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Telocytes in cardiac regeneration and repair

Telocytes (TCs) are a novel type of stromal cells reported by Popescu’s group as a case of serendipity in 2010. The unique morphological feature of TCs which distinguishes them from “classical” stromal cells is their extremely thin and long telopodes (Tps). Additionally, TCs are completely different from other stromal cells according to their distinctive immunophenotypes, gene expressions, proteomics, and microRNA profiles.

As evidenced by transmission electron microscopy and electron tomography, TCs were found to be widely distributed in almost all organs and tissues including heart. Recently, the focused ion beam scanning electron microscopy (FIB SEM)tomography further provides a three-dimensional(3D) reconstruction and a spatial view of human cardiac and skin TCs.

Based on their long and dichotomous Tps, TCs contribute to establish a 3D interstitial network and participate in the intercellular communication via homocellular and heterocellular junctions or shed vesicles. Meanwhile, TCs were indicated to be involved in tissue homeostasis, development, and immunosurveillance.

The number, distribution, and ultrastructural changes of TCs were also reported in many pathologies and diseases. Interestingly, increasing evidence suggests that TCs might play an important role in tissue regeneration and repair. TCs have their strategic location in the stem cell niches of different organs and tissues.

General aspects of telocytes

The distinct morphology of TCs is characterized with a small cell body (9.39 ± 3.26 m in diameter) and extremely thin (0.10 ± 0.05 m in thickness) and long Tps (up to 1000 m in length). The cell body contains a nucleus and a small amount of cytoplasm accommodating mitochondria, endoplasmic reticulum, Golgi complex, and cytoskeletal elements.

Tps have dichotomous and moniliform aspects, which are featured by alternate thin segments (podomers) and dilated segments (podoms). Moreover, multiple functional cell organelles including mitochondria and endoplasmic reticulum, as well as caveolae could be found at the level of dilations. Tps could form a labyrinthine network in the interstitial space, which indeed facilitates intercellular communication between TCs and other cells.

To date, the electron microscopy is still the unique golden standard method to identify TCs, which provides evidence for the widely distribution of TCs in almost all organs including heart. Despite the fact that no specific immunophenotype for TCs has yet been identified, the immunolabellings for TCs remain a useful tool to make semi-quantitative analysis for TCs.

It has been reported that the PDGFR- positive but CD34 negative TCs are located in the suburothelium, while the PDGFR- negative but CD34 positive ones are present in the submucosa and detrusor of human urinary bladder. Thus, TC immunophenotype could be varied in different types of organs and tissues.

Moreover, TCs could undergo immunophenotype changes as evidenced by a gradual gain of CD34 phenotype during cardiac development, which was however initially negative in embryo. Cellular junctions of telocytes with other types of cells With the advance oftransmission electron microscopy and electrontomography,ithas beenrevealedthat TCs, especially withtheir long and dichotomous Tps, can form both homocellular and heterocellular junctions with neighboring cells, vessels, and nerve endings.

Telocyte identification in the heart

Since the discovery of TCs in 2010, the scientists have been increasing their focus on the functional roles of TCs in the heart. TCs are widely distributed in the epicardium, myocardium, endocardium, and even cardiac valves, as evidenced by electron microscopy. TCs make a supportive interstitial network and act as important regulators in intercellular signaling with the surrounding cells (cardiomyocytes, stem/progenitor cells, endothelial cells, pericytes, fibroblasts, and immunoreactive cells), blood vessels, nerve endings, and extracellular matrix elements.

In adult hearts, TCs have CD34, c-kit, vimentin, It has recently been demonstrated that cardiac TCs also express CD34/PDGFR, although the latter was previously considered as a specific marker for TCs in gastrointestinal system. In primary culture, the TC proliferative ability and dynamics of Tp extension depend on the culture condition and matrix proteins. Cardiac TCs are easily to be distinguished from fibroblasts, pericytes, and bone mesenchymal stem cells (BMSC) due to differentimmunophenotypes.

The CD34/c-kit cardiac TCs have lower telomerase activity than BMSC, cardiac fibroblasts, and cardiomyocytes. Analyzed with isobaric tag for relative and absolute quantification (iTRAQ) and automated 2-D nano-ESI LC–MS/MS, the protein profiles of cardiac TCs were foundto bedifferentfromendothelial cells.Inaddition, miR-193 has been reported to be differentially expressed by TCs and other stromal cells.

Heterocellular communication of cardiac telocytes: cell basis for regenerative medicine

With the electron microscopy, the direct connections TCscardiomyocytes could be easily found within the heart and even in primary culture or engineered heart tissue. The dot contacts and small electron-dense nanostructures are common junctions between TCs and cardiomyocytes. The electron tomography revealed that the nanocontacts (10–15 nm) or even more complex and atypical junctions (e.g., molecular interactions) existed between TCs and cardiomyocytes.

In primary culture of TCs from neonatal rat cardiac tissues, TCs extend their Tps to the surrounding cardiomyocytes, which might contribute to the synchronous beating of cardiac cells. Moreover, the direct connections or shed vesicles between TCs and cardiomyocytes in the 3D engineered heart tissue strongly support the concept that TCs play a vital role in the architectural organization of the heart.

Telocytes in myocardial infarction and heart failure

Myocardial infarction (MI) is a major cause of death and disability worldwide. The ischemic myocardium suffers a loss ofcardiomyocytes via necrosis and apoptosis, and undergoes neoangiogenesis and fibrotic changes, leading to pathological cardiac remodeling and probably end-stage heart failure.

In a rat experimental myocardial infarction model, TCs were found to be significantly increased in the border zone of infarction in the angiogenesisprocess (30days afterMI) comparedtonormalmyocardium . Based on transmission electron microscopy, the direct nanocontacts, as well as the shed vesicles, were detected between TCs and the abluminal face of endothelium of neo-formed or preexisting capillaries.

Moreover, cardiac TCs secret VEGF and NOS2, and express angiogenic microRNAs,further suggesting a role of cardiac TCs in angiogenesis process after MI. As evidenced by immunofluorescent staining and semiquantification of TCs, cardiac TCs were decreased and undetectable in the infarct zone at 4 days after MI.

Telocytes in cardiac development and aging

Itis generally accepted thatthe cardiac epicardium-derived cells (EPDCs) play important roles in cardiac development, which could differentiate into smooth muscle cells, fibroblasts, and endothelial cells, and also participate in the form of subepicardial mesenchyme, blood vessels, atrioventricular valves, and purkinjefibers. Being considered as a subpopulation of EPDCs, TCs were found to be constantly present in the embryonic, newborn, and adult mice hearts. With sustained immunoreactivity to vimentin, cardiac TCs were initially CD34 negative (Embryonic 14), but gradually CD34 positive from late embryonic (Embryonic 17) to adult life.

On the other hand,the c-kitimmunoreactivity of cardiac TCs decreased along with cell differentiation. As evidenced by transmission electron microscopy, TCs were widely distributed in the subepicardial layer, embracing the surrounding growing cardiomyocytes and endothelial cells via Tps or interacting with these cells via releasing exosomes.

Telocyte-derived exosomes in cardiac regeneration and repair

Transmission electron microscopy and electron tomography provide direct evidence that cardiac TCs can secret extracellular vesicles, which serve as important transporters for biological signalings between cells. Cardiac TCs release three types of extracellular vesicles: exosomes (45 ± 8 nm), ectosomes (128 ± 28 nm), and multivesicular cargos (1 ± 0.4 m), depending on their mechanisms of biogenesis and secretion.

Extracellular vesicles can carry a variety of molecules, including lipids, proteins, DNA, mRNA, and non-coding RNA, thus also acting as vehicles for genetic information exchanges between cells. Growing evidence suggests that exosomes play an important role in the cross talk of different intramyocardial cells, contributing to cardiac physiology and responses to injury.

Interestingly, intramyocardial delivery of ESC-, MSC-, iPSC-, or CPC-derived exosomes were associated with reduced myocardial apoptosis and fibrosis, enhanced neovascularization, and improved cardiac function after experimental MI. The enriched exosomal angiogenic or anti-apoptotic microRNAs (e.g., miR-21, miR-210, miR-132, and miR-146a-3p) were thought to be responsible for the cardioprotective effects of exosomes.

Future directions

Increasing evidence shows the vital roles of TCs in cardiac homeostasis and adaptive responses of the heart to injury. Importantly, TCs are considered as critical contributors to cardiac regeneration and repair. However, direct evidence of the functional impact of cardiac TCs on cardiomyocytes and non-myocytes (e.g., endothelial cells, fibroblasts, and stem/progenitor cells) is still lacking.

Meanwhile, the intercellular signaling and communication mechanism between TCs and other cells, either via direct contacts or shed vesicles, remains a topic for further investigation. Also, it will be of greatinterestto clarify the role of TCs in exercise-induced physiological cardiac growth as well as in aging-related myocar- dial dysfunction. Overall, TC-based therapy or TC-derived exosome delivery might be novel strategies to promote cardiac regeneration and repair.

Author: Yihua Bei, Qiulian Zhou, Qi Sun, Junjie Xiao

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