Erythropoietin (EPO) has been shown to protect neurons from ischemic stroke, but can also increase thrombotic events and mortality rates in patients with ischemic heart disease. We reasoned that benefits of EPO might be offset by increases in hematocrit and evaluated the direct effects of EPO in the ischemic heart. We show that preconditioning with EPO protects H9c2 myoblasts in vitro and cardiomyocytes in vivo against ischemic injury. EPO treatment leads to significantly improved cardiac function following myocardial infarction. This protection is associated with mitigation of myocyte apoptosis, translating into more viable myocardium and less ventricular dysfunction. EPO-mediated myocyte survival appears to involve Akt activation. Importantly, cardioprotective effects of EPO were seen without an increase in hematocrit (eliminating oxygen delivery as an etiologic factor in myocyte survival and function), demonstrating that EPO can directly protect the ischemic and infarcted heart. Conflict of interest:The authors have declared that no conflict of interest exists. Nonstandard abbreviations used: myocardial infarction (MI); erythropoietin (EPO); erythropoietin receptor (EPO-R); Janus-associated kinase-2 (Jak2); trichloroacetic acid (TCA); left circumflex coronary artery (LCx); reactive oxygen species (ROS); left ventricle (LV); triphenyltetrazolium chloride (TTC); β-adrenergic receptor (βAR); LV end-diastolic pressure (LVEDP).

Isolation of hepatic progenitor cells is a promising approach for cell replacement therapy of chronic liver disease. The winged helix transcription factor Foxl1 is a marker for progenitor cells and their descendants in the mouse liver in vivo. Here, we purify progenitor cells from Foxl1-Cre; RosaYFP mice and evaluate their proliferative and differentiation potential in vitro. Treatment of Foxl1-Cre; RosaYFP mice with a 3,5-diethoxycarbonyl-1, 4-dihydrocollidine diet led to an increase of the percentage of YFP-labeled Foxl1 + cells. Clonogenic assays demonstrated that up to 3.6% of Foxl1 + cells had proliferative potential. Foxl1 + cells differentiated into cholangiocytes and hepatocytes in vitro, depending on the culture condition employed. Microarray analyses indicated that Foxl1 + cells express stem cell markers such as Prom1 as well as differentiation markers such as Ck19 and Hnf4a. Thus, the Foxl1-Cre; RosaYFP model allows for easy isolation of adult hepatic progenitor cells that can be expanded and differentiated in culture.

BackgroundValidation of physiologic miRNA targets has been met with significant challenges. We employed HITS-CLIP to identify which miRNAs participate in liver regeneration, and to identify their target mRNAs.ResultsmiRNA recruitment to the RISC is highly dynamic, changing more than five-fold for several miRNAs. miRNA recruitment to the RISC did not correlate with changes in overall miRNA expression for these dynamically recruited miRNAs, emphasizing the necessity to determine miRNA recruitment to the RISC in order to fully assess the impact of miRNA regulation. We incorporated RNA-seq quantification of total mRNA to identify expression-weighted Ago footprints, and developed a microRNA regulatory element (MRE) prediction algorithm that represents a greater than 20-fold refinement over computational methods alone. These high confidence MREs were used to generate candidate ‘competing endogenous RNA’ (ceRNA) networks.ConclusionHITS-CLIP analysis provide novel insights into global miRNA:mRNA relationships in the regenerating liver.

Background-Mechanical assistance of the failing left ventricle (LV) can lead to functional recovery after a period of unloading, including restoration of ␤-adrenergic receptor (␤AR) inotropic reserve. We tested whether prolonged LV unloading of failing rabbit hearts by use of a heterotopic transplantation technique could lead to recovery and whether adenoviral gene transfer of a ␤ 2 AR transgene (Adv-␤ 2 AR) could alter this process. Methods and Results-Heart failure was induced by coronary artery ligation in adult New Zealand White rabbits. After 4 weeks, failing hearts were heterotopically transplanted into recipient rabbits, allowing normal coronary perfusion but complete LV unloading. We also placed an LV latex balloon for remote access and in vivo physiological analysis. We found that there was reversal of signaling and functional abnormalities after 30 days of unloading. In another set of failing hearts, we randomly delivered, at the time of transplantation, either 2ϫ10 11 viral particles of Adv-␤ 2 AR or saline via the coronary arteries. Sham-operated animals with nonfailing hearts served as controls. After 5 days of unloading, in vivo LV contractility (LV dP/dt max ) and relaxation (LV dP/dt min ) were significantly decreased in saline-treated failing hearts compared with control nonfailing hearts (PϽ0.05). In failing hearts treated with Adv-␤ 2 AR, however, LV dP/dt max and LV dP/dt min were improved in response to higher preloads (PϽ0.05) and ␤AR stimulation (PϽ0.01). Conclusions-Heterotopic transplantation in the rabbit does allow recovery of the failing heart, and ␤ 2 AR overexpression acutely enhances this functional improvement. Accordingly, genetic manipulation of ␤AR signaling may represent a novel molecular adjunct to mechanical assistance to facilitate functional myocardial recovery.

A decrease in vascular density in peripheral skeletal muscle has been associated with exercise intolerance in humans with congestive heart failure (CHF). The purpose of this study was to determine whether CHF results in a reduction in vascular density in peripheral skeletal muscle. In this established model, CHF was induced by coronary artery ligation in New Zealand White rabbits and sham rabbits that underwent an identical surgical procedure without ligation of the coronary artery. At study termination, rabbits underwent hemodynamic testing and skeletal muscle analysis. The first series of rabbits was divided into sham (n = 6) and CHF (n = 6) 21 days postoperatively. Ten CHF rabbits were then examined 3 (n = 3), 7 (n = 3), and 14 days (n = 4) postoperatively. Vascular density in sham tibialis anterior muscle was 347 +/- 41 capillaries/mm2 or 1.20 +/- 0.11 capillaries/muscle fiber. In 21-day CHF rabbits, the capillary density was significantly lower, 236 +/- 14 capillaries/mm2 or 0.84 +/- 0.04 capillaries/muscle fiber (both P < 0.00001 vs. sham); PECAM protein was 2-fold lower (P < 0.0001) in muscle protein lysates; the fraction of apoptotic cells was greater, 3.8 +/- 2.2 vs. 0.69 +/- 0.56 (P < 0.02 vs. sham) with many TdT-mediated dUTP-biotin nick-end labeling-positive endothelial cells; and Bax protein was 2.8-fold greater (P < 0.0001). By regression analysis, vascular density tended to decrease over time (r2 = 0.572, P < 0.0001). Vascular rarefaction and endothelial apoptosis develop after experimental CHF and may contribute to the skeletal muscle abnormalities in this disease. Modulating vascular density may provide new approaches to treat exercise intolerance in CHF.