July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
TRIB3 ablation attenuates hypoxia-induced angiogenesis in mouse retinas
Author Affiliations & Notes
  • Priyamvada M Pitale
    University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Yvonne Adu-Agyeiwaah
    University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Sergio Li Calzi
    University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Takashi Satoh
    Osaka University, Osaka, Japan
  • Shizuo Akira
    Osaka University, Osaka, Japan
  • Maria B Grant
    University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Marina S Gorbatyuk
    University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Footnotes
    Commercial Relationships   Priyamvada Pitale, None; Yvonne Adu-Agyeiwaah, None; Sergio Li Calzi, None; Takashi Satoh, None; Shizuo Akira, None; Maria Grant, None; Marina Gorbatyuk, None
  • Footnotes
    Support  R01 EY027763
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 6428. doi:
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      Priyamvada M Pitale, Yvonne Adu-Agyeiwaah, Sergio Li Calzi, Takashi Satoh, Shizuo Akira, Maria B Grant, Marina S Gorbatyuk; TRIB3 ablation attenuates hypoxia-induced angiogenesis in mouse retinas. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6428.

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

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Purpose : Retinal neovascularization and vascular obliteration are critical pathological hallmarks of proliferative diabetic retinopathy. Vascular endothelial growth factor (VEGF) is a key factor promoting neovascularization and tufts formation. The metabolic stress marker, pseudokinase tribbles 3 (TRIB3) has been shown to be upregulated and promote VEGF expression in tumor hypoxia. We hypothesized that downregulation of TRIB3 would reprogram VEGF expression and the rate of angiogenesis in the oxygen-induced retinopathy (OIR) mouse model.

Methods : C57BL6 and TRIB3KO pups were exposed to high oxygen (75%) from postnatal (P) day 7 to 12. Pups then were returned to room air (21%) and euthanized at P13 or P17. At P17, eyes were enucleated and fixed. Retinal flat mounts and cryosections were used to perform immunohistochemical analyses to determine areas of neovascularization and reactive gliosis by Isolectin staining and GFAP/Vimentin immunostaining, respectively. Neovascularization was calculated as the percentage of neovascular area to total retinal area using Image J software. Inner nuclear layer (INL) nuclei were counted in H&E stained cryosections. INL nuclei were counted by masked investigator to avoid bias. Retinal VEGF expression at P13 was estimated by western blot analysis. We used one-way ANOVA for statistics.

Results : We found that, as expected, at P13, VEGF expression was significantly increased in the retina of C57BL6-OIR (*p<0.05) compared to normoxia C57BL6 pups; however, ablation of TRIB3 in OIR pups demonstrated dramatic decline in VEGF expression as compared to C57BL6 OIR pups (**p<0.01). These findings correlated with neovascularization data. Thus, in P17 C57BL6-OIR pups, we observed 37.47% of the area with retinal neovascularization, while in TRIB3KO-OIR pups we detected only 6.08%. In addition, we found that TRIB3 ablation in OIR pups resulted in 22.67% increase in the number of nuclei of the INL in retina as compared to the C57BL6-OIR pups. Interestingly, we found GFAP/vimentin immunostaining in retinal astrocytes and Müller cells of both C57BL6-OIR and TRIB3KO-OIR pups suggesting reactive gliosis.

Conclusions : Our results suggest that TRIB3 ablation could decrease the levels of neovascularization and VEGF expression in hypoxic retina. Future experiments will help to validate TRIB3 as a therapeutic target for aberrant retinal vascularization.

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


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