Amino Acid Restriction Triggers Angiogenesis via GCN2/ATF4 Regulation of VEGF and H2S Production

Abstract

Angiogenesis, the formation of new blood vessels by endothelial cells (ECs), is an adaptive response to oxygen/nutrient deprivation orchestrated by vascular endothelial growth factor (VEGF) upon ischemia or exercise. Hypoxia is the best-understood trigger of VEGF expression via the transcription factor HIF1α. Nutrient deprivation is inseparable from hypoxia during ischemia, yet its role in angiogenesis is poorly characterized. Here, we identified sulfur amino acid restriction as a proangiogenic trigger, promoting increased VEGF expression, migration and sprouting in ECs in vitro, and increased capillary density in mouse skeletal muscle in vivo via the GCN2/ATF4 amino acid starvation response pathway independent of hypoxia or HIF1α. We also identified a requirement for cystathionine-γ-lyase in VEGF-dependent angiogenesis via increased hydrogen sulfide (H₂S) production. H₂S mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport and oxidative phosphorylation, resulting in increased glucose uptake and glycolytic ATP production.

Figure 1. SAA restriction induces endothelial VEGF expression in vitro and functional angiogenesis in vivo

(A) VEGF mRNA levels (left, n=4 experiments/group) and secreted protein concentration in the media (right, n=6 experiments/group) of HUVEC cultured in control (Ctrl) or SAA deficient (- M&C) media for 16hr; error bars indicate SEM. (B) Migration assay: Representative migration across a scratch (left, 10X mag at t=20hr; dotted lines indicate boundary of the scratch at t= 0hr) and area under the curve (AUC, right, n=7–10 data points/condition, with each data point representing the mean of multiple measures within a single well in a representative experiment) from HUVEC cultured in the indicated media. (C) Tube formation assay: Representative capillary-like structures (left, 40X mag) and quantification of tube length/field in arbitrary units (AU, right; n=8–10 data points/condition) in HUVEC incubated in the indicated media +/−SIRT1 inhibitor Ex-527 for 18hr. (D) Spheroid assay: Representative images (left, 40X mag) and quantification (right, in triplicate) of sprouting HUVEC spheroids in the indicated media +/− VEGFR2 inhibitor SU5416 for 24hr; blue, DNA (DAPI); red, F-actin (phalloidin). (E) Representative transverse sections (left, 40X mag) and quantification (right) of gastrocnemius muscle stained for endothelial marker CD31 in mice fed 2wk on Ctrl or MR diet +/−VEGFR2 inhibitor axitinib; n=6–8 mice/group. (F) Longitudinal Doppler imaging of blood flow in WT mice preconditioned 1mo on Ctrl or MR diet prior to femoral artery ligation (I, ischemic; NI, non-ischemic). Left: representative infrared images on the indicated day after ligation. Right: quantification of blood flow recovery with individual animal AUCs used for statistical comparison; n=7–8 mice/group. (G) Representative transverse sections (left, 40X mag) and quantification (right) of CD31-stained gastroc 10d after ligation from (F); n=4 mice/group. Error bars indicate SD unless otherwise noted; asterisks indicate the significance of the difference by Student’s T test or 1-way ANOVA with Sidak’s MCT between diets in vivo or SAA deprivation in vitro; *P<0.05, **P<0.01, ***P<0.001. See also Fig. S1.

Figure 2. GCN2-dependent, hypoxia-independent regulation of VEGF and angiogenesis upon SAA restriction

(A) Relative VEGF mRNA expression in HUVEC 2d after transfection with HIF1α siRNA or control scrambled (Sble) siRNA and cultured in control (Ctrl) or SAA deficient (-M&C) media for 16hr; n=5 experiments/group; error bars indicate SEM. (B) Immunoblots of HIF1α, eIF2α (p- Ser51, total) and ATF4 in HUVEC cultured as indicated for 16hr. (C) Relative VEGF mRNA expression in HUVEC 2d after transfection with ATF4 or Sble siRNA and cultured as indicated for 16hr; n=4 experiments/group; SEM. (D, E) Relative HUVEC VEGF mRNA expression (D, n=3 experiments/group; SEM) and secreted VEGF protein concentration in media (E, n=3–6 experiments/group; SEM) 2d after transfection with ATF4 overexpression (ATF4^OE) or control construct (Empty). (F, G) VEGF mRNA expression (F) and spheroid formation (G) in WT and GCN2KO primary mouse EC from n=3 mice/genotype cultured as indicated for 16hr. For sprouting assay (G), representative images (left, 40X mag) and quantification (right) of WT and GCN2KO EC spheroids cultured in the indicated media for 24hr; blue, DNA (DAPI); red, F-actin (phalloidin). (H) Representative transverse sections (left, 40X mag) and quantification (right) of CD31-stained gastroc in WT or GCN2KO mice fed for 2–4wk on Ctrl or MR diets; n=5–6 mice/group. (I) VEGF mRNA in MDF, MEF or C2C12 myotubes cultured as indicated for 16hr; n=4–6 experiments/group; SEM. (J) VEGF mRNA expression in WT and GCN2KO primary mouse skeletal myotubes (n=5 mice/genotype tested at 2 different passages) cultured as indicated for 16hr. (K) Immunoblots of HIF1α, PGC1α, eIF2α (p-Ser51, total) and ATF4 in C2C12 myotubes cultured as indicated for 16hr. Error bars indicate SD unless otherwise noted; asterisks indicate the significance of the difference by Student’s T test or 1-way ANOVA with Sidak’s MCT between diets in vivo or SAA deprivation in vitro; *P<0.05, **P<0.01, ***P<0.001. See also Fig. S2.

Figure 3. VEGF signalling and AASR converge on endothelial H₂S production by CGL

(A) Representative H₂S production capacity as indicated by black lead sulfide formation from HUVEC cultured in media +/−M&C or VEGF (50ng/mL) in the presence or absence of the CGL inhibitor PAG (100μM) as indicated for 16hr. (B) Representative (left) endogenous H₂S levels (blue, H₂S (P3 fluorescence); red, DNA (DRAQ5)) and quantification of P3 intensity (right) in HUVEC upon VEGF or -M&C treatment; n=4 wells/treatment with 4–6 images/well; 1-way ANOVA with Sidak’s MCT vs. Control (asterisks) or +/−PAG within treatment (carets). (C) CGL mRNA expression in WT and GCN2KO primary mouse EC cultured from n=3 mice/genotype in control (Ctrl) or -M&C media for 16hr. (D) CGL mRNA expression in HUVEC 2d after transfection with ATF4 or control scrambled (Sble) siRNA and cultured in the indicated media for 16hr; n=4 experiments/group; SEM. (E) CGL mRNA expression in HUVEC 2d after transfection with ATF4 overexpression or control (empty) plasmid; n=3 experiments/group; SEM. (F, G) Representative images (left, 40X mag) and quantification (right, in triplicate) of spheroids cultured from (F) HUVEC +/−M&C for 24hr in the presence of vehicle (Veh) or PAG, and (G) WT or CGLKO primary EC sprouts in control or -M&C media for 24hr; blue, DNA (DAPI); red, F-actin (phalloidin). Unless otherwise indicated, error bars indicate SD, and asterisks indicate the significance of the difference between diets in vivo or SAA levels in vitro by Student’s T test or 1-way ANOVA with Sidak’s MCT; *P<0.05, **P<0.01, ***^/^^^P<0.001. See also Fig. S3.

Figure 4. CGL required for angiogenesis in vivo

(A) Representative transverse sections (20X mag) of gastrocnemius muscle from WT and CGLKO mice stained for CD31 and endogenous H₂S. (B) Quantification of endogenous H₂S in CD31⁺ EC in the gastrocnemius muscle of WT and CGLKO mice fed for 2wk on Ctrl or MR diets as indicated; n=4–5 mice/group, with quantification of 3–10 images/mouse. (C) Representative transverse sections (left, 40X mag) and quantification (right) of CD31-stained gastroc from WT and CGLKO mice fed for 2wk on Ctrl or MR diets as indicated; n=4 mice/group. (D) Representative transverse sections of CD31-stained gastroc (left, 40X mag) and quantification (right) 2wk after Ad-Null or Ad-CGL injections; n=3–4 mice/group; Student’s T test. (E) Representative transverse sections (left, 40X mag) and quantification (right) of CD31-stained gastroc from WT and CGLKO mice subjected to low intensity running (exercised) vs. control (sedentary) for 1mo; n=4–5 mice/group. (F) Representative transverse sections of CD31-stained gastroc (left, 40X mag) and quantification (right) from WT and CGLKO mice 6d after the final intramuscular injection of control (Ad-Null) or VEGF₁₆₅ -expressing (Ad-VEGF) adenovirus; n=4 mice/group. Error bars indicate SD; asterisks indicate the significance of the difference between diets or treatments within genotype by 1-way ANOVA with Sidak’s MCT unless otherwise noted; *P<0.05, **P<0.01, ***P<0.001. See also Fig. S4.

Figure 5. H₂S promotes glucose uptake and ATP generation by glycolysis for EC migration

(A) Representative migration across scratch (left, 10X mag) and quantification (right) of HUVEC +/−100μM NaHS in the presence of vehicle or mitomycin C (MitoC, 1μg/mL) to inhibit proliferation; n=12 wells each from cells at 2 different passages; 1-way ANOVA with Sidak’s MCT between control and NaHS within vehicle or MitoC treatment group. (B, C) Representative images (B) and quantification (C) of migration speed (left, n=5–7 cells/condition) and distance (right, n=5–7 cells/condition in x and y directions) from time-lapse video imaging of GFP+ HUVEC infected with control (Ad-Null) or CGL adenovirus (Ad-CGL) as indicated; Student’s T test. (D) Relative glucose uptake in HUVEC pretreated with NaHS or 50ng/mL VEGF for 1hr; n=3–6 experiments/group; 1-way ANOVA with Dunnett’s MCT. (E) Extracellular acidification rate (ECAR) in WT and CGLKO primary mouse EC pretreated for 1hr with VEGF or NaHS; 10 technical replicates from EC pooled from 6 mice/genotype; 1-way ANOVA with Sidak’s MCT as indicated. (F) Glycolytic flux in HUVEC pretreated for 3hr with NaHS or VEGF; 1-way ANOVA with Dunnett’s MCT. Representative experiment of 6 with n=3/group; 1-way ANOVA with Sidak’s MCT. (G) Time dependent ATP production in HUVEC pretreated with NaHS or 1mM 2DG at t=0; n=4 experiments each for NaHS and VEGF and 2 for 2DG; error bars indicate SEM; 2-way ANOVA with Dunnett’s MCT relative to t=0 (asterisk, NaHS; caret, VEGF; pound sign, 2DG). (H, I) Representative migration (left, 10X mag) and quantification (right, AUC) of HUVEC treated +/−NaHS (H, n=11 technical replicates/condition) or infected with a control (Ad-Null) or CGL adenovirus (Ad-CGL) at a multiplicity of infection of 50 (I, n=5–6 technical replicates/condition), in the presence of vehicle or 2DG; 1-way ANOVA with Sidak’s MCT between control and NaHS within 2DG or vehicle treatment group. (J) Log2 fold change of C13- labelled metabolites in HUVEC measured by mass spectrometry after 1hr of NaHS pretreatment compared to control; red dots, metabolites with FDR adjusted P<0.05 and absolute value of log2 fold change>1.2; blue dots, metabolites with FDR adjusted P>0.05 and/or absolute value of log2 fold change<1.2. (K) Plot of the first two components of orthogonal partial least squares discriminant analysis on unlabeled metabolite levels in HUVEC after 15min, 2hr or 4hr treatment with NaHS or -M&C. Ellipses represent 99% confidence bound for treatment groups. (L) Average metabolite log2 fold changes after 15min of NaHS or -M&C. All metabolites that significantly (P<0.05) changed in the same direction in both treatment groups are shown. Error bars indicate SD unless otherwise indicated; *^/^P<0.05, **P<0.01, ***^/###P<0.001, ^####P<0.0001. See also Fig. S5.

Figure 6. H₂S shifts oxidative/glycolytic balance concomitant with inhibition of mitochondrial OXPHOS

(A) Basal oxygen consumption rate (OCR) in WT and CGLKO primary mouse EC; n=10 technical replicates from EC pooled from 6 mice/genotype; Student’s T test. (B) OCR (left) and extracellular acidification rate (ECAR, right) in HUVEC pretreated for 2hr with 100μM NaHS followed by oligomycin (oligo, 2.5μM) injection at the indicated time; representative experiment with n=10 technical replicates/treatment. (C) Mitochondrial complex activity in permeabilized HUVEC pretreated for 1hr with NaHS or 10μM KCN ; representative experiment with n=6 technical replicates; 1-way ANOVA with Sidak’s MCT within complex activity group. (D, E) Immunoblots of ACC (pSer79, total) and AMPK (Thr172, total) in HUVEC treated with NaHS (D) or KCN (E) for the indicated time. (F) ECAR in HUVEC pretreated for 1hr with NaHS +/−10μM Compound C (Comp C, an AMPK inhibitor) as indicated; n=10 technical replicates from EC pooled from 6 mice/genotype; 1-way ANOVA with Sidak’s MCT between NaHS treatment within Comp C treatment group. (G) Relative glucose uptake in HUVEC pretreated with the indicated agent for 1–3hr; n=2–8 experiments/group; error bars indicate SEM; 1-way ANOVA with Dunnett’s MCT. (H) ECAR in HUVEC pretreated for 2hr with KCN, 2μM oligo or phenformin (Phen, 500μM) expressed as a percent of control over time after addition of 10mM glucose; n=12–19 technical replicates; 1-way ANOVA with Dunnett’s multiple comparison test. (I) Migration across scratch expressed as fold change relative to control in HUVEC treated with oligomycin (2μM), phenformin or KCN +/−2DG (1mM) as indicated; n=5–12 AUC values/group each from cells at different passages; 1-way ANOVA with Sidak’s MCT vs. control without 2DG treatment. Error bars indicate SD unless otherwise noted; *P<0.05, **P<0.01, ***P<0.001. See also Fig. S6.

Figure 7. Model for regulation of angiogenesis by AA restriction

GCN2/ATF4 pathway activation in EC by AA restriction induces VEGF expression as well as CGL-mediated H₂S production with effects on glucose uptake and utilization via glycolysis and PPP required for EC migration and proliferation.