Cardioprotective factors against myocardial infarction selected in vivo from an AAV secretome library.

Therapies for patients with myocardial infarction and heart failure are urgently needed, in light of the breadth of these conditions and lack of curative treatments. To systematically identify previously unidentified cardioactive biologicals in an unbiased manner in vivo, we developed cardiac FunSel, a method for the systematic, functional selection of effective factors using a library of 1198 barcoded adeno-associated virus (AAV) vectors encoding for the mouse secretome. By pooled vector injection into the heart, this library was screened to functionally select for factors that confer cardioprotection against myocardial infarction. After two rounds of iterative selection in mice, cardiac FunSel identified three proteins [chordin-like 1 (Chrdl1), family with sequence similarity 3 member C (Fam3c), and Fam3b] that preserve cardiomyocyte viability, sustain cardiac function, and prevent pathological remodeling. In particular, Chrdl1 exerted its protective activity by binding and inhibiting extracellular bone morphogenetic protein 4 (BMP4), which resulted in protection against cardiomyocyte death and induction of autophagy in cardiomyocytes after myocardial infarction. Chrdl1 also inhibited fibrosis and maladaptive cardiac remodeling by binding transforming growth factor-ß (TGF-ß) and preventing cardiac fibroblast differentiation into myofibroblasts. Production of secreted and circulating Chrdl1, Fam3c, and Fam3b from the liver also protected the heart from myocardial infarction, thus supporting the use of the three proteins as recombinant factors. Together, these findings disclose a powerful method for the in vivo, unbiased selection of tissue-protective factors and describe potential cardiac therapeutics.

In Vivo Functional Selection Identifies Cardiotrophin-1 as a Cardiac Engraftment Factor for Mesenchymal Stromal Cells.

BACKGROUND: Transplantation of cells into the infarcted heart has significant potential to improve myocardial recovery; however, low efficacy of cell engraftment still limits therapeutic benefit. Here, we describe a method for the unbiased, in vivo selection of cytokines that improve mesenchymal stromal cell engraftment into the heart both in normal conditions and after myocardial infarction. METHODS: An arrayed library of 80 secreted factors, including most of the currently known interleukins and chemokines, were individually cloned into adeno-associated viral vectors. Pools from this library were then used for the batch transduction of bone marrow-derived mesenchymal stromal cells ex vivo, followed by intramyocardial cell administration in normal and infarcted mice. Three weeks after injection, vector genomes were recovered from the few persisting cells and identified by sequencing DNA barcodes uniquely labeling each of the tested cytokines. RESULTS: The most effective molecule identified by this competitive engraftment screening was cardiotrophin-1, a member of the interleukin-6 family. Intracardiac injection of mesenchymal stromal cells transiently preconditioned with cardiotrophin-1 preserved cardiac function and reduced infarct size, parallel to the persistence of the transplanted cells in the healing hearts for at least 2 months after injection. Engraftment of cardiotrophin-1-treated mesenchymal stromal cells was consequent to signal transducer and activator of transcription 3-mediated activation of the focal adhesion kinase and its associated focal adhesion complex and the consequent acquisition of adhesive properties by the cells. CONCLUSIONS: These results support the feasibility of selecting molecules in vivo for their functional properties with adeno-associated viral vector libraries and identify cardiotrophin-1 as a powerful cytokine promoting cell engraftment and thus improving cell therapy of the infarcted myocardium.

Unacylated Ghrelin Reduces Skeletal Muscle Reactive Oxygen Species Generation and Inflammation and Prevents High-Fat Diet-Induced Hyperglycemia and Whole-Body Insulin Resistance in Rodents.

Excess reactive oxygen species (ROS) generation and inflammation may contribute to obesity-associated skeletal muscle insulin resistance. Ghrelin is a gastric hormone whose unacylated form (UnAG) is associated with whole-body insulin sensitivity in humans and may reduce oxidative stress in nonmuscle cells in vitro. We hypothesized that UnAG 1) lowers muscle ROS production and inflammation and enhances tissue insulin action in lean rats and 2) prevents muscle metabolic alterations and normalizes insulin resistance and hyperglycemia in high-fat diet (HFD)-induced obesity. In 12-week-old lean rats, UnAG (4-day, twice-daily subcutaneous 200-µg injections) reduced gastrocnemius mitochondrial ROS generation and inflammatory cytokines while enhancing AKT-dependent signaling and insulin-stimulated glucose uptake. In HFD-treated mice, chronic UnAG overexpression prevented obesity-associated hyperglycemia and whole-body insulin resistance (insulin tolerance test) as well as muscle oxidative stress, inflammation, and altered insulin signaling. In myotubes, UnAG consistently lowered mitochondrial ROS production and enhanced insulin signaling, whereas UnAG effects were prevented by small interfering RNA-mediated silencing of the autophagy mediator ATG5. Thus, UnAG lowers mitochondrial ROS production and inflammation while enhancing insulin action in rodent skeletal muscle. In HFD-induced obesity, these effects prevent hyperglycemia and insulin resistance. Stimulated muscle autophagy could contribute to UnAG activities. These findings support UnAG as a therapeutic strategy for obesity-associated metabolic alterations.

AAV-mediated in vivo functional selection of tissue-protective factors against ischaemia.

Functional screening of expression libraries in vivo would offer the possibility of identifying novel biotherapeutics without a priori knowledge of their biochemical function. Here we describe a procedure for the functional selection of tissue-protective factors based on the in vivo delivery of arrayed cDNA libraries from the mouse secretome using adeno-associated virus (AAV) vectors. Application of this technique, which we call FunSel, in the context of acute ischaemia, revealed that the peptide ghrelin protects skeletal muscle and heart from ischaemic damage. When delivered to the heart using an AAV9 vector, ghrelin markedly reduces infarct size and preserves cardiac function over time. This protective activity associates with the capacity of ghrelin to sustain autophagy and remove dysfunctional mitochondria after myocardial infarction. Our findings describe an innovative tool to identify biological therapeutics and reveal a novel role of ghrelin as an inducer of myoprotective autophagy.

Intracellular function of interleukin-1 receptor antagonist in ischemic cardiomyocytes.

BACKGROUND: Loss of cardiac myocytes due to apoptosis is a relevant feature of ischemic heart disease. It has been described in infarct and peri-infarct regions of the myocardium in coronary syndromes and in ischemia-linked heart remodeling. Previous studies have provided protection against ischemia-induced cardiomyocyte apoptosis by the anti-inflammatory cytokine interleukin-1 receptor-antagonist (IL-1Ra). Mitochondria triggering of caspases plays a central role in ischemia-induced apoptosis. We examined the production of IL-1Ra in the ischemic heart and, based on dual intra/extracellular function of some other interleukins, we hypothesized that IL-1Ra may also directly inhibit mitochondria-activated caspases and cardiomyocyte apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: Synthesis of IL-1Ra was evidenced in the hearts explanted from patients with ischemic heart disease. In the mouse ischemic heart and in a mouse cardiomyocyte cell line exposed to long-lasting hypoxia, IL-1Ra bound and inhibited mitochondria-activated caspases, whereas inhibition of caspase activation was not observed in the heart of mice lacking IL-1Ra (Il-1ra-/-) or in siRNA to IL-1Ra-interfered cells. An impressive 6-fold increase of hypoxia-induced apoptosis was observed in cells lacking IL-1Ra. IL-1Ra down-regulated cells were not protected against caspase activation and apoptosis by knocking down of the IL-1 receptor, confirming the intracellular, receptor-independent, anti-apoptotic function of IL-1Ra. Notably, the inhibitory effect of IL-1Ra was not influenced by enduring ischemic conditions in which previously described physiologic inhibitors of apoptosis are neutralized. CONCLUSIONS/SIGNIFICANCE: These observations point to intracellular IL-1Ra as a critical mechanism of the cell self-protection against ischemia-induced apoptosis and suggest that this cytokine plays an important role in the remodeling of heart by promoting survival of cardiomyocytes in the ischemic regions.