Skip to main content

Advertisement

SpringerLink
Log in

RNA-Seq reveals placental growth factor regulates the human retinal endothelial cell barrier integrity by transforming growth factor (TGF-β) signaling

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

Placental growth factor (PlGF or PGF) is a member of the VEGF (vascular endothelial growth factor) family. It plays a pathological role in inflammation, vascular permeability, and pathological angiogenesis. The molecular signaling by which PlGF mediates its effects in non-proliferative diabetic retinopathy (DR) remains elusive. This study aims to characterize the transcriptome changes of human retinal endothelial cells (HRECs) with the presence and the absence of PlGF signaling. Primary HRECs were treated with the PlGF antibody (ab) to block its activity. The total RNA was isolated and subjected to deep sequencing to quantify the transcripts and their changes in both groups. We performed transcriptome-wide analysis, gene ontology, pathway enrichment, and gene–gene network analyses. The results showed that a total of 3760 genes were significantly differentially expressed and were categorized into cell adhesion molecules, cell junction proteins, chaperone, calcium-binding proteins, and membrane traffic proteins. Functional pathway analyses revealed that the TGF-β pathway, pentose phosphate pathway, and cell adhesion pathway play pivotal roles in the blood-retina barrier and antioxidant defense system. Collectively, the data provide new insights into the molecular mechanisms of PlGF’s biological functions in HRECs relevant to DR and diabetic macular edema (DME). The newly identified genes and pathways may act as disease markers and target molecules for therapeutic interventions for the patients with DR and DME refractory to the current anti-VEGF therapy.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Data availability

All data generated and analyzed during this study are included in this published article and its supplementary information. RNA-Seq datasets were submitted to the NCBI-SRA (Sequence Read Archive (SRA) with accession numbers, SRA No: SRP239417; BioProject: PRJNA598727.

References

  1. Lee R, Wong TY, Sabanayagam C (2015) Epidemiology of diabetic retinopathy, diabetic macular edema and related vision loss. Eye Vis (Lond) 2:17. https://doi.org/10.1186/s40662-015-0026-2

    Article  Google Scholar 

  2. Curtis TM, Gardiner TA, Stitt AW (2009) Microvascular lesions of diabetic retinopathy: clues towards understanding pathogenesis? Eye (Lond) 23:1496–1508. https://doi.org/10.1038/eye.2009.108

    Article  CAS  Google Scholar 

  3. Friedlander M (2007) Fibrosis and diseases of the eye. J Clin Invest 117:576–586. https://doi.org/10.1172/JCI31030

    Article  CAS  Google Scholar 

  4. Diabetic Retinopathy Clinical Research N, Elman MJ, Aiello LP, Beck RW, Bressler NM, Bressler SB, Edwards AR, Ferris FL III, Friedman SM, Glassman AR, Miller KM, Scott IU, Stockdale CR, Sun JK (2010) Randomized trial evaluating ranibizumab plus prompt or deferred laser or triamcinolone plus prompt laser for diabetic macular edema. Ophthalmology 117(1064–1077):e35. https://doi.org/10.1016/j.ophtha.2010.02.031

    Article  Google Scholar 

  5. Storkebaum E, Carmeliet P (2004) VEGF: a critical player in neurodegeneration. J Clin Invest 113:14–18. https://doi.org/10.1172/JCI20682

    Article  CAS  Google Scholar 

  6. Aiello LP (2005) Angiogenic pathways in diabetic retinopathy. N Engl J Med 353:839–841. https://doi.org/10.1056/NEJMe058142

    Article  CAS  Google Scholar 

  7. Maglione D, Guerriero V, Viglietto G, Delli-Bovi P, Persico MG (1991) Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor. Proc Natl Acad Sci USA 88:9267–9271

    Article  CAS  Google Scholar 

  8. Saddala MS, Lennikov A, Grab DJ, Liu GS, Tang S, Huang H (2018) Proteomics reveals ablation of PlGF increases antioxidant and neuroprotective proteins in the diabetic mouse retina. Sci Rep 8:16728. https://doi.org/10.1038/s41598-018-34955-x

    Article  CAS  Google Scholar 

  9. Ohno-Matsui K, Uetama T, Yoshida T, Hayano M, Itoh T, Morita I, Mochizuki M (2003) Reduced retinal angiogenesis in MMP-2-deficient mice. Invest Ophthalmol Vis Sci 44:5370–5375. https://doi.org/10.1167/iovs.03-0249

    Article  Google Scholar 

  10. Autiero M, Waltenberger J, Communi D, Kranz A, Moons L, Lambrechts D, Kroll J, Plaisance S, De Mol M, Bono F, Kliche S, Fellbrich G, Ballmer-Hofer K, Maglione D, Mayr-Beyrle U, Dewerchin M, Dombrowski S, Stanimirovic D, Van Hummelen P, Dehio C, Hicklin DJ, Persico G, Herbert JM, Communi D, Shibuya M, Collen D, Conway EM, Carmeliet P (2003) Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Flt1 and Flk1. Nat Med 9:936–943. https://doi.org/10.1038/nm884

    Article  CAS  Google Scholar 

  11. Carmeliet P, Moons L, Luttun A, Vincenti V, Compernolle V, De Mol M, Wu Y, Bono F, Devy L, Beck H, Scholz D, Acker T, DiPalma T, Dewerchin M, Noel A, Stalmans I, Barra A, Blacher S, VandenDriessche T, Ponten A, Eriksson U, Plate KH, Foidart JM, Schaper W, Charnock-Jones DS, Hicklin DJ, Herbert JM, Collen D, Persico MG (2001) Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat Med 7:575–583. https://doi.org/10.1038/87904

    Article  CAS  Google Scholar 

  12. Huang H, He J, Johnson D, Wei Y, Liu Y, Wang S, Lutty GA, Duh EJ, Semba RD (2015) Deletion of placental growth factor prevents diabetic retinopathy and is associated with Akt activation and HIF1alpha-VEGF pathway inhibition. Diabetes 2015;64:200–212. Diabetes 64:1067. https://doi.org/10.2337/db15-er03

    Article  CAS  Google Scholar 

  13. Huang H, Lennikov A, Saddala MS, Gozal D, Grab DJ, Khalyfa A, Fan L (2019) Placental growth factor negatively regulates retinal endothelial cell barrier function through suppression of glucose-6-phosphate dehydrogenase and antioxidant defense systems. FASEB J 33:13695–13709. https://doi.org/10.1096/fj.201901353R

    Article  CAS  Google Scholar 

  14. Saddala MS, Lennikov A, Mukwaya A, Huang H (2020) Transcriptome-wide analysis of CXCR5 deficient retinal pigment epithelial (RPE) cells reveals molecular signatures of RPE homeostasis. Biomedicines 8:147. https://doi.org/10.3390/biomedicines8060147

    Article  Google Scholar 

  15. Saddala MS, Lennikov A, Bouras A, Huang H (2020) RNA-Seq reveals differential expression profiles and functional annotation of genes involved in retinal degeneration in Pde6c mutant Danio rerio. BMC Genom 21:132. https://doi.org/10.1186/s12864-020-6550-z

    Article  CAS  Google Scholar 

  16. Saddala MS, Lennikov A, Mukwaya A, Fan L, Hu Z, Huang H (2019) Transcriptome-wide analysis of differentially expressed chemokine receptors, SNPs, and SSRs in the age-related macular degeneration. Hum Genom 13:15. https://doi.org/10.1186/s40246-019-0199-1

    Article  Google Scholar 

  17. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. https://doi.org/10.1093/bioinformatics/btu170

    Article  CAS  Google Scholar 

  18. Huang DW, Sherman BT, Tan Q, Kir J, Liu D, Bryant D, Guo Y, Stephens R, Baseler MW, Lane HC, Lempicki RA (2007) DAVID bioinformatics resources: expanded annotation database and novel algorithms to better extract biology from large gene lists. Nucleic Acids Res 35:W169–W175. https://doi.org/10.1093/nar/gkm415

    Article  Google Scholar 

  19. Rivals I, Personnaz L, Taing L, Potier MC (2007) Enrichment or depletion of a GO category within a class of genes: which test? Bioinformatics 23:401–407. https://doi.org/10.1093/bioinformatics/btl633

    Article  CAS  Google Scholar 

  20. Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA (2003) DAVID: database for annotation, visualization, and integrated discovery. Genome Biol 4:P3

    Article  Google Scholar 

  21. Saddala MS, Lennikov A, Huang H (2020) Placental growth factor regulates the pentose phosphate pathway and antioxidant defense systems in human retinal endothelial cells. J Proteomics 217:103682. https://doi.org/10.1016/j.jprot.2020.103682

    Article  CAS  Google Scholar 

  22. Yoshimura A, Wakabayashi Y, Mori T (2010) Cellular and molecular basis for the regulation of inflammation by TGF-beta. J Biochem 147:781–792. https://doi.org/10.1093/jb/mvq043

    Article  CAS  Google Scholar 

  23. Derynck R, Zhang YE (2003) Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature 425:577–584. https://doi.org/10.1038/nature02006

    Article  CAS  Google Scholar 

  24. Behzadian MA, Wang XL, Windsor LJ, Ghaly N, Caldwell RB (2001) TGF-beta increases retinal endothelial cell permeability by increasing MMP-9: possible role of glial cells in endothelial barrier function. Invest Ophthalmol Vis Sci 42:853–859

    CAS  Google Scholar 

  25. Close JL, Gumuscu B, Reh TA (2005) Retinal neurons regulate proliferation of postnatal progenitors and Muller glia in the rat retina via TGF beta signaling. Development 132:3015–3026. https://doi.org/10.1242/dev.01882

    Article  CAS  Google Scholar 

  26. Yi JJ, Barnes AP, Hand R, Polleux F, Ehlers MD (2010) TGF-beta signaling specifies axons during brain development. Cell 142:144–157. https://doi.org/10.1016/j.cell.2010.06.010

    Article  CAS  Google Scholar 

  27. Brionne TC, Tesseur I, Masliah E, Wyss-Coray T (2003) Loss of TGF-beta 1 leads to increased neuronal cell death and microgliosis in mouse brain. Neuron 40:1133–1145. https://doi.org/10.1016/s0896-6273(03)00766-9

    Article  CAS  Google Scholar 

  28. ten Dijke P, Arthur HM (2007) Extracellular control of TGFbeta signalling in vascular development and disease. Nat Rev Mol Cell Biol 8:857–869. https://doi.org/10.1038/nrm2262

    Article  CAS  Google Scholar 

  29. Neubauer K, Kruger M, Quondamatteo F, Knittel T, Saile B, Ramadori G (1999) Transforming growth factor-beta1 stimulates the synthesis of basement membrane proteins laminin, collagen type IV and entactin in rat liver sinusoidal endothelial cells. J Hepatol 31:692–702. https://doi.org/10.1016/s0168-8278(99)80350-x

    Article  CAS  Google Scholar 

  30. Obermeier B, Daneman R, Ransohoff RM (2013) Development, maintenance and disruption of the blood-brain barrier. Nat Med 19:1584–1596. https://doi.org/10.1038/nm.3407

    Article  CAS  Google Scholar 

  31. Song L, Yan Y, Marzano M, Li Y (2019) Studying heterotypic cell–cell interactions in the human brain using pluripotent stem cell models for neurodegeneration. Cells 8:299. https://doi.org/10.3390/cells8040299

    Article  CAS  Google Scholar 

  32. Hammes HP (2005) Pericytes and the pathogenesis of diabetic retinopathy. Horm Metab Res 37(Suppl 1):39–43. https://doi.org/10.1055/s-2005-861361

    Article  CAS  Google Scholar 

  33. Braunger BM, Pielmeier S, Demmer C, Landstorfer V, Kawall D, Abramov N, Leibinger M, Kleiter I, Fischer D, Jagle H, Tamm ER (2013) TGF-beta signaling protects retinal neurons from programmed cell death during the development of the mammalian eye. J Neurosci 33:14246–14258. https://doi.org/10.1523/JNEUROSCI.0991-13.2013

    Article  CAS  Google Scholar 

  34. Shen W, Li S, Chung SH, Zhu L, Stayt J, Su T, Couraud PO, Romero IA, Weksler B, Gillies MC (2011) Tyrosine phosphorylation of VE-cadherin and claudin-5 is associated with TGF-beta1-induced permeability of centrally derived vascular endothelium. Eur J Cell Biol 90:323–332. https://doi.org/10.1016/j.ejcb.2010.10.013

    Article  CAS  Google Scholar 

  35. Lan Y, Liu B, Yao H, Li F, Weng T, Yang G, Li W, Cheng X, Mao N, Yang X (2007) Essential role of endothelial Smad4 in vascular remodeling and integrity. Mol Cell Biol 27:7683–7692. https://doi.org/10.1128/MCB.00577-07

    Article  CAS  Google Scholar 

  36. Masuda T, Shimazawa M, Hara H (2017) Retinal Diseases Associated with Oxidative Stress and the Effects of a Free Radical Scavenger (Edaravone). Oxid Med Cell Longev 2017:9208489. https://doi.org/10.1155/2017/9208489

    Article  CAS  Google Scholar 

  37. Saddala MS, Lennikov A, Huang H (2020) Discovery of small-molecule activators for glucose-6-phosphate dehydrogenase (G6PD) using machine learning approaches. Int J Mol Sci 21:1523. https://doi.org/10.3390/ijms21041523

    Article  CAS  Google Scholar 

  38. Pinna A, Carru C, Solinas G, Zinellu A, Carta F (2007) Glucose-6-phosphate dehydrogenase deficiency in retinal vein occlusion. Invest Ophthalmol Vis Sci 48:2747–2752. https://doi.org/10.1167/iovs.06-1064

    Article  Google Scholar 

  39. Chidlow G, Wood JP, Knoops B, Casson RJ (2016) Expression and distribution of peroxiredoxins in the retina and optic nerve. Brain Struct Funct 221:3903–3925. https://doi.org/10.1007/s00429-015-1135-3

    Article  CAS  Google Scholar 

  40. Rhee SG, Woo HA (2011) Multiple functions of peroxiredoxins: peroxidases, sensors and regulators of the intracellular messenger H2O2, and protein chaperones. Antioxid Redox Signal 15:781–794. https://doi.org/10.1089/ars.2010.3393

    Article  CAS  Google Scholar 

  41. Pieragostino D, Agnifili L, Fasanella V, D’Aguanno S, Mastropasqua R, Di Ilio C, Sacchetta P, Urbani A, Del Boccio P (2013) Shotgun proteomics reveals specific modulated protein patterns in tears of patients with primary open angle glaucoma naive to therapy. Mol Biosyst 9:1108–1116. https://doi.org/10.1039/c3mb25463a

    Article  CAS  Google Scholar 

  42. Immenschuh S, Baumgart-Vogt E (2005) Peroxiredoxins, oxidative stress, and cell proliferation. Antioxid Redox Signal 7:768–777. https://doi.org/10.1089/ars.2005.7.768

    Article  CAS  Google Scholar 

  43. Fischer C, Jonckx B, Mazzone M, Zacchigna S, Loges S, Pattarini L, Chorianopoulos E, Liesenborghs L, Koch M, De Mol M, Autiero M, Wyns S, Plaisance S, Moons L, van Rooijen N, Giacca M, Stassen JM, Dewerchin M, Collen D, Carmeliet P (2007) Anti-PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels. Cell 131:463–475. https://doi.org/10.1016/j.cell.2007.08.038

    Article  CAS  Google Scholar 

  44. Van de Veire S, Stalmans I, Heindryckx F, Oura H, Tijeras-Raballand A, Schmidt T, Loges S, Albrecht I, Jonckx B, Vinckier S, Van Steenkiste C, Tugues S, Rolny C, De Mol M, Dettori D, Hainaud P, Coenegrachts L, Contreres JO, Van Bergen T, Cuervo H, Xiao WH, Le Henaff C, Buysschaert I, Kharabi Masouleh B, Geerts A, Schomber T, Bonnin P, Lambert V, Haustraete J, Zacchigna S, Rakic JM, Jimenez W, Noel A, Giacca M, Colle I, Foidart JM, Tobelem G, Morales-Ruiz M, Vilar J, Maxwell P, Vinores SA, Carmeliet G, Dewerchin M, Claesson-Welsh L, Dupuy E, Van Vlierberghe H, Christofori G, Mazzone M, Detmar M, Collen D, Carmeliet P (2010) Further pharmacological and genetic evidence for the efficacy of PlGF inhibition in cancer and eye disease. Cell 141:178–190. https://doi.org/10.1016/j.cell.2010.02.039

    Article  CAS  Google Scholar 

  45. Yao YG, Yang HS, Cao Z, Danielsson J, Duh EJ (2005) Upregulation of placental growth factor by vascular endothelial growth factor via a post-transcriptional mechanism. FEBS Lett 579:1227–1234. https://doi.org/10.1016/j.febslet.2005.01.017

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors wish to acknowledge the contribution of the Division of Information Technology (UM system) University of Missouri (Columbia, MO, USA) for High-Performance Computing (HPC) facilities. The early version of the manuscript was deposited to www.preprints.org (https://www.preprints.org/manuscript/201907.0140/v1) under the title “Pathway-Focused Gene Interaction Analysis Reveals the Regulation of TGFβ, Pentose Phosphate and Antioxidant Defense System by Placental Growth Factor in Retinal Endothelial Cell Functions: Implication in Diabetic Retinopathy.” And was assigned https://doi.org/10.20944/preprints201907.0140.v1.

Funding

This work was supported by MU start-up funds and NIH grant (EY027824 to H.H.).

Author information

Authors and Affiliations

  1. The University of Missouri School of Medicine, Columbia, MO, USA

    Hu Huang, Madhu Sudhana Saddala, Anton Lennikov & Lijuan Fan

  2. Department of Ophthalmology, Institute for Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden

    Anthony Mukwaya

Authors
  1. Hu Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Madhu Sudhana Saddala
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anton Lennikov
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anthony Mukwaya
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lijuan Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization, HH and MSS; methodology, MSS, HH, and LF; software, MSS; formal analysis, MS; investigation, AL, AM, HH, and MSS; resources, HH; data curation, HH; writing—original draft preparation, MSS, HH, AL, and AM; writing—review and editing, HH, AL, and AM; visualization, MSS, AL and AM; supervision, HH; project administration, HH; funding acquisition, HH”.

Corresponding author

Correspondence to Hu Huang.

Ethics declarations

Conflicts of interest

The authors have no conflict of interest to disclose in relation to this paper. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; in the decision to publish the results.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 2421 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Huang, H., Saddala, M.S., Lennikov, A. et al. RNA-Seq reveals placental growth factor regulates the human retinal endothelial cell barrier integrity by transforming growth factor (TGF-β) signaling. Mol Cell Biochem 475, 93–106 (2020). https://doi.org/10.1007/s11010-020-03862-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-020-03862-z

Keywords

Search

Navigation