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.
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
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
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
Friedlander M (2007) Fibrosis and diseases of the eye. J Clin Invest 117:576–586. https://doi.org/10.1172/JCI31030
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
Storkebaum E, Carmeliet P (2004) VEGF: a critical player in neurodegeneration. J Clin Invest 113:14–18. https://doi.org/10.1172/JCI20682
Aiello LP (2005) Angiogenic pathways in diabetic retinopathy. N Engl J Med 353:839–841. https://doi.org/10.1056/NEJMe058142
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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.
Rights and permissions
About this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11010-020-03862-z