Andrew H. Baker

Inhibition of Late Vein Graft Neointima Formation in Human and Porcine Models by Adenovirus-Mediated Overexpression of Tissue Inhibitor of Metalloproteinase-3

BACKGROUND Autologous saphenous vein coronary artery bypass graft surgery is complicated by late graft failure due to neointima formation and subsequent atherosclerosis. Growth factors and metalloproteinases (MMPs) act in concert to promote neointima formation. Tissue inhibitor of metalloproteinase-3 (TIMP-3), an extracellular matrix-associated MMP inhibitor, uniquely promotes apoptosis of isolated vascular smooth muscle cells. Here, we overexpressed TIMP-3 at the luminal surface of human saphenous veins before organ culture and in pig saphenous veins before interposition grafting into carotid arteries in vivo to assess neointima formation. METHODS AND RESULTS In both models, high TIMP-3 immunoreactivity occurred in the luminal and upper medial extracellular matrix after adenovirus delivery. MMP activity measured by in situ zymography was reduced throughout the veins, confirming a bystander effect. By use of 3 independent techniques, apoptosis levels in the neointima and medial layer were significantly elevated by TIMP-3 overexpression. Neointima formation was reduced by 84% in 14-day human organ cultures and by 58% in 28-day pig vein grafts (both P<0.05). In contrast, TIMP-2 overexpression had no effect on neointima formation in vivo. CONCLUSIONS Our results highlight the potential therapeutic benefit for TIMP-3 overexpression to reduce neointima formation associated with late vein graft failure.

Upregulation of basement membrane-degrading metalloproteinase secretion after balloon injury of pig carotid arteries.

Basement membrane-degrading metalloproteinases (gelatinases) appear necessary for vascular smooth muscle cell migration and proliferation in culture and for intimal migration of cells after balloon injury to the rat carotid artery. We investigated in the present study the secretion of gelatinases from pig carotid artery tissue after balloon injury. Segments of injured artery and segments proximal and distal to the area of injury were removed 3, 7, and 21 days after balloon dilatation. Medial explants from these segments were then cultured for 3 days, and the serum-free conditioned media were subjected to gelatin zymography. Production of 72- and 95-kD gelatinases was quantified by densitometry. Balloon-injured segments secreted significantly more 72- and 95-kD gelatinase than did paired distal segments at all time points. Release of both gelatinase activities was increased at 3 and 7 days relative to segments from uninjured arteries but declined again by 21 days after balloon injury. Similar results were found for gelatinase levels in extracts of arterial tissue. Consistent with the protein secretion data, in situ hybridization demonstrated that the mRNAs for both gelatinases were upregulated after balloon injury. Expression was prominent in medial smooth muscle cells, particularly around foci of necrosis, and in neointimal cells 3 and 7 days after balloon injury; 72-kD gelatinase mRNA persisted after 21 days and was prominent in regrown endothelial cells. The upregulation of gelatinase activity paralleled the time course of smooth muscle cell migration and proliferation in this model. We conclude that increased gelatinase production occurs in response to balloon injury and may play a role in permitting migration and proliferation of vascular smooth muscle cells.

Wild-type p53 gene transfer inhibits neointima formation in human saphenous vein by modulation of smooth muscle cell migration and induction of apoptosis

Patency of autologous human saphenous vein coronary artery bypass grafts (CABG) is compromised by intimal thickening and superimposed atherosclerosis, caused by migration of vascular smooth muscle cells (SMC) to the intima where they proliferate. Here, using adenoviral transfer, we have targeted SMCs using wild-type p53 (wt p53) overexpression. Initial in vitro analyses demonstrated that wt p53 overexpression had no effect on SMC proliferation but promoted apoptosis, which was inhibited by co-expression of bcl2 or crmA. Wt p53 inhibited SMC invasion through reconstituted matrices, a phenotype not affected by bcl2 or crmA. Overexpression of wt p53 in human saphenous vein before organ culture significantly induced apoptosis (P < 0.01, Students t test) without affecting proliferation rates either in the media or in the intima. SMC migration was, however, significantly reduced by wt p53 (P < 0.01, Students t test). Intimal thickening and the number of neointimal cells were reduced by 89% and 73%, respectively, after 14 days (P < 0.01 and P < 0.001, respectively, Students t test). This study demonstrates that overexpression of wt p53 promotes apoptosis and inhibits migration of SMC leading to reduced intimal thickening. This maybe a useful approach for increasing patency rates in CABG procedures in the clinic.

Arteriolar Genesis and Angiogenesis Induced by Endothelial Nitric Oxide Synthase Overexpression Results in a Mature Vasculature

Background—Generation of physiologically active vascular beds by delivery of combinations of growth factors offers promise for vascular gene therapy. Methods and Results—In a mesenteric model of physiological angiogenesis, combining endothelial nitric oxide synthase (eNOS) (and hence NO production) with VEGF and angiopoietin-1 overexpression resulted in a more functional vascular phenotype than growth factor administration alone. eNOS gene delivery upregulated eNOS, VEGF, and Ang-1 to similar levels as gene transfer with VEGF or Ang-1. eNOS overexpression resulted in neovascularization to a similar extent as VEGF and Ang-1 combined, but not by sprouting angiogenesis. Whereas combining Ang-1 and VEGF increased both exchange vessels and conduit vessels, neither growth factor nor eNOS alone resulted in vessels with smooth muscle cell (SMC) coverage. In contrast, combining all three generated microvessels with SMCs (arteriolar genesis) and further increased functional vessels. Use of a vasodilator, prazosin, in combination with Ang1 and VEGF, but not alone, also generated SMC-positive vessels. Conclusion—Coexpression of eNOS, VEGF, and Ang-1 results in a more mature vascularization of connective tissue, and generates new arterioles as well as new capillaries, and provides a more physiological therapeutic approach than single growth factor administration, by combining hemodynamic forces with growth factors.

Simple methods for preparing recombinant adenoviruses for high-efficiency transduction of vascular cells

Adenoviruses are icosahedral viruses 70-90 nm in diameter with a double-stranded, linear DNA genome of approx 36 kb. They are widely utilized in gene transfer protocols owing to their relative ease of genetic manipulation, ability to grow to high titres (10(9)-10(12) plaque forming units [pfu]/mL), and their ability to infect both dividing and nondividing cells efficiently. The latter reason makes them extremely suitable for investigations in vascular systems where the low proliferative indices of cells is a limiting factor for retroviral gene transfer.

Potential applications of tissue inhibitor of metalloproteinase (TIMP) overexpression for cancer gene therapy.

Invasion and metastasis of malignant cells is a multistep process which involves detachment of cells from primary tumor, degradation of structural barriers such as basement membrane and collagenous extracellular matrix (ECM), as well as migration of cells through the degraded matrix (see MacDougal and Matrisian, 1995; Johnsen et al., 1998). Tumor invasion in vivo involves interplay between tumor cells, stromal cells and inflammatory cells, all of which can express a variable set of proteolytic enzymes and contribute to degradation of peritumoral stromal matrix. Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases collectively capable of degrading essentially all components of the ECM (see Birkedal-Hansen et al., 1993). Upregulation of MMP expression has been documented in a number of malignant tumors and has been associated with invasive capacity of tumors. In addition, MMPs apparently play a role in tumor-induced angiogenesis. The activity of MMPs is inhibited specifically by tissue inhibitors of metalloproteinases (TIMPs). Here we discuss the role of MMPs

Biological consequences of a point mutation at codon 969 of the FMS gene

The FMS proto-oncogene encodes the cell surface receptor for colony stimulating factor-1 (CSF-1). Mutations of the FMS gene at codon 969, in the C-terminal region of the gene, have been detected in haematological malignancies. To ascertain the biological significance of a mutation at this codon, we have used a murine haematopoietic cell line, FDC-P1, containing a mutation at codon 969 that results in a phenylalanine replacing a tyrosine. FMS 969 mutant cells and v-fms transfected cells conferred interleukin 3 (IL-3) independent stimulation of FDC-P1 cells, whereas cells transfected with a wild-type FMS construct required exogenous IL-3 for growth. FDC-P1 cells containing a FMS 969 mutation and v-fms transfected cells were tumorigenic in nude mice. Binding studies with radioidonated CSF-1 revealed saturable specific binding in FMS wild-type cells with a Km of 0.9 mM; however, mutant FMS-containing cells did not display saturation kinetics, but instead exhibited a linear relationship between ligand concentration and amount bound. Constitutive expression of FOS was detected in 969 mutant cells in the absence of exogenous CSF-1, a phenotype that was only inducible in wild-type cells in response to CSF-1. FOS and JUNB expression by v-FMS transfected cells showed a similar pattern to FMS wild-type cells. This mutation has been detected in patients with haematological malignancies, and illustrates that the pathway of FMS 969 phenylalanine mutations and v-fms induced pathogenesis can be distinguished. These data indicate that there is a biological role for FMS codon 969 phenylalanine mutation which results in transformation of FDC-P1 cells.

Delivery of Recombinant Adenoviruses to Human Saphenous Vein

The human saphenous vein is the most commonly used conduit for coronary artery bypass grafting owing to its ready availability, ease of harvesting, and favorable surgical handling (1). However, it suffers from a progressive decline in patency, resulting in a graft failure rate of 50% after 10 yr (2,3). This high failure rate is caused by either early thrombosis occlusion, which occurs in the first year after graft implantation, or the later development of intimal thickening and superimposed atherosclerosis (1-3).