Methodology

Effect of Collagen Fiber Structure on Cell-Matrix Interactions

Benjamin Albert1, Jeffrey Tonniges2, Gunjan Agarwal1,3

1Biomedical Engineering, 2Biophysics, The Ohio State University, 3Davis Heart and Lung Research

Institute

Introduction Results Conclusions

Future Work

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AcknowledgementsSpecial thanks to Dr. Gunjan Agarwal, Jeff Tonniges, and David Yeung.

Work on this project was supported by the Choose Ohio First for Bioinformatics research scholarship and the OSU Second Year Transformational Experience Program

Collagen is the main structural biomaterial of many different types of human tissue. It plays a significant role in the mechanical properties of tissue. Discoidin Domain Receptor 1 (DDR1) is a cell surface protein that binds to collagen in the extracellular matrix.

Collagen is very prevalent in the adventitial layer of the aorta, the main artery of the body. The adventitial layer plays an important role in maintaining shape and elasticity of the aorta. Changes in collagen structure can have adverse effects. Previous studies in my mentor’s lab have shown that DDR1 modulates collagen structure in-vitro (Flynn et. al., 2010).

Glycoprotein VI (GPVI) is a blood platelet receptor protein that is known to bind to collagen. Platelet binding to collagen is one of the mechanisms that can lead to several different cardiovascular pathologies such as thrombosis.

Aims: Determine the effect of DDR1 on collagen ultrastructure and GPVI binding in-vivo

Collagen has a characteristic banded periodicity that is visible in AFM imaging. This periodicity results from the arrangement and overlap of collagen fibrils that bind together to form fibers. The result of this binding pattern is the formation of “peaks” and “troughs” along the backbone of each collagen fiber. The depth of the troughs in each of the periods were measured using AFM image analysis software. The measurements for the two groups of mice were compared through a Student’s t-test and it was shown that the KO mice had a significantly deeper trough than the WT mice .

Amplitude (A and B) and height (C and D)images taken of adventitial collagen in mouse aorta through atomic force microscopy. Profile view of depth of periodicity along collagen backbone (C and D insets). Box and whisker plot of depth measurements (E). Geometrical analysis of the periodicity (F).

Six month old DDR1 Knockout (KO) mice and their wild type (WT) littermates were used for this study. The KO genotype was generated by Lexicon Pharmaceuticals using exon replacement methods.

For atomic force microscopy (AFM), sections were frozen in a substrate and cryosectioned onto prepared, glass coverslips. AFM imaging was done in tapping mode and analyzed in Nanoscope Analysis image processing software.

In-vitro collagen was prepared polymerizing harvested bovine collagen. Collagen was incubated overnight at 37⁰C to allow the fibers to form. GPVI was diluted from a stock solution. In-vitro samples were spread onto freshly cleaved mica, dip washed in water, and left to dry overnight before imaging.

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GPVI was imaged on mica by itself through AFM. The result shows the relative structure of the protein so that it can be compared to the images of GPVI binding to fibrillar collagen. GPVI is known to dimerize and tetromerize when in proximity with itself. This may explain the degree of clumping shown in the image.Polymerized collagen and GPVI were incubated together and imaged. Binding of the protein to the backbone of collagen was visible at an image size of 1 micron.

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E

AFM amplitude image of GPVI on mica (A) . AFM height image of same region (B). Arrows show possible oligomerization of the proteins on both images. Binding of GPVI to polymerized collagen fiber (C). Arrows show GPVI binding sites along the collagen backbone.

These experiments combine to set a base for future experiments involving the differences in GPVI binding to collagen binding to KO and WT mice. The establishment of a protocol to show the binding of GPVI to collagen was a very important step. Now we may be able to form binding in KO and WT collagen fibers in-vitro. Hopefully, the results will build upon themselves and allow imaging of the binding in in-vivo adventitial collagen.

The results of the depth of periodicity experiment show that there is an increased depth in the periodicity of KO mice. Other imaging analysis in the lab of KO and WT collagen show that KO mice have a significantly smaller length of periodicity. When considered together, these results seem to show that the overall collagen structure in KO mice is compressed compared to WT. This may mean that the presence of DDR1 changes the way that fibrils bind together to form full fibers.

Simplified triple helix structure of collagen

The visible binding of GPVI to polymerized collagen allows us to now move on to samples that represent collagen from KO and WT mice. Any significant difference s in the binding of GPVI to the collagen would confirm the altered structure of collagen and also show that the presence of DDR1 may have a significant effect on platelet binding.

Platelet binding to aortic collagen can cause very important changes to the flow of blood throughout the body. Vascular injury is a significant pathology that uses this platelet aggregation to its advantage, and individuals with decreased GPVI binding may be less likely to heal properly.

Atomic force microscopysample analysis

DDR1 Protein