Belamkar : The profibrotic phenotype in COL4A3 mouse model of Alport Syndrome
The profibrotic phenotype in COL4A3 mouse model of Alport Syndrome
Indiana University Bloomington
Ameya Belamkar1, Qianyi Luo2, Neha Mahajan2, Surabhi Abhyankar2, Bryce Jones3, Padmanabhan Pattabiraman2, Moshe Levi3, and Ashay D. Bhatwadekar2
1. Indiana University Bloomington, Bloomington, IN
2. Department of Ophthalmology, Eugene and Marilyn Glick Eye Institute, Indianapolis, IN
3. Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC
The mechanism of ocular pathology in Alport Syndrome (AS) is poorly understood. This study characterizes inflammatory and fibrotic changes between the eyes of wild-type (WT) and COL4A3 knockout (KO) mice.
Methods: Col4a3tm1Dec mice were euthanized at 25 weeks, and eyes were processed for paraffin sectioning. Sections were stained with 1:200 anti-α3 (IV) antibody with 1:2000 secondary antibody to evaluate the localization of the α3 (IV) in WT eyes. Staining for inflammatory markers included 1:200 anti-TGF-ß2 with 1:500 secondary antibody, 1:100 anti-CTGF with 1:800 secondary antibody, and 1:100 anti-ß-catenin with 1:800 secondary antibody. All sections were also stained with DAPI. Photomicrographs visualizing protein distribution were taken under a confocal microscope. mRNA levels of pro-fibrotic genes were assessed using real-time quantitative PCR (qPCR).
Retinal staining of the α3 (IV) protein revealed no positive staining, while corneal staining showed positive staining in Descemet’s membrane. An upregulation of TGF-ß2 was observed in both the retina and cornea. While there was an increase in CTGF and ß-catenin in the cornea, there was no observable difference in the retina.
qPCR showed a significant increase in the levels of FSP1, αSMA, Col1a1, SNAIL, and SLUG in KO retinas. There was a moderate increase in FSP1 and SLUG in the corneas of KO mice.
The observed increase in inflammatory cytokines in the corneas of KO mice may be due to degradation of corneal endothelium integrity in KO mice caused by an absence of the α3 (IV) protein. However, a similar increase in the transcription of pro-fibrotic genes was not clearly observed in KO corneas. Surprisingly, there was a clear trend in increasing levels of pro-fibrotic mRNA in the retina despite finding no α3 (IV) protein in WT retinas.
Translational/Human Health Impact:
Ocular abnormalities are often observed in patients with AS. Yet, the pathology causing these abnormalities is not currently well understood. This study aims to evaluate the role of inflammation and fibrosis in the ocular pathogenesis of AS.