Vein Graft Disease

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Autologous vein grafts remain the most commonly used conduits for surgical revascularization of the heart and lower extremities. Given the failure of traditional therapies at improving long-term vein graft function, gene therapy offers a new opportunity for reducing the morbidity and increased costs associated with the current limitations on functional graft survival. The vein graft offers an unusual opportunity for combining intact tissue in vivo gene-transfer techniques with the increased safety of an ex vivo application of the transfection medium. Manipulation of transfection conditions, including increased exposure time and controlling components of the transfection medium, also can be more easily achieved. Some researchers have begun to explore the possibility of ex vivo virus-mediated gene transfer in autologous vein grafts. Chen et al.49 demonstrated the expression of the marker gene P-galactosidase along the luminal surface and in the adventitia of porcine vein grafts infected with a replication-deficient adenoviral vector at the time of surgery. In this same study, short-term expression of soluble VCAM-1 was documented after transfection of vein grafts.

The Dzau laboratory hypothesized that genetic engineering could alter the ability of the grafts to mount a hyperplastic response to acute injury while leaving intact their ability to respond to chronic hemodynamic stress via a hypertrophic response, such as that seen in arteries exposed to hypertension. This group used HVJ liposomes to deliver a combination of ASOs to cdc2/PCNA to rabbit veins at the time of grafting into the carotid artery and observed a greater than 90 percent inhibition of smooth muscle cell (SMC) proliferation during the first postoperative week63 (Fig. 83). This blockade of cell cycle progression resulted in a near-complete inhibition of neointimal hyperplasia. Instead, the vein graft wall was shifted to an adaptive process of medial hypertrophy. Having redirected the genetically engineered grafts away from neointimal hyperplasia and toward medial hypertrophy as an adaptation to the arterial environment, the susceptibility of these ASO-treated grafts to accelerated atherogenesis was tested. Control ASO-treated and untreated grafts placed in cholesterol-fed rabbits developed significant foam cell lesions and plaque within 6 weeks after surgery. ASO-treated grafts that had remained free of neointima formation, however, resisted macrophage invasion and the development of macroscopic plaque. This inhibition of cell cycle progression is likely to have effects on the phenotypes of the vascular cells undergoing remodeling after vein grafting, and these changes are likely to affect the proatherogenic environment of the normal graft wall. For example, the endothelium of ASO-treated grafts retained more of its capacity to produce nitric oxide and resist monocyte adhesion in comparison with untreated or control ASO-treated grafts.64

Coronary Artery Rabbit

Figure 8-3: Control oligonucleotide-treated (A and B) and ASO (against cdc2 kinase/PCNA)-treated vein grafts (C and D) in hypercholesterolemic rabbits 6 weeks after surgery (x70). Sections of 5 mm were stained with hematoxylin/van Gieson (A and C) and a monoclonal antibody against rabbit macrophages (B and D). Arrows indicate the location of the internal elastic

Figure 8-3: Control oligonucleotide-treated (A and B) and ASO (against cdc2 kinase/PCNA)-treated vein grafts (C and D) in hypercholesterolemic rabbits 6 weeks after surgery (x70). Sections of 5 mm were stained with hematoxylin/van Gieson (A and C) and a monoclonal antibody against rabbit macrophages (B and D). Arrows indicate the location of the internal elastic lamina. (From Mann et al.,63 with permission.)

Gene Transfer and Vascular Remodeling

Molecular cardiovascular research has resulted in significant gains in the knowledge of disease processes at cellular and molecular levels and has led to the characterization of expressed genes in diseased blood vessels. These gene products play autocrine and/or paracrine roles in vascular pathophysiology.

Nabel et al.65 overexpressed an expression vector encoding a secreted form of fibroblast growth factor 1 (FGF-1) in porcine arteries. FGF-1 expression was associated with intimal thickening of the transfected vessels together with neocapillary formation in the expanded intima. These findings suggest that FGF-1 induces intimal hyperplasia in the arterial wall in vivo, and through its ability to stimulate angiogenesis in the neointima, FGF-1 could stimulate neovascularization of atherosclerotic plaques. In the same porcine model, the overexpression of transforming growth factor Pi (TGF-f-'l) in normal arteries resulted in substantial production of extracellular matrix accompanied by intimal and medial hyperplasia.05 These findings demonstrated that TGF-ftl differentially modulates extracellular matrix production and cellular proliferation in the arterial wall and plays a reparative role in response to arterial injury. The increased production of extracellular matrix that accompanied the intimal and medial hyperplasia was not observed following expression of other growth factor genes in the vessel wall, including genes for platelet-derived growth factor (PDGF-BB)27,66 or the secreted form of FGF-1.62 Porcine arteries transfected with human PDGF-BB demonstrated intimal hyperplasia with increased numbers of intimal smooth muscle cells. An increased deposition of procollagen, however, as seen in TGF-ftl-transfected vessels, was not observed. By stimulating the formation of extracellular matrix, it is possible that TGF-ftl could promote healing following vascular injury, limiting the extensive cellular intimal hyperplasia observed with PDGF-BB.66

The pathogenesis of vascular diseases such as hypertension involves a process of vascular remodeling associated with increased vascular hypertrophy and activation of the local angiotensin system. Angiotensin II has been shown to stimulate the growth and proliferation of vascular smooth muscle as well as collagen biosynthesis in vitro. Its in vivo role has been inferred from experiments using angiotensin converting enzyme (ACE) inhibitors. Since these drugs produce hemodynamic effects, a direct role of local angiotensin in vascular remodeling was not clear. To study the local effects of angiotensin, Morishita et al.67 overexpressed ACE within the vascular wall. Immunoreactive ACE activity was noted in medial vascular smooth muscle cells as well as in intimal endothelial cells. Vascular ACE activity was associated with increased DNA synthesis and vascular protein content via the local production and action of vascular angiotensin II without changes in systemic blood pressure. Parallel to these biochemical changes, medial thickening of ACE-transfected vessel segments was noted, without changes in luminal diameters, implying medial wall hypertrophy by local production of angiotensin II. In a subsequent study, Nakajima et al.68 demonstrated that overexpression of the type 2 angiotensin II (AT2) receptor in balloon-injured rat carotid arteries exerts an antiproliferative effect, counteracting the growth action of AT1 receptors.

One approach to the treatment of vascular diseases that are characterized by excessive cell proliferation is to overexpress a gene that inhibits cellular proliferation. It is important that expression of the gene proceed during the time period when intimal cells undergo proliferation following vascular injury. This may vary between animal models, and it is likely that in humans, cell proliferation following angioplasty, stent placement, or bypass graft surgery may proceed over a longer period of time than in animal models. Nonetheless, several gene products have proven efficacious in appropriate animal models of vascular injury. Most of these approaches are based on arresting vascular cells in G1 or S phase of the cell cycle (Fig. 8-4). One approach is to express the herpes simplex virus thymidine kinase gene (HSVtk). HSVtk encodes for the enzyme thymidine kinase that phosphorylates the nucleoside analog ganciclovir or acyclovir into a metabolite that disrupts replication of DNA during S phase of the cell cycle. A by-product of this biochemical reaction is diffusible to adjacent cells, where it is incorporated in replicating cells, leading to inhibition of cell replication. This property is termed a bystander effect and allows for inhibition of replication in a greater number of cells than transfected. This model was established initially in a pig peripheral artery model of vascular injury, where intimal hyperplasia was decreased by 50 percent.69 Subsequent investigations in the rat, rabbit, and a transplant model also demonstrated reductions in cell proliferation and lesion formation by about 50 percent.70-73 This approach is currently being investigated in a model of in-stent restenosis. Chang et al.74 demonstrated that localized arterial infection with a replication-defective adenovirus encoding a nonphosphorylatable, constitutively active form of the retinoblastoma gene product at the time of angioplasty significantly reduced SMC proliferation and neointima formation in both the rat carotid and porcine femoral artery models of restenosis. The cyclin-dependent kinase inhibitors p21 and p27 that arrest smooth muscle cells in G1 phase of the cell cycle are also potent negative regulators of lesion formation after vascular injury.75-78 Ras proteins are key transducers of mitogenic signals from the cell membrane to the nucleus. The local delivery of vectors expressing ras transdominant negative mutants, which interfere with ras function, reduced neointimal lesion formation in a rat carotid artery balloon-injury model.79

Cell Injury Cycle

Figure 8-4: Regulation of the cell cycle. Progression through the G1 phase of the cell cycle is regulated by the assembly and phosphorylation of cyclins and cyclin-dependent kinases (CDKs). The cyclin-CDK complexes are inhibited by cyclin-dependent kinase inhibitors (CKIs), of which p21 and p27 are examples. These CKIs lead to G1 arrest. Inhibition of Rb phosphorylation, inactiviation of E2F, or inhibition of cyclin A and B also lead to disruption of DNA synthesis and inhibition of cell proliferation. (From Tanner et al.,96 with permission.)

Figure 8-4: Regulation of the cell cycle. Progression through the G1 phase of the cell cycle is regulated by the assembly and phosphorylation of cyclins and cyclin-dependent kinases (CDKs). The cyclin-CDK complexes are inhibited by cyclin-dependent kinase inhibitors (CKIs), of which p21 and p27 are examples. These CKIs lead to G1 arrest. Inhibition of Rb phosphorylation, inactiviation of E2F, or inhibition of cyclin A and B also lead to disruption of DNA synthesis and inhibition of cell proliferation. (From Tanner et al.,96 with permission.)

To assess the effect of endothelial cell nitric oxide synthase (ecNOS) on vessel lesion formation, a DNA vector encoding ecNOS was expressed in a rat model of arterial injury80 (Fig. 8-5). Four methods were used to verify ecNOS expression: transgene protein expression by Western blot, localization of enzyme expression by in situ histochemical staining, enzymatic activity of the transgene product, and vascular reactivity in response to the transgene. Overexpression of ecNOS led to vasorelaxation and 70 percent inhibition of neointima formation after balloon injury. This same approach has been investigated in a pig coronary model.8! The loss of ecNOS may play a fundamental role in the pathogenesis of vascular diseases, including atherosclerosis. The overexpression of ecNOS may be useful for gene therapy of neointimal hyperplasia and associated local vasospasm after vascular injury.

Corelation Coronary Corotid Artaryl

Figure 8-5: Inhibition of neointimal hyperplasia by in vivo gene transfer of endothelial cell nitric oxide synthase (ecNOS) in balloon-injured rat carotid arteries. A. Normal artery. B. Injured, untransfected artery. C. Injured, control vector-transfected artery. D. Injured, ecNOS-transfected artery. M, media; N, neointima. (From von der Leyen et al.,80 with permission.)

Figure 8-5: Inhibition of neointimal hyperplasia by in vivo gene transfer of endothelial cell nitric oxide synthase (ecNOS) in balloon-injured rat carotid arteries. A. Normal artery. B. Injured, untransfected artery. C. Injured, control vector-transfected artery. D. Injured, ecNOS-transfected artery. M, media; N, neointima. (From von der Leyen et al.,80 with permission.)

Angiogenesis

Angiogenic growth factors may be useful to augment collateral artery development in animal models of myocardial and hind limb ischemia. Initial studies using intramuscular injections of angiogenic proteins, including basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF) into the hind limbs of rabbits with surgically induced ischemia lead to increased capillary densities and augmented blood flow.82,83 These findings have been extended to genetransfer approaches using vascular endothelial growth factor (VEGF). Following gene transfer of VEGF via a hydrogel balloon, increased numbers of capillary vessels also were observed in a rabbit model of hind limb ischemia, and improvement of resting and maximum flow was achieved that was comparable with that of a single administration of VEGF protein.84 Intracoronary delivery of a recombinant adenovirus that encodes FGF-5 has been shown to induce collateral blood flow and restore myocardial function in a pig model of myocardial ischemia.85 While the studies are encouraging, our understanding of the process by which VEGF, angiopoietin, and other angiogenic proteins lead to blood vessel formation and maturation is still incomplete.

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