Abstract
ObjectiveRegenerative therapy using mesenchymal stem cells (MSC) is a promising therapeutic method for critical limb ischemia (CLI). To understand how the cells are involved in the regenerative process of limb ischemia locally, we proposed a metabolic protein labeling method to label cell proteomes in situ and then decipher the proteome dynamics of MSCs in ischemic hind limb.Methods and ResultsIn this study, we overexpressed mutant methionyl-tRNA synthetase (MetRS), which could utilize azidonorleucine (ANL) instead of methionine (Met) during protein synthesis in MSCs. Fluorescent non-canonical amino-acid tagging (FUNCAT) was performed to detect the utilization of ANL in mutant MSCs. Mice with hindlimb ischemia (HLI) or Sham surgery were treated with MetRSmut MSCs or PBS, followed by i.p. administration of ANL at days 0, 2 6, and 13 after surgery. FUNCAT was also performed in hindlimb tissue sections to demonstrate the incorporation of ANL in transplanted cells in situ. At days 1, 3, 7, and 14 after the surgery, laser doppler imaging were performed to detect the blood reperfusion of ischemic limbs. Ischemic tissues were also collected at these four time points for histological analysis including HE staining and vessel staining, and processed for click reaction based protein enrichment followed by mass spectrometry and bioinformatics analysis. The MetRSmut MSCs showed strong green signal in cell culture and in HLI muscles as well, indicating efficient incorporation of ANL in nascent protein synthesis. By 14 days post-treatment, MSCs significantly increased blood reperfusion and vessel density, while reducing inflammation in HLI model compared to PBS. Proteins enriched by click reaction were distinctive in the HLI group vs. the Sham group. 34, 31, 49, and 26 proteins were significantly up-regulated whereas 28, 32, 62, and 27 proteins were significantly down-regulated in HLI vs. Sham at days 1, 3, 7, and 14, respectively. The differentially expressed proteins were more pronounced in the pathways of apoptosis and energy metabolism.ConclusionIn conclusion, mutant MetRS allows efficient and specific identification of dynamic cell proteomics in situ, which reflect the functions and adaptive changes of MSCs that may be leveraged to understand and improve stem cell therapy in critical limb ischemia.
Highlights
Peripheral artery disease (PAD) is caused by limited or restricted blood flow in peripheral arteries that can lead to limb pain, disability, or mortality
Bone marrow-derived mesenchymal stem cells (BMSCs), which were derived from C57BL/6J mice, was purchased from Cyagen Biosciences (Sunnyvale, CA, United States, Catalog Number: MUBMX-01001). (L274G) mutant-mCherry fusion protein was inserted into the cloning site of pMaRSC plasmid (Addgene, Plasmid #89189)
Nascent proteins with ANL incorporation in mutant MSCs can be detected by click reaction with alkyne conjugated with Alexa Fluor 488, while no proteins were labeled with Alexa Fluor 488 in control groups, indicating a high labeling efficiency and a good sensitivity of this method (Figure 1B)
Summary
Peripheral artery disease (PAD) is caused by limited or restricted blood flow in peripheral arteries that can lead to limb pain, disability, or mortality. Critical limb ischemia (CLI) is the most serious form of PAD, which is characterized by a severe blockage in the arteries of the lower extremities and markedly reduced blood-flow (Dua and Lee, 2016; Campia et al, 2019). MSCs are multipotent stem cells with the capacity of selfrenewal and multilineage differentiation including mesodermal lineage, endodermal lineage and ectodermal lineage. They are easy to be obtained and expanded in vitro, marking its importance in cell therapy, regenerative medicine and tissue repair (Samsonraj et al, 2017; Trohatou and Roubelakis, 2017; Fu et al, 2019; Zhao et al, 2019). The specific mechanisms how MSCs respond to and improve the ischemic microenvironment have yet to be fully characterized
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