TY - JOUR
T1 - Transforming Growth Factor Beta 3 Modifies Mechanics and Composition of Extracellular Matrix Deposited by Human Trabecular Meshwork Cells
AU - Raghunathan, Vijay Krishna
AU - Morgan, Joshua T.
AU - Chang, Yow Ren
AU - Weber, Darren
AU - Phinney, Brett
AU - Murphy, Christopher J
AU - Russell, Paul
PY - 2015/2/9
Y1 - 2015/2/9
N2 - Pseudoexfoliation syndrome is a systemic disorder of the extracellular matrix (ECM) with ocular manifestations in the form of chronic open angle glaucoma. Elevated levels of TGFβ3 in the aqueous humor of individuals with pseudoexfoliation glaucoma (PEX) have been reported. The influence of TGFβ3 on the biochemical composition and biomechanics of ECM of human trabecular meshwork (HTM) cells was investigated. HTM cells from eye bank donor eyes were isolated, plated on aminosilane functionalized glass substrates and cultured in the presence or absence of 1 ng/mL TGFβ3 for 4 weeks. After incubation, samples were decellularized and decellularization was verified by immunostaining. The mechanics of the remaining ECM that was deposited by the treated or the control cells were measured by atomic force microscopy (AFM). Imaged by AFM, the surface features of the ECM from both sets of samples had a similar roughness/topography (as determined by RMS values) suggesting surface features of the ECM were similar in both cases; however, the ECM from the HTM cells treated with TGFβ3 was between 3- and 5-fold stiffer than that produced by the control HTM cells. Proteins present in the ECM were solubilized and analyzed using liquid chromatography tandem mass spectroscopy (LC-MS/MS). Data indicate that multiple proteins previously reported to be altered in glaucoma were changed in the ECM as a result of the presence of TGFβ3, including inhibitors of the BMP and Wnt signaling pathways. Gremlin1and 4, SERPINE1 and 2, periostin, secreted frizzled related protein (SFRP) 1 and 4, and ANGPTL4 were among those proteins that were overexpressed in the ECM after TGFβ3 treatment.
AB - Pseudoexfoliation syndrome is a systemic disorder of the extracellular matrix (ECM) with ocular manifestations in the form of chronic open angle glaucoma. Elevated levels of TGFβ3 in the aqueous humor of individuals with pseudoexfoliation glaucoma (PEX) have been reported. The influence of TGFβ3 on the biochemical composition and biomechanics of ECM of human trabecular meshwork (HTM) cells was investigated. HTM cells from eye bank donor eyes were isolated, plated on aminosilane functionalized glass substrates and cultured in the presence or absence of 1 ng/mL TGFβ3 for 4 weeks. After incubation, samples were decellularized and decellularization was verified by immunostaining. The mechanics of the remaining ECM that was deposited by the treated or the control cells were measured by atomic force microscopy (AFM). Imaged by AFM, the surface features of the ECM from both sets of samples had a similar roughness/topography (as determined by RMS values) suggesting surface features of the ECM were similar in both cases; however, the ECM from the HTM cells treated with TGFβ3 was between 3- and 5-fold stiffer than that produced by the control HTM cells. Proteins present in the ECM were solubilized and analyzed using liquid chromatography tandem mass spectroscopy (LC-MS/MS). Data indicate that multiple proteins previously reported to be altered in glaucoma were changed in the ECM as a result of the presence of TGFβ3, including inhibitors of the BMP and Wnt signaling pathways. Gremlin1and 4, SERPINE1 and 2, periostin, secreted frizzled related protein (SFRP) 1 and 4, and ANGPTL4 were among those proteins that were overexpressed in the ECM after TGFβ3 treatment.
KW - atomic force microscopy
KW - biomechanics
KW - extracellular matrix
KW - glaucoma
KW - pseudoexfoliation syndrome
KW - trabecular meshwork
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U2 - 10.1021/ab500060r
DO - 10.1021/ab500060r
M3 - Article
AN - SCOPUS:84969190216
VL - 1
SP - 110
EP - 118
JO - ACS Biomaterials Science and Engineering
JF - ACS Biomaterials Science and Engineering
SN - 2373-9878
IS - 2
ER -