnetic resonance imaging data were used to obtain regional brain values for 1q21.1 distal deletion (n =30) and duplication (n = 27) and 15q11.2 BP1-BP2 deletion (n = 170) and duplication (n = 243) carriers and matchednoncarriers (n = 2350). Regional intra-deviation scores, i.e., the standardized difference between an individual’sregional difference and global difference, were used to test for regional differences that diverge from the globaldifference.RESULTS: For the 1q21.1 distal deletion carriers, cortical surface area for regions in the medial visual cortex, posterior cingulate, and temporal pole differed less and regions in the prefrontal and superior temporal cortex differedmore than the global difference in cortical surface area. For the 15q11.2 BP1-BP2 deletion carriers, cortical thicknessin regions in the medial visual cortex, auditory cortex, and temporal pole differed less and the prefrontal andsomatosensory cortex differed more than the global difference in cortical thickness.CONCLUSIONS: We find evidence for regional effects beyond differences in global brain measures in 1q21.1 distaland 15q11.2 BP1-BP2 copy number variants. The results provide new insight into brain profiling of the 1q21.1 distaland 15q11.2 BP1-BP2 copy number variants, with the potential to increase understanding of the mechanismsinvolved in altered neurodevelopment

Genetic Studies of Leptin Concentrations Implicate Leptin in the Regulation of Early Adiposity.

Leptin influences food intake by informing the brain about the status of body fat stores. Rare LEP mutations associated with congenital leptin deficiency cause severe early-onset obesity that can be mitigated by administering leptin. However, the role of genetic regulation of leptin in polygenic obesity remains poorly understood. We performed an exome-based analysis in up to 57,232 individuals of diverse ancestries to identify genetic variants that influence adiposity-adjusted leptin concentrations. We identify five novel variants, including four missense variants, in LEP, ZNF800, KLHL31, and ACTL9, and one intergenic variant near KLF14. The missense variant Val94Met (rs17151919) in LEP was common in individuals of African ancestry only, and its association with lower leptin concentrations was specific to this ancestry (P = 2 × 10-16, n = 3,901). Using in vitro analyses, we show that the Met94 allele decreases leptin secretion. We also show that the Met94 allele is associated with higher BMI in young African-ancestry children but not in adults, suggesting that leptin regulates early adiposity

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Symptom-based stratification of patients with primary Sjögren's syndrome: multi-dimensional characterisation of international observational cohorts and reanalyses of randomised clinical trials

Background Heterogeneity is a major obstacle to developing effective treatments for patients with primary Sjögren's syndrome. We aimed to develop a robust method for stratification, exploiting heterogeneity in patient-reported symptoms, and to relate these differences to pathobiology and therapeutic response. Methods We did hierarchical cluster analysis using five common symptoms associated with primary Sjögren's syndrome (pain, fatigue, dryness, anxiety, and depression), followed by multinomial logistic regression to identify subgroups in the UK Primary Sjögren's Syndrome Registry (UKPSSR). We assessed clinical and biological differences between these subgroups, including transcriptional differences in peripheral blood. Patients from two independent validation cohorts in Norway and France were used to confirm patient stratification. Data from two phase 3 clinical trials were similarly stratified to assess the differences between subgroups in treatment response to hydroxychloroquine and rituximab. Findings In the UKPSSR cohort (n=608), we identified four subgroups: Low symptom burden (LSB), high symptom burden (HSB), dryness dominant with fatigue (DDF), and pain dominant with fatigue (PDF). Significant differences in peripheral blood lymphocyte counts, anti-SSA and anti-SSB antibody positivity, as well as serum IgG, κ-free light chain, β2-microglobulin, and CXCL13 concentrations were observed between these subgroups, along with differentially expressed transcriptomic modules in peripheral blood. Similar findings were observed in the independent validation cohorts (n=396). Reanalysis of trial data stratifying patients into these subgroups suggested a treatment effect with hydroxychloroquine in the HSB subgroup and with rituximab in the DDF subgroup compared with placebo. Interpretation Stratification on the basis of patient-reported symptoms of patients with primary Sjögren's syndrome revealed distinct pathobiological endotypes with distinct responses to immunomodulatory treatments. Our data have important implications for clinical management, trial design, and therapeutic development. Similar stratification approaches might be useful for patients with other chronic immune-mediated diseases. Funding UK Medical Research Council, British Sjogren's Syndrome Association, French Ministry of Health, Arthritis Research UK, Foundation for Research in Rheumatology