This study aimed to investigate the effect of calcitonin gene-related peptide (CGRP) on the temporal alteration of macrophage phenotypes and macrophage-regulated angiogenesis during early bone healing and preliminarily elucidate the mechanism.

In vivo, the rat mandibular defect models were established with inferior alveolar nerve transection (IANT) or CGRP receptor antagonist injection. Radiographic and histologic assessments for osteogenesis, angiogenesis, and macrophage phenotypic alteration within bone defects were performed. In vitro, the effect and mechanism of CGRP on macrophage polarization and phenotypic alteration were analyzed. Then the conditioned medium (CM) from CGRP-treated M1 or M2 macrophages was used to culture human umbilical vein endothelial cells (HUVECs), and the CGRP’s effect on macrophage-regulated angiogenesis was detected.

Comparable changes following IANT and CGRP blockade within bone defects were observed, including the suppression of early osteogenesis and angiogenesis, the prolonged M1 macrophage infiltration and the prohibited transition toward M2 macrophages around vascular endothelium. In vitro experiments showed that CGRP promoted M2 macrophage polarization while upregulating the expression of interleukin 6 (IL-6), a major cytokine that facilitates the transition from M1 to M2-dominant stage, in M1 macrophages via the activation of Yes-associated protein 1. Moreover, CGRP-treated macrophage-CM showed an anabolic effect on HUVECs angiogenesis compared with macrophage-CM and might prevail over the direct effect of CGRP on HUVECs.

Collectively, our results reveal the effect of CGRP on M1 to M2 macrophage phenotypic alteration possibly via upregulating IL-6 in M1 macrophages, and demonstrate the macrophage-regulated pro-angiogenic potential of CGRP in early bone healing.