SAN FRANCISCO—Research inspired by the mapping of the human genome has already sparked important discoveries in pulmonary medicine. By studying gene and protein expression in pulmonary hypertension, pulmonary fibrosis, and endotoxin-induced lung injury, for example, experts have gained knowledge that could someday lead to gene therapies. Investigators highlighted their findings at the American Thoracic Society 97th International Conference.[1]

PPH: A FAMILY DISEASE

Inherited mutations in the bone morphogenetic protein receptor 2 gene are clearly associated with primary pulmonary hypertension (PPH), new research has demonstrated. “Much of the time—perhaps even most of the time—[PPH] may have a familial basis [although] it is not recognized,” said James E. Loyd, MD, a Professor in the Division of Allergy, Pulmonary, and Critical Care Medicine at the Vanderbilt University Medical Center in Nashville, Tennessee.

Although the mutated PPH gene is as common in men as it is in women, the disease develops twice as often in women, Dr. Loyd noted. However, it is not clear whether male gender is protective or whether a factor related to female gender increases the likelihood that the disorder develops. Overall penetrance is very low, he added. PPH develops in only about one in five of those who have the gene.

Familial PPH has been associated with other genetic mutations, such as those involving several transforming growth factor ß–pathway participants. The disease also seems to show genetic anticipation—that is, penetrance, age of onset, severity, and/or rate of survival worsen with successive generations. However, this aspect of familial PPH remains highly controversial, said Dr. Loyd, because it is still unproven whether genetic anticipation in familial PPH has a biologic explanation or whether it is a result of ascertainment bias.

GENES AND PROTEINS

Unique patterns of gene and protein expression have been identified in both primary and secondary forms of pulmonary hypertension, reported Mark W. Geraci, MD. Patients have shown prostacyclin and thromboxane metabolite imbalances in their urine, for example, and increased endothelin-1 expression in their lungs, he noted.

Also, patients with PPH appear to over-express 5-lipoxygenase and 5-lipoxygenase–activating protein in the endothelia of plexiform lesions and inflammatory cells, remarked Dr. Geraci, an Associate Professor in the Division of Pulmonary Sciences and Critical Care Medicine at the University of Colorado Health Sciences Center in Denver. These patients have also shown decreased prostacyclin synthase in lung tissue.

Such discoveries have prompted animal research that may result in human gene therapies. Mice that are genetically altered to over-express prostacyclin synthase, for example, have been found less likely than unaltered mice to develop pulmonary hypertension after exposure to chronic hypobaric hypoxia. Targeted disruption of 5-lipoxygenase seems to prevent right ventricular hypertrophy in rodent hypoxia models.

Gene micro-array technology has made it possible to analyze gene expression in lung tissue from PPH patients and compare it with that in normal tissue, Dr. Geraci pointed out. This has led to the discovery of mutational differences in sporadic and familial PPH, suggesting that the two diseases are mechanistically distinct, he continued.

GENETICS AND THE RESPONSE TO ENDOTOXIN

Genetics strongly influence the response to endotoxin, a ubiquitous and important mediator of acute and chronic airway disease. Indeed, some mutations of the toll-like receptor 4 gene (TLR4) appeared to increase endotoxin susceptibility in animal and human studies, said David A. Schwartz, MD, MPH.

These studies linked the mutation to a poor immune response and significant decreases in forced expiratory volume in one second (FEV1) in study subjects experimentally challenged with endotoxin. They also found that patients with this mutation have a greater susceptibility to gram-negative infection. Among patients in the surgical intensive care unit (ICU), for example, the TLR4 mutation has been found to multiply the risk of gram-negative septic complications by about 8.5-fold. These patients also had higher APACHE III scores throughout their ICU stay than did the patients without the mutation, although their length of stay and mortality were not increased.

“However, our results also suggest [TLR4] is not the only gene that is important in determining endotoxin responsiveness,” said Dr. Schwartz, Director of the Division of Pulmonary and Critical Care Medicine at Duke University in Durham, North Carolina. The response to endotoxin is normal in some individuals with the TLR4 mutation but impaired in others without the mutation, he explained.

GENE EXPRESSION IN PULMONARY FIBROSIS

Using oligonucleotide micro-array analysis, Naftali Kaminski, MD, has identified patterns of gene expression in pulmonary fibrosis. Dr. Kaminski is head of the Functional Genomics Unit at the Sheba Medical Center in Israel.

Dr. Kaminski used advanced bioinformatic approaches in his experiments and demonstrated that two distinct transcriptional programs (inflammatory and fibrotic) were active in bleomycin-induced lung fibrosis. He subsequently analyzed gene expression in 13 human lung tissue samples. Five of these had histologic evidence of fibrosis consistent with usual interstitial pneumonia; the other eight samples had normal histologic findings.

Comparison of expression in the samples’ 7,129 genes revealed those genes usually expressed in pulmonary fibrosis, such as collagen 1 and 3, in the fibrotic lung tissue. “What surprised us,” commented Dr. Kaminski, “was an unexpected increase in metalloproteinases” in the fibrotic tissue: Matrix metalloproteinase (MMP)-1, -2, -9, and, in particular, -7 were clearly over-expressed.

The phosphoprotein osteopontin also is significantly over-expressed in fibrotic lung tissue from mice and humans, he added. Both of the genes that express osteopontin and MMP-7 may have a role in the development of pulmonary fibrosis, the results of further mouse experiments suggest.

THERAPY THAT TARGETS THE LUNGS

Delivering gene therapy to the lungs requires a vector, pointed out Paul N. Reynolds, MD, PhD, a Research Assistant Professor of Medicine in the Division of Human Gene Therapy at the University of Alabama at Birmingham. The adenovirus is a potentially excellent vector, he said, because of its systemic stability and high in vivo transfection efficiency, which is a hundred times greater than that of nonviral vectors.

The problem with the adenovirus, animal studies suggest, is that uptake occurs primarily in the liver. However, Dr. Reynolds and his research team have developed a method to redirect the adenovirus to the lungs. They first identified a target for the virus on the pulmonary endothelium, selecting angiotensin-converting enzyme (ACE) because of its normally high level of expression at that site. ACE is up-regulated in pulmonary hypertension, making it a useful target for future PPH gene therapies.

The researchers then made a bi-specific antibody using a monoclonal antibody that has an affinity for ACE and cross-reacts with multiple species, including humans. Binding the adenovirus to the bi-specific antibody and injecting it into the tail vein of rats resulted in high levels of transgene expression in the lungs while significantly decreasing such expression in the liver and spleen. Subsequent refinements of the technique, using an endothelial-specific promoter to restrict the expression of the delivered genes to the endothelium, further improved the selectivity of the approach for the lungs.

Preliminary assessment of the technique’s inflammatory consequences was also performed. “There was no significant inflammation noted by histology, at least on day 4,” reported Dr. Reynolds. “But we need to look at this further.”

—Timothy Begany

Reference
1. Loyd JE, Geraci MW, Schwartz DA, et al. Implications of the Human Genome Project for pulmonary vascular disease. Presented at: American Thoracic Society 97th International Conference; May 23, 2001; San Francisco.

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