Mihaela Gherghiceanu

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.

Cardioprotection by resveratrol: a novel mechanism via autophagy involving the mTORC2 pathway

AIMS On the basis of our previous reports that cardioprotection induced by ischaemic preconditioning induces autophagy and that resveratrol, a polyphenolic antioxidant present in grapes and red wine induces preconditioning-like effects, we sought to determine if resveratrol could induce autophagy. METHODS AND RESULTS Resveratrol at lower doses (0.1 and 1 microM in H9c2 cardiac myoblast cells and 2.5 mg/kg/day in rats) induced cardiac autophagy shown by enhanced formation of autophagosomes and its component LC3-II after hypoxia-reoxygenation or ischaemia-reperfusion. The autophagy was attenuated with the higher dose of resveratrol. The induction of autophagy was correlated with enhanced cell survival and decreased apoptosis. Treatment with rapamycin (100 nM), a known inducer of autophagy, did not further increase autophagy compared with resveratrol alone. Autophagic inhibitors, wortmannin (2 microM) and 3-methyladenine (10 mM), significantly attenuated the resveratrol-induced autophagy and induced cell death. The activation of mammalian target of rapamycin (mTOR) was differentially regulated by low-dose resveratrol, i.e. the phosphorylation of mTOR at serine 2448 was inhibited, whereas the phosphorylation of mTOR at serine 2481 was increased, which was attenuated with a higher dose of resveratrol. Although resveratrol attenuated the activation of mTOR complex 1, low-dose resveratrol significantly induced the expression of Rictor, a component of mTOR complex 2, and activated its downstream survival kinase Akt (Ser 473). Resveratrol-induced Rictor was found to bind with mTOR. Furthermore, treatment with Rictor siRNA attenuated the resveratrol-induced autophagy. CONCLUSION Our results indicate that at lower dose, resveratrol-mediated cell survival is, in part, mediated through the induction of autophagy involving the mTOR-Rictor survival pathway.

Cardiomyocyte precursors and telocytes in epicardial stem cell niche: electron microscope images

A highly heterogeneous population of stem and progenitor cells has been described by light immunohistochemistry in the mammalian adult heart, but the ultrastructural identity of cardiac stem cells remains unknown. Using electron microscopy, we demonstrate the presence of cells with stem features in the adult mouse heart. These putative cardiac stem cells are small (6–10 μm), round cells, with an irregular shaped nucleus, large nucleolus, few endoplasmic reticulum cisternae and mitochondria, but numerous ribosomes. Stem cells located in the epicardial stem cell niche undergo mitosis and apoptosis. Cells with intermediate features between stem cells and cardiomyocyte progenitors have also been seen. Moreover, electron microscopy showed that cardiomyocyte progenitors were added to the peripheral working cardiomyocytes. Telocytes make a supportive interstitial network for stem cells and progenitors in the stem cell niche. This study enhances the hypothesis of a unique type of cardiac stem cell and progenitors in different stages of differentiation. In our opinion, stem cells, cardiomyocyte progenitors and telocytes sustain a continuous cardiac renewal process in the adult mammalian heart.

Novel type of interstitial cell (Cajal‐like) in human fallopian tube

We describe here ‐ presumably for the first time‐a Cajal‐like type of tubal interstitial cells (t‐ICC), resembling the archetypal enteric ICC. t‐ICC were demonstrated in situ and in vitro on fresh preparations (tissue cryosections and primary cell cultures) using methylene‐blue, crystal‐violet, Janus‐Green B or Mito Tracker‐Green FM Probe vital stainings. Also, t‐ICC were identified in fixed specimens by light microscopy (methylene‐blue, Giemsa, trichrome stainings, Gomori silver‐impregnation) or transmission electron microscopy (TEM). The positive diagnosis of t‐ICC was strengthened by immunohistochemistry (IHC; CD117/c‐kit+ and other 14 antigens) and immunofluorescence (IF; CD117/c‐kit+ and other 7 antigens). The spatial density of t‐ICC (ampullar‐segment cryosections) was 100–150 cells/mm2. Non‐conventional light microscopy (NCLM) of Epon semithin‐sections revealed a network‐like distribution of t‐ICC in lammina propria and smooth muscle meshwork. t‐ICC appeared located beneath of epithelium, in a 10–15μ thick ‘belt’, where 18±2% of cells were t‐ICC. In the whole lamina propria, t‐ICC were about 9%, and in muscularis ∼7%. In toto, t‐ICC represent ∼8% of subepithelial cells, as counted by NCLM. In vitro, t‐ICC were 9.9±0.9% of total cell population.

Identification of telocytes in skeletal muscle interstitium: implication for muscle regeneration

Skeletal muscle interstitium is crucial for regulation of blood flow, passage of substances from capillaries to myocytes and muscle regeneration. We show here, probably, for the first time, the presence of telocytes (TCs), a peculiar type of interstitial (stromal) cells, in rat, mouse and human skeletal muscle. TC features include (as already described in other tissues) a small cell body and very long and thin cell prolongations—telopodes (Tps) with moniliform appearance, dichotomous branching and 3D‐network distribution. Transmission electron microscopy (TEM) revealed close vicinity of Tps with nerve endings, capillaries, satellite cells and myocytes, suggesting a TC role in intercellular signalling (via shed vesicles or exosomes). In situ immunolabelling showed that skeletal muscle TCs express c‐kit, caveolin‐1 and secrete VEGF. The same phenotypic profile was demonstrated in cell cultures. These markers and TEM data differentiate TCs from both satellite cells (e.g. TCs are Pax7 negative) and fibroblasts (which are c‐kit negative). We also described non‐satellite (resident) progenitor cell niche. In culture, TCs (but not satellite cells) emerge from muscle explants and form networks suggesting a key role in muscle regeneration and repair, at least after trauma.

Telocytes in Human Term Placenta: Morphology and Phenotype

In the last few years, a new cell type – interstitial Cajal-like cell (ICLC) – has been described in digestive and extra-digestive organs. The name has recently been changed to telocytes (TC) and their typical thin, long processes have been named telopodes (TP). To support the hypothesis that TC may also be present in human placenta and add to the information already available, we provide evidence on the ultrastructure, immunophenotype, distribution, and interactions with the surrounding stromal cells of TC in the villous core of human term placenta. We used phase-contrast microscopy, light microscopy of semithin sections, transmission electron microscopy, immunohistochemistry, and immunofluorescence of tissue sections or cell cultures, following a pre-established diagnostic algorithm. Transmission electron microscopy showed cells resembling TC, most (∼76%) having 2–3 very thin, longprocesses (tens to hundreds of micrometers), with an uneven calibre(≤0.5 µm thick) and typical branching pattern. The dilations of processes accommodate caveolae, endoplasmic reticulum cisternae, and mitochondria. These TC have close contacts with perivascular SMC in stem villi. In situ, similar cells are positive for c-kit, CD34, vimentin, caveolin-1, vascular endothelial growth factor (VEGF), and inducible nitric oxide synathase (iNOS). The c-kit-positive cells inconsistently co-express CD34, CD44, αSMA, S100, neuron-specific enolase, and nestin. Among cells with a morphologic TC profile in cell cultures, about 13% co-express c-kit, vimentin, and caveolin-1; 70% of the c-kit-positive cells co-express CD34 and 12% co-express iNOS or VEGF. In conclusion, this study confirms the presence of TC in human term placenta and provides their ultrastructural and immunophenotypic characterization.

Experimental acute myocardial infarction: telocytes involvement in neo-angiogenesis.

We used rat experimental myocardial infarction to study the ultrastructural recovery, especially neo‐angiogenesis in the infarction border zone. We were interested in the possible role(s) of telocytes (TCs), a novel type of interstitial cell very recently discovered in myocardim (see http://www.telocytes.com). Electron microscopy, immunocytochemistry and analysis of several proangiogenic microRNAs provided evidence for TC involvement in neo‐angiogenesis after myocardial infarction. Electron microscopy showed the close spatial association of TCs with neoangiogenetic elements. Higher resolution images provided the following information: (a) the intercellular space between the abluminal face of endothelium and its surrounding TCs is frequently less than 50 nm; (b) TCs establish multiple direct nanocontacts with endothelial cells, where the extracellular space seems obliterated; such nanocontacts have a length of 0.4–1.5 μm; (c) the absence of basal membrane on the abluminal face of endothelial cell. Besides the physical contacts (either nanoscopic or microscopic) TCs presumably contribute to neo‐angiognesis via paracrine secretion (as shown by immunocytochemistry for VEGF or NOS2). Last but not least, TCs contain measurable quantities of angiogenic microRNAs (e.g. let‐7e, 10a, 21, 27b, 100, 126‐3p, 130a, 143, 155, 503). Taken together, the direct (physical) contact of TCs with endothelial tubes, as well as the indirect (chemical) positive influence within the ‘angiogenic zones’, suggests an important participation of TCs in neo‐angiogenesis during the late stage of myocardial infarction.

C-kit immunopositive interstitial cells (Cajal-type) in human myometrium.

Previous reports describing Cajal‐like interstitial cells in human uterus are contradictory in terms of c‐kit immunoreactivity: either negative (but vimentin‐positive) in pregnant myometrium, or positive, presumably in the endometrium. The aim of this study was to verify the existence of human myometrial Cajal‐like interstitial cells (m‐CLIC). Six different, complementary approaches were used: 1) methylene‐blue supravital staining of tissue samples (cryosections), 2) methylene blue and Janus green B vital staining (m‐CLIC and mitochondrial markers, respectively), and 3) extracellular single‐unit electrophysiological recordings in cell cultures, 4) non‐conventional light microscopy on glutaraldehyde/osmium fixed, Epon‐embedded semi‐thin sections (less than 1μm) stained with toluidine blue (TSM), 5) transmission electron microscopy (TEM), and 6) immunofluorescence (IF). We found m‐CLIC in myometrial cryosections and in cell cultures. In vitro, m‐CLIC represented ∼7% of the total cell number. m‐CLIC had 2–3 characteristic processes which were very long (∼ 60 μm), very thin (±0.5μm) and moniliform. The dilated portions of processes usually accomodated mitochondria. In vitro, m‐CLIC exhibited spontaneous electrical activity (62.4 ± 7.22 mV field potentials, short duration: 1.197 ± 0.04ms). Moreover, m‐CLIC fulfilled the usual TEM criteria, the so‐called ‘gold’ or ‘platinum’ standards (e.g. the presence of discontinuos basal lamina, caveolae, endoplasmic reticulum, and close contacts between each other, with myocytes, nerve fibers and/or capillaries etc.). IF showed that m‐CLIC express CD117/c‐kit, sometimes associated with CD34 and with vimentin along their processes.

Cardiac renewing: interstitial Cajal‐like cells nurse cardiomyocyte progenitors in epicardial stem cell niches

Recent studies suggested that various cell lineages exist within the subepicardium and we supposed that this area could host cardiac stem cell niches (CSCNs). Using transmission electron microscopy, we have found at least 10 types of cells coexisting in the subepicardium of normal adult mice: adipocytes, fibroblasts, Schwann cells and nerve fibres, isolated smooth muscle cells, mast cells, macrophages, lymphocytes, interstitial Cajal‐like cells (ICLCs) and cardiomyocytes progenitors (CMPs). The latter cells, sited in the area of origin of coronary arteries and aorta, showed typical features of either very immature or developing cardiomyocytes. Some of these cells were connected to each other to form columns surrounded by a basal lamina and embedded in a cellular network made by ICLCs. Complex intercellular communication occurs between the ICLCs and CMPs through electron‐dense nanostructures or through shed vesicles. We provide here for the first time the ultrastructural description of CSCN in the adult mice myocardium, mainly containing ICLCs and CMPs. The existence of resident CMPs in different developmental stages proves that cardiac renewing is a continuous process. We suggest that ICLCs might act as supporting nurse cells of the cardiac niches and may be responsible for activation, commitment and migration of the stem cells out of the niches. Briefly, not only resident cardiac stem cells but also ICLCs regulate myocyte turnover and contribute to both cardiac cellular homeostasis and endogenous repair/remodelling after injuries.

Cardioprotection by adaptation to ischaemia augments autophagy in association with BAG-1 protein.

Autophagy is an intracellular process in which a cell digests its own constituents via lysosomal degradative pathway. Though autophagy has been shown in several cardiac diseases like heart failure, hypertrophy and ischaemic cardiomyopathy, the role and the regulation of autophagy is still largely unknown. Bcl‐2‐associated athanogene (BAG‐1) is a multifunctional pro‐survival molecule that binds with Hsp70/Hsc70. In this study, myocardial adaptation to ischaemia by repeated brief episodes of ischaemia and reperfusion (I/R) prior to lethal I/R enhanced the expression of autophagosomal membrane specific protein light chain 3 (LC3)‐II, and Beclin‐1, a molecule involved in autophagy and BAG‐1. Autophagosomes structures were found in the adapted myocardium through electron microscopy. Co‐immunoprecipitation and co‐immunofluorescence analyses revealed that LC3‐II was bound with BAG‐1. Inhibition of autophagy by treating rats with Wortmannin (15 μg/kg; intraperitoneally) abolished the ischaemic adaptation‐induced induction of LC3‐II, Beclin‐1, BAG‐1 and cardioprotection. Intramyocardial injection of BAG‐1 siRNA attenuated the induction of LC3‐II, and abolished the cardioprotection achieved by adaptation. Furthermore, hypoxic adaptation in cardiac myoblast cells induced LC3‐II and BAG‐1. BAG‐1 siRNA treatment attenuated hypoxic adaptation‐induced LC3‐II and BAG‐1, and abolished improvement in cardiac cell survival and reduction of cell death. These results clearly indicate that myocardial protection elicited by adaptation is mediated at least in part via up‐regulation of autophagy in association with BAG‐1 protein.