| Part ?Background:Acute myocardial infarction (AMI) is the leading cause of death in patients with coronary heart disease. Timely, effectively and sustainedly open the infarct-related coronary artery is the key to save the myocardium at risk, reduce infarct size, prevent ventricular remodeling, improve clinical outcomes and reduce mortality. Thrombolytic therapy or percutaneous coronary intervention (PCI) can make recanalization of the infarct-related coronary artery happen and reduce cardiac mortality greatly, which is widely used in clinical.Large numbers of animal experiments and clinical evidences suggest that the process of restoring blood flow to the ischemic myocardium itself can induce additional myocardial injury, which can paradoxically reduce the beneficial effects of myocardial reperfusion. This phenomenon is termed myocardial ischemia-reperfusion injury (MIRI). MIRI includes reversible reperfusion arrhythmias, myocardial stunning, and irreversible microvascular obstruction, lethal reperfusion injury. Microvascular obstruction and lethal reperfusion injury can expand infarct area, reduce ventricular systolic and diastolic function, and then induce the ventricular remodeling, which lead to heart failure. Therefore, reducing myocardial ischemia-reperfusion injury, limiting infarct size expansion and preventing ventricular remodeling will have important clinical significance for improving the treatment effect of acute myocardial infarction and the long-term prognosis of patients with myocardial infarction.Thermosensitive dextran- poly(e- caprolactone)-2-hydroxylethyl methacrylate-poly(N-isopropylacrylamide) (Dex-PCL-HEMA/PNIPAAm) hydrogel is a novel biocompatible and biodegradable biomaterial which can make a sol gel transition in a particular temperature. After gelation, it can provide not only a local mechanical support, but also a three-dimensional porous network structure which is similar with extracellular matrix. In this study, the hydrogel was injected into the ischemic border zones of rats following ischemia-reperfusion injury to observed the treatment effect and investigate the mechanism.Objective:This study was designed to investigate whether intramyocardial injection of Dex-PCL-HEMA/PNIPAAm hydrogel have the cardioprotective effects after rat myocardial ischemia-reperfusion.Methods:Rats were randomly assigned to three groups. While one group was subjected to sham surgery, the other two groups received ischemia/reperfusion (IR) surgery (30 min. coronary artery ligation followed by reperfusion), with multi-point injection into the perimeter of ischemic zone of the following:100?l phosphate buffered saline (PBS group); 100?l Dex-PCL-HEMA/PNIPAAm hydrogel (gel group). At 28 days after treatment, left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD) and left ventricular ejection fraction (LVEF) were evaluated by transthoracic echocardiography. Infarct size and apoptosis were analyzed by Masson staining and histological TUNEL staining.Results:28 days after myocardial ischemia-reperfusion, compared with PBS group, direct intramyocardial injection of hydrogel significantly decreased LVESD and LVEDD, and increased LVEF(P<0.05). What is more, direct intramyocardial injection of hydrogel significantly reduced the cell apoptosis and infarct size compared with PBS group(P<0.05).Conclusion:Direct intramyocardial injection of Dex-PCL-HEMA/PNIPAAm hydrogel into rats after myocardial ischemia-reperfusion can inhibit cardiomyocyte apoptosis, reduce infarct size, inhibit ventricular remodeling and improve cardiac function.Part ?Background:The renin-angiotensin system(RAS) activation during myocardial ischemia-reperfusion plays an important role in ventricular remodeling after myocardial infarction. Angiotensin-converting enzyme(ACE) convert angiotensin I (Ang I) into the activated angiotensin II (Ang II). Ang II is the most important enzyme in RAS, its over-activation related to the cardiac tissue repairing and remodeling after myocardial infarction. Thus inhibiting the conversion from Ang I to Ang II by angiotensin converting enzyme inhibitors (ACEI) to regulate the level of RAS system, particularly Ang II is vital to prevent cardiac remodeling. However, ACEI may bring side effects like irritating cough and blood pressure dropping. Moreover, the dose selection and many other issues restrict its application clinically in cardiovascular diseases.RNA interference (RNAi) is a novel gene silencing technology which can specially degrade the target messenger RNAs(mRNA) to silence the target genes with endogenous or exogenous double-stranded RNA (dsRNA). To silence ACE with RNAi might be superior to ACEI to protect the heart. Currently small interfering RNA (siRNA)is used to induce RNAi in vivo, while the synthesized siRNA tends to degrade, resulting in unstable reaction duration. Non-viral vectors represented by plasmid DNA and liposome/DNA complexes are safer, easier to prepare, more convenient to storage compared to viral vectors, and not restricted by the inserting gene fragment size, but its low transfection efficiency and short silencingduration are great limitations for its application. Therefore, how to construct efficient, stable and accessible expression vectors is a pivotal issue within the RNA interference technology applications.Thermosensitive hydrogels Dex-PCL-HEMA/PNIPAAm is flexible, biocom-patible, which will increase the reaction time of siRNA to enhance the gene silencing effect. In part I we have proved that Dex-PCL-HEMA/PNIPAAm hydrogel has cardioprotective effects on myocardial ischemia-reperfusion injury. In this part we will further investigate wherther the ACE silence induced by RNA interference with the hydrogel as a carrier will result in superior cardiac protection effects.Objective:To investigate whether intramyocardial injection of plasmid containing the ACE-shRNA with the same hydrogel enhances the cardioprotective effects superior to either alone after rat myocardial ischemia-reperfusion.Methods:Rats were randomly assigned to four groups. All groups received ischemia/reperfusion (IR) surgery (30 min. coronary artery ligation followed by reperfusion), with multi-point injection into the perimeter of ischemic zone of the following:100?l phosphate buffered saline (PBS group); 100?l phosphate buffered saline containing 10?l ACE-shRNA plasmid (pDNA group); 100?l hydrogel containing lO?l negative control ACE-shRNA plasmids (NC group); 100?l hydrogel containing 10?l ACE-shRNA plasmid (pDNA+Gel group). At 28 days after treatment, fluorescence microscopy was used to detect enhanced green fluorescent protein (EGFP) expression in cells around the injection sites. left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD) and left ventricular ejection fraction (LVEF) were evaluated by transthoracic echocardiography. Respectively, infarct size and apoptosis were analyzed by Masson staining and histological TUNEL staining. The mRNA and protein expression of ACE were detected by real-time RT-PCR and western blot analysis.Results:28 days after myocardial ischemia-reperfusion, EGFP was detected in the pDNA +Gel group, while not in the pDNA group.Direct intramyocardial injection of negative control ACE-shRNA plasmid-loaded hydrogel significantly decreased LVESD and LVEDD, increased LVEF,inhibited the expression of local ACE expression, reduced cell apoptosis and infarct size compared with the injection of PBS 28d after myocardial ischemia-reperfusion in rats (P<0.05), and the effects were significantly enhanced by injection of ACE-shRNA plasmid-loaded hydrogel. However, these results had not significant difference between PBS group and pDNA group (P>0.05).Conclusion:Dex-PCL-HEMA/PNIPAAm hydrogel-mediated ACE-shRNA plasmid transfer significantly extended the duration of gene expression in heart. Moreover, direct intramyocardial injection of Dex-PCL-HEMA/PNIPAAm hydrogel loaded with ACE-shRNA plasmid may have synergistic benefits superior to either alone in rats after myocardial ischemia-reperfusion. |
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