July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Alpha-1 Antitrypsin Decreases Cx43 Expression Through PI3K/AKT Pathway in ARPE-19 Cells Exposed to High Glycemia
Author Affiliations & Notes
  • MARÍA CONSTANZA POTILINSKI
    IIMT-CONICET-Austral, Buenos Aires, Buenos Aires, Argentina
  • Gustavo Ortiz
    IIMT-CONICET-Austral, Buenos Aires, Buenos Aires, Argentina
  • Juan Pablo Salica
    IIMT-CONICET-Austral, Buenos Aires, Buenos Aires, Argentina
    Oftalmología, Hospital Universitario Austral, Buenos Aires, Argentina
  • Emiliano López
    IIMT-CONICET-Austral, Buenos Aires, Buenos Aires, Argentina
    Oftalmología, Hospital Universitario Austral, Buenos Aires, Argentina
  • Eduardo Chuluyán
    Farmacología, Universidad de Buenos Aires, Buenos Aires, Argentina
    Oftalmología, Hospital Universitario Austral, Buenos Aires, Argentina
  • Juan E Gallo
    IIMT-CONICET-Austral, Buenos Aires, Buenos Aires, Argentina
    Oftalmología, Hospital Universitario Austral, Buenos Aires, Argentina
  • Footnotes
    Commercial Relationships   MARÍA CONSTANZA POTILINSKI, None; Gustavo Ortiz, None; Juan Salica, None; Emiliano López, None; Eduardo Chuluyán, None; Juan Gallo, None
  • Footnotes
    Support  Universidad Austral
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 196. doi:https://doi.org/
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      MARÍA CONSTANZA POTILINSKI, Gustavo Ortiz, Juan Pablo Salica, Emiliano López, Eduardo Chuluyán, Juan E Gallo; Alpha-1 Antitrypsin Decreases Cx43 Expression Through PI3K/AKT Pathway in ARPE-19 Cells Exposed to High Glycemia. Invest. Ophthalmol. Vis. Sci. 2018;59(9):196. doi: https://doi.org/.

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

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Abstract

Purpose : Diabetic retinopathy (DR) is associated with persistent inflammation and with damage to the vascular bed. The ophthalmic therapy for this retinal pathology is focused on severe stages of the disease. Previous results obtained by our group show that Alpha-1 Antitrypsin (A1AT) acts like an anti-inflammatory agent that could play a role on DR treatment. However, it is important to know the effect of A1AT on proteins that are relevant to retinal function and the molecular mechanisms modified by this serine protein. The retinal pigment epithelium (RPE) forms the epithelial component of the blood-retinal barrier. Connexin43 is a major gap junction protein expressed in RPE cells. Cx43 upregulation and hemichannel opening have been implicated in all aspects of secondary damage, edema and loss of vascular integrity, leading to neuronal death.
We evaluated Cx43 expression and proteins implicated in different signaling pathways in an in vitro diabetic retinopathy cell model.

Methods : ARPE-19 cells (ATCC® CRL-2302TM, Manassas, Virginia, USA) were maintained in DMEM/F12 (Invitrogen, Carlsbad, California, USA) containing 2 μM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum. ARPE-19 cells (passages 9 to 12) were incubated 16h with DMEM 5,5 mM glucose (Control), DMEM 5,5 mM glucose + 4.5 mg/ml A1AT (Control + A1AT), DMEM 30 mM glucose (Diabetic), DMEM 30 mM glucose + 4.5 mg/ml A1AT (Diabetic + A1AT). Cells were harvested with RIPA buffer for Western blot assay or fixed for immunohistochemistry.

Results : A1AT diminished levels of Cx43 overexpressed in high glucose conditions, A1AT also reduces AKT and pAKT1/2/3 Ser473 expression levels, indicating PI3K/AKT pathway participation, and a possible crosstalk and activation of Wnt signaling. Therefore, we could also observe a lower expression of NFkB p65 and iNOS, both proteins involved in the inflammatory response.

Conclusions : Results support the hypothesis that A1AT regulates Cx43 expression through PI3K/AKT and Wnt signaling pathway. This is a novel aspect about Cx43 expression modulation. Taking together with results obtained on inflammatory response, A1AT is a promising molecule to treat DR.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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