No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Gene Therapy as a Therapeutic Intervention for Vascular Disease
Abstract
Gene therapy for the treatment of many medical problems, including vascular disease, has become the subject of increasing discussion in both the scientific literature and the national press over the past decade. This review will examine the history and current status of gene therapy for vascular proliferative disorders and advanced chronic peripheral and cardiac ischemia.
... . In another review, Meyerson et al. (43) discuss advances in gene therapy for vascular proliferative disorders and chronic peripheral and cardiac ischemia. ...
Somatic gene therapy of vascular diseases is a promising new field in modern medicine. Recent advancements in gene transfer technology have greatly evolved our understanding of the pathophysiologic role of candidate disease genes. With this knowledge, the expression of selective gene products provides the means to test the therapeutic use of gene therapy in a multitude of medical conditions. In addition, with the completion of genome sequencing programs, gene transfer can be used also to study the biologic function of novel genes in vivo.
Novel genes are delivered to targeted tissue via several different vehicles. These vectors include adenoviruses, retroviruses, plasmids, plasmid/liposomes, and oligonucleotides. However, each one of these vectors has inherent limitations. Further investigations into developing delivery systems that not only allow for efficient, targeted gene transfer, but also are stable and nonimmunogenic, will optimize the clinical application of gene therapy in vascular diseases. This review further discusses the available mode of gene delivery and examines six major areas in vascular gene therapy, namely prevention of restenosis, thrombosis, hypertension, atherosclerosis, peripheral vascular disease in congestive heart failure, and ischemia. Although we highlight some of the recent advances in the use of gene therapy in treating vascular disease discovered primarily during the past two years, many excellent studies published during that period are not included in this review due to space limitations.
The following is a selective review of practical uses of gene transfer therapy in vascular diseases. This review primarily covers work performed in the last 2 years. For earlier work, the reader may refer to several excellent review articles. For instance, Belalcazer et al. (6) reviewed general aspects of somatic gene therapy and the different vehicles used for the delivery of therapeutic genes. Gene therapy in restenosis and stimulation of angiogenesis in the cardiac muscle are discussed in reviews by several investigators (13,26,57, 74,83). In another review, Meyerson et al. (43) discuss advances in gene therapy for vascular proliferative disorders and chronic peripheral and cardiac ischemia.
... Gene therapy is under investigation for various kinds of cardiovascular diseases [Meyerson, 1999, Morishita et al., 2001,Kozarsky, 2001,McKay, 2001,Nikol, 2001,Isner, 2002, using many different therapeutic genes [Tio et al., 1998,Nakamura, 2002. For several prevalent disease conditions, e.g. ...
Numerous gene therapy vectors, both viral and non-viral, are taken into the cell by endocytosis, and for efficient gene delivery the therapeutic genes carried by such vectors have to escape from endocytic vesicles so that the genes can further be translocated to the nucleus. Since endosomal escape is often an inefficient process, release of the transgene from endosomes represents one of the most important barriers for gene transfer by many such vectors. To improve endosomal escape we have developed a new technology, named photochemical internalisation (PCI). In this technology photochemical reactions are initiated by photosensitising compounds localised in endocytic vesicles, inducing rupture of these vesicles upon light exposure. The technology constitutes an efficient light-inducible gene transfer method in vitro, where light-induced increases in transfection or viral transduction of more than 100 and 30 times can be observed, respectively. The method can potentially be developed into a site-specific method for gene delivery in vivo. This article will review the background for the PCI technology, and several aspects of PCI induced gene delivery with synthetic and viral vectors will be discussed. Among these are: (i) The efficiency of the technology with different gene therapy vectors; (ii) use of PCI with targeted vectors; (iii) the timing of DNA delivery relative to the photochemical treatment. The prospects of using the technology for site-specific gene delivery in vivo will be thoroughly discussed, with special emphasis on the possibilities for clinical use. In this context our in vivo experience with the PCI technology as well as the clinical experience with photodynamic therapy will be treated, as this is highly relevant for the clinical use of PCI-mediated gene delivery. The use of photochemical treatments as a tool for understanding the more general mechanisms of transfection will also be discussed.
During the past decade, gene therapy for the treatment of many inherited and acquired medical problems has become the subject of increasing focus in both the scientific literature and the lay press. This review examines the history and currant status of gene therapy for advanced chronic peripheral and myocardial ischemia.
The traditional risk factors associated with the development of cardiovascular disease (CVD) account for approximately 50% of the variability in the risk for developing heart disease. Research in the genetic basis of CVD has helped scientists begin to quantify the nature of the additional unexplained variance. CVD may result from a variety of genetic causes, including single-gene mutations, the combined effect of more than one mutation, and the interaction of multiple genes and environmental factors. This article describes genetic influences on risk factors for CVD, discusses three genetic CVD disorders, and highlights recent developments in genetic therapy for the treatment of CVD.
Due to differences in their knowledge base, scientists, health care professionals, and the general public have varying perceptions of the expectations and risks of using gene therapy for the treatment of cardiovascular disease. Gene therapists are aware of the importance of selecting the right vector, the right gene, and the right administration procedure for a specific application and for characterization of the heterogeneity of the physiologic response to gene expression. They also recognize the need for large-scale clinical trials to demonstrate short- and long-term safety, in addition to efficacy, for widespread acceptance. Interventional cardiologists are more likely to judge a therapy primarily on its ability to improve cardiovascular pathophysiology and function, although they too will demand safety data. Once convinced of the benefits of a gene therapy, interventional cardiologists will rely on regulatory and reimbursement authorities to approve the procedure. Attitudes of less-specialized physicians toward gene therapy and, specifically, therapeutic angiogenesis will be influenced by the level of clinical trial activity in their country and opinions expressed in the media. However, all will want safety and efficacy data as well as clear guidance on the type of patients who should be referred for treatment. Nurses' roles are set to expand with the introduction of gene-based therapies; most will need further genetics education to rise to these challenges with confidence. The success of a gene therapy will also rely on patients' perceptions and their willingness to receive the treatment. Any misconceptions based on unreliable information sources need to be addressed.
The mainstay of treatment for long-segment small-vessel chronic occlusive disease not amenable to endovascular intervention remains surgical bypass grafting using autologous vein. The procedure is largely successful and the immediate operative results almost always favorable. However, the lifespan of a given vein graft is highly variable, and less than 50% will remain primarily patent after 5 years. The slow process of graft malfunction is a result of the vein's chronic maladaptive response to the systemic arterial environment, its primary component being the uncontrolled proliferation of vascular smooth muscle cells (SMCs). It has recently been suggested that this response might be attenuated through pre-implantation genetic modification of the vein, so-called gene therapy for the extension of vein graft patency. Gene therapy seems particularly well suited for the prevention or postponement of vein graft failure since: (1) the stimulation of SMC proliferation appears to largely be an early and transient process, matching the kinetics of current gene transfer technology; (2) most veins are relatively normal and free of disease at the time of bypass allowing for effective gene transfer using a variety of systems; and (3) the target tissue is directly accessible during operation because manipulation and irrigation of the vein is part of the normal workflow of the surgical procedure. This review briefly summarizes the current knowledge of the incidence and basic mechanisms of vein graft failure, the vector systems and molecular targets that have been proposed as possible pre-treatments, the results of experimental genetic modification of vein grafts, and the few available clinical studies of gene therapy for vascular proliferative disorders.
Influenza virosomes are reconstituted influenza virus envelopes that may be used as vaccines or as carrier systems for cellular delivery of therapeutic molecules. Here we present a procedure to generate influenza virosomes as a stable dry-powder formulation by freeze-drying (lyophilization) using an amorphous inulin matrix as a stabilizer. In the presence of inulin the structural integrity and fusogenic activity of virosomes were fully preserved during freeze-drying. For example, the immunological properties of the virosomes, i.e. the HA potency in vitro and the immunogenic potential in vivo, were maintained during lyophilization in the presence of inulin. In addition, compared to virosomes dispersed in buffer, inulin-formulated virosomes showed substantially prolonged preservation of the HA potency upon storage. Also the capacity of virosomes to mediate cellular delivery of macromolecules was maintained during lyophilization in the presence of inulin and upon subsequent storage. Specifically, when dispersed in buffer, virosomes with encapsulated plasmid DNA lost their transfection activity completely within 6 weeks, whereas their transfection activity was fully preserved for at least 12 weeks after incorporation in an inulin matrix. Thus, in the presence of inulin as a stabilizing agent, the shelf-life of influenza virosomes with and without encapsulated macromolecules was considerably prolonged. Formulation of influenza virosomes as a dry-powder is advantageous for storage and transport and offers the possibility to develop needle-free dosage forms, e.g. for oral, nasal, pulmonal, or dermal delivery.
Virtually every subspecialty in medicine in one way or another deals with angiogenesis-associated physiological or pathological processes and, without exception, every organ system in the body has many diseases in which angiogenesis is an important component. This in itself makes the study of angiogenesis mandatory, in both basic science and clinical settings. Yet the study of angiogenesis does not require this justification. As a biological process it is extraordinarily rich, touching on virtually every aspect of modern cell biology, making it almost impossible for molecular biologists, biochemists and morphologists to ignore. Considerable therapeutic benefit can now be obtained through positive or negative manipulation of the angiogenic process, and this is due in large part to the rapid transfer to the clinical setting of knowledge acquired through a cell biological approach.
The possibility of using an exclusively percutaneous strategy to deliver foreign DNA to normal and balloon-dilated atherosclerotic arteries was studied by analysis of transfection efficiency in a rabbit model. A total of 22 external iliac arteries from 22 rabbits (10 normal and 12 atherosclerotic) were transfected with a solution of luciferase expression vector plasmid and liposome, using a dual balloon-catheter system. Analysis of the transfected segments revealed luciferase activity in 10 of the 22 arteries (4/10 normal vs 6/12 balloon-injured atherosclerotic, P = NS); no activity could be detected in the contralateral limb arterial segments used as controls. Luciferase activity levels in successfully transfected segments measured 4.10 +/- 1.19 (m +/- SEM) Turner light units (TLU), with 3.03 +/- 1.16 TLU found in normals vs 4.81 +/- 1.87 TLU in balloon-injured atherosclerotic arteries (P = NS). In situ hybridization of successfully transfected atherosclerotic sections showed expression of the luciferase gene mRNA from rare cells (less than 1/1,000) limited to the neointimal lesion. Thus, expression of new genetic material may be achieved in both normal and balloon-dilated atherosclerotic arteries following an exclusively percutaneous approach. The low efficiency of the current delivery strategy, however, represents a potential limitation that must be improved if this strategy is to be applied as a therapeutic approach to human vascular disease.
Previous reports have shown that retrovirus infection is inhibited in nonreplicating (stationary-phase [hereafter called stationary])
cells. Infection of stationary cells was shown to occur when the cells were allowed to replicate at times up to a week after
infection, suggesting that an unintegrated retrovirus could persist in a form that was competent to integrate after release
of the block to replication. However, those studies were complicated by the use of replication-competent virus, which can
spread in the infected cells. We have used a replication-defective retrovirus vector to compare the efficiency of gene transfer
in stationary and replicating rat embryo fibroblasts. In agreement with previous results, gene transfer was inhibited 100-fold
in stationary versus replicating cells. In contrast to previously reported results, the block to infection could not be relieved
by stimulating stationary cells to divide at times from 6 h to 10 days after infection. Thus, for successful retroviral infection,
the infected cells must be replicating at the time of infection. These results have important implications for the use of
retroviral vectors for gene transfer.
A recombinant plasmid encoding rat preproinsulin I was encapsulated in large liposomes and intravenously injected in rats. Glycemia and blood, splenic, and hepatic insulin were assayed at various times beginning 6 hr after inoculation. The results were compared with controls that had received (i) empty liposomes, (ii) liposomes carrying the Escherichia coli pBR322 plasmid, (iii) the free rat insulin I gene, and (iv) no injection at all. Whereas all controls showed unchanged glucose and insulin levels, the treated animals had, 6 hr after inoculation, a blood glucose level of 72 +/- 5 mg of glucose/100 ml of blood as compared with 107 +/- 2 mg/ml for controls. Radioimmunoassay of blood insulin gave 61 +/- 8 microunits/ml as compared with 43 +/- 5 microunits/ml for controls and the spleen and liver values were 242 +/- 22 and 204 +/- 20 microunits/g of tissue, respectively, as compared with 112 +/- 20 and 87 +/- 15 microunits/g in controls. External gamma-camera imaging of the organ uptake of 111In-labeled liposomes permitted study of the kinetics and extent of uptake of the liposomes by spleen and liver, results that support the findings concerning insulin synthesis in the two organs.
It is postulated that vascular disease involves a disturbance in the homeostatic balance of factors regulating vascular tone and structure. Recent developments in gene transfer techniques have emerged as an exciting therapeutic option to treat vascular disease. Several studies have established the feasibility of direct in vivo gene transfer into the vasculature by using reporter genes such as beta-galactosidase or luciferase. To date no study has documented therapeutic effects with in vivo gene transfer of a cDNA encoding a functional enzyme. This study tests the hypothesis that endothelium-derived nitric oxide is an endogenous inhibitor of vascular lesion formation. After denudation by balloon injury of the endothelium of rat carotid arteries, we restored endothelial cell nitric oxide synthase (ec-NOS) expression in the vessel wall by using the highly efficient Sendai virus/liposome in vivo gene transfer technique. ec-NOS gene transfection not only restored NO production to levels seen in normal untreated vessels but also increased vascular reactivity of the injured vessels. Neointima formation at day 14 after balloon injury was inhibited by 70%. These findings provide direct evidence that NO is an endogenous inhibitor of vascular lesion formation in vivo (by inhibiting smooth muscle cell proliferation and migration) and suggest the possibility of ec-NOS transfection as a potential therapeutic approach to treat neointimal hyperplasia.
Proliferation of smooth muscle cells of the arterial wall in response to local injury is an important aetiologic factor of vascular proliferative disorders such as atherosclerosis and restenosis after angioplasty. Ras proteins are key transducers of mitogenic signals from membrane to nucleus in many cell types. We investigated the role of ras proteins in the vascular response to arterial injury by inactivating cellular ras of rats in which the common carotid artery was subjected to balloon injury. DNA vectors expressing ras transdominant negative mutants, which interfere with ras function, reduced neointimal formation after injury. Our results indicate a key role for ras in smooth muscle cell proliferation and show that the local delivery of transdominant negative mutants of ras in vivo might prevent some of the acute vascular injury caused by balloon injury.
The application of DNA technology to regulate the transcription of disease-related genes in vivo has important therapeutic potentials. The transcription factor E2F plays a pivotal role in the coordinated transactivation of cell cycle-regulatory genes such as c-myc, cdc2, and the gene encoding proliferating-cell nuclear antigen (PCNA) that are involved in lesion formation after vascular injury. We hypothesized that double-stranded DNA with high affinity for E2F may be introduced in vivo as a decoy to bind E2F and block the activation of genes mediating cell cycle progression and intimal hyperplasia after vascular injury. Gel mobility-shift assays showed complete competition for E2F binding protein by the E2F decoy. Transfection with E2F decoy inhibited expression of c-myc, cdc2, and the PCNA gene as well as vascular smooth muscle cell proliferation both in vitro and in the in vivo model of rat carotid injury. Furthermore, 2 weeks after in vivo transfection, neointimal formation was significantly prevented by the E2F decoy, and this inhibition continued up to 8 weeks after a single transfection in a dose-dependent manner. Transfer of an E2F decoy can therefore modulate gene expression and inhibit smooth muscle proliferation and vascular lesion formation in vivo.
Previously, researchers have speculated that genetic engineering can improve the long-term function of vascular grafts which are prone to atherosclerosis and occlusion. In this study, we demonstrated that an intraoperative gene therapy approach using antisense oligodeoxynucleotide blockage of medial smooth muscle cell proliferation can prevent the accelerated atherosclerosis that is responsible for autologous vein graft failure. Selective blockade of the expression of genes for two cell cycle regulatory proteins, proliferating cell nuclear antigen and cell division cycle 2 kinase, was achieved in the smooth muscle cells of rabbit jugular veins grafted into the carotid arteries. This alteration of gene expression successfully redirected vein graft biology away from neointimal hyperplasia and toward medial hypertrophy, yielding conduits that more closely resembled normal arteries. More importantly, these genetically engineered grafts proved resistant to diet-induced atherosclerosis. These findings establish the feasibility of developing genetically engineered bioprostheses that are resistant to failure and better suited to the long-term treatment of occlusive vascular disease.
Adeno-associated viral (AAV) vectors are non-pathogenic, integrating DNA vectors in which all viral genes are removed and helper virus is completely eliminated. To evaluate this system in the post-mitotic cells of the brain, we found that an AAV vector containing the lacZ gene (AAVlac) resulted in expression of beta-galactosidase up to three months post-injection in vivo. A second vector expressing human tyrosine hydroxylase (AAVth) was injected into the denervated striatum of unilateral 6-hydroxydopamine-lesioned rats. Tyrosine hydroxylase (TH) immunoreactivity was detectable in striatal neurons and glia for up to four months and we also found significant behavioural recovery in lesioned rats treated with AAVth versus AAVlac controls. Safe and stable TH gene transfer into the denervated striatum may have potential for the genetic therapy of Parkinson's disease.
Moloney Murine Leukemia Virus (MoMuLV) causes T cell neoplasms in rodents but is not known to be a pathogen in primates. The core protein and enzyme genes of the MoMuLV genome together with an amphotropic envelope gene are utilized to engineer the cell lines that generate retroviral vectors for use in current human gene therapy applications. We developed a producer clone that generates a very high concentration of retroviral vector particles to optimize conditions for gene insertion into pluripotent hematopoietic stem cells. This producer cell line also generates a much lower concentration of replication-competent virus that arose through recombination. Stem cells from rhesus monkeys were purified by immunoselection with an anti-CD34 antibody, incubated in vitro for 80-86 h in the presence of retroviral vector particles with accompanying replication-competent virus and used to reconstitute recipients whose bone marrow had been ablated by total body irradiation. The retroviral vector genome was detected in circulating cells of five of eight transplant recipients of CD34+ cells and in the circulating cells of two recipients of infected, unfractionated bone marrow mononuclear cells. Three recipients of CD34+ cells had a productive infection with replication-competent virus. Six or seven mo after transplantation, each of these animals developed a rapidly progressive T cell neoplasm involving the thymus, lymph nodes, liver, spleen, and bone marrow. Lymphoma cells contained 10-50 copies of the replication-competent virus, but lacked the retroviral vector genome. We conclude that replication-competent viruses arising from producer cells making retroviral vectors can be pathogenic in primates, which underscores the importance of carefully screening retroviral producer clones used in human trials to exclude contamination with replication-competent virus.
Over the last four decades, improvements in surgical technique and vascular grafts have permitted the reconstruction of both large and small vessels. Although such reconstructions may be technically satisfactory, they may fail for several reasons: spontaneous thrombosis, restenosis, or infection. This chapter focuses on the problem of restenosis as it applies to vascular grafts and other forms of arterial reconstruction. Over the last five years, substantial progress has been made in defining the various factors contributing to stenosis. Perhaps the most important is intimal smooth muscle hyperplasia. During the next decade, a detailed understanding of molecular mechanisms that regulate smooth muscle function should permit the development of pharmacological strategies to prevent restenosis (1).
1. From Type III pneumococci a biologically active fraction has been isolated in highly purified form which in exceedingly
minute amounts is capable under appropriate cultural conditions of inducing the transformation of unencapsulated R variants
of Pneumococcus Type II into fully encapsulated cells of the same specific type as that of the heat-killed microorganisms
from which the inducing material was recovered.
2. Methods for the isolation and purification of the active transforming material are described.
3. The data obtained by chemical, enzymatic, and serological analyses together with the results of preliminary studies by
electrophoresis, ultracentrifugation, and ultraviolet spectroscopy indicate that, within the limits of the methods, the active
fraction contains no demonstrable protein, unbound lipid, or serologically reactive polysaccharide and consists principally,
if not solely, of a highly polymerized, viscous form of desoxyribonucleic acid.
4. Evidence is presented that the chemically induced alterations in cellular structure and function are predictable, type-specific,
and transmissible in series. The various hypotheses that have been advanced concerning the nature of these changes are reviewed.
Angiogenic cytokines constitute a potentially novel form of therapy for patients with cardiovascular disease. The feasibility of using recombinant formulations of angiogenic growth factors to expedite and/or augment collateral artery development in animal models of myocardial and hindlimb ischemia--'therapeutic angiogenesis'--has now been well established. These studies have suggested that two angiogenic growth factors in particular--basic fibroblast growth factor and vascular endothelial growth factor--are sufficiently potent to merit further investigation. More recently, experiments performed in our laboratory have indicated that, in the case of vascular endothelial growth factor--a secreted protein--similar results may be achieved by percutaneous arterial gene transfer. Further laboratory and clinical studies may yield promising insights into the fundamental basis for native as well as therapeutic angiogenesis, and at the same time more explicitly define the manner in which therapeutic angiogenesis may be successfully incorporated into clinical practice.
Preclinical studies have indicated that angiogenic growth factors can stimulate the development of collateral arteries, a concept called "therapeutic angiogenesis." The objectives of this phase 1 clinical trial were (1) to document the safety and feasibility of intramuscular gene transfer by use of naked plasmid DNA encoding an endothelial cell mitogen and (2) to analyze potential therapeutic benefits in patients with critical limb ischemia.
Gene transfer was performed in 10 limbs of 9 patients with nonhealing ischemic ulcers (n=7/10) and/or rest pain (n=10/10) due to peripheral arterial disease. A total dose of 4000 microg of naked plasmid DNA encoding the 165-amino-acid isoform of human vascular endothelial growth factor (phVEGF165) was injected directly into the muscles of the ischemic limb. Gene expression was documented by a transient increase in serum levels of VEGF monitored by ELISA. The ankle-brachial index improved significantly (0.33+/-0.05 to 0.48+/-0.03, P=.02); newly visible collateral blood vessels were directly documented by contrast angiography in 7 limbs; and magnetic resonance angiography showed qualitative evidence of improved distal flow in 8 limbs. Ischemic ulcers healed or markedly improved in 4 of 7 limbs, including successful limb salvage in 3 patients recommended for below-knee amputation. Tissue specimens obtained from an amputee 10 weeks after gene therapy showed foci of proliferating endothelial cells by immunohistochemistry. PCR and Southern blot analyses indicated persistence of small amounts of plasmid DNA. Complications were limited to transient lower-extremity edema in 6 patients, consistent with VEGF enhancement of vascular permeability.
These findings may be cautiously interpreted to indicate that intramuscular injection of naked plasmid DNA achieves constitutive overexpression of VEGF sufficient to induce therapeutic angiogenesis in selected patients with critical limb ischemia.
Objectives:
Recombinant viral vectors based on the nonpathogenic parvovirus, adeno-associated virus (AAV), have a number of attractive features for gene therapy, including the ability to transduce non-dividing cells and its long-term transgene expression. In this study, an AAV vector containing bacterial beta-galactosidase gene (lacZ) was used to transduce cultured rat vascular smooth muscle cells (VSMC) in vitro and rat thoracic aortas ex vivo.
Methods:
VSMC were transduced with AAV-lacZ at multiplicities of infection (MOI) ranging from 5.0 x 10(5) to 1.0 x 10(7). Expression of beta-galactosidase (beta-gal) in VSMC was evaluated by X-gal staining and a beta-gal ELISA method. Excised rat aortas were incubated with medium containing AAV-lacZ. Expression of beta-gal in the aortic segments was evaluated by X-gal staining.
Results:
With increasing MOI, up to 50% of cultured VSMC were positive by X-gal staining and the beta-gal expression increased up to 15 ng/mg protein. The expression gradually decreased during the culture but was detectable for at least 1 month. In the ex vivo study, AAV vectors transduced endothelial and adventitial cells in rat aortic segments, while no expression was seen in medial VSMC.
Conclusions:
AAV vectors can efficiently transduce rat VSMC in vitro. AAV-mediated ex vivo gene transfer into the normal aorta resulted in efficient gene transfer into endothelial and adventitial cells but not into medial VSMC. These findings suggest that AAV-based vectors are promising for use in cardiovascular gene therapy.
Coronary restenosis is a reparative response to arterial injury during angioplasty, and remains a major clinical problem. The reasons for treatment failures likely stem from our incomplete understanding of the cellular mechanisms in restenotic neointimal formation. Restenosis is thought to result from migration and replication of medial smooth muscle cells to form an obstructive neointima, a concept neither observed nor demonstrated in humans. An alternative hypothesis for restenosis is based on observations in the porcine coronary injury model. In this model, there are three cellular stages in neointimal formation: thrombotic (stage I), cellular recruitment (stage II) and proliferative (stage III). The thrombotic stage occurs early and consists of platelets, fibrin and red blood cells accumulating at the vessel injury site. In the recruitment stage, the mural thrombus itself develops an endothelium, followed by a mononuclear leukocytic infiltrate beginning on the lumen side of the vessel. In the proliferative stage, a "cap" of actin-positive cells forms on the lumen surface and progressively thickens. These cells do not arise from media at the injury site. Extracellular matrix secretion and additional recruitment likely add to neointimal volume during this phase. Thrombus assumes a major role in restonosis by providing an absorbable matrix into which smooth muscle cells proliferate. Further studies are needed to validate or modify this hypothesis.
To test the feasibility of percutaneous deployment of intracoronary polymeric stents, a prototype polyethylene terephthalate (PET) stent and a catheter-based delivery system were developed.
Polymeric stents were deployed in the coronary arteries of 11 Yucatan swine: six stents were placed in the left anterior descending coronary artery, four stents were placed in the circumflex artery, and one stent was placed in the right coronary artery. Stent deployment was achieved by withdrawal of an outer delivery sheath, thus allowing the PET stent to self-expand to a preformed configuration. Two animals died during surgery, one during stent placement and the other several hours after implantation due to intracoronary thrombus formation. Two animals were electively sacrificed within 24 hours of stent implant to examine the adequacy of stent deployment within the coronary vessel. The remaining seven animals survived until the termination of the study 4-6 weeks later. Light microscopic examination of the stented vessels showed an extensive neointimal proliferative response with vessel occlusion in all animals who survived initial stent placement. There were two distinct types of histological responses to the PET stent--a chronic foreign body inflammatory response around the stent tines and a neointimal proliferative response in the center of the occluded vessel lumen. The histological response seen in the central area of the vessel was morphologically similar to that seen in patients with restenosis after successful percutaneous transluminal coronary angioplasty, whereas the morphological response seen at the periphery of the stent tine was similar to that exhibited by a chronic foreign body reaction and was not typical of that seen in a restenosis lesion. A ventricular aneurysm also developed in the area of myocardium that was previously supplied by the occluded vessel.
This study demonstrates that percutaneous deployment of polymeric stents in the coronary arteries is technically feasible. The use of PET polymer was associated with an intense proliferative neointimal response that resulted in complete vessel occlusion. Histological examination of the stented segments of the vessel revealed no evidence that dissection of the vessel wall had occurred at the time of initial stent deployment. Although the PET polymer was of similar quality to that used in the manufacture of balloon angioplasty catheters, a toxic chemical or contaminant effect cannot be completely excluded as the stimulus to intimal proliferation. This finding may have relevance to the selection of materials for use as intravascular devices.
Genetic manipulation of the vasculature may offer insights into the pathogenesis of coronary artery disease and may lead to gene therapy for disorders such as restenosis after percutaneous coronary angioplasty. The goal of this study was to develop a percutaneous method for gene transfer into coronary arteries of intact animals. Liposomes were used to facilitate transfection in coronary arteries with a plasmid containing the cDNA encoding luciferase. This reporter was chosen since it is not expressed in mammalian cells, and it can be quantified using a sensitive assay (light production). Mongrel dogs were catheterized, and DNA was delivered to coronary arteries via a porous perfusion balloon system. Luciferase expression was measured 3-5 days after the procedure, when the dogs were killed. Luciferase activity in control arteries (n = 12) was no higher than average background activity. Eight of 12 transfected arteries exhibited gene expression, averaging 4.3 +/- 2.1 pg luciferase (p less than 0.01, transfected versus control arteries). In addition, the ability to transfect DNA into femoral arteries without a transfection vehicle was tested. Five dogs were subjected to surgical transfection attempts in their femoral arteries with either DNA alone or DNA plus liposomes. Luciferase was expressed in all 10 femoral arteries; those treated with DNA alone expressed 35.6 +/- 8 pg luciferase, and those treated with DNA plus liposomes expressed 42.3 +/- 14 pg luciferase (p = 0.70). These results demonstrate the use of a percutaneous catheter to achieve gene transfer and expression in coronary arteries of intact dogs and suggest that the efficiency of intra-arterial gene transfer may be similar whether or not a transfection vehicle is used.
Coronary revascularization in patients with persistent angina after myocardial infarction reduces the incidence of recurrent angina pectoris and myocardial infarction and improves left ventricular function. The results of revascularization after a Q wave myocardial infarction when there is no residual ischemia may depend on myocardial viability.
To determine whether there was viable myocardium in the infarct area in the absence of clinical and scintigraphic evidence of myocardial ischemia, 15 asymptomatic patients with a Q wave myocardial infarction, no redistribution on stress 201Tl test, and single-vessel disease (greater than 70% stenosis) with persistent anterograde blood flow were randomized to percutaneous transluminal coronary artery angioplasty (PTCA) or conservative medical treatment. After 2 months of follow-up, mean coronary blood flow measured by Doppler catheter in the infarct-related artery was higher in the PTCA treatment group (33 +/- 6 ml/min, n = 8) than in the conservative treatment group (16 +/- 4 ml/min, n = 7; p less than 0.05 between groups). The 201Tl pathological-to-normal ratios measured on postexercise images did not change in patients treated conservatively during the follow-up period (delta = +1.1 +/- 2.2%; NS from baseline) but increased significantly in patients treated by PTCA (delta = +8.5 +/- 2.3%; p less than 0.01 from baseline; p less than 0.05 between groups). Segmental wall motion improved on left ventricular angiography 2 months after PTCA (delta = +11.5 +/- 2.2%; p less than 0.001 from baseline) significantly more than in the conservative treatment group (delta = +4.1 +/- 1.4%; p less than 0.05 between both groups). Improvements of 201Tl ratios and segmental wall motion indexes correlated significantly (r = 0.73, p = 0.002). The mild improvement of global left ventricular ejection fraction measured in the PTCA treatment group did not differ significantly from changes in the conservative treatment group.
Successful angioplasty of the stenotic infarct artery in patients with a Q wave myocardial infarction and no residual ischemia improved coronary flow, 201Tl uptake in the infarct area, and regional wall motion. Therefore, myocardial viability may last several weeks, as long as residual blood flow persists in the infarct-related artery. Optimal assessment of viability by imaging techniques should identify patients who are most likely to benefit from revascularization.
Gene transfer into vascular smooth muscle cells in animals was examined by using recombinant retroviral vectors containing an Escherichia coli beta-galactosidase gene or a human adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) gene. Direct gene transfer by infusion of virus into rat carotid arteries was not observed. However, gene transfer by infection of smooth muscle cells in culture and seeding of the transduced cells onto arteries that had been denuded of endothelial cells was successful. Potentially therapeutic levels of human adenosine deaminase activity were detected over 6 months of observation, indicating the utility of vascular smooth muscle cells for gene therapy in humans.
Vascular smooth muscle cells contribute to the formation of atherosclerotic plaques by proliferating in response to vascular injury and releasing growth-promoting factors. Because their autocrine and paracrine effects are not fully understood, expression of such growth factor genes in specific cell types in vivo would help to determine their mechanism of action. We describe a method to transfer vascular smooth muscle cells expressing recombinant gene products to localized segments of the arterial wall. Vascular smooth muscle cells from the inbred Yucatan minipig were infected in vitro with an amphotropic, replication-defective retrovirus transducing the gene for Escherichia coli beta-galactosidase. Vascular smooth muscle cells expressing this recombinant gene were implanted, using a catheter, into denuded iliofemoral artery segments of pigs in vivo. These arteries subsequently demonstrated beta-galactosidase activity in cells of the intima and media. This method, which provides for the introduction of genetically modified smooth muscle cells, can be used to define the biological effects of recombinant genes in the vessel wall and potentially to provide alternative treatments of vascular diseases.
Gene therapy approaches have been suggested for the treatment of cardiovascular disease. Recently, direct transfer of the gene encoding beta-galactosidase into peripheral arteries of the pig has been demonstrated. To determine whether this approach is applicable to other arterial beds and to other species, we first evaluated the use of beta-galactosidase as a marker protein in the canine model. We demonstrate that variable but substantial endogenous beta-galactosidase-like activity is induced by manipulation of canine peripheral arteries, which precludes the use of this marker protein in evaluating the efficiency of gene transfer in this model. A marker gene encoding firefly luciferase was then evaluated, and background luciferase activity was found to be low in the dog even after arterial manipulation. Using the luciferase gene, we then demonstrated lipid-mediated gene transfer directly into both coronary and peripheral arteries of the intact dog. These results indicate the feasibility of in vivo gene transfer into coronary arteries and demonstrate the use of the luciferase marker protein in quantifying recombinant protein expression following gene transfer in canine models. This simple and effective method for direct in vivo gene transfer into coronary and peripheral arteries may be applicable to the localized production of therapeutically important proteins for the treatment of cardiovascular diseases.
Despite substantial basic and clinical efforts to address the problem of restenosis after percutaneous coronary intervention, effective preventive therapies have not yet been developed. Nevertheless, the accumulated information has provided much insight into the process of restenosis in addition to allowing standards to be developed for adequate clinical trials. The pathophysiology of restenosis increasingly appears to be distinct from that of primary atherosclerosis. Restenosis involves elastic recoil, incorporation of thrombus into the lesion and fibrocellular proliferation in varying degrees in different patients. Lack of an animal model that satisfactorily mimics restenosis is a major impediment to further understanding of the process. Clinical studies are hampered by difficulties in finding a single unifying definition of restenosis and by variable methods of reporting follow-up. Reporting of clinical outcomes of all patients in angiographic substudies would allow a more satisfactory interpretation of the results of clinical trials. Current noninvasive test results are not accurate enough to substitute for angiographic and clinical outcome data in intervention trials. In the majority of observational studies, only diabetes and unstable angina have emerged as consistently associated with restenosis; whereas most of the standard risk factors for atherosclerosis have a less consistent relation. Disappointingly, the new atherectomy and laser technologies have not affected restenosis rates. The one possible exception is coronary stenting, as a result of the larger luminal diameter achieved by the placement of the stent. In conclusion, although substantial continued effort is necessary to explore the basic aspects of cellular proliferation and mechanical alteration of atherosclerotic vessels, attention to the principles of clinical trials and observation are required to detect the impact of risk factors and interventions on the multifactorial problem of restenosis. Adequate sample sizes, collection of clinical and angiographic outcomes and factorial study designs hold promise for unraveling this important limitation of percutaneous intervention.
A recombinant beta-galactosidase gene has been expressed in a specific arterial segment in vivo by direct infection with a murine amphotropic retroviral vector or by DNA transfection with the use of liposomes. Several cell types in the vessel wall were transduced, including endothelial and vascular smooth muscle cells. After retroviral infection, a recombinant reporter gene was expressed for at least 5 months, and no helper virus was detected. Recombinant gene expression achieved by direct retroviral infection or liposome-mediated DNA transfection was limited to the site of infection and was absent from liver, lung, kidney, and spleen. These results demonstrate that site-specific gene expression can be achieved by direct gene transfer in vivo and could be applied to the treatment of such human diseases as atherosclerosis or cancer.
A technique for the transfer of endothelial cells and expression of recombinant genes in vivo could allow the introduction
of proteins of therapeutic value in the management of cardiovascular diseases. Porcine endothelial cells expressing recombinant
beta-galactosidase from a murine amphotropic retroviral vector were introduced with a catheter into denuded iliofemoral arteries
of syngeneic animals. Arterial segments explanted 2 to 4 weeks later contained endothelial cells expressing beta-galactosidase,
an indication that they were successfully implanted on the vessel wall.
Restenosis after successful PTCA remains a major problem limiting the efficacy of the procedure. The pathophysiologic mechanism of restenosis has been enigmatic so far, but accumulated evidence strongly suggests that intimal hyperplasia is the major mechanism. Based on current understanding of the process of intimal hyperplasia, one unifying concept may be that there are at least two major local biologic determinants influencing this process, lesion characteristics and regional flow dynamics. Lesion characteristics include the plaque structure and the quantity of smooth muscle. These may provide the anatomic substrate that determines the extent of injury and the degree of smooth muscle cell proliferation. The amount of smooth muscle cells in the stenotic lesion activated by injury to undergo proliferation may determine the eventual bulk of the restenotic lesion. In addition, low wall shear stress could promote intimal hyperplasia and cause structural change of vessels to decrease the lumen, whereas high wall shear stress exerts the opposite effects. Intimal hyperplasia after balloon injury is a complex process involving platelets, growth factors, endothelial cells, smooth muscle cells, mechanical injury, wall shear stress, and probably other unknown factors. Platelets not only contribute growth factors such as PDGF but also cause organized thrombus. Different growth factors may be involved in initiating smooth muscle cell proliferation and may come from many different sources, including smooth muscle cells, endothelial cells, and macrophages. Intact confluent endothelial cells may produce heparin sulfates and inhibit intimal proliferation; however, regenerating endothelial cells may have the opposite effect. Thus, the proliferative potential of smooth muscle cells, endothelial recovery, extent of injury, wall shear stress, and other unknown factors may all influence this process. Based on these concepts concerning the biology of restenosis, some research directions concerning potential forms of therapy are proposed.
Introduction of an exogenous retinoblastoma (RB) gene in RB-deficient retinoblastoma or osteosarcoma cells has been shown to suppress their neoplastic phenotype. In experiments designed to explore the potential mechanism of RB tumor suppression, we report here that the phosphorylation state of RB protein is modulated during normal cellular events. In resting cells, RB protein is present in its least phosphorylated form; in rapidly proliferating cells, RB protein is highly phosphorylated. Maximal phosphorylation is associated with S phase of the cell cycle. Induction of differentiation in several human leukemia cell lines by treatment with phorbol ester or retinoic acid leads to dephosphorylation of RB. Time course studies indicate that RB dephosphorylation precedes the total arrest of cell growth during differentiation. These observations strongly suggest that the function of RB protein is modulated by a phosphorylation/dephosphorylation mechanism during cell proliferation and differentiation.
Serial arteriograms were obtained in 501 patients after coronary bypass grafting. Study I within 5 years of operation (mean interval 15 months) and Study II more than 5 years after (mean interval 88 months, range 60 to 147 months). One hundred patients received both internal mammary artery and saphenous vein grafts: 37, mammary artery grafts only, and 364, vein grafts only. In Study I, 645 (82%) of 786 vein grafts were patent, 42 (5%) stenotic or irregular, and 99 (13%) occluded. Of 140 mammary artery grafts, 136 (97%) were patent, two (2%) stenotic, and two (2%) occluded. Of the 645 vein grafts patent in Study I, 357 (55%) remained patent in Study II, 119 (18%) were stenotic or irregular, and 169 (26%) were occluded. Of 136 mammary artery grafts patent in Study I, 130 (96%) were unchanged, one was stenotic, and five (4%) were occluded in Study II. Early vein graft patency was influenced by the coronary artery grafted and by angina. Progression of vein grafts patent at Study I to stenosis or occlusion at Study II was associated with increasing postoperative interval (p less than 0.00001), interval myocardial infarction (p less than 0.001), angina (p less than 0.001), diabetes (p less than 0.004), hypercholesterolemia (p less than 0.006), and hypertriglyceridemia (p less than 0.02); it was not influenced by the coronary artery grafted. Within 5 years of operation, mammary artery graft patency exceeded vein graft patency. Between 5 and 12 years after operation, the attrition rate of vein grafts greatly exceeded that of mammary artery grafts (p less than 0.0001).
We investigated the sites of integration of exogenous DNA fragments introduced by DNA-mediated gene transfer. Mouse Ltk- cells
were transformed with the herpes simplex virus thymidine kinase gene and pBR322 DNA by the calcium phosphate precipitation
method. Some of the integrated exogenous DNA sequences were recovered from the stable tk+ transformants in the form of plasmids
that were capable of propagation in bacteria. Four plasmids derived from two cloned cell lines were analyzed in detail by
nucleotide sequencing and hybridization techniques. These plasmids contained a total of seven cellular-exogenous DNA junctions.
In all cases, there was no sequence homology between the exogenous and cellular DNA sequences adjacent to the joining sites,
and no specific exogenous or cellular sequences occurred at the junctions. Rearrangement or deletion of Ltk- DNA was always
associated with the integration of exogenous DNA. All of the assignable cellular sequences at the junctions were repetitive
sequences. Two of these sequences were from the MIF-1 repetitive sequence family, and a third consisted of a 40-base pair
simple copolymer of alternating deoxyadenosine-deoxythymidine. Our results suggest that repetitive sequences are relatively
favorable sites for the integration of exogenous DNA.
We have developed methods for covalently joining duplex DNA molecules to one another and have used these techniques to construct circular dimers of SV40 DNA and to insert a DNA segment containing lambda phage genes and the galactose operon of E. coli into SV40 DNA. The method involves: (a) converting circular SV40 DNA to a linear form, (b) adding single-stranded homodeoxypolymeric extensions of defined composition and length to the 3' ends of one of the DNA strands with the enzyme terminal deoxynucleotidyl transferase (c) adding complementary homodeoxypolymeric extensions to the other DNA strand, (d) annealing the two DNA molecules to form a circular duplex structure, and (e) filling the gaps and sealing nicks in this structure with E. coli DNA polymerase and DNA ligase to form a covalently closed-circular DNA molecule.
This editorial argues the cause-and-effect relationship between in utero exposure to diethylstilbestrol (DES) and incidence of vaginal adenocarcinoma among young women. It is conjectured that the mechanism by which DES induces tumors is a result of transplacental carcinogenesis; i.e., DES causes a malignant change in any fetal cell of future vaginal tissue, resulting in a genetic defect which may not be realized until puberty, when endogenous hormone production acts as a promoter of the malignancy initiated by DES exposure during gestation. If this theory is correct, local progestational therapy may arrest future adenocarcinoma cases. Though it is obvious that DES therapy must be avoided in the future in the population of pregnant women, of more concern is the presence of residual DES in foodstuffs, particularly livestock meat. A ban of such diet supplementation of human foodstuffs is called for.
The efficacy of aorto-coronary vein grafting is limited by early graft thrombosis and accelerated graft atherosclerosis. Direct adenovirus-mediated transfer of genes encoding inhibitory proteins may prevent or slow progression of vein graft disease.
Recombinant adenoviruses containing the cDNA for the marker gene lacZ (Ad.CMVlacZ) or soluble vascular cell adhesion molecule (sVCAM) (Ad.CB-sVCAM) were used to infect segments of porcine jugular vein or human saphenous vein. Ex vivo testing showed expression of the introduced genes after incubation with Ad.CMVlacZ or Ad.CBsVCAM for periods from 1 to 24 hours, with an increase in transfection efficiency with increasing incubation time. Porcine jugular veins were then interposed as vascular grafts in the carotid arteries of four juvenile farm pigs after ex vivo gene transfer by incubation for 90 to 120 minutes with Ad.CMVlacZ or Ad.CBsVCAM. sVCAM-transfected carotid vein grafts were placed on one side and lacZ transfected veins were placed contralaterally as controls. Three days later, the vein graft segments were resected. Expression of the lacZ gene was confirmed by X-Gal chromagen staining and visualization by light and transmission electron microscopy. Gene expression was apparent in all layers of the vein graft wall, with prominent staining in the adventitia. sVCAM expression was confirmed by immunohistochemistry and in situ hybridization.
We conclude that ex vivo gene transfer before vein grafting is feasible using a replication-deficient recombinant adenovirus and results in a high level of gene expression in vivo. The potential for this approach to prevent early vein graft thrombosis or accelerated vein graft atherosclerosis requires further evaluation.
### Peripheral Artery Disease: Primary Pharmacological Therapy Is Ineffective for Patients With Critical Limb Ischemia
The prognosis for patients with chronic critical leg ischemia, ie, rest pain and/or established lesions that jeopardize the integrity of the lower limbs, is often poor. Psychological testing of such patients has typically disclosed quality-of-life indexes similar to those of patients with cancer in critical or even terminal phases of their illness.1 It has been estimated that in toto,2 150 000 patients per year require lower-limb amputations for ischemic disease in the United States. Their prognosis after amputation is even worse3 : the perioperative mortality for below-knee amputation in most series is 5% to 10% and for above-knee amputation 15% to 20%. Even when they survive, nearly 40% will have died within 2 years of their first major amputation; a major amputation is required in 30% of cases; and full mobility is achieved in only 50% of below-knee and 25% of above-knee amputees.
These grim statistics are compounded by the lack of efficacious drug therapy. As concluded in the Consensus Document of the European Working Group on Critical Leg Ischemia,3 “. . .there presently is inadequate evidence from published studies to support the routine use of primary pharmacological treatment in patients with critical leg ischemia. . . .” Evidence for the utility of medical therapy in the treatment of claudication is no better.4 5 Consequently, the need for alternative treatment strategies in such patients is compelling.
### Therapeutic Angiogenesis Is a Novel Strategy for the Treatment of Critical Limb Ischemia
The therapeutic implications of angiogenic growth factors were identified by the pioneering work of Folkman6 and other workers more than two decades ago. Beginning a little more than a decade ago,7 a series of polypeptide growth factors were purified, sequenced, and demonstrated to be responsible for natural as well as pathological angiogenesis.
More recent investigations have established the feasibility of using recombinant formulations of such angiogenic growth …
In 1990, a clinical trial was started using retroviral-mediated transfer of the adenosine deaminase (ADA) gene into the T cells of two children with severe combined immunodeficiency (ADA- SCID). The number of blood T cells normalized as did many cellular and humoral immune responses. Gene treatment ended after 2 years, but integrated vector and ADA gene expression in T cells persisted. Although many components remain to be perfected, it is concluded here that gene therapy can be a safe and effective addition to treatment for some patients with this severe immunodeficiency disease.
The specific targeting of signal transduction components implicated in vascular disease may be accomplished more efficiently with genes than with drugs (pages 541–545).
To evaluate the concept that localized delivery of angiogenic factors via virus-mediated gene transfer may be useful in the treatment of ischemic disorders, the replication-deficient adenovirus (Ad) vector AdCMV.VEGF165 (where CMV is cytomegalovirus and VEGF is vascular endothelial growth factor) containing the cDNA for human VEGF165, a secreted endothelial cell-specific angiogenic growth factor, was constructed. Human umbilical vein endothelial cells (HUVECs) and rat aorta smooth muscle cells (RASMCs) infected with AdCMV.VEGF165 (5 and 20 plaque-forming units [pfu] per cell) demonstrated VEGF mRNA expression and protein secretion into the supernatant. Furthermore, the conditioned medium from these cells enhanced vascular permeability in vivo. In contrast, neither VEGF mRNA nor secreted protein was found in uninfected HUVECs or RASMCs or in cells infected with the control vector AdCMV.beta gal (where beta gal is beta-galactosidase). Assessment of starved HUVECs at 14 days demonstrated sixfold more cells for AdCMV.VEGF165-infected HUVECs (20 pfu per cell) than for either infected or uninfected control cells. RASMC proliferation was unaffected by infection with AdCMV.VEGF165. When plated in 2% serum on dishes precoated with reconstituted basement membrane (Matrigel), HUVECs infected with AdCMV.VEGF165 (20 pfu per cell) differentiated into capillary-like structures. Under similar conditions, both uninfected HUVECs and HUVECs infected with AdCMV.beta gal did not differentiate. To evaluate the ability of AdCMV.VEGF165 to function in vivo, either AdCMV. VEGF165 or AdCMV.beta gal (2 x 10(10) pfu) was resuspended in 0.5 mL Matrigel and injected subcutaneously into mice. Immunohistochemical staining demonstrated VEGF in the tissues surrounding the Matrigel plugs containing AdCMV.VEGF165 up to 3 weeks after injection, whereas no VEGF was found in the control plugs with AdCMV.beta gal. Two weeks after injection, there was histological evidence of neovascularization in the tissues surrounding the Matrigel containing AdCMV.VEGF165, whereas no significant angiogenesis was observed in response to AdCMV.beta gal. Furthermore, the Matrigel plugs with AdCMV.VEGF165 demonstrated hemoglobin content fourfold higher than the plugs with AdCMV.beta gal. Together, these in vitro and in vivo studies are consistent with the concept that Ad vectors may provide a useful strategy for efficient local delivery of VEGF165 in the treatment of ischemic diseases.
Vascular smooth muscle cell (VSMC) proliferation after arterial injury is important in the pathogenesis of a number of vascular proliferative disorders, including atherosclerosis and restenosis after balloon angioplasty. Thus, a better understanding of the molecular mechanisms underlying VSMC proliferation in response to arterial injury would have important therapeutic implications for patients with atherosclerotic vascular disease. The p21 protein is a negative regulator of mammalian cell cycle progression that functions both by inhibiting cyclin dependent kinases (CDKs) required for the initiation of S phase, and by binding to and inhibiting the DNA polymerase delta co-factor, proliferating cell nuclear antigen (PCNA). In this report, we show that adenovirus-mediated over-expression of human p21 inhibits growth factor-stimulated VSMC proliferation in vitro by efficiently arresting VSMCs in the G1 phase of the cell cycle. This p21-associated cell cycle arrest is associated both with significant inhibition of the phosphorylation of the retinoblastoma gene product (Rb) and with the formation of complexes between p21 and PCNA in VSMCs. In addition, we demonstrate that localized arterial infection with a p21-encoding adenovirus at the time of balloon angioplasty significantly reduced neointimal hyperplasia in the rat carotid artery model of restenosis. Taken together, these studies demonstrate the important role of p21 in regulating Rb phosphorylation and cell cycle progression in VSMC, and suggest a novel cytostatic gene therapy approach for restenosis and related vascular proliferative disorders.
The high affinity of even relatively short sequences of DNA for their target mRNA suggests that antisense agents represent an ideal method of suppressing specific gene products both in vitro and in vivo. In experiments performed thus far, an effect on the target mRNA in cultured vascular cells and in the vessel wall can be documented. The in vitro activity, toxicity, and pharmacokinetic data of antisense oligonucleotides are encouraging, and the in vivo animal experiments demonstrating suppression of neointimal formation are very promising. If animals trials presently under way show continued suppression not only of intimal formation but also of loss of lumen caliber after a single application, then effective delivery of antisense oligonucleotides is a realistic possibility. Nevertheless, some words of caution regarding the use of antisense oligonucleotides are warranted. Potential nonspecific effects of antisense oligonucleotides should be carefully considered in studies in which antisense agents are used to define biological functions of specific genes. In particular, demonstration that the target mRNA has been suppressed does not prove that other sequences within the mRNA pool have not also been suppressed. Critical control measures include adding back the target mRNA or protein and demonstrating similar biological effects with antisense sequences, which also suppress target gene expression directed at different regions of the target mRNA. At the clinical level, the systemic effects of antisense oligonucleotides, the dosage required, the timing of administration compared with mechanical intervention, and the toxicity of breakdown products all need to be established. In addition, the most appropriate targets for antisense use in restenosis remain largely obscure. Indiscriminate suppression of cell-cycle genes or proto-oncogenes may be as acutely toxic as current anticancer chemotherapy if the site delivery is not completely localized. Furthermore, much of the clinical evidence suggests that restenosis is a chronic process, continuing to develop weeks to months after the procedure. If this is the case, then the current approaches that rely on a transient, local application of an antisense agent may fail. If, however, a target gene is identified that is specific to vascular tissue, then repeated administration of an antisense agent may be tolerated via a systemic route. This approach has proved successful in targeting mutated genes with little suppression of closely related genes and with minimal systemic toxicity. An alternative approach is to transfect the target tissue with a gene that makes it susceptible to systemic delivery of a drug that is not normally toxic to mammalian cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Within the first year, 15-20% of coronary artery saphenous bypass vein grafts (SVGs) occlude because of thrombosis or progressive intimal hyperplasia. One potential new strategy to reduce this complication would be to introduce antithrombotic or antiproliferative genes in vein grafts before implantation. The success of this approach requires an efficient DNA delivery system. In the present study we tested the feasibility of using adenovirus-transferrin/polylysine-DNA complexes (TfAdpl/DNA) to achieve high-efficiency gene transfer into vascular interposition vein grafts. All studies used the Escherichia coli LacZ (beta-galactosidase [beta-Gal]) reporter gene under the control of the cytomegalovirus (CMV) earlier promoter and enhancer (pCMV/LacZ). Autologous rabbit jugular vein segments were incubated ex vivo for 60 min in a solution of TfAdpl/DNA complexes (1.2 x 10(10) biotinylated adenovirus particles, 2,430 ng of streptavindylated polylysine. 10 micrograms of plasmid DNA, and 9 micrograms of transferrin-polylysine per ml), and then reimplanted across the ligated right carotid artery. Control veins were incubated in TfAdpl solution in which DNA was omitted. A total of six grafts were treated with TfAdpl/DNA, and two grafts were treated with TfAdpl. Veins were harvested 3 (n = 3) and 7 (n = 3) days later and beta-Gal activity was determined by X-Gal chromogen staining. All six TfAdpl/DNA-treated grafts stained intensely blue, whereas control grafts were negative. Microscopic examination of serial sections revealed intracellular blue granules consistent with beta-Gal activity to be present in all of the endothelial cells and in numerous medial and advential cells.(ABSTRACT TRUNCATED AT 250 WORDS)
The authors determined the impact of an intensive surveillance program of autogenous vein bypasses on patency and limb salvage.
Surveillance protocols of vein bypasses can identify graft-threatening lesions to permit elective revisions before thrombosis. The authors compared follow-up based on clinically indicated procedures with intensive surveillance.
From 1985 to 1994, 615 autogenous vein bypasses (454 in situ, 161 reversed/composite) to popliteal (n = 169) and tibial (n = 446) arteries were performed for critical limb ischemia (n = 507), claudication (n = 88), and popliteal aneurysm (n = 20). Intensive surveillance of autogenous vein bypasses consisted of ankle brachial index and duplex scan with graft velocities measured at 1 month, 3 months, 6 months, and every 6 months subsequently. After surgery 317 bypasses had intensive surveillance, 222 bypasses were clinically indicated for follow-up, and 76 bypasses were excluded because follow-up or patency was less than 31 days.
Primary patency at 5 years was similar for bypasses treated by intensive surveillance (56%) and those treated with clinically indicated procedures (67%). Secondary patency and limb salvage at 5 years was significantly improved (p < 0.02) for bypasses followed by intensive surveillance (80% and 94%) compared with clinically indicated procedures (67% and 73%). Revision of patent bypasses was higher (p < 0.000001) for bypasses treated by intensive surveillance (61 of 70, 87%) compared with those treated with clinically indicated procedures (9 of 34, 26%). Secondary patency at 2 years was significantly higher (p < 0.02) for revision of patent bypasses (79%) compared with thrombosed bypasses (55%).
Long-term autogenous vein bypass patency and limb salvage is significantly improved by intensive surveillance, permitting identification and correction of graft threatening lesions before thrombosis.
Most strategies designed to reduce restenosis by the use of pharmacological or biological reagents involve direct inhibition of vascular smooth muscle cell (SMC) proliferation. Alternatively, SMC proliferation might be indirectly inhibited if reendothelialization could be specifically facilitated at sites of balloon-induced arterial injury. Accordingly, we investigated the hypothesis that application of an endothelial cell (EC)-specific mitogen to a freshly denuded intimal surface could accelerate reendothelialization and thereby attenuate intimal hyperplasia.
The left carotid artery of 31 Sprague-Dawley rats was subjected to balloon injury, after which 16 rats were treated with a 30-minute incubation with 100 micrograms of vascular endothelial growth factor (VEGF), an EC-specific mitogen. Control animals (n = 15) received a 30-minute incubation with 0.9% saline. At 2 weeks after balloon injury, carotid artery reendothelialization was markedly superior in the VEGF-treated group compared with the control group (14.59 +/- 1.12 versus 7.96 +/- 0.51 mm2, P < 0.005). The extent of reendothelialization measured at 4 weeks after balloon injury remained superior for arteries treated with VEGF (18.04 +/- 0.90 mm2) versus saline (13.42 +/- 0.84 mm2, P < .005). Neointimal thickening was correspondingly attenuated to a statistically significant degree in arteries treated with VEGF versus the control group at both the 2-week and 4-week time points. Immunostaining for proliferating cell nuclear antigen (PCNA) disclosed a threefold increase in PCNA-positive cells in the neointima of control arteries versus VEGF-treated arteries at 2 weeks after injury.
Application of VEGF, an EC-specific growth regulatory molecule, may be effectively used in vivo to promote reendothelialization and thereby indirectly attenuate neointimal thickening due to SMC proliferation.
Vascular smooth muscle cell (SMC) proliferation in response to injury is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis after balloon angioplasty. The retinoblastoma gene product (Rb) is present in the unphosphorylated and active form in quiescent primary arterial SMCs, but is rapidly inactivated by phosphorylation in response to growth factor stimulation in vitro. A replication-defective adenovirus encoding a nonphosphorylatable, constitutively active form of Rb was constructed. Infection of cultured primary rat aortic SMCs with this virus inhibited growth factor-stimulated cell proliferation in vitro. Localized arterial infection with the virus at the time of balloon angioplasty significantly reduced SMC proliferation and neointima formation in both the rat carotid and porcine femoral artery models of restenosis. These results demonstrate the role of Rb in regulating vascular SMC proliferation and suggest a gene therapy approach for vascular proliferative disorders associated with arterial injury.
Restenosis after percutaneous coronary balloon angioplasty remains a significant problem. Despite success with a variety of agents in animal models, no agent has proved clearly successful in reducing restenosis in humans. There are many potential reasons for this, but one possibility is that because of our incomplete understanding of the restenotic process, therapy has been directed at the wrong target. Arterial remodeling (changes in total vessel area or changes in area circumscribed by the internal elastic lamina) is well described in de novo atherosclerosis, and there is increasing evidence that this process occurs after angioplasty. Thus, restenosis can be thought of not merely as neointimal formation in response to balloon injury, but as arterial remodeling in response to balloon injury and neointimal formation. Arterial remodeling may consist of actual constriction of the artery, as has been described in some animal models and in preliminary fashion in humans, or of compensatory enlargement as has been described in de novo atherosclerosis and in the hypercholesterolemic rabbit iliac artery model. Arterial constriction can result in restenosis with minimal neointimal formation. Compensatory enlargement accommodates significant amounts of neointimal formation, with preservation of lumen area despite an increase in neointimal area adequate to cause restenosis in a noncompensated artery. This expanded paradigm of arterial remodeling and intimal formation may in part account for the lack of success in clinical trials to date, and therapy directed at arterial remodeling as well as intimal formation may be required to reduce restenosis after coronary interventions.
The potential for catheter-based in vivo delivery of genetic material to the arterial wall is incompletely explored. We evaluated the level of recombinant protein production as well as the anatomic distribution and duration of gene expression following adenoviral vector-mediated gene transfer into sheep arteries via a double balloon catheter. Catheters were positioned in the carotid or femoral arteries of 20 sheep via a combined percutaneous and surgical approach, and virions infused over a 30-min period. Three days later, recombinant gene expression was identified in approximately 30% (range 0-80%) of the luminal endothelial cells within the targeted area of the artery. Persistent recombinant protein expression was identified histochemically for up to 4 weeks, although the number of positive cells decreased steadily. High levels of both beta-galactosidase (beta-Gal) activity and protein (mean 20 mU and 44 ng per vessel) were measured in vessel extracts 3 days after gene transfer, again decreasing significantly over a 4-week period. Transgene expression was limited almost entirely to the intima and adventitia; adventitial gene transfer occurred virtually exclusively along the vasa vasorum. In comparison to previous studies of catheter-based gene transfer, adenoviral vectors delivered by double balloon catheter resulted in a particularly high efficiency of endothelial cell gene transfer. The efficiency and amount of recombinant gene expression achieved in this study suggest that catheter-based gene delivery may eventually be applicable to the treatment of focal human arterial disease.
Prosthetic vascular grafts containing retrovirally transduced autologous vascular smooth muscle cells were studied as a model for introduction of human genes into baboons. Retroviral vectors encoding beta-galactosidase (beta-Gal) (LNPoZ) or human purine nucleoside phosphorylase (LPNSN-2), a control gene, were used for ex vivo transduction of autologous baboon smooth muscle cells obtained from vein biopsies. Transduced cells were placed into a collagen solution and seeded into the interstices of polytetrafluoroethylene vascular grafts. Endothelial cells were then seeded onto the luminal surface of the grafts to reduce thrombus formation. One LNPoZ-seeded graft and one LPNSN-2-seeded control graft were implanted bilaterally into the aorto-iliac circulation of each of 4 animals. All grafts remained patent until they were removed after 3-5 weeks and examined histochemically for vector-expressing cells. All histological cross-sections from the beta-Gal vector seeded grafts contained cells staining blue with the X-Gal chromogen. For the four grafts, the mean fraction of LNPoZ expressing cells was 10%, with a range of 2-20%, while no sections from the control grafts contained stainable cells. Smooth muscle cells expressing the reporter gene were localized within the graft wall but not in the newly forming intima or outer capsule of fibrous tissue. Implantation of transduced cells within this type of vascular graft may provide a useful approach for long-term local and systemic gene therapy.
Gene therapy has been proposed as a possible solution to the problem of restenosis after coronary angioplasty. The current study was undertaken to assess conventional methods of gene transfer and to develop percutaneous techniques for introducing genes directly into the coronary arteries of large mammals. Since the anticipated targets of gene therapy against restenosis include atherosclerotic and previously instrumented arteries, we also evaluated gene transfer in atherosclerotic coronary arteries and in two porcine models of restenosis: one using intracoronary stents and a second using balloon overstretch angioplasty.
The conventional method of using perforated balloon catheters to deliver Lipofectin-DNA complexes directly into the coronary arteries of intact animals was applied to 18 porcine coronary arteries including normal arteries, hypercholesterolemic arteries, and those simulating restenosis. The results of this study were consistent with previously published results indicating that only low levels of luciferase gene expression could be obtained by Lipofectin-mediated gene transfer. We therefore undertook a second, parallel study to evaluate percutaneous transluminal in vivo gene transfer using a replication-deficient adenoviral vector. A comparison of the two studies revealed that the mean level of reporter gene expression in the cohort undergoing adenoviral infection was 100-fold higher than in the cohort undergoing Lipofection. Analysis of luciferase activity over time in normal arteries revealed that recombinant gene expression was half-maximal after 1 day, peaked within 1 week, was still half-maximal at 2 weeks, and declined to low levels by 4 weeks. Histochemical analysis of coronary arteries treated with a second adenovirus expressing a nuclear-localized beta-galactosidase gene demonstrated gene transfer to a limited number of cells in the media and adventitia. Immunohistochemical analysis of Ad5-infused arteries using a monoclonal antibody directed against CD44 identified a periadventitial infiltrate composed of leukocytes.
The recombinant adenoviral vectors proved to be far more effective than Lipofectin at delivering foreign genes directly into the coronary arteries of living mammals. Furthermore, the influences of hypercholesterolemia and arterial injury appeared to have little effect on the levels of gene expression obtained using either method. The results demonstrate that low-level recombinant gene expression, the major obstacle impeding gene therapy for the prevention of restenosis, can potentially be overcome by using adenoviral vectors to mediate coronary gene transfer in vivo. The duration of gene expression provided by these vectors and their effective deployment in atherosclerotic, balloon-overstretched, and stented coronary arteries suggest that recombinant adenovirus may have potential for evaluating gene therapy in the clinically informative porcine models of coronary restenosis.
First-generation recombinant adenoviruses that lack E1 sequences have shown tremendous promise in animal and human models of gene therapy. Important limitations of these vectors are that recombinant gene expression is transient and inflammation occurs at the site of gene transfer. Our hypothesis for generating vectors with increased persistence is that present recombinant adenoviruses express viral proteins that stimulate cellular immune responses leading to destruction of the infected cells and repopulation of the organ with non-transgene-containing cells. This model predicts that further crippling of the virus will improve persistence and diminish pathology. We describe in this report second-generation recombinant adenoviruses harboring a beta-galactosidase-expressing transgene in which a temperature-sensitive mutation has been introduced into the E2A gene of an E1-deleted recombinant. At nonpermissive temperature, this virus fails to express late gene products, even when E1 is expressed in trans. The biology of this recombinant was studied in vivo in the context of mouse liver, a setting that is permissive for adenovirus type 5 replication. Animals that received the second-generation virus expressed the transgene for at least 70 days, whereas expression of the first-generation virus was no longer than 14 days. In addition, the inflammatory response, as measured by infiltration of CD8+ T cells, was blunted and delayed in livers infected with second-generation virus. These studies illustrate that modifications that disrupt structural protein expression in recombinant adenoviruses may be useful in enhancing their utility for gene therapy.