Bone-marrow-derived nerve growth factor receptor-positive dendritic cells contribute to the suppression of arterial remodelling via apoptosis
Authors: Takashima S , Usui S , Matsuura S , Goten C , Inoue O , Takeda Y , Yamaguchi K , Hashimuko D , Ohtani K , Kubota K , Sakata K , Kaneko S , Takamura M 
Published: November 2023

Abstract

Background
Bone marrow (BM) contributes to the pathogenesis of arteriosclerosis. Nerve growth factor receptor (NGFR) is expressed in BM stroma and is present in peripheral blood and the ischemic coronary artery. NGFR transduces signals towards cell survival or apoptosis in different cell types according to the coupling co-receptors. The previous clinical study demonstrated that low gene expression of NGFR in peripheral leucocytes in patients with acute coronary syndrome predicted 5-year repetitive coronary interventions at a de novo lesion.

Purpose
To investigate the hypothesis that BM-derived NGFR-positive (NGFR+) cells are associated with arterial remodelling.

Methods
Adult C57BL/6J male NGFR-wild-type (WT) or -knockout (KO) mice were subjected to unilateral carotid artery ligation after BM transplantation (BMT) from green fluorescent protein-positive (GFP+) NGFR-WT or -KO mice (N = 4-6). The ligated and the opposite-sided, sham-operated arteries were assessed by immunohistology and gene expression analysis using a PCR on day 28. Also, human peripheral blood NGFR+ mononuclear cells (MNCs) from a healthy volunteer were sorted using FACS Aria II and co-cultured with smooth muscle cells (SMCs) to examine early apoptosis (AnnexinV+7AAD-) in response to the NGFR ligands; proNGF and NGF. Also, human NGFR+MNCs from three healthy volunteers underwent RNA sequencing analysis and were immunophenotypically characterised by flow cytometry.

Results
In WT mice, NGFR+ cells accumulated in the neointima after ligation. The neointimal area was significantly larger in the mice with BM-specific depletion of NGFR than that in WT mice. After BMT from GFP+NGFR-WT mice, NGFR+GFP+ cells accumulated in the neointima, exhibited apoptosis (TUNEL+ and cleaved caspase-3+), and promoted F4/80+GFP+ macrophage accumulation. By contrast, in the BM-specific NGFR-KO model, medial SMCs (αSMA+GFP-) occupied the neointima with augmented proliferation, and GFP+ cells were rare. Notably, apoptotic NGFR+ cells were accompanied by the accumulation of macrophages (F4/80+NGFR-) and expressed IL-10, which were abolished by BM-specific depletion of NGFR. Furthermore, human NGFR+, but not NGFR- cells, co-cultured with SMCs were susceptible to apoptosis in response to proNGF. Additionally, PDGF stimulated NGF expression in SMCs in vitro, and proNGF was expressed in a ligated artery, implying that local proNGF might cause NGFR+ cell apoptosis. Meanwhile, RNA sequencing and flow cytometric analysis demonstrated that human NGFR+MNCs resemble dendritic cells (DCs) and predominantly express the plasmacytoid DCs markers. Consistently, NGFR+ cells in the neointima were positive for HLA-DR and CD11c.

Conclusions
BM-derived NGFR+ cells expressed dendritic cell markers and contributed to the suppression of arterial remodelling mediated by apoptosis. Apoptotic NGFR+ cells promoted macrophage chemotaxis and coordinately inhibited medial SMC migration to, and proliferation in, the neointima.