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Nebivolol is a selective β1-blocker with nitric oxide–enhancing effects. MicroRNAs are small noncoding RNA molecules that downregulate gene expression. We compared the effects of nebivolol and atenolol, a first generation β1-selective blocker, on left ventricular hypertrophy, fibrosis, and function and microRNA expression in a rodent model of hypertension. Dahl salt-sensitive rats received either low-salt chow (control) or AIN-76A high-salt (8% NaCl) diet and randomized to vehicle (high-salt), nebivolol (20 mg/kg per day), or atenolol (50 mg/kg per day) for 8 weeks. High-salt induced left ventricular hypertrophy and fibrosis and decreased the expression of miR-27a, -29a, and -133a. Nebovolol attenuated deterioration of left ventricular systolic function, remodeling, and fibrosis more than atenolol, despite similar effects on heart rate and blood pressure. Nebivolol, but not atenolol, prevented the decrease in miR-27a and -29a induced by high-salt. Nebivolol and atenolol equally attenuated the decrease in miR-133a. In vitro overexpression of miR-27a,-29a, and -133a inhibited cardiomyocyte hypertrophy and reduced collagen expression. Both miR-27a and -29a target Sp1, and miR-133a targets Cdc42. Pharmacological inhibition of Sp1 and Cdc42 decreased myocardial fibrosis and hypertrophy. Our data support a differential microRNAs expression profile in salt-induced hypertension. Nebivolol substantially attenuated cardiac remodeling, hypertrophy, and fibrosis more than atenolol. These effects are related to attenuation of the hypertension-induced decrease in miR-27a and -29a (with a subsequent decrease in Sp1 expression) and miR-133a (with a subsequent decrease in Cdc42).

Purpose:To assess the effects of Aleglitazar on hyperglycaemia-induced apoptosis.Methods:We incubated human cardiomyocytes, cardiomyocytes from cardiac-specific peroxisome proliferator-activated receptor-γ knockout or wild-type mice in normoglycaemic or hyperglycaemic conditions (glucose 25 mM). Cells were treated with different concentrations of Aleglitazar for 48 h. We measured viability, apoptosis, caspase-3 activity, cytochrome-C release, total antioxidant capacity and reactive oxygen species formation in the treated cardiomyocytes. Human cardiomyocytes were transfected with short interfering RNA against peroxisome proliferator-activated receptor-α or peroxisome proliferator-activated receptor-γ.Results:Aleglitazar attenuated hyperglycaemia-induced apoptosis, caspase-3 activity and cytochrome-C release and increased viability in human cardiomyocyte, cardiomyocytes from cardiac-specific peroxisome proliferator-activated receptor-γ knockout and wild-type mice. Hyperglycaemia reduced the antioxidant capacity and Aleglitazar significantly blunted this effect. Hyperglycaemia-induced reactive oxygen species production was attenuated by Aleglitazar in both human cardiomyocyte and wild-type mice cardiomyocytes. Aleglitazar improved cell viability in cells exposed to hyperglycaemia. The protective effect was partially blocked by short interfering RNA against peroxisome proliferator-activated receptor-α alone and short interfering RNA against peroxisome proliferator-activated receptor-γ alone and completely blocked by short interfering RNA to both peroxisome proliferator-activated receptor-α and peroxisome proliferator-activated receptor-γ.Conclusion:Aleglitazar protects cardiomyocytes against hyperglycaemia-induced apoptosis by combined activation of both peroxisome proliferator-activated receptor-α and peroxisome proliferator-activated receptor-γ in a short-term vitro model.

Ticagrelor inhibits the equilibrative-nucleoside-transporter-1 and thereby, adenosine cell re-uptake. Ticagrelor limits infarct size (IS) in non-diabetic rats and the effect is adenosine-dependent. Statins, via ecto-5′-nucleotidase activation, also increase adenosine levels and limit IS. Ticagrelor and rosuvastatin have additive effects on myocardial adenosine levels, and therefore, on IS and post-reperfusion activation of the NLRP3-inflammasome. Diabetic ZDF rats received via oral gavage; water (control), ticagrelor (150 mg/kg/d), prasugrel (7.5 mg/kg/d), rosuvastatin (5 mg/kg/d), ticagrelor + rosuvastatin and prasugrel + rosuvastatin for 3d. On day 4, rats underwent 30 min coronary artery occlusion and 24 h of reperfusion. Two additional groups received, ticagrelor + rosuvastatin or water in combination with CGS15943 (CGS, an adenosine receptor antagonist, 10 mg/kg i.p. 1 h before ischemia). Both ticagrelor and rosuvastatin increased myocardial adenosine levels with an additive effect of the combination whereas prasugrel had no effect. Similarly, both ticagrelor and rosuvastatin significantly reduced IS with an additive effect of the combination whereas prasugrel had no effect. The effect on IS was adenosine dependent as CGS15943 reversed the effect of ticagrelor + rosuvastatin. The ischemia-reperfusion injury increased myocardial mRNA levels of NLRP3, ASC, IL-1β and IL-6. Ticagrelor and rosuvastatin, but not prasugrel, significantly decreased these pro-inflammatory mediators with a trend to an additive effect of the combination. The combination also increased the levels of anti-inflammatory 15-epilipoxin A4. Ticagrelor and rosuvastatin when given in combination have an additive effect on local myocardial adenosine levels in the setting of ischemia reperfusion. This translates into an additive cardioprotective effect mediated by adenosine-induced effects including downregulation of pro- but upregulation of anti-inflammatory mediators.

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have potent anti-inflammatory, vasodilatory and anti-platelet effects that are independent of the lipid-lowering effects. These non–lipid-lowering or pleiotropic effects are dependent on HMG-CoA reductase inhibition in tissues other than the liver. In animal models, high-dose statins upregulate cytosolic phospholipase A2 and cyclooxygenase-2, leading to increased production of prostacyclin and 15-deoxy-PGJ2. In addition, statins activate protein kinase A, which phosphorylates 5-lipoxygenase, resulting in decreased production of the pro-inflammatory leukotrienes and increased production of 15-epi-lipoxin A4, an eicosanoid with potent anti-inflammatory and inflammation-resolution properties. It is unclear, however, whether these effects occur in the clinical setting and whether these effects (partially) explain the anti-inflammatory effects of statins in patients.

MicroRNAs (miRNAs) are small (∼22 nt) noncoding single-stranded RNA molecules that downregulate gene expression. Studies have shown that miRNAs control diverse aspects of heart disease, including hypertrophy, remodeling, heart failure, and arrhythmia. Recently, several studies have suggested that miRNAs contribute to ischemia-reperfusion injury by altering key signaling elements, thus making them potential therapeutic targets. By altering the expression of various key elements in cell survival and apoptosis [such as phosphoinositide 3-kinase (PI3K), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), Bcl-2, Mcl-1, heat shock protein (HSP)60, HSP70, HSP20, programmed cell death 4 (Pdcd4), LRRFIP1, Fas ligand (FasL), Sirt-1, etc.], miRNAs alter the response to ischemia-reperfusion injury. Studies using various in vivo, ex vivo, and in vitro models have suggested the possible involvement of miR-1, miR-21, miR-29, miR-92a, miR-133, miR-199a, and miR-320 in ischemia-reperfusion injury and/or remodeling after myocardial infarction. Thus miRNAs could be potential therapeutic targets for the treatment of heart disease. Inhibiting miRNAs by antisense strategies or pharmacological approaches is likely to emerge as an alternative and safe method for conferring short- and intermediate-term protection against ischemia-reperfusion injury.

Pioglitazone (PIO), a PPAR-γ agonist, limits myocardial infarct size by activating Akt and upregulating cytosolic phospholipase A(2) (cPLA(2)) and cyclooxygenase (COX)-2. However, PIO has several PPAR-γ-independent effects. We assessed whether PIO limits myocardial infarct size in PPAR-γ-knockout mice, attenuates hypoxia-reoxygenation injury and upregulates P-Akt, cPLA(2), and COX-2 expression in PPAR-γ-knockout cardiomyocytes. Cardiac-specific inducible PPAR-γ knockout mice were generated by crossing αMHC-Cre mice to PPAR-γ(loxp/loxp) mice. PPAR-γ deletion was achieved after 7 days of intraperitoneal tamoxifen (20 mg/kg/day) administration. Mice received PIO (10 mg/kg/day), or vehicle, for 3 days and underwent coronary occlusion (30 min) followed by reperfusion (4 h). We assessed the area at risk by blue dye and infarct size by TTC. Cultured adult cardiomyocytes of PPAR-γ(loxp/loxp/cre) mice without or with pretreatment with tamoxifen were incubated with or without PIO and subjected to 2 h hypoxia/2 h reoxygenation. Cardiac-specific PPAR-γ knockout significantly increased infarct size. PIO reduced infarct size by 51% in PPAR-γ knockout mice and by 55% in mice with intact PPAR-γ. Deleting the PPAR-γ gene increased cell death in vitro. PIO reduced cell death in cells with and without intact PPAR-γ. PIO similarly increased myocardial Ser-473 P-Akt, cPLA(2), and COX-2 levels after hypoxia/reoxygenation in cells with and without intact PPAR-γ. PIO limited infarct size in mice in a PPAR-γ-independent manner. PIO activated Akt, increased the expression of cPLA(2) and COX-2, and protected adult cardiomyocytes against the effects of hypoxia/reoxygenation independent of PPAR-γ activation.

Purpose Statins protect against ischemia-reperfusion injury and limit myocardial infarct size (IS). This effect is dependent on increased generation of adenosine by ecto-5′ nucleotidase and downstream activation of cyclooxygenase-2 (COX2). Dipyridamole (DIP) augments the IS-limiting effects of statins by blocking the cellular reuptake of adenosine; whereas aspirin (ASA) attenuates the effect by inhibiting COX2. We studied the effect of acute administration of DIP, ASA and their combination on the IS-limiting effect of simvastatin (SIM). Methods Rats received oral SIM (10 mg/kg/d) or vehicle for 3 days. Rats underwent 30 min of coronary artery occlusion and 4 h reperfusion. After 5 min of ischemia rats received i.v. DIP (5 mg/kg), ASA (20 mg/kg or 2 mg/kg) or DIP+ASA (2 mg/kg) or vehicle alone. Ischemia area at risk (AR) was assessed by blue dye and IS by TTC. Myocardial samples were analyzed for the activation of Akt, ERK 1/2, endothelial nitric oxide synthase (eNOS), and cyclic-AMP-response-element-binding-protein (CREB). Results SIM limited IS. High- or low-dose ASA alone had no effect on IS. DIP alone or with low-dose ASA significantly reduced IS. Low-dose ASA did not attenuate the SIM effect, whereas high-dose ASA completely blocked the effect. The combination of DIP+low-dose ASA+SIM resulted in the smallest IS. Both SIM and DIP+low-dose ASA augmented Akt phosphorylation and their effect was additive. Both SIM and DIP+low-dose ASA augmented eNOS, ERK 1/2 and CREB phosphorylation. Conclusions During acute myocardial ischemia, DIP alone or with low-dose ASA limits IS and does not attenuate the IS-limiting effect of SIM as high-dose ASA.

Statins and antiplatelet agents are currently used as therapeutic agents for patients with acute myocardial infarction. Statins limit myocardial infarct size by activating phosphatidylinositol-3-kinase (PI3K), ecto-5'-nucleotidase, Akt/endothelial nitric oxide synthase (eNOS), and the downstream effectors inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Inhibition of PI3K, adenosine receptors, eNOS, iNOS, or COX-2 abrogates the protective effects of statins. At >5 mg/kg, aspirin attenuates the myocardial infarct-size-limiting effect of statins. In contrast, the combination of low-dose atoravastatin with either the phosphodiesterase-III inhibitor cilostazol or the adenosine reuptake inhibitor dipyridamole synergistically limits infarct size. Low-dose aspirin with dipyridamole started during ischemia augmented the infarct-size-limiting effects of simvastatin. In contrast, high-dose aspirin blocked the protective effect of simvastatin. The combination of dipyridamole with low-dose aspirin and simvastatin resulted in the smallest infarct size. According to the most current data available, we believe that antiplatelet regimens may require modification for patients who are receiving statins.

The purpose of the present study was to assess whether resolvin E1 (RvE1), an anti-inflammatory mediator derived from eicosapentaenoic acid, would limit myocardial infarct size in the rat. The H9c2 cell line was used to assess whether RvE1 has direct protective effects on cardiomyocytes. In in vivo experiments, Male Sprague-Dawley rats underwent 30 min of ischemia/4 h of reperfusion. Before reperfusion, rats received intravenous RvE1 (0, 0.03, 0.1, or 0.3mg/kg). In in vitro experiments, H9c2 cells were incubated with RvE1 (0, 1, 10, 100, or 1000 nM). Cells were subjected to 18 h of incubation under normoxic conditions, 16 h of hypoxia, or 16 h of hypoxia and 2 h of reoxygenation. In vivo, RvE1 dose dependently reduced infarct size (30.7 ± 1.7% of the area at risk in the control group and 29.1 ± 1.6%, 14.7 ± 1.3%, and 9.0 ± 0.6% in the 0.03, 0.1, and 0.3 mg/kg groups, respectively, P < 0.001). In vitro, RvE1 increased viability and decreased apoptosis in a dose-dependent fashion in cells exposed to hypoxia or hypoxia/reoxygenation. A maximal effect was achieved at a concentration of 100 nM. RvE1 augmented phosphoinositide 3-kinase activity, attenuated caspase-3 activity, and augmented calcium-dependent nitric oxide synthase activity in cells exposed to hypoxia or hypoxia/reoxygenation. RvE1 increased Akt, ERK1/2, and endothelial nitric oxide synthase phosphorylation and attenuated the levels of activated caspase-3 and phosphorylated p38 levels. AG-1478, an EGF receptor tyrosine kinase inhibitor, blocked the protective effect of RvE1 both in vivo and in vitro and attenuated the RvE1-induced increase in Akt and ERK1/2 phosphorylation. In conclusion, RvE1, an anti-inflammatory mediator derived from eicosapentaenoic acid, has a direct protective effect on cardiomyocytes against ischemia-reperfusion injury and limits infarct size when administered intravenously before reperfusion.

The 5-lipoxygenase (5LO) produces leukotriene B4 and 15-epilipoxin-A4 (15-epi-LXA4). Phosphorylation at Ser523 by protein kinase A (PKA) prevents 5LO shift to the perinuclear membrane. Atorvastatin and pioglitazone up-regulate 15-epi-LXA4 production in the heart. We assessed whether phosphorylation of 5LO by PKA determines whether 5LO interacts with the membranous cytosolic phospholipase A2 (cPLA2) to produce leukotriene B4 or with cyclooxygenase-2 (COX2) to produce 15-epi-LXA4. Rats received either pioglitazone, atorvastatin, pioglitazone plus atorvastatin, vehicle, or LPS. Rat myocardial cells were incubated with pioglitazone plus atorvastatin, pioglitazone plus atorvastatin plus H-89 (PKA inhibitor), H-89, or vehicle for 8 h. Pioglitazone and atorvastatin did not affect total 5LO expression. However, both increased 5LO levels in the cytosolic fraction. H-89 caused a shift of 5LO to the membranous fraction in atorvastatin- and pioglitazone-treated rats. Pioglitazone and atorvastatin increased phospho-5LO levels. H-89 attenuated this increase. Both pioglitazone and atorvastatin increased COX2 levels in the cytosolic fraction and the membranous fraction. H-89 prevented this increase. Pioglitazone and atorvastatin increased cPLA2 expression in the membranous fraction. This effect was not attenuated by H-89. Pioglitazone plus atorvastatin increased 15-epi-LXA4 levels. H-89 attenuated the effect of pioglitazone plus atorvastatin. Pioglitazone plus atorvastatin plus H-89 increased leukotriene B4 levels. Coimmunoprecipitation showed that without H-89, atorvastatin and pioglitazone induced an interaction between 5LO and COX2 in the cytosolic fraction, whereas when H-89 was added, 5LO interacted with cPLA2 on the membranous fraction. The 5LO phosphorylation determines whether 15-epi-LXA4 (anti-inflammatory) or leukotriene B4 (inflammatory mediator) is produced.

We asked whether caffeinated coffee (CC) blunts the infarct size (IS)-limiting effects of atorvastatin (ATV).Adenosine receptor activation is essential for mediating the IS-limiting effects of statins. Caffeine is a nonspecific adenosine receptor blocker, and thus drinking CC may block the myocardial protective effects of statins.Rat received 3-day ATV (10 mg/kg/day) or water by oral gavage once daily. Drinking water was replaced by water + sugar (7.5 g/100 ml), CC with sugar, or decaffeinated coffee (DC) with sugar. On the 4th day, rats were anesthetized and underwent 30 min of coronary artery occlusion and 4 h reperfusion. Area at risk was assessed by blue dye and infarct size by TTC.Body weight and area at risk was comparable among groups. IS was 25.1 +/- 3.9% of the area at risk in the control group. In rats not receiving ATV, CC (25.5 +/- 3.1%) and DC (34.0 +/- 2.8%) did not affect IS. IS was significantly reduced by ATV in the water + sugar (11.7 +/- 0.7%, p = 0.015) and DC (11.5 +/- 1.0%; p0.001) groups, but not in the CC group (32.3 +/- 3.0%; p = 0.719). ATV increased myocardial levels of Ser-473 phosphorylated Akt in the water + sugar and DC groups, but not in the CC group.CC, but not DC, abrogated the IS-limiting effects of ATV by blocking the adenosine receptors and preventing the phosphorylation of Akt. CC did not affect IS in rats not receiving ATV.

Statins reduce infarct size by upregulating nitric oxide synthases and PGI2 production. In this article, the infarct size-limiting effect of low-dose simvastatin + ezetimibe, ezetimibe, and high-dose statins were compared. Rats received 3-day water, atorvastatin (10 mg/kg/d), simvastatin (10 mg/kg/d), simvastatin (2 mg/kg/d), simvastatin (2 mg/kg/d) + ezetimibe (1 mg/kg/d), or ezetimibe. Rats underwent 30-minute coronary artery occlusion and 4-hour reperfusion. Atorvastatin and simvastatin 10 reduced infarct size, whereas simvastatin 2, ezetimibe, and simvastatin 2 + ezetimibe had no effect. Atorvastatin and simvastatin 10 increased nitric oxide synthases activity, whereas simvastatin-2, ezetimibe, and simvastatin-2 + ezetimibe had only a small effect. Atorvastatin and simvastatin 10 significantly increased myocardial 6-ketoprostaglandin F1α levels, whereas simvastatin 2, ezetimibe, and simvastatin 2 + ezetimibe had no effect. High-dose statin is required to decrease infarct size, upregulate myocardial nitric oxide synthases activities, and increase 6-keto prostaglandin F1α levels. Combination of ezetimibe and low-dose statin is ineffective in modulating myocardial biochemical changes associated with cardioprotection.

To evaluate whether aleglitazar (Ale), a dual PPARα/γ agonist, has additive effects on myocardial protection against ischemia-reperfusion injury. Human cardiomyocytes (HCMs), cardiomyocytes from cardiac-specific PPARγ knockout (MCM-PPARγ CKO ) or wild type (MCM-WT) mice were incubated with different concentrations of Ale, and subjected to simulated ischemia-reperfusion (SIR) or normoxic conditions (NSIR). Cell viability, apoptosis and caspase-3 activity were determined. HCMs were transfected with siRNA against PPARα (siPPARα) or PPARγ (siPPARγ) followed by incubation with Ale. PPARα/γ DNA binding capacity was measured. Cell viability, apoptosis and levels of P-AKT and P-eNOS were assessed. Infarct size following 30 min coronary artery occlusion and 24 h reperfusion were assessed in WT and db/db diabetic mice following 3-day pretreatment with vehicle, Ale or glimeperide. Ale (at concentrations of 150–600 nM) increased cell viability and reduced apoptosis in HCMs, MCM-WT and MCM-PPAR CKO exposed to SIR. In HCM, the protective effect was partially blocked by siPPARα alone or siPPARγ alone, and completely blocked by siPPARα+siPPARγ. Ale increased P-Akt/P-eNOS in HCMs. P-Akt or P-eNOS levels were decreased when PPARα alone, PPARγ alone and especially when both were knocked down. Peritoneal GTTs revealed that db/db mice had developed impaired glucose tolerance and insulin sensitivity, which were normalized by Ale or glimepiride treatment. Ale, but not glimepiride, limited infarct size in both WT and diabetic mice after ischemia-reperfusion. Ale protects against myocardial apoptosis caused by hypoxia-reoxygenation in vitro and reduces infarct size in vivo.

Stimulation of cardiac β2-adrenergic receptor (β2-AR) or δ-opioid receptor (DOR) exerts a similar degree of cardioprotection against myocardial ischemia in experimental models. We hypothesized that δ-opioid-initiated cardioprotection is mediated by the intrinsic cardiac adrenergic (ICA) cell via enhanced epinephrine release. Using immunohistochemical and in situ hybridization methods, we detected in situ tyrosine hydroxylase (TH) mRNA and TH immunoreactivity that was colocalized with DOR immunoreactivity in ICA cells in human and rat hearts. Western blot analysis detected DOR protein in ICA cells isolated from rat ventricular myocytes. The physiology of DOR expression was examined by determining changes of cytosolic Ca2+ concentration ([Ca2+]i) transients in isolated rat ICA cells using fluorescence spectrophotometry. Exposing the selective δ-opioid agonist d-[Pen2,5]enkephalin (DPDPE) to ICA cells increased [Ca2+]i transients in a concentration-dependent manner. Such an effect was abolished by the Ca2+ channel blocker nifedipine. HPLC-electrochemical detection demonstrated a 2.4-fold increase in epinephrine release from ICA cells following DPDPE application. The significance of the ICA cell and its epinephrine release in δ-opioid-initiated cardioprotection was demonstrated in the rat myocardial infarction model and ICA cell-ventricular myocyte coculture. DPDPE administered before coronary artery occlusion or simulated ischemia-reperfusion reduced left ventricular infarct size by 54 ± 15% or myocyte death by 26 ± 4%, respectively. β2-AR blockade markedly attenuated δ-opioid-initiated infarct size-limiting effect and abolished δ-opioid-initiated myocyte survival protection in rat ICA cell-myocyte coculture. Furthermore, δ-opioid agonist exerted no myocyte survival protection in the absence of cocultured ICA cells during ischemia-reperfusion. We conclude that δ-opioid-initiated myocardial infarct size reduction is primarily mediated via endogenous epinephrine/β2-AR signaling pathway as a result of ICA cell activation.

Atorvastatin (ATV) limits infarct size (IS) by activating Akt and ecto-5-nucleotidase, which generates adenosine. Activated Akt and adenosine activate endothelial nitric oxide synthase (eNOS). When given orally, high doses (10 mg/kg) are needed to achieve full protection. We determined whether dipyridamole (DIP), by preventing the reuptake of adenosine, has a synergistic effect with ATV in reducing myocardial IS. In this study, rats received 3-days of the following: water, ATV (2 mg·kg−1·day−1), DIP (6 mg·kg−1·day−1), or ATV + DIP. In addition, rats received 3-days of the following: aminophylline (Ami; 10 mg·kg−1·day−1) or Ami + ATV + DIP. Rats underwent 30 min of myocardial ischemia followed by 4 h of reperfusion (IS protocol), or hearts were explanted for immunoblotting. As a result, IS in the controls was 34.0 ± 2.8% of the area at risk. ATV (33.1 ± 2.1%) and DIP (30.5 ± 1.5%) did not affect IS, whereas ATV + DIP reduced IS (12.2 ± 0.5%; P < 0.001 vs. each of the other groups). There was no difference in IS between the Ami alone (48.1 ± 0.8%) and the Ami + ATV + DIP (45.8 ± 2.9%) group ( P = 0.422), suggesting that Ami completely blocked the protective effect. Myocardial adenosine level in the controls was 30.6 ± 3.6 pg/μl. ATV (51.0 ± 4.9 pg/μl) and DIP (51.5 ± 6.8 pg/μl) caused a small increase in adenosine levels, whereas ATV + DIP caused a greater increase in adenosine levels (66.4 ± 3.1 pg/μl). ATV and DIP alone did not affect myocardial Ser473 phosphorylated-Akt and Ser1177 phosphorylated-eNOS levels, whereas ATV + DIP significantly increased them. In conclusion, low-dose ATV and DIP had synergistic effects in reducing myocardial IS and activation of Akt and eNOS. This combination may have a potential benefit in augmenting the eNOS-mediated pleiotropic effects of statins.

Aspirin (ASA) inhibits cycloxygenase-1 and modifies cycloxygenase-2 (COX2) by acetylation at Ser(530), leading to a shift from production of PGH(2), the precursor of prostaglandin, to 15-R-HETE which is converted by 5-lipoxygenase to 15-epi-lipoxin A(4) (15-epi-LXA4), a potent anti-inflammatory mediator. Both atorvastatin (ATV) and pioglitazone (PIO) increase COX2 expression. ATV activates COX2 by S-nitrosylation at Cys(526) to produce 15-epi-LXA4 and 6-keto-PGF(1alpha) (the stable metabolite of PGI(2)). We assessed the effect of ASA on the myocardial production of 15-epi-LXA4 and PGI(2) after induction by lipopolysaccharide (LPS) or PIO+ATV. Sprague-Dawley rats were pretreated with: control; ASA 10 mg/kg; ASA 50 mg/kg; LPS alone; LPS+ASA 10 mg/kg; LPS+ASA 50 mg/kg; LPS+ASA 200 mg/kg; PIO (10 mg/kg/d)+ATV (10 mg/kg/d); PIO+ATV+ASA 10 mg/kg; PIO+ATV+ASA 50 mg/kg; PIO+ATV+ASA 50 mg/kg+1400 W, a specific iNOS inhibitor; or PIO+ATV+1400 W. ASA alone had no effect on myocardial 15-epi-LXA4. LPS increased 15-epi-LXA4 and 6-keto-PGF(1alpha) levels. ASA (50 mg/kg and 200 mg/kg, but not 10 mg/kg) augmented the LPS effect on 15-epi-LXA4 but attenuated the effect on 6-keto-PGF(1alpha). PIO+ATV increased 15-epi-LXA4 and 6-keto-PGF(1alpha) levels. ASA and 1400 W attenuated the effects of PIO+ATV on 15-epi-LXA4 and 6-keto-PGF(1alpha). However, when both ASA and 1400 W were administered with PIO+ATV, there was a marked increase in 15-epi-LXA4, whereas the production of 6-keto-PGF(1alpha) was attenuated. In conclusion, COX2 acetylation by ASA shifts enzyme from producing 6-keto-PGF(1alpha) to 15-epi-LXA4. In contrast, S-nitrosylation by PIO+ASA augments the production of both 15-epi-LXA4 and 6-keto-PGF(1alpha). However, when COX2 is both acetylated and S-nitrosylated, it is inactivated. We suggest potential adverse interactions among statins, thiazolidinediones, and high-dose ASA.

Background: Clopidogrel (C) and Ticagrelor (T) are P2Y12 ADP receptor antagonists. In addition, ticagrelor inhibits adenosine cell uptake. In PLATO trial T reduced the incidence of the primary composite endpoint myocardial infarction, stroke or cardiovascular death over C in patients with acute coronary syndromes. Previous data show that 7d pretreatment with T limits infarct size (IS) in rats. We compared the effects of C and T, administered just before reperfusion on IS. We also assessed the effect of T and C, administered just before reperfusion and/or 6w oral treatment on cardiac remodeling. Methods: Rats underwent 30min coronary artery ligation. 1) At 25min of ischemia rats received intraperitoneal (IP) vehicle, T (10 or 30mg/kg), or C (12.5mg/kg). Area at risk (AR) was assessed by blue dye and IS by TTC staining 24h after reperfusion. 2) Rats received vehicle without (sham) or with (control) coronary ischemia, T (30mg/kg) IP (TIP), T (300mg/kg/d) oral for 6w, started a day after reperfusion (TPO), TIP+PO (TIPPO), or C (12.5mg/kg IP +62.5mg/kg/d PO for 6w). LV dimensions and function was assessed by echo at 6w. Results: 1) AR was comparable among groups. IS was 45.3±1.7% of the AR in the control group. T10 (31.5±1.8%; p=0.001) and T30 (21.4±2.6% p

We assessed 1) whether pretreatment before ischemia with pioglitazone (Pio) limits infarct size (IS) and whether this protective effect is due to nitric oxide synthase (NOS) and/or prostaglandin production, as has been shown for atorvastatin (ATV); and 2) whether Pio and ATV have synergistic effects on myocardial protection. Sprague-Dawley rats received oral ATV (10 mg·kg−1·day−1), Pio (10 mg·kg−1·day−1), their combination (Pio + ATV), or water alone for 3 days. Additional rats received Pio (10 mg·kg−1·day−1) for 3 days and intravenous SC-58125 [a cyclooxygenase-2 (COX-2) inhibitor] or SC-560 (a COX-1 inhibitor) 15 min before ischemia. Rats underwent 30 min of myocardial ischemia and 4 h of reperfusion, or hearts were harvested for analysis. IS in the Pio and in the ATV groups was significantly smaller than in the sham-treated group. IS in the Pio + ATV group was smaller than in all other groups ( P < 0.001 vs. each group). The protective effect of Pio was abrogated by SC-58125 but not by SC-560. Pio, ATV, and Pio + ATV increased the expression and activity of cytosolic phospholipase A2 (cPLA2) and COX-2. ATV increased phosphorylated-Akt, phosphorylated-endothelial NOS (P-eNOS), inducible NOS, and COX-2 levels. In contrast, Pio caused an insignificant increase in myocardial levels of phosphorylated-Akt but did not change P-eNOS and iNOS expression. In conclusion, the IS-limiting effects of Pio and ATV involve COX-2. However, the upstream steps differ. ATV induced eNOS phosphorylation and iNOS, cPLA2, and COX-2 expression, whereas Pio induced mainly the expression and activity of cPLA2. The effects of Pio and ATV were additive.

Conjugates of the anti-CanAg humanized monoclonal antibody huC242 with the microtubule-formation inhibitor DM1 (a maytansinoid), or with the DNA alkylator DC1 (a CC1065 analogue), have been evaluated for their ability to eradicate mixed cell populations formed from CanAg-positive and CanAg-negative cells in culture and in xenograft tumors in mice. We found that in culture, conjugates of either drug killed not only the target antigen-positive cells but also the neighboring antigen-negative cells. Furthermore, we showed that, in vivo, these conjugates were effective in eradicating tumors containing both antigen-positive and antigen-negative cells. The presence of antigen-positive cells was required for this killing of bystander cells. This target cell–activated killing of bystander cells was dependent on the nature of the linker between the antibody and the drug. Conjugates linked via a reducible disulfide bond were capable of exerting the bystander effect whereas equally potent conjugates linked via a nonreducible thioether bond were not. Our data offer a rationale for developing optimally constructed antibody-drug conjugates for treating tumors that express the target antigen either in a homogeneous or heterogeneous manner. (Cancer Res 2006; 66(6): 3214-21)

The development of breast cancer is thought to occur through a multi-step process. The majority of breast cancers likely develop over extended periods of time arising from early, pre-invasive lesions such as atypical ductal hyperplasia (ADH) and carcinoma in situ (DCIS), progressing to invasive carcinoma and culminating in metastatic disease. However, the molecular mechanisms underlying this process are still poorly understood. The molecular analysis of this multi-step process in human patients is hampered by the difficulty in obtaining tissue samples at all tumor stages, especially from the same patient. In contrast, mouse models of mammary cancer progression are amenable to pathological, genetic and biochemical analyses at all tumor stages. Global gene expression profiling allows for simultaneous interrogation of the expression of thousands of genes and provides important opportunities to identify molecular signatures of tumor progression. This approach provides a means to define networks of cancer-related genes and their potential role in tumor progression. In this review, we discuss mouse models that have contributed substantially to understanding the molecular mechanisms of breast cancer progression and insights gained from gene expression profiling of mouse mammary cancer models and human breast cancer.

Statins increase the incidence of new onset diabetes. Prolonged statin therapy upregulates PTEN expression. PTEN levels are also elevated in diabetic animals. Activation of protein kinase A by cAMP decreases PTEN expression. We assessed whether prolonged treatment with rosuvastatin (ROS) induces glucose intolerance by upregulating Phosphatase and Tensin Homologue on Chromosome 10 (PTEN) in mice receiving normal (ND) or Western Diet (WD) and whether concomitant treatment with cilostazol (CIL, a phosphodiesterase-3 inhibitor) attenuates the effects.PTEN(loxp/cre) or PTEN(+/-) mice received ND or WD without or with ROS (10 mg/kg/day). Wild-type mice received ND or WD without or with ROS, CIL (10 mg/kg/day), or ROS+CIL for 30 days. Fasting insulin and glucose tolerance test were measured as well as PTEN and P-AKT levels in skeletal muscle.Serum glucose after intraperitoneal injection of glucose was higher in PTEN(loxp/cre) mice receiving WD or ROS and especially WD+ROS. Levels were lower in PTEN(+/-) mice compared to PTEN(loxp/cre) in each treatment group. CIL decreased glucose levels in mice receiving WD, ROS and their combination. Insulin levels were higher in the WD+ROS group. CIL decreased insulin in mice receiving WD+ROS. WD, ROS and especially their combination increased PTEN and decreased P-AKT levels. CIL attenuated the effect of WD, ROS and their combination.Long-term ROS can induce diabetes by upregulating PTEN. CIL attenuates these changes. Partial knockdown of PTEN also ameliorates ROS-induced insulin resistance. Further studies are needed to assess the effects of increasing cAMP levels to prevent the induction of diabetes by statins.

Diabetes mellitus is a major risk factor for cardiovascular events and patient death. Many animal and clinical studies are now being conducted exploring the potential of antidiabetic drugs such as glucagon-like peptide 1 (GLP-1) agonists and dipeptidyl peptidase IV (DPP-IV) inhibitors to improve cardiovascular outcomes. This review summarizes the effect of DPP-IV inhibitors on myocardial ischemia–reperfusion injury in animal models. The DPP-IV inhibitors prevent the rapid degradation and inactivation of incretins and lead to the accumulation of GLP-1 and other chemokines and cytokines, which appear to have both GLP-1 receptor-dependent and -independent cardioprotective, antiapoptotic, and anti-inflammatory effects. Conflicting results, however, have been reported regarding the effect of DPP-IV inhibitors on infarct size in nondiabetic and diabetic animal models. Some studies suggest that DPP-IV inhibitors given as part of preconditioning can decrease infarct size while others found no difference in infarct size compared to placebo. As postconditioning, one study suggested it does provide cardioprotection. No clinical trials have yet been conducted addressing the effect of DPP-IV inhibitors on infarct size. Thus far, clinical trials have not demonstrated improvement in cardiovascular events or mortality from any cause in high cardiovascular risk, type 2 diabetic patients with the use of DPP-IV inhibitors. Although further experiments and clinical trials will be warranted to confirm the results of these studies, the myocardial protection afforded by DPP-IV inhibitors in preclinical animal studies poses a potential breakthrough role for antidiabetic medications in attenuation of ischemia–reperfusion injury that occurs with cardiovascular disease.

We used the rat intestinal cell line, IEC-6, to study potential effects of overexpression of PTH-related protein (PTHrP) on apoptosis. A clonal line of PTHrP-overexpressing cells was established by stably transfecting parental cells with PTHrP complementary DNA in a sense orientation (sense). A similarly transfected line stably, transfected with empty vector, served as control (vector). Immunoreactive PTHrP, measured in culture medium, showed that sense cells secreted approximately 30 times as much PTHrP as did vector control cells. Apoptosis induced by serum withdrawal was evaluated by several methods. DNA laddering was demonstrable in sense-transfected cells as early as 12 h after serum withdrawal but not until later time points in vector-transfected control cells. Flow cytometric analysis of propidium iodide-stained cells showed a greater increase in the sub-G1 (apoptotic) population in sense cells, compared with vector. Fluorescent microscopy with Hoechst 33258 dye showed increased nuclear fragmentati...

Background Dysregulation of microRNAs (miRNAs) in arterial dysfunction and hypertension has not been extensively investigated yet. This project determined the effects of two anti-hypertensive β1 adrenergic selective blockers on miRNA expression in the Dahl Salt Sensitive (DSS) hypertensive rat model. Methods and results Microarray analysis showed that a set of miRNAs is differently expressed in the aorta of high salt (HS) treated rats with miR-320 increased and miR-26b and -21 decreased. All of these changes were reverted to normal by nebivolol (NEB, a β1 selective-blocker and β3 activator). The selective β3-adrenoceptor antagonist S-(−)-cyanopindolol (Syc) counteracted the effect of NEB on these miRNAs. Atenolol (ATN, a pure β1-blocker) combined with specific β3 agonist BRL37344 restored the expression of all three miRNAs, similar to NEB, while ATN alone had only a partial effect on miR-320 expression. Computational analysis found Insulin Growth Factor-1 Receptor (IGF1R) as a putative target of miR-320, and Phosphatase and tensin homolog on chromosome ten (PTEN) as a putative target of miR-26b and -21. The targets were verified by luciferase reporter assays. Inhibition of miR-320 by an antisense inhibitor or NEB increased IGF1R expression, while miR-320 overexpression reversed the effect of NEB. Overexpression of miR-26b or -21 or NEB decreased PTEN levels, while inhibition of miR-26b or -21 attenuated the effect of NEB. HS diet induced downregulation of IGF1R and upregulation of PTEN in the aorta. NEB normalized the aberrant expression of IGF1R and PTEN and also improved the impairment of vascular AKT/eNOS signaling. Moreover, both NEB and ATN showed to have protective effects on salt-induced hypertension, oxidative stress, and vascular remodeling. NEB had a greater effect than ATN. Conclusions Our data supports a differential miRNA expression profile in salt-induced hypertension. Manipulation of dysregulated miRNAs by β-blockers may substantially induce alterations of gene expression and prevent arterial dysfunction and remodeling.

Competitive endogenous RNAs (ceRNAs) regulate mRNA transcripts containing common microRNA (miRNA) recognition elements (MREs) through sequestration of shared miRNAs. Interactions of ceRNA have been demonstrated in cancerous cells. However, a paucity of information is available relative to the interactions of ceRNAs interaction in diabetes mellitus and the myocardium. The purpose of this study is to assess the potential role of DKK1 and PTEN in ceRNA regulation utilizing their common miRNAs in diabetic cardiomyocytes. The interactions' regulation between PTEN and DKK1 were determined in two diabetic models in vivo (streptozotocin-induced type-1 DM mice and db/db mice) and in vitro (human cardiomyocytes cells exposed to hyperglycemia). The levels of DKK1 and PTEN (mRNA and protein) were upregulated in parallel in all three diabetic models. DKK1 modulates PTEN protein levels in a miRNA and 3'UTR-dependent manner. RNAi-mediated DKK1 gene silencing resulted in a decreased PTEN expression and vice versa. The effect was blocked when Dicer was inhibited. Silencing either PTEN or DKK1 resulted in an increase of the availabilities of shared miRNAs. The silencing of either PTEN or DKKI resulted in a suppression end of the luciferase-PTEN 3'UTR activity. However, the over expression of DKK1 3'UTR or PTEN 3'UTR resulted in an increase in the activity. The attenuation of DKK1 increased AKT phosphorylation, improved glucose uptake and decreased apoptosis in HCMs exposed to hyperglycemia. The effects were blocked by PI3K inhibition. DKK1 and PTEN transcripts are co-upregulated in DM and hyperglycemia. DKK1 and PTEN serve as ceRNA, affecting the expression of each other via competition for miRNAs binding.

glucagon-like peptide-1 (GLP1) receptor agonists have gained popularity as a second-line treatment (after diet and metformin) for patients with type 2 diabetes mellitus (T2DM) ([9][1]). With therapy, a 1–2% reduction in HbA1c levels is reported, which is compatible with the magnitude of effect of

Glucagon-like peptide (GLP)-1 receptor activation increases intracellular cAMP with downstream activation of PKA. Cilostazol (CIL), a phosphodiesterase-3 inhibitor, prevents cAMP degradation. We assessed whether CIL amplifies the exenatide (EX)-induced increase in myocardial cAMP levels and PKA activity and augments the infarct size (IS)-limiting effects of EX in db/ db mice. Mice fed a Western diet received oral CIL (10 mg/kg) or vehicle by oral gavage 24 h before surgery. One hour before surgery, mice received EX (1 μg/kg sc) or vehicle. Additional mice received H-89, a PKA inhibitor, alone or with CIL + EX. Mice underwent 30 min of coronary artery occlusion and 24 h of reperfusion. Both EX and CIL increased myocardial cAMP levels and PKA activity. Levels were significantly higher in the EX + CIL group. Both EX and CIL reduced IS. IS was the smallest in the CIL + EX group. H-89 completely blocked the IS-limiting effects of EX + CIL. EX + CIL decreased phosphatase and tensin homolog on chromosome 10 upregulation and increased Akt and ERK1/2 phosphorylation after ischemia-reperfusion. These effects were blocked by H-89. In conclusion, EX and CIL have additive effects on IS limitation in diabetic mice. The additive effects are related to cAMP-induced PKA activation, as H-89 blocked the protective effect of CIL + EX.

We assessed whether phosphodiesterase-III inhibition with cilostazol (Cil) augments the infarct size (IS)-limiting effects of MK0626 (MK), a dipeptidyl-peptidase-4 (DPP4) inhibitor, by increasing intracellular cAMP in mice with type-2 diabetes.Db/Db mice received 3-day MK (0, 1, 2 or 3 mg/kg/d) with or without Cil (15 mg/kg/d) by oral gavage and were subjected to 30 min coronary artery occlusion and 24 h reperfusion.Cil and MK at 2 and 3 mg/kg/d significantly reduced IS. Cil and MK had additive effects at all three MK doses. IS was the smallest in the MK-3+Cil. MK in a dose dependent manner and Cil increased cAMP levels (p0.001). cAMP levels were higher in the combination groups at all MK doses. MK-2 and Cil increased PKA activity when given alone; however, PKA activity was significantly higher in the MK-2+Cil group than in the other groups. Both MK-2 and Cil increased myocardial levels of Ser(133) P-CREB, Ser(523) P-5-lipoxygenase, Ser(473)P-Akt and Ser(633) P-eNOS. These levels were significantly higher in the MK-2+Cil group. Myocardial PTEN (Phosphatase and tensin homolog on chromosome ten) levels were significantly higher in the Db/Db mice compared to nondiabetic mice. MK-2 and Cil normalized PTEN levels. PTEN levels tended to be lower in the combination group than in the MK and Cil alone groups.MK and Cil have additive IS-limiting effects in diabetic mice. The additive effects are associated with an increase in myocardial cAMP levels and PKA activity with downstream phosphorylation of Akt, eNOS, 5-lipoxygenase and CREB and downregulation of PTEN expression.

Arachidonic acid-derived eicosanoids (lipoxins and 15-epilipoxins) have a major role in resolution of inflammation. 15-epi-lipoxin A(4) (15-epi-LXA(4)) is a lipid mediator with strong anti-inflammatory and inflammation-resolving effects. We examined the effect of pioglitazone therapy on plasma 15-epi-LXA(4) in patients with type 2 diabetes (T2DM).T2DM patients (Age = 56 ± 2 y, BMI = 33 ± 1.8, HbA1c = 7.8 ± 0.3%) not on thiazolidinedione therapy for at least 12 months were randomized to receive either pioglitazone 15 mg/daily for two months (PIO 15) or pioglitazone 15 mg/day for one month followed by a dose escalation to 30 mg/day for an additional one month (PIO 30).PIO 15 increased plasma 15-epi-LXA(4) levels (0.63 ± 0.06-1.05 ± 0.08 ng/mL, p0.01) and adiponectin levels (6.4 ± 0.3-10.1 ± 0.7 μg/mL, p0.001) and decreased fasting plasma glucose (125 ± 8-106 ± 9 mg/dL, p0.05), free fatty acids (FFA) (414 ± 46-320 ± 38 μmol/l, p0.05) and HOMA-IR (5.3 ± 0.4 to 4.0 ± 0.4, p0.05). Body weight (Δ = 0.2 kg) and HbA1c (7.4 ± 0.2-7.1 ± 0.2%) did not change significantly. PIO 30 treated patients had similar increase in plasma 15-epi-LXA(4) (0.64 ± 0.10-1.08 ± 0.09 ng/mL, p0.01), and decrease in plasma FFA (423 ± 42-317 ± 40 μmol/l, p0.05) despite a greater increase in plasma adiponectin (6.5 ± 0.4-15.5 ± 0.7 ug/mL, p0.001) and a greater reduction in HbA1c (8.7 ± 0.5-7.4 ± 0.3%, p0.01), FPG (159 ± 16-120 ± 10 mg/dL, p0.01), and HOMA-IR (6.6 ± 0.8-4.4 ± 0.4, p0.005). Furthermore, PIO 30 treated patients had a significant increase in body weight (Δ = 1.7 kg, p0.02).In T2DM, low dose pioglitazone (15 mg/day) increases 15-epi-LXA(4) and adiponectin levels in the absence of significant changes in body weight. Dose escalation of pioglitazone to 30 mg/day is associated with a similar increase in 15-epi-LXA(4) despite a greater increase in plasma adiponectin concentrations.

Phosphatase and tensin homolog on chromosome 10 (PTEN) is downregulated during hypertrophic and cancerous cell growth, leading to activation of the prosurvival Akt pathway. However, PTEN regulation in cardiac myocytes upon exposure to hypoxia remains unclear. We explored the role of PTEN in response to hypoxia/ischemia in the myocardium. We validated that PTEN is a transcriptional target of activating transcription factor 2 (ATF-2) and is positively regulated via a p38/ATF-2 signaling pathway. Accordingly, hypoxia-induced upregulation of phosphorylation of ATF-2 and PTEN were reversed by a dominant negative mutant p38. Inhibition of PTEN in cardiomyocytes attenuated hypoxia-induced cell death and apoptosis. Cardiac-specific knockout of PTEN resulted in increased phosphorylation of Akt and forkhead box O 1 (forkhead transcription factors), limited infarct size in animals exposed to ischemia-reperfusion injury, and ameliorated deterioration of left ventricular function and remodeling following permanent coronary artery occlusion. In addition, the activation of Bim, FASL, and caspase was coupled with PTEN activation, all of which were attenuated by PTEN inhibition. In conclusion, cardiomyocyte-specific conditional PTEN deletion limited myocardial infarct size in an in vivo model of ischemia-reperfusion injury and attenuated adverse remodeling in a model of chronic permanent coronary artery ligation.

We assessed the ability of Aliskiren (AL), a direct renin inhibitor, and Valsartan (VA), an angiotensin receptor blocker, to limit myocardial infarct size (IS) in mice with type-2 diabetes mellitus.Db/Db mice, fed Western Diet, received 15-day pretreatment with: 1) vehicle; 2) AL 25 mg/kg/d; 3) AL 50 mg/kg/d; 4) VA 8 mg/kg/d; 5) VA 16 mg/kg/d; 6) AL 25+VA 16 mg/kg/d; or 7) AL 50+VA 16 mg/kg/d. Mice underwent 30 min coronary artery occlusion and 24 h reperfusion. Area at risk (AR) was assessed by blue dye and IS by TTC staining. Protein expression was assessed by immunobloting.IS in the control group was 42.9 ± 2.1% of the AR. AL at 25 (21.9 ± 2.9%) and 50 mg/kg/d (15.5 ± 1.3%) reduced IS. VA at 16 mg/kg/d (18.8 ± 1.2%), but not at 8 mg/kg/d (35.2 ± 4.0%), limited IS. IS was the smallest in the AL50+VA16 group (6.3 ± 0.9%). Both AL and VA reduced myocardial AT1R levels, without affecting AT2R levels, and increased the expression of Sirt1 and PGC-1α with increased phosphorylation of Akt and eNOS.AL, dose dependently limited myocardial IS in mice with type-2 diabetes mellitus. At doses shown to limit IS in non-diabetic animals, VA failed to reduce IS in Db/Db mice. However, at higher dose (16 mg/kg/d), VA reduced IS. Both drugs reduced the expression of AT1R and increased myocardial levels of the longevity genes Sirt1 and PGC-1α along with increased Akt and eNOS phosphorylation.

PURPOSE: Micro(mi)RNAs negatively regulate a wide variety of genes through degradation or posttranslational inhibition of their target genes. The purpose of this study was to investigate the role of miR-23a in modulating RPE cell survival and gene expression in response to oxidative damage. METHODS: The expression level of miR-23a was measured in macular retinal pigment epithelial (RPE) cells of donor eyes with aged-related macular degeneration (AMD) and age-matched normal eyes by using qRT-PCR. Cultured human ARPE-19 cells were transfected with miR-23a mimic or inhibitor. Cell viability was assessed by the MTT assay. Apoptosis was determined by incubating cells with hydrogen peroxide (H(2)O(2)) or t-butylhydroperoxide (tBH). Caspase-3 activity and DNA fragmentation were measured by enzyme-linked immunosorbent assays. The protein relevant to apoptosis, such as Fas expression level, was analyzed by Western blot analysis. RESULTS: miR-23a expression was significantly downregulated in macular RPE cells from AMD eyes. H(2)O(2)-induced ARPE-19 cell death and apoptosis were increased by an miR-23a inhibitor and decreased by an miR-23a mimic. Computational analysis found a putative target site of miR-23a in the 3'UTR of Fas mRNA, which was verified by a luciferase reporter assay. Forced overexpression of miR-23a decreased H(2)O(2) or tBH-induced Fas upregulation, and this effect was blocked by downregulation of miR-23a. CONCLUSIONS: The protection of RPE cells against oxidative damage is afforded by miR-23a through regulation of Fas, which may be a novel therapeutic target in retinal degenerative diseases.

In addition to cholesterol-lowering effect, HMG-CoA reductase inhibition by statins has been shown to have protective effect in many cells type. The loss of vision in retinal degeneration disease associates with oxidative stress and apoptosis in retinal pigment epithelium (RPE) cell. This study was designed to examine the effect of statins on oxidant-induced damage in human RPE cells. Cultured human ARPE-19 (ARPE) cells were challenged with hydrogen peroxide (H(2) O(2) ) plus tumor necrosis factor alpha (TNFα) in the presence or absence of statins or various stress signaling inhibitors, including anti-oxidants N-acetyl cysteine (NAC), the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI), and the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580. Apoptosis was evaluated by TUNEL analysis and cell viability was determined by MTT assay. Reactive oxygen species (ROS) were detected by 2',7'-dichlorodihydrofluorescein diacetate (H(2) DCFH-DA). Expression of p-p38 MAPK protein was measured by Western blot analysis. Our findings indicate that statins treatment significantly suppressed oxidant-induced ROS accumulation and RPE apoptosis. Statins increased cell viability in a dose-dependent manner. In addition, statins treatment prevented the activation of NADPH oxidase and p38 MAPK signaling induced by oxidative stress. These results suggest that statins protects ARPE cells from oxidative stress via an NADPH oxidase and/or p38 MAPK-dependent mechanisms, which may contribute to statins-induced beneficial effects on RPE function.

We assessed the effects of TAK-491 (a newly designed potent and selective ARB) alone and in combination with pioglitazone (PIO) on myocardial infarct size (IS).Rats received TAK-491 (0.3, 1, 3, or 10 mgkg(-1)d(-1)), PIO (1.0 or 2.5 mgkg(-1)d(-1)), or PIO 2.5 mgkg(-1)d(-1) with TAK-491 (1 or 3 mgkg(-1)d(-1)) for 4 days. On day 5 rats underwent 30-minute coronary artery occlusion and 4-hour reperfusion. Area at risk (AR) was assessed by blue dye and IS by TTC. Left ventricular (LV) dimensions and function was assessed by echocardiography 35 days after infarction.TAK (1.0-10 mgkg(-1)d(-1)), PIO (1.0 to 2.5 mgkg(-1)d(-1)), PIO2.5+TAK1.0, and PIO2.5+TAK3.0 significantly reduced IS. IS was the smallest in the TAK 10.0, followed by PIO+TAK 3.0. The protective effects of TAK and PIO were additive, as IS was smaller in the PIO2.5+TAK1.0 than in PIO 2.5 alone (p = 0.008) or TAK1.0 alone (p = 0.002); and in PIO2.5+TAK3.0 than in PIO alone (p0.001) or TAK3.0 alone (p0.001). TAK, PIO and their combination tended to attenuate LV remodeling and improved LV function 35 days after infarction; however, the differences among individual groups were not statistically significant. Both TAK-491 and PIO increased calcium-dependent nitric oxide synthase activity, whereas only PIO increased COX2 expression and activity. Both PIO and TAK-491 increased Akt, ERK 1/2 and eNOS phosphorylation and inhibited BAX activation.TAK-491 and PIO independently limited myocardial IS in a dose-dependent fashion; and the effects were additive. The mechanism of protection and the role of TAK-491 in this clinical setting should be further investigated.

Pioglitazone (PIO) and glucagon-like peptide-1 (GLP-1) analogs limit infarct size (IS) in experimental models. The effects of the dipeptidyl-peptidase-IV inhibitors, which increase the endogenous levels of GLP-1, on myocardial protection, are unknown. We studied whether sitagliptin (SIT) and PIO have additive effects on IS limitation in the mouse. Mice received 3-day or 14-day oral SIT (300 mg·kg−1·day−1), PIO (5 mg·kg−1·day−1), SIT + PIO, or vehicle. In addition, mice received intravenous H-89 [20 mg/kg, a protein kinase A (PKA) inhibitor] or vehicle 1 h before ischemia. Rats underwent 30 min myocardial ischemia and 4 h reperfusion. SIT, PIO, and SIT + PIO for 3 days significantly reduced IS (24.3 ± 2.7, 23.0 ± 0.8, and 14.7 ± 0.9%) compared with controls (46.2 ± 2.8%). H-89 completely blocked the effect of SIT and partially blocked the PIO effect. SIT, but not PIO, increased cAMP levels. PKA activity was increased by PIO and to a greater extent by SIT. PIO, but not SIT, increased cytosolic phospholipase A2 and cyclooxygenase-2 activity. Accordingly, 6-keto-PGF1α and 15-deoxy-PGJ2 increased by PIO but not SIT. In contrast, SIT, and to a lesser extent PIO, increased 15-epi-lipoxin A4 levels. H-89 completely blocked the effect of SIT and PIO on 15-epi-lipoxin A4 levels. PIO, and to a greater extent SIT, increased endothelial nitric oxide synthase and cAMP response element-binding protein phosphorylation, an effect that was blocked by H-89. With a 14-day pretreatment experiment, IS was 46.4 ± 1.0% in the control group, 16.9 ± 0.6% in SIT ( P < 0.001), 19.1 ± 1.1% in PIO ( P = 0.014), and 12.9 ± 0.7% in SIT + PIO ( P < 0.001). We found that SIT and PIO limit IS using different pathways. The protective effect of SIT is via cAMP-dependent PKA activation, whereas PIO mediates its effects via both PKA-dependent and -independent pathways.

Aims MicroRNAs (miRNAs) regulate various cardiac processes including cell proliferation and apoptosis. Pioglitazone (PIO), a peroxisome proliferator-activated receptor (PPAR)-γ agonist, protects against myocardial ischaemia–reperfusion (IR) injury. We assessed the effects of PPAR-γ activation on myocardial miRNA levels and the role of miRNAs in IR injury. Methods and results We evaluated the expression changes of miRNAs in the rat heart after PIO administration using miRNA arrays and then confirmed the result by northern blot. miR-29a and c levels decreased remarkably after 7-day treatment with PIO. In H9c2 cells, the effects of PIO and rosiglitazone on miR-29 expression levels were blocked by a selective PPAR-γ inhibitor GW9662. Downregulation of miR-29 by antisense inhibitor or by PIO protected H9c2 cells from simulated IR injury, indicated as increased cell survival and decreased caspase-3 activity. In contrast, overexpressing miR-29 promoted apoptosis and completely blocked the protective effect of PIO. Antagomirs against miR-29a or -29c significantly reduced myocardial infarct size and apoptosis in hearts subjected to IR injury. Western blot analyses demonstrated that Mcl-2, an anti-apoptotic Bcl-2 family member, was increased by miR-29 inhibition. Conclusion Downregulation of miR-29 protected hearts against IR injury. The modulation of miRNAs can be achieved by pharmacological intervention. These findings provide a rationale for the development of miRNA-based strategies for the attenuation of IR injury.

Phosphoinositide 3-kinase (PI3K) mediates myocardium protective signaling through phosphorylation of phosphatidylinositol (Ptdins) to produce Ptdins(3,4,5)P3. Lipid phosphatase and tensin homolog on chromosome 10 (PTEN) antagonizes PI3K activity by dephosphorylating Ptdins(3,4,5)P3; therefore, the inhibition of PTEN enhances PI3K/Akt signaling and could prevent myocardium from ischemia-reperfusion (I/R) injury. Here we studied 1) whether the pharmacological inhibition of PTEN by bisperoxovanadium molecules [BpV(HOpic)] attenuates simulated I/R (SIR) injury in vitro and 2) whether the administration of BpV(HOpic) either before or after ischemia limits myocardial infarct size (IS) and ameliorates cardiodysfunction caused by infarction. First, adult rat cardiomyocytes were treated with or without BpV(HOpic) and then exposure to SIR. Second, anesthetized rats received BpV(HOpic) either before or after ischemia. IS was assessed at 4 h reperfusion, and left ventricular function was evaluated by echocardiography at 28 days postreperfusion. As a result, BpV(HOpic) decreased cell death, improved 3-[4,5-yl]-2,5-diphenyltetrazolium bromide (MTT) viability, and reduced apoptosis in cells exposed to SIR. These protective effects of BpV(HOpic) are associated with increased phospho-Akt and the repression of caspase-3 activity. Second, the administration of BpV(HOpic) significantly reduced IS and suppressed caspase-3 activity following I/R injury and consequentially improved cardiac function at 28 day postinfarction. These beneficial effects of BpV(HOpic) are attributed to increases in myocardial levels of phosphorylation of Akt/endothelial nitric oxide synthase (eNOS), ERK-1/2, and calcium-dependent nitric oxide synthase activity. In conclusion, the pharmacological inhibition of PTEN protects against I/R injury through the upregulation of the PI3K/Akt/eNOS/ERK prosurvival pathway, suggesting a new therapeutic strategy to combat I/R injury.

The authors proposed a novel structure representation strategy __ “Interaction-Distance” theory to study the quantitative structure-activity relationships (QSARs) of peptide. The “Interaction-Distance” theory stated that: I. In a peptide chain, there exists interaction between any two amino acids; II. The value of interaction lies on not only the types the two amino acids, but also the distance between them; III. The structure of the peptide may be represented by TIV (total interaction value) value__ the sum of all of the interaction values in peptide. The merit of the strategy is that the structures of all peptides comprising of different numbers of amino acids may be represented by the TIV score. The strategy is helpful of studying the peptides QSARs conveniently. In the end, QSARs of several peptides were studied by the strategy.

Endothelial nitric oxide synthase (eNOS) activation with subsequent inducible NOS (iNOS), cytosolic phospholipase A2 (cPLA2), and cyclooxygenase-2 (COX2) activation is essential to statin inhibition of myocardial infarct size (IS). In the rat, the peroxisome proliferator-activated receptor-γ agonist pioglitazone (Pio) limits IS, upregulates and activates cPLA2 and COX2, and increases myocardial 6-keto-PGF1α levels without activating eNOS and iNOS. We asked whether Pio also limits IS in eNOS−/− and iNOS−/− mice. Male C57BL/6 wild-type (WT), eNOS−/−, and iNOS−/− mice received 10 mg·kg−1·day−1 Pio (Pio+) or water alone (Pio−) for 3 days. Mice underwent 30 min coronary artery occlusion and 4 h reperfusion, or hearts were harvested and subjected to ELISA and immunoblotting. As a result, Pio reduced IS in the WT (15.4 ± 1.4% vs. 39.0 ± 1.1%; P < 0.001), as well as in the eNOS−/− (32.0 ± 1.6% vs. 44.2 ± 1.9%; P < 0.001) and iNOS−/− (18.0 ± 1.2% vs. 45.5 ± 2.3%; P < 0.001) mice. The protective effect of Pio in eNOS−/− mice was smaller than in the WT ( P < 0.001) and iNOS−/− ( P < 0.001) mice. Pio increased myocardial Ser633 and Ser1177 phosphorylated eNOS levels in the WT and iNOS−/− mice. iNOS was undetectable in all six groups. Pio increased cPLA2, COX2, and PGI2 synthase levels in the WT, as well as in the eNOS−/− and iNOS−/−, mice. Pio increased the myocardial 6-keto-PGF1α levels and cPLA2 and COX2 activity in the WT, eNOS−/−, and iNOS−/− mice. In conclusion, the myocardial protective effect of Pio is iNOS independent and may be only partially dependent on eNOS. Because eNOS activity decreases with age, diabetes, and advanced atherosclerosis, this effect may be relevant in a clinical setting and should be further characterized.

Many patients with type 2 diabetes mellitus receive several oral hypoglycemic agents, including sulfonylurea drugs. Intravenous glyburide (Glyb), a sulfonylurea agent, blocks the protective effects of “ischemic” and pharmacologic preconditioning in various animal models without affecting myocardial infarct size when administered alone. However, there are conflicting results when other sulfonylurea drugs are used. Pioglitazone (PIO) reduces infarct size in the rat. We asked whether oral Glyb and glimepiride (Glim) affect the infarct size-limiting effects of PIO. Sprague–Dawley rats received 3-day oral treatment with: PIO (5 mg/kg/day); PIO + Glyb (10 mg/kg/day); PIO + Glim (4 mg/kg/day) or water alone (experiment 1) or PIO (5 mg/kg/day) with or without 5-hydroxydecanoate (5HD, 10 mg/kg), a specific mitochondrial ATP-sensitive K+ channels inhibitor, administered intravenously 30 min before coronary artery ligation. PIO, Glyb and Glim were administered by oral gavage. Sugar 5% was added to water to prevent hypoglycemia. Rats underwent 30 min coronary artery occlusion and 4 h reperfusion (n = 6 in each group). Ischemic area at risk was assessed by blue dye and infarct size by triphenyl-tetrazolium-chloride. Body weight and the size of the area at risk were comparable among groups. Infarct size (% of the area at risk) was significantly smaller in the PIO (14.3 ± 1.1%; p

Pretreatment with atorvastatin (ATV) reduces infarct size (IS) and increases myocardial expression of phosphorylated endothelial nitric oxide synthase (p-eNOS), inducible NOS (iNOS), and cyclooxygenase-2 (COX2) in the rat. Inhibiting COX2 abolished the ATV-induced IS limitation without affecting p-eNOS and iNOS expression. We investigated 1) whether 3-day ATV pretreatment limits IS in eNOS−/− and iNOS−/− mice and 2) whether COX2 expression and/or activation by ATV is eNOS, iNOS, and/or NF-κB dependent. Male C57BL/6 wild-type (WT), University of North Carolina eNOS−/− and iNOS−/− mice received ATV (10 mg·kg−1·day−1; ATV+) or water alone (ATV−) for 3 days. Mice underwent 30 min of coronary artery occlusion and 4 h of reperfusion, or hearts were harvested and subjected to ELISA, immunoblotting, biotin switch, and electrophoretic mobility shift assay. As a result, ATV reduced IS only in the WT mice. ATV increased eNOS, p-eNOS, iNOS, and COX2 levels and activated NF-κB in WT mice. It also increased myocardial COX2 activity. In eNOS−/− mice, ATV increased COX2 expression but not COX2 activity or iNOS expression. NF-κB was not activated by ATV in the eNOS−/− mice. In the iNOS−/− mice, eNOS and p-eNOS levels were increased but not iNOS and COX2 levels; however, NF-κB was activated. In conclusion, both eNOS and iNOS are essential for the IS-limiting effect of ATV. The expression of COX2 by ATV is iNOS, but not eNOS or NF-κB, dependent. Activation of COX2 is dependent on iNOS.

Statins activate phosphatidylinositol-3-kinase, which activates ecto-5′-nucleotidase and phosphorylates 3-phosphoinositide-dependent kinase-1 (PDK-1). Phosphorylated (P-)PDK-1 phosphorylates Akt, which phosphorylates endothelial nitric oxide synthase (eNOS). We asked if the blockade of adenosine receptors (A1, A2A, A2B, or A3 receptors) could attenuate the induction of Akt and eNOS by atorvastatin (ATV) and whether ERK1/2 is involved in the ATV regulation of Akt and eNOS. In protocol 1, mice received intraperitoneal ATV, theophylline (TH), ATV + TH, or vehicle. In protocol 2, mice received intraperitoneal injections of ATV, U0126 (an ERK1/2 inhibitor), ATV + U0126, or vehicle; 8 h later, hearts were assessed by immunoblot analysis. In protocol 3, mice received intraperitoneal ATV alone or with 8-sulfophenyltheophylline (SPT); 1, 3, and 6 h after injection, hearts were assessed by immunoblot analysis. In protocol 4, mice received intraperitoneal ATV alone or with SPT, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC), alloxazine, or MRS-1523; 3 h after injection, hearts were assessed by immunoblot analysis. ATV increased P-ERK, P-PDK-1, Ser473 P-Akt, Thr308 P-Akt, and P-eNOS levels. TH blocked ATV-induced increases in P-ERK, Ser473 P-Akt, Thr308 P-Akt, and P-eNOS levels without affecting the induction of P-PDK-1 by ATV. U0126 blocked the ATV induction of Ser473 P-Akt and Thr308 P-Akt while attenuating the induction of P-eNOS. A detectable increase in P-ERK, Ser473 P-Akt and P-eNOS was seen 3 and 6 h after injection but not at 1 h. DPCPX, CSC, and alloxazine partially blocked the ATV induction of P-ERK, Ser473 P-Akt, and P-eNOS. In conclusion, blockade of adenosine A1, A2A, and A2B receptors but not A3 receptors inhibited the induction of Akt and eNOS by statins. Adenosine was required for ERK1/2 activation by statins, which resulted in Akt and eNOS phosphorylation.

We assessed whether aspirin (acetylsalicylic acid, ASA), administered before reperfusion, abrogates the infarct size (IS)-limiting effect of atorvastatin (ATV). Statins reduce IS. This dose-dependent effect is mediated by upregulation of cycloxygenase-2 (COX2) and PGI2 production. Administration of selective COX2-inhibitors either with ATV for 3 days or immediately before coronary occlusion blocks the IS-limiting effect of ATV. Sprague-Dawley rats received 3-day ATV (10 mg·kg−1·day−1) or water alone. Rats underwent 30 min coronary artery occlusion and 4 h reperfusion (IS protocol, n = 8 in each group), or rats underwent 30 min coronary artery occlusion and 10 min reperfusion (enzyme expression and activity protocol, n = 4 in each group). Immediately before reperfusion rats received intravenous ASA (5, 10, or 20 mg/kg) or saline. Area-at-risk (AR) was assessed by blue dye and IS by triphenyltetrazolium chloride. ATV reduced IS (10.1 ± 1.4% of the AR) compared with controls (31.0 ± 2.2%). Intravenous ASA alone did not affect IS (29.0 ± 2.6%); however, ASA dose dependently (5, 10, and 20 mg/kg) attenuated the protective effect of ATV on IS (15.8 ± 0.9%, 22.0 ± 1.6%, and 23.7 ± 3.8%, respectively). ASA dose dependently blocked the upregulation of COX2 by ATV. COX2 activity was as follows: control, 8.93 ± 0.90 pg/mg; ATV, 75.85 ± 1.08 pg/mg; ATV + ASA5, 34.39 ± 1.48 pg/mg; ATV + ASA10, 19.87 ± 1.10 pg/mg; and ATV + ASA20, 9.36 ± 0.94 pg/mg. ASA, administered before reperfusion in doses comparable to those used in the clinical setting, abrogates the IS-limiting effect of ATV in a model with mechanical occlusion of the coronary artery. This potential adverse interaction should be further investigated in the clinical setting of acute coronary syndromes.