Giuseppe Vassalli

Extracellular vesicles from human cardiac progenitor cells inhibit cardiomyocyte apoptosis and improve cardiac function after myocardial infarction

AIMS Recent evidence suggests that cardiac progenitor cells (CPCs) may improve cardiac function after injury. The underlying mechanisms are indirect, but their mediators remain unidentified. Exosomes and other secreted membrane vesicles, hereafter collectively referred to as extracellular vesicles (EVs), act as paracrine signalling mediators. Here, we report that EVs secreted by human CPCs are crucial cardioprotective agents. METHODS AND RESULTS CPCs were derived from atrial appendage explants from patients who underwent heart valve surgery. CPC-conditioned medium (CM) inhibited apoptosis in mouse HL-1 cardiomyocytic cells, while enhancing tube formation in human umbilical vein endothelial cells. These effects were abrogated by depleting CM of EVs. They were reproduced by EVs secreted by CPCs, but not by those secreted by human dermal fibroblasts. Transmission electron microscopy and nanoparticle tracking analysis showed most EVs to be 30-90 nm in diameter, the size of exosomes, although smaller and larger vesicles were also present. MicroRNAs most highly enriched in EVs secreted by CPCs compared with fibroblasts included miR-210, miR-132, and miR-146a-3p. miR-210 down-regulated its known targets, ephrin A3 and PTP1b, inhibiting apoptosis in cardiomyocytic cells. miR-132 down-regulated its target, RasGAP-p120, enhancing tube formation in endothelial cells. Infarcted hearts injected with EVs from CPCs, but not from fibroblasts, exhibited less cardiomyocyte apoptosis, enhanced angiogenesis, and improved LV ejection fraction (0.8 ± 6.8 vs. -21.3 ± 4.5%; P < 0.05) compared with those injected with control medium. CONCLUSION EVs are the active component of the paracrine secretion by human CPCs. As a cell-free approach, EVs could circumvent many of the limitations of cell transplantation.

Methods for evaluating endothelial function: a position statement from the European Society of Cardiology Working Group on Peripheral Circulation

The endothelium holds a pivotal role in cardiovascular health and disease. Assessment of its function was until recently limited to experimental designs due to its location. The advent of novel techniques has facilitated testing on a more detailed basis, with focus on distinct pathways. This review presents available in-vivo and ex-vivo methods for evaluating endothelial function with special focus on more recent ones. The diagnostic modalities covered include assessment of epicardial and microvascular coronary endothelial function, local vasodilation by venous occlusion plethysmography and flow-mediated dilatation, arterial pulse wave analysis and pulse amplitude tonometry, microvascular blood flow by laser Doppler flowmetry, biochemical markers and bioassays, measurement of endothelial-derived microparticles and progenitor cells, and glycocalyx measurements. Insights and practical information on the theoretical basis, methodological aspects, and clinical application in various disease states are discussed. The ability of these methods to detect endothelial dysfunction before overt cardiovascular disease manifests make them attractive clinical tools for prevention and rehabilitation.

Adeno-associated virus (AAV) vectors achieve prolonged transgene expression in mouse myocardium and arteries in vivo: a comparative study with adenovirus vectors

Plasmid DNA and adenovirus vectors currently used in cardiovascular gene therapy trials are limited by low efficiency and short-lived transgene expression, respectively. Recombinant adeno-associated virus (AAV) has recently emerged as an attractive vector for cardiovascular gene therapy. In the present study, we have compared AAV and adenovirus vectors with respect to gene transfer efficiency and the duration of transgene expression in mouse hearts and arteries in vivo. AAV vectors (titer: 5 x 10(8) transducing units (TU)/ml) and adenovirus vectors (1.2 x 10(10) TU/ml) expressing a green fluorescent protein (EGFP) gene were injected either intramyocardially (n=32) or intrapericardially (n=3) in CD-1 mice. Hearts were harvested at varying time intervals (3 days to 1 year) after gene delivery. After intramyocardial injection of 5 microl virus stock solution, cardiomyocyte transduction rates with AAV vectors were 4-fold lower than with adenovirus vectors (1.5% (range: 0.5-2.6%) vs. 6.2% (range: 2.7-13.7%); P<0.05), but similar to titer-matched adenovirus vectors (0.7%; range: 0.2-1.2%). AAV-mediated EGFP expression lasted for at least 1 year. AAV vectors instilled into the pericardial space transduced epicardial myocytes. Arterial gene transfer was studied in mouse carotids (n=26). Both vectors selectively transduced endothelial cells after luminal instillation. Transduction rates with AAV vectors were 8-fold lower than with adenovirus vectors (2.0% (range: 0-3.2%) vs. 16.2% (range: 8.5-20.2%); P<0.05). Prolonged EGFP expression was observed after AAV but not adenovirus-mediated gene transfer. In conclusion, AAV vectors deliver and express genes for extended periods of time in the myocardium and arterial endothelium in vivo. AAV vectors may be useful for gene therapy approaches to chronic cardiovascular diseases.

A review of methods for assessment of coronary microvascular disease in both clinical and experimental settings

Obstructive disease of the large coronary arteries is the prominent cause for angina pectoris. However, angina may also occur in the absence of significant coronary atherosclerosis or coronary artery spasm, especially in women. Myocardial ischaemia in these patients is often associated with abnormalities of the coronary microcirculation and may thus represent a manifestation of coronary microvascular disease (CMD). Elucidation of the role of the microvasculature in the genesis of myocardial ischaemia and cardiac damage-in the presence or absence of obstructive coronary atherosclerosis-will certainly result in more rational diagnostic and therapeutic interventions for patients with ischaemic heart disease. Specifically targeted research based on improved assessment modalities is needed to improve the diagnosis of CMD and to translate current molecular, cellular, and physiological knowledge into new therapeutic options.

Multiply Attenuated, Self-Inactivating Lentiviral Vectors Efficiently Deliver and Express Genes for Extended Periods of Time in Adult Rat Cardiomyocytes In Vivo

Background—Among retroviral vectors, lentiviral vectors are unique in that they transduce genes into both dividing and nondividing cells. However, their ability to provide sustained myocardial transgene expression has not been evaluated. Methods and Results—Multiply attenuated, self-inactivating lentivectors based on human immunodeficiency virus-1 contained the enhanced green fluorescent protein (EGFP) gene under the transcriptional control of either the cytomegalovirus (CMV) immediate-early enhancer/promoter, the elongation factor-1&agr; (EF-1&agr;) promoter, or the phosphoglycerate-kinase (PGK) promoter. Lentivectors transduced adult rat cardiomyocytes in a dose-dependent manner (transduction rates, >90%; multiplicity of infection, ≈5). The CMV promoter achieved higher EGFP expression levels than the EF-1&agr; and PGK promoters. Insertion of the central polypurine tract pol sequence improved gene transfer efficiency by ≈2-fold. In vivo gene transfer kinetics was studied by measuring the copy number of integrated lentivirus DNA and EGFP concentrations in cardiac extracts by real-time polymerase chain reaction and ELISA, respectively. With CMV promoter-containing lentivectors, vector DNA peaked at day 3, declined by ≈4-fold at day 14, but then remained stable up to week 10. Similarly, EGFP expression peaked at day 7, decreased by ≈7-fold at day 14, but was essentially stable thereafter. In contrast, vector DNA and EGFP expression declined rapidly with EF-1&agr; promoter–containing lentivectors. Peak EGFP expression with titer-matched adenovectors was ≈35% higher than with CMV lentivectors but was lost rapidly over time. Conclusions—Lentivectors efficiently transduce and express genes for extended periods of time in cardiomyocytes in vivo. Lentivectors provide a useful tool for studying myocardial biology and a potential system for gene heart therapy.

Effect of NO Donors on LV Diastolic Function in Patients With Severe Pressure-Overload Hypertrophy

BACKGROUND Previous experimental studies have shown that nitric oxide (NO) modulates cardiac function by an abbreviation of systolic contraction and an enhancement of diastolic relaxation. However, the response to NO donors of patients with severe pressure-overload hypertrophy and diastolic dysfunction is unknown. METHODS AND RESULTS Intracoronary NO donors were given to 17 patients with severe aortic stenosis. A dose-response curve was obtained with nitroglycerin (30, 90, and 150 microg) in 11 patients and sodium nitroprusside (1, 2, and 4 microg/min) in 6. Left ventricular (LV) high-fidelity pressure measurements with simultaneous LV angiograms were performed at baseline and after the maximal dose of NO. The dose-response curve for intracoronary NO donors showed a marked fall in LV end-diastolic pressure, from 23 to 14 mm Hg (-39%; P<0.0001), whereas LV peak systolic pressure fell only slightly, from 206 to 196 mm Hg (-4%; P<0.01). End-diastolic chamber stiffness decreased from 0.12 to 0.07 mm Hg/mL (P<0.0001) and end-systolic stiffness from 1.6 to 1.3 mm Hg/mL (P<0.01). Heart rate, right atrial pressure, LV ejection fraction, the time constant of isovolumic pressure decay (tau), and LV filling rates remained unchanged. CONCLUSIONS In patients with severe pressure-overload hypertrophy, intracoronary NO donors exert a marked decrease in LV end-diastolic pressure without affecting LV systolic pump function. Thus, the hypertrophied myocardium appears to be particularly susceptible to NO donors, with a marked improvement in diastolic function.

Adult human adipose tissue contains several types of multipotent cells.

Multipotent mesenchymal stromal cells (MSCs) are a type of adult stem cells that can be easily isolated from various tissues and expanded in vitro. Many reports on their pluripotency and possible clinical applications have raised hopes and interest in MSCs. In an attempt to unify the terminology and the criteria to label a cell as MSC, in 2006 the International Society for Cellular Therapy (ISCT) proposed a standard set of rules to define the identity of these cells. However, MSCs are still extracted from different tissues, by diverse isolation protocols, are cultured and expanded in different media and conditions. All these variables may have profound effects on the selection of cell types and the composition of heterogeneous subpopulations, on the selective expansion of specific cell populations with totally different potentials and ergo, on the long-term fate of the cells upon in vitro culture. Therefore, specific molecular and cellular markers that identify MSCs subsets as well as standardization of expansion protocols for these cells are urgently needed. Here, we briefly discuss new useful markers and recent data supporting the rapidly emerging concept that many different types of progenitor cells are found in close association with blood vessels. This knowledge may promote the necessary technical improvements required to reduce variability and promote higher efficacy and safety when isolating and expanding these cells for therapeutic use. In the light of the discussed data, particularly the identification of new markers, and advances in the understanding of fundamental MSC biology, we also suggest a revision of the 2006 ISCT criteria.

Exosomes: Therapy delivery tools and biomarkers of diseases

&NA; Virtually all cells in the organism secrete extracellular vesicles (EVs), a heterogeneous population of lipid bilayer membrane‐enclosed vesicles that transport and deliver payloads of proteins and nucleic acids to recipient cells, thus playing central roles in cell‐cell communications. Exosomes, nanosized EVs of endosomal origin, regulate many pathophysiological processes including immune responses and inflammation, tumour growth, and infection. Healthy subjects and patients with different diseases release exosomes with different RNA and protein contents into the circulation, which can be measured as biomarkers. The discovery of exosomes as natural carriers of functional small RNA and proteins has raised great interest in the drug delivery field, as it may be possible to harness these vesicles for therapeutic delivery of miRNA, siRNA, mRNA, lncRNA, peptides, and synthetic drugs. However, systemically delivered exosomes accumulate in liver, kidney, and spleen. Targeted exosomes can be obtained by displaying targeting molecules, such as peptides or antibody fragments recognizing target antigens, on the outer surface of exosomes. Display of glycosylphosphatidylinositol (GPI)‐anchored nanobodies on EVs is a novel technique that enables EV display of a variety of proteins including antibodies, reporter proteins, and signaling molecules. However, naturally secreted exosomes show limited pharmaceutical acceptability. Engineered exosome mimetics that incorporate desirable components of natural exosomes into synthetic liposomes or nanoparticles, and are assembled using controllable procedures may be more acceptable pharmaceutically. In this communication, we review the current understanding of physiological and pathophysiological roles of exosomes, their potential applications as diagnostic markers, and current efforts to develop improved exosome‐based drug delivery systems.

Heterogeneity of Human Monocytes: An Optimized Four-Color Flow Cytometry Protocol for Analysis of Monocyte Subsets

Monocytes are central mediators in the development of atherosclerotic plaques. They circulate in blood and eventually migrate into tissue including the vessel wall where they give rise to macrophages and dendritic cells. The existence of monocyte subsets with distinct roles in homeostasis and inflammation suggests specialization of function. These subsets are identified based on expression of the CD14 and CD16 markers. Routinely applicable protocols remain elusive, however. Here, we present an optimized four-color flow cytometry protocol for analysis of human blood monocyte subsets using a specific PE-Cy5–conjugated monoclonal antibody (mAb) to HLA-DR, a PE-Cy7-conjugated mAb to CD14, a FITC-conjugated mAb to CD16, and PE-conjugated mAbs to additional markers relevant to monocyte function. Classical CD14+CD16− monocytes (here termed “Mo1” subset) expressed high CCR2, CD36, CD64, and CD62L, but low CX3CR1, whereas “nonclassical” CD14loCD16+ monocytes (Mo3) essentially showed the inverse expression pattern. CD14+CD16+ monocytes (Mo2) expressed high HLA-DR, CD36, and CD64. In patients with stable coronary artery disease (n = 13), classical monocytes were decreased, whereas “nonclassical” monocytes were increased 90% compared with healthy subjects with angiographically normal coronary arteries (n = 14). Classical monocytes from CAD patients expressed higher CX3CR1 and CCR2 than controls. Thus, stable CAD is associated with expansion of the nonclassical monocyte subset and increased expression of inflammatory markers on monocytes. Flow cytometric analysis of monocyte subsets and marker expression may provide valuable information on vascular inflammation. This may translate into the identification of monocyte subsets as selective therapeutic targets, thus avoiding adverse events associated with indiscriminate monocyte inhibition.

Ultrastructural Evidence of Exosome Secretion by Progenitor Cells in Adult Mouse Myocardium and Adult Human Cardiospheres

The demonstration of beneficial effects of cell therapy despite the persistence of only few transplanted cells in vivo suggests secreted factors may be the active component of this treatment. This so-called paracrine hypothesis is supported by observations that culture media conditioned by progenitor cells contain growth factors that mediate proangiogenic and cytoprotective effects. Cardiac progenitor cells in semi-suspension culture form spherical clusters (cardiospheres) that deliver paracrine signals to neighboring cells. A key component of paracrine secretion is exosomes, membrane vesicles that are stored intracellularly in endosomal compartments and are secreted when these structures fuse with the cell plasma membrane. Exosomes have been identified as the active component of proangiogenic effects of bone marrow CD34+ stem cells in mice and the regenerative effects of embryonic mesenchymal stem cells in infarcted hearts in pigs and mice. Here, we provide electron microscopic evidence of exosome secretion by progenitor cells in mouse myocardium and human cardiospheres. Exosomes are emerging as an attractive vector of paracrine signals delivered by progenitor cells. They can be stored as an “off-the-shelf” product. As such, exosomes have the potential for circumventing many of the limitations of viable cells for therapeutic applications in regenerative medicine.