We isolated and subjected wild-type (WT) and RIP2^–/– mouse hearts to 30 min of global ischaemia and 120 min of reperfusion with or without perfusion of MDP (10 µg/mL) before or after the ischaemic period and determined the infarct size. We examined activation of the TAK1/nuclear factor B (NFB) signalling pathway. The effect of TAK1 inhibition on MDP-induced cardioprotection was also evaluated. Exposure to MDP during reperfusion significantly reduced infarct size in WT hearts (from 51.7 ± 5.6% in control to 38.1 ± 6.7%, P < 0.05), but not in RIP2^–/– hearts or in WT hearts with coincident pharmacological inhibition of TAK1. MDP treatment significantly increased the levels of p-TAK1 and p-JNK (Jun N-terminal kinase) and led to NFB activation via phosphorylation and degradation of IkappaB in the WT, but not in the RIP2^–/–, myocardium.

These results indicate that MDP at reperfusion induced cardioprotection through an RIP2/TAK1-dependent mechanism.

Isolated rat cardiomyocytes were stimulated with exogenous S1P, the selective S1P1 receptor agonist SEW2871, or the S1P1/3 receptor agonist FTY720. Western blot analysis was performed to analyse downstream signalling pathways. Ischaemia–reperfusion studies were conducted in rat cardiomyocytes, isolated Langendorff-perfused rat hearts, and in human myocardial muscle strip preparations to evaluate the effect of S1P receptor agonists on cell death and recovery of mechanical function. All S1P receptor agonists were able to activate Akt. This was associated with transactivation of the epidermal growth factor receptor. In isolated cardiomyocytes, selective stimulation of the S1P1 receptor by SEW2871 induced protection against cell death when administered either before or after ischaemia–reperfusion. In isolated rat hearts, treatment with FTY720 during reperfusion attenuated the rise in left ventricular end-diastolic pressure (LVEDP) and improved the recovery of left ventricular developed pressure without limiting infarct size. However, selective S1P1 receptor stimulation did not improve functional recovery but rather increased LVEDP. Additional experiments employing a human myocardial ischaemia–reperfusion model also demonstrated improved functional recovery induced by FTY720 treatment during reperfusion.

Pharmacological S1P receptor agonists have distinct effects on ischaemia–reperfusion injury. Their efficacy when applied during reperfusion makes them potential candidates for pharmaceutical postconditioning therapy after cardiac ischaemia.

Adult mouse cardiomyocytes were cultured in normal-glucose (NG, 5.5 mM) or high-glucose (HG, 25 mM) medium. Twelve hours after NG or HG pre-culture, cardiomyocytes were subjected to 3 h of simulated ischaemia (SI), followed by 3 h of reperfusion (R) in NG medium. Prior to and after SI/R, the following were determined: cardiomyocyte death and apoptosis, sustained oxidative/nitrative stress and thioredoxin (Trx) activity, expression, and nitration. Compared with NG-cultured cardiomyocytes, 12 h HG culture significantly increased superoxide and peroxynitrite production, increased Trx-1 nitration, and reduced Trx activity (P < 0.01). Despite being subject to identical SI/R procedures and conditions, cells pre-cultured in HG sustained greater injury, evidenced by elevated lactate dehydrogenase release and caspase-3 activation (P < 0.01). Moreover, SI/R induced greater superoxide/peroxynitrite overproduction and greater Trx-1 nitration and inactivation in HG pre-cultured cardiomyocytes than in NG pre-cultured cardiomyocytes. Finally, the supplementation of human Trx-1, superoxide scavenger, or peroxynitrite decomposition catalyst in HG pre-cultured cells reduced Trx-1 nitration, preserved Trx-1 activity, and normalized SI/R injury to levels observed in NG pre-cultured cardiomyocytes.

High glucose sensitized cardiomyocytes to ischaemia/reperfusion injury through nitrative Trx-1 inactivation. Interventions restoring Trx-1 activity in the diabetic heart may represent novel therapies attenuating cardiac injury in diabetic patients.

Reverse transcriptase–polymerase chain reaction analysis showed GHRH receptor (GHRH-R) mRNA in adult rat ventricular myocytes (ARVMs) and in rat heart H9c2 cells. In ARVMs, GHRH prevented cell death and caspase-3 activation induced by serum starvation and by the β-adrenergic receptor agonist isoproterenol. The GHRH-R antagonist JV-1-36 abolished GHRH survival action under both experimental conditions. GHRH-induced cardiac cell protection required extracellular signal-regulated kinase (ERK)1/2 and phosphoinositide-3 kinase (PI3K)/Akt activation and adenylyl cyclase/cAMP/protein kinase A signalling. Isoproterenol strongly upregulated the mRNA and protein of the pro-apoptotic inducible cAMP early repressor, whereas GHRH completely blocked this effect. Similar to ARVMs, in H9c2 cardiac cells, GHRH inhibited serum starvation- and isoproterenol-induced cell death and apoptosis through the same signalling pathways. Finally, GHRH improved left ventricular recovery during reperfusion and reduced infarct size in Langendorff-perfused rat hearts, subjected to ischaemia–reperfusion (I/R) injury. These effects involved PI3K/Akt signalling and were inhibited by JV-1-36.

Our findings suggest that GHRH promotes cardiac myocyte survival through multiple signalling mechanisms and protects against I/R injury in isolated rat heart, indicating a novel cardioprotective role of this hormone.

Hearts from C57BL/6J and three groups of leptin-deficient Ob/Ob mice (food-restricted, ad libitum, and leptin-repleted) were examined 4 weeks after permanent left coronary artery ligation or sham operation. Inflammatory and apoptotic cell number was determined in cardiac sections by immunostaining. Expression of cardiac bcl-2, survivin, and pro and active caspase-3 was determined and correlated with in vitro caspase-3 activity. In the absence of MI, both lean and obese leptin-deficient mice exhibited increased cardiac apoptosis compared with wild-type mice. After MI, the highest rates of apoptosis were seen in the infarcted tissue of lean and obese Ob/Ob mice. Further, leptin-deficient hearts, as well as hearts from wild-type mice treated with the STAT-3 inhibitor WP1066, exhibited blunted anti-apoptotic bcl-2 and survivin gene expression, and increased caspase-3 protein expression and activity. The increased caspase-3 activity and apoptosis in hearts of leptin-deficient mice after MI was significantly attenuated in Ob/Ob mice replete with leptin, reducing apoptosis to levels comparable to that observed in wild-type mice after MI.

These results demonstrate that intact leptin signalling post-MI acts through STAT-3 to increase anti-apoptotic bcl-2 and survivin gene expression and reduces caspase-3 activity, consistent with a cardioprotective role of leptin in the setting of chronic ischaemic injury.

We assessed the protective role of parstatin in an in vivo and in vitro rat model of myocardial ischaemia–reperfusion injury. Parstatin treatment before, during, and after ischaemia decreased infarct size by 26%, 23%, and 18%, respectively, in an in vivo model of ischaemia–reperfusion injury. Parstatin treatment immediately before ischaemia decreased infarct size by 65% and increased recovery in ventricular function by 23% in an in vitro model. We then assessed whether parstatin induced cardioprotection by activation of a Gi-protein-dependent pathway. Gi-protein inactivation by pertussis toxin completely abolished the cardioprotective effects. The cardioprotective effects were also abolished by inhibition of nitric oxide synthase (NOS), extracellular signal-regulated kinases 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK), and K_ATP channels in vitro. Furthermore, parstatin increased coronary flow and decreased perfusion pressure in the isolated heart. The vasodilatory properties of parstatin were confirmed in rat coronary arterioles.

A single treatment of parstatin administered prior to ischaemia confers immediate cardioprotection by recruiting the Gi-protein activation pathway including p38 MAPK, ERK1/2, NOS, and K_ATP channels. Parstatin exerts effects on both the cardiomyocytes and the coronary circulation to induce cardioprotection. This suggests a potential therapeutic role of parstatin in the treatment of cardiac injury resulting from ischaemia and reperfusion.

We created an adenovirus carrying short-interfering RNA (siRNA) that inactivates CypD. Transduction of CypD-siRNA in rat cardiomyocytes achieved a 61% reduction in CypD mRNA and a 63% reduction in protein levels as well as protection against oxidant-induced _m loss and cytotoxicity. To further investigate the effects in vivo, we monitored the spatio-temporal changes of _m in perfused rat hearts subjected to ischaemia/reperfusion using two-photon imaging. Adult rats received direct intramyocardial injections of the adenovirus. Two to three days after injection, rat hearts were perfused by the Langendorff method and _m levels of individual cells were monitored. The progressive loss of _m during ischaemia/reperfusion was significantly suppressed in CypD-siRNA-transduced cells compared with non-transduced cells. Furthermore, the protective effect of CypD-siRNA was dose-dependent.

Therapeutic interventions designed to inactivate CypD may be a promising strategy for reducing cardiac injury against myocardial ischaemia/reperfusion. The two-photon imaging technique provides deeper insight into cardioprotective therapy that targets mitochondria.

Rabbits were chronically instrumented with a coronary artery occluder and myocardial ultrasonic crystals for assessment of segment length-shortening. Two weeks later they were re-anaesthetized and underwent either a normothermic 30-min coronary artery occlusion (CAO) (Control group, n = 7) or a comparable CAO with cooling initiated by a 10-min hypothermic TLV and maintained by a cold blanket placed on the skin. Cooling was initiated after 5 or 15 min of CAO (Hypo-TLV and Hypo-TLV_15' groups, n = 6 and 5, respectively). A last group underwent normothermic TLV during CAO (Normo-TLV group, n = 6). Wall motion was measured in the conscious state over three days of reperfusion before infarct size evaluation and histology. Additional experiments were done for myocardial sampling in anaesthetized rabbits for mitochondrial studies. The Hypo-TLV procedure induced a rapid decrease in myocardial temperature to 32–34°C. Throughout reperfusion, segment length-shortening was significantly increased in Hypo-TLV and Hypo-TLV_15' vs. Control and Normo-TLV (15.1 ± 3.3%, 16.4 ± 2.3%, 1.8 ± 0.6%, and 1.1 ± 0.8% at 72 h, respectively). Infarct sizes were also considerably attenuated in Hypo-TLV and Hypo-TLV_15' vs. Control and Normo-TLV (4 ± 1%, 11 ± 5%, 39 ± 2%, and 42 ± 5% infarction of risk zones, respectively). Mitochondrial function in myocardial samples obtained at the end of ischaemia or after 10 min of reperfusion was improved by Hypo-TLV with respect to ADP-stimulated respiration and calcium-induced opening of mitochondrial permeability transition pores (mPTP). Calcium concentration opening mPTP was, e.g., increased at the end of ischaemia in the risk zone in Hypo-TLV vs. Control (157 ± 12 vs. 86 ± 12 µM). Histology and electron microscopy also revealed better preservation of lungs and of cardiomyocyte ultrastructure in Hypo-TLV when compared with Control.

Institution of rapid cooling by TLV during ischaemia reduces infarct size as well as other sequelae of ischaemia, such as post-ischaemic contractile and mitochondrial dysfunction.

Treatment with 0.5 µmol/L MG132 for 48 h proved to be non-toxic and protected neonatal rat cardiac myocytes against H₂O₂-mediated oxidative stress in lactate dehydrogenase assays. This correlated with reduced levels of intracellular reactive oxygen species as determined by loading myocytes with dichlorofluorescein. Immunoblots showed significant upregulation of superoxide dismutase 1 (SOD1), haem oxygenase 1, and catalase upon proteasome inhibition. Luciferase assays using a reporter driven by the SOD1 promoter revealed proteasome inhibitor-mediated induction of luciferase activity. Deletion and mutation analyses identified an antioxidant response element (ARE) in the SOD1 promoter to be not only essential but also sufficient for transcriptional upregulation by proteasome inhibition. An essential role for the antioxidative transcription factor NF-E2-related factor 2 (Nrf2)—which was stabilized by proteasome inhibition—in ARE-mediated transcriptional activation was revealed in cardiac myocytes from Nrf2 wild-type and knockout mice: proteasome inhibition upregulated antioxidative enzymes and conferred protection against H₂O₂-mediated oxidative stress in Nrf2 wild-type cells. In contrast, the induction of antioxidative enzymes and cytoprotection were completely abolished in cardiac myocytes from Nrf2 knockout mice.

Non-toxic proteasome inhibition upregulates antioxidative enzymes via an Nrf2-dependent transcriptional activation of AREs and confers cardioprotection.

Isolated mouse hearts were perfused in the Langendorff mode and subjected to global no-flow ischaemia followed by reperfusion. Hearts were analysed for recovery of left ventricular developed pressure (LVDP), mRNA levels, and protein expression. Naïve hearts from sEH null mice had similar expression of preproBNP (Nppb) mRNA compared with wild-type (WT) hearts. However, significant increases in Nppb mRNA and BNP protein expression occurred during post-ischaemic reperfusion and correlated with improved post-ischaemic recovery of LVDP. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid prior to ischaemia reduced the preproBNP mRNA in sEH null hearts. Inhibitor studies demonstrated that perfusion with the natriuretic peptide receptor type-A (NPR-A) antagonist, A71915, limited the improved recovery in recombinant full-length mouse BNP (rBNP)- and 11,12-EET-perfused hearts as well as in sEH null mice. Increased expression of phosphorylated protein kinase C and Akt were found in WT hearts perfused with either 11,12-EET or rBNP, while mitochondrial glycogen synthase kinase-3β was significantly lower in the same samples. Furthermore, treatment with the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin abolished improved LVDP recovery in 11,12-EET-treated hearts but not did significantly inhibit recovery of rBNP-treated hearts.

Taken together, these data indicate that EET-mediated cardioprotection involves BNP and PI3K signalling events.

Acute left anterior descending CAO was induced by a balloon catheter in pigs after 12 weeks of high-cholesterol (HC) diet, renovascular hypertension (HTN), or normal control. Cardiac structure, myocardial perfusion, and functional response to iv adenosine and CAO were studied in vivo using electron beam computed tomography (CT). The intra-myocardial microvessels were subsequently evaluated ex vivo using micro-CT, and myocardial expression of growth factors using immunoblotting. Basal myocardial perfusion and microvascular permeability were similar among the groups, whereas their responses to adenosine were attenuated in HC and HTN. A significant decline in myocardial perfusion in normal pigs during acute CAO was attenuated in HC and abolished in HTN. CAO also elicited an increase in normal anterior wall microvascular permeability (+202 ± 59%, P < 0.05), which was attenuated in HC and HTN (+55 ± 9 and +31 ± 8%, respectively, P < 0.05 vs. normal). Microvascular (<200 µm) spatial density was significantly elevated in HC and HTN, accompanied by increased myocardial growth factor expression.

This study demonstrates that early exposure to the cardiovascular risk factors HC and HTN protects the heart from decreases in myocardial perfusion during acute subtotal CAO. This protective effect is associated with and potentially mediated by pre-emptive development of intra-myocardial microvessels that might serve as recruitable CCA.

Experiments were performed with cultured coronary endothelial monolayers and isolated saline-perfused rat hearts. HMR1766 (1 µmol/L) or DEAnonoate (0.5 µmol/L) were used to activate sGC. After exposure to simulated ischaemic conditions, reperfusion of endothelial cells led to a pronounced increase in cytosolic calcium levels and intercellular gaps. Stimulation of cGMP signalling during reperfusion increased Ca²⁺ sequestration in the endoplasmic reticulum (ER) and attenuated the reperfusion-induced increase in cytosolic [Ca²⁺]. Phosphorylation of phospholamban was also increased, indicating a de-inhibition of the ER Ca²⁺ pump (SERCA). Reperfusion-induced intercellular gap formation was reduced. Reduction of myosin light chain phosphorylation indicated inactivation of the endothelial contractile machinery. Effects on cytsolic Ca²⁺ and gaps were abrogated by inhibition of cGMP-dependent protein kinase (PKG) with KT5823. In reperfused hearts, stimulation of cGMP signalling led to decreased oedema development.

sGC/PKG activation during reperfusion reduces reperfusion-induced endothelial intercellular gap formation by attenuation of cytosolic calcium overload and reduction of contractile activation in endothelial cells. This mechanism protects the heart against reperfusion-induced oedema.

Isolated guinea pig hearts were perfused with Krebs–Henseleit buffer (KHB). Antithrombin was applied to achieve physiological levels (1 U/mL) before inducing 20 min of ischaemia (37°C). Hearts were reperfused for 20 min at constant flow (baseline perfusion pressure 70 cmH₂O) with KHB or KHB plus 2 g% hydroxyethyl starch (130 kDa). Coronary net fluid filtration was assessed directly by measuring transudate formation on the epicardial surface. Post-ischaemic coronary release of syndecan-1 and heparan sulfate was quantified by ELISA. Hearts were perfusion-fixed to visualize the glycocalyx by electron microscopy. Ischaemia/reperfusion caused degradation of the glycocalyx, enhanced coronary perfusion pressure, and increased vascular permeability. Antithrombin significantly reduced post-ischaemic glycocalyx shedding, coronary perfusion pressure, coronary leak, and tissue oedema formation compared to untreated hearts. Additional application of colloid augmented these actions of antithrombin. Electron microscopy revealed a mostly intact glycocalyx after antithrombin treatment.

Antithrombin preserves the endothelial glycocalyx, sustaining the vascular barrier function and reducing interstitial oedema. The potentiated effect of colloid in these hearts suggests that the prevention of shedding should be of functional benefit also in vivo.

Hearts from male Tuck Ordinary mice were perfused with Krebs solution (modified to contain low-normal K⁺, 3 mmol/L, and high Ca²⁺, 2.4 mmol/L) containing different combinations of catecholamines (epinephrine 313 nmol/L plus norepinephrine 75 nmol/L) and/or angiotensin II (100 pmol/L) designed to mimic the in vivo milieu. VF was absent during 30 min regional ischaemia (and during 10 min reperfusion) in Krebs-perfused hearts. Catecholamines unmasked ischaemia-induced VF (50%; P < 0.05) and reperfusion-induced VF (50%; P < 0.05). Co-perfusion with angiotensin II did not facilitate VF. Supraventricular pacing (600 b.p.m.) stabilized pre-ischaemic sinus rhythm and partially mimicked the VF-unmasking effect of catecholamines. Arrhythmia susceptibility was greatest with supraventricular pacing plus catecholamines (57% VF during ischaemia and 71% during reperfusion).

For the first time, regional ischaemia-induced VF was consistently evoked in a mouse Langendorff preparation, unmasked by simple periphysiological manipulation of the perfusion conditions. The model is suitable for functional genomic studies.

We determined the influence of 50 nM AAP10 (H₂N-Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH₂) on macroscopic gap junction conductance by dual whole-cell voltage clamping in human and rat cardiomyocytes. Cells were partially uncoupled by CO₂-mediated acidosis (pH 6.3) or kept at ‘normal’ conditions (pH 7.4, T 36°C). Furthermore, we investigated effects of AAP10 in HeLa cells stably transfected with connexin 40, 43, or 45 and on metabolic coupling determined by dye transfer (Lucifer yellow). AAP10 (50 nM)-enhanced gap-junctional intercellular coupling in human and rat cardiomyocytes, completely prevented CO₂-acidosis-induced uncoupling and improved metabolic coupling. The coupling effect of AAP10 was significantly enhanced in previously uncoupled cells. Regarding the connexin isoforms, AAP10-enhanced electrical and metabolic coupling in HeLa cells expressing Cx43 or Cx45, but not in HeLa cells expressing Cx40.

We conclude that the antiarrhythmic peptide AAP10, which improves gap-junctional intercellular coupling and prevents uncoupling by acidification in human cardiomyocytes, might be useful for antiarrhythmic strategies regarding arrhythmias caused by uncoupling of Cx43 and Cx45, but not Cx40.

Human EPCs were expanded ex vivo, treated with a specific GSKi, and then assessed for both yield and functional characteristics by in vitro assays for adherence, apoptosis, and survival. In vivo functionality of treated human EPCs was assessed in immune-tolerant mice subjected to femoral artery wire injury. Re-endothelialization was assessed at 72 h and neointima formation at 7 and 14 days following injury. GSKi treatment resulted in an improvement in the yield of EPCs and a reduction in apoptosis in cells derived from both healthy controls and patients with coronary artery disease. Treatment also increased vascular endothelial growth factor secretion, up-regulated expression of mRNA for the -4 integrin subunit, and improved adhesion, an effect which could be abrogated with an -4 integrin blocking antibody. EPCs without or with ex vivo GSKi treatment enhanced re-endothelialization 72 h following injury as well as reduced neointima formation at 7 days (e.g. endothelial coverage: 7.2 ± 1.7% vs. 70.7 ± 5.8% vs. 87.2 ± 4.1%; intima to media ratios: 1.05 ± 0.19 vs. 0.39 ± 0.08 vs. 0.14 ± 0.02; P < 0.05 for all comparisons), an effect that was persistent at 14 days.

GSKi improves the functional profile of EPCs and is associated with improved re-endothelialization and reduced neointima formation following injury.

Permeability coefficients were measured using the Landis–Michel technique (in frog and rat mesenteric microvessels) and an oncopressive permeability technique (in glomeruli). Ang1 decreased water permeability (L_P: hydraulic conductivity) in continuous and fenestrated microvessels and increased the retention of albumin (: reflection coefficient) in continuous microvessels. Endothelial glycocalyx is common to these anatomically distinct microvascular beds, and contributes to the magnitude of both L_P and . Ang1 treatment increased the depth of endothelial glycocalyx in intact microvessels and increased the content of glycosaminoglycan of cultured microvascular endothelial cell supernatant. Ang1 also prevented the pronase-induced increase in L_P (attributable to selective removal of endothelial glycocalyx by pronase) by restoration of glycocalyx at the endothelial cell surface. The reduction in permeability was inhibited by a cell transport inhibitor, Brefeldin.

Ang1 modifies basal microvessel permeability coefficients, in keeping with previous reports demonstrating reduced solute flux in inflamed vessels. Anatomical, biochemical, and physiological evidence indicates that modification of endothelial glycocalyx is a novel mechanism of action of Ang1 that contributes to these effects.

Heart function and morphology were examined in AQP7-knockout (KO) mice under basal conditions and during pressure overload [isoproterenol infusion and transverse aortic constriction (TAC)]. Glycerol uptake and glycerol-dependent ATP production were measured in AQP7-knockdown cardiac cells. Cardiac glycerol consumption was analysed in ex vivo beating hearts. Cardiac morphology and function in KO mice were similar to those of wild-type (WT) mice under basal conditions, although low glycerol and ATP content were noted in hearts of KO mice. In H9c2 cardiomyotubes, knockdown of AQP7 was associated with a significant reduction of glycerol uptake. The ex vivo heart study demonstrated that cardiac glycerol consumption levels in KO mice were significantly lower than those of WT mice. Furthermore, isoproterenol challenge induced severe left ventricular hypertrophy in KO mice, and TAC resulted in a higher mortality rate in KO mice than in WT mice.

The results indicate that AQP7 acts as a glycerol facilitator in cardiomyocytes and that glycerol is a substrate for cardiac energy production.

Apolipoprotein E-deficient (apoE^–/–) mice fed a high-fat, high-cholesterol diet were treated daily with a CD36 ligand, EP 80317 (300 µg/kg), or 0.9% NaCl for 6 or 12 weeks. Forty-eight hours before sacrifice, mice were injected iv with ¹¹¹Indium-labelled macrophages. A 65% (P < 0.001) reduction of labelled macrophage accumulation at aortic lesions was observed in EP 80317-treated mice, mainly at the level of the aortic arch and iliac arteries, correlating with a 43% reduction of atherosclerotic lesion areas. This was associated with reduced phosphorylation of the focal adhesion kinase Pyk2 following stimulation with oxidized phospholipid in a Src kinase- and CD36-dependent manner. At the vascular level, EP 80317 treatment reduced the expression of pro-inflammatory proteins, including NADPH oxidase, inducible nitric oxide synthase, vascular endothelial cell adhesion molecule-1, and CCL2 chemokine. Plasma IL-6 levels were also reduced by 40% (P < 0.05). In contrast, none of these proteins was modulated in EP 80317-treated apoE/CD36 double knockout (apoE^–/–/CD36^–/–) mice.

Our results support a role for CD36 signalling in the regulation of mononuclear phagocyte trafficking to atherosclerotic-prone sites and in the associated vascular wall inflammation.

Cx43 and Scn5a^1798insD/+ heterozygous (HZ) mice were crossbred to create a mixed offspring: wild-type (WT, n = 15), Cx43 HZ (n = 14), Scn5a^1798insD/+ (Scn5a) HZ (n = 17), and Cx43/Scn5a^1798insD/+ (Cx43/Scn5a) HZ (n = 15) mice. After ECG recording, epicardial activation mapping (208 recording sites) was performed on Langendorff-perfused hearts. Arrhythmia inducibility was tested by one to three premature stimuli and burst pacing. Conduction velocity longitudinal (CV_L) and transverse (CV_T) to fibre orientation and dispersion of conduction were determined during S1-S1 pacing (150 ms). Connexin43 (Cx43) and sodium channel Nav1.5 protein expression and myocardial tissue collagen content were determined by immunohistology. Compared with WT animals, P, QRS, and QTc intervals were prolonged in Scn5a HZ and Cx43/Scn5a HZ, but not in Cx43 HZ animals. Scn5a HZ mice showed decreased CV_L in right ventricle (RV) but not in left ventricle compared with WT. In the RV of Cx43/Scn5a HZ, CV_T was reduced, but CV_L was not different from WT. Arrhythmia inducibility was low and not increased in either single- or double-mutant mice.

Reduction of both electrical coupling and excitability results in normal conduction velocity parallel to fibre orientation but in pronounced conduction slowing transverse to fibre orientation in RV only, although this does not affect arrhythmogeneity.

Male C57/Bl6 mice were injected with the specific PPARβ agonists GW0742 or GW501516, or vehicle. Cardiomyocyte size and vascularisation were determined at different time points. Expression differences were investigated by quantitative reverse transcriptase–polymerase chain reaction and western blotting. In addition, the effects of PPARβ stimulation were compared with hearts of mice undergoing long-term voluntary exercise or pharmacological PPAR activation. Five hours after GW0742 injection, we detected an enhanced angiogenesis compared with vehicle-injected controls. After 24 h, the heart-to-body weight ratios were higher in mice injected with either GW0742 or GW501516 vs. controls. The increased heart size was due to cardiomyocyte enlargement. No signs of pathological cardiac hypertrophy (i.e. apoptosis, fibrosis, or deteriorated cardiac function) could be detected. The effects are mediated via calcineurin A (CnA) activation as: (i) CnA was upregulated, (ii) GW0742 administration or co-transfection of PPARβ significantly stimulated the activity of the CnA promoter, (iii) PPARβ protein bound directly to the CnA promoter, (iv) the CnA target genes NFATc3, Hif-1, and Cdk 9 were upregulated in response to PPARβ stimulation, and (v) the inhibition of CnA activity by cyclosporine A abolished the hypertrophic and angiogenic responses to PPARβ stimulation.

Our data suggest PPARβ pharmacological activation as a novel approach to increase cardiac vascularization and cardiac muscle mass.

In cultured adult rat cardiomyocytes, LPS (1 µg/mL) induced calpain and caspase-3 activity, and up-regulated TNF- expression. These effects of LPS were abrogated by over-expression of calpastatin, an endogenous calpain inhibitor, transfection of calpain-1 siRNA, or various pharmacological calpain inhibitors. Furthermore, blocking gp91^phox-NADPH oxidase prevented calpain and caspase-3 activation and decreased TNF- expression in LPS-stimulated cardiomyocytes. To investigate the role of calpastatin in endotoxaemia, transgenic mice with calpastatin over-expression (CAST-Tg) and wild-type mice were treated with LPS (4 mg/kg, i.p.) or saline in the presence of calpain inhibitor-III (10 mg/kg, i.p.) for 4 h, and their heart function was measured with a Langendorff system. Over-expression of calpastatin significantly attenuated myocardial dysfunction (P < 0.05). Consistently, calpain activity, caspase-3 activity, and TNF- expression were also reduced in CAST-Tg and calpain inhibitor-III compared with wild-type and vehicle-treated hearts, respectively.

gp91^phox-NADPH oxidase-mediated calpain-1 activation induces caspase-3 activation and TNF- expression in cardiomyocytes during LPS stimulation. Over-expression of calpastatin inhibits calpain activation and improves myocardial function in endotoxaemia. The present study suggests that targeting calpain/calpastatin system may be a potential therapeutic intervention for septic hearts.

The yeast-two-hybrid method was used to identify the protein bound to a domain encoded by exon 4 of LDB3. Interaction of ZASP/Cypher with the binding protein was investigated in relation to the functional alterations caused by LDB3 mutations. Localization of the ZASP/Cypher-binding protein was examined at the cellular level in rat cardiomyocytes. Phosphoglucomutase 1 (PGM1), a metabolic enzyme involved in glycolysis and gluconeogenesis, was identified as a protein interacting with ZASP/Cypher. PGM1 bound to ZASP/Cypher at the domains encoded by exons 4 and 10. Two LDB3 mutations in exon 4 (Ser189Leu and Thr206Ile) and another mutation in exon 10 (Ile345Met) reduced the binding to PGM1. PGM1 showed diffuse localization in the cytoplasm of rat cardiomyocytes under standard culture conditions, and distribution at the Z-discs was observed under stressed culture conditions. Binding of endogenous PGM1 and ZASP/Cypher was found to be enhanced by stress in rat cardiomyocytes.

ZASP/Cypher anchors PGM1 to Z-disc under conditions of stress. The impaired binding of PGM1 to ZASP/Cypher might be involved in the pathogenesis of DCM.

Leakage from SR in mouse permeabilized preparations was assessed using exogenous ATP, ATP + phosphocreatine (activation of bound creatine kinase, CK), ATP + mitochondrial substrates (mitochondrial activation), or with all of these together (optimal energetic conditions) in Ca²⁺-free solution. In ventricular fibres caffeine-induced tension transients under optimal energetic conditions were used to estimate SR [Ca²⁺]. In cardiomyocytes, intra-SR Ca²⁺ was monitored by use of the fluorescent marker Mag-fluo 4. In fibres, SR Ca²⁺ content after 5 min incubation strongly depended on energy supply (100%—optimal energetic conditions; 27 ± 5%—exogenous ATP only, 52 ± 5%—endogenous CK activation; 88 ± 8%—mitochondrial activation, P < 0.01 vs. CK system). The significant loss with only exogenous ATP was not inhibited by the ryanodine receptor blockers tetracaine or ruthenium red. However, the SR Ca²⁺-ATPase (SERCA) inhibitors cyclopiazonic acid or 2,5-di(tert-butyl)-1,4-benzohydroquinone significantly decreased Ca²⁺ loss. At 100 nM external [Ca²⁺], the SR Ca²⁺ loss was also energy dependent and was not significantly inhibited by tetracaine. In cardiomyocytes, the decline in SR [Ca²⁺] at zero external [Ca²⁺] was almost two times slower under optimal energetic conditions than in the presence of exogenous ATP only.

At low extra-reticular [Ca²⁺], the main leak pathway is an energy-sensitive backward Ca²⁺ pump, and direct mitochondrial-SERCA ATP channelling is more effective in leak prevention than local ATP generation by bound CK.

Cytosolic pH (pH_i) and Ca²⁺ were measured using fluorescent dyes, BCECM/AM (pH-indicator) and fura-2/AM (Ca²⁺-indicator), respectively. EDRFs, nitric oxide (NO) and prostaglandin I₂ (PGI₂) were assessed using DAF-FM/DA (NO-indicator dye) fluorometry and 6-keto PGF₁ enzyme immunoassay, respectively. HEPES buffers titrated to pH 6.4, 6.9, and 7.4 were used to alter extracellular pH (pH_o), and propionate (20 mmol/L) was applied to cause intracellular acidosis. Extracellular acidosis strongly suppressed bradykinin (BK, 10 nmol/L)- and thapsigargin (TG, 1 µmol/L)-induced Ca²⁺ responses by 30 and 23% at pH_o 6.9, and by 80 and 97% at pH_o 6.4, respectively. During the examinations, there were no significant differences in pH_i among the three groups at pH_o 7.4, 6.9, and 6.4. Extracellular acidosis also inhibited BK-stimulated PGI₂ production by 55% at pH_o 6.9 and by 77% at pH_o 6.4, and NO production by 38% at pH_o 6.9 and by 91% at pH_o 6.4. The suppressive effects of extracellular acidosis on Ca²⁺ responses and NO production were reversible. Propionate changed pH_i from 7.3 to 6.9, without altering pH_o (7.4). Intracellular acidosis had no effect on BK- and TG-induced Ca²⁺ responses or NO production.

These results indicate that extracellular, but not intracellular, acidosis causes endothelial dysfunction by inhibiting store-operated Ca²⁺ entry, so helping to clarify the vascular pathophysiology of conditions such as ischaemia, hypoxia, acidosis, and ischaemia-reperfusion.

In C57/Bl6 mice, development of cardiac hypertrophy induced by transaortic constriction (TAC) for 5 weeks was reduced by ramipril, 5 mg/kg p.o., independent of blood pressure lowering. Ramipril prevented TAC-induced apoptosis of cardiac myocytes and endothelial cells. On day 1 after TAC, upregulation of Sca-1^pos/KDR^pos EPC was observed, which was further increased by ramipril. EPC were persistently elevated in the TAC mice receiving vehicle treatment but not in the ramipril group after 5 weeks. These effects were independent of hypoxia-inducible factor-1 mRNA and protein expression. The ACE inhibitor but not TAC improved the migratory capacity of DiLDL^pos EPC. Increased cardiac afterload induced upregulation of extracardiac neoangiogenesis. This effect was enhanced by ACE inhibition. Ramipril but not TAC markedly increased cardiac capillary density determined by the ratio of CD31^pos cells to cardiomyocytes. Bone marrow transplantation studies revealed that TAC increased the percentage of bone marrow-derived GFP^pos endothelial cells in the myocardium, and ramipril made this effect more pronounced.

ACE inhibition prevents pressure-induced maladaptive cardiac hypertrophy and increases intra- and extracardiac neoangiogenesis associated with the upregulation of EPC and amelioration of EPC migration. The regulation of progenitor cells from the bone marrow identifies a novel effect of ACE inhibitors during cardiac remodelling.

The expression of ZO-2 and Stat1 upon vascular injury was studied using ex vivo organ culture of coronary arteries combined with immunohistochemistry. ZO-2 silencing in human primary VSMC was achieved by means of lentiviral gene transfer. Cell proliferation was assessed by analysing DNA synthesis and by cell counting. Stat1 expression was examined using immunoblotting, immunocytochemistry, TaqMan, and fluorescence activated cell sorting (FACS) analysis. Functional relevance of Stat1 up-regulation was studied using a Stat1 promoter-luciferase reporter assay and intracellular microinjections of a Stat1 specific antibody. ZO-2 was highly expressed in the media and neointima of dilated but not of control arteries, whereas expression of the transcription factor Stat1 was inversely regulated upon injury. Analysis of VSMC with down-regulated ZO-2 revealed increased expression of Stat1 in these cells, whereas Stat1 phosphorylation was not affected. Stat1 up-regulation in VSMC with ZO-2 silencing resulted in a coordinate activation of Stat1-specific genes and consequently led to inhibition of cell proliferation. This effect was restored by microinjection of a Stat1 neutralising antibody.

Our data suggest that the tight junction protein ZO-2 is involved in regulation of VSMC growth control upon vascular injury that is mediated by the transcription factor Stat1. Our findings point to a novel function of ZO-2 in VSMC and implicate ZO-2 as a novel important molecular target in pathological states of vascular remodelling in cardiovascular diseases.

Platelets isolated from the blood of healthy human subjects were exposed in vitro to vehicle or aspirin at different concentrations (5 µmol/L–4 mmol/L). Changes in intraplatelet Ca²⁺ concentration were determined from fura-2 fluorescence. Following immunoprecipitation of NOS-3 from platelet lysates, its activity was determined from l-[³H]arginine to l-[³H]citrulline conversion, and its serine phosphorylation quantified by western blotting. Acetylation of NOS-3 in platelets was assessed by the incorporation of radioactivity into the immunoprecipitated enzyme from [acetyl-¹⁴C]aspirin. Following transfection of HeLa cells with NOS-3, NO biosynthesis in response to aspirin was determined from cyclic GMP measurement, and sites of NOS-3 acetylation were ascertained by liquid chromatography–tandem mass spectrometry. At all concentrations tested, aspirin increased the activity of NOS-3 from platelets. This was not associated with any measurable change in intraplatelet Ca²⁺ concentration. Serine phosphorylation of NOS-3 in platelets was decreased, and this was especially marked for serine-1177 phosphorylation, whereas acetylation of NOS-3 was increased, by aspirin incubation. HeLa cells transfected with NOS-3 exhibited an increase in NO biosynthesis following aspirin exposure, and this was associated with acetylation of the enzyme on both serine-765 and serine-771.

Aspirin acetylates NOS-3 acutely in platelets, and this causes an increase in its activity as well as a decrease in its phosphorylation. It is also possible that aspirin indirectly affects NOS-3 activity by acetylating other substrates within the platelet, but this remains to be determined.

Microarrays were used to analyse the global expression of microRNAs in oxLDL-stimulated human primary peripheral blood monocytes. Expression profiles of the microRNAs were verified using TaqMan real-time PCR. Five microRNAs (microRNA-125a-5p, microRNA-9, microRNA-146a, microRNA-146b-5p, and microRNA-155) were aberrantly expressed after oxLDL treatment of human primary monocytes. Bioinformatics analysis suggested that microRNA-125a-5p is related to a protein similar to ORP9 (oxysterol binding protein-like 9) and this was confirmed by a luciferase reporter assay. MicroRNA-125a-5p was found to mediate lipid uptake and to decrease the secretion of some inflammatory cytokines (interleukin-2, interleukin-6, tumour necrosis factor-, transforming growth factor-beta) in oxLDL-stimulated monocyte-derived macrophages.

MicroRNA-125a-5p may partly provide post-transcriptional regulation of the proinflammatory response, lipid uptake, and expression of ORP9 in oxLDL-stimulated monocyte/macrophages.

Vascular CRP3/MLP expression was mainly observed in arterial samples from both human and rat. Using quantitative real time RT–PCR, we demonstrated that the CRP3/MLP expression was 10 times higher in smooth muscle cells (SMCs) from human mammary artery (h-MA) vs. saphenous vein (h-SV). In endothelial cells (ECs), CRP3/MLP was scarcely detected in either h-MA or h-SV. Using an ex vivo flow through system that mimics arterial condition, we observed induction of CRP3/MLP expression in arterialized h-SV. Interestingly, the upregulation of CRP3/MLP was primarily dependent on stretch stimulus in SMCs, rather than shear stress in ECs. Finally, using a rat vein in vivo arterialization model, early (1–14 days) CRP3/MLP immunostaining was observed predominantly in the inner layer and later (28–90 days) it appeared more scattered in the vessel layers.

Here we provide evidence that CRP3/MLP is primarily expressed in arterial SMCs and that stretch is the main stimulus for CRP3/MLP induction in veins exposed to arterial haemodynamic conditions.

In 12-week-old, male db/db mice (with homozygote mutation in leptin receptor), systolic blood pressure was significantly elevated, compared with control heterozygotes. Isolated, pressurized gracilis muscle arterioles (~90 µm) of db/db mice exhibited an enhanced pressure- and angiotensin II (0.1–10 nM)-induced tone, which was reduced by the selective EP1 receptor antagonist, AH6809 (10 µM), to the level observed in arterioles of control mice. Exogenous application of PGE₂ (10 pM–100 nM) or the selective agonist of the EP1 receptor, 17-phenyl-trinor-PGE₂ (10 pM–100 nM), elicited arteriolar constrictions that were significantly enhanced in db/db mice (max: 31 ± 4 and 29 ± 5%), compared with controls (max: 20 ± 2 and 14 ± 3%, respectively). In the aorta of db/db mice, an increased protein expression of EP1, but not EP4, receptor was also detected by western immunoblotting. Moreover, we found that oral administration of the EP1 receptor antagonist, AH6809 (10 mg/kg/day, for 4 days), significantly reduced the systolic blood pressure in db/db, but not in control mice.

Activation of EP1 receptors increases arteriolar tone, which could contribute to the development of hypertension in the db/db mice.

Rat aortic or mesenteric arterial rings were used to test the vascular effect of salicylates and other NSAIDs. RhoA translocation and the phosphorylation of MYPT1, the regulatory subunit of myosin light chain phosphatase, were measured by western blot, as evidenced for RhoA/Rho-kinase activation. Salicylates, but not other NSAIDs, relaxed contraction induced by most tested constrictors except for calyculin A, indicating that RhoA/Rho-kinase-mediated calcium sensitization is involved. The involvement of RhoA/Rho kinase in vasodilation by salicylates was confirmed by measurements of RhoA translocation and MYPT1 phosphorylation. The calculated half maximal inhibitory concentration (IC₅₀) of vasodilation was apparently higher than that of cyclooxygenase inhibition, but comparable to that of proline-rich tyrosine kinase 2 (PYK2) inhibition. Over-expression of PYK2 induced RhoA translocation and MYPT1 phosphorylation, and these effects were markedly inhibited by sodium salicylate treatment. Consistent with the ex vitro vascular effects, sodium salicylate acutely decreased blood pressure in spontaneous hypertensive rats but not in Wistar Kyoto rats.

Salicylates dilate blood vessels through inhibiting PYK2-mediated RhoA/Rho-kinase activation and thus lower blood pressure.

We packaged the Sgcd cDNA under transcriptional control of a myosin light chain-promoter fused with a cytomegalovirus enhancer into AAV-9 capsids. Vectors carrying either the Sgcd cDNA or an enhanced green fluorescent protein (EGFP) reporter gene were intravenously injected into adult Sgcd knockout mice. After 6 months, immunohistochemistry revealed almost complete reconstitution of the sarcoglycan subcomplex in heart but not skeletal muscle of mice with the Sgcd vector. Furthermore, Sgcd gene transfer resulted in prevention of cardiac fibrosis and significantly increased running distance measured by voluntary wheel running. Left ventricular function remained stable in mice expressing Sgcd while it deteriorated in EGFP controls within 6 months, paralleled by increased expression of brain natriuretic peptide, a molecular marker of heart failure.

Our study establishes an approach to specifically treat hereditary cardiomyopathies by targeting gene expression into the myocardium upon systemic application of AAV vectors.

A significant increase in the expression of cardiac markers was observed after 5 days of exposure to Ca²⁺-ICR in both human CSps and CDCs, as evidenced at transcriptional, translational, and phenotypical levels. Ca²⁺ mobilization among intracellular storages was observed and confirmed by compartmentalized analysis of Ca²⁺ fluorescent probes.

These results suggest that ELF-EMFs tuned at Ca²⁺-ICR could be used to drive cardiac-specific differentiation in adult cardiac progenitor cells without any pharmacological or genetic manipulation of the cells that will be used for therapeutic purposes.

Mouse bone marrow-derived c-kit⁺ cells differentiate in endothelial-like cells when plated on fibronectin (BM-derived EPCs). We found that cell culture in the presence of HG up-regulated p66^ShcA protein expression and that HG exposure markedly decreased the number of BM-derived EPCs. Conversely, p66^ShcA ko BM-derived EPCs were not sensitive to HG inhibition. Indeed, the resistance of p66^ShcA ko BM-derived EPCs to HG was associated with reduced levels of both apoptosis and oxidative stress. To functionally link the HG response to ROS production, p66^ShcA ko BM-derived EPCs were reconstituted either with p66^ShcA wild-type (wt) or with a p66^ShcA allele (p66^ShcA qq) that was devoid of its ROS-generating function. We found that only p66^ShcA wt and not the qq mutant rescued p66^ShcA ko cell sensitivity to HG. One major feature of oxidative stress is its ability to reduce the bio-availability of nitric oxide (NO) that, in turn, plays a crucial role in endothelial differentiation and function. We found that the p66^ShcA deletion prevented the HG-induced increase of nitrotyrosine, and that the resistance to HG of p66^ShcA ko BM-derived EPCs was prevented by NO synthase inhibition. With a reciprocal approach, the treatment of p66^ShcA wt cells with a NO donor prevented the HG-induced deficit. Finally, using a Matrigel plug angiogenesis assay, we demonstrated that p66^ShcA ko prevented diabetic impairment of angiogenesis in vivo.

p66^ShcA deletion rescues the BM-derived EPCs defect induced by HG, indicating p66^ShcA as a potential therapeutic target in diabetic vasculopathy.

Whole-cell patch-clamping, confocal immunofluorescence microscopy, antibody feeding, biotin feeding, fluorescent transferrin feeding, and protein biochemistry techniques were applied to COS-7 cells heterologously expressing KCNQ1 with wild-type or mutant MinK and dynamin 2 and to native I_Ks channels in guinea-pig myocytes. KCNQ1–MinK complexes, but not homomeric KCNQ1 channels, were found to undergo clathrin- and dynamin 2-dependent internalization (DDI). Three sites on the MinK intracellular C-terminus were, in concert, necessary and sufficient for DDI. Gating kinetics and sensitivity to XE991 indicated that DDI decreased cell-surface KCNQ1–MinK channels relative to homomeric KCNQ1, decreasing whole-cell current but increasing net activation rate; inhibiting DDI did the reverse.

The data redefine MinK as an endocytic chaperone for KCNQ1 and present a dynamic mechanism for controlling net surface Kv channel subunit composition—and thus current density and gating kinetics—that may also apply to other –β type Kv channel complexes.

In order to investigate the effects of β-adrenergic activity and to identify sources of ambiguity in earlier studies, we developed a computational model incorporating the effects of β-agonists and β-blockers into an LQT3 mutant guinea pig ventricular myocyte model. β-Activation suppressed two arrhythmogenic phenomena, transmural dispersion of repolarization and early after depolarizations, in a dose-dependent manner. However, the ability of β-activation to prevent cardiac conduction block was pacing-rate-dependent. Low-dose β-blockade by propranolol reversed the beneficial effects of β-activation, while high dose (which has off-target sodium channel effects) decreased arrhythmia susceptibility.

These results demonstrate that β-activation may be protective in LQT3 and help to reconcile seemingly conflicting results from different experimental models. They also highlight the need for well-controlled clinical investigations re-evaluating the use of β-blockers in LQT3 patients.

We generated a mouse strain in which the gene encoding MnSOD was exchanged against a cassette containing the SOD cDNA under the control of the tetracycline response element. After breeding with mice carrying the tetracycline receptor, compound mice express MnSOD depending on the presence of tetracycline. Without tetracycline receptor the MnSOD gene is fully inactivated, and animals show an MnSOD-deficient phenotype. Using echocardiographic recordings, we found an impairment of left ventricular functions: MnSOD^+/– mice displayed a decrease in fraction shortening and ejection fraction and an increase in left ventricular internal diameter in systole. Furthermore, MnSOD^+/– mice developed heart hypertrophy with accompanying fibrosis and necrosis revealed by immunhistochemical analysis. Although we did not find an increase in apoptosis in MnSOD^+/– hearts under normal conditions, we observed an increase of the number of apoptotic cells and vascular senescence after treatment with doxorubicin.

Our study demonstrates that lifelong reduction of MnSOD activity has a negative effect on normal heart function. This animal model presents a valuable tool to investigate the mechanism of heart pathology reported in patients bearing different polymorphic variants of the MnSOD gene and to develop new therapeutic strategies through manipulation of the antioxidative defence system.

HUVECs (passage 2) were used for experiments. Total and hENT1-mediated adenosine transport was measured in the absence or presence of TGF-β1, N^G-nitro-l-arginine methyl ester (l-NAME, NO synthase inhibitor), S-nitroso-N-acetyl-l,d-penicillamine (SNAP, NO donor), and/or KT-5823 (protein kinase G inhibitor) in control cells and cells expressing a truncated form of TGF-β1 receptor type II (TTβRII). Western blot and real-time PCR were used to determine hENT1 protein abundance and mRNA expression. SLC29A1 gene promoter and specific protein 1 (Sp1) transcription factor activity was assayed. Vascular reactivity was assayed in endothelium-intact or -denuded umbilical vein rings. TGF-β1 reduced hENT1-mediated adenosine transport, hENT1 protein abundance, hENT1 mRNA expression, and SLC29A1 gene promoter activity, but increased Sp1 binding to DNA. TGF-β1 effect was blocked by l-NAME and KT-5823 and mimicked by SNAP in control cells. However, TGF-β1 was ineffective in cells expressing TTβRII or a mutated Sp1 consensus sequence. Vasodilatation in response to TGF-β1 and S-(4-nitrobenzyl)-6-thio-inosine (an ENT inhibitor) was endothelium-dependent and blocked by KT-5823 and ZM-241385.

hENT1 is down-regulated by activation of TβRII by TGF-β1 in HUVECs, a phenomenon where NO and Sp1 play key roles. These findings comprise physiological mechanisms that could be important in diseases where TGF-β1 plasma level is increased as in gestational diabetic mothers or patients with diabetes mellitus.

NO production was examined by Griess reagent assay, DAF-2 DA fluorescence staining and cGMP ELISA kits. Protein interaction was determined by western blotting and immunoprecipitation. Treating bovine or human aortic ECs with valsartan increased NO production, as evidenced by elevated level of stable NO metabolites and intracellular cGMP. Valsartan increased the phosphorylation but not the protein level of endothelial NO synthase (eNOS). Inhibition of phosphoinositide-3 kinase (PI3K)/Akt and Src pathways by specific inhibitors suppressed valsartan-induced NO release. In addition, valsartan increased the tyrosine residue phosphorylation of AT₁R, which was attenuated by inhibition of Src but not PI3K activities. Valsartan also suppressed the interaction of eNOS and AT₁R, which was blocked by Src or PI3K inhibition.

Valsartan-induced NO production in ECs is mediated through Src/PI3K/Akt-dependent phosphorylation of eNOS. Valsartan-induced AT₁R phosphorylation depends on Src but not PI3K, whereas valsartan-induced suppression of AT₁R–eNOS interaction depends on Src/PI3K/Akt signalling. These results indicate a novel vasoprotective mechanism of valsartan in upregulating NO production in ECs.

We assessed agonist-induced and conducted dilations as well as endothelial hyperpolarization in the cremaster microcirculation of K_Ca3.1-deficient (K_Ca3.1–/–) and wild-type mice (wt) in vivo after blockade of NO and prostaglandins. Compared with wt, resting tone was enhanced by ~25% in arterioles of K_Ca3.1–/– mice. ACh-induced dilations in K_Ca3.1–/– mice were virtually abolished at low and intermediate concentrations and a remaining dilation at 10 µmol/L ACh was abrogated by blockade of K_Ca2.3 with UCL1684. Sodium nitroprusside- and adenosine-induced dilations were similar in wt and K_Ca3.1–/–. Focal application of ACh induced dilations at the local site in both genotypes, which conducted along the vessel. However, the amplitude of the dilation decreased with distance only in K_Ca3.1–/–. Blockade of K_Ca2.3 in wt did not affect conducted dilations. A K_Ca3.1 opener induced a conducting dilation in wt but not in K_Ca3.1–/–. Membrane potential recordings in vivo demonstrated endothelial hyperpolarization in response to ACh in both genotypes; however, the hyperpolarization was severely impaired in K_Ca3.1–/– ( membrane potential: –3 ± 1 vs. –14 ± 2 mV).

We conclude that K_Ca3.1 is of major importance for endothelial hyperpolarization and EDHF-type responses in skeletal muscle arterioles, and its deficiency is not compensated by K_Ca2.3. Sole activation of K_Ca3.1 is capable of initiating conducted responses, and K_Ca3.1 may contribute to the propagation of the signal, although its presence is not mandatory.

In both cultured endothelial cells and blood vessels isolated from rat aorta, berberine concentration dependently enhanced phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser¹¹⁷⁷ and promoted the association of eNOS with heat shock protein 90 (HSP90), leading to an increased production of nitric oxide. Furthermore, berberine attenuated high glucose-induced generation of reactive oxygen species, cellular apoptosis, nuclear factor-B activation, and expression of adhesion molecules, thus suppressing monocyte attachment to endothelial cells. In mouse aortic rings, berberine elicited endothelium-dependent vasodilatations and alleviated high glucose-mediated endothelial dysfunction. All these beneficial effects of berberine on the endothelium were abolished by either pharmacological inhibition of adenosine monophosphate-activated protein kinase (AMPK) or adenovirus-mediated overexpression of a dominant negative version of AMPK.

Berberine protects against endothelial injury and enhances the endothelium-dependent vasodilatation, which is mediated in part through activation of the AMPK signalling cascade. Berberine or its derivatives may be useful for the treatment and/or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease.

Relaxation of mesenteric arteries was measured using a wire myograph. Compared with the control group, mice overexpressing LOX-1 on a high-fat diet (FD) had preserved vascular smooth muscle relaxation, but impaired endothelium-dependent relaxation via NO. Vascular NO availability was decreased by exaggerated formation of reactive oxygen species and decreased endothelial NO synthase expression. Endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation via cytochrome P450 metabolites was increased in LOX-1 + FD animals, but did not completely compensate for the loss of NO. Currents of calcium-activated potassium channels with large conductance (BK_Ca channels) were measured by the voltage-clamp method. The BK_Ca current amplitudes were not altered in endothelial cells, but highly increased in vascular smooth muscle cells from resistance arteries of LOX-1-overexpressing mice on FD. BK_Ca currents were activated by low-dose H₂O₂ and cytochrome P450 metabolites 11,12-EET and 14,15-EET as EDHF in control mice.

LOX-1 overexpression and FD caused functional changes in endothelial and vascular smooth muscle cells of small resistance arteries.

Rats were infused intravenously with ANG II (10 ng/kg per min) or saline for 4 weeks. These rats received either vehicle or losartan (LOS, 20 µg/h), an angiotensin II type 1 receptor (AT1-R) antagonist; pyrrolidine dithiocarbamate (PDTC, 5 µg/h), a NF-B inhibitor; tempol (TEMP, 80 µg/h), a superoxide scavenger; LOS (20 µg/h), and PDTC (5 µg/h); or TEMP (80 µg/h) and PDTC (5 µg/h), given intracerebroventricularly (ICV) via osmotic minipump. ANG II infusion resulted in increased mean arterial pressure, renal sympathetic nerve activity, plasma proinflammatory cytokines (PIC), norepinephrine, and aldosterone. These rats also had higher levels of Fra-LI (an indicator of chronic neuronal activation), PIC, phosphorylated IKKβ, NF-B subunits, AT1-R, superoxide, and gp91^phox (a subunit of NADP(H) oxidase) and lower levels of IB in the PVN than control animals. ICV treatment with LOS, PDTC, or TEMP attenuated these changes, and combined treatment with ICV LOS and PDTC, or ICV TEMP and PDTC prevented these ANG II-induced hypertensive responses.

These findings suggest that an ANG II-induced increase in the brain renin–angiotensin system activates NF-B in the PVN and contributes to sympathoexcitation in hypertension. The increased superoxide in the PVN contributes to NF-B activation and neurohumoral excitation in hypertension.

The antagonistic activities of SDF-1βP2G against CXCR4 were evaluated in vitro and in vivo and compared with phosphate-buffered saline and AMD3100 (a small bicyclam antagonist of SDF-1). Angiogenesis, muscle regeneration and the expression of pro-angiogenic factors were evaluated in ischaemic gastrocnemius muscles. Distant toxic effects of SDF-1βP2G were evaluated by inflammatory and apoptotic markers. SDF-1βP2G induced CXCR4 internalization and competitively inhibited the chemotaxis of SDF-1β but did not mediate migration, calcium influx, or the phosphorylation of Akt and extracellular signal-regulated kinase in cultured T-lymphoblastic leukaemia cells or H9C2 cells. SDF-1βP2G enhanced blood flow, angiogenesis, and muscle regeneration in ischaemic hind limbs, and the enhancement was significantly better than that of AMD3100. Markers of angiogenesis and progenitor cell migration, including phosphorylated Akt, vascular endothelial growth factor (VEGF), SDF-1 and CXCR4, were up-regulated by SDF-1βP2G and co-localized with CD31-positive cells. Neutralization of VEGF with its specific antibody abolished SDF-1βP2G-induced blood reperfusion and angiogenesis. No apparent inflammatory and apoptotic effects were found in heart, liver, kidneys, and testes after SDF-1βP2G administration.

Our findings indicate that the novel CXCR4 antagonist, SDF-1βP2G, can efficiently enhance ischaemic angiogenesis, blood flow restoration, and muscle regeneration without apparent adverse effects, most likely through a VEGF-dependent pathway.

Western blotting, promoter assay, RT–PCR, and PGE₂ immunoassay revealed that ATPS induced expression of COX-2 and prostaglandin (PGE₂) synthesis through the activation of p42/p44 MAPK (mitogen-activated protein kinase), p38 MAPK, and nuclear factor-B (NF-B). These responses were attenuated by inhibitors of MAPK/ERK kinase (MEK1/2; U0126), p38 MAPK (SB202190), and NF-B (helenalin), or by tranfection with dominant negative mutants of p42, p38, IB kinase (IKK), and IKKβ. Furthermore, the ATPS-stimulated translocation of NF-B into the nucleus and degradation of IB was blocked by U0126 and helenalin. In addition, the ATPS-stimulated cPLA₂ expression was inhibited by U0126, SB202190, helenalin, celecoxib (a selective COX-2 inhibitor), and PGE₂ receptor antagonists (AH6809, GW627368X, and SC-19220). However, the inhibitory effect of celecoxib on cPLA₂ expression was reversed by addition of exogenous PGE₂.

Our results suggest that in VSMCs, activation of p42/p44 MAPK, p38 MAPK, and NF-B is essential for ATPS-induced COX-2 expression and PGE₂ synthesis. Newly synthesized PGE₂ was observed to act as an autocrine signal contributing to cPLA₂ expression, which may be implicated in inflammatory responses. Collectively, our findings provide insights into the correlation between COX-2 and cPLA₂ expression in ATPS-stimulated VSMCs with similar molecular mechanisms and functional coupling to amplify the occurrence of vessel disease-related vascular inflammation.

Conditioned media from luminal, intermediate, and abluminal layers of 29 human ILTs were analysed for neutrophil markers [elastase/₁-antitrypsin and MMP9/NGAL complexes, myeloperoxidase (MPO), and -defensin peptides], RANTES, platelet factor 4 (PF4), and interleukin-8 (IL-8). Their time-dependent release into serum from clots generated in vitro and their plasma concentrations in AAA patients and controls were determined. Immunohistochemistry for neutrophils, platelets, IL-8, PF4, and RANTES on AAA sections was performed; and molecules involved in ILT neutrophil chemotactic function were analysed in vitro. Neutrophils and platelets colocalized in the luminal layer of the thrombus. Consistently, neutrophil markers and platelet-derived RANTES and PF4 were released predominantly by the luminal thrombus pole, where their concentrations were significantly correlated. The luminal ILT layer was also the main source of IL-8, whose immunostaining colocalized with neutrophils. All were also released time dependently from clots and were increased in plasma of AAA patients. Luminal ILT layers displayed potent neutrophil chemotactic activity in vitro, which was inhibited by RANTES- and IL-8-blocking antibodies as well as by reparixin, an antagonist of the IL-8 receptors CXCR1 and CXCR2.

Taken together, these results suggest that platelet-derived RANTES and neutrophil-derived IL-8 are involved in attracting neutrophils to the luminal layer of AAA ILT.

We found that leoligin potently inhibits vascular smooth muscle cell (SMC) proliferation by inducing cell cycle arrest in the G1-phase. Leoligin induced cell death neither in SMCs nor, more importantly, in endothelial cells. In a human saphenous vein organ culture model for graft disease, leoligin potently inhibited intimal hyperplasia, and even reversed graft disease in pre-damaged vessels. Furthermore, in an in vivo mouse model for venous bypass graft disease, leoligin potently inhibited intimal hyperplasia.

Our data suggest that leoligin might represent a novel non-toxic, non-thrombogenic, endothelial integrity preserving candidate drug for the treatment of vein graft disease.

SSZ was found to have pro-apoptotic and anti-proliferative activity in cultured VSMCs. Unexpectedly, these effects were not mediated by nuclear factor kappa B (NF-B) inhibition, which has been suggested to be the anti-inflammatory mechanism associated with the effects of SSZ. Instead, cell-cycle arrest of the VSMCs was observed, which was mediated by induction of haem oxygenase-1 (HO-1) followed by an increased expression of p21^waf1/Cip1. The underlying mechanism for SSZ-induced HO-1 expression was by reactive oxygen species (ROS)-dependent nuclear translocation and activation of nuclear factor erythroid-2-related factor 2 (Nrf2). In a rat carotid artery balloon injury model, administration of SSZ significantly suppressed neointimal growth. In a series of reverse experiments, inhibition of HO-1 by shRNA, ROS by N-acetylcysteine (NAC) or Nrf2 by dominant-negative Nrf2 abrogated the beneficial effects of SSZ.

Our data demonstrate that SSZ inhibits VSMC proliferation in vitro and in vivo through a novel signalling pathway and may be a promising therapeutic option for the treatment of proliferative vascular disease.