Hypoxia-induced P4HA1 overexpression promotes post-ischemic angiogenesis by enhancing endothelial glycolysis through downregulating FBP1

(2024) 22:74 Xu et al. Journal of Translational Medicine https://doi.org/10.1186/s12967-024-04872-x Journal of Translational Medicine Open Access RESEARCH Hypoxia-induced P4HA1 overexpression promotes post-ischemic angiogenesis by enhancing endothelial glycolysis through downregulating FBP1 Yating Xu1,4, Di Xia1,2, Kai Huang1,2,3,4* and Minglu Liang1,2,3*    Abstract Background Angiogenesis is essential for tissue repair in ischemic diseases, relying on glycolysis as its primary energy source. Prolyl 4-hydroxylase subunit alpha 1 (P4HA1), the catalytic subunit of collagen prolyl 4-hydroxylase, is a glycolysis-related gene in cancers. However, its role in glycolysis-induced angiogenesis remains unclear. Methods P4HA1 expression was modulated using adenoviruses. Endothelial angiogenesis was evaluated through 5-ethynyl-2′-deoxyuridine incorporation, transwell migration, and tube formation assays in vitro. In vivo experiments measured blood flow and capillary density in the hindlimb ischemia (HLI) model. Glycolytic stress assays, glucose uptake, lactate production, and quantitative reverse transcription-polymerase chain reaction (RT-PCR) were employed to assess glycolytic capacity. Transcriptome sequencing, validated by western blotting and RT-PCR, was utilized to determine underlying mechanisms. Results P4HA1 was upregulated in endothelial cells under hypoxia and in the HLI model. P4HA1 overexpression promoted angiogenesis in vitro and in vivo, while its knockdown had the opposite effect. P4HA1 overexpression reduced cellular α-ketoglutarate (α-KG) levels by consuming α-KG during collagen hydroxylation. Downregulation of α-KG reduced the protein level of a DNA dioxygenase, ten–eleven translocation 2 (TET2), and its recruitment to the fructose-1,6-biphosphatase (FBP1) promoter, resulting in decreased FBP1 expression. The decrease in FBP1 enhanced glycolytic metabolism, thereby promoting endothelial angiogenesis. Conclusions Hypoxia-induced endothelial P4HA1 overexpression enhanced angiogenesis by promoting glycolytic metabolism reprogramming through the P4HA1/α-KG/TET2/FBP1 pathway. The study’s findings underscore the significance of P4HA1 in post-ischemic angiogenesis, suggesting its therapeutic potential for post-ischemic tissue repair. Keywords P4HA1, α-Ketoglutarate, TET2-FBP1 pathway, Glycolysis, Angiogenesis *Correspondence: Kai Huang Minglu Liang Full list of author information is available at the end of the article © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Xu et al. Journal of Translational Medicine (2024) 22:74 Background Angiogenesis, the process of forming new blood vessels from pre-existing ones, plays a crucial role in various biological processes such as wound healing, ischemia, and cancer metastasis. Timely angiogenesis is essential for protecting tissues from injury in ischemia-related diseases, such as peripheral arterial diseases, stroke, and myocardial infarction [1]. Therefore, a comprehensive understanding of the mechanisms underlying angiogenesis is essential for identifying potential therapeutic targets for these diseases. Angiogenic growth factors, including fibroblast growth factors, vascular endothelial growth factors, and angiopoietin, play pivotal roles in regulating angiogenesis. While many studies currently focus on these growth factors and associated signaling pathways [2], treatments targeting them still face challenges in terms of efficacy and specificity. Several angiogenic growth factors have been identified to induce angiogenesis by modulating cellular metabolism to ensure an adequate energy supply [3]. Recent research has highlighted the close relationship between metabolism and endothelial cell (EC) function [4, 5]. Therefore, targeting endothelial metabolism holds promise for enhancing the effectiveness of pro-angiogenic therapy. Despite the higher ATP production capacity of oxidative metabolism compared to glycolysis, ECs predominantly rely on glycolysis, accounting for up to 85% of their total ATP. This preference is attributed to the rapid ability of glycolysis to produce ATP and shield ECs from oxidative stress. Additionally, glycolysis becomes more significant under hypoxic conditions, making ECs more resilient to oxygen deprivation [4, 5]. Moreover, glycolysis plays a critical role in promoting EC migration, extension, and proliferation [6–8]. Knockdown of the rate-limiting enzyme in glycolysis, 6-phosphofructo2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), has been shown to impair blood vessel sprouting [9]. Despite the increasing focus on glycolysis in angiogenesis, further investigation is needed to elucidate the underlying mechanisms of glycolysis-induced angiogenesis. Collagen prolyl 4-hydroxylase (P4H), an α-ketoglutarate (α-KG)-dependent dioxygenase, facilitates proline hydroxylation in collagen, promoting collagen synthesis by utilizing α-KG as a substrate and releasing succinate as a product [10]. P4H consists of two identical catalytic subunits and two identical β subunits (i.e., P4HB). The major isoform among the catalytic subunits is P4H subunit alpha 1 (P4HA1). Han et al. reported that P4HA1 promotes angiogenesis in glioblastoma multiforme by driving the transition of stem-like cells into tumor ECs through the P4HA1/ COL6A1/CD31 pathway [11]. P4HA1 also enhances glioma neovascularization by facilitating the transition Page 2 of 19 of glioma stem cells into ECs and the formation of vascular basement membrane [12]. While existing research on the relationship between P4HA1 and angiogenesis mainly focuses on its role in the transformation of tumor stem cells into ECs, the role of P4HA1 in postischemic angiogenesis and the underlying mechanisms remains unclear. Bioinformatics analysis has identified P4HA1 as a glycolysis-related gene in various cancers [13, 14], suggesting its potential involvement in glycolysis-induced angiogenesis. Therefore, there is a need to elucidate the role (...truncated)