Ultrasound-Targeted Microbubble Destruction Mediated Microrna-21 Transfection Regulated PDCD4/NF-κB/TNF-α Pathway To Prevent Coronary Microembolization-Induced Myocardial Injury

Coronary microembolization (CME) is a frequent clinical event which could be caused by both the spontaneous and procedure-related atherosclerotic plaque rupture in patients with acute coronary syndromes (ACS), particularly during the percutaneous coronary intervention (PCI) process. The transient "no blood flow" or "slow flow" caused by the CME has been recognized to be an independent predictor of long-term poor prognosis in patients with acute myocardial infarction. Studies have shown that CME-induced localized myocardial inflammation is the major cause of progressive cardiac dysfunction, and that TNF-α is an important inflammatory mediator of myocardial contractile dysfunction. However, the The exact roles of TNF-α in myocardial dysfunction in the CME is unknowed.PDCD4 is known as a critical mediator for cancer cell apoptosis. Interestingly, the role of PDCD4 in cardiac dysfunction has also been described in recently studies. As a target gene of miR-21, cardiac protective role of PDCD4 was verified in the cultured cardiac cells and cardiac I/R model. Sheedy et al. reported that PDCD4 promoted the activation of NF-κB and inhibited IL-10 upregulation, thus promoting inflammation. Moreover, intensive atorvastatin treatment reduced the post-PCI myocardial inflammatory response in patients with unstable angina, which may relate to the inhibition of PDCD4 expression in CD4+T lymphocytes. In earlier studies, we found that myocardial PDCD4 levels were significantly higher after CME, implicating that PDCD4-mediated signaling pathway might play an important role in CME-induced inflammation.MicroRNA is a class of the small molecule RNA about 18 to 25 nucleotides. After transcription as primary miRNAs, they are processed into pre-miRNAs by the enzyme Drosha, exported from the nucleus by exportin-5 and further processed into their mature form by Dicer. Recent studies have revealed that its expression is deregulated in heart and vasculature under cardiovascular disease conditions such as proliferative vascular disease, cardiac hypertrophy and heart failure, and ischemic heart disease.It has been recently reported that microRNA-21 exerts a protective effect on myocardium mainly through the regulation of PDCD4-mediated pathways involved in anti-apoptotic and anti-inflammatory processes. Therefore, we speculated that transfection of microRNA-21 into myocardium to regulate PDCD4 expression could potentially inhibit CME-induced activation of PDCD4/NF-KB/TNF-α pathway and protect the myocardium. Although microRNA-21 has great potential in CME prevention and treatment, naked microRNAs are not stable in blood. Hence naked microRNA-21 cannot be used for treatment. How to achieve safe and efficient microRNA transfection and expression remains-unsolved. Ultrasound-targeted microbubble destruction (UTMD) is an efficient, safe and targeted nonviral delivery method that has been widely used in recent years. It faciliates the targeted gene to enter the tissues and cells more easily and enhances the transfection and expression efficiency.Therefore, we proposed to establish a pig model for CME by microcatheter, clone microRNA-21 eukaryotic expression vector for UTMD-mediated transfection and examine the activation of PDCD4/NF-κB/TNF-α pathway after CME in order to further elucidate the mechanisms involved in microRNA-21-mediated protection of CME-induced myocardial injury.Part I:Experimental Study on the Dynamic Changes of PDCD4 and Its Association with Myocardial Function after Coronary MicroembolizationObjective:Coronary microembolization (CME) has been related to the inflammatory response and apoptosis of the myocardium. This study aims to investigate the role of the apoptotic protein, PDCD4, in the myocardium after CME in pigs.Methods:Sixty Bama miniature pigs were randomly divided into two groups, sham group and CME group,30 pigs for each group. The CME group was induced by injecting polyethylene microspheres into the left anterior descending; the sham group was injected with saline. HE and HBFP staining of the myocardial tissues was used to evaluate the infarction of the operated pigs. Serum troponin I (cTnI) was detected by electrochemiluminescence (ECL). Echocardiography, q-PCR and Western blotting for PDCD4 and TNF-a were used to evaluate cardiac function, expression of PDCD4 and TNF-α at Oh,3h,6h, 9h,12h, and 24h hours after operation.Results:Echocardiographic parameters displayed that cardiac function significantly decreased compared with the sham group (P<0.05). The expression of cTnI, PDCD4 and TNF-a of the CME group increased significantly (P<0.05) at all the time points, while the infarct area was not different among all the groups and time points (P>0.05).Conclusion:In summary, these data suggest that significant upregulation of PDCD4 which results in myocardial injury in myocardial tissues in CME pigs could be by regulating the expression of TNF-a. This could be a potential mechanism for left ventricular dysfunction in CME heart. used to evaluate cardiac function, expression of PDCD4 and TNF-α at Oh,3h,6h, 9h,12h, and 24h hours after operation.Part Ⅱ:Mechanism of PDCD4/NF-κB Signaling Pathway in Porcine Coronary Micro-embolization-induced Myocardial InjuryObjective:The aim of this study was to investigate the role of the programmed cell death factor 4 (PDCD4)/nuclear factor-κB (NF-κB) signaling pathway in coronary micro-embolism (CME)-induced inflammatory responses and myocardial injury in a porcine model.Methods:Bama miniature pigs were randomly divided into four groups (n = 5 per group). Micro-embolization balls or saline were infused through a microcatheter in the left anterior descending (LAD) artery in the CME and Sham groups, respectively. PDCD4 siRNA or Control siRNA mixed with transfection reagent was infused via the LAD artery 72 h before CME induction in the CME +siRNA-PDCD4 and siRNA-control groups, respectively. Cardiac function was evaluated with ultrasound. Serum troponin I (cTnI) was detected by electrochemiluminescence (ECL). Tissue biopsy was stained with hematoxylin-eosin (HE) and hematoxylin basic fuchsin picric acid (HBFP) to measure infarction area. Myocardial PDCD4 and tumor necrosis factor-a (TNF-a) mRNA and protein expression were analyzed by quantitative PCR and Western blotting. NF-κB activity was evaluated in gel electrophoretic mobility shift assay.Results:Echocardiographic parameters showed that compared with the sham group, the CME group had impaired heart function, manifested as systolic dysfunction and left ventricular dilatation (reduced left ventricular ejection fraction (LVEF), left ventricular fractional shortening (FS) and cardiac output (CO) (P<0.05) and increased left ventricular end-diastolic diameter (LVEDd) (P <0.05)). Compared with the CME group, the CME+siRNA-PDCD4 group had attenuated CME-induced cardiac function damage (increased LVEF, FS and CO (P<0.05) and reduced LVEDd (P<0.05)). Compared with the sham group, the CME group had significantly increased PDCD4 and TNF-a mRNA and protein expression, elevated the level of cTnI and increased NF-κB activity (P<0.05). These effects were significantly inhibited in the CME+siRNA-PDCD4 group (P<0.05).Conclusion:PDCD4/NF-κB signaling pathway activation is an important mechanism for CME-induced myocardial injury, suggesting that inhibition of PDCD4/NF-κB signaling pathway may be a potential target for the prevention and treatment of CME.Part Ⅲ:Ultrasound-targeted microbubble destruction mediated microRNA-21 transfection regulated PDCD4/NF-κB/TNF-α pathway to prevent coronary Coronary Microembolization-induced Myocardial InjuryObjective:Coronary microembolization (CME)-induced inflammation is a major cause of myocardial injury. PDCD4/NF-KB/TNF-a signaling pathway plays an important role in CME-induced myocardial inflammatory responses. microRNA-21 protects myocardium mainly via regulation of its target gene PDCD4. Therefore, in this study we explore the effect of ultrasound-targeted microbubble destruction-mediated microRNA-21 transfection on myocardial injury in CME porcine model and the potential mechanisms involved.Methods:Bama miniature pigs were randomly divided into sham-operated, CME, CME plus gene and CME plus ultrasound mediated gene groups (n= 5 per group). Micro-embolization balls or saline were infused through a microcatheter in the left anterior descending (LAD) artery in the CME and Sham groups, respectively. The CME plus ultrasound mediated gene group was injected with plasmid-microbubble mixture through the marginal ear vein 4 days before CME treatment. Meanwhile, ultrasound treatment was given to the myocardium through chest. The CME plus gene group was injected with plasmid-microbubble mixture through the marginal ear vein 4 days before CME but received no ultrasdund treatment. Cardiac function indices were examined by cardiac ultrasound; Serum troponin I (cTnl) was detected by electrochemiluminescence (ECL); Infarct area was measured by HE and HBFP staining in tissue pathological sections; Green fluorescent protein (GFP)-labeled gene expression levels were evaluated by fluorescent microscopy in frozen sections; myocardial PDCD4 and Tumor Necrosis Factor a (TNF-a) mRNA levels were measured by fluorescent quantitative PCR and protein levels were measured by western blotting; NF-κB activity was tested by electrophoretic mobility shift assay (EMSA).Results:(1) Compare to CME plus gene group, the CME plus ultrasound mediated gene group had over 8 times higher expression of exogenous genes in myocardium (P<0.05); (2) Echocardiography showed that compared to Sham group, the CME group had significantly lower cardiac function represented by systolic dysfunction and left ventricular dilatation, as indicated by the decreased left ventricular ejection fraction (LVEF), left ventricular fractional shortening (FS), cardiac output (CO) (P<0.05) and increased left ventricular end-diastolic diameter (LVEDd) (P<0.05); compared to CME group, the CME plus ultrasound mediated gene group demonstrated improved CME-induced cardiac dysfunction as indicated by increased LVEF, FS and CO (P<0.05) as well as decreased LVEDd (P<0.05); (3) Compared to Sham group, the mRNA and protein levels of PDCD4 and TNF-a as well as NF-κB activity and serum cTnI level were significantly increased (P<0.05) in CME group; compared with CME group, the CME plus ultrasound mediated gene group showed significantly lower mRNA and protein expression of PDCD4 as well as NF-κB activity and serum cTnI level (P<0.05).Conclusion:Ultrasound microbubble-mediated microRNA-21 transfection effectively improved CME-induced myocardial injury mainly through the inhibition of PDCD4/NF-κB/TNF-α pathway to reduce myocardial inflammation.