Changes in vasoactive pathways in congenital diaphragmatic hernia associated pulmonary hypertension explain unresponsiveness to pharmacotherapy

Human lung samples were retrieved from the archives of the Department of Pathology of the Erasmus Medical Center, Rotterdam. In our high-volume, leading center of the EURO consortium [4], approximately 15 to 20 CDH patients a year are born, which ensures a large experience in the treatment of this disease. Paraffin-embedded lung samples, lacking severe hemorrhage or necrosis, were selected of controls, of CDH patients and of patients with lung hypoplasia or pulmonary hypertension unrelated to CDH. Only lung material of patients with a severe left-sided CDH and a survival of less than 7 h were selected to prevent secondary sequelae. Patient characteristics are described in Table 3.

The well-established animal model was used, where in short pregnant Sprague-Dawley rats received either 100 mg nitrofen dissolved in 1 ml olive oil or just 1 ml olive oil by gavage on gestational age day E9.5 [27]. Nitrofen induces left-sided CDH in approximately 70% of the offspring, while all pups have pulmonary hypertension. At day E21 pups were delivered by caesarean section and euthanized by lethal injection of pentobarbital.

Immunohistochemistry (IHC) was performed on 5 μm thick paraffin sections of lungs of both rats and humans according to standard protocols, using the Envision™ detection system (Dako Cytomatic, Glostrup, Denmark) [28]. Briefly, sections were deparaffinized with xylene and rehydrated in gradual series of ethanol, after which antigen retrieval was performed by boiling samples in 10 mM Tris (pH 9.0), 1 mM EDTA. Primary and Horse Redox Peroxidase conjugated secondary antibodies were diluted in antibody dilution buffer (DAKO) with 0,5% Tween-20, and the peroxidase was detected with diamino-benzidine tetrahydrochloride (Fluka, Buchs, Switzerland). Validated primary antibodies used for IHC were Endothelin receptor A (ETA; 1:5000 (rat) 1:100 (human); Alamone, Jerusalem, Israel), Endothelin receptor B (ETB; 1:2500 (rat) 1:500 (human); Alamone), Endothelin Converting Enzyme (ECE-1; 1:500 (human); Abcam, Cambridge, MA, USA), endothelial nitric oxide synthase (eNOS; 1:400 (rat); Thermo Fisher Scientific, Waltham, MA, USA) and prostaglandin-I₂ receptor (Ptgir; 1:1000 (rat) 1:500 (human); Cayman Chemical, Ann Arbor, Michigan, USA). Negative controls were performed by omitting the primary antibody.

RNA isolation of whole lungs of rat pups, cDNA synthesis and subsequent qPCR analysis was performed as previously described [28]. The gene-specific primers used are listed in Table 4. Actb was used as reference gene.

Data are presented as means (SD) for normally distributed variables. Univariate analyses were performed using independent samples t-tests for normally distributed variables. The analyses were performed using SPSS 21.0 for Windows (Armonk, NY, USA: IBM Corp.). All statistical tests were two-sided and used a significance level of 0.05.

Besides the use of inhaled NO (iNO), current treatment is based on targeting the receptors in both the prostacyclin and endothelin pathway. Therefore, we analyzed the expression of the critical proteins of both pathways in human lung samples of control and CDH patients by immunohistochemistry. Human control lung samples showed little expression of the main target of the prostacyclin therapy, the important prostacyclin receptor PTGIR, in the fetal period. This sharply increased later during gestation at the preterm and term age. However this significant increase was absent in CDH (Fig. 2). The ETA receptor, which induces vasoconstriction and cell proliferation, was expressed in the small (25–50 μm) and larger (>50 μm) vessels as well as in the very small capillaries (<25 μm) in CDH. In contrast, the distribution of the ETA receptor in control lungs was limited to the small and larger vessels only (Arrowheads in Fig. 3a). The ETB receptor, involved in vasodilation through the release of NO and PGI₂ (Fig. 1), was expressed both in the bronchial epithelium and in some of the larger vessels (>50 μm) in CDH (Arrowheads in Fig. 3a). However, the expression of ETB in control lungs was found only in the bronchial epithelium (Fig. 3a). Next, we analyzed the expression of ECE-1, a membrane-bound metalloprotease that converts big-endothelin into the biologically active compound ET1 and it is a rate-limiting factor in the ET pathway. Early during gestation, in the fetal period, ECE-1 is minimally expressed in the vessels of the human control lung samples, with an increase at preterm and term age. Increased expression of this enzyme at both fetal, preterm and term age was observed in CDH (Fig. 3b), indicating a potential increased bio-availability of active ET-1 already at the fetal stage of development.

To exclude that the differences in expression patterns of the crucial prostacyclin – and endothelin receptors and the rate-limiting factor ECE-1 was solely an effect of lung hypoplasia (LH) or PH, we performed immunohistochemistry on lungs of patients with LH and PH with other cause than CDH. The PTGIR receptor expression was reduced in both LH and PH (Fig. 4a). Increased expression of ETA was detected in the smallest vessels in lungs of both LH and PH (Arrowheads in Fig. 4b), whereas increased expression of ETB was only observed in both small and very small vessels of lungs of PH patients (Arrowheads in Fig. 4c). ECE-1 was not expressed differently in both LH and PH lung samples (Fig. 4d).

In order to validate these interesting human data, we evaluated the expression patterns of the proteins of these three pathways in the nitrofen rat model. This was supplemented with RNA and protein expression analysis of related factors. Real-time qPCR showed that the mRNA expression of both the Eta and Etb receptors was significantly higher in lungs of E21 pups with CDH compared to those of control pups. We also analyzed the expression of the ETA and ETB ligand, Et-1, but no significant differences were found between the groups. However, the mRNA encoding the rate-limiting factor Ece-1 was significantly increased in CDH compared to control, confirming the human data (Fig. 5a). Next, we analyzed the protein expression pattern of the ET receptors with immunohistochemistry. The ETA receptor was expressed in the small capillaries of both groups at E15 until E21 with a stronger expression level in CDH. At E21 only CDH lungs showed expression of the ETA receptor in the larger vessels (>50 μm) (Fig. 5b). The ETB receptor was expressed in the bronchial epithelium of all lungs without significant differences between control and CDH at all ages (Fig. 5c). There was a significant higher mRNA expression of eNos in CDH rats compared to control in relation to all cells as well as in relation to only the smooth muscles cells (Fig. 6a) or endothelial cells (data not shown). This increased expression was clearly detectable with immunostaining in the larger and smaller (<50 μm) vessels at E21. However, no obvious differences were noted earlier during development (E15 till E19) (Fig. 6b). Although there was no difference in expression of prostaglandin-I₂ synthase (Ptgis) between control and CDH rat pups, there was a slight increase in the expression of Ptgir and the prostaglandin-E₁ receptor (Ptger1) in CDH at the mRNA level in both the whole lung as well as compared to the number of smooth muscle cells. In contrast, the expression of thromboxane synthase (Tbxas1), the enzyme converting prostaglandin H2 into thromboxane A2, which is in turn critical for vasoconstriction, was clearly reduced in CDH, whereas the thromboxane receptor (Tbxa2r) did not show significant differences (Fig. 7a). However, immunostaining showed no clear differences in expression of PTGIR in the vessels between both groups (Arrowheads in Fig. 7b).