Identification of a pro-angiogenic functional role for FSP1-positive fibroblast subtype in wound healing

Fig. 1. FSP1 and αSMA mark distinct populations after multiple types of tissue injury. a Confocal analysis of FSP1+ and αSMA+ cell populations from heart 2, 8, 12, 20, and 30 days post myocardial infarction (MI; ×40). Uninjured heart was used as control (n = 3). b Confocal analysis of representative 1 µM histologic sections from murine skin stained for FSP1+ and αSMA+ fibroblast populations 4 and 7 days after excisional full-thickness cutaneous wound. Uninjured skin was used as control (×40) (n = 3). c Confocal analysis of representative histologic sections of murine kidney stained for FSP1+ and αSMA+ fibroblast populations 7 and 14 days after unilateral ureteral ligation. Contralateral uninjured kidney was used as control (×40) (n = 3). d FSP1 and αSMA staining on histological sections of uninjured (n = 1) and post myocardial infarction human hearts (n = 3)

Fig. 2. FSP1+ and αSMA+ cells exhibit the molecular phenotype and functionality of fibroblasts. a Schematic of activated fibroblast isolation 10 days post myocardial infarction (MI) from murine hearts. b, c Representative dot plot for fluorescence assisted cell sorting (FACS) of FSP1-expressing (GFP+/CD31−/CD45−) and αSMA-expressing (GFP+) fibroblasts from FSP1-GFP and αSMA-GFP mice left ventricle 10 days after MI. Uninjured fibroblasts were used as unstained negative control. CD45 and CD31 antibodies were used to gate out hematopoietic and endothelial cells, respectively. d Immunofluorescence staining of GFP+ populations (P0) from left ventricle of uninjured, FSP1-GFP, or αSMA-GFP sorted by FACS showed FSP1+ and αSMA+ as unique fibroblast populations, which express fibroblasts markers, such as COL1α1, periostin, and vimentin, but not hematopoietic marker CD45 or the endothelial marker CD31. Nuclei were stained with DAPI (n = 3). Scale bar = 100 µM; ×10 magnification. e Representative FACS overlay histogram of uninjured, FSP1+, and αSMA+ fibroblasts (P3–P5) to evaluate expression of MEF-SK4. Rat IgG was used as negative control (n = 2). f Representative picture of collagen gel…

Fig. 3. FSP1+ fibroblasts do not express pericyte marker, AN2/NG2. a Staggered FACS histogram representing AN2/NG2 expression in FSP1+ and uninjured fibroblasts (P3–P5). Human retinal pericytes were used as positive control and secondary only antibody was used as a negative control. b Relative expression of An2/Ng2 in cultured uninjured, FSP1+, and αSMA+ fibroblasts (P3–P5). p < 0.05 was calculated by one-way ANOVA, n = 3 experiments were performed independently; bar represents mean ± SD. Mesenchymal stem cells (MSCs) were used as a positive control. c Confocal analysis of FSP1 (green), AN2/NG2 (red), and DAPI (blue) staining on post myocardial infarcted human heart; n = 2

Fig. 4. FSP1+ fibroblasts do not differentiate into αSMA-expressing fibroblasts. a. Immunofluorescence staining for αSMA protein in fibroblasts (P0) isolated from uninjured and FSP1-GFP mice hearts 10 days post injury and cultured for 72 h in the presence or absence of TGFβ. Nuclei were stained with DAPI (n = 3). αSMA+ fibroblasts isolated from αSMA-GFP mice post injury were used a positive control (P0). Scale bar = 100 µM; ×10 magnification b Relative fold change of αSMA expression in both uninjured fibroblast and FSP1+ fibroblasts (P3–P5) cultured with or without TGFβ, *p < 0.05; **p < 0.005; nsp > 0.05 was calculated by two-way ANOVA, n = 2 experiments for uninjured fibroblasts and n = 3 experiments with FSP1+ and αSMA+ fibroblasts were performed; bar represents mean ± SD; ns = not significant

Fig. 5. Distinct molecular signatures of uninjured, FSP1+, and αSMA+ fibroblasts identified by fibrosis array and RNA sequencing. a Heat map from fibrosis array representing magnitude of gene expression of genes expressed in freshly isolated uninjured, FSP1+, and αSMA+ fibroblasts (n = 3 for uninjured and αSMA+ fibroblasts, n = 4 for FSP1+ fibroblasts; cell isolation and FACS were performed at least three independent times. For each sorting, cells were isolated from pooled homogenates from three to four injured murine hearts 10 days post MI). FB = fibroblasts b Relative fold change of selected highest expressing genes identified by fibrosis array expressed by FSP1+ and αSMA+ fibroblasts compared with uninjured fibroblasts. c Relative fold change of upregulated genes expressed in FSP1+ fibroblasts compared with αSMA fibroblasts by fibrosis array. d Relative fold change of downregulated genes expressed by FSP1+ fibroblasts compared to αSMA+ fibroblasts by fibrosis array. e Hierarchical clustering (partial heat map representing log10 values) of RNA transcript reads per kilobase per million mapped reads for genes with significant differential expression (fold difference > 2, p value < 0.05)…

Fig. 6. FSP1+ fibroblasts exhibit pro-angiogenic protein signature in vitro. a Relative pixel density of selected angiogenic proteins significantly high in FSP1+ vs. αSMA+ fibroblast lysates (P3–P5) identified using mouse angiogenesis proteome profiler array. *p < 0.01 and ***p < 0.0001 were calculated by two-way ANOVA using Bonferroni post-test, n = 2 technical replicates were performed; bar represents mean ± SD. b Relative pixel density of selected angiogenic proteins significantly high in αSMA+ vs. FSP1+ fibroblast lysates (P3–P5) identified using mouse angiogenesis proteome profiler array. **p < 0.001 and ***p < 0.0001 were calculated by two-way ANOVA using Bonferroni post-test, n = 2 technical replicates were performed; bar represents mean ± SD. c (left panel) Quantification of secreted vascular endothelial growth factor (VEGF) by Quantikine VEGF ELISA in the conditioned media (CM) obtained from FSP1+ (n = 5) and αSMA+ (n = 4) fibroblasts cultured for 72 h at 37 °C. **p < 0.001 was calculated by unpaired t test; bar represents mean ± SD. (Right panel) Quantification of Gremlin 1 by ELISA in cell lysates (n = 2) and CM (n = 4) obtained from FSP1+ and αSMA+ fibroblasts cultured…

Fig. 7. FSP1+ fibroblasts are pro-angiogenic in vitro and in vivo. a Representative images from an endothelial cell assembly assay using HUVECs treated with conditioned media (CM) collected from fibroblasts (P3–P5) or FSP1 recombinant protein. In the top row, the endothelial cells were stained with cell tracker orange and the second row contains corresponding bright field images (×20). Scale bar = 100 nM. b Quantification from the endothelial cell assembly assay based on the number of intersecting branch points per field of view (×10). Data represent an average of HUVEC cell assembly from n = 16 for NT, n = 10 for positive control, n = 15 for FSP1+ FB CM, n = 7 for FSP1 protein, and n = 10 for αSMA+ FB CM images from three independent biological replicates; bar represents mean ± SD. c BrdU proliferation assay performed on HUVECs (P5) in response to either FSP1+ fibroblasts conditioned media (with 0.2% serum; n = 3) or FSP1 recombinant protein (10 nM; n = 4). HUVECs cultured in full serum (2%; n = 4) were used as positive control. d Representative images of FSP1+ and αSMA+ fibroblast soak loaded sponges stained with CD31 to analyze vascular density. SP = sponge matrix, arrows point at…