COMP-Ang1，一种血管生成素 1 的嵌合形式，增强 BMP2 诱导的成骨细胞分化和骨形成。

COMP-Ang1, a chimeric form of Angiopoietin 1, enhances BMP2-induced osteoblast differentiation and bone formation.

机构信息

Dental Science Research Institute, BK21 Project, School of Dentistry, Chonnam National University, Gwangju 500-757, Korea.

出版信息

Bone. 2010 Feb;46(2):479-86. doi: 10.1016/j.bone.2009.09.019. Epub 2009 Sep 25.

DOI:10.1016/j.bone.2009.09.019

PMID:19782780

Abstract

INTRODUCTION

Angiogenesis is closely associated with bone formation, especially endochondral ossification. Angiopoietin 1 (Ang1) is a specific growth factor functioning to generate a stable and matured vasculature through the Tie2 receptor/PI3K/AKT pathway. Recently cartilage oligomeric matrix protein (COMP)-Ang1, an Ang1 variant which is more potent than native Ang1 in phosphorylating Tie2 receptor and AKT, was developed. This study was designed to examine the effects of angiogenic COMP-Ang1 on BMP2-induced osteoblast differentiation and bone formation.

METHODS

Expression of endogenous Ang-1 and its binding receptor Tie 2 mRNA was examined in osteoblast-like cells and primary mouse calvarial cells by RT-PCR analysis, and was also monitored during osteoblast differentiation induced by BMP-2 and/or ascorbic acid and beta-glycerophosphate. Effects of COMP-Ang-1 on osteoblast differentiation and mineralization were evaluated by alkaline phosphatase (ALP) activity and osteocalcin (OC) production, and Alizarin red stain. For a molecular mechanism, Western blot and OG2 and 6xOSE promoter assays were done. For in vivo evaluation, adenoviral (Ad) vectors containing COMP-Ang-1 or BMP-2 gene were administered into thigh muscle of mice, and after 2 weeks bone formation was analyzed by micro-computed tomography and histology. Angiogenic event of COMP-Ang1 was confirmed by immunofluorescence analysis with anti-CD31 antibody.

RESULTS

Expression of Tie2 receptor was significantly increased in the course of osteoblast differentiation. Treatment or overexpression of COMP-Ang1 enhanced BMP2-induced ALP activity, OC production, and mineral deposition in a dose-dependent manner. In addition, COMP-Ang1 synergistically increased OG2 and 6xOSE promoter activities of BMP2, and sustained p38, Smad and AKT phosphorylation of BMP2. Notably, in vivo intramuscular injection of COMP-Ang1 dose-dependently enhanced BMP2-induced ectopic bone formation with increases in CD31 reactivity.

CONCLUSIONS

These results suggest that COMP-Ang1 synergistically enhanced osteoblast differentiation and bone formation through potentiating BMP2 signaling pathways and angiogenesis. Combination of BMP2 and COMP-Ang1 should be clinically useful for therapeutic application to fracture and destructive bone diseases.

摘要

简介

血管生成与骨形成密切相关，特别是骺软骨内骨化。血管生成素 1（Ang1）是一种特异性生长因子，通过 Tie2 受体/PI3K/AKT 通路生成稳定和成熟的血管。最近开发了软骨寡聚基质蛋白（COMP）-Ang1，这是一种比天然 Ang1 更能磷酸化 Tie2 受体和 AKT 的 Ang1 变体。本研究旨在研究血管生成 COMP-Ang1 对 BMP2 诱导的成骨细胞分化和骨形成的影响。

方法

通过 RT-PCR 分析检测成骨细胞样细胞和原代小鼠颅骨细胞中内源性 Ang-1 及其结合受体 Tie2 mRNA 的表达，并在 BMP-2 和/或抗坏血酸和β-甘油磷酸诱导的成骨细胞分化过程中进行监测。通过碱性磷酸酶（ALP）活性和骨钙素（OC）产生以及茜素红染色评估 COMP-Ang1 对成骨细胞分化和矿化的影响。为了研究分子机制，进行了 Western blot 和 OG2 和 6xOSE 启动子测定。为了进行体内评估，将含有 COMP-Ang1 或 BMP-2 基因的腺病毒（Ad）载体注入小鼠大腿肌肉中，2 周后通过微计算机断层扫描和组织学分析评估骨形成。通过抗 CD31 抗体的免疫荧光分析证实了 COMP-Ang1 的血管生成事件。

结果

Tie2 受体的表达在成骨细胞分化过程中显著增加。COMP-Ang1 的处理或过表达以剂量依赖性方式增强 BMP2 诱导的 ALP 活性、OC 产生和矿化沉积。此外，COMP-Ang1 协同增强了 BMP2 的 OG2 和 6xOSE 启动子活性，并持续磷酸化 BMP2 的 p38、Smad 和 AKT。值得注意的是，体内肌肉内注射 COMP-Ang1 剂量依赖性地增强了 BMP2 诱导的异位骨形成，并增加了 CD31 反应性。