Periluminal expression of a secreted transforming growth factor-ß type II receptor inhibits in-stent neointima formation following adenovirus-mediated stent-based intracoronary gene transfer.

Transforming growth factor-ß1 (TGF-ß1) has been shown unequivocally to enhance neointima formation in carotid and ileo-femoral arteries. In our previous studies, however, TGF-ß1 expression in coronary arteries actually reduced neointima formation without affecting luminal loss postangioplasty, while expression of a TGF-ß1 antagonist (RIIs) in balloon-injured coronary arteries reduced luminal loss without affecting neointima formation. These observed effects may be a consequence of the mode of coronary artery gene transfer employed, but they may also represent differences in the modes of healing of coronary, carotid, and ileo-femoral arteries after endoluminal injury. To help clarify whether a gene therapy strategy to antagonize TGF-ß might have application within the coronary vasculature, we have investigated the effect of high-level periluminal expression of RIIs using stent-based adenovirus-mediated intracoronary gene transfer. Porcine coronary arteries were randomized to receive a custom-made CoverStent preloaded with saline only, or with 1×10(9) infectious units of adenovirus expressing RIIs or ß-galactosidase (lacZ). Vessels were analyzed 28 days poststenting, at which time angiographic in-stent diameter was significantly greater in RIIs-treated arteries, and in-stent luminal loss significantly reduced. Computerized morphometric minimum in-stent lumen area was ~300% greater in RIIs-exposed vessels than in lacZ or saline-only groups. This was because of significantly reduced neointima formation in the RIIs group. RIIs had no demonstrable effect on cellular proliferation or apoptosis, but greater normalized neointimal/medial collagen content was observed in RIIs-exposed arteries. These data highlight the qualitatively similar effect of TGF-ß antagonism on neointima formation in injured coronary and noncoronary arteries, and suggest that since cellular proliferation is unaffected, TGF-ß1 antagonism might prevent in-stent restenosis without the delayed healing that is associated with drug-eluting stents in current clinical use.

Beta-adrenoceptor blockade markedly attenuates transgene expression from cytomegalovirus promoters within the cardiovascular system.

OBJECTIVE: The major immediate-early cytomegalovirus enhancer/promoter (MIECMV), widely used in cardiovascular gene therapy, contains several positively regulatory cAMP response elements (CRE). Catecholamine signaling via beta-adrenoceptors might increase transgene expression from MIECMV, and if so, beta-blockers may have a detrimental effect on the efficacy of clinical cardiovascular gene therapy strategies. METHODS AND RESULTS: Cultured smooth muscle cells were exposed to isoprenaline, atenolol, or propranolol, alone and in combination before infection with adenoviruses expressing beta-galactosidase. beta-galactosidase expression was assayed 72 hours later. Isoprenaline increased transgene expression from MIECMV up to 8-fold (P<0.001), but had no effect on a promoter containing no CRE. The effect of isoprenaline was inhibited by beta-blockade and by specific CRE-decoy oligonucleotides. Beta-blockers did not reduce transgene expression below basal levels. After adenovirus-mediated porcine intracoronary gene transfer, however, beta-blockade reduced beta-galactosidase expression by up to 250-fold compared with non-beta-blocked animals (P<0.01). CONCLUSIONS: Enhancement of promoter activity by endogenous catecholamines is essential for high-level transgene expression from MIECMV within the vasculature. Beta-blocker-mediated suppression of transgene expression from MIECMV in vascular tissues has a significant bearing on clinical studies of cardiovascular gene transfer. This is the first described interaction to our knowledge between widely prescribed pharmaceuticals and a commonly used promoter of clinical transgene expression.

Covalently-bound heparin makes collagen thromboresistant.

OBJECTIVE: Blood compatibility of artificial surfaces depends on their immunogenic and thrombogenic properties. Collagen's weak antigenicity makes it an attractive candidate for stent coatings or fabrication of vascular grafts. However, the thrombogenic nature of collagen limits its application. We examined whether heparinization can make collagen more thromboresistant. METHODS AND RESULTS: Collagen was heparinized by crosslinking collagen with extensively periodate oxidized heparin and/or by covalently bonding of mildly periodate oxidized heparin. Both ways of heparinization have no effect on platelet adhesion and could not abolish induction of platelet procoagulant activity. However, thrombin generation was completely prevented under static and flow conditions. The functionality of immobilized heparin was confirmed by specific uptake of antithrombin, 13.5+/-4.7 pmol/cm2 and 1.95+/-0.21 pmol/cm2 for mildly and heavily periodated heparin, respectively. CONCLUSIONS: These results indicate that immobilization of heparin on collagen, even as a crosslinker, is a very effective way to prevent surface thrombus formation. These data encourage the application of heparinized collagen as stent-graft material in animal and eventually human studies.