Gene Therapy For Vascular Grafts

A. Vein Graft Engineering with E2F Decoy ODN

The long-term success of surgical revascularization in the lower extremity and coronary circulations has been limited by significant rates of autologous vein graft failure. No pharmacological approach has been successful at preventing long-term graft diseases such as neointimal hyperplasia or graft atherosclerosis. Gene therapy offers a new avenue for the modification of vein graft biology that might lead to a reduction in clinical morbidity from graft failures. Intra-operative transfection of the vein graft also offers an opportunity to combine intact tissue DNA transfer techniques with the increased safety of ex vivo transfection, and a number of studies have documented the feasibility of ex vivo gene transfer into vein grafts using viral vectors.

The vast majority of vein graft failures have been linked to the neointimal disease is part of graft remodeling after surgery (66). Although neointimal hyperplasia contributes to the reduction of wall stress in vein grafts after bypass, this process can also lead to luminal narrowing of the graft conduit during the first years after operation (67). Furthermore, the abnormal neointimal layer, with its production of proinflammatory proteins, is believed to form the basis for an accelerated form of atherosclerosis that causes late graft failure (68).

* Porcine ameroid model of / chronic ischemia

V o / with CO2 laser

• Injection of plasmid adjacent to TMR site-VEGF vs. Bgal

Similar to observations made in the arterial balloon injury model, it was found that a combination of antisense ODN that inhibit expression of at least two cell cycle regulatory genes could significantly block neointimal hyperplasia in vein grafts

(69). Additionally, E2F decoy ODN yielded similar efficacy in the vein graft when compared to the arterial injury model

(70). In contrast to arterial balloon injury, however, vein grafts are not only subjected to a single injury at the time of operation, but are also exposed to chronic hemodynamic stimuli for remodeling. Despite these chronic stimuli, a single, intraoperative ODN treatment of vein grafts resulted in a resistance to neointimal hyperplasia that lasted for at least 6 months in the rabbit model (69). During that time period, the grafts treated with antisense ODN were able to adapt to arterial conditions via hypertrophy of the medial layer. Furthermore, these genetically engineered conduits proved resistant to diet-induced graft atherosclerosis (Fig. 4) and were associated with preserved endothelial function (71).

Figure 4 Control oligonucleotide-treated (A and B) and antisense oligonucleotide (against cdc2 kinase/PCNA)-treated vein grafts (C and D) in hypercholesterolemic rabbits, 6 weeks after surgery (X 70). Sections were stained with hematoxylin/van Gie-son (A and C) and a monoclonal antibody against rabbit macrophages (B and D). Arrows indicate the location of the internal elastic lamina.

Figure 4 Control oligonucleotide-treated (A and B) and antisense oligonucleotide (against cdc2 kinase/PCNA)-treated vein grafts (C and D) in hypercholesterolemic rabbits, 6 weeks after surgery (X 70). Sections were stained with hematoxylin/van Gie-son (A and C) and a monoclonal antibody against rabbit macrophages (B and D). Arrows indicate the location of the internal elastic lamina.

A large-scale, prospective, randomized, double-blind trial of human vein graft treatment with E2F decoy ODN has been initiated (18). Efficient delivery of the ODN is accomplished within 15 min during the operation by placement of the graft after harvest in a device that exposes the vessel to ODN in physiological solution and creates a nondistending, pressurized environment of 300 mm Hg. Preclinical findings indicated ODN delivery to greater than 80% of graft cells, and effective blockade of target gene expression. This study will measure the effect of cell-cycle gene blockade on primary graft failure rates, and represents 1 of the first attempts to definitively determine the feasibility of clinical genetic manipulation in the treatment of a common cardiovascular disorder.

B. Vein Graft Gene Transfer

With the development of viral-mediated gene delivery methods, some investigators have begun to explore the possibility of using these systems ex vivo in autologous vein grafts. Chen et al. (72) demonstrated the expression of the marker gene b-galactosidase along the luminal surface and in the adventitia of 3-day porcine vein grafts infected with a replication-deficient adenoviral vector at the time of surgery. The vein segments were incubated in a high viral titer suspension for approximately 2 h prior to implantation. Although these researchers documented expression of a soluble vascular cell adhesion molecule-1 (sVCAM-1) isomer on the luminal and abluminal surfaces of 3-day grafts infected with an adenoviral vector encoding this protein, no long-term expression or functional effect of this gene was reported. In a study previously alluded to, Kupfer et al. (73) explored the use of a novel adenovirus-based transduction system, in which adenoviral particles were linked to plasmid DNA via biotin/streptavidin-transferrin/ polylysine complexes. p-galactosidase expression was documented 3 and 7 days after surgery in rabbit vein grafts that had been incubated for 1 h with complexes prior to grafting. Expression was again greatest on the luminal surfaces of the grafts, although then presence of transfected cells in the medial and adventitial layers was also reported.

The feasibility of gene transfer in vein grafts has subsequently led to the investigation of potential therapeutic endpoints such as neointima formation. George et al. (74), using a replication-deficient adenovirus expressing tissue inhibitor of mettaloproteinasee-2 (TIMP-2), was able to demonstrate a dramatic decrease in neointimal formation in a saphenous vein organ culture model. In vivo gene transfer has also been shown to effectively reduce neointima formation in experimental vein grafts. Bai et al. (75) performed intra-operative transfec-tion of the senescent cell-derived inhibitor (sdi-1) gene, a downstream mediator of the tumor suppresser gene p53, using the HVJ-liposome system, and was once again able to demonstrate a significant reduction in neointima formation. The use of gene transfer in vein grafts may reach beyond the treatment of the graft itself. The expression of therapeutic proteins by transduced grafts can lead to the treatment of diseases in tissues downstream to the location of graft implantation, further expanding the versatility of this bypass conduit.

C. Bioprosthetic Grafts

Prosthetic materials, such as PTFE or Dacron, often used as small-caliber arterial substitutes or in the construction of arteriovenous grafts, have been limited in their long-term use due to their thrombogenic surfaces. A bioengineering, cell-based strategy for decreasing or eliminating this thrombogeni-city may therefore yield a prosthetic graft capable of maintaining normal flow. Successful isolation of autologous endothe-lial cells and their seeding onto prosthetic grafts in animal models has been well characterized (76). Furthermore, it has been hypothesized that one can enhance the function of these endothelial cells via the transfer of genes prior to seeding of the cells on the graft surface. Such a bioprosthesis could be useful for delivering genetically engineered factors that would enhance graft function and survival or even provide an avenue for intravascular drug delivery. First indication for the possible use of this strategy was presented by Wilson at al. (10), who demonstrated successful endothelialization of a prosthetic vascular graft with autologous endothelial cells transduced with a recombinant retrovirus encoding the lacZ gene. Additionally, seeding of transduced vascular smooth muscle cells into the interstices of a PTFE graft then luminally seeded with untreated endothelial cells revealed stable expression of the reporter gene after 3-5 weeks (77).

Successful clinical applications of these concepts, however, have not been reported. In an attempt to decrease graft thrombogenicity, Dunn et al. (78) seeded 4-mm Dacron grafts with retrovirally transduced endothelial cells encoding the gene for human tissue plasminogen activator (TPA) and implanted them into the femoral and carotid circulation of sheep. The proteolytic action of TPA resulted in a decrease in seeded endothelial cell adherence, with no improvement in surface thrombogenicity. The use of VEGF in this context also has potentially significant clinical applications. VEGF, a potent endothelial cell mitogen, when transduced into a limited number of endothelial cells and placed on the graft surface, may promote endothelial survival and replication, and yield improved and more rapid graft coverage with a nonthrombogenic endothelial layer. Additionally, secretion of VEGF could lead to angiogenesis distal to the grafted area in what is likely to be an ischemic tissue bed. Yamamoto et al. (79) has demonstrated successful seeding of PTFE grafts with VEGF-transduced, adipose-derived, endothelial cells and expression of the transgene after several weeks. Further studies are needed to determine the local effect of VEGF secretion on endothelial cell proliferation along with distant angiogenic stimuli.

Was this article helpful?

0 0

Post a comment