Secretory Protein NBL1 In The Body - Pulmonary Shunt Pulmonary Hypertension In Rats

Part I:An original rat model of highkinetic unilateral pulmonary hypertension surgically induced by combined surgeryBackground:Characteristic morphological lesions observed in lungs of patients with congenital cardiac anomalies have not been closely generated in rat shunt-related models except reversible grade one change. This study presented an original rat model of unilateral pulmonary arterial hypertension (PAH) surgically induced by combined surgery to reproduce more advanced pulmonary vascular lesions.Methods:Right pulmonary artery was ligated through a right posterolateral thoracotomy; subsequently, a cervical shunt was established one week later. Immediate and chronic effects on pulmonary hemodynamics were evaluated through right heart catheterization immediately after and an interval of8,12weeks. Morphological changes in pulmonary vasculature were analyzed after staining with hematoxylin-eosin and modified Weighert’s method. Right ventricular hypertrophy index and artery blood gas analysis were also calculated and performed.Results:Pulmonary hypertensive status was successfully induced immediately after cervical surgery and progressively aggravated into a borderline state with the course advancing. Pulmonary vasculopathy demonstrated a transition from reversibility (muscularization, intimal proliferation-grade one, two) at the8th week to irreversibility (intimal fibrosis, entirely luminal occlusion-grade three) at the12week. Conspicuous right ventricular hypertrophy and descending partial arterial pressure of oxygen were also observed.Conclusions:This shunt-related model successfully simulated a hypertensive status in pulmonary circulation and reproduced the characteristic transition of pulmonary vasculopathy from reversibility to irreversibility within a relatively short period. Thus, this model may offer an alternative with low mortality and high reproducibility for investigations on the underling mechanisms of shunt-related PAH. PartⅡ:Secreted protein NBL1exerts a critical role in pulmonary arterial hypertension associated with systemic-to-pulmonary shuntsBackground:Proteins mainly expressed in normal lungs and characteristically changed in lungs suffering from systemic-to-pulmonary shunts will be useful research targets for pulmonary arterial hypertension in patients with congenital heart diseases(PAH/CHD). Thus, this study aimed to identify secreted proteins that significantly altered during the genesis and progression of PAH/CHD.Methods:An antibody microarrary procedure was performed to detect proteins whose plasma levels specifically changed in patients with congenital intracardiac shunts. Then, significantly changed proteins were identified and the target protein NBL1was determined. Real-time quantitative PCR (RT-PCR), western-blot analysis and immunohistochemistry were performed to further examine the expression changes and location of NBL1in lungs from patients and rats with systemic-to-pulmonary shunts; the potentially biological role of NBL1on HPASMCs and HPAECs proliferation were also explored. The plasma NBL1concentration in a set of120patients with or without PAH was assessed by a commercially available enzyme-linked immunosorbent assay.Results:The antibody microarrary procedure derived several proteins with s tatistically significant differences between patients with or without PAH. Secreted protein NBLl, which appeared as a valuable candidate for molecular markers of PAH, was selected for validation. Quantitative RT-PCR analysis revealed that NBL1was expressed with much higher specificity in normal lung tissues than in other systemic organ tissues, and the mRAN level was downregulated in a time-related modus in lungs suffering from systemic-to-pulmonary shunts; Western blot analysis revealed that NBL1was highly expressed in normal lung tissues and similarly a time-related reduction was also observed in pulmonary hypertensive lungs; Immunohistochemical analysis showed that NBL1was highly detected in normal lung tissues and was significantly down-regulated with the progression of PAH. Furthermore, NBL1could specially reverse the inhibitory effect of BMP2/4on HPASMCs and HPAECs proliferation; Elisa-kit also determined a detectable level of NBL1in the supernatants of culture media of the HPASMCs and HPAECs. Finally, plasma NBL1concentration was significantly downregulated according to the PAH/CHD stage.Conclusion:NBLl is a secreted protein that is highly and mainly expressed in lungs. Downregulation of NBL1correlated with the severity of PAH. NBL1might be a candidate biomarker and a novel therapeutic target for PAH/CHD.