Congenital diaphragmatic hernia (CDH) is a life-threatening disease that occurs in 1:2500 live births with 40% of mortality. The pathogenesis of CDH is controversial and complex as it involves, in severely affected newborn infants, lung hypoplasia and pulmonary hypertension. Microscopically, the lungs in CDH show decreased airway branching, decreased alveolarization and thickened muscular layer of the pulmonary arterioles that is related to the degree of pulmonary hypertension.

Vascular endothelial growth factor (VEGF) and its receptors, VEGFR1 (flt1) and VEGFR2 (flk1), play a central role in lung development. VEGF is a vascular endothelial cell-specific mitogen essential to vasculogenesis, angiogenesis, and type II pneumocyte differentiation. Moreover, vascular growth and alveolarization are coupled processes so that disruption of the VEGF pathway in experimental models cause both decreased vascular growth with pulmonary hypertension as well as decreased alveolarization.

Studies on the expression of VEGF in the nitrofen-induced CDH rat model have been conflicting, showing either a decrease in the concentration of the protein or an increase in the mRNA. In contrast, analysis of VEGFR1 and VEGFR2 mRNA content in the lung of animals with nitrofen-induced CDH have not demonstrated any difference to controls. However, the protein content of VEGFR1 and VEGFR2 has not been assessed in this model.

Even though pulmonary hypoplasia and hypertension are the main determinants of the severity of CDH, most of the current available treatments fail to directly address these problems. Steroids improve lung maturity by increasing surfactant production, increasing air space and thinning the alveolar walls, but do not have a clear role in the treatment of CDH yet. More recently, maternal administration of steroids has been shown to cause thinning of the wall of pulmonary arterioles and decrease the muscularization of pulmonary vessels in rabbit fetuses in a dose dependent fashion. In the same study, it was observed a concomitant increase in the VEGFR2 expression, suggesting that the effects of steroids in pulmonary arterial structure may be mediated by the VEGF.

In contrast, fetal tracheal occlusion (TO) promotes lung distention and growth and is a treatment successfully used in some centers for severe CDH. By accumulating liquid inside the lungs and distending them, TO partially reverses pulmonary hypoplasia in CDH leading to increased alveolar spaces and also partially reverses the vascular structural abnormalities observed in CDH. In addition, TO has been shown to increase VEGF-A mRNA levels in the lung of fetal rats without changing the VEGFR1 or VEGFR2 mRNA content. However, the effect of TO in the expression of VEGF and its receptors in CDH has not been investigated. Interestingly, TO reduces cell proliferation and the number of type II pneumocytes, decreasing surfactant production, which are deleterious effects that can be partially reversed by antenatal steroids administration.

Therefore, we hypothesized that the deleterious effects of CDH on the muscularization of the vessels could be related to a defect in the VEGF signaling through its receptors and that the beneficial effects of steroids and TO on lung maturation and vascularization could be related to a restoration of the VEGF signaling. To test this hypothesis, we investigated the expression of VEGFR1 and VEGFR2 in the nitrofen-induced rat model of CDH with antenatal steroid treatment and TO.

Materials and Methods

Animals

The study was approved by our Institution’s Ethics Committee on Animal Experimentation. Female and male Sprague-Dawley rats were kept in a controlled dark-night cycle with ad libitum chow and water. Animals were mated overnight, and a sperm-positive vaginal smear confirmed mating and was designated as gestational day 0. Fetuses were harvested at term at 20.5 days of gestation through a median maternal laparotomy and hysterotomy, weighted (body weight [BW]) and dissected. Their left lung weight (LLW) and total lung weight (TLW) were measured, then the lung-to-body weight ratio (LBWR) was calculated. Afterward, the left lung was either frozen or fixated in formaldehyde 10%.

Study groups

The study design is shown below with the respective coding for each group ( Figure 1 ).

Study design and fetal groups

Schmidt. VEGF receptors in CDH rat model. Am J Obstet Gynecol 2010.

Nitrofen administration

To induce CDH, timed-pregnant rats were given 100 mg nitrofen (2,4-dichloro-4′-nitrodiphenyl ether, Maybridge; Tintagel, Cornwall, UK) dissolved in 1 mL of olive oil at 9.5 days of gestation by gavage. In the placebo group we administered 1 ml of olive oil without nitrofen at the same gestational age (olive oil group). This dosage of nitrofen has been shown to cause left-sided in 42% of the litter.

Steroid administration

For treatment with steroids, timed-pregnant rats were given 0.4 mg/kg dexamethasone dissolved in 1 mL normal saline at 18.5 days of gestation intraperitoneally.

TO

On 18.5 days of gestation, timed-pregnant rats were anesthethized and TO was performed in the fetuses using a microclip. Briefly, a median laparotomy was performed and a section of the uterine horn exposed. A purse string suture was placed in the uterine wall near the fetal head, a small hysterostomy was made and the fetal head and neck were exposed. The neck was dissected to expose the fetal trachea and a titanium microclip (Teleflex Medical, Research Triangle Park, NC) was placed with an appropriate clip applier. The fetus was then returned to the amniotic cavity and the purse string tightened. In each uterine horn the first and last fetus were not considered, then TO was performed in the second fetus, the next fetus was undisturbed and used as control, and the following fetuses were exposed through a hysterostomy, had their necks dissected but did not have the TO performed and were considered sham fetuses. This sequence—TO, control, sham—was repeated as many times as possible in each uterine horn. The maternal abdomen was then closed in 2 layers.

Immunohistochemistry

Fetal lungs fixed in formaldehyde, 10% were dehydrated, dyaphanized, and embedded in paraffin. Then, 5 μm slices were mounted on slides for the immunohistochemical reaction. The slides were treated with sodium borate (0.1 M, pH 7.4) for 1 hour at room temperature, followed by sodium citrate (0.01 M, pH 6.0) for antigen exposition. Then, they were treated with 1% H ₂ O ₂ and washed in phosphate-buffered saline solution (PBS) (0.01 M, pH 7.4). Sections were incubated with blocking solution of bovine serum albumin (BSA) 1% for 1 hour at room temperature to block nonspecific binding sites. Sections were incubated with anti-flt1 (VEGFR1) diluted 1:100 in 0.1% PBS or anti-flk1 (VEGFR2) diluted 1:25 in 0.1% PBS (sc-316 and sc-6251, respectively; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. After washing, sections were incubated with biotin-conjugated antirabbit IgG (1:200 in 1% BSA) for 1 hour at room temperature for VEGFR1 and with biotin-conjugated antimouse IgG (1:200 in 1% BSA) for 2 hours for VEGFR2. As negative controls, we omitted the primary antibody. For visualization of histochemical reaction, we used the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and DAB (3,3′-diaminobenzidine tetrahydrochloride hydrated; Sigma-Aldrich, St. Louis, MO). Sections were counterstained with Harris hematoxylin and mounted. Slides were analyzed by 2 independent and blinded investigators. VEGFR1 and VEGFR2 expression was analyzed semiquantitatively, at the same magnification, using a visual scale ranging from 0 to 3: grade 0 = no staining; grade 1 = faint staining; grade 2 = moderate staining; and grade 3 = strong staining.

Western blotting

Whole lungs from 6 animals per group were homogenized in 1 mL per lung of extraction buffer containing (mm): Tris 100 (pH 7.4), sodium pyrophosphate 100, sodium fluoride 100, EDTA 10, sodium vanadate 10, and phenylmethylsulfonyl fluoride (PMSF) 2, and 0.1 mg aprotinin mL ⁻¹ , and 1%Triton-X100 at 4°C with a tissue homogenizer (Tecnal, Paulinia, Brazil) operated at maximum speed for 30 seconds. The extracts were centrifuged at 14,000 rpm (9000 g ) at 4°C in a Mikro 200R centrifuge (Hettich, Tuttlingen, Germany) for 30 minutes to remove insoluble material, and the supernatants of these tissues were used for protein quantification using the Bradford method. Then proteins were denaturated, run on SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were blocked for 1 hour in 1% BSA solution and incubated with anti-flt1 (VEGFR1) diluted 1:200 in 0.1% PBS or anti-flk1 (VEGFR2) diluted 1:200 in 0.1% PBS (sc-316 and sc-6251, respectively; Santa Cruz Biotechnology) at 4°C overnight. The following day, after washing, sections were incubated with biotin-conjugated antirabbit IgG (1:10000 in 1% BSA) for 2 hours at room temperature for VEGFR1 and with biotin-conjugated antimouse IgG (1:5000 in 1% BSA) for 2 hours for VEGFR2. Afterward, membranes were probed with Supersignal Chemiluminescence kit (Pierce, Rockville, IL), exposed to radiographic films (Kodak, Rochester, NY), and developed.

Statistical analysis

Morphometric data were compared using the analysis of variance analysis with Tukey’s posttest. VEGFR1 and VEGFR2 staining scores were analyzed using the Kruskal-Wallis test with Dunn’s posttest. A P value < .05 was considered significant.