Targeting cardiac fibroblasts: The pressure is on

Heart failure is a major health issue, with its incidence on the rise. It is well established that fibroblast accumulation in stressed myocardium causes arrhythmias and drives adverse remodeling that underlies heart failure. Hence, defining mechanisms responsible for this surge in cardiac fibroblast numbers represents a key clinical issue. Recent studies have proposed that endothelial-to-mesenchymal transition (EndoMT) and recruitment of circulating fibroblast progenitors generate significant numbers of fibroblasts and could be targeted to alleviate fibrosis.^1,2 Here, we discuss limitations of these studies arising from markers utilized for cardiac fibroblasts, as well as our recently reported findings on cardiac fibroblast markers and origins³ and their clinical impact.

Fibroblasts are abundant interstitial cells principally known for secreting extracellular matrix, particularly collagen type I. Markers used to identify fibroblasts include CD90 (Thy1), DDR2, and fibroblast specific protein 1 (FSP1), although none are fibroblast-specific.² To label cardiac fibroblasts in heart at baseline, we used a Collagen1a1-GFP reporter line⁴ that we found was not expressed in non-fibroblast lineages including endothelium, pericytes, and haematopoietic lineages. The mesenchymal marker PDGFRα, expressed by fibroblasts,⁵ was coincident with Collagen1a1-GFP. In contrast, a marker commonly used to label fibroblasts, Fibroblast Specific Protein 1 (FSP1), was expressed in leukocytes and was absent from Collagen1a1-GFP⁺ fibroblasts.

Following pressure overload, Collagen1a1-GFP⁺ fibroblasts proliferated, and constituted the major cell population in fibrotic lesions as identified by excess collagen deposition. Interestingly, α smooth muscle actin (αSMA), used to identify activated fibroblasts, termed “myofibroblasts,” was expressed in only approximately 15% of fibroblasts in interstitial lesions, and not in perivascular lesions, suggesting that many fibroblasts would be overlooked when using this marker, at least in the context of pressure overload. Following pressure overload, FSP1 marked a significant proportion of CD45⁺ immune cells, and labeled only 50% of fibroblasts in perivascular lesions, but did not label fibroblasts in interstitial lesions, demonstrating the limitations of this marker for identifying fibrotic fibroblasts (Fig. 1). Observed heterogeneity of fibroblast gene expression in interstitial and perivascular lesions is consistent with work of others⁶ and could be further analyzed by approaches such as single cell transcriptomics and clonal analysis.

Figure 1.

Cardiac fibroblast markers and lineages following pressure-overload. At baseline and following pressure overload, all fibroblasts are marked by Collagen1a1-GFP and PDGFRα. FSP1 labels most haematopoietic cells and a subset of fibroblasts (50%) in perivascular areas. αSMA labels a small subset of fibroblasts (15%) in interstitial lesions. Spatially complementary endocardial- and epicardial-derived lineages behave similarly following pressure overload.

EndoMT of subsets of endocardial cells of the atrioventricular canal and outflow tract during midgestation generates mesenchymal valve progenitor cells. It has been suggested that an analogous process is reactivated following pressure overload, generating fibroblasts from the microvasculature.¹ However, our lineage tracing of adult endothelium with an inducible endothelial specific Cre, VEcadherin-CreERT2 did not reveal evidence that EndoMT had occurred.³ A previous study performed to assess the occurrence of EndoMT utilized an endothelial Cre, Tie1-Cre, which labels both endothelial and haematopoietic lineages.¹ It has emerged from work of others,⁷ and our work, that a majority of FSP1 expressing cells in fibrotic lesions are immune cells, not fibroblasts. These FSP1 expressing immune cells are labeled by most endothelial Cres. Thus, the assumption that FSP1 is a specific marker for fibroblasts would lead to the mistaken conclusion that fibroblasts were derived from endothelial or haematopoietic lineages.¹ Utilizing a pan-haematopoietic Cre, Vav-Cre, in conjunction with Collagen1a1-GFP and PDGFRα, we could not find evidence for any fibroblasts of haematopoietic origin in fibrotic lesions following pressure overload.³

Fibroblasts have been shown to derive from epicardial EMT during mid-gestation, although other developmental origins have not been excluded.² Using epicardial-specific Cre drivers, we observed that most, but not all, cardiac fibroblasts were of epicardial origin. We identified a second cardiac fibroblast lineage residing mainly in the septum that we show likely derives from endocardial EndoMT associated with endocardial cushion formation. In this manner, our study demonstrated that resident cardiac fibroblasts develop from 2 distinct lineages that are differentially distributed within myocardium (Fig. 1). Combined epicardial and endothelial lineage tracing accounted for approximately 95% of fibroblasts in fibrotic hearts. Although we could detect no EndoMT, it remains a possibility that other, non-fibroblast lineages labeled by epicardial Cres may also contribute to fibroblasts during fibrosis. In this regard, pericytes, derived from epicardial lineages in heart, become fibrogenic in various other organs, notably liver and kidney.^4,5

In the pressure overload mouse model, both interstitial and perivascular fibrotic lesions develop. The 2 developmentally-derived fibroblast lineages we identified contributed to both perivascular and interstitial fibrotic lesions, located within myocardial regions reflecting their original developmental distribution. Intriguingly, our analyses demonstrated that both lineages responded very similarly to pressure overload in terms of proliferation and transcriptional regulation, despite their distinct developmental origins and highlight the possibility of identifying common therapeutic targets.

Hence, our study predicts that targeting proliferation of resident cardiac fibroblasts will be critical for mitigation of fibrosis. This hypothesis is in agreement with previous clinical observations on the anti-fibrotic action of Losartan, an Angiotensin II receptor blocker, likely mediated by its action on fibroblast proliferation. However, as fibrosis occurs rapidly, developing approaches to resolve established scarring will be important. In this context, much may be learned from models of fibrosis in the liver, where fibroblasts undergo apoptosis and quiescence during scar resolution.⁴ Interestingly, we identified upregulated genes associated with “negative regulation of cell death” in cardiac fibroblasts from hypertrophic hearts, suggesting cardiac fibroblasts could be particularly resistant to apoptosis.