人源调节性巨噬细胞（Mreg）来源的大型细胞外囊泡（L-EV）的特征：在创伤愈合和血管生成中的新型介质？
Characterization of large extracellular vesicles (L-EV) derived from human regulatory macrophages (Mreg): novel mediators in wound healing and angiogenesis?
机构信息
Department of Anesthesiology and Intensive Care Medicine, University Hospital of Schleswig-Holstein, Schwanenweg 21, 24105, Kiel, Germany.
Clinic of Cardiovascular Surgery, University Hospital of Schleswig-Holstein, Kiel, Germany.
出版信息
J Transl Med. 2023 Jan 30;21(1):61. doi: 10.1186/s12967-023-03900-6.
BACKGROUND
Large extracellular vesicles (L-EV) with a diameter between 1 and 10 µm are released by various cell types. L-EV contain and transport active molecules which are crucially involved in cell to cell communication. We have shown that secretory products of human regulatory macrophages (Mreg) bear pro-angiogenic potential in-vitro and our recent findings show that Mreg cultures also contain numerous large vesicular structures similar to L-EV with so far unknown characteristics and function.
AIM OF THIS STUDY
To characterize the nature of Mreg-derived L-EV (L-EV) and to gain insights into their role in wound healing and angiogenesis.
METHODS
Mreg were differentiated using blood monocytes from healthy donors (N = 9) and L-EV were isolated from culture supernatants by differential centrifugation. Characterization of L-EV was performed by cell/vesicle analysis, brightfield/transmission electron microscopy (TEM), flow cytometry and proteome profiling arrays. The impact of L-EV on wound healing and angiogenesis was evaluated by means of scratch and in-vitro tube formation assays.
RESULTS
Mreg and L-EV show an average diameter of 13.73 ± 1.33 µm (volume: 1.45 ± 0.44 pl) and 7.47 ± 0.75 µm (volume: 0.22 ± 0.06 pl) respectively. Flow cytometry analyses revealed similarities between Mreg and L-EV regarding their surface marker composition. However, compared to Mreg fewer L-EV were positive for CD31 (P < 0.01), CD206 (P < 0.05), CD103 (P < 0.01) and CD45 (P < 0.05). Proteome profiling suggested that L-EV contain abundant amounts of pro-angiogenic proteins (i.e. interleukin-8, platelet factor 4 and serpin E1). From a functional point of view L-EV positively influenced in-vitro wound healing (P < 0.05) and several pro-angiogenic parameters in tube formation assays (all segment associated parameters, P < 0.05; number of meshes, P < 0.05).
CONCLUSION
L-EV with regenerative and pro-angiogenic potential can be reproducibly isolated from in-vitro cultured human regulatory macrophages. We propose that L-EV could represent a putative therapeutic option for the treatment of chronic wounds and ischemia-associated diseases.
背景
直径在 1 到 10 微米之间的大型细胞外囊泡（L-EV）由各种细胞类型释放。L-EV 包含并运输活性分子，这些分子在细胞间通讯中起着至关重要的作用。我们已经证明，人类调节性巨噬细胞（Mreg）的分泌产物在体外具有促血管生成潜力，我们最近的研究结果表明，Mreg 培养物中还含有许多类似于 L-EV 的大型囊泡结构，其特征和功能尚不清楚。
本研究的目的
表征 Mreg 衍生的 L-EV（L-EV）的性质，并深入了解它们在伤口愈合和血管生成中的作用。
方法
使用来自健康供体的血液单核细胞分化 Mreg（N=9），并通过差速离心从培养上清液中分离 L-EV。通过细胞/囊泡分析、明场/透射电子显微镜（TEM）、流式细胞术和蛋白质组谱分析对 L-EV 进行表征。通过划痕和体外管形成测定评估 L-EV 对伤口愈合和血管生成的影响。
结果
Mreg 和 L-EV 的平均直径分别为 13.73±1.33µm（体积：1.45±0.44pl）和 7.47±0.75µm（体积：0.22±0.06pl）。流式细胞术分析显示，Mreg 和 L-EV 在表面标志物组成方面具有相似性。然而，与 Mreg 相比，L-EV 中 CD31 的阳性率较低（P<0.01），CD206（P<0.05），CD103（P<0.01）和 CD45（P<0.05）。蛋白质组谱分析表明，L-EV 富含促血管生成蛋白（即白细胞介素 8、血小板因子 4 和丝氨酸蛋白酶 E1）。从功能角度来看，L-EV 可促进体外伤口愈合（P<0.05）和管形成测定中的几种促血管生成参数（所有节段相关参数，P<0.05；网格数，P<0.05）。
结论
具有再生和促血管生成潜力的 L-EV 可从体外培养的人类调节性巨噬细胞中重复分离。我们提出，L-EV 可能是治疗慢性伤口和缺血相关疾病的潜在治疗选择。
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