PDGF-AB Reduces Myofibroblast Differentiation Without Increasing Proliferation After Myocardial Infarction

Abstract

After myocardial infarction (MI), fibroblasts progress from proliferative to myofibroblast states, resulting in fibrosis. Platelet-derived growth factors (PDGFs) are reported to induce fibroblast proliferation, myofibroblast differentiation, and fibrosis. However, we have previously shown that PDGFs improve heart function post-MI without increasing fibrosis. We treated human cardiac fibroblasts with PDGF isoforms then performed RNA sequencing to show that PDGFs reduced cardiac fibroblasts myofibroblast differentiation and downregulated cell cycle pathways. Using mouse/pig MI models, we reveal that PDGF-AB infusion increases cell-cell interactions, reduces myofibroblast differentiation, does not affect proliferation, and accelerates scar formation. RNA sequencing of pig hearts after MI showed that PDGF-AB reduces inflammatory cytokines and alters both transcript variants and long noncoding RNA expression in cell cycle pathways. We propose that PDGF-AB could be used therapeutically to manipulate post-MI scar maturation with subsequent beneficial effects on cardiac function.

Keywords: myocardial infarction; platelet-derived growth factor.

Graphical abstract

Figure 1

PDGFs Reduce Fibroblast-Myofibroblast Differentiation and Increase Migration Without Increasing Proliferation of human cFib Human cardiac fibroblasts (cFib) were treated with platelet-derived growth factor (PDGF) subunits AA, AB, or BB ± transforming growth factor (TGF)-β1. (A) cFib were imaged using brightfield microscopy and immunostained with α-smooth muscle actin (SMA) (green). (B) cFib Western blotting probed for αSMA. (C) cFib Western blotting α-SMA densitometry analysis. (D) cFib quantitative polymerase chain reaction (qPCR) for actin alpha 2, smooth muscle (ACTA2) fold change normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2^-ΔΔCt). (E) cFib immunostained for vimentin (red) and α-SMA (green). (F) cFib transwell assays showing 4’,6-diamidino-2-phenylindole (DAPI)–stained migratory cells. (G) Analysis of transwell assays, each graph representing a different cFib donor-derived cell line. (H) cFib immunostaining for Ki67 (red) positive DAPI (blue). (I) Image analysis of proliferating (Ki67⁺) cFib. Statistical analyses were performed using a 1-way analysis of variance with a Tukey’s multiple comparisons test. ∗P < 0.05. ∗∗∗P < 0.001. n.s. = not significant.

Figure 2

PDGFs Reduce Myofibroblast and Cell Cycle Gene Expression and Increase Migration and ECM Gene Expression of cFib cFib were treated with PDGF-AA, PDGF-AB, or PDGF-BB ± TGFβ1 followed by RNA sequencing (RNAseq). (A) Principal component analysis (PCA). (B) Differential gene expression (DE) analysis heatmap of myofibroblast markers (scale bar = log₂fold change). (C) Gene set enrichment analysis (GSEA) of PDGF-AB–treated samples showing the top and bottom 10 most effected gene sets. (D) Summary of RNAseq pathway analyses for PDGF-AB samples. (E to H) cFib qPCR for extracellular matrix (ECM) genes normalized to GAPDH (2^-ΔΔCt, n.s., ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). (I) Heatmap of cFib RNAseq ECM-related genes with hierarchical clustering (scale bar = log₂fold change). (J) Reactfoam pathway analysis of PDGF-AB-treated cFib RNAseq gene expression showing upregulated pathways (yellow) highlighting ECM-related pathways (red inset). Abbreviations as in Figure 1.

Figure 3

PDGF-AB Increases Cell-Cell Interactions Within Infarct Scar (A) Schematic of post–myocardial infarction (MI) mouse model. (B) Schematic of IMC. (C, D) Analysis of IMC for αSMA⁺ and αSMA⁺col1α1⁺ cells in the remote zone (RZ), border zone (BZ) and infarct zone (IZ) of sham, vehicle control, and PDGF-AB–treated post-MI mice (∗P < 0.05). (E) t-distributed stochastic neighbor embedding (tSNE) unbiased clustering analysis of IMC data with sample types labelled. (F) Heatmap of IMC marker (x-axis) expression for each cell subpopulation (y-axis) derived from tSNE unbiased clustering. (G) tSNE clustering analysis of IMC data with unbiased clusters labelled. (H) Expression plots of IMC markers. (I) Proportion of fibroblast 1 IMC cluster in RZ, BZ, and IZ in sham, vehicle control, and PDGF-AB–treated mice (∗P < 0.05). (J) IMC spatial analysis showing significantly reduced average distances of fibroblast 5 (green) and fibroblast 3 (red) IMC clusters following PDGF-AB treatment within the IZ. (K) Volcano plot of IMC spatial analysis. Red dots represent comparisons that met both P value and fold change cutoffs. IMC = imaging mass cytometry; other abbreviations as in Figure 1.

Figure 4

PDGF-AB Reduces Fibroblast-Myofibroblast Differentiation and Does Not Increase Proliferation in Pigs Post-MI (A) Schematic of post-MI pig model. (B) Coronary angiogram pre- and post-left anterior descending (LAD) artery balloon occlusion to induce MI. Arrowhead indicates balloon inflation point in LAD. (C) Short-axis cMRI view of the heart in end-diastole and end-systole at the mid-papillary muscle level. (D) Change in left ventricular ejection fraction (ΔLVEF) from day 2 to day 9 post-MI. (E to K) Immunohistochemistry (IHC) and analyses for fibroblasts (vimentin⁺), myofibroblasts (vimentin⁺αSMA⁺) and proliferating (Ki67⁺) cells within pig post-MI IZ (scale bar = 100 μm). (L to N) IHC and analyses of αSMA⁺ cells with myofibroblast morphology within col1α1^high areas in pig IZs. cMRI = cardiac magnetic resonance imaging; other abbreviations as in Figures 1 and 3.

Figure 5

De Novo Genome Assembly Reveals Novel Pig Genes, Transcripts, and Infarct Biology and Shows That PDGF-AB Accelerates Scar Maturation and Reduces Inflammatory Cytokines in Pigs Post-MI (A, B) Number of identified genes and transcripts for current pig genome annotation (SScrofa11.1) and our de novo library assembly. (C) DE genes for control pig 11 days post-MI cardiac tissue vs shams using our de novo library assembly. (D) Volcano plots showing DE genes in control pigs (vs shams). Red dots represent genes that met both P value and log₂(fold change) cutoffs. (E) GSEA for control DE genes in control pigs (vs shams). (F) Heatmap of PDGF-AB–treated pig RNAseq for DE ECM-related genes. (G) Picro-Sirius red histological staining of collagen fibers (red) in pig post-MI infarct zone (IZ, scale bar = 50 μm). (H) IHC of col1α1 scar (grey), CD3⁺ T cells (red, arrows), and FXIIIa⁺ macrophages (blue, arrowheads) in pigs post-MI. Scale bar = 2 mm (top) and 100 μm (bottom). (I to K) IHC image analyses of pig infarct zones (∗P < 0.05). (L) Heatmap of PDGF-AB–treated pig RNAseq for DE immune-related genes. Abbreviations as in Figures 1, 2, 3, and 4.