Extracellular matrix permits the expression of von Willebrand's factor, uptake of di-I-acetylated low density lipoprotein and secretion of prostacyclin in cultures of endothelial cells from rat brain microvessels.
Microvascular endothelial cells from the adult rat brain were cultured on Matrigel and found to express many differentiated properties including secretion of prostacyclin (PGI2) and von Willebrand's factor (vWF). Brain microvascular endothelial cells (BMECs) were purified by dextran and percoll gradients after enzymatic treatment and cultured under various conditions. BMECs that were plated on Matrigel stained positively for factor VIII-related antigen and incorporated Di-I-acetylated low density lipoprotein, whereas BMEC plated on fibronectin, gelatin, or uncoated dishes did not express any of the above properties which are characteristic of endothelial cells. vWF was measured by a sensitive ELISA in the culture media of BMECs plated on different types of matrices. Specificity of the anti-human vWF antibodies for the rat vWF was verified by immunoabsorption on a solid phase, sodium dodecyl sulfate, and Western blot analysis. BMECs also secreted vWF into the culture media only when the cells were plated on Matrigel, and this secretion was augmented after a 6 h incubation with an interleukin-1 tumor necrosis factor-alpha mixture, but not by lipopolysaccharide. From different matrices tested, only Matrigel permitted the secretion of PGI2 by BMECs. Cells also proved to be sensitive to mechanical stimulation and became refractory to secretagogue if the mechanical stimulation was serially repeated. Under the best conditions, stimulation of the cells with bradykinin (1 microM) substantially increased PGI2 secretion. These data indicate that growth of BMECs on Matrigel in vitro permits the expression of classical endothelial cell markers in a manner similar to the behavior of these cells in situ.
Doron
D A
DA
Department of Neurology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814.
Jacobowitz
D M
DM
Heldman
E
E
Feuerstein
G
G
Pollard
H B
HB
Hallenbeck
J M
JM
D000107:Acetylation; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D001920:Bradykinin / Q000494:pharmacology; D001921:Brain / Q000098:blood supply*; D003094:Collagen / Q000032:analysis; D004338:Drug Combinations; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D011464:Epoprostenol / Q000378:metabolism*; D005109:Extracellular Matrix / Q000737:chemistry / Q000502:physiology*; D005353:Fibronectins / Q000032:analysis; D005780:Gelatin / Q000032:analysis; D007375:Interleukin-1 / Q000494:pharmacology; D007797:Laminin / Q000032:analysis; D008077:Lipoproteins, LDL / Q000493:pharmacokinetics*; D008833:Microcirculation / Q000378:metabolism; D010812:Physical Stimulation; D011509:Proteoglycans / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014841:von Willebrand Factor / Q000032:analysis / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D004338:Drug Combinations; D005353:Fibronectins; D007375:Interleukin-1; D007797:Laminin; D008077:Lipoproteins, LDL; D011509:Proteoglycans; D014409:Tumor Necrosis Factor-alpha; D014841:von Willebrand Factor; C059485:matrigel; D005780:Gelatin; D003094:Collagen; D011464:Epoprostenol; D001920:Bradykinin