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Development of a Pericyte-Based Cell-Free Strategy to Recover Aged Skeletal Muscle After Disuse

ABSTRACT: Our laboratory previously demonstrated that perivascular stem/stromal cells (CD146+ pericytes) can effectively recover muscle mass after a period of immobilization in young adult mice. However, cell-based therapies are problematic in aged mouse models due to lack of viability upon transplantation. Therefore, the purpose of this study was to develop a pericyte-based, cell-free strategy to recover muscle mass after disuse in aged mice. Single-cell RNA sequencing (scRNA-Seq) was performed on adult mouse skeletal muscle after two weeks of unilateral hindlimb immobilization, which revealed that muscle-resident pericytes uniquely upregulate the long noncoding RNA Malat1, a negative regulator of Nrf2, and fail to induce antioxidant gene expression in response to reactive oxygen species (ROS; H2O2). This information was used to guide the design of a strategy in which healthy donor pericytes were stimulated with ROS to produce small extracellular vesicles (EVs) that were subsequently transplanted into 4- and 24-26-month-old C57BL/6 mice after two weeks of unilateral hindlimb immobilization. H2O2-primed healthy muscle-derived pericytes produced EVs in culture that effectively reduced restored myofiber CSA in both adult (p=0.009) and aged (p=0.006) muscle after disuse. In contrast, unprimed pericyte-derived EVs did not influence myofiber size. Neither primed, nor unprimed EVs recovered capillary density, yet both stimulated collagen turnover. Healthy ROS-primed pericyte-derived small EVs effectively improve skeletal muscle recovery after immobilization, representing a novel cell-free approach to rebuild muscle mass in older adults after a period of disuse.

ORGANISM(S): Mus musculus

PROVIDER: GSE185560 | GEO | 2022/09/28

REPOSITORIES: GEO

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Project description:Background: Pericytes regulate vessel stabilization and function and their loss is associated with diseases such as diabetic retinopathy or cancer. Despite their physiological importance, pericyte function and molecular regulation during angiogenesis remain poorly understood. Methods: To decipher the transcriptomic programs of pericytes during angiogenesis, we crossed the Pdgfrb(BAC)-CreERT2 into the RiboTagflox/flox mice. Pericyte morphological changes were assessed in mural cell-specific R26-mTmG reporter mice, in which low doses of tamoxifen allowed labeling of single cell pericytes at high resolution. To study the role of phosphoinositide 3-kinase (PI3K) signaling in pericyte biology during angiogenesis, we used genetic mouse models which allow selective inactivation of PI3Kα and PI3Kβ isoforms and their negative regulator PTEN (phosphate and tensin homologue deleted on chromosome ten, PTEN) in mural cells. Results: At the onset of angiogenesis, pericytes exhibit molecular traits of cell proliferation and activated PI3K signaling, whereas during vascular remodeling pericytes upregulate genes involved in mature pericyte cell function, together with a remarkable decrease in PI3K signaling. Immature pericytes showed stellate shape and high proliferation, and mature pericytes were quiescent and elongated. Unexpectedly, we demonstrate that the PI3Kβ, but not PI3Kα, regulates pericyte proliferation and maturation during vessel formation. Genetic PI3Kβ inactivation in pericytes triggered early pericyte maturation. Conversely, unleashing PI3K signaling by means of PTEN deletion delayed pericyte maturation. Pericyte maturation was necessary to undergo vessel remodeling during angiogenesis. Conclusions: Our results identify new molecular and morphological traits associated to pericyte maturation and uncover PI3Kβ activity as a checkpoint to ensure appropriate vessel formation. In turn, our results may open new therapeutic opportunities to regulate angiogenesis in pathological processes through the manipulation of pericyte PI3Kβ activity.

Project description:Pericytes have been implicated in regulation of inflammatory, reparative, fibrogenic and angiogenic responses in several different organs and pathologic conditions. Although the adult mammalian heart contains abundant pericytes, their fate and involvement in myocardial disease remains unknown. We used NG2Dsred;PDGFRaEGFP pericyte-fibroblast dual reporter mice and inducible NG2CreER mice to study the fate and phenotypic modulation of pericytes in a model of myocardial infarction. The transcriptomic profile of pericyte-derived fibroblasts was studied using PCR arrays. The transcriptomic profile of NG2 lineage cells (pericytes) was studied in control and infarcted hearts using single cell RNA-sequencing analysis. The role of TGF-b signaling in regulation of pericyte phenotype in vivo was investigated using pericyte-specific Tgfbr2 knockout mice. In vitro, the effects of TGF-b were studied in cultured human placental pericytes.In normal mouse hearts, NG2 and PDGFRa identified distinct non-overlapping populations of pericytes and fibroblasts respectively. Following myocardial infarction, a population of cells expressing both pericyte and fibroblast markers emerged. These cells expressed large amounts of extracellular matrix (ECM) genes. Lineage tracing demonstrated that in the infarcted region, a subpopulation of pericytes underwent fibroblast conversion. Single cell RNA-seq experiments demonstrated expansion and diversification of pericyte-derived cells in the infarct, associated with emergence of subpopulations exhibiting accentuated matrix gene synthesis. In vitro studies and the profile of pericyte-derived fibroblasts identified TGF-b as a potentially causative mediator in fibrogenic activation of infarct pericytes. However, pericyte-specific Tgfbr2 disruption had no significant effects on myofibroblast infiltration and collagen deposition in the infarct. Pericyte-specific TGF-b signaling was involved in vascular maturation, mediating formation of a mural cell coat investing infarct neovessels. These reparative effects of infarct pericytes protected the infarcted heart from dilative remodeling.

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Development of a Pericyte-Based Cell-Free Strategy to Recover Aged Skeletal Muscle After Disuse

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