Pulmonary Expression of Vascular Endothelial Growth Factor atid Myosin Isoforms in Rats With Congenital Diaphragmatic Hernia By Tadaharu Okazaki, Hari S. Sharma, Masanori Aikawa, Atsuyuki Yamataka, Ryozo Nagai, Takeshi Miyano, and Dick Tibboel Rotterdam, The Netherlands; Tokyo and Gunma, *Japan l Abnormalities of the pulmonary vasculature are well documented in cases of congenital diaphragmatic hernia (CDH). Vascular endothelial growth factor (VEGF), an angio- genie factor, is a recently described endothelial cell-specific growth factor. Myosin heavy chain (MHC) isoforms such as SMemb, SMI and SM2 are important molecular markers used to study vascular smooth muscle cell differentiation. SMemb is predominantly expressed in immature smooth muscle cells (SMCI, and SM2 is expressed in mature SMCs. The authors investigated the expression of VEGF and SMC differentiation in pulmonary vessels in CDH rat lungs and in controls. The lungs of nitrofen-induced CDH rat fetuses (n = 16, gestational age 16,18,20, and 22 days) were stained immunohistochemically using antibodies against VEGF, SMemb and SM2, while alpha-actin was used as a general marker of vascular smooth muscle cells. In the CDH group VEGF expression was negative in pulmonary vessels before birth, and in the control group VEGF was positive in smooth muscle cells in vessel walls from 20 days both in vessels at the hilum and in pulmonary parenchyma. In both control and CDH groups, SMemb expression was positive from 16 days’ gestation. SM2 expression was negative in vessel walls during the prenatal period in both groups, Alpha-actin was localized in both lungs obtained from control and CDH groups in the lung hilum from 16 days and around peripheral vessels from 18 days. Differences in vascular smooth muscle cell differentiation were not observed between control and CDH lung. These findings suggest that differences in pulmo- nary vascular development exist between control and CDH rats for VEGF expression, and maturational differences in smooth muscle cell differentiation are not present. This role of altered endothelial cell growth might be related to the different pulmonary vascular reactivity present in CDH lungs. Copyright Q 1997 by W B. Saunders Company INDEX WORDS: Congenital diaphragmatic hernia, vascular endothelial growth factor, myosin heavy chain isoforms, pulmonary hypertension. C ONGENITAL diaphragmatic hernia (CDH) is a malformation associated with high mortality and morbidity caused by pulmonary hypoplasia and pulmo- nary hypertension. lo The mortality rate of 40% to 50% has not changed significantly during recent years despite changing concepts in treatment such as delayed surgery, nitric oxide, and extracorporeal membrane oxygenation (ECM0).3,4 As pulmonary vessel abnormalities are well documented in CDH2, recent reports have examined vasoactive factors such as endothelin5 and nitric oxide synthase’j in CDH. The natural history of pulmonary vascular development, especially in the embryonic and fetal phase, has not been reported. Vascular endothelial growth factor (VEGF), an angio- Journal offediatric Surgery, Vol32, No 3 (March), 1997: pp 391-394 genie growth factor, described in 1989 as a specific endothelial cell growth promoteq7,* it is an important regulator of endothelial cell proliferation during the extensive tissue growth and remodeling that occurs in utero.9 Myosih heavy chain (MHC) isoforms, such as SMemb, SMl and SM2, are important molecular markers used to study vascular smooth muscle cell differentia- tion.‘O-iz SMemb is predominantly expressed in immature smooth muscle cells (SMC), and SM2 is expressed in mature SMCs. SMI is considered a general marker of SMCs because it is expressed at all stages of cell differentiation. Because the interrelationship of endothelial cell growth and SMC differentiation in pulmonary vessel develop- ment has not been studied in CDH so far, we examined the expression of VEGF and MHC isoforms immunohis- tochemically in a rat model of CDH. MATERIALS AND METHODS Adult female Sprague-Dawley rats were mated overnight. Observa- tion of positive smears was considered proof of pregnancy (day 0 of pregnancy). To induce CDH, 100 mg of 2,4-dichloro-phenyl-p- nitrophenylether (nitrofen) dissolved in 1 mL olive oil was given on day 10 of gestation.t3 In control animals, the same dose of olive oil was given without nitrofen. Water and food were supplied ad libitum during the entire period of the experiment. At gestational age 16. l&20, and 22 days (term) the mother was anesthesized by inhalation of ether and cesarean section was performed. The fetuses were removed and killed before any breathing occurred. Age-matched normal fetuses were obtained from control animals. The lungs from CDH and control fetuses (n = 16 in each gestational age group) were fixed in a mixed solution of 95 ~01% ethanol and 1% acetic acid. They were embedded in pa&tin and 3-pm thick sections were prepared. Immunohistochemical studies were performed with an enzymatic color development method. To reduce nonspecific reactions, sections were preincubated with 0.3% hydrogen peroxidase and normal goat serum. Antibodies (Ab) against VEGF (diluted l:lOO), SMemb From the Department of Pediatric Surgery, Sophia Children’s Hospital and Department of Pharmacology, Erasmus University, Rotter- dam, The Netherlands; the Departments of Pediatric Surgery and Cardiology, Jwzfendo U~~iwrsity School of Medic&e, Tokyo, Japan; and the Second Department oflntental Medicine, Gunma University School of Medicine, Gunma, Japan. Presented at the 13rd Armual International Congress of the British Association of Paediatric Surgeons, St Helier; Jersey. Channel Islands, July 16-19, 1996. Address reprint requests to Professor Dr Dick Tibboel, MD, PhD, Department of Pediatric Surgery, Sophia Children S Hospital, Erasmus University Rotterdam, Dr Molewatevplein 60, 3015 GJ Rotterdam, The Netherlands. Copyright o 1997 by WB. Saunders Company 0002.3468/97/3203-0002$03.00/O 391