Abstract
Angiogenic programming in the vascular endothelium is a tightly regulated process for maintaining tissue homeostasis and is activated in tissue injury and the tumor microenvironment. The metabolic basis of how gas signaling molecules regulate angiogenesis is elusive. Here, we report that hypoxic upregulation of ·NO in endothelial cells reprograms the transsulfuration pathway to increase biogenesis of hydrogen sulfide (H2S), a proangiogenic metabolite. However, decreased H2S oxidation due to sulfide quinone oxidoreductase (SQOR) deficiency synergizes with hypoxia, inducing a reductive shift and limiting endothelial proliferation that is attenuated by dissipation of the mitochondrial NADH pool. Tumor xenografts in whole-body (WBCreSqorfl/fl) and endothelial-specific (VE-cadherinCre-ERT2Sqorfl/fl) Sqor-knockout mice exhibit lower mass and angiogenesis than control mice. WBCreSqorfl/fl mice also exhibit decreased muscle angiogenesis following femoral artery ligation compared to control mice. Collectively, our data reveal the molecular intersections between H2S, O2 and ·NO metabolism and identify SQOR inhibition as a metabolic vulnerability for endothelial cell proliferation and neovascularization.
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Data availability
All data generated and analyzed in this study are included in the main text and Supplementary Information file. Source data are provided with this paper.
Code availability
The code used to analyze tip cells in the microfluidic device is available for download at https://github.com/hirakih/Sulfide-oxidation-promotes-hypoxic-angiogenesis-and-neovascularization-MATLAB-Code.
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Acknowledgements
This work was supported, in part, by grants from the National Institutes of Health (GM130183 to R.B., R01CA248160 to C.A.L. and R01CA148828 and R01CA245546 to Y.M.S.), the American Heart Association (826245 to R.K. and 19POST34380588 to S.S.), the American Physiology Society and Crohn’s and Colitis Foundation (1003279 to R.S. and 623914 to S.S.) and the National Institute of Dental and Cranofacial Research (T32DE00705745 to H.L.H.) and by T32 GM 132046 (National Institutes of Health) support to S.A. We acknowledge A. Landry and W. Huang (University of Michigan) for their technical help with generating the SQOR KD in EA.hy926 cells and with collecting mouse samples, respectively. We acknowledge S. Whitesall in the Physiology Phenotyping Core at the University of Michigan for hind limb ischemia surgery and laser doppler perfusion imaging. We thank M. Mattea at the University of Michigan Center for Gastrointestinal Research for histology studies.
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R.K., Y.M.S. and R.B. conceptualized the study, and R.K. performed and analyzed the majority of the experiments, with assistance from V.V. ([35S]methionine flux, glucose consumption assays and NAD+:NADH estimation), A.S. (proliferation assays), R.S. (HIF-1/HIF-2 western blots, YUMM5.2 culture, lung endothelial cell isolation FACS and real-time quantitative PCR), S.S. (tumor xenograft (experiment 2) and immunohistochemistry), S.A. (GSH, GSSG, cysteine quantitation and HIF-1 stabilization in KD cells), H.L.H. and B.M.B. (tip sprouting assay), H.N.B. (tumor xenograft (experiment 1)) and A.A. and C.A.L. (metabolomics data generation and analysis). R.K. and R.B. drafted the manuscript, and all authors edited and approved the final version.
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C.A.L. has received consulting fees from Astellas Pharmaceuticals, Odyssey Therapeutics and T-Knife Therapeutics and is an inventor on patents pertaining to Kras-regulated metabolic pathways, redox control pathways in pancreatic cancer and targeting the GOT1 pathway as a therapeutic approach (US Patent number 2015126580-A1, 5 July 2015; US Patent number 20190136238, 9 May 2019; International Patent number WO2013177426-A2, 23 April 2015). The remaining authors declare no competing interests.
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Supplementary Figs. 1–8 and uncropped blots for Supplementary Fig. 3.
Supplementary Table 1
Comparison of metabolite levels in normoxia (N; 21% O2) versus hypoxia (H; 2% O2) in quadruplicates.
Supplementary Table 2
Comparison of metabolite levels in normoxia (21% O2) versus hypoxia (2% O2) in quadruplicates in scrambled and SQOR-KD EA.hy926 cells.
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Kumar, R., Vitvitsky, V., Sethaudom, A. et al. Sulfide oxidation promotes hypoxic angiogenesis and neovascularization. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01583-8
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DOI: https://doi.org/10.1038/s41589-024-01583-8