Marine lipids
Introduction
Vascular endothelium is a monolayer epithelium which is characterized
by a polarized structure and by its intercellular junctions allowing
several types of barrier between intravascular and interstitial compartments. Endothelial permeability may be modified in several pathophysiological conditions. In inflammatory conditions, transvascular
fluid and solute exchange can be increased by raises in filtration pressure and by generation of cell-derived mediators that can influence the
permeability of the endothelial barrier.
A major pathway increasing vascular endothelial permeability is related to protein kinase C (PKC) activation resulting in phosphorylation
of linking proteins at the cell-cell and cell-matrix junctions (Lum &
Malik, 1994). PKC is a family of protein kinases whose most subtypes
are activated by 1,2-diacyl-sn-glycerol (DACi) in the presence or absence
of calcium. DAG is a phospholipase product and a major second messenger for receptor-coupled cell triggering.
The 1-O-alkylglycerols (alkyl-Gro) are naturally occurring compounds,
abundant in shark liver oil, that have therapeutic properties: inhibition
of tumour growth (Brohult et al., 1978) and protection against side effects
of radiotherapy (Brohult, 1963; Brohult et al., 1977 ). Their structures
allow them to incorporate into cellular phospholipids (PL) and then
modify quantitatively or qualitatively the production of lipidic mediators (Hichami et al., 1997) or second messengers such as DAG (Daniel
et al., 1988).
In this study, we addressed the questions whether alkyl-Gro incorporate into endothelial PL in our model of porcine aortic endothelial cells
(PAEC) and if so, would incorporated alkyl-Gro modify vascular permeability under resting and stimulated conditions, possibly through
the production of altered lipidic second messengers.
Materials and methods
Materials
Fetal bovine serum (FBS), antibiotics-antimycotics (10,000 units/ml
penicillin G sodium, 10,000 pg/ml streptomycin sulfate, and 25 pg/ml
amphotericin B as Fungizone®), Fungizone® (250 pg/ml amphotericin B), trypsin (0.25%), and culture medium M199 with phenol red,
Hepes (25 mM) and Earle’s salt medium with glutamin (100 mg/1)
were from Gibco (Cergy-Pontoise, France). Bovine serum albumin (BSA,
V fragment), collagen type I (from rat tail) clissolved in an acetic acid
solution (0.5%, v/v) at a concentration of 3 mg/ml, phorbol-12-myristate 13-acetate (PMA) and Evans blue dye were from Sigma (La Verpillière, France). Evans blue dye-labelled albumin was prepared by
adding 13.4 pg of Evans blue clye per millilitre to the albumin solution
(0.5%, w/v). Millicell® -CM tissue culture plate inserts ( 12 mm diameter,
0.4 pm pore size) were purchased from Millipore (Saint-Quentin-en148
Introduction
Vascular endothelium is a monolayer epithelium which is characterized
by a polarized structure and by its intercellular junctions allowing
several types of barrier between intravascular and interstitial compartments. Endothelial permeability may be modified in several pathophysiological conditions. In inflammatory conditions, transvascular
fluid and solute exchange can be increased by raises in filtration pressure and by generation of cell-derived mediators that can influence the
permeability of the endothelial barrier.
A major pathway increasing vascular endothelial permeability is related to protein kinase C (PKC) activation resulting in phosphorylation
of linking proteins at the cell-cell and cell-matrix junctions (Lum &
Malik, 1994). PKC is a family of protein kinases whose most subtypes
are activated by 1,2-diacyl-sn-glycerol (DACi) in the presence or absence
of calcium. DAG is a phospholipase product and a major second messenger for receptor-coupled cell triggering.
The 1-O-alkylglycerols (alkyl-Gro) are naturally occurring compounds,
abundant in shark liver oil, that have therapeutic properties: inhibition
of tumour growth (Brohult et al., 1978) and protection against side effects
of radiotherapy (Brohult, 1963; Brohult et al., 1977 ). Their structures
allow them to incorporate into cellular phospholipids (PL) and then
modify quantitatively or qualitatively the production of lipidic mediators (Hichami et al., 1997) or second messengers such as DAG (Daniel
et al., 1988).
In this study, we addressed the questions whether alkyl-Gro incorporate into endothelial PL in our model of porcine aortic endothelial cells
(PAEC) and if so, would incorporated alkyl-Gro modify vascular permeability under resting and stimulated conditions, possibly through
the production of altered lipidic second messengers.
Materials and methods
Materials
Fetal bovine serum (FBS), antibiotics-antimycotics (10,000 units/ml
penicillin G sodium, 10,000 pg/ml streptomycin sulfate, and 25 pg/ml
amphotericin B as Fungizone®), Fungizone® (250 pg/ml amphotericin B), trypsin (0.25%), and culture medium M199 with phenol red,
Hepes (25 mM) and Earle’s salt medium with glutamin (100 mg/1)
were from Gibco (Cergy-Pontoise, France). Bovine serum albumin (BSA,
V fragment), collagen type I (from rat tail) clissolved in an acetic acid
solution (0.5%, v/v) at a concentration of 3 mg/ml, phorbol-12-myristate 13-acetate (PMA) and Evans blue dye were from Sigma (La Verpillière, France). Evans blue dye-labelled albumin was prepared by
adding 13.4 pg of Evans blue clye per millilitre to the albumin solution
(0.5%, w/v). Millicell® -CM tissue culture plate inserts ( 12 mm diameter,
0.4 pm pore size) were purchased from Millipore (Saint-Quentin-en148