Department of Cardiology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai, China.
Department of Endocrinology, Xinhua Hospital, Shanghai Jiaotong University, School of Medicine, Shanghai, China.
Theranostics. 2021 Mar 4;11(9):4483-4501. doi: 10.7150/thno.54473. eCollection 2021.
Angiogenesis is a critical step in repair of tissue injury. The pattern recognition receptors (PRRs) recognize pathogen and damage associated molecular patterns (DAMPs) during injury and achieve host defense directly. However, the role of NLR family CARD domain containing 5 (NLRC5), an important member of PPRs, beyond host defense in angiogenesis during tissue repair remains unknown. western blot and real-time PCR (RT-PCR) were used to detect the expression of NLRC5 in endothelial cells (ECs). Immunofluorescence microscopy was used to reveal the subcellular location of NLRC5 in ECs. Cell proliferation, wound healing, tube formation assays of ECs were performed to study the role of NLRC5 in angiogenesis. By using Tie2Cre-NLRC5 mice and bone marrow transplantation studies, we defined an EC-specific role for NLRC5 in angiogenesis. Mechanistically, co-immunoprecipitation studies and RNA sequencing indicated that signal transducer and activator of transcription 3 (STAT3) was the target of NLRC5 in the nucleus. And Co-IP was used to verify the specific domain of NLRC5 binding with STAT3. ChIP assay determined the genes regulated by interaction of STAT3 and NLRC5. Knockdown of NLRC5 or inhibited pathological angiogenesis, but had no effect on physiological angiogenesis. NLRC5 was also identified to bind to STAT3 in the nucleus required the integrated death-domain and nucleotide-binding domain (DD+NACHT domain) of NLRC5. And the interaction of STAT3 and NLRC5 could enhance the transcription of angiopoietin-2 (Ang2) and cyclin D1 (CCND1) to participate in angiogenesis. In the ischemic microenvironment, NLRC5 protein accumulates in the nucleus of ECs and enhances STAT3 transcriptional activity for angiogenesis. These findings establish NLRC5 as a novel modulator of VEGFA signaling, providing a new target for angiogenic therapy to foster tissue regeneration.
血管生成是组织损伤修复的关键步骤。模式识别受体（PRRs）在损伤过程中直接识别病原体和损伤相关分子模式（DAMPs），从而实现宿主防御。然而，NLR 家族 CARD 结构域包含 5（NLRC5）作为 PRR 的重要成员，在组织修复过程中的血管生成中，除了宿主防御之外，其作用尚不清楚。Western blot 和实时 PCR（RT-PCR）用于检测内皮细胞（ECs）中 NLRC5 的表达。免疫荧光显微镜用于揭示 NLRC5 在 ECs 中的亚细胞定位。通过进行 EC 增殖、伤口愈合和管形成测定，研究 NLRC5 在血管生成中的作用。通过使用 Tie2Cre-NLRC5 小鼠和骨髓移植研究，我们定义了 NLRC5 在血管生成中的 EC 特异性作用。从机制上讲，共免疫沉淀研究和 RNA 测序表明，信号转导和转录激活因子 3（STAT3）是 NLRC5 在核内的靶标。并且 Co-IP 用于验证 NLRC5 与 STAT3 结合的特定结构域。ChIP 测定确定了由 STAT3 和 NLRC5 相互作用调节的基因。NLRC5 的敲低或抑制病理性血管生成，但对生理性血管生成没有影响。还确定 NLRC5 与 STAT3 结合在核内需要 NLRC5 的完整死亡域和核苷酸结合域（DD+NACHT 域）。STAT3 和 NLRC5 的相互作用可以增强血管生成素-2（Ang2）和细胞周期蛋白 D1（CCND1）的转录以参与血管生成。在缺血性微环境中，NLRC5 蛋白在 EC 核内积累，并增强 STAT3 的转录活性以促进血管生成。这些发现确立了 NLRC5 作为 VEGFA 信号的新型调节剂，为促进组织再生的血管生成治疗提供了新的靶点。
