Endothelial cells are progenitors of cardiac pericytes and vascular smooth muscle cells

Abstract

Mural cells of the vessel wall, namely pericytes and vascular smooth muscle cells, are essential for vascular integrity. The developmental sources of these cells and molecular mechanisms controlling their progenitors in the heart are only partially understood. Here we show that endocardial endothelial cells are progenitors of pericytes and vascular smooth muscle cells in the murine embryonic heart. Endocardial cells undergo endothelial–mesenchymal transition and convert into primitive mesenchymal progenitors expressing the platelet-derived growth factor receptors, PDGFRα and PDGFRβ. These progenitors migrate into the myocardium, differentiate and assemble the wall of coronary vessels, which requires canonical Wnt signalling involving Frizzled4, β-catenin and endothelial cell-derived Wnt ligands. Our findings identify a novel and unexpected population of progenitors for coronary mural cells with potential relevance for heart function and disease conditions.

Introduction

Ischaemic heart disease, which is typically caused by dysfunction of the coronary vasculature, is the leading cause of death worldwide1,2. The integrity, perfusion and function of blood vessels inside and outside of the heart critically rely on the interaction of different cell types3,4,5,6. While a monolayer of endothelial cells (ECs) encloses the vessel lumen, mural cells, namely pericytes, are associated with the abluminal surface of capillaries. Vascular smooth muscle cells (vSMCs), that is, mural cells covering larger calibre arteries and veins, are thought to be closely related to pericytes and, in heart, are even derived from pericytes7,8,9. Mural cells stabilize vessels through physical and molecular interactions with adjacent ECs, and absence of mural cells leads to vascular leakage and haemorrhaging3,4,7. Pericytes and their progenitors have high clinical relevance and, accordingly, several studies have explored the potential of these cells for cardiac regeneration and heart tissue engineering10,11,12,13,14,15. Remarkably, mural cells expressing the markers platelet-derived growth factor receptor β (PDGFRβ), CD146 and NG2/Cspg4 have been proposed to function as mesenchymal stem cells in multiple organs and act as myofibroblast progenitors during injury-induced fibrosis16,17,18.

Despite the great importance of mural cells, the precise properties and developmental sources of these cells remain poorly understood. In the heart, previous studies have shown that progenitor cells derived from the embryonic epicardium invade into the myocardium and give rise to cardiomyocytes and mural cells19,20,21. It was also shown that these cardiac mural cell progenitors express PDGFRβ and require PDGFRβ-driven phosphoinositide 3′ kinase (PI3K) signalling for their migration21. In addition to PDGFRβ, the related receptor PDGFRα is expressed by epicardial cells. Combined tissue-specific inactivation of the genes for both PDGF receptors disrupted the migration of epicardial progenitors into the myocardium, while it had no effect on the proliferation or survival of these cells. Furthermore, it was also shown that PDGFRα is specifically required for the formation of cardiac fibroblast, whereas only PDGFRβ is indispensable for mural cell development22. However, genetic lineage tracing indicated that not all cardiac mural cells are derived from epicardial cells19,20,21. Likewise, inactivation of the Pdgfrbgene (encoding PDGFRβ) in epicardial cells did not eliminate all cardiac mural cells21 arguing for additional, so far unknown developmental sources of pericytes and vSMCs in the heart.

In this study, we have identified endocardial ECs as novel progenitors for mural cells in the heart with the help of genetic lineage tracing and gene inactivation experiments. While endothelial and mural cells belong to distinct lineages in most tissues and model systems, our work also establishes that this separation is not maintained in the developing cardiac vasculature. Thus, mural and endothelial cells develop from a common progenitor population during early stages of heart development.

Results

Molecular markers of cardiac mural cells

As mural cells are known to show heterogeneous expression of molecular markers7, we first characterized mural cells in sections of murine heart at postnatal day (P) 6. In these experiments, Tg(Cspg4-dsRed.T1)1Akik reporter mice were used to identify the in vivo expression pattern of NG2. In Pdgfratm11(EGFP)Sor knockin reporter mice, PDGFRα expression is detected via a nuclear green fluorescent protein (H2B-GFP) reporter. PDGFRβ+ cells and their progeny were stably labelled with Pdgfrb(BAC)-CreERT2 transgenic mice, which were recently generated by our group. These mouse lines (Supplementary Table 1) in combination with immunostaining showed that the majority of mural cells associated with coronary capillaries were positive for platelet-derived growth factor receptor β (PDGFRβ) and the proteoglycan NG2 but lacked PDGFRα expression (Supplementary Fig. 1a–e). Only few cardiac mural cells expressed CD13 or desmin (Supplementary Fig. 1d,f), which have been used as pericyte markers in other organs. Desmin was also prominently expressed by cardiomyocytes (Supplementary Fig. 1f). On the basis of this analysis, we defined capillary-associated mural cells as PDGFRβ+ NG2+ PDGFRα- cells.

Identification of putative cardiac mural cell progenitors

In contrast to postnatal heart, PDGFRβ+ cells at midgestation were not associated with myocardial capillaries, but were instead confined to large clusters located in atrioventricular canal (AVC) and outflow tract (OFT; Fig. 1a–c; Supplementary Fig. 2a). Expression of PDGFRβ protein was absent in epicardial cells at embryonic day (E) 10.5, and, likewise, PDGFRβ expression was not detectable in cells of the proepicardial organ at E9.5 (Supplementary Fig. 2a). In addition to the large clusters in the AVC and heart valves, some PDGFRβ+ cells were detected in the myocardium, ventricular septum and developing valves at E12.5 (Fig. 1d–f). From E14.5, PDGFRβ+ cells were abundant in myocardium and closely associated with ECs (Fig. 1g–i).

Figure 1: Developmental distribution and molecular properties of PDGFRβ+ cells.

(a–i) Distribution of PDGFRβ (green) immunostained cells from E10.5 to E14.5. Heart sections from wild-type mice were stained for PDGFRβ (green) and isolectinB4 (blue). Arrows indicate PDGFRβ+ cells, arrowheads mark PDGFRβ- epicardial cells at the indicated stages. Panels at the bottom (b,c,e,f,h,i) are higher magnifications of insets in (a,d,g), respectively. A, atrium; V, ventricle; AVC, atrioventricular canal; Epi, epicardium; Myo, myocardium; VS, ventricular septum. (j–l) Transient co-expression of PDGFRβ (red) and PDGFRα (Pdgfratm11(EGFP)Sor reporter; nuclear H2B-GFP; green) during heart development. ECs, isolectinB4 (blue). Arrows indicate GFP+ PDGFRβ+ double-positive cells in the E10.5 AVC (j) and E14.5 myocardium (k). Double-positive cells were very rare in the E16.5 myocardium (l), whereas GFP- PDGFRβ+ mural cells (arrowheads) and GFP+ PDGFRβ- interstitial cells (arrows in l) were abundant.