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Introduction While the optimal care of patients with massive pulmonary embolism (PE) is unclear, the general goal of therapy is to rapidly correct the physiologic derangements propagated by obstructive clot. Extracorporeal membrane oxygenation (ECMO) in this setting is promising, however the paucity of data limits its routine use. Our institution expanded the role of ECMO as an advanced therapy option in the initial management of massive PE. The purpose of this project was to evaluate ECMO-treated patients with massive PE at an academic medical center and report shortterm mortality outcomes. Methods Thirty-two patients placed on ECMO for confirmed, massive PE from January 2012 to December 2015 were retrospectively analyzed. All patients had PE confirmed by computerized tomography and/or invasive pulmonary angiography. Results In our population of patients managed with ECMO, 21 (65.6%) patients survived to decannulation and 17 (53.1%) survived index hospitalization. Baseline characteristics and clinical variables showed no difference in age, gender, right ventricular-to-left ventricular ratios, or peak troponin-T between survivors and non-survivors. Non-survivors tended to have a previous history of malignancy. Cardiac arrest prior to ECMO cannulation was associated with worse outcomes. All 5 patients who received concomitant systemic thrombolysis died, while 11 of 15 patients who received catheter-directed thrombolysis survived. A lactic acid level ≤6 mmol/L had an 82.4% sensitivity and 84.6% specificity for predicting survival to discharge. Conclusion The practical approach of utilizing ECMO for massive PE is to reserve it for those who would receive the greatest benefit. Patients with poor perfusion, for example from cardiac arrest, may gain less benefit from ECMO. Our findings indicate that a serum lactate > 6 mmol/L may be an indicator of worse prognosis. Finally, in our patient population, catheter-directed thrombolytics was effectively combined with ECMO.

Introduction The multifunctional serine protease thrombin exerts proinflammatory and profibrotic cellular effects that may contribute to cardiac remodeling. This study was designed to investigate whether direct thrombin inhibition with dabigatran attenuates myocardial injury in the setting of pressure overload-induced heart failure. Material and methods Transverse aortic constriction (TAC) surgery was performed on C57Bl/6J male mice to elicit cardiac hypertrophy. TAC, or sham, mice were randomly assigned to receive chow supplemented with the oral anticoagulant, dabigatran etexilate, or placebo. Results Dabigatran did not affect cardiac hypertrophy, as measured by heart weight-to-body weight or the heart weight-to-tibia length, although a non-significant reduction in myocardial hypertrophic markers (ANP, BNP and MHC) occurred. Dabigatran reduced perivascular fibrosis by 25%, interstitial fibrosis by 54%, and the expression of myocardial fibrosis markers collagen I & III, MMP9, SMA, and PAR-1. These changes were associated with significant improvement in both coronary flow reserve and global left ventricular function. In cultured cardiac fibroblasts, dabigatran decreased thrombin and PAR-1-mediated collagen deposition by 30% and 37%, respectively. Conclusions Dabigatran attenuates cardiac fibrosis in the setting of pressure overload and improves coronary flow reserve and global cardiac function possibly by inhibiting thrombin activity and down-regulating PAR-1 expression in the absence of an effect on cardiomyocyte hypertrophy.

Pneumothorax (PTX) is a potential complication of vascular access during cardiac implantable electronic device (CIED) procedures and is being scrutinized as a health care-acquired condition.The purpose of this study was to determine the trends in PTX incidence in the United Stated over a 16-year period and to determine whether PTX is associated with increased mortality after adjustment for other factors.Using weighted sampling in the largest inpatient health database in the United States (National Inpatient Sample), we evaluated data from patients with a primary procedure of CIED implantation from 1998 to 2013 who had at least 1 new vascular access (new or upgrade of prior CIED). The unadjusted and adjusted associations of PTX with mortality and other parameters were examined.Among 3,764,703 CIED procedures, PTX occurred in 47,839 cases (1.3%). The apparent incidence of PTX peaked at 1.6% in 2012 and 2013, although this result may have been affected by a concomitant decrease of inpatient (vs outpatient) CIED. PTX was significantly associated with pulmonary complications, chest tube insertion, length of stay, and costs. Mortality was statistically higher in patients with PTX (1.2% vs 0.7%; P.001), a relationship that remained significant in a multivariate logistic regression analysis (odds ratio 1.50, 95% confidence interval 1.36-1.65; P.001). Age80 years, female gender, Caucasian race, chronic obstructive pulmonary disease, and dual-chamber (vs single-chamber) device were all associated with higher odds for PTX occurrence. Placement of a chest tube was a major determinant of worse outcomes and higher costs.PTX remains an important complication of CIED procedures and is associated with increased morbidity, mortality, and costs.

Managing Cushing's disease remains a challenge. Surgery is the first option of treatment and it offers a high success rate. Even in cases where biochemical remission is not achieved, it is crucial to obtain surgical tissue for morphological diagnosis because the therapeutic approach can be modified according to the findings.A literature search was performed using PubMed for information regarding pathology and Cushing's disease.The histopathological features found in the pituitary gland of patients with Cushing's disease are presented.Different subtypes of ACTH-producing pituitary tumors are recognized and characterized. The significance of finding a normal pituitary gland with or without Crooke's changes is also discussed.

In patients with acute coronary syndromes (ACS), early therapy with high-dose statins may reduce short-term adverse clinical outcomes. The mechanisms responsible are not known but could involve anti-inflammatory or anti-thrombotic effects. Compelling evidence from experimental models and clinical studies suggests that the interplay between inflammatory and thrombotic systems, typified by platelet–monocyte and platelet–neutrophil interactions, might be a key regulator of ischemic vascular events. The study sought to determine if early, high-dose administration of the HMG-CoA reductase inhibitor rosuvastatin in the setting of ACS exerts beneficial vascular effects by reducing, and inhibiting biomarkers of thromboinflammation, such as platelet-monocyte and platelet-neutrophil interactions, and biomarkers of myocardial necrosis. A total of 54 patients presenting with ACS within 8 h of symptom onset were randomized to rosuvastatin 40 mg or placebo. Rosuvastatin significantly reduced interactions between platelets and circulating neutrophils (P = 0.015) and monocytes (P = 0.009) within 24 h. No significant effects were observed on platelet aggregation or plasma levels of PF4, sP-selectin, or sCD40L, whereas significant reductions of RANTES occurred over time in both treatment groups. Plasma levels of myeloperoxidase (MPO) declined more rapidly with rosuvastatin therapy than placebo. In a subset of patients with normal cardiac necrosis biomarkers at randomization, rosuvastatin therapy was associated with less myocardial damage as measured by troponin-I or CK-MB. Early administration of high-dose statin therapy in patients with ACS appears to improve biomarkers of inflammation within 8 h, which may translate into fewer ischemic events. Electronic supplementary material The online version of this article (doi:10.1007/s11239-014-1142-x) contains supplementary material, which is available to authorized users.

Genome-wide association studies (GWAS) have linked genes encoding several soluble NSF attachment protein receptor (SNARE) regulators to cardiovascular disease risk factors. Because these regulatory proteins may directly affect platelet secretion, we used SNARE-containing complexes to affinity purify potential regulators from human platelet extracts. Syntaxin-binding protein 5 (STXBP5; also known as tomosyn-1) was identified by mass spectrometry, and its expression in isolated platelets was confirmed by RT-PCR analysis. Coimmunoprecipitation studies showed that STXBP5 interacts with core secretion machinery complexes, such as syntaxin-11/SNAP23 heterodimers, and fractionation studies suggested that STXBP5 also interacts with the platelet cytoskeleton. Platelets from Stxbp5 KO mice had normal expression of other key secretory components; however, stimulation-dependent secretion from each of the 3 granule types was markedly defective. Secretion defects in STXBP5-deficient platelets were confirmed via lumi-aggregometry and FACS analysis for P-selectin and LAMP-1 exposure. Interestingly, STXBP5-deficient platelets had altered granule cargo levels, despite having normal morphology and granule numbers. Consistent with secretion and cargo deficiencies, Stxbp5 KO mice showed dramatic bleeding in the tail transection model and defective hemostasis in the FeCl3-induced carotid injury model. Transplantation experiments indicated that these defects were due to loss of STXBP5 in BM-derived cells. Our data demonstrate that STXBP5 is required for normal arterial hemostasis, due to its contributions to platelet granule cargo packaging and secretion.

Objective— Platelet–neutrophil interactions play a key role in cardiovascular disease and inflammatory processes. Src family kinases mediate P-selectin glycoprotein ligand-1–Mac-1 cross talk necessary for firm platelet–neutrophil adhesion. Because Src family kinase activity can be regulated by cAMP-dependent pathways, in this work, we evaluated the role of phosphodiesterases in the signaling events that are required to sustain platelet–neutrophil interactions and neutrophil recruitment at the site of vascular injury. Approach and Results— In neutrophils exposed to P-selectin, selective phosphodiesterase 4 (PDE4) inhibition prevented Src family kinase–mediated phosphorylation of the proline-rich tyrosine kinase 2 on Tyr579/Tyr580. The effects of PDE4 inhibition required protein kinase A, likely through protein kinase A–mediated activation of COOH-terminal Src kinase, a major negative regulator of Src family kinases. PDE4, but not other phosphodiesterase inhibitors, reduced platelet–neutrophil conjugates as well as neutrophil firm adhesion on spread platelets under flow conditions. The effect of PDE4 inhibition on neutrophil adhesion was primarily mediated by downregulation of P-selectin–induced activation of Mac-1. In a murine model of endovascular injury, selective inhibition of PDE4 significantly reduced neutrophil recruitment at the site of vascular damage. Conclusions— This study identifies PDE4 as a central node in the signaling network that mediates platelet–neutrophil adhesion and suggests that pharmacological inhibition of PDE4 may represent a novel therapeutic avenue for the treatment of cardiovascular disease.

Objective— Lipid phosphate phosphatase 3 (LPP3), encoded by the PPAP2B gene, is an integral membrane enzyme that dephosphorylates, and thereby terminates, the G-protein–coupled receptor–mediated signaling actions of lysophosphatidic acid (LPA) and sphingosine-1-phosphate. LPP3 is essential for normal vascular development in mice, and a common PPAP2B polymorphism is associated with increased risk of coronary artery disease in humans. Herein, we investigate the function of endothelial LPP3 to understand its role in the development and human disease. Approach and Results— We developed mouse models with selective LPP3 deficiency in endothelial and hematopoietic cells. Tyrosine kinase Tek promoter–mediated inactivation of Ppap2b resulted in embryonic lethality because of vascular defects. LPP3 deficiency in adult mice, achieved using a tamoxifen-inducible Cre transgene under the control of the Tyrosine kinase Tek promoter, enhanced local and systemic inflammatory responses. Endothelial, but not hematopoietic, cell LPP3 deficiency led to significant increases in vascular permeability at baseline and enhanced sensitivity to inflammation-induced vascular leak. Endothelial barrier function was restored by pharmacological or genetic inhibition of either LPA production by the circulating lysophospholipase D autotaxin or of G-protein–coupled receptor–dependent LPA signaling. Conclusions— Our results identify a role for the autotaxin/LPA-signaling nexus as a mediator of endothelial permeability in inflammation and demonstrate that LPP3 limits these effects. These findings have implications for therapeutic targets to maintain vascular barrier function in inflammatory states.

The structurally simple glycero- and sphingo-phospholipids, lysophosphatidic acid (LPA) and sphingosine-1-phosphate, serve as important receptor-active mediators that influence blood and vascular cell function and are positioned to influence the events that contribute to the progression and complications of atherosclerosis. Growing evidence from preclinical animal models has implicated LPA, LPA receptors, and key enzymes involved in LPA metabolism in pathophysiologic events that may underlie atherosclerotic vascular disease. These observations are supported by genetic analysis in humans implicating a lipid phosphate phosphatase as a novel risk factor for coronary artery disease. In this review, we summarize current understanding of LPA production, metabolism, and signaling as may be relevant for atherosclerotic and other vascular disease.

Venous thromboembolism (VTE), including pulmonary embolism (PE) and deep vein thrombosis (DVT), is one of the most common causes of cardiovascular death worldwide. Monocytic cells (including monocytes/macrophages) and their derived tissue factor have been reported to play important roles in the development of DVT. However, the mechanism by which monocytes contribute to the development DVT is not well elucidated. In this study, we reported a critical role of inflammasome activation and pyroptosis in the development of DVT. Using a flow restriction-induced mouse DVT model in the inferior vena cava, we show that deficiency of caspase-1 protected against flow restriction-induced DVT. Inflammasome activation leads to interleukin (IL)-1b and IL-18 maturation/release and pyroptosis. Recent studies show that caspases-1 cleaves Gasdermin D (GSDMD) and triggers pyroptosis-a form of programmed cell death with similar morphology to necrosis. We tested the hypothesis that GSDMD-dependent pyroptosis drives inflammasome-induced coagulation and DVT using Gsdmd-/- mice. Indeed, flow restriction-induced DVT was inhibited by GSDMD deficiency. After induction of DVT, fibrin was deposited in the vein as detected by Western blot with a monoclonal antibody that specifically recognizes mouse fibrin, which were inhibited in the caspase-1 deficient mice and GSDMD deficient mice. IL-1b was also increased in tissue of vein following induction of DVT, which was inhibited by caspase-1 or GSDMD deficiency. Following inflammasome activation, pyroptotic macrophages release tissue factor (TF), an essential initiator of coagulation cascades. Consistently, flow restriction-induced DVT was flow restriction-induced DVT inhibited in inducible TF deficient mice. Our data reveal a critical role of inflammasome activation and pyroptosis in the development of DVT. Disclosures No relevant conflicts of interest to declare.

Tricuspid valve surgery (TVS) has been the standard treatment for tricuspid valve endocarditis (TVE) refractory to medical therapy, however many patients are not candidates for a surgical intervention due to prohibitive surgical risk. Percutaneous tricuspid vegetation debulking (PTVD) with the

Atrial septal defect is a common congenital heart disease. We designed a propensity-match analysis to describe the outcomes of acute PE in patients with ASD. Our primary outcome of interest was embolic cerebrovascular events (stroke/TIA). Using the National Inpatient Sample database, we extracted

Several studies for outcomes data regarding cardiac arrest use ICD-9 codes to establish study populations; however, the accuracy of this mode of data collection is unclear. This study evaluates institutional data for patients with cardiac arrest to determine precision and accuracy of the cardiac

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.

Venous thrombosis is a common medical disorder affecting nearly one million Americans each year. This review will focus primarily on the formation of venous thrombosis as well as current and future treatment options. While the full pathophysiology of venous thrombosis is not known, recent evidence points to a role for von Willebrand Factor, platelets, and neutrophils in thrombus formation. Many laboratory and imaging tests may be used for the diagnosis of venous thrombosis (VTE), but risk factor identification and clinical examination should not be overlooked as they are vital in assuring accurate treatment and patient identification. Historically heparin followed by a vitamin K antagonist has been the standard of care for treatment of VTE, but increasing data involving factor Xa inhibitors and direct thrombin inhibitors may mean a shift in first-line therapy in the very near future. Invasive therapies such as catheter-directed thrombolysis have also shown promise in the treatment of venous thrombosis and will likely see increased use in the future.

Transcatheter aortic valve implantation (TAVI) has emerged as a therapeutic alternative for patients with symptomatic aortic stenosis at high or prohibitive surgical risk. However, patients undergoing TAVI are also at high risk for both bleeding and stroke complications, and specific mechanical aspects of the procedure itself can increase the risk of these complications. The mechanisms of periprocedural bleeding complications seem to relate mainly to vascular/access site complications (related to the use of large catheters in a very old and frail elderly population), whereas the pathophysiology of cerebrovascular events remains largely unknown. Further, although mechanical complications, especially the interaction between the valve prosthesis and the native aortic valve, may play a major role in events that occur during TAVI, post-procedural events might also be related to a prothrombotic environment or state generated by the implanted valve, the occurrence of atrial arrhythmias, and associated comorbidities. Antithrombotic therapy in the setting of TAVI has been empirically determined, and unfractionated heparin during the procedure followed by dual antiplatelet therapy with aspirin (indefinitely) and clopidogrel (1 to 6 months) is the most commonly recommended treatment. However, bleeding and cerebrovascular events are common; these may be modifiable with optimization of periprocedural and post-procedural pharmacology. Further, as the field of antiplatelet and anticoagulant therapy evolves, potential drug combinations will multiply, introducing variability in treatment. Randomized trials are the best path forward to determine the balance between the efficacy and risks of antithrombotic treatment in this high risk-population.

Autotaxin (ATX) is a secreted lysophospholipase D (lysoPLD) that binds to integrin adhesion receptors. We dissected the roles of integrin binding and lysoPLD activity in stimulation of human breast cancer and mouse aortic vascular smooth muscle cell migration by ATX. We compared effects of wild-type human ATX, catalytically inactive ATX, an integrin binding-defective ATX variant with wild-type lysoPLD activity, the isolated ATX integrin binding N-terminal domain, and a potent ATX selective lysoPLD inhibitor on cell migration using transwell and single-cell tracking assays. Stimulation of transwell migration was reduced (18 or 27% of control, respectively) but not ablated by inactivation of integrin binding or inhibition of lysoPLD activity. The N-terminal domain increased transwell migration (30% of control). ATX lysoPLD activity and integrin binding were necessary for a 3.8-fold increase in the fraction of migrating breast cancer cell step velocities >0.7 μm/min. ATX increased the persistent directionality of single-cell migration 2-fold. This effect was lysoPLD activity independent and recapitulated by the integrin binding N-terminal domain. Integrin binding enables uptake and intracellular sequestration of ATX, which redistributes to the front of migrating cells. ATX binding to integrins and lysoPLD activity therefore cooperate to promote rapid persistent directional cell migration.—Wu, T., Kooi, C. V., Shah, P., Charnigo, R., Huang, C., Smyth, S. S., Morris, A. J. Integrin-mediated cell surface recruitment of autotaxin promotes persistent directional cell migration.

Rationale: Bioactive lipid mediators, derived from membrane lipid precursors, are released into the airway and airspace where they bind high-affinity cognate receptors and may mediate asthma pathogenesis. Lysophosphatidic acid (LPA), a bioactive lipid mediator generated by the enzymatic activity of extracellular autotaxin (ATX), binds LPA receptors, resulting in an array of biological actions on cell proliferation, migration, survival, differentiation, and motility, and therefore could mediate asthma pathogenesis.Objectives: To define a role for the ATX-LPA pathway in human asthma pathogenesis and a murine model of allergic lung inflammation.Methods: We investigated the profiles of LPA molecular species and the level of ATX exoenzyme in bronchoalveolar lavage fluids of human patients with asthma subjected to subsegmental bronchoprovocation with allergen. We interrogated the role of the ATX-LPA pathway in allergic lung inflammation using a murine allergic asthma model in ATX-LPA pathway–specific geneticall...

Lysophosphatidic acid (LPA) is a bioactive lipid mediator. Concentrations of the major LPA species in mouse plasma decreased uniformly following administration of a potent selective inhibitor of the LPA-generating lysophospholipase D autotaxin, identifying an active mechanism for removal of LPA from the circulation. LPA, akylglycerol phosphate (AGP), sphingosine 1-phosphate (S1P), and a variety of structural mimetics of these lipids, including phosphatase-resistant phosphonate analogs of LPA, were rapidly eliminated (t1/2 < 30 s) from the circulation of mice following intravenous administration of a single bolus dose without significant metabolism in situ in the blood. These lipids accumulated in the liver. Elimination of intravenously administered LPA was blunted by ligation of the hepatic circulation, and ∼90% of LPA administered through the portal vein was accumulated by the isolated perfused mouse liver at first pass. At early times following intravenous administration, more LPA was associated with a nonparenchymal liver cell fraction than with hepatocytes. Primary cultures of nonparenchymal liver cells rapidly assimilated exogenously provided LPA. Our results identify hepatic uptake as an important determinant of the bioavailability of LPA and bioactive lysophospholipid mimetics and suggest a mechanism to explain changes in circulating LPA levels that have been associated with liver dysfunction in humans.

Acute myocardial infarction (AMI) triggers mobilization of stem cells from bone marrow (BM) into peripheral blood (PB). Based on our observation that the bioactive sphingophospholipids, sphingosine-1 phosphate (S1P), and ceramide-1 phosphate (C1P) regulate trafficking of hematopoietic stem cells (HSCs), we explored whether they also direct trafficking of non-hematopoietic stem cells (non-HSCs). We detected a 3–6-fold increase in circulating CD34+, CD133+, and CXCR4+ lineage-negative (Lin−)/CD45− cells that are enriched in non-HSCs [including endothelial progenitors (EPCs) and very small embryonic-like stem cells (VSELs)] in PB from AMI patients (P

Lipid phosphate phosphatases (LPP) are integral membrane proteins with broad substrate specificity that dephosphorylate lipid substrates including phosphatidic acid, lysophosphatidic acid, ceramide 1-phosphate, sphingosine 1-phosphate, and diacylglycerol pyrophosphate. Although the three mammalian enzymes (LPP1-3) demonstrate overlapping catalytic activities and substrate preferences in vitro, the phenotypes of mice with targeted inactivation of the Ppap2 genes encoding the LPP enzymes reveal nonredundant functions. A specific role for LPP3 in vascular development has emerged from studies of mice lacking Ppap2b. A meta-analysis of multiple, large genome-wide association studies identified a single nucleotide polymorphism in PPAP2B as a novel predictor of coronary artery disease. In this review, we will discuss the evidence that links LPP3 to vascular development and disease and evaluate potential molecular mechanisms. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

See accompanying article on page 2058 In addition to their role in transporting cholesterol and triglycerides, plasma lipoprotein particles have been long known to promote signaling responses in many blood and vascular cell types. Some of these effects have been attributed to minor but potent bioactive lipid constituents. For example, oxidized phosphatidylcholines are inflammatory mediators that likely contribute to the development and progression of atherosclerosis that is driven by subendothelial accumulation of low-density lipoproteins.1 Similarly, some of the athero- and vasoprotective effects of high-density lipoproteins may be accounted for by the bioactive lipid sphingosine 1 phosphate that is carried on this class of lipoprotein particles in association with apolipoprotein M.2 A growing body of evidence implicates another bioactive lipid, lysophosphatidic acid (LPA) as a regulator of vascular development and pathologies. LPA is present in plasma bound to both lipoproteins and serum albumin. LPA-specific G-protein–coupled receptors expressed on vascular endothelial and smooth muscle cells regulate vascular permeability, inflammation, intimal hyperplasia (in the setting of vascular injury), and atherosclerosis.3 Although LPA is a ubiquitous intermediate in intracellular phospholipid synthesis, extracellular signaling LPA is primarily generated by a secreted lysophospholipase D called autotaxin.4 In this issue of the …

In the majority of cases, Cushing’s disease is the result of a small basophilic corticotroph microadenoma with an average size of less than 5 mm. Transsphenoidal microsurgery can cure patients with Cushing’s disease; however, selective removal of the lesion requires precise preoperative localization. In this article, we present the pathological findings and clinical outcomes of four patients who underwent inferior petrosal sinus sampling (IPSS) for ACTH, pituitary imaging and subsequent transsphenoidal surgery for the diagnosis and treatment of Cushing’s disease. All patients fulfilled accepted biochemical criteria for the diagnosis of ACTH-dependent Cushing’s syndrome. Histological examination revealed a basophilic corticotroph adenoma in two patients. In one other patient, only Crooke’s hyalinization was found; however, the patient achieved a complete clinical and biochemical remission following a hemihypophysectomy based on IPSS findings. Thus, a microadenoma was assumed or proven in three patients, of whom two were cured by surgery alone. In the third patient, cortisol excess persisted following transsphenoidal surgery because of a coexistent functioning adrenal adenoma. The fourth patient developed recurrent nodular corticotroph hyperplasia following a 17-yr remission. The second transsphenoidal procedure failed to ameliorate cortisol excess, necessitating a subsequent bilateral adrenalectomy. IPSS accurately localized the site of the lesion in all four cases. Although magnetic resonance imaging (MRI) identified a distinct lesion in three cases, two of these represented false positives (a cyst in one case and a prolactinoma in the other), whereas in only one did MRI correctly match the site of the lesion. In each case, conflicting test results and/or difficult management decisions posed a challenge. Thus, successful resolution of disease requires a multidisciplinary approach to validate clinical, biochemical, and radiographic data based on morphologic findings.

Obesity is an established risk factor for cardiovascular disease (CVD) and stroke, and the cardiovascular complications of obesity are a leading cause of potentially preventable death. Recent evidence suggests that increased cardiovascular mortality in patients with obesity may not be fully explained by associated risk factors such as dyslipidemia, hyperglycemia, insulin resistance and hypertension. In addition to contributing to traditional CVD risk factors, obesity is also characterized by a chronic sub-acute inflammatory state termed “metainflammation” involving increased circulating inflammatory cytokines. Autotaxin (ATX), encoded by the ecto-nucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) gene, is a potent cell motility-stimulating factor that is secreted by adipose tissue. ATX hydrolyzes lysophosphatidylcholine (LPC), a lipid that is abundant in the circulation, to lysophosphatidic acid (LPA). The main objective of this study was to explore whether adipose ATX contributes to systemic inflammatory responses in obesity. We established two animal models with reduced ATX expression: MX-1 Cre-mediated deletion of the ENPP2 gene to generate a global ATX loss (Mx1) and Adipoq-Cre mediated loss of ATX expression in adipocytes (AdipoQ). Mice were fed a high fat diet for up to 20 weeks. No differences in body weight between the ENPP2 loss and littler mate controls were found. RNA and proteins were extracted, qPCR and western blots were performed for ATX and inflammatory cytokines; MCP, TNF and IFN. Results demonstrate that mice lacking global or adipose-derived ATX display approx 2 fold lower adipose inflammation. Furthermore, LPS was used to provoke a systemic response in mice that had been on a HFD for 10 weeks. Six hours after LPS injection, RNA was extracted and qPCR performed for IL6, IL1beta and IFN gamma. In response to a LPS challenge, AdipoQ mice showed a 2 fold reduction in inflammatory response in adipose tissue. The data generated from these experiments will provide important insight into potential mechanisms of how ATX and adipose tissue influence obesity induced inflammation and results from this and future projects may identify potential therapeutic targets to prevent and treat obesity induced inflammation and CVD.

Diet-dependent obesity is a risk factor for coronary artery disease and a powerful risk factor for some cancers particularly in women. One mechanism that has been proposed to link diet-dependent obesity to disease is that diets high in fats and lipids promote the formation of bioactive lipid metabolites that increase disease risk. Lysophosphatidic acid (LPA) is a family of bioactive lysophospholipids that elicit broadly mitogenic and proinflammatory responses in multiple cell types, primarily through actions at cell surface G-protein coupled receptors. LPA promotes vascular smooth muscle cell differentiation and migration, decreases vascular barrier endothelial function, activates platelets and myeloid cells all of which could contribute to the development of atherosclerosis. In mice, genetic and pharmacological targeting of LPA receptors inhibits atherosclerosis. We conducted studies to investigate the impact of fasting and high fat feeding on circulating levels, metabolic sources and distribution of plasma LPA in both humans and mice. After overnight fasting, blood was drawn from human volunteers to obtain baseline LPA levels before being given a drink supplemented with fats to provide approximately 50% of daily energy requirement. Blood was subsequently drawn at intervals to prepare plasma. In mice, after overnight fasting, the animals were gavaged with olive oil either with or without a tracer phosphatidylcholine. These studies were extended to mice with genetically induced hyperlipidemia fed models of diet. Changes in plasma LPA levels were measured by using HPLC ESI multistage mass spectrometry. We found that plasma LPA levels in mice and humans are acutely sensitive to fasting and high fat feeding. In mice plasma LPA levels are dramatically increased by a combination of feeding a high fat atherogenic diet and genetic induction of hyperlipidemia by inactivation of the LDLr or apoE genes. In addition, in mice, plasma LPA can be formed from dietary phosphatidylcholine. These results provide support for the concept that high fat atherogenic diets promote the formation of circulating LPA and have led to development and characterization of mouse models that can be used to define the role of LPA in obesity in other disease processes including cancer.

Transcatheter aortic valve replacement (TAVR) has been increasingly used to treat patients with symptomatic aortic stenosis. Despite the evolution of the valves and their deployment, patients that have undergone TAVR continue to be at high risk for major adverse events following the procedure. We recently reported that TAVR elicits a thromboinflammatory state that may contribute to the observed post-procedural thrombocytopenia associated with worse clinical outcomes. With the approval of the newer generation valves and delivery systems, we have now compared thromboinflammatory responses in patients receiving the Medtronic CoreValve and Edward’s new SAPIEN XT valve. Blood cell numbers, platelet function, and biomarkers of systemic inflammation and thrombosis were analyzed in 34 sequential patients undergoing transcatheteraortic valve replacement with the Edward’s SAPIEN XT valve or the Medtronic CoreValve Following valve deployment, platelet count, platelet activity, and platelet factor 4 levels decreased, while the inflammatory marker interleukin-6 increased. There were no significant differences in any of the parameters between the two valve types. Interestingly, baseline inflammation status predicted the extent of thrombocytopenia observed in overall cohort. A comparions of the SAPIENT XT valve and the Medtronic CoreValve reveals that the new generation valves generate a lower inflammatory response than that observed in the older SAPIEN valce. Importantly, our results add to the growing body of literature that suggests the thromboinflammatory changes that occur early after TAVR may predict long-term outcomes and suggest potential beneficial effects of an anti-inflammatory strategic.

Lipid phosphate phosphatase3 (LPP3) encoded by the PPAP2B gene, is an integral membrane enzyme that degrades bioactive lysophospholipids including lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P) and related bioactive lipids. These bioactive lipids regulate a wide variety of cellular functions such as proliferation, apoptosis and differentiation. We suggest that LPP3 hydrolysis of LPA is a critical step in regulating pathophysiological important responses in vascular cells. To test this hypothesis, we generated mice lacking LPP3 by breeding Ppap2B fl / fl mice (fl/fl) animals expressing Cre recombinase under control of the SM22 promoter on the LDLr-/- background. At weaning, littermate SM22-Δ and fl/fl mice were fed Western Diet (WD) for 12 weeks. LPP3 expression in aortic SMCs was reduced (0.139 ± 0.0246 vs 1.0 ± 0; P < 0.001) and LPA content was significantly higher (9.32 ± 4.66 vs 5.66 ± 2.65; P < 0.05) in the aortic arch in SM22-Δ mice compared to control fl/fl mice. SM22-Δ mice demonstrated increased atherosclerotic lesions (18.98 ± 7.11% vs 6.06 ± 4.01%; P < 0.001), measured by en face analysis, of the aorta and significantly higher IL-6 expression (18.83 ± 2.87 vs 3.59 ± 8.92; P < 0.05) in the aortic arch. Sections of aorta at the level of the aortic root indicated that the loss of LPP3 upregulate CD68 and α-SMA staining. These results reveal that LPP3 normally functions to suppress atherosclerosis. LPP3 deficiency increases vascular LPA content, may promote the infiltration of inflammatory cells as macrophages into lesions and smooth muscle cell proliferation to accelerate the development of atherosclerosis.

Primary control of the staphylococcal virulon is mediated by the accessory gene regulator (agr) quorum-sensing system [1]. agr-defective mutants are attenuated for virulence in animal models of acute infection, and agents that block agr and quorum-sensing exhibit antiinfective properties [2]. However, agr-defective mutants are frequently recovered from patients with bacteremia, where the mutants are associated with persistent infection and poor outcome [3, 4]. The frequent recovery of agr− and mixtures of agr+ and agr− phenotypes from patients supports the idea that agr variation is selected for in vivo. However, it is unknown whether and to what extent loss-of-function mutations arise during the initial invasive stage of blood infection in human hosts. In the present work, we sought to determine the frequency of a within-host shift in agr functionality during infection by screening for mutational changes in the agr locus among 158 Staphylococcus aureus clone pairs obtained from blood and nasal sites of individual patients [5]. Additionally, when a clonal S. aureus isolate was recovered from the clinically presumed focus of infection, strains were characterized to determine their role as a reservoir for agr-defective mutants. To address the directionality of colonization and infection, we included in the analysis carriage strains isolated both before and after the detection of bacteremia. Finally, to determine whether fully virulent, agr+ strains were more likely to cause invasive disease than agr− strains, we compared the frequency of agr dysfunction in colonizing strains from bacteremic patients and uninfected controls. We report that agr− strains were frequently recovered from patients with S. aureus bacteremia and that clones having the same inactivating agr mutation were recovered from colonizing sites within the same individual. Furthermore, agr− strain frequencies were similar among carriers who developed bacteremia and controls who did not, providing evidence that variation in agr was not correlated with progression to disease. Taken together, these results support the importance of variables other than agr functionality, such as host risk factors, the mode of bloodstream invasion, and possibly inoculum size, in determining the invasiveness of S. aureus in hospitalized patients.

Objective:To evaluate the outcomes of patients with giant pituitary tumours (GPTs) who underwent a purely binasal endoscopic transsphenoidal surgery (BETS) and compare their outcomes with those achieved through craniotomy and microscopic transsphenoidal surgery (MTS).Methods:Seventy-two consecutive patients with GPTs (greater than 10 cm3 in volume) who were treated surgically with BETS, craniotomy, or MTS from October 1994 to July 2009 were reviewed for clinical outcomes, degree of tumor resection, recurrence rates, and surgical complications.Results:The BETS group had significantly better mean reduction of tumor volume (91%) than the craniotomy (63%, p = 0.001), and the MTS (63%, p = 0.010) groups. Gross total resection rates were also higher for BETS patients than for craniotomy patients (p = 0.010). Improvements in vision and headaches were noted in 96% and 100% of patients in the BETS group, respectively; these rates were similar to those in the craniotomy and MTS groups. Of the four patients with hormone-secreting tumours in the BETS group, three remained in remission. The median length-of-stay (four days) for the BETS group was shorter (p = 0.010), and surgical complications were less frequent (p = 0.037) and less severe compared to the craniotomy group. There were no differences in the recurrence rates: 79% percent of patients in the BETS group, 69% in the craniotomy group, and 79% in the MTS group were recurrence free at last follow-up (p = 0.829).Conclusions:Treatment of GPT with BETS offers excellent oncologic and clinical outcomes and can frequently obviate the need for craniotomy in these patients.

Cardiac complications following stroke or acute cerebrovascular accidents (CVA) are common; however, many of these complications are asymptomatic and do not cause adverse cardiac effects. Symptomatic events (such as acute myocardial infarction after CVA) rarely occur and are often the result of an underlying cardiac embolic source, such as a left ventricular thrombus. We report a case of spontaneous coronary thrombosis following thrombolytic therapy for acute CVA, and discuss the implication that an underlying systemic pro-thrombotic state may predispose individuals to thrombosis in disparate vascular beds.

Brown adipose tissue is a thermogenic organ that dissipates stored energy as heat to maintain body temperature. This process may also provide protection from development of diet-induced obesity. We report that the bioactive lipid mediator lysophosphatidic acid (LPA) markedly decreases differentiation of cultured primary brown adipocyte precursors, whereas potent selective inhibitors of the LPA-generating enzyme autotaxin (ATX) promote differentiation. Transgenic mice overexpressing ATX exhibit reduced expression of brown adipose tissue-related genes in peripheral white adipose tissue and accumulate significantly more fat than wild-type controls when fed a high-fat diet. Our results indicate that ATX and its product LPA are physiologically relevant negative regulators of brown fat adipogenesis and are consistent with a model in which a decrease in mature peripheral brown adipose tissue results in increased susceptibility to diet-induced obesity in mice.

Women are underrepresented in cardiovascular studies, even as their preponderance in the aging population steadily increases. Although concerns have been raised about the differential benefit of antiplatelet medications for women, the propensity for increased bleeding among women has also been recognized. A better understanding of the factors contributing to the observed sex-related differences in platelet biology is warranted. These factors include differences in the frequency and expression of genetic polymorphisms affecting platelet responsiveness to agonists (with and without antiplatelet therapies), which might be obtained through population-based studies and in large controlled clinical trials; inflammatory marker levels and their influence on atherothrombotic risk, and the role of specific hormones in mediating platelet activation and function. Knowledge gained about these mechanistic factors might inform the development of sex-specific antithrombotic treatment regimens that confer optimized safety and efficacy.

Rap1b is activated by platelet agonists and plays a critical role in integrin α(IIb)β(3) inside-out signaling and platelet aggregation. Here we show that agonist-induced Rap1b activation plays an important role in stimulating secretion of platelet granules. We also show that α(IIb)β(3) outside-in signaling can activate Rap1b, and integrin outside-in signaling-mediated Rap1b activation is important in facilitating platelet spreading on fibrinogen and clot retraction. Rap1b-deficient platelets had diminished ATP secretion and P-selectin expression induced by thrombin or collagen. Importantly, addition of low doses of ADP and/or fibrinogen restored aggregation of Rap1b-deficient platelets. Furthermore, we found that Rap1b was activated by platelet spreading on immobilized fibrinogen, a process that was not affected by P2Y(12) or TXA(2) receptor deficiency, but was inhibited by the selective Src inhibitor PP2, the PKC inhibitor Ro-31-8220, or the calcium chelator demethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis. Clot retraction was abolished, and platelet spreading on fibrinogen was diminished in Rap1b-deficient platelets compared with wild-type controls. The defects in clot retraction and spreading on fibrinogen of Rap1b-deficient platelets were not rescued by addition of MnCl(2), which elicits α(IIb)β(3) outside-in signaling in the absence of inside-out signaling. Thus, our results reveal two different activation mechanisms of Rap1b as well as novel functions of Rap1b in platelet secretion and in integrin α(IIb)β(3) outside-in signaling.

Autotaxin (ATX) is a secreted lysophospholipase D that generates the bioactive lipid mediator lysophosphatidic acid (LPA). We and others have reported that ATX binds to integrins, but the function of ATX-integrin interactions is unknown. The recently reported crystal structure of ATX suggests a role for the solvent-exposed surface of the N-terminal tandem somatomedin B-like domains in binding to platelet integrin αIIbβ(3). The opposite face of the somatomedin B-like domain interacts with the catalytic phosphodiesterase (PDE) domain to form a hydrophobic channel through which lysophospholipid substrates enter and leave the active site. Based on this structure, we hypothesize that integrin-bound ATX can access cell surface substrates and deliver LPA to cell surface receptors. To test this hypothesis, we investigated the integrin selectivity and signaling pathways that promote ATX binding to platelets. We report that both platelet β1 and β3 integrins interact in an activation-dependent manner with ATX via the SMB2 domain. ATX increases thrombin-stimulated LPA production by washed platelets ~10-fold. When incubated under conditions to promote integrin activation, ATX generates LPA from CHO cells primed with bee venom phospholipase A(2), and ATX-mediated LPA production is enhanced more than 2-fold by CHO cell overexpression of integrin β(3). The effects of ATX on platelet and cell-associated LPA production, but not hydrolysis of small molecule or detergent-solubilized substrates, are attenuated by point mutations in the SMB2 that impair integrin binding. Integrin binding therefore localizes ATX activity to the cell surface, providing a mechanism to generate LPA in the vicinity of its receptors.

Lung cell migration is a crucial step for re-epithelialization that in turn is essential for remodelling and repair after lung injury. In the present paper we hypothesize that secreted ATX (autotaxin), which exhibits lysoPLD (lysophospholipase D) activity, stimulates lung epithelial cell migration through LPA (lysophosphatidic acid) generation-dependent and -independent pathways. Release of endogenous ATX protein and activity was detected in lung epithelial cell culture medium. ATX with V5 tag overexpressed conditional medium had higher LPA levels compared with control medium and stimulated cell migration through Gαi-coupled LPA receptors, cytoskeleton rearrangement, phosphorylation of PKC (protein kinase C) δ and cortactin at the leading edge of migrating cells. Inhibition of PKCδ attenuated ATX–V5 overexpressed conditional medium-mediated phosphorylation of cortactin. In addition, a recombinant ATX mutant, lacking lysoPLD activity, or heat-inactived ATX also induced lung epithelial cell migration. Extracelluar ATX bound to the LPA receptor and integrin β4 complex on A549 cell surface. Finally, intratracheal administration of LPS (lipopolysaccharide) into the mouse airway induced ATX release and LPA production in BAL (bronchoalveolar lavage) fluid. These results suggested a significant role for ATX in lung epithelial cell migration and remodelling through its ability to induce LPA production-mediated phosphorylation of PKCδ and cortactin. In addition we also demonstrated assocation of ATX with the epithelial cell-surface LPA receptor and integrin β4.

Anaemia and RBC (red blood cell) transfusion may be associated with worse clinical outcomes, especially with longer blood storage duration prior to transfusion. The mechanisms underlying these harmful effects are unknown. RBCs have been proposed to buffer plasma S1P (sphingosine 1-phosphate), a lysophospholipid essential for the maintenance of endothelial integrity and important in the regulation of haematopoietic cell trafficking. The present study examined the effect of anaemia, RBC transfusion and RBC storage duration on plasma S1P levels. Plasma S1P from 30 individuals demonstrated a linear correlation with Hct (haematocrit; R2=0.51, P

Staphylococcal chromosomal cassette mec (SCCmec) is a mobile genetic element that carries resistance genes for beta-lactam antibiotics. Coagulase-negative staphylococci, such as S. epidermidis, are thought to be a reservoir of diverse SCCmec elements that can spread to the most virulent staphylococcal species, S. aureus, but very little is known about the extent of cross-species spread of these elements in natural populations or their dynamics in different species. We addressed these questions by using a sample of 86 S. aureus and S. epidermidis isolates with SCCmec type IV that were collected from a single hospital over a period of six months. To subtype SCCmec IV, we used multiplex PCR to detect structural variations and we used sequences from a fragment of the ccrB gene and from the dru repeats to detect additional variations. Multiplex PCR had significantly lower typeability than ccrB:dru sequencing, due to more nontypeable isolates among S. epidermidis. No statistically significant differences in diversity were detected by subtyping method or species. Interestingly, while only 4 of 24 subtypes (17%) were shared between species, these so-called shared subtypes represented 58 of 86 isolates (67%). The shared subtypes differed significantly between species in their frequencies. The shared subtypes were also significantly more concordant with genetic backgrounds in S. aureus than in S. epidermidis. Moreover, the shared subtypes had significantly higher minimum inhibitory concentrations to oxacillin in S. aureus than in S. epidermidis. This study has identified particular SCCmec IV subtypes with an important role in spreading beta-lactam resistance between species, and has further revealed some species differences in their abundance, linkage to genetic background, and antibiotic resistance level.

Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.

Lipid phosphate phosphatases (LPPs, encoded by the PPAP2 genes) are broad specificity enzymes that can dephosphorylate lipid phosphate esters such as phosphatidic acid (PA), lysophosphatidic acid (LPA), and sphingosine- 1- phosphate (S1P) that serve as critical intermediates in intracellular pathways of lipid metabolism and signaling (Fig. 1) (1). Of these substrates, LPA and S1P are also bioactive mediators that can be released or generated extracellularly and/or act on cell surface receptors to initiate a broad range of cellular responses (2). LPPs can localize to the cell surface with their active site facing the extracellular space allowing them to function as ecto enzymes (3, 4). Accordingly, it has been attractive to speculate that one function of these enzymes is to terminate the receptor-mediated signaling actions of LPA and S1P by dephosphorylating them to generate the corresponding alcohols, which are not receptor-active. In support of this concept, overexpression of LPP1 or LPP3 increases the LPP activity of several intact cells, and this activity is decreased by silencing or genetic inactivation of the corresponding PPAP2A and PPAP2B genes (5–7). These manipulations are broadly associated with reciprocal effects on LPA responsiveness of stereotypic cellular responses to this stimulus, particularly cell growth and migration. Mice that are genetically hypomorphic for the PPAP2A gene encoding LPP1 exhibit increased circulating LPA levels (8) whereas inducible postnatal and/or tissue-restricted inactivation of the PPAP2B gene encoding LPP3 alters S1P gradients that control lymphocyte egress from the lymphatic system to the peripheral circulation (9) and renders vascular smooth muscle cells more responsive to LPA (6). Fig. 1. Regulation of extra- and intra-cellular lipid metabolism and signaling by LPPs. Cell surface localized LPPs can act on extracellular or plasma membrane-associated substrates to limit their actions on cell surface receptors. LPPs can also act intracellularly ... In Drosophila, LPP homologs encoded by the Wunen genes regulate the migration of germ cells in the developing embryo (10). Ectopic expression of these genes in somatic tissues that are normally permissive for germ cell migration results in repulsion of the migrating germ cells. This nonautonomous function of the Wunen genes is consistent with the idea that a diffusible Wunen substrate regulates germ cell migration (11). However, Wunens are also expressed in the germ cells themselves from maternally inherited mRNA and inactivation of Wunen in the female germline also decreases germ cell migration identifying a cell autonomous function for these genes (12). LPPs also localize to intracellular membranes. Manipulations of LPP expression have also been shown to alter levels of cell-associated LPP substrates and products including PA and its dephosphorylation product diacylglycerol (DG) that are well established to regulate intracellular signaling pathways. These include pathways mediated by the Raf and mTOR protein kinases and conventional protein kinases C (13, 14). Evidence that LPP3 has an intracellular role as a regulator of vesicular transport between the endoplasmic reticulum and Golgi apparatus has also been reported (15). It is also important to note that not all studies of the effects of LPP overexpression have revealed phenotypes in cell and animals that can be explained simply by attenuation of LPA signaling (16). Accordingly, although there is a broad consensus that manipulating LPP expression can alter cellular signaling responses to bioactive lipid phosphate mediators in some systems and often in a manner that depends on catalytic activity of the enzymes, these effects could be mediated by directly limiting the agonist actions of the mediators by degradation of extracellular or cell-surface associated lipids, by postreceptor effects on downstream lipid signaling pathways, or a combination of both (Fig. 1). In the current issue of the Journal of Lipid Research, Tang et al. (17) generated breast cancer cell lines with inducible expression of wild-type and catalytically inactive LPP1. Increased ecto LPA and S1P phosphatase activity in these cells was associated with decreased growth and migration in response to LPA and serum (a rich source of LPA). Migration responses of these cells to LPA but not epidermal growth factor (EGF) were selectively attenuated by increased LPP1 expression, and these effects are likely mediated, at least in part, by decreased activation of Rho family guanosine triphosphate hydrolases (GTPases). Phosphatase resistant LPA analogs with agonist activities at LPA receptors (phosphonates and thiophosphates) offer an elegant approach to determine the extent to which observed decreases in responses to bioactive lipid phosphate agonists in LPP-overexpressing cells can be explained by increased dephosphorylation and consequent inactivation of these mediators (5–7). Tang et al. found that mobilization of intracellular Ca2+ in response to LPA receptor activation by a metabolically stable LPA analog or to an agonist acting at protease activated receptor 1 was preserved in LPP1-overexpressing cells but still decreased in comparison to the response observed in cells overexpressing a catalytically inactive LPP1 variant. While consistent with observations that LPP-resistant LPA analogs can circumvent decreases in LPA signaling responses observed in LPP-overexpressing cells, these observations provide further evidence that overexpression of LPP1 has postreceptor effects on signaling responses to both LPA and a nonlipid agonist, at least on the Ca2+ mobilization response studied. These authors reported that overexpression of LPP1 dramatically attenuates metastasis and tumor growth when these cells were studied in a mouse xenograft model, providing further evidence that targeting LPA metabolism and signaling could be a beneficial strategy for breast cancer therapy (18). Understanding how LPPs regulate lipid metabolism and signaling has broad relevance to human disease because a common variant of the PPAP2B gene encoding LPP3 that appears to predict lower expression of the enzyme is associated with increased risk of coronary artery disease (19). LPA is present in oxidized low density lipoprotein and elicits responses that are broadly inflammatory and pro-atherogenic in multiple blood and vascular cell types (20, 21). While PPAP2B is essential for embryonic development (22) (and must therefore function nonredundantly with the other PPAP2 genes during this process), mice with restricted or inducible postnatal inactivation of LPP3 in vascular smooth muscle cells and vascular endothelial cells exhibit increased injury-induced intimal hyperplasia and vascular permeability, respectively (6, 23). Because LPA receptor deficient mice are protected in models of aortic atherosclerosis, the most parsimonious explanation of these data would be that LPP3 normally suppresses atherosclerosis by opposing LPA signaling. However, this idea needs to be tested experimentally. Taken together with previous reports, this new study by Tang et al. focuses attention on the possibility that LPPs can also regulate signaling responses to LPA and potentially other agonists through postreceptor effects on intracellular signaling pathways. These observations suggest that the range and impact of alterations in LPP expression extend beyond inactivation of bioactive lipids at the cell surface or in the extra-cellular space. Postreceptor effects of LPP expression could be associated with human heritable cardiovascular disease risk and with breast and ovarian cancers.

Fibrinolytic therapy (FT) is recommended as an alternative reperfusion strategy in patients with ST elevation myocardial infarction (STEMI), when percutaneous coronary intervention (PCI) is not readily accessible. This study describes trends and outcomes of this strategy specifically in an older

Fibrinolytic therapy (FT) is recommended for the urgent management of ST elevation myocardial infarction (STEMI) among selected patients who cannot undergo percutaneous coronary intervention (PCI) in a timely manner. Hemorrhagic stroke (HS) is a known and feared complication of FT in these patients

A polybasic motif in the metabolic regulator lipin1 is both a membrane anchor and a nuclear localization sequence required for lipin1 function in phospholipid metabolism and adipogenesis., Lipins are phosphatidic acid phosphatases with a pivotal role in regulation of triglyceride and glycerophospholipid metabolism. Lipin1 is also an amplifier of PGC-1α, a nuclear coactivator of PPAR-α responsive gene transcription. Lipins do not contain recognized membrane-association domains, but interaction of these enzymes with cellular membranes is necessary for access to their phospholipid substrate. We identified a role for a conserved polybasic amino acid motif in an N-terminal domain previously implicated as a determinant of nuclear localization in selective binding of lipin1β to phosphatidic acid, using blot overlay assays and model bilayer membranes. Studies using lipin1β polybasic motif variants establish that this region is also critical for nuclear import and raise the possibility that nuclear/cytoplasmic shuttling of lipin1β is regulated by PA. We used pharmacological agents and lipin1β polybasic motif mutants to explore the role of PA-mediated membrane association and nuclear localization on lipin1β function in phospholipid metabolism and adipogenic differentiation. We identify a role for the lipin1 polybasic motif as both a lipid binding motif and a primary nuclear localization sequence. These two functions are necessary for full expression of the biological activity of the protein in intracellular lipid metabolism and transcriptional control of adipogenesis.

Summary. Background: The serine-threonine kinase Akt plays an important role in regulating platelet activation. Stimulation of platelets with various agonists results in Akt activation as indicated by Akt phosphorylation. However, the mechanisms of Akt phosphorylation in platelets are not completely understood. Objectives and Methods: We used P2Y12 knockout mice to address the role of P2Y12 in Akt phosphorylation in response to thrombin receptors in platelets. Results: Thrombin or the PAR4 thrombin receptor peptide AYPGKF at high concentrations stimulated substantial phosphorylation of Akt residues Thr308 and Ser473 in P2Y12-deficient platelets. AYPGKF-induced Akt phosphorylation is enhanced by expression of recombinant human PAR4 cDNA in Chinese hamster ovary (CHO) cells. P2Y12-independent Akt phosphorylation was not inhibited by integrin inhibitor peptide RGDS or integrin β3 deficiency. Akt phosphorylation induced by thrombin or AYPGKF in P2Y12-deficient platelets was inhibited by the calcium chelator dimethyl-BAPTA, the Src family kinase inhibitor PP2, and PI3K inhibitors, respectively. Conclusions: Our results reveal a novel P2Y12-independent signaling pathway mediating Akt phosphorylation in response to thrombin receptors.

A novel composite transposon (Tn 6072 ) resembling staphylococcal cassette chromosome mercury (SCC Hg ) was identified in a collection of sequence type (ST) 239 methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates from Romanian hospitals. Tn 6072 is homologous to the 5′ region of SCC Hg found in staphylococcal cassette chromosome mec (SCC mec ) type III prototype strain 85/2082 but lacks the characteristic mer operon. SCC Hg has previously been reported to integrate downstream of orfX , at the same chromosomal location as SCC mec . Tn 6072 , by contrast, is demarcated by two IS 431 elements, flanked by 8-bp direct repeats, and inserted upstream of the origin of replication, within an open reading frame homologous to SAR2700 of S. aureus strain MRSA252. Analysis of a geographically and temporally diverse collection of 111 strains from the ST239 clonal group uncovered 11 additional strains harboring Tn 6072 , demonstrating a lineage-specific insertion pattern. Complete sequence analysis of the SCC mec regions of two representative Romanian strains (BK16704, BK16691) revealed two additional novel structures derived from a type III SCC mec background. BK16704 possesses an SCC mec 3A.1.4 structure, with an IS 256 insertion downstream of the right chromosomal junction. In contrast, the SCC mec element of BK16691 is truncated downstream of the mec gene complex, with a 24-kb deletion encompassing the right chromosomal junction and an inverted downstream IS 256 element. This structure, tentatively named “ψSCC mec 16691 ,” confers methicillin resistance but lacks most of the J1/J2 region, including the ccr gene complex. Taken together, these findings provide evidence for the continuing evolution of SCC elements, as well as the ST239 clonal group.