The Secretome of Hydrogel-Coembedded Endothelial Progenitor Cells and Mesenchymal Stem Cells Instructs Macrophage Polarization in Endotoxemia

Abstract

We previously reported the delivery of endothelial progenitor cells (EPCs) embedded in hyaluronic acid-based (HA)-hydrogels protects renal function during acute kidney injury (AKI) and promotes angiogenesis. We attempted to further ameliorate renal dysfunction by coembedding EPCs with renal mesenchymal stem cells (MSCs), while examining their paracrine influence on cytokine/chemokine release and proinflammatory macrophages. A live/dead assay determined whether EPC-MSC coculturing improved viability during lipopolysaccharide (LPS) treatment, and HA-hydrogel-embedded delivery of cells to LPS-induced AKI mice was assessed for effects on mean arterial pressure (MAP), renal blood flow (RBF), circulating cytokines/chemokines, serum creatinine, proteinuria, and angiogenesis (femoral ligation). Cytokine/chemokine release from embedded stem cells was examined, including effects on macrophage polarization and release of proinflammatory molecules. EPC-MSC coculturing improved stem cell viability during LPS exposure, an effect augmented by MSC hypoxic preconditioning. The delivery of coembedded EPCs with hypoxic preconditioned MSCs to AKI mice demonstrated additive improvement (compared with EPC delivery alone) in medullary RBF and proteinuria, with comparable effects on serum creatinine, MAP, and angiogenesis. Exposure of proinflammatory M1 macrophages to EPC-MSC conditioned medium changed their polarization to anti-inflammatory M2. Incubation of coembedded EPCs-MSCs with macrophages altered their release of cytokines/chemokines, including enhanced release of anti-inflammatory interleukin (IL)-4 and IL-10. EPC-MSC delivery to endotoxemic mice elevated the levels of circulating M2 macrophages and reduced the circulating cytokines/chemokines. In conclusion, coembedding EPCs-MSCs improved their resistance to stress, impelled macrophage polarization from M1 to M2 while altering their cytokine/chemokines release, reduced circulating cytokines/chemokines, and improved renal and vascular function when MSCs were hypoxically preconditioned. Significance This report provides insight into a new therapeutic approach for treatment of sepsis and provides a new and improved strategy using hydrogels for the delivery of stem cells to treat sepsis and, potentially, other injuries and/or diseases. The delivery of two different stem cell lines (endothelial progenitor cells and mesenchymal stem cells; delivered alone and together) embedded in a protective bioengineered scaffolding (hydrogel) offers many therapeutic benefits for the treatment of sepsis. This study shows how hydrogel-delivered stem cells elicit their effects and how hydrogel embedding enhances the therapeutic efficacy of delivered stem cells. Hydrogel-delivered stem cells influence the components of the overactive immune system during sepsis and work to counterbalance the release of many proinflammatory and prodamage substances from immune cells, thereby improving the associated vascular and kidney damage.

The cell secretome, a mediator of cell-to-cell communication

“…Our own observations (10) supplement this data with findings obtained in septic mice receiving a combination therapy with mesenchymal stem and endothelial progenitor cells, each acting via the secretome to elicit beneficial effect. The cyto-/chemokine release from embedded stem cells was examined including effects on modulating the polarization and release of proinflammatory molecules from macrophages (Fig.…”

“…Exposure of proinflammatory M1 macrophages to EPC-MSC conditioned culture medium changed their polarization to anti-inflammatory M2 macrophages, while EPC-MSC delivery to endotoxemic mice elevated levels of circulating M2 macrophages. Incubation of co-embedded EPC-MSC with macrophages altered their release of cyto-/chemokines including enhanced release of anti-inflammatory IL-10 from macrophages (10). …”

Dental Follicle Stem Cells Ameliorate Lipopolysaccharide-Induced Inflammation by Secreting TGF-β3 and TSP-1 to Elicit Macrophage M2 Polarization

“…Various soluble factors secreted by MSCs, including IL-4 [50], IL-13 [51], prostaglandin E2 (PGE2) [52], and tumor necrosis factor-α-induced gene/protein 6 (TSG-6) [53], have been shown to play a critical role in modulating inflammatory processes and macrophage polarization towards the M2 phenotype in animal models of sepsis [54], acute kidney injury [17], experimental colitis [18], spinal cord injury [19], and skin wounds [1]. To determine the key factors responsible for macrophage polarization towards the M2 phenotype induced by DFSC-CM, we performed a protein array, which revealed that rDFSCs could release many secreted proteins, including immunomodulatory factors, growth factors, cytokines, chemokines and neurotrophic factors, thus providing useful information for future studies.…”

“…Recently, MSCs have been shown to regulate immune responses by suppressing the proliferation and activation of T and B cells, and promote the generation of regulatory T cells, manifesting the immunosuppressive functions [13,14]. In addition, MSCs elicit a phenotypic switch from M1 to M2 macrophages in vitro [15,16] as well as in animal models of acute kidney injury [17], experimental colitis [18], spinal cord injury [19], and skin wounds [1]. MSCs affect neighboring immune cells primarily through direct cell-to-cell contact and/or various soluble factors [12][13][14]20].…”

The Secretome-Modified Endothelial Cell Microparticles: Novel Player in the Endothelium Reparation: The Role of Endothelial Cell-Derived Microparticles?

“…Indeed, the cell-toMPs' cooperation is depended on presentation of the surface of the vesicles and target cells appropriate recognizing antigens, which interact with secreted proteins and consequently regulate in different manner the tissue response via switching specific transmembrane signal systems to nuclear transcriptional factors (i.e., NK-kB). Recently it has been shown that the components of the secretome might be attributes of overactive immune system during antigen stimulation and probably act to counterbalance the release of many inflammatory and damageable substances, thereby improving the associated vascular injury and tissue damages [23].…”