Active Wnt signaling in response to cardiac injury

Although the contribution of Wnt signaling in infarct healing is suggested, its exact role after myocardial infarction (MI) still needs to be unraveled. We evaluated the cardiac presence of active Wnt signaling in vivo following MI, and investigated in which cell types active Wnt signaling was present by determining Axin2 promoter-driven LacZ expression. C57BL/6 Axin2-LacZ reporter mice were sacrificed at days 0, 1, 3, 7, 14, and 21 after LAD ligation. Hearts were snap-frozen for immunohistochemistry (IHC) or enzymatically digested to obtain a single cell suspension for flow cytometric analysis. For both FACS and IHC, samples were stained for β-galactosidase and antibodies against Sca-1, CD31, ckit, and CD45. Active Wnt signaling increased markedly in the myocardium, from 7 days post-MI onwards. Using Sca-1 and CD31, to identify progenitor and endothelial cells, a significant increase in LacZ+ cells was found at 7 and 14 days post-MI. LacZ+ cells also increased in the ckit+ and CD45+ cell population. IHC revealed LacZ+ cells co-expressing Sca, CD31, CD45, vWF, and αSMA in the border zone and the infarcted area. Wnt signaling increased significantly after MI in Sca+- and CD31+-expressing cells, suggesting involvement of Wnt signaling in resident Sca+ progenitor cells, as well as endothelial cells. Moreover, active Wnt signaling was present in ckit+ cells, leukocytes, and fibroblast. Given its broad role during the healing phase after cardiac injury, additional research seems warranted before a therapeutic approach on Wnt to enhance cardiac regeneration can be carried out safely

Caveolin-1 Influences Vascular Protease Activity and Is a Potential Stabilizing Factor in Human Atherosclerotic Disease

Caveolin-1 (Cav-1) is a regulatory protein of the arterial wall, but its role in human atherosclerosis remains unknown. We have studied the relationships between Cav-1 abundance, atherosclerotic plaque characteristics and clinical manisfestations of atherosclerotic disease.We determined Cav-1 expression by western blotting in atherosclerotic plaques harvested from 378 subjects that underwent carotid endarterectomy. Cav-1 levels were significantly lower in carotid plaques than non-atherosclerotic vascular specimens. Low Cav-1 expression was associated with features of plaque instability such as large lipid core, thrombus formation, macrophage infiltration, high IL-6, IL-8 levels and elevated MMP-9 activity. Clinically, a down-regulation of Cav-1 was observed in plaques obtained from men, patients with a history of myocardial infarction and restenotic lesions. Cav-1 levels above the median were associated with absence of new vascular events within 30 days after surgery [0% vs. 4%] and a trend towards lower incidence of new cardiovascular events during longer follow-up. Consistent with these clinical data, Cav-1 null mice revealed elevated intimal hyperplasia response following arterial injury that was significantly attenuated after MMP inhibition. Recombinant peptides mimicking Cav-1 scaffolding domain (Cavtratin) reduced gelatinase activity in cultured porcine arteries and impaired MMP-9 activity and COX-2 in LPS-challenged macrophages. Administration of Cavtratin strongly impaired flow-induced expansive remodeling in mice.This is the first study that identifies Cav-1 as a novel potential stabilizing factor in human atherosclerosis. Our findings support the hypothesis that local down-regulation of Cav-1 in atherosclerotic lesions contributes to plaque formation and/or instability accelerating the occurrence of adverse clinical outcomes. Therefore, given the large number of patients studied, we believe that Cav-1 may be considered as a novel target in the prevention of human atherosclerotic disease and the loss of Cav-1 may be a novel biomarker of vulnerable plaque with prognostic value

Increased local delivery of antagomir therapeutics to the rodent myocardium using ultrasound and microbubbles

Recent developments in microRNA (miRNA) research have identified these as important mediators in the pathophysiological response upon myocardial infarction (MI). Specific miRNAs can inhibit the translation of entire groups of mRNAs, which are involved in specific processes in the pathophysiology after MI, e.g. the fibrotic, apoptotic or angiogenic response. By modulating miRNAs in the heart, these processes can be tuned to improve cardiac function. Antagomirs are effective miRNA-inhibitors, but have a low myocardial specificity and cardiac antagomir treatment therefore requires high doses, which causes side effects. In the present study, ultrasound-triggered microbubble destruction (UTMD) was studied to increase specific delivery of antagomir to the myocardium. Healthy control mice were treated with UTMD and sacrificed at 30min, 24h and 48h, after which antagomir delivery in the heart was analyzed, both qualitatively and quantitatively. Additionally, potential harmful effects of treatment were analyzed by monitoring ECG, analyzing neutrophil invasion and cell death in the heart, and measuring troponin I after treatment. Finally, UTMD was tested for delivery of antagomir in a model of ischemia-reperfusion (I/R) injury. We found that UTMD can significantly increase local antagomir delivery to the non-ischemic heart with modest side-effects like neutrophil invasion without causing apoptosis. Delivered antagomirs enter cardiomyocytes within 30min after treatment and remains there for at least 48h. Interestingly, after I/R injury antagomir already readily enters the infarcted zone and we observed no additional benefit of UTMD for antagomir delivery. This study is the first to explore cardiac antagomir delivery using UTMD. In addition, it is the first to study tissue distribution of short RNA based therapeutics (~22 base pairs) at both the cellular and organ levels after UTMD to the heart in general. In summary, UTMD provides a myocardial delivery strategy for non-vascular permeable cardiac conditions later in the I/R response or chronic conditions like cardiac hypertrophy

BLT1 antagonist LSN2792613 reduces infarct size in a mouse model of myocardial ischaemia-reperfusion injury

AIMS: Restoration of coronary blood flow is crucial in the treatment of acute myocardial infarction. Reperfusion, however, induces ischaemia-reperfusion (IR) injury, which further deteriorates myocardial function. The innate immune system plays an important role in this process, mediating rapid influx of immune cells into the reperfused myocardium. Leukotriene B4 is an important leucocyte chemoattractant, performing its actions through binding to its specific receptor BLT1. We hypothesized that treatment with LSN2792613, a selective BLT1 antagonist, reduces infarct size (IS) in a mouse model of myocardial IR injury. METHODS AND RESULTS: Male C57Bl/6J mice were subjected to myocardial ischaemia for 30 min by surgical coronary artery ligation, followed by reperfusion. Mice received either LSN2792613 or vehicle, three times daily (orally) for up to 72 h after reperfusion. BLT1 inhibition with LSN2792613 reduced IS compared with vehicle treatment (26.9 ± 2.7 vs. 34.9 ± 2.2%, P = 0.030) at 24 h after reperfusion. The levels of IL-6 and keratinocyte chemoattractant were reduced in the infarcted tissue of LSN2792613-treated mice. Reduced apoptosis in LSN2792613-treated mice was suggested by increased levels of phosphorylated JNK and GSK3α/β, and confirmed by flow cytometric analysis showing less apoptotic and necrotic cardiomyocytes in the infarcted myocardium. Echocardiography at 4 weeks after myocardial IR showed a slightly higher ejection fraction and stroke volume in mice treated with LSN2792613 compared with vehicle-treated mice, whereas left ventricular volumes were comparable. CONCLUSION: Selective BLT1 inhibition with LSN2792613 reduces inflammation and apoptosis following IR, resulting in reduced IS, and therefore might be a promising strategy to prevent myocardial IR injury

Leucocyte expression of complement C5a receptors exacerbates infarct size after myocardial reperfusion injury

Aims Early reperfusion is mandatory for the treatment of acute myocardial infarction. This process, however, also induces additional loss of viable myocardium, called ischaemia-reperfusion (IR) injury. Complement activation plays an important role in IR injury, partly through binding of C5a to its major receptor (C5aR). We investigated the role of C5aR on infarct size and cardiac function in a model for myocardial IR injury