CLINICAL TRANSLATION PIPELINE FOR DETECTING SENESCENCE IN OSTEOARTHRITIS USING THE Β-GALACTOSIDASE RESPONSIVE GD-CHELATE

V. Suryadevara , R. von Kruechten , J.H. Tang , A.M. Dreisbach , Z. Shokri Varniab , S.B. Singh , A. Lubke , T. Liang , J. Wong , J. Wang , R. Duwa , J. Wang , M. Barbieri , F. Kogan , S.B. Goodman , L. Chou , D. Oji , J. Chan , T.J. Meade , H.E. Daldrup-Link
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Abstract

INTRODUCTION

Cellular senescence is one of the key mechanisms implicated in the development and progression of OA. The identification of senescence-mediated molecular mechanisms in OA needs novel imaging tools to detect senescence and monitor the efficacy of new senolytic therapies. Progress in molecular imaging techniques has led to the creation of a novel β-gal responsive Gd-chelate for identifying senescence using MRI, the widely used imaging modality for OA.

OBJECTIVE

The hypothesis is that β-gal responsive Gd-chelate can detect senescence in vitro, in vivo and in human OA specimens.

METHODS

Senescence was induced in mesenchymal stem cells (MSCs) using 400nM doxorubicin over 5 days. Control and senescent cell suspensions incubated with 0.25 mM β-gal responsive Gd-chelate underwent MRI on a 3T MRI scanner (Bruker BioSpec, Billerica, MA). Further cartilage defects created in pig knees were implanted with control and senescent cells, followed by MRI after intraarticular injection of 2.5 mM β-gal responsive Gd-chelate. As a first step of clinical translation, human OA specimens were obtained from 30 patients undergoing hip/knee/ankle replacement. The fresh specimens were incubated with 2.5 mM of β-gal responsive Gd-chelate for an hour, before and after which MRI was performed using the following parameters: fat-saturated PD-weighted fast spin-echo sequence (TR=1500ms, matrix size=512 × 512pixels, slice thickness(=1mm, FOV=15cm, and NEX=2); SMART1 MAP sequence (TR = 40, 75, 150, 300, 500, 700, and 2,000 ms, matrix size=160 × 160 pixels SL=6mm, FOV=15 cm, and NEX=1) and T1 weighted fast SE sequence (TR=500ms, matrix size=512 × 512 pixels, SL=1mm, FOV=15cm, and NEX=2). T1 maps were generated to calculate the T1 relaxation times.

RESULTS

Senescence was first confirmed with immunohistochemistry for senescence markers including p16, p21 and β-gal. In vitro studies indicated that senescent MSCs demonstrated a notable increase in MRI signal after being incubated with the β-gal responsive Gd-chelate probe, compared to control cells (Fig. 1A). In vivo, the probe was injected intraarticularly into pig knee joints, and a marked decrease in T1 relaxation times indicated the retention of the probe and it’s activation by senescent cells in cartilage defects (Fig. 1B). The Wilcoxon ranksum test was used to determine the significance between control and senescence group. In human OA specimens, areas with severe cartilage damage as graded by a radiologist using Outerbridge score demonstrated higher number of senescent cells seen on immunohistochemistry. MRI indicated that there is pronounced hyperintense signal in the T1-SE images upon incubation with the β-gal responsive Gd-chelate probe, compared to MRI of the specimens before incubation. This was further quantified on T1 maps and indicated a significant reduction in T1 relaxation times, which also correlated with the Outerbridge score (Fig. 1C). The ordinal logistic regression indicated a significant negative correlation between T1 relaxation times and Outerbridge score (p<0.0001).

CONCLUSIONS

This study demonstrates the clinical translation of the β-gal responsive Gd-chelate for detecting senescent cells in vitro, in vivo and in human OA specimens.
利用Β-galactosidase反应性gd -螯合物检测骨关节炎衰老的临床翻译管道
细胞衰老是骨性关节炎发生和发展的关键机制之一。确定OA中衰老介导的分子机制需要新的成像工具来检测衰老并监测新的抗衰老疗法的疗效。分子成像技术的进步导致了一种新的β-gal反应性gd螯合物的产生,用于使用MRI识别衰老,这是OA广泛使用的成像方式。目的:假设β-gal反应性gd -螯合物在体外、体内和人体OA标本中具有检测衰老的作用。方法采用400nM阿霉素诱导间充质干细胞(MSCs)衰老5 d。对照和衰老细胞悬液用0.25 mM β-gal反应性gd -螯合物孵育,在3T MRI扫描仪上进行MRI (Bruker BioSpec, Billerica, MA)。用对照细胞和衰老细胞植入猪膝关节软骨缺损,关节内注射2.5 mM β-gal反应性gd -螯合物后进行MRI检查。作为临床转化的第一步,从30例髋关节/膝关节/踝关节置换术患者中获得人类OA标本。新鲜标本与2.5 mM β-gal反应性gd -螯合物孵育1小时,前后采用以下参数进行MRI:脂肪饱和pd加权快速自旋回波序列(TR=1500ms,基质尺寸=512 × 512pixels,切片厚度(=1mm, FOV=15cm, NEX=2);SMART1 MAP序列(TR = 40、75、150、300、500、700和2000 ms,矩阵大小=160 × 160像素SL=6mm, FOV=15cm, NEX=1)和T1加权快速SE序列(TR=500ms,矩阵大小=512 × 512像素,SL=1mm, FOV=15cm, NEX=2)。生成T1映射以计算T1弛豫时间。结果p16、p21、β-gal等衰老标志物首次被免疫组化证实衰老。体外研究表明,与对照细胞相比,衰老的MSCs与β-gal反应性gd螯合探针孵育后,MRI信号明显增加(图1A)。在体内,探针被注射到猪膝关节关节内,T1松弛时间明显减少,表明探针被软骨缺损中的衰老细胞保留和激活(图1B)。采用Wilcoxon秩和检验确定对照组与衰老组之间的显著性。在人类OA标本中,放射科医生使用Outerbridge评分分级的严重软骨损伤区域在免疫组织化学上显示出更多的衰老细胞。MRI显示β-gal反应性gd -螯合探针孵育后T1-SE图像较孵育前的MRI有明显的高信号。这在T1图上进一步量化,表明T1松弛时间显著减少,这也与Outerbridge评分相关(图1C)。有序逻辑回归显示T1松弛时间与Outerbridge评分呈显著负相关(p<0.0001)。结论β-gal反应性gd -螯合物在体外、体内和人体OA标本中检测衰老细胞具有临床翻译作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Osteoarthritis imaging
Osteoarthritis imaging Radiology and Imaging
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