Fig 2: FAPs are the major source of IL-33, which activates muscle ILC2s(A and B) The expression (A) and quantification cycle (Cq) values (B) of ILC2 activators in WT and mdx hamstring determined by qRT-PCR. n = 8–10.(C) Expression of human IL-33 (hIL-33) in HC and DMD muscle biopsies. n = 6–8.(D) Linear regression analysis comparing ??Cq and patient age. HC, gray; DMD, blue. n = 6–8.(E) Gating strategy used to sort CD45+ immune cells (I), CD31+ endothelial cells (II), PDGFRa+ FAPs (III), ITGa7+ satellite cells (V), and PDGFRa- and ITGa7- double-negative (IV) cells from mdx skeletal muscle.(F) Quantification of IL-33 expression by qRT-PCR of cell populations sorted in (E). n = 5 pooled mice.(G) Expression of IL-33 in WT and mdx FAPs isolated by FACS. n = 4.(H) Immunofluorescence staining of mdx quadriceps showing PDGFRa, laminin, and IL-33 expression. Nuclei were stained with DAPI.(I) The number of muscle ILC2s in mdx mice treated with exogenous cytokines. n = 6–11. IL-2c, IL-2 complex; IL-2c/IL-33, IL-2c + IL-33; IL-2c/IL-25, IL-2c + IL-25.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using an unpaired Welch’s t test (A and B) or one-way ANOVA with Bonferroni correction (F). All mice were analyzed at 4 weeks of age. Error bars indicate SEM.

Fig 3: FACS gating strategy for sorting retinal CD157-positive VESCs. Representative FACS plots of the percentage of parent gates for each population. The retinal cell population was selected (A), dead cells were excluded (B), and doublets were excluded (C, D). (E) CD31-BV421-A versus CD45-FITC-A dot plot was generated to identify vascular endothelial cells (ECs) positive for the endothelial marker CD31 and negative for the hematopoietic marker CD45 (CD31+CD45-) in the retina. The proportion of retinal ECs shown in red squares was 3.3% in this experiment. The orange square shows hematopoietic cells (CD31+CD45-), and the blue squares indicate others (CD31-CD45-). (F) CD31-BV421-A versus CD157-APC-A dot plot was generated to detect CD157-positive vascular endothelial stem cells (VESCs). The proportion of VESCs was 1.6% among all retinal ECs.

Fig 4: RNA sequencing (RNA-seq) assay shows mesenchymal-endothelial transition (MEndoT)-derived cells are more endothelial-like after cardiac hypertrophy but may be a different cell sub-population. Non-myocytes were isolated from hearts of 14 day post-sham or -transverse aortic constriction (TAC) in Col1a2-CreERT: R26RtdTomato mice and native fibroblasts (tdTomato+-labeled fibroblasts after sham-injury), MEndoT-derived cells (tdTomato+CD31+-labeled cells) were collected using flow cytometry for RNA-seq. The tdTomato+-labeled endothelial cells from heart tissue of Tek-CreERT: R26RtdTomato mice after sham injury were collected and served as native coronary endothelial cells. (a) Principal component analysis clustering show MEndoT-derived cells are a subset cell population with characteristics that were closer to native endothelial cells than native fibroblasts. (b) p53 is specifically and significantly upregulated in MEndoT-derived cells. Heatmap show significantly expressed genes indicative of (c) fibroblast lineage commitment and (d) endothelial lineage commitment (all genes in b-d have a q-value <0.05 compared with native fibroblast, n = 3 animals/group).

Fig 5: CD157 expressed in the vascular endothelial cells (ECs) of large choroidal vessels. (A) CD31 (white) and CD157 (red) were stained using the choroidal flat mount of a wild-type mouse. CD157-positive ECs were located in the large vessels in the choroid, but not in the capillaries. Scale bars represents 100 µm. (B) CD157 positive ECs were not detected in the choroid of CD157-KO (CD157-/-) mice, indicating that the CD157 antibody used in A specifically recognized CD157 in choroidal vasculature. Scale bars represents 100 µm.