Cquiescence

c-myc c-myh edc2 kinase PCMA

cyclin A

c-myc c-myb cdc2 kinase PCNA

Figure 1 Principle of E2F ''decoy'' strategy. TTTCGCGC, consensus sequence for the E2F binding site. (A) In quiescent cell state, the transcription factor E2F is complexed Rb (retinoblastoma gene product), cyclin A, and cyclin-dependent kinase cdK2. (B) Phosphorylation of releases free E2F, which binds to cis elements of the cell-cycle regulatory genes, resulting in the transactivation of these genes. (C) The E2F decoy cis-element double-stranded oligonucleotide binds to free E2F, preventing E2F-mediated transactivation of cell-cycle regulatory genes. See the color insert for a color version of this figure.

Figure 1 Principle of E2F ''decoy'' strategy. TTTCGCGC, consensus sequence for the E2F binding site. (A) In quiescent cell state, the transcription factor E2F is complexed Rb (retinoblastoma gene product), cyclin A, and cyclin-dependent kinase cdK2. (B) Phosphorylation of releases free E2F, which binds to cis elements of the cell-cycle regulatory genes, resulting in the transactivation of these genes. (C) The E2F decoy cis-element double-stranded oligonucleotide binds to free E2F, preventing E2F-mediated transactivation of cell-cycle regulatory genes. See the color insert for a color version of this figure.

Figure 2 Inhibition of neointimal hyperplasia by in vivo gene transfer of endothelial cell-nitric oxide synthase (ecNOS) in balloon-injured rat carotid arteries. (A) uninjured control artery (CTRL); (B) injured, untransfected artery (INJ); (C) injured, control vector transfected artery (INJ + CV); (D) injured, ecNOS transfected artery (INJ + NOS).

Figure 2 Inhibition of neointimal hyperplasia by in vivo gene transfer of endothelial cell-nitric oxide synthase (ecNOS) in balloon-injured rat carotid arteries. (A) uninjured control artery (CTRL); (B) injured, untransfected artery (INJ); (C) injured, control vector transfected artery (INJ + CV); (D) injured, ecNOS transfected artery (INJ + NOS).

dominant noncellular components of complex atherosclerotic plaque in humans.

An ex vivo gene transfer approach, involving implantation of genetically modified endothelial cells or VSMCs at sites of arterial injury, is also being investigated. Conte et al. successfully demonstrated efficient repopulation of denuded rabbit arteries with genetically modified autologous endothelial cells (52). The need to harvest autologous donor tissue for target cells, coupled with the increased costs and complexity of tissue culture, have greatly dampened the enthusiasm for this strategy in favor of more direct methods. Nonetheless, it remains clinically feasible and, in the case of endothelial cells, the implanted cells alone may confer beneficial properties to the healing arterial wall. Application of these cell transplantation approaches would be greatly facilitated by the development of ''universal donor'' cell lines in which major histocompatibility antigens have been ''knocked out.'' Such a development, while clearly years or decades away, may no longer be merely science fiction fantasy.

In summary, it would appear that the application of gene therapy for post angioplasty restenosis may be somewhat premature (53). In addition to major obstacles in delivering gene transfer agents to the atherosclerotic vessel wall, the fundamental biological process remains incompletely understood. Nonetheless, continued progress on each of these fronts warrants an optimistic view for genetic approaches to control the arterial injury response. These developments will undoubtedly yield important corollaries for the surgical treatment of arterial occlusive diseases as well.

B. Gene Therapy vs. Molecular Therapy for Angiogenesis

The vascularization observed in neoplastic tissue led researchers such as Judah Folkman in the 1970s to investigate the role of molecular factors in the induction of new blood vessel growth (54). The subsequent identification and characterization of ''angiogenic'' growth factors created an opportunity not only to target the growth of solid tumors, but also to attempt the therapeutic ''neovascularization'' of tissue rendered ischemic by occlusive disease in the native arterial bed. An-giogenesis has come to refer more strictly to the sprouting of new capillary networks from preexisting vascular structures, whereas vasculogenesis is the de novo development of both simple and complex vessels during embryonic development. Although it has been clearly established in a number of animal models that angiogenic factors can, in fact, stimulate the growth of capillary networks in vivo, it is less certain that these molecules can induce the development of larger, more complex vessels in adult tissues that would be capable of carrying significantly increased bulk blood flow. Nevertheless, the possibility of an improvement even of just the microvascular collateralization as a ''biological'' approach to the treatment of tissue ischemia has sparked the beginning of human clinical trials in neovascularization therapy.

After the first description of the angiogenic effect of fibro-blast growth factors (FGFs), an abundance of ''proan-giogenic'' factors were discovered to stimulate either endothe-lial cell proliferation, enhanced endothelial cell migration, or both. Many of these factors possess heparin-binding domains, which not only increase their retention in heparin-rich extracellular matrix, but also play critical roles in mediating the interaction of the factors with cell surface receptors. Although the list of angiogenic factors includes such diverse molecules as insulin-like growth factor, hepatocyte growth factor, angio-poeitin and platelet-derived endothelial growth factor, the molecules that have received the most attention as potential therapeutic agents for neovascularization are vascular endo-thelial growth factor (VEGF) and two members of the FGF family, acidic FGF (FGF-1) and basic FGF (FGF-2).

Whereas all angiogenic factors share some ability to stimulate capillary growth in classical models such as the chick allantoic membrane, much debate persists regarding the optimum agent and the optimum route of delivery for angiogenic therapy in the ischemic human myocardium or lower extremity. VEGF may be the most selective agent for stimulating endothelial cell proliferation, although VEGF receptors are also expressed on a number of inflammatory cells, including members of the monocyte-macrophage lineage (55). This selectivity has been viewed as an advantage, since the unwanted stimulation of fibroblasts and VSMCs in native arteries might exacerbate the growth of neointimal or atherosclerotic lesions. Despite this theoretical selectivity, however, the experimental use of VEGF in animal models has been associated not only with capillary growth, but also the development of more complex vessels involving these other cell types (56). The FGFs are believed to be even more potent stimulators of endothelial cell proliferation, but, as their name implies, are much less selective in their proproliferative action (57).

Optimizing the route of drug delivery depends heavily on the pharmacokinetic properties of the agent. Angiogenesis, however, is a very complex biological process involving multiple cell types engaged in multiple activities, including extracellular tissue dissolution and remodeling, cell proliferation, cell migration, cell recruitment, and programmed cell death. The role of any single agent must be understood within the complicated orchestration of multiple signaling agents and effectors. Despite the large amount of data that has become available in the past two decades, details of the cellular and molecular mechanisms of angiogenesis remain poorly understood. Still, it is believed that many of the known angiogenic factors, including VEGF and the FGFs, are exquisitely potent and would not, therefore, require large or prolonged dosing regimens. These conclusions are partly based on the results of in vivo experiments in which a broad range of dosing strategies, ranging from implantation of sustained release formulations to single intra-arterial boluses, have been reported to induce similarly successful increases in tissue perfusion (55).

The contribution of gene therapy to the potential development of therapeutic neovascularization is primarily one of drug delivery. The availability of the genetic sequences encoding these paracrine peptide agents provides an opportunity for the establishment of local tissue factories for drug production. Both intravascular as well as extravascular modes of gene product delivery are feasible, as gene transfer can be attempted either in the walls of vessels feeding the ischemic tissue or in the target myocardial or skeletal tissue itself. In fact, muscle tissue of both myocardial and skeletal origin are among the most receptive for gene transfer with the simplest of agents, pure plasmid DNA (58). Adenoviral vectors are also effective at achieving transgene expression in these muscle cells. A number of reports have suggested that plasmid injections can result in long-term gene expression in these muscle tissues, whereas the higher levels of expression associated with adenoviral vectors is likely limited to 1 or 2 weeks (18,59).

Preclinical studies of angiogenic gene therapy have utilized a number of models of chronic ischemia. An increase in capillary density was reported in an ischemic rabbit hind limb model after VEGF administration, and these results did not differ significantly regardless of whether VEGF was delivered as a single intra-arterial bolus of protein, plasmid DNA applied to surface of an upstream arterial wall, or direct injection of the plasmid into the ischemic limb (55). Direct injection of an adenoviral vector encoding VEGF also succeeded in improving regional myocardial perfusion and ventricular fractional wall thickening at stress in a model of chronic myocar-dial ischemia induced via placement of a slowly occluding Ameroid constrictor around the the circumflex coronary artery in pigs (60).

Unlike VEGF, FGF-1 and -2 do not possess signal sequences that facilitate secretion of the protein, so that transfer of these genetic sequences is less likely to yield an adequate supply of growth factor to target endothelial cells. To overcome this limitation, Tabata and associates constructed a plasmid encoding a modified FGF-1 molecule onto which a hydrophobic leader sequence had been added to enhance secretion (61). Delivery of this plasmid to the femoral artery wall, even at very low transfection efficiencies, was found to improve capillary density and reduce vascular resistance in the ischemic rabbit hind limb. Applying a similar strategy, Giordano et al. employed intracoronary infusion of 1011 viral particles of an adenoviral vector encoding human FGF-5, which does contain a secretory signal sequence at its amino terminus, to achieve enhanced wall thickening with stress and a higher number of capillary structures per myocardial muscle fiber 2 weeks after gene transfer (62).

Another novel approach to molecular neovascularization has been the combination of growth factor gene transfer with a potentially synergistic method of angiogenic stimulation: transmyocardial laser therapy. The formation of transmural laser channels, though not yet fully established as an effective means of generating increased collateral flow, has had documented, clinical success in reducing angina scores and improving myocardial perfusion in otherwise untreatable patients. In a porcine Ameroid model, Sayeed-Shah et al. found that direct injection of plasmid DNA encoding VEGF in the region surrounding laser channel formation yielded better normalization of myocardial function than either therapy alone (63), and this therapeutic strategy can now be delivered either through minimally invasive thoracotomy or a percutaneous catheter-based approach (Fig. 3).

A number of Phase I safety studies have already been reported in which angiogenic factors or the genes encoding these factors have been administered to patients in small numbers (64,65). These studies have involved either the use of an-giogenic factors in patients with peripheral vascular or coronary artery disease who were not candidates for conventional revascularization therapies, or the application of proan-

* Porcine ameroid model of / chronic ischemia

V o / with CO2 laser

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

Figure 3 Combined gene transfer and transmyocardial laser revascularization (TMR). Schematic representation of chronic ischemia induced by placement of Ameroid constrictor around the circumflex coronary artery in pigs. Ischemic hearts that underwent TMR, followed by injection of plasmid encoding VEGF, demonstrated better normalization of myocardial function than either therapy alone. See the color insert for a color version of this figure.

giogenic factors as an adjunct to conventional revasculariza-tion. The modest doses of either protein factors or genetic material delivered in these studies were not associated with any acute toxicities. Concerns remain, however, regarding the safety of potential systemic exposure to molecules known to enhance the growth of possible occult neoplasms, or that can enhance diabetic retinopathy and potentially even occlusive arterial disease itself. Despite early enthusiasm, there is also little experience with the administration of live viral vectors in extremely large numbers to a large number of patients, and it is uncertain whether potential biological hazards of reversion to replication-competent states or mutation and recombination will eventually become manifest.

In addition to issues of safety, it is also unclear whether the clinical success of conventional revascularization, which has involved the resumption of lost bulk blood flow through larger conduits, will be reproduced via biological strategies that primarily involve increased microscopic collateral networks. It must also be remembered that neovascularization is itself a naturally occurring process, and that the addition of a single factor may not overcome conditions that have resulted in an inadequate endogenous neovascularization response in patients suffering from myocardial and lower limb ischemia. Despite these limitations, angiogenic gene therapy may provide an alternative not currently available to a significant number of patients suffering from untreatable disease, and may offer an adjunct to traditional therapies that improves their long-term outcomes.

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