December 2024
Volume 65, Issue 14
Open Access
Physiology and Pharmacology  |   December 2024
Characterization of the Ocular Phenotype in a Col4a3 Knockout Mouse Model of Alport Syndrome
Author Affiliations & Notes
  • Ameya Belamkar
    Indiana University of School of Medicine, Indiana, United States
  • Qianyi Luo
    Department of Ophthalmology, Eugene and Marilyn Glick Eye Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Neha Mahajan
    Department of Ophthalmology, Eugene and Marilyn Glick Eye Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Surabhi Abhyankar
    Department of Ophthalmology, Eugene and Marilyn Glick Eye Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Bryce A. Jones
    Department of Pharmacology & Physiology, Georgetown University Medical Center, Washington, DC, United States
  • Rupinder Kaur Sodhi
    Department of Ophthalmology, Eugene and Marilyn Glick Eye Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Padmanabhan P. Pattabiraman
    Department of Ophthalmology, Eugene and Marilyn Glick Eye Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Moshe Levi
    Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC, United States
  • Ashay D. Bhatwadekar
    Department of Ophthalmology, Eugene and Marilyn Glick Eye Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Correspondence: Ashay D. Bhatwadekar, Department of Ophthalmology, Eugene and Marilyn Glick Eye Institute, Indiana University School of Medicine, 1160 West Michigan Street, Indianapolis, IN 46202, USA; [email protected]
Investigative Ophthalmology & Visual Science December 2024, Vol.65, 29. doi:https://doi.org/10.1167/iovs.65.14.29
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      Ameya Belamkar, Qianyi Luo, Neha Mahajan, Surabhi Abhyankar, Bryce A. Jones, Rupinder Kaur Sodhi, Padmanabhan P. Pattabiraman, Moshe Levi, Ashay D. Bhatwadekar; Characterization of the Ocular Phenotype in a Col4a3 Knockout Mouse Model of Alport Syndrome. Invest. Ophthalmol. Vis. Sci. 2024;65(14):29. https://doi.org/10.1167/iovs.65.14.29.

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

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Abstract

Purpose: Alport syndrome (AS) is a genetic condition caused by a dysfunctional collagen (IV) α3α4α5 heterotrimer, leading to basement membrane instability and, ultimately, abnormalities in the kidney, inner ear, and eyes. This study aimed to characterize ocular pathology of AS by focusing on inflammatory and fibrotic markers.

Methods: Col4a3 tm1Dec knockout (KO) mice eyes were evaluated for the localization of collagen (IV) α3 and collagen (IV) α4, then stained for transforming growth factor-β1 (TGF-β1), TGF-β2, connective tissue growth factor (CTGF), and β-catenin. mRNA levels of the profibrotic genes S100a4, Acta2, Col1a1, Snai1, Snai2, and Twist1 were assessed using real-time reverse transcription quantitative PCR (RT-qPCR).

Results: Collagen (IV) α3 and collagen (IV) α4 were co-expressed in Descemet's and Bruch's membrane but not in the retina, lens, or other corneal substructures. Immunofluorescence quantitation revealed upregulation of TGF-β1 in the anterior lens and TGF-β2 in the retina of KO eyes. Conversely, CTGF and β-catenin were shown to be elevated in the corneal epithelium but not the retina or lens. RT-qPCR showed an increase in the transcription of Acta2, Col1a1, and Snai2 in the retinas and Snai2 in anterior segments of KO mice.

Conclusions: Col4a3 KO mice exhibited a differential inflammatory and profibrotic response in the cornea, retina, and lens, which may play a role in the ocular pathology of AS.

Alport syndrome (AS) is a rare genetic disorder characterized by kidney disease, ocular abnormalities, and loss of hearing. AS is caused by mutations in the genes encoding collagen type IV α3, α4, or α5, collectively comprising the collagen (IV) α3α4α5 heterotrimer. AS may be inherited in an autosomal recessive or dominant pattern due to mutations in the COL4A3 or COL4A4 gene or an X-linked pattern due to mutations in the COL4A5 gene.1 In embryonic membranes, the collagen (IV) α1α1α2 heterotrimer predominates but is replaced by the α3α4α5 heterotrimer through isotype switching in the basement membranes of the kidney and eye.24 However, isotype switching has been shown to be developmentally arrested in the glomerular basement membrane of patients with AS, thus causing a perpetuation of fetal α1α1α2 and an absence of α3α4α5.5 The α1α1α2 heterotrimer is less structurally sound and more susceptible to proteolysis and biomechanical strain, producing abnormal membranes and clinical pathologies observed in patients with AS.6 
Progressive loss of kidney function is common in patients with AS due to ultrastructural changes in the glomerular basement membrane (GBM). Type IV collagen is critical for GBM stability, and loss of the α3 (IV) protein leads to GBM thinning and variable segmental GBM thickening and splitting. The dysfunctional GBM found in patients with AS causes hematuria and proteinuria, ultimately resulting in chronic inflammation and fibrosis.79 
Retinal abnormalities are the most common ocular manifestation of AS. In a study evaluating 15 patients with autosomal recessive AS, Wang et al.10 found that 87% had perimacular fleck retinopathy or peripheral coalescing fleck retinopathy. Perimacular fleck retinopathy can range from scattered dots and flecks in the temporal retina to a perimacular annulus of densely packed dots.11 This may cause a dull macular reflex or lozenge sign sharply contrasting with the normal retina; however, visual acuity is not affected by retinopathy. Macular holes are a rare complication of AS and may be caused by accelerated fluid flow through an abnormal Bruch's membrane. Temporal retinal thinning is observed in most patients with autosomal recessive AS.6 Specifically, the internal limiting membrane/nerve fiber layer (ILM/NFL) and Bruch's membrane are thinned. Thinning of these structures has been implicated as the primary cause of retinopathy, temporal macular thinning and lozenge, and the development of macular holes in patients with AS.12 
Corneal disease occurs infrequently in patients with AS. Recurrent corneal erosions, caused by an abnormal Bowman's membrane of the corneal subepithelium, have been noted to occur in about 20% of patients with AS and renal failure.13,14 Symptoms of erosions are unilateral or bilateral ocular pain accompanied by watering, photophobia, and blurred vision. Posterior polymorphous dystrophy, caused by an abnormal Descemet's membrane of the corneal endothelium, is a much rarer and more serious manifestation of AS. Patients may be asymptomatic or have recurrent episodes of grittiness, watering, and photophobia. Patients with corneal disease frequently present with corneal opacities due to scarring.6 
Lenticonus is present in about half of males with X-linked AS and is common in both men and women with autosomal recessive AS.6 AS patients typically present with bilateral anterior lenticonus, and posterior lenticonus is less common.15 Several studies have utilized electron microscopy to demonstrate pronounced thinning and vertical dehiscence of the anterior lens capsule.1619 Anterior lenticonus results from the conical protrusion of the lens through the thinnest and weakest part of the anterior lens capsule. Furthermore, spontaneous anterior lens capsule rupture has been reported in patients with AS, suggesting that capsular strength is deficient.20,21 Cataracts develop following the healing of small partial splits that rupture from the lenticonus.15 
Several inflammatory biomarkers have been linked to the rapid progression of chronic kidney disease in patients with AS.22 Furthermore, there is evidence that the progression of AS is determined by the quality and quantity of proinflammatory and profibrotic proteins in the urine.23 Thus, it has been shown that the abnormal glomerular basement membrane caused by a lack of α3 (IV) leads to inflammation and fibrosis, ultimately leading to chronic kidney disease. Although it is known that the absence of the collagen (IV) α3α4α5 heterotrimer produces abnormal membranes and, ultimately, the ocular pathology observed in patients with AS, the underlying mechanism by which this pathology manifests is not well understood. Furthermore, no surgical or gene therapy approaches were shown to alleviate the ocular symptoms of AS in a large study.15 As such, it is critical to characterize the biochemical alterations underlying the ocular defects in AS to inform future therapeutic options. 
Col4a3 knockout (KO) mice have been shown to successfully replicate many of the renal pathological features observed in patients with AS.2426 However, the ocular features of these KO mice have not yet been well explored. While our paper was under revision, a study by Wang et al.27 characterized the ocular morphology of Col4a3 KO mice, concluding that these mice exhibit ocular abnormalities comparable to patients with AS. The authors utilized transmission electron microscopy (TEM) to demonstrate abnormalities in the ILM of the retina, retinal pigment epithelium (RPE) basement membrane, corneal epithelium basement membrane, and an anterior lenticonus-like phenotype in a subset of the KO mice. Furthermore, they confirmed retinal thinning in KO mice, specifically caused by thinning of the inner plexiform layer (IPL). Finally, they demonstrated activation of Müller cells in KO mice, which they speculated was due to damage to the ILM caused by abnormal collagen chain organization. Therefore, their work gives credence to the idea that an aberrant ocular microenvironment caused by collagen (IV) abnormalities may lead to secondary phenotypes that warrant further research. In the present study, we hope to characterize the abnormal ocular microenvironment found in Col4a3 mice by investigating the role inflammation and fibrosis play in the ocular pathogenesis of AS. 
Methods
Animals
Animal studies were performed at Georgetown University with approval from the Institutional Animal Care and Use Committee. Col4a3tm1Dec mice on the C57BL/6J background were obtained and housed as previously described.28 Mice were euthanized with carbon dioxide at 25 weeks of age. Enucleated eyes were lanced and drop-fixed (15 minutes) in 4% paraformaldehyde (AAJ19943K2; Thermo Fisher Scientific, Waltham, MA, USA). They were washed (three times, 5 minutes each) in phosphate-buffered saline (BP24384; Thermo Fisher Scientific) and stored at 4°C for downstream processing. For RNA studies, the eyes were stored in RNAlater Stabilization Solution (AM7020; Thermo Fisher Scientific) and processed for anterior segment and retinal separation as described under real-time reverse transcription quantitative PCR (RT-qPCR) studies. 
Immunofluorescence
The eyes were processed for paraffin sectioning. For the anti-α3 (IV) antibody, epitope retrieval was performed by heating slides in 0.2-M HCl, pH = 0.9, in the 2100 Antigen Retriever (Aptum Biologics, Southampton, UK), followed by soaking in 0.1-M glycine/6-M urea solution, pH = 3.2, for 30 minutes at room temperature. For all other antibodies, epitope retrieval was performed using 1× Citrate Plus buffer, pH = 6, at 56°C overnight. Sections were blocked in 3% goat serum in staining buffer for 2 hours at room temperature. Primary antibodies—anti-α3 (IV) (1:200, 7076; Chondrex, Redmond, WA, USA), anti-α4 (IV) (1:200, 7073; Chondrex), anti-transforming growth factor-β1 (TGF-β1; 1:200, 21898-1-AP; Proteintech, Rosemont, IL, USA), anti-TGF-β2 (1:200, 19999-1-AP; Proteintech), anti-connective tissue growth factor (CTGF; 1:100, ab6992; Abcam, Cambridge, UK), and anti-β-catenin (1:100, 9582; Cell Signaling Technology, Danvers, MA, USA)—were incubated overnight at 4°C. The secondary antibody—Alexa Fluor 555 Goat Anti-Rat (A-21434; Thermo Fisher Scientific) and Alexa Fluor 555 Goat Anti-Rabbit (1:800, A-21428; Thermo Fisher Scientific)—was incubated for 2 hours. VECTASHIELD Antifade Mounting Medium with DAPI (VectorLabs, Burlingame, CA, USA) was added, and a coverslip was placed and sealed with nail varnish. 
For picrosirius red staining, sections were soaked in picrosirius red solution (0.5 g Sirius red F3B mixed with 500 mL of saturated aqueous picric acid solution) for 60 minutes, then quickly dipped in two changes of 0.5% acidified water. Slides were then serially dehydrated and mounted with resinous medium. Photomicrographs of the retina, cornea, and lens were taken under a confocal microscope (Zeiss LSM 510 META; ZEISS Microscopy, Jena, Germany). 
Immunofluorescence Quantification Analysis
Ocular sections were visualized using a fluorescence lamp illuminator (X-Cite 120Q; Excelitas, Pittsburgh, PA, USA), and photomicrographs of the retina, cornea, and anterior lens were taken at 200× under a fluorescent microscope (AXIO Observer.A1 Inverted Fluorescence Microscope; ZEISS Microscopy). A protocol outlined by Shihan et al.29 was followed to measure mean fluorescence intensity (MFI) in the retina and corneal epithelium using ImageJ Fiji (National Institutes of Health, Bethesda, MD, USA). Images were split by channel, and the red channel was segmented into regions of interest using an external drawing pen and the thresholding function on Fiji. The net MFI of a particular tissue was calculated by measuring the MFI of the region of interest and subtracting the MFI of the background. 
Real-Time qRT-PCR
Animals were euthanized, and the eyes were enucleated. A small incision was made in front of the eyeball using a fine scalpel blade (#11), followed by a cut behind the ora serrata using vannas scissors separating the anterior and posterior portions of the eye. Finally, the lens was removed, and the retinas were carefully separated from the posterior segments of the eye. RNA was extracted from the retina and anterior segment of the eye using the TRIzol (Thermo Fisher Scientific) chloroform method. Briefly, the samples were homogenized in the TRIzol, followed by layer separation using chloroform. The top layer containing the RNA was precipitated using ice-cold isopropanol. The RNA pellet was washed with 70% ethanol and eluted in nuclease-free water. The RNA was then purified with the RNeasy MinElute Cleanup Kit (QIAGEN, Hilden, Germany); 1 µg of purified RNA (average A260/280 ratio was 2.03) was used to prepare cDNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). Real-time RT-qPCR (ViiA 7; Thermo Fisher Scientific) was performed using a master mix (TaqMan Fast Universal; Thermo Fisher Scientific) with gene-specific primers for the following markers: S100a4 (gene for FSP-1; Mm00803372_g1), Acta2 (gene for αSMA; Mm00725412_s1), Col1a1 (Mm00801666_g1), Snai1 (gene for Snail; Mm00441533_g1), Snai2 (gene for Slug; Mm00441531_m1), and Twist1 (Mm00442036_m1). All genes were normalized to Tbp (gene for TATA-box binding protein; Mm00446973_m1). 
Statistical Analysis
All data are expressed as mean ± SEM, and statistical analysis was performed using Prism 10.0.2 for Mac (GraphPad, Boston, MA, USA). All datasets were tested for normality using a Shapiro–Wilk test. Statistical significance for datasets that passed the Shapiro–Wilk tests was tested using a two-tailed unpaired t-test. Statistical significance for datasets that did not pass the Shapiro–Wilk test was tested using a two-tailed Mann–Whitney U test. Data were considered statistically significant for P < 0.05. 
Results
Histological Characteristics of Col4a3 KO Eyes
A pathologist evaluated ocular sections stained with hematoxylin and eosin (H&E) independently for histological changes in the retina, cornea, and lens (Fig. 1). There were no observable trends within each group. Furthermore, we observed no evident histological changes in Col4a3 KO eyes, including lenticonus or cataracts, although these findings have been reported previously in human15 and mouse27 studies. 
Figure 1.
 
H&E staining of WT and KO mice eyes. Representative photomicrographs taken at 20× magnification of WT and KO retinas, corneas, and lenses show no evident histological differences between WT and KO mice eyes (n = 7). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PR, photoreceptor layer; RPE, retinal pigment epithelium; ALC, anterior lens capsule; LEC, lens epithelial cells; LF, lens fibers.
Figure 1.
 
H&E staining of WT and KO mice eyes. Representative photomicrographs taken at 20× magnification of WT and KO retinas, corneas, and lenses show no evident histological differences between WT and KO mice eyes (n = 7). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PR, photoreceptor layer; RPE, retinal pigment epithelium; ALC, anterior lens capsule; LEC, lens epithelial cells; LF, lens fibers.
Distribution of Collagen (IV) α3 and α4 in Mice Eyes
To investigate the distribution of the collagen (IV) α3 protein in the eye, we stained wild-type (WT) and KO eyes with anti-α3 (IV) antibodies. The specificity of antibodies was tested using a positive control of kidney sections (Supplementary Fig. S1). Similar to previous reports,30,31 collagen (IV) α3 stained strongly in the glomerular basement membrane, validating the selection of our antibodies. Although it has been previously reported that collagen (IV) α3 is found in the corneal endothelium, Bruch's membrane, and the anterior lens capsule in mice,32,33 our immunofluorescence staining detected the α3 (IV) protein in Bruch's membrane (Fig. 2A) and the corneal endothelium (Fig. 2C), but not in the anterior lens capsule of WT mice (Fig. 2E). Specifically, the thin band of labeling between the corneal endothelium and stroma indicates Descemet's membrane, and the single, tight line of labeling in Bruch's membrane indicates the RPE basement membrane. Staining confirmed that the α3 (IV) protein is absent in Col4a3 KO eyes. To investigate collagen turnover in Col4a3 KO mice, we further stained for the α4 (IV) protein (Figs. 2B, 2D, 2F), which colocalizes exclusively with the α3 (IV) protein as part of the collagen (IV) α3α4α5 heterotrimer. The pattern of labeling was identical to that of α3 (IV), and labeling was completely absent in KO eyes, which is consistent with previous findings showing that the absence of one α-chain leads to the complete absence of the heterotrimer.34 Although collagen (IV) α5 is another interesting target to explore, we limited our studies to collagen α3 (IV) and α4 (IV) because collagen α5 (IV) is also present in the collagen (IV) α5α5α6 heterotrimer.32,35 
Figure 2.
 
Immunofluorescence of collagen (IV) α3 and collagen (IV) α4 in WT and KO mice eyes. Ocular sections were stained with DAPI (blue) and collagen (IV) α3 (red; n = 5) or collagen (IV) α4 (red, n = 4). (A, B) Representative photomicrographs of α3 (IV) in the retina (A) and α4 (IV) in the retina (B) show a positive staining in the Bruch's membrane of the WT mice. (C, D) Corneal staining of α3 (IV) (C) and α4 (IV) (D) revealed a positive staining in Descemet's membrane, which anchors the endothelium to the rest of the cornea. (E, F) There was no labeling of α3 (IV) (E) or α4 (IV) (F) in the anterior lens of WT mice. We confirmed an absence of both isoforms in KO mice. Arrows indicate regions of positive staining; staining in regions not indicated by arrows is due to nonspecific binding of the secondary antibody.
Figure 2.
 
Immunofluorescence of collagen (IV) α3 and collagen (IV) α4 in WT and KO mice eyes. Ocular sections were stained with DAPI (blue) and collagen (IV) α3 (red; n = 5) or collagen (IV) α4 (red, n = 4). (A, B) Representative photomicrographs of α3 (IV) in the retina (A) and α4 (IV) in the retina (B) show a positive staining in the Bruch's membrane of the WT mice. (C, D) Corneal staining of α3 (IV) (C) and α4 (IV) (D) revealed a positive staining in Descemet's membrane, which anchors the endothelium to the rest of the cornea. (E, F) There was no labeling of α3 (IV) (E) or α4 (IV) (F) in the anterior lens of WT mice. We confirmed an absence of both isoforms in KO mice. Arrows indicate regions of positive staining; staining in regions not indicated by arrows is due to nonspecific binding of the secondary antibody.
Upregulation of Proinflammatory Cytokines in Col4a3 Eyes
Previous reports have demonstrated a proinflammatory phenotype in the kidneys of patients with AS.22,23 Thus, we decided to study the distribution of inflammatory markers in ocular sections by measuring the MFI of immunofluorescence stains. We first investigated the distribution of TGF-β1 and TGF-β2, both well-known markers of fibrosis in various fibrotic ocular diseases.3639 There was a significant increase in the level of TGF-β2 (Figs. 3B, 4B, 5B) in the retina (P = 0.0111), as well as a nonsignificant increase in TGF-β1 (Figs. 3A, 4A, 5A) in the lens (P = 0.0570) of KO mice. Because CTGF and β-catenin have both been shown to be downstream mediators of TGF-β signaling, we further stained ocular sections with anti-CTGF and anti-β-catenin antibodies.4043 Histological examination revealed an increase in immunopositive cells for CTGF (P = 0.0252) (Figs. 4C, 4E) and β-catenin (P = 0.0439) (Figs. 4D, 4E) in the corneas of KO mice, and no apparent changes in the retina (Figs. 3C, 3D) or anterior lens (Figs. 5C, 5D). Specifically, these inflammatory markers were observed to be upregulated in the corneal epithelium. 
Figure 3.
 
Immunofluorescence quantitation of inflammatory markers in WT and KO mice retinas. Representative photomicrographs of KO and WT mice retinas show staining patterns of DAPI (blue). (A) TGF-β1 (nWT = 6, nKO = 6); (B) TGF-β2 (nWT = 5, nKO = 5); (C) CTGF (nWT = 3, nKO = 3); (D) β-catenin (nWT = 4, nKO = 3) (red). Arrows indicate relative changes in staining. (E) Bar chart compares the MFI of these inflammatory markers between WT and KO mice. *P < 0.05, **P < 0.01, two-tailed unpaired t-test.
Figure 3.
 
Immunofluorescence quantitation of inflammatory markers in WT and KO mice retinas. Representative photomicrographs of KO and WT mice retinas show staining patterns of DAPI (blue). (A) TGF-β1 (nWT = 6, nKO = 6); (B) TGF-β2 (nWT = 5, nKO = 5); (C) CTGF (nWT = 3, nKO = 3); (D) β-catenin (nWT = 4, nKO = 3) (red). Arrows indicate relative changes in staining. (E) Bar chart compares the MFI of these inflammatory markers between WT and KO mice. *P < 0.05, **P < 0.01, two-tailed unpaired t-test.
Figure 4.
 
Immunofluorescence quantitation of inflammatory markers in WT and KO mice corneas. Representative photomicrographs of KO and WT mice retinas show staining patterns of DAPI (blue). (A) TGF-β1 (nWT = 6, nKO = 5); (B) TGF-β2 (nWT = 5, nKO = 5); (C) CTGF (nWT = 3, nKO = 4); (D) β-catenin (nWT = 4, nKO = 4) (red). Arrows indicate relative changes in staining. (EG) Bar chart compares the MFI of these inflammatory markers between WT and KO mice in the corneal epithelium (E), corneal stroma (F), and corneal endothelium (G). *P < 0.05, **P < 0.01, two-tailed unpaired t-test.
Figure 4.
 
Immunofluorescence quantitation of inflammatory markers in WT and KO mice corneas. Representative photomicrographs of KO and WT mice retinas show staining patterns of DAPI (blue). (A) TGF-β1 (nWT = 6, nKO = 5); (B) TGF-β2 (nWT = 5, nKO = 5); (C) CTGF (nWT = 3, nKO = 4); (D) β-catenin (nWT = 4, nKO = 4) (red). Arrows indicate relative changes in staining. (EG) Bar chart compares the MFI of these inflammatory markers between WT and KO mice in the corneal epithelium (E), corneal stroma (F), and corneal endothelium (G). *P < 0.05, **P < 0.01, two-tailed unpaired t-test.
Figure 5.
 
Immunofluorescence quantitation of inflammatory markers in WT and KO mice lenses. Representative photomicrographs of KO and WT mice lenses show staining patterns of DAPI (blue). (A) TGF-β1 (nWT = 6, nKO = 5); (B) TGF-β2 (nWT = 5, nKO = 5); (C) CTGF (nWT = 4, nKO = 3); (D) β-catenin (nWT = 3, nKO = 3) (red). Arrows indicate relative changes in staining. (E) Bar chart compares the MFI of these inflammatory markers between WT and KO mice. *P < 0.05, **P < 0.01, two-tailed unpaired t-test.
Figure 5.
 
Immunofluorescence quantitation of inflammatory markers in WT and KO mice lenses. Representative photomicrographs of KO and WT mice lenses show staining patterns of DAPI (blue). (A) TGF-β1 (nWT = 6, nKO = 5); (B) TGF-β2 (nWT = 5, nKO = 5); (C) CTGF (nWT = 4, nKO = 3); (D) β-catenin (nWT = 3, nKO = 3) (red). Arrows indicate relative changes in staining. (E) Bar chart compares the MFI of these inflammatory markers between WT and KO mice. *P < 0.05, **P < 0.01, two-tailed unpaired t-test.
Figure 6.
 
mRNA levels of profibrotic genes in the eye. (A, B) Bar chart shows mRNA levels of the profibrotic genes S100a4, Acta2, Col1a1, Snai1, Snai2, and Twist1, which encode FSP-1, αSMA, collagen (I) α1, Snail, Slug, and Twist1, respectively, relative to Tbp in the retina (A) and the anterior segments (B) (nWT = 4, nKO = 3). *P < 0.05, **P < 0.01, two-tailed unpaired t-test and two-tailed Mann–Whitney U test.
Figure 6.
 
mRNA levels of profibrotic genes in the eye. (A, B) Bar chart shows mRNA levels of the profibrotic genes S100a4, Acta2, Col1a1, Snai1, Snai2, and Twist1, which encode FSP-1, αSMA, collagen (I) α1, Snail, Slug, and Twist1, respectively, relative to Tbp in the retina (A) and the anterior segments (B) (nWT = 4, nKO = 3). *P < 0.05, **P < 0.01, two-tailed unpaired t-test and two-tailed Mann–Whitney U test.
Upregulation of Profibrotic Gene Expression in Col4a3 KO Eyes
TGF-β1, TGF-β2, CTGF, and β-catenin are cytokines known to induce fibrosis in various tissues in vivo.36,37,44,45 To better understand the changes in the distribution of these profibrotic markers in Col4a3 KO eyes, we tested if these changes were at the transcriptional or mRNA level. Therefore, we utilized real-time RT-qPCR to evaluate the expression of a panel of profibrotic genes (Fig. 6Tables 1 and 2). In the retinas of KO mice, there was a significant increase in the transcription of Acta2 (P = 0.0023, 6.8-fold increase normalized to Tbp), Col1a1 (P = 0.0117, 1.8-fold increase), and Snai2 (P = 0.0241, 2.1-fold increase), as well as a nonsignificant increase in the transcription of S100a4 (P = 0.0571, 18.4-fold increase), Snai1 (P = 0.0638, 1.9-fold increase), and Twist1 (P = 0.1143, 3.6-fold increase). In the corneas of KO mice, there was a significant increase in the transcription Snai2 (P = 0.0444, 2.2-fold increase) and a nonsignificant increase in the transcription of S100a4 (P = 0.0600, 2.8-fold increase). 
Table 1.
 
Relative RT-qPCR Gene Expression Changes in the Retina
Table 1.
 
Relative RT-qPCR Gene Expression Changes in the Retina
Table 2.
 
Relative RT-qPCR Gene Expression Changes in the Anterior Segment
Table 2.
 
Relative RT-qPCR Gene Expression Changes in the Anterior Segment
Fibrotic Changes in Col4a3 KO Mice
To determine the downstream fibrotic consequences of an upregulation of profibrotic cytokines and gene expression in Col4a3 KO mice eyes, we utilized picrosirius red staining to visualize collagen deposition (Fig. 7). We found an increase in collagen deposition in the corneal epithelium of KO mice (P = 0.0117), but not the retina, anterior lens, or other corneal substructures (Fig. 7B). This mimics the changes observed in the expression of CTGF and β-catenin in KO mice, implying that these cytokines may, individually or synergistically, play a role in mediating fibrosis in ocular tissue. 
Figure 7.
 
Quantitation of picrosirius red staining in WT and KO mice eyes. (A) Representative photomicrographs were taken at 20× magnification of WT and KO retinas, corneas, and lenses stained with picrosirius red. (B) Bar chart compares the MFI of picrosirius red between WT and KO mice (nWT = 5, nKO = 5). *P < 0.05, **P < 0.01, two-tailed unpaired t-test.
Figure 7.
 
Quantitation of picrosirius red staining in WT and KO mice eyes. (A) Representative photomicrographs were taken at 20× magnification of WT and KO retinas, corneas, and lenses stained with picrosirius red. (B) Bar chart compares the MFI of picrosirius red between WT and KO mice (nWT = 5, nKO = 5). *P < 0.05, **P < 0.01, two-tailed unpaired t-test.
Discussion
In this study, we report that Col4a3 KO mice exhibited distinct profibrotic changes in the retina, cornea, and lens. Together, these defects may contribute to the ocular defects associated with AS. In contrast to findings by Wang et al.,27 H&E staining revealed no evident histological changes in Col4a3 KO mice. However, our study did not utilize TEM to characterize structural changes in Col4a3 KO mice, so microstructural changes may be present in our tissue but not evident via light microscopy. Additionally, although their study observed lenticonus in two animals, we did not observe such changes. Furthermore, they found abnormal ILM structure, thicker and shorter folds of the RPE basement membrane, and thinning of the retina and anterior lens capsule in the KO group, which were not readily observed in our study. Whereas their study was a time course (8–21 weeks) with a smaller sample size (n = 4), our study evaluated mice at 25 weeks of age, with seven mice in each group. Regardless, there is a compelling rationale for studying microscopic changes, including optical coherence tomography on live animals; these studies pave the way for future exploration. 
Our immunofluorescence staining confirmed that the α3 (IV) protein is absent in Col4a3 KO eyes. Previously, Saito et al.33 performed detailed staining of collagen (IV) in ocular anterior segments of adult mice, and Bai et al.32 performed a similar study to understand the developmental distribution of collagen (IV) isoforms in mice eyes. Our studies align with these groups’ findings regarding a lack of collagen (IV) α3 staining in the epithelial basement membrane. Additionally, we also validated our findings using positive control showing staining in the glomerular basement membrane, where collagen (IV) α3 is known to be located. Wang et al.27 reported the presence of Col4a3 mRNA in the cornea, lens, and retina of WT mice; however, there is no information on collagen (IV) α3 distribution. In addition to collagen (IV) α3 protein, we elected to stain Col4a3 KO sections with α4 (IV) protein because it exclusively colocalizes with the α3 (IV) protein as part of the collagen (IV) α3α4α5 heterotrimer; however, we did not observe any difference in KO sections. It is worth noting that the study by Wang et al.27 observed a markedly reduced collagen (IV) α1 expression in KO mice; although there is no precise rationale for studying collagen (IV) α1, perhaps the staining was used to investigate collagen turnover and supplement lenticonus findings in their mouse model. 
Our study demonstrates that the collagen (IV) α3 heterotrimer is localized to Descemet's and Bruch's membranes on immunofluorescence. Descemet's membrane anchors the endothelium to the cornea and thus is important in maintaining corneal integrity.46 Furthermore, Descemet's membrane serves as a barrier that modulates the bidirectional passage of nutrients, cytokines, and growth factors between the aqueous humor and corneal stroma. Abnormalities in Descemet's membrane may impair its ability to modulate the entry of growth factors, particularly TGF-β, into the corneal stroma. Damage to Descemet's membrane has been shown to induce the development and proliferation of posterior myofibroblasts and ultimately lead to fibrosis.47,48 Bruch's membrane is a pentalaminar structure located in between the RPE and choriocapillaris. It provides physical support for the RPE, serves as a barrier between the RPE and choriocapillaris, and regulates the diffusion of molecules between these structures.49 Damage to Bruch's membrane leads to ECM degeneration, accumulation of deposits, and angiogenesis.50 Furthermore, degradation of Bruch's membrane is a prerequisite for neovascular age-related macular degeneration, which may eventually lead to macular fibrosis.51 Savige et al.12,52 reported that Bruch's membrane is thinner in patients with AS, and the impaired nutritional regulation and retinal integrity may contribute to overall retinal thinning, the appearance of a retinal lozenge, and development of bull's eye maculopathy or a macular hole. Thus, we hypothesized that the loss of collagen (IV) α3 in these critical membranes may play a role in the observed inflammatory and profibrotic changes in Col4a3 KO mice. 
Quantitation of MFI of immunofluorescence stains demonstrated an upregulation of TGF-β1 in (P = 0.0570) the anterior lens and TGF-β2 in the retina. TGF-β plays an integral role in the pathogenesis of fibrosis and has been shown to be upregulated and activated in fibrotic conditions.53 TGF-β is perhaps the most important modulator of fibrosis and wound healing in the eye.38,54 TGF-β1 has been shown to increase the expression of profibrotic genes and induce epithelial–mesenchymal transition (EMT) in the lens epithelium.5557 Additionally, the lens-specific expression of TGF-β1 was demonstrated to induce cataracts in transgenic mice.58 However, we did not observe any change in the levels of CTGF or β-catenin, and there was no evidence of fibrosis or development of cataracts. 
TGF-β2 is the predominant isoform in the eye, and the concentration of TGF-β2 has been shown to increase concomitantly with the progression of retinal fibrosis.39 Due to its role as a central mediator of fibrosis, it is not surprising that an elevation of TGF-β2 in the retina is accompanied by an increase in the transcription of the profibrotic genes S100a4, Acta2, Col1a1, Snai1, Snai2, and Twist1, which encode FSP-1, αSMA, collagen (I) α1, Snail, Slug, and Twist1, respectively. FSP-1 and αSMA have been implicated in endothelial to mesenchymal transition (EndMT).59,60 EndMT has been observed in the pathogenesis of fibrotic diseases in various tissues, including the retinas of diabetic patients.59,61 TGF-β signaling pathways are the most important regulators of EndMT in most physiological and pathological conditions, and the EndMT process is mediated by a number of transcriptional regulators, including Snail, Slug, and Twist1.59 Zeisberg et al.60 found that EndMT may substantially contribute to the accumulation of fibroblasts in kidney fibrosis in Col4a3 KO mice. Furthermore, EndMT has been implicated in pathological fibrotic conditions of the retina, including diabetic retinopathy and age-related macular degeneration.62,63 However, despite an elevation of TGF-β2 and increased transcription of profibrotic genes, quantitation of picrosirius red staining showed no difference in collagen deposition between KO and WT mice retinas. Therefore, the increase in TGF-β2 levels and profibrotic gene expression may be insufficient to induce fibrosis in the retina. This study is limited by its cross-sectional nature, as the expression of profibrotic genes may not represent the levels of their protein products. Of translational significance, the distribution of collagen (IV) α3 in the eye differs between mice and humans. In humans, the collagen (IV) α3α4α5 heterotrimer is present in the ILM and Bruch's membrane of the retina and Descemet's membrane and Bowman's membrane of the cornea.3,6 Due to the absence of staining in immunofluorescence in the ILM and Bowman's membrane in our mouse model, these findings may not be generalizable to the retinal pathology of AS in humans and are another limitation of our study. 
In addition to EndMT, TGF-β modulates fibrosis via a combination of its canonical Smad signaling pathway, non-canonical pathways such as activation of various mitogen-activated protein kinases (MAPKs), and crosstalk between various downstream mediators.3638 One such downstream mediator is CTGF, a mitogenic heparin-binding protein that acts on connective tissue cells to stimulate cell proliferation and extracellular matrix synthesis.40,42 Another downstream mediator of TGF-β function is β-catenin, a transcription factor that is translocated to the nucleus in the canonical Wnt signaling pathway, where it serves as an important regulator of fibrosis.41,43 Wu et al.64 demonstrated that TGF-β treatment of old bovine nucleus pulposus cells causes rapid accumulation of β-catenin, which recruits cytoplasmic CTGF to the membrane for secretion to the extracellular matrix, where it induces fibrogenesis. Clearly, there is a significant interplay among TGF-β, CTGF, and β-catenin, and each of these proteins plays an important role in modulating fibrosis. Based on the elevation of TGF-β2 in the retinas of Col4a3 KO mice, we decided to stain for the downstream mediators CTGF and β-catenin. 
Interestingly, we found no change in the levels of CTGF and β-catenin in the retina or lens of KO mice, but an increase in both markers in the corneal epithelium of Col4a3 KO mice despite no change in the level of TGF-β1 or TGF-β2. CTGF levels have been demonstrated to be increased in several pathological fibrotic conditions, including idiopathic pulmonary fibrosis, post-COVID-19 pulmonary fibrosis, hepatic fibrosis, cardiac fibrosis, and renal fibrosis.65 CTGF also plays a critical role in pathological corneal fibrosis. Blalock et al.44 showed that CTGF is induced by TGF-β in the cornea, mediates the effects of TGF-β on collagen synthesis, and is likely a key regulator in corneal wound healing and fibrosis. β-Catenin has also been implicated in a number of fibrotic conditions, and inhibition of β-catenin has been shown to mitigate dermal fibrosis, renal fibrosis, hepatic fibrosis, and fibrosis in the lens and cornea.45,6669 Jeon et al.70 demonstrated that reduction in β-catenin signaling in corneal fibroblasts prevents these cells from adopting a scarring phenotype, implying a fundamental role of Wnt/β-catenin signaling in corneal fibrosis. The mechanism by which CTGF and β-catenin induce fibrosis in ocular pathologies has not been well established. In our study, we found that the upregulation of CTGF and β-catenin in the corneal epithelium of KO mice was accompanied by increased collagen deposition. Furthermore, we found an upregulation in the transcription of S100a4 and Snai2 in the corneas of KO mice. Wang et al.71 found that S100a4 silencing protected rabbit corneal stromal cells from fibrosis, whereas S100a4 overexpression led to a proliferation of corneal fibroblasts. Overexpression of Slug has been shown to mediate EMT in the corneal epithelium.72,73 Therefore, overexpression of these profibrotic genes may be a downstream consequence of elevated CTGF and β-catenin levels and may contribute to fibrosis in the cornea by promoting EMT. However, Snail and Twist1 are also important regulators of EMT in the cornea, and a lack of elevation of Snai1 and Twist1 transcription makes it unclear if EMT is a likely pathological condition in Col4a3 KO mice. Of note, RT-qPCR was performed on RNA extracted from the whole anterior segment, so the measured mRNA levels may not be representative of the corneal epithelium. 
Moreover, there is substantial evidence suggesting that the profibrotic function of CTGF and β-catenin is a consequence of upstream TGF-β signaling.44,64,70,74 An elevation of CTGF and β-catenin levels combined with increased collagen deposition in the corneal epithelium despite no change in TGF-β levels suggests that these cytokines may be able to induce fibrosis independently of TGF-β. On the other hand, an increase in TGF-β2 in the retina without an increase in CTGF or β-catenin suggests that TGF-β may induce the transcription of various profibrotic genes independently of these central downstream mediators. In summary, these findings add to the evidence suggesting that TGF-β, CTGF, and β-catenin function in a complex and tissue-specific manner. 
In conclusion, our studies demonstrate a differential inflammatory and profibrotic response in the eyes of Col4a3 KO mice, which may play a role in ocular pathology observed in AS. Loss of the collagen (IV) α3α4α5 heterotrimer in Bruch's membrane and Descemet's membrane may play a role in these observed profibrotic changes. 
Acknowledgments
The authors thank George Sandusky, PhD, Professor of Pathology and Laboratory Medicine at Indiana University School of Medicine, and his lab manager, Amanda Werner, for their help with the assessment of H&E sections. 
Supported by grants from the National Eye Institute, National Institutes of Health (R01EY027779, R01EY027779-S1, and R01EY032080 to ADB; R01EY029320 to PPP); National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (F30DK129003 to BAJ); and a Challenge Grant from Research to Prevent Blindness to the Department of Ophthalmology. 
Disclosure: A. Belamkar, None; Q. Luo, None; N. Mahajan, None; S. Abhyankar, None; B.A. Jones, None; R.K. Sodhi, None; P.P. Pattabiraman, None; M. Levi, None; A.D. Bhatwadekar, CVS Health/Aetna (F) 
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Figure 1.
 
H&E staining of WT and KO mice eyes. Representative photomicrographs taken at 20× magnification of WT and KO retinas, corneas, and lenses show no evident histological differences between WT and KO mice eyes (n = 7). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PR, photoreceptor layer; RPE, retinal pigment epithelium; ALC, anterior lens capsule; LEC, lens epithelial cells; LF, lens fibers.
Figure 1.
 
H&E staining of WT and KO mice eyes. Representative photomicrographs taken at 20× magnification of WT and KO retinas, corneas, and lenses show no evident histological differences between WT and KO mice eyes (n = 7). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PR, photoreceptor layer; RPE, retinal pigment epithelium; ALC, anterior lens capsule; LEC, lens epithelial cells; LF, lens fibers.
Figure 2.
 
Immunofluorescence of collagen (IV) α3 and collagen (IV) α4 in WT and KO mice eyes. Ocular sections were stained with DAPI (blue) and collagen (IV) α3 (red; n = 5) or collagen (IV) α4 (red, n = 4). (A, B) Representative photomicrographs of α3 (IV) in the retina (A) and α4 (IV) in the retina (B) show a positive staining in the Bruch's membrane of the WT mice. (C, D) Corneal staining of α3 (IV) (C) and α4 (IV) (D) revealed a positive staining in Descemet's membrane, which anchors the endothelium to the rest of the cornea. (E, F) There was no labeling of α3 (IV) (E) or α4 (IV) (F) in the anterior lens of WT mice. We confirmed an absence of both isoforms in KO mice. Arrows indicate regions of positive staining; staining in regions not indicated by arrows is due to nonspecific binding of the secondary antibody.
Figure 2.
 
Immunofluorescence of collagen (IV) α3 and collagen (IV) α4 in WT and KO mice eyes. Ocular sections were stained with DAPI (blue) and collagen (IV) α3 (red; n = 5) or collagen (IV) α4 (red, n = 4). (A, B) Representative photomicrographs of α3 (IV) in the retina (A) and α4 (IV) in the retina (B) show a positive staining in the Bruch's membrane of the WT mice. (C, D) Corneal staining of α3 (IV) (C) and α4 (IV) (D) revealed a positive staining in Descemet's membrane, which anchors the endothelium to the rest of the cornea. (E, F) There was no labeling of α3 (IV) (E) or α4 (IV) (F) in the anterior lens of WT mice. We confirmed an absence of both isoforms in KO mice. Arrows indicate regions of positive staining; staining in regions not indicated by arrows is due to nonspecific binding of the secondary antibody.
Figure 3.
 
Immunofluorescence quantitation of inflammatory markers in WT and KO mice retinas. Representative photomicrographs of KO and WT mice retinas show staining patterns of DAPI (blue). (A) TGF-β1 (nWT = 6, nKO = 6); (B) TGF-β2 (nWT = 5, nKO = 5); (C) CTGF (nWT = 3, nKO = 3); (D) β-catenin (nWT = 4, nKO = 3) (red). Arrows indicate relative changes in staining. (E) Bar chart compares the MFI of these inflammatory markers between WT and KO mice. *P < 0.05, **P < 0.01, two-tailed unpaired t-test.
Figure 3.
 
Immunofluorescence quantitation of inflammatory markers in WT and KO mice retinas. Representative photomicrographs of KO and WT mice retinas show staining patterns of DAPI (blue). (A) TGF-β1 (nWT = 6, nKO = 6); (B) TGF-β2 (nWT = 5, nKO = 5); (C) CTGF (nWT = 3, nKO = 3); (D) β-catenin (nWT = 4, nKO = 3) (red). Arrows indicate relative changes in staining. (E) Bar chart compares the MFI of these inflammatory markers between WT and KO mice. *P < 0.05, **P < 0.01, two-tailed unpaired t-test.
Figure 4.
 
Immunofluorescence quantitation of inflammatory markers in WT and KO mice corneas. Representative photomicrographs of KO and WT mice retinas show staining patterns of DAPI (blue). (A) TGF-β1 (nWT = 6, nKO = 5); (B) TGF-β2 (nWT = 5, nKO = 5); (C) CTGF (nWT = 3, nKO = 4); (D) β-catenin (nWT = 4, nKO = 4) (red). Arrows indicate relative changes in staining. (EG) Bar chart compares the MFI of these inflammatory markers between WT and KO mice in the corneal epithelium (E), corneal stroma (F), and corneal endothelium (G). *P < 0.05, **P < 0.01, two-tailed unpaired t-test.
Figure 4.
 
Immunofluorescence quantitation of inflammatory markers in WT and KO mice corneas. Representative photomicrographs of KO and WT mice retinas show staining patterns of DAPI (blue). (A) TGF-β1 (nWT = 6, nKO = 5); (B) TGF-β2 (nWT = 5, nKO = 5); (C) CTGF (nWT = 3, nKO = 4); (D) β-catenin (nWT = 4, nKO = 4) (red). Arrows indicate relative changes in staining. (EG) Bar chart compares the MFI of these inflammatory markers between WT and KO mice in the corneal epithelium (E), corneal stroma (F), and corneal endothelium (G). *P < 0.05, **P < 0.01, two-tailed unpaired t-test.
Figure 5.
 
Immunofluorescence quantitation of inflammatory markers in WT and KO mice lenses. Representative photomicrographs of KO and WT mice lenses show staining patterns of DAPI (blue). (A) TGF-β1 (nWT = 6, nKO = 5); (B) TGF-β2 (nWT = 5, nKO = 5); (C) CTGF (nWT = 4, nKO = 3); (D) β-catenin (nWT = 3, nKO = 3) (red). Arrows indicate relative changes in staining. (E) Bar chart compares the MFI of these inflammatory markers between WT and KO mice. *P < 0.05, **P < 0.01, two-tailed unpaired t-test.
Figure 5.
 
Immunofluorescence quantitation of inflammatory markers in WT and KO mice lenses. Representative photomicrographs of KO and WT mice lenses show staining patterns of DAPI (blue). (A) TGF-β1 (nWT = 6, nKO = 5); (B) TGF-β2 (nWT = 5, nKO = 5); (C) CTGF (nWT = 4, nKO = 3); (D) β-catenin (nWT = 3, nKO = 3) (red). Arrows indicate relative changes in staining. (E) Bar chart compares the MFI of these inflammatory markers between WT and KO mice. *P < 0.05, **P < 0.01, two-tailed unpaired t-test.
Figure 6.
 
mRNA levels of profibrotic genes in the eye. (A, B) Bar chart shows mRNA levels of the profibrotic genes S100a4, Acta2, Col1a1, Snai1, Snai2, and Twist1, which encode FSP-1, αSMA, collagen (I) α1, Snail, Slug, and Twist1, respectively, relative to Tbp in the retina (A) and the anterior segments (B) (nWT = 4, nKO = 3). *P < 0.05, **P < 0.01, two-tailed unpaired t-test and two-tailed Mann–Whitney U test.
Figure 6.
 
mRNA levels of profibrotic genes in the eye. (A, B) Bar chart shows mRNA levels of the profibrotic genes S100a4, Acta2, Col1a1, Snai1, Snai2, and Twist1, which encode FSP-1, αSMA, collagen (I) α1, Snail, Slug, and Twist1, respectively, relative to Tbp in the retina (A) and the anterior segments (B) (nWT = 4, nKO = 3). *P < 0.05, **P < 0.01, two-tailed unpaired t-test and two-tailed Mann–Whitney U test.
Figure 7.
 
Quantitation of picrosirius red staining in WT and KO mice eyes. (A) Representative photomicrographs were taken at 20× magnification of WT and KO retinas, corneas, and lenses stained with picrosirius red. (B) Bar chart compares the MFI of picrosirius red between WT and KO mice (nWT = 5, nKO = 5). *P < 0.05, **P < 0.01, two-tailed unpaired t-test.
Figure 7.
 
Quantitation of picrosirius red staining in WT and KO mice eyes. (A) Representative photomicrographs were taken at 20× magnification of WT and KO retinas, corneas, and lenses stained with picrosirius red. (B) Bar chart compares the MFI of picrosirius red between WT and KO mice (nWT = 5, nKO = 5). *P < 0.05, **P < 0.01, two-tailed unpaired t-test.
Table 1.
 
Relative RT-qPCR Gene Expression Changes in the Retina
Table 1.
 
Relative RT-qPCR Gene Expression Changes in the Retina
Table 2.
 
Relative RT-qPCR Gene Expression Changes in the Anterior Segment
Table 2.
 
Relative RT-qPCR Gene Expression Changes in the Anterior Segment
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