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Two-photon microscopy of aorta fibers shows proteolysis induced by LDL hydroperoxides
1Istituto di Medicina Sperimentale, CNR, Roma, Italy. tiziana@biocell.irmkant.rm.cnr.it
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-butyl-hydroperoxide 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.
Insights
Oxidatively modified LDL damages arterial wall matrix proteins via a redox-sensitive proteolytic process. Antioxidants and protease inhibitors prevent this damage, offering potential therapeutic targets for atherosclerosis.
Area of Science:
- Cardiovascular Biology
- Atherosclerosis Research
- Biochemistry
Background:
- Oxidatively modified low-density lipoprotein (LDL) plays a key role in atherogenesis.
- Matrix protein degradation is a hallmark of atherosclerosis.
- A direct link between oxidized LDL and matrix degradation in the arterial wall needs further elucidation.
Purpose of the Study:
- To investigate the relationship between matrix protein degradation and oxidatively modified LDL using an innovative ex vivo approach.
- To visualize the dynamic changes in arterial matrix proteins upon exposure to oxidized LDL.
Main Methods:
- Utilized two-photon excitation fluorescence microscopy on fresh rat aorta cross-sections.
- Employed an ex vivo model maintaining tissue viability in a supplemented buffer.
- Visualized LDL adhesion and internalization using lipid fluorophores.
- Analyzed fiber autofluorescence properties (polarization, lifetime) via image spectroscopy.
Main Results:
- Oxidatively modified LDL and tert-butyl-hydroperoxide induced fragmentation and curvature of extracellular matrix proteins.
- LDL adhesion and internalization into the arterial wall were directly observed.
- Antioxidants (ascorbate, Trolox C) and protease inhibitors completely prevented matrix damage.
- Image spectroscopy indicated increased cross-link mobility in damaged fibers.
- Similar matrix damage was observed in aorta from apolipoprotein E knock-out mice.
Conclusions:
- Lipid hydroperoxides in LDL directly trigger a rapid, redox-sensitive proteolytic process within the arterial wall.
- This process leads to the degradation of extracellular matrix proteins, contributing to atherogenesis.
- The findings highlight the critical role of oxidized LDL in arterial wall remodeling and suggest potential therapeutic interventions targeting this pathway.