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

Abstract

Background: Patients with congenital diaphragmatic hernia (CDH) have structural and functional different pulmonary vessels, leading to pulmonary hypertension. They often fail to respond to standard vasodilator therapy targeting the major vasoactive pathways, causing a high morbidity and mortality. We analyzed whether the expression of crucial members of these vasoactive pathways could explain the lack of responsiveness to therapy in CDH patients.

Methods: The expression of direct targets of current vasodilator therapy in the endothelin and prostacyclin pathway was analyzed in human lung specimens of control and CDH patients.

Results: CDH lungs showed increased expression of both ETA and ETB endothelin receptors and the rate-limiting Endothelin Converting Enzyme (ECE-1), and a decreased expression of the prostaglandin-I₂ receptor (PTGIR). These data were supported by increased expression of both endothelin receptors and ECE-1, endothelial nitric oxide synthase and PTGIR in the well-established nitrofen-CDH rodent model.

Conclusions: Together, these data demonstrate aberrant expression of targeted receptors in the endothelin and prostacyclin pathway in CDH already early during development. The analysis of this unique patient material may explain why a significant number of patients do not respond to vasodilator therapy. This knowledge could have important implications for the choice of drugs and the design of future clinical trials internationally.

Keywords: Development; Endothelin; Lung; Nitric oxide; Prostacyclin; Vasculature; Vasodilation.

Fig. 1

Three major pathways involved in vasodilation and vasoconstriction. a Schematic overview of the major pathways and the key proteins involved in vasodilation and vasoconstriction. b Aberrant expression of key factors in the three pathways in both human and rat congenital diaphragmatic hernia (CDH). Solid arrows represent up- or downregulation in human CDH, dashed arrows represent up- or downregulation in rat CDH. ECE-1 = endothelin converting enzyme 1, ETA = endothelin A, ETB = endothelin B, eNOS = endothelial nitric oxide synthase, sGC = soluble guanylate cyclase, COX = cyclooxygenase, PGIS = prostaglandin synthase, PGI₂ = prostaglandin I₂, AC = adenylate cyclase, TBXAS1 = thromboxane synthase, TXA₂ = thromboxane

Fig. 2

Suppressed progression of prostaglandin-I₂ receptors during gestation in human CDH. Representative images show progressive expression of PTGIR in the vessels during gestation in human control lungs, which is reduced in lungs of human CDH patients. Scale bars represent 20 μm. Patients: GA 18 + 0, 33 + 0 and 38 + 0 (control), GA 21 + 4, 36 + 2 and 37 + 2 (CDH)

Fig. 3

Increased expression of ECE-1 and both ET receptors in human CDH. a Representative images show increased expression of ETA in the smaller vessels and clear expression of the ETB receptor in vessels in lungs of CDH patients compared to control. b ECE-1 is increasingly expressed in the vessels in human control patients during gestation, whereas the expression is already high in the fetal stage of development in CDH patients. Arrows indicate vessels, A indicates airways. Scale bars represent 100 μm (low power) and 20 μm (high power). Patients: GA 38 + 3 (control), GA 38 + 0 and 37 + 2 (CDH) (A + B). Patients: GA 18 + 0, 26 + 5 and 38 + 0 (control), GA 21 + 4, 36 + 2 and 37 + 2 (CDH) (C + D)

Fig. 4

Expression of prostaglandin and endothelin factors in human LH and PH patients. Representative images show expression of PTGIR (a), ETA (b), ETB (c) and ECE-1 (d) in patients with lung hypoplasia (LH) or pulmonary hypertension (PH) unrelated to CDH. The expression of ETA is increased in the smaller vessels of patients with LH and PH (b), and the expression of ETB is only increased in the vessels of patients with PH (c). ECE-1 is not differently expressed in the vessels both LH and PH lung samples (d) Scale bars represent 20 μm. Arrows indicate very small vessels. Patients: GA 38 + 3 (control), GA 41 + 0 (LH), GA 34 + 3 (PH)

Fig. 5

Upregulation of ET-receptors in lungs of rat CDH. a Relative expression of the ETA receptor (Eta) and ETB receptor (Etb) shows a significant increase in rat CDH pups (p < 0.001 and p < 0.05, respectively), whereas RNA expression of ET-1 shows no differences between control and CDH and Ece-1 is significantly increased (p < 0.05). b Representative images show increased expression of the Eta receptor in the parenchyma of CDH lungs at all stages of development and in the larger vessels at E21. c Representative images show expression of the Etb receptor in the bronchial epithelium of both control and CDH lungs at all stages of development. Inserts represent higher magnifications of a pulmonary vessel. *p < 0.05, ***p < 0.001. Error bars represent SD. Arrows indicate vessels, A indicates airways. Scale bars represent 100 μm (low power) and 20 μm (high power)

Fig. 6

Increased eNOS expression in lungs of CDH rats. a eNOS expression compared to total rat lungs (left) or to the pulmonary Sma⁺ smooth muscle cells fraction (right) shows increased expression in CDH lungs. b Representative images show increased expression of eNOS in the vessels of the lungs of CDH rat pups at E21, but not at other stages of gestation. **p < 0.01, ***p < 0.001. Error bars represent SD. Arrows indicate vessels, A indicates airways. Scale bars represent 100 μm (low power) and 20 μm (high power) and 50 μm

Fig. 7

Prostacyclin expression in rat pups. a Relative gene expression of prostaglandin I synthase (Ptgis), thromboxane synthase (Tbxas1), prostaglandin-I₂ receptor (Ptgir) and prostaglandin-E1 receptor (Ptger1) in lungs of control and CDH rat pups. b Representative images showing the protein expression of PTGIR in the pulmonary vessels in control and CDH lungs.**p < 0.01, ***p < 0.001. Error bars represent SD. Arrows indicate vessels, A indicates airways. Scale bars represent 100 μm (low power) and 20 μm (high power)