Article, see p 1647

Fibroblasts are traditionally defined as connective tissue cells that populate most mammalian organs and are involved in formation, maintenance, and degradation of the extracellular matrix (ECM) scaffold, thereby contributing to the mechanical properties of tissues. This definition, however, fails to convey the remarkable heterogeneity of fibroblasts residing in mammalian tissues and their extensive functional repertoire in homeostasis and disease.

After injury, fibroblasts secrete a wide range of mediators beyond proteins involved in ECM synthesis and metabolism and have been suggested to serve inflammatory,¹ angiogenic,² and cytoprotective³ roles in a wide range of pathological conditions. In addition to effects mediated through secretion of ECM proteins, bioactive mediators, or microRNA-containing exosomes, injury-site fibroblasts have also been suggested to exhibit phenotypic plasticity, undergoing conversion to endothelial cells through a process called mesenchymal to endothelial transition. In a model of myocardial ischemia/reperfusion, genetic fate mapping experiments suggested that fibroblasts can convert to endothelial cells through a p53-dependent pathway, thus contributing to neovascularization.⁴ However, these findings were challenged by a systematic lineage tracing study that used 3 endothelial inducible Cre lines and 6 different genetic fibroblast tracing tools to demonstrate that fibroblasts do not undergo endothelial conversion and that preexisting endothelial cells are almost exclusively responsible for neovascularization in reperfused myocardial infarction.⁵ Thus, whether fibroblasts can acquire endothelial cell characteristics in injured hearts or other organs remains a hotly debated question.

In the current issue of Circulation, Meng et al⁶ report that a subpopulation of interstitial cells with fibroblast-like features contributes to angiogenesis and improves recovery of ischemic skeletal muscle by converting to endothelial cells. The authors used lineage tracing with an FSP (fibroblast-specific protein)–1 Cre line to identify a nonmyeloid, CD144⁺ population of cells derived from FSP-1⁺ progeny that expanded after hindlimb ischemia and expressed endothelial cell genes. Generation of these angiogenic cells, neovascularization, and recovery of the ischemic skeletal muscle were dependent on Toll-like receptor–3 signaling and on a nuclear factor κB–mediated inflammatory cascade.

Single-cell RNA sequencing of the cells derived from FSP-1⁺ progeny identified several distinct clusters of fibroblast-like cells, 1 of which had similar characteristics to the CD144⁺ endothelioid population, and formed endothelial cell–like networks in Matrigel. The authors interpret the findings as suggestive of a critical contribution of fibroblasts to the postischemic angiogenic response through their direct conversion to endothelial cells that is mediated via innate immune signaling. The study highlights the phenotypic and functional heterogeneity of fibroblasts in vivo, based on a superbly designed combination of lineage tracing and single cell transcriptomic data. However, considering the controversies in the field and the limitations of the mouse lines used for fibroblast labeling, does this study provide definitive in vivo proof of fibroblast to endothelial conversion?

In the early years of cell-specific in vivo targeting, FSP-1/S100A4 was used as a fibroblast-specific marker in many high-profile studies.⁷ However, over the last 10 years, extensive evidence has emerged challenging its specificity. In addition to its expression by fibroblast-like cells, FSP-1 is localized in myeloid cells and plays an important role in regulation of macrophage migration.⁸ In the injured liver, FSP-1 predominantly labels a subpopulation of inflammatory macrophages.⁹ In normal, infarcted, and remodeling hearts, the majority of FSP-1–positive cells express markers of hematopoietic or endothelial cells.¹⁰ Moreover, a recently published study using single cell transcriptomics in mouse skeletal muscle did not identify FSP-1 as a fibroblast-enriched gene.¹¹

Thus, the exclusive use of the FSP-1 Cre driver limits the conclusions of the current study on the fibroblast origin of the angiogenic endothelial cells in ischemic muscle. However, the use of single cell transcriptomics overcomes many of the shortcomings of the lineage tracing approach. The subsets of FSP-1–derived cells that expressed endothelial genes also expressed several fibroblast-associated genes, including Vim (vimentin), Lamc1, Tcf4, Nav1, and Igfbp6. Expression of ECM genes, the major defining characteristic of fibroblasts, was less consistent: The angiogenic cells expressed Fn1 and type IV collagen genes but had negligible levels of type I collagen genes and of the myofibroblast-associated genes Acta2 (α–smooth muscle actin) and Postn (periostin). Moreover, expression of PDGFRa, a commonly used fibroblast marker, was not detected. Thus, the transcriptomic data suggest that a population of FSP-1⁺ “fibroblast-like” cells may contribute to endothelial cell expansion in ischemic angiogenesis.

Are these cells truly fibroblasts? The answer to this question depends on how we define the fibroblast. Under a rigid definition that requires synthesis of significant amounts of fibrillar collagens, the cells identified by Meng et al may not be considered mature fibroblasts. However, this is a semantic argument. In multicellular organisms, classification of cells into specific types satisfies our need to bring order into the chaos of cellular diversity, which is necessary to understand complex biological responses. For well-differentiated cells with unique characteristics or functional properties (such as cardiomyocytes and neurons), the criteria used for identification are clear and unambiguous. In other cases (such as in the biology of immune cells), decades of research have contributed to identification of functionally distinct and well-defined subpopulations. In contrast, identification criteria for fibroblasts have remained vague since the first description of these cells more than 150 years ago, and characterization of specific subpopulations with distinct functional properties is lacking. This may reflect, at least in part, the absence of polarized subpopulations of fibroblasts, which could be characterized by unique and distinct markers. Tissue fibroblast populations may represent a palette of nuanced phenotypes, rather than cells that can be unambiguously subclassified on the basis of a few specific markers (Figure [A]). The diversity of fibroblast subpopulations is amplified in injured tissues, as damage-associated molecular patterns released by injured cells activate innate immune pathways, and infiltrating leukocytes secrete cytokines and growth factors that alter the cellular microenvironment. Single cell transcriptomics have revealed interesting patterns of fibroblast activation in injured tissues^12,13 that may correspond to distinct functional properties.

Regardless of the terminology used to describe the angiogenic cells of FSP-1 lineage, the findings of the study add to a growing body of evidence suggesting an important role for fibroblast-like interstitial cells in angiogenesis (Figure [B]). Considering their predominant role as matrix-producing and matrix-remodeling cells, it is not surprising that fibroblasts have been suggested to contribute to angiogenesis through modulation of the ECM network that is necessary for formation of a lumen and via secretion of specialized matrix proteins that regulate endothelial and mural cell responses.² Fibroblasts can also serve as a source of angiogenic mediators, such as vascular endothelial growth factor¹⁴ and angiogenic chemokines. Studies in a model of myocardial infarction identified an FSP-1⁺ fibroblast-like subpopulation with proangiogenic properties that is functionally distinct from matrix-secreting α–smooth muscle actin⁺ myofibroblasts and expresses the angiogenic chemokines CXCL12 and CXCL16.¹⁵

Figure. The functional pluralism of fibroblasts in injured and healing tissues. A, The palette of fibroblast phenotypes in normal and injured tissues. Fibroblasts exhibit remarkable heterogeneity. Although characterization of tissue fibroblasts remains incomplete (especially in humans), their diversity does not appear to involve polarized phenotypes but rather a palette of nuanced profiles. Fibroblast diversity is amplified after injury, as innate immune signals triggered by damage-associated molecular patterns (DAMPs), cytokines, and specialized matrix proteins trigger expansion and activation of certain subsets and emergence of additional subpopulations with distinct transcriptomic and functional characteristics. The role of fibroblasts in injured tissues extends beyond the secretion, processing, and remodeling of the extracellular matrix (ECM). Important functions of fibroblast subpopulations in injured and remodeling tissues may involve regulation of inflammation, cytoprotective and regenerative actions, and modulation of angiogenic responses. B, Angiogenic actions of fibroblasts. Injury may be associated with generation of angiogenic fibroblasts that stimulate neovessel formation through several different mechanisms, including secretion of angiogenic cytokines, chemokines, and growth factors, secretion of matricellular proteins with angiogenic properties, and release of exosomes containing microRNAs. The current study suggests that in the ischemic skeletal muscle, a subpopulation of fibroblast-like cells derived from FSP (fibroblast-specific protein)–1⁺ progeny acquire characteristics of endothelial cells (EC) and contribute to angiogenesis and functional recovery. The findings support the controversial concept of mesenchymal to endothelial transition (MendoT).

Whether fibroblasts significantly contribute to angiogenesis by directly converting to endothelial cells through mesenchymal to endothelial transition remains unclear. Although the current study supports this concept, the relative significance of this cellular process is not established. Experiments ablating the angiogenic FSP-1 lineage cells were not performed. Moreover, the critical role of nuclear factor κB signaling in FSP-1⁺ cells in mediating angiogenesis, suggested by the genetic loss-of-function experiments using the FSP1-Cre deleter, may be independent of fibroblast to endothelial conversion, reflecting actions of nuclear factor κB on FSP-1–expressing myeloid cells or effects on secretion of fibroblast-derived angiogenic mediators. The origin of endothelial cells in injury-related angiogenesis and the relative contribution of nonendothelial cell types are best addressed through systematic study of the relative contribution of preexisting endothelial cells and nonendothelial cell types, rather than by exclusively focusing on the fate of a specific subpopulation.

Dr Frangogiannis’s laboratory is supported by National Institutes of Health grants R01HL76246, R01HL85440, and R01HL149407 and by Department of Defense grants PR151029, PR151134, and PR181464.

None.

The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.

https://www.ahajournals.org/journal/circ