June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
SARS-CoV-2 Spike (S) protein activates retinal endothelial and innate immune cells to cause thrombosis in retinal blood vessels
Author Affiliations & Notes
  • Sneha Singh
    Ophthalmology, Visual and Anatomical Sciences (OVAS), Wayne State University School of Medicine, Detroit, Michigan, United States
  • Ashok Kumar
    Ophthalmology, Visual and Anatomical Sciences (OVAS), Wayne State University School of Medicine, Detroit, Michigan, United States
  • Footnotes
    Commercial Relationships   Sneha Singh None; Ashok Kumar None
  • Footnotes
    Support  R01EY026964, R01EY027381, R01EY032149, R21 AI149385, P30EY004068
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3797. doi:
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    • Get Citation

      Sneha Singh, Ashok Kumar; SARS-CoV-2 Spike (S) protein activates retinal endothelial and innate immune cells to cause thrombosis in retinal blood vessels. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3797.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : We recently reported the presence of SARS-CoV-2 RNA, Spike, and Envelope proteins in the corneas/conjunctiva of COVID-19-affected donors. Clinical studies have also reported cotton wool spots, retinal vessel dilation, and hemorrhages in COVID-19 patients, indicating the involvement of the retina. Studies have also reported possible retinal thrombotic complications in COVID-19. This study aims to investigate the role of S protein in triggering retinal vascular inflammation and causing thrombotic complications.

Methods : Human retinal vascular endothelial cells (HRvEC) and primary human neutrophils were used. The endothelial cell junction integrity, cell activation, and inflammation markers were assessed by qPCR and ELISA. The integrity of the interendothelial junction was assessed by transwell FITC-Dextran permeability assay as well as a western blot for tight junction protein, ZO-1. The activation of NETosis was assessed by Myeloperoxidase (MPO) and Elastase staining. Real-time imaging of Sytox green staining for NET-DNA in primary human neutrophils was performed using the Sartorius Incucyte system. The thromboembolic events in the wild-type mice eyes were assessed by fluorescein angiography and fundus imaging using the Micron IV system.

Results : The exposure of primary human neutrophils to S protein led to their activation and induction of NETosis with increased expression of elastase, MPO, and inflammatory markers (TNFa, IL-6, IL-8, and IL1b). The cells also showed increased secretion of NET-DNA on real time-basis which was diminished with DNAase treatment. The treatment of retinal endothelial cells with S protein induced the expression of ACE2 at both transcript and protein levels along with increased the expression of inflammatory and cell activation (ICAM-1, VCAM-1) markers. S protein exposure of HRvEC reduced the expression of tight junction protein ZO-1 along with an increase in FITC-dextran permeability, indicating the loss of barrier integrity. Interestingly, in the wild-type mice eyes, the treatment with Spike protein led to blockage of retinal capillaries evident by fundus imaging and fluorescein angiography.

Conclusions : Our study indicates that the SARS-CoV-2 S protein can directly activate retinal endothelial cells and neutrophils and increased NETosis and inflammatory response could trigger thromboembolic events in the retinal vessels during COVID-19.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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