Cardiovascular malformations in congenital diaphragmatic hernia: Human and experimental studies

Cardiovascular malformations in congenital diaphragmatic hernia: Human and experimental studies

Cardiovascular By Lucia Malformations in Congenital Diaphragmatic Human and Experimental Studies Migliazza, Christian Otten, Huimin and Juan Mad...

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Cardiovascular By Lucia

Malformations in Congenital Diaphragmatic Human and Experimental Studies

Migliazza,

Christian

Otten,

Huimin and

Juan Madrid,

Be&ground/Purpose: Cardiovascular malformations (CVM) associated with congenital diaphragmatic hernia (CDH) account in part for the high mortality caused by this defect. The aim of this study is to examine the nature of these malformations in a large series of autopsies and to assess if similar defects are also present in rat fetuses with experimental CDH. Methods: The incidence of CVM and their nature were examined in the autopsy records of 136 stillborns and neonates with CDH admitted to our institution in the last 30 years. Experimental CDH was induced in rat fetuses by giving 100 mg of nitrofen to their mothers on gestational day 9.5, and the fetuses were harvested on day 21 (near full term). The presence of CDH and the anatomy of the heart and great vessels were studied under dissecting microscope after formalin fixation. Unexposed fetuses were used as controls. Results: Thirty-three newborns with CDH (24%) had CVM, either isolated or associated with other defects, and 7 had heart hypoplasia. Most CVM (ventricular septal defect, tetralogy of Fallot, transposition of the great vessels, double-outlet right ventricle) involved the outflow tract. In our animal experiments, no malformations were found in 21 control pups. Conversely, 80 of 130 nitrofen-exposed fetuses (61%)

B

ABIES WITH CONGENITAL diaphragmatic hernia (CDH) have herniation of abdominal viscera into the thorax accompanied by lung hypoplasia, biochemical immaturity, abnormal pulmonary arterioles, and often by malformations of various other organs, particularly congenital heart defects (CVM). The frequent coincidence of both types of anomalies suggests that the same mechanisms might be involved in their pathogenesis. The availability of a rodent model of CDH has allowed morphological and biochemical demonstration of lung hypoplasia and immaturity very similar to those present in human CDH and has permitted investigation on various aspects of this malformation. However, no anaFrom the Departments of Surgery and Pathology, Hospital Infantil ‘La Paz, “Madrid, Spain. Supported by FIS Grant #96/0059-01. Address reprint requests to Juan A. Tovar; MD, PhD, Department of Surgery, Hospital Infantil Universitario “‘LA Paz, ” P de la Castellana 261,28046, Madrid, Spain. Copyright 0 1999 by WB. Saunders Company 0022-3468/99/3409-0011$03.00/0

1352

Xia,

Jose

I. Rodriguez,

Juan

Hernia:

A. Diez-Pardo,

A. Tovar Spain

had CDH, and 59 of them (74%) had CVM. A significant association (Fisher’s Exact test, P< .Ol) was found between CDH and CVM because only 25 of the 50 exposed animals without CDH (50%) had CVM. Again, most defects involved the outflow tract and were similar to those seen in human CDH (tetralogy of Fallot, persistent truncus, ventricular septal defect, double-outlet right ventricle, aberrant right subclavian artery, agenetic ductus, and interrupted aortic arch). Animals with CDH had significantly decreased heart weight to fetal weight ratio in comparison with controls and with those without CDH. Conc/usions:The similar nature of the cardiovascular defects found in babies succumbing to CDH and in nitrofen-exposed rats suggests that a similar disturbance of the regional organogenesis related to the neural crest might be involved in both settings, and further validates the use of this animal model for clarifying the cellular and molecular pathogenetic mechanisms. J Pediatr Surg 34:1352-1358. Copyright o 1999 by W.B. Saunders Company. INDEX WORDS: rat, heart, great

Congenital diaphragmatic vessels, malformations,

neural

hernia, nitrofen, crest.

tomic study of the heart and great vessels in this experimental setting has been performed, and we felt it necessary to examine whether any CVM were present in this model and, if so, whether they are similar to those observed in babies with CDH. For this purpose we studied the cardiovascular anatomy in a large series of human CDH autopsies and in rat fetuses prenatally exposed to nitrofen. MATERIALS

AND

METHODS

The autopsy records of 136 stillboms and newborns with posterolateral CDH admitted to our institution between 1966 and 1997 were reviewed retrospectively to determine the incidence and the nature of associated cardiovascular defects. Simultaneously, we carried out animal experiments directed to induce CDH in fetal rats and to examine the heart and great vessels. For this purpose, 2 groups of time-mated Sprague-Dawley pregnant rats received either 100 mg of nitrofen (2,4-dichloro-4’-nitrodiphenyl-ether) in 1 mL of olive oil by gavage on day 9.5 of gestation (nitrofen group, II = lo), or I mL of olive oil alone (control group, n = 3). Day 0 was that on which, after overnight mating, a vaginal smear showed the presence of spermatozoa. On day 21 of gestation (full term = 22) 130 fetuses from the nitrofen group and 21 from the control group were recovered by cesarean section and

Journal

of Pediatric

Surgery,

Vol34,

No 9 (September),

1999: pp 1352-1358

CARDIOVASCULAR

MALFORMATIONS

IN CDH

1353

groups were analyzed by contingency values of less than .05 were considered as proportions or as means -C SD.

processed for study. All experiments were performed after approval by the institutional research Committee in compliance with the current European Union regulations for animal care (E.C. 86iL 609). The presence of congenital diaphragmatic hernia was assessed with a binocular surgical microscope through a wide thoraco-abdominal incision. The whole fetus was then immersed in 10% buffered formalin for 7 days and maintained at 18” to ‘20°C for adequate fixation of the heart and great vessels. The specimens subsequently were weighed, the chest wall was removed, and the cardiovascular anatomy was carefully exammed in situ. The heart and great vessels were dissected, the heart was weighed. and the thickness and shape of the aortrc and pulmonary valve cusps were assessed. Anomalies of the interatrial septum and of the atrioventricular valves were inspected. Finally, the heart was transversally transected through the equatorial plane midway between the apex and the aortic root and the interventricular septum, the right outflow tract and the subaortic portion were examined. Measurements were taken when necessary with an eye-piece micrometer, and all anomalies found were photographed. Analysis of variance (ANOVA) and post-hoc Ftsher’s testing were used for comparison of continuous numerical variables among groups. The differences in the proportions of malformations in the different Table

No 1

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Total

Left Right CDH CDH

+ + + + + + + + + + + + + + + + + + +

+ + + + + + + + -

1. Cardiovascular

Malformations

Bilateral Fallot Single CDH VSD ASD Tetral DORV GVT Ventricle

+ + + -

+ -

-

+ +

20

8

5

+ + + + + + + + + + + + +

-

subclavian

artery;

+

-

+ + + + + + + + +

+ + -

+ +

9

3

2

13

Abbreviations: VSD, ventricular aortic arch; CoA, aortic coarctation; right

Found

T18, trisomy

-

-

+ -

-

-

-

+ -

+ + -

at Autopsy

RESULTS In our series of autopsies, 33 of 136 individuals (24%) had CDH with cardiovascular malformations, 51 (38%) had isolated diaphragmatic defects, and the remaining 52 (38%) had CDH with other malformations but without cardiovascular involvement. In this series there were 14 polymalformed babies and 11 cases of known syndromes including Fryns’, trisomy 13, trisomy 18, and limb-body wall complex. As shown in Table 1 the most frequent cardiac defect was ventricular septal defect (VSD) followed by septum primum atrial septal defect (ASD) and tetralogy of Fallot (TOF). Other defects less frequently found were double outlet right ventricle (DORV), transposition of the great vessels (TGV), narrow outflow pulmoin 33 of 136 Newborns

Right PersEt AA CoA LSVC

+ +

+

t

-

-

-

-

2

2

2

tables and Fisher’s Exact test. P significant. Values are expressed

2

Aberrant NOPT PTA HSA

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

t t 2

and Stillborns

-

-

-

t -

-

-

-

-

-

-

-

-

+ -

-

-

-

-

-

1

t -

Hypop Heart

+ t t + -

-

-

+ + +

1

1

7

Other

+ t t t t t t + 8

With

CDH (1966 to 1997)

Poly LBW Malf T18 T13 Syndrome

t

-

+

- + -

t

-

t

- + t - -

t

-

+ -

t t t +

-

-

-

- + -

+

-

-

t + t t -

t t

-

14

5

2

-

13; LSW, limb body

wall syndrome.

Poly Splen

-

-

-

t -

t -

t -

-

2

1

septal defect; ASD, atrial septal defect; DORV, double-outlet right ventricle; GVT, great vessel LSVC, left superior vena cava; NOPT, narrow outflow pulmonary tract; PTA, persistent truncus 18; T13, trisomy

FVllS Syndrome

-

trasposition; arteriosus;

AA, RSA,

MIGLIAZZA

1354

nary tract (NOPT), and persistent truncus arteriosus (PTA). The anomalies of the great vessels observed were right aortic arch (RAA), coarctation of the aorta (CoA), persistent left superior vena cava (PLSVC), and aberrant right subclavian artery (ARSA). In 7 individuals a marked ventricular hypoplasia was observed. In regard to the animal studies, all 21 control fetuses had normal diaphragms and no cardiovascular defects could be detected in them (Table 2). Conversely, 80 of the 130 nitrofen-exposed animals (61.5%) had posterolateral CDH (left sided 72 times, right sided 4 times, and bilateral 4 times). More than half the animals exposed to nitrofen had some heart or great vessel malformation or a combination of them. There was a significant association (Fisher’s Exact test, P < .OS)between the occurrence of CDH and that of cardiovascular defects that were present in 74% of animals with CDH and only in 50% of those without it. Fetal and heart weights after fixation were smaller in nitrofen-exposed pups, and the percentage of the body weight represented by the heart in animals with CDH was significantly decreased showing heart hypoplasia (Table 3). Table 4 shows that the most common heart defect found in nitrofen-exposed animals was NOPT with normal infundibulum and pulmonary valve. TOF with large VSD was present in 16% of fetuses. In these, the defect was overrided by an aortic root displaced to the right and dysplastic pulmonary valve or narrow pulmonary infundibulum. PTA with left aortic arch was seen in 15.4% of fetuses; 2 of them had complete absence of the pulmonary valve with a single arterial trunk giving origin to pulmonary and systemic arteries (type II persistent truncus arteriosus), whereas the remaining 18 pups had the main pulmonary arteries separated from the truncus by a severely hypoplastic pulmonary trunk arising from an atretic pulmonary valve (type IV persistent truncus arteriosus). This malformation often was associated with tetralogy of Fallot. Perimembranous VSD was found in Table 2. Heart

and Great Vessel Nitrofen-Exposed

Malformations Fetal Rats

in Control

and

Nitrofen No-CDH (n = 50)

Control

(n = 21) Heart malformations*

0

13 (26%)

Great vessel formationst

0

0

Total (n = 130)

Normal cardlovascular system

21 (100%)

Control In = 21)

4.591 2 0.5* 0.034 k 0.005’t

Heart weight to body weight ratio (%)

0.893

0.838

0.749 + 0.1*t

rt 0.083

tP<

.Ol compared

with

no-CDH

group.

14% of treated fetuses and isolated aortic or pulmonary valve dysplasia (thickened and distorted cusps but without commissural fusion) or DORV in smaller proportions. Large ASD (common atrium type) were found 5 times always in association with great vessel malformations (RAA, right ductus arteriosus, ARSA). Milder types of ASD were disregarded because the foramen ovale usually is patent in rat fetuses on the 21st day of gestation. Figure 1 depicts the anatomy of some of these defects. Ductal agenesis, right-sided ductus or left or right subclavian artery arising from the ductus alone or in association with other defects TOF, ASD, or VSD frequently were observed. When the ductus was on the right side, it formed a tracheal vascular ring with the normal left-sided aortic arch. Six animals presented an upwarddisplaced aortic arch (cervical aortic arch). The aortic arch was right sided in 7 fetuses and in 5 it was interrupted beyond the emergence of the left common carotid artery. In all these cases, a left ductus arteriosus was in continuity with the left subclavian artery and with Table 4. Heart and Great Vessel Defects in Nitrofen-Exposed Rat Fetuses No-CDH (n = 50)

CDH In = 80)

Total (n = 130)

Heart Narrow outflow pulmonary Tetralogy of Fallot Persistent Ventricular Pulmonary Aortic valve Atrial septal Double-outlet

tract

truncus arteriosus septal defect valve dysplasia dysplasia defect right ventricle

Great vessels Ductal right subclavian Retroesophageal right

artery subclavian

14 (11%)

artery Right aortic arch Absent ductus erteriosus

8 (16%)

14 (17.5%)

22 (17%)

Cervical aortic arch Interrupted aortic arch Right ductus arteriosus Ductal left subclavian artery Right carotid-subclavia aberrant junction

great vessel malformations. heart malformations.

k 0.15

NOTE: Data are expressed as mean 2 SD. *PC .Ol compared with control group.

10 (12.5%)

46 (35%)

Nitrofen-CDH (n = 80)

4.786 k 0.7’ 0.04 2 0.006’

4 (8%)

21 (26%)

Nitrofen-No CDH (n = 50)

and

5.621 2 0.6 0.05 2 0.007

48 (37%)

25 (50%)

in Control

Fetal weight(g) Heart weight(g)

35 (44%)

mal-

Heart and great vessel malformations

*Without twithout

CDH (n = 80)

Table 3. Postfixation Fetal and Heart Weights Nitrofen-Exposed Fetal Rats

ET AL

19 (23.75%)

24 (19%)

14 (17.5%) 13 (16.2%) 14 (17.5%)

21 (16%) 20 (15%) 18 (14%)

6 (7.5%) 3 (3.75%) 2 (2.5%)

8 (6%) 5 (4%) 5 (4%)

2 (2.5%)

4 (3%)

1 (2%)

11 (13.75%)

12 (9%)

4 (8%) 4 (8%)

6 (7.5%) 3 (3.75%) 2 (2.5%)

10 (8%) 7 (5%) 7 (5%)

5 (10%) 7 (14%) 7 (14%) 4 (8%) 2 (4%) 2 (4%) 3 (6%) 2 (4%)

5 (10%) 1 (2%)

1 (2%)

5 2 4 2

0

2 (2.5%)

3 (6%) 0

(6.25%) (2.5%) (5%) (2.5%)

6 5 4 3

(5%) (4%) (3%) (2%)

2 (1%)

CARDIOVASCULAR

MALFORMATIONS

IN CDH

1355

Fig 1. (A) View of the heart from above (atria removed) in a control fetus shows the relationship between the (B) Similar view of the heart of a nitrofen-treated fetus wlth tetralogy of Fallot. The pulmonary ring is small and (C) Membranous ventricular septal defect (arrow) seen from within the left ventricle in a nitrofen-treated fetus. (arrows) in a nitrofen-treated fetus seen from within the right ventricle (apical portion of the heart removed). a, R, right ventricle; L, left ventricle.

the descending aorta, and all had heart defects like VSD, ASD, NOPT, and DORV. ARSA (“retroesophageal” when it arose as the most distal element of the aortic arch or “ductal” when it took its origin from the junction between the ductus and the pulmonary trunk) was found in 22 fetuses. Two of them had an anomalous vessel that joined the right carotid and the right subclavian artery, and most had other associated cardiovascular defects. In Fig 2 some examples of great vessels malformations are shown. DISCUSSION

A recent epidemiological investigation on 24,005 live neonates and stillborns aimed at identifying possible specific patterns of CDH-associated defects found cardiovascular anomalies in 10.32% of infants with CDH and only in 3.48% of those without it when known syndromes like trisomies 13 and 18, Fryns’ syndrome, or limb-body

aortic and the pulmonary valves. the aortic valve right positioned. (D) Double outlet right ventricle aortic valve; p, pulmonary valve;

wall complex were excluded from the analysis; in individuals with such syndromes, the respective figures were 50% and 13.43% and the differences between CDH and non-CDH populations were significant in both cases showing a clear cut predominance of cardiovascular anomalies above 17 other congenital defects.’ Several studies on the nature of the congenital heart diseases associated with CDH2-9 and our own series show a clear predominance of outflow tract anomalies: VSD involving the subaortic portion of the septum, TOF, TGV, DORV, and PTA (Table 5). Inflow tract anomalies (aberrant tricuspid valve, atrio-ventricular septal defect), anomalies of the aortic arch (CoA, interrupted aortic arch) and heart hypoplasia were also found. The overall incidence of cardiovascular malformations associated with CDH varies among investigators according to the modality of data collection: if only autopsy records are considered, as in ours and other series4Jjthe frequency is greater than

1356

MIGLIAZZA

ET AL

.I

Fig 2. Anterior and of the origin subclavian artery left-sided ductus truncus arteriosus a, aortic arch; Is, trachea.

view of the great vessels in nitrofen-treated fetuses. (A) of the right subclavian artery. (B) Interrupted aortic arch arises. (C) Right-sided aortic arch with right-sided ductus arteriosus that continues with an aberrant left subclavian with retroesophageal right subclavian artery. (F) Type IV left subclavian artery; rs, right subclavian artery; d, ductus

25%, but the proportions are smaller if only survivors are taken into account, suggesting that the presence of congenital heart disease influences the outcome. Greenwood et al2 reported that CDH mortality rate in infants with cardiovascular defects is higher (73% as opposed to 27%) than in those without them. The current study demonstrates a significant association between the diaphragmatic defect and the abnormal cardiovascular shaping in fetal rats with CDH and also that cardiac defects (NOFT, TOF, PTA, VSD, aortic or pulmonary valve dysplasia, or DORV) in this model involve most frequently, like in human CDH, the outflow tract. Furthermore, the great vessel malformations ob-

Cervical aortic arch: upward displaced position of the left aortic arch with a right-sided ductus arteriosus from which an aberrant right arteriosus. (D) Right-sided aortic arch forming a vascular ring with a artery. (E) Type II persistent truncus arteriosus: single left-sided persistent truncus arteriosus: severely hypoplastic pulmonary trunk. arteriosus; t, persistent truncus arteriosus; p, pulmonary trunk; tr,

served (aberrant subclavian artery, abnormal-rightsided, cervical, or interrupted-aortic arch, and abnormal ductus arteriosus) also are the same occasionally found in human CDH. Finally, and also like some CDH babies, CDH rats bear severe heart hypoplasia. The formation of the primitive cardiac tube begins in human embryos on day 20 by the committment of progenitor cells within the anterior lateral plate mesoderm to a cardiogenic lineage. On day 23 the cardiac tube undergoes a rightward looping, first indication of the left-right embryonic asymmetry, and acquires an anteroposterior polarity with chamber specification: atria, left ventricle, right ventricle, conotruncus (outflow tract), and

CARDIOVASCULAR

MALFORMATIONS

Table

IN CDH

5. Cardiovascular

Malformations

Study

Greenwood

et al (1976)*

VSD

n = 48 (A, C)

series

-

Abbreviations: vessel

n = 130 (A, C)

(1998)

transposition;

n = 136 (A)

A, autopsy

records;

DORV, double-outlet

Hypopl Heart

ASD

3

Sweed and Puri (1993j6n = 64 (A) Fauza and Wilson (1994P n = 166 (A, C) Alan et al (1996)s n = 93 (C) Losty et al (1998P

Found

in Human

Fallot Tetral

Individuals

With

CoA

GVT

Persist LSVC

3

1 3

-

Congenital

Diaphragmatic

DORV

Aberrant Valve

1 -

--I I--

-

5 -

-

PTA

Hernia

lnterr AA

AVSD

Other

CDH Infants With CVM (%I

43-311--2---4

David and lllingworth (1976Pn = 143(A,C) Benjamin et al (1988j4 n = 60 (A) Sharland et al (1992P n = 55 (A, C)

Current

1357

C, clinical

23

4

1

3 2 ,---

-

-

4 5 9 5

10 -

24-*-----13 * 3

2

3 1

-

3 -

1 -

-

-

-?

9

2

-

1 3

7 12 -

5

3

5

1

3

1

-

-

1

1

2

-

4

13

9

7

3

2

2

2

2

1

1

-

-

13

records;

right ventricle;

VSD, ventricle PTA, persistent

aortic that gives origin to the aorta and the pulmonary arteries. Subsequently, the left and right atrioventricular canals undergo septation, and the conotruncus also septates and rotates for a proper orientation of the great vessels. At the same time, after a process of formation and regression in crania-caudal sequence, six paired pharyngeal aortic arches extending from the aortic sac to the dorsal aortae develop; the third, fourth, and sixth arches give origin to the definitive aortic arch, the subclavian and the carotid arteries, the ductus arteriosus, and the proximal pulmonary arteries.lO The neural crest has a crucial role in these processes: ectomesenchymal cells from the cardiac neural crest, that extends between the midotic placode and the caudal limit of the third somite, migrate and populate the cardiac outflow tract, the cusps of the arterial valves, and the pharyngeal arches third, fourth, and sixth. Ablation of the crest before migration in chick embryos results in a variety of malformations that depend on its extent: outflow tract anomalies (subaortic VSD, TOF, PTA, TGV, DORV), aortic arch anomalies (interrupted aortic arch, abnormal position of the aortic arch, persistence of some vessels that should disappear), and, less frequently, inflow tract anomalies (abnormal tricuspid valve and atrioventricular septal defect). Interestingly, cardiac neural crest removal induces not only structural defects, but also functional changes in the developing myocardium leading to markedly depressed contractility index and severe cardiac dysfunction.11,12 Pharyngeal arch development and conal septation take place in the rat between gestational day 10.5 and 12.5 and neural crest cell migration starts shortly before that time. Nitrofen exposure on day 9.5 could, therefore, exert a strong teratogenic influence precisely at that moment. Bilateral mesenchymal anomalies of the cervical somites 2,3, and 4 were observed in 10.5day-old rat embryos 24 hours after nitrofen exposure and more laterally in the intermediate mesoderm adjacent to the same somites after 48 hours, and this embryonic mesodermal perturba-

septal truncus

defect;

ASD, atrial

arteriosus;

septal

AA, aortic

defect;

CoA, aortic

coarction;

arch; AVSD, atrioventricular

13 42 16 25 25 15 9 24 GVT, great septal

defect.

tion has been interpreted as being most likely related to the subsequent appearance of left-sided CDH and pulmonary hypoplasia because the increased incidence of somitic cell death correlated closely with the incidence of left-sided CDH seen in full-term fetuses.13 The relationship between these phenomena and the origin of cardiac anomalies is unclear, but the regional vicinity of the involved mesoderm and the coincidence in time cannot be ignored. The practically identical pattern of human and murine cardiovascular malformations associated with CDH suggests that similar pathogenic mechanisms might be at play in both settings and that a disorder of neural crest cell migration very likely is involved. Very little is known about the mechanisms of nitrofen teratogenesis. Costlow and Manson14 were the first to demonstrate that the heart is, with the diaphragm, the main target organ for this chemical in rodents and that their malformations accounted for 75% of the neonatal mortality associated with in utero exposure. But other teratogens have similar effects: Momma et all5 induced CDH accompanied by lung and heart hypoplasia in rat fetuses by prenatal exposure to bis-diamine. Interestingly, they also found cardiovascular anomalies corresponding to defects of the outflow tract (VSD, NOPT, pulmonary valve dysplasia, DORV, TOE PTA) and of the aortic arch system (double or right aortic arch, aberrant subclavian artery, right or agenetic ductus arteriosus, and anomalous origin of pulmonary arteries). A variable degree of myocardial fiber disarray has been observed by other investigators after prenatal bis-diamine exposure.16 According to these investigators, the mechanism of the malformations could be sequential: the basic defect would be a disorder of neural crest cell migration to the aortic arches leading to an abnormal ductus arteriosus (defective development of the sixth aortic arch) able to cause a perimembranous VSD, pulmonary valve dysplasia, and other outflow tract defects as a consequence of a right ventricular pressure

1358

MIGLIAZZA

overload. This change in the hemodynamic status also could contribute to the abnormal myocardial architecture. Molenaar et a1,17 reviewing the history of CDH, hypothesized that it might be part of a more basic disturbance involving many organs and suggested that the diaphragmatic one could be the less relevant and that it is clearly wrong, when dealing with these patients, to focus our attention exclusively on the surgically correctable diaphragmatic defect. l7 There must be a common pathogenic link among the malformations clustered in both patients and animals with CDH, and it has to act necessarily at the time of the para-axial mesenchymal organization and of the mesenchymal-epithelial interactions leading to organogenesis. The molecular information necessary for these processes is mainly transmitted by the Hox group of genes that participate in cell migration and development of neural crest-derived tissues,18and their possible involvement in the pathogenesis

ET AL

of these and other congenital malformations is most likely. Mutations of the homeodomain protein HOXa3, in the retinoic acid receptors (RAR) that modulate Hex gene expression and in the homeobox gene Pan3 are able to induce in the mouse cardiac outflow tract and aortic arch defects as well as hypoplasia of the ventricular wall suggesting that appropriate sets of Hex genes regulate cardiac neural crest development and that abnormal expression of these genes could be the pathogenetic process leading to these defects.lO The results of the current investigation further reinforce the concept of the nitrofen rat model being a powerful research tool capable of reproducing the pattern of CDH-associated anomalies and most likely the timing and the level of the embryonic insult resulting in the human condition. Further investigation on morphogenesis, cellular signaling, and gene regulation causing these congenital defects are warranted.

REFERENCES 1. Martinez-Frias ML, Prieto L, Urioste M, et al: Clinical/ epidemiological analysis of congenital anomalies associated with diapbragmatic hernia. Am J Med Genet 62:71-76,1996 2. Greenwood RD, Rosenthal A, Nadas AS: Cardiovascular abnormalities associated with congenital diaphragmatic hernia. Pediatrics 57:92-97,1976 3. David TJ, Illingworth CA: Diaphragmatic hernia in the south-west of England. J Med Genet 13:253-262,1976 4. Benjamin DR, Juul S, Siebert JR: Congenital posterolateral diapbragmatic hernia: Associated malformations. J Pediatr Surg 23:899903,1988 5. Sharland GK, Lockhart SM, Heward AJ, et al: Prognosis in fetal diaphragmatic hernia. Am J Obstet Gynecol 166:9-13,1992 6. Sweed Y, Puri P: Congenital diaphragmatic hernia: Influence of associated malformations on survival. Arch Dis Child 69:68-70, 1993 7. Fauza DO, Wilson JM: Congenital diaphragmatic hernia and associated anomalies: Their incidence, identification, and impact on prognosis. J Pediatr Surg 29:1113-1117,1994 8. Allan LD, Irish MS, Glick PL: The fetal heart in diapbragmatic hernia. Clin Perinatol23:795-812,1996 9. Losty PD, Vanamo K, Rintala RJ, et al: Congenital diapbragmatic hernia-Does the side of the defect influence the incidence of associated malformations? J Pediatr Surg 33:507-510, 1998

10. Olson ER, Srivstava D: Molecular pathways controlling heart development. Science 272:671-676, 1996 11. Kirby ML, Waldo KL: Role of neural crest in congenital heart disease. Circulation 82:332-340, 1990 12. Kirby ML, Waldo KL: Neural crest and cardiovascular patteming. Circ Res 77:211-215, 1995 13. Alles A.J, Losty PD, Danahoe PK, et al: Embryonic cell death patterns associated with nitrofen-induced congenital diaphragmatic hernia. .I Pediatr Surg 30:353-360, 1995 14. Costlow RD, Manson JM: The heart and diaphragm: Target organs in the neonatal death induced by nitrofen (2,4-dichlorophenyl-pnitrophenyl ether). Toxicology 20:209-227,198l 15. Momma K, Ando M, Mori Y, et al: Hypoplasia of the lung and heart in fetal rats with diaphragmatic hernia. Fetal Diagn Ther 7:46-52, 1992 16. Kmibayashi T, Roberts WC: Tetralogy of Fallot, truncus arteriosus, abnormal myocardial architecture and anomalies of the aortic arch system induced by bis-diamine in rat fetuses. J Am Co11 Cardiol 21:768-776,1993 17. Molenaar JC, Bos AP, Hazebroek FWJ, et al: Congenital diaphragmatic hernia, What defect? J Pediatr Surg 26248-254, 1991 18. Mark M, Rijli FM, Chambon P: Homeobox genes in embryogenesis and pathogenesis. Pediatr Res 42:421-429, 1997