Giulia Ruozi

Cardiomyocyte VEGFR-1 activation by VEGF-B induces compensatory hypertrophy and preserves cardiac function after myocardial infarction

Mounting evidence indicates that the function of members of the vascular endothelial growth factor (VEGF) family extends beyond blood vessel formation. Here, we show that the prolonged intramyocardial expression of VEGF‐A165 and VEGF‐B167 on adeno‐associated virus‐mediated gene delivery determined a marked improvement in cardiac function after myocardial infarction in rats, by promoting cardiac contractility, preserving viable cardiac tissue, and preventing remodeling of the left ventricle (LV) over time. Consistent with this functional outcome, animals treated with both factors showed diminished fibrosis and increased contractile myocardium, which were more pronounced after expression of the selective VEGF receptor‐1 (VEGFR‐1) ligand VEGF‐B, in the absence of significant induction of angiogenesis. We found that cardiomyocytes expressed VEGFR‐1, VEGFR‐2, and neuropilin‐1 and that, in particular, VEGFR‐1 was specifically up‐regulated in hypoxia and on exposure to oxidative stress. VEGF‐B exerted powerful antiapoptotic effect in both cultured cardiomyocytes and after myocardial infarction in vivo. Finally, VEGFR‐1 activation by VEGF‐B was found to elicit a peculiar gene expression profile proper of the compensatory, hypertrophic response, consisting in activation of αMHC and repression of βMHC and skeletal α‐actin, and an increase in SERCA2a, RYR, PGC1α, and cardiac natriuretic peptide transcripts, both in cultured cardiomyocytes and in infarcted hearts. The finding that VEGFR‐1 activation by VEGF‐B prevents loss of cardiac mass and promotes maintenance of cardiac contractility over time has obvious therapeutic implications.—Zentilin, L., Puligadda, U., Lionetti, V., Zacchigna, S., Collesi, C., Pattarini, L., Ruozi, G., Camporesi, S., Sinagra, G., Pepe, M., Recchia, F. A., Giacca, M. Cardiomyocyte VEGFR‐1 activation by VEGF‐B induces compensatory hypertrophy and preserves cardiac function after myocardial infarction. FASEB J. 24, 1467–1478 (2010). www.fasebj.org

In vivo therapeutic potential of mesenchymal stromal cells depends on the source and the isolation procedure.

Summary Over the last several years, mesenchymal stromal cells (MSCs) have been isolated from different tissues following a variety of different procedures. Here, we comparatively assess the ex vivo and in vivo properties of MSCs isolated from either adipose tissue or bone marrow by different purification protocols. After MSC transplantation into a mouse model of hindlimb ischemia, clinical and histological analysis revealed that bone marrow MSCs purified on adhesive substrates exerted the best therapeutic activity, preserving tissue viability and promoting formation of new arterioles without directly transdifferentiating into vascular cells. In keeping with these observations, these cells abundantly expressed cytokines involved in vessel maturation and cell retention. These findings indicate that the choice of MSC source and purification protocol is critical in determining the therapeutic potential of these cells and warrant the standardization of an optimal MSC isolation procedure in order to select the best conditions to move forward to more effective clinical experimentation.

Neuropilin-1 Identifies a Subset of Bone Marrow Gr1− Monocytes That Can Induce Tumor Vessel Normalization and Inhibit Tumor Growth

Improving tumor perfusion, thus tempering tumor-associated hypoxia, is known to impair cancer progression. Previous work from our laboratory has shown that VEGF-A165 and semaphorin 3A (Sema3A) promote vessel maturation through the recruitment of a population of circulating monocytes expressing the neuropilin-1 (Nrp1) receptor (Nrp1-expressing monocytes; NEM). Here, we define the characteristics of bone marrow NEMs and assess whether these cells might represent an exploitable tool to induce tumor vessel maturation. Gene expression signature and surface marker analysis have indicated that NEMs represent a specific subset of CD11b+ Nrp1+ Gr1- resident monocytes, distinctively recruited by Sema3A. NEMs were found to produce several factors involved in vessel maturation, including PDGFb, TGF-β, thrombospondin-1, and CXCL10; consistently, they were chemoattractive for vascular smooth muscle cells in vitro. When directly injected into growing tumors, NEMs, isolated either from the bone marrow or from Sema3A-expressing muscles, exerted antitumor activity despite having no direct effects on the proliferation of tumor cells. NEM inoculation specifically promoted mural cell coverage of tumor vessels and decreased vascular leakiness. Tumors treated with NEMs were smaller, better perfused and less hypoxic, and had a reduced level of activation of HIF-1α. We conclude that NEMs represent a novel, unique population of myeloid cells that, once inoculated into a tumor, induce tumor vessel normalization and inhibit tumor growth.

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.

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.

Unacylated ghrelin normalizes skeletal muscle oxidative stress and prevents muscle catabolism by enhancing tissue mitophagy in experimental chronic kidney disease

Unacylated ghrelin (UnAG) may lower skeletal muscle oxidative stress, inflammation, and insulin resistance in lean and obese rodents. UnAG‐induced autophagy activation may contribute to these effects, likely involving removal of dysfunctional mitochondria (mitophagy) and redox state maintenance. In chronic kidney disease (CKD) oxidative stress, inflammation and insulin resistance may negatively influence patient outcome by worsening nutritional state through muscle mass loss. Here we show in a 5/6 nephrectomy (Nx) CKD rat model that 4 d s.c. UnAG administration (200 μg twice a day) normalizes CKD‐induced loss of gastrocnemius muscle mass and a cluster of high tissue mitochondrial reactive oxygen species generation, high proinflammatory cytokines, and low insulin signaling activation. Consistent with these results, human uremic serum enhanced mitochondrial reactive oxygen species generation and lowered insulin signaling activation in C2C12 myotubes while concomitant UnAG incubation completely prevented these effects. Importantly, UnAG enhanced muscle mitophagy in vivo and silencing RNA‐mediated autophagy protein 5 silencing blocked UnAG activities in myotubes. UnAG therefore normalizes CKD‐induced skeletal muscle oxidative stress, inflammation, and low insulin signaling as well as muscle loss. UnAG effects are mediated by autophagy activation at the mitochondrial level. UnAG administration and mitophagy activation are novel potential therapeutic strategies for skeletal muscle metabolic abnormalities and their negative clinical impact in CKD.—Gortan Cappellari, G., Semolic, A., Ruozi, G., Vinci, P., Guarnieri, G., Bortolotti, F., Barbetta, D., Zanetti, M., Giacca, M., Barazzoni, R. Unacylated ghrelin normalizes skeletal muscle oxidative stress and prevents muscle catabolism by enhancing tissue mitophagy in experimental chronic kidney disease. FASEB J. 31, 5159–5171 (2017). www.fasebj.org

Acylated ghrelin treatment normalizes skeletal muscle mitochondrial oxidative capacity and AKT phosphorylation in rat chronic heart failure

Chronic heart failure (CHF) is associated with skeletal muscle abnormalities contributing to exercise intolerance, muscle loss, and negative impact on patient prognosis. A primary role has been proposed for mitochondrial dysfunction, which may be induced by systemic and tissue inflammation and further contribute to low insulin signalling. The acylated form of the gastric hormone ghrelin (AG) may improve mitochondrial oxidative capacity and insulin signalling in both healthy and diseased rodent models.

Unacylated Ghrelin Enhances Satellite Cell Function and Relieves the Dystrophic Phenotype in Duchenne Muscular Dystrophy mdx Model

Muscle regeneration depends on satellite cells (SCs), quiescent precursors that, in consequence of injury or in pathological states such as muscular dystrophies, activate, proliferate, and differentiate to repair the damaged tissue. A subset of SCs undergoes self‐renewal, thus preserving the SC pool and its regenerative potential. Unacylated ghrelin (UnAG) is a circulating hormone that protects muscle from atrophy, promotes myoblast differentiation, and enhances ischemia‐induced muscle regeneration. Here we show that UnAG increases SC activity and stimulates Par polarity complex/p38‐mediated asymmetric division, fostering both SC self‐renewal and myoblast differentiation. Because of those activities on different steps of muscle regeneration, we hypothesized a beneficial effect of UnAG in mdx dystrophic mice, in which the absence of dystrophin leads to chronic muscle degeneration, defective muscle regeneration, fibrosis, and, at later stages of the pathology, SC pool exhaustion. Upregulation of UnAG levels in mdx mice reduces muscle degeneration, improves muscle function, and increases dystrophin‐null SC self‐renewal, maintaining the SC pool. Our results suggest that UnAG has significant therapeutic potential for preserving the muscles in dystrophies. Stem Cells 2017;35:1733–1746

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.

Gut-Derived Hormones—Cardiac Effects of Ghrelin and Glucagon-Like Peptide-1

The effects of gut-derived hormones on the cardiovascular system have aroused the interest of numerous experimental and clinical investigators for decades. Among these hormones, ghrelin and glucagon-like peptide-1 (GLP-1) are certainly the most promising for their therapeutic potential. Ghrelin is a potent growth hormone secretogogue mainly produced by the stomach. It regulates feeding and metabolism and exerts also a number of beneficial actions on the cardiovascular system, such as cardiomyocyte cytoprotection, mitigation of inflammatory processes, and improvement of cardiac function. GLP-1 is an incretin hormone secreted by endocrine L-cells. It enhances glucose-dependent insulin secretion after food ingestion and, similar to ghrelin, exerts a number of actions on different organs, including the heart. GLP-1 administration to type 2 diabetic patients improves body weight and glycemic control, reduces inflammation, and protects the heart. In this chapter we will review the existing evidence supporting important cardiovascular effects of these two hormones and their potential therapeutic use.