June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
K2000 is involved in pathological neovascularization
Author Affiliations & Notes
  • Guillaume Blot
    Department of Ophtalmology, Maisonneuve-Rosemont Hospital Research Center, Universite de Montreal Faculte de Medecine, Montreal, Quebec, Canada
    Department of Biochemistry and molecular medicine, Maisonneuve-Rosemont Hospital Research Center, Universite de Montreal Faculte de Medecine, Montreal, Quebec, Canada
  • Gabrielle Girouard
    Department of Biomedical Sciences, Maisonneuve-Rosemont Hospital Research Center, Universite de Montreal Faculte de Medecine, Montreal, Quebec, Canada
  • Vera Guber
    Department of Ophtalmology, Maisonneuve-Rosemont Hospital Research Center, Universite de Montreal Faculte de Medecine, Montreal, Quebec, Canada
  • Agnieszka Dejda
    Department of Ophtalmology, Maisonneuve-Rosemont Hospital Research Center, Universite de Montreal Faculte de Medecine, Montreal, Quebec, Canada
  • Ariel Wilson
    Department of Ophtalmology, Maisonneuve-Rosemont Hospital Research Center, Universite de Montreal Faculte de Medecine, Montreal, Quebec, Canada
  • Przemyslaw Mike Sapieha
    Department of Ophtalmology, Maisonneuve-Rosemont Hospital Research Center, Universite de Montreal Faculte de Medecine, Montreal, Quebec, Canada
    Department of Biochemistry and molecular medicine, Maisonneuve-Rosemont Hospital Research Center, Universite de Montreal Faculte de Medecine, Montreal, Quebec, Canada
  • Footnotes
    Commercial Relationships   Guillaume Blot None; Gabrielle Girouard None; Vera Guber None; Agnieszka Dejda None; Ariel Wilson None; Przemyslaw Mike Sapieha None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3728. doi:
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      Guillaume Blot, Gabrielle Girouard, Vera Guber, Agnieszka Dejda, Ariel Wilson, Przemyslaw Mike Sapieha; K2000 is involved in pathological neovascularization. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3728.

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

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Abstract

Purpose : In a quest to elucidate pathways involved in pathological angiogenesis, we identified a gene highly regulated in a mouse model of oxygen-induced retinopathy (OIR) we named K2000. K2000 and its human homolog have unknown function and its reported expression is restricted to testes and tumor environment. We therefore genetically engineered a mouse harboring K2000 deletion (K2000-/-) and studied its potential role in pathological retinal angiogenesis.

Methods : Bulk RNA sequencing analysis (RNAseq) was performed on retinas from P14, P17 and P30 OIR (P7-P12 75% O2; P12-P17 21% O2) and normoxic C57BL/6J mice using Torrent suite software. CRISPER/Cas9 genetic engineering technology was used to produce K2000-/-. In OIR, retinal avascular area and neovascularization was assessed at P17. Choroidal neovascularization (CNV) was induced on 8-week-old male by rupture of Bruch’s membrane using argon laser. 14 days after CNV induction, mice were perfused with fluorescein isocyanate-dextran (FITC). Fixed choroids were additionally labeled with Isolectin-B4 (IB4). Statistical analysis for categorical frequency data and continuous data were performed with exact Fisher’s test and unpaired t-test with Welch’s correction, respectively.

Results : RNAseq revealed downregulation of K2000 expression (log2 FC -3.4, p = 0.02) in OIR WT mice at P30. Engineered K2000-/- were viable, their generation from K2000+/- x K2000+/- breeding did not significantly deviate from expected Mendelian inheritance (p = 0.18; n = 38 pups). Compared to wild type mice, when K2000-/- mice were bred, the mean number of pups per litter per male genitor was slightly reduced (6.2 SEM ± 0.3 vs 6.8 SEM ± 0.2, p = 0.04; n = 45 male genitors) and the frequency of female pups was decreased (46.1% vs 52.4%, odds ratio 0.77 CI95% = 0.62-0.97, p = 0.03; n = 1578 sexed pups). Neonatal mortality was not significantly different (p = 0.38; n = 2002 born pups). In OIR, K2000-/- showed reduced avascular area and neovascularization at P17 (15.4% SEM ± 0.7 vs 17,4% SEM ± 0.6, p = 0.03 and 5.3% SEM ± 0.2 vs 6.6% SEM ± 0.2, p ≤ 0.01, respectively; n 12 mice per group). In contrast in CNV the neovascular area (FITC), burn scare area (IB4) or ratio of FITC/IB4 did not significantly change (p = 0.93, p = 0.52 and p = 0.89, respectively; n ≥ 7 mice per group).

Conclusions : K2000 may contribute to the angiogenic response to hypoxia. Structural and functional analyses of the K2000 gene product are ongoing.

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

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