Costanza Emanueli

Methodological guidelines to study extracellular vesicles

Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, in this review, we will focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research.

Concise Review: MicroRNAs as Modulators of Stem Cells and Angiogenesis

MicroRNAs (miRs) are highly conserved, short noncoding RNA molecules that negatively regulate messenger RNA (mRNA) stability and/or translational efficiency. Since a given miR can control the expression of many mRNAs, their importance in governing gene expression in specific cell types including vascular cells and their progenitor cells has become increasingly clear. Understanding how the expression of miRs themselves is regulated and how miRs exert their influence on post‐transcriptional gene control provides novel opportunities to dissect gene regulatory networks in clinically relevant cell types. A multitude of miRs have been identified with key roles in vascular development, homeostasis, function, disease, and regeneration. In this review, we will describe the impact of miRs on angiogenesis and their capacity to modulate the behavior of stem and progenitor cells which may be utilitarian for promoting vascular growth in ischemic tissue. Moreover, we summarize these strategies available for modulating miR expression and function and future therapeutic applications. Stem Cells 2014;32:1059–1066

Expansion and Characterization of Neonatal Cardiac Pericytes Provides a Novel Cellular Option for Tissue Engineering in Congenital Heart Disease

Background Living grafts produced by combining autologous heart-resident stem/progenitor cells and tissue engineering could provide a new therapeutic option for definitive correction of congenital heart disease. The aim of the study was to investigate the antigenic profile, expansion/differentiation capacity, paracrine activity, and pro-angiogenic potential of cardiac pericytes and to assess their engrafting capacity in clinically certified prosthetic grafts. Methods and Results CD34pos cells, negative for the endothelial markers CD31 and CD146, were identified by immunohistochemistry in cardiac leftovers from infants and children undergoing palliative repair of congenital cardiac defects. Following isolation by immunomagnetic bead-sorting and culture on plastic in EGM-2 medium supplemented with growth factors and serum, CD34pos/CD31neg cells gave rise to a clonogenic, highly proliferative (>20 million at P5), spindle-shape cell population. The following populations were shown to expresses pericyte/mesenchymal and stemness markers. After exposure to differentiation media, the expanded cardiac pericytes acquired markers of vascular smooth muscle cells, but failed to differentiate into endothelial cells or cardiomyocytes. However, in Matrigel, cardiac pericytes form networks and enhance the network capacity of endothelial cells. Moreover, they produce collagen-1 and release chemo-attractants that stimulate the migration of c-Kitpos cardiac stem cells. Cardiac pericytes were then seeded onto clinically approved xenograft scaffolds and cultured in a bioreactor. After 3 weeks, fluorescent microscopy showed that cardiac pericytes had penetrated into and colonized the graft. Conclusions These findings open new avenues for cellular functionalization of prosthetic grafts to be applied in reconstructive surgery of congenital heart disease.

Copper Transport Protein Antioxidant-1 Promotes Inflammatory Neovascularization via Chaperone and Transcription Factor Function.

Copper (Cu), an essential micronutrient, plays a fundamental role in inflammation and angiogenesis; however, its precise mechanism remains undefined. Here we uncover a novel role of Cu transport protein Antioxidant-1 (Atox1), which is originally appreciated as a Cu chaperone and recently discovered as a Cu-dependent transcription factor, in inflammatory neovascularization. Atox1 expression is upregulated in patients and mice with critical limb ischemia. Atox1-deficient mice show impaired limb perfusion recovery with reduced arteriogenesis, angiogenesis, and recruitment of inflammatory cells. In vivo intravital microscopy, bone marrow reconstitution, and Atox1 gene transfer in Atox1−/− mice show that Atox1 in endothelial cells (ECs) is essential for neovascularization and recruitment of inflammatory cells which release VEGF and TNFα. Mechanistically, Atox1-depleted ECs demonstrate that Cu chaperone function of Atox1 mediated through Cu transporter ATP7A is required for VEGF-induced angiogenesis via activation of Cu enzyme lysyl oxidase. Moreover, Atox1 functions as a Cu-dependent transcription factor for NADPH oxidase organizer p47phox, thereby increasing ROS-NFκB-VCAM-1/ICAM-1 expression and monocyte adhesion in ECs inflamed with TNFα in an ATP7A-independent manner. These findings demonstrate a novel linkage between Atox1 and NADPH oxidase involved in inflammatory neovascularization and suggest Atox1 as a potential therapeutic target for treatment of ischemic disease.

Extracellular Vesicles at the Cross-line between Basic Science and Clinical Needs

MiRNAs are small noncoding RNAs vital for protein regulation and gene expression. Since their discovery in the early nineties, many of their intracellular roles have been characterized. However, it is only recently that EVs loaded with miRNAs and other molecular types have started to be appreciated for their substantial involvement in cell‐to‐cell communication and signaling in physiological and pathological processes. EVs cell‐to‐cell signaling functions are complex and largely unknown, which still hampers the direct use of endogenous engineered EVs as therapeutics. However, ad hoc engineered synthetic EVs could represent new therapeutics. The potential of EV‐inspired delivery carriers has now attracted the interest of the pharmaceutical industry and has challenged drug delivery researchers with new questions. This review will focus on EVs and EV‐inspired drug delivery carriers, on their potential and on the challenges involved in the use of EV‐inspired drug delivery systems.

You can teach an old dog new tricks: angiopoietin‐1 instructs Tie2pos myeloid cells to promote neovascularization in ischemic limbs

See related articles in EMBO Molecular Medicine http://dx.doi.org/10.1002/emmm.201302695 and http://dx.doi.org/10.1002/emmm.201302752