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Adipose Stem Cells Mitigate Profibrotic, Proinflammatory Vascular Endothelial Cell Phenotype And Reduce Innate Immune Activation
Alexander G. Stavros, BS1, Hengyun Sun, MD, PhD2, Jeffrey A. Gusenoff, MD3, J. Peter Rubin, MD3, Lauren E. Kokai, PhD3.
1University of Pittsburgh School of Medicine, Pittsburgh, PA, USA, 2Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China, 3University of Pittsburgh Department of Plastic Surgery, Pittsburgh, PA, USA.

Purpose: CD47 is a ubiquitously expressed surface-membrane protein that acts as a marker of self and serves as a ligand for the myeloid immunoreceptor signal regulatory protein alpha (SIRPα). Compelling literature suggests that upon SIRPα ligation, the conformation of the extracellular IgG region of CD47 initiates either phagocytic activation or inactivation in macrophages, relaying an "eat me" / "don't eat me" signal. This is a well-established mechanism through which aged erythrocytes, which highly express the 2D3 pro-phagocytic CD47 conformation, are cleared by macrophages in the spleen. The goal of this study was to determine if endothelial cells also undergo CD47 conformational changes with cell stress and express the pro-phagocytic 2D3 epitope of CD47, which has never been demonstrated before. Further, adipose stem cells reside in a vascular niche and are often shown as immunomodulators with potent anti-inflammatory effects. Therefore, we compared indirect and direct in vitro co-culture of adipose stem cells (ASCs) with stress-induced, CD47-2D3 expressing endothelial cells and measured matrix protein secretion indicative of fibrosis and SIRPα binding. The long term goal for this research is to determine if the CD47- SIRPα signaling mechanism could be therapeutically switched into the "off" position during graft ischemia/reperfusion to reduce macrophage binding and fibrosis in free-flap vasculature.
Methods: Human umbilical vein endothelial cells (HUVECs) were cultured and treated with ionomycin and PMA to simulate an oxidative environment; the consequence of this environment on CD47 conformation, THSP1 protein secretion, and SIRPα binding capacity was analyzed. Additionally, stromal vascular fraction (SVF) was isolated from adipose tissue of donors, and flow cytometry was performed to quantify the CD45-CD31-CD34+ ASC cell population, as well as the CD47 conformation-independent and -dependent positivity on CD45- CD31+ cells. These cell populations were compared to donor demographic characteristics.
Results: In vitro, HUVECs cultured in an oxidative environment were found to express conformation-dependent CD47-2D3 at a higher rate than HUVECs in an untreated culture environment (p < 0.05). Moreover, THSP1 expression and SIRPα binding capacity was increased (p <0.05) as a consequence of oxidative stress. In human adipose, donor age was found to correlate with an increase in the CD31+CD47-2D3 pro-inflammatory cell population (p 0.4513). Furthermore, it was found that the size of the ASC cell population yielded from donor SVF was inversely correlated with age (p 0.1717). Together, ASCs were shown to be inversely correlated with the overall population of CD31+CD47-2D3 cells (p 0.0795).
Conclusion: Inflammation induces a conformational change in the CD47 signaling complex (CD47-2D3) that enhances SIRPα binding while also facilitating binding of thrombospondin-1 (THSP1), a proinflammatory extracellular matrix protein. Together, this CD47-THSP1/SIRPα complex promotes macrophage activation toward a pro-phagocytic phenotype. In endothelial cells in-vitro, oxidative stress potentiates the expression of pro-inflammatory markers via the CD47-2D3/THSP1/SIRPα complex. In human adipose, increasing age is associated with increased CD47-2D3 conformation and a decreased total ASC population. Therefore, adipose stem cells may have important immune functions in fat by decreasing CD47-2D3 facilitated inflammation leading to a ‘healthier' adipose tissue microenvironment.


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