Mark D. Moore

Gene Expression Patterns in the Lungs of Patients With Primary Pulmonary Hypertension A Gene Microarray Analysis

Abstract— Primary pulmonary hypertension (PPH) is a disease of unknown etiology characterized by lumen-obliterating endothelial cell proliferation and vascular smooth muscle hypertrophy of the small precapillary pulmonary arteries. Because the vascular lesions are homogeneously distributed throughout the entire lung, we propose that a tissue fragment of the lung is representative of the whole lung. RNA extracted from the fragments is likely to provide meaningful information regarding the changes in gene expression pattern in PPH when compared with structurally normal lung tissue. We hypothesize that the lung tissue gene expression pattern of patients with PPH has a characteristic profile when compared with the gene expression pattern of structurally normal lungs and that this characteristic gene expression profile provides new insights into the pathobiology of PPH. Using oligonucleotide microarray technology, we characterized the expression pattern in the lung tissue obtained from 6 patients with primary pulmonary hypertension (PPH)—including 2 patients with the familial form of PPH (FPPH)—and from 6 patients with histologically normal lungs. For the data analysis, gene clusters were generated and the gene expression pattern differences between PPH and normal lung tissue and between PPH and FPPH lung tissue were compared. All PPH lung tissue samples showed a decreased expression of genes encoding several kinases and phosphatases, whereas several oncogenes and genes coding for ion channel proteins were upregulated in their expression. Importantly, we could distinguish by pattern comparison between sporadic PPH and FPPH, because alterations in the expression of transforming growth factor-&bgr; receptor III, bone morphogenic protein 2, mitogen-activated protein kinase kinase 5, RACK 1, apolipoprotein C-III, and the gene encoding the laminin receptor 1 were only found in the samples from patients with sporadic PPH, but not in FPPH samples. We conclude that the microarray gene expression technique is a new and useful molecular tool that provides novel information pertinent to a better characterization and understanding of the pathobiology of the distinct clinical phenotypes of pulmonary hypertension.

Pulmonary prostacyclin synthase overexpression in transgenic mice protects against development of hypoxic pulmonary hypertension

Prostacyclin synthase (PGIS) is the final committed enzyme in the metabolic pathway leading to prostacyclin (PGI2) production. Patients with severe pulmonary hypertension have a PGIS deficiency of their precapillary vessels, but the importance of this deficiency for lung vascular remodeling remains unclear. We hypothesized that selective pulmonary overexpression of PGIS may prevent the development of pulmonary hypertension. To study this hypothesis, transgenic mice were created with selective pulmonary PGIS overexpression using a construct of the 3.7-kb human surfactant protein-C (SP-C) promoter and the rat PGIS cDNA. Transgenic mice (Tg+) and nontransgenic littermates (Tg-) were subjected to a simulated altitude of 17,000 ft for 5 weeks, and right ventricular systolic pressure (RVSP) was measured. Histology was performed on the lungs. The Tg+ mice produced 2-fold more pulmonary 6-keto prostaglandin F1alpha (PGF1alpha) levels than did Tg- mice. After exposure to chronic hypobaric hypoxia, Tg+ mice have lower RVSP than do Tg- mice. Histologic examination of the lungs revealed nearly normal arteriolar vessels in the Tg+ mice in comparison with vessel wall hypertrophy in the Tg- mice. These studies demonstrate that Tg+ mice were protected from the development of pulmonary hypertension after exposure to chronic hypobaric hypoxia. We conclude that PGIS plays a major role in modifying the pulmonary vascular response to chronic hypoxia. This has important implications for the pathogenesis and treatment of severe pulmonary hypertension.

HOX Genes in Human Lung : Altered Expression in Primary Pulmonary Hypertension and Emphysema

HOX genes belong to the large family of homeodomain genes that function as transcription factors. Animal studies indicate that they play an essential role in lung development. We investigated the expression pattern of HOX genes in human lung tissue by using microarray and degenerate reverse transcriptase-polymerase chain reaction survey techniques. HOX genes predominantly from the 3 end of clusters A and B were expressed in normal human adult lung and among them HOXA5 was the most abundant, followed by HOXB2 and HOXB6. In fetal (12 weeks old) and diseased lung specimens (emphysema, primary pulmonary hypertension) additional HOX genes from clusters C and D were expressed. Using in situ hybridization, transcripts for HOXA5 were predominantly found in alveolar septal and epithelial cells, both in normal and diseased lungs. A 2.5-fold increase in HOXA5 mRNA expression was demonstrated by quantitative reverse transcriptase-polymerase chain reaction in primary pulmonary hypertension lung specimens when compared to normal lung tissue. In conclusion, we demonstrate that HOX genes are selectively expressed in the human lung. Differences in the pattern of HOX gene expression exist among fetal, adult, and diseased lung specimens. The altered pattern of HOX gene expression may contribute to the development of pulmonary diseases.

Human herpesvirus-8 infection of primary pulmonary microvascular endothelial cells.

Human herpesvirus-8 (HHV-8) is the causative agent of Kaposis sarcoma and is associated with the angioproliferative disorders primary effusion lymphoma and multicentric Castlemans disease. Evidence of HHV-8 infection within the pulmonary vasculature of patients with idiopathic pulmonary arterial hypertension (IPAH) has been described. We hypothesize that HHV-8 infection of pulmonary microvascular endothelial cells results in an apoptotic-resistant phenotype characteristic of severe pulmonary arterial hypertension. Our objective was to investigate the ability of HHV-8 to infect human pulmonary microvascular endothelial cells in vitro and characterize the phenotypic effect of this infection. Human pulmonary microvascular endothelial cells were exposed to HHV-8 using two methods (direct virus and co-culture technique). The presence of lytic and latent infection was confirmed. Changes in endothelial cell gene and protein expression and effects on cellular apoptosis were measured. HHV-8 can both lytically and latently infect primary human pulmonary microvascular endothelial cells in vitro. HHV-8 infection results in significant changes in gene expression, including alterations of pathways important to cellular apoptosis. HHV-8 infection also alters expression of genes integral to the bone morphogenic protein pathway, including down-regulation of bone morphogenic protein-4. Other genes previously implicated in the development of PAH are affected by HHV-8 infection, and cells infected with HHV-8 are resistant to apoptosis.

Mutational inactivation of aminoacylase‐1 in a small cell lung cancer cell line

Small cell lung cancer (SCLC) cell lines and tumors invariably exhibit loss of heterozygosity (LOH) or, in rare cases, homozygous deletions involving part or all of chromosome arm 3p, suggesting the presence of 1 or more tumor‐suppressor genes in this region. The gene encoding aminoacylase‐1 (ACY1) is localized on chromosome segment 3p21.1. ACY1 enzymatic activity, protein, and mRNA have been demonstrated to be expressed at either undetectable or very low levels in a group of SCLC cell lines and tumors. The demonstration of mutational inactivation of ACY1 would support the hypothesis that ACY1 inactivation in SCLC confers a selective growth advantage. One of four SCLC cell lines with undetectable Acylenzymatic activity and protein exhibited a compound mutation: nonconservative missense point mutations at codons 195 and 254. No wildtype sequence transcripts were identified in the cell line. Although nonmutational mechanisms for low or undetectable ACY1 enzymatic activity, protein, and mRNA expression are most frequently operant in SCLC, the demonstration of a mutation supports selection for ACY1 inactivation. Analysis of normal liver and a liver metastasis from the same patient from whom the NCI‐H711 cell line was derived demonstrated that the mutation was neither germline nor an early event in the development of SCLC. It is of interest that several genes involved in the regulation of intracellular protein degradation are encoded by chromosome band 3p21 and display unusual expression in SCLC. The presence of other loci involved in protein degradation on chromosome band 3p21 and their aberrant expression in SCLC suggest that a variety of mechanisms involved in the normal degradation of intracellular proteins may be perturbed in this neoplasm. Genes Chromosomes Cancer 21:320–325, 1998. Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Pulmonary Prostacyclin Synthase Overexpression Improves Survival Following Butylated Hydroxytoluene Exposure

Lung cancer is the leading cause of cancer death in men and women in North America [1]. Although primary prevention of tobacco smoking and smoking cessation are the most potent interventions available, most lung cancers are currently diagnosed in former smokers [1], underscoring the need for effective chemoprevention strategies. Mouse models of lung carcinogenesis display both histologic and molecular genetic similarities to adenocarcinoma [2], the most common histologic type of human lung cancer. In these models, modulation of inflammation with either nonselective COX-1 and COX-2 or selective COX-2 inhibition, cause reductions in lung tumor multiplicity [3,4]. To date, large-scale interventional trials of COX inhibition in human lung cancer chemoprevention have not been performed.