Association of Placental Growth Factor and Angiopoietin in Human Retinal Endothelial Cell-Pericyte co-Cultures and iPSC-Derived Vascular Organoids

Abstract

Purpose

Placental growth factor (PlGF) and Angiopoietin (Ang)-1 are two proteins that are involved in the regulation of endothelial cell (EC) growth and vasculature formation. In the retina and endothelial cells, pericytes are the major source of both molecules. The purpose of this study is to examine the association of PlGF and Ang-1 with human EC/pericyte co-cultures and iPSC-derived vascular organoids.

Methods

In this study, we used co-cultures of human primary retinal endothelial cells (HREC) and primary human retinal pericytes (HRP), western blotting, immunofluorescent analysis, TUNEL staining, LDH-assays, and RNA seq analysis, as well as human-induced pluripotent stem cells (iPSC), derived organoids (VO) to study the association between PlGF and Ang-1.

Results

Inhibition of PlGF by PlGF neutralizing antibody in HREC-HRP co-cultures resulted in the increased expression of Ang-1 and Tie-2 in a dose-dependent manner. This upregulation was not observed in HREC and HRP monocultures but only in co-cultures suggesting the association of pericytes and endothelial cells. Furthermore, Vascular endothelial growth factor receptor 1 (VEGFR1) inhibition abolished the Ang-1 and Tie-2 upregulation by PlGF inhibition. The pericyte viability in high-glucose conditions was also reduced by VEGFR1 neutralization. Immunofluorescent analysis showed that Ang-1 and Ang-2 were expressed mainly by perivascular cells in the VO. RNA seq analysis of the RNA isolated from VO in high glucose conditions indicated increased PlGF and Ang-2 expressions in the VO. PlGF inhibition increased the expression of Ang-1 and Tie-2 in VO, increasing the pericyte coverage of the VO microvascular network.

Conclusion

Combined, these results suggest PlGF's role in the regulation of Ang-1 and Tie-2 expression through VEGFR1. These findings provide new insights into the neovascularization process in diabetic retinopathy and new targets for potential therapeutic intervention.

Keywords:

• PlGF
• angiopoietin
• diabetic retinopathy
• pericyte
• vascular organoids

Acknowledgments

The authors like to acknowledge the contributions of a lab technician Ms. Lijuan Fan for assistance with iPSC-derived vascular organoid cultures.

Author contributions

The study was conceived and designed by HH. HH and AL performed in vitro experiments and evaluations. MSS has performed RNA seq datasets analysis. MSS and HH have conducted DESeq2 and R program analysis, figure design, and statistical analysis. The manuscript was written by HH, MSS, AM, RM, and AL and critically revised by HH, AM, and AL. All authors reviewed and accepted the final version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. RNA-Sequencing raw data (Bioproject: PRJNA352279) originally produced by Wimmer et al. 2019 is available at: https://www.ncbi.nlm.nih.gov/sra/?term=SRP092491.

Additional information

Funding

This work was supported by NIH R01 grant [grant number EY027824] to HH; Missouri University start-up fund (to HH); and Missouri University Postdoctoral Research Grant (to AL).