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D.C. Paik, L.Y. Saito, J.W. Holmes; Non–Enzymatic Nitration Alters Remodeling and Mechanical Properties of Collagenous Engineered Tissues . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3235.
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© ARVO (1962-2015); The Authors (2016-present)
Reactions that result in the age–related accumulation of increasingly insoluble, undigestible collagen in human Bruch's membrane (BM) are only partially known. Non–enzymatic glycation is one such process and has been linked to diabetic–related complications and aging. A novel mechanism particularly relevant to smoking– and inflammation–related processes is non–enzymatic nitration (NEN). BM contains meshwork collagen IV in RPE and choriodal basement membranes as well as fibrillar collagen I in the inner and outer collagen layers. We have recently shown that NEN of basement membrane proteins can impart deleterious effects on overlying RPE cells. The present study was undertaken in order to examine the effects of NEN of fibrillar collagen I on cell–mediated remodeling and biomechanical properties of tissues.
We used an engineered tissue analog (previously developed by our group) for studying the remodeling and mechanics of collagenous tissues. Adult rat cardiac fibroblasts were harvested and seeded into collagen type I gels (1.92 mg/ml) at a cell density of 200,000 cells/ml. The collagen was reacted with sodium nitrite (0–100mM) either pre– or post– fibroblast seeding. The gels were allowed to polymerize and anchor to porous polyethylene bars which were used for biomechanical testing as well as uni– or biaxial constraints during remodeling. TiO4 paint was spotted into the central region of the gel and was used to monitor changes in gel contraction during remodeling as well as changes in gel deformation related to mechanical loading tests. Confocal reflectance microscopy was used to determine changes in collagen fiber orientation.
Pre–treatment with 100mM NaNO2 reduced vertical remodeling strain by 16% in uniaxial, horizontally constrained gels but did not measurably alter the resulting collagen fiber alignment. In the same gels, NEN reduced vertical deformation during equibiaxial loading, shifting the load–strain curves leftward and significantly reducing vertical strain at 1g equibiaxial load. Unexpectedly, NEN also reduced the amount of permanent vertical and horizontal deformation that resulted from preconditioning prior to mechanical testing.
The results show that NEN impairs both cell–mediated remodeling and mechanical deformability in collagenous engineered tissues, indicating that these reactions stiffen tissues. Thus, NEN may contribute to alterations in the biomechanical properties of collagen containing tissues. These findings have relevance to AMD since type I collagen is a major component of human Bruch's membrane.
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