Poster X1 - Targeted Antagonism of Sphingosine-1-Phosphate Receptor as a Regenerative Immunotherapy for Volumetric Muscle Loss Injury

Introduction: Skeletal muscle has a remarkable capacity for repair following acute injuries, however in the case of volumetric muscle loss (VML) injuries the endogenous healing mechanisms are overwhelmed, leading to failed regeneration, gross fibrosis, and ultimately loss of function. Post injury, myeloid and lymphoid cell trafficking within the injury milieu is highly correlated with the stages of muscle regeneration; thus, regulation of the immune response is crucial to functional healing. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that is produced upon disturbance of tissue homeostasis and acts as a potent chemoattractant for immune cells towards inflammatory sites. S1P acts directly on macrophages and therefore plays a vital role in modulating their pro-inflammatory vs pro-regenerative functions in response to injury. During chronic inflammation, such as with VML, S1P gradients are perturbed which can lead to S1P-mediated secretion of pro-fibrotic factors, particularly in macrophages, which advances to tissue fibrosis. VML defects were shown to express increased M2 macrophages co-expressing TNFα and TGFβ, indicative of perturbed macrophage transitions which also induce fibrosis. S1P signals through 5 known G protein-coupled receptors (S1PR1-5) and therefore S1P-dependent immune cell responses are dependent on their S1PR profile. It has been shown that upregulated S1PR3 expression is implicated in TGFβ-driven fibrosis and impaired myogenesis, suggesting that S1PR3 may be a therapeutic target to enhance healing. Thus, the objective of this work is to modulate local S1PR signaling using biomaterial strategies to attenuate pro-longed immune cell retention to reduce pro-fibrotic signaling notable of VML injuries and enhance muscle regeneration and recovery.

Materials and

Methods: 13-week-old male C57BL6 mice received either a full thickness, subcritical (2mm) or critically sized (3mm) VML injury through the quadriceps muscle using a biopsy punch. Hyaluronic acid nanofibrous hydrogels, hydrogel precursors of 4-arm PEG norbornene macromers, thiolated HA, RGD, and PEG dithiol crosslinker were combined with sacrificial poly(ethylene oxide) and a photoinitiator in deionized water to facilitate thiol-norbornene photoclick reaction chemistry to form fibrous matrices. The electrospinning solution was prepared in a 1:200 drug:polymer ratio for loading of VPC01091. To provide a control for the fibrous architecture, bulk hydrogels were made from the same hydrogel precursors but were not electrospun prior to ultraviolet polymerization. Systemic injections were performed intraperitoneally the day of injury at a dose of 5mg/kg. Flow cytometric analysis was performed on injured quadriceps (days 1, 3, 7 post injury) to assess immune cell infiltration and intracellular cytokine expression. Single cell suspensions from injured muscles were stained with a panel of fluorescent antibodies and analyzed on a FACS-AriaIIIu flow cytometer. Data was analyzed using FlowJo software in addition to utilizing high dimensionality reduction and clustering algorithms including Spanning-tree Progression Analysis of Density-normalized Events (SPADE). Mice were euthanized 7 days after injury and vasculature was perfused with saline followed by PFA to fix tissues. Tissues were incubated overnight in Alexa Fluor 488 antidesmin and Alexa Fluor 647 anti-CD68 for macrophage visualization. Animal Procedure Approval. All animal procedures were conducted according to protocols approved by Georgia Institute of Technology Institutional Animal Care and Use Committee.

Results, Conclusions, and Discussions: Systemic delivery of S1PR3 antagonist VPC01091 showed striking muscle regeneration following critically sized VML. Inspired by these results, we directed our efforts to converting this approach into a local therapeutic strategy to achieve targeted delivery and reduced potential for side effects associated with systemic immunomodulation. To assess the efficacy of pharmacologically inhibiting S1PR3 in critically sized VML injuries, we systemically delivered VPC01091 at day 0 and day 6 following injury. Our results show that VPC01091 reduced circulating numbers of immune cells up to 3 days post injection with a particular effect on lymphocytes. We rationalized that this is primarily a consequence of VPC01091 acting as an agonist of S1PR1 and likely causing S1PR1 to irreversibly internalize and degrade, similar to the therapeutic action of FTY720. Compared to untreated VML, VPC01091 delivery led to substantially decreased remaining defect areas as well as significant increases in myofibers expressing eMHC and myofibers with centrally located nuclei. Additionally, vascular density was increased and myofibers around the defect areas were larger in size, indicating their maturity over smaller myofibers attempting to regenerate. We qualitatively observed decreased collagen deposition when mice received VPC01091 injections and importantly, numerous studies have elucidated the role of S1PR3 in promoting fibrosis. This implies that S1PR3 antagonism may be directly affecting muscle cells to influence regeneration. The success of systemic treatments suggested the possible efficacy of local release. Total immune cell numbers in the blood were significantly reduced by both fibrous and bulk hydrogel groups. While to some extent, implantation of either bulk or nanofibrous gels imparted a systemic effect, it is apparent that hydrogels with nanofibrous structure did so more robustly. Both nanofibrous gel groups significantly decreased total circulating T cells and cytotoxic T cells specifically compared to untreated controls. In assessing the muscle regenerative effects of our local hydrogels with or without VPC01091, we found that VPC nanofibrous treatment was the most effective at promoting regeneration. VPC NF treatment led to qualitative improvements in myofiber bridging over the defect, reduced collagen deposition, and significantly more myofibers with central myonuclei compared to either bulk group.

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