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Albert Lee, Dimitris Karamichos, Celeste Rich, James D. Zieske, Vickery Trinkaus-Randall; Hypoxia Modulates the Expression of Extracellular Matrix Proteins in a 3-D Primary Human Fibroblast Construct. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3425.
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Previously, we demonstrated that 3-dimensional multilayer constructs formed using primary human corneal fibroblasts deposited an organized extracellular matrix (ECM) composed of collagens and proteoglycans. Here we used the construct to test our hypothesis that hypoxia is an early event in corneal pathology, which leads to changes in the ECM. Our goal was to determine how the matrix was altered at the transcriptional and translational level.
Constructs were cultured for 1, 2 and 4 weeks. At each end time point the constructs were incubated under normoxic or hypoxic conditions (1% O2) for 48 hours. The expression of ECM proteins was analyzed by immunofluorescence, confocal microscopy and real-time PCR. Total RNA was extracted from the construct, cDNA synthesized, and real-time analysis performed using TaqMan probes. Data analysis used the ddCt method to compare gene expression. Values are given as mean +/- SEM with statistical comparisons made using Student’s t test (P<0.05). All samples were performed in triplicate.
Short-term exposure to hypoxia did not change the integrity or thickness of the construct; however the organization of actin filaments was lessened. The decrease in oxygen resulted in no detectable change in mRNA or protein expression of smooth muscle cell alpha-actin at any time point. However immunofluorescence revealed a significant decrease in Type III collagen expression under hypoxic conditions when compared to normoxic conditions. In addition, mRNA expression was decreased in agreement with immunofluoresence data. In contrast Types I and V did not change. Furthermore, the expression of proteoglycans was significantly altered under hypoxia; with a decrease in the mRNA of Small Leucine-Rich repeats (SLRPs), (lumican, decorin and keratacan) and an increase in perlecan mRNA expression. Moreover, mRNA expression of lysyl oxidase (LOX), a potential marker of apoptosis, was significantly increased under hypoxic conditions at all times.
Our results demonstrated that hypoxia rapidly alters the expression of ECM components in 3-D stromal constructs. This may be related to the upregulation of LOX expression. The dramatic change in Type III collagen and specific proteoglycans suggest that hypoxia has the ability to rapidly modify the stromal matrix. Together these suggest that ECM alterations may lead to potential changes in interactions between growth factors and ECM molecules, potentially leading to degradation of the corneal matrix.
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