PERSPECTIVE
Pulmonary Vascular Gene Transfer
Prospects for Successful Therapy of Pulmonary Hypertension

Brian Fouty and David M. Rodman

University of Colorado Health Sciences Center, Denver, Colorado

In this issue of the Journal, Campbell and coworkers report studies of cell-based gene transfer to the pulmonary circulation (1). This is the first demonstration that this technique could hold promise for therapy of pulmonary vascular disease. It has been nearly ten years since the first report of successful in vivo gene transfer. Subsequently, "gene therapy" has been proposed as a therapeutic modality for a variety of diseases, and proof of principle has been demonstrated in several animal models. However, the lung remains a complex target for gene therapy, and the majority of work in this area has focused on the airway and the disease paradigm of cystic fibrosis (2). Although the pulmonary circulation has received much less attention, it is an equally intriguing target for gene transfer.

Overview of the Problem

Normal pulmonary circulation is a low-pressure, high- capacitance vascular bed exposed to 100% of cardiac output, and it functionally matches ventilation with perfusion to maintain adequate arterial oxygenation. Disorders of the pulmonary circulation that could be addressed by gene transfer include primary pulmonary hypertension (PPH), persistent pulmonary hypertension of the neonate, secondary pulmonary hypertension as occurs in progressive systemic sclerosis and other connective tissue diseases, acute lung injury (acute respiratory distress syndrome), and metastatic neoplasms. Lung transplantation presents several interesting possibilities for gene transfer as well, including interventions designed to mitigate reperfusion injury in the acute phase and reduce allograft rejection chronically.

Pulmonary hypertension (PH), the disease process addressed by Campbell and associates, presents a compelling initial target because of the paucity of effective therapeutic options. The disease represents the sum of increased vascular tone and vascular remodeling in the injured pulmonary circulation, and most patients with PH present with advanced disease characterized by marked vascular remodeling and limited reversibility with vasodilators. Any successful approach to treating PH must address the mechanism of vascular remodeling.

Lessons from the Systemic Circulation

Clues on how to approach the problem of pulmonary vascular remodeling can be found in research being done in the systemic circulation. Restenosis after angioplasty and bypass grafting is an important clinical problem that has been intensively studied. Three general gene-transfer strategies have been used to minimize the cellular proliferation after injury: (1) modulation of cell-cycle proteins; (2) conversion of prodrugs to cytotoxic agents by transfection of viral thymidine kinase into the vessel wall; and (3) local overexpression of genes with paracrine antiproliferative potential (such as nitric oxide synthase [NOS]).

Cellular proliferation after stimulation or injury requires the coordinated interaction of cell-cycle proteins known as cyclins and cyclin-dependent kinases (cdk). Inhibitors of these cyclin-cdk complexes, such as p21, p27, and p16, are important in controlling cell proliferaton in cell culture as well as in animal models of injury (3). Overexpression of the cdk inhibitor p21 at the time of balloon injury significantly decreased neointimal formation in a rat carotid artery model of balloon angioplasty (4). An alternative approach aimed at disrupting the cell cycle involves introduction of a constitutively active retinoblastoma protein into injured vessels. Retinoblastoma is a critical protein involved in controlling cell proliferation. In its hypophosphorylated (active) state, retinoblastoma binds to the transcriptional factor E2F (3). When retinoblastoma is phosphorylated by the sequential action of cdk4 and cdk2 (5), E2F is released and can bind to the promoter region of proproliferative genes, such as proliferating cell nuclear antigen, cdk2 kinase, c-myc, and c-myb, and activate their transcription. Transfection of this mutated retinoblastoma into veins grafted into the carotid circulation significantly decreased neointimal proliferation compared with controls (6). Rather than prevent E2F release, E2F binding to the promoter region of target genes can be blocked by introducing into cells double-stranded oligonucleotide sequences containing the consensus E2F binding site (E2F decoy) (7). Introduction of these oligonucleotide decoys into (cultured) vascular smooth muscles inhibited serum-stimulated growth, whereas transfection into rat carotid artery reduced neointimal proliferation after balloon angioplasty. This strategy is currently in early-phase human trials to prevent vein-graft restenosis. Although these three approaches target different components of the cell cycle, each was found to be beneficial in reducing neointimal proliferation.

The major limitation of the strategies based on interruption of the cell cycle is the requirement that nearly 100% of replicating cells be transduced. The problem is circumvented somewhat by the thimidine-kinase strategy. Herpes simplex virus thymidine kinase (HSV-TK) gene can phosphorylate the nucleoside analogue, gancyclovir, transforming it into a cytotoxic form. Local transfection of thymidine kinase at the time of injury after systemic infusion of gancyclovir will kill rapidly dividing cells that have been transfected (7). A major advantage of this strategy is a significant bystander effect, meaning that adjacent cells not transduced with HSV-TK will still be killed by the toxic metabolites. Using this approach, a number of investigators have demonstrated a reduction in neointimal formation in previously normal, injured vessels (8), as well as in atherosclerotic vessels (9).

However, the bystander effect of the HSV-TK strategy is still limited by a requirement for relatively high transfection efficiency. This limitation can potentially be overcome by transduction of genes with even broader paracrine activity. NOS has been tested as such a candidate. Von der Leyen and coworkers (10) first demonstrated that transfection of NOS3 into injured carotid vessels reduced neointimal formation. Campbell and associates extend those studies to the pulmonary circulation, demonstrating similar paracrine effects after cell-based gene transfer to the monocrotaline-injured pulmonary circulation.

Other Potential Therapeutic Genes

One of the most significant limitations to gene therapy for pulmonary vascular disease is identification of potential therapeutic genes. NOS3 and NOS2 are potentially useful candidates for PPH and secondary PH, as is prostacyclin synthase (11). Recently, vascular endothelial growth factor and atrial natriuetic peptide have also been demonstrated as potential therapeutic genes for PH (12, 13). For other disease targets, such as metastatic neoplasms, strategies designed to reduce tumor vascularization or to transfer HSV-TK may prove useful. In the lung injury and transplantation area, genes that reduce inflammation and white blood cell adhesion may be effective. And reduction of allograft rejection may result from strategies that selectively deplete clones of T lymphocytes, such as overexpression of Fas-ligand in the engrafted lung. As the human genome project progresses and additional human genes are identified and their functions delineated, more therapeutic genes will likely be identified.

Methods of Gene Delivery

Two other significant limitations to pulmonary vascular gene therapy are gene delivery and duration of expression. Campbell and colleagues use the technique of cell-based gene delivery, wherein syngeneic cells or cells from the recipient are removed, expanded, transduced in vitro, and re-infused. Their study demonstrates that vascular smooth-muscle cells injected into the systemic venous circulation are filtered by the lung, and a subpopulation migrate into the pulmonary artery medial layer. It is not certain if this technique is suitable for human therapy, but the study is encouraging. Other vectors that have been demonstrated to have some efficacy in the pulmonary circulation include the recombinant adenovirus and cationic lipids. To date both have significant limitations, including inflammation and relatively low efficiency (14). Other viral and nonviral vector systems have been described but their activity has not yet been reported in the pulmonary circulation.

Duration of expression is the other major limitation of gene-transfer strategies. Host response to both the vector and gene product can limit duration of expression. In addition, promoter downregulation can turn off transgene expression despite prolonged retention of the vector. Recent advances in the development of integrating vectors, such as lentivirus and adeno-associated virus, may obviate this problem. In addition, newer adenoviral vectors known as helper-dependent or "gutless" appear to have much less inflammation and more prolonged expression. Similarly, the use of cell-specific promoters, rather than viral promoters, may result in prolonged transgene expression. While cell-based gene transfer can be accomplished after stably transducing the donor cells, the duration of retention of those cells in the pulmonary circulation has not yet been established.

Challenges for the Future

Successful application of gene-transfer technology to the pulmonary circulation requires that the following four challenges be met: (1) identification of appropriate therapeutic genes, (2) improved vector efficiency, (3) specific pulmonary vascular targeting, and (4) elimination of the host-immune response to the vector and transgene. Although these challenges may seem daunting, the gene-transfer field is rapidly advancing. In the next few years these advances, in concert with our expanding knowledge of the human genome, should provide an excellent opportunity to develop novel strategies for the therapy of pulmonary vascular disease.

	Footnotes

Address correspondence to: Brian W. Fouty, M.D., Univ. of CO Health Sci. Ctr., 4200 E. 9th Ave., Denver, CO 80262. E-mail: brian.fouty{at}uchsc.edu

(Received in original form July 30, 1999).

	References

1. Campbell, A. I. M., M. A. Kuliszewski, and D. J. Stewart. 1999. Cell-based gene transfer to the pulmonary vasculature: endothelial nitric oxide synthase overexpression inhibits monocrotaline-induced pulmonary hypertension. Am. J. Respir. Cell Mol. Biol. 21: 567-575 [Abstract/Free Full Text].

2. K. L. Brigham, editor. 1997. Gene Therapy for Diseases of the Lung. Marcel Dekker, New York.

3. Sherr, C. J., and J. M. Roberts. 1999. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Devel. 13: 1501-1512 [Free Full Text].

4. Chang, M. W., E. Barr, M. M. Lu, K. Barton, and J. M. Leiden. 1995. Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21, inhibits vascular smooth muscle cell proliferation, and neointimal formation in the rat carotid artery model of balloon angioplasty. J. Clin. Invest. 96: 2260-2268 .

5. Lundberg, A. S., and R. A. Weinberg. 1998. Functional inactivation of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes. Mol. Cell Biol. 18: 753-761 [Abstract/Free Full Text].

6. Schwartz, L. B., J. Moawad, E. C. Svensson, R. L. Tufts, S. L. Meyerson, D. Baunoch, and J. M. Leiden. 1999. Adenoviral-mediated gene transfer of a constitutively active form of the retinoblastoma gene product attennuates neointimal thickening in experimental vein grafts. J. Vasc. Surg. 29: 874-881 [Medline].

7. Morishita, R., G. H. Gibbons, M. Horiuchi, K. E. Willison, M. Nakajima, L. Zhang, Y. Kaned, T. Ogihara, and V. J. Dzau. 1995. A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo. Proc. Natl. Acad. Sci. USA 92: 5855-5859 [Abstract/Free Full Text].

8. Ohno, T., D. Gordon, H. San, V. J. Pompili, M. J. Imperiale, G. J. Nabel, and E. G. Nabel. 1994. Gene therapy for vascular smooth muscle cell proliferation after arterial injury. Science 265: 781-784 [Abstract/Free Full Text].

9. Simari, R. D., H. San, M. Rekhter, T. Ohno, D. Gordon, G. J. Nabel, and E. G. Nabel. 1996. Regulation of cellular proliferation and intimal formation following balloon injury in atherosclerotic rabbit arteries. J. Clin. Invest. 98: 225-235 [Medline].

10. Von der Leyen, H., G. H. Gibbons, R. Morishita, N. P. Lewis, L. Zhang, M. Nakajima, Y. Kaneda, J. P. Cooke, and V. J. Dzau. 1995. Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene. Proc. Natl. Acad. Sci. USA 92: 1137-1141 [Abstract/Free Full Text].

11. Geraci, M. W., B. Gao, D. C. Shephherd, M. D. Moore, J. Y. Westcott, K. A. Fagan, L. A. Alger, R. M. Tuder, and N. F. Voelkel. 1999. Pulmonary prostacyclin synthase overexpression in transgenic mice protects against development of hypoxic pulmonary hypertension. J. Clin. Invest. 103: 1509-1515 [Medline].

12. Partovian, C., S. Adnot, B. Raffestin, M. Levame, P. Lemarchand, and S. Eddahibi. 1998. Adenoviral-mediated VEGF overexpression reduces chronic hypoxic pulmonary hypertension in rats. Am. J. Respir. Crit. Care Med. 159: A167 .

13. Louzier, V., S. Eddahibi, M. Hira, O. Desprez, I. Pham, M. Adam, M. Eloit, B. Raffestin, and S. Adnot. 1998. Adenovirus-mediated atrial natriuetic peptide (ANP) gene transfer in rat lungs protects against hypoxic pulmonary hypertension. Am. J. Respir. Crit. Care Med. 159: A165 .

14. Rodman, D. M., H. San, R. Simari, D. Stephan, F. Tanner, Z. Yang, G. J. Nabel, and E. G. Nabel. 1997. In vivo gene delivery to the pulmonary circulation in rats: transgene distribution and vascular inflammatory response. Am. J. Respir. Cell Mol. Biol. 16: 640-649 [Abstract].