Resident fibroblast lineages mediate pressure overload-induced cardiac fibrosis

Abstract

Activation and accumulation of cardiac fibroblasts, which result in excessive extracellular matrix deposition and consequent mechanical stiffness, myocyte uncoupling, and ischemia, are key contributors to heart failure progression. Recently, endothelial-to-mesenchymal transition (EndoMT) and the recruitment of circulating hematopoietic progenitors to the heart have been reported to generate substantial numbers of cardiac fibroblasts in response to pressure overload-induced injury; therefore, these processes are widely considered to be promising therapeutic targets. Here, using multiple independent murine Cre lines and a collagen1a1-GFP fusion reporter, which specifically labels fibroblasts, we found that following pressure overload, fibroblasts were not derived from hematopoietic cells, EndoMT, or epicardial epithelial-to-mesenchymal transition. Instead, pressure overload promoted comparable proliferation and activation of two resident fibroblast lineages, including a previously described epicardial population and a population of endothelial origin. Together, these data present a paradigm for the origins of cardiac fibroblasts during development and in fibrosis. Furthermore, these data indicate that therapeutic strategies for reducing pathogenic cardiac fibroblasts should shift from targeting presumptive EndoMT or infiltrating hematopoietically derived fibroblasts, toward common pathways upregulated in two endogenous fibroblast populations.

Figure 1. CF markers in adult murine myocardium.

(A) Confocal analysis of ventricular myocardium shows that collagen1a1-GFP labels CFs (arrows) that expressed the mesenchymal marker PDGFRα and vimentin. Collagen1a1-GFP⁺ fibroblasts were negative for markers of endothelium (PECAM1), blood lineages (CD45), pericytes (PDGFRβ), and smooth muscle (αSMA). (B) Flow cytometry analysis of dissociated LV and IVS, showing overlap of collagen1a1-GFP and PDGFRα signals. (C) Flow cytometry analysis of LV and IVS, showing that collagen1a1-GFP cells are mostly, but not all, Thy1.2 positive. (D) Collagen1a1-GFP⁺ cells were PECAM1^– and CD45^–. (E) Quantitative real-time PCR showing fold enrichment of DDR2, prolyl-4-hydroxylase (p4h), and PECAM1 in flow cytometry–sorted collagen1a1-GFP⁺ and Pecam1⁺ cells (n = 3 hearts, average ± SEM). (F) Confocal images showing colocalization of FSP1 CD45⁺ leukocytes (arrows) but not with collagen1a1-GFP fibroblasts. Images are representative of at least 3 hearts. Scale bars: 20 μm; 5 μm (inset).

Figure 2. Fibroblast markers in pressure overload associated with fibrosis.

(A) Collagen1a1-GFP⁺ fibroblast accumulation was associated with collagen type I deposition, as evidenced by immunofluorescence staining against collagen type I and trichrome staining in adjacent sections. Similar results were observed following 28 days of TAC. (B) Double-positive CD45⁺FSP1⁺ cells within interstitial pathologic fibrotic lesions (arrows). The pathologic lesion is evident as an area rich in collagen-GFP⁺ cells and CD45⁺ leukocytes on the left of the images. No double-positive collagen1a1-GFP⁺CD45⁺ cells were observed. (C) Percentages of the total number of cells in interstitial fibrotic areas expressing collagen1a1-GFP, FSP1, and CD45 following 7 days of TAC. (D) αSMA expression in some collagen1a1-GFP⁺ fibroblasts (arrows) following 7 days of TAC. (E) Percentages of the total number of cells in interstitial fibrotic areas expressing collagen1a1-GFP, αSMA, and CD45 following 7 days of TAC. CD45⁺ cells were never collagen1a1-GFP⁺. (F) Overlap of collagen1a1-GFP and PDGFRα signals in sham and following 7 and 28 days of TAC. Histograms represent mean ± SD of 12 fields from 3 mice. Scale bars: 100 μm (A); 20 μm (B, D, and F).

Figure 3. Complementary distribution patterns of Wt1-Cre and Tie2-Cre CF lineages.

(A) Representative 4-chamber view of adult Wt1-Cre^+/– collagen1a1-GFP^+/– Rosa-tdT^+/– heart. IVS (A1, A1′) contained few lineage-traced fibroblasts, whereas fibroblasts in the LVFW (A2, A2′) were predominantly lineage traced (lineage traced, arrows; nonlineage traced, arrowheads). Labeled myocytes were abundant in the IVS (asterisks). (B) Representative Tie2-Cre^+/– collagen1a1-GFP^+/– Rosa-tdT^+/– heart showing Tie2-Cre lineage-traced fibroblasts were predominant in the IVS (B1, B1′), but most fibroblasts were nonlineage traced in the LVFW (B2, B2′) (lineage traced, arrows; nonlineage traced, arrowheads). (C) Flow cytometry plots of dissociated LV and IVS from Tie2-Cre, Wt1-Cre, Wt1-Cre + Tie2-Cre, and Vav-Cre lineage-traced mice. Tie2-Cre and Wt1-Cre, but not Vav-Cre, labeled collagen1a1-GFP⁺ CF populations. (D) Quantification of the relative numbers of lineage-traced fibroblasts (n = 3 per group). Combining Wt1-Cre and Tie2-Cre resulted in labeling of approximately 95% of fibroblasts. Histograms represent average ± SD. Data from Wt1-Cre and Wt1-Cre + Tie2-Cre groups were compared using unpaired Student’s t test. Scale bars: 1 mm (A and B); 250 μm (A1, A2, B1, B2); and 20 μm (A1′, A2′, B1′, B2′).

Figure 4. Fibroblasts accumulate by proliferation of resident lineages.

(A) Confocal images showing Wt1-Cre and Tie2-Cre lineage-traced cells in sham-operated mice (7 days) and following 7 and 28 days pressure overload. The differential distribution of Wt1-Cre and Tie2-Cre fibroblast lineages in the IVS and LVFW was similar in sham-operated and fibrotic hearts. (B) Flow cytometry analysis of LV and IVS showing Wt1-Cre and Tie2-Cre lineage-traced collagen1a1-GFP⁺ fibroblasts in sham-operated animals (7 days) and following pressure overload (7 and 28 days). (C) Quantitative analysis of FACS data from Wt1-Cre and Tie2-Cre mice demonstrated that the relative numbers of lineage-traced fibroblasts did not significantly vary between sham- and TAC-operated animals, except for a small decrease in Wt1-Cre lineage-traced cells at 7 days of TAC. In double Wt1-Cre^+/– Tie2-Cre^+/– collagen1a1-GFP^+/– Rosa-tdT^+/– mice, 94% ± 0.8% of all fibroblasts were labeled following 28 days of TAC. S, sham-operated mice. (D) Quantification of proliferation rates of Tie2-Cre lineage-traced fibroblasts in the IVS and Tie2-Cre nonlineage-traced fibroblasts in the LVFW 4, 7 and 28 days after surgery (trans-stenotic systolic pressure gradient [PG] day 4, 76.7 ± 8.2 mmHg, n = 3). Cells were counted in n = 3 mice per group, 6 fields per heart. NS, not significant (P ≥ 0.05). P = 0.01, *P < 0.001, compared with other groups, determined by ANOVA followed by Bonferroni’s test. Data are shown as mean ± SD. Scale bars: 20 μm.

Figure 5. CFs do not derive from the hematopoietic lineage following pressure overload.

(A) Confocal images showing a lack of collagen1a1-GFP⁺ Vav-Cre lineage-traced cells in sham-operated hearts and following 7 and 28 days of TAC. (B) Flow cytometry plots showing that Vav-Cre lineage-traced cells were collagen1a1-GFP^–. Images/plots are representative of at least 3 individuals. Scale bars: 20 μm.

Figure 6. Lineage tracing of adult cardiac endothelium following TAC.

(A) Confocal analysis of LV and IVS in sham-operated mice or following 7 and 28 days of pressure overload. Lineage-traced cells (red) were PECAM1⁺ endothelial cells (gray) and were never collagen1a1-GFP⁺ fibroblasts (green) (representative of 3 mice per group). (B) Flow cytometry analysis of dissociated LV and IVS from VE-cadherin-CreERT2^+/– collagen1a1-GFP^+/– Rosa-tdT^+/– lineage-traced sham-operated hearts and following 7 and 28 days of TAC. Plots are representative of 2 to 3 individuals. Cell populations are identified with Rosa-tdT (VE-cadherin-CreERT2 lineage traced), collagen1a1-GFP (fibroblasts), PECAM1 (endothelium), and CD45 (leukocytes). In fibrotic hearts as well as in sham-operated hearts, VE-cadherin-CreERT2 lineage-traced cells (Rosa-tdT⁺, top gates) were PECAM1⁺ or CD45⁺, indicating that they had not adopted a fibroblast fate. Accordingly, VE-cadherin-CreERT2 Rosa-tdT⁺ cells were collagen1a1-GFP^–. The collagen1a1-GFP⁺ fibroblasts were PECAM^– and CD45^– (bottom gates). In all mice, a small percentage of collagen1a1-GFP⁺ (∼1%) were PECAM1⁺, due to collagen expression in some endocardial cells. Scale bars: 20 μm.

Figure 7. Epicardium does not give rise to fibroblasts following pressure overload.

(A) Confocal images showing labeling in epicardium (arrows) but not in collagen1a1-GFP⁺ fibroblasts of sham-operated Wt1-CreERT2^+/– collagen1a1-GFP^+/– Rosa-tdT^+/– mice and following 7 and 28 days of pressure overload. Rare lineage-traced collagen1a1-GFP^– cells (arrowheads) were present in the interstitium of sham-operated and hypertrophic hearts. Scale bars: 50 μm. (B) No collagen1a1-GFP⁺ fibroblasts were labeled in fibrotic areas following 7 days of TAC. Similar results were observed following 28 days of TAC. Scale bars: 20 μm; 5 μm (insets).

Figure 8. Analysis of the fate Tie2-Cre and of VE-cadherin-CreERT2 lineage-traced cells during early development.

(A) Time course showing Tie2-Cre^+/– collagen1a1-GFP^+/– Rosa-tdT^+/– embryonic hearts at E12.5, E15.5, E18.5, and P7. Tie2-Cre lineage-traced fibroblasts were first visible within the myocardium close to the AV canal cushion at E12.5 (arrows, image A1) and subsequently populated the IVS. (B) Timing of tamoxifen inductions for VE-cadherin-CreERT2 embryos. (C) Inductions at E7.5 resulted in labeling of collagen1a1-GFP⁺/PDGFRα⁺ valve mesenchyme (V) and interstitial fibroblasts (arrows) in the upper septum by E17.5. Many VE-cadherin-CreERT2 lineage-traced fibroblasts were also found migrating into the lower septum (arrows). Far less abundant VE-cadherin-CreERT2 lineage-traced PECAM1^– fibroblasts were found in the ventricular free walls (arrows). (D) Induction at E10.5 resulted in labeling of endocardium (arrows) but not valve mesenchyme or interstitial CFs. Scale bars: 500 μm (A, 4-chamber images); 100 μm (A, bottom 2 rows, C, and D).

Figure 9. Gene profiling of fibroblast lineages and endothelium following pressure overload.

(A) Heat plot representing all possible pairwise HOPACH correlation (euclidean) values. Darkest values indicate a high level of correlation (=1 across the diagonal), and lighter color indicates lower correlation. A clear segregation of fibroblast and endothelial expression profiles is evident. (B) Hierarchical clustering of genes up or down 2-fold in at least one group revealed clusters of genes specific to fibroblasts following TAC (cluster 1), fibroblasts (cluster 2), endothelial cells (cluster 3), and fibroblasts and endothelial cells following pressure overload (cluster 4). (C) Proposed model for fibroblast origins based on findings from this study. Fibroblast accumulation during fibrosis resulting from pressure overload results from the proliferation of distinctly distributed resident epicardial- and endothelial-derived fibroblast populations. Fibrosis does not result from EndoMT, epicardial EMT, or recruitment of hematopoietic fibroblast progenitors. The similar genetic profiles and behavior of these fibroblast populations suggests that they could be targeted in a similar way to alleviate fibrosis.