Contribution of macromolecular structure to the retention of low-density lipoprotein at arterial branch points

Abstract

Background: Extracellular deposition of low-density lipoprotein (LDL) in the arterial wall is an essential early step in atherosclerosis. This process preferentially occurs at arterial branch points, reflecting a regional variation in lipoprotein-arterial wall interactions. In this study, we characterized the submicron microstructure of arterial wall collagen and elastin to evaluate its potential role in regional LDL deposition.

Methods and results: With 2-photon microscopy, we used the intrinsic optical properties of collagen and elastin to determine the arterial wall macromolecular microstructure in fresh porcine and murine arteries. This optical approach generated unique nondestructive en face 3-dimensional views of the wall. The collagen/elastin microstructure was found to vary with the topology of the arterial bed. A nearly confluent elastin surface layer was present throughout but was missing at atherosclerosis-susceptible branch points, exposing dense collagen-proteoglycan complexes. In LDL binding studies, this luminal elastin layer limited LDL penetration, whereas its absence at the branches resulted in extensive LDL binding. Furthermore, LDL colocalized with proteoglycans with a sigmoidal dose dependence (inflection point, approximately 130 mg LDL/dL). Ionic strength and competing anions studies were consistent with the initial interaction of LDL with proteoglycans to be electrostatic in nature.

Conclusions: This optical sectioning approach provided a robust 3-dimensional collagen/elastin microstructure of the arterial wall in fresh samples. At atherosclerosis-susceptible vascular branch points, the absence of a luminal elastin barrier and the presence of a dense collagen/proteoglycan matrix contribute to increased retention of LDL.

Figure 1

Collagen and elastin microstructure of the mouse aorta. A, Two-photon z-series images through the thoracic aortic wall, starting from the luminal surface (4.00 μm) through to the adventitia (70.00 μm). Elastin auto-fluorescence is red and the collagen SHG is green. A nearly confluent, wavy sheet of elastin envelops the luminal surface of the aortic wall. Collagen fibrils are radially arranged within the inner folds of the elastin lamellae as well as between lamellar units. Further out from the lumen, tortuous collagen fibrils make up the adventitia. Scale bar, 50 μm. This series demonstrates that both the intima-media interface, classically marked by the internal elastic lamina, as well as the media-adventitia interface, marked by the collagen rich connective tissue layer, can be effectively demarcated using this approach B, Detailed image of the aortic wall showing bunched-up individual collagen fibrils radially arranged within elastin folds. Scale bar, 10 μm. C, Three dimensional surface reconstructions of atherosclerosis susceptible intervertebral branch points with a ring of exposed collagen immediately surrounding the ostia. Scale bar, 50 μm. D, Aortic arch branch point composed of a dense, knotted sheet of collagen. Scale bar, 50 μm.

Figure 2

Collagen and elastin microstructure of the porcine carotid artery. Selected two-photon z-series images through the porcine carotid intima, starting from the luminal surface (11.80 mm) through to the first lamellar unit. Elastin auto-fluorescence is in red and the collagen SHG is in green. A wavy sheet of elastin with circular holes throughout envelops the luminal surface of the artery. Individual, bunched-up collagen fibrils are radially arranged within the inner folds of the elastin lamellae. Scale bar, 50 μm.

Figure 3

Collagen and elastin microstructure of the porcine coronary artery. A, Selected two-photon z-series images from the luminal surface (12.00 μm) through to the deeper layers of the arterial media (115.00 μm). Elastin auto-fluorescence is in red and collagen SHG is in green. A net-like IEL covers the luminal surface of the coronary free wall. Diagram illustrates the areas at which images were taken along a coronary arterial branch point. B, Images were taken from the edge of the free wall. C, Densely knotted collagen structure is not covered by the IEL surrounds the ostia. D, Portion of the cut branching artery. Scale bar, 50 μm.

Figure 4

LDL binding along the porcine coronary arterial wall. A, Selected z-series two-photon images of a pig coronary arterial branch point. LDL (red) bound extensively along the surface of the exposed collagen fibrils (green). Scale bar, 50 μm. B, A minimal amount of LDL (red) bound to the surface of the IEL (blue). Scale bar, 50 μm. C, Saturation curve of LDL binding to branch points at various incubation times with 2 mg/mL LDL. LDL binding reached saturation after about two hours of incubation with the artery (n = 4). D, Steady-state LDL binding studies in PBS showed a sigmoid pattern where (n = 3): $$\text{Y}=1/(1+{\text{e}}^{-(\text{x}-103)/301)})$$

Figure 5

LDL binding along the mouse aorta. A, Mouse aorta prior to incubation with LDL. Autofluorescence was low at 680 nm. Paired intervertebral branch points along the aortic wall became visible at a high intensity and increased exposure time (Intensity = 100%, exposure = 680 ms and gain = 165). Scale bar, 500 μm. B, Mouse aorta after incubation with LDL. LDL bound extensively to a circular region around intervertebral branch points. LDL bound lightly along the rest of the arterial wall (Intensity = 7.72%, exposure = 31 ms, Gain = 114). Scale bar, 500 μm. Zoom scale bar, 100 μm.

Figure 6

LDL binding to pig coronary arterial branch points in various buffered solutions. A, LDL binding in dH₂O was very high and LDL formed insoluble complexes along the surface of the exposed collagen. B, LDL binding in normal saline was lower than that seen in dH₂O above or in bicarbonate buffer B. C, LDL in a highly anionic buffer composed of 1X PBS with 2 mM azide showed very low binding to the surface of the branch point. Scale bar, 50 μm.

Figure 7

Co-localization analysis of LDL binding at porcine coronary arterial branch points. A, Quantification of colocalization between collagen and LDL. Pixel codistribution was calculated for blue (collagen) and red (LDL) channels for z-series two-photon data sets. Two-dimensional histogram (fluorogram) show the distribution of pixel intensities for collagen vs. LDL with poor co-localization (n = 3). B, Quantification of co-localization between proteoglycans and LDL. Pixel co-distribution was calculated for green (proteoglycan) and red (LDL) channels. Two-dimensional histogram showed that the distribution of pixel intensities for proteoglycans vs. LDL revealing a positive spatial correlation. Pearson correlation coefficient demonstrates that there is significantly greater co-localization between LDL and proteoglycans (0.469 ± 0.030) compared with LDL and collagen (0.056 ± 0.075). (1 = perfect correlation, 0 = no correlation, and −1 = perfect inverse correlation). C, Two-photon image of LDL (red) colocalization with immunolabeled proteoglycans (green).

Figure 8

Surface rendering of the porcine arterial beds. Elastin is pseudo colored red while the collagen in green. A) Aorta sample. B) Carotid sample. C) Coronary sample. Surface rendering was performed using the software resident in the Zeiss 510 imaging system.