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/

The natural cardioprotective particle HDL modulates connexin43 gap junction channels

Aims High-density lipoprotein (HDL) is known for its cardioprotective properties independent from its cholesterol transport activity. These properties are mediated by activation of kinases such as protein kinase C (PKC). Connexin43 (Cx43) is a gap junction protein present in ventricular cardiomyocytes. PKC-dependent phosphorylation modifies Cx43 gap junction channel properties and is involved in cardioprotection. We hypothesized that cardioprotective properties of HDL may be mediated in part by affecting Cx43 gap junction channels. Methods and results Neonatal rat cardiomyocytes were treated with HDL and Cx43 phosphorylation was evaluated by western blotting and immunofluorescence. We found that HDL promoted phosphorylation of Cx43 with a maximal induction at 5 min, which was inhibited by pre-treatment with various PKC inhibitors. Sphingosine-1-phosphate (S1P), a component of HDL, induced effects that were similar to those of HDL. These compounds significantly reduced diffusion of fluorescent dye among cardiomyocytes (∼50%) which could be prevented by PKC inhibition. As observed during optical recordings of transmembrane voltage, HDL and S1P depressed impulse conduction only minimally (<5%). Moreover, 5 min of HDL and S1P treatment at the onset of reperfusion significantly reduced infarct size (∼50%) in response to 30 min ischaemia in ex vivo experiments. Conclusion Short-term treatment with HDL or S1P induces phosphorylation of Cx43 by a PKC-dependent pathway. HDL-induced phosphorylation of Cx43 reduced the diffusion of large tracer molecules between cells, whereas impulse conduction was maintained. Moreover, 5 min treatment with HDL confers cardioprotection against ischaemia/reperfusion injury. These results link Cx43 for the first time to the short-term cardioprotective effects of HD

Anti-ApoA-1 IgGs in Familial Hypercholesterolemia Display Paradoxical Associations with Lipid Profile and Promote Foam Cell Formation

Anti-Apolipoprotein A-1 autoantibodies (anti-ApoA-1 IgG) promote atherogenesis via innate immune receptors, and may impair cellular cholesterol homeostasis (CH). We explored the presence of anti-ApoA-1 IgG in children (5-15 years old) with or without familial hypercholesterolemia (FH), analyzing their association with lipid profiles, and studied their in vitro effects on foam cell formation, gene regulation, and their functional impact on cholesterol passive diffusion (PD)

Neuroinflammation Plays a Critical Role in Cerebral Cavernous Malformation Disease

BackgroundCerebral cavernous malformations (CCMs) are neurovascular lesions caused by loss of function mutations in 1 of 3 genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3). CCMs affect ≈1 out of 200 children and adults, and no pharmacologic therapy is available. CCM lesion count, size, and aggressiveness vary widely among patients of similar ages with the same mutation or even within members of the same family. However, what determines the transition from quiescent lesions into mature and active (aggressive) CCM lesions is unknown.MethodsWe use genetic, RNA-sequencing, histology, flow cytometry, and imaging techniques to report the interaction between CCM endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils (CCM endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils interaction) during the pathogenesis of CCMs in the brain tissue.ResultsExpression profile of astrocytes in adult mouse brains using translated mRNAs obtained from the purification of EGFP (enhanced green fluorescent protein)-tagged ribosomes (Aldh1l1-EGFP/Rpl10a) in the presence or absence of CCM lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) identifies a novel gene signature for neuroinflammatory astrocytes. CCM-induced reactive astrocytes have a neuroinflammatory capacity by expressing genes involved in angiogenesis, chemotaxis, hypoxia signaling, and inflammation. RNA-sequencing analysis on RNA isolated from brain endothelial cells in chronic Pdcd10BECKO mice (CCM endothelium), identified crucial genes involved in recruiting inflammatory cells and thrombus formation through chemotaxis and coagulation pathways. In addition, CCM endothelium was associated with increased expression of Nlrp3 and Il1b. Pharmacological inhibition of NLRP3 (NOD [nucleotide-binding oligomerization domain]-' LRR [leucine-rich repeat]- and pyrin domain-containing protein 3) significantly decreased inflammasome activity as assessed by quantification of a fluorescent indicator of caspase-1 activity (FAM-FLICA [carboxyfluorescein-fluorochrome-labeled inhibitors of caspases] caspase-1) in brain endothelial cells from Pdcd10BECKO in chronic stage. Importantly, our results support the hypothesis of the crosstalk between astrocytes and CCM endothelium that can trigger recruitment of inflammatory cells arising from brain parenchyma (microglia) and the peripheral immune system (leukocytes) into mature active CCM lesions that propagate lesion growth, immunothrombosis, and bleedings. Unexpectedly, partial or total loss of brain endothelial NF-κB (nuclear factor κB) activity (using Ikkbfl/fl mice) in chronic Pdcd10BECKO mice does not prevent lesion genesis or neuroinflammation. Instead, this resulted in a trend increase in the number of lesions and immunothrombosis, suggesting that therapeutic approaches designed to target inflammation through endothelial NF-κB inhibition may contribute to detrimental side effects.ConclusionsOur study reveals previously unknown links between neuroinflammatory astrocytes and inflamed CCM endothelium as contributors that trigger leukocyte recruitment and precipitate immunothrombosis in CCM lesions. However, therapeutic approaches targeting brain endothelial NF-κB activity may contribute to detrimental side effects

People living with HIV display increased anti-apolipoprotein A1 auto-antibodies, inflammation, and kynurenine metabolites: a case–control study

ObjectiveThis study aimed to study the relationship between auto-antibodies against apolipoprotein A1 (anti-apoA1 IgG), human immunodeficiency virus (HIV) infection, anti-retroviral therapy (ART), and the tryptophan pathways in HIV-related cardiovascular disease.DesignThis case–control study conducted in South Africa consisted of control volunteers (n = 50), people living with HIV (PLWH) on ART (n = 50), and untreated PLWH (n = 44). Cardiovascular risk scores were determined, vascular measures were performed, and an extensive biochemical characterisation (routine, metabolomic, and inflammatory systemic profiles) was performed.MethodsAnti-apoA1 IgG levels were assessed by an in-house ELISA. Inflammatory biomarkers were measured with the Meso Scale Discovery® platform, and kynurenine pathway metabolites were assessed using targeted metabolomic profiling conducted by liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS).ResultsCardiovascular risk scores and vascular measures exhibited similarities across the three groups, while important differences were observed in systemic inflammatory and tryptophan pathways. Anti-apoA1 IgG seropositivity rates were 15%, 40%, and 70% in control volunteers, PLWH ART-treated, and PLWH ART-naïve, respectively. Circulating anti-apoA1 IgG levels were significantly negatively associated with CD4+ cell counts and positively associated with viremia and pro-inflammatory biomarkers (IFNγ, TNFα, MIPα, ICAM-1, VCAM-1). While circulating anti-apoA1 IgG levels were associated with increased levels of kynurenine in both control volunteers and PLWH, the kynurenine/tryptophan ratio was significantly increased in PLWH ART-treated.ConclusionHIV infection increases the humoral response against apoA1, which is associated with established HIV severity criteria and kynurenine pathway activation