Zhang Hui, Wang Daren, Li Min, Plecitá-Hlavatá Lydie, D'Alessandro Angelo, Tauber Jan, Riddle Suzette, Kumar Sushil, Flockton Amanda, McKeon B Alexandre, Frid Maria G, Reisz Julie A, Caruso Paola, El Kasmi Karim C, Ježek Petr, Morrell Nicholas W, Hu Cheng-Jun, Stenmark Kurt R
Cardiovascular Pulmonary Research Laboratories, Departments of Pediatrics and Medicine (H.Z., D.W., M.L., S.R., S.K., A.F., B.A.M., M.G.F., K.R.S.).
University of Colorado Anschutz Medical Campus, Aurora. Department of Mitochondrial Physiology, Institute of Physiology, Czech Academy of Sciences, Prague (L.P.-H., J.T., P.J.).
Circulation. 2017 Dec 19;136(25):2468-2485. doi: 10.1161/CIRCULATIONAHA.117.028069. Epub 2017 Sep 26.
An emerging metabolic theory of pulmonary hypertension (PH) suggests that cellular and mitochondrial metabolic dysfunction underlies the pathology of this disease. We and others have previously demonstrated the existence of hyperproliferative, apoptosis-resistant, proinflammatory adventitial fibroblasts from human and bovine hypertensive pulmonary arterial walls (PH-Fibs) that exhibit constitutive reprogramming of glycolytic and mitochondrial metabolism, accompanied by an increased ratio of glucose catabolism through glycolysis versus the tricarboxylic acid cycle. However, the mechanisms responsible for these metabolic alterations in PH-Fibs remain unknown. We hypothesized that in PH-Fibs microRNA-124 (miR-124) regulates PTBP1 (polypyrimidine tract binding protein 1) expression to control alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2, resulting in an increased PKM2/PKM1 ratio, which promotes glycolysis and proliferation even in aerobic environments.
Pulmonary adventitial fibroblasts were isolated from calves and humans with severe PH (PH-Fibs) and from normal subjects. PTBP1 gene knockdown was achieved via PTBP1-siRNA; restoration of miR-124 was performed with miR-124 mimic. TEPP-46 and shikonin were used to manipulate PKM2 glycolytic function. Histone deacetylase inhibitors were used to treat cells. Metabolic products were determined by mass spectrometry-based metabolomics analyses, and mitochondrial function was analyzed by confocal microscopy and spectrofluorometry.
We detected an increased PKM2/PKM1 ratio in PH-Fibs compared with normal subjects. PKM2 inhibition reversed the glycolytic status of PH-Fibs, decreased their cell proliferation, and attenuated macrophage interleukin-1β expression. Furthermore, normalizing the PKM2/PKM1 ratio in PH-Fibs by miR-124 overexpression or PTBP1 knockdown reversed the glycolytic phenotype (decreased the production of glycolytic intermediates and byproducts, ie, lactate), rescued mitochondrial reprogramming, and decreased cell proliferation. Pharmacological manipulation of PKM2 activity with TEPP-46 and shikonin or treatment with histone deacetylase inhibitors produced similar results.
In PH, miR-124, through the alternative splicing factor PTBP1, regulates the PKM2/PKM1 ratio, the overall metabolic, proliferative, and inflammatory state of cells. This PH phenotype can be rescued with interventions at various levels of the metabolic cascade. These findings suggest a more integrated view of vascular cell metabolism, which may open unique therapeutic prospects in targeting the dynamic glycolytic and mitochondrial interactions and between mesenchymal inflammatory cells in PH.
一种新出现的肺动脉高压（PH）代谢理论认为，细胞和线粒体代谢功能障碍是该疾病病理生理的基础。我们和其他研究人员先前已证明，从人和牛的高血压肺动脉壁中分离出的增殖性增强、抗凋亡、促炎性外膜成纤维细胞（PH-Fibs）存在糖酵解和线粒体代谢的组成性重编程，同时糖酵解途径与三羧酸循环相比，葡萄糖分解代谢的比例增加。然而，PH-Fibs中这些代谢改变的机制仍不清楚。我们推测，在PH-Fibs中，微小RNA-124（miR-124）调节多嘧啶序列结合蛋白1（PTBP1）的表达，以控制丙酮酸激酶M1和M2（PKM）亚型的可变剪接，导致PKM2/PKM1比例增加，即使在有氧环境中也能促进糖酵解和细胞增殖。
从患有严重PH的小牛和人类（PH-Fibs）以及正常受试者中分离出肺外膜成纤维细胞。通过PTBP1小干扰RNA（PTBP1-siRNA）实现PTBP1基因敲低；用miR-124模拟物恢复miR-124。使用TEPP-46和紫草素调节PKM2的糖酵解功能。使用组蛋白去乙酰化酶抑制剂处理细胞。通过基于质谱的代谢组学分析测定代谢产物，并通过共聚焦显微镜和荧光分光光度法分析线粒体功能。
与正常受试者相比，我们检测到PH-Fibs中的PKM2/PKM1比例增加。PKM2抑制可逆转PH-Fibs的糖酵解状态，降低其细胞增殖，并减弱巨噬细胞白细胞介素-1β的表达。此外，通过miR-124过表达或PTBP1敲低使PH-Fibs中的PKM2/PKM1比例正常化，可逆转糖酵解表型（减少糖酵解中间体和副产物即乳酸的产生），挽救线粒体重编程，并降低细胞增殖。用TEPP-46和紫草素对PKM2活性进行药理学调节或用组蛋白去乙酰化酶抑制剂处理产生了类似的结果。
在PH中，miR-124通过可变剪接因子PTBP1调节PKM2/PKM1比例、细胞的整体代谢、增殖和炎症状态。通过对代谢级联反应不同水平的干预可挽救这种PH表型。这些发现提示了对血管细胞代谢的更全面的认识，这可能为靶向PH中动态的糖酵解与线粒体相互作用以及间充质炎症细胞之间的相互作用开辟独特的治疗前景。
