Developmental Heterogeneity of Cardiac Fibroblasts Does Not Predict Pathological Proliferation and Activation

Abstract

Rationale: Fibrosis is mediated partly by extracellular matrix–depositing fibroblasts in the heart. Although these mesenchymal cells are reported to have multiple embryonic origins, the functional consequence of this heterogeneity is unknown. Objective: We sought to validate a panel of surface markers to prospectively identify cardiac fibroblasts. We elucidated the developmental origins of cardiac fibroblasts and characterized their corresponding phenotypes. We also determined proliferation rates of each developmental subset of fibroblasts after pressure overload injury. Methods and Results: We showed that Thy1 + CD45 − CD31 − CD11b − Ter119 − cells constitute the majority of cardiac fibroblasts. We characterized these cells using flow cytometry, epifluorescence and confocal microscopy, and transcriptional profiling (using reverse transcription polymerase chain reaction and RNA-seq). We used lineage tracing, transplantation studies, and parabiosis to show that most adult cardiac fibroblasts derive from the epicardium, a minority arises from endothelial cells, and a small fraction from Pax3-expressing cells. We did not detect generation of cardiac fibroblasts by bone marrow or circulating cells. Interestingly, proliferation rates of fibroblast subsets on injury were identical, and the relative abundance of each lineage remained the same after injury. The anatomic distribution of fibroblast lineages also remained unchanged after pressure overload. Furthermore, RNA-seq analysis demonstrated that Tie2-derived and Tbx18-derived fibroblasts within each operation group exhibit similar gene expression profiles. Conclusions: The cellular expansion of cardiac fibroblasts after transaortic constriction surgery was not restricted to any single developmental subset. The parallel proliferation and activation of a heterogeneous population of fibroblasts on pressure overload could suggest that common signaling mechanisms stimulate their pathological response.

The extracellular matrix in myocardial injury, repair, and remodeling

“…Mechanical stress due to pressure overload activates the renin-angiotensin-aldosterone system (RAAS), triggering inflammatory signaling and leading to downstream stimulation of TGF-β cascades. Neurohumoral mediators, cytokines, and growth factors directly stimulate a fibrogenic program, triggering myofibroblast conversion and stimulating synthesis of large amounts of structural matrix proteins (103)(104)(105)(106). Stress-induced fibroblast activation in the pressure-overloaded myocardium may also be indirect, at least in part, requiring stimulation of fibrogenic cascades in cardiomyocytes and immune cells.…”

Developmental origin and lineage plasticity of endogenous cardiac stem cells

“…It is generally thought that cardiac precursor cells represent multipotent lineage progenitors for cardiomyocytes, the endocardium and smooth muscle cells, a view that has been much influenced by studies of differentiating embryonic stem cells (ESCs) (summarized in Box 1). However, recent lineage-tracing studies have shown that although FHF and SHF cells arise from a common progenitor, probably in the pre-gastrulation embryo, their territories become geographically separate (Ali et al, 2014;Buckingham et al, 2005;Meilhac et al, 2004). FHF progenitors that form the primary heart tube and future left ventricle (Buckingham et al, 2005;Lescroart et al, 2014) are unipotent for cardiomyocytes or endothelial cell fates (Cohen-Gould and Mikawa, 1996;Lescroart et al, 2014).…”

“…An early CRE-based lineage-tracing experiment suggested that a subset of cardiac stromal fibroblasts arise de novo via endothelial-tomesenchymal transition (EndMT) from adult endothelial cells (Zeisberg et al, 2007). However, more recent lineage-tracing studies have shown that the proliferation and expansion of resident fibroblasts during cardiac hypertrophy accounts for almost all cardiac fibroblasts (Ali et al, 2014;Moore-Morris et al, 2014). Around 80% of the fibroblasts had their origins in the epicardium, with the remainder derived from the endocardium via EndMT (Ali et al, 2014;Moore-Morris et al, 2014), although a limited number of adult fibroblasts in the right atrium and outflow tract region might derive from neural crest cells (Ali et al, 2014).…”

“…However, more recent lineage-tracing studies have shown that the proliferation and expansion of resident fibroblasts during cardiac hypertrophy accounts for almost all cardiac fibroblasts (Ali et al, 2014;Moore-Morris et al, 2014). Around 80% of the fibroblasts had their origins in the epicardium, with the remainder derived from the endocardium via EndMT (Ali et al, 2014;Moore-Morris et al, 2014), although a limited number of adult fibroblasts in the right atrium and outflow tract region might derive from neural crest cells (Ali et al, 2014). Importantly, the developmental origin of adult stromal fibroblasts does not appear to affect the cellular fibroblast phenotype, transcriptional profile, response to apoptotic stimuli or proliferation rate (Ali et al, 2014).…”

“…Around 80% of the fibroblasts had their origins in the epicardium, with the remainder derived from the endocardium via EndMT (Ali et al, 2014;Moore-Morris et al, 2014), although a limited number of adult fibroblasts in the right atrium and outflow tract region might derive from neural crest cells (Ali et al, 2014). Importantly, the developmental origin of adult stromal fibroblasts does not appear to affect the cellular fibroblast phenotype, transcriptional profile, response to apoptotic stimuli or proliferation rate (Ali et al, 2014). Based on the current literature, therefore, it appears likely that de novo EndMT makes a modest contribution to the myofibroblast population in the adult heart.…”