Human ESC-derived vascular cells promote vascular regeneration in a HIF-1α dependent manner

Abstract

Hypoxia-inducible factor (HIF-1α), a core transcription factor responding to changes in cellular oxygen levels, is closely associated with a wide range of physiological and pathological conditions. However, its differential impacts on vascular cell types and molecular programs modulating human vascular homeostasis and regeneration remain largely elusive. Here, we applied CRISPR/Cas9-mediated gene editing of human embryonic stem cells and directed differentiation to generate HIF-1α-deficient human vascular cells including vascular endothelial cells, vascular smooth muscle cells, and mesenchymal stem cells (MSCs), as a platform for discovering cell type-specific hypoxia-induced response mechanisms. Through comparative molecular profiling across cell types under normoxic and hypoxic conditions, we provide insight into the indispensable role of HIF-1α in the promotion of ischemic vascular regeneration. We found human MSCs to be the vascular cell type most susceptible to HIF-1α deficiency, and that transcriptional inactivation of ANKZF1, an effector of HIF-1α, impaired pro-angiogenic processes. Altogether, our findings deepen the understanding of HIF-1α in human angiogenesis and support further explorations of novel therapeutic strategies of vascular regeneration against ischemic damage.

Key words： HIF-1; human ESC; vascular cell; regeneration

References

1 Altin JG, Sloan EK. The role of CD45 and CD45-associated molecules in T cell activation. Immunol Cell Biol 1997;75:430–45.

2 Amsden BG. Delivery approaches for angiogenic growth factors in the treatment of ischemic conditions. Expert Opin Drug Deliv 2011;8:873–90.

3 Anders S, Pyl PT, Huber W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 2015;31:166–9.

4 Annex BH, Cooke JP. New directions in therapeutic angiogenesis and arteriogenesis in peripheral arterial disease. Circ Res 2021;128:1944–1957.

5 Bailey TL, Elkan C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol 1994;2:28–36.

6 Bian X, Ma K, Zhang C et al. Therapeutic angiogenesis using stem cell-derived extracellular vesicles: an emerging approach for treatment of ischemic diseases. Stem Cell Res Ther 2019;10:158.

7 Cai Y, Ji Z, Wang S et al. Genetic enhancement: an avenue to combat aging-related diseases. Life Med 2022;1:307–318.

8 Capitano ML, Mohamad SF, Cooper S et al. Mitigating oxygen stress enhances aged mouse hematopoietic stem cell numbers and function. J Clin Invest 2021;131:e140177.

9 Castro-Mondragon JA, Riudavets-Puig R, Rauluseviciute I et al. JASPAR 2022: the 9th release of the open-access data-base of transcription factor binding profiles. Nucleic Acids Res 2022;50:D165–D173.

10 Chen J, Lopez JA. Interactions of platelets with subendothelium and endothelium. Microcirculation 2005;12:235–46.

11 Cheng F, Wang S, Song M et al. DJ-1 is dispensable for human stem cell homeostasis. Protein Cell 2019;10:846–853.

12 Contreras-Baeza Y, Sandoval PY, Alarcon R et al. Monocarboxylate transporter 4 (MCT4) is a high affinity transporter capable of exporting lactate in high-lactate microenvironments. J Biol Chem 2019;294:20135–20147.

13 Cowman SJ, Koh MY. Revisiting the HIF switch in the tumor and its immune microenvironment. Trends Cancer 2022;8:28–42.

14 De Bock K, Georgiadou M, Schoors S et al. Role of PFKFB3-driven glycolysis in vessel sprouting. Cell 2013;154:651–63.

15 Dor Y, Keshet E. Ischemia-driven angiogenesis. Trends Cardiovasc Med 1997;7:289–94.

16 Du W, Ren L, Hamblin MH et al. Endothelial cell glucose metabolism and angiogenesis. Biomedicines 2021;9:147

17 Eltzschig HK, Carmeliet P. Hypoxia and inflammation. N Engl J Med 2011;364:656–65.

18 Ergun S, Tilki D, Klein D. Vascular wall as a reservoir for different types of stem and progenitor cells. Antioxid Redox Signal 2011;15:981–995.

19 Feng H, Jiang B, Xing W et al. Skeletal stem cells: origins, definitions, and functions in bone development and disease. Life Med 2022;1:276–293.

20 Geng L, Zhang B, Liu H et al. A comparative study of metformin and nicotinamide riboside in alleviating tissue aging in rats. Life Med 2022;2. https://doi.org/10.1093/lifemedi/lnac045.

21 Ghaffari-Makhmalbaf P, Sayyad M, Pakravan K et al. Docosahexaenoic acid reverses the promoting effects of breast tumor cell-derived exosomes on endothelial cell migration and angiogenesis. Life Sci 2021;264:118719.

22 Golledge J. Update on the pathophysiology and medical treatment of peripheral artery disease. Nat Rev Cardiol 2022;19:456–474.

23 Ha G, Roth A, Lai D et al. Integrative analysis of genomewide loss of heterozygosity and monoallelic expression at nucleotide resolution reveals disrupted pathways in triple-negative breast cancer. Genome Res 2012;22:1995–2007.

24 van Haaften-Visser DY, Harakalova M, Mocholi E et al. Ankyrin repeat and zinc-finger domain-containing 1 mutations are associated with infantile-onset inflammatory bowel disease. J Biol Chem 2017;292:7904–7920.

25 Hu H, Ji Q, Song M et al. ZKSCAN3 counteracts cellular senescence by stabilizing heterochromatin. Nucleic Acids Res 2020;48:6001–6018.

26 Ivan M, Kondo K, Yang H et al. HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 2001;292:464–8.

27 Jaakkola P, Mole DR, Tian YM et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 2001;292:468–72.

28 Kalogeris T, Baines CP, Krenz M et al. Cell biology of ischemia/reperfusion injury. Int Rev Cell Mol Biol 2012;298:229–317.

29 Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods 2015;12:357–360.

30 Klein D. Improved isolation of human vascular wall-resident mesenchymal stem cells. Methods Mol Biol 2020;2155:71–81.

31 Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods 2012;9:357–9.

32 Lee JW, Bae SH, Jeong JW et al. Hypoxia-inducible factor (HIF-1)alpha: its protein stability and biological functions. Exp Mol Med 2004;36:1–12.

33 Lei J, Wang S, Kang W et al. FOXO3-engineered human mesenchymal progenitor cells efficiently promote cardiac repair after myocardial infarction. Protein Cell 2021;12:145–151.

34 Liang C, Liu Z, Song M et al. Stabilization of heterochromatin by CLOCK promotes stem cell rejuvenation and cartilage regeneration. Cell Res 2021;31:187–205.

35 Ling C, Liu Z, Song M et al. Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells. Protein Cell 2019;10:249–271.

36 Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014;15:550.

37 Martinez VG, Ontoria-Oviedo I, Ricardo CP et al. Overexpression of hypoxia-inducible factor 1 alpha improves immunomodulation by dental mesenchymal stem cells. Stem Cell Res Ther 2017;8:208.

38 Masoud GN, Li W. HIF-1alpha pathway: role, regulation and intervention for cancer therapy. Acta Pharm Sin B 2015;5:378–89.

39 Maxwell PH, Wiesener MS, Chang GW et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 1999;399:271–5.

40 Pertea M, Pertea GM, Antonescu CM et al. StringTie enables improved reconstruction of a transcriptome from RNAseq reads. Nat Biotechnol 2015;33:290–5.

41 Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med 2003;9:677–84.

42 Pyagay P, Heroult M, Wang Q et al. Collagen triple helix repeat containing 1, a novel secreted protein in injured and diseased arteries, inhibits collagen expression and promotes cell migration. Circ Res 2005;96:261–8.

43 Rouillard AD, Gundersen GW, Fernandez NF et al. The harmonizome: a collection of processed datasets gathered to serve and mine knowledge about genes and proteins. Database (Oxford) 2016;2016:baw100.

44 Stabile E, Burnett MS, Watkins C et al. Impaired arteriogenic response to acute hindlimb ischemia in CD4-knockout mice. Circulation 2003;108:205–10.

45 Stapf C, Cartwright E, Bycroft M et al. The general definition of the p97/valosin-containing protein (VCP)-interacting motif (VIM) delineates a new family of p97 cofactors. J Biol Chem 2011;286:38670–38678.

46 Valvona CJ, Fillmore HL, Nunn PB et al. The regulation and function of lactate dehydrogenase A: therapeutic potential in brain tumor. Brain Pathol 2016;26:3–17.

47 Van Nguyen TT, Vu NB, Van Pham P. Mesenchymal stem cell transplantation for ischemic diseases: mechanisms and challenges. Tissue Eng Regen Med 2021;18:587–611.

48 Vimalraj, S. A concise review of VEGF, PDGF, FGF, Notch, angiopoietin, and HGF signalling in tumor angiogenesis with a focus on alternative approaches and future directions. Int J Biol Macromol 2022;221:1428–1438.

49 Vono R, Spinetti G, Gubernator M et al. What’s new in regenerative medicine: split up of the mesenchymal stem cell family promises new hope for cardiovascular repair. J Cardiovasc Transl Res 2012;5:689–99.

50 Vrselja Z, Daniele SG, Silbereis J et al. Restoration of brain circulation and cellular functions hours post-mortem. Nature 2019;568:336–343.

51 Wahlberg E. Angiogenesis and arteriogenesis in limb ischemia. J Vasc Surg 2003;38:198–203.

52 Wang D, Wang X, Ba H et al. Chimeric blood vessels sustained development of the xenogeneic antler: a unique model for xenogeneic organ generation. Life Med 2022a;2:lnac021.

53 Wang H, Jiang C, Cai J et al. Nestin prevents mesenchymal stromal cells from apoptosis in LPS-induced lung injury via inhibition of unfolded protein response sensor IRE1α. Life Med 2022b;1:359–371.

54 Wang P, Liu Z, Zhang X et al. CRISPR/Cas9-mediated gene knockout reveals a guardian role of NF-kappaB/RelA in maintaining the homeostasis of human vascular cells. Protein Cell 2018;9:945–965.

55 Wang S, Cheng F, Ji Q et al. Hyperthermia differentially affects specific human stem cells and their differentiated derivatives. Protein Cell 2022c;13:615–622.

56 Wang Y, Qin J. Advances in human organoids-on-chips in biomedical research. Life Med 2023;2:lnad007.

57 Worsdorfer P, Mekala SR, Bauer J et al. The vascular adventitia: an endogenous, omnipresent source of stem cells in the body. Pharmacol Ther 2017;171:13–29.

58 Yan P, Li Q, Wang L et al. FOXO3-engineered human ESC-derived vascular cells promote vascular protection and regeneration. Cell Stem Cell 2019;24:447–461.e8.

59 Yang J, Li J, Suzuki K et al. Genetic enhancement in cultured human adult stem cells conferred by a single nucleotide recoding. Cell Res 2017;27:1178–1181.

60 Zhang F, Qiu H, Dong X et al. Single-cell atlas of multilineage cardiac organoids derived from human induced pluripotent stem cells. Life Med 2022a;1:179–195.

61 Zhang Q, Liu W, Zhang HM et al. hTFtarget: a comprehensive database for regulations of human transcription factors and their targets. Genomics Proteomics Bioinformatics 2020a;18:120–128.

62 Zhang W, Zhang S, Yan P et al. A single-cell transcriptomic landscape of primate arterial aging. Nat Commun 2020b;11:2202.

63 Zhang S, Wu Z, Shi Y et al. FTO stabilizes MIS12 and counteracts senescence. Protein Cell 2022b;13:54–960.

64 Zhao H, Yang K, Zhang Y et al. APOE-mediated suppression of the lncRNA MEG3 protects human cardiovascular cells from chronic inflammation. Protein Cell. 2023;14:908–913.

65 Zheng YL, Wang WD, Cai PY et al. Stem cell-derived exosomes in the treatment of acute myocardial infarction in preclinical animal models: a meta-analysis of randomized controlled trials. Stem Cell Res Ther 2022;13:151.

66 Zhou X, Shang YN, Lu R et al. High ANKZF1 expression is associated with poor overall survival and recurrence-free survival in colon cancer. Future Oncol 2019a;15:2093–2106.

67 Zhou Y, Zhou B, Pache L et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat Commun 2019b;10:1523.

68 Zimna A, Kurpisz, M. Hypoxia-inducible factor-1 in physiological and pathophysiological angiogenesis: applications and therapies. Biomed Res Int 2015;2015:549412.