Tiny dancers: the integrin–growth factor nexus in angiogenic signaling

A vital step in growth factor–driven angiogenesis is the coordinated engagement of endothelial integrins with the extracellular matrix. The molecular mechanisms that partner growth factors and integrins are being elucidated, revealing an intricate interaction of surface receptors and their signaling pathways

Synergistic Effect of Anemia and Red Blood Cells Transfusion on Inflammation and Lung Injury

Anemia and resultant red blood cell transfusion may be associated with adverse long-term clinical outcomes. To investigate the mechanism(s) responsible, we profiled inflammatory biomarkers and circulating levels of the bioactive lysophospholipid mediator sphingosine-1-phosphate (S1P) in control and anemic mice with or without LPS-induced systemic inflammation. Acute anemia or lipopolysaccharide (LPS) challenge alone triggered an increase of circulating levels of the inflammatory markers IL-6 and keratinocyte-derived chemokine (CXCL1/KC). Moreover, administration of LPS to anemic mice reduced circulating S1P levels and augmented lung injury and pulmonary vascular permeability. Transfusion of aged, but not fresh, red blood cells (RBCs) worsened pulmonary vascular leak. S1P levels decline markedly during storage of mouse RBCs. Loading stored murine RBCs with S1P prior to transfusion partially attenuated anemia-associated acute pulmonary vascular leak. Taken together, our results indicate that anemia and systemic inflammation can alter the S1P buffering capacity of RBCs, suggesting possible strategies for alleviating transfusion-related lung injury in clinical practice

Phosphorylation of human platelet glycoprotein IIIa (GPIIIa) : dissociation from fibrinogen receptor activation and phosphorylation of GPIIIa in vitro

Glycoprotein IIb-IIIa (GPIIb-IIIa) is the fibrinogen receptor on activated platelets. GPIIIa is phosphorylated in resting platelets and the incorporation of 32Pi increases with platelet activation. To address the functional significance of this modification, the stoichiometry of GPIIIa phosphorylation was determined in resting and activated platelets by estimating the specific activity of metabolic [gamma-32P]ATP from the specific activity of phosphatidic acid. Approximately 0.01 mol of P/mol of GPIIIa was phosphorylated in resting platelets and 0.03 mol of P/mol of GPIIIa was phosphorylated in thrombin-, phorbol ester-, or U46619-treated platelets. Myosin light chain (MLC) phosphorylation served as a positive control for this method (1.2 mol of P/mol of MLC). Phosphorylation of purified GPIIb-IIIa by human platelet protein kinase C (PKC) resulted in levels of GPIIIa phosphorylation similar to that in platelets (0.05 mol of P/mol of GPIIIa). However, while GPIIIa in platelets was phosphorylated primarily on threonine, purified GPIIIa treated with PKC was phosphorylated primarily on serine. These results suggest that PKC may not directly phosphorylate GPIIIa in intact platelets. Ca2+/calmodulin-dependent kinase II phosphorylated purified GPIIIa to higher levels (0.5 mol of P/mol of GPIIIa) with phosphorylation on both threonine and serine. The limited phosphorylation of GPIIIa in intact platelets suggests that this event is unlikely to affect functions involving large populations of GPIIb-IIIa, such as its conversion to a fibrinogen receptor. However, these results may suggest the existence of a more readily phosphorylated subpopulation of GPIIb-IIIa with potentially distinct structural or functional properties

Fibrinogen binding to purified platelet glycoprotein IIb-IIIa (integrin alpha IIb beta 3) is modulated by lipids.

Soluble fibrinogen binding to the glycoprotein IIb-IIIa complex (integrin alpha IIb beta 3) requires platelet activation. The intracellular mediator(s) that convert glycoprotein IIb-IIIa into an active fibrinogen receptor have not been identified. Because the lipid composition of the platelet plasma membrane undergoes changes during activation, we investigated the effects of lipids on the fibrinogen binding properties of purified glycoprotein IIb-IIIa. Anion exchange chromatography of lipids extracted from platelets exposed to thrombin or other platelet agonists resolved an activity that increased fibrinogen binding to glycoprotein IIb-IIIa. A monoester phosphate was important for activity, and phosphatidic acid coeluted with the peak of activity. Purified phosphatidic acid dose-dependently promoted a specific interaction between glycoprotein IIb-IIIa and fibrinogen which possessed many but not all of the properties of fibrinogen binding to activated platelets. Phosphatidic acid appeared to increase the proportion of fibrinogen binding-competent glycoprotein IIb-IIIa complexes without altering their affinity for fibrinogen. The effects of phosphatidic acid were a result of specific structural properties of the lipid and were not mimicked by other phospholipids. Lysophosphatidic acid, however, was a potent inducer of fibrinogen binding to glycoprotein IIb-IIIa. These results demonstrate that specific lipids can affect fibrinogen binding to purified glycoprotein IIb-IIIa and suggest that the lipid environment has the potential to influence fibrinogen binding to its receptor

Planning Implementation Success of Syncope Clinical Practice Guidelines in the Emergency Department Using CFIR Framework

Background and Objectives: Overuse and inappropriate use of testing and hospital admission are common in syncope evaluation and management. Though guidelines are available to optimize syncope care, research indicates that current clinical guidelines have not significantly impacted resource utilization surrounding emergency department (ED) evaluation of syncope. Matching implementation strategies to barriers and facilitators and tailoring strategies to local context hold significant promise for a successful implementation of clinical practice guidelines (CPG). Our team applied implementation science principles to develop a stakeholder-based implementation strategy. Methods and Materials: We partnered with patients, family caregivers, frontline clinicians and staff, and health system administrators at four health systems to conduct quantitative surveys and qualitative interviews for context assessment. The identification of implementation strategies was done by applying the CFIR-ERIC Implementation Strategy Matching Tool and soliciting stakeholders’ inputs. We then co-designed with patients and frontline teams, and developed and tested specific strategies. Results: A total of 114 clinicians completed surveys and 32 clinicians and stakeholders participated in interviews. Results from the surveys and interviews indicated low awareness of syncope guidelines, communication challenges with patients, lack of CPG protocol integration into ED workflows, and organizational process to change as major barriers to CPG implementation. Thirty-one patients and their family caregivers participated in interviews and expressed their expectations: clarity regarding their diagnosis, context surrounding care plan and diagnostic testing, and a desire to feel cared about. Identifying change methods to address the clinician barriers and patients and family caregivers expectations informed development of the multilevel, multicomponent implementation strategy, MISSION, which includes patient educational materials, mentored implementation, academic detailing, Syncope Optimal Care Pathway and a corresponding mobile app, and Lean quality improvement methods. The pilot of MISSION demonstrated feasibility, acceptability and initial success on appropriate testing. Conclusions: Effective multifaceted implementation strategies that target individuals, teams, and healthcare systems can be employed to plan successful implementation and promote adherence to syncope CPGs

Adipose-Derived Autotaxin Regulates Inflammation and Steatosis Associated with Diet-Induced Obesity

Autotaxin (ATX) is a secreted enzyme that generates the bioactive lipid lysophosphatidic acid (LPA). We generated mice with global inducible post-natal inactivation or adipose-specific loss of the Enpp2 gene encoding ATX. The animals are phenotypically unremarkable and exhibit differences in adipocyte size and adipose tissue expression of inflammatory genes after high fat feeding without gross differences in fat distribution or body mass. Surprisingly, both models of Enpp2- deficiency exhibited marked protection from high fat diet-induced hepatic steatosis. This phenotype was not associated with differences in dietary fat absorption but may be accounted for by differences in hepatic expression of genes involved in de novo synthesis of triglycerides. These findings suggest that pharmacological inhibition of ATX might be protective against hepatic steatosis

The P2Y(12) antagonists, 2MeSAMP and cangrelor, inhibit platelet activation through P2Y(12)/G(i)-dependent mechanism

BACKGROUND: ADP is an important physiological agonist that induces integrin activation and platelet aggregation through its receptors P2Y(1) (Gα(q)-coupled) and P2Y(12) (Gα(i)-coupled). P2Y(12) plays a critical role in platelet activation and thrombosis. Adenosine-based P2Y(12) antagonists, 2-methylthioadenosine 5\u27-monophosphate triethylammonium salt hydrate (2MeSAMP) and Cangrelor (AR-C69931MX) have been widely used to demonstrate the role of P2Y(12) in platelet function. Cangrelor is being evaluated in clinical trials of thrombotic diseases. However, a recent study reported that both 2MeSAMP and Cangrelor raise intra-platelet cAMP levels and inhibit platelet aggregation through a P2Y(12)-independent mechanism. METHODOLOGY/PRINCIPAL FINDINGS: The present work, using P2Y(12) deficient mice, sought to clarify previous conflicting reports and to elucidate the mechanisms by which 2MeSAMP and Cangrelor inhibit platelet activation and thrombosis. 2MeSAMP and Cangrelor inhibited aggregation and ATP release of wild-type but not P2Y(12) deficient platelets. 2MeSAMP and Cangrelor neither raised intracellular cAMP concentrations nor induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in washed human or mouse platelets. Furthermore, unlike the activators (PGI(2) and forskolin) of the cAMP pathway, 2MeSAMP and Cangrelor failed to inhibit Ca(2+) mobilization, Akt phosphorylation, and Rap1b activation in P2Y(12) deficient platelets. Importantly, while injection of Cangrelor inhibited thrombus formation in a FeCl(3)-induced thrombosis model in wild-type mice, it failed to affect thrombus formation in P2Y(12) deficient mice. CONCLUSIONS: These data together demonstrate that 2MeSAMP and Cangrelor inhibit platelet function through the P2Y(12)-dependent mechanism both in vitro and in vivo

Acute Effects of Implantable Cardioverter-Defibrillator Shocks on Biomarkers of Myocardial Injury, Apoptosis, Heart Failure, and Systemic Inflammation

Background: Implantable cardioverter‐defibrillator (ICD) shocks are potentially associated with myocardial injury, altered hemodynamics, apoptosis, and inflammatory signaling. Their precise cellular impact can be explored after defibrillation testing (DFT) via biomarkers. We evaluated changes in biomarkers after ICD shocks during DFT. Methods: We prospectively enrolled outpatients presenting for first implantation of a cardiac device. Biomarkers indicative of myocardial injury, inflammation, and apoptosis were measured before and after implantation, and compared between patients receiving DFT (DFT+) to those not (DFT−). Results: Sixty‐three patients were enrolled, 40 in the DFT+ group and 23 in the DFT− group. Average levels of troponin I, hsCRP, Calprotectin, N‐terminal pro B‐type natriuretic peptide (NTproBNP), and sFas increased by \u3e50% after cardiac device implantation compared to baseline. Increase in troponin never exceeded the 50‐fold upper limit of normal (2 ng/mL). Troponin trended higher in the DFT+ group at 8 hours (median 0.18 ng/mL, interquartile range [IQR] 0.11–0.48) versus the DFT− group (0.10 ng/mL, IQR 0.06–0.28, P = 0.0501); NTproBNP had a similar trend (P = 0.0581). sFas significantly increased in the DFT+ group from baseline (median 4663 pg/mL, IQR 2908–5679) to 24 hours (5039 pg/mL, IQR 3274–6261; P = 0.0338) but not in the DFT− group (P = 0.4705). Conclusion: DFT testing is associated with acutely increased plasma levels of troponin and sFas, a biomarker of apoptosis, along with a trend toward higher NTproBNP

Lipid-Induced Epigenomic Changes in Human Macrophages Identify a Coronary Artery Disease-Associated Variant that Regulates \u3cem\u3ePPAP2B\u3c/em\u3e Expression through Altered C/EBP-Beta Binding

Genome-wide association studies (GWAS) have identified over 40 loci that affect risk of coronary artery disease (CAD) and the causal mechanisms at the majority of loci are unknown. Recent studies have suggested that many causal GWAS variants influence disease through altered transcriptional regulation in disease-relevant cell types. We explored changes in transcriptional regulation during a key pathophysiological event in CAD, the environmental lipid-induced transformation of macrophages to lipid-laden foam cells. We used a combination of open chromatin mapping with formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) and enhancer and transcription factor mapping using chromatin immuno-precipitation (ChIP-seq) in primary human macrophages before and after exposure to atherogenic oxidized low-density lipoprotein (oxLDL), with resultant foam cell formation. OxLDL-induced foam cell formation was associated with changes in a subset of open chromatin and active enhancer sites that strongly correlated with expression changes of nearby genes. OxLDL-regulated enhancers were enriched for several transcription factors including C/EBP-beta, which has no previously documented role in foam cell formation. OxLDL exposure up-regulated C/EBP-beta expression and increased genomic binding events, most prominently around genes involved in inflammatory response pathways. Variants at CAD-associated loci were significantly and specifically enriched in the subset of chromatin sites altered by oxLDL exposure, including rs72664324 in an oxLDL-induced enhancer at the PPAP2B locus. OxLDL increased C/EBP beta binding to this site and C/EBP beta binding and enhancer activity were stronger with the protective A allele of rs72664324. In addition, expression of the PPAP2B protein product LPP3 was present in foam cells in human atherosclerotic plaques and oxLDL exposure up-regulated LPP3 in macrophages resulting in increased degradation of pro-inflammatory mediators. Our results demonstrate a genetic mechanism contributing to CAD risk at the PPAP2B locus and highlight the value of studying epigenetic changes in disease processes involving pathogenic environmental stimuli