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Title:Optimization of a pericyte therapy to improve muscle recovery following hindlimb immobilization
Author(s):Lapp, Samuel Christian
Advisor(s):Boppart, Marni D
Contributor(s):Konopka, Adam R
Department / Program:Kinesiology & Community Health
Discipline:Kinesiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:M.S.
Genre:Thesis
Subject(s):pericyte
skeletal muscle
immobilization
disuse atrophy
Tbx18
recovery
Abstract:Extended bed rest or single limb immobilization can significantly reduce skeletal muscle mass and function. Recovery may be severely delayed or incomplete due to the inability to effectively engage in physical therapy. Thus, alternative strategies are needed to enhance recovery and prevent long-term disability, particularly in individuals prone to muscle loss (older adults, neuromuscular disease). Our lab recently demonstrated that pericyte quantity is decreased in skeletal muscle following immobilization and appropriate replacement via intramuscular transplantation can effectively recover myofiber size in mice. These data suggest that pericytes positively influence skeletal muscle growth and may represent a novel approach to the prevention and treatment of disability following disuse. However, one of the challenges in developing a pericyte therapy is the lack of a unique marker for identification and isolation of pericytes. Investigators have established that pericytes express a wide variety of cell surface markers, such as neural/glial antigen 2 (Cspg4/NG2), melanoma cell adhesion molecule (Mcam/CD146), and platelet-derived growth factor receptor β (PDGFRβ). Currently, none of these markers are uniquely expressed by pericytes; therefore, cell heterogeneity likely exists when isolating based on one pericyte marker. Tbx18 (T-box18) was recently proposed as a transcription factor uniquely expressed in pericytes, providing the opportunity to examine surface marker expression using the Tbx18CreERT2;Rosa26:tdTomato(Ai9) mouse line. PURPOSE: Aim 1: To compare the capacity for NG2+ and CD146+ pericytes to recover skeletal muscle mass following a period of disuse. Aim 2: To characterize cell surface marker expression of muscle-resident Tbx18+ cells using multiplex flow cytometry. METHODS: Adult, 4-month-old C57BL/6 mice (n=24) were randomly divided into four groups (n=6/group). Mice hindlimbs were immobilized in full dorsiflexion via a surgical staple inserted through the center of the foot and body of the gastrocnemius for 2 weeks. At 2 weeks post immobilization, staples were removed and either pericytes (NG2+CD45-CD31-[Lin-], CD146+Lin-, or CD146+NG2-Lin-) or saline (control) were injected into the tibialis anterior (TA) muscle. TA muscles were excised for analysis after 2 weeks of remobilization and the extent of recovery was assessed. To characterize Tbx18+ cells, 5-month-old, Tbx18CreERT2:Ai9 mice (n=4) were injected with tamoxifen, and tdTomato expressing cells from the TA muscle were assessed for CD146 and NG2 using multiplex flow cytometry. Lineage tracing was also performed to determine pericyte fate following a period of immobilization, as well as following a period of immobilization and remobilization. RESULTS: There was no difference between myofiber CSA or muscle wet weight between groups; however, CD146+Lin-pericytes demonstrated the greatest capacity for recovery based on a trend toward an increase in myofiber cross-sectional area (p=0.071), increased collagen remodeling (p=0.048), and significant improvements in capillarization (p=0.043). CD146+NG2-Lin- also improved capillarization (p=0.012) and a trend toward an increase in collagen remodeling (p=0.053) compared to the saline control group. There were no significant benefits to NG2+Lin- pericyte transplantation in any measure. In adult mice, the total relative percentage of Tbx18+ cells was unexpectedly low (~2%), yet the majority of cells expressed CD146 (>95%). Tbx18+ cells were present in the interstitium between muscle fibers following immobilization and remobilization, and no evidence of Tbx18+ cell contribution to myogenesis was observed. CONCLUSION: CD146+Lin- and CD146+NG2-Lin- pericyte transplantation effectively recovered capillary quantity and collagen remodeling following a period disuse compared to controls, with a trend in myofiber size also observed. NG2+Lin- pericytes likely do not contribute to regrowth following a period of disuse. The relatively low percentage of Tbx18+ pericytes in adult muscle suggest that Tbx18 is a developmentally regulated transcription factor that is suppressed during the postnatal period.
Issue Date:2020-04-30
Type:Thesis
URI:http://hdl.handle.net/2142/108287
Rights Information:Copyright 2020 Samuel Lapp
Date Available in IDEALS:2020-08-27
Date Deposited:2020-05


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