Model Of Coronary Artery Occlusion Research Articles

A canine acute coronary occlusion model using thrombus implantation by percutaneous coronary intervention

AbstractObjectiveTo establish an acute canine coronary artery occlusion model by implanting a mixed thrombus prepared in vitro into the coronary artery by percutaneous coronary intervention (PCI).MethodsThis study included 14 healthy Beagle dogs aged 10–13 months. First, 5 ml of canine venous blood was drawn to prepare the coronary artery thrombus in vitro. The thrombus was then implanted into the canine coronary artery by PCI to establish an animal model of acute coronary artery occlusion. Dogs were randomly divided into two experimental groups: group A, in which dogs were assessed for 2 h after coronary occlusion, and group B, in which dogs were assessed for 4 h after surgery. Baseline and postoperative assessment included cardiac troponin T (cTnT) levels, electrocardiography (ECG), plain and contrast echocardiography, and large‐scale digital subtraction angiography (DSA).ResultsCanine venous blood was successfully used to prepare a mixed thrombus with reticular fibrin structure and activated platelets in vitro. After the mixed thrombus was implanted into the coronary artery, large‐scale DSA showed no contrast medium perfusion, confirming complete occlusion of the coronary artery. After the coronary artery occlusion, dogs in both groups showed ST segment elevation >0.5 MV, a 0.9 μg/L increase in CTnT (p < 0.001), and increased EDV and ESV. Dogs also showed decreased left ventricular EF and FS and decreased MBF in the infarcted area (p < 0.05).ConclusionsAn experimental model of acute coronary artery occlusion in dogs was successfully established by implanting a mixed thrombus developed in vitro into the coronary artery using minimally invasive PCI.

Multidimensional immunoimaging to characterize the inflammatory reaction after permanent and temporary coronary artery occlusion in mice

Abstract Background The immune response following acute myocardial infarction encompasses a delicate balance between inflammatory and reparative programs. Our knowledge of these complex mechanisms is mainly derived from studies using a murine model of permanent coronary artery occlusion. In this study we developed, validated and implemented multiparametric imaging methods to investigate cardiac function and the systemic immune response in transient or permanent coronary artery occlusion mouse models. Methods The myocardial infarction models encompassed either transient (40 min) or permanent LAD occlusion in C57BL/6 mice, and non-infarcted mice were used as controls. Two or seven days later, the animals subjected to systemic immunoimaging of the bone marrow, spleen and myocardium with late gadolinium enhancement cardiac MRI (LGE cMRI), 18F-fluorodeoxyglucose (18F-FDG) PET, 18F-Fluorothymidine (18F-FLT) PET, 64Cu-CCR2 PET targeting inflammatory monocytes, 89Zr-CD11b nanobody PET and 19F-HDL-PERFECTA MRI, both targeting myeloid cells. In addition, the same myocardial infarction models were applied to atherosclerosis-prone Apoe−/− and systemic inflammation and plaque progression were assessed by flow cytometry and immunohistochemistry four weeks after infarction. Results Through LGE cMRI and 18F-FDG PET, we observed that temporary coronary occlusion resulted in a smaller infarct size, better cardiac function and viability compared to permanent occlusion. Multiparametric immunoimaging targeting CD11b+ cells by 89Zr-CD11b nanobody PET and Ly6Chi inflammatory monocytes by 64Cu-CCR2 PET demonstrated that mice subjected to transient coronary occlusion had less immune cell influx to the ischemic myocardium. This finding was confirmed by flow cytometry analysis of the infarct zone. In contrast, both myocardial infarction models cause a similar systemic immune response in the bone marrow and spleen as observed with multimodal imaging with subsequent similar numbers of CD11b+ cells in the blood. Both permanent and temporary coronary artery occlusion aggravate atherosclerosis in Apoe−/− mice with higher macrophage and Ly6Chi monocyte numbers in aortas and larger plaque size compared to Apoe−/− mice without myocardial infarction. Conclusions We developed and employed multimodal, multiparametric imaging protocols to characterize the immune response in the heart, bone marrow and spleen in two models of myocardial infarction. While cardiac function was superior in the ischemia reperfusion model, both types of myocardial infarction accelerated atherosclerosis. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): NIH and DFG

Isosteviol improves cardiac function and promotes angiogenesis after myocardial infarction in rats.

Isosteviol has been indicated as a cardiomyocyte protector. However, the underlying mechanism remains unclear. Thus, we sought to confirm the protective effect of isosteviol after myocardial infarction in a model of permanent coronary artery occlusion and investigate the potential proangiogenic activity in vitro and in vivo. A 4-week permanent coronary artery occlusion rat model was generated, and the protective effect of isosteviol was evaluated by echocardiographic imaging and hemodynamics assays. The coronary capillary density was tested by immunochemistry and micro-computed tomography (μCT) imaging. The effect of isosteviol on endothelial cells was determined in human umbilical vein endothelial cells (HUVECs) in vitro and Tg (kdrl: EGFP) zebrafish in vivo. We also examined the expression of related transcription factors by real-time polymerase chain reaction (RT-qPCR). Isosteviol increased ejection fraction (EF), fractional shortening (FS), cardiac systolic index (CI), maximum rate of increase of left ventricular pressure (Max dp/dt), and left ventricular systolic pressure (LVSP) by 32%, 40%, 25%, 26%, and 10%, respectively, in permanent coronary artery occlusion rats. Interestingly, it also promoted coronary capillary density by 2.5-fold. In addition, isosteviol promoted the proliferation and branching of HUVECs in vitro. It also rescued intersegmental vessel (ISV) development and improved endothelial cell proliferation by approximately fivefold (4-6) in zebrafish embryos in vivo. Isosteviol also upregulated the expression of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor A (VEGFA) in zebrafish by fourfold and 3.5-fold, respectively. Our findings suggest that isosteviol is a proangiogenic agent and that this activity is related to its protective effects against myocardial ischemia. After using the permanent coronary artery occlusion model, we demonstrated that isosteviol promotes angiogenesis directly and increases capillary density in myocardial ischemia rats. Isosteviol promotes angiogenesis in zebrafish in vivo and increases vascular endothelial cell proliferation in HUVECs and zebrafish. The angiogenesis activity of isosteviol may be correlated with VEGFA and HIF-1α signaling.

Excess ischemic tachyarrhythmias trigger protection against myocardial infarction in hypertensive rats.

Increased level of C-reactive protein (CRP) is a risk factor for cardiovascular diseases, including myocardial infarction and hypertension. Here, we analyzed the effects of CRP overexpression on cardiac susceptibility to ischemia/reperfusion (I/R) injury in adult spontaneously hypertensive rats (SHR) expressing human CRP transgene (SHR-CRP). Using an in vivo model of coronary artery occlusion, we found that transgenic expression of CRP predisposed SHR-CRP to repeated and prolonged ventricular tachyarrhythmias. Excessive ischemic arrhythmias in SHR-CRP led to a significant reduction in infarct size (IS) compared with SHR. The proarrhythmic phenotype in SHR-CRP was associated with altered heart and plasma eicosanoids, myocardial composition of fatty acids (FAs) in phospholipids, and autonomic nervous system imbalance before ischemia. To explain unexpected IS-limiting effect in SHR-CRP, we performed metabolomic analysis of plasma before and after ischemia. We also determined cardiac ischemic tolerance in hearts subjected to remote ischemic perconditioning (RIPer) and in hearts ex vivo. Acute ischemia in SHR-CRP markedly increased plasma levels of multiple potent cardioprotective molecules that could reduce IS at reperfusion. RIPer provided IS-limiting effect in SHR that was comparable with myocardial infarction observed in naïve SHR-CRP. In hearts ex vivo, IS did not differ between the strains, suggesting that extra-cardiac factors play a crucial role in protection. Our study shows that transgenic expression of human CRP predisposes SHR-CRP to excess ischemic ventricular tachyarrhythmias associated with a drop of pump function that triggers myocardial salvage against lethal I/R injury likely mediated by protective substances released to blood from hypoxic organs and tissue at reperfusion.

Adenosine stress CMR perfusion imaging of the temporal evolution of perfusion defects in a porcine model of progressive obstructive coronary artery occlusion

Adenosine stress CMR perfusion imaging can quantify absolute perfusion and myocardial perfusion reserve (MPR) in coronary artery disease (CAD) with higher spatial resolution than positron emission tomography, the only clinically available technique for quantitative myocardial perfusion imaging. While porcine models of CAD are excellent for studying perfusion abnormalities in chronic CAD, to date there are a limited number of studies that use quantitative perfusion for evaluation. Therefore, we developed an adenosine stress CMR protocol to evaluate the temporal evolution of perfusion defects in a porcine model of progressive obstructive CAD. 10 Yucatan minipigs underwent placement of an ameroid occluder around the left circumflex artery (LCX) to induce a progressive chronic coronary obstruction. Four animals underwent a hemodynamic dose range experiment to determine the adenosine dose inducing maximal hyperemia. Each animal had a CMR examination, including stress/rest spiral quantitative perfusion imaging at baseline and 1, 3, and 6weeks. Late gadolinium enhancement images determined the presence of myocardial infarction, if any existed. Pixelwise quantitative perfusion maps were generated using Fermi deconvolution. The results were statistically analyzed with a repeated mixed measures model to block for physiological variation between the animals. Five animals developed myocardial infarction by 3weeks, while three developed ischemia without an infarction. The perfusion defects were located in the inferolateral myocardium in the perfusion territory of the LCX. Stress perfusion values were higher in remote segments than both the infarcted and ischemic segments (p<0.01). MPR values were significantly greater in the remote segments than infarcted and ischemic segments (p<0.01). While the MPR decreased in all segments, the MPR recovered by the sixth week in the remote regions. We developed a model of progressive CAD and evaluated the temporal evolution of the development of quantitative perfusion defects. This model will serve as a platform for understanding the development of perfusion abnormalities in chronic occlusive CAD.

Nitroglycerin application and coronary arteriogenesis.

BackgroundIn the presence of a coronary occlusion, pre-existing small collateral vessels (arterioles) develop into much larger arteries (biological bypasses) that have the potential to allow a certain level of perfusion distal to the blockage. Termed arteriogenesis, this phenomenon proceeds via a complex combination of events, with nitric oxide (NO) playing an essential role. The aim of this study was to investigate the effects of supplemental administration of NO donors, i.e., short-acting nitroglycerin (NTG) or slow-release pelleted isosorbide dinitrate (ISDN), on collateral development in a repetitive coronary artery occlusion model in rats.MethodsCoronary collateral growth was induced via a repetitive occlusion protocol (ROP) of the left anterior descending coronary artery (LAD) in rats. The primary endpoints were the histological evaluation of rat heart infarct size and ST-segment elevation (ECG-analysis) upon final permanent occlusion of the LAD (experimentally induced myocardial infarction). The effects of NTG or ISDN were also evaluated by administration during 5 days of ROP. We additionally investigated whether concomitant application of NTG can compensate for the anti-arteriogenic effect of acetylsalicylic acid (ASA).ResultsAfter 5 days of ROP, the mean infarct size and degree of ST-elevation were only slightly lower than those of the SHAM group; however, after 10 days of the protocol, the ROP group displayed significantly less severe infarct damage, indicating enhanced arteriogenesis. Intermittent NTG application greatly decreased the ST-elevation and infarct size. The ISDN also had a positive effect on arteriogenesis, but not to the same extent as the NTG. Administration of ASA increased the infarct severity; however, concomitant dosing with NTG somewhat attenuated this effect.ConclusionIntermittent treatment with the short-acting NTG decreased the size of an experimentally induced myocardial infarct by promoting coronary collateral development. These new insights are of great relevance for future clinical strategies for the treatment of occlusive vascular diseases.

Abstract 337: Long Term Safety and Efficacy of a Cardiac Graft in a Swine Model of Heart Failure

Introduction: We have shown efficacy with cardiac grafts in the rat coronary ligation model of chronic heart failure (CHF) and feasibility of implantation and assessment in a swine model. Now we propose testing its long term safety and efficacy in a swine model of CHF. Our aims are: 1) Evaluate long term (6 months) left ventricular functional improvements after implantation of the graft in a swine coronary artery occlusion model of CHF, 2) Evaluate quality of life and functional capacity improvements of swine treated with the graft, and 3) Evaluate long term safety of the graft. Hypothesis: An engineered tissue comprised of differentiated cardiac myocytes and fibroblasts will effectively integrate into the host tissue, and restore cardiac function in swine with CHF. Methods: Male Yucatan mini swine 30-40kg were infarcted using percutaneous methods. Balloon occlusions (90 minute) are performed via catheter, distal to the first diagonal branch of the left anterior descending (LAD) coronary artery and animals. Four weeks after myocardial infarction (MI) cardiac grafts engineered with bio absorbable knitted mesh are implanted on the infarcted epicardium. Cardiac magnetic resonance imaging (MRI) is performed prior to MI, 1 month after MI, 1 month after patch placement, again at 3 month and 6 months. All swine are implanted with continuous event recorders to acquire surface electrocardiogram during the entire study. In addition, quality of life/functional capacity are assessed using FitBark collar activity monitors and treadmill exertion testing. Infarct size is determined using MRI images and 2,3,5-triphenyltetrazolium chloride staining at study end. Results: Medium to large transmural infarcts are generated with 90min balloon occlusion of the LAD which demonstrate compensatory remodeling of the LV as assessed via cardiac MRI. Upscale cardiac matrix grafts to 6cm diameter are easily handled by the surgeon and can be surgically implanted on the epicardium using sub-xiphoid median sternotomy. No adverse arrhythmic activity has been observed. Conclusion: Our cardiac graft is well suited for upscale to clinical size, can be easily handled and implanted in a swine model of CHF.

Insights into coronary collateral formation from a novel porcine semiacute infarction model

For patients with severe ischemic heart disease, complete revascularization by a percutaneous coronary intervention or coronary artery bypass grafting is often not achieved and may still cause residual angina. In case of progressive coronary artery occlusions, therapeutic arteriogenesis constitutes a promising strategy for increasing blood supply to the ischemic myocardium. Whether the formation of collaterals in the hypofused myocardium is angiogenetic in nature or based on preformed coronary artery anastomoses remains debatable. The objectives of this research were (i) the development of an appropriate research methodology to study a humanoid animal semiacute infarction model with low mortality and (ii) to answer the question of whether collateral revascularization follows a pre-existing 'blueprint'. A porcine model was chosen in which a step-wise vessel occlusion was performed by implantation of a copper stent into the distal left anterior descending artery. Vessel occlusion and collateral development were confirmed in vivo every 14 days up to day 56 by repeated coronary angiography and myocardial perfusion measurement using cardiac MRI. After the completion of the in-vivo imaging studies, animals were euthanized and collateral growth was evaluated using microcomputer tomography. Our porcine model of semiacute noninvasive coronary artery occlusion confirmed the existence of preformed coronary anastomoses and the proliferation of functional vessels in hypoperfused myocardium. Repetitive intra-animal MRIs showed the functional impact of these growing collaterals. The confirmation of preformed coronary anastomoses during the process of collateralization (natural bypasses) offers a preclinical avenue to carry out arteriogenetic pharmaceutical research in patients with ischemic heart disease.

Molecular Imaging of Apoptosis in Ischemia Reperfusion Injury With Radiolabeled Duramycin Targeting Phosphatidylethanolamine: Effective Target Uptake and Reduced Nontarget Organ Radiation Burden

ObjectivesThe purpose of this study was to evaluate the feasibility of imaging apoptosis in experimental ischemia-reperfusion model by technetium-99m (99mTc)-labeled Duramycin, and compare it to an established tracer, 99mTc-labeled Annexin-V, which has a relative disadvantage of high radiation burden to nontarget organs. BackgroundDuring apoptosis, the cell membrane phospholipids-phosphatidylserine (PS) and phosphatidylethanolamine (PE) are exposed and can be targeted by Annexin-V and Duramycin, respectively, for in vivo imaging. Identification of a reversible cell death process should permit therapeutic intervention to help reduce myocyte loss and left ventricle dysfunction. MethodsIn a 40-min left coronary artery ischemia-reperfusion model in 17 rabbits, 7 mCi of 99mTc-labeled Duramycin (n = 10), 99mTc-linear Duramycin (a negative tracer control; n = 3), or 99mTc-Annexin-V (a positive tracer-control; n = 4) were intravenously administered 30 min after reperfusion. Of the 10 Duramycin group animals, 4 animals were treated with an antiapoptotic agent, minocycline at the time of reperfusion. In vivo and ex vivo micro–single-photon emission computed tomography (μSPECT) and micro-computed tomography (μCT) imaging was performed 3 h after reperfusion, followed by quantitative assessment of tracer uptake and pathological characterization. Fluorescent Duramycin and Annexin-V were injected in 4 rats to visualize colocalization in infarct areas in a 40-min left coronary artery occlusion and 30-min reperfusion model. ResultsIntense uptake of Duramycin and Annexin-V was observed in the apical (infarcted) areas. The percent injected dose per gram uptake of Duramycin in apical region (0.751 ± 0.262%) was significantly higher than remote area in same animals (0.045 ± 0.029%; p < 0.01). Duramycin uptake was insignificantly lower than Annexin-V uptake (1.23 ± 0.304%; p > 0.01) but demonstrated substantially lower radiation burden to kidneys (0.358 ± 0.210% vs. 1.58 ± 0.316%, respectively; p < 0.001). Fluorescence studies with Duramycin and Annexin V showed colocalization in infarct areas. Minocycline treatment substantially resolved Duramycin uptake (0.354% ± 0.0624%; p < 0.01). ConclusionsDuramycin is similarly effective in imaging apoptotic cell death as Annexin-V with lower nontarget organ radiation. Clinical feasibility of apoptosis imaging with a PE-seeking tracer should be tested.

Defining the sham environment for post-myocardial infarction studies in mice.

The purpose of this study was to evaluate the effect of sham surgery in a minimally invasive surgical model of permanent coronary artery occlusion used to generate myocardial infarction (MI) in mice. Adult male C57BL/6J mice (3-6 mo old) were divided into five groups: day (D) 0 (no surgical operation), D1 Sham, D1 MI, D7 Sham, and D7 MI. A refined MI surgery technique was used to approach the coronary artery without the ribs being cut. Both sham and MI mice had the left ventricle (LV) exposed through a small incision. To test the effects of surgery alone, the suture was passed around the coronary artery but not ligated. The MI mice were subjected to permanent coronary artery ligation. The mice were killed at D1 or D7 postsurgical procedure. Compared with D0 no surgery controls, the D1 and D7 sham groups exhibited no surgical mortality and similar necropsy and echocardiographic variables. Surgery alone did not induce an inflammatory cell response, as evidenced by the lack of leukocyte infiltration in the sham groups. Analysis of 165 inflammatory cytokines and extracellular matrix factors in sham revealed that a minor gene response was initiated but not translated to protein levels. Collagen deposition did not occur in the absence of MI. In contrast, the D1 and D7 MI groups showed the expected robust inflammatory and scar formation responses. When a minimally invasive procedure to generate MI in mice was used, the D0 (no surgical operation) control was an adequate replacement for the use of sham surgery groups.

Liquid Ventilation for the Induction of Ultrafast Hypothermia in Resuscitation Sciences: A Review

Liquid ventilation was initially proposed for lung lavage and respiratory support. More recently, it was also investigated as an experimental strategy for ultrafast cooling or organ preservation during ischemic disorders. The goal of this article is to identify and review the studies that investigated liquid ventilation in the field of resuscitation sciences. An exhaustive analysis of the literature was performed using the Medline database up to 15th September 2015. Articles were selected according to their relevance. All articles focusing on respiratory support were excluded. On the basis of 76 retrieved studies from the Medline database, 29 were included in this review. All studies were experimental reports and most of them investigated the cooling properties of liquid ventilation in animal models of experimental cardiac arrest or coronary artery occlusion in rabbits or pigs. Animal studies demonstrated a wide range of potential applications of total liquid ventilation in resuscitation sciences. This strategy is able to provide ultrafast cooling, independent of the body weight. In animal models of cardiopulmonary resuscitation, it was shown to provide potent benefits widely linked to cooling rapidity.

Abstract 19684: Nitric Oxide Protects From Cardiac Ischemia/reperfusion Damage by Blocking Activation of Extracellular Matrix Metalloproteinase Inducer EMMPRIN Through Binding to Caveolin3

Introduction: Nitric Oxide (NO) induces cardiac protection by still unknown mechanisms. We previously found that Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) is a target of NO during ischemia/reperfusion (IR). Hypothesis: EMMPRIN is glycosylated at different locations, and high glycosylated forms of EMMPRIN are required for matrix metalloproteinase downstream activation in several cell types. In hearts subject to IR NO may inhibit EMMPRIN glycosylation. Methods: A surgical model of coronary artery occlusion. Results: We found that iNOS null mice show high glycosylated (active) forms of EMMPRIN, whereas NO producing mice also show low glycosylated EMMPRIN. Likewise, NO producing mice exhibit a significant level of cardiac protection, as shown by increased left ventricle ejection fraction detected by high frequency ultrasound, and decreased area of infarction as shown by ex vivo TTC staining. To test whether low glycosylated forms of EMMPRIN may play a role in iNOS WT cardiac protection, we found that EMMPRIN binds to caveolin-3 in healthy hearts, however, I/R prompted the disruption of low glycosylated EMMPRIN from caveolin-3 in iNOS null mice in a time dependent manner whereas a significant low glycosylated portion of EMMPRIN remained bound to caveolin-3 in iNOS WT mice. To study this effect in more detail, we found that caveolin-3 binding to low glycosylated forms of EMMPRIN, prevents downstream EMMPRIN-mediated extracellular matrix degradation through matrix metalloproteinase activation. I/R damage resulted more severe in caveolin-3 null mice, where no low glycosylated EMMPRIN was detected after reperfusion. Conclusions: We show for the first time that caveolin-3 induces nitric oxide mediated cardiac protection by preventing glycosylation of EMMPRIN in mouse hearts.

The cardioprotective compound cloxyquin uncouples mitochondria and induces autophagy.

Mitochondrial quality control mechanisms have been implicated in protection against cardiac ischemia-reperfusion (IR) injury. Previously, cloxyquin (5-chloroquinolin-8-ol) was identified via phenotypic screening as a cardioprotective compound. Herein, cloxyquin was identified as a mitochondrial uncoupler in both isolated heart mitochondria and adult cardiomyocytes. Additionally, cardiomyocytes isolated from transgenic mice expressing green fluorescent protein-tagged microtubule-associated protein light chain 3 showed increased autophagosome formation with cloxyquin treatment. The autophagy inhibitor chloroquine abolished cloxyquin-induced cardioprotection in both cellular and perfused heart (Langendorff) models of IR injury. Finally, in an in vivo murine left anterior descending coronary artery occlusion model of IR injury, cloxyquin significantly reduced infarct size from 31.4 ± 3.4% to 16.1 ± 2.2%. In conclusion, the cardioprotective compound cloxyquin simultaneously uncoupled mitochondria and induced autophagy. Importantly, autophagy appears to be required for cloxyquin-induced cardioprotection.

Setting Up an Efficient Therapeutic Hypothermia Team in Conscious ST Elevation Myocardial Infarction Patients: A UK Heart Attack Center Experience.

Patients presenting with ST elevation myocardial infarction (STEMI) are routinely treated with percutaneous coronary intervention to restore blood flow in the occluded artery to reduce infarct size (IS). However, there is evidence to suggest that the restoration of blood flow can cause further damage to the myocardium through reperfusion injury (RI). Recent research in this area has focused on minimizing damage to the myocardium caused by RI. Therapeutic hypothermia (TH) has been shown to be beneficial in animal models of coronary artery occlusion in reducing IS caused by RI if instituted early in an ischemic myocardium. Data in humans are less convincing to date, although exploratory analyses suggest that there is significant clinical benefit in reducing IS if TH can be administered at the earliest recognition of ischemia in anterior myocardial infarction. The Essex Cardiothoracic Centre is the first UK center to have participated in administering TH in conscious patients presenting with STEMI as part of the COOL-AMI case series study. In this article, we outline our experience of efficiently integrating conscious TH into our primary percutaneous intervention program to achieve 18 minutes of cooling duration before reperfusion, with no significant increase in door-to-balloon times, in the setting of the clinical trial.

Abstract 12: EMMPRIN-Targeted Magnetic Nanoparticles for in vivo Visualization and Regression of Acute Myocardial Infarction in a Model of Acute Coronary Artery Occlusion

Extracellular matrix metalloproteinase inducer EMMPRIN, is highly expressed in patients with acute myocardial infarction (AMI), and induces activation of several matrix metalloproteinases (MMPs), including MMP-9 and MMP-13. To prevent Extracellular matrix degradation and cardiac cell death we targeted EMMPRIN with paramagnetic/fluorescent micellar nanoparticles with an EMMPRIN binding peptide AP9 conjugated (NAP9), or an AP9 scramble peptide as a negative control (NAPSC). NAP9 binds to endogenous EMMPRIN as detected by confocal microscopy of cardiac myocytes and macrophages incubated with NAP and NAPSC in vitro, and in vivo in mouse hearts subjected to left anterior descending coronary artery occlusion (IV injection 50mγ/Kg NAP9 or NAP9SC). Administration of NAP9 at the same time or 1 hour after AMI reduced infarct size over a 20% respect to untreated and NAPSC injected mice, recovered left ventricle ejection fraction (LVEF) similar to healthy controls, and reduced EMMPRIN downstream MMP9 expression. In magnetic resonance scans of mouse hearts 2 days after AMI and injected with NAP9, we detected a significant gadolinium enhancement in the left ventricle respect to non-injected mice and to mice injected with NAPSC. Late gadolinium enhancement assays exhibited NAP9-mediated left ventricle signal enhancement as early as 30 minutes after nanoprobe injection, in which a close correlation between the MRI signal enhancement and left ventricle infarct size was detected. Taken together, these results point EMMPRIN targeted nanoprobes as a new tool for the treatment of AMI.

Preventive effect of yuzu and hesperidin on left ventricular remodeling and dysfunction in rat permanent left anterior descending coronary artery occlusion model.

Left ventricular (LV) remodeling, which includes ventricular dilatation and increased interstitial fibrosis after myocardial infarction (MI), is the critical process underlying the progression to heart failure. Therefore, a novel approach for preventing LV remodeling after MI is highly desirable. Yuzu is a citrus plant originating in East Asia, and has a number of cardioprotective properties such as hesperidin. However, no study has proved whether yuzu can prevent LV remodeling. The aim of this study was to determine the effects of yuzu on heart failure (HF) and its potential impact on the LV remodeling process after MI. Our in vivo study using the permanent left anterior descending coronary artery (LAD) occlusion model demonstrate that one week pre-treatment with yuzu or its major metabolite hesperidin before LAD occlusion significantly attenuated cardiac dysfunction, myocyte apoptosis and inflammation. Not only yuzu but also hesperidin inhibited caspase-3 activity, myeloperoxidase expression, α-smooth muscle actin expression, and matrix metalloproteinase-2 activity in a permanent LAD occlusion rat model. To our knowledge, our findings provide the first evidence that yuzu and hesperidin prevent MI-induced ventricular dysfunction and structural remodeling of myocardium.

ASSA14-03-23 CREG over-expression in BMSC protect against myocardial infarction via VHL/HIF-1α/VEGF pathway mediated vascularisation

<h3>Background</h3> We hypothesised that mesenchymal stem cells (MSCs) overexpressing Cellular Repressor of E1A-stimulated Genes (CREG) showed improved survival and engraftment in the infarcted heart and promoted vascularisation through paracrine release of vascular endothelial growth factor (VEGF) and activation of the HIF-1α signal pathway. <h3>Methods</h3> Mononuclear cells derived from rat bone marrow were isolated by density-gradient centrifugation and were cultured on fibronectin-coated plates, supplied with bovine pituitary extract. After the marrow-derived mesenchymal stem cells over-expressed CREG, we compare with the production of VEGF by ELISA and the protein expression of HIF-1α in different groups by western blot under hypoxia. Rat bone marrow–derived MSCs were used as nontransduced (^NormMSCs) or transduced with adenoviral-GFP vector (^GFPMSCs) or vector encoding for GFP-CREG (^CREGMSCs). <h3>Results</h3> For <i>in vivo</i> studies, 50 μl of DMEM without cells (group 1) or containing 1.5×10^6NormMSCs (group 2) or ^GFPMSCs (group 3) or ^CREGMSCs (group 4) were implanted intramyocardially in rat model of permanent coronary artery occlusion. one week later, immunoblot on rat heart tissue (n = 4 per group) showed highest survival of ^CREGMSCs (p &lt; 0.06 vs ^NormMSCs and ^GFPMSCs)(n = 6 per group). Confocal imaging after immunostaining for CD31 showed extensive angiomyogenesis in the infarcted heart. Infarction size was significantly reduced in cell transplanted groups compared with the control. Indices of left ventricular function, including ejection fraction and fractional shortening, were improved in group 4 as compared with group 1 (p &lt; 0.05). <i>In vitro</i>, under hypoxia the secretion of VEGF are most in the supernatant from the MSCs over-expression CREG. And the supernatants from BMSC over-expressed CREG exhibited a significant increase in angiogenic tube formation. In addition, the expression of CREG begins to appear after hypoxia. Molecular studies revealed that under hypoxia CREG active HIF-1α, but not HIF-2α, HIF-1β and we use the inhibition of HIF-1α confirm it. Further, we found that CREG can not influence the HIF-1α mRNA synthesis but it can inhibit the expression of pVHL which is the key protein regulation degradation of HIF-1α. <h3>Conclusions</h3> The strategies of CREG transgene expression inhibit pVHL and induced HIF-1α signalling and culminated in extensive angiomyogenesis in the infarcted heart.

Rapid Induction of Hypothermia by the ThermoSuit System Profoundly Reduces Infarct Size and Anatomic Zone of No Reflow Following Ischemia-Reperfusion in Rabbit and Rat Hearts.

Although hypothermia reduces myocardial infarct size, noninvasive and rapid cooling methods are lacking. This study tests the effectiveness of a novel cooling apparatus on myocardial infarct size and no-reflow area in 2 models of coronary artery occlusion (CAO). Animals were randomized to normothermic (N) or hypothermic (H) groups after isolation of a proximal coronary artery. Animals were subjected to 30 minutes CAO and 3 hours reperfusion. In protocol 1 (rabbit, n = 8 per group), hypothermia was initiated, using the ThermoSuit apparatus (convective-immersion cooling), 5 minutes after the onset of CAO to a goal temperature of ∼32°C. In protocol 2 (rat, n = 5 per group), hypothermia was initiated 2 minutes after the onset of CAO to a goal temperature of ∼30°C. Goal temperature was reached in ∼20 minutes. In protocol 1, hypothermia caused an 82% reduction in infarct size as a percentage of the ischemic risk zone (N, 44% ± 5%; H; 8% ± 2%, P < 0.001) and an 89% reduction in the no-reflow area (N, 44% ± 4%; H, 5% ± 1%, P < 0.001). In protocol 2, hypothermia caused a 73% infarct size reduction (N, 51% ± 5%; H, 14% ± 6%, P < 0.01) and a 99% reduction in the no-reflow area (N, 33% ± 5%; H, 0.4% ± 0.3%, P < 0.01). The ThermoSuit device induced rapid hypothermia and limited infarct size and no reflow to the greatest extent ever observed in this laboratory with a single intervention.

Abstract 330: Endothelial JNK Is Critically Required for Native Collateral Artery Development

Arterial occlusive diseases are major causes of morbidity and mortality in industrialized countries and represent a huge economic burden. The extent of the native collateral circulation is considered to be the most important determinant of the magnitude of tissue damage and subsequent functional impairment that ensues following an arterial occlusive event. Therefore, understanding the mechanisms responsible for collateral artery development may provide avenues for the design of strategies to enhance the collateral circulation in patients at risk for arterial occlusive diseases. Here, we identify a critical requirement for cJun-NH2-Terminal Kinase (JNK) in collateral artery development. Indeed, using immunofluorescence, microfil perfusion and three dimensional high resolution micro computed tomography (μCT) imaging techniques, we demonstrate that mice with compound JNK-deficiency in the vascular endothelium display abnormal collateral arteries, which are unable to restore blood perfusion following occlusion of a major artery leading to severe tissue necrosis in animal models of femoral and coronary artery occlusion. Furthermore, employing constitutive and inducible conditional deletion strategies, we demonstrate that JNK acts during the initial development of collateral arteries to ensure proper patterning and connectivity, but is, for the most part, dispensable for angiogenic and arteriogenic responses in adult mice. The defects in collateral artery development may reflect an enhanced sensitivity of JNK-deficient endothelial cells to cell death in specific contexts. This study introduces JNK as a major regulator of vascular development and highlights the crucial importance of the collateral circulation as the main defining factor in reducing the severity of tissue damage and functional impairment following arterial occlusive events.

Rapid induction of hypothermia by the Thermosuit system profoundly reduces infarct size and anatomic zone of no‐reflow following ischemia/reperfusion in rabbit and rat hearts (547.12)

Background: While hypothermia reduces myocardial infarct size, non‐invasive and rapid cooling methods are lacking. This study tests the effectiveness of a novel cooling apparatus on myocardial infarct size and no‐reflow area in two models of coronary artery occlusion (CAO).Methods and Results: Anesthetized rabbits (protocol 1) and anesthetized rats (protocol 2) were randomized to Normothermic (N) or Hypothermic (H) groups after isolation of a proximal coronary artery. Animals were subjected to 30 minutes CAO and 3 hours reperfusion. In protocol 1, hypothermia was initiated, using the Thermosuit apparatus (convective‐immersion cooling), 5 minutes after the onset of CAO to a goal temperature of ~32°C. In protocol 2, hypothermia was initiated 2 minutes after the onset of CAO to a goal temperature of ~30°C. Goal temperature was reached in ~20 minutes. In protocol 1, hypothermia caused an 82% reduction of infarct size as a percentage of the ischemic risk zone (N, 44±5%; H; 8±2%, P&lt;0.001) and an 89% reduction of the no‐reflow area (N, 44±4%; H, 5±1%, P&lt;0.001). In protocol 2, hypothermia caused a 73% infarct size reduction (N, 51%±5%; H, 14±6%, P&lt;0.001) and a 99% reduction of the no‐reflow area (N, 33±5%; H, 0.4±0.3%, P&lt;0.001).Conclusion: The Thermosuit device induced rapid hypothermia and limited infarct size and no‐reflow to the greatest extent ever observed in this laboratory with a single intervention.