RNA sequence of the subcutaneous microenvironment surrounding the rabbit's engineered cartilage

Purpose: To identify how axitinib stabilized the rabbit's engineered cartilage through the niche Methods: Rabbit's engineered cartilage was encapsulated in the PCL/collagen nanofibrous membrane containing no axitinib (0%-Axitinib group) or 3% axitinib (3%-Axitinib group) and implanted into nude mice. After 12 weeks of implantation, samples from the 0%-Axitinib group and 3%-Axitinib group were harvested, with the murine tissues entangled. Transcriptional profiles were generated by deep sequencing, in triplicate, using an Illumina HiSeqTM2000 machine, and filtered using Fastp software. The trimmed reads were mapped to the mouse genome (mm10, genome.UCSC.edu) using the STAR software to identify the transcriptional information in the niche of engineered cartilage. Then, the DESeq2 “counts” function of the R package was used to normalize gene expression levels based on the number of total reads of each sample. Differentially expressed genes between the 0%-Axitinib group and 3%-Axitinib group were identified as meeting the criteria of fold change ≥ 1.5 and P-value＜0.05. Lastly, we used Metascape (http://metascape.org) for gene ontology analysis. Results: Using an optimized data analysis workflow, we mapped about 20 to 30 million sequence reads per sample to the mouse genome (build mm10) and identified 20318 transcripts in the 0%-Axitinib and 3%-Axitinib groups. 837 genes were differentially expressed by more than 1.5-fold, p-value <0.05, with 313 genes downregulated and 524 genes upregulated in the 3%-Axitinib group. Gene ontology (GO) analysis revealed that the genes downregulated in the 3%-Axitinib group were mainly enriched in endothelial development, angiogenesis, and immune response. Genes upregulated in the 3%-Axitinib group were mainly enriched in response to hypoxia, Hif-1 signaling pathway, cartilage differentiation and development, and muscle structure development. Altered expression of 15 genes was further illustrated by heatmap, demonstrating the inhibited angiogenesis and improved chondrogenesis. Especially, Timp1, as a response to hypoxia, was remarkably upregulated, which also in turn inhibited the activity of matrix metalloproteinases (MMPs) and played an important role in preventing ossification. Conclusion: In this study, we proposed the first detailed transcriptional analysis of the effects of the sustainable and localized release of axitinib on the subcutaneous niche of engineered cartilage. The sustainable and localized release of axitinib created a relatively avascular, hypoxia, and low immune response niche in the 3%-Axitinib group. In this niche, Timp1 was remarkably upregulated, which inhibited the activity of MMPs and prevented endochondral ossification. mRNA profiles of murine tissues surrounding the rabbit's engineered cartilage in 0%-Axitinib and 3%-Axitinib groups after 12-week subcutaneous implantation

研究目的：阐明阿昔替尼（axitinib）如何通过微环境（niche）稳定兔工程化软骨。
实验方法：将兔工程化软骨封装于不含阿昔替尼的聚己内酯/胶原蛋白纳米纤维膜（PCL/collagen nanofibrous membrane）（0%-阿昔替尼组）或含3%阿昔替尼的聚己内酯/胶原蛋白纳米纤维膜（3%-阿昔替尼组）中，植入裸鼠皮下。植入12周后，收获0%-阿昔替尼组与3%-阿昔替尼组的样本，此时样本已与小鼠宿主组织缠绕结合。采用Illumina HiSeqTM2000平台进行三次重复深度测序以获取转录组谱，通过Fastp软件对测序数据进行过滤。将修剪后的测序reads比对至小鼠基因组（mm10，genome.UCSC.edu），使用STAR软件以鉴定工程化软骨微环境中的转录信息。随后利用R包DESeq2的“counts”函数，基于每个样本的总reads数对基因表达水平进行标准化。以倍数变化≥1.5且P值＜0.05为筛选标准，鉴定0%-阿昔替尼组与3%-阿昔替尼组间的差异表达基因。最后，使用Metascape（http://metascape.org）进行基因本体（gene ontology, GO）富集分析。
实验结果：通过优化的数据分析流程，我们将每个样本约2000万至3000万条序列reads比对至小鼠基因组（版本mm10），在0%-阿昔替尼组与3%-阿昔替尼组中共鉴定到20318条转录本。共有837个基因的表达差异倍数超过1.5倍且P值＜0.05，其中3%-阿昔替尼组中313个基因表达下调，524个基因表达上调。基因本体分析显示，3%-阿昔替尼组下调的基因主要富集于内皮发育、血管生成及免疫应答相关通路；而上调的基因主要富集于缺氧应答、缺氧诱导因子-1（Hif-1）信号通路、软骨分化与发育以及肌肉结构发育相关通路。我们通过热图进一步展示了15个差异表达基因的表达变化，证实阿昔替尼可抑制血管生成并促进软骨形成。尤为重要的是，作为缺氧应答相关基因的Timp1表达显著上调，其可通过抑制基质金属蛋白酶（matrix metalloproteinases, MMPs）的活性，在阻止软骨内骨化过程中发挥关键作用。
研究结论：本研究首次对可持续局部释放阿昔替尼对兔工程化软骨皮下微环境的影响进行了详细的转录组分析。3%-阿昔替尼组中，可持续局部释放的阿昔替尼构建了相对无血管、缺氧且低免疫应答的微环境。在此微环境中，Timp1表达显著上调，通过抑制基质金属蛋白酶活性阻止了软骨内骨化。本数据集包含兔工程化软骨植入裸鼠皮下12周后，0%-阿昔替尼组与3%-阿昔替尼组中小鼠宿主周围组织的mRNA表达谱。