1511 - Disruption of CD47 Signaling PromotesEndothelial Cell Proliferation but Inhibits Mesenchymal Stem Cell Expansion Leading to Delayed Fracture Healing.
Year: 2019
Type: other
Abstract: INTRODUCTION: CD47 is an integrin-associated protein that can interact with thrombospondin-1 (TSP1) and thrombospondin-2 (TSP2) which are extracellular matrix proteins known to have anti-angiogenic effects. CD47-null mice show enhanced recovery from various soft tissue injuries (e.g., ischemia, ischemic reperfusion injuries, radiation injuries). However, in addition to regulating angiogenesis, CD47 is implicated in regulating cell proliferation, motility, adhesion, and differentiation. Our previous findings of decreased callus formation in CD47-null mice with ischemic fracture were surprising and led us to speculate that there could be some dysfunction in the proliferation and/or differentiation of mesenchymal stem cells (MSC). To explore the role of CD47 in fracture healing in greater depth we disrupted CD47 at the time of surgeryu2014rather than whole body knockoutu2014and also investigated the cell autonomous role of CD47 by isolating MSC and endothelial cells (EC). We hypothesized that disruption of CD47 would negatively impact mesenchymal stem cells, while increasing EC proliferation.METHODS: All animal procedures were approved by the University of Michigan Institutional Animal Care and Use Committee. An ischemic fracture model was used to investigate the effect of disrupting CD47 on callus formation. The femoral artery was surgically ligated, and a simple transverse fracture was created in the distal third of the tibia using a custom guillotine device. At days 2 and 5 post-fracture, mice (n=4-8 per group) were injected with a morpholino (GeneTools) to disrupt the expression of CD47 (M-CD47) or a control morpholino (M-Ctrl). Mice tibia were harvested at day 10, 15, 20, and 40 post-fracture and scanned using u00b5CT. u00b5CT images were analyzed (MicroView) for callus length, callus volume, bone volume, bone volume fraction, bone mineral content, and bone mineral density. Following in vivo fracture experiments, the effect of disrupting CD47 in EC and MSC was studied. ECs and MSC were harvested from the lungs and long bones of 6-week-old wild type (WT) and CD47-null mice (n=8), respectively. EC cells were grown on 2% gelatin coated T75 flasks in angiogenic media (VascuLife EnGS). MSC were grown on uncoated T75 flasks in maintenance media (uf061MEM, 10% FBS, 1% L-glutamine, 1% Anti-Anti). Cells were allowed to proliferate for two weeks then trypsinized, passaged, and seeded at equal densities to assess viability, ALP activity, apoptosis, and gene expression. Cell viability was quantified in ECs and MSCs using an MTT assay (Millipore Sigma u2013 CT02) at day 1, 6, and 9 post-passage. In MSCs, ALP activity was quantified by calculating percent area positive ALP and at day 3 and 6 post-passage. Apoptosis was measured in MSCs at day 6 post-passage by immune staining for caspase3 and calculating percent positive cells. Gene expression for stem markers (OCT4, KLF, SOX2, MYC), proliferation markers (FOXm1, PLK1, E2F1), apoptosis (Caspase3), and ALP were quantified using qPCR at day 6. Statistical analysis was performed using open-source R. Within and between group differences were tested using students T-test. A significance level was set at p=0.05.RESULTS: In vivo, ischemic surgery decreased perfusion to the hind limb by 89.2% and 85.2% in the M-Ctrl and M-CD47 mice, respectively (p =0.31). At 10 days post-fracture, perfusion had improved similarly by 50.1% and 61.9% in M-Ctrl and M-CD47 mice, respectively (p=0.233). Four independent experiments were conducted with the morpholino. There was no difference in callus morphology at day 10 and 40, but there was a significant decrease in callus size at day 15 and 20. Here we show day 15 data relative to M-Ctrl, the M-CD47 group developed 79.1% less callus volume (p=0.024) and 67.2% less bone mineral (p=0.017). In vitro, disruption of CD47 significantly reduced MSC viability at day 1, 6, and 9 post-passage (p < 0.01) compared to WT controls. However, disruption of CD47 significantly increased EC viability at day 1 post-passage (p=0.011) with no difference seen at day 6 and 9 when compared to WT controls. In MSCs, there was no difference in caspase3 activity between WT and CD47-null cells. Alkaline phosphatase activity was 65.1% and 72.2% lower in the CD47-null cells compared to WT after 3 (p=0.003) and 6 (p=0.001) days post-passage, respectively (Fig. 3). Compared to WT MSCs, CD47-null cells demonstrated transcriptional downregulation of the proliferation markers FOXm1, PLK1, and E2F1 (p<0.05). Caspase3, ALP, and the stem marker MYC, were also downregulated in CD47-null cells (p<0.05). However, at day 6 post-passage, the stem marker KLF-4 was upregulated in CD47-null cells (p=0.001). There was no significant difference in the two other stem genes, OCT4 and SOX2.DISCUSSION: Results from this study suggest disruption of CD47 has a contradictory proliferative effect on MSCs and ECs. Disruption of CD47 has been used to improve revascularization in soft tissue wound models; however, in fractures, the beneficial effects of disrupting CD47 on angiogenesis are limited by its inhibitory proliferative effect on MSCs. Temporal disruption of CD47 during early ischemic fracture healing significantly disrupts the formation of a callus, but this difference recovers by day 40 post-fracture. Based on in vitro data, the lack of callus formation appears to result from a disruption in MSC proliferation rather than an increase in apoptosis. Exploiting pathways to promote revascularization of wounds and fractures should continue to be explored; however, the multifactorial role of CD47 in angiogenesis and proliferation in different cell types suggest that it is a suboptimal target, without significant further understanding of mechanism. Future studies should explore independent downstream CD47 signaling pathways (e.g., TSP/CD47 & SIRPuf061/CD47) that contribute to angiogenesis and MSC proliferation to identify potential therapeutic targets for improving fracture healing.SIGNIFICANCE/CLINICAL RELEVANCE: Disruption of CD47 has been shown to improve revascularization and healing in many tissues; however, its therapeutic use in fracture healing is contraindicated due to its inhibitory effect on MSC proliferation. ACKNOWLEDGEMENTS: RLZ is supported by NIH F30AR071201, KDH, RSM, JSI, JA are supported by NIH R01AR066028
Author: Robert Zondervan
Institutions: University of Michigan, Michigan State University
Language: English
Subfield: Epidemiology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W4391553261