Zhu T, Chiacchia S, Kameny RJ, Garcia De Herreros A, Gong W, Raff GW, Boehme JB, Maltepe E, Lasheras JC, Black SM, Datar SA, Fineman JR.Pulm Circ. 2020 May 14;10(2):2045894020922118. doi: 10.1177/2045894020922118. eCollection 2020 Apr-Jun.PMID: 32489641 Free PMC article.
The risk and progression of pulmonary vascular disease in patients with congenital heart disease is dependent on the hemodynamics associated with different lesions. However, the underlying mechanisms are not understood. Endothelin-1 is a potent vasoconstrictor that plays a key role in the pathology of pulmonary vascular disease. We utilized two ovine models of congenital heart disease: (1) fetal aortopulmonary graft placement (shunt), resulting in increased flow and pressure; and (2) fetal ligation of the left pulmonary artery resulting in increased flow and normal pressure to the right lung, to investigate the hypothesis that high pressure and flow, but not flow alone, upregulates endothelin-1 signaling. Lung tissue and pulmonary arterial endothelial cells were harvested from control, shunt, and the right lung of left pulmonary artery lambs at 3-7 weeks of age. We found that lung preproendothelin-1 mRNA and protein expression were increased in shunt lambs compared to controls. Preproendothelin-1 mRNA expression was modestly increased, and protein was unchanged in left pulmonary artery lambs. These changes resulted in increased lung endothelin-1 levels in shunt lambs, while left pulmonary artery levels were similar to controls. Pulmonary arterial endothelial cells exposed to increased shear stress decreased endothelin-1 levels by five-fold, while cyclic stretch increased levels by 1.5-fold. These data suggest that pressure or an additive effect of pressure and flow, rather than increased flow alone, is the principal driver of increased endothelin signaling in congenital heart disease. Defining the molecular drivers of the pathobiology of pulmonary vascular disease due to differing mechanical forces will allow for a more targeted therapeutic approach.
Fig. 1. Lung preproET-1 mRNA and protein expression were increased in shunt lambs compared to controls (a and c) (p < 0.05). PreproET-1 mRNA expression was unchanged in LPA lambs, and protein was modestly increased (a and c). In both shunt and LPA lambs, ECE-1 mRNA and protein expression were increased compared to controls (b and d) (p < 0.05). In addition, shunt ECE-1 mRNA and protein expression were increased compared to LPA (b and d). Representative images of western blots (c and d) are shown. In all panels, shunt and LPA values are normalized to control. Values are mean ± SD, n = 5 for each group, *vs. control; †vs. LPA p < 0.05. LPA: left pulmonary artery; ET-1: endothelin-1; qPCR: quantitative real-time PCR.
Fig. 2. Peripheral lung tissue levels of ET-1 (ELISA) are increased in shunt lambs, but not LPA lambs (a). n = 5 for each group, * vs. control p < 0.05. Control PAECs exposed to 8 h of shear stress (20 dyn/cm) decreases ET-1 levels; 8 h of cyclic stretch (CS, 20%) increases ET-1 levels (b). n = 5, *vs. static; †vs. LPA p < 0.05. LPA: left pulmonary artery; ET-1: endothelin-1.
Fig. 3. EDN1 transcriptional characterization of control, LPA and shunt PAECs. n = 4 control, n = 3 LPA, n = 2 shunt. Gene expression of the 136 significant DEGs exhibiting expression patterns similar to EDN1 with respect to animal model hemodynamics (q-value <0.05) are represented here in a heatmap. Expression is quantified by log fragments per kilobase million (FPKM) where green indicates relatively higher levels of expression and red represents lower levels. Heat map of PAEC RNA-Seq data confirm clustering of gene expression by model. LPA: left pulmonary artery.
Fig. 4. IPA Pathway Analysis (Qiagen, Inc) was utilized to generate networks of differentially expressed genes (DEG) (FDR <0.05; FE >2) downstream of EDN1 (a and c). DEGs shown in orange are predicted to be activated, while those in blue are predicted to be inhibited. We then utilized IPA knowledge database to highlight terms associated with either angiogenesis or apoptosis as indicated by light blue dashed lines. We searched our PAEC dataset of differentially expressed genes for transcripts that adhere to EDN1’s unique expression pattern (i.e. elevated in shunt, depressed in LPA). These unique 136 DEGs were submitted to the Gene Ontology (GO) database for pathway enrichment analysis which revealed a total of 152 significantly enriched biological processes (FDR <0.05; fold enrichment (FE) >2). Among the most significant pathways associated with PVD physiology, were associated angiogenesis (b) and negative regulation of apoptotic process (d). For each pathway, mean expressions of candidate effector genes are displayed. LPA: left pulmonary artery.
Fig. 5. PAECs angiogenic capacity was assess using Matrigel assay. Over a 72-h period, both shunt PAECs formed tubes of longer length that PAECs isolated from control lambs, while shunt PAECs from LPA animals had a similar tube length to controls. The addition of ET-1 (1 µM/mL) increased tube length in cells harvested from LPA lambs, but did not change tube length in cells harvested from control or shunt lambs. The addition of BQ788 (1 µM/mL, an ETB receptor antagonist) decreased tube length in shunt PAECs but did not change tube length in control or LPA PAECs. n = 5 per group. (a) representative photos; (b) quantification. *p < 0.05 vs. baseline controls; †p < 0.05 vs. baseline LPA; ϕp < 0.05 vs. corresponding baseline cell model. LPA: left pulmonary artery; ET-1: endothelin-1.
Fig. 6. PAECs apoptosis susceptibility was assessed using TUNEL assay following TNF-α treatment to induce apoptosis. Over a 24-h period, both LPA and shunt PAECs had less apoptosis than did PAECs isolated from control lambs, while shunt PAECs had even less apoptosis than did PAECs derived from LPA lambs. The addition of ET-1 (1 µM/mL) decreased apoptosis in cells from each animal model. The addition of BQ788 (1 µM/mL, an ETB receptor antagonist) increased apoptosis in shunt PAECs. n = 5 per group. (a) representative photos; (b) quantification. *p < 0.05 vs. baseline controls; †p < 0.05 vs. baseline LPA; ϕp < 0.05 vs. corresponding baseline cell model. LPA: left pulmonary artery; ET-1: endothelin-1.