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Published on: November 12, 2015
Loss of alpha3(IV) collagen expression associated with corneal keratocyte activation
Emily Guerriero1, Jian Chen, Yoshikazu Sado
1UPMC Eye Center, Ophthalmology and Visual Science Research Center, Eye and Ear Institute, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
Purpose:
To determine whether changes in the expression of type IV alpha1, alpha2, or alpha3 collagen isoforms are stringently associated with corneal stromal cell activation.
Methods:
Keratocytes isolated from rabbit corneal stroma by collagenase digestion were plated in serum-free or insulin-, bFGF/heparin sulfate (HS)-, TGF-beta1-, or fetal bovine serum (FBS)-supplemented DMEM/F12 medium. Expression of type IV collagen isoforms and keratan sulfate proteoglycans (KSPGs) was evaluated by immunocytochemical analysis, Western blot analysis, or both. Concentrations of mRNAs were estimated by quantitative RT-PCR using SYBR Green RT-PCR reagents.
Results:
Immunohistochemical analysis indicated that type IV alpha1, alpha2, and alpha3 collagens were expressed in normal rabbit corneal stroma and in keratocytes cultured in serum-free and insulin-supplemented media. However, alpha3(IV) collagen was not detectable in the regenerating stroma after photorefractive keratectomy (PRK) in rabbit or in corneal stromal cells cultured in media supplemented with FBS, bFGF/HS, or TGF-beta1. alpha3(IV) collagen mRNA levels were also diminished in the stromal cells cultured in these growth factor-supplemented media. KSPGs (lumican and keratocan) were expressed and secreted in serum-free medium. Although the expression of KSPGs was promoted by insulin, the expression and intracellular levels of lumican and keratocan mRNAs were downregulated by TGF-beta1 and FBS. bFGF/HS promoted the downregulation of intracellular keratocan but not lumican mRNA levels.
Conclusions:
The loss in the expression of alpha3(IV) collagen is a stringent phenotypic change associated with activation of keratocytes in vivo and in vitro. This phenotypic change in activated corneal stromal cells is induced by bFGF/HS and by TGF-beta1, and it accompanies the downregulation of keratocan expression.
Insights
The expression of alpha3(IV) collagen is lost when corneal stromal cells activate. This change, induced by growth factors like TGF-beta1, is a key marker of cell activation in the cornea.
Area of Science:
- Ophthalmology
- Cell Biology
- Biochemistry
Background:
- Corneal stromal cells, known as keratocytes, maintain the cornea's transparency and structure.
- Cellular activation in the corneal stroma is associated with wound healing and disease, involving phenotypic changes.
- Type IV collagen isoforms are crucial components of basement membranes, but their specific roles in corneal stromal cell activation are not fully understood.
Purpose of the Study:
- To investigate the association between the expression of type IV alpha1, alpha2, and alpha3 collagen isoforms and the activation state of corneal stromal cells.
- To determine if specific growth factors induce changes in these collagen isoforms during cell activation.
Main Methods:
- Keratocytes were isolated from rabbit corneal stroma and cultured in various media conditions, including serum-free, insulin, bFGF/heparin sulfate (HS), TGF-beta1, and fetal bovine serum (FBS).
- Expression of type IV collagen isoforms and keratan sulfate proteoglycans (KSPGs) was assessed using immunocytochemistry and Western blot analysis.
- Messenger RNA (mRNA) levels were quantified using SYBR Green RT-PCR.
Main Results:
- Type IV alpha1, alpha2, and alpha3 collagens were detected in normal corneal stroma and quiescent keratocytes.
- alpha3(IV) collagen expression and mRNA levels were significantly reduced in activated keratocytes cultured with FBS, bFGF/HS, or TGF-beta1, and in regenerating stroma post-PRK.
- Keratan sulfate proteoglycans (lumican and keratocan) were expressed in quiescent cells, with insulin promoting their expression, while TGF-beta1 and FBS downregulated their mRNA levels.
Conclusions:
- Loss of alpha3(IV) collagen expression is a specific marker for corneal stromal cell activation, both in vivo and in vitro.
- Transforming growth factor-beta1 (TGF-beta1) and bFGF/HS are key inducers of this alpha3(IV) collagen downregulation.
- This phenotypic shift is accompanied by altered expression of keratan sulfate proteoglycans, such as keratocan.
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