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Human Keratoconus Cell Contractility is Mediated by Transforming Growth Factor-Beta Isoforms.

Lyon D', McKay TB, Sarkar-Nag A, Priyadarsini S, Karamichos D - J Funct Biomater (2015)

Bottom Line: HKCs showed delayed contractility with decreased Collagen I:Collagen V ratios.We also found that HKCs have significantly decreased Collagen I:Collagen III ratios suggesting a potential link to altered collagen isoform expression in KC.Our findings show that HKCs have significant variations in collagen secretion in a 3D collagen gel and have delayed contraction of the matrix compared to HCFs.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology/Dean McGee Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. desiree-lyon@ouhsc.edu.

ABSTRACT
Keratoconus (KC) is a progressive disease linked to defects in the structural components of the corneal stroma. The extracellular matrix (ECM) is secreted and assembled by corneal keratocytes and regulated by transforming growth factor-β (TGF-β). We have previously identified alterations in the TGF-β pathway in human keratoconus cells (HKCs) compared to normal corneal fibroblasts (HCFs). In our current study, we seeded HKCs and HCFs in 3D-collagen gels to identify variations in contractility, and expression of matrix metalloproteases (MMPs) by HKCs in response the TGF-β isoforms. HKCs showed delayed contractility with decreased Collagen I:Collagen V ratios. TGF-β1 significantly increased ECM contraction, Collagen I, and Collagen V expression by HKCs. We also found that HKCs have significantly decreased Collagen I:Collagen III ratios suggesting a potential link to altered collagen isoform expression in KC. Our findings show that HKCs have significant variations in collagen secretion in a 3D collagen gel and have delayed contraction of the matrix compared to HCFs. For the first time, we utilize a collagen gel model to characterize the contractility and MMP expression by HKCs that may contribute to the pathobiology of KC.

No MeSH data available.


Related in: MedlinePlus

Quantification of the contraction of the collagen matrix in HCFs and HKCs from day 0 to 26. (A) control, (B) TGF-β1, (C) TGF-β2, and (D) TGF-β3 samples. A significant reduction in area of the collagen matrix correlates with increased contractility. Rate of contraction from day 0 to day 26 for (E) HCFs and (F) HKCs. n = 3, error bars represent standard error of the mean (SEM). (**** denotes p < 0.0001, *** denotes p < 0.001, ** denotes p < 0.01, and * denotes p < 0.05.)
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jfb-06-00422-f002: Quantification of the contraction of the collagen matrix in HCFs and HKCs from day 0 to 26. (A) control, (B) TGF-β1, (C) TGF-β2, and (D) TGF-β3 samples. A significant reduction in area of the collagen matrix correlates with increased contractility. Rate of contraction from day 0 to day 26 for (E) HCFs and (F) HKCs. n = 3, error bars represent standard error of the mean (SEM). (**** denotes p < 0.0001, *** denotes p < 0.001, ** denotes p < 0.01, and * denotes p < 0.05.)

Mentions: In order to define the role of the surrounding matrix on contractility, we utilized a pre-assembled 3D collagen gel with seeded HKCs and measured rate of contraction compared to normal HCFs. We measured changes in the area of the gel matrix biweekly for 4 weeks in control, TGF-β1, -2, and -3 treated samples using light microscopy, as shown in Figure 1 for representative control samples. At day 1, we identified a 57 mm2 (16%) reduction in gel area by HCF controls compared to a 12 mm2 reduction (7%) in matrix area in HKCs (Figure 2A, p < 0.0001). By day 12, HCFs had contracted the matrix at an average rate of 20 mm2/day compared to a contraction rate of 15 mm3/day by HKCs (Figure 2E,F). The initial delay in contractility by HKCs corresponded to an incremental delay in shrinkage of the matrix area compared to HCFs, both of which reached maximal contraction by day 26. However, the average rate of contraction from day 0 to day 26 were comparable between HCFs and HKCs (10 mm2/day and 9 mm2/day, respectively) showing that though HKCs have an initial delayed contractility compared to HCFs, the KC cells eventually reach similar HCF average contraction rate (Figure 2E,F).


Human Keratoconus Cell Contractility is Mediated by Transforming Growth Factor-Beta Isoforms.

Lyon D', McKay TB, Sarkar-Nag A, Priyadarsini S, Karamichos D - J Funct Biomater (2015)

Quantification of the contraction of the collagen matrix in HCFs and HKCs from day 0 to 26. (A) control, (B) TGF-β1, (C) TGF-β2, and (D) TGF-β3 samples. A significant reduction in area of the collagen matrix correlates with increased contractility. Rate of contraction from day 0 to day 26 for (E) HCFs and (F) HKCs. n = 3, error bars represent standard error of the mean (SEM). (**** denotes p < 0.0001, *** denotes p < 0.001, ** denotes p < 0.01, and * denotes p < 0.05.)
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493522&req=5

jfb-06-00422-f002: Quantification of the contraction of the collagen matrix in HCFs and HKCs from day 0 to 26. (A) control, (B) TGF-β1, (C) TGF-β2, and (D) TGF-β3 samples. A significant reduction in area of the collagen matrix correlates with increased contractility. Rate of contraction from day 0 to day 26 for (E) HCFs and (F) HKCs. n = 3, error bars represent standard error of the mean (SEM). (**** denotes p < 0.0001, *** denotes p < 0.001, ** denotes p < 0.01, and * denotes p < 0.05.)
Mentions: In order to define the role of the surrounding matrix on contractility, we utilized a pre-assembled 3D collagen gel with seeded HKCs and measured rate of contraction compared to normal HCFs. We measured changes in the area of the gel matrix biweekly for 4 weeks in control, TGF-β1, -2, and -3 treated samples using light microscopy, as shown in Figure 1 for representative control samples. At day 1, we identified a 57 mm2 (16%) reduction in gel area by HCF controls compared to a 12 mm2 reduction (7%) in matrix area in HKCs (Figure 2A, p < 0.0001). By day 12, HCFs had contracted the matrix at an average rate of 20 mm2/day compared to a contraction rate of 15 mm3/day by HKCs (Figure 2E,F). The initial delay in contractility by HKCs corresponded to an incremental delay in shrinkage of the matrix area compared to HCFs, both of which reached maximal contraction by day 26. However, the average rate of contraction from day 0 to day 26 were comparable between HCFs and HKCs (10 mm2/day and 9 mm2/day, respectively) showing that though HKCs have an initial delayed contractility compared to HCFs, the KC cells eventually reach similar HCF average contraction rate (Figure 2E,F).

Bottom Line: HKCs showed delayed contractility with decreased Collagen I:Collagen V ratios.We also found that HKCs have significantly decreased Collagen I:Collagen III ratios suggesting a potential link to altered collagen isoform expression in KC.Our findings show that HKCs have significant variations in collagen secretion in a 3D collagen gel and have delayed contraction of the matrix compared to HCFs.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology/Dean McGee Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. desiree-lyon@ouhsc.edu.

ABSTRACT
Keratoconus (KC) is a progressive disease linked to defects in the structural components of the corneal stroma. The extracellular matrix (ECM) is secreted and assembled by corneal keratocytes and regulated by transforming growth factor-β (TGF-β). We have previously identified alterations in the TGF-β pathway in human keratoconus cells (HKCs) compared to normal corneal fibroblasts (HCFs). In our current study, we seeded HKCs and HCFs in 3D-collagen gels to identify variations in contractility, and expression of matrix metalloproteases (MMPs) by HKCs in response the TGF-β isoforms. HKCs showed delayed contractility with decreased Collagen I:Collagen V ratios. TGF-β1 significantly increased ECM contraction, Collagen I, and Collagen V expression by HKCs. We also found that HKCs have significantly decreased Collagen I:Collagen III ratios suggesting a potential link to altered collagen isoform expression in KC. Our findings show that HKCs have significant variations in collagen secretion in a 3D collagen gel and have delayed contraction of the matrix compared to HCFs. For the first time, we utilize a collagen gel model to characterize the contractility and MMP expression by HKCs that may contribute to the pathobiology of KC.

No MeSH data available.


Related in: MedlinePlus