Molecular Mechanisms Linking Diabetes with Increased Risk of Thrombosis.

This review will provide an overview of what is currently known about mechanisms linking poor glycaemic control with increased thrombotic risk. The leading causes of death in people with diabetes are strokes and cardiovascular disease. Significant morbidity is associated with an increased risk of thrombosis, resulting in myocardial infarction, ischaemic stroke, and peripheral vascular disease, along with the sequelae of these events, including loss of functional ability, heart failure, and amputations. While the increased platelet activity, pro-coagulability, and endothelial dysfunction directly impact this risk, the molecular mechanisms linking poor glycaemic control with increased thrombotic risk remain unclear. This review highlights the complex mechanisms underlying thrombosis prevalence in individuals with diabetes and hyperglycaemia. Post-translational modifications, such as O-GlcNAcylation, play a crucial role in controlling protein function in diabetes. However, the role of O-GlcNAcylation remains poorly understood due to its intricate regulation and the potential involvement of multiple variables. Further research is needed to determine the precise impact of O-GlcNAcylation on specific disease processes.

Self-sampling as the principal modality for population based cervical screening: Five-year follow-up of the PaVDaG study.

Self-sampling provides a powerful means to engage women in cervical screening. In the original Papillomavirus Dumfries and Galloway study (PaVDaG), we demonstrated cross-sectional similarity of high-risk human papillomavirus (Hr-HPV) testing on self-taken vaginal vs clinician-taken samples for the detection of cervical intraepithelial neoplasia 2 or worse (CIN2+). Few data exist on the longitudinal performance of self-sampling; we present longitudinal outcomes of PaVDaG. Routinely screened women provided a self-taken and a clinician-collected sample. Ninety-one percent of 5136 women from the original cohort completed a further screening round. Sensitivity, specificity, positive predictive value and complement of the negative predictive value of the Hr-HPV test on self-samples for detection of CIN2+ and CIN3+ up-to 5 years after testing were determined. Additionally, clinical accuracy of Hr-HPV testing on vaginal and clinician-collected samples was assessed. A total of 183 CIN2+ and 102 CIN3+ lesions were diagnosed during follow-up. Risk of CIN2+ and CIN3+ following an Hr-HPV negative self-sample was 0.6% and 0.2%, respectively, for up to 5 years after testing. The relative sensitivity for CIN3+ and specificity for ≤CIN1 of Hr-HPV testing on self-taken specimens was slightly lower vs clinician-collected samples: 0.95 (95% CI: 0.90-0.99; PMcN = .0625) and 0.98 (95% CI: 0.95-1.00; PMcN = <.0000), respectively. The low risk of CIN2+ in women with Hr-HPV-self-sample(s) suggests, that the 3 to 5-year recall interval implemented in several cervical screening settings, based on clinician-taken samples, may be safe for self-samples. Future assessment will show if "universal" 5-year screening is appropriate for programs based on self-sampling.

Nutrient regulation of inflammatory signalling in obesity and vascular disease.

Despite obesity and diabetes markedly increasing the risk of developing cardiovascular diseases, the molecular and cellular mechanisms that underlie this association remain poorly characterised. In the last 20 years it has become apparent that chronic, low-grade inflammation in obese adipose tissue may contribute to the risk of developing insulin resistance and type 2 diabetes. Furthermore, increased vascular pro-inflammatory signalling is a key event in the development of cardiovascular diseases. Overnutrition exacerbates pro-inflammatory signalling in vascular and adipose tissues, with several mechanisms proposed to mediate this. In this article, we review the molecular and cellular mechanisms by which nutrients are proposed to regulate pro-inflammatory signalling in adipose and vascular tissues. In addition, we examine the potential therapeutic opportunities that these mechanisms provide for suppression of inappropriate inflammation in obesity and vascular disease.

Emerging roles of protein O-GlcNAcylation in cardiovascular diseases: Insights and novel therapeutic targets.

Cardiovascular diseases (CVDs) are the leading cause of death globally. While the major focus of pharmacological and non-pharmacological interventions has been on targeting disease pathophysiology and limiting predisposing factors, our understanding of the cellular and molecular mechanisms underlying the pathogenesis of CVDs remains incomplete. One mechanism that has recently emerged is protein O-GlcNAcylation. This is a dynamic, site-specific reversible post-translational modification of serine and threonine residues on target proteins and is controlled by two enzymes: O-linked ß-N-acetylglucosamine transferase (OGT) and O-linked ß-N-acetylglucosaminidase (OGA). Protein O-GlcNAcylation alters the cellular functions of these target proteins which play vital roles in pathways that modulate vascular homeostasis and cardiac function. Through this review, we aim to give insights on the role of protein O-GlcNAcylation in cardiovascular diseases and identify potential therapeutic targets in this pathway for development of more effective medicines to improve patient outcomes.

Revascularisation of type 2 diabetics with coronary artery disease: Insights and therapeutic targeting of O-GlcNAcylation.

AIM: Coronary artery bypass graft (CABG) using autologous saphenous vein continues to be a gold standard procedure to restore the supply of oxygen-rich blood to the heart muscles in coronary artery disease (CAD) patients with or without type 2 diabetes mellitus (T2DM). However, CAD patients with T2DM are at higher risk of graft failure. While failure rates have been reduced through improvements in procedure-related factors, much less is known about the molecular and cellular mechanisms by which T2DM initiates vein graft failure. This review gives novel insights into these cellular and molecular mechanisms and identifies potential therapeutic targets for development of new medicines to improve vein graft patency. DATA SYNTHESIS: One important cellular process that has been implicated in the pathogenesis of T2DM is protein O-GlcNAcylation, a dynamic, reversible post-translational modification of serine and threonine residues on target proteins that is controlled by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Protein O-GlcNAcylation impacts a range of cellular processes, including trafficking, metabolism, inflammation and cytoskeletal organisation. Altered O-GlcNAcylation homeostasis have, therefore, been linked to a range of human pathologies with a metabolic component, including T2DM. CONCLUSION: We propose that protein O-GlcNAcylation alters vascular smooth muscle and endothelial cell function through modification of specific protein targets which contribute to the vascular re-modelling responsible for saphenous vein graft failure in T2DM.

Extracellular adenosine sensing-a metabolic cell death priming mechanism downstream of p53.

Tumor cells undergo changes in metabolism to meet their energetic and anabolic needs. It is conceivable that mechanisms exist to sense these changes and link them to pathways that eradicate cells primed for cancer development. We report that the tumor suppressor p53 activates a cell death priming mechanism that senses extracellular adenosine. Adenosine, the backbone of ATP, accumulates under conditions of cellular stress or altered metabolism. We show that its receptor, A2B, is upregulated by p53. A2B expression has little effect on cell viability, but ligand engagement activates a caspase- and Puma-dependent apoptotic response involving downregulation of antiapoptotic Bcl-2 proteins. Stimulation of A2B also significantly enhances cell death mediated by p53 and upon accumulation of endogenous adenosine following chemotherapeutic drug treatment and exposure to hypoxia. Since extracellular adenosine also accumulates within many solid tumors, this distinct p53 function links programmed cell death to both a cancer- and therapy-associated metabolic change.

Identification of myeloid cells in the human enthesis as the main source of local IL-23 production.

OBJECTIVE: We investigated whether the normal human spinal enthesis contained resident myeloid cell populations, capable of producing pivotal proinflammatory cytokines including tumour necrosis factor (TNF) and interleukin (IL)-23 and determined whether these could be modified by PDE4 inhibition. METHODS: Normal human enthesis soft tissue (ST) and adjacent perientheseal bone (PEB) (n=15) were evaluated using immunohistochemistry (IHC), digested for myeloid cell phenotyping, sorted and stimulated with different adjuvants (lipopolysaccharide and mannan). Stimulated enthesis fractions were analysed for inducible production of spondyloarthropathy disease-relevant mediators (IL-23 full protein, TNF, IL-1ß and CCL20). Myeloid populations were also compared with matched blood populations for further mRNA analysis and the effect of PDE4 inhibition was assessed. RESULTS: A myeloid cell population (CD45+ HLADR+ CD14+ CD11c+) phenotype was isolated from both the ST and adjacent PEB and termed 'CD14+ myeloid cells' with tissue localisation confirmed by CD14+ IHC. The CD14- fraction contained a CD123+ HLADR+ CD11c- cell population (plasmacytoid dendritic cells). The CD14+ population was the dominant entheseal producer of IL-23, IL-1ß, TNF and CCL20. IL-23 and TNF from the CD14+ population could be downregulated by a PDE4I and other agents (histamine and 8-Bromo-cAMP) which elevate cAMP. Entheseal CD14+ cells had a broadly similar gene expression profile to the corresponding CD14+ population from matched blood but showed significantly lower CCR2 gene expression. CONCLUSIONS: The human enthesis contains a CD14+ myeloid population that produces most of the inducible IL-23, IL-1ß, TNF and CCL20. This population has similar gene expression profile to the matched blood CD14+ population.

Is there a role for prostanoid-mediated inhibition of IL-6 trans-signalling in the management of pulmonary arterial hypertension?

Inflammation has been highlighted as a key factor in pulmonary arterial hypertension (PAH) development, particularly interleukin-6 (IL-6). IL-6 activates JAK-STAT signalling to induce transcription of pro-inflammatory and pro-angiogenic genes, enabling PAH progression, as well as the transcription of suppressor of cytokine signalling 3 (SOCS3) which limits IL-6 signalling. Current PAH therapies include prostanoid drugs which induce vasodilation via stimulating intracellular 3',5'-cyclic adenosine monophosphate (cAMP) levels. cAMP can also inhibit IL-6-mediated endothelial dysfunction via the induction of SOCS3. Thus, we propose that an important mechanism by which cAMP-mobilising prostanoid drugs limit PAH is by inhibiting IL-6-mediated pulmonary inflammation and remodelling via SOCS3 inhibition of IL-6 signalling. Further clarification may result in effective strategies with which to target the IL-6/JAK-STAT signalling pathway in PAH.

Linking energy sensing to suppression of JAK-STAT signalling: A potential route for repurposing AMPK activators?

Exaggerated Janus kinase-signal transducer and activator of transcription (JAK-STAT) signalling is key to the pathogenesis of pro-inflammatory disorders, such as rheumatoid arthritis and cardiovascular diseases. Mutational activation of JAKs is also responsible for several haematological malignancies, including myeloproliferative neoplasms and acute lymphoblastic leukaemia. Accumulating evidence links adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), an energy sensor and regulator of organismal and cellular metabolism, with the suppression of immune and inflammatory processes. Recent studies have shown that activation of AMPK can limit JAK-STAT-dependent signalling pathways via several mechanisms. These novel findings support AMPK activation as a strategy for management of an array of disorders characterised by hyper-activation of the JAK-STAT pathway. This review discusses the pivotal role of JAK-STAT signalling in a range of disorders and how both established clinically used and novel AMPK activators might be used to treat these conditions.

A769662 Inhibits Insulin-Stimulated Akt Activation in Human Macrovascular Endothelial Cells Independent of AMP-Activated Protein Kinase.

Protein kinase B (Akt) is a key enzyme in the insulin signalling cascade, required for insulin-stimulated NO production in endothelial cells (ECs). Previous studies have suggested that AMP-activated protein kinase (AMPK) activation stimulates NO synthesis and enhances insulin-stimulated Akt activation, yet these studies have largely used indirect activators of AMPK. The effects of the allosteric AMPK activator A769662 on insulin signalling and endothelial function was therefore examined in cultured human macrovascular ECs. Surprisingly, A769662 inhibited insulin-stimulated NO synthesis and Akt phosphorylation in human ECs from umbilical veins (HUVECs) and aorta (HAECs). In contrast, the AMPK activators compound 991 and AICAR had no substantial inhibitory effect on insulin-stimulated Akt phosphorylation in ECs. Inhibition of AMPK with SBI-0206965 had no effect on the inhibition of insulin-stimulated Akt phosphorylation by A769662, suggesting the inhibitory action of A769662 is AMPK-independent. A769662 decreased IGF1-stimulated Akt phosphorylation yet had no effect on VEGF-stimulated Akt signalling in HUVECs, suggesting that A769662 attenuates early insulin/IGF1 signalling. The effects of A769662 on insulin-stimulated Akt phosphorylation were specific to human ECs, as no effect was observed in the human cancer cell lines HepG2 or HeLa, as well as in mouse embryonic fibroblasts (MEFs). A769662 inhibited insulin-stimulated Erk1/2 phosphorylation in HAECs and MEFs, an effect that was independent of AMPK in MEFs. Therefore, despite being a potent AMPK activator, A769662 has effects unlikely to be mediated by AMPK in human macrovascular ECs that reduce insulin sensitivity and eNOS activation.

Protein kinase C phosphorylates AMP-activated protein kinase α1 Ser487.

The key metabolic regulator, AMP-activated protein kinase (AMPK), is reported to be down-regulated in metabolic disorders, but the mechanisms are poorly characterised. Recent studies have identified phosphorylation of the AMPKα1/α2 catalytic subunit isoforms at Ser487/491, respectively, as an inhibitory regulation mechanism. Vascular endothelial growth factor (VEGF) stimulates AMPK and protein kinase B (Akt) in cultured human endothelial cells. As Akt has been demonstrated to be an AMPKα1 Ser487 kinase, the effect of VEGF on inhibitory AMPK phosphorylation in cultured primary human endothelial cells was examined. Stimulation of endothelial cells with VEGF rapidly increased AMPKα1 Ser487 phosphorylation in an Akt-independent manner, without altering AMPKα2 Ser491 phosphorylation. In contrast, VEGF-stimulated AMPKα1 Ser487 phosphorylation was sensitive to inhibitors of protein kinase C (PKC) and PKC activation using phorbol esters or overexpression of PKC-stimulated AMPKα1 Ser487 phosphorylation. Purified PKC and Akt both phosphorylated AMPKα1 Ser487 in vitro with similar efficiency. PKC activation was associated with reduced AMPK activity, as inhibition of PKC increased AMPK activity and phorbol esters inhibited AMPK, an effect lost in cells expressing mutant AMPKα1 Ser487Ala. Consistent with a pathophysiological role for this modification, AMPKα1 Ser487 phosphorylation was inversely correlated with insulin sensitivity in human muscle. These data indicate a novel regulatory role of PKC to inhibit AMPKα1 in human cells. As PKC activation is associated with insulin resistance and obesity, PKC may underlie the reduced AMPK activity reported in response to overnutrition in insulin-resistant metabolic and vascular tissues.

Human Papillomavirus Prevalence and Herd Immunity after Introduction of Vaccination Program, Scotland, 2009-2013.

In 2008, a national human papillomavirus (HPV) immunization program using a bivalent vaccine against HPV types 16 and 18 was implemented in Scotland along with a national surveillance program designed to determine the longitudinal effects of vaccination on HPV infection at the population level. Each year during 2009-2013, the surveillance program conducted HPV testing on a proportion of liquid-based cytology samples from women undergoing their first cervical screening test for precancerous cervical disease. By linking vaccination, cervical screening, and HPV testing data, over the study period we found a decline in HPV types 16 and 18, significant decreases in HPV types 31, 33, and 45 (suggesting cross-protection), and a nonsignificant increase in HPV 51. In addition, among nonvaccinated women, HPV types 16 and 18 infections were significantly lower in 2013 than in 2009. Our results preliminarily indicate herd immunity and sustained effectiveness of the bivalent vaccine on virologic outcomes at the population level.

ß1-adrenergic receptor and sphingosine-1-phosphate receptor 1 (S1PR1) reciprocal downregulation influences cardiac hypertrophic response and progression to heart failure: protective role of S1PR1 cardiac gene therapy.

BACKGROUND: The sphingosine-1-phosphate receptor 1 (S1PR1) and ß1-adrenergic receptor (ß1AR) are G-protein-coupled receptors expressed in the heart. These 2 receptors have opposing actions on adenylyl cyclase because of differential G-protein coupling. Importantly, both of these receptors can be regulated by the actions of G-protein-coupled receptor kinase-2, which triggers desensitization and downregulation processes. Although classic signaling paradigms suggest that simultaneous activation of ß1ARs and S1PR1s in a myocyte would simply result in opposing action on cAMP production, in this report we have uncovered a direct interaction between these 2 receptors, with regulatory involvement of G-protein-coupled receptor kinase-2. METHODS AND RESULTS: In HEK (human embryonic kidney) 293 cells overexpressing both ß1AR and S1PR1, we demonstrated that ß1AR downregulation can occur after stimulation with sphingosine-1-phosphate (an S1PR1 agonist), whereas S1PR1 downregulation can be triggered by isoproterenol (a ß-adrenergic receptor agonist) treatment. This cross talk between these 2 distinct G-protein-coupled receptors appears to have physiological significance, because they interact and show reciprocal regulation in mouse hearts undergoing chronic ß-adrenergic receptor stimulation and in a rat model of postischemic heart failure. CONCLUSIONS: We demonstrate that restoration of cardiac plasma membrane levels of S1PR1 produces beneficial effects that counterbalance the deleterious ß1AR overstimulation in heart failure.

Cavin-1: caveolae-dependent signalling and cardiovascular disease.

Caveolae are curved lipid raft regions rich in cholesterol and sphingolipids found abundantly in vascular endothelial cells, adipocytes, smooth muscle cells and fibroblasts. They are multifunctional organelles with roles in clathrin-independent endocytosis, cholesterol transport, mechanosensing and signal transduction. Caveolae provide an environment where multiple receptor signalling components are sequestered, clustered and compartmentalized for efficient signal transduction. Many of these receptors, including cytokine signal transducer gp130 (glycoprotein 130), are mediators of chronic inflammation during atherogenesis. Subsequently, disruption of these organelles is associated with a broad range of disease states including cardiovascular disease and cancer. Cavin-1 is an essential peripheral component of caveolae that stabilizes caveolin-1, the main structural/integral membrane protein of caveolae. Caveolin-1 is an essential regulator of eNOS (endothelial nitric oxide synthase) and its disruption leads to endothelial dysfunction which initiates a range of cardiovascular and pulmonary disorders. Although dysfunctional cytokine signalling is also a hallmark of cardiovascular disease, knowledge of caveolae-dependent cytokine signalling is lacking as is the role of cavin-1 independent of caveolae. The present review introduces caveolae, their structural components, the caveolins and cavins, their regulation by cAMP, and their potential role in cardiovascular disease.

Proposed Screening for Congenital Hyperinsulinism in Newborns: Perspective from a Neonatal-Perinatal Medicine Group.

This perspective work by academic neonatal providers is written specifically for the audience of newborn care providers and neonatologists involved in neonatal hypoglycemia screening. Herein, we propose adding a screen for congenital hyperinsulinism (CHI) by measuring glucose and ketone (i.e., ß-hydroxybutyrate (BOHB)) concentrations just prior to newborn hospital discharge and as close to 48 h after birth as possible, at the same time that the mandated state Newborn Dried Blood Spot Screen is obtained. In the proposed protocol, we do not recommend specific metabolite cutoffs, as our primary objective is to simply highlight the concept of screening for CHI in newborns to newborn caregivers. The premise for our proposed screen is based on the known effect of hyperinsulinism in suppressing ketogenesis, thereby limiting ketone production. We will briefly discuss genetic CHI, other forms of neonatal hypoglycemia, and their shared mechanisms; the mechanism of insulin regulation by functional pancreatic islet cell membrane KATP channels; adverse neurodevelopmental sequelae and brain injury due to missing or delaying the CHI diagnosis; the principles of a good screening test; how current neonatal hypoglycemia screening programs do not fulfill the criteria for being effective screening tests; and our proposed algorithm for screening for CHI in newborns.

Detection of microplastics in human saphenous vein tissue using µFTIR: A pilot study.

Microplastics (MPs) are ubiquitous in the environment, in the human food chain, and have been recently detected in blood and lung tissues. To undertake a pilot analysis of MP contamination in human vein tissue samples with respect to their presence (if any), levels, and characteristics of any particles identified. This study analysed digested human saphenous vein tissue samples (n = 5) using µFTIR spectroscopy (size limitation of 5 µm) to detect and characterise any MPs present. In total, 20 MP particles consisting of five MP polymer types were identified within 4 of the 5 vein tissue samples with an unadjusted average of 29.28 ± 34.88 MP/g of tissue (expressed as 14.99 ± 17.18 MP/g after background subtraction adjustments). Of the MPs detected in vein samples, five polymer types were identified, of irregular shape (90%), with alkyd resin (45%), poly (vinyl propionate/acetate, PVAc (20%) and nylon-ethylene-vinyl acetate, nylon-EVA, tie layer (20%) the most abundant. While the MP levels within tissue samples were not significantly different than those identified within procedural blanks (which represent airborne contamination at time of sampling), they were comprised of different plastic polymer types. The blanks comprised n = 13 MP particles of four MP polymer types with the most abundant being polytetrafluoroethylene (PTFE), then polypropylene (PP), polyethylene terephthalate (PET) and polyfumaronitrile:styrene (FNS), with a mean ± SD of 10.4 ± 9.21, p = 0.293. This study reports the highest level of contamination control and reports unadjusted values alongside different contamination adjustment techniques. This is the first evidence of MP contamination of human vascular tissues. These results support the phenomenon of transport of MPs within human tissues, specifically blood vessels, and this characterisation of types and levels can now inform realistic conditions for laboratory exposure experiments, with the aim of determining vascular health impacts.

Anxiety, Depression, and Behavioral and/or Conduct Disorder in Adolescence Among Former Preterm and Term Infants of Different Race and Ethnicities.

OBJECTIVE: To compare parental reports of recent diagnoses of anxiety, depression, and/or behavioral/conduct disorder among former preterm (PT) and term adolescents by race/ethnicity and evaluate receipt of mental healthcare within the past year among those adolescents with any of these conditions. STUDY DESIGN: A total of 20,871 Non-Hispanic white (NHW), Non-Hispanic black (NHB), and Hispanic adolescents were evaluated using data from the 2017/2018 National Survey of Children's Health. PT birth and race/ethnicity disparity in the diagnosis of these emotional/behavioral problems and receipt of mental healthcare among adolescents with any of these diagnoses were analyzed using logistic regression. RESULTS: The unadjusted prevalence (95% CI) of these diagnoses was significantly higher among former PT (0.19 [0.17-0.22]) compared to term (0.15 [0.14-0.16]) adolescents. Despite having higher rates of adverse socioeconomic measures, former PT and term NHBs and Hispanics had lower unadjusted prevalence of these diagnoses in comparison to NHWs. After adjusting for differences in demographic, clinical, and socioeconomic characteristics, NHBs (0.47 [0.36-0.64]) and Hispanics (0.40 [0.30-0.54]) remain at lower odds of the composite measure of the emotional and/or behavioral problems compared to NHWs, while PT birth did not have a significant impact on this outcome measure. Only 53% of adolescents with these diagnoses received recent mental healthcare. No significant differences in the adjusted odds of receipt of mental healthcare were noted across the groups based on PT birth or race/ethnicity. CONCLUSIONS: In contrast to PT birth, race/ethnicity had a significant impact on the adjusted odds of emotional/behavioral disorders during adolescence. Among adolescents with these diagnoses, PT birth and race/ethnicity did not significantly influence the adjusted odds of receipt of mental healthcare.

Clinical potential of microdystrophin as a surrogate endpoint.

Accelerated approval based on a likely surrogate endpoint can be life-changing for patients suffering from a rare progressive disease with unmet medical need, as it substantially hastens access to potentially lifesaving therapies. In one such example, antisense morpholinos were approved to treat Duchenne muscular dystrophy (DMD) based on measurement of shortened dystrophin in skeletal muscle biopsies as a surrogate biomarker. New, promising therapeutics for DMD include AAV gene therapy to restore another form of dystrophin termed mini- or microdystrophin. AAV-microdystrophins are currently in clinical trials but have yet to be accepted by regulatory agencies as reasonably likely surrogate endpoints. To evaluate microdystrophin expression as a reasonably likely surrogate endpoint for DMD, this review highlights dystrophin biology in the context of functional and clinical benefit to support the argument that microdystrophin proteins have a high probability of providing clinical benefit based on their rational design. Unlike exon-skipping based strategies, the approach of rational design allows for functional capabilities (i.e. quality) of the protein to be maximized with every patient receiving the same optimized microdystrophin. Therefore, the presence of rationally designed microdystrophin in a muscle biopsy is likely to predict clinical benefit and is consequently a strong candidate for a surrogate endpoint analysis to support accelerated approval.