Role of Notch Signaling in Pulmonary Hypertension

Pulmonary Hypertension (PH) is a disease condition characterized by excessive pulmonary vascular remodeling leading to increased vascular resistance and ultimately death due to right heart failure. Notch Signaling is an evolutionarily conserved mechanism, functioning during various developmental and physiological processes. Notch signaling molecules are not only instrumental in physiological processes, but also key mediators in pathological conditions, including chronic lung diseases. Hence, we hypothesized that Notch may be involved in pathogenesis of PH.

Expression of Notch family genes was investigated in lung homogenates from 3 different animal models of PH: (i) Monocrotaline (MCT) – induced PH in rats, (ii) Hypoxia (HOX) – induced PH in mice and (iii) Hypoxia + Su5416- induced PH in rats. Expression was also compared in idiopathic pulmonary arterial hypertension (IPAH) patients- and donors- lung homogenates. To analyze the role of Notch receptors in vitro, BrdU proliferation assays were performed using gamma secretase inhibitor (DBZ) on human pulmonary arterial smooth muscle cells (hPASMCs) and human pulmonary arterial endothelial cells (hPAECs). Notch1 over expression and knock down were carried out in hPAECs to study their effect on proliferation and apoptosis.

MCT induced PH rats showed significantly lower expression levels for the Notch receptors (Notch1, 4) and ligands (Jagged1, DLL1, 4), at both mRNA and protein level, compared to controls. Down regulation of DLL4 was observed in HOX mice compared to controls. Importantly, Notch 3 was significantly increased in both the above-mentioned models. Hypoxia+Su5416 rats as well as IPAH samples, exhibited a significant increase in expression of Notch receptors (Notch1, 3) and ligands (DLL1, Jagged 1). DBZ, in vitro, was able to significantly reduce notch signaling and effectively attenuated proliferation of hPASMCs and hPAECs. Over-activation of Notch1 signaling pathway via N1ICD transfection increased proliferation of human PAECs while knock down attenuated proliferation significantly. This effect was associated with the modulation of cell cycle markers. Interestingly, Notch1 activation also showed anti-apoptotic effect on hPAECs which was antagonized by Notch1 knock down.

In conclusion, our results suggest that Notch signaling might play an important role in pulmonary vascular remodeling observed in pulmonary arterial hypertension.