Two-photon microscopy of aorta fibers shows proteolysis induced by LDL hydroperoxides

Abstract

Abstract-Oxidatively modified LDL mimics several aspects of atherogenesis. In this disease, degradation of the matrix proteins' network also occurs. By a new morphological ex vivo approach, not requiring sample processing, we explored the relationship between the degradation of matrix protein and oxidatively modified LDL. Two-photon excitation fluorescence microscopy images of fresh cross-section rings of rat aorta, acquired while the sample was maintained in a glucose-and oxygen-supplemented buffer, showed straight, parallel, thick, long extracellular matrix proteins. Traditional microscopic examination, requiring sample fixation and staining, shows smaller and curved fibers. Instead, we observed curved and broken fibers after a 30-min incubation of aorta with either LDL containing lipid hydroperoxides, or tert-butyl-hydroperoxide. The adhesion of LDL to the endothelium and its internalization was directly visualized by using a lipid fluorophore. The damage to aorta matrix proteins induced by LDL and tert-butylhydroperoxide was fully prevented by antioxidants, such as ascorbate or Trolox C, or inhibitors of proteases. The image spectroscopy of the fibers' autofluorescence (polarization and lifetime) revealed an increased mobility of the fluorescent cross-link in fibers. Damaged matrix proteins were also imaged in aorta samples from apolipoprotein E knock-out mice. Our ex vivo images directly visualized the activation of a fast redox-sensitive proteolytic process in the arterial wall triggered by lipid hydroperoxides in LDL.

Two-Photon Microscopy for Imaging of the (Atherosclerotic) Vascular Wall: A Proof of Concept Study

“…1d). Such autofluorescence is in agreement with earlier findings for elastin [22]and collagen [25, 26]. Note that collagen and elastin fibers appeared slightly thicker than in figure 1c.…”

“…These studies also indicate that penetration depth of TPLSM varies between tissues. However, in atherosclerosis research TPLSM has only found very limited, and primarily ex vivo, applications [22, 23]. To our knowledge, penetration depth has not been determined in blood vessels so far.…”

Two-photon lifetime imaging of blood and blood vessels

“…The fluorescence lifetime found for the elastine autofluorescence is τ = 2.05 ± 0.05 ns. This corresponds with values found before 27 . For the SYTO in the smooth muscle cells a lifetime of τ = 3.4 ± 0.05 ns is found.…”

Two-Photon Microscopy of Vital Murine Elastic and Muscular Arteries

“…In addition, photobleaching, -damage and -toxicity are drastically reduced [8], enabling imaging of delicate structures in viable tissue. Recently, TPLSM was established as a valuable tool for imaging of blood vessels [4,9,10,11], skeletal muscle arterioles [12], and microvasculature of the human uterus [13]. However, the blood vessels were very small [12], still treated with a fixative, freeze-thawed and sliced, or casted in hot (>40°C) agarose gel, resulting in structural alterations and loss of functionality [14].…”