Exercise-induced angiogenesis is dependent on metabolically primed ATF3/4+ endothelial cells

Abstract

Exercise is a powerful driver of physiological angiogenesis during adulthood, but the mechanisms of exercise-induced vascular expansion are poorly understood. We explored endothelial heterogeneity in skeletal muscle and identified two capillary muscle endothelial cell (mEC) populations that are characterized by differential expression of ATF3/4. Spatial mapping showed that ATF3/4⁺ mECs are enriched in red oxidative muscle areas while ATF3/4^low ECs lie adjacent to white glycolytic fibers. In vitro and in vivo experiments revealed that red ATF3/4⁺ mECs are more angiogenic when compared with white ATF3/4^low mECs. Mechanistically, ATF3/4 in mECs control genes involved in amino acid uptake and metabolism and metabolically prime red (ATF3/4⁺) mECs for angiogenesis. As a consequence, supplementation of non-essential amino acids and overexpression of ATF4 increased proliferation of white mECs. Finally, deleting Atf4 in ECs impaired exercise-induced angiogenesis. Our findings illustrate that spatial metabolic angiodiversity determines the angiogenic potential of muscle ECs.

Keywords: amino acid metabolism; endothelial heterogeneity; endothelial metabolism; exercise; muscle angiogenesis; single-cell RNA-seq.

Graphical abstract

Figure 1

scRNA-seq reveals EC subpopulations in skeletal muscle (A) Visualization of mEC populations on a t-SNE plot, color-coded for the identified subpopulations, including venous ECs (vEC), arterial ECs (aEC), arteriolar ECs (arlEC), capillary ECs (CapEC 1 and CapEC 2), and an unknown EC population (xEC). (B) Heatmap with Z scores showing top 10 enriched marker genes per cell type relative to other subpopulations. (C) Violin plots of normalized scRNA expression profiles of known EC subtype markers in each mEC cluster. (D–F) Representative images of GAS cross-sections, stained for (D) an EC marker (CD31, red) combined with α smooth muscle actin (αSMA) (orange), SELP (green), and VWF (gray) (enriched in venous ECs); (E) EFNB2 (green) (enriched in arteries and arterioles) and AQP1 (gray) (low expression in arteries and arterioles; and (F) CAR4 (green) and AQP1 (gray) (enriched in capillaries). Arrows indicate SELP⁺VWF⁺ veins in (D) and EFNB2⁺AQP1⁻ arteries in (E). Scale bars, 50 μm. See also Figure S1.

Figure 2

Muscle contains two capillary EC populations characterized by distinct Atf3/4 (A) Left: heatmap with log fold changes of 50 top marker genes of CapEC 1 relative to CapEC 2. Right: violin plot showing the distribution of Atf3 and Atf4 expression within different mEC populations. (B) Representative fluorescence images showing ATF3 (gray) and CD31 (red) combined with type I (MHCI, green), type IIa (MHCIIa, blue), and type IIb (MHCIIb, yellow) fiber-type staining on cross-sections in oxidative and glycolytic areas of GAS. Arrows indicate ATF3⁺CD31⁺ ECs. Scale bar, 50 μm. (C) Quantification of the percentage of ATF3⁺ vessels in oxidative and glycolytic areas of GAS (n = 6). (D) Representative fluorescence images showing ATF3 (gray) and CD31 (red) combined with type I (MHCI, green), type IIa (MHCIIa, blue), and type IIb (MHCIIb, yellow) fiber-type staining on thick longitudinal sections of GAS. Arrows indicate ATF3⁺CD31⁺ ECs. Scale bar, 20 μm. (E) Representative images of CD31 (red), ATF3 (green), and Hoechst (blue) staining of RmECs and WmECs that were cultured for 7 days. Scale bar, 50 μm. (F) Quantification of the percentage of ATF3^high ECs in RmECs versus WmECs (n = 3). (G) Heatmap of the top 75 most highly variable genes in RmECs versus WmECs. (H) TF motif enrichment analysis of upregulated genes in RmECs over WmECs. (I) ATF4-dependent biological process analysis of upregulated genes in RmECs over WmECs. Two-tailed unpaired Student’s t test in (C) and (F) (^∗p < 0.05). Bar graphs represent mean ± SEM. See also Figure S2 and Data S1.

Figure 3

ATF3/4^low WmECs have a lower angiogenic potential (A) Representative images of EdU incorporation (gray) and EdU intensity in RmECs and WmECs combined with CD31 (red) and ERG (green) staining. Scale bar, 100 μm. (B) Quantification of the percentage of EdU⁺ ECs (CD31⁺) in RmECs versus WmECs (n = 6). (C) Quantification of (per mEC) EdU median intensity of RmECs versus WmECs (n = 5). (D and E) Representative bright field pictures (D) and quantification (E) of number of sprouts and average and total sprout length in spheroids composed of RmECs and WmECs (n = 30). Scale bar, 50 μm. (F and G) Representative images (F) and quantification of the length (G) of vascular sprouts from collagen embedded muscle explants from red oxidative (top row) and white glycolytic (bottom row) muscles or muscle areas. Explants were stained for isolectin B4 (IB4) (red), phalloidin (green), and Hoechst (blue). Scale bar, 100 μm. (H and I) Representative fluorescence images (H) and quantification (I) of EdU incorporation (red) and Hoechst (blue) in scr versus Atf3/4^KD HUVECs (n = 4). Scale bar, 100 μm. (J and K) Representative pictures (J) and quantifications (K) of scratch wound width in mitomycin C (mitoC)-treated WT (scr) and Atf3/4^KD HUVECs (n = 3). (L and M) Representative pictures (L) and quantifications (M) of total and average sprout length and number of sprouts in WT (scr) and Atf3/4^KD HUVEC spheroids (n = 30), with or without mitomycin C (mitoC) treatment. Scale bar, 50 μm. (N and O) Representative images (N) and quantification (O) of EdU incorporation (gray) in GFP or Atf4 (Atf4^OE) overexpressing RmECs and WmECs combined with CD31 (red) and ERG (green). Scale bar, 100 μm. Two-tailed unpaired Student’s t test in (B), (C), (E), (G), and (I) (^∗p < 0.05). One-way ANOVA with Tukey’s multiple comparisons test in (M) and (O) (^∗p < 0.05; n.s., not significant). Two-way ANOVA with Sidak’s multiple comparisons test in (K). Bar graphs represent mean ± SEM. See also Figure S3.

Figure 4

ATF3/4 rewires AA metabolism and biomass synthesis to metabolically prime RmECs for angiogenesis (A and B) Gene expression analysis of neutral AA transporters and metabolic genes in RmECs and WmECs (A), as well as WT (scr) and Atf3/4^KD HUVECs (B) (n = 3–6). (C) Leucine and glutamine uptake assay in WT (scr) and Atf3/4^KD HUVECs (n = 6). (D and E) Incorporation of [U-¹³C]-glucose carbon into serine and glycine in RmECs versus WmECs (D), as well as WT (scr) versus Atf3/4^KD HUVECs (E) (n = 4). (F and G) Intracellular free AA abundance in RmECs versus WmECs (F) (n = 10), and WT (scr) versus Atf3/4^KD HUVECs (G) (n = 5). (H and I) Protein synthesis rate in RmECs versus WmECs (H), as well as WT (scr) versus Atf3/4^KD HUVECs (I) (n = 8–12). (J and K) Intracellular purine levels in RmECs versus WmECs (J), and WT (scr) versus Atf3/4^KD HUVECs (K) (n = 5). (L and M) Total levels and labeling of ADP from [U-¹³C] glucose in RmECs versus WmECs (L) as well as WT (scr) versus Atf3/4^KD HUVECs (M) (n = 4–5). (N and O) Representative fluorescence images (N) and quantification (O) of EdU incorporation (red) in WT (scr) and Atf3/4^KD HUVECs cultured in control conditions (ctrl) and upon supplementation with NEAAs or EAAs (n = 6). Scale bar, 100 μm. (P and Q) Representative pictures (P) and quantifications (Q) of total sprout length and number of sprouts in WT and Atf3/4^KD HUVEC spheroids cultured under control conditions (ctrl) and upon supplementation with NEAAs or EAAs (n = 25). Scale bar, 50 μm. (R and S) Representative fluorescence images (R) and quantification (S) of EdU incorporation (gray) in RmECs and WmECs (CD31, red; ERG, green) cultured in control (ctrl) and upon supplementation with NEAA (n = 12). Scale bar, 100 μm. Two-tailed unpaired Student’s t test in (A)–(M) (^∗p < 0.05; n.s., not significant). One-way ANOVA with Tukey’s multiple comparisons test in (O), (Q), and (S) (^∗p < 0.05). Bar graphs represent mean ± SEM. See also Figure S4.

Figure 5

Exercise leads to selective expansion of RmECs (A) Representative fluorescence image showing IB4 (red) combined with type I (MHCI, green), type IIa (MHCIIa, blue), and type IIb (MHCIIb, yellow) fiber-type staining on QUAD cross-sections. Oxidative, peri-oxidative, and glycolytic areas are marked. Scale bar, 400 μm. (B) Representative fluorescence images of IB4 (red) and ERG (green) staining in oxidative and glycolytic areas of QUAD from sedentary versus exercised (14 days voluntary running) mice. Scale bar, 50 μm. (C and D) Quantification of IB4⁺ area (% of total area, C) and number of ERG⁺ ECs (D) within oxidative and glycolytic areas of sedentary versus exercised mice (n = 3–4). (E) t-SNE plot derived from scRNA-seq of mECs from sedentary versus exercised mice (14 days voluntary running). Pie charts show the fraction of each population in sedentary (top) and exercised (bottom) mice. (F) RNA velocity analysis of mECs from sedentary (top) and exercised (bottom) mice (14 days voluntary running). Arrows indicate extrapolated future states of ECs. (G and H) Representative images of staining of serial sections of QUAD for type I (MHCI, green), type IIa (MHCIIa, blue), and type IIb (MHCIIb, yellow) fibers (first section) and EdU incorporation (gray) co-stained with IB4 (red) and ERG (green) (second section) after 14 days of exercise (G). Scale bar, 200 μm. Magnification of white box (90° rotated) is shown in (H); arrows indicate EdU⁺ERG⁺ ECs. Scale bar, 50 μm. (I) Quantification of % EdU⁺ERG⁺ ECs in oxidative and glycolytic areas of QUAD from sedentary versus exercised mice (n = 3). (J) Representative fluorescence images showing EdU incorporation (gray) and IB4 (red) on thick longitudinal QUAD sections. Arrows indicate EdU⁺IB4⁺ ECs. Scale bar, 20 μm. (K) Quantification of spatial distribution of EdU⁺ERG⁺ proliferating ECs in QUAD of sedentary versus exercised mice (n = 6). Two-tailed unpaired Student’s t test in (C), (D), and (I) (^∗p < 0.05; n.s., not significant). Two-way ANOVA with Sidak’s multiple comparisons test in (K) (^∗p < 0.05; n.s., not significant). Bar graphs represent mean ± SEM. See also Figure S5.

Figure 6

Deletion of Atf4 in ECs impairs exercise-induced EC proliferation and vascular expansion (A) Scheme showing the generation of Pdgfb-Cre × Atf4^Loxp/Loxp (Atf4^ΔEC) mice and experimental plan. (B) Left: heatmap of the top 75 most highly variable genes in WT RmECs versus WT WmECs. Right: relative expression in Atf4^ΔEC/ΔEC RmECs versus Atf4^ΔEC/ΔEC WmECs. (C) Left: heatmap showing relative expression of ATF4-dependent anabolic gene set in WT RmECs versus WT WmECs. Right: relative expression in Atf4^ΔEC/ΔEC RmECs versus Atf4^ΔEC/ΔEC WmECs. Bold gene names refer to DEGs between WT RmECs and WT WmECs (log fold change > 1 and adjusted p value < 0.05). (D) PCA showing sample distances between RmECs and WmECs (WT and Atf4^ΔEC/ΔEC). (E and F) Representative fluorescence images of serial sections of QUAD from WT and Atf4^ΔEC/ΔEC mice stained for type I (MHCI, green), type IIa (MHCIIa, blue), and type IIb (MHCIIb,yellow) fibers (first section) and EdU incorporation (gray) co-stained with IB4 (red) and ERG (green) (second section) after 14 days of exercise (E). Scale bar, 100 μm. Magnification of white box is shown in (F); arrows indicate EdU⁺ERG⁺ ECs. Scale bar, 50 μm. (G) Representative fluorescence images of IB4 vascular staining in glycolytic and oxidative areas of QUAD from WT and Atf4^ΔEC/ΔEC mice in sedentary and exercised conditions. Scale bar, 50 μm. (H and I) Quantification of IB4⁺ area (% of total area) (H) and the percentage of EdU⁺ERG⁺ ECs (I) in oxidative versus glycolytic areas in QUAD of WT versus Atf4^ΔEC/ΔEC mice in sedentary and after exercise (n = 6). One-way ANOVA with Tukey’s multiple comparisons test (^∗p < 0.05; n.s., not significant). Each dot represents a single mouse. Bar graphs represent mean ± SEM. See also Figure S6.