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RNA sequencing reveals BMP4 as a basis for the dual-target treatment of diabetic retinopathy

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Abstract

Background

Diabetic retinopathy (DR), currently considered as a neurovascular disease, has become the major cause of blindness. More and more scholars believe that DR is no longer just a kind of microvascular disease, but accompanied by retinal neurodegenerative changes. Intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs is a classic treatment for DR; however, anti-VEGF drugs can exacerbate fibrosis and eventually lead to retinal detachment. The aim of this study was to explore the pathogenesis of DR and identified new treatments that can provide dual-target intervention for angiogenesis and fibrosis.

Methods

We explored changes in gene expression in high glucose–induced vascular endothelial cells using RNA sequencing (RNA-seq) technology. We identified bone morphogenetic protein 4 (BMP4) and SMAD family member 9 (SMAD9) among 449 differentially expressed genes from RNA-seq data and confirmed the expression of these two genes in the blood of diabetes patients by RT-PCR and in streptozotocin-induced rat retinas by RT-PCR, immunofluorescence, and western blot. Moreover, considering that DR is a multifactorial and multicellular disease, we used hydrogen peroxide (H2O2), advanced glycation end products (AGEs), CoCl2, 4-hydroxynonenal (4-HNE), and hypoxia to induce three human retinal cell types (Müller, retinal pigment epithelium, and human retinal capillary endothelial cells) to simulate the pathogenesis of DR, and MTT experiment, scratch experiment, Transwell experiment, and lumen formation experiment were used to test whether the model was successfully established. Then, we verified the overexpression of these two genes in the cell models by RT-PCR, immunofluorescence, and western blot. We further tested the effects of BMP4 on retinal cells. We use BMP4 to stimulate retinal cells and observe the effect of BMP4 on retinal cells by MTT experiment, scratch experiment, and RT-PCR.

Results

The results demonstrated that BMP4 and SMAD9 were highly expressed in both in vivo and in vitro models, while BMP4 could significantly upregulate the expression of SMAD9 and promote the expression of VEGF and fibrosis factors.

Conclusions

This study is the first to analyze the mechanism by which high glucose levels affect retinal vascular endothelial cells through RNA transcriptome sequencing and indicates that BMP4 may be a potential target for the dual-target treatment (anti-VEGF and anti-fibrosis) of DR.

Key messages

• High-glucose effect on vascular endothelial cell was analyzed by RNA-seq.

• KEGG analysis revealed enrichment of TGF-beta signaling pathway.

• SMAD9 and BMP4 expression was upregulated in all samples.

• Dual-target therapy of PDR by antagonizing BMP4.

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Data availability

All data generated or analyzed during this study are included in this published article.

Abbreviations

DAPI:

4′,6-diamidino-2-phenylindole

DR:

diabetic retinopathy

GO:

Gene Ontology (GO)

HRCECs:

human retinal capillary endothelial cells

KEGG:

Kyoto Encyclopedia of Genes and Genomes

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NEB:

New England Biolabs (NEB)

nr:

nonredundant

PFA:

paraformaldehyde

RNA-seq:

sequencing

RT-qPCR:

reverse transcription quantitative PCR

SD:

Sprague Dawley

STZ:

streptozotocin

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Acknowledgments

The authors thank all clinical laboratory colleagues for their assistance with the regular blood sample collection. We are also thankful for the support and assistance from the Tianjin Medical University Eye Institute Biobank.

Funding

This study was supported by the National Natural Science Funds (81570872), China; National Natural Science Foundation of China (81670875), Tianjin Municipal Health and Family Planning Commission young medical talents project, Tianjin, China; Tianjin Medical University Eye Hospital young innovative talents project (YDYYRCXM-C2018-02), Tianjin, China; Tianjin Education Commission research project (2018KJ063), Tianjin, China; Bethune Merck Diabetes Research Foundation; Tianjin clinical key discipline construction project (TJLCZDXKM010), Tianjin, China; and Tianjin Medical University Eye Hospital clinical research fund (2016LCKY005).

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Authors and Affiliations

  1. Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin International Joint Research and Development Center of Ophthalmology and Vision Science, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin Medical University Eye Institute, 251 Fukang Road, Nankai, Tianjin, 300384, People’s Republic of China

    Lijie Dong, Zhe Zhang, Xun Liu, Qiong Wang, Yaru Hong, Xiaorong Li & Juping Liu

  2. Department of Ophthalmology and Optometry, Eye and ENT Hospital, Fudan University, Shanghai, China

    Zhe Zhang

  3. NHC Key Laboratory of Myopia (Fudan University), Shanghai, China

    Zhe Zhang

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  1. Lijie Dong
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  2. Zhe Zhang
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  4. Qiong Wang
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Contributions

Li Jiedong designed the research; Xiaorong Li provided advice and guidance; Zhe Zhang and Xun Liu performed the research; Zhe Zhang and Juping Liu wrote the paper. All authors read and approved the final manuscript.

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Correspondence to Lijie Dong, Xiaorong Li or Juping Liu.

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The authors declare that they no conflicts of interests.

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All human studies have been approved by the Ethics Committee of Tianjin Medical University Eye Hospital, and animal experimentation conforms to protocols were approved by the Animal Ethical and Welfare Committee of Tianjin Medical University Eye Institute.

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Dong Lijie and Zhang Zhe as the co-first authors, contributed equally to this manuscript.

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Dong, L., Zhang, Z., Liu, X. et al. RNA sequencing reveals BMP4 as a basis for the dual-target treatment of diabetic retinopathy. J Mol Med 99, 225–240 (2021). https://doi.org/10.1007/s00109-020-01995-8

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  • DOI: https://doi.org/10.1007/s00109-020-01995-8

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