Collagen Fibril Ultrastructure in Mice Lacking Discoidin Domain Receptor 1

Abstract

The quantity and quality of collagen fibrils in the extracellular matrix (ECM) have a pivotal role in dictating biological processes. Several collagen-binding proteins (CBPs) are known to modulate collagen deposition and fibril diameter. However, limited studies exist on alterations in the fibril ultrastructure by CBPs. In this study, we elucidate how the collagen receptor, discoidin domain receptor 1 (DDR1) regulates the collagen content and ultrastructure in the adventitia of DDR1 knock-out (KO) mice. DDR1 KO mice exhibit increased collagen deposition as observed using Masson's trichrome. Collagen ultrastructure was evaluated in situ using transmission electron microscopy, scanning electron microscopy, and atomic force microscopy. Although the mean fibril diameter was not significantly different, DDR1 KO mice had a higher percentage of fibrils with larger diameter compared with their wild-type littermates. No significant differences were observed in the length of D-periods. In addition, collagen fibrils from DDR1 KO mice exhibited a small, but statistically significant, increase in the depth of the fibril D-periods. Consistent with these observations, a reduction in the depth of D-periods was observed in collagen fibrils reconstituted with recombinant DDR1-Fc. Our results elucidate how DDR1 modulates collagen fibril ultrastructure in vivo, which may have important consequences in the functional role(s) of the underlying ECM.

Keywords: aorta; atomic force microscopy; collagen; discoidin domain receptor 1; electron microscopy.

Figure 1

Genotyping of discoidin domain receptor 1 (DDR1) knock-out (KO) mice. a: Schematic shows generation of the DDR1 KO mouse through homologous recombination, by replacing exons 1–3 with a LacZ/Neo cassette. The genotype of the mice was determined using polymerase chain reaction of tail lysates. Presence of DDR1, Neo3a, or both alleles indicated homozygous positive (DDR1^+/+), homozygous negative (DDR1^−/−), and heterozygous (DDR1^+/−) mice, respectively. b: Brain, skin, and heart extracted from DDR1 KO mice exhibited strong x-gal staining, whereas wild-type (WT) tissues did not. The brain and heart have been cut in half to illustrate staining. Aortic longitudinal sections, viewed with white light microscopy from the luminal side, revealed punctate x-gal staining in DDR1 KO aortas (black arrows), whereas no staining was observed in WT aortas. Scale bar is 30 µm.

Figure 2

Schematic illustrating geometric analysis of collagen fibril profiles from atomic force microscopy height images to determine the length and depth of D-periodicity. This geometric analysis accounts for fibril inclinations departing from the horizontal plane. The lengths (a´, b´, and a, b) and angles (α₁, α₂), corresponding to the width and height of an individual D-period were directly measured using WSxM software. The segment c and angle, α₃, in red were calculated. The length of a D-period (bold black line) was calculated using a´ and cosα₁. The D-period depth (bold blue line) was defined as the line perpendicular to D-period length and extending up to the highest point in the profile for the overlap. The depth was calculated as c sin α₃.

Figure 3

Deletion of discoidin domain receptor 1 (DDR1) enhanced collagen deposition in aortic adventitia. Masson’s trichrome staining shows adventitial collagen (blue stain) in the thoracic aorta from (a) wild-type (WT) and (b) DDR1 knock-out (KO) mice. Brackets indicate the adventitial (red) and medial (green) layers. c: The mean adventitial thickness for each genotype is displayed in boxplots. Average of the means is indicated by a dot. The adventitial thickness for DDR1 KO aortas was significantly greater than the WT (p = 0.039). Scale bar is 100 µm.

Figure 4

Collagen fibril diameter distribution in the mouse tunica adventitia. Representative transmission electron microscopy images of typical collagen fibril cross-sections from (a) discoidin domain receptor 1 (DDR1) knock-out (KO) and (b) wild-type (WT) are displayed. In addition to regular fibrils, both genotypes exhibited large irregular-shaped collagen fibrils (c,d), which made up <10% of the total fibrils measured and were only observed near the adventitia-media interface. Scale bar is 100 nm. e: Average percentage of fibrils in each diameter range across eight mice of each genotype. The χ² test showed that the percent of fibrils >50 nm was significantly higher in the DDR1 KO mice (p < 0.0001).

Figure 5

Length of D-periods in collagen fibrils from discoidin domain receptor 1 (DDR1) knock-out (KO) mice examined by atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Representative AFM (amplitude), SEM, and TEM images of adventitial collagen fibrils from DDR1 KO and wild-type (WT) aortas are displayed. Higher magnification images are shown as insets. Boxplots are shown for each imaging technique. Scale bar is 225 nm (inset scale bars are 100 nm).

Figure 6

Increased D-period depth in collagen fibrils from discoidin domain receptor 1 (DDR1) knock-out (KO) mice. Representative atomic force microscopy topographic images from the adventitia of DDR1 KO and wild-type (WT) aortas show collagen fibrils. Section profiles, measured along the axis of the fibril, are shown next to the corresponding image. Boxplots exhibit mean depth of D-periodicity for each genotype. Collagen fibrils from DDR1 KO mice exhibited a significantly greater depth compared with WT (p = 0.030).

Figure 7

Representative atomic force microscopy (AFM) topographic images of collagen fibrils reconstituted in the absence and presence of discoidin domain receptor 1 (DDR1)-Fc. Collagen fibrils with DDR1-Fc exhibited no banded structure or measurable D-period depth. Line profiles of a collagen fibril are displayed adjacent to the corresponding AFM images. Scale bar is 200 nm.

Figure 8

Effect of fibril orientation on the D-period depth. Fibrils from atomic force microscopy images were selected for each wild-type/knock-out (WT/KO) littermate pair, which were at identical orientations (±15°) for that set. WT/KO littermate pairs are indicated by a gray line connecting the discoidin domain receptor 1 KO (red circle) and the WT (blue circle). The fibril D-depth is plotted as a function of the mean fibril orientation for that pair. D-period depth from in vitro reconstituted collagen is shown as black squares.