De Biasi Sara, Cerri Stefania, Bianchini Elena, Gibellini Lara, Persiani Elisa, Montanari Gloria, Luppi Fabrizio, Carbonelli Cristiano Matteo, Zucchi Luigi, Bocchino Marialuisa, Zamparelli Alessandro Sanduzzi, Vancheri Carlo, Sgalla Giacomo, Richeldi Luca, Cossarizza Andrea
Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia School of Medicine, via Campi, 287-41125, Modena, Italy.
Department of Medical and Surgical Sciences for Children and Adults, University of Modena and Reggio Emilia, Modena, Italy.
BMC Med. 2015 Nov 9;13:277. doi: 10.1186/s12916-015-0515-0.
It has been suggested that circulating fibrocytes and endothelial cells actively participate in the intense remodelling of the pulmonary vasculature in patients with idiopathic pulmonary fibrosis (IPF). Indeed, fibrotic areas exist that have fewer blood vessels, whereas adjacent non-fibrotic tissue is highly vascularized. The number of circulating endothelial cells (CEC) and endothelial progenitor cells (EPC) might reflect the balance between vascular injury and repair. Thus, fibrocytes as well as endothelial cells could potentially be used as biomarkers of disease progression and treatment outcome.
Peripheral blood samples were collected from 67 patients with a multidisciplinary diagnosis of IPF and from 45 age-matched and sex-matched healthy volunteers. Buffy coat was isolated according to standard procedures and at least 20 million cells were stained with different monoclonal antibodies for the detection of CEC, EPC and circulating fibrocytes. For the detection of CEC and EPC, cells were stained with anti-CD45, anti-CD34, anti-CD133, anti-CD14, anti-CD309 and with the viability probe Far-Red LIVE/DEAD. For the detection of circulating fibrocytes, cells were first stained with LIVE/DEAD and the following monoclonal antibodies: anti-CD3, anti-CD19, anti-CD45, anti-CD34 and anti-CD14, then cells were fixed, permeabilized and stained with fluorochrome-conjugated anti-collagen I monoclonal antibodies.
Patients with IPF displayed almost undetectable levels of circulating fibrocytes, low levels of CEC, and normal levels of EPC. Patients treated with nintedanib displayed higher levels of CEC, but lower levels of endothelial cells expressing CD309 (the type II receptor for vascular endothelial growth factor). Treatment with both nintedanib and pirfenidone reduced the percentage of CEC and circulating fibrocytes.
Levels of CEC were reduced in patients with IPF as compared to healthy individuals. The anti-fibrotic treatments nintedanib and pirfenidone further reduced CEC levels. These findings might help explain the mechanism of action of these drugs and should be explored as predictive biomarkers in IPF.
有研究表明，循环纤维细胞和内皮细胞积极参与特发性肺纤维化（IPF）患者肺血管的强烈重塑过程。实际上，纤维化区域的血管较少，而相邻的非纤维化组织血管高度丰富。循环内皮细胞（CEC）和内皮祖细胞（EPC）的数量可能反映血管损伤与修复之间的平衡。因此，纤维细胞以及内皮细胞有可能用作疾病进展和治疗结果的生物标志物。
从67例经多学科诊断为IPF的患者以及45例年龄和性别匹配的健康志愿者中采集外周血样本。按照标准程序分离血沉棕黄层，并用不同的单克隆抗体对至少2000万个细胞进行染色，以检测CEC、EPC和循环纤维细胞。为检测CEC和EPC，细胞用抗CD45、抗CD34、抗CD133、抗CD14、抗CD309以及活力探针远红活/死染料进行染色。为检测循环纤维细胞，细胞先用活/死染料以及以下单克隆抗体进行染色：抗CD3、抗CD19、抗CD45、抗CD34和抗CD14，然后固定、通透处理，并用荧光素偶联的抗I型胶原单克隆抗体进行染色。
IPF患者的循环纤维细胞水平几乎检测不到，CEC水平较低，EPC水平正常。接受尼达尼布治疗的患者CEC水平较高，但表达CD309（血管内皮生长因子II型受体）的内皮细胞水平较低。尼达尼布和吡非尼酮联合治疗可降低CEC和循环纤维细胞的百分比。
与健康个体相比，IPF患者的CEC水平降低。抗纤维化治疗药物尼达尼布和吡非尼酮可进一步降低CEC水平。这些发现可能有助于解释这些药物的作用机制，应作为IPF的预测生物标志物进行探索。
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