Notch3 signaling activation in smooth muscle cells promotes extrauterine growth restriction-induced pulmonary hypertension

https://doi.org/10.1016/j.numecd.2019.03.004Get rights and content

Highlights

  • Extrauterine growth restriction male rats developed increased pulmonary artery pressure and pulmonary arteriolar remodeling.

  • Elevated cell proliferation and migration of pulmonary artery smooth muscle cells in EUGR rats were found for the first time.

  • Notch3 and its downstream effector Hey1 were upregulated in EUGR rat models.

  • Pharmacological and genetic ablation of Notch3 attenuated PAH features in vitro and in vivo.

Abstract

Background and aims

Early postnatal life is a critical developmental period that affects health of the whole life. Extrauterine growth restriction (EUGR) causes cardiovascular development problems and diseases, including pulmonary arterial hypertension (PAH). PAH is characterized by proliferation, migration, and anti-apoptosis of pulmonary artery smooth muscle cells (PASMCs). However, the role of PASMCs in EUGR has not been studied. Thus, we hypothesized that PASMCs dysfunction played a role in EUGR-induced pulmonary hypertension.

Methods and results

Here we identified that postnatal nutritional restriction-induced EUGR rats exhibited an elevated mean pulmonary arterial pressure and vascular remodeling at 12 weeks old. PASMCs of EUGR rats showed increased cell proliferation and migration features. In EUGR-induced PAH rats, Notch3 signaling was activated. Relative mRNA and protein expression levels of Notch3 intracellular domain (Notch3 ICD), and Notch target gene Hey1 in PASMCs were upregulated. We further demonstrated that pharmacological inhibition of Notch3 activity by using a γ-secretase inhibitor DAPT, which blocked the cleavage of Notch proteins to ICD peptides, could effectively inhibit PASMC proliferation. Specifically knocked down of Notch3 in rat PASMCs by shRNA restored the abnormal PASMC phenotype in vitro. We found that administration of Notch signaling inhibitor DAPT could successfully reduce mean pulmonary arterial pressure in EUGR rats.

Conclusions

The present study demonstrated that upregulation of Notch3 signaling in PASMCs was crucial for the development of EUGR-induced PAH. Blocking Notch3-Hey1 signaling pathway in PASMCs provides a potential therapeutic target for PAH.

Introduction

Early postnatal life is a critical developmental period for determining organ function and long-term metabolic status [1]. Extrauterine growth restriction (EUGR) remains a serious problem in very low birth weight infants and is a manifestation of severe malnutrition in the first few weeks of life [2]. EUGR is associated with a higher risk of long-term cardiovascular diseases including pulmonary hypertension [3], [4], [5], [6], [7]. Dysfunction of pulmonary vascular endothelial cells has been identified in EUGR-induced pulmonary hypertension in adulthood [8], [9]. However, the role and mechanism of pulmonary artery smooth muscle cells (PASMCs) of EUGR and pulmonary hypertension are still unclear.

Pulmonary arterial hypertension (PAH) is a severe and progressive disease. The remodeling of pulmonary arterioles results in increased pulmonary vascular resistance, elevated mean pulmonary artery pressure, right ventricular failure or death [10]. It is pathologically characterized by proliferation, migration, anti-apoptosis, or phenotype switching of pulmonary arterial endothelial cells (PAEC), PASMCs, and fibroblasts [11], [12], [13]. Multiple etiologies account for PAH, but the mechanism of pulmonary hypertension due to developmental lung diseases remains unclear [14]. Thus, we explore the mechanism of PAH from the perspective of EUGR-induced pulmonary arterial hypertension, and this might provide a new potential solution for PAH.

Increasing evidence suggests that Notch3 is activated in pulmonary hypertension (PH) [15], [16]. Notch3 is the major Notch receptor in vascular smooth muscle cells, and is critical in determining the lineage fate of the of vascular smooth muscle cells (SMC) in late embryonic development [17]. Notch receptors (Notch1–4) are single-pass transmembrane proteins that receive signals from ligands encoded by the Jag (Jag1, Jag2) and Delta-like (Dll1, Dll3, and Dll4) gene families [18]. After ligand binding, Notch receptors undergo several proteolytic events mediated by a succession of proteases (including γ-secretases), result in releasing of the intracellular domains (ICD) of these receptors [19]. The Notch ICD translocates to the nucleus to form an active transcriptional complex with transcription factor to activate the transcription of effector genes. The key downstream genes of Notch signaling are the Hes/Hey families, which have been shown to function as downstream of notch receptors in many organs [20]. Blockade of the Notch pathway using the γ-secretase inhibitor N-[N-(3,5-diflurophenacetyl)-L-alanyl]-S-phenylglycinetbutyl ester (DAPT) which blocked the cleavage of Notch proteins to ICD peptides could be a valuable approach for the therapy of PAH [6].

Since the function of PASMCs in EUGR has not been studied, we hypothesize that EUGR induced pulmonary hypertension is related to dysfunction of PASMCs, possibly through Notch pathway. Here we show that Notch3 is implicated in the development of PAH in vitro and in vivo EUGR models. Inhibition or knockdown of Notch3 leads to reversing of the hyperproliferative phenotype of PASMCs in PAH. Thus, our findings identify that Notch3 is centrally involved in EUGR-induced PAH, and provide new insights into its role in pulmonary hypertension.

Section snippets

Extrauterine growth restriction rat model

This study was carried out in accordance with the recommendations of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health. The procedures and protocols were approved by the Animal Care and Use Committee of Zhejiang University (No. ZJU20160215). All surgery was carried out under anesthesia, and all efforts were aimed at reducing pain. Sprague–Dawley rats obtained from Zhejiang Chinese Medical University Laboratory Animal

Extrauterine growth restriction male rats developed increased pulmonary artery pressure and pulmonary arteriolar remodeling

EUGR rat model was set up as Fig. 1a. On the first day, there was no significant difference of body weight between EUGR and control group. Then after the group was assigned, the weight of EUGR group was much lower than that of control group (Fig. 1b), which showed that the model of EUGR had been successfully constructed. To explore whether EUGR induces pulmonary vascular dysfunction, we assessed hemodynamics and pulmonary vascular remodeling of EUGR rats. Compared with control, EUGR rats

Discussion

Our goal has been to understand the molecular basis by which EUGR causes smooth muscle hyperplasia in pulmonary vasculature leading to the occluding of the distal pulmonary arterial tree and the development of PAH. The present study provides strong evidence that upregulation of Notch3 signaling in the medial layer of small pulmonary arteries is centrally involved in the adult-onset EUGR-induced PAH. This concept is based on the findings that (i) EUGR caused development of an increased mPAP in

Author contributions

Y.W., S.X.D, L.L.Y, J.H., Q.H., X.Y.C. and Y.L. acquired the data. Y.W., S.X.D, J.H., Y.L. and L.Z.D. analyzed and interpreted the data. Y.W., S.X.D and L.Z.D. conceived and designed research. Y.W., E.P. and L.Z.D. drafted and revised the manuscript.

Conflicts of interest

The authors declare no competing interests.

Acknowledgments

This work was supported by Zhejiang Key Laboratory for Diagnosis and Therapy of Neonatal Diseases, Key Laboratory of Reproductive Genetics (Zhejiang University), Ministry of Education, and by grants from the National Natural Science Foundation of China (No. 81471480, 81630037 and 81501293).

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