Arterial extracellular matrix: a mechanobiological study of the contributions and interactions of elastin and collagen

Abstract

The complex network structure of elastin and collagen extracellular matrix (ECM) forms the primary load bearing components in the arterial wall. The structural and mechanobiological interactions between elastin and collagen are important for properly functioning arteries. Here, we examined the elastin and collagen organization, realignment, and recruitment by coupling mechanical loading and multiphoton imaging. Two-photon excitation fluorescence and second harmonic generation methods were performed with a multiphoton video-rate microscope to capture real time changes to the elastin and collagen structure during biaxial deformation. Enzymatic removal of elastin was performed to assess the structural changes of the remaining collagen structure. Quantitative analysis of the structural changes to elastin and collagen was made using a combination of two-dimensional fast Fourier transform and fractal analysis, which allows for a more complete understanding of structural changes. Our study provides new quantitative evidence, to our knowledge on the sequential engagement of different arterial ECM components in response to mechanical loading. The adventitial collagen exists as large wavy bundles of fibers that exhibit fiber engagement after 20% strain. The medial collagen is engaged throughout the stretching process, and prominent elastic fiber engagement is observed up to 20% strain after which the engagement plateaus. The fiber orientation distribution functions show remarkably different changes in the ECM structure in response to mechanical loading. The medial collagen shows an evident preferred circumferential distribution, however the fiber families of adventitial collagen are obscured by their waviness at no or low mechanical strains. Collagen fibers in both layers exhibit significant realignment in response to unequal biaxial loading. The elastic fibers are much more uniformly distributed and remained relatively unchanged due to loading. Removal of elastin produces similar structural changes in collagen as mechanical loading. Our study suggests that the elastic fibers are under tension and impart an intrinsic compressive stress on the collagen.

Figure 1

The custom built tissue stretching device allowing for multiphoton microscopy to be performed while tissue undergoes equal and unequal biaxial strain. To see this figure in color, go online.

Figure 2

Multiphoton images of adventitial collagen (top), medial collagen (middle), and medial elastin (bottom) during equal biaxial strain. Images are 110 × 110 μm.

Figure 3

Fractal (left column) and FFT (right column) analysis of equal biaxial strain images of adventitial collagen, medial collagen, and medial elastin. Fractal analysis is plotted with the mean of the normalized absolute difference in fractal number ± SE along with increasing strain. FFT analysis is plotted with the fiber angle on the x axis (fibers oriented at 0° and 90° are in the circumferential and longitudinal anatomic directions of the artery, respectively), increasing strain on the y axis, and amount of fibers on the z axis. To see this figure in color, go online.

Figure 4

Straightness parameter of adventitial collagen fibers as measured with NeuronJ. Compared with the fractal analysis in Fig. 3, both methods consistently show a lack of structural change initially and then a decrease in fiber waviness at higher strains.

Figure 5

Average normalized distributions of fiber orientation with biaxial strains of 0%C-0%L, 30%C-30%L, 15%C-30%L, and 30%C-15%L applied to the tissue. Fibers oriented at 0° and 90° are in the circumferential and longitudinal anatomic directions of the artery, respectively. C and L represent the circumferential and longitudinal directions, respectively.

Figure 6

Ratio of circumferential to longitudinal distributed fibers, defined as the number of fibers oriented between 0° ± 20° divided by the number of fibers oriented at 90° ± 20° from Fig. 5, of medial collagen, medial elastin, and adventitial collagen during biaxial deformation. The 0, 15, and 30 denote strain levels; and C and L represent the circumferential and longitudinal directions, respectively. Comparisons between the stretched states and the unloaded (0C-0L) condition are shown (^∗p < 0.05).

Figure 7

Multiphoton images of adventitial collagen (top) and medial collagen (bottom) during elastin removal from the ECM. The average elastin content was reported as μg elastin/mg wet tissue weight. Images are 110 × 110 μm.

Figure 8

FFT analysis of elastin degradation images with the fiber angle on the x axis (fibers oriented at 0° and 90° are in the circumferential and longitudinal anatomic directions of the artery, respectively), decreasing elastin content on the y axis, and amount of fibers on the z axis. To see this figure in color, go online.