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E G Nabel

Publications and source records attributed to E G Nabel.

At least 55 records · Page 3Linked to original sources
PubMed1996

A new cationic liposome DNA complex enhances the efficiency of arterial gene transfer in vivo.

An important goal of gene therapy for cardiovascular diseases and cancer is the development of effective vectors for catheter-based gene delivery. Although adenoviral vectors have proven effective for this purpose in animal models, the ability to achieve comparable gene transfer with nonviral vectors would provide potentially desirable safety and toxicity features for clinical studies. In this report, we describe the use of a new cationic DNA-liposome complex using an improved expression vector and lipid, N-(3-aminopropyl)-N, N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminium bromide/dioleyl phosphatidylethanolamine (GAP-DL-RIE/DOPE) to optimize catheter-mediated gene transfer in porcine arteries. The efficiency of this vector was compared to DNA alone, DNA with a previously described cationic liposome complex, (+/-)-N-(2-hydroxyethyl)-N, N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE/DOPE), and a replication-defective adenoviral vector in a porcine artery gene transfer model. When used in optimal ratios, GAP-DL-RIE/DOPE liposomes provided a 15-fold higher level of gene expression in arteries compared to DNA alone or DMRIE/DOPE. Gene expression was observed in intimal and medial cells. However, when compared to adenoviral vectors (10(10) pfu/ml), gene expression following GAP-DLRIE/DOPE transfection was approximately 20-fold lower. Following intravenous injection of GAP-DLRIE/DOPE in mice, biochemical, hematological, and histopathological abnormalities were not observed. Significant improvements in the efficacy of arterial gene expression can be achieved by optimization of transfection condition with DNA-liposome complexes in vivo that may prove useful for arterial gene delivery in cardiovascular diseases and cancer.

Animals↗
PubMed1996

Role of the p21 cyclin-dependent kinase inhibitor in limiting intimal cell proliferation in response to arterial injury.

Arterial injury induces a series of proliferative, vasoactive, and inflammatory responses that lead to vascular proliferative diseases, including atherosclerosis and restenosis. Although several factors have been defined which stimulate this process in vivo, the role of specific cellular gene products in limiting this response is not well understood. The p21 cyclin-dependent kinase inhibitor affects cell cycle progression, senescence, and differentiation in transformed cells, but its expression in injured blood vessels has not been investigated. In this study, we report that p21 protein is induced in porcine arteries following balloon catheter injury and suggest that p21 is likely to play a role in limiting arterial cell proliferation in vivo. Vascular endothelial and smooth muscle cell growth was arrested through the ability of p21 to inhibit progression through the G1 phase of the cell cycle. Following injury to porcine arteries, p21 gene product was detected in the neointima and correlated inversely with the location and kinetics of intimal cell proliferation. Direct gene transfer of p21 using an adenoviral vector into balloon injured porcine arteries inhibited the development of intimal hyperplasia. Taken together, these findings suggest that p21, and possibly related cyclin-dependent kinase inhibitors, may normally regulate cellular proliferation following arterial injury, and strategies to increase its expression may prove therapeutically beneficial in vascular diseases.

Adenoviridae↗
PubMed1996

Regulation of cellular proliferation and intimal formation following balloon injury in atherosclerotic rabbit arteries.

Injury to atherosclerotic arteries induces the expression of growth regulatory genes that stimulate cellular proliferation and intimal formation. Intimal expansion has been reduced in vivo in nonatherosclerotic balloon-injured arteries by transfer of genes that inhibit cell proliferation. It is not known, however, whether vascular cell proliferation can be inhibited after injury in more extensively diseased atherosclerotic arteries. Accordingly, the purpose of this study was to investigate whether expression of recombinant genes in atherosclerotic arteries after balloon injury could inhibit intimal cell proliferation. To test this hypothesis, we examined the response to balloon injury in atherosclerotic rabbit arteries after gene transfer of herpesvirus thymidine kinase gene (tk) and administration of ganciclovir. Smooth muscle cells from hyperlipidemic rabbit arteries infected with adenoviral vectors encoding tk were sensitive to ganciclovir, and bystander killing was observed in vitro. In atherosclerotic arteries, a human placental alkaline phosphatase reporter gene was expressed in intimal and medial smooth muscle cells and macrophages, identifying these cells as targets for gene transfer. Expression of tk in balloon-injured hyperlipidemic rabbit arteries followed by ganciclovir treatment resulted in a 64% reduction in intimal cell proliferation 7 d after gene transfer (P = 0.004), and a 35-49% reduction in internal area 21 d after gene transfer, compared with five different control groups (P < 0.05). Replication of smooth muscle cells and macrophages was inhibited by tk expression and ganciclovir treatment. These findings indicate that transfer of a gene that inhibits cellular proliferation limits the intimal area in balloon-injured atherosclerotic arteries. Molecular approaches to the inhibition of cell proliferation in atherosclerotic arteries constitute a possible treatment for vascular proliferative diseases.

Adenoviridae↗
PubMed1996

Direct transfer of a foreign MHC gene into human melanoma alters T cell receptor V beta usage by tumor-infiltrating lymphocytes.

The direct introduction of foreign genes into tumors shows promise as a therapeutic modality to enhance tumor immunogenicity. Hence, melanoma nodules were directly injected with a vector encoding an allogeneic MHC class I molecule, HLA-B7. Tumor-infiltrating lymphocytes (TIL) were isolated from cutaneous melanoma biopsies before and after HLA-B7 gene transfer. TIL were expanded in interleukin-2 (IL-2) by standard techniques for approximately 4 weeks, then analyzed for T cell receptor V beta usage by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Prior to gene transfer. TIL V beta usage was found to be highly restricted, the only one to four V beta families being expressed and one or two of these families representing more than 90% of the repertoire. As anticipated, TIL V beta usage varied among patients expressing different HLA types. However, V beta 13 was over-represented in that six of eight patients utilized V beta 13 as a dominant family, regardless of HLA type. Following HLA-B7 gene transfer, TIL V beta usage was markedly altered: (1) V beta families that dominated following gene transfer differed from the V beta families utilized by TIL prior to treatment, and (2) introduction of the HLA-B7 gene resulted in a more diverse repertoire with an increase in the number of V beta families represented. In two patients, TIL were evaluated before treatment and from multiple, distinct melanoma nodules following gene transfer. In these two patients, a comparison was made between TIL V beta profiles obtained after treatment from nodules that had been injected with the HLA-B7 gene or left untreated. Interestingly, the V beta repertoires of TIL from uninjected nodules following gene transfer were similar to that of TIL from injected nodules, rather than pretreatment TIL. These data demonstrate a direct immunological effect of foreign MHC gene transfer into human melanoma, and suggest that local expression of an allogeneic MHC molecule generates systemic alterations in the antitumor immune response.

CD4 Antigens↗
PubMed1996

Genetic therapy.

Vascular gene transfer is the introduction of foreign DNA into host cells within the vessel wall. Expression of recombinant genes within vascular cells has been demonstrated in normal, injured and atherosclerotic arteries. Transfer of genes with biological activity has provided insights into the role of specific genes in normal and pathological states. The development of gene transfer as a form of vascular therapy will require safe and effective vectors and intravascular delivery systems. Viral and non-viral vectors and several delivery catheters are being evaluated to determine their clinical applicability. The current applications of vascular gene transfer to cardiovascular diseases are the prevention of restenosis following arterial injury and induction of angiogenesis in occlusive vascular disease.

Animals↗
PubMed1996

Gene transfer approaches to the regulation of vascular cell proliferation.

Considerable progress has been made in identifying potential targets for treating vascular proliferative diseases. In this review, we discuss gene transfer approaches to regulating smooth muscle cell proliferation after vascular injury using the cell cycle specific proteins, p21, delta Rb and HSV-tk. Results from these studies suggest that replicating smooth muscle cells and macrophages are inhibited in vivo in several animal models of restenosis, including hyperlipidaemic vessels. Identification of appropriate vascular diseases and improvements in gene delivery and vectors will require careful optimization in order to develop effective molecular therapies for human vascular diseases.

Animals↗
PubMed1995

Direct gene transfer for treatment of human cancer.

Genetic instability within malignant cells gives rise to mutant proteins which can be recognized by the immune system. Recognition of tumor-associated antigens by T lymphocytes could thus contribute to the elimination of neoplastic cells. We have developed a molecular genetic intervention for the treatment of malignancies based upon the knowledge that foreign major histocompatibility complex (MHC) proteins expressed on the cell surface are efficient at stimulating an immune response. Expression of this foreign MHC gene within tumors induced a cytotoxic T cell response to the introduced gene. More importantly, the immune system recognized tumor-specific antigens on unmodified tumor cells as foreign. Growth of the tumors diminished, and in many cases, there was complete regression. This research provides evidence that direct gene transfer in vivo can induce cell-mediated immunity against specific gene products, and offers the potential for effective immunotherapy for the treatment of cancer and infectious diseases in man. Our laboratory conducted a phase I clinical trial to determine the safety and efficacy of this treatment in humans. These studies suggest that direct gene transfer provides a safe and feasible approach for the treatment of human cancer. More recent developments using combination gene therapy and the initiation of a second human trial with improvements on this technology have been implemented. Finally, we have begun to define mechanisms of resistance to immune recognition by established malignancies.

Animals↗
PubMed1995

Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product.

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.

Adenoviridae↗
PubMed1995

The p21 cyclin-dependent kinase inhibitor suppresses tumorigenicity in vivo.

The p21 gene encodes a cyclin-dependent kinase inhibitor that affects cell-cycle progression, but the potential of this gene product to serve as a tumour suppressor in vivo has not been established. In this report, we show that the growth of malignant cells in vitro and in vivo is inhibited by expression of p21. Expression of p21 resulted in an accumulation of cells in G0/G1, altered morphology, and cell differentiation, but apoptosis was not induced. Introduction of p21 with adenoviral vectors into malignant cells completely suppressed their growth in vivo and also reduced the growth of established pre-existing tumours. Gene transfer of p21 may provide a molecular genetic approach to arresting cancer cell growth by committing malignant cells irreversibly to a pathway of terminal differentiation.

3T3 Cells↗
PubMed1995

Expression and function of a recombinant PDGF B gene in porcine arteries.

Platelet-derived growth factor (PDGF) B is a mitogen and chemoattractant for smooth muscle cells in vitro, and expression of a recombinant PDGF B gene in porcine arteries stimulates intimal thickening. To define the mechanisms by which PDGF B gene expression induces intimal thickening in vivo, we examined its effects on smooth muscle cell proliferation and migration, extracellular matrix synthesis, and inflammatory cell infiltration in intimal lesions of pig arteries after direct gene transfer of a recombinant PDGF B gene. PDGF B gene expression was associated with rapid formation of an intima, including 3- to 10-fold increases in intimal thickness and intima-to-media area ratio 4 to 21 days after gene transfer compared with control transfected arteries. Intimal smooth muscle cell proliferation was detected at 2 days, peaked at 7 days (P < .01), and declined by 14 days, although the total number of intimal nuclei progressively increased to 21 days (P < .01). Calculations of expected-to-observed ratios of intimal cells, based on BrdC proliferation indexes, demonstrated that the increases in intimal cell number on days 2 through 7 could not be accounted for by proliferation alone, suggesting that recombinant PDGF BB acts to stimulate cell proliferation and migration of smooth muscle cells into the intima. Extracellular matrix deposition and procollagen synthesis were observed after 7 days (P < .01) and were associated with a decline in cell density in the intima, suggesting that extracellular matrix synthesis may contribute to progressive intimal thickening in response to PDGF B gene expression. There was minimal accumulation of inflammatory cells, including macrophages and CD3(+) lymphocytes, in transfected arteries. These data suggest that PDGF B gene expression promotes intimal expansion by both proliferation and migration of smooth muscle cells followed by synthesis of extracellular matrix and therefore acts through several mechanisms to play a role in the pathogenesis of intimal lesions in vivo.

Animals↗
PubMed1995

Fibroblast growth factor stimulates angiotensin converting enzyme expression in vascular smooth muscle cells. Possible mediator of the response to vascular injury.

Angiotensin converting enzyme (ACE) activity contributes to the vascular response to injury because ACE inhibition limits neointima formation in rat carotid arteries after balloon injury. To investigate the mechanisms by which ACE may contribute to vascular smooth muscle cell (VSMC) proliferation, we studied expression of ACE in vivo after injury and in vitro after growth factor stimulation. ACE activity 14 d after injury was increased 3.6-fold in the injured vessel. ACE expression, measured by immunohistochemistry, became apparent at 7 d in the neointima and at 14 d was primarily in the most luminal neointimal cells. To characterize hormones that induce ACE in vivo, cultured VSMC were exposed to steroids and growth factors. Among steroids, only glucocorticoids stimulated ACE expression with an 8.0 +/- 2.1-fold increase in activity and a 6.5-fold increase in mRNA (30 nM dexamethasone for 72 h). Among growth factors tested, only fibroblast growth factor (FGF) stimulated ACE expression (4.2 +/- 0.7-fold increase in activity and 1.6-fold increase in mRNA in response to 10 ng/ml FGF for 24 h). Dexamethasone and FGF were synergistic at the indicated concentrations inducing 50.6 +/- 12.4-fold and 32.5-fold increases in activity and mRNA expression, respectively. In addition, when porcine iliac arteries were transfected with recombinant FGF-1 (in the absence of injury), ACE expression increased in neointimal VSMC, to the same extent as injured, nontransfected arteries. The data suggest a temporal sequence for the response to injury in which FGF induces ACE, ACE generates angiotensin II, and angiotensin II stimulates VSMC growth in concert with FGF.

Animals↗
PubMed1995

Adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene inhibits vascular smooth muscle cell proliferation and neointima formation following balloon angioplasty of the rat carotid artery.

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation following arterial injury plays a critical role in a variety of vascular proliferative disorders, including atherosclerosis and restenosis after balloon angioplasty. In this study, we tested the hypothesis that localized arterial infection at the time of balloon angioplasty with an adenovirus (ADV-tk) encoding the herpes simplex virus thymidine kinase gene (HSV-tk), followed by systemic ganciclovir administration, can inhibit VSMC proliferation and neointima formation in a well-characterized model of arterial injury and restenosis. MATERIALS AND METHODS: The left carotid arteries of 31 male Sprague-Dawley rats were subjected to balloon angioplasty and immediately infected with 2 x 10(9) pfu of either ADV-tk or a control adenovirus that does not encode a recombinant protein (ADV-delta E1). Twenty-four hours after injury, animals from each experimental group were randomized to receive a course of systemic ganciclovir (ADV-tk/+GC, ADV delta E1/+GC) or saline (ADV-tk/-GC, ADV-delta E1/-GC). VSMC DNA synthesis was measured by 5'-bromodeoxuridine (BrdU) incorporation 2-4 days after balloon injury. The extent of restenosis, expressed as the neointima to media (I/M) area ratio was determined by digital planimetry 20 days after balloon injury in each of the four treatment groups. Immunohistochemistry using a mAb to von Willebrand factor (vWF) was used to determine the effects of ADV-tk infection and ganciclovir treatment on re-endothelialization of the carotid arteries 20 days following balloon angioplasty. RESULTS: Forty-one percent of the medial VSMCs in the ADV-tk/-GC arteries were labeled with BrdU 4 days after balloon injury. In contrast, ADV-tk infected animals that were treated with systemic ganciclovir (ADV-tk/+GC) displayed a 40% reduction in BrdU-staining medial VSMCs (p < 0.03). I/M area ratios of the three control groups were 1.17 +/- 0.18 (ADV-tk/-GC, n = 5), 1.15 +/- 0.10 (ADV-delta E1/+GC, n = 6), and 0.91 +/- 0.08 (ADV-delta E1/-GC, n = 6). These differences were not statistically significant (p > 0.05). In contrast, the ADV-tk/+GC animals (n = 6) displayed an I/M area ratio of 0.49 +/- 0.13 which was significantly lower than that seen in each of the three control groups (p < 0.02). None of the treated animals showed evidence of significant organ toxicity at autopsy. A regenerated endothelium was observed in the ADV-tk/+GC animals 20 days after balloon injury. CONCLUSIONS: Localized arterial infection with ADV-tk at the time of balloon angioplasty followed by systemic ganciclovir therapy reduces VSMC proliferation and neointimal expansion in the rat carotid artery injury model. Moreover, combined treatment with ADV-tk and systemic ganciclovir does not result in systemic toxicity and appears to selectively eliminate proliferating VSMCs, while preserving the capacity of the injured arterial segments to re-endothelialize within 3 weeks of injury. Taken together, these results support the feasibility of using this gene therapy approach for the treatment of human vascular proliferative disorders.

Adenoviridae↗
PubMed1995

Gene therapy for vascular diseases.

Arterial injury induces the synthesis of gene products that stimulate smooth muscle cell (SMC) migration and proliferation, leading to intimal hyperplasia. This process contributes to the pathogenesis of many cardiovascular diseases, including vascular proliferative diseases and atherosclerosis. Molecular approaches to the inhibition of SMC proliferation could potentially limit intimal expansion following vascular injury. During the past five years, there has been considerable interest in developing methods for the introduction and expression of recombinant genes in the vasculature. Gene transfer offers novel approaches to the study and treatment of vascular diseases. To date, gene transfer to the vasculature has been employed largely for two purposes: (1) examination of the expression and function of recombinant genes in vivo; and (2) development of potential new therapies for vascular diseases.

Animals↗
PubMed1994

Gene therapy for vascular smooth muscle cell proliferation after arterial injury.

Accumulation of vascular smooth muscle cells as a consequence of arterial injury is a major feature of vascular proliferative disorders. Molecular approaches to the inhibition of smooth muscle cell proliferation in these settings could potentially limit intimal expansion. This problem was approached by introducing adenoviral vectors encoding the herpesvirus thymidine kinase (tk) into porcine arteries that had been injured by a balloon on a catheter. These smooth muscle cells were shown to be infectable with adenoviral vectors, and introduction of the tk gene rendered them sensitive to the nucleoside analog ganciclovir. When this vector was introduced into porcine arteries immediately after a balloon injury, intimal hyperplasia decreased after a course of ganciclovir treatment. No major local or systemic toxicities were observed. These data suggest that transient expression of an enzyme that catalyzes the formation of a cytotoxic drug locally may limit smooth muscle cell proliferation in response to balloon injury.

Adenoviridae↗
PubMed1994

Direct gene transfer for the understanding and treatment of human disease.

Direct gene transfer has been used to develop molecular genetic interventions for acquired diseases in several animal models. Through the use of intravascular catheters or anatomically localized injection of DNA liposome complexes, specific tissues can be transduced with recombinant genes. Several promising applications of this method for the study of vascular biology have been demonstrated by direct gene transfer into arteries in vivo. Delivery, via catheter, of genes that modulate the thrombogenic or proliferative properties of vascular cells may someday provide therapy for stenotic lesions of atherosclerosis or following angioplasty. Cancer is another acquired disorder in which direct gene transfer may improve the efficacy of treatment. Introduction of class I MHC or cytokine genes with antitumor or immunostimulatory effects have demonstrated promise in animal models. Direct transfer of an allogeneic class I MHC gene into tumors in vivo induces a CD8+ CTL response against weak antigens on poorly immunogenic tumors. The efficacy of this antitumor response can be augmented to induce regression of actively growing established tumors. Additional strategies, such as intratumoral delivery of combinations of multiple cytokine and MHC genes, may serve to improve the antitumor response. A clinical gene therapy protocol is underway to analyze the safety and efficacy of DNA liposome-mediated gene transfer in humans. Development of improved gene delivery systems and introduction of recombinant genes into visceral tumors by intravascular catheter will extend the application of direct gene transfer to immunotherapy of malignancies. These clinical trials of direct gene transfer will help to develop new treatment strategies for human diseases.

Animals↗
PubMed1994

Recombinant growth factor gene expression in vascular cells in vivo.

Several issues are important to the use of direct gene transfer as an investigative tool and as a potential therapeutic modality (Table 3). Transfection efficiencies of different vectors must be improved and optimized. Retroviral vectors and DNA liposome conjugates currently used in animal models are low-efficiency vectors. Adenoviruses and adenoviral conjugates appear promising, but issues related to gene persistence, germ-line transmission, and stability of expression must be explored. Second, the pharmacology or dose-response properties of recombinant gene expression have not been investigated. It is not currently known how many cells must be transfected in an arterial segment in order to produce a desired biological effect. Our studies suggest that only a small population of cells is required to secrete a recombinant gene product into the local milieu. This gene product may then have local paracrine effects with amplification of the biological response, suggesting a "gain of function." Third, methods must be developed to target recombinant genes specifically to endothelial cells or smooth muscle cells using cell-specific promoters. Finally, gene expression should be regulated through inducible or repressible promoters. Nonetheless, during the past ten years a dramatic expansion in the fields of gene transfer and gene therapy has occurred. We have entered a new era in which molecular genetic techniques are being increasingly used to investigate the pathophysiology of cardiovascular disorders and to design potential therapies for these diseases. Although technical hurdles related to optimization of vectors and regulated gene expression must be solved, molecular genetic approaches will be increasingly used to study and treat cardiovascular diseases.

Animals↗