Short-Chain Fatty Acids and Palmitate Induce Distinct Metabolic and Phenotypic Signatures in Normal and Ischemic Skeletal Muscle Microvascular Endothelial Cells

Figures

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  Figure 1. Differential effects of palmitate and short-chain fatty acids on normal vs. ischemic EC proliferation and survival. (A,B) CCK8 cell proliferation/survival assay in (A) normal, (B) hypoxia serum-starved (HSS) mSkVECs treated with BSA (vehicle control), palmitate (PA: 100 µM, 250 µM, 500 µM), or short-chain fatty acids (SCFAs: 100 µM, 250 µM, 500 µM) dose-dependently for 24 h. n ≥ 7 biological replicates. One-way ANOVA with Dunnett’s post-test. Mean ± SEM.

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  Figure 2. Angiogenic effects of palmitate and short-chain fatty acids on normal vs. ischemic ECs. In vitro tube formation assay of (A) normal, (B) HSS mSkVECs treated with BSA, palmitate (100 µM, 250 µM, 500 µM), or short-chain fatty acids (SCFAs: 100 µM, 250 µM, 500 µM) dose-dependently for 24 h on growth factor-reduced Matrigel. n = 4 biological replicates. One-way ANOVA with Dunnett’s post-test. Mean ± SEM.

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  Figure 3. Vascular permeability effects of palmitate and short-chain fatty acids on normal vs. ischemic ECs. (A,B) Trans-endothelial electrical resistance (TEER, Ohms/cm2, presented as % change (denoted as 1) from 0 h timepoint of specific group. 0 h b: 0 h base line (before treatment), 0 h tx: 0 h timepoint immediately after treatment) of (A) normal, (B) HSS mSkVECs treated with BSA, 250 µM-palmitate, or SCFAs 500 µM for 24 h. n = 4 biological replicates. Repeated measures ANOVA with Bonferroni select pair comparison. p < 0.05 is considered significant. Mean ± SEM.

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  Figure 4. Effects of palmitate and short-chain fatty acids on mitochondrial respiration in normal vs. ischemic ECs. (A,B) Seahorse mitostress test in (A) normal, (B) HSS mSkVECs treated with BSA (n = 5 biological replicates), palmitate (PA-250 µM, n = 6 biological replicates), or short-chain fatty acids (SCFAs-500 µM, n = 6 biological replicates) for 24 h. One-way ANOVA with Dunnett’s post-test. Mean ± SEM.

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  Figure 5. Effects of palmitate and SCFAs on normal vs. ischemic EC β-oxidation. (A,B) Seahorse basal fatty acid oxidation test (that uses CPT1 inhibitor without exogenous/additional palmitate in the Seahorse assay medium) in (A) normal, (B) HSS mSkVECs treated with BSA (n = 5 biological replicates), palmitate (PA-250 µM, n = 6 biological replicates), or short-chain fatty acids (SCFAs 500 µM, n = 6 biological replicates) for 24 h. One-way ANOVA with Dunnett’s post-test. p < 0.05 is considered significant. Mean ± SEM.

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  Figure 6. Effect of palmitate and short-chain fatty acids on normal and ischemic EC glycolysis. (A,B) Seahorse glycolysis stress test in (A) normal, (B) HSS mSkVECs treated with BSA (n = 5 biological replicates), palmitate (PA-250 µM, n = 5 biological replicates), or short-chain fatty acids (SCFAs 500 µM, n = 6 biological replicates) for 24 h. One-way ANOVA with Dunnett’s post-test. Mean ± SEM.