Fig 1: 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 aSMA+ 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 aSMA+ fibroblast soak loaded sponges stained with CD31 to analyze vascular density. SP = sponge matrix, arrows point at positive stain. Scale bar = 500 µM. e Vascular density graphed as percentage of immunopositive CD31 area/total tissue area in histologic sections from granulation tissue. Data represent averages of multiple 40× fields from unpaired samples (n = 6 for aSMA+ fibroblast loaded sponges and n = 5 for FSP1+ fibroblast loaded sponges). f BrdU proliferation assay performed on HUVECs (P5) in response to NT (n = 4), FSP1+ (n = 4), aSMA+ (n = 4), and uninjured (n = 3) fibroblast (P3–P5) conditioned media. g Immunofluorescence staining of GFP+ FACS sorted fibroblasts (P3–P5) isolated from left ventricle of FSP1-GFP or aSMA-GFP mice indicate that FSP1+ and aSMA+ cell populations do not express endothelial markers such as vWF or Flk-1 (VEGFR2). HUVECs were used as positive control (n = 3). nsp > 0.05, *p < 0.05, **p < 0.001, and ***p < 0.0001 was calculated by one-way ANOVA, n = 3 experiments were performed; bar represents mean ± SD
Fig 2: FSP1+ fibroblasts exhibit pro-angiogenic protein signature in vitro. a Relative pixel density of selected angiogenic proteins significantly high in FSP1+ vs. aSMA+ 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 aSMA+ 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 aSMA+ (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 aSMA+ fibroblasts cultured (P3–P5) for 72 h at 37 °C. ***p < 0.0001 was calculated by one-way ANOVA; bar represents mean ± SD. d Relative fold change of Vegfa (n = 3 for uninjured, n = 4 for FSP1+, and n = 2 for aSMA+ fibroblasts), Vegfb (n = 2 for uninjured, n = 4 for FSP1+, and n = 2 for aSMA+ fibroblasts), Grem1 (n = 3 for uninjured, n = 2 for FSP1+, and n = 3 for aSMA+ fibroblasts), Angpt1 ((n = 3 for uninjured, n = 2 for FSP1+, and n = 3 for aSMA+ fibroblasts), and Fgf1 (n = 4 for uninjured, n = 5 for FSP1+, and n = 4 for aSMA+ fibroblasts) transcripts measured by real-time RT-PCR in uninjured, FSP1+, and aSMA+ fibroblasts (P0–P5) . *p < 0.01, **p < 0.001, and ***p < 0.0001 was calculated by one-way ANOVA; bar represents mean ± SD
Fig 3: Distinct molecular signatures of uninjured, FSP1+, and aSMA+ 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 aSMA+ fibroblasts (n = 3 for uninjured and aSMA+ 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 aSMA+ fibroblasts compared with uninjured fibroblasts. c Relative fold change of upregulated genes expressed in FSP1+ fibroblasts compared with aSMA fibroblasts by fibrosis array. d Relative fold change of downregulated genes expressed by FSP1+ fibroblasts compared to aSMA+ 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) between uninjured, FSP1+, and aSMA+ fibroblasts using CLC Genomics Workbench 8.0. f Relative fold change of genes representing key pathways identified by RNA sequencing in aSMA+ vs. FSP1+ fibroblasts. Genes downregulated in aSMA+ fibroblasts are mentioned as genes upregulated in FSP1+ fibroblasts. KEGG pathway analysis was performed on the RNA sequencing data on DAVID bioinformatics Resource 6.7 platform
Fig 4: FSP1 and aSMA mark distinct populations after multiple types of tissue injury. a Confocal analysis of FSP1+ and aSMA+ 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 aSMA+ 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 aSMA+ fibroblast populations 7 and 14 days after unilateral ureteral ligation. Contralateral uninjured kidney was used as control (×40) (n = 3). d FSP1 and aSMA staining on histological sections of uninjured (n = 1) and post myocardial infarction human hearts (n = 3)
Fig 5: FSP1+ fibroblasts do not differentiate into aSMA-expressing fibroblasts. a. Immunofluorescence staining for aSMA 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 TGFß. Nuclei were stained with DAPI (n = 3). aSMA+ fibroblasts isolated from aSMA-GFP mice post injury were used a positive control (P0). Scale bar = 100 µM; ×10 magnification b Relative fold change of aSMA expression in both uninjured fibroblast and FSP1+ fibroblasts (P3–P5) cultured with or without TGFß, *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 aSMA+ fibroblasts were performed; bar represents mean ± SD; ns = not significant
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