Icon Legend

This session is not in your schedule.

This session is in your schedule. Click again to remove it.

Back

Immunoengineering

Shifting the balance of inflammatory and pro-resolving lipid mediators in volumetric muscle loss (VML) injury

(J-366) Shifting the balance of inflammatory and pro-resolving lipid mediators in volumetric muscle loss (VML) injury

Friday, October 13, 2023

3:30 PM – 4:30 PM PDT

Location: Exhibit Hall - Row J - Poster # 366

Presenting Author(s)

FP

Frank Pittman

Graduate Student
University of Oregon
Eugene, Oregon, United States

Co-Author(s)

TT

Thomas Turner

Graduate Student
Georgia Tech, United States
LH

Lauren Hymel

Graduate Student
Georgia Tech, United States
MB

Monica Behara

Graduate Student
Georgia Tech, United States
SA

Shannon Anderson

Graduate Student
Georgia Tech, United States
KM

Kristal Maner-Smith

Research Scientist
Emory University, United States
HL

Hong Lim

Graduate Student
Georgia Tech, United States
PQ

Peng Qiu

Professor
Georgia Institute of Technology, United States
YJ

Young Jang

Principal Investigator
Emory University, United States
AG

Andrés J. García

Executive Director, Parker H. Petit Institute for Bioengineering & Bioscience, Professor in Mech E
Georgia Institute of Technology, United States
NW

Nick Willett, PhD

Associate Professor
University of Oregon, Knight Campus, United States

Primary Investigator(s)

EB

Ed Botchwey

Principal Investigator
Georgia Tech, United States

Introduction:: Volumetric Muscle Loss (VML) is the extensive loss of skeletal muscle tissue that overwhelms the body’s capacity for repair; these injuries can often result in permanent physical disability, chronic pain, and the onset of many comorbid diseases. VML injuries are typically sustained through trauma such as car accidents, military combat, or sarcoma excision. The gold-standard treatment for VML is either an autologous muscle flap or free tissue transfer to salvage the limb. Patients then will undergo long term functional rehabilitation to strengthen surrounding intact muscle. Even if the treatment salvages the limb, patients often experience negligible functional improvements due to a heightened and persistent inflammatory response that can impair growth of new muscle, cause collateral tissue damage, and drive fibrosis and adiposis within the injury site. The mechanisms behind this immune dysregulation following VML are not fully characterized. However, in smaller injuries and other conditions presenting with acute inflammation, it is known that the production of pro-resolving lipid mediators in the injury milieu is necessary to activate inflammation resolution which results in tissue regeneration and a return to homeostasis. Therefore, we hypothesized that immune dysregulation following VML is caused by impaired lipid mediator metabolism characterized by a lack of pro-resolving mediator accumulation, which can be improved via local delivery of an exogeneous specialized pro-resolving mediator. Thus, we sought to mimic the short-range paracrine signaling of SPMs within a smaller injury by locally delivering resolvin D1’s aspirin-triggered isoform (AT-RvD1) in a 4-arm poly(ethylene-glycol)-maleimide hydrogel (PEG-4MAL) to a critically-sized VML injury.

Materials and Methods:: All hydrogels were 20uL 4.5% (w/v) four-arm poly(ethylene glycol)-maleimide (PEG-4MAL) functionalized with the cell adhesion ligand RGD and crosslinked with the protease-degradable peptide VPM. For loaded hydrogels, 4ug/mL AT-RvD1 was thoroughly mixed with PEG-4MAL before crosslinking.

All animal procedures used 8-12 week-old male C57BL6/J mice and were conducted according to protocols approved by Georgia Tech’s IACUC. Full-thickness unilateral quadriceps injuries were created using a 2mm or 3mm biopsy punch for sub-critical or VML injuries, respectively. Animals were euthanized after 1, 3 ,5, 7, or 14 days post-injury (dpi), and injured and contralateral quadriceps were dissected for lipidomics. Local AT-RvD1 treatment was performed in a murine spinotrapezius model of VML. Briefly, a 2mm biopsy punch was used to create a full-thickness defect, and a 20uL PEG-4MAL hydrogel was injected into the defect site with or without AT-RvD1. Animals were euthanized 1, 3, or 7 dpi for flow cytometry, or at 3 and 7 dpi for lipidomics.

For lipidomics, muscle samples were homogenized, then oxylipins were extracted using solid phase extraction (SPE) followed by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Spinotrapezius samples were dissected, digested, stained for immune cells, satellite cells (MuSCs) and fibroadipogenic progenitors (FAPs), and run on a BD FACSAria II. The dimensionality reduction algorithm Spanning-tree Progression of Density-normalized Events (SPADE) was used to quantitate cell populations.

Statistics were two-tailed unpaired t-tests for two-group comparisons and one-way ANOVA with Tukey’s post-hoc was used for multi-group comparisons. Two-way ANOVA with Sidak’s post-hoc was used for grouped analyses with two independent variables.

Results, Conclusions, and Discussions:: We used LC/MS-MS to perform a differential lipidomic analysis of the Arachidonic Acid (AA), Docosahexaenoic Acid (DHA), and Eicosapentaenoic Acid (EPA) metabolic pathways before and after a 3mm quadriceps VML injury. Figure 1A shows changes in absolute concentration of select DHA metabolites over a 14-day time period post-injury. There was an overall increase in AA-derived prostaglandins and leukotrienes 1 dpi compared to uninjured control, which returned to uninjured levels by day 3. Despite a significant increase in total DHA at 1 and 3 dpi compared to pre-injury, the specialized pro-resolving mediators (SPMs) resolvin D3, maresin 1, and 17R-resolvin D3 had a significantly decreased concentration in the muscle at 7 and/or 14 dpi (Figure 1A). These data suggest that while pro-inflammatory mediator biosynthesis can resolve after VML similarly to smaller injuries, there is impaired enzymatic conversion of DHA into its respective SPMs, which can help drive the sustained presence of pro-inflammatory immune cells in the injury niche .

Lipidomic analysis of AA, DHA, and EPA metabolites was performed 3 and 7 days after VML injury and subsequent treatment with either an unloaded or AT-RvD1-loaded PEG-4MAL hydrogel. We detected significant increases in the total concentration of SPMs (Figure 1C), including D- and E-series resolvins, maresins, and lipoxins 3 days post-VML in the AT-RvD1-treated group compared to the control hydrogel group. Flow cytometry data was analyzed via SPADE. These results show that local AT-RvD1 treatment significantly reduces the total population of CD11b+LY6G+ neutrophils (Figure 1D), while also significantly decreasing the frequency of CD62Lhi aged neutrophils in the muscle 3 days post-VML compared to control hydrogel. Furthermore, pro-resolving hydrogel treatment was found to significantly decrease the concentration of CD206-Ly6Chi M1 macrophages at day 3 and significantly increase the concentration of CD206+Ly6Clo M2 macrophages (Figure 1E) and CD11b+CD64+Mertk-Ly6Clo non-classical monocytes at day 7 post-injury versus the control hydrogel group.

These results suggest that early treatment of a VML injury with AT-RvD1 locally released from a PEG-4MAL hydrogel stimulates both molecular and cellular pathways of inflammation resolution, which could improve functional outcomes for patients by reducing fibrosis and improving myogenesis within the injured muscle.

Acknowledgements (Optional): :

References (Optional): :