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Nerve Growth Factor Derives From Pericytes And Smooth Muscle Cells After Extremity Trauma
Charles Hwang, BS1, Simone Marini, PhD1, Amanda K. Huber, PhD1, Seungyong Lee, PhD2, David M. Stepien, MD, PhD1, Carrie A. Kubiak, MD1, Carolyn Meyers, BS2, Michael Sorkin, MD1, Chase A. Pagani, BA1, Talis Rehse, BS1, Noelle D. Visser, MS1, Mohamed Ali Garada1, Husain Rasheed1, Joseph A. Greenstein1, Zaid N. Khatib, BS1, Prasanth Kotha1, Kaetlin Vasquez1, Jeffrey Lisiecki, MD1, Paul S. Cederna, MD1, Stephen W.P. Kemp, PhD1, Aaron W. James, MD, PhD2, Benjamin Levi, MD1.
1University of Michigan, Ann Arbor, MI, USA, 2Johns Hopkins University, Baltimore, MD, USA.

PURPOSE: Neurotrophic factors like nerve growth factor (NGF) have been described in the literature as a crucial regulator in developmental biology and inflammation of musculoskeletal tissues. While extremity trauma has been shown to elicit re-engagement of developmental programs like NGF upregulation and invasion of new sensory nerve endings in a mature organism, the source of NGF has remained largely unknown. In a validated extremity trauma mouse model, we have previously demonstrated a significant attenuation of injury site re-innervation by sensory fibers following inhibiting of afferent neural signaling and NGF binding of its receptor TrkA. Given this significant impact of NGF on sensory reinnervation of injured soft tissues and downstream bone formation, we examined the source of NGF after extremity trauma using single cell transcriptomic and reporter proteomic technologies. METHODS: A 30% dorsal burn and Achilles transection was performed. The tendon site tissues were harvested from baseline (t0) and day 3, 7, and 21 (n=3-4/group) after induction. Samples were prepared for library generation on a 10x Genomics Chromium Controller, sequenced on a Illumina HiSeq 4000, and analyzed with Cell Ranger Software for pre-processing and alignment to the mm10 genome. Downstream analyses including unsupervised clustering and canonical correlation analyses were performed with Seurat. Immunofluorescent (IF) labeling of cellular markers including αSMA was performed using NGF-eGFP reporter mice at baseline, and at 1,3 and 9 weeks after injury (n=2-3/group). RESULTS: To localize cell specific Ngf expression from injured soft tissue, 9 cell clusters were defined across all timepoints: mesodermal (Prrx1) populations including Acta2+ pericyte/vascular smooth muscle (SMC) and Pdgfra+ mesenchymal cells, two Pecam1+ endothelium, and four inflammatory cell populations (mixed, B cell, T cell, and neutrophil) (Fig. A,B). Ngf was found uniquely enriched in the pericyte/SMC cluster in a composite view (Fig. C). These pericytes/SMCs were found in increasing number and Ngf expression across timepoints, peaking at day 21 (Fig. D). To further characterize this joint cluster (1273 cells), the pericyte/SMC cluster was isolated and blindly re-clustered to produce new sub-clusters, distinguishing Pdgfrb (platelet derived growth factor receptor beta)high Prrx1high Abcc9+ pericytes from Acta2high Pln+ Myh11high SMCs; an uncharacterized cluster with 24 cells was discarded from analysis (Fig. E,F). Of these two sub-clusters, vascular SMCs demonstrated the highest Ngf expression at baseline (mean normalized Ngf expression of 0.52 [in 41% of cells] vs 0.28 [in 23% of cells], Fig. G). IF of NGF-eGFP reporter mice show robust NGF co-localization with αSMA+ SMCs (Fig. H). CONCLUSIONS: This is the first work characterizing the pericyte and vascular SMC as a major contributor to NGF signaling at an extremity injury site. The fine resolution defining the cellular source of NGF provides insight into potential mechanisms correlating nascent nerve and vascular growth given the intersection of these pathways at the level of pericytes and myofibroblastic SMCs that will inform future candidate therapeutics to improve extremity trauma healing and re-innervation.


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