The dynamic transcriptional programs unfolding from midgestion (E9.5) to adulthood (P21) were studied using spatiotemporal RNA-seq analyses of single cells isolated from mouse hearts and captured using microfluidics, to delineate lineage specific transcriptional regulation, including CM maturation, during heart development (143). Classification of different cell types at the single cell level with spatiotemporal resolution allowed the definition of time and chamber lineage-specific gene programs underlying normal cardiac development. Further post-natal maturation occurred by P21 with downregulation of calmodulin-interacting proteins, Bex1 and Bex4, previously shown to promote muscle regeneration (144). Albeit, one should consider the potential bias introduced by size limits inherent to the microfluidics system used to select large mature CMs, these datasets are useful to define precise developmental stages of human and mouse embryonic stem cells-derived CMs, prompting the utility for characterization of induced pluripotent stem cells-derived CMs that are becoming the gold standard in cardiac drug discovery (27, 145). Notably, analysis of single cells from Nkx2.5+/<sup>−</sup> murine hearts were used as a model for congenital heart disease and allowed to define lineage-specific maturation defects, including expected changes in the CM lineage but also in the endothelial lineage compartment. This supports an instructive role of CMs on endocardium development and predicting underlying mechanisms associated with human heart malformations.

The experiments herein described prove the advantage of using single cell transcriptomics in enabling the study of cellular states at the moment when fate decisions are executed, contributing to the precise deconvolution of lineage decisions previously masked by bulk analysis and, especially in early stages, difficult to study given the paucity of cells available.

# Adult Heart and Cardiovascular Disease Models From a Single Cell Perspective

Compared to other areas of research—until very recently—the cardiac field has infrequently taken advantage of single cell technologies to study adult heart cells' gene expression and, even less, for epigenetic studies. Although, cells of the adult heart are not readily accessible, a number of protocols have been historically described to dissociate single CMs and single stromal cells. If some skepticism can be raised by the fact that isolation protocols are lengthy and potential side effects on gene expression are a deterrent, the use of whole tissue as comparison and validation experiments obviates any doubt. The only technical impediment remains the size of adult mature CMs (∼150µm in length) that limits the use of microfluidic apparatuses with upper size limits usually around 50µM (except for ICELL8).

Albeit the regenerative capacity of the adult heart is meager, there is some evidence that induction of CM proliferation could be a targetable mechanism to induce myocardial selfrenewal and repair after injury (146, 147). The proliferative potential, especially the capacity to undergo cytokinesis of CMs in physiological conditions as well as in response to stress, remains unclear. Confounding factors could be the methods of analysis but, most importantly, also the absence or presence of external stimuli that can enhance CMs proliferation and self-renewal (146, 148–150). Recently, to understand the proliferative capacity of CMs, single nuclear RNA-seq was performed in single CMs from healthy and diseased hearts, including pressure overload failing murine hearts and human dilated cardiomyopathy failing hearts (151). Heterogeneity of the myocardial environment in response to stress was uncovered (151). Through weighted gene co-expression network analysis, several subgroups of CMs were characterized, including ones with a gene signature indicative of dedifferentiation and cell cycle entry. Further, two long intergenic non-coding RNAs, Gas5 and Sghrt, were found in highly interconnected nodal hubs of gene regulatory networks and, subsequently, confirmed as essential regulator of CMs' cell cycle entry and de-differentiation programmes (151). These data are a first and encouraging step toward answering additional fundamental questions: the functional implications of specific gene expression patterns in the various CMs subtypes; which subtypes are elicited or depressed in diverse pathological conditions, including early stages of dilated cardiomyopathies, hypertrophic cardiomyopathies and ischemic disease; the physical localization of CMs subpopulations and how the cellular demographics change in response to stress; finally the concasual relationships with neighboring non-CMs.

A complementary study focused on the cardiac stromal compartment and used a 10X Genomics platform to capture and sequence just over 10,000 non-myocyte cells (26). With a mean of 1,900 genes detected/cell, an expected overall complexity in the composition of the cardiac non-myocyte cellular compartment was revealed and a few additional markers and subpopulations were identified in the fibroblasts compartment and in the lymphocytes and myeloid lineages (26). Interestingly, clustering patterns for female and male cells were largely overlapping but within individual cell types, small levels of sexual dimorphism was detected for selected genes' expression, implying a diverse predisposition of the cardiac stroma response to stress (26).

A further level of complexity was obtained by comparing scRNA-seq results from healthy vs. post-ischemia mouse hearts using the SORT-seq protocol, which is essentially an integration of FACS for single cell capturing and highthroughput multiplexed linear amplification by CEL-seq2 (29, 48). In this study, cell capturing has two limitations: (1) large mature CMs have dimensions above the size of the sorting nozzle, (2) the utilized flow cytometry gates were biased toward large live cells (29). Thus, the representation of the cell types was biased, with 71% of CMs vs. the 30% expected, plus a putative selection of myocytes subtypes due the upper size limits of the sorter outlet (29). Nevertheless, all main cardiac cell types were recognized as seen using differential gene expression analysis and clustering methods. In addition, identification of new putative markers including ones for specific subpopulations were pinpointed. Amongst these, Ckap4 was defined as a novel marker for activated fibroblasts post-ischemia in mouse, and subsequently confirmed as a marker in human ischemic hearts. CKAP4 function remains to be clarified, although knock-down experiments suggested it controls the expression of activatedfibroblasts genes. Interestingly, in single CMs' sequencing, up to 84% of reads were coming from mitochondrial genes (29). This is surely in part dependent on the high enrichment of mitochondria in CMs; however, while enrichment of mitochondrial related gene categories are amongst the features characteristic for low quality cells in a number of independent cellular models, this is not the case for myocytes (93). Thus, it could be necessary to establish a specific set of features to determine the quality of single heart myocytes based on their gene expression.

Single cell transcriptomics analysis was used to characterize the changes occurring at the single cell level in a mouse model of cardiac fibrosis (28). Co-expression analysis inherent in single cell transcriptomics studies allowed to highlight that both IL-11 and its receptor IL11RA are expressed in activated fibroblasts, the latter defined by a transcriptional profile typical of TGFβ stimulated cells and by induction of extracellular matrix encoding genes (28).

Tissue regeneration using exogenous products, including iPS-CMs, is a highly sought-after therapeutic approach but much remains to be understood regarding the level of differentiation, purity, genomic, and phenotypic stability of induced CMs especially because, until recently, gene expression studies were almost exclusively performed on bulk populations. Reprogramming fibroblasts into induced cardiomyocytes (iCMs) using combinations of cardiogenic transcription factors (152– 154) offers an interesting alternative to the use of iPS-CMs to generate newly formed CMs in vitro and then used as cell therapy, but also for in vivo direct reprograming. To study and characterize the non-conventional differentiation mechanisms driving cell conversion to iCMs, single cell approaches are a suitable option allowing to unpick the heterogeneity of conversion events and the level of differentiation in single cells. Single cell transcriptomics analysis of the early stages during the reprogramming of mouse fibroblasts into iCMs uncovered a heterogeneous group of cells differentiating at unsynchronized pace (155). The definition of different cell clusters based on dimensionality reduction and unsupervised clustering algorithms was the critical step to make functional inferences. Some of these were anticipated, like the reconstruction of differentiation trajectories, the correlation between the expression of each reprogramming factor and the progress of individual cells through the reprogramming process, and the discovery of new surface markers for iCMs (155). Among other discoveries, the downregulation of factors involved in mRNA processing and splicing was found to be critical in reprogramming; interestingly, a specific splicing factor, Ptbp1, was identified and subsequently functionally validated to be a critical barrier for the acquisition of CM-specific splicing patterns in fibroblasts (155). Thus, in this settings scRNA-seq enabled the identification of targets to enhance reprogramming efficiency and new markers for the prospective isolation of iCMs but also demonstrated the immense utility of these methods in studying further models of programming and reprogramming.

An in vitro system was used to study the dedifferentiation potential of adult CMs to cardiac progenitor-like cells, using a genetic cell fate mapping system plus single cells transcriptome and further methylome analysis (156, 157). The authors reported a correlation between hyper-methylation of promotor regions and suppression of cardiac specific genes related with maturation, while cell cycle and stemness genes were upregulated suggesting that CMs' de-differentiation depends on epigenomic regulation of the cells transformed into progenitors. Nevertheless, this study relies on very few single cells and confirmation of the specific gene expression regulation via epigenetic modulation would benefit from analysis of the transcriptome and methylome from the same single cell.

Within the cardiac regenerative medicine space, c-kit+ adult cardiac progenitors have been proposed as a cell product albeit controversial data were reported (158). Recently, single freshly isolated adult c-kit+ CPCs were compared with short-term expanded cultures by 10X Genomics and SmartSeq2, in parallel with the meta-analysis of multiple scRNA-Seq datasets, finding substantial transcriptome alterations in the in vitro expanded cells (159). This increase in the transcriptome diversity included the induction of thousands of genes related to cell cycle and metabolism, and loss of expression of identity genes, suggesting a marked change in functional characteristics of in vitro expanded cells, with important implications to consider when developing cell therapies than involve previous in vitro expansion.

Altogether, these initial explorative studies both in healthy and disease hearts demonstrate how single cell transcriptomics alone has already contributed to uncover novel cell types, further our understating of candidate cell products for cardiac cell therapies and most importantly discover novel functional pathways regulating heart function.

# Predicting Achievable Outcomes and Influencing Future Clinical Applications

Thanks to the immense technological advancements of highthroughput single cell gene expression analysis and the highresolution achieved with scRNA-seq, we can now obtain information on prevalence, heterogeneity and co-expression at the individual cell level for virtually the entire transcriptome. This enables us to get a new perspective on cellular demographics of the heart and improve intracellular and intercellular pathways definition, otherwise unthinkable with bulk analysis and in circumstances where cellular material available is limited. The almost immensurable amount of data being produced requires tight integration and interaction of biological and clinical experts with computational biologists starting from the initial experimental design. In fact, a dynamic and continued assessment of data generation and results is necessary to reach solid conclusions of any single cell 'omic study.

The complete exploitation of the value of single cell transcriptomic results comes from the integration and validation of these data by orthogonal methods. Validation of the expression of subsets of genes can be performed by standard techniques with emphasis on single cell resolution approaches ranging from gene expression validation with or without spatial resolution (e.g., single molecule in situ hybridization, single cell qRT-PCR, respectively), to testing the expression of the proteins encoded by the genes of interest, by flow cytometry that allows a higher bandwidth for multiplexing or immunostaining to acquire localization information. Of note, spatial transcriptomics was developed in order to retain spatial information related to the transcriptome of isolated single cells and was elected Method of the Year 2017 by Nature Methods (160). The protocol requires tissue sections that are then divided in a mosaic of tiles of ∼100µm of diameter, and uses spatial barcodes permitting the localization of specific cells and their transcripts by overlaying haematoxylin & eosin stained sections onto microarrays spotted with the barcoded topographies (132, 161). The first reported application of single cell RNA-seq and spatial transcriptomics was to identify in vivo genes relevant to endocardial epithelialto-mesenchymal transformation (162). More recently, spatial resolution proved to be essential in the identification of SOX9 as a key regulator of cardiac fibrosis in a mouse model of ischemic injury and injured human heart (163). Critically, rather than just allowing to localize the cell types identified by scRNAseq, spatial transcriptomics will expedite the reconstruction of the dynamic architecture of the heart (134). Indeed, changes of gene expression and especially of transcriptomic profiles can depend on the reciprocal interactions with adjacent cells, the migration to a specific tissue site of cells' subsets and by locationspecific variation of cell "states." Thus, starting from a single cells and functional micro-niches of few cells, one can envision to reconstruct the changes occurring in the cellular landscape of whole tissues and organs in 3D during specific processes including development, aging, and disease (134).

Despite the recent explorative studies, the cellular cardiac landscape, especially human, remains largely unknown (**Figure 4**). Most significantly, the integration of single cell transcriptomic and spatial transcriptomic provides the platform to reconstruct the cellular demographics of the heart with spatial resolution. For smaller animals, this will require a shorter period of time, and although the reconstruction of the entire human cardiac cellular landscape in 3D will require many iterations, every step will provide new strategic information. For example, cardiac muscle scRNA-seq will alone contribute to the clarification of CMs' heterogeneities within microniches, on the epicardial vs. endocardial fronts; as well as chamber specificities that remain largely unknown. Notably, one of the therapeutic targets for cardiac regeneration is CMs' proliferation, but for clinical translation we still need to: (1) clarify the true endogenous and spontaneous capacity of human CMs to re-enter cell cycle and undergo mitosis since results in mouse remain controversial and (2) define the stimuli that can trigger or improve their proliferation. Importantly, transcriptomic analysis of single mononucleated vs. binucleated CMs and comparison with single nuclei transcriptomics will provide inferences on their proliferative and differentiation status (146, 148–151, 164).

The identity and role of cardiac progenitors in the adult heart remains elusive with a dozen different identifiers already described, and their relationship and function still unclear (136). It is expected that an unbiased 'omic approach will allow to define their molecular signatures and likely deconvolute their ontogeny and function in the healthy and diseased heart.

The heterogeneity of vascular cells across organs and within the same organ or system has been hypothesized for a long time but the advancements in uncovering of subpopulations with distinct functions has been slow because of bulk analysis and largely limited to hypothesis driven studies that can focus on one gene/pathway at the time. The availability of single cell transcriptomics and spatial transcriptomics is providing the right tools to study endothelial cells, smooth muscle cells and pericytes' subtypes and functional changes across vessels of different diameter and function. A recent study proved feasibility and produced outstanding results by sequencing the vascular cells of the mouse brain (165) uncovering the transcriptional basis of the gradual phenotypic change along the arteriovenous axis and revealing unpredicted differences in the cells' molecular signatures with a seamless continuum for endothelial cells vs. a punctuated pattern for mural ones (165). Importantly, brain specific features were highlighted for pericytes in comparison to the lung. These important data provide the evidence for a higher organizational structure of the vasculature, providing a healthy reference for subsequent disease studies, but also provides an invaluable hypothesis-generating data source.

Although initial scRNA-seq studies in the adult heart have already contributed to highlighting the role of specific genes in the fibrotic response after TGFβ1 stimuli (28) and ischemic injury (29), much remains to be learnt about the identity of fibroblasts, their function, heterogeneity, anatomical specificity and role in cardiac homeostasis and disease.

In recent years, a number of studies have investigated the adaptive and innate immune response following cardiac injury beyond the response to microbial causes, how these affect repair and regeneration and their potential manipulation as therapeutic targets [as reviewed in Prabhu and Frangogiannis (166), Sattler et al. (167), Toldo and Abbate (168), and Wysoczynski et al. (169)]. Single cell transcriptomics focusing on immune cells will provide an invaluable support to decode higher organizational networks triggered in cardiac disease and in physiological conditions. Finally, single cell transcriptomics will also contribute to our understanding on the conductive system of the heart, both in unbiased studies and targeted analysis.

Studying the developing heart at the single cell level has an inherent huge power, not only to further our understanding of the perfect orchestration needed for cells to go from a primordial linear tube to a four-chamber heart and uncover maps and trajectories of cellular differentiation but also, importantly, to discover how lineage-specific gene programs are altered in congenital heart disease (24, 27, 43, 51, 138). This implies a need to study and build a developing atlas of the healthy developing heart in animal models but most importantly in human, and use this as reference to study and understand the genesis of cardiac malformations within single cells, and between them.

Two studies have generated independent atlases for mouse models, providing an initial compendium of single cell transcriptome profiling representing a new resource for cell biology (170, 171). Providing gene expression data from 20 anatomical locations, the atlases allow the comparison across tissues of cell types present in every tissue such as immune cells and endothelial cells enabling hypothesis generation. Strikingly the Human Cell Atlas (HCA) initiative has triggered widespread excitement with its ambitious goals aiming to describe and define the complete cellular landscape of the human body (https://www.humancellatlas.org/) (172, 173). This will be achieved by mapping cells of the human body using a

## REFERENCES

combination of single cell and spatial transcriptomics, resulting in datasets that go beyond descriptive features and will guide functional studies toward the identification of previously unpredictable subtleties of disease. Effectively, the HCA plans to integrate leading-edge technologies to: (1) defining the histological and anatomical position of newly identified cell types/states; (2) outlining lineage decisions with topographic and functional insights; (3) recapitulating precise and specific cell-to-cell interactions including autocrine and paracrine pathways.

Urgent clinical questions, that would immediately benefit from an integration of single cell transcriptome studies and spatially resolved gene expression profiling, include the definition of the changes occurring within the cellular landscape of the border zone following myocardial ischemia; the deconvolution of atherosclerotic plaque's single cell transcriptomic profiles. Inferences in ethiopathogenetic mechanisms that are invisible using standard low resolution and bulk techniques include proliferation, differentiation, survival as well as ligand/receptor pairs to infer functional networks. Additionally, transcriptomic profiling of single cells will undoubtedly enable the definition of novel biomarkers, including parameters such as frequency and pattern of gene expression within tissues and/or specific cell types.

Altogether, these new technologies will not only contribute to address hypothesis-driven questions but, most importantly, will broaden our perspective, providing hypothesis generating platforms to further our understanding on heart function in health and disease. The auspicable future of cardiac disease monitoring is based on low-risk, minimally invasive and disruptive approaches, where multi-'omics single cell approaches will play a leading role.

# AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

# ACKNOWLEDGMENTS

This study was supported by British Heart Foundation grants (BHF PG/16/47/32156 and RG/15/1/31165) and a BHF studentship to SS (BHF FS/14/62/31288).

as a new modulator of fibroblasts activation. Circulation (2018). 138:166–80. doi: 10.1161/CIRCULATIONAHA.117.030742

reveals alternative tuft cell origins in the gut. Cell Syst. (2018) 6:37–51 e9. doi: 10.1016/j.cels.2017.10.012

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling Editor declared a shared affiliation, though no other collaboration, with several of the authors MN, AM, PC, SS at the time of the review.

Copyright © 2018 Massaia, Chaves, Samari, Miragaia, Meyer, Teichmann and Noseda. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Extracellular Vesicles Secreted by Hypoxic AC10 Cardiomyocytes Modulate Fibroblast Cell Motility

Imelda Ontoria-Oviedo1,2†, Akaitz Dorronsoro1,2†, Rafael Sánchez 1,2, Maria Ciria1,2 , Marta Gómez-Ferrer 1,2, Marc Buigues <sup>1</sup> , Elena Grueso1,2, Sandra Tejedor 1,2 , Francisco García-García<sup>3</sup> , Hernán González-King1,2, Nahuel A. Garcia<sup>1</sup> , Esteban Peiró-Molina<sup>1</sup> and Pilar Sepúlveda1,2 \*

<sup>1</sup> Regenerative Medicine and Heart Transplantation Unit, Instituto de Investigación Sanitaria La Fe, Valencia, Spain, <sup>2</sup> Associated Unit for Cardiovascular Repair, Instituto de Investigación Sanitaria La Fe-Centro de Investigación Príncipe Felipe, Valencia, Spain, <sup>3</sup> Bioinformatics and Biostatistics Unit, Centro de Investigación Principe Felipe, Valencia, Spain

#### Edited by:

Gabor Foldes, Imperial College London, United Kingdom

#### Reviewed by:

Zoltán Giricz, Semmelweis University, Hungary Lucio Barile, Cardiocentro Ticino, Switzerland

#### \*Correspondence:

Pilar Sepúlveda<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Cardiovascular Biologics and Regenerative Medicine, a section of the journal Frontiers in Cardiovascular Medicine

> Received: 01 June 2018 Accepted: 05 October 2018 Published: 25 October 2018

#### Citation:

Ontoria-Oviedo I, Dorronsoro A, Sánchez R, Ciria M, Gómez-Ferrer M, Buigues M, Grueso E, Tejedor S, García-García F, González-King H, Garcia NA, Peiró-Molina E and Sepúlveda P (2018) Extracellular Vesicles Secreted by Hypoxic AC10 Cardiomyocytes Modulate Fibroblast Cell Motility. Front. Cardiovasc. Med. 5:152. doi: 10.3389/fcvm.2018.00152 Extracellular vesicles (EVs) are small membrane vesicles secreted by most cell types with important roles in cell-to-cell communication. To assess their relevance in the context of heart ischemia, EVs isolated from the AC10 ventricular cardiomyocyte cell line (CM-EVs), exposed to normoxia (Nx) or hypoxia (Hx), were incubated with fibroblasts (Fb) and endothelial cells (EC). CM-EVs were studied using electron microscopy, nanoparticle tracking analysis (NTA), western blotting and proteomic analysis. Results showed that EVs had a strong preference to be internalized by EC over fibroblasts, suggesting an active exosome-based communication mechanism between CM and EC in the heart. In Matrigel tube-formation assays, Hx CM-EVs were inferior to Nx CM-EVs in angiogenesis. By contrast, in a wound-healing assay, wound closure was faster in fibroblasts treated with Hx CM-EVs than with Nx CM-EVs, supporting a pro-fibrotic effect of Hx CM-EVs. Overall, these observations were consistent with the different protein cargoes detected by proteomic analysis under Nx and Hx conditions and the biological pathways identified. The paracrine crosstalk between CM-EVs, Fb, and EC in different physiological conditions could account for the contribution of CM-EVs to cardiac remodeling after an ischemic insult.

Keywords: cardiomyocytes, fibroblasts, endothelial cells, extracellular vesicles, hypoxia, cellular communication

# INTRODUCTION

Cardiac muscle is a highly adaptable tissue that responds to physiological challenges, in part, by releasing paracrine factors including extracellular vesicles (EVs) (1). Exosomes are small nanomembranous extracellular vesicles (30–150 nm in diameter) that contain a variety of molecules including cytokines, membrane trafficking molecules, chemokines, heat shock proteins, and also several types of RNA molecules. Due to their diverse cargo, exosomes play a central role in cell-to-cell communication and we and others have previously examined the role of cardiomyocyte-derived exosomes (CM-Exo) in different stress conditions, such as glucose starvation (2), inflammation (3, 4) or ethanol treatment (5). In response to ischemia, different cardiac cell types, including cardiomyocytes, release high amounts of exosomes and other types of EVs with different cargoes (6, 7). To characterize the changes in CM-EV cargo during ischemia, we performed a comparative proteomic analysis of EVs isolated from cardiomyocytes cultured in normoxia (Nx) or hypoxia (Hx), and mapped the biological processes activated in both conditions. We found that Hx increased the secretion of CM-EVs containing proteins related to wound healing. Consistent with this, we observed that Hx CM-EVs stimulated fibroblast motility. Our results shed more light on the contribution of CM-EVs to cardiac remodeling.

# MATERIALS AND METHODS

All procedures were approved by national and local ethical committees (reference number 2016/0192).

# Cell Culture

The human ventricular cardiomyocyte cell line AC10 (8) was cultured in Dulbecco's Modified Eagle's Medium-F-12 (DMEM-F12, Gibco-Invitrogen <sup>R</sup> ) supplemented with 10% fetal bovine serum (FBS, Gibco-Invitrogen <sup>R</sup> ) and 1% penicillin/streptomycin (P/S, Millipore). Primary cultures of human umbilical cord vein endothelial cells (HUVEC) were obtained from Lonza and were grown in Endothelial Cell Growth Medium-2 (EGM-2) BulletKitTM (Lonza). Human coronary microvasculature (HCAEC; ATCC) endothelial cells were grown in Vascular Cell Basal Medium supplemented with the Endothelial Cell Growth Kit-VEGF (ATCC). Fibroblasts were obtained from skin biopsies after informed consent and were cultured in high-glucose DMEM (Thermo Fisher Scientific) supplemented with 10% FBS and 1% P/S. Cell were maintained under control conditions (Nx) in a humidified atmosphere at 37◦C containing 5% CO2. AC10 cells cultured in Hx were incubated at 2% O<sup>2</sup> for 48 h with glucose deprivation during the first 4 h and DMEM/F12 with depleted FBS during the next 44 h at 2% O2. Depleted FBS was generated by ultracentrifugation of an FBS and DMEM/F12 (1:1) mix at 100,000 g for 16 h. The induction of Hx in cell cultures was monitored by stabilization of hypoxia inducible factor-1 alpha (HIF-1α) expression and cell viability reduction (**Figure S1**).

# Extracellular Vesicle Purification

We used ultracentrifugation without sucrose-gradient centrifugation step to isolate EVs from cardiomyocytes. Accordingly, we refer to the isolated pool of vesicles as CM-EVs and not CM-exosomes. Approximately 150 mL of culture media was collected and EVs were isolated by several ultracentrifugation steps as described (9). Briefly, supernatants were centrifuged first at 2,000 g for 20 min (Eppendorf 5804 benchtop centrifuge, A-4-62 rotor), 10,000 g for 70 min (Hitachi CP100NX centrifuge, Beckman Coulter 50.2 Ti rotor) and subsequently filtered manually through a 0.22µm filter to eliminate cell debris using a syringe. Then, EVs were pelleted by ultracentrifugation at 110,000 g for 120 min (Hitachi CP100NX centrifuge, Beckman Coulter 50.2 Ti rotor), filtered through a 0.22µm filter to maintain sterility and ultracentrifuged again at 110,000 g for 120 min (Hitachi CP100NX centrifuge, Beckman Coulter 50.2 Ti rotor). The manipulation of EV extracts was performed in a laminar flow hood to preserve sterility. EV protein concentration was determined with the Pierce BCA Protein Assay Kit (ThermoFisher Scientific) to ensure equal amounts of protein samples. EVs were suspended in RIPA buffer [1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate in Tris-buffered saline (TBS), Sigma-Aldrich] for western blotting or phosphate buffered saline (PBS) for nanoparticle tracking, electron microscopy, flow cytometry, proteomic and functional analysis.

# Extracellualar Vesicle Incorporation Experiments

Capture of labeled EVs by fibroblasts and EC was performed using procedures modified from previous reports (10). EVs were fluorescently stained with carboxyfluorescein diacetate succinimidyl diester (CFSE; 5µM) (ThermoFisher Scientific) for 15 min at 37◦C, and unincorporated dye was removed via ultracentrifugation. As a negative control to normalize, the same amount of PBS was added to CFSE to monitor unincorporated dye carried over after the staining steps (PBS control). Cells were seeded at 50% confluency in 24-well plates and incubated with CFSE-labeled EVs at 2µg/mL (5.00 E + 08 particles/mL). After 3 h of incubation, cells were washed twice in cold PBS, trypsinized and analyzed by flow cytometry using a FACS Canto II at the Cytomics Unit of the Instituto de Investigación Sanitaria, La Fe.

# Western Blot Analysis

EVs or cells were lysed in 100 µL of RIPA buffer containing protease (Complete, Sigma-Aldrich) and phosphatase (PhosSTOP, Sigma-Aldrich) inhibitors. Equal amounts of protein samples were suspended in non-reducing Laemmli sample buffer (BioRad) and denatured at 100◦C for 5 min. Proteins were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore). Membranes were blocked with TBS containing 5% (w/v) nonfat dry milk powder with 0.1% Tween-20. Antibodies used were anti-CD9 (Ab 92726, Abcam), anti-TGS 101 (Santa Cruz. Sc-7964), anti-Alix (Santa Cruz. Sc-53538) and HIF-1α (610958, BD biosciences). Detection was carried out using peroxidase-conjugated secondary antibodies with the ECL Plus Reagent (Amersham, GE Healthcare, Munich, Germany). Proteins were visualized using an Amershan Imager 600 (GE Healthcare) and quantified with ImageJ software (NIH).

# Nanoparticle Tracking Analysis

EV size distribution and quantification of vesicles was analyzed by Nanoparticle tracking analysis (NTA) using a NanoSight NS3000 System (Malvern Instruments, UK) Samples were suspended in 0.22µm pre-filtered PBS and dilutions between 1:1,000 and 1:10,000 were used to achieve a particle count between 2 × 10<sup>8</sup> and 2 × 10<sup>9</sup> per mL. Measurement of the diameter was performed on 3 independent experiments and showed as mode ± standard deviation.

# Electron Microscopy

Electron microscopy was performed as described previously (11). Briefly, isolated EVs were diluted in PBS, loaded onto Formwar carbon-coated grids, contrasted with 2% uranyl acetate and finally examined with a FEI Tecnai G2 Spirit transmission electron microscope. Images were acquired using a Morada CCD Camera (Olympus Soft Image Solutions GmbH). The size of the EVs was quantified using ImageJ. The diameter of a minimum of 25 EVs in different fields was measured in 3 independent experiments and the median ± standard deviation showed.

# Cell Proliferation

To test whether purified EVs from AC10 cells grown under Nx and Hx conditions altered cell viability, AC10 cells, fibroblasts and HUVEC were cultured at a density of 5 × 10<sup>3</sup> per well of a 96-well plate. Cells were incubated for 48 h with 30µg/mL (7.00 E + 09 particles/mL) of purified EVs and the Cell Counting Kit-8 (CCK-8) assay was used to measure proliferation following the manufacturer's instructions. The optical density of the cultures was measured at 450 nm in each well 4 h after incubation with the CCK-8 assay solution.

# Tube Formation Assay

Capillary-like tube formation, as a readout of angiogenesis, was measured using the tube formation assay as previously described (12). In total, 1.2 × 10 <sup>4</sup> HUVEC were seeded per well into 96-well plates precoated with 50 µl of growth factor-reduced Matrigel (BD Biosciences). Cells were incubated for 6 h with 30µg/mL (7.00 E + 09 particles/mL) of purified EVs from Nx and Hx conditions to evaluate formation of tube-like structures. The day after, images from three different viewing fields per sample were taken using an inverted microscope (Leica DM6000) with a 10× magnification. Images were analyzed with WimTube online software (WimTube: Tube Formation Assay Image Analysis Solution. Release 4.0. https://www.wimasis.com/en/WimTube).

# In vitro Scratch Assay

The scratch assay is a good and valid method to measure cell migration in vitro (13). Human fibroblasts were seeded in 12 well culture plates at a density of 1.2 × 10<sup>5</sup> cells/well. The day after, a straight line was created on the monolayer with a pipette tip. Cells were washed once with PBS to remove the debris and treatments were added in 1 mL of corresponding medium. In total, 30µg/mL (7.00 E + 09 particles/mL) of EVs obtained from AC10 cells cultured in Nx and Hx conditions were added to the cells. The capacity of the cells to migrate and invade the denuded area was tracked using time-lapse microscopy during 24 h. Image analysis was performed using ImageJ software.

# Proteomic Analysis

Proteomic analysis was carried out as described previously (11). Briefly, CM-EVs were harvested from 75 mL of culture medium, suspended in RIPA buffer, and protein concentration was determined using the Qubit <sup>R</sup> Protein Assay Kit (Invitrogen). A total of 30 µg of protein extract in Laemmli buffer was subjected to 12.5% acrylamide 1D SDS-PAGE electrophoresis and proteins were digested with trypsin. Digestion was stopped with 1% trifluoroacetic acid and 5 µL of each sample was loaded for liquid chromatography and tandem mass spectrometry (LC-MS/MS). Data were analyzed following the ProteinPilot default parameters (ProteinPilot v4.5. search engine, ABSciex). Proteins were arranged by the Unused Protein Score, which is a measure of the protein confidence calculated from the peptide confidence for peptides from spectra.

Functional enrichment analysis (14, 15) was performed to detect associations of Gene Ontology (GO) terms (16) for each experimental group. This functional characterization method allowed us to determine whether a set of functions was over-represented in a subset of proteins. Significant biological processes were summarized and represented by TreeMaps using the REVIGO webtool (17), which enabled the comparison of results between groups and is especially useful in revealing functional patterns.

# Statistical Analysis

Data are represented as mean ± standard error (SE) unless otherwise is specified. Comparisons between experimental conditions were performed with Student's unpaired t-test. Analyses were conducted with GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA). Differences were considered statistically significant at p < 0.05 with a 95% confidence interval.

# RESULTS

Analysis of CM-EVs by western blotting for the assessment of the common exosomal markers CD9, TSG101, and ALIX (**Figure 1B**), showed no difference between EVs isolated from Nx or Hx conditions. In addition morphological analysis (**Figure 1A**) and size measurement by electron microscopy showed no significant neither difference (Nx: 102.9 ± 19.81µm vs. Hx: 88.49 ± 24.35µm). Further characterization using the NanoSight instrument revealed that the majority of EVs under both conditions were ∼150 nm in size (Nx: 147.9 ± 14µm vs. Hx: 136.75 ± 13.78µm; **Figure 1C**). Total protein analysis of the EV extracts showed that Hx significantly increased the secretion of CM-EVs (**Figure 1D**), as previously described (7). To functionally characterize the protein content of CM-EVs after Nx or Hx, we used LC-MS/MS. Protein contaminants from the cell culture medium (serum albumin and keratin) were removed from the analysis. Although an equivalent amount of total protein was used (30 µg), a broader range of proteins was observed in EVs recovered from Hx cultures (**Tables S1**, **S2**): 55 proteins were identified in Nx and 99 in Hx conditions. Among the identified proteins, we found abundant extracellular matrix (ECM) proteins including collagens and laminins, and also integrins, tetraspanins, ribosomal proteins and cardiac-specific proteins. Of note, multiple relevant proteins were found only under Hx conditions, including cardiogenic and cardioprotective proteins (Dickkopf-related protein 1, neuropilin 1 and netrin 4) and proteins related to cellular stress (ATP-citrate synthase, fatty acid synthase, X-ray repair cross-complementary protein 5 and aminopeptidase N). As described previously, Hx can induce the release of cardiac EVs that contain proteins with opposing activities (6). We further mapped the biological processes and metabolic pathways represented by CM-EVs proteins in Nx and Hx, finding 101 biological processes enriched in Nx and 124 in Hx (P < 0.05; **Tables S3**, **S4**). The most significant biological processes identified in both conditions were visualized in a Treemap diagram using REVIGO (**Figure 1E**). CM-EVs secreted in Nx conditions contained protein cargo involved in ECM organization, integrin-signaling, collagen catabolism, and cell adhesion and cell migration, whereas additional biological processes were over-represented in Hx CM-EVs, such as protein folding and peptide cross-linking, including pathways involved in chaperone activity and protein synthesis (**Figure 1E**).

To evaluate the functional activity of CM-EVs, we used CFSE to stain equal amounts of CM-EVs from both conditions and added them to fibroblast or HUVEC cultures to explore their ability to capture vesicles. Fluorescent cells resulting from the internalization of EVs were then quantified by flow cytometry. Results showed that CM-EVs had a stronger preference to be internalized in HUVEC than in fibroblasts, which was independent of culture conditions (**Figures 2A,B**), suggesting an active exosome-based communication mechanism between these cell types in the heart. To further explore this phenomenon, we added CM-EVs from Nx and Hx conditions to EC and evaluated their functional impact in a tube formation assay. We found that tube length and total number of loops in EC was lower when CM-EVs from Hx conditions were used than when Nx CM-EVs were used (**Figures 2C,D**), although no significant differences were found in the other parameters analyzed, such as total number of tubes and number of branching points in vascular nets (**Figures 2C,D**). Similar results were obtained when CM-EVs were added to HCAEC, in which tube length, number of total tubes and also branching points were significantly lower when Hx CM-Evs were used (**Figure S2**). To analyze the intercellular cross-talk between cardiomyocytes and fibroblasts mediated by CM-EVs, we evaluated the ability of these vesicles to modify the motility of fibroblasts in a wound healing assay. We observed accelerated wound closure when CM-EVs isolated under Hx conditions were added to fibroblasts as compared with Nx CM-EVs counterparts (**Figures 2E,F**). This occurred in the absence of any pro-mitotic effects of the CM-EVs (**Figure S3**). These responses of fibroblasts and EC to Hx CM-EVs might account for the cellular events observed during post-ischemic remodeling of the heart.

# DISCUSSION

Cardiac cells, including cardiomyocytes, fibroblasts and EC, act in a coordinated fashion to support cardiac function; hence, an efficient intra-cardiac communication system is needed to control heart integrity in health and disease (6). Similar to many other eukaryotic cells, cardiac cells can release EVs, and changes in the amount and composition of these vesicles have been related to changes in physiological or pathological conditions (6, 18). The interplay between different cell populations mediated by EVs, including cardiac resident progenitor cells or cardiac progenitors, in homeostasis and pathological conditions is currently the object of intense investigation (19–21). A recent study exploring the functional activity of EVs isolated from iPSCderived cardiovascular progenitors showed that these vesicles improved the survival of cardiac myoblasts and promoted tube formation in HUVEC in vitro, and also enhanced cardiac function in vivo in a murine model of chronic heart failure (22). In the present study, we sought to assess these biological processes by functionally analyzing EVs secreted by an adult cardiomyocyte-derived cell line (AC10) cultured in Nx and Hx conditions. We found that both Nx- and Hx-derived CM-EVs were loaded with ECM components including different types of collagens and laminins, which are typically secreted by interstitial cells to modulate normal cardiac growth and remodeling. Hx-derived CM-EVs also contained proteins related to chaperone activity and aminoacylation of proteins including T-complex protein 1 subunits α and γ and the aminoacyl-tRNA synthetase multienzyme complex. Another protein detected in Hx-derived CM-EVs was the tissue factor pathway inhibitor TFPI1, which has been shown to correlate with circulating fibrinogen levels and coronary artery disease (23), suggesting that CM-EVs might contribute to blood coagulation under similar in vivo conditions. GO analysis revealed the enrichment of terms associated with ECM organization, integrin-mediated signaling pathways and cell adhesion common to both Nx and Hx culture. Also, we found a major association of Hxderived CM-EVs with two biological processes, peptide crosslinking and chaperone-mediated protein folding, which have been described in other cell types in response to pro-apoptotic stress (24). Nevertheless, our proteomic assay failed to find significant amounts of apoptotic proteins or biological processes related to apoptotic cascades, suggesting residual presence of apoptosis related vesicles in our preparations. However, since the presence of the mentioned vesicles has not been discarded, it is possible that observed effects on fibroblast and endothelial cells are partially trigger by apoptosis related vesicles. Further characterization of the secreted EVs will clarify the signaling processes involve in the effect of these EVs on the cardiac cells.

Low levels of oxygen following myocardial infarction account for the massive destruction of cardiomyocytes and EC in the myocardium. We show here a preferential EV-mediated communication between cardiomyocytes and EC, both in Nx and Hx conditions. Using a tube-forming assay, we observed lower tube formation in EC treated with Hx CM-EVs when compared with Nx CM-EVs. This result suggests that CM-EVs play a role in angiogenesis. Proteomic analysis revealed that both Nx- and Hx-derived EVs carry pro-angiogenic proteins, such as TGF-β and VEGF-C, but GO analysis indicated that Hx-derived EVs are less enriched in these factors than Nx EVs. This correlates with the results of the tube formation assay and could be related to the stress conditions of the parental cells. In this regard, the addition of CM-EVs from diabetic rats to EC cultures inhibited proliferation, migration and tubelike formation (25). Nevertheless, it has recently been described that rat CM-EVs obtained under Hx promote angiogenesis (26). The differences in regenerative potential between human and murine cells could account for these discrepancies, and further experiments are needed to elucidate these processes in detail.

To analyze the functional activity of CM-EVs on fibroblasts, we used a scratch assay to measure cell migration, finding that CM-EVs isolated from Hx cultures promoted fibroblast motility. This accelerated wound closure is consistent with the pro-fibrotic reaction in the heart after an ischemic

insult. Similar deleterious effects exerted by CM through paracrine mechanisms have been described in other pathological conditions. For example, hypertrophic remodeling and fibrosis occurs in mice subjected to aortic constriction, which could be attenuated by cardiac-specific over-expression of the β3 adrenergic receptor (β3AR). The analysis of the secretome of β3AR-stimulated cardiac myocytes identified connective tissue growth factor as the main pro-fibrotic paracrine factor, which was reduced under β3AR stimulation (27). In another study, it was reported that diabetic (db/db) mice subjected to acute exercise increased their number of EVs in the heart containing miRNAs that reduce the content of MMP9, a metalloproteinase involved in cardiac fibrosis, with the consequent mitigation of this process (28). Similarly, in our proteomic analysis, we found disintegrin, metalloproteinase domain-containing protein 9 (ADAM9), laminin subunit gamma-1, TGFβI, fibronectin, laminin subunit alpha-5 and several subtypes of collagenalpha protein in CM-EVs from Hx conditions; all of which are related to cell adhesion, cell-matrix interactions and cell motility, and that could account for the pro-fibrotic effects of CM-EVs. Overall, the results presented here contribute to the emerging picture of how CM-EVs function in cardiac remodeling after an ischemic insult. The differences between human and animal model systems should, however, be taken in consideration for the functional analysis of these vesicles.

images taken after 24 h culture normalized to initial wound area (n = 6, \*\*P < 0.01).

# LIMITATIONS OF THE STUDY

The major limitation of this study is the use of the AC10 cell line rather than primary cardiomyocyte cultures. However, the enormous number of cells needed for the isolation of EVs to perform the experiments would not have been feasible using primary cardiomyocytes and thus we decided to use AC10 cells. Another limitation of the study is that we did not verify the presence of proteins detected in the proteomic analysis by immunogold electron microscopy or alternative techniques, and so we cannot be sure that the proteins detected by LC-MS/MS are internalized in EVs and not non-specifically attached to isolated EVs. Finally, the low but unavoidable contamination of EV extracts with EVs originating from FBS used in cell culture should be considered. However, since we compared EVs derived from cell cultures in Nx and Hx conditions using the same batch of FBS, the differences observed in this study cannot be due to contamination with EVs from serum.

# AUTHOR CONTRIBUTIONS

IO-O and AD: conception and design of the study, acquisition of data, and analysis and interpretation of data; RS, MC, MG-F, MB, EG, ST, HG-K, NG, and FG-G: acquisition and analysis of data; IO-O, AD, NG, and EP-M: critical revision of the manuscript; PS: manuscript drafting, critical revision, and final approval of the version to be submitted.

# FUNDING

This work was supported by grants PI16/0107 and RETICS Program (RD16/0011/0004) from Instituto de Salud Carlos III cofunded by the European Regional Development Fund–ERDF una manera de hacer Europa. The proteomic studies were carried out in the University of Valencia Proteomics Unit, a member of the ISCIII ProteoRed Proteomics Platform. The bioinformatics analysis was performed in the Bioinformatic and Biostatistics Unit of the Principe Felipe Research Center using the computational infrastructure supported by ERDF.

# ACKNOWLEDGMENTS

The authors acknowledge the Cytomics Unit at Instituto de Investigación Sanitaria La Fe for technical support.

# REFERENCES

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcvm. 2018.00152/full#supplementary-material

Figure S1 | (A) Representative western blot images of proteins extracted from AC10 cell cultures in normoxia or hypoxia (2% O2). Lanes were loaded with equal amounts of protein and incubated with a hypoxia inducible factor-1 antibody. Signals were quantified by densitometry (n = 3). (B) Quantification of viable AC10 by CCK8 after different culture conditions used to isolate EVs.

Figure S2 | Tube formation assay of human coronary artery endothelial cells (HCAEC). (A) Representative images of tube formation after 6 h of culture in the presence of normoxia (Nx) or hypoxia (Hx) cardiomyocyte-derived extracellular vesicles. (B) Quantification of total loops, tube length, total tubes and number of branching points from images taken after 6 h culture. Results are expressed in arbitrary units (n = 3, <sup>∗</sup>P < 0.05).

Figure S3 | Cell proliferation assay of AC10, fibroblasts and human umbilical cord vein cells (HUVEC). Representative example of cell proliferation measured by CCK-8 assay after 48 h of culture in presence of Nx and Hx CM-EVs. Results are presented in arbitrary units (n = 3).

Table S1 | Protein identification in cardiomyocyte-derived extracellular vesicles in normoxia.

Table S2 | Protein identification in cardiomyocyte-derived extracellular vesicles in hypoxia.

Table S3 | Gene ontology biological processes for proteins identified in extracellular vesicles derived in normoxia.

Table S4 | Gene ontology biological processes for proteins identified in extracellular vesicles derived in hypoxia.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Ontoria-Oviedo, Dorronsoro, Sánchez, Ciria, Gómez-Ferrer, Buigues, Grueso, Tejedor, García-García, González-King, Garcia, Peiró-Molina and Sepúlveda. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# It Takes Two: Endothelial-Perivascular Cell Cross-Talk in Vascular Development and Disease

#### Mark Sweeney <sup>1</sup> \* and Gabor Foldes 1,2

*<sup>1</sup> Cardiovascular Division, National Heart and Lung Institute, Imperial College London, London, United Kingdom, <sup>2</sup> Heart and Vascular Center, Semmelweis University, Budapest, Hungary*

The formation of new blood vessels is a crucial step in the development of any new tissue both during embryogenesis and *in vitro* models as without sufficient perfusion the tissue will be unable to grow beyond the size where nutrition and oxygenation can be managed by diffusion alone. Endothelial cells are the primary building block of blood vessels and are capable of forming tube like structures independently however they are unable to independently form functional vasculature which is capable of conducting blood flow. This requires support from other structures including supporting perivascular cells and the extracellular matrix. The crosstalk between endothelial cells and perivascular cells is vital in regulating vasculogenesis and angiogenesis and the consequences when this is disrupted can be seen in a variety of congenital and acquired disease states. This review details the mechanisms of vasculogenesis *in vivo* during embryogenesis and compares this to currently employed *in vitro* techniques. It also highlights clinical consequences of defects in the endothelial cell—pericyte cross-talk and highlights therapies which are being developed to target this pathway. Improving the understanding of the intricacies of endothelial—pericyte signaling will inform pathophysiology of multiple vascular diseases and allow the development of effective *in vitro* models to guide drug development and assist with approaches in tissue engineering to develop functional vasculature for regenerative medicine applications.

#### Edited by:

*Andrea Caporali, University of Edinburgh, United Kingdom*

#### Reviewed by:

*Elisa Avolio, Bristol Medical School, University of Bristol, United Kingdom Xuechong Hong, Harvard Medical School, United States*

> \*Correspondence: *Mark Sweeney<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Cardiovascular Biologics and Regenerative Medicine, a section of the journal Frontiers in Cardiovascular Medicine*

> Received: *13 July 2018* Accepted: *10 October 2018* Published: *30 October 2018*

#### Citation:

*Sweeney M and Foldes G (2018) It Takes Two: Endothelial-Perivascular Cell Cross-Talk in Vascular Development and Disease. Front. Cardiovasc. Med. 5:154. doi: 10.3389/fcvm.2018.00154*

Keywords: cell-cell interaction, vascular development, endothelial, pericytes, perivascular, vascular dysfunction

# INTRODUCTION

The processes of vasculogenesis and angiogenesis play vital roles in embryonic development and vascular homeostasis during adulthood. The endothelial cell is the most basic building block of blood vessels and the growth factor mediated proliferation and migration of these cells is responsible for forming the complex vascular networks within the body. Endothelial cells do not perform these tasks in isolation and their interaction with supporting perivascular cells is one of the key processes during the formation of new blood vessels to ensure that durable vessels are formed which can support blood flow. This review will discuss the embryological origins and interactions of endothelial cells and perivascular cells and compare this with current in vitro approaches used to model this interaction. In addition, it will discuss how interruption of this interaction causes a variety of genetic and acquired diseases and how novel approaches to co-culture may help to develop our understanding of this area and provide potential therapeutic options in the future.

# MULTICELLULAR INTERACTIONS IN EMBRYOGENESIS

## Building Blocks for New Vessels

The process of creating vascular networks involves two sequential steps: vasculogenesis, the de novo formation of blood vessels from progenitor cells, and angiogenesis the migration, branching, and pruning of existing blood vessels to form complex vascular networks and capillary beds (1). The endothelial cell is the most basic building block of new blood vessels and the processes of angiogenesis and vasculogenesis both require the proliferation and migration of these cells to under perfused tissues. This must be followed by the formation of strong connections between adjacent cells and the extracellular matrix (ECM) to create a durable conduit which can support blood flow. In the developing embryo there are multiple interactions between the cell and its environment responsible for controlling this process (2). This includes interactions between neighboring endothelial cells, between endothelial cells and surrounding support cells as well as the paracrine effects of growth factors released into the ECM. In addition, these newly developing vessels respond to changes in the extracellular environment including the composition of the ECM and relative levels of hypoxia or nutritional deficiencies of surrounding cells (3).

### Endothelial Cells

During embryogenesis the first recognizable blood vessels occur in the yolk sac as groups of cells expressing endothelial markers including vascular endothelial growth factor receptor (VEGFR), VE-cadherin and CD31 (1, 4). These primitive endothelial cells are derived from the mesodermal layer of the embryo. They migrate to form aggregates of cells known as blood islands which are capable differentiating toward either haematopoietic or angioblastic lineages (5). As these cells begin to differentiate they align with angioblastic cells on the outside of the blood islands and haematopoietic cells in the central core. Angioblasts in the outer lining flatten and form intercellular connections to create a circumferential layer of primitive endothelial cells which is the first stage in vessel formation (1).

The formation of these blood islands in the mesoderm is controlled by growth factors released from the endodermal layer. Hedgehog signaling via the bone morphogenic protein-4 (BMP-4) pathway is one of the earliest steps that initiates endothelial differentiation from multipotent mesodermal cells and is vital in early vascular development (6–8). Fibroblast growth factors (FGF) stimulation of these cells induces the expression of early endothelial markers. The FGF driven expression of VEGFR (9– 11) is an essential step in sensitizing the cells to the potent angiogenic growth factor vascular endothelial growth factor (VEGF) which is one of the key growth factors in promoting angiogenesis (12, 13).

As the blood vessel matures the endothelial layer forms a confluent monocellular layer in contact with the blood. This functions as barrier to prevent the widespread extravasation of blood and fluid however also needs to be sufficiently permeable to enable the passage of required gases, nutrient and leukocytes into the perivascular space when required. VE-cadherin one of the earliest markers expressed on the surface of developing endothelial cells. It forms part of the adherens junctions between endothelial cells to begin the formation of the monolayer. Further control of the permeability is mediated by the formation of tight junctions which are formed from claudins, occludins and junctional adhesion molecules which are upregulated as the endothelial cell matures (14).

### Perivascular Cells

Perivascular or mural cells were first described histologically as cells closely associated to the endothelial layer of blood vessel and are found in all organs throughout the body. They are a phenotypically diverse family of cells with a variety of roles depending upon the anatomical location and function of the vessel (15). They can be divided into two main categories: vascular smooth muscle cells and pericytes although much heterogeneity exists within these groups.

Vascular smooth muscle cells are associated with larger conduit vessels such as arteries and veins and are separated from the endothelial layer by the basement membrane and the inner elastic lamina. They have greater expression of contractile proteins such as α-smooth muscle actin (α-SMA), adopt a stellate shape and line up in a circumferential pattern around the vessel. Pericytes, in contrast, are associated with small caliber capillaries where they are embedded in the same basement membrane as the endothelial layer. They have an elongated and flattened shape and are orientated along the long axis of the vessel with multiple finger-like projection extending through the basement membrane to make direct contact with the endothelial layer (15). These connections have a peg and socket arrangement that enables signals to be passed directly between the pericyte and endothelial layers. A single pericyte can interact with multiple endothelial cell and the relative density of pericytes surrounding a blood vessel depends upon anatomical location and function of the vessel. In skeletal muscle there is approximately a 1:100 relationship between pericytes and endothelial cells (16) whereas in the brain and the retina there is a 1:1 relationship where it is thought to play a role in maintaining the blood brain barrier (17–19). Their position in contact with multiple endothelial cells can be advantageous in co-ordinating the response of a population of endothelial cells along the vessel as intercellular signaling can be propagated to multiple cells in the endothelial layer through a single pericyte (15, 17).

Debate exists regarding the characteristics which define a perivascular cell and thus a precise definition remains elusive. Consequently, there is no single specific marker to define this population. Instead, a combination of cell surface markers are typically used and these may have variable expression depending upon the anatomical location and angiogenic state of the cell (20, 21). Common markers which are expressed in perivascular cells include neuron-glial antigen-2 (NG2), CD146, α-smooth muscle actin (α-SMA), and platelet derived growth factor receptor-β (PDGFR-β). Endothelial and haematopoietic specific markers such as CD31, VE-cadherin, von Willebrand factor and CD45 should be absent (15, 22–25).

The precise origin of perivascular cells is more controversial than that of endothelial cells and there appears to be more than a single origin for these cells (**Figure 1**). Perivascular cells in the bowel, lung and liver have been found to have an endodermal lineage which initially develops toward an epithelial fate and subsequently undergo epithelial to mesenchymal transition into pericytes (16). In the brain, they are derived from neural crest cells (26) and vessel such as the aorta have pericytes of multiple different origins within a single vessel (16).

Perivascular cells were once thought to only provide mechanical support to the vessel however it is now clear that it has a variety of functions within the vasculature. The contractile properties of perivascular cells enable regulation of blood supply by altering the vessel diameter in response to vasoactive substances (21). In the central nervous system, where they are significantly more abundant, they are thought to play a role in the control of cerebral perfusion and restricting the permeability of the blood brain barrier (15, 21, 27). They may also play a role in tissue repair as they have the ability to transdifferentiate into fibroblast in response to injury to the surrounding tissue (16, 28) Finally, they also have an important role in intercellular signaling both to endothelial cells and to the tissues surrounding the blood vessels.

# Crosstalk Between Perivascular and Endothelial Cells Is Key During Early Vasculogenesis

Signaling between the endothelial and perivascular cell layers is mediated by the release of growth factors, direct cell-cell contact at peg and socket connections and modulation of the ECM (**Figure 2**). Perivascular cell are recruited to the developing vessel as the vessel matures. Perivascular cells coating the vessel have an anti-angiogenic effect which stabilizes the vessel and limits further proliferation and migration of the endothelial cells. This is mediated by a number of signaling molecules released by the perivascular cells which act on the endothelial layer including Angiopoietins, sphingosine-1-phosphate as well as contact inhibition largely mediated by the Notch family.

### VEGF

The expression of VEGF receptor on primitive endothelial cells is one of the most important steps in vasculogenesis as this enables cells to respond to the potent mitogenic and

Cadherin, and Notch mediated signals are prerequisites of endothelial -pericyte cross-talk, cell recruitment and subsequent vessel stabilization. chemotactic effects of VEGF (10, 29). The importance VEGF signaling pathway in early vasculogenesis is highlighted by VEGF knockout mice which die early in development without any organized vasculature present (30, 31). VEGF-A is the prototypical member of the VEGF family and is coded for by the VEGFA gene on chromosome 6. It is released by multiple cells throughout the body typically in response to hypoxia or hypoperfusion of a tissue (32).

VEGF receptors are transmembrane tyrosine kinase receptors denoted VEGFR1-R3. The most important signaling receptor in vasculogenesis and angiogenesis is the VEGFR2 which is a relatively low affinity receptor with high intracellular tyrosine kinase activity when activated. Knockout of this receptor has the same effects as VEGF-A knockouts with early embryonic lethality due to lack of organized vasculature. In contrast, VEGFR1 has a much higher affinity for VEGF-A however minimal intracellular tyrosine kinase activity upon activation (32). Knockout of the VEGFR1 receptor causes unchecked endothelial hyperplasia (33) however selective knockout of the tyrosine kinase domain of the VEGFR1 has minimal effect on vascular development. This demonstrates that this receptor acts largely as ligand trap to sequester VEGF and reduce VEGFR2 signaling and therefore angiogenesis (33).

Intracellularly activation of VEGFR2 is propagated by a number of downstream signaling pathways. This is classically via ERK signaling, however, a variety of non-canonical pathways are also known to play a role (34). Stimulation of this pathway induces endothelial proliferation and differentiation including the expression of cell adhesion molecules such as VE-cadherin which form junctional complexes between the endothelial cells and creates the tubular structure from which further blood vessel development can occur (35, 36). Control of VEGF receptor expression is maintained by exogenous signaling including concentration of FGF and transforming growth factor-β (TGF-β) as well as by negative feedback loops within the cell to internalize and degrade receptors to prevent overstimulation of the VEGFR2 pathway and excessive neovascularisation with immature and permeable vessels as is seen in VEGFR1 knockout mice.

As perivascular cells differentiate they begin to express VEGF which is expressed in response to hypoxia and TGF-β signaling (28, 37). The isoform produced by perivascular cells is most commonly VEGF-A<sup>165</sup> which binds to heparin sulfate proteoglycans on the cell surface, therefore, remaining associated with the cells (37). This local release of VEGF is important in promoting endothelial cell survival and stabilizing newly formed vessels, however, does not set up a concentration gradient to encourage endothelial cell migration as occurs when more soluble isoforms are released (37, 38). Pericytes also have VEGFR1 on the cell surface which binds VEGF and sequesters it from the VEGFR2 on the endothelial cells, therefore, prevents the initiation of angiogenesis in mature, quiescent vessels (39).

### TGF-β

The transforming growth factor-β family of signaling molecules consists of more than thirty molecules which have a diverse range of targets. TGF-β is the most well characterized member and is expressed by multiple cells throughout the body including both endothelial and perivascular cells. When secreted by these cells it is associated with specific TGF-β binding proteins which maintain it in an inactivated form bound to the extracellular matrix (ECM) (40). Various mechanisms can release and activate TGF-β1 including the action of proteases such as matrix metalloproteinases (MMPs) which are produced by endothelial and perivascular cells when they come into contact. Once activated TGF-β has a variety of receptors on the surface of endothelial cells and pericytes (23, 41, 42).

The effect of TGF-β is highly context dependent and varies with the local concentration of TGF-β as well as the relative expression of receptors on the cell surface of the target cells (43, 44). The two most important TGF- β receptors in angiogenesis are Activin like kinase-1 (ALK-5) which is expressed on both endothelial cells and perivascular cells and Activin like kinase-1 (ALK-1) which is restricted mainly to endothelial cells. These two receptors have opposing effects on the response of the cell to TGF-β. ALK-5 stimulation causes down regulation of proangiogenic VEGFR-2 and upregulation of the antiangiogenic VEGFR-1 leading to reduced proliferation and increased differentiation of endothelial cells (45, 46). Similarly, ALK-5 activation causes differentiation of the perivascular cells and promotes release of ECM proteins such as fibronectin from both cell types (40). Overall this encourages formation of quiescent mature vessels with significant perivascular cell coating and a well-developed stable ECM. In contrast, ALK-1 signaling which is preferentially activated at lower concentrations of TGF-β increases proliferation of endothelial cells and encourages angiogenesis to proceed (45, 47, 48).

### Platelet Derived Growth Factor

Platelet derived growth factor (PDGF) is released mainly by endothelial cells and binds to the PDGFR-β on perivascular cells. Activation of this receptor stimulates proliferation of perivascular cells and acts as a potent chemoattractant causing cells to migrate toward expanding endothelial cell populations (49, 50). Absence of PDGF or PDGFR- β in mice is lethal in late gestation due to large, permeable, dilated vessels with microaneurysm formation, microvascular leakage and oedema throughout the embryo (49). Histologically these mice have widespread deficiency of perivascular cells coating the blood vessels and there is endothelial cell hyperplasia at the site of microaneurysms formation. This demonstrates the role that recruitment of perivascular cells to the perivascular niche plays in stabilizing developing vessels and reducing proliferation and angiogenesis in fully formed mature vessels (24).

VEGF signaling on perivascular cells via the VEGFR2 supresses the response of the cell to stimulation by PDGF-B by phosphorylation of the PDGFR-β receptor. This reduces the maturation and migration of perivascular cells to the site blood vessels undergoing of active angiogenesis and enables endothelial cell proliferation and vasculogenesis to continue without the inhibitory effect of pericytes (51).

#### Angiopoietins

Angiopoietins are a group of glycoproteins which act as ligands for the tyrosine kinase receptors TIE (Tyrosine kinase with immunoglobulin-like and EGF-like domains). The TIE-2 receptor is exclusively expressed on the surface of endothelial cells (52). Angiopoietin-1 (ANG-1) is a ligand for the TIE receptors which is expressed by perivascular cells in response to PDGF-B stimulation (53). Activation of TIE-2 receptors causes stabilization of the vessel through the inhibition of apoptosis in endothelial cells, enhancing inter-endothelial connections and reducing endothelial layer permeability (54– 56). ANG-2 is released mainly by endothelial cells and acts as an antagonist at the TIE-2 receptors preventing the anti-angiogenic activity of ANG-1 and thereby promoting angiogenesis (57).

### Sphingosine-1-Phosphate

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite released by perivascular cells (58) which acts on the endothelial differentiation gene-1 (EDG-1) receptor on the endothelial cell surface. S1P specifically promotes membrane expression and trafficking of the cell-cell adhesion protein N-cadherins to the cell membrane. N-cadherins localize to the peg and socket contacts between the endothelial and pericyte where it enhances these contacts (54, 59). Similar to PDGFR-β knockouts, loss of S1P results in absence of pericyte association with the vessels and subsequent vessel dilation and hemorrhage (54). Binding of the S1P also upregulates the expression of the inter-endothelial cell adhesion molecule VE-cadherin and down-regulates the expression of the pro-angiogenic ANG-2 thereby strengthening tight junctions between cells and increasing the stability of the developing vessel (36, 58–61).

## Notch Receptors and Ligands

Notch receptors are transmembrane proteins which binds to Notch ligands which are also typically membrane bound structures, therefore, having an important role in mediating signaling between adjacent cells in direct contact. Binding of a Notch ligand causes the intracellular effector domain of the Notch receptor to be cleaved which travels to the nucleus where it acts as a transcription factor (62). Endothelial cells express the Notch ligands delta-like-ligand 4 (DLL-4), Jagged-1 (JAG-1), and Jagged-2 (JAG-2) and Notch receptors 1–4 while perivascular cells express Jagged-1 and Notch receptors 1–3 (63).

Notch receptors on the endothelial cells binds JAG-1 on the perivascular cells mediating contact inhibition by downregulating expression of VEGFR2 and upregulating VEGFR1 therefore desensitizing the cell to VEGF activation, (64, 65) reducing endothelial proliferation (66–68) and encouraging the formation of a mature endothelial cell phenotype (69). The expression integrin αvβ3 is increased and which causes adhesion of endothelial cells to von Willebrand factor in the basement membrane and strengthens the structure of the developing vessel (69).

Activation of Notch receptors in the perivascular cells upregulate the expression of PDGFR-β which promotes maturation of perivascular cells with increased expression of α-SMA (68, 70). It also enhances recruitment and attachment of perivascular cells to the endothelial layer forming more stable and mature vessels (71). Notch knockout mice have early embryonic lethality due to widespread vascular abnormalities (72).

# Extracellular Matrix for Vessel Formation

In embryogenesis, the ECM and particularly the vascular basement membrane plays a vital role in controlling the development of the primitive vasculature by recruiting cells to the surrounding area and modifying their transcription profile depending upon the composition of the surrounding ECM (73). The vascular basement membrane is a structure of highly crosslinked insoluble materials including collagens, laminins, and fibronectin in which endothelial cells are embedded (74). This is formed by synthesis and deposition of these substances from both endothelial cells and perivascular cells which is upregulated following contact between these two cell types (2).

The basement membrane is not a static structure; it is continually being remodeled depending upon the conditions of the vessel and surrounding tissue. The changes in composition affect the endothelial cell behavior through interaction with integrins on the cell surface. MMPs and tissue inhibitors of metalloproteinases (TIMPs) catalyze this remodeling and are in constantly shifting equilibrium (75). Close interaction of endothelial cells with perivascular cells cause TIMP expression to predominate leading to inhibition of MMP and a more stable, type IV collagen rich and highly cross-linked basement membrane (76). In this state growth factors and signaling molecules including VEGF and TGF-β remain embedded in the basement membrane and are not available for the cell to access causing it to remain in a state of quiescence. In contrast, when the basement membrane is being assembled or disassembled by MMPs these growth factors are released and activate endothelial cells by exposing them to provisional matrix components, such as vitronectin, fibronectin, type I collagen, and thrombin. Additionally, growth factors including TGF-β and VEGF which are released from their latent bound states within the ECM and encourages endothelial proliferation and migration leading to sprouting and growth of the vessel (75, 77).

## Cadherins

The cadherins, N-cadherin and VE-cadherin mediate the interaction between endothelial cells and surrounding cells and structures by linking the extracellular environment to the cytoskeletal framework of the cell (78). VE-cadherin is concentrated at adherens junctions, its extracellular component mediates cell-cell interaction with cadherins on other endothelial cells (79). The inhibition of this interaction with monoclonal antibodies results in increased permeability of the endothelial layer and apoptosis of endothelial cells (80). Congenital absence of VE-cadherin causes early embryonic lethality due to a lack of organized vasculature (81).

The intracellular components of the VE-cadherin complex; β-catenin, p120-catenin and plakoglobin act on various pathways to control gene expression, proliferation, and the cytoskeleton. Binding of VE-cadherin causes VEGFR2 to be internalized and degraded therefore mediating the contact inhibition seen in confluent endothelial cell culture (82). Interaction with catenin molecules causes alteration in the cytoskeleton on binding of VE-cadherin molecules which causes the endothelial cells to change shape in response to contact with neighboring cells. The expression of VE-cadherin is under the control of multiple growth factors including VEGF causing the phosphorylation of VE-cadherin and destabilization of the adherens junction (83, 84). This results in increased permeability of the endothelial cell layer and increased mobility of endothelial cells during angiogenesis.

N-cadherin molecules are positioned on the abluminal side of the endothelial cells mediating its contact with pericytes and disruption of this interaction with N-cadherin blocking antibodies impairs the pericyte-endothelial interaction. Although pericytes are recruited to the perivascular niche, they are weakly associated with the endothelial layer. Large extravascular cavities form and there is rupture of the endothelial layer, loss of polarity and extensive hemorrhage (85, 86).

# DYSFUNCTIONAL MULTICELLULAR INTERACTIONS IN DISEASE

# Congenital Disease

The failure of cell-cell interactions between endothelial cells and perivascular cells have been implicated in a variety of congenital and acquired diseases (**Table 1**). Approximately 80 per cent of rare diseases have a genetic basis, so identifying the specific mutations in underlying cell-autonomous pathways may provide insight into more common developmental and disease processes.

### Hereditary Haemorrhagic Telangiectasia

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominantly inherited condition caused by mutations in the ALK-1 receptors or Endoglin receptor (ENG) which is a co-receptor in the TGF-β signaling pathway (94, 95). It is characterized by large arteriovenous malformations (AVMs) in major organs including the lungs, liver, brain and mucosa. The large fragile vessels formed in AVMs are prone to bleeding resulting in presentations with epistaxis or intracranial hemorrhage and causing shunting between the arterial and venous circulation which can present as paradoxical emboli or high output heart failure (95). The loss of TGF-β signaling in endothelial cells reduced expression of N-cadherin which impaired the formation of heterotopic cell contacts with perivascular cells. This lack of cell-cell contacts results in further reduction in TGF-β activation from the surrounding ECM and therefore reduced TGF-β signals for the perivascular cell to encourage differentiation and attachment to the endothelial layer. This impaired recruitment of perivascular cells to the developing vessel and overactive proliferation of endothelial cells results in endothelial hyperplasia. These large dilated vessels with little perivascular cell coating develop into the arteriovenous malformations which characterize the disease (95–97). Similar phenotypes are seen in spontaneous cerebral AVMs with histological analysis demonstrating dilated vessels with segmental loss of smooth muscle cells and the internal elastic lamina. This phenotype is reproducible in mice by knockout of the ALK-1 and TGF-β receptor (98).

Currently emerging treatment for HHT is targeted at increasing the number of pericytes associated with the endothelial layer. Thalidomide is one such treatment which reduces the frequency and severity of epistaxis in patients with HHT (99). In mice, thalidomide has been noted to increase the number of pericytes surrounding vessels and improves their attachment to the endothelial cell layer thereby stabilizing the vessel which is likely to play a role in the reduced bleeding from AVMs. Thalidomide treated mouse retinas have increased

expression of PDGF-B by endothelial cells (94) providing a potent chemotactic and maturation signal to neighboring pericytes.

### Idiopathic Basal Ganglia Calcification

Idiopathic basal ganglia calcification is a rare disorder, most commonly inherited in an autosomal dominant fashion. It presents with neurological and psychiatric abnormalities at a young age with associated areas of calcification within the basal ganglia of the brain. A loss of function mutations in PDGF-B is associated with this condition in humans (100). This is thought to cause reduced pericyte recruitment to cerebral vasculature which increases the permeability of the blood-brain barrier. Mouse models of the disease involving knockdown of PDGF-B function result in similar reduction in perivascular coverage of the cerebral vasculature and progressive brain calcification (100).

### Notch Mutations

Mutations in Notch receptor genes or ligands lead to a variety of congenital disorders in humans. Adams-Oliver syndrome is a rare inherited disorder, with varying degrees of defective vasculature defects including hypoplastic aortic arch, middle cerebral artery, and pulmonary arteries. The spectrum of vascular defects has been thought to be due to a disorder of vasculogenesis, perivascular dysfunction and abnormal vascular coverage during development (88, 101, 102). To support this idea, transient inhibition of Notch signaling in perivascular cells inhibited their differentiation and led to localized hemorrhages in newly forming vasculature during embryonic development (103). Alagille syndrome is a multi-systems inherited disease caused by loss of function mutations in JAG1 or NOTCH2 (91). Pulmonary artery involvement including hypoplastic pulmonary arteries and pulmonary stenosis are the common vascular manifestation of the disease. There is also a high rate of intracranial hemorrhage up to 16% suggesting the presence of fragile intracranial vessels (91).

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is a degenerative disorder characterized by early-onset strokes and dementia; it is caused by loss of Jag1 in the endothelium or defective Notch3 in vascular smooth muscle cells. This results in alterations of vascular smooth muscle cells; NOTCH1 and NOTCH3 mutant mice develop arteriovenous malformations and show hallmarks of CADASIL (68).

### TIE Mutations

Hereditary cutaneomucosal venous malformation is a rare condition which is inherited in an autosomal dominant fashion due to abnormalities in the TIE-2 receptor gene (TEK). This causes small and usually asymptomatic venous malformations mostly in the mucous membranes and face and much less common involving the internal organs (104). The mutation is a hyperphosphorylating mutation within the TIE-2 receptor which results in a patchy coating of perivascular cells along the vessel wall associated with hyperplasia of the endothelial layer producing large and dilated vessels (105–107).

# Acquire Disease

#### Retinopathy

Diabetic retinopathy is a common condition affecting over one third of patients with diabetes worldwide (108). Chronic hyperglycaemia causes endothelial cells to release reactive oxygen species and inflammatory cytokines and reduces the expression of growth factors including PDGF-B (109). These changes coupled with direct effects of hyperglycaemia on the basement membrane and pericytes cause the vessel to become denuded of pericytes (110–112). This is one of the earliest histological findings in diabetic retinopathy which is followed by microaneurysm formation and oedema which can be detected clinically as the fragile neovasculature ruptures or leaks (113).

As described previously physical contact between pericytes and endothelial cells is required for activation of TGF-β and deficiency of TGF-β signaling impairs vessel maturity and increases permeability (114, 115). Additionally, ANG-1 released by pericytes has an important role in maintaining quiescence of the endothelial layer and reducing the permeability of the vessel. Blockage of the ANG-1 pathway using the competitive antagonist ANG-2 in normoglycaemic mice induces pericyte loss similar to diabetic retinopathy and subsequent endothelial hyperplasia. Models of diabetic retinopathy can also be induced in mice by reducing PDGF-B and TGF-β signaling in the absence of hyperglycaemia (114–117).

Pathological neovascularization also occurs in the setting of age-related macular degeneration (AMD). AMD is a condition where there is neovascularization of the macula of the retina which causes progressive blindness. Wet AMD is associated with macular oedema as the fragile and permeable vessels allow the leakage of fluid into the subretinal space (118).

The mainstay of treatment to date has been VEGF inhibition with monoclonal antibodies against VEGF which reduces the permeability of these vessel and induces regression of the vasculature. This treatment has significantly reduced the rates of progression and blindness associated with this condition (119). However, a proportion of patients become resistant to anti-VEGF treatment after an initial period of success. Pericytes have been implicated in providing survival signal to the developing blood vessels overcoming the inhibition of VEGF leading to ongoing angiogenesis within the macula. Combination of monoclonal antibodies against VEGF and PDGF have shown promise in pre-clinical trials to reduce the extent of neovascularization (120) and currently a trial of combination therapy is ongoing (121, 122). Similarly, treatments targeting other combinations of signaling molecules involved in this pathway are currently being trialed in diabetic retinopathy. This includes antibodies targeting both VEGF and Angiopoietin-2 (Clinicaltrials.gov-BOULEVARD, Clinicaltrials.gov-RUBY) and treatments targeted at VEGF, PDGF-B and FGF (clinicaltrials.gov-squalamine).

### Cancer

One of the hallmarks of cancer is the ability to induce angiogenesis to augment the blood supply to the rapidly dividing cancerous cells (123). Therefore, targeting this pathway with inhibitors of angiogenesis has become an important target in oncological therapy. Monoclonal antibodies blocking VEGF Sweeney and Foldes Endothelial–Pericyte Cross-Talk

and inhibitors of tyrosine kinase receptors have become a mainstay in cancer regimes for multiple indications (124). However, after a period of treatment, a proportion of cancers become resistant to the anti-VEGF treatment and escape this inhibition. Thus, further efforts have been invested in developing therapies to target the pericyte-endothelial cell interaction to prevent this resistance developing. Less selective tyrosine kinase inhibitors with action against multiple tyrosine kinases has shown promise in this area. Sunitinib targets the VEGF and PDGF receptors and is a potent inhibitor of angiogenesis. It reduces the coverage of pericytes along the developing vessel causing reduction in angiogenesis overall and therefore improved survival in renal cell cancers (125, 126). Preventing migration of perivascular cells to the perivascular niche of developing cancer vasculature reduces the survival signal to endothelial cells leading to apoptosis and preventing further angiogenesis (127).

Manipulation of TIE2/Angiopoietin pathway has also been targeted in preclinical models of renal cell cancer by using antibodies which selectively block the effect ANG-2, therefore, potentiating the effects of ANG-1 which will stabilize the vasculature and prevent further angiogenesis and therefore limit further growth of the tumor (128). This is currently being trialed in phase 1 human trials (129).

# MULTICELLULAR INTERACTIONS IN CULTURE

Modeling rare vascular genetic disease in vitro is possible using pluripotent stem cell technology which provides insights into the mechanisms involved in these diseases. The use of multicellular constructs provides a more analogous environment to the in vivo environment than can be replicated by the exogenous application of growth factors as it is able to model and regulate a greater variety of interaction between cells in a more physiological manner.

# Endothelial Cells

The earliest stage of in vitro blood vessel formation involves expansion of endothelial cells in culture to form a network of tubular structures replicating the early steps of vasculogenesis in the embryo. Multiple cell types and sources have been used to provide endothelial cells for this purpose. Fully differentiated primary endothelial cells have been used, most commonly these are sourced from the human umbilical vein (HUVEC). These cells already have a fully differentiated endothelial phenotype and therefore will immediately begin forming tubular structures in culture (35). However, use of these cells is limited by replicative senescence which develops after limited passages in culture making them sub-optimal for large-scale replication. Less well differentiated cells including late outgrowth endothelial cells (OEC) or multipotent cells such as mesenchymal stem cells (130) show greater replicative potential and are also partially differentiated toward vascular lineage, therefore, require less manipulation of growth factors to produce fully differentiated endothelial cells.

Pluripotent cells including embryonic stem cells or inducible pluripotent stem cells have attracted most interest recently (131, 132). The ability to produce more than one cell type from the a single cell source enables the development of complex vascular structures incorporating endothelial cells and supporting cells from the same cell source. This raises the possibility of individualized regenerative medicine approaches being possible in the future and additionally, makes it possible to create models of inherited genetic disease states in vitro by inducing pluripotency in cells of patients with specific genetic diseases and creating models of vasculogenesis using these cells.

There are three main methods used to differentiate pluripotent cells toward endothelial phenotype (131). Each of these methods attempt to replicate parts of the embryonic development of endothelial cells and vascular networks. The first method requires embryoid body formation which are three dimensional aggregates of pluripotent stem cells which form in suspended culture. These replicate the blastocyst formation seen in vivo (133, 134) and begin to differentiate with all three primitive germ layers represented; ectoderm, mesoderm and endoderm. Aggregates of cells expressing early endothelial markers developed within the embryoid body resembling blood islands (135) and these can be expanded and selected for by plating on suitable matrix with the addition of exogenous growth factors (136, 137).

The second commonly used method is co-culture of pluripotent stem cell with a feeder layer of mesenchymal cells such as bone marrow stromal cells. The feeder later provides growth factors to the stem cells encouraging differentiation toward mesoderm and expression of key endothelial markers (8, 138, 139). This also recapitulates the embryonic development of endothelial cells by providing Indian hedgehog (IHH) signaling which acts through BMP4 to upregulate endothelial markers such as VE-cadherin, CD31 and VEGFR2. These effects are abolished by neutralizing either IHH or BMP4 signaling and IHH signaling is rescued by exogenous BMP4 (8).

The third method is by 2D culture of inducible pluripotent stem cells on culture plates coated with protein substrate and culture media enriched sequentially with specific growth factors to direct differentiation toward mesodermal lineage and then endothelial lineage. The two most common protocols used to differentiate pluripotent cells toward endothelial lineage have numerous similarities and both attempt to recreate the embryonic development in the blastocyst. Mesodermal differentiation in the embryo relies on BMP-4, nodal and Wnt signaling pathways (132). In vitro supplementation of the media with BMP-4 and inhibitors of GSK3β, which upregulates the Wnt/B-catenin pathway, or Activin A which acts on nodal receptors are used to achieve this mesodermal transition (29, 140–142).

Following mesodermal induction, the addition of VEGF to the media is needed to start endothelial differentiation. Activators of the protein kinase A pathway such as forskolin or 8-bromocAMP appears to enhance the effects of VEGF and produce a greater purity of endothelial cells (29, 143, 144). The endothelial cells formed from these differentiation procedures have multiple markers of endothelial lineage including VE-cadherin, CD31 and

von Willebrand factor, however, there remain a proportion of cells which differentiate toward other lineages and therefore to obtain a pure culture of endothelial cells requires cell sorting and re-plating of endothelial cells.

# Perivascular Cells

The source of perivascular cells used in multicellular culture also varies, however, these are typically mesenchymal in origin. This includes aortic smooth muscle cells, adipose derived stem cells, osteoblasts (145) and mesenchymal stem cells (146). Pericytes have also been developed using embryoid body method from pluripotent stem cells. Similarly, to the endothelial cell protocols following formation of the embryoid bodies in suspended culture they can be dissociated and sorted to using cell surface markers to identify a population of pericytes which can then be plated and expanded (147).

It is also possible to produce perivascular cells from iPSC by manipulation of growth factors in the growth media. As with endothelial cells, the first step requires induction of mesoderm which is followed by supplementation with PDGF-B along with TGF- β or Activin A to direct differentiation toward a perivascular cell fate. This is highly efficient at creating a culture of perivascular cells which can be used in co-culture experiments with endothelial cells. (140, 148) Orlova et al used a single source of hiPSC and exposed them to a short period of supplementation with VEGF to begin vascular specification which yielded a mixed population of CD31+ and CD31- cells. Following cell sorting the endothelial and perivascular cells were able to be produced from the CD31+ and CD31- fractions, respectively (149). In co-culture, the perivascular cells were able to control sprouting and proliferation of the endothelial cells in a TGF-β dependent manner similar to that seen in the developing embryo.

# Co-culture

Combining endothelial cells and pericytes to create multicellular models of vasculogenesis can recreate a greater degree of the complexity of intracellular signaling seen in vivo than is provided by supplementing the media with supra-physiological levels of growth factors. Intrinsic intercellular regulatory mechanisms which are as yet incompletely understood and therefore difficult to replicate may provide a more physiological environment in which to model vasculogenesis.

Culture of mesenchymal stem cells (MSC) alongside the developing vascular network of endothelial cells increases the survival of vessels and helps to maintain these vessels in longterm culture (150). This coculture has been shown to upregulate VE-cadherin on the cell surface of endothelial cells and reduce the rate of proliferation and apoptosis of endothelial cells (151, 152). This results more stable, less permeable cells which form a more durable vascular network. Chen et al used coculture of HUVEC and mature perivascular cells obtained from skeletal muscle to create vascular networks in Matrigel plugs (28). The inclusion of perivascular cells supported the formation of complex capillary structures within the plugs which was not seen with culture of endothelial cells alone (28).

Additionally, Koike and colleagues produced a model of vasculogenesis by combining HUVEC with mesenchymal stem cells in a 3D type 1 collagen and fibronectin construct and implanting these into mice. The implantation of HUVEC alone formed tubules initially but it was unable to support perfusion and subsequently, these tubules regressed. In contrast during coculture experiments, the mesenchymal precursor cells migrated to a perivascular position started to express perivascular cell markers including α-SMA and produced stable long-lasting blood vessels which were able to support flow for over 60 days (150).

Melero-Martin and colleagues have produced similarly robust vascular networks in Matrigel plugs by using blood and cord blood derived OEC and MSC derived from the bone marrow. OEC and MSC have a greater replicative capacity than primary cell types and can be harvested from the same donor from blood and bone marrow. This enables sufficient quantities of genetically identical cells to be produced which enables more accurate modeling of inherited diseases and has potentially be used in regenerative medicine to vascularize tissue engineered grafts (153).

The development of inducible pluripotent stem cells (iPSC) by Takahashi and colleagues (154) provided further tools to build multicellular constructs which could now be derived from a single cell without many of the ethical difficulties which surrounded embryonic stem cells. Samuel et al developed endothelial and mesenchymal precursor cells in parallel from human iPSCs and by combining these cells in culture were able to form robust long-lasting functional blood vessels when implanted in mice (155). This parallel development of endothelial cells and the supporting perivascular cells was a step forward, however, still requires cells to be dissociated and sorted using flow cytometry before being plated and expanded.

Kusuma et al have started to overcome this difficulty by creating a bipotent population of cells from hiPSC which are able to differentiate toward endothelial and perivascular cell lineage (156). The difficulty with this approach in vitro is that proliferation is inhibited when perivascular cells and endothelial cells come into contact. Therefore, they initially focused on developing VE-cadherin positive endothelial cells. They used high concentrations of VEGF and inhibited TGFβ signaling which causes perivascular cell differentiation and mediates contact inhibition of endothelial cells (15, 157, 158). This created two populations of early vascular cells both of which expressed CD146<sup>+</sup> and CD105<sup>+</sup> and were negative for the haematopoietic marker CD45. However, the endothelial precursors expressed VE -cadherin while the perivascular precursors expressed PDGFRβ. When plated in hydrogel these cells were able to self-assemble into tubular-like structures of endothelial cells with perivascular cells surrounding these vessel forming a multicellular vascular network (156). Although these networks were not durable and typically regressed within approximately 2 weeks it provided a significant step forward in developing engineered multicellular vascular networks (159).

Use of patient-specific hiPSC-derived endothelial cells is now being used for disease modeling some of the inherited vascular disorders. The model of CADASIL is one of the first attempts to gain a better understanding of the developmental biology and cross-talk of endothelial cells and perivascular cells with NOTCH3 mutations. Similarly, NOTCH1 mutations have also been studied with pluripotent stem cell platforms (160). This in vitro approach may also facilitate the development of therapeutic gene editing interventions for vascular malformations and arteriopathies in the future.

The use of three cells together in culture has recently introduced and this extra complexity appears to have further benefits to promote stable vasculature. Caspi and colleagues used coculture of HUVEC, embryonic fibroblasts and embryonic stem cell derived cardiomyocytes on a biodegradable scaffold. The embryonic fibroblasts differentiate into perivascular cells and produced increased complexity of vascular networks. Additionally, there was upregulation of several growth factors in this model including VEGF, PDGF-B and ANG-1 compared to single cell culture and the endothelial cell survival was higher (161). Similarly, bone grafts have been produced by combining HUVEC and CD146+ perivascular cells with human inducible pluripotent mesenchymal stem cells in a calcium phosphate cement. This triculture demonstrated more complex vascular networks compared to culture with mesenchymal stem cell and endothelial cells alone. There was more rapid induction of VEGF signaling and there was improved bone mineral density all of which suggested benefits from combining multiple cell types (162).

# CONCLUSIONS

Development of functional blood vessels requires precise interaction between the tubule forming endothelial cells and the surrounding environment including growth factors, ECM and perivascular cells. The crosstalk between perivascular and endothelial cells is complex and incompletely understood. This cross talk plays a vital role in normal blood vessel development and homeostasis while abnormalities in this relationship have been implicated in multiple congenital and acquired diseases.

# REFERENCES

Knowledge of the molecular process involved in vasculogenesis and angiogenesis provides insight into the pathophysiological mechanism multiple vascular diseases including diabetic retinopathy and cancer. Therapies targeting the interaction between endothelial cells and neighboring perivascular cells are already in use or in late stages of development (94, 121). However, an in-depth understanding of the crosstalk between these cells will allow the development of more accurate vascular models to enable the identification of novel drug targets and further guide drug development.

Improved understanding of vascular development will also provide useful in the field of regenerative medicine where ensuring perfusion of stem cell derived tissue is one of the key challenges in producing viable implantable grafts. The development of vascular networks using multiple cell types is being used increasingly commonly and appears to provide greater complexity, stability and durability of the vasculature. Approaches using pluripotent cells are particularly intriguing given the possibility to develop these structures from a single cell source (156). This has the potential to develop more accurate models of vascular disease as well as personalized regenerative medicine applications.

# AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work and approved it for publication.

# ACKNOWLEDGMENTS

We gratefully acknowledge funding support from the Medical Research Council (MR/R025002/1), the British Heart Foundation Center of Regenerative Medicine, and the Hungarian National Research, Development and Innovation Fund (NVKP\_16-1- 2016-0017, NKFI-6 K128444).

formation during vasculogenesis. Development (2004) 131:4371–80. doi: 10.1242/dev.01304

and zebrafish xenografts. Arteriosclerosis, Thrombosis, Vascu Biol. (2014) 34:177–86. doi: 10.1161/ATVBAHA.113.302598

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Sweeney and Foldes. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Changing Metabolism in Differentiating Cardiac Progenitor Cells—Can Stem Cells Become Metabolically Flexible Cardiomyocytes?

Sophia Malandraki-Miller † , Colleen A. Lopez † , Heba Al-Siddiqi and Carolyn A. Carr\*

*Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom*

#### Edited by:

*Gabor Foldes, Imperial College London, United Kingdom*

#### Reviewed by:

*Silvia Martin-Puig, Centro Nacional De Investigaciones Cardiovasculares (CNIC), Spain Praphulla C. Shukla, Indian Institute of Technology Kharagpur, India*

\*Correspondence:

*Carolyn A. Carr<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Cardiovascular Biologics and Regenerative Medicine, a section of the journal Frontiers in Cardiovascular Medicine*

Received: *30 April 2018* Accepted: *10 August 2018* Published: *19 September 2018*

#### Citation:

*Malandraki-Miller S, Lopez CA, Al-Siddiqi H and Carr CA (2018) Changing Metabolism in Differentiating Cardiac Progenitor Cells—Can Stem Cells Become Metabolically Flexible Cardiomyocytes? Front. Cardiovasc. Med. 5:119. doi: 10.3389/fcvm.2018.00119* The heart is a metabolic omnivore and the adult heart selects the substrate best suited for each circumstance, with fatty acid oxidation preferred in order to fulfill the high energy demand of the contracting myocardium. The fetal heart exists in an hypoxic environment and obtains the bulk of its energy via glycolysis. After birth, the "fetal switch" to oxidative metabolism of glucose and fatty acids has been linked to the loss of the regenerative phenotype. Various stem cell types have been used in differentiation studies, but most are cultured in high glucose media. This does not change in the majority of cardiac differentiation protocols. Despite the fact that metabolic state affects marker expression and cellular function and activity, the substrate composition is currently being overlooked. In this review we discuss changes in cardiac metabolism during development, the various protocols used to differentiate progenitor cells to cardiomyocytes, what is known about stem cell metabolism and how consideration of metabolism can contribute toward maturation of stem cell-derived cardiomyocytes.

Keywords: heart, progenitor cells, substrate metabolism, mitochondria, differentiation, cardiomyocytes

# STEM CELL THERAPY FOR THE HEART

Myocardial infarction (MI) is the primary cause of disease-related death in the world with no reliable therapy (1). Acute coronary syndromes, like MI, account for half of all cardiovascular deaths in the industrialized world, with around 20% of patients developing heart failure (HF) and having a 1-year mortality rate (2, 3). Current therapeutic strategies focus on reperfusion, thrombolysis and reducing the workload of the heart using pharmacological agents or surgical procedures (4–6). Recent advances in treatment have improved time to reperfusion, but progress in identifying efficient therapies to offer more than symptom alleviation and support the surviving myocardium is yet to result in substantial clinical benefit (7, 8). The only current long-term solution is heart transplantation but with the limited numbers of donors, and the need for chronic immunosuppressants (9), the search to find an alternative solution to the problem of end stage HF is becoming increasingly urgent. MI can lead to a loss of up to 1 billion cardiomyocytes, which cannot be replaced due to the insufficient degree of regeneration in the adult heart (10). Although the heart is no longer considered a post-mitotic organ, the turnover of cardiomyocytes in the adult heart is around 1% per year (11) which is insufficient to counter the loss caused by MI. Stem cell therapy (SCT) has the potential to regenerate the damaged tissue and restore its contractility, harnessing the self-renewal and differentiation potential of stem cells (SCs) (12). In vivo, the transplanted cells can act via a combination of the following mechanisms; (a) replicate themselves and/or differentiate to mature cardiomyocytes; (b) stimulate the endogenous cardiac cells to regenerate; (c) exert a beneficial effect via paracrine mechanisms of action (13) (**Figure 1**).

# TYPES OF STEM CELLS FOR THERAPY

A wide range of cells have been tested both in animal models or early-stage human clinical trials in order to find the appropriate source for SCT (14, 15). These include bone-marrow derived cells (16–18), cardiac stem or progenitor cells (19–25), human embryonic stem cell-derived cardiomyocytes (26–29) and human inducible-pluripotent stem cell-derived cardiomyocytes (30, 31).

Bone marrow-derived stem cells were claimed to differentiate into cardiomyocytes that spontaneously beat after 2 weeks in culture (17) or into myotubules that, when injected into infarcted hearts, stimulated angiogenesis and generated cardiaclike cells (16). In addition, it was reported that when bone marrow-derived stem cell growth factor receptor-positive/linage negative (c-kit+/lin-) cells were injected into infarcted tissue, they generated new cardiac cells and blood vessels and remuscularised the damaged region (18). However, later studies showed that bone marrow-derived cells do not trans-differentiate into cardiomyocytes and that retained transplanted cells adopted a mature haematopoetic fate (32, 33). Bone-marrow derived mesenchymal cells have also been shown to improve cardiac function following MI, although repair is now thought to result from the delivery of a cocktail of beneficial cytokines which induce angiogenesis, limit scar fibrosis and may activate endogenous cardiac progenitors (34–36). Other key types of mesenchymal stem cells (MSCs) such as umbilical cord MSCs (37, 38), adipose-derived MSCs (39–41) and amniotic fluid MSCs (42), chosen for their ease of isolation and differentiation, have also been tested for therapeutic potential after infarction. As with bone marrow cells, any beneficial effect was deemed to be paracrine.

In 2003, a population of cardiac progenitor cells called stem cell growth factor receptor-positive (c-kit+) cells were identified (19). In vitro, these proliferative cells can self-renew and differentiate into myocytes. When injected into infarcted hearts, c-kit+ cells were shown to differentiate into cardiomyocytes resulting in myocardium regeneration and improved heart function (19). Subsequent studies supported the beneficial effect after cardiac injury, but suggested that the c-kit+ cells were of bone marrow origin (43) or were mast cells (44), endothelial cells (45) or maybe a mixed population of both (46). Studies looking at the issue from a developmental perspective suggested that c-kit+ cells contribute to new cardiomyocytes after injury in the neonate, but they were unable to do so in the adult heart (47), in line with previous observations regarding the role of c-kit+ cells in the neonatal heart (48). In 2011, the Anversa group used c-kit+ cardiac progenitor cells (CPCs) in a phase-I Stem Cell Infusion in Patients with Ischemic cardiomyopathy (SCIPIO) clinical trial showing encouraging results for post-MI treatment (49). However, in 2014 the results were questioned for their integrity (50). In 2013, Ellison et al. showed that ckit+ CPCs were necessary and sufficient for cardiac recovery in rodent models of diffuse myocardial damage causing acute heart failure (51). This was subsequently challenged by studies using ckit+ lineage tracing mouse models, and reporter lines, where it was shown that cardiac c-kit+ cells contribute to cardiomyocytes only minimally, but mainly and substantially generate cardiac endothelial cells (52–54). The debate continues, with recent publications showing that selected c-kit+ cardiac cells contain a low level (about 1%) of clonal cells that can be expanded and differentiated into spontaneously beating cardiomyocytes (55) and that c-kit expression can be identified on both CPCs and a subpopulation of cardiomyocytes and is upregulated in response to pathological stress (56).

Also in 2003, another population of cardiac progenitor cells was identified, the stem cell antigen 1 (sca-1+) cells, in the mouse heart, having stem-like self-renewal characteristics and the ability to home to the injured myocardium (20). Later it was shown that this cell population led to increased ejection fraction and neoangiogenesis, after injection into the acutely infarcted mouse heart (57). It was also shown that sca-1+ CPCs contribute to the generation of cardiomyocytes during normal aging and after injury sca-1+ cells were induced to differentiate to three cardiac cell types (44). In line with these observations, a genetic deletion of sca-1 caused primary cardiac defects in heart contractility, an impaired damage response and reduced CPC proliferation (58). Although the human sca-1 isoform does not exist, a sca-1-like cell population has been isolated from the human heart using the murine antibody and has been extensively studied by the Goumans group (59). In a similar manner to the isolation of a clonal c-kit+ population, clonal cells have been identified within selected sca-1+, PDGRFα+ mouse cells and have been shown to improve cardiac function after MI, but again by a largely paracrine mechanism (60).

In addition, a population of progenitor cells were derived, via the formation of cardiospheres, from cells migrating from adult human and murine heart explants, called cardiospherederived cells (CDCs) (21). It was reported that human CDCs have the ability to self-renew in culture and express the endothelial kinase insert domain receptor (KDR) and other known stem cell markers (CD-31, CD-34, c-kit, and sca-1) (21). This heterogeneous cell population has also been shown to have beneficial effects, both in animal models (22, 61) and in a clinical trial (62), but again via the release of paracrine factors.

CPCs have been also identified by markers of embryonic origin, like Insulin gene enhancer protein 1 (Isl1) and NK2 homeobox 5 (Nkx2.5). Isl1 is a cardiac transcription factor expressed in second heart field progenitors and cardiac neural crest cells, involved in cardiovascular development, and leading to severely deformed hearts in rodents after genetic deletion (24, 63). Nkx2.5, a homeobox-containing transcription factor, has been identified via its involvement in cardiac looping (64, 65). Isl1+ and Nkx2.5+ CPCs have been shown to differentiate into

major cardiac lineages, mainly contributing to proepicardium during development (25).

Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of blastocysts from donated fertilized eggs (66). Human ESCs have the ability to continuously proliferate in an undifferentiated state and, when given the appropriate signals, will differentiate into any cell type. An interesting tool for cell therapy that originated from this field, are the in vitro–generated, stem cell–derived cardiomyocytes (SC-CMs). These have been shown to integrate structurally and functionally with healthy host cardiac tissue in vivo in various studies (26, 67, 68). These cells show great promise, but there are ethical concerns using hESCs in the clinic and the risk of teratoma formation (69). In 2007, Yamanaka's group were the first to report the reprogramming of human somatic cells into induced pluripotent stem cells (iPSCs), by overexpression of the transcription factors: Oct4, Sox2, KLF4, and c-myc (70). The reprogrammed hiPSCs resembled hESCs and had the ability to self-renew while maintaining pluripotency (70). Human iPSCs can be produced from patientspecific somatic cells, therefore overcoming the problem of immune rejection and the ethical concerns of using hESCs (69). hiPSCs have been shown to improve cardiac function, albeit with limited donor cell retention (30, 31) and used extensively as in vitro human-cell-based models to study basic biology and development (71), to model diseases (72) and to screen for drugs (73, 74). This is particularly important for the heart, since adult cardiomyocytes do not survive in vitro, as morphological and functional changes occur in long-term culture and so there has been no easy way to determine whether the effect of genetic mutations or of drug compounds that were observed in animal models would also be seen in a human cardiomyocyte. However, despite the promising in vivo results, the initiation of beating in SC-derived cardiomyocytes does not mean that these cells have the maturity or metabolic characteristics of mature cardiomyocytes found in the healthy heart (75). Studies have shown that SC-derived cardiomyocytes have immature calcium handling (76) and a response to drugs more akin to cardiomyocytes from the failing heart (77).

The effect of the transplantation environment on enhancing the maturation of human pluripotent SC-derived cardiomyocytes has been studied in rats. Despite their capacity to survive and form grafts, they failed to improve adverse remodeling or overall cardiac function after chronic MI (28). Approaches to enhance their efficacy, via preconditioning the cells and host environment, are currently being investigated [reviewed here (78)].

## CARDIAC METABOLISM

The heart is a fascinating organ that beats 100,000 times a day and pumps 7,200 L of blood through the body, in the same period using 35 L of O<sup>2</sup> for energy production. It requires about 6 kg of adenosine triphosphate (ATP), which it utilizes at a rate of 30 mg per second to sustain myocardial contraction and maintain ion homeostasis (79, 80). Since the heart has a low capacity for energy storage (81), an array of metabolic networks guides ATP production rates, based on demand. The heart has been characterized as a metabolic omnivore, being able to use a variety of substrates for energy production [see reviews (82, 83)]. Glucose, pyruvate, triglycerides, glycogen, lactate, ketone bodies, fatty acids (FAs) of different chainlengths and certain amino acids are among the energy-providing substrates of the heart. It is responsible for almost 10% of the whole body fuel consumption; with FAs accounting for 70% of ATP production and carbohydrates for the remaining 30%.

Energy, in the form of ATP, can be produced in the cytosol via glycolysis (**Figure 2**); catabolism of glucose derived from carbohydrates. The end-product of glycolysis is pyruvate, which can be further reduced to produce lactate. In case of carbohydrate shortage, gluconeogenesis of pyruvate, reoxygenation of lactate or glycerol metabolism, can be used as sources of glucose synthesis (82, 84). Alternatively, pyruvate can enter the mitochondria in the form of acetyl-coenzyme A (Acetyl-CoA) and be oxidized in the TCA cycle (also known as the Krebs cycle), in a process called oxidative phosphorylation (85). The reducing equivalents of this chained reaction act as hydrogen carriers (Nicotinamide Adenine Dinucleotide Hydrogen; NADH and Flavin Adenine Dinucleotide Hydrogen; FADH2) and enter the electron transport chain (ETC). There the coupled-transfer of electrons and H<sup>+</sup> creates an electrochemical proton gradient that leads to the production of ATP.

ATP can also be generated by the degradation of lipids (including triglycerides) into FAs, which are metabolized in the mitochondria via beta-oxidation (**Figure 2**), which converts Fatty Acyl-CoA to Acetyl-CoA for flux into the TCA cycle and ATP synthesis as above (86). Under aerobic conditions, more than 95% of ATP production comes from oxidative phosphorylation (86) and in the healthy heart 50–80% of the energy is generated via beta-oxidation (87). Oxidative phosphorylation yields 36 ATP/glucose molecule, being more efficient than glycolysis (2 ATP/glucose). Lipids, due to their reduced state are more oxygen-demanding than glucose (producing 2.8 ATP/O2, vs. 3.7 ATP/O2), but they are more energy-dense with a much higher yield of ATP/carbon (depending on the chain length of the parent FA) (79, 88).

The heart has a remarkable ability to adapt to changes in its physiological state by selecting the most efficient substrate, depending on the conditions of its environment (82). For example, as FAs require more oxygen than glucose, to generate the same amount of ATP (82, 89), upregulation of the hypoxiainducible factor (HIF) under hypoxia has been shown to increase

glycolysis and suppress mitochondrial oxidative metabolism (83, 90), shifting toward the more oxygen-efficient fuel: glucose. A network of interrelated signaling pathways control the flux of glucose and fatty acid metabolism to enable the heart to switch substrates rapidly. This was first described as the glucose-fatty acid cycle by Randle in 1963 (91), but the complexity of this network is yet to be fully explored (92). Under conditions of starvation, acetyl-CoA in the liver can form ketone bodies, that are water-soluble forms of this metabolite, and can be efficiently used by the brain and other oxidative organs (82) (**Figure 2**).

# Cardiac Metabolism During Development—Metabolic Switch (1)

The fetal heart is adapted to an environment of low oxygen and low fatty acid content, so fetal cardiomyocytes are highly dependent on glycolysis for ATP production (93). In addition, lactate availability allows for energy production via lactate oxidation. During development, the heart undergoes a major metabolic alteration; the main physiological changes during the transition to the post-natal stage are the increased workload, and the demand for growth, that cannot be met by glucose and lactate consumption alone (94, 95). Interestingly, immediately after birth some studies suggest that the main energy biogenesis mechanism is still glycolysis (96). The post-natal increase in both circulating levels of free FAs (due to dietary alteration and lipid content in maternal milk) and in O<sup>2</sup> levels mediates a switch from glycolysis-dependence, to predominantly relying on oxidative metabolism as mature cardiomyocytes (93, 97) (**Figure 3**). After birth, the "fetal switch" to oxidative metabolism of glucose and fatty acids has been linked to the loss of the regenerative phenotype (98). Neonatal mouse hearts can regenerate in the first postnatal week but this is lost after day 7 (99). Puente et al. investigated exposure of neonatal mice to hyperoxia (100% oxygen) or mild hypoxia (15% oxygen) and found that hyperoxia induced cardiomyocyte cell cycle arrest after birth whereas hypoxia prolonged the regenerative window (98). They hypothesized that this effect resulted from increased oxidative stress that accompanied the induction of oxidative metabolism and showed that treatment with an antioxidant resulted in a significant increase in cardiomyocyte mitosis in the first weeks after birth. During the early postnatal period, as cardiac energy demands increase, the number of mitochondria in cardiomyocytes increases dramatically (100, 101). A recent study has shown that the HIF1 signaling localisation pattern controls the embryonic switch toward oxidative metabolism, disruption of which affects cardiac maturation. The cardiac compartment where HIF was absent, the trabeculae, has increased oxidative metabolism, as well as higher mitochondrial content (102). Several studies demonstrated an increase in PPAR coactivator 1α (PGC-1), as well as Peroxisome Proliferator-Activated Receptor α (PPARα), mRNA levels in mice or rats during development (103, 104). The regulatory mechanism involves genes encoding several key mitochondrial ETC proteins, specifically the transcription factors nuclear respiratory factors-1 and−2 (NRF-1 and−2) (103, 105). Disruption of the ETC function, during cardiac development, leads to disrupted mitochondrial organization in the cardiomyocytes, resulting in perturbed sarcomere formation and contraction (106). Various

mouse models have revealed that disruption in mtDNA processes leads to embryonic lethality (107). In addition, signaling pathways involving the Nuclear factor of activated T-cell (NFAT) family of transcription factors play a role in mitochondrial biogenesis and cardiac function. Mice with nfatc3 and nfatc4 deletion had abnormal mitochondrial structure and reduced oxidative capacity, eventually dying after E10.5 (108). The ERRα/ERRγ axis plays a crucial role in mitochondrial energy production, as well, and their deletion leads to premature lethality in mice (109). Disruption of the ERRγ gene leads to impaired mitochondrial function, eventually blocking the switch from glycolysis to oxidative phosphorylation and leading to early prenatal death in mice (110).

# Cardiac Metabolism in MI and HF—Metabolic Switch (2)