July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Novel blockers of the L-VGCC play a protective role in microglia-related ocular inflammation and angiogenesis disorders
Author Affiliations & Notes
  • Madhu Sudhana Saddala
    Ophthalmology, University of Missouri , Columbia, Missouri, United States
    Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Anton Lennikov
    Ophthalmology, University of Missouri , Columbia, Missouri, United States
    Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Anthony Mukwaya
    Ophthalmology, Linköping University, Linköping, Linköping, Sweden
  • Hu Huang
    Ophthalmology, University of Missouri , Columbia, Missouri, United States
    Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Madhu Sudhana Saddala, None; Anton Lennikov, None; Anthony Mukwaya, None; Hu Huang, None
  • Footnotes
    Support  This work is supported by NIH grant (R01 EY027824) and Missouri University internal start-up funds (Hu Huang research group)
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 254. doi:
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    • Get Citation

      Madhu Sudhana Saddala, Anton Lennikov, Anthony Mukwaya, Hu Huang; Novel blockers of the L-VGCC play a protective role in microglia-related ocular inflammation and angiogenesis disorders. Invest. Ophthalmol. Vis. Sci. 2019;60(9):254.

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

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Abstract

Purpose : In this study, we investigated blockade of L-type voltage-gated calcium channel (L-VGCC) by novel compounds identified using in silico, in vitro and in vivo evaluations.

Methods : We have screened five novel compounds (1a, 1b, 1c, 1d and 1e) based on binding energies and ADMET profiles by using computational structure-based pharmacophore modeling, virtual screening, and molecular docking. BV-2 cells were used in vitro experiments. The selective blockers of L-VGCC were verified using Fura-2 fluorescent dye and Electric cell-substrate impedance sensing (ECIS) array. BV-2 cell migration and Actin-F cytoskeleton were assayed by Calcein-AM and phalloidin staining respectively. Expression of COX-2 and Arg-1 in microglia cells under the effects of L-VGCC blockers were evaluated by RT-PCR and IHC. Choroidal neovascularization (CNV) was induced by laser injury to the RPE and Bruch’s membrane in 6~8-week-old C57BL/6J and CX3CR1gfp/wt mice. The compound 10 µg of 1a the most effect compound was delivered into the mouse eye by sub-conjunctival injection daily.

Results : Substances have low cell toxicity in concentrations as high as 5 µg/ml (p>0.05) in a number of dead cells when compared to water. All compounds have demonstrated high efficacy in blockade significantly decreasing resistance (p<0.01) and increasing capacitance (p<0.01) of BV-2 cells, this data was further supported by Fura-2 staining. Migration assay and Actin-F staining have detected significant (p<0.01) reduction in migration of BV-2 cells with disruption of Actin-F. RT-PCR analysis indicated a significant reduction of COX-2 (p<0.01) and Arg-1 (p<0.01) expression when stimulated with LPS (1µg/ml) and IL-4 (20 ng/ml) respectively. In Laser CNV mouse model,. In vivo imaging indicated a marked reduction of laser CNV lesson sizes (1.27 cm) and significant decrease of vascular leakage (p<0.01) in 1a compound treated animals, these results were further corroborated by ex vivo staining with a significant reduction of lesion size (p<0.01) and GSA-lectin staining (p<0.01). CX3CR1gfp/wt mice demonstrated a reduction of lesion size and infiltration with GFP positive cells when treated with 1a compound.

Conclusions : These findings indicate that microglia [Ca2+]i signaling plays a crucial role in neuro-inflammation and L-VGCCblockage leads to reduced microglia activity.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Neuroinflammatory signaling without blocker and with blocker

Neuroinflammatory signaling without blocker and with blocker

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