TY - JOUR
T1 - Hypoxia modulates the development of a corneal stromal matrix model
AU - Lee, Albert
AU - Karamichos, Dimitrios
AU - Onochie, Obianamma E.
AU - Hutcheon, Audrey E.K.
AU - Rich, Celeste B.
AU - Zieske, James D.
AU - Trinkaus-Randall, Vickery
N1 - Funding Information:
This work was supported by the National Institutes of Health [ EY06000 (V.T-R), [ S-EY06000 (O.O./V.T-R)], Massachusetts Lions Eye Research Fund and the New England Corneal Transplant Fund to Boston University ; National Institutes of Health [ EY05665 (J.D.Z.)] and Grant P30 EY03790 (Core at Schepens).
Funding Information:
We thank YoonJoo Lee for assistance with imaging and the Boston University School of Medicine Confocal Facility. In addition, we acknowledge the use of tissues procured by the National Disease Research Interchange (NDRI) with support from NIH grant 2 U42 OD011158 .
Publisher Copyright:
© 2018 Elsevier Ltd
PY - 2018/5
Y1 - 2018/5
N2 - Deposition of matrix proteins during development and repair is critical to the transparency of the cornea. While many cells respond to a hypoxic state that can occur in a tumor, the cornea is exposed to hypoxia during development prior to eyelid opening and during the diurnal sleep cycle where oxygen levels can drop from 21% to 8%. In this study, we used 2 three-dimensional (3-D) models to examine how stromal cells respond to periods of acute hypoxic states. The first model, a stromal construct model, is a 3-D stroma-like construct that consists of human corneal fibroblasts (HCFs) stimulated by a stable form of ascorbate for 1, 2, and 4 weeks to self-assemble their own extracellular matrix. The second model, a corneal organ culture model, is a corneal wound-healing model, which consists of wounded adult rat corneas that were removed and placed in culture to heal. Both models were exposed to either normoxic or hypoxic conditions for varying time periods, and the expression and/or localization of matrix proteins was assessed. No significant changes were detected in Type V collagen, which is associated with Type I collagen fibrils; however, significant changes were detected in the expression of both the small leucine-rich repeating proteoglycans and the larger heparan sulfate proteoglycan, perlecan. Also, hypoxia decreased both the number of Cuprolinic blue-positive glycosaminoglycan chains along collagen fibrils and Sulfatase 1, which modulates the effect of heparan sulfate by removing the 6-O-sulfate groups. In the stromal construct model, alterations were seen in fibronectin, similar to those that occur in development and after injury. These changes in fibronectin after injury were accompanied by changes in proteoglycans. Together these findings indicate that acute hypoxic changes alter the physiology of the cornea, and these models will allow us to manipulate the conditions in the extracellular environment in order to study corneal development and trauma.
AB - Deposition of matrix proteins during development and repair is critical to the transparency of the cornea. While many cells respond to a hypoxic state that can occur in a tumor, the cornea is exposed to hypoxia during development prior to eyelid opening and during the diurnal sleep cycle where oxygen levels can drop from 21% to 8%. In this study, we used 2 three-dimensional (3-D) models to examine how stromal cells respond to periods of acute hypoxic states. The first model, a stromal construct model, is a 3-D stroma-like construct that consists of human corneal fibroblasts (HCFs) stimulated by a stable form of ascorbate for 1, 2, and 4 weeks to self-assemble their own extracellular matrix. The second model, a corneal organ culture model, is a corneal wound-healing model, which consists of wounded adult rat corneas that were removed and placed in culture to heal. Both models were exposed to either normoxic or hypoxic conditions for varying time periods, and the expression and/or localization of matrix proteins was assessed. No significant changes were detected in Type V collagen, which is associated with Type I collagen fibrils; however, significant changes were detected in the expression of both the small leucine-rich repeating proteoglycans and the larger heparan sulfate proteoglycan, perlecan. Also, hypoxia decreased both the number of Cuprolinic blue-positive glycosaminoglycan chains along collagen fibrils and Sulfatase 1, which modulates the effect of heparan sulfate by removing the 6-O-sulfate groups. In the stromal construct model, alterations were seen in fibronectin, similar to those that occur in development and after injury. These changes in fibronectin after injury were accompanied by changes in proteoglycans. Together these findings indicate that acute hypoxic changes alter the physiology of the cornea, and these models will allow us to manipulate the conditions in the extracellular environment in order to study corneal development and trauma.
KW - Confocal fluorescence microscopy
KW - Cornea
KW - Corneal organ culture
KW - Extracellular matrix
KW - Stroma
UR - http://www.scopus.com/inward/record.url?scp=85042635574&partnerID=8YFLogxK
U2 - 10.1016/j.exer.2018.02.021
DO - 10.1016/j.exer.2018.02.021
M3 - Article
C2 - 29496505
AN - SCOPUS:85042635574
SN - 0014-4835
VL - 170
SP - 127
EP - 137
JO - Experimental Eye Research
JF - Experimental Eye Research
ER -