Prostacyclin production in rat aortic smooth muscle cells: role of protein kinase C, phospholipase D and cyclooxygenase-2 expression

Objective: The present study was designed to investigate the role of protein kinase C (PKC) and phospholipase D (PLD) in angiotensin II (AngII)- and phorbol ester (PMA)-induced cyclooxygenase-2 (COX-2) expression and prostacyclin (PGI2) production in rat aortic smooth muscle cells (VSMC). Methods: Prostacyclin production in cultured VSMC was determined by radioimmunoassay. PKC activity was examined by measuring the transfer of 32P from (γ-32P)ATP to histone III-S. COX-2 expression was determined by Western blotting. To measure PLD activity, thin layer chromatography was used. Results: AngII (50 nM) and PMA (100 nM) promoted the translocation of PKC activity from the cytosol to the membranes within 30 min, followed by a strong increase in PLD activity as well as COX-2 expression and PGI2 production. After 48 h exposure to PMA, PKC was downregulated resulting in a complete suppression of its activity. PKC-downregulation and the PKC inhibitor CGP41251 abolished PMA- and AngII-induced PLD activation, suppressed the stimulatory effect of PMA on COX-2 expression and PGI2 production and strongly inhibited that of AngII. Furthermore, AngII- and PMA-induced PGI2 production depended on protein synthesis and COX-2 but not COX-1 activity. Inhibition of PLD-mediated phosphatidic acid (PA) formation by 1% 1-butanol abolished AngII-induced COX-2 expression and PGI2 secretion, while dioctanoyl PA increased COX-2 expression and PGI2 production in a time- and concentration-dependent manner. Conclusion: Our results indicate that in VSMC, AngII promotes PGI2 production to a large extent through a rise in COX-2 expression which is mediated by PA generated from increased PKC-dependent PLD activit

Native and reconstituted HDL protect cardiomyocytes from doxorubicin-induced apoptosis

Aims We analysed the impact of native and reconstituted HDL on doxorubicin-induced cardiomyocyte apoptosis. While it is an effective anti-cancer agent, doxorubicin has serious cardiotoxic side effects. HDL has been shown to protect cardiomyocytes, notably against oxidative stress. Methods and results Cultured neonatal rat ventricular cardiomyocytes were subjected to doxorubicin-induced stress, monitored as caspase3 activation, apoptotic DNA fragmentation and cell viability. The protective effects of HDL and sphingosine-1-phosphate (S1P) were investigated using native HDL, reconstituted HDL of varied composition and agonists and antagonists of S1P receptors. Anti-apoptotic signalling pathways were identified with specific inhibitors. Native and reconstituted HDL significantly decreased doxorubicin-induced cardiomyocyte apoptosis, essentially due to the S1P component of HDL. The latter was mediated by the S1P2 receptor, but not the S1P1 or S1P3 receptors. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) signalling pathway was required for the anti-apoptotic effects of HDL and S1P. The transcription factor Stat3 also played an important role, as inhibition of its activity compromised the protective effects of HDL and S1P on doxorubicin-induced apoptosis. Conclusion HDL and its sphingosine-1-phosphate component can protect cardiomyocytes against doxorubicin toxicity and may offer one means of reducing cardiotoxic side effects during doxorubicin therapy. The study identified anti-apoptotic pathways that could be exploited to improve cardiomyocyte surviva

Prostaglandin E2 activates Stat3 in neonatal rat ventricular cardiomyocytes: A role in cardiac hypertrophy

Objective: The purpose of this study was to investigate whether prostaglandin E2 (PGE2) induces Signal transducer and activator of transcription 3 (Stat3) activation in neonatal rat ventricular cardiomyocytes and if so to determine the possible role of this activation in PGE2-induced hypertrophic responses. Methods: Stat3 activation and its nuclear phosphorylation were determined by electrophoretic mobility shift assay (EMSA) and by Western blots, respectively. Protein synthesis was assessed by [3H]-leucine incorporation into total protein and cell surface was quantified by microscopic analysis. Results: We found that PGE2 induces a concentration- (1-100nM) and time-dependent increase in Stat3 activation, reaching maximal values after 90min of stimulation. Experiments with agonists and antagonists of the PGE2 receptor subtypes EP1-EP4 indicate that PGE2 activates Stat3 mainly through the EP4 receptor. We further observed that the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor U0126 abolishes PGE2-induced Stat3 activation whereas the p38 MAP kinase blocker SB203580 has no effect. Nuclear Stat3 phosphorylation induced by PGE2 is also suppressed by the translation and transcription inhibitors, cycloheximide and actinomycin D, respectively. Transfecting ventricular cardiomyocytes with a small interfering RNA (siRNA) targeting rat Stat3, we obtained an approximately 70% reduction in Stat3 expression, 24 and 48h after electroporation. In these Stat3-silenced cells, the PGE2-induced increase in protein synthesis and cell surface is strongly inhibited. Conclusion: In ventricular cardiomyocytes, PGE2 induces the activation of Stat3 which plays an essential role in PGE2-induced increase in cell size and protein synthesis. The activation of Stat3 occurs mainly through EP4 and involves ERK1/2 as well as newly synthesized protein(s

Native and reconstituted HDL activate Stat3 in ventricular cardiomyocytes via ERK1/2: Role of sphingosine-1-phosphate

Aims High-density lipoprotein (HDL) has been reported to have cardioprotective properties independent from its cholesterol transport activity. The influence of native HDL and reconstituted HDL (rHDL) on Stat3, the transcription factor playing an important role in myocardium adaptation to stress, was analysed in neonatal rat ventricular cardiomyocytes. We have investigated modulating the composition of rHDL as a means of expanding its function and potential cardioprotective effects. Methods and results Stat3 phosphorylation and activation were determined by western blotting and electrophoretic mobility shift assay (EMSA). In ventricular cardiomyocytes, HDL and the HDL constituent sphingosine-1-phosphate (S1P) induce a concentration- and time-dependent increase in Stat3 activation. They also enhance extracellular signal-regulated kinases (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. U0126, a specific inhibitor of MEK1/2, the upstream activator of ERK1/2, abolishes HDL- and S1P-induced Stat3 activation, whereas the p38 MAPK blocker SB203580 has no significant effect. Inhibition of the tyrosine kinase family Src (Src) caused a significant reduction of Stat3 activation, whereas inhibition of phosphatidylinositol 3-kinase (PI3K) had no effect. S1P and rHDL containing S1P have a similar strong stimulatory action on Stat3, ERK1/2, and p38 MAPK comparable to native HDL. S1P-free rHDL has a much weaker effect. Experiments with agonists and antagonists of the S1P receptor subtypes indicate that HDL and S1P activate Stat3 mainly through the S1P2 receptor. Conclusion In ventricular cardiomyocytes, addition of S1P to rHDL enhances its therapeutic potential by improving its capacity to activate Stat3. Activation of Stat3 occurs mainly via the S1P constituent and the lipid receptor S1P2 requiring stimulation of ERK1/2 and Src but not p38 MAPK or PI3K. The study underlines the therapeutic potential of tailoring rHDL to confront particular clinical situation

The PGE2-Stat3 interaction in doxorubicin-induced myocardial apoptosis

Aims Both cyclooxygenase-2 (COX-2) and the transcription factor signal transducer and activator of transcription 3 (Stat3) are involved in adaptive growth and survival of cardiomyocytes. In ventricular cardiomyocytes, prostaglandin E2 (PGE2), a major COX-2 product, leads to adaptive growth via Stat3 activation, but whether this transcription factor acts as a signalling molecule in PGE2-induced cell survival is unknown. Therefore, the purpose of this study was to determine whether PGE2 counteracts cardiac apoptosis induced by doxorubicin (DOX), and if so, whether Stat3 plays a critical role in this cardioprotective effect. Methods and results Neonatal rat ventricular cardiomyocytes were incubated with DOX (0.5 µM) and/or PGE2 (1 µM). Apoptosis was assessed by determining caspase3 activation and apoptotic DNA fragmentation. The role of Stat3 was evaluated in vitro and in vivo by transfecting cardiomyocytes with siRNA targeting rat Stat3 and by using cardiomyocyte-restricted Stat3 knockout (Stat3 KO) mice, respectively. Incubation of ventricular cardiomyocytes with PGE2 led to a time-dependent decrease in the DOX-induced caspase3 activation, reaching a maximal inhibition of 70 ± 5% after 4 h. Similarly, PGE2 inhibited DOX-induced DNA fragmentation by 58 ± 5% after 24 h. This antiapoptotic action of PGE2 was strongly reduced by the ERK1/2 inhibitor, U0126, whereas the p38 MAP kinase inhibitor, SB203580, had no effect. Depleting Stat3 expression by 50-60% in isolated ventricular cardiomyocytes markedly reduced the protective effect of PGE2 on DOX-induced caspase3 activation and DNA fragmentation. Likewise, the stable PGE2 analogue, 16,16-dimethyl-PGE2, was unable to counteract cardiac apoptosis induced by DOX in Stat3 KO mice. Conclusion Our results demonstrate that PGE2 prevents myocardial apoptosis induced by DOX. This protection requires the activation of the prosurvival pathways of Stat3 and ERK1/

Suppression of Exosomal PD-L1 Induces Systemic Anti-tumor Immunity and Memory.

PD-L1 on the surface of tumor cells binds its receptor PD-1 on effector T cells, thereby suppressing their activity. Antibody blockade of PD-L1 can activate an anti-tumor immune response leading to durable remissions in a subset of cancer patients. Here, we describe an alternative mechanism of PD-L1 activity involving its secretion in tumor-derived exosomes. Removal of exosomal PD-L1 inhibits tumor growth, even in models resistant to anti-PD-L1 antibodies. Exosomal PD-L1 from the tumor suppresses T cell activation in the draining lymph node. Systemically introduced exosomal PD-L1 rescues growth of tumors unable to secrete their own. Exposure to exosomal PD-L1-deficient tumor cells suppresses growth of wild-type tumor cells injected at a distant site, simultaneously or months later. Anti-PD-L1 antibodies work additively, not redundantly, with exosomal PD-L1 blockade to suppress tumor growth. Together, these findings show that exosomal PD-L1 represents an unexplored therapeutic target, which could overcome resistance to current antibody approaches

High sensitivity troponin T and I reflect mitral annular plane systolic excursion being assessed by cardiac magnetic resonance imaging

Purpose: This study aims to evaluate the association between high sensitivity troponins (hsTn) and mitral annular plane systolic excursion (MAPSE) in patients undergoing cardiac magnetic resonance imaging (cMRI). Methods: Patients undergoing cMRI were prospectively enrolled. Patients with right ventricular dysfunction(< 50%) were excluded. Blood samples for measurements of hsTn and amino-terminal pro-brain natriuretic peptide (NT-proBNP) were collected at the time of cMRI. Results: 84 patients were included. Median left ventricular ejection fraction was 59% (IQR 51–64%). HsTn were correlated inversely with MAPSE within multivariable linear regression models (hsTnI: Beta − 0.19; T − 1.96; p = 0.05; hsTnT: Beta − 0.26; T − 3.26; p = 0.002). HsTn increased significantly according to decreasing stages of impaired MAPSE (p < 0.003). HsTn discriminated patients with impaired MAPSE < 11 mm (hsTnT: AUC = 0.67; p = 0.008; hsTnI: AUC = 0.64; p = 0.03) and < 8 mm (hsTnT: AUC = 0.79; p = 0.0001; hsTnI: AUC = 0.75; p = 0.001) and were still significantly associated in multivariable logistic regression models with impaired MAPSE < 11 mm (hsTnT: OR = 4.71; p = 0.002; hsTnI: OR = 4.22; p = 0.009). Conclusions: This study demonstrates that hsTn are able to reflect MAPSE being assessed by cMRI

Ischemic biomarker heart-type fatty acid binding protein (hFABP) in acute heart failure - diagnostic and prognostic insights compared to NT-proBNP and troponin I

Background: To evaluate diagnostic and long-term prognostic values of hFABP compared to NT-proBNP and troponin I (TnI) in patients presenting to the emergency department (ED) suspected of acute heart failure (AHF). Methods: 401 patients with acute dyspnea or peripheral edema, 122 suffering from AHF, were prospectively enrolled and followed up to 5 years. hFABP combined with NT-proBNP versus NT-proBNP alone was tested for AHF diagnosis. Prognostic value of hFABP versus TnI was evaluated in models predicting all-cause mortality (ACM) and AHF related rehospitalization (AHF-RH) at 1 and 5 years, including 11 conventional risk factors plus NT-proBNP. Results: Additional hFABP measurements improved diagnostic specificity and positive predictive value (PPV) of sole NT-proBNP testing at the cutoff <300 ng/l to “rule out” AHF. Highest hFABP levels (4th quartile) were associated with increased ACM (hazard ratios (HR): 2.1–2.5; p = 0.04) and AHF-RH risk at 5 years (HR 2.8–8.3, p = 0.001). ACM was better characterized in prognostic models including TnI, whereas AHF-RH was better characterized in prognostic models including hFABP. Cox analyses revealed a 2 % increase of ACM risk and 3–7 % increase of AHF-RH risk at 5 years by each unit increase of hFABP of 10 ng/ml. Conclusions: Combining hFABP plus NT-proBNP (<300 ng/l) only improves diagnostic specificity and PPV to rule out AHF. hFABP may improve prognosis for long-term AHF-RH, whereas TnI may improve prognosis for ACM. Trial registration: ClinicalTrials.gov identifier: NCT00143793

Cultivating Everyday Life: Yards, Nature and Time in the City

University of Minnesota Ph.D. dissertation. 2015. Major: Geography. Advisors: Vinay Gidwani, Helga Leitner. 1 computer file (PDF); 393 pages.This dissertation focuses on everyday practices in residential yards, in the context of recent shifts towards urban sustainability policies and projects. Yards, and the variegated access to these private landscapes, are deeply political, and shaped by fundamentally racialized histories of home ownership and urbanization in US cities and suburbs. Yards are also an arena in which people are confronted with an array of contemporary social and environmental issues. Through qualitative and ethnographic fieldwork with residents in three diverse Minneapolis neighborhoods, I studied how yards are inhabited, experienced, and cultivated. I also analyzed municipal sustainability policies and environmental advocacy projects, to situate residents’ experiences within regimes of urban governance. I found yards are experienced and understood by residents in much more diverse and complex ways than is generally considered from scholarly and policy perspectives. Engagement with yards often involves decades of maintenance, cultivation, and care. I have found a surprisingly diverse range of informal property arrangements and sharing economies, with varying forms and meanings across and within study areas. Engagement with yards also depends on embodied skills, socioeconomic positions, and capacities to pause and attune to more-than-human rhythms. I argue yards and yard practices contribute to the reinforcement of certain fundamental urban logics such as private property and the production of a discrete and manageable nature. But everyday yard practices also provide disruptions to these logics and create the conditions for new social relations to emerge, such as urban commons in variegated forms. Furthermore, cultivating yards entail affective attunements between human practice and encounters with more-than-human organisms, within the context of sociopolitical relations at multiple scales. Thus, the research contributes to debates about urban environmentalisms by considering sustainability in terms of experiential and affective registers beyond best practices and measure. The research also reveals diverse and collective practices of property ownership and stewardship, in the midst of what is often considered the most iconic landscape of American private property – neighborhoods of single family houses. Finally, the research contributes to recent calls within geography about the possibilities and limitations of a renovated phenomenology in the ways geographers study and represent diverse human experiences

Comparative analysis of high-sensitivity cardiac troponin I and T for their association with coronary computed tomography-assessed calcium scoring represented by the Agatston score

Background: This study evaluates the association between high-sensitivity cardiac troponin I (hs-cTnI) and T (hs-cTnT) and coronary calcium concentration (CAC) detected by coronary computed tomography (CCT) and evaluated with the Agatston score in patients with suspected coronary artery disease (CAD). Methods: Patients undergoing CCT during routine clinical care were enrolled prospectively. CCT was indicated for patients with a low to intermediate pretest probability for CAD. Within 24 h of CCT examination, peripheral blood samples were taken to measure cardiac biomarkers hs-cTnI and hs-cTnT. Results: A total of 76 patients were enrolled including 38% without detectable CAC, 36% with an Agatston score from 1 to 100, 17% from 101 to 400, and 9% with values ≥ 400. hs-cTnI was increasing alongside Agatston score and was able to differentiate between different groups of Agatston scores. Both hs-cTn discriminated values greater than 100 (hs-cTnI, AUC = 0.663; p = 0.032; hs-cTnT, AUC = 0.650; p = 0.048). In univariate and multivariate logistic regression models, hs-cTnT and hs-cTnI were significantly associated with increased Agatston scores. Patients with hs-cTnT ≥ 0.02 µg/l and hs-cTnI ≥ 5.5 ng/l were more likely to reveal values ≥ 400 (hs-cTnT; OR = 13.4; 95% CI 1.545–116.233; p = 0.019; hs-cTnI; OR = 8.8; 95% CI 1.183–65.475; p = 0.034). Conclusion: The present study shows that the Agatston score was significantly correlated with hs cardiac troponins, both in univariable and multivariable linear regression models. Hs-cTnI is able to discriminate between different Agatston values. The present results might reveal potential cut-off values for hs cardiac troponins regarding different Agatston values. Trial registration Cardiovascular Imaging and Biomarker Analyses (CIBER), NCT03074253 https://clinicaltrials.gov/ct2/show/record/NCT0307425