DNA delivery from an intravascular stent with a denatured collagen-polylactic-polyglycolic acid-controlled release coating: mechanisms of enhanced transfection.
We previously demonstrated that DNA-polylactic-polyglycolic acid (PLGA)-coated stents can deliver genes to the arterial wall with reporter expression involving 1% of neointimal cells. The present study investigated a novel formulation utilizing denatured collagen in DNA-stent coatings; denatured collagen was hypothesized to enhance gene transfer due to adhesion molecule interactions and actin-related mechanisms. Arterial smooth muscle cells (SMCs) cultivated on denatured collagen had significantly greater plasmid DNA (beta-galactosidase) transfection than SMC grown on native collagen (18.3+/-1.2 vs 1.0+/-0.1%, P<0.001). The denatured-collagen effect was completely blocked with anti-alpha(v)beta(3) integrin antibody. SMCs cultivated on native collagen supplemented with tenascin-C (TN-C), a protein recognized by alpha(v)beta(3) integrins, showed a 33-fold increase in transfection compared to control (P<0.001); this effect was also blocked with anti-alpha(v)beta(3) antibody. We observed that cells grown on denatured collagen had marked F-actin-enriched stress fibers and intense perinuclear G actin, compared to those grown on native collagen, which demonstrated F-actin-enriched focal adhesions without perinuclear G-actin localization. Cytochalasin-D, an F actin depolymerizing agent, caused significantly increased SMC transfection in cells cultivated on native collagen compared to control cells (18.0+/-1.8 vs 3.02+/-0.9%, P<0.001) further supporting the view that actin-related cytoskeletal changes influence transfection. A denatured-collagen-PLGA composite vascular stent coating similarly resulted in increased plasmid DNA green fluorescent protein (GFP) expression compared to controls (P<0.001) in SMC cultures; the increased transfection was blocked by anti-alpha(v)beta(3) antibody. Pig coronary studies comparing denatured-collagen-PLGA-coated stents containing plasmid DNA (encoding GFP) to coated stents without DNA demonstrated 10.8% of neointimal cells transfected; this level of expression was almost an order of magnitude greater than previously reported with a DNA delivery stent. It is concluded that denatured collagen incorporated into plasmid DNA-stent coating formulations may increase the level of gene expression in vitro and in vivo because of integrin-related mechanisms and associated changes in the arterial smooth muscle cell actin cytoskeleton.
Perlstein
I
I
Division of Cardiology, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
Connolly
J M
JM
Cui
X
X
Song
C
C
Li
Q
Q
Jones
P L
PL
Lu
Z
Z
DeFelice
S
S
Klugherz
B
B
Wilensky
R
R
Levy
R J
RJ
D000199:Actins / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D003331:Coronary Vessels / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004247:DNA / Q000008:administration & dosage*; D003692:Delayed-Action Preparations; D015316:Genetic Therapy / Q000379:methods*; D039302:Integrin alphaVbeta3; D019344:Lactic Acid; D008856:Microscopy, Fluorescence; D011091:Polyesters; D011100:Polyglycolic Acid; D011108:Polymers; D051381:Rats; D015607:Stents; D013552:Swine; D014162:Transfection / Q000379:methods*
D000199:Actins; D003692:Delayed-Action Preparations; D039302:Integrin alphaVbeta3; D011091:Polyesters; D011108:Polymers; D011100:Polyglycolic Acid; D019344:Lactic Acid; C033616:poly(lactide); D003094:Collagen; D004247:DNA