Kate G. Ackerman

Multi-pronged inhibition of airway hyper-responsiveness and inflammation by lipoxin A 4

The prevalence of asthma continues to increase and its optimal treatment remains a challenge. Here, we investigated the actions of lipoxin A4 (LXA4) and its leukocyte receptor in pulmonary inflammation using a murine model of asthma. Allergen challenge initiated airway biosynthesis of LXA4 and increased expression of its receptor. Administration of a stable analog of LXA4 blocked both airway hyper-responsiveness and pulmonary inflammation, as shown by decreased leukocytes and mediators, including interleukin-5, interleukin-13, eotaxin, prostanoids and cysteinyl leukotrienes. Moreover, transgenic expression of human LXA4 receptors in murine leukocytes led to significant inhibition of pulmonary inflammation and eicosanoid-initiated eosinophil tissue infiltration. Inhibition of airway hyper-responsiveness and allergic airway inflammation with a stable LXA4 analog highlights a unique counter-regulatory profile for the LXA4 system and its leukocyte receptor in airway responses. Moreover, our findings suggest that lipoxin and related pathways offer novel multi-pronged therapeutic approaches for human asthma.

Fog2 Is Required for Normal Diaphragm and Lung Development in Mice and Humans

Congenital diaphragmatic hernia and other congenital diaphragmatic defects are associated with significant mortality and morbidity in neonates; however, the molecular basis of these developmental anomalies is unknown. In an analysis of E18.5 embryos derived from mice treated with N-ethyl-N-nitrosourea, we identified a mutation that causes pulmonary hypoplasia and abnormal diaphragmatic development. Fog2 (Zfpm2) maps within the recombinant interval carrying the N-ethyl-N-nitrosourea-induced mutation, and DNA sequencing of Fog2 identified a mutation in a splice donor site that generates an abnormal transcript encoding a truncated protein. Human autopsy cases with diaphragmatic defect and pulmonary hypoplasia were evaluated for mutations in FOG2. Sequence analysis revealed a de novo mutation resulting in a premature stop codon in a child who died on the first day of life secondary to severe bilateral pulmonary hypoplasia and an abnormally muscularized diaphragm. Using a phenotype-driven approach, we have established that Fog2 is required for normal diaphragm and lung development, a role that has not been previously appreciated. FOG2 is the first gene implicated in the pathogenesis of nonsyndromic human congenital diaphragmatic defects, and its necessity for pulmonary development validates the hypothesis that neonates with congenital diaphragmatic hernia may also have primary pulmonary developmental abnormalities.

Infants with Bochdalek diaphragmatic hernia: Sibling precurrence and monozygotic twin discordance in a hospital‐based malformation surveillance program

Congenital diaphragmatic hernia (CDH) is a common and often devastating birth defect. In order to learn more about possible genetic causes, we reviewed and classified 203 cases of the Bochdalek hernia type identified through the Brigham and Womens Hospital (BWH) Active Malformation Surveillance Program over a 28‐year period. Phenotypically, 55% of the cases had isolated CDH, and 45% had complex CDH defined as CDH in association with additional major malformations or as part of a syndrome. When classified according to likely etiology, 17% had a Recognized Genetic etiology for their CDH, while the remaining 83% had No Apparent Genetic etiology. Detailed analysis using this largest cohort of consecutively collected cases of CDH showed low precurrence among siblings. Additionally, there was no concordance for CDH among five monozygotic twin pairs. These findings, in conjunction with previous reports of de novo dominant mutations in patients with CDH, suggest that new mutations may be an important mechanism responsible for CDH. The twin data also raise the possibility that epigenetic abnormalities contribute to the development of CDH.

Congenital diaphragmatic hernia and pulmonary hypoplasia: New insights from developmental biology and genetics†

Congenital diaphragmatic hernia (CDH), a common birth defect affecting as many as 1/3000–1/4000 live born infants has long been recognized as a major congenital anomaly, with drawings and written descriptions dating back to 1700 [Irish et al., 1996]. During the past 50 years, surgical and medical management techniques have greatly improved but so has our understanding of the complexities of CDH including the facts that CDH:(a) does not designate a single or specific type of diaphragmatic defect; (b) almost always co-occurs with serious and often life-threatening abnormalities of pulmonary airway and vascular development; (c) is accompanied by additional major malformations in as many as half of cases; and (d) appears to be etiologically heterogeneous. These factors contribute to the wide discrepancy in outcomes. While some survivors are healthy and have normal development, others either die or have long-term morbidity. The mortality of CDH is persistently high and approaches 50% when all cases of CDH are considered [Stege et al., 2003; Colvin et al., 2005]. The particularly deleterious impact of cardiovascular malformations (CVMs) which commonly co-occur with CDH is well-described in the article by Lin et al.; the authors also provide management guidelines pertaining to clinical and genetic aspects of this potentially life-threatening combination of major malformations. Improved outcomes for individuals with either isolated CDH (e.g., CDH is the only major malformation), or those with complex CDH (e.g., CDH plus one or more additional malformations) will most likely come after greater understanding of basic biology and of pathophysiology have been achieved. There is increasing evidence that mutations in genes belonging to one or more important developmental pathways contribute to CDH and its accompanying defects. Current knowledge of the genes and pathways associated with CDH and lung development in both humans and model organisms are presented in this issue. For example, Fog2, Gata4, and COUP-TFII likely function in the same developmental pathway, and all three of these genes have been implicated in diaphragm development in mouse models. In humans, a FOG2 mutation has been associated with a posterior diaphragmatic defect while GATA4 and COUP-TFII are strong CDH candidate genes based on their locations at cytogenetic hot spots [Ackerman et al., 2005; Klaassens et al., 2005; Slavotinek et al., 2005; You et al., 2005; Ackerman et al., 2006; Jay et al., 2006]. Numerous perturbations in the retinoic acid pathways are associated with CDH; these include mutations in genes that are part of, or interact with members of, this pathway (such as RXR, RAR, STRA6, COUP-TFII, GATA4, and FOG2) [Mendelsohn et al., 1994; Mascrez et al., 1998; Malpel et al., 2000; Clabby et al., 2003; Scribner et al., 2006; Pasutto et al., 2007] as well as teratogenic exposures such as vitamin A deficiency and the herbicide nitrofen [Andersen, 1941; Kluth et al., 1990]. Although not proven, these findings suggest that genetic abnormalities in the vitamin A pathway may contribute to the most common form of CDH, namely isolated “Bochdalek” hernia and even more intriguingly raise the possibility of a role for preventative supplementation. However, these articles also point out the likely genetic heterogeneity underlying CDH and indicate that a variety of newer techniques (including molecular cytogenetics, gene sequencing, SNP arrays, and increasingly sophisticated analyses of genetic/teratogenic model organisms) will be required to dissect this complex birth defect. Standard G-banding chromosome studies identify aneuploidy in ~10% of all CDH cases. Array-based comparative genomic hybridization (aCGH) at the 1 Mb resolution has already identified small CDH-associated deletions below the resolution of standard karyotyping [Slavotinek et al., 2005; Kantarci et al., 2006], and it is likely that new higher resolutions platforms will detect additional even smaller areas of anueploidy serving to narrow critical regions that, in turn, will pinpoint genes key for normal diaphragm development. Since pulmonary hypoplasia and pulmonary hypertension are the major determinants of survival in the neonatal period and predictors of long-term morbidity, several articles (Dr. Kinane, Dr. Khan and co-authors, and Dr. Keller) in this issue pay particular attention to normal and abnormal lung development, to pulmonary vascular development, and to potential pulmonary rescue therapies. The data presented therein make it abundantly clear that CDH-associated pulmonary hypoplasia is not SOLELY due to mechanical compression of the developing lung from herniation of abdominal contents into the chest cavity. Rather, intrinsic pulmonary developmental arrest accounts for at least some degree of the pulmonary hypoplasia seen at birth. Evidence for this hypothesis, coined the “two hit hypothesis,” is based on the discovery that both pulmonary development and diaphragm development are primarily affected by the chemical nitrofen [Keijzer et al., 2000]. There is now genetic evidence supporting this hypothesis (as discussed in the article in this issue by Ackerman and Greer) in that several genes have been identified which play an important role in the development of the lung and of the diaphragm, such as Fog2 and Gata4. At this time, the percentage of CDH patients with primary defects of both lung and diaphragm development versus the percentage of patients with only secondary pulmonary hypoplasia, due to lung compression and abnormal diaphragmatic function, remains unknown. It is also likely, that some of the co-morbidity that occurs with CDH in the gastrointestinal system such as gastroesophageal reflux and dysmotility is not simply secondary to anatomic aberrations caused by herniation. Development of the esophagus and stomach occurs in close proximity to the lung, heart, and diaphragm, and some transcriptional regulatory pathways required for the development of these organs likely also play a role in upper gastrointestinal development [Muratore et al., 2001; Jacobsen et al., 2005; Jay et al., 2006]. The exciting new therapeutic possibility of tracheal occlusion (TO) is discussed in great detail by Khan and co-authors. Although TO appears to promote pulmonary development in model organisms by mechano-transduction (e.g., stretch-induced) acceleration of Type I pneumocyte cell division, the authors cautiously point out not only the lack of long-term outcome studies but also the immediate effect of diminished Type II pneumocytes with consequent diminished surfactant production. Identification of TO-induced genetic changes may ultimately lead to gene therapy targets that can stimulate lung development in utero or shortly after birth. The rationale, implementation, and outcomes for TO in the human population are discussed in the article by Dr. Keller.

Timing of Delivery and Survival Rates for Infants With Prenatal Diagnoses of Congenital Diaphragmatic Hernia

OBJECTIVES. The goal of the study was to test the hypothesis that infants with known congenital diaphragmatic hernias born at early term gestation (37–38 weeks) rather than later (39–41 weeks) had greater survival rates and less extracorporeal membrane oxygenation use. Primary outcomes were survival to hospital discharge or transfer and extracorporeal membrane oxygenation use. METHODS. A retrospective cohort study of term infants with prenatal diagnoses of congenital diaphragmatic hernia was performed with the Congenital Diaphragmatic Hernia Study Group Registry of patients with congenital diaphragmatic hernias who were treated between January 1995 and December 2006. RESULTS. Among 628 term infants at 37 to 41 weeks of gestation who had prenatal diagnoses of congenital diaphragmatic hernia and were free of major associated anomalies, early term birth (37 vs 39–41 weeks) and greater birth weight were associated independently with survival, whereas black race was related inversely to survival. Infants born at early term with birth weights at or above the group mean (3.1 kg) had the greatest survival rate (80%). Among infants born through elective cesarean delivery, infants born at 37 to 38 weeks of gestation, compared with 39 to 41 weeks, had less use of extracorporeal membrane oxygenation (22.0% vs 35.5%) and a trend toward a greater survival rate (75.0% vs 65.8%). CONCLUSIONS. The timing of delivery is an independent, potentially important factor in the consideration of elective delivery for infants diagnosed prenatally as having congenital diaphragmatic hernias. Among fetuses with prenatally diagnosed congenital diaphragmatic hernias and without major associated anomalies, early term delivery may confer advantage.

Fog2 is critical for cardiac function and maintenance of coronary vasculature in the adult mouse heart

Aberrant transcriptional regulation contributes to the pathogenesis of both congenital and adult forms of heart disease. While the transcriptional regulator friend of Gata 2 (FOG2) is known to be essential for heart morphogenesis and coronary development, its tissue-specific function has not been previously investigated. Additionally, little is known about the role of FOG2 in the adult heart. Here we used spatiotemporally regulated inactivation of Fog2 to delineate its function in both the embryonic and adult mouse heart. Early cardiomyocyte- restricted loss of Fog2 recapitulated the cardiac and coronary defects of the Fog2 germline murine knockouts. Later cardiomyocyte-restricted loss of Fog2 (Fog2MC) did not result in defects in cardiac structure or coronary vessel formation. However, Fog2MC adult mice had severely depressed ventricular function and died at 8-14 weeks. Fog2MC adult hearts displayed a paucity of coronary vessels, associated with myocardial hypoxia, increased cardiomyocyte apoptosis, and cardiac fibrosis. Induced inactivation of Fog2 in the adult mouse heart resulted in similar phenotypes, as did ablation of the FOG2 interaction with the transcription factor GATA4. Loss of the FOG2 or FOG2-GATA4 interaction altered the expression of a panel of angiogenesis-related genes. Collectively, our data indicate that FOG2 regulates adult heart function and coronary angiogenesis.

Development of the diaphragm and genetic mouse models of diaphragmatic defects

Improving our understanding of diaphragmatic development is essential to making progress in defining the pathogenesis and genetic etiologies of congenital diaphragmatic defects in humans. As mouse genetic technology has given us new tools to manipulate and observe development, a number of mouse models have recently emerged that provide valuable insight to this field. In this article, we review our current understanding of diaphragmatic embryogenesis including the origin of diaphragmatic tissue. We use rodent models to review the muscularization of the diaphragm and review selected genetic models of abnormal muscularization. We also review models of posterior diaphragmatic defects and discuss evidence for the pleuroperitoneal fold (PPF) tissue contributing to the diaphragm. Finally, we discuss models of anterior and central hernias. It may be simplistic to subdivide this review based on anatomic regions of the diaphragm, as evidence is emerging that defects in different regions of the diaphragm in humans and in mice may be etiologically related. However, at this time we do not have enough knowledge to make more mechanistic or genetic classifications though with time, genetic progress in the field of diaphragm development will allow us to do this.

Congenital diaphragmatic hernia candidate genes derived from embryonic transcriptomes

Congenital diaphragmatic hernia (CDH) is a common (1 in 3,000 live births) major congenital malformation that results in significant morbidity and mortality. The discovery of CDH loci using standard genetic approaches has been hindered by its genetic heterogeneity. We hypothesized that gene expression profiling of developing embryonic diaphragms would help identify genes likely to be associated with diaphragm defects. We generated a time series of whole-transcriptome expression profiles from laser captured embryonic mouse diaphragms at embryonic day (E)11.5 and E12.5 when experimental perturbations lead to CDH phenotypes, and E16.5 when the diaphragm is fully formed. Gene sets defining biologically relevant pathways and temporal expression trends were identified by using a series of bioinformatic algorithms. These developmental sets were then compared with a manually curated list of genes previously shown to cause diaphragm defects in humans and in mouse models. Our integrative filtering strategy identified 27 candidates for CDH. We examined the diaphragms of knockout mice for one of the candidate genes, pre–B-cell leukemia transcription factor 1 (Pbx1), and identified a range of previously undetected diaphragmatic defects. Our study demonstrates the utility of genetic characterization of normal development as an integral part of a disease gene identification and prioritization strategy for CDH, an approach that can be extended to other diseases and developmental anomalies.

CC chemokine receptor-2 is not essential for the development of antigen-induced pulmonary eosinophilia and airway hyperresponsiveness.

Monocyte chemoattractant proteins-1 and -5 have been implicated as important mediators of allergic pulmonary inflammation in murine models of asthma. The only identified receptor for these two chemokines to date is the CCR2. To study the role of CCR2 in a murine model of Ag-induced asthma, we compared the pathologic and physiological responses of CCR2−/− mice with those of wild-type (WT) littermates following immunization and challenge with OVA. OVA-immunized/OVA-challenged (OVA/OVA) WT and CCR2−/− mice developed significant increases in total cells recovered by bronchoalveolar lavage (BAL) compared with their respective OVA-immunized/PBS-challenged (OVA/PBS) control groups. There were no significant differences in BAL cell counts and differentials (i.e., macrophages, PMNs, lymphocytes, and eosinophils) between OVA/OVA WT and CCR2−/− mice. Serologic evaluation revealed no significant difference in total IgE and OVA-specific IgE between OVA/OVA WT mice and CCR2−/− mice. Lung mRNA expression and BAL cytokine protein levels of IL-4, IL-5, and IFN-γ were also similar in WT and CCR2−/− mice. Finally, OVA/OVA CCR2−/− mice developed increased airway hyper-responsiveness to a degree similar to that in WT mice. We conclude that following repeated airway challenges with Ag in sensitized mice, the development of Th2 responses (elevated IgE, pulmonary eosinophilia, and lung cytokine levels of IL-4 and IL5) and the development of airway hyper-responsiveness are not diminished by a deficiency in CCR2.

Characterization of the chromosome 1q41q42.12 region, and the candidate gene DISP1, in patients with CDH.

Cytogenetic and molecular cytogenetic studies demonstrate association between congenital diaphragmatic hernia (CDH) and chromosome 1q41q42 deletions. In this study, we screened a large CDH cohort (N = 179) for microdeletions in this interval by the multiplex ligation‐dependent probe amplification (MLPA) technique, and also sequenced two candidate genes located therein, dispatched 1 (DISP1) and homo sapiens H2.0‐like homeobox (HLX). MLPA analysis verified deletions of this region in two cases, an unreported patient with a 46,XY,del(1)(q41q42.13) karyotype and a previously reported patient with a Fryns syndrome phenotype [Kantarci et al., 2006 ]. HLX sequencing showed a novel but maternally inherited single nucleotide variant (c.27C>G) in a patient with isolated CDH, while DISP1 sequencing revealed a mosaic de novo heterozygous substitution (c.4412C>G; p.Ala1471Gly) in a male with a left‐sided Bochdalek hernia plus multiple other anomalies. Pyrosequencing demonstrated the mutant allele was present in 43%, 12%, and 4.5% of the patients lymphoblastoid, peripheral blood lymphocytes, and saliva cells, respectively. We examined Disp1 expression at day E11.5 of mouse diaphragm formation and confirmed its presence in the pleuroperitoneal fold, as well as the nearby lung which also expresses Sonic hedgehog (Shh). Our report describes the first de novo DISP1 point mutation in a patient with complex CDH. Combining this finding with Disp1 embryonic mouse diaphragm and lung tissue expression, as well as previously reported human chromosome 1q41q42 aberrations in patients with CDH, suggests that DISP1 may warrant further consideration as a CDH candidate gene.