Riedmann Kyle J, Meegan Jamie E, Afzal Aqeela, Cervantes-Cruz Yatzil, Obeidalla Sarah, Bogart Avery M, Ware Lorraine B, Shaver Ciara M, Bastarache Julie A
Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee, USA.
Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
Microcirculation. 2025 May;32(4):e70012. doi: 10.1111/micc.70012.
Cell-free hemoglobin (CFH) is released into the circulation during sepsis where it can redox cycle from the ferrous 2+ to ferric 3+ and disrupt endothelial function, but the mechanisms of CF-mediated endothelial dysfunction are unknown. We hypothesized that oxidized CFH induces mitochondrial dysfunction via the mitochondrial permeability transition pore (mPTP) in pulmonary endothelial cells, leading to the release of mitochondrial DNA (mtDNA).
Human lung microvascular endothelial cells were treated with CFH2+/CFH3+. We measured mitochondrial mPTP activation (flow cytometry), network and mass (immunostaining), structure (electron microscopy), mtDNA release (PCR), and oxygen consumption rate (OCR; Seahorse). Plasma from critically ill patients and conditioned cell media were quantified for mtDNA and CFH.
CFH3+ disrupted the mitochondrial network, activated the mPTP (1434 (874-1642) vs. 2302 (1729-2654) mean fluorescent intensity, p = 0.02), increased the spare respiratory capacity (30.61 (29.36-37.78) vs. 7.83 (3.715-10.63) OCR, p = 0.004), and caused the release of mtDNA. CFH was associated with circulating mtDNA (R = 0.1912, p = 0.0077) in plasma from critically ill patients.
CFH3+, not CFH2+, is the primary driver of CFH-induced lung microvascular mitochondrial dysfunction. Activation of the mPTP and the release of mtDNA are a feature of CFH3+ mediated injury.
在脓毒症期间，无细胞血红蛋白（CFH）释放到循环系统中，它可以从亚铁离子（Fe2+）氧化还原循环为铁离子（Fe3+），并破坏内皮功能，但CF介导的内皮功能障碍的机制尚不清楚。我们假设氧化型CFH通过肺内皮细胞中的线粒体通透性转换孔（mPTP）诱导线粒体功能障碍，导致线粒体DNA（mtDNA）释放。
用人肺微血管内皮细胞与CFH2+/CFH3+进行处理。我们测量了线粒体mPTP激活（流式细胞术）、网络和质量（免疫染色）、结构（电子显微镜）、mtDNA释放（PCR）以及耗氧率（OCR；海马分析仪）。对重症患者的血浆和条件培养基中的mtDNA和CFH进行定量。
CFH3+破坏了线粒体网络，激活了mPTP（平均荧光强度为1434（874 - 1642）对2302（1729 - 2654），p = 0.02），增加了备用呼吸能力（OCR为30.61（29.36 - 37.78）对7.83（3.715 - 10.63），p = 0.004），并导致mtDNA释放。CFH与重症患者血浆中的循环mtDNA相关（R = 0.1912，p = 0.0077）。
CFH3+而非CFH2+是CFH诱导的肺微血管线粒体功能障碍的主要驱动因素。mPTP的激活和mtDNA的释放是CFH3+介导损伤的特征。
