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Background and Aims: The ecto-nucleoside triphosphate diphosphohydrolases of the CD39 family degrade ATP and ADP into AMP, which is converted into adenosine by the extracellular CD73/ecto-5-nucleotidase. This pathway has been explored in antithrombotic treatments but little in myocardial protection. We have investigated whether the administration of solCD39L3 (AZD3366) confers additional cardioprotection to that of ticagrelor alone in a pre-clinical model of myocardial infarction (MI)., Methods: Ticagrelor-treated pigs underwent balloon-induced MI (90 min) and, before reperfusion, received intravenously either vehicle, 1 mg/kg AZD3366 or 3 mg/kg AZD3366. All animals received ticagrelor twice daily for 42 days. A non-treated MI group was run as a control. Serial cardiac magnetic resonance (baseline, Day 3 and Day 42 post-MI), light transmittance aggregometry, bleeding time, and histological and molecular analyses were performed., Results: Ticagrelor reduced oedema formation and infarct size at Day 3 post-MI vs. controls. A 3 mg/kg AZD3366 provided an additional 45% reduction in oedema and infarct size compared with ticagrelor and a 70% reduction vs. controls (P < .05). At Day 42, infarct size declined in all ticagrelor-administered pigs, particularly in 3 mg/kg AZD3366-treated pigs (P < .05). Left ventricular ejection fraction was diminished at Day 3 in placebo pigs and worsened at Day 42, whereas it remained unaltered in ticagrelor ± AZD3366-administered animals. Pigs administered with 3 mg/kg AZD3366 displayed higher left ventricular ejection fraction upon dobutamine stress at Day 3 and minimal dysfunctional segmental contraction at Day 42 (χ2P < .05 vs. all). Cardiac and systemic molecular readouts supported these benefits. Interestingly, AZD3366 abolished ADP-induced light transmittance aggregometry without affecting bleeding time., Conclusions: Infusion of AZD3366 on top of ticagrelor leads to enhanced cardioprotection compared with ticagrelor alone., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Objectives: We performed a comprehensive assessment of the effect of myocardial ischemia duration on cardiac structural and functional parameters by serial cardiac magnetic resonance (CMR) and characterized the evolving scar., Background: CMR follow-up on the cardiac impact of time of ischemia in a closed-chest animal model of myocardial infarction with human resemblance is missing., Methods: Pigs underwent MI induction by occlusion of the left anterior descending (LAD) coronary artery for 30, 60, 90 or 120 min and then revascularized. Serial CMR was performed on day 3 and day 42 post-MI. CMR measurements were also run in a sham-operated group. Cellular and molecular changes were investigated., Results: On day 3, cardiac damage and function were similar in sham and pigs subjected to 30 min of ischemia. Cardiac damage (oedema and necrosis) significantly increased from 60 min onwards. Microvascular obstruction was extensively seen in animals with ≥90 min of ischemia and correlated with cardiac damage. A drop in global systolic function and wall motion of the jeopardized segments was seen in pigs subjected to ≥60 min of ischemia. On day 42, scar size and cardiac dysfunction followed the same pattern in the animals subjected to ≥60 min of ischemia. Adverse left ventricular remodelling (worsening of both LV volumes) was only present in animals subjected to 120 min of ischemia. Cardiac fibrosis, myocyte hypertrophy and vessel rarefaction were similar in the infarcted myocardium of pigs subjected to ≥60 min of ischemia. No changes were observed in the remote myocardium., Conclusion: Sixty-minute LAD coronary occlusion already induces cardiac structural and functional alterations with longer ischemic time (120 min) causing adverse LV remodelling., (© 2022 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.)

Background and Aims: Myocardial infarction (MI) is the clinical manifestation of atherosclerotic coronary artery disease. Spirulina is an algae known to ameliorate cardiometabolic disorders and with proven anti-inflammatory and anti-oxidant effects. We investigated, in a highly translatable animal model, whether oral supplementation with spirulina protects against the deleterious effects triggered by ST-elevation MI (STEMI). Methods: Pigs were fed a regular diet supplemented with spirulina (1 g/animal/bid) or placebo-control for 10 days. Thereafter, animals were subjected to 1.5 h percutaneous balloon-induced coronary occlusion (STEMI) followed by 2.5 h reperfusion and then sacrificed. We assessed infarct size and cardiac function. Blood samples and infarcted and remote myocardial tissue were obtained. Results: Spirulina supplementation reduced infarct size by 64%, increased myocardial salvage by 18%, and improved cardiac function by 30% vs. controls ( p < 0.05). These benefits were associated with attenuation in DNA-oxidative damage and apoptotic markers and increased iNOS in the infarcted myocardium, higher AMPK activation in the remote myocardium, and lower myocardial MCP-1 expression. Systemically, spirulina attenuated Cox-2 expression in STEMI-activated peripheral blood mononuclear cells and enhanced TNF-α release acutely post-STEMI. Additionally, spirulina decreased weight gain progression over time ( p < 0.05) without changes in lipids, glucose, liver or kidney parameters. Conclusion: A 10-day supplementation with spirulina exerts cardioprotection in a preclinical setting of STEMI by limiting cardiac damage and improving ventricular contractility through anti-oxidative, anti-inflammatory, and anti-apoptotic mechanisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vilahur, Sutelman, Ben-Aicha, Mendieta, Radiké, Schoch, Casaní, Borrell-Pagés, Padro and Badimon.)

Background and Aims: Silybum marianum (SM) is an herbal product with cytoprotective and antioxidant properties. We have previously demonstrated that SM ameliorates ventricular remodeling and improves cardiac performance. Here, we evaluated whether SM could exert beneficial effects against cardiac lipotoxicity in a pig model of closed-chest myocardial infarction (MI)., Methods: Study 1 investigated the effect of SM administration on lipid profile and any potential SM-related adverse effects. Animals received SM or placebo during 10 days and were afterward sacrificed. Study 2 evaluated the effectiveness of SM daily administration in reducing cardiac lipotoxicity in animals subjected to a 1.5h myocardial infarction (MI), who were subsequently reperfused for 2.5h and euthanized or kept under study for three weeks and then sacrificed., Results: Animals administered a 10-day SM regime presented a sharp decline in plasma triglyceride levels vs. controls, with no other modifications in lipid profile. The decrease in triglyceride concentration was accompanied by a marked reduction in triglyceride intestinal absorption and glycoprotein-P expression. Three weeks post-MI the triglyceride content in the ischemic myocardium of the SM-treated animals was significantly lower than in the ischemic myocardium of placebo-controls. This effect was associated with an enhanced cardiac expression of PPARγ and triglyceride clearance receptors. This long-term SM-administration induced a lower expression of lipid receptors in subcutaneous adipose tissue. No SM-related side-effects were registered., Conclusion: SM administration reduces plasma triglyceride levels through attenuation of triglyceride intestinal absorption and modulates cardiac lipotoxicity in the ischemic myocardium, likely contributing to improve ventricular remodeling., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objective: HDL (high-density lipoprotein) role in atherosclerosis is controversial. Clinical trials with CETP (cholesterylester transfer protein)-inhibitors have not provided benefit. We have shown that HDL remodeling in hypercholesterolemia reduces HDL cardioprotective potential. We aimed to assess whether hypercholesterolemia affects HDL-induced atherosclerotic plaque regression. Approach and Results: Atherosclerosis was induced in New Zealand White rabbits for 3-months by combining a high-fat-diet and double-balloon aortic denudation. Then, animals underwent magnetic resonance imaging (basal plaque) and randomized to receive 4 IV infusions (1 infusion/wk) of HDL isolated from normocholesterolemic (NC-HDL; 75 mg/kg; n=10), hypercholesterolemic (HC-HDL; 75 mg/Kg; n=10), or vehicle (n=10) rabbits. Then, animals underwent a second magnetic resonance imaging (end plaque). Blood, aorta, and liver samples were obtained for analyses. Follow-up magnetic resonance imaging revealed that NC-HDL administration regressed atherosclerotic lesions by 4.3%, whereas, conversely, the administration of HC-HDLs induced a further 6.5% progression ( P <0.05 versus basal). Plaque characterization showed that HC-HDL administered animals had a 2-fold higher lipid and cholesterol content versus those infused NC-HDL and vehicle ( P <0.05). No differences were observed among groups in CD31 levels, nor in infiltrated macrophages or smooth muscle cells. Plaques from HC-HDL administered animals exhibited higher Casp3 (caspase 3) content ( P <0.05 versus vehicle and NC-HDL) whereas plaques from NC-HDL infused animals showed lower expression of Casp3, Cox1 (cyclooxygenase 1), inducible nitric oxide synthase, and MMP (metalloproteinase) activity ( P <0.05 versus HC-HDL and vehicle). HDLs isolated from animals administered HC-HDL displayed lower antioxidant potential and cholesterol efflux capacity as compared with HDLs isolated from NC-HDL-infused animal and vehicle or donor HDL ( P <0.05). There were no differences in HDL-ApoA1 content, ABCA1 (ATP-binding cassette transporter A1) vascular expression, and SRB1 (scavenger receptor B1) and ABCA1 liver expression., Conclusions: HDL particles isolated from a hypercholesterolemic milieu lose their ability to regress and stabilize atherosclerotic lesions. Our data suggest that HDL remodeling in patients with co-morbidities may lead to the loss of HDL atheroprotective functions.

Aims: High-density lipoproteins (HDLs) are circulating micelles that transport proteins, lipids, and miRNAs. HDL-transported miRNAs (HDL-miRNAs) have lately received attention but their effects on vascular cells are not fully understood. Additionally, whether cardiovascular risk factors affect HDL-miRNAs levels and miRNA transfer to recipient cells remains equally poorly known. Here, we have investigated the changes induced by hypercholesterolaemia on HDL-miRNA levels and its effect on recipient endothelial cells (ECs)., Methods and Results: Pigs were kept on a high-fat diet (HC; n = 10) or a normocholesterolaemic chow (NC; n = 10) for 10 days reaching cholesterol levels of 321.0 (229.7-378.5) mg/dL and 74.0 (62.5-80.2) mg/dL, respectively. HDL particles were isolated, purified, and quantified. HDL-miRNA profiling (n = 149 miRNAs) of HC- and NC-HDLs was performed by multipanel qPCR. Cell cultures of porcine aortic ECs were used to determine whether HDL-miRNAs were delivered to ECs. Potential target genes modulated by miRNAs were identified by bioinformatics and candidate miRNAs were validated by molecular analysis. In vivo effects in the coronary arteries of normocholesterolaemic swine administered HC- or NC-HDLs were analysed. Among the HDL-miRNAs, four were found in different amounts in HC- and NC-HDL (P < 0.05). miR-126-5p and -3p and miR-30b-5p (2.7×, 1.7×, and 1.3×, respectively) were found in higher levels and miR-103a-3p and miR-let-7g-5p (-1.6×, -1.4×, respectively) in lower levels in HC-HDL. miR-126-5p and -3p were transferred from HC-HDL to EC (2.5×; P < 0.05), but not from NC-HDL, by a SRB1-mediated mechanism. Bioinformatics revealed that HIF1α was the miR-126 target gene with the highest predictive value, which was accordingly found to be markedly reduced in HC-HDL-treated ECs and in miR126 mimic transfected ECs. In vivo validation confirmed that HIF1α was diminished in the coronary endothelial layer of NC pigs administered HC-HDL vs. those administered NC-HDL (P < 0.05)., Conclusion: Hypercholesterolaemia induces changes in the miRNA content of HDL enhancing miR126 and its delivery to ECs with the consequent down-regulation of its target gene HIF1α., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.)

A high fat diet (HFD) intake is crucial for the development and progression of metabolic syndrome (MtS). Increasing evidence links gut dysbiosis with the metabolic and vascular alterations associated with MtS. Here we studied the use of a combination of various probiotic strains together with a prebiotic (synbiotic) in a commercially available Prodefen ® Plus . MtS was induced by HFD (45%) in male Wistar rats. Half of the MtS animals received Prodefen ® Plus for 4 weeks. At 12 weeks, we observed an increase in body weight, together with the presence of insulin resistance, liver steatosis, hypertriglyceridemia and hypertension in MtS rats. Prodefen ® Plus supplementation did not affect the body weight gain but ameliorated all the MtS-related symptoms. Moreover, the hypertension induced by HFD is caused by a diminished both nitric oxide (NO) functional role and release probably due to a diminished neuronal nitric oxide synthase (nNOS) activation by protein kinase A (PKA) pathway. Prodefen ® Plus supplementation for 4 weeks recovered the NO function and release and the systolic blood pressure was returned to normotensive values as a result. Overall, supplementation with Prodefen ® Plus could be considered an interesting non-pharmacological approach in MtS.

Aims: Myocardial infarction induces myocardial injury and tissue damage. During myocardial infarction strong cellular response is initiated to salvage the damaged tissues. This response is associated with the induction of different signaling pathways. Of these, the canonical Wnt signaling is increasingly important for its prosurvival cellular role, making it a good candidate for the search of new molecular targets to develop therapies to prevent heart failure in infarcted patients., Methods: Herein we report that GSK3β regulates the canonical Wnt signaling in C57Bl6 mice hearts. GSK3β is a canonical Wnt pathway inhibitor. Using GSK3β inhibitors and inducing myocardial injury (MI) in Lrp5-/- mice model we show that GSK3β phosphorylation levels regulate downstream canonical Wnt pathway genes in the ischemic heart. In the setting of MI, myocardial damage assessment usually correlates with functional and clinical outcomes. Therefore, we measured myocardial injury size in Wt and Lrp5-/- mice in the presence and absence of two different GSK3 inhibitors prior to MI. Myocardial injury was independent of GSK3 inhibitor treatments and GSK3β expression levels., Results: These studies support a central role for GSK3β in the activation of the canonical Wnt pathway in the Wt heart. Although LRP5 is protective against myocardial injury, GSK3β expression levels do not regulate heart damage., Competing Interests: The authors have declared that no competing interests exist.

Aims: Milk thistle (Silybum marianum; SM) is an herb commonly used for hepatoprotection with antioxidant and antifibrotic properties. We investigated in pigs the cardiac effects of SM intake during the acute phase of myocardial infarction (MI) and remodeling period post-MI., Methods: Study-1 tested the effect of SM use on the acute phase of MI. Hence, animals were distributed to a control group or to receive SM prior infarction (1.5 h ischemia). Animals were sacrificed after 2.5 h of reperfusion. Study-2 tested the effect of SM use in the cardiac remodeling phase. Accordingly, animals received for 10 d diet ± SM prior MI and followed the same regime for 3 weeks and then sacrificed. Study-3 tested the effect of SM in a non-infarcted heart; therefore, animals received for 10 d diet ± SM and then sacrificed., Results: Animals taking SM before MI showed a reduction in cardiac damage (decreased oxidative damage, ROS production and xanthine oxidase levels; preserved mitochondrial function; and increased myocardial salvage; p < 0.05) versus controls. Animals that remained on chronic SM intake post-MI improved left ventricular remodeling. This was associated with the attenuation of the TGFß 1 /TßRs/SMAD2/3 signaling, lower myofibroblast transdifferentiation and collagen content in the border zone (p < 0.05 vs. all other groups). Cardiac contractility improved in animals taking SM (p < 0.05 vs. post-MI-control). No changes in cardiac function or fibrosis were detected in animals on SM but without MI., Conclusion: Intake of SM protects the heart against the deleterious effects of an MI and favors cardiac healing. These benefits may be attributed to the antioxidant and antifibrotic properties of SM., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Myocardial microvascular loss after myocardial infarction (MI) remains a therapeutic challenge. Autologous stem cell therapy was considered as an alternative; however, it has shown modest benefits due to the impairing effects of cardiovascular risk factors on stem cells. Allogenic adipose-derived stem cells (ASCs) may overcome such limitations, and because of their low immunogenicity and paracrine potential may be good candidates for cell therapy. In the present study we investigated the effects of allogenic ASCs and their released products on cardiac rarefaction post MI., Methods: Pig subcutaneous adipose tissue ASCs were isolated, expanded and GFP-labeled. ASC angiogenic function was assessed by the in-vivo chick chorioallantoic membrane (CAM) model. Pigs underwent MI induction and 7 days after were randomized to receive: allogenic ASCs (intracoronary infusion); conditioned media (CM; intravenous infusion); ASCs + CM; or PBS/placebo (control). Cardiac damage and function were monitored by 3-T cardiac magnetic resonance imaging upon infusion (baseline CMR) and 1 and 3 weeks thereafter. We assessed in the myocardium: microvessel density; angiogenic markers (CD105, CD31, TF, VEGFR2, VEGFR1, vWF, eNOS, CD62); collagen deposition; and reparative fibrosis (TGFβ/TβRII/collagen). Differential proteomics of ASCs and CM was performed to characterize the ASC protein signature., Results: CAM indicated a significant ASC proangiogenic capacity. In pigs after MI, only PBS/placebo animals displayed an impaired cardiac function 3 weeks after infusion (p < 0.05 vs baseline). Administration of ASCs + CM significantly enhanced neovessel formation and favored cardiac repair post MI (p < 0.05 vs the other groups). Molecular markers of angiogenesis were significantly upregulated both at transcriptional and protein levels (p < 0.05). The in-silico bioinformatics analysis of the ASC and CM proteome (interactome) indicated activation of a coordinated protein network involved in the formation of microvessels and the resolution of rarefaction., Conclusion: Coadministration of allogenic ASCs and their CM synergistically contribute to the neovascularization of the infarcted myocardium through a coordinated upregulation of the proangiogenic protein interactome.

Aims: Molecular chaperones constitute protectors of intracellular protein integrity and seem to confer short-term defence against various cell insults. Myocardial damage is associated to a loss of protective chaperones. Ischemic post-conditioning (IPost-Co) is a procedure that seems to protect against reperfusion injury. However, little is known on alpha-crystallin-B-chain (cryab/HspB5) evolution in IPost-Co. Here we have investigated cryab in myocardial ischemia and IPost-Co., Methods and Results: Pigs underwent closed-chest 1.5h mid-left anterior descending (LAD) balloon occlusion and were either sacrificed without reperfusion (I;N=10), subjected to 2.5h of reperfusion and sacrificed (I/R; N=5); or subjected to IPost-Co before reperfusion and sacrificed 2.5h afterwards (IPost-Co; N=5). A sham-operated group was included (N=6). Proteomic analysis (2-D-electrophoresis/MALDI-TOF/TOF) revealed cryab as a single spot (20kDa; pI7.6). Myocardial cryab-20-protein and cryab-gene expression levels were decreased after ischemia and I/R(P<0.05). After IPost-Co, cryab-20-protein and cryab-gene expression levels were similar to those found in the heart of sham-operated animals (P<0.05). There was a direct correlation between LVEF-improvement after IPost-Co and myocardial cryab-20-protein levels. In a mice proof-of-principle study, cryab-20-peptide was synthesized and administered 1h before LAD-ligation and ECG-proven MI. A 59% reduction in infarct size was achieved in cryab-20-treated animals (P<0.05)., Conclusions: Ischemia and reperfusion induce a decrease in myocardial cryab-20-protein levels together with a clinical impairment of cardiac function. IPost-Co induces a clinical improvement of cardiac function and a preservation of cryab-20 levels. Intervention studies on a mice-MI model showed that cryab-20-peptide administration reduces infarct size. All together our results show a significant cardioprotective effect of cryab., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Intake of tomatoes has been linked with healthy diets (eg, Mediterranean diet). However, it remains unknown whether tomato intake exerts protective effects on the vasculature. The aim of this study was to determine whether medium-term supplementation with cooked tomato sauce (CTS) Mediterranean style (sofrito) attenuates diet-induced coronary endothelial dysfunction in an animal model with clinical impact and explore the mechanisms behind the effects. Pigs (N = 18) were fed a 10-day hypercholesterolemic diet. Half of the animals were given a supplement of 100 g/d of CTS (21.5 mg lycopene per day). Coronary responses to escalating doses of vasoactive drugs (acetylcholine, calcium ionophore, and sodium nitroprusside) and L-NG-monomethylarginine (endothelial nitric oxide synthase [eNOS] inhibitor) were measured using flow Doppler. In the coronary arteries, we investigated eNOS gene expression and activation, monocyte chemoattractant protein 1 (MCP-1) expression, and oxidative DNA damage. In the circulation, we investigated lipoprotein resistance to oxidation and the differential proteomic protein profile. In dyslipidemic animals, CTS intake prevented diet-induced impairment of receptor-operated and nonreceptor-operated endothelial-dependent coronary vasodilation. These beneficial effects were associated with enhanced eNOS transcription and activation and diminished DNA damage in the coronary arteries. CTS-fed animals showed lower lipid peroxidation, higher high-density lipoprotein (HDL) antioxidant potential and plasma lycopene levels of 0.16 mg/L. Interestingly, improved HDL functionality was associated with protein profile changes in apolipoprotein A-I and apolipoprotein J. Lipids levels and MCP-1 expression were not affected by CTS. We report that CTS intake protects against low-density lipoprotein-induced coronary endothelial dysfunction by reducing oxidative damage, enhancing eNOS expression and activity, and improving HDL functionality., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Drug-induced vascular injury (DIVI) is commonly associated with phosphodiesterase (PDE) inhibitors. Despite histological characterization, qualified biomarkers for DIVI detection are lacking. We investigated whether a single administration of roflumilast (PDE-IV inhibitor) induces vascular damage and identified novel surrogate biomarkers of acute vascular injury. Pigs received postoperative 250, 375, or 500 μg of roflumilast or placebo/control. After 1.5 hr, coronary reactivity was determined by catheter-based administration of acetylcholine and sodium nitroprusside (SNP) in the coronary sinus. Immunohistochemical analysis of vessel integrity (von Willebrand factor [vWF]) and fibrin(ogen) deposition was performed in the coronary artery and aorta. Peripheral blood was collected for differential proteomics and microparticles analysis. Circulating interleukin (IL)-6 was analyzed. Roflumilast-treated animals displayed higher vasodilation to acetylcholine and SNP versus controls (p < .05). Roflumilast-treated animals showed a dose-dependent (p < .05) decrease in vessel integrity and dose-dependent increase in fibrin deposition forming a continuous layer at roflumilast-500 μg. Peripheral blood of roflumilast-500-μg-treated animals showed increased levels of total and endothelial-derived microparticles and exhibited a coordinated change in proteins kininogen-1, endothelin-1, gelsolin, apolipoprotein A-I, and apolipoprotein-J associated with vascular injury (p < .05 vs. controls). IL-6 remained unaltered. Roflumilast-induced vascular injury can be detected by novel markers in peripheral blood. Validation of these surrogate markers in human samples seems required., (© 2014 by The Author(s).)

Introduction and Objectives: The Mediterranean diet, rich in polyphenols, has shown to be cardioprotective. However the mechanisms involved remain unknown. We investigated whether supplementation with a pomegranate extract rich in polyphenols renders beneficial effects on coronary function in a clinically relevant experimental model and characterized the underlying mechanisms., Methods: Pigs were fed a 10-day normocholesterolemic or hypercholesterolemic diet. Half of the animals were given a supplement of 625 mg/day of a pomegranate extract (Pomanox; 200 mg punicalagins/day). Coronary responses to escalating doses of vasoactive drugs (acetylcholine, calcium ionophore, and sodium nitroprusside) and L-NG-monomethylarginine (endothelial nitric oxide-synthase inhibitor) were measured using flow Doppler. Akt/endothelial nitric oxide-synthase axis activation, monocyte chemoattractant protein-1 expression, oxidative deoxyribonucleic acid damage in the coronary artery, and lipoprotein resistance to oxidation were evaluated., Results: In dyslipidemic animals, Pomanox supplementation prevented diet-induced impairment of endothelial relaxation, reaching vasodilatory values comparable to normocholesterolemic animals upon stimulation with acetylcholine and/or calcium ionophore. These beneficial effects were associated with vascular Akt/endothelial nitric oxide-synthase activation and lower monocyte chemoattractant protein-1 expression. Pomanox supplementation reduced systemic oxidative stress (higher high-density lipoprotein-antioxidant capacity and higher low-density lipoprotein resistance to oxidation) and coronary deoxyribonucleic acid damage. Normocholesterolemic animals elicited similar drug-related vasodilation regardless of Pomanox supplementation. All animals displayed a similar vasodilatory response to sodium nitroprusside and L-NG-monomethylarginine blunted all vasorelaxation responses except for sodium nitroprusside., Conclusions: Pomanox supplementation hinders hyperlipemia-induced coronary endothelial dysfunction by activating the Akt/endothelial nitric oxide-synthase pathway and favorably counteracting vascular inflammation and oxidative damage., (Copyright © 2014 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.)

Background: Adipose tissue-derived progenitor cells (ATDPCs) isolated from human cardiac adipose tissue are useful for cardiac regeneration in rodent models. These cells do not express cardiac troponin I (cTnI) and only express low levels of PECAM-1 when cultured under standard conditions. The purpose of the present study was to evaluate changes in cTnI and PECAM-1 gene expression in cardiac ATDPCs following their delivery through a fibrin patch to a murine model of myocardial infarction using a non-invasive bioluminescence imaging procedure., Methods and Results: Cardiac and subcutaneous ATDPCs were doubly transduced with lentiviral vectors for the expression of chimerical bioluminescent-fluorescent reporters driven by constitutively active and tissue-specific promoters (cardiac and endothelial for cTnI and PECAM-1, respectively). Labeled cells mixed with fibrin were applied as a 3-D fibrin patch over the infarcted tissue. Both cell types exhibited de novo expression of cTnI, though the levels were remarkably higher in cardiac ATDPCs. Endothelial differentiation was similar in both ATDPCs, though cardiac cells induced vascularization more effectively. The imaging results were corroborated by standard techniques, validating the use of bioluminescence imaging for in vivo analysis of tissue repair strategies. Accordingly, ATDPC treatment translated into detectable functional and morphological improvements in heart function., Conclusions: Both ATDPCs differentiate to the endothelial lineage at a similar level, cardiac ATDPCs differentiated more readily to the cardiomyogenic lineage than subcutaneous ATDPCs. Non-invasive bioluminescence imaging was a useful tool for real time monitoring of gene expression changes in implanted ATDPCs that could facilitate the development of procedures for tissue repair., (© 2013.)

The extent of cardiac remodeling determines survival after acute MI. However, the mechanisms driving cardiac remodeling remain unknown. We examined the effect of ischemia and reperfusion (R) on myocardial changes up to 6 days post-MI. Pigs underwent 1.5h or 4h mid-LAD balloon occlusion and sacrificed or 1.5h occlusion followed by R and sacrificed at 2.5h, 1 day, 3 days, and 6 days. Ischemic- (IM) and non-ischemic myocardium (NIM) was obtained for molecular analysis of: 1) apoptosis (P-Bcl2, Bax, P-p53, active-caspase-3); 2) the TLR-4-MyD88-dependent and independent pathways; 3) Akt/mTOR/P70(S6K) axis activation; and, 4) fibrosis (TGF-β, collagen1-A1/A3). Histopathology for inflammation, collagen, and fibroblast content, TUNEL staining, and metalloproteinase activity was performed. Apoptosis is only detected upon R in IM cardiomyocytes and progresses up to 6 days post-R mainly associated with infiltrated macrophages. The Akt/mTOR/P70(s6K) pathway is also activated upon R (IM) and remains elevated up to 6 days-R (P<0.05). Ischemia activates the TLR-4-MyD88-dependent (cytokines/chemokines) and -independent (IRF-3) pathways in IM and NIM and remains high up to 6 days post-R (P<0.05). Accordingly, leukocytes and macrophages are progressively recruited to the IM (P<0.05). Ischemia up-regulates pro-fibrotic TGF-β that gradually rises collagen1-A1/-A3 mRNA with subsequent increase in total collagen fibrils and fibroblasts from 3 days-R onwards (P<0.005). MMP-2 activity increases from ischemia to 3 days post-R (P<0.05). We report that there is a timely coordinated cellular and molecular response to myocardial ischemia and R within the first 6 days after MI. In-depth understanding of the mechanisms involved in tissue repair is warranted to timely intervene and better define novel cardioprotective strategies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objectives: Coronary occlusion and revascularization leads to myocardial damage and heart function deterioration. Statins can regress atherosclerosis and modulate platelet function, but their effect on post-acute myocardial infarction (AMI) injury remains to be fully determined. We sought to examine whether rosuvastatin (R) exerts any effect on the RISK/apoptosis pathway when administered early after coronary reperfusion., Methods: Pigs were fed 10 days a hypercholesterolemic diet before AMI induction and thereafter for 7 days randomly distributed to receive R or placebo (C) with the same diet. At sacrifice, hearts were sliced and alternatively collected for MI size and molecular analysis (gene and protein expression) in the peri-infarcted and remote myocardium. The RISK components (PKC, Erk2, and Akt/PKB) and downstream targets (HIF-1alpha and VEGF), and cell survival/apoptosis markers (Bcl-2, Bax, and Caspase-3) were analyzed. Annexin-V, Mito-Tracker staining, and inflammatory infiltration were also evaluated., Results: R enhanced PKC, Erk2, Akt/PKB and its downstream effectors, and attenuated inflammation and cardiomyocyte apoptosis in the peri-infarcted zone (p<0.05). No changes were detected in the remote myocardium. Infarct size was smaller in R than in C pigs (7% absolute reduction; 36% relative reduction; p<0.05) and was associated with an absolute 12% recovery of LVEF (24% relative restoration; p<0.05 vs. post-AMI)., Conclusions: HMG-CoA inhibition early after reperfusion activates RISK kinases, reduces the extent of damaged myocardium, and improves heart function.

Acute thrombosis is a threat in patients undergoing percutaneous coronary intervention with stent implantation. Our objective was to determine if stent-induced thrombus formation could be inhibited by oral treatment with a thromboxane A(2)/prostaglandin H(2) receptor antagonist (TPr; S18886) as an alternative to standard therapy. Pigs were allocated in the following treatment (p.o) groups: I) clopidogrel (CLOP); II) ASA; III) S18886; IV) ASA+CLOP; and V) placebo-control. Damaged vessel was placed in the Badimon chamber containing a stent and perfused at 212/s. Antithrombotic effects were assessed as (111)In-platelet deposition (PD) in two series (60 and 180 min after drug intake). Fibrin(ogen) deposition, light transmittance aggregometry (LTA; collagen, U46619, and ADP), and bleeding time (BT) were also evaluated. After 60 min S18886 reduced PD < or =48%, 40%, and 35% vs placebo, CLOP-, and ASA-treated animals, respectively (P < 0.05), while ASA+CLOP showed a 58% reduction versus placebo (P < 0.01). After 3 hours, ASA+CLOP decreased PD by 55%, S18886 by 40%, CLOP alone by 28% (P < 0.05), and ASA showed no inhibition versus placebo. Similar effects were found in S18886- and ASA+CLOP-treated animals at both times. Fibrin(ogen) deposition followed the same pattern. Collagen-induced LTA was significantly reduced by ASA, ASA+CLOP, and S18886; S18886 abolished U46619-induced LTA; and, CLOP +/- ASA reduced ADP-induced LTA in a time-dependent manner. TPr blockade did not prolong BT, whereas CLOP +/- ASA significantly did (P < 0.0001). In conclusion, blockade of the TPr provided a fast and potent platelet inhibitory effect in a porcine model of in-stent thrombosis comparable to that of blocking both the ADP receptor and cyclooxygenase activation; in addition, TPr provided a more favorable bleeding risk profile.

Platelets play a primary role in thrombus formation after plaque rupture. Platelets recognize the exposed collagen via Von Willebrand factor (VWF) and become activated. Saratin, an inhibitor of the VWF-dependent binding of platelets to collagen, may reduce the thrombotic risk associated to atherosclerosis. Our objective was to evaluate the antithrombotic effects of local treatment with saratin on human atherosclerotic lesions. Thrombus formation was assessed by the deposition of (111)In-platelets on different human atherosclerotic lesions under three local shear conditions (800,1700 and 3400/s) with blood derived from catheterized pigs. Human atherosclerotic lesions were locally treated with saratin (30 microg/ml) at 37 degrees C for 5 min and placed in the chamber. Under stenotic shear conditions of 800/s, saratin significantly (p<0.05) reduced platelet deposition triggered by human denuded vessel wall (44%), fatty streaks (47%), severely damaged vessel (50%) and atherosclerotic plaque (57%). Thrombus characterization by immunohistochemistry showed also a reduction in fibrin deposition in treat-ed vessels. These results suggest that the local site-specific treatment with saratin inhibits atherosclerotic plaque thrombogenicity at haemodynamic conditions typical of moderately stenotic coronary arteries.

Platelets are involved in the clinical presentations of ischemic heart disease. Our objective was to study the antithrombotic effects of a new nitric oxide donor (LA419), a neutral sugar organic nitrate with a protected thiol group in its molecular structure. Animals were randomly distributed in three groups: I) oral administration of LA419 (0.9-1.8-3.6-5 mg/kg/d, 10 days); II) oral administration of standard IS-5-MN (0.9-1.8 mg/kg/d, 10 days); III) non-treated group (control). In catheterized pigs, thrombosis was studied under controlled rheological conditions by radioisotopic evaluation of deposited platelets on damaged vessel wall, placed in an extracorporeal perfusion chamber. Changes in blood pressure, heart rate, and platelet aggregation were evaluated. Results have shown that LA419 significantly decreased thrombus formation according to the degree of vascular damage, and shear rate conditions in a dose-dependent manner (p<0.005), without significant modifications on blood pressure and/or elevation of liver enzymes. In contrast, IS-5-MN only showed a significant reduction on platelet deposition at the high dose, that was associated to hypotension and elevation of liver enzymes. Therefore, we conclude that this new anti-ischemic NO-donor (NOd) LA419 that inhibits platelet function without modifying blood pressure may be a highly efficacious strategy to passivate platelet activation induced by a damaged vessel wall.

Objective: Hypercholesterolemia induces endothelial dysfunction, a hallmark of the atherosclerotic process, modulating the expression of key genes in vascular endothelial cells., Methods and Results: By differential display analysis, we have studied the effect of high concentrations of native low density lipoprotein (LDL) on endothelial gene expression. mRNA levels of lysyl oxidase (LOX), an enzyme involved in collagen and elastin cross-linking, were downregulated by LDL treatment in endothelial cells in a dose- and time-dependent manner (80% of inhibition by 180 mg/dL LDL for 24 hours). This reduction of LOX expression was associated with a decrease in LOX activity (40% and 54% of inhibition after 24 and 48 hours of LDL treatment, respectively). LOX mRNA half-life was not modified by LDL, but transcriptional inhibition blocked the effect of LDL. Inhibition of LOX activity by either LDL or beta-aminopropionitrile, an inhibitor of LOX, increased endothelial permeability (192+/-0.19- and 3.37+/-0.74-fold, respectively). Interestingly, a reduction in LOX expression (3.5-fold) was observed in vivo in the vascular wall of hypercholesterolemic pigs., Conclusions: These findings suggest that LDL downregulation of LOX could contribute to the endothelial dysfunction caused by hypercholesterolemia, thus contributing to atherosclerotic plaque formation.