Ketone body oxidation increases cardiac endothelial cell proliferation

Abstract

Blood vessel formation is dependent on metabolic adaption in endothelial cells. Glucose and fatty acids are essential substrates for ATP and biomass production; however, the metabolism of other substrates remains poorly understood. Ketone bodies are important nutrients for cardiomyocytes during starvation or consumption of carbohydrate-restrictive diets. This raises the question whether cardiac endothelial cells would not only transport ketone bodies but also consume some of these to achieve their metabolic needs. Here, we report that cardiac endothelial cells are able to oxidize ketone bodies and that this enhances cell proliferation, migration, and vessel sprouting. Mechanistically, this requires succinyl-CoA:3-oxoacid-CoA transferase, a key enzyme of ketone body oxidation. Targeted metabolite profiling revealed that carbon from ketone bodies got incorporated into tricarboxylic acid cycle intermediates as well as other metabolites fueling biomass production. Elevation of ketone body levels by a high-fat, low-carbohydrate ketogenic diet transiently increased endothelial cell proliferation in mouse hearts. Notably, in a mouse model of heart hypertrophy, ketogenic diet prevented blood vessel rarefication. This suggests a potential beneficial role of dietary intervention in heart diseases.

Keywords: angiogenesis; endothelial cell; heart; ketogenic diet; ketone bodies.

Figure 1. Key enzymes of ketone body oxidation are expressed in cardiac endothelial cells

1. A

   Oxidation of ketone bodies requires the enzymes SCOT and BDH1 and yields acetyl‐CoA which enters the tricarboxylic acid (TCA) cycle.

2. B

   Immunoblot of SCOT, BDH1, and the loading control VCP across various organs obtained from adult C57Bl/6J mice.

3. C, D

   Quantification of SCOT and BDH1 expression levels relative to VCP in various murine organs.

4. E

   Immunoblot of SCOT, BDH1, CD31, and the loading control VCP in murine endothelial cells isolated from heart, muscle, lung, and adipose tissue.

5. F

   Immunoblot of SCOT, BDH1, CD31, and the loading control VCP in human cardiac ECs, HUVECs, and adipose tissue ECs.

6. G

   Immunoblot of SCOT, BDH1, CD31, and VCP in cardiac endothelial cells (EC) and non‐endothelial cell fraction (non‐EC) isolated from adult C57Bl/6J mice.

7. H

   Immunoblot of SCOT, BDH1, and β‐actin in immortalized murine cardiac endothelial cells (MCEC).

8. I, J

   Ketone body concentration in cell culture medium of MCEC treated with 2 mM D‐β‐hydroxybutyrate (β‐OHB) or 2 mM acetoacetate (AcAc) after 24 h. Data are presented as mean ± SD. n = 3; *P < 0.05; ***P < 0.001 unpaired Student’s t‐test.

Source data are available online for this figure.

Figure 2. Ketone body treatment increases TCA cycle intermediates in MCEC

1. A, B

   Murine cardiac endothelial cells (MCEC) were treated with 2 mM D‐β‐hydroxybutyrate (β‐OHB) or 2 mM acetoacetate (AcAc) for 24 h. Quantification of concentrations of TCA cycle intermediates compared to control treatment.

2. C

   Scheme of ketone body oxidation and the tricarboxylic acid (TCA) cycle. MCEC were incubated with 1 mM ¹³C₄‐β‐hydroxybutyrate or 1 mM ¹³C₄‐acetoacetate for 24 h or the same concentration of unlabeled ketone bodies ([¹²C] isotope). The [¹³C]‐labeled fractions of the TCA cycle intermediates citrate, α‐ketoglutarate and malate (marked with blue star) were quantified with an untargeted metabolomics approach indicating that ¹³C carbon from ketone bodies was incorporated into TCA cycle intermediates.

3. D

   Quantifications of [¹³C]‐labeled fractions of amino acids (asparagine, l‐aspartate, l‐glutamate, l‐proline), putative lipid species (nervonic acid, 1‐palmitoylglycerol, ceramide (d18:1/16:0)) and uridine diphosphate (UDP) species (uridine monophosphate, uridine 5'‐diphosphate, UDP‐N‐acetylglucosamine) in MCEC treated with 1 mM ¹³C₄‐β‐hydroxybutyrate or 1 mM ¹³C₄‐acetoacetate for 24 h or the same concentration of unlabeled ketone bodies ([¹²C] isotope). Data are presented as mean ± SD. n ≥ 4; ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 unpaired Student’s t‐test.

Figure EV1. Effects of ketone bodies on mitochondrial respiration

Mitochondrial function of MCECs stimulated with ketone bodies was characterized using the Seahorse Bioanalyzer by measuring the rate of oxygen consumption (OCR) following sequential additions of oligomycin, FCCP and antimycin/rotenone (A) to the cells to determine non‐mitochondrial oxygen consumption (B), basal respiration (C), maximal respiration (D), proton leakage (E), ATP production (F), spare respiratory capacity (G), and the bioenergetic health index (H), respectively.

1. A–H

   MCECs were starved for 1 h before addition of 2 mM R‐β‐hydroxybutyrate (βOHB) compared to H₂O and acetoacetate (AcAc) compared to ethanol for 24 h.

2. I–L

   MCECs were cultured in basal medium before addition of ketone bodies (2 mM) and solvent control. (I, J) Cellular ATP contents after 12 and 24 h. (K, L) Targeted metabolomics using UPLC to determine cellular nucleotides upon treatment with ketone bodies for 24 h. Data are presented as mean ± SD. One‐way ANOVA using nonparametric (Kruskal–Wallis) test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, NS > 0.05.

Figure 3. Ketone body treatment promotes endothelial cell proliferation, migration, and sprouting capacity

1. A, B

   Murine cardiac endothelial cells (MCEC) were treated with different concentrations of D‐β‐hydroxybutyrate (βOHB) or acetoacetate (AcAc) for 24 h. Relative BrdU absorbance was quantified compared to control treatment (H₂O for βOHB and EtOH for AcAc).

2. C, D

   MCEC were treated with 10 mM D‐β‐hydroxybutyrate (βOHB) or 10 mM acetoacetate (AcAc) for 24 h. Electrical impedance was measured compared to control treatment (dashed line; H₂O for βOHB and EtOH for AcAc).

3. E, F

   MCEC were seeded onto Boyden chambers and treated with (E) 10 mM D‐β‐hydroxybutyrate (βOHB) or (F) 10 mM acetoacetate (AcAc) for 24 h. Electrical impedance was measured in the lower chamber compared to control treatment (dashed line; H₂O for βOHB and EtOH for AcAc).

4. G, H

   Representative images of MCEC spheroids treated with H₂O, 30 mM R‐β‐hydroxybutyrate (βOHB), ethanol (EtOH) or 30 mM acetoacetate (AcAc) for 72 h; scale bar: 50 µm.

5. I–M

   Angiogenic capacity of MCEC in response to ketone body treatment was analyzed using a spheroid‐based sprouting assay. Spheroids were treated with media containing 30 mM D‐β‐hydroxybutyrate (βOHB), 30 mM acetoacetate (AcAc), 10 mM acetate, 1 mM octanoate, 1 mM butyrate, 30 mM L‐β‐hydroxybutyrate (L‐ βOHB), 1 mM niacin or the respective controls (final concentration of reagents is diluted to approximately 10%) and analyzed after 48 h. The (I, L) average number of sprouts per spheroid, the (J, M) accumulated total sprout length and (K) average sprout length were quantified. Data are presented as mean ± SD. n ≥ 3; *P < 0.05; **P < 0.01; ***P < 0.001 unpaired Student’s t‐test.

Figure EV2. Effects of ketone body supplementation on sprouting potential

1. A–C

   Cell counts of MCECs treated with 0.1, 1 or 10 mM R‐β‐hydroxybutyrate (βOHB) for 24, 48, or 72 h compared to treatment with H₂O (control).

2. D–F

   Cell counts of MCECs treated with 0.1, 1 or 10 mM acetoacetate (AcAc) for 24, 48, or 72 h compared to treatment with ethanol (control).

3. G

   Representative images of HUVEC spheroids treated with H₂O (control), recombinant VEGF‐A165, 30 mM R‐β‐hydroxybutyrate (βOHB) for 72 h; scale bar: 50 μm.

4. H

   Quantification of the accumulated total sprout length per spheroid. n = 4.

5. I

   Relative absorbance of BrdU incorporated into DNA of HUVEC treated with 1, 4 or 10 mM R‐β‐hydroxybutyrate (βOHB), 0.1, 1 or 10 mM acetoacetate (AcAc) or a combination of both for 24 h. Data are presented as mean ± SD. n ≥ 2. One‐way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4. Ketone body treatment does not increase sprouting capacity in SCOT‐deficient cardiac endothelial cells

1. A

   SCOT‐deficient murine cardiac endothelial cells were generated using the CRISPR/Cas9 technology by targeting the SCOT‐encoding gene Oxct1. A non‐targeting control construct was used to generate the non‐targeting control cells (nt ctrl). Immunoblot of SCOT and β‐actin in non‐targeting control cells (nt ctrl) and Oxct1 knockout cells (Oxct1 ko).

2. B, C

   Angiogenic capacity of Oxct1 knockout cells in response to ketone body treatment was analyzed using a spheroid‐based sprouting assay. Spheroids were treated with media containing 30 mM D‐β‐hydroxybutyrate (βOHB), 30 mM acetoacetate (AcAc) or the respective controls (final concentration of reagents is diluted to approximately 10%) for 48 h. The average number of sprouts per spheroid and the accumulated sprout length was quantified. Data are presented as mean ± SD. n = 4; *P < 0.05; **P < 0.01; unpaired Student’s t‐test.

Source data are available online for this figure.

Figure 5. Ketogenic diet alters gene expression in cardiac endothelial cells towards a more proliferative signature

1. A

   Ketone body concentrations were measured in serum of adult C57Bl/6J mice that were fed a ketogenic diet for the indicated time periods.

2. B

   RNA sequencing was performed on isolated cardiac endothelial cells of male C57Bl/6J mice that were fed a ketogenic diet or control diet for 2 weeks. Volcano plot showing differentially expressed genes as genes with a log2 fold change ≤ −0.5 or ≥ 0.5 (vertical dashed lines) and P ≤ 0.01 (horizontal dashed line).

3. C, D

   Heatmaps showing expression levels of genes involved in KEGG pathways (C) fatty acid degradation and (D) ketone body metabolism in cardiac endothelial cells isolated from male C57Bl/6J mice that were fed a ketogenic diet or control diet for 2 weeks.

4. E

   Gene set enrichment analysis of hallmark (GSEA gene set h, blue) and gene ontology (GO)‐derived (GSEA gene set c5, green) gene sets enriched in cardiac endothelial cells of C57Bl/6J mice kept on a ketogenic diet compared to littermate animals kept on control diet.

5. F, G

   Enrichment plots of the hallmark gene sets fatty acid metabolism and G2M checkpoint comparing expression pattern of cardiac endothelial cells isolated from C57Bl/6J mice kept on a ketogenic diet compared to littermate animals kept on control diet. Data are presented as mean ± SD. n ≥ 4; ***P < 0.001; unpaired Student’s t‐test.

Figure EV3. Cardiac endothelial cells isolated from mice fed a ketogenic diet

1. Relative mRNA levels of cardiomyocyte, fibroblast, and smooth muscle cell marker genes in anti‐CD31‐isolated cells compared to levels in total heart lysates of C57Bl/6J mice.

2. Flow cytometry analysis of anti‐CD31‐isolated cells from hearts of C57Bl/6J mice using the endothelial cell marker CD31 and the immune cell marker CD45.

3. Relative fold changes of Oxct1 (Scot) and Bdh1 expression levels in mice fed a ketogenic diet relative to a control diet were obtained from RNAseq analyses.

4. Relative mRNA levels of cardiac ECs isolated from mice fed a ketogenic or control diet confirming increased levels of pro‐proliferative genes Hmgcs2 and Pdk4.

5. Heat‐map showing expression levels of genes involved in regulation of cell cycle (KEGG). Data are presented as mean ± SD. n ≥ 3. Two‐tailed unpaired Student’s t‐test; **P < 0.01; ***P < 0.001.

Figure 6. Ketogenic diet induces proliferation of cardiac endothelial cells in mice

1. A, B

   Representative images of heart sections of animals kept on a ketogenic diet or control diet for 2 weeks stained against ERG and Ki67 or EdU. Double‐positive cells are indicated by arrowheads. Quantification is of double‐positive cells in heart sections per high power field (HPF).

2. C, D

   Quantification of Ki67⁺/ERG⁺ cells in heart sections of mice kept on a ketogenic diet or control diet for 4 or 6 weeks.

3. E

   Quantification of CD31⁺ area in heart sections of mice kept on a ketogenic diet or control diet for 4 months.

4. F

   Quantification of Ki67⁺ endothelial cells in several organs of mice kept on a ketogenic diet or control diet for 4 weeks (gastrocnemius and soleus muscle) or 2 weeks (brain, lungs, subcutaneous adipose tissue, liver). Scale bar: 50 μm. Data are presented as mean ± SD. n ≥ 3; *P < 0.05; **P < 0.01; ***P < 0.001 unpaired Student’s t‐test.

Figure EV4. Endothelial cell apoptosis in hearts of mice receiving a ketogenic diet

1. A, B

   Quantification of cleaved caspase 3⁺/CD31⁺ double‐positive cells per high power field (HPF) in heart sections of C57Bl/6J mice kept on a control diet or a ketogenic for (A) 2 weeks or (B) 4 weeks.

2. C

   Representative images of heart sections of animals kept on the control or ketogenic diet for 4 weeks stained against CD31.

3. D–G

   Quantification of CD31‐positive area per high power field (HPF) in heart sections of mice kept on control diet (ctrl diet) or ketogenic diet (keto diet) for 2 weeks / 4 weeks / 6 weeks / 4 months. Scale bar: 50 µm.

4. H

   Quantification of Ki67⁺/CD31⁺ double‐positive cells per high power field (HPF) in B16F10 tumor sections of C57Bl/6J mice kept on a control diet or a ketogenic 10 days after tumor inoculation.

5. I

   Quantification of CD31⁺ vessels per high power field (HPF) in B16F10 tumor sections of C57Bl/6J mice kept on a control diet or a ketogenic 10 days after tumor inoculation.

6. J

   Tumor volume of C57Bl/6J mice kept on a control diet or ketogenic diet. Data are presented as mean ± SD. n ≥ 3; statistical significance determined using unpaired Student’s t‐test.

Figure EV5. Schematic overview of experimental animal groups for transverse aortic constriction study and aortic flow rates

1. Male and female C57Bl/6J mice were randomly assigned to a diet group at 8 weeks old. After 4 weeks on the respective diet, mice in each group underwent either transverse aortic constriction (TAC) or sham surgery. Mice were afterward kept on the respective diet for eight more weeks.

2. Quantification of aortic blood flow rates within the stenosis of C57Bl/6J mice after TAC or sham surgery. Data are presented as mean n ≥ 2.

3. Trichrome staining of heart sections from sham and TAC‐operated animals kept either on a control diet or ketogenic diet 8 weeks after surgery. Scale bar: 1 mm.

4. Representative images of heart vasculature (CD31⁺ vessels) of mice kept on a control or ketogenic diet 8 weeks after TAC surgery. Scale bar: 50 µm. Data are presented as mean ± SD. Two‐tailed unpaired Student’s t‐test; **P < 0.01; ***P < 0.001.

Figure 7. Ketogenic diet prevents vascular rarefaction in mice after transverse aortic constriction

1. Parameters of cardiac function (fractional shortening, left ventricular end‐diastolic anterior wall thickness (LVAW;d), ratio of left ventricular (LV) mass to body mass) 8 weeks after transverse aortic constriction (TAC) in male and female C57Bl/6J mice receiving a control diet or ketogenic diet compared to sham animals kept on the same diet.

2. Representative images of heart sections of mice kept on a control or ketogenic diet 8 weeks after TAC surgery stained against Ki67 and ERG.

3. Quantification of Ki67⁺/ERG⁺ cells per high power field (HPF) in heart sections of mice kept on a control diet or a ketogenic diet 8 weeks after TAC surgery.

4. Quantification of isolectin IB4‐positive vessels per high power field (HPF) in heart sections of mice kept on a control diet or a ketogenic diet 8 weeks after TAC surgery relative to sham animals from the respective diet group. Scale bar: 50 µm. Data are presented as mean ± SD. n ≥ 4; *P < 0.05; ***P < 0.001 unpaired Student’s t‐test.