Clonal and diverse: revisiting cardiac endothelial cells after myocardial infarction

This editorial refers to ‘Single-cell transcriptome analyses reveal novel targets modulating cardiac neovascularization by resident endothelial cells following myocardial infarction’^†, by Z. Li et al., on page 2507.

Acute myocardial infarction (MI) is among the most frequent life-threatening medical emergencies worldwide. Ischaemic myocardial damage frequently leads to chronic heart failure, the leading cause of morbidity and mortality in industrialized countries.¹ At the time of diagnosis, long-term survival of patients suffering from severe heart failure is worse than for most types of cancer.² Following MI, the process of myocardial repair represents a critical determinant for transition to heart failure. Therefore, development of novel therapeutic strategies aimed at limiting residual myocardial damage and preserving long-term cardiac anatomy and function after MI are vital.

Over the past decade of research, endothelial cells emerged as key cells involved in cardiac repair. Endothelial cells actually represent the largest non-cardiomyocyte cell population within myocardial tissue.³ Following MI, rapid restoration of myocardial blood vessels is essential for prolonging survival of the injured myocardium. Efficient perfusion provided by microvessels is required to prevent cardiomyocyte death, which contributes to infarct expansion, left ventricular dilation, and adverse cardiac remodelling. In fact, de novo formation of blood vessels (angiogenesis) has the potential to salvage ischaemic myocardium at early stages after MI, and is also essential for preventing the long-term transition to heart failure.⁴ The main cytokine regulating endothelial cell proliferation is vascular endothelial growth factor (VEGF), which is secreted by monocytes and macrophages, and induces angiogenesis via VEGF receptor 2. Newly sprouting vessels then augment oxygen and nutrient availability in the infarcted heart. However, clinical trials supplementing VEGF have not yielded the expected results so far,⁵ indicating that our understanding of the mechanisms underlying post-MI angiogenesis and repair is incomplete.

In this issue of the European Heart Journal, Li et al. demonstrate that the structural integrity of murine adult cardiac blood vessels after MI is maintained through clonal proliferation of resident endothelial cells.⁶ Taking advantage of an endothelial cell-specific multispectral lineage-tracing mouse (Pdgfb-iCreERt2-R26R-Brainbow2.1), the authors found significantly more single fluorophore-labelled endothelial patches in the infarct border at 7 days post-MI when compared with healthy hearts. As clone size did not differ between colours in either group, each vascular patch was most probably formed through clonal expansion of a single confetti-positive endothelial cell and not the merger of multiple smaller clones of the same colour. The functionality of new blood vessels formed by clonal endothelial cell expansion was confirmed by intravenous infusion with isolectin-B4.

The authors went on to isolate endothelial cells from healthy and ischaemic murine hearts using FACS (fluorescence-activated cell sorting) at 7 days post-MI for single cell RNA-sequencing. Principal component analysis followed by 3D t-distributed stochastic neighbour embedding (t-SNE) revealed 10 transcriptionally discrete cell states in the healthy and post-ischaemic heart. Subsequently, Gene Ontology term enrichment analysis was used to predict the clusters’ functional identities. Of note, one cluster (#10), comprised predominantly of cells from the MI group, displayed a high expression of genes involved in proliferation and cell cycle regulation. On the single-gene level, the authors followed up on plasmalemma vesicle-associated protein (PLVAP), as Plvap expression was found to be highest in clusters predominantly comprised of endothelial cells from the MI group. Using immunofluorescence histological staining, Li et al. confirmed increased PLVAP abundance in diseased vs. healthy murine and, importantly, also human myocardium. Co-localization of PLVAP with fluorophore-labelled endothelial cells in Pdgfb-iCreERt2-R26R-Brainbow2.1 mice post-MI suggested high Plvap expression in clonally expanding endothelial cells. Finally, small interfering RNA (siRNA)-mediated gene silencing of Plvap in cell culture of human umbilical vein endothelial cells resulted in significantly diminished proliferation.

The authors’ observation that clonal expansion of pre-existing cardiac endothelial cells represents the predominant mechanism for angiogenesis in the post-MI myocardium expands on the findings of two previous studies.^7,8 Viewed together with previous data investigating the source of new vascular endothelium,⁹ a relevant contribution of bone marrow- or circulation-derived progenitor cells to the pool of adult myocardial endothelial cells (in fact, to any cardiac cells except leucocytes) appears extremely unlikely. On the other hand, bone marrow-derived cells, most probably innate immune cells, influence cardiac neovascularization via paracrine mechanisms such as VEGF.¹⁰ Clinical trials investigating transplantation of bone marrow-derived cells into the myocardium have failed.¹¹ This indicates that we need to rethink strategies for optimizing myocardial repair; we believe that shaping endogenous immune responses,¹² enhancing angiogenesis, and in situ cell reprogramming¹³ are among the most exciting yet feasible strategies.

Of note, the founding number of recombined events in the cardiac vasculature did not change before or after ischaemic injury, leading to the authors’ conclusion that ischaemia-induced clonal proliferation might be initiated by a subpopulation of endothelial cells with progenitor-like properties. Indeed, further characterization of this hypothetical type of cell with enhanced proliferative and clonogenic potential in response to organ damage could open up a whole new avenue of research. However, the reason behind one endothelial cell staying quiescent while another clonally expands in response to MI might also lie in the locally diverse micromilieu and the properties of surrounding cells, possibly explaining only moderately altered transcriptome data in samples isolated by laser capture microdissection.⁸ Proper lineage-tracing experiments would help to identify the endothelial cell hierarchy in adult myocardium, similar to previous work performed in the bone marrow.¹⁴

The second major finding of Li and colleagues lies in the identification of 10 myocardial endothelial cell subsets with distinct gene expression signatures in the healthy and post-MI myocardium as assessed by single-cell RNA-sequencing. Even though not the first study to apply this technology to cardiac endothelial cells,^15,16 the new data provide the first in-depth characterization of endothelial cell heterogeneity and plasticity in response to MI. Although the richness of this resource is unquestionable, it raises a number of questions. Where is each of the 10 subsets localized within the vascular network of the myocardium? For instance, are proliferating cell clusters located in specific niches or adjacent to VEGF-rich macrophages? Given that only day 7 day post-MI has been investigated, how would the clustering of cardiac endothelail cells evolve at earlier and later time points? This study was performed in a mouse with healthy blood vessels and a normal healing response in the absence of any comorbidities—what happens in mice or humans with high cardiovascular risk, atherosclerosis, and dysfunctional endothelial cells? Can similar clusters be found in other ischaemic organs, i.e. the brain and skeletal muscle? Could endothelial cells with enhanced proliferative and clonogenic potential be identified, and what factors give rise to their preferential expansion? Which clusters would comprise the proliferative vs. quiescent confetti-positive cells described in the first part of the manuscript?

Taken together, the work by Li et al. expands our current understanding of clonal dynamics and transcriptional heterogeneity of myocardial endothelial cells in response to ischaemic injury. Further in-depth mechanistic research on the molecular mechanisms underlying cardiac neovascularization has the potential to foster the development of novel therapeutic approaches aimed at improving angiogenesis and myocardial repair.

Funding

This work was funded in part by federal funds from the National Institutes of Health NS108419, HL139598, and HL125428; from the European Union’s Horizon 2020 research and innovation programme 667837; and the MGH Research Scholar Program. D.R. was supported by the German Research Foundation RO5071/1-1.

Conflict of interest: M.N. has been a paid consultant or received research support from Novartis, GSK, Medtronic, Verseaux, Sigilon, Alnylam, IFM therapeutics, and Molecular Imaging Inc. The authors declare no conflicts of interest, financial or otherwise.

The opinions expressed in this article are not necessarily those of the Editors of the European Heart Journal or of the European Society of Cardiology.