Perturbations in Endothelial Dysfunction-Associated Pathways in the Nitrofen-Induced Congenital Diaphragmatic Hernia Model

Abstract

Although it is well known that nitrofen induces congenital diaphragmatic hernia (CDH), including CDH-associated lung hypoplasia and pulmonary hypertension (PH) in rodents, the mechanism of pathogenesis remains largely unclear. It has been reported that pulmonary artery (PA) endothelial cell (EC) dysfunction contributes to the development of PH in CDH. Thus, we hypothesized that there is significant alteration of endothelial dysfunction-associated proteins in nitrofen-induced CDH PAs. Pregnant SD rats received either nitrofen or olive oil on gestational day 9.5. The newborn rats were sacrificed and divided into a CDH (n = 81) and a control (n = 23) group. After PA isolation, the expression of PA endothelial dysfunction-associated proteins was assessed on Western blot and immunostaining. We demonstrate that the expression of C-reactive protein and endothelin-1 and its receptors, ET_A and ET_B, were significantly increased in the CDH PAs. Levels of phosphorylated myosin light chain were significantly elevated, but those of phosphorylated endothelial nitric oxide synthase, caveolin-1, and mechanistic target of rapamycin were significantly decreased in the CDH PAs. In this work, we elucidate alterations in the expression of endothelial dysfunction-associated proteins specific to nitrofen-induced CDHrodent PAs, thereby advancing our understanding of the critical role of endothelial dysfunction-associated pathways in the pathogenesis of nitrofen-induced CDH.

Pulmonary hypertension secondary to congenital diaphragmatic hernia: factors and pathways involved in pulmonary vascular remodeling

“…68 The C-reactive protein (CRP) is a well-known marker of inflammation and a risk factor for endothelial dysfunction and, when present in the vessel wall, induces the expression of the vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) by ECs and serves as a chemoattractant for monocytes. 9 CRP and both adhesion molecules were reported as significantly increased in pulmonary arteries of newborn pups with CDH. 9,69 Another trigger for expression of these adhesion molecules by ECs is tumor necrosis factor-α (TNFα), which was increased in the lungs of nitrofen-exposed pups with CDH.…”

“…In pulmonary hypoplasia secondary to CDH, the impaired homeostasis of the respiratory epithelium results in decreased signaling to ECs. 9,12 Vascular endothelial growth factor (VEGF) is a signal protein released by the epithelial cells to regulate differentiation of mesenchymal cells into ECs, EC proliferation, and vascular development. 13 ECs express two receptors for VEGF: VEGFR-2 that is essential for angiogenesis in early stages and has a proliferative role in ECs, and VEGFR-1 that is a negative regulator of EC division in later embryonic stages, and allows for EC maturation, tube formation, and integrity of the vessel wall.…”

“…8 In the nitrofen model, fetuses with CDH have decreased distal vessel density, increased muscularization of small arteries, resulting in increased pulmonary vascular wall thickness. 7,9 In fact, both medial and adventitial layers of the vascular walls are thickened in the experimental and human CDH. 10 Over the years, the nitrofen model has been extensively employed, but to the best of our knowledge, no study has synthesized the accumulated data on the pathogenesis of PHTN in this experimental model of CDH.…”

Extracellular vesicles influence the pulmonary arterial extracellular matrix in congenital diaphragmatic hernia

“…The nitrofen-induced CDH rodent model is well-established. 19,20 The animal protocol was approved by the Animal Welfare Committee The anatomic and pulmonary vascular cell/tissue consequences have been described previously. [20][21][22] Litters were born on GD 22, and pups were immediately euthanized via thoracotomy.…”

“…19,20 The animal protocol was approved by the Animal Welfare Committee The anatomic and pulmonary vascular cell/tissue consequences have been described previously. [20][21][22] Litters were born on GD 22, and pups were immediately euthanized via thoracotomy. Around 70% of pups from nitrofen-exposed dams will be born with CDH, which is iden- Images were captured using a Leica DMIL LED microscope with a digital camera (DMC 2900) then quantitatively analyzed using Image-J.…”