Nozawa Akifumi, Abe Taiki, Niihori Tetsuya, Ozeki Michio, Aoki Yoko, Ohnishi Hidenori
Department of Pediatrics, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan.
Department of Medical Genetics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Sendai 980-8574, Japan.
Hum Mol Genet. 2024 Aug 6;33(16):1420-1428. doi: 10.1093/hmg/ddae080.
Generalized lymphatic anomaly (GLA) and kaposiform lymphangiomatosis (KLA) are rare congenital disorders that arise through anomalous embryogenesis of the lymphatic system. A somatic activating NRAS p.Q61R variant has been recently detected in GLA and KLA tissues, suggesting that the NRAS p.Q61R variant plays an important role in the development of these diseases. To address this role, we studied the effect of the NRAS p.Q61R variant in lymphatic endothelial cells (LECs) on the structure of the lymphatics during embryonic and postnatal lymphangiogenesis applying inducible, LEC-specific NRAS p.Q61R variant in mice. Lox-stop-Lox NrasQ61R mice were crossed with Prox1-CreERT2 mice expressing tamoxifen-inducible Cre recombinase specifically in LECs. Whole-mount immunostaining of embryonic back skin using an antibody against the LEC surface marker VEGFR3 showed considerably greater lymphatic vessel width in LEC-specific NRAS p.Q61R mutant embryos than in littermate controls. These mutant embryos also showed a significant reduction in the number of lymphatic vessel branches. Furthermore, immunofluorescence staining of whole-mount embryonic back skin using an antibody against the LEC-specific nuclear marker Prox1 showed a large increase in the number of LECs in LEC-specific NRAS p.Q61R mutants. In contrast, postnatal induction of the NRAS p.Q61R variant in LECs did not cause abnormal lymphatic vessel morphogenesis. These results suggest that the NRAS p.Q61R variant in LECs plays a role in development of lymphatic anomalies. While this model does not directly reflect the human pathology of GLA and KLA, there are overlapping features, suggesting that further study of this model may help in studying GLA and KLA mechanisms.
泛发性淋巴管异常（GLA）和卡波西样淋巴管瘤病（KLA）是罕见的先天性疾病，由淋巴系统异常胚胎发育引起。最近在GLA和KLA组织中检测到一种体细胞激活型NRAS p.Q61R变异，提示NRAS p.Q61R变异在这些疾病的发生发展中起重要作用。为了阐明这一作用，我们通过在小鼠中应用可诱导的、淋巴管内皮细胞（LEC）特异性的NRAS p.Q61R变异，研究了胚胎期和出生后淋巴管生成过程中LEC中的NRAS p.Q61R变异对淋巴管结构的影响。Lox-stop-Lox NrasQ61R小鼠与在LEC中特异性表达他莫昔芬诱导型Cre重组酶的Prox1-CreERT2小鼠杂交。使用针对LEC表面标志物VEGFR3的抗体对胚胎背部皮肤进行全组织免疫染色显示，LEC特异性NRAS p.Q61R突变胚胎中的淋巴管宽度比同窝对照明显更宽。这些突变胚胎的淋巴管分支数量也显著减少。此外，使用针对LEC特异性核标志物Prox1的抗体对胚胎背部皮肤全组织进行免疫荧光染色显示，LEC特异性NRAS p.Q61R突变体中的LEC数量大幅增加。相比之下，出生后在LEC中诱导NRAS p.Q61R变异并未导致淋巴管形态发生异常。这些结果表明，LEC中的NRAS p.Q61R变异在淋巴管异常的发生发展中起作用。虽然该模型不能直接反映GLA和KLA的人类病理学情况，但存在一些重叠特征，提示对该模型的进一步研究可能有助于研究GLA和KLA的发病机制。
