Laurentiu M. Popescu

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.

Signature microRNA Expression Profile of Essential Hypertension and Its Novel Link to Human Cytomegalovirus Infection

Background— Essential hypertension has been recognized as a disease resulting from a combination of environmental and genetic factors. Recent studies demonstrated that microRNAs (miRNAs) are involved in cardiac hypertrophy and heart failure. However, little is known about the roles of miRNAs in essential hypertension. Methods and Results— Using microarray-based miRNA expression profiling, we compared the miRNA expressions in plasma samples from 13 hypertensive patients and 5 healthy control subjects. Twenty-seven miRNAs were found to be differentially expressed. The expressions of selected miRNAs (miR-296–5p, let-7e, and a human cytomegalovirus [HCMV]–encoded miRNA, hcmv-miR-UL112) were validated independently in plasma samples from 24 hypertensive patients and 22 control subjects. The absolute expression levels of hcmv-miR-UL112, miR-296–5p, and let-7e were further determined in 127 patients and 67 control subjects (fold changes are 2.5, 0.5, and 1.7 respectively; all P<0.0001). Additionally, we demonstrated that interferon regulatory factor 1 is a direct target of hcmv-miR-UL112. Increased HCMV seropositivity and quantitative titers were found in the hypertension group compared with the control group (52.7% versus 30.9%, P=0.0005; 1870 versus 54 copies per 1 mL plasma, P<0.0001). Seropositivity, log-transformed copies of HCMV, and hcmv-miR-UL112 were independently associated with an increased risk of hypertension (odds ratio, 2.48; 95% confidence interval, 1.48 to 4.15; P=0.0005; odds ratio, 1.97; 95% confidence interval, 1.58 to 2.46; P<0.0001; and odds ratio, 2.55; 95% confidence interval, 1.98 to 3.27; P<0.0001, respectively). Conclusions— We report for the first time a circulating miRNA profile for hypertensive patients and demonstrate a novel link between HCMV infection and essential hypertension. These findings may reveal important insights into the pathogenesis of essential hypertension. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00420784.

Interstitial Cajal-like cells in human uterus and fallopian tube.

Abstract:  Recently, parallels have been drawn between enteric interstitial cells of Cajal (ICC) and similar cells outside the gut—interstitial Cajal‐like cells (ICLC). This article reviews our laboratory findings on ICLC in the female reproductive tract. Since the morphology and function of ICLC are still a subject of debate, our purpose was to investigate whether ICLC are present in the fallopian tube and/or uterus, and if they share ultrastructural and immunohistochemical (IHC) features and/or functional roles. We studied ICLC presence in the human fallopian tube and myometrium primarily by light microscopy, and then by transmission electron microscopy (TEM), in tissue samples and at a single cell level. Taking advantage of our ICLC studies of several organs (pancreas, mammary gland, myocardium), we assembled a set of criteria, derived from ultrastructural features of ICLC, called “platinum standard.” Besides the putative pacemaker function, ICLC might have other physiological roles, depending on tissue type (e.g., intercellular signaling, immune surveillance, steroid sensors). Consequently, there is a great urge for a conceptual framework that could allow a better understanding, from a functional point of view, and more so, as the ICLC processes are the longest cellular prolongations (except neurons).

Heterocellular communication in the heart: electron tomography of telocyte–myocyte junctions

Myocardium is composed of two main cell populations: cardiomyocytes (CMs) and interstitial cells (e.g. fibroblasts, immunoreactive cells, capillaries). However, very recently we have showed that a novel type of interstitial cell called telocytes (TCs) does exist in epi‐, myo‐ and endocardium. They have very long and thin telopodes (Tp) formed by alternating podomeres and podoms. Heterocellular communication between TCs and CMs it is supposed to occur by shed vesicles and close apposition. If TCs have to play a role in cardiac physiology it is expected to develop direct and unambiguous contacts with CMs. Because a clear membrane‐to‐membrane junction has not been reported by electron microscopy we have investigated the heterocellular communication in the mouse heart by electron tomography. This advanced technique showed that small dense structures (10–15 nm nanocontacts) directly connect TCs with CMs. More complex and atypical junctions could be observed between TCs and CMs at the level of intercalated discs. This study proves that TCs and CMs are directly connected and might represent a ‘functional unit’.

Human myometrium – the ultrastructural 3D network of telocytes

Telocytes (TCs), a novel type of interstitial cells, were recently described in the interstitial space of tissues (www.telocytes.com). Telocytes TCs have several very long, moniliform extensions, namely telopodes (Tps). However, the functional role(s) of TCs is not yet understood. Successive photomicrographs of ultrathin sections were concatenated to capture the entire length of Tps which usually measure tens to hundreds of micrometres. Besides the podoms (dilations) and podomers (thin segments), ultrastructural features of Tps include the dichotomous branching and establishing homo‐ and heterocellular contacts. Telopodes make a labyrinthine system by 3D convolution and overlapping, their number being roughly estimated at approximately 20 per 1000 μm2. Moreover, the presence of extracellular vesicles (shedding vesicles/exosomes) along the Tps suggests an active intercellular signalling (micro‐ and macromolecules), with possible significance in regulating uterine contractility.

Extracellular vesicles release by cardiac telocytes: electron microscopy and electron tomography.

Telocytes have been reported to play an important role in long‐distance heterocellular communication in normal and diseased heart, both through direct contact (atypical junctions), as well as by releasing extracellular vesicles (EVs) which may act as paracrine mediators. Exosomes and ectosomes are the two main types of EVs, as classified by size and the mechanism of biogenesis. Using electron microscopy (EM) and electron tomography (ET) we have found that telocytes in culture release at least three types of EVs: exosomes (released from endosomes; 45 ± 8 nm), ectosomes (which bud directly from the plasma membrane; 128 ± 28 nm) and multivesicular cargos (MVC; 1 ± 0.4 μm), the latter containing tightly packaged endomembrane‐bound vesicles (145 ± 35 nm). Electron tomography revealed that endomembrane vesicles are released into the extracellular space as a cargo enclosed by plasma membranes (estimated area of up to 3 μm2). This new type of EV, also released by telocytes in tissue, likely represents an essential component in the paracrine secretion of telocytes and may consequently be directly involved in heart physiology and regeneration.

Comparative proteomic analysis of human lung telocytes with fibroblasts

Telocytes (TCs) were recently described as interstitial cells with very long prolongations named telopodes (Tps; www.telocytes.com). Establishing the TC proteome is a priority to show that TCs are a distinct type of cells. Therefore, we examined the molecular aspects of lung TCs by comparison with fibroblasts (FBs). Proteins extracted from primary cultures of these cells were analysed by automated 2‐dimensional nano‐electrospray ionization liquid chromatography tandem mass spectrometry (2D Nano‐ESI LC‐MS/MS). Differentially expressed proteins were screened by two‐sample t‐test (P < 0.05) and fold change (>2), based on the bioinformatics analysis. We identified hundreds of proteins up‐ or down‐regulated, respectively, in TCs as compared with FBs. TC proteins with known identities are localized in the cytoskeleton (87%) and plasma membrane (13%), while FB up‐regulated proteins are in the cytoskeleton (75%) and destined to extracellular matrix (25%). These identified proteins were classified into different categories based on their molecular functions and biological processes. While the proteins identified in TCs are mainly involved in catalytic activity (43%) and as structural molecular activity (25%), the proteins in FBs are involved in catalytic activity (24%) and in structural molecular activity, particularly synthesis of collagen and other extracellular matrix components (25%). Anyway, our data show that TCs are completely different from FBs. In conclusion, we report here the first extensive identification of proteins from TCs using a quantitative proteomics approach. Protein expression profile shows many up‐regulated proteins e.g. myosin‐14, periplakin, suggesting that TCs might play specific roles in mechanical sensing and mechanochemical conversion task, tissue homoeostasis and remodelling/renewal. Furthermore, up‐regulated proteins matching those found in extracellular vesicles emphasize TCs roles in intercellular signalling and stem cell niche modulation. The novel proteins identified in TCs will be an important resource for further proteomic research and it will possibly allow biomarker identification for TCs. It also creates the premises for understanding the pathogenesis of some lung diseases involving TCs.

Telocytes transfer extracellular vesicles loaded with microRNAs to stem cells.

Telocytes (TCs) are cells ubiquitously distributed in the body and characterized by very long and thin prolongations named telopodes (Tps). Cardiac TCs are the best characterized TCs for the moment. Tps release extracellular vesicles (EVs) in vivo and in vitro suggesting that TCs regulate the activity of other cells by vesicular paracrine signals. TCs have been found within the stem cell niche of several organs. Electron microscopy or electron tomography has shown that Tps are located in close vicinity of stem cells (SC). Since stem cell regulation by niche components involves paracrine signalling, we have investigated if TCs could be part of this mechanism. Using fluorescent labelling of cells and EVs with calcein and Cy5‐miR‐21 oligos, we provide evidence that TCs can modulate SC through EVs loaded with microRNAs. TCs deliver microRNA to cardiac stem cells (CSCs), as well as to other types of SCs (e.g. hematopoietic SC) indicating that this mechanism is not restricted to cardiac tissue. We also found that CSCs deliver microRNA loaded EVs to TCs, suggesting that there is a continuous, post‐transcriptional regulatory signal back and forth between TCs and SC. In conclusion, our data reveal the existence of a reciprocal (bidirectional) epigenetic signalling between TCs and SC.

MicroRNA-134 as a potential plasma biomarker for the diagnosis of acute pulmonary embolism

BackgroundAcute pulmonary embolism (APE) remains a diagnostic challenge due to a variable clinical presentation and the lack of a reliable screening tool. MicroRNAs (miRNAs) regulate gene expression in a wide range of pathophysiologic processes. Circulating miRNAs are emerging biomarkers in heart failure, type 2 diabetes and other disease states; however, using plasma miRNAs as biomarkers for the diagnosis of APE is still unknown.MethodsThirty-two APE patients, 32 healthy controls, and 22 non-APE patients (reported dyspnea, chest pain, or cough) were enrolled in this study. The TaqMan miRNA microarray was used to identify dysregulated miRNAs in the plasma of APE patients. The TaqMan-based miRNA quantitative real-time reverse transcription polymerase chain reactions were used to validate the dysregulated miRNAs. The receiver-operator characteristic (ROC) curve analysis was conducted to evaluate the diagnostic accuracy of the miRNA identified as the candidate biomarker.ResultsPlasma miRNA-134 (miR-134) level was significantly higher in the APE patients than in the healthy controls or non-APE patients. The ROC curve showed that plasma miR-134 was a specific diagnostic predictor of APE with an area under the curve of 0.833 (95% confidence interval, 0.737 to 0.929; P < 0.001).ConclusionsOur findings indicated that plasma miR-134 could be an important biomarker for the diagnosis of APE. Because of this finding, large-scale investigations are urgently needed to pave the way from basic research to clinical utilization.

Platelet‐derived growth factor receptor‐β‐positive telocytes in skeletal muscle interstitium

Telocytes (TCs) represent a new cell type recently described in mammalian skeletal muscle interstitium as well as in other organs. These have a specific morphology and phenotype, both in situ and in vitro. Telocytes are cells with long and slender cell prolongations, in contact with other interstitial cells, nerve fibres, blood capillaries and resident stem cells in niches. Our aim was to investigate the potential contribution of TCs to micro‐vascular networks by immunofluorescent labelling of specific angiogenic growth factors and receptors. We found that in human skeletal muscle TCs were constantly located around intermediate and small blood vessels and endomysial capillaries. Epi‐fluorescence and laser confocal microscopy showed that TCs express c‐kit, platelet‐derived growth factor receptor (PDGFR)‐β and VEGF, both in situ and in vitro. Telocytes were constantly located in the perivascular or pericapillary space, as confirmed by double staining of c‐kit/CD31, PDGFR‐β/CD31 and PDGFR‐β/α‐smooth muscle actin, respectively. Electron microscopy (EM) differentiated between pericytes and other cell types. Laminin labelling showed that TCs are not enclosed or surrounded by a basal lamina in contrast to mural cells. In conclusion, a) PDGFR‐β could be used as a marker for TCs and b) TCs are presumably a transitional population in the complex process of mural cell recruitment during angiogenesis and vascular remodelling.