ENTRY AND DISTRIBUTION OF FLUORESCENT ANTIPROLIFERATIVE HEPARIN DERIVATIVES INTO RAT VASCULAR SMOOTH-MUSCLE CELLS - COMPARISON BETWEEN HEPARIN-SENSITIVE AND HEPARIN-RESISTANT CULTURES
T. Barzu et al., ENTRY AND DISTRIBUTION OF FLUORESCENT ANTIPROLIFERATIVE HEPARIN DERIVATIVES INTO RAT VASCULAR SMOOTH-MUSCLE CELLS - COMPARISON BETWEEN HEPARIN-SENSITIVE AND HEPARIN-RESISTANT CULTURES, Journal of cellular physiology, 167(1), 1996, pp. 8-21
We studied the binding and entry of fluorescein (FITC)-labeled heparin
derivatives into rat aortic smooth muscle cells (SMC) by confocal mic
roscopy. FITC-labeled heparin fractions or FITC-labeled SR 80037A, a p
otent antiproliferative heparin derivative (Barzu et al., Eur. J. Phar
macol., 219:225-233 1992), were prepared and their antiproliferative a
ctivity was confirmed. By incubating SMC with FITC-labeled heparins, a
specific cell-associated fluorescence was found. Cellular fluorescenc
e was mostly located around the nucleus and at the level of cell conta
cts or cell adhesion. The fluorescence was displaced neither by chasin
g with excess of unlabeled heparins nor by washing with 1 M NaCl, whic
h proved that labeled heparins had been internalized by SMC. Kinetics
of internalization of FITC-heparins suggested receptor-mediated endocy
tosis of heparins by SMC. Double labeling of SMC with biotinylated Con
canavalin A and FITC-SR 80037A also indicated that heparin derivative
enters the endocytic pathway. The process was accelerated when serum w
as present in the incubation medium. Treatment of cells with chloroqui
ne (50 mu M) induced accumulation of FITC-SR 80037A in the late endoso
mes, around the nucleus. No fluorescence labeling could be evidenced i
nside the nucleus. Neither electron microscopy nor cell fractionation
experiments performed with SMC previously incubated with [H-3]-heparin
were able to ascertain nuclear uptake of heparin, as proposed by othe
r workers (Busch et al., Cell Biol., 116:31-42; 1992; Sing et al., Dru
g Dev. Res., 29:129-136 1993). The cell-associated fluorescence was ve
ry weak in SMC resistant to the antiproliferative activity of heparin,
selected by longterm heparin treatment (HT-SMC) as previously shown [
Barzu et al., J. Cell. Physiol., 160:239-248, 1994]. The HT-SMC differ
ed from control SMC with regard to expression of extracellular matrix
proteins. These cells exhibited very low expression of fibronectin and
prevalent expression of laminin and synthesized less cell-associated
glycosaminoglycans. From our results, the following conclusions can be
drawn: (1) the antiproliferative heparins are bound and internalized
by SMC without being taken up into the nucleus; (2) there is a correla
tion between the binding and/or the internalization process and the se
nsitivity of SMC to the antiproliferative activity of heparins; and (3
) selection of heparin-resistant SMC by long treatment with heparin re
sults in particular growth pattern of SMC (absence of focal overgrowth
), associated with changes In the expression of the extracellular matr
ix components (finbronectin, laminin, and cell-bound glycosaminoglycan
s). (C) 1996 Wiley-Liss, Inc.