Calcitonin gene-related peptide-modulated macrophage phenotypic alteration regulates angiogenesis in early bone healing.

OBJECTIVES: This study aimed to investigate the effect of calcitonin gene-related peptide (CGRP) on the temporal alteration of macrophage phenotypes and macrophage-regulated angiogenesis duringearlybonehealing and preliminarily elucidate the mechanism. METHODS: In vivo, the rat mandibular defect models were established with inferior alveolar nerve transection (IANT) or CGRP receptor antagonist injection. Radiographicandhistologic 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. RESULTS: 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. CONCLUSIONS: 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.

Zoledronic acid affects the process of Porphyromonas gingivalis infecting oral mucosal epithelial barrier: An in-vivo and in-vitro study.

Zoledronic acid (ZA), one of the commonly used bisphosphonates, is mainly used for bone-metabolic diseases. Studies proved that ZA has adverse effects on oral soft tissues. As the first line of innate immunity, the gingival epithelium could be infected by periodontal pathogens, which is a key process of the initiation of periodontal diseases. Yet, how ZA affects the periodontal pathogens infecting the epithelial barrier remains unclear. This study aimed to investigate the influences of ZA on the process of Porphyromonas gingivalis (P. gingivalis) infecting the gingival epithelial barrier via in-vitro and in-vivo experiments. In the in-vitro experiments, under the condition of different concentrations of ZA (0, 1, 10, and 100 µM), P. gingivalis was used to infect human gingival epithelial cells (HGECs). The infections were detected by transmission electron microscope and confocal laser scanning microscope. Besides, the internalization assay was applied to quantify the P. gingivalis, which infected the HGECs, in the different groups. To evaluate the expression levels of pro-inflammatory cytokines, including interleukin (IL)-1ß, IL-6, and IL-8, by infected HGECs, real-time quantitative reverse transcription-polymerase chain reactions were applied. In the in-vivo experiments, rats were given ZA solution (ZA group) or saline (control group) by tail intravenous injection for 8 weeks. Subsequently, we put ligatures around the maxillary second molars of all the rats and inoculated P. gingivalis to the gingiva every other day from day 1 to day 13. The rats were sacrificed on days 3, 7, and 14 for micro-CT and histological analyses. The in-vitro results manifested that the quantity of P. gingivalis that had infected HGECs increased with the ZA concentrations. Pro-inflammatory cytokines expression by HGECs were significantly increased by 100 µM ZA. In the in-vivo study, compared to the control group, more P. gingivalis was detected in the superficial layer of gingival epithelium in the ZA group. Besides, ZA significantly increased the expression level of IL-1ß on day 14 and IL-6 on days 7 and 14 in gingival tissues. These findings suggest that the oral epithelial tissues of patients who receive high-dose ZA treatment may be more susceptible to periodontal infections, resulting in severe inflammatory conditions.

High Expression of Tomm34 and Its Correlations With Clinicopathology in Oral Squamous Cell Carcinoma.

Tomm34, as a member of the outer mitochondrial membrane proteins, is evenly distributed between the cytoplasm and the outer mitochondrial membrane. It is up-regulated in a variety of tumors and correlates with poor prognosis. This study aimed to investigate expression of Tomm34 and its correlations with clinicopathology in oral squamous cell carcinoma (OSCC). Oncomine database and UALCAN database were utilized to predict the expression and prognosis values of Tomm34 in head and neck squamous cell carcinoma (HNSCC). By immunohistochemistry, a retrospective study was performed to verify the bioinformatics results to evaluate the Tomm34 expression and clinicopathological variables in both HPV-positive OSCC and HPV-negative OSCC. Immunohistochemistry of our cohort revealed that 48 cases fulfilled the Tomm34 high expression judgment criteria, and the overall positive rate was 60% (48/80), and 27 cases fulfilled the p16 expression judgment criteria (33.75%, 27/80). The high expression of Tomm34 was closely related with the TNM classification of OSCC (p < 0.01) and tumor size (p < 0.01) both in HPV-negative OSCC and HPV-positive OSCC, while related with lymph node metastasis (p = 0.001) in HPV-negative OSCC and drinking history (p = 0.044) in HPV-positive OSCC. In addition, the Kaplan-Meier curves indicated that higher level of Tomm34 was correlated with poorer overall survival (OS) and disease-free survival (DFS) in HPV-negative OSCC (OS, p = 0.046; DFS, p = 0.020) but not in HPV-positive OSCC (OS, p = 0.824; DFS, p = 0.782). In conclusion, Tomm34 is highly expressed in OSCC and may be a useful factor to provide prognostic information, especially in HPV-negative OSCC group.