Human pulmonary microvascular endothelial cell DDAH1-mediated nitric oxide production promotes pulmonary smooth muscle cell apoptosis in co-culture.
Human pulmonary microvascular endothelial cell DDAH1-mediated nitric oxide production promotes pulmonary smooth muscle cell apoptosis in co-culture.
复制标题
人肺微血管内皮细胞 DDAH1 介导的一氧化氮产生促进共培养中的肺平滑肌细胞凋亡。
DOI：
10.1152/ajplung.00433.2021
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Trittmann,JenniferK
中科院分区：
文献类型：
--
作者：
Almazroue,Hanadi;Jin,Yi;Nelin,LeifD;Barba2nd,JohnC;Milton,AvanteD;Trittmann,JenniferK
支气管肺发育不良（BPD）是早产儿最常见的慢性肺部疾病，25%-40%的BPD患者发生肺动脉高压（PH），增加了发病率和死亡率。BPD-PH以血管收缩和血管重构为特征。一氧化氮（NO）是一氧化氮合酶（eNOS）在肺内皮中产生的肺血管扩张剂和凋亡介质。不对称二甲基精氨酸（ADMA）是一种内源性eNOS抑制剂，主要由二甲基精氨酸二甲氨基水解酶-1（DDAH 1）代谢。我们的假设是，DDAH 1敲低人肺微血管内皮细胞（hPMVEC）将导致较低的NO产生，减少凋亡，和人肺动脉平滑肌细胞（hPASMC）的增殖，而DDAH 1过表达将有相反的效果。用靶向DDAH 1的小干扰RNA（siDDAH 1）/乱序或含有DDAH 1的腺病毒载体（AdDDAH 1）/AdGFP转染hPMVEC 24 h，并与hPASMC共培养24 h。分析包括裂解的和总的半胱天冬酶-3、半胱天冬酶-8、半胱天冬酶-9、β-肌动蛋白的蛋白质印迹;活细胞数的台盼蓝排除;末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）;和BrdU掺入。靶向DDAH 1的小干扰RNA（siDDAH 1）转染到hPMVEC中导致较低的培养基亚硝酸盐，裂解的caspase-3和caspase-8蛋白表达，和TUNEL染色;和更大的活细胞数和BrdU掺入共培养的hPASMC中。腺病毒介导的DDAH 1基因（AdDDAH 1）转染hPMVEC后，共培养的hPASMC中caspase-3和caspase-8蛋白表达增加，活细胞数减少。当用血红蛋白处理培养基以隔离NO时，观察到AdDDAH 1-hPMVEC转染后hPASMC活细胞数的部分恢复。总之，hPMVEC-DDAH 1介导的NO产生正调节hPASMC凋亡，这可能预防/减弱BPD-PH中异常的肺血管增殖/重塑。NO是由eNOS在肺内皮中产生的凋亡介质。ADMA是一种内源性eNOS抑制剂，由DDAH 1代谢。EC-DDAH 1过表达导致更大的切割caspase-3和caspase-8蛋白表达和更低的活细胞数在共培养的SMC。NO封存后，尽管EC-DDAH 1过表达，SMC活细胞数量仍部分恢复。EC-DDAH 1介导的NO产生正调节SMC凋亡，这可能预防/减轻BPD-PH中异常的肺血管增殖/重塑。
Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease in preterm infants, and pulmonary hypertension (PH) develops in 25%–40% of patients with BPD, increasing morbidity and mortality. BPD-PH is characterized by vasoconstriction and vascular remodeling. Nitric oxide (NO) is a pulmonary vasodilator and apoptotic mediator made in the pulmonary endothelium by NO synthase (eNOS). Asymmetric dimethylarginine (ADMA) is an endogenous eNOS inhibitor, primarily metabolized by dimethylarginine dimethylaminohydrolase-1 (DDAH1). Our hypothesis is that DDAH1 knockdown in human pulmonary microvascular endothelial cells (hPMVEC) will result in lower NO production, decreased apoptosis, and greater proliferation of human pulmonary arterial smooth muscle cells (hPASMC), whereas DDAH1 overexpression will have the opposite effect. hPMVECs were transfected with small interfering RNA targeting DDAH1 (siDDAH1)/scramble or adenoviral vector containing DDAH1 (AdDDAH1)/AdGFP for 24 h and co-cultured for 24 h with hPASMC. Analyses included Western blot for cleaved and total caspase-3, caspase-8, caspase-9, β-actin; trypan blue exclusion for viable cell numbers; terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL); and BrdU incorporation. Small interfering RNA targeting DDAH1 (siDDAH1) transfected into hPMVEC resulted in lower media nitrites, cleaved caspase-3 and caspase-8 protein expression, and TUNEL staining; and greater viable cell numbers and BrdU incorporation in co-cultured hPASMC. Adenoviral-mediated transfection of theDDAH1gene (AdDDAH1) into hPMVEC resulted in greater cleaved caspase-3 and caspase-8 protein expression and lower viable cell numbers in co-cultured hPASMC. Partial recovery of hPASMC viable cell numbers after AdDDAH1-hPMVEC transfection was observed when media were treated with hemoglobin to sequester NO. In conclusion, hPMVEC-DDAH1-mediated NO production positively regulates hPASMC apoptosis, which may prevent/attenuate aberrant pulmonary vascular proliferation/remodeling in BPD-PH.NEW & NOTEWORTHYBPD-PH is characterized by vascular remodeling. NO is an apoptotic mediator made in the pulmonary endothelium by eNOS. ADMA is an endogenous eNOS inhibitor metabolized by DDAH1. EC-DDAH1 overexpression resulted in greater cleaved caspase-3 and caspase-8 protein expression and lower viable cell numbers in co-cultured SMC. After NO sequestration, SMC viable cell numbers partially recovered despite EC-DDAH1 overexpression. EC-DDAH1-mediated NO production positively regulates SMC apoptosis, which may prevent/attenuate aberrant pulmonary vascular proliferation/remodeling in BPD-PH.