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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

(A) Collagen I (Col I), (B) Collagen III (Col III), and (C) Collagen V (Col V) secretion measured from conditioned media by Western blot from week 1 to week 4. Data reported as ratios of (D) Col I/Col V and (E) Col I/Col III. n = 3. Error bars represent standard error of the mean. (*** denotes p < 0.001, ** denotes p < 0.01, and * denotes p < 0.05.)
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jfb-06-00422-f003: (A) Collagen I (Col I), (B) Collagen III (Col III), and (C) Collagen V (Col V) secretion measured from conditioned media by Western blot from week 1 to week 4. Data reported as ratios of (D) Col I/Col V and (E) Col I/Col III. n = 3. Error bars represent standard error of the mean. (*** denotes p < 0.001, ** denotes p < 0.01, and * denotes p < 0.05.)

Mentions: Corneal ECM organization and composition provides the structural, mechanical, and physiochemical properties that define the integrity and function of the tissue. KC is characterized by a thin corneal stroma that leads to corneal protrusion and disruption of visual acuity. The major components of the corneal stroma include collagen fibrils and the resident cell, corneal keratocytes, which secrete and assemble the surrounding matrix. The TGF-β pathway is a primary regulator of ECM production by stromal keratocytes. Several studies have identified significant defects in TGF-β signaling and ECM composition [9,43,50,51]. Col I is the dominant structural component of the corneal stroma [52]. Col V is a known regulator of collagen fibrillogenesis and is present at 20% of total collagen composition within the cornea [26,53], whereas Col III is not normally expressed in the uninjured cornea [54,55]. In order to determine the effect of the 3D-collagen gel on ECM secretion by HCFs and HKCs, we measured the amount of Col I, Col III, and Col V secreted into the media by HCFs and HKCs (Figure 3A–C). Basal secretion of Col I was reduced in HKCs by 12% compared to HCFs (Figure 3A). TGF-β1, -2, and -3 increased Col I secretion by 32%, 35%, and 52%, respectively, in HCFs, compared to an increase of 51%, 17%, and 17% by HKCs, respectively (Figure 3A, p < 0.05). Col III secretion did not increase significantly in HCFs following treatment with the TGF-β isoform, while HKCs showed increased Col III secretion by 49% following TGF-β2 stimulation (Figure 3B, p < 0.05). Col V secretion was not significantly different between the two cell types with or without TGF-β treatment suggesting a significant role for Col I and III regulation between HCFs and HKCs (Figure 3C).


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)

(A) Collagen I (Col I), (B) Collagen III (Col III), and (C) Collagen V (Col V) secretion measured from conditioned media by Western blot from week 1 to week 4. Data reported as ratios of (D) Col I/Col V and (E) Col I/Col III. n = 3. Error bars represent standard error of the mean. (*** 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-f003: (A) Collagen I (Col I), (B) Collagen III (Col III), and (C) Collagen V (Col V) secretion measured from conditioned media by Western blot from week 1 to week 4. Data reported as ratios of (D) Col I/Col V and (E) Col I/Col III. n = 3. Error bars represent standard error of the mean. (*** denotes p < 0.001, ** denotes p < 0.01, and * denotes p < 0.05.)
Mentions: Corneal ECM organization and composition provides the structural, mechanical, and physiochemical properties that define the integrity and function of the tissue. KC is characterized by a thin corneal stroma that leads to corneal protrusion and disruption of visual acuity. The major components of the corneal stroma include collagen fibrils and the resident cell, corneal keratocytes, which secrete and assemble the surrounding matrix. The TGF-β pathway is a primary regulator of ECM production by stromal keratocytes. Several studies have identified significant defects in TGF-β signaling and ECM composition [9,43,50,51]. Col I is the dominant structural component of the corneal stroma [52]. Col V is a known regulator of collagen fibrillogenesis and is present at 20% of total collagen composition within the cornea [26,53], whereas Col III is not normally expressed in the uninjured cornea [54,55]. In order to determine the effect of the 3D-collagen gel on ECM secretion by HCFs and HKCs, we measured the amount of Col I, Col III, and Col V secreted into the media by HCFs and HKCs (Figure 3A–C). Basal secretion of Col I was reduced in HKCs by 12% compared to HCFs (Figure 3A). TGF-β1, -2, and -3 increased Col I secretion by 32%, 35%, and 52%, respectively, in HCFs, compared to an increase of 51%, 17%, and 17% by HKCs, respectively (Figure 3A, p < 0.05). Col III secretion did not increase significantly in HCFs following treatment with the TGF-β isoform, while HKCs showed increased Col III secretion by 49% following TGF-β2 stimulation (Figure 3B, p < 0.05). Col V secretion was not significantly different between the two cell types with or without TGF-β treatment suggesting a significant role for Col I and III regulation between HCFs and HKCs (Figure 3C).

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