Stress or injury induces cellular plasticity in salivary gland acinar cells.

Salivary gland function is severely disrupted by radiation therapy used to treat patients diagnosed with head and neck cancer and by Sjögren's syndrome. The resulting condition, which results in xerostomia or dry mouth, is due to irreversible loss of the secretory acinar cells within the major salivary glands. There are presently no treatments for the resolution of xerostomia. Cell-based approaches could be employed to repopulate acinar cells in the salivary gland but investigations into potential therapeutic strategies are limited by the challenges of maintaining and expanding acinar cells in vitro. We investigate the encapsulation of salivary gland cell aggregates within PEG hydrogels as a means of culturing secretory acinar cells. Lineage tracing was used to monitor the fate of acinar cells isolated from murine submandibular gland (SMG). Upon initial formation in vitro, SMG aggregates comprise both acinar and duct cells, with the majority cells of acinar origin. With longer culture times, acinar cells significantly decreased the expression of specific markers and activated the expression of keratins normally found in duct cells. A similar acinar-to-duct cell transition was also observed in vivo, following duct ligation injury. These results indicate that under conditions of stress (mechanical and enzymatic isolation from glands) or injury (duct ligation), salivary gland acinar cells exhibit plasticity to adopt a duct cell phenotype.

Encapsulation of primary salivary gland cells in enzymatically degradable poly(ethylene glycol) hydrogels promotes acinar cell characteristics.

Radiation therapy for head and neck cancers leads to permanent xerostomia due to the loss of secretory acinar cells in the salivary glands. Regenerative treatments utilizing primary submandibular gland (SMG) cells show modest improvements in salivary secretory function, but there is limited evidence of salivary gland regeneration. We have recently shown that poly(ethylene glycol) (PEG) hydrogels can support the survival and proliferation of SMG cells as multicellular spheres in vitro. To further develop this approach for cell-based salivary gland regeneration, we have investigated how different modes of PEG hydrogel degradation affect the proliferation, cell-specific gene expression, and epithelial morphology within encapsulated salivary gland spheres. Comparison of non-degradable, hydrolytically-degradable, matrix metalloproteinase (MMP)-degradable, and mixed mode-degradable hydrogels showed that hydrogel degradation by any mechanism is required for significant proliferation of encapsulated cells. The expression of acinar phenotypic markers Aqp5 and Nkcc1 was increased in hydrogels that are MMP-degradable compared with other hydrogel compositions. However, expression of secretory acinar proteins Mist1 and Pip was not maintained to the same extent as phenotypic markers, suggesting changes in cell function upon encapsulation. Nevertheless, MMP- and mixed mode-degradability promoted organization of polarized cell types forming tight junctions and expression of the basement membrane proteins laminin and collagen IV within encapsulated SMG spheres. This work demonstrates that cellularly remodeled hydrogels can promote proliferation and gland-like organization by encapsulated salivary gland cells as well as maintenance of acinar cell characteristics required for regenerative approaches. Investigation is required to identify approaches to further enhance acinar secretory properties. STATEMENT OF SIGNIFICANCE: Regenerative strategies to replace damaged salivary glands require the function and organization of acinar cells. Hydrogel-based approaches have shown promise to control cell function and phenotype. However, little is known about how specific parameters, such as the mechanism of hydrogel degradation (e.g., hydrolytic or enzymatic), influence the viability, proliferation, organization, and phenotype of salivary gland cells. In this work, it is shown that hydrogel-encapsulated primary salivary gland cell proliferation is dependent upon hydrogel degradation. Hydrogels crosslinked with enzymatically degradable peptides promoted the expression of critical acinar cell markers, which are typically downregulated in primary cultures. Furthermore, salivary gland cells encapsulated in enzymatically- but not hydrolytically-degradable hydrogels displayed highly organized and polarized salivary gland cell markers, which mimics characteristics found in native gland tissue. In sum, results indicate that salivary gland cells respond to cellularly remodeled hydrogels, resulting in self-assembly and organization akin to acini substructures of the salivary gland.

Influence of PECAM-1 ligand interactions on PECAM-1-dependent cell motility and filopodia extension.

Platelet endothelial cell adhesion molecule (PECAM-1) has been implicated in angiogenesis through processes that involve stimulation of endothelial cell motility. Previous studies suggest that PECAM-1 tyrosine phosphorylation mediates the recruitment and then activation of the tyrosine phosphatase SHP-2, which in turn promotes the turnover of focal adhesions and the extension of filopodia, processes critical to cell motility. While these studies have implicated PECAM-1-dependent signaling in PECAM-1-mediated cell motility, the involvement of PECAM-1 ligand binding in cell migration is undefined. Therefore to investigate the role of PECAM-1 binding interactions in cell motility, mutants of PECAM-1 were generated in which either homophilic or heparin/glycosaminoglycan (GAG)-mediated heterophilic binding had been disabled and then expressed in an endothelial cell surrogate. We found that the ability of PECAM-1 to stimulate cell migration, promote filopodia formation and trigger Cdc42 activation were lost if PECAM-1-dependent homophilic or heparin/GAG-dependent heterophilic ligand binding was disabled. We further observed that PECAM-1 concentrated at the tips of extended filopodia, an activity that was diminished if homophilic, but not heparin/GAG-mediated heterophilic binding had been disrupted. Similar patterns of activities were seen in mouse endothelial cells treated with antibodies that specifically block PECAM-1-dependent homophilic or heterophilic adhesion. Together these data provide evidence for the differential involvement of PECAM-1-ligand interactions in PECAM-1-dependent motility and the extension of filopodia.