Skip to main content

Advertisement

Springer Nature Link
Log in

The role of MDM2 in angiogenesis: implications for endothelial tip cell formation

  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Abstract

In the present study, we examined the role of MDM2 in the angiogenesis process and its potential association with the sprouting of endothelial tip cells. To address this, we performed hypoxia-treated gastric cancer cells (HGC-27) to quantitative RT-PCR and Western blot analysis to measure the levels of MDM2 and VEGF-A mRNA and protein expression. Subsequently, we employed siRNA to disrupt MDM2 expression, followed by hypoxia treatment. The expression levels of MDM2 and VEGF-A mRNA and protein were subsequently reassessed. Additionally, ELISA was utilized to quantify the secretion levels of VEGF-A in each experimental group. A conditioned medium derived from HGC-27 cells treated with different agents was employed to assess its influence on the formation of EA.hy926 endothelial tip cells, using various techniques including Transwell plates migration assays, wound healing experiments, vascular formation assays, scanning electron microscopy, and immunofluorescence staining. These findings demonstrated that the in vitro knockdown of MDM2 in the conditioned medium exhibited significant inhibitory effects on endothelial cell migration, wound healing, and vascular formation. Additionally, the intervention led to a reduction in the presence of CD34+ tip cells and the formation of filopodia in endothelial cells, while partially restoring the integrity of tight junctions. Subsequent examination utilizing RNA-seq revealed that the suppression of MDM2 in HGC-27 cells resulted in the downregulation of the PI3K/AKT signaling pathway. Consequently, this downregulation led to an elevation in angiogenic effects induced by hypoxia.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.

Similar content being viewed by others

Data availability

All data supporting the findings of this study are available within the paper.

References

  • Bond GL, Hu W, Bond EE, Robins H, Lutzker SG, Arva NC, Bargonetti J, Bartel F, Taubert H, Wuerl P, Onel K, Yip L, Hwang SJ, Strong LC, Lozano G, Levine AJ (2004) A single nucleotide polymorphism in the MDM2 promoter attenuates the p53 tumor suppressor pathway and accelerates tumor formation in humans. Cell 119:591–602

    Article  PubMed  CAS  Google Scholar 

  • Cao H, Chen X, Wang Z, Wang L, Xia Q, Zhang W (2020) The role of MDM2–p53 axis dysfunction in the hepatocellular carcinoma transformation. Cell Death Discovery 6:53

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Carmeliet P, Jain RK (2000) Angiogenesis in cancer and other diseases. Nature 407:249–257

    Article  PubMed  CAS  Google Scholar 

  • Cathcart JM, Banach A, Liu A, Chen J, Goligorsky M, Cao J (2016) Interleukin-6 increases matrix metalloproteinase-14 (MMP-14) levels via down-regulation of p53 to drive cancer progression. Oncotarget 7:61107

    Article  PubMed  PubMed Central  Google Scholar 

  • Chekhonin VP, Shein SA, Korchagina AA, Gurina OI (2013) VEGF in tumor progression and targeted therapy. Curr Cancer Drug Targets 13:423–443

    Article  PubMed  CAS  Google Scholar 

  • Chen W, Xia P, Wang H, Tu J, Liang X, Zhang X, Li L (2019) The endothelial tip-stalk cell selection and shuffling during angiogenesis. J Cell Commun Signal 13:291–301

    Article  PubMed  PubMed Central  Google Scholar 

  • Davies EM, et al (2023) PI(4,5)P2-dependent regulation of endothelial tip cell specification contributes to angiogenesis. Sci Adv 9(13):eadd6911. https://doi.org/10.1126/sciadv.add6911

  • De Smet F, Segura I, De Bock K, Hohensinner PJ, Carmeliet P (2009) Mechanisms of vessel branching: filopodia on endothelial tip cells lead the way. Arterioscler Thromb Vasc Biol 29:639–649

    Article  PubMed  Google Scholar 

  • Delgado-Bellido D, Serrano-Saenz S, Fernández-Cortés M, Oliver FJ (2017) Vasculogenic mimicry signaling revisited: focus on non-vascular VE-cadherin. Mol Cancer 16:1–14

    Article  Google Scholar 

  • FarhangGhahremani M, Goossens S, Nittner D, Bisteau X, Bartunkova S, Zwolinska A, Hulpiau P, Haigh K, Haenebalcke L, Drogat B (2013) p53 promotes VEGF expression and angiogenesis in the absence of an intact p21-Rb pathway. Cell Death Differ 20:888–897

    Article  CAS  Google Scholar 

  • Folkman J (2006) Angiogenesis. Annu Rev Med 57:1–18. https://doi.org/10.1146/annurev.med.57.121304.131306

    Article  PubMed  CAS  Google Scholar 

  • Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D, Betsholtz C (2003) VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 161:1163–1177

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Graupera M, Potente M (2013) Regulation of angiogenesis by PI3K signaling networks. Exp Cell Res 319:1348–1355

    Article  PubMed  CAS  Google Scholar 

  • Hultgren NW, Fang JS, Ziegler ME, Ramirez RN, Phan DTT, Hatch MMS, Welch-Reardon KM, Paniagua AE, Kim LS, Shon NN, Williams DS, Mortazavi A, Hughes CCW (2020) Slug regulates the Dll4-Notch-VEGFR2 axis to control endothelial cell activation and angiogenesis. Nat Commun 11:5400

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Joshi S, Singh AR, Durden DL (2014) MDM2 regulates hypoxic hypoxia-inducible factor 1α stability in an E3 ligase, proteasome, and PTEN-phosphatidylinositol 3-kinase-AKT-dependent manner. J Biol Chem 289:22785–22797

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Kim D-H, Chung J-K (2002) Akt: versatile mediator of cell survival and beyond. BMB Rep 35:106–115

    Article  CAS  Google Scholar 

  • Konisti S, Kiriakidis S, Paleolog EM (2012) Hypoxia—a key regulator of angiogenesis and inflammation in rheumatoid arthritis. Nat Rev Rheumatol 8:153–162

    Article  PubMed  CAS  Google Scholar 

  • Liu LX, Lu H, Luo Y, Date T, Belanger AJ, Vincent KA, Akita GY, Goldberg M, Cheng SH, Gregory RJ (2002) Stabilization of vascular endothelial growth factor mRNA by hypoxia-inducible factor 1. Biochem Biophys Res Commun 291:908–914

    Article  PubMed  CAS  Google Scholar 

  • Liu ZJ, Xiao M, Balint K, Soma A, Pinnix CC, Capobianco AJ, Velazquez OC, Herlyn M (2006) Inhibition of endothelial cell proliferation by Notch1 signaling is mediated by repressing MAPK and PI3K/Akt pathways and requires MAML1. FASEB J 20:1009–1011

    Article  PubMed  CAS  Google Scholar 

  • Martini M et al (2014) PI3K/AKT signaling pathway and cancer: an updated review. Ann Med 46(6):372–383. https://doi.org/10.3109/07853890.2014.912836

    Article  PubMed  CAS  Google Scholar 

  • Onel K, Cordon-Cardo C (2004) MDM2 and prognosis. Mol Cancer Res 2:1–8

    Article  PubMed  CAS  Google Scholar 

  • Patterson DM, Gao D, Trahan DN, Johnson BA, Ludwig A, Barbieri E, Chen Z, Diaz-Miron J, Vassilev L, Shohet JM (2011) Effect of MDM2 and vascular endothelial growth factor inhibition on tumor angiogenesis and metastasis in neuroblastoma. Angiogenesis 14:255–266

    Article  PubMed  CAS  Google Scholar 

  • Saharinen P et al (2011) VEGF and angiopoietin signaling in tumor angiogenesis and metastasis. Trends Mol Med 17(7):347–362. https://doi.org/10.1016/j.molmed.2011.01.015

    Article  PubMed  CAS  Google Scholar 

  • Siemerink MJ, Klaassen I, Vogels IM, Griffioen AW, Van Noorden CJ, Schlingemann RO (2012) CD34 marks angiogenic tip cells in human vascular endothelial cell cultures. Angiogenesis 15:151–163

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Stoletov KV (2003) Role of Nck and Crk adapter proteins in the VEGF promoted endothelial cell migration Yeshiva University

  • Suchting S, Freitas C, le Noble F, Benedito R, Bréant C, Duarte A, Eichmann A (2007) The Notch ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching. Proc Natl Acad Sci U S A 104:3225–3230

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Tezel G, Wax MB (2004) Hypoxia-inducible factor 1α in the glaucomatous retina and optic nerve head. Arch Ophthalmol 122:1348–1356

    Article  PubMed  CAS  Google Scholar 

  • Tokunaga CC, Mitton KP, Dailey W, Massoll C, Roumayah K, Guzman E, Tarabishy N, Cheng M, Drenser KA (2014) Effects of anti-VEGF treatment on the recovery of the developing retina following oxygen-induced retinopathy. Invest Ophthalmol vis Sci 55:1884–1892

    Article  PubMed  CAS  Google Scholar 

  • Usui J, Glezerman IG, Salvatore SP, Chandran CB, Flombaum CD, Seshan SV (2014) Clinicopathological spectrum of kidney diseases in cancer patients treated with vascular endothelial growth factor inhibitors: a report of 5 cases and review of literature. Hum Pathol 45:1918–1927

    Article  PubMed  CAS  Google Scholar 

  • Van Maerken T, Vandesompele J, Rihani A, De Paepe A, Speleman F (2009) Escape from p53-mediated tumor surveillance in neuroblastoma: switching off the p14ARF-MDM2-p53 axis. Cell Death Differ 16:1563–1572

    Article  PubMed  Google Scholar 

  • Wojdasiewicz P, Wajda A, Haładyj E, Romanowska-Próchnicka K, Felis-Giemza A, Nałęcz-Janik J, Walczyk M, Olesińska M, Tarnacka B, Paradowska-Gorycka A (2019) IL-35, TNF-α, BAFF, and VEGF serum levels in patients with different rheumatic diseases. Reumatologia 57:145–150

    Article  PubMed  PubMed Central  Google Scholar 

  • Xiong J et al (2014) Effects of MDM2 inhibitors on vascular endothelial growth factor-mediated tumor angiogenesis in human breast cancer. Angiogenesis 17(1):37–50. https://doi.org/10.1007/s10456-013-9376-3

    Article  PubMed  CAS  Google Scholar 

  • Zhang L, Hill RP (2004) Hypoxia enhances metastatic efficiency by up-regulating Mdm2 in KHT cells and increasing resistance to apoptosis. Cancer Res 64:4180–4189

    Article  PubMed  CAS  Google Scholar 

  • Zhou S, Gu L, He J, Zhang H, Zhou M (2011) MDM2 regulates vascular endothelial growth factor mRNA stabilization in hypoxia. Mol Cell Biol 31:4928–4937

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Funding

This research had been supported by Qinghai Province Applied Basic Research Fund (2023-ZJ-932 M), and by the Natural Science Foundation of China (32360242).

Author information

Authors and Affiliations

  1. Department of Basic Medicine Science, Qinghai University Medical College, Xining, 810001, China

    Yi Yi, Lina Suo, Shaoqian Zhai, Haiyan Wang & Zhanhai Su

  2. Research Center for High Altitude Medicine, Key Laboratory for High Altitude Medicine, Ministry of Education, Qinghai University, 16# Kunlun Road, Xining, 810016, Qinghai, China

    Haixiu Ma, Ronghua Ma & Jing Zhao

Authors
  1. Yi Yi
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Lina Suo
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Haixiu Ma
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Ronghua Ma
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Jing Zhao
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Shaoqian Zhai
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Haiyan Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Zhanhai Su
    View author publications

    You can also search for this author inPubMed Google Scholar

Corresponding authors

Correspondence to Haiyan Wang or Zhanhai Su.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yi, Y., Suo, L., Ma, H. et al. The role of MDM2 in angiogenesis: implications for endothelial tip cell formation. In Vitro Cell.Dev.Biol.-Animal 60, 983–995 (2024). https://doi.org/10.1007/s11626-024-00946-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-024-00946-8

Keywords