Rom J Morphol Embryol 2016, 57(2 Suppl):767–774 R J M E RIGINAL APER Romanian Journal of O P Morphology & Embryology http://www.rjme.ro/

Subsets of telocytes: the progenitor cells in the human endocardial niche

FLORENTINA GRIGORIU1), SORIN HOSTIUC2), ALEXANDRA DIANA VRAPCIU1), MUGUREL CONSTANTIN RUSU1,3)

1)Division of Anatomy, Department 1, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania 2)Division of Legal Medicine and Bioethics, Department 2 – Morphological Sciences, Faculty of Medicine, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania 3)MEDCENTER, Center of Excellence in Laboratory Medicine and Pathology, Bucharest, Romania

Abstract Telocytes (TCs) are cells defined by their long and moniliform processes termed telopodes. They were previously identified in the endocardium and express neural markers, such as nestin and neuron-specific enolase (NSE). Previous studies found a positive expression of neuro- filaments in endocardial endothelial cells, and a positive expression of nestin in cardiac side population (CSP) progenitor cells, which allowed us to hypothesize that TCs in the endocardial stem niche could be in fact progenitors that express nestin. Materials and Methods: We used cardiac samples from 10 human adult donor cadavers. Endocardial endothelial cells expressed CD146, alpha- (α-SMA), smooth muscle (SMM), nestin and, scarcely, neurofilaments. Within atrial and ventricular samples, we found an endocardial discontinuous smooth muscle layer expressing, similar to pericytes and vascular smooth muscle cells, α-SMA, nestin, SMM, and CD146. We assessed a similar phenotype in the subendocardial muscle layer, which also expressed neuron-specific enolase. The expression of nestin and CD146 strongly indicates a progenitor phenotype, which, in turn, supports the hypothesis that, in humans, an endocardial stem niche supplied by an endothelial–mesenchymal transition should be considered, although it mimics a primitive supply from the cardiac neural crest with dormant cardiac side population progenitor cells. Nevertheless, the endocardial niche could indeed harbor precursor cells that are morphologically similar to TCs. Keywords: cardiac stem cells, pericytes, nestin, CD146, endothelial–mesenchymal transition.

 Introduction The structure of endocardium was analyzed in a paper dealing with endocardial telocytes (TCs) [4]. The A paper by Rusu et al. (2012) showed a positive authors discussed that the endocardium has just two layers, expression of 200 kDa neurofilaments (NFs) in endocardial the endocardial and the subendothelial loose and microvascular endothelial cells in human adult cardiac – the subendocardium [4]. However, samples [1]. That evidence was regarded with caution, a long time ago, Nagayo (1909) proposed two different, and further nestin labeling was recommended to evaluate ventricular and atrial, models for the normal endocardial whether or not the endothelial expression of NFs is related to neo-vessels formation [1]. structure, documented and cited by Okada, in 1961 [5]. In a review on the cardiac side population (CSP) cells As presented by Okada (1961), the left ventricular endo- [2], which are a subset of cardiac progenitors, Unno et al. cardium model of Nagayo consists of: (1) endothelial layer; (2012) discussed that “the developmental origins of CSP (2) inner connective tissue layer; (3) elastic layer; (4) cells and molecular cues that dictate CSP development smooth muscle layer; (5) subendocardial connective tissue remain to be determined” and indicate a study of Tomita layer which contains fibers of the conduction system [5]. et al. (2005) to document that CSP cells express neural The left atrial endocardium model of Nagayo consists of precursor markers [3]. Tomita et al. (2005) performed four layers only: (1) endothelial layer; (2) inner connective in vitro experiments using tissues from rodents and found tissue layer; (3) median elastic connective tissue layer; (4) that at day 0 almost all cardiosphere-derived cells expressed outer connective tissue layer [5]. For avoiding confusions, nestin, thus being regarded as undifferentiated neural we shall use here the term “subendothelial layer” that precursor cells; at day 14, the expression of nestin was corresponds to all three structural models of endocardium, lost, and the cells were positive for myosin and GFAP, although the authors who indicated a bi-layered endo- indicating a differentiated status [3]. Those results were cardial structure present images [4] in which the layers interpreted as suggestive for cardiac neural crest-derived indicated by Nagayo are quite evident. cells that populate the heart to remain there as dormant History of telocytes, for which the term was established multipotent stem cells that are further able to differentiate in 2010 by Popescu & Faussone-Pellegrini in an editorial into various cells types, such as cardiac myocytes, glial material [6] began in 2005 when the group of Popescu cells, neurons and smooth muscle cells [3]. described a new cell type in various tissues, which they

ISSN (print) 1220–0522 ISSN (online) 2066–8279 768 Florentina Grigoriu et al. first termed “interstitial Cajal-like cell” (ICLC) [7]. (NSE, mouse monoclonal, clone BBS/NC/VI-H14, Dako, Actually, TCs indicate a peculiar morphology: “cells with Glostrup, Denmark, 1:200); neurofilaments (mouse mono- telopodes” [8–10]. Regarding the specific markers for TCs clonal, clone RT97, Novocastra-Leica, Leica Biosystems identification, Faussone Pellegrini & Popescu indicate Newcastle Ltd., Newcastle Upon Tyne, UK, 1:50). that “TC might show different immunohistochemical For CD146, c-erbB2, SMM and α-SMA labeling, profiles among organs and even in the same organ tissues were deparaffinized and rehydrated, then endo- examined”. As TCs could not yet be identified as a genous peroxidase was blocked using Peroxidazed 1 distinctive cell type it seems reasonable, as Varga et al. (Biocare Medical, Concord, CA, USA). For the heat- (2016) observed already, that TCs are not referenced in induced epitope retrieval was used the Decloaking Chamber internationally accepted [11]. (Biocare Medical, Concord, CA, USA) and retrieval solution Various studies attempted to identify suitable markers pH 6 (Biocare Medical, Concord, CA, USA), the latter for TCs. Although (see above) they are tissue-specific, being a buffer specially formulated for superior pH stability there were indicated to be useful various hematopoietic at high temperatures. Background Blocker (Biocare and endothelial markers, such as c-kit, CD34, endoglin, Medical, Concord, CA, USA) was used to reduce non- neural markers, such as nestin, neuron-specific enolase specific background staining. The primary antibody was (NSE) and neuronal nitric oxide synthase (NOS), or myoid applied at specific dilutions (see above). As detection markers, such as alpha-smooth muscle actin (α-SMA) [12]. system was used MACH 4 (Biocare Medical, Concord, Therefore, a TC morphology of a progenitor cell or a CA, USA), which is a two-step (probe/polymer) universal neural phenotype for TCs cannot yet be rejected. horseradish peroxidase (HRP) detection method. A HRP- We, therefore, hypothesized that TCs in the endocardial compatible chromogen (3,3’-diaminobenzidine, DAB) was stem niche could be in fact progenitors expressing nestin applied. Sections were counterstained with Hematoxylin which, in turn, is not indicating a neural crest origin of and rinsed with deionized water. For washing steps was dormant subendocardial progenitor CSP cells but it rather used tris-buffered saline (TBS) solution, pH 7.6. points to an endocardial origin of these. For nestin, NSE and neurofilaments labeling, tissues It was thus aimed an immunohistochemical study on were deparaffinized and rehydrated, then endogenous human samples to test the in situ molecular phenotypes peroxidase was blocked using Peroxidazed 1 (Biocare within the endocardial stem niche. Medical, Concord, CA, USA). For the heat-induced epitope retrieval was used the Decloaking Chamber (Biocare Medical, Concord, CA, USA) and retrieval solution pH 6  Materials and Methods (Biocare Medical, Concord, CA, USA). Background Blocker For the immunohistochemical studies, we used ade- (Biocare Medical, Concord, CA, USA) was used to reduce quately preserved human postmortem tissue, obtained non-specific background staining. The primary antibody during the autopsy of 10 cadavers (1:1 sex ratio) with was applied at specific dilutions (see above). As detection ages varying between 28 and 82 years. system was used MACH 2 rabbit HRP polymer detection The study was conducted according to the national (Biocare Medical, Concord, CA, USA), which consists of laws regarding the use of cadaveric material for research a single reagent applied after the primary antibody. An purposes (including Law No. 104/2003 relating to the HRP-compatible chromogen (DAB) was applied. Sections manipulation of human cadavers), and the general principles were counterstained with Hematoxylin and rinsed with of the Declaration of Helsinki, Rio de Janeiro revision. deionized water. For washing steps was used TBS solution, Tissue samples were adequately preserved in buffered pH 7.6. formalin (8%), being further embedded in paraffin and The microscopic slides were analyzed and micrographs prepared for immunohistochemistry. Paraffin blocks were were acquired and scaled using a calibrated Zeiss working processed with an automatic histoprocessor (Diapath, station consisting of an AxioImager M1 microscope Martinengo, BG, Italy). Sections were cut manually at equipped with an AxioCam HRc camera and operated 3 μm and mounted on SuperFrost® electrostatic slides for with the digital microscopic image processing software immunohistochemistry (Thermo Scientific, Menzel-Gläser, AxioVision (Carl Zeiss, Oberkochen, Germany). Braunschweig, Germany). Histological evaluations used 3–4 μm thick sections stained with Hematoxylin and  Results Eosin. of endocardium was evaluated in atrial, We used primary antibodies for c-erbB2 (HER-2, ventricular and papillary muscle samples and was found mouse monoclonal, clone CB11, Biocare Medical, that the endocardial smooth muscle layer presence, although Concord, CA, USA, 1:50); nestin (mouse monoclonal, not exclusive, was not restricted to ventricular endocardium, clone 10c2, Santa Cruz Biotechnology, Santa Cruz, CA, being also present in atrial endocardium. It was also noted USA, 1:500); CD146 (mouse monoclonal, clone N1238, that thickness of endocardium was not uniform in individual Novocastra-Leica, Leica Biosystems Newcastle Ltd., samples: areas lacking the endocardial smooth muscle layer Newcastle Upon Tyne, UK, 1:50); α-smooth muscle had rather a bi-layered endocardium. It was also noted actin (α-SMA, mouse monoclonal, clone 1A4, Biocare that the endocardial endothelium was heterogeneous, as Medical, Concord, CA, USA, 1:50); smooth muscle there were endocardial areas with flattened, spindle-shaped myosin heavy chain (SMM, mouse monoclonal, clone endothelial cells, other areas had hypertrophied endothelial S131, Novocastra-Leica, Leica Biosystems Newcastle Ltd., cells, being also found endocardial areas denuded of Newcastle Upon Tyne, UK, 1:100); neuron-specific enolase endotheliocytes.

Subsets of telocytes: the progenitor cells in the human endocardial niche 769 Immunohistochemistry with antibodies against neural- the endocardium smooth muscle layer were occasionally specific intermediate filaments scarcely found the positive found myoid cords and vascular channels expressing expression of nestin and neurofilaments in spindle-shaped α-SMA. Moreover, expression of α-SMA was encountered endocardial endothelial cells (Figure 1), as well as in sub- in subendocardium, as well as in myoid cords included in endothelial layers (Figure 1). Hypertrophied endocardial the subjacent myocardium, which, in turn, was α-SMA- endothelial cells were rarely expressing these markers, negative. Pericytes and vascular smooth muscle cells were which, on other hand, were also expressed in vascular also α-SMA-positive (Figures 2 and 5). endothelial cells. Expression of CD146 was also found in smooth muscle Expression of α-SMA was also found (Figure 2) in tissues: pericytes, vascular smooth muscle cells, and focal areas in endocardial endothelial cells, usually in endocardial smooth muscle layer. In subendocardium, those spindle-shaped, and scarcely in those hypertrophied. the muscle fibers of the conduction system were also Similarly, the spindle-shaped endocardial endothelial cells expressing CD146 (Figures 3–5). expressed CD146 (Figure 3) and SMM (Figure 4). Vascular Expression of SMM was found in the endocardial endothelial cells expressed CD146 but were negative for and subendocardial muscle layers, cords and vascular α-SMA and SMM. channels (Figure 4). Pericytes and smooth muscle cells The anti-α-SMA antibody labeled the endocardial also expressed SMM (Figure 5). smooth muscle layer, which appeared discontinuous, of We found the positive expression of NSE in the variable thickness, consisting either of a single compact endocardial smooth muscle layer and, occasionally, in layer, or of multiple loosely arranged thin layers built-up pericytes and vascular smooth muscle cells (Figure 6). by spindle-shaped cells with TCs morphology. Within

Figure 1 – Expression of neural markers in human adult samples of right atrium (A and B) and left ventricular papillary muscle (C and D). Endocardial endothelial cells express neurofilaments (NFs) (A, arrowheads), as well as nestin (C, arrowhead). Immediate subendothelial endocardial cells are also found expressing NFs (B, arrow). Spindle- shaped cells projecting moniliform processes are identified (D, arrow) within the endocardial smooth muscle layer of Nagayo. n: Nerve.

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Figure 2 – Expression of alpha-smooth muscle actin (α-SMA) in human adult samples of left ventricles (A–D) and right ventricle (E and F). Positive expression of α-SMA is found in endocardial endothelial cells (arrowheads), as well as in the endocardial smooth muscle layer of Nagayo (arrows) which, in several instances, had a loose multilayered appearance (*). Myoid cords are identified embedded within the endocardial smooth muscle layer (D, double-headed arrow) but also beneath it, thus in subendocardium (B, double-headed arrow), and myocardial-embedded (C, double- headed arrow).

Subsets of telocytes: the progenitor cells in the human endocardial niche 771

Figure 3 – Expression of CD146 in left ventricular samples (A–D). Endocardial endothelial cells (A and C, arrowheads), as well as the endocardial smooth muscle layer of Nagayo (A and C, arrows) and subendocardial cells of Purkinje (B and C, double-headed arrows). CD146 is also expressed in vascular endothelial cells and perivascular cells (D, triple-headed arrow).

Figure 4 – Expression of SMM (A–C) and nestin (D) in samples of left ventricular papillary muscle. Positive expression of SMM is scarce in endocardial endothelial cells (A, arrowhead) and is also found in subendocardial cords (B, double-headed arrow), vascular channels (triple-headed arrow), as well as in the endocardial smooth muscle layer (C, arrow), which also expresses nestin (D, arrow). Endocardial endothelial cells expressing nestin are indicated in (D) (arrowhead).

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Figure 5 – Expression of markers in left ventricular papillary muscle (A–C) and left anterior ventricular wall (D) samples. Vascular smooth muscle cells (arrows) express α-SMA (A), nestin (B), SMM (C), and CD146 (D).

Figure 6 – Positive expression of neuron-specific enolase (NSE) in the posterior wall of left atrium is found in the endocardial smooth muscle layer (arrow) and, occasionally, in vascular smooth muscle cells (arrowhead).

 Discussion We indicated in the Introduction the study of Tomita et al. (2005), in which the authors observed CSP cells Different progenitor populations were described within characterized by expression of nestin; in differentiation the heart: cardiac stem cells (CSCs), epicardial-derived stages, expression of nestin was lost, and cells gained progenitor cells, cardiosphere-derived cells [13]. The neuronal, glial, smooth muscle or cardiomyocyte phenotypes differentiation of adult cardiac stem cells that is able of [3]. The authors then reasonably speculated that CSP renewing cardiac cells, reproduce the stages observed cells could have a neural crest origin. We brought here during the embryonic development [13]. It is known supportive evidence of an endocardial origin of nestin- that, during development, endocardial cells undergo an expressing progenitor cells that expresses smooth muscle endothelial–mesenchymal transformation and provides (α-SMA, SMM) and neural (NSE) markers. It appears that mesenchymal cells to adjacent tissue [14]. the endocardial muscle layer, poorly indicated in studies,

Subsets of telocytes: the progenitor cells in the human endocardial niche 773 is equally present in atrial and ventricular walls, and it transformation of endocardium [27]. In a simplistic way, should be viewed as a progenitor layer, which is weakly one should not discard the cell lineage sequence by which contractile [15] and has a positive expression of nestin from endocardial endothelial cells could derive vascular and smooth muscle markers (α-actin and myosin). endothelial cells, this hypothesis being reinforced by the Our attention was raised by the expression of the known expression of nestin in newly-formed vascular same markers in the endocardial endothelium and smooth endothelia [28–30]. muscle layer, and in vascular smooth muscle cells and A single study evaluated, in transmission electron pericytes. This indicates an anatomical similarity between: microscopy, the endocardial TCs, in mice [4]. It was then (a) the endocardial endothelium and the smooth muscle stated they are “the dominant cell population in sub- layer on its abluminal side and (b) the vascular endothe- endothelial layer of endocardium” [4]. Therefore, we can lium and the myoid layer coating it abluminally. These, assume that the population of spindle-shaped progenitor in turn, lead to two possible consequences. The first one cells, which populate the endocardial niche corresponds, is that perivascular/periendothelial cells, including those at least in part, to the endocardial TCs, this being supported in the cardiac wall, belong to the universal perivascular by previous evidence of expression of nestin and NSE stem niche of the adult [16–19]; therefore, this concept in TCs [12]. could be extended to include the endocardial stem niche. The second one is that the cardiac endothelium should be  Conclusions able of endothelial–mesenchymal transition, similar to Although it is actually accepted that the heart is not the vascular endothelium [20, 21]. In this regard, it is of at all a postmitotic organ, and that cardiac regeneration is utmost importance a recent study by Chen et al. (2016) driven by adult stem cells, the endocardial stem/progenitor who demonstrated in mice that endocardial cells undergo niche is still poorly explored. Specific studies could shed endothelial–mesenchymal transition to generate pericytes light on cardiac tissues, such as the endocardial endothelium, and vascular smooth muscle cells in the cardiac wall [22]. able to supply the heart with cardiac stem cells, being We have evidence that supports this novel mechanism of these cardiomyogenic or vasculogenic. generating cardiac progenitors in humans. Moreover, it Conflict of interests seems that the endocardium gives rise not only to peri- All authors declare that they have no conflict of vascular cells, but also to an individually and morpho- interests. logically variable endocardial smooth muscle layer with contractile properties, as proven by expression of myosin, References which could interfere with the normal cardiac functions. [1] Rusu MC, Jianu AM, Pop F, Hostiuc S, Leonardi R, Curcă GC. Further studies should test this last hypothesis. This Immunolocalization of 200 kDa neurofilaments in human cardiac hypothesis is supported by another recent study, published endothelial cells. Acta Histochem, 2012, 114(8):842–845. by Chabot et al. (2016), who demonstrated that nestin [2] Unno K, Jain M, Liao R. Cardiac side population cells: moving toward the center stage in cardiac regeneration. 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Corresponding author Mugurel Constantin Rusu, Professor, MD, PhD, Dr. Hab., Division of Anatomy, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy, 8 Eroilor Sanitari Avenue, 050474 Bucharest, Romania; Phone +40722–363 705, e-mail: [email protected]

Received: January 21, 2016

Accepted: August 31, 2016