Christine M. Costello

Genetic variation in DLG5 is associated with inflammatory bowel disease

Crohn disease and ulcerative colitis are two subphenotypes of inflammatory bowel disease (IBD), a complex disorder resulting from gene-environment interaction. We refined our previously defined linkage region for IBD on chromosome 10q23 and used positional cloning to identify genetic variants in DLG5 associated with IBD. DLG5 encodes a scaffolding protein involved in the maintenance of epithelial integrity. We identified two distinct haplotypes with a replicable distortion in transmission (P = 0.000023 and P = 0.004 for association with IBD, P = 0.00012 and P = 0.04 for association with Crohn disease). One of the risk-associated DLG5 haplotypes is distinguished from the common haplotype by a nonsynonymous single-nucleotide polymorphism 113G→A, resulting in the amino acid substitution R30Q in the DUF622 domain of DLG5. This mutation probably impedes scaffolding of DLG5. We stratified the study sample according to the presence of risk-associated CARD15 variants to study potential gene-gene interaction. We found a significant difference in association of the 113A DLG5 variant with Crohn disease in affected individuals carrying the risk-associated CARD15 alleles versus those carrying non-risk-associated CARD15 alleles. This is suggestive of a complex pattern of gene-gene interaction between DLG5 and CARD15, reflecting the complex nature of polygenic diseases. Further functional studies will evaluate the biological significance of DLG5 variants.

Inhibition of Rho-kinase attenuates hypoxia-induced angiogenesis in the pulmonary circulation.

Pulmonary hypertension (PH) is a common complication of chronic hypoxic lung diseases, which increase morbidity and mortality. Hypoxic PH has previously been attributed to structural changes in the pulmonary vasculature including narrowing of the vascular lumen and loss of vessels, which produce a fixed increase in resistance. Using quantitative stereology, we now show that chronic hypoxia caused PH and remodeling of the blood vessel walls in rats but that this remodeling did not lead to structural narrowing of the vascular lumen. Sustained inhibition of the RhoA/Rho-kinase pathway throughout the period of hypoxic exposure attenuated PH and prevented remodeling in intra-acinar vessels without enlarging the structurally determined lumen diameter. In chronically hypoxic lungs, acute Rho kinase inhibition markedly decreased PVR but did not alter the alveolar to arterial oxygen gap. In addition to increased vascular resistance, chronic hypoxia induced Rho kinase–dependent capillary angiogenesis. Thus, hypoxic PH was not caused by fixed structural changes in the vasculature but by sustained vasoconstriction, which was largely Rho kinase dependent. Importantly, this vasoconstriction had no role in ventilation-perfusion matching and optimization of gas exchange. Rho kinase also mediated hypoxia-induced capillary angiogenesis, a previously unrecognized but potentially important adaptive response.

Evaluation of AGR2 and AGR3 as candidate genes for inflammatory bowel disease.

Linkage analyses have implicated chromosome 7p21.3 as a susceptibility region for inflammatory bowel disease (IBD). Recently, the mouse phenotype with diarrhea and goblet cell dysfunction caused by anterior gradient protein 2 dysfunction was reported (European patent WO2004056858). The genes encoding for the human homologues AGR2 and AGR3 are localized on chromosome 7p21.3. The gene structures were verified and mutation detection was performed in 47 IBD patients. A total of 30 single nucleotide polymorphisms (SNPs) were tested for association to ulcerative colitis (UC, N=317) and Crohns disease (CD, N=631) in a German cohort and verified in a UK cohort of 384 CD and 311 UC patients. An association signal was identified in the 5′ region of the AGR2 gene (most significant SNP hcv1702494, nominal PTDT=0.011, Pcase/control=0.0007, OR=1.34, combined cohort). The risk haplotype carried an odds ratio of 1.43 in the German population (P=0.002). AGR2 was downregulated in UC patients as compared to normal controls (P<0.001) and a trend toward lower expression was seen in carriers of the risk alleles. Luciferase assays of the AGR2 promoter showed regulation by the goblet cell-specific transcription factors FOXA1 and FOXA2. In summary, AGR2 represents an interesting new avenue into the etiopathophysiology of IBD and the maintenance of epithelial integrity.

Gremlin Plays a Key Role in the Pathogenesis of Pulmonary Hypertension

Background— Pulmonary hypertension occurs in chronic hypoxic lung diseases, significantly worsening morbidity and mortality. The important role of altered bone morphogenetic protein (BMP) signaling in pulmonary hypertension was first suspected after the identification of heterozygous BMP receptor mutations as the underlying defect in the rare heritable form of pulmonary arterial hypertension. Subsequently, it was demonstrated that BMP signaling was also reduced in common forms of pulmonary hypertension, including hypoxic pulmonary hypertension; however, the mechanism of this reduction has not previously been elucidated. Methods and Results— Expression of 2 BMP antagonists, gremlin 1 and gremlin 2, was higher in the lung than in other organs, and gremlin 1 was further increased in the walls of small intrapulmonary vessels of mice during the development of hypoxic pulmonary hypertension. Hypoxia stimulated gremlin secretion from human pulmonary microvascular endothelial cells in vitro, which inhibited endothelial BMP signaling and BMP-stimulated endothelial repair. Haplodeficiency of gremlin 1 augmented BMP signaling in the hypoxic mouse lung and reduced pulmonary vascular resistance by attenuating vascular remodeling. Furthermore, gremlin was increased in the walls of small intrapulmonary vessels in idiopathic pulmonary arterial hypertension and the rare heritable form of pulmonary arterial hypertension in a distribution suggesting endothelial localization. Conclusions— These findings demonstrate a central role for increased gremlin in hypoxia-induced pulmonary vascular remodeling and the increased pulmonary vascular resistance in hypoxic pulmonary hypertension. High levels of basal gremlin expression in the lung may account for the unique vulnerability of the pulmonary circulation to heterozygous mutations of BMP type 2 receptor in pulmonary arterial hypertension.

HIV‐disease and bone marrow changes: A study of 60 cases

We report our experience of peripheral blood and bone marrow changes in patients with HIV disease. Abnormalities were most commonly seen in patients with advanced disease. In AIDS group IV patients (CDC classification) anaemia (92%) neutropenia (85%) monocytopenia (75%) and thrombocytopenia (61%) have their highest incidence, the reason being a combination of factors such as infection, myelosuppressive drugs and HIV infection itself. Bone marrow examinations were performed most commonly for microbiological culture (25%) and the investigation of anaemia (16%). Morphological changes in the bone marrow were non‐specific and not pathognomic; however erythroid hypoplasia was found to be a distinctive feature associated with MAI infection. The procedure provided a high yield for microbiological culture, particularly in MAI infection.

Lung-selective gene responses to alveolar hypoxia: potential role for the bone morphogenetic antagonist gremlin in pulmonary hypertension.

Pulmonary hypoxia is a common complication of chronic lung diseases leading to the development of pulmonary hypertension. The underlying sustained increase in vascular resistance in hypoxia is a response unique to the lung. Thus we hypothesized that there are genes for which expression is altered selectively in the lung in response to alveolar hypoxia. Using a novel subtractive array strategy, we compared gene responses to hypoxia in primary human pulmonary microvascular endothelial cells (HMVEC-L) with those in cardiac microvascular endothelium and identified 90 genes (forming 9 clusters) differentially regulated in the lung endothelium. From one cluster, we confirmed that the bone morphogenetic protein (BMP) antagonist, gremlin 1, was upregulated in the hypoxic murine lung in vivo but was unchanged in five systemic organs. We also demonstrated that gremlin protein was significantly increased by hypoxia in vivo and inhibited HMVEC-L responses to BMP stimulation in vitro. Furthermore, significant upregulation of gremlin was measured in lungs of patients with pulmonary hypertensive disease. From a second cluster, we showed that CXC receptor 7, a receptor for the proangiogenic chemokine CXCL12, was selectively upregulated in the hypoxic lung in vivo, confirming that our subtractive strategy had successfully identified a second lung-selective hypoxia-responsive gene. We conclude that hypoxia, typical of that encountered in pulmonary disease, causes lung-specific alterations in gene expression. This gives new insights into the mechanisms of pulmonary hypertension and vascular loss in chronic lung disease and identifies gremlin 1 as a potentially important mediator of vascular changes in hypoxic pulmonary hypertension.

Hypoxia Selectively Activates the CREB Family of Transcription Factors in the In Vivo Lung

RATIONALE Pulmonary hypertension is a common complication of chronic hypoxic lung diseases and is associated with increased morbidity and reduced survival. The pulmonary vascular changes in response to hypoxia, both structural and functional, are unique to this circulation. OBJECTIVES To identify transcription factor pathways uniquely activated in the lung in response to hypoxia. METHODS After exposure to environmental hypoxia (10% O(2)) for varying periods (3 h to 2 wk), lungs and systemic organs were isolated from groups of adult male mice. Bioinformatic examination of genes the expression of which changed in the hypoxic lung (assessed using microarray analysis) identified potential lung-selective transcription factors controlling these changes in gene expression. In separate further experiments, lung-selective activation of these candidate transcription factors was tested in hypoxic mice and by comparing hypoxic responses of primary human pulmonary and cardiac microvascular endothelial cells in vitro. MEASUREMENTS AND MAIN RESULTS Bioinformatic analysis identified cAMP response element binding (CREB) family members as candidate lung-selective hypoxia-responsive transcription factors. Further in vivo experiments demonstrated activation of CREB and activating transcription factor (ATF)1 and up-regulation of CREB family-responsive genes in the hypoxic lung, but not in other organs. Hypoxia-dependent CREB activation and CREB-responsive gene expression was observed in human primary lung, but not cardiac microvascular endothelial cells. CONCLUSIONS These findings suggest that activation of CREB and AFT1 plays a key role in the lung-specific responses to hypoxia, and that lung microvascular endothelial cells are important, proximal effector cells in the specific responses of the pulmonary circulation to hypoxia.

Role of Gremlin in the Lung: Development and Disease

Gremlin is an extracellular glycoprotein that was first identified over a decade ago through its important role in embryonic development, in which it acts as an antagonist of bone morphogenetic protein actions. It plays a critical role in the development of normal airways and the pulmonary circulation in the embryo. More recently, considerable evidence has been presented for a role for gremlin in the pathogenesis of lung diseases, particularly pulmonary hypertension and idiopathic pulmonary fibrosis. The purpose of this article is to review this evidence, consider the potential mechanisms and multicellular actions by which gremlin contributes to disease pathogenesis, and suggest future avenues of research.

L-Arginine promotes angiogenesis in the chronically hypoxic lung: a novel mechanism ameliorating pulmonary hypertension

Chronic alveolar hypoxia, whether due to residence at high altitude or lung disease, leads to a sustained increase in pulmonary vascular resistance and pulmonary hypertension (PH). Strategies that augment endogenous nitric oxide production or activity, including l-arginine supplementation, attenuate the development of PH. This action has been attributed to inhibition of vessel wall remodeling, thus preventing structural narrowing of the vascular lumen. However, more recent evidence suggests that structural changes are not responsible for the elevated vascular resistance observed in chronic hypoxic PH, calling into question the previous explanation for the action of l-arginine. We examined the effect of dietary l-arginine supplementation on pulmonary vasoconstriction, structurally determined maximum vascular lumen diameter, and vessel length in rats during 2 wk of exposure to hypoxia. l-Arginine attenuated the development of hypoxic PH by preventing increased arteriolar resistance. It did not alter mean maximal vascular lumen diameter, nor did it augment nitric oxide-mediated vasodilatation, in chronically hypoxic lungs. However, the total length of vessels within the gas exchange region of the hypoxic lungs was significantly increased after l-arginine supplementation. These findings suggest that dietary l-arginine ameliorated hypoxic PH, but not by an effect on the structurally determined lumen diameter of pulmonary blood vessels. l-Arginine enhanced angiogenesis in the hypoxic pulmonary circulation, which may attenuate hypoxic PH by producing new parallel vascular pathways through the lung.

Effect of nebulised recombinant DNase on neutrophil elastase load in cystic fibrosis.

BACKGROUND: DNA released by degenerating inflammatory neutrophils contributes to mucous plugging of airways in patients with cystic fibrosis. Neutrophil elastase, a major effector of tissue destruction in the lungs of patients with cystic fibrosis, is a highly cationic molecule which is bound and inhibited by negatively charged polyanions such as mucin and DNA in purulent sputum. Thus, the solubilisation of DNA in the airways by aerosolised recombinant DNase may remove a source of neutrophil elastase inhibition, effectively increasing elastase load. The aim of this study was to assess the effect of rhDNase therapy on neutrophil elastase load in patients with cystic fibrosis. METHODS: Blood and sputum were collected from 15 patients with cystic fibrosis before initiation of nebulised DNase therapy and at 12 weeks following therapy. The long term effects of continuous rhDNase administration were evaluated at 52 weeks for 11 of these patients. Plasma was analysed for neutrophil elastase, interleukin (IL)-8 and neutrophil elastase in complex with alpha 1-protease inhibitor (alpha 1PI). Sputum was assessed for neutrophil elastase, IL-8, and active elastase. At each visit spirometric measurements were carried out. RESULTS: Sputum elastase activity decreased at 12 weeks and was maintained at 52 weeks when a decline in total plasma elastase was also observed. Although, as expected, there was a correlation between plasma levels of total elastase and neutrophil elastase/alpha 1PI complex, the decrease in the levels of the complex at 52 weeks did not reach statistical significance. CONCLUSIONS: This study indicates that prolonged daily administration of rhDNase results in a reduction in elastase load in patients with cystic fibrosis.