SMAD7 prevents heterotopic ossification by regulating endothelial-mesenchymal transition after Achilles tendon imjury

doi:10.3969/j.issn.2095-4344.2017.08.006

Abstract

Abstract:

BACKGROUND: The endothelial-mesenchymal transition is known to play a central role in the pathological process of heterotopic ossification.
OBJECTIVE: To verify the inhibitory effect of SMAD7 on endothelial-mesenchymal transition such as myofibroblast transformation and to explore whether it is a potential target for heterotopic ossification.
METHODS: A lentivirus overexpressing SMAD7 was contructed and the optimal titre and transfection efficiency in rat aortic endothelial cells were determined. The lentivirus was then injected into a rat model of Achilles tendon injury, while the controls were given the injection of normal saline. Expressions of endothelial and mesenchymal markers at the injured site were analyzed by quantitative PCR and western blot assay. The heterotopic ossification was observed radiologically and histologically.
RESULTS AND CONCLUSION: Local injection of SMAD7-delivering lentivirus resulted in an upregulation of CD31 and VE-cadherin, and a downregulation of N-cadherin and vimentin, suggesting that the endothelial-mesenchymal transition is blocked due to local SMAD7 overexpression. The inhibitory effect became more evident at 10 weeks than at 6 weeks after modeling. Radiology and histological staining further confirmed that the ossified structures in the tendon tissue disappeared after injection of SMAD7-delivering lentivirus, as opposed to the control group. These data suggest that local overexpression of SMAD7 can prevent postoperative heterotopic ossification with no effect on wound healing.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Osteogenesis, Heterotopic, Stromal Cells, Lentivirus, Smad7 Protein, Tissue Engineering

CLC Number:

R318

Zhang Chi1, 2, Ji Fang1. SMAD7 prevents heterotopic ossification by regulating endothelial-mesenchymal transition after Achilles tendon imjury[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(8): 1178-1185.

Figures/Tables 2

2.1 pCDH1-MCS1-EF1-copGFP-SMAD7慢病毒载体的构建有效性评估为了测试SMAD7过表达是否能阻断异位骨化，构建了慢病毒载体，用于目标细胞中及时稳定表达SMAD7。载体构建基于pCDH1-MCS1-EF1-copGFP，SMAD7被插入到EcoRI和BamHI位点之间(图1A)。瞬时转染pCDH1-MCS1-EF1-copGFP(PLV-对照)和pCDH1- SMAD7-EF1-copGFP(pLv-SMAD7)到293T细胞后，2组GPF表达率均达约90%，提示SMAD7被正确插入到载体中(图1B)。作定量PCR(qPCR)和蛋白印迹，评估瞬时转染后载体的表达水平。qPCR及蛋白印迹显示，转染pLv-SMAD7的细胞中，SMAD7的表达强度显著高于pLv-Control(qPCR，4.8倍，P < 0.0001；蛋白印迹，3.5倍，吸光度法)(图1C-E)。 2.2 SMAD转染慢病毒与大鼠主动脉内皮细胞共培养结果验证表达质粒后，使用293T细胞包装并收集慢病毒。通过终点稀释法测定慢病毒滴度，将病毒稀释至1× 104 (IFU)/μL。该慢病毒对大鼠主动脉内皮细胞的最佳感染复数(MOI)为10，该感染复数下，刚好能达到100%的转导效率。确定Lv-SMAD7-GFP对大鼠主动脉内皮细胞的转导效率后，细胞经Lv-control-GFP和Lv-SMAD7-GFP转导后，于首次传代(P1)及感染后48，72及96 h收集细胞(图2A)。使用qPCR及蛋白印迹检测SMAD7的表达(图2B、C、D)；两者均显示SMAD7的表达随时间增加，并于96 h达到高峰，且在P1传代时保持稳定。为了确定SMAD7高表达对表型的影响，使用10 mg/L的转化生长因子β1刺激大鼠主动脉内皮细胞48 h(图2E、F)以模拟细胞水平上，转化生长因子β1对局部EndMT和异位骨形成的影响。蛋白印迹分析显示，一般情况下，转化生长因子β1会下调内皮细胞标志物CD31和VE-cadherin，并上调间质标志物N-cadherin和vimentin。相比Lv-对照组，SMAD-7过表达会上调CD31和VE-cadherin的表达，其幅度几乎可以抵消转化生长因子β1处理造成的下调。同时，SMAD7过表达也还下调了N-cadherin和vimentin的表达。 2.3 对跟腱损伤大鼠模型的慢病毒载体基因治疗结果为了评估SMAD7基因疗法在体内实验中的疗效，通过手术切断跟腱的方式建立了大鼠跟腱损伤模型，并对局部注射了载有SMAD7基因的慢病毒。实验设置3组：生理盐水组(n=12)，Lv-对照组(n=12)以及Lv-SMAD7组(n=12)。每组中均于第2，6和10周分别取4只做组织学和基因表达分析。相比Lv-对照组，Lv-Smad组中，损伤的肌腱中VE-cadherin和CD31上调，而N-cadherin和vimentin下调，第6和第10周，变化尤其明显(图3A-C)。这些数据与体外大鼠主动脉内皮细胞实验中基因表达分析的结果一致。 2.4 过表达SMAD7对大鼠跟腱损伤模型表型的影响为了确认基因表达改变后对表型的影响，通过X射线光成像观察了潜在的异位骨结构。大鼠于第10周，在麻醉状态下作X射光摄片(图4A-K)。注射盐水或Lv-对照的大鼠跟腱手术区表现出高密度阴影，提示存在骨化结构，而注射Lv-SMAD7的手术区则未见任何高密度阴影，类似于未受伤肢体(图4I-K)。该过程结束时，切取跟腱组织作组织学分析(图4L-O)。生理盐水和Lv-对照组中，组织切片显示典型的异位骨化组织结构，存在致密骨化小梁区域及广泛的胶原蛋白表达(图4L，M)，而Lv-SMAD7组仅出现小的不连贯的小梁结构(图4N，O)，提示Lv-SMAD7处理的肢体中，异位骨形成相比对照组明显减轻。3组大鼠中，跟腱组织以及伤口均得到愈合。"

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