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Theranostics 2021; 11(9):4483-4501. doi:10.7150/thno.54473 This issue Cite

Research Paper

The subcellular redistribution of NLRC5 promotes angiogenesis via interacting with STAT3 in endothelial cells

Xu Xu^1*, Yefei Shi^1*, Peipei Luan¹, Wenxin Kou¹, Bo Li¹, Ming Zhai¹, Shuangjie You¹, Qing Yu¹, Jianhui Zhuang¹, Weixia Jian^2✉, Mark W. Feinberg^3✉, Wenhui Peng^1✉

1. Department of Cardiology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai, China.
2. Department of Endocrinology, Xinhua Hospital, Shanghai Jiaotong University, School of Medicine, Shanghai, China.
3. Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
*Co-first authors.

✉ Corresponding authors: Wenhui Peng, Department of Cardiology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, 301 Middle Yanchang Road, Shanghai, 200072, China. E-mail: pwenhuiedu.cn; or Mark W. Feinberg, Division of Cardiovascular Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 02115, USA. Phone: 617-525-4381, E-mail: mfeinbergharvard.edu; or Weixia Jian, Department of Endocrinology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, 1665 Kongjiang Road, Shanghai, 200092, China. E-mail: jianweixiacom.cn. More

Abstract

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.

Methods: In vitro, 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^flox/flox 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.

Results: Knockdown of NLRC5 in vitro or in vivo 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.

Conclusions: 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.

Keywords: angiogenesis, NLRC5, STAT3, signal transduction, endothelial cell

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