Skip to Main content Skip to Navigation
Theses

Interactions des lipoprotéines de faible densité avec les cellules endothéliales vasculaires humaines : influence des contraintes de cisaillement : étude in vitro par microscopie de fluorescence confocale

Abstract : Numerous studies on the pathogenesis of atherosclerosis suggest that several steps are involved, such as lipid accumulation in the artery wall resulting from transendothelial uptake of LDL-Cholesterol, followed by LDL oxidation. In this work, we developed spectroscopic and microscopic methods to study the effect of shear stress on the kinetics of internalization and degradation of native LDL in human vascular endothelial cells. FRET and FCS techniques were performed by using confocal microscopy. Native LDL were labeled with two carbocyanine dyes, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiO) as donor and 3,3'- dioctadecyloxa-carbocyanine perchlorate (DiI) as receptor. FCS techniques allowed us to evaluate the yield of the double labeling of the LDL with DiI and DiO, where as the average lifetime of the both fluorophore was determined by FLIM techniques, and the FRET measurements enable us to follow the degradation of the LDL in the living single cells. Our studies show: 1) - that the yield of the LDL double-labeling with DiI and DiO was about 30%, and 82,84% of DiI carried by LDL are subject in FRET process with DiO. 2) - the average lifetime of DiO coupled with LDL in HUVEC was about 2 100 ps [more or less] 800 by adjustment of order 1 following the excitation of molecules in multiphoton mode ( [lambda] ex = 800 nm), whereas the average lifetime of DiO coupled with DiI-LDL measured under the same conditions was about 320 ps [more or less] 400, this decreasing of the lifetime of the donor DiO (from 2 100 ps to 320 ps) in the presence of DiI reflects the energy transfer dependent on the molecular proximity of the two partners (DiO, DiI) on LDL molecules ; 3) - the possibility to evaluate the kinetics of LDL endocytosis in living single cells and the degradation of LDL was depending on the shear conditions (shear stress induced changes in the kinetics of uptake and degradation). In accordance with increasing knowledge and understanding, and regarding all available data, we conclude that confocal microscopic study, FRET, FCS and FLIM could be useful methods to establish the basic and clinical studies of LDL uptake in atherosclerosis. However, further studies need to be performed to elucidate the molecular mechanism by which shear stress modulates the expression of LDL receptors.
Document type :
Theses
Complete list of metadata

Cited literature [53 references]  Display  Hide  Download

https://hal.univ-lorraine.fr/tel-01748387
Contributor : Thèses Ul <>
Submitted on : Thursday, March 29, 2018 - 11:33:47 AM
Last modification on : Monday, March 29, 2021 - 4:03:15 PM
Long-term archiving on: : Friday, September 14, 2018 - 6:29:46 AM

File

SCD_T_2008_0123_TRAORE.pdf
Files produced by the author(s)

Identifiers

  • HAL Id : tel-01748387, version 1

Collections

Citation

Mariama Traoré. Interactions des lipoprotéines de faible densité avec les cellules endothéliales vasculaires humaines : influence des contraintes de cisaillement : étude in vitro par microscopie de fluorescence confocale. Biologie cellulaire. Université Henri Poincaré - Nancy 1, 2008. Français. ⟨NNT : 2008NAN10123⟩. ⟨tel-01748387⟩

Share

Metrics

Record views

53

Files downloads

461