Mitochondrial respiration controls the Prox1-Vegfr3 feedback loop during lymphatic endothelial cell fate specification and maintenance

Abstract

Recent findings indicate that mitochondrial respiration regulates blood endothelial cell proliferation; however, its role in differentiating lymphatic endothelial cells (LECs) is unknown. We hypothesized that mitochondria could work as a sensor of LECs' metabolic specific needs by determining their functional requirements according to their differentiation status and local tissue microenvironment. Accordingly, we conditionally deleted the QPC subunit of mitochondrial complex III in differentiating LECs of mouse embryos. Unexpectedly, mutant mice were devoid of a lymphatic vasculature by mid-gestation, a consequence of the specific down-regulation of main LEC fate regulators, particularly Vegfr3, leading to the loss of LEC fate. Mechanistically, this is a result of reduced H3K4me3 and H3K27ac in the genomic locus of key LEC fate controllers (e.g., Vegfr3 and Prox1). Our findings indicate that by sensing the LEC differentiation status and microenvironmental metabolic conditions, mitochondrial complex III regulates the critical Prox1-Vegfr3 feedback loop and, therefore, LEC fate specification and maintenance.

Fig. 1. QPC^f/f;PdpnGFPCre embryos show edema and lack LECs.

(A and B) E14.5 QPC^f/f;PdpnGFPCre mutant embryos exhibit severe dermal edema (white arrow) (n = 5). (C and D) Whole-mount immunostaining of the dermal lymphatics of those embryos using Prox1 and VEcad antibodies (n = 4) shows that lymphatic vessels (red) are missing from the skin of the mutant embryos. Whole-mount immunostaining of control and mutant E16.5 mesenteric (E and F) and cardiac (dorsal view) (G and H) lymphatics (n = 4) revealed that lymphatics were also absent in these organs. Dermal lymphatic vessel length (I), blood vessel density (J), mesenteric lymphatic vessel length (K), and cardiac lymphatic vessel length (L) are quantified by ImageJ and shown as a percentage of the mean of total vessel length in the control group. All results are presented as means ± SEM and analyzed with a two-tailed Student’s t test. Scale bars, 2 mm (A and B), 200 μm (C and D), 75 μm (E and F), and 500 μm (G and H).

Fig. 2. Expression of LEC markers is reduced in E11.5 QPC^f/f;PdpnGFPCre embryos.

(A and B) Transverse sections at the level of the anterior CV show that expression of Vegfr3 is severely reduced in differentiating Prox1-expressing LECs outside the CV. Similarly, other LEC markers such as Pdpn (C and D) and Lyve1 (E and F) are also down-regulated, whereas expression of the pan-endothelial markers VEcad (G and H) and CD31 (I and J) is normal. White box insets correspond to higher magnification of the dotted box region in each panel. Arrows indicate dorsal (D) and lateral (L) orientations. Mean fluorescence intensity of each staining was quantified and shown as percentage of mean of control group in (K) and (L). Each data point represents a biological replicate. All results are presented as means ± SEM and analyzed by multiple t test. Scale bar, 50 μm (n = 6).

Fig. 3. QPC^f/f;PdpnGFPCre mutant embryos show severely enlarged lymph sacs.

(A to J) At E12.5, expression of Prox1 still remains in the mutant embryos; however, expression of most other LEC markers is barely detected or is severely down-regulated. Expression of the pan-endothelial marker CD31seems normal. White arrows in control embryo (E) point to the dermal lymphatic vasculature; mutant LECs fail to sprout from the lymph sacs such that lymph sacs (LS) become abnormally enlarged and mutant embryos lack dermal lymphatics. White box insets correspond to higher magnifications of the regions inside the dotted boxes in each panel. Mean fluorescence intensity of each staining was quantified and shown as percentage of mean of control group in (K). Each data point represents a biological replicate. Area of the CV and lymph sac was quantified and shown as fold change of lymph sac area compared to CV in each genotype (L). All results are presented as means ± SEM and analyzed by multiple t test for (K) and two-tailed Student’s t test for (L). Scale bar, 50 μm (n = 3 to 8).

Fig. 4. QPC-deficient LECs gradually up-regulate the expression of BEC markers.

(A and B) At E11.5, QPC^f/f;PdpnGFPCre LECs show severely reduced Vegfr3 levels, but similar to controls, Prox1⁺ LECs do not coexpress BEC markers such as CD34 (n = 3). (C and D) At around E12.5, Vegfr3 expression becomes undetectable in mutant LECs and Prox1⁺(low) LECs abnormally up-regulate the expression of CD34, an indication that they are losing LEC fate and regaining BEC fate. White arrows indicate Prox1⁺ CD34⁺ LECs. Blood cells are abnormally seen inside the mutant lymph sacs (D), most likely a consequence of defective lymphovenous valves. White box insets correspond to higher magnification of the regions in dotted boxes in each panel. CD34⁺ Prox1⁺ cells were quantified and shown as percentage of Prox1⁺ in each group (E). Each data point represents a biological replicate. All results are presented as means ± SEM and analyzed by two-tailed Student’s t test. Scale bar, 50 μm (n = 6).

Fig. 5. LEC fate is lost in QPC null embryos.

(A to B′′′) Lineage tracing analysis using Prox1CreERT2 mice crossed to QPC^f/f;RosamTmG. A low dose of tamoxifen is injected at E9.5, and embryos are harvested at E12.5. As expected, in QPC^f/+embryos, few GFP⁺ cells colocalize with Prox1 (A to A′′′); however, in QPC^f/f mutant littermates, some of the GFP⁺ cells were negative for Prox1 (arrows), confirming their loss of LEC fate (B to B′′′). GFP⁺ Prox1⁻ cells were quantified and shown as percentage of GFP⁺ cells in each group (C). Each data point represents a biological replicate. All results are presented as means ± SEM and analyzed by two-tailed Student’s t test. Scale bar, 50 μm (n = 3).

Fig. 6. Mitochondrial complex III respiration is required for Vegfr3 and Prox1 expression maintenance in cultured LECs.

(A) Antimycin A (Anti) treatment reduces Prox1 (n = 9), Vegfr3 (n = 9), and Nrp2 (n = 5) mRNA levels but not those of VEcad (n = 3). (B) Myxothiazol (Myx) treatment also reduces Prox1 (n = 4), Vegfr3 (n = 6), and Nrp2 (n = 3) mRNA levels but not those of VEcad (n = 3). (C) Representative Western blot shows that antimycin A and myxothiazol treatment reduces VEGFR3 (n = 6) and PROX1 (n = 6) levels but not those of VECAD (n = 5 to 6). (D) Quantification of the densitometry of each protein normalized to GAPDH and shown as fold change compared to controls. Each data point represents a biological replicate. All results are presented as means ± SEM and analyzed by multiple t test in (A) and (B) or two-way analysis of variance (ANOVA) in (D). (E and F) RNA-seq of LECs treated with vehicle or antimycin A shows that expression of most LEC genes in the dataset is reduced, while almost half of the BEC genes are up-regulated, including Nrp1 and ICAM1 (n = 3).

Fig. 7. LECs expressing AOX restore oxygen consumption, NAD⁺/NADH ratio, proliferation, and Vegfr3 levels in the presence of antimycin.

Antimycin A inhibits OCR (A), NAD⁺/NADH ratio (B), and cell proliferation (C) in LECs transduced with EV-GFP, while AOX rescues OCR (n = 3 to 4) (A), NAD⁺/NADH ratio (n = 3) (B), and cell proliferation (n = 3) (C) in the presence of antimycin A. AOX also rescued the Vegfr3, Prox1, and Nrp2 levels in the presence of antimycin A (D) (n = 5 to 6). Each data point represents a biological replicate. All results are presented as means ± SEM and analyzed by one-way ANOVA for (A) to (C) or two-way ANOVA for (D).

Fig. 8. Mitochondrial respiration is required for epigenetic regulation of Vegfr3 and Prox1.

Mitochondrial complex III inhibition with antimycin A alters TCA cycle metabolites (A) and 2HG levels (B). Values are normalized to mean of control group. (C) ChIP-seq analysis of H3K27ac, H3K4me3, and H3K4me1 histone modifications in LEC cultures treated with vehicle control (Ctrl) or antimycin A (Anti) for 48 hours. Track examples for LEC-specific genes (Prox1 and Vegfr3) and BEC genes (Nrp1 and ICAM1) are shown. Antimycin A–treated LECs show a marked reduction in the H3K4me3 and H3K27ac signal at the Vegfr3 locus and a reduction in H3K4me3 in the Prox1 locus, while H3K27ac peaks are increased in the Nrp1 and ICAM1 locus. (D) ChIP-seq analysis of the same modifications in (C) in LECs and BECs reveals that H3K27ac and H3K4me3 peaks are elevated in the Prox1 and Vegfr3 loci in LECs, whereas those of Nrp1 and ICAM1 are reduced in LECs and BECs. Scale bar, 5 kb. Each data point represents a biological replicate. All results are presented as means ± SEM and analyzed by multiple t test or two-tailed Student’s t test.

Fig. 9. Schematic representation of the functional role of mitochondrial respiration in LEC fate maintenance.

Venous ECs (VECs) inside the CV are glycolytic. Starting at around E9.5, CoupTFII and Sox18 induce Prox1 expression in some VECs inside the CV to give rise to Prox1⁺ LEC progenitors. Specification of LEC fate, LEC budding, and maintenance of LEC fate is regulated by a Prox1-Vegfr3 feedback loop. Prox1 also regulates CPT1a, which, in turn, increases FAO in LECs. FAO-derived acetyl-CoA promotes histone acetylation by the histone acetyltransferase P300 and Prox1 complex at lymphangiogenic genes such as Vegfr3. In addition, ketone body oxidation mediated by 3-oxoacid-CoA-transferase-1 (OXCT1) also generates acetyl-CoA, regulates TCA cycle metabolites and aspartate and dNTP levels, and is required for LEC proliferation. We demonstrate that mitochondrial respiratory chain is required for LEC fate maintenance. When complex III is blocked, mutant embryos lack LECs as a consequence of loss of Vegfr3 expression and LEC fate. Mitochondrial respiration is required for nucleotide synthesis and the maintenance of H3K4me3 and H3K27ac histone modifications at the Vegfr3 and Prox1 promoters. Blockage of mitochondrial respiration reduced Vegfr3 and disrupted the Vegfr3-Prox1 feedback loop.