EFFECTS OF RETROVIRAL-MEDIATED TISSUE-PLASMINOGEN ACTIVATOR GENE-TRANSFER AND EXPRESSION ON ADHERENCE AND PROLIFERATION OF CANINE ENDOTHELIAL-CELLS SEEDED ONTO EXPANDED POLYTETRAFLUOROETHYLENE
Ts. Huber et al., EFFECTS OF RETROVIRAL-MEDIATED TISSUE-PLASMINOGEN ACTIVATOR GENE-TRANSFER AND EXPRESSION ON ADHERENCE AND PROLIFERATION OF CANINE ENDOTHELIAL-CELLS SEEDED ONTO EXPANDED POLYTETRAFLUOROETHYLENE, Journal of vascular surgery, 22(6), 1995, pp. 795-803
Purpose: Seeding prosthetic arterial grafts with genetically modified
endothelial cells (ECs) has the potential to substantially improve gra
ft function. However, preliminary applications suggest that grafts see
ded with retrovirally transduced ECs yield a significantly lower perce
nt surface coverage than those seeded with nontransduced ECs. The obje
ctive of this study was to test the hypothesis that canine ECs transdu
ced with the human tissue plasminogen activator (tPA) gene would have
a lower rate of adherence to pretreated expanded polytetrafluoroethyle
ne (ePTFE) both in vitro and in vivo and that they would proliferate a
t a slower rate on pretreated ePTFE in vitro. Methods: Early passage E
Cs derived from canine external jugular vein were transduced with the
retroviral MFG vector containing the gene for human tPA. ECs exposed t
o media alone served as controls. Iodine 125-labeled ECs were seeded i
n vitro onto ePTFE graft segments pretreated with canine whole blood,
fibronectin (50 mu g/ml), or media alone, and the percent of ECs adher
ent at 1 hour were determined (n = 3). Additional tPA-transduced and -
nontransduced ECs were grown for 10 days on either fibronectin (50 mu
g/ml)-pretreated ePTFE wafers or tissue culture plastic pretreated wit
h gelatin (1%) or fibronectin (50 mu g/ml), and the EC proliferation r
ates were determined (n = 3). furthermore, I-125-labeled ECs were seed
ed onto fibronectin (50 mu g/ml)-pretreated ePTFE graft segments impla
nted as carotid and femoral artery interposition grafts (n = 3). The g
rafts were harvested after 1 hour, and the percent of ECs adherent was
determined. Results: Human tPA was detected by immunohistochemical st
aining in 61% +/- 5% of the transduced ECs and was expressed at 35.4 /- 12.9 ng/hr/10(6) cells. Fibronectin and whole blood pretreatment of
the ePTFE grafts led to greater EC adherence in vitro than did media
alone (90.9% +/- 5.3% vs 77.8% +/- 5.8% vs 4.7% +/- 1.1%, p less than
or equal to 0.05). No significant difference in the rates of adherence
or proliferation was seen in vitro between the transduced and nontran
sduced ECs. No significant difference in proliferation was found for t
he transduced ECs on the three matrices tested in vitro. In contrast,
adherence of the transduced ECs in vivo was significantly lower than t
hat of nontransduced ECs(64.7% +/- 2.1% vs 73.7% +/- 4.1%, p less than
or equal to 0.05) 1 hour after implantation. Conclusions: Lower rates
of surface endothelialization by genetically modified ECs in vivo do
not appear to be due to an impaired capacity to initially adhere or pr
oliferate on the synthetic graft but may result from decreased adheren
ce after exposure to in vivo hemodynamic forces.