FAK, vinculin, and talin control mechanosensitive YAP nuclear localization.

Focal adhesions (FAs) are nanoscale complexes containing clustered integrin receptors and intracellular structural and signaling proteins that function as principal sites of mechanotransduction in part via promoting the nuclear translocation and activation of the transcriptional coactivator yes-associated protein (YAP). Knockdown of FA proteins such as focal adhesion kinase (FAK), talin, and vinculin can prevent YAP nuclear localization. However, the mechanism(s) of action remain poorly understood. Herein, we investigated the role of different functional domains in vinculin, talin, and FAK in regulating YAP nuclear localization. Using genetic or pharmacological inhibition of fibroblasts and human mesenchymal stem cells (hMSCs) adhering to deformable substrates, we find that disruption of vinculin-talin binding versus talin-FAK binding reduces YAP nuclear localization and transcriptional activity via different mechanisms. Disruption of vinculin-talin binding or knockdown of talin-1 reduces nuclear size, traction forces, and YAP nuclear localization. In contrast, disruption of the talin binding site on FAK or elimination of FAK catalytic activity did not alter nuclear size yet still prevented YAP nuclear localization and activity. These data support both nuclear tension-dependent and independent models for matrix stiffness-regulated YAP nuclear localization. Our results highlight the importance of vinculin-talin-FAK interactions at FAs of adherent cells, controlling YAP nuclear localization and activity.

Resolvin E1 is a pro-repair molecule that promotes intestinal epithelial wound healing.

Resolution of intestinal inflammation and wound repair are active processes that mediate epithelial healing at mucosal surfaces. Lipid molecules referred to as specialized proresolving mediators (SPMs) play an important role in the restorative response. Resolvin E1 (RvE1), a SPM derived from omega-3 fatty acids, has been reported to dampen intestinal inflammation by promoting anti-inflammatory responses including increased neutrophil spherocytosis and macrophage production of IL-10. Despite these observations, a role for RvE1 in regulating intestinal epithelial cell migration and proliferation during mucosal wound repair has not been explored. Using an endoscopic biopsy-based wound healing model, we report that RvE1 is locally produced in response to intestinal mucosal injury. Exposure of intestinal epithelial cells to RvE1 promoted wound repair by increasing cellular proliferation and migration through activation of signaling pathways including CREB, mTOR, and Src-FAK. Additionally, RvE1-triggered activation of the small GTPase Rac1 led to increased intracellular reactive oxygen species (ROS) production, cell-matrix adhesion, and cellular protrusions at the leading edge of migrating cells. Furthermore, in situ administration of RvE1-encapsulated synthetic targeted polymeric nanoparticles into intestinal wounds promoted mucosal repair. Together, these findings demonstrate that RvE1 functions as a prorepair lipid mediator by increasing intestinal epithelial cell migration and proliferation, and highlight potential therapeutic applications for this SPM to promote mucosal healing in the intestine.

Desmocollin-2 promotes intestinal mucosal repair by controlling integrin-dependent cell adhesion and migration.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a tight barrier, separating luminal antigens and microbes from underlying tissue compartments. Mucosal damage results in a compromised epithelial barrier that can lead to excessive immune responses as observed in inflammatory bowel disease. Efficient wound repair is critical to reestablish the mucosal barrier and homeostasis. Intestinal epithelial cells (IEC) exclusively express the desmosomal cadherins, Desmoglein-2 and Desmocollin-2 (Dsc2) that contribute to mucosal homeostasis by strengthening intercellular adhesion between cells. Despite this important property, specific contributions of desmosomal cadherins to intestinal mucosal repair after injury remain poorly investigated in vivo. Here we show that mice with inducible conditional knockdown (KD) of Dsc2 in IEC (Villin-CreERT2; Dsc2 fl/fl) exhibited impaired mucosal repair after biopsy-induced colonic wounding and recovery from dextran sulfate sodium-induced colitis. In vitro analyses using human intestinal cell lines after KD of Dsc2 revealed delayed epithelial cell migration and repair after scratch-wound healing assay that was associated with reduced cell-matrix traction forces, decreased levels of integrin ß1 and ß4, and altered activity of the small GTPase Rap1. Taken together, these results demonstrate that epithelial Dsc2 is a key contributor to intestinal mucosal wound healing in vivo.

Integrin-specific hydrogels modulate transplanted human bone marrow-derived mesenchymal stem cell survival, engraftment, and reparative activities.

Stem cell therapies are limited by poor cell survival and engraftment. A hurdle to the use of materials for cell delivery is the lack of understanding of material properties that govern transplanted stem cell functionality. Here, we show that synthetic hydrogels presenting integrin-specific peptides enhance the survival, persistence, and osteo-reparative functions of human bone marrow-derived mesenchymal stem cells (hMSCs) transplanted in murine bone defects. Integrin-specific hydrogels regulate hMSC adhesion, paracrine signaling, and osteoblastic differentiation in vitro. Hydrogels presenting GFOGER, a peptide targeting α2ß1 integrin, prolong hMSC survival and engraftment in a segmental bone defect and result in improved bone repair compared to other peptides. Integrin-specific hydrogels have diverse pleiotropic effects on hMSC reparative activities, modulating in vitro cytokine secretion and in vivo gene expression for effectors associated with inflammation, vascularization, and bone formation. These results demonstrate that integrin-specific hydrogels improve tissue healing by directing hMSC survival, engraftment, and reparative activities.

TNFα promotes mucosal wound repair through enhanced platelet activating factor receptor signaling in the epithelium.

Pathobiology of several chronic inflammatory disorders, including ulcerative colitis and Crohn's disease is related to intermittent, spontaneous injury/ulceration of mucosal surfaces. Disease morbidity has been associated with pathologic release of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). In this report, we show that TNFα promotes intestinal mucosal repair through upregulation of the GPCR platelet activating factor receptor (PAFR) in the intestinal epithelium. Platelet activating factor (PAF) was increased in healing mucosal wounds and its engagement with epithelial PAFR leads to activation of epidermal growth factor receptor, Src and Rac1 signaling to promote wound closure. Consistent with these findings, delayed colonic mucosal repair was observed after administration of a neutralizing TNFα antibody and in mice lacking PAFR. These findings suggest that in the injured mucosa, the pro-inflammatory milieu containing TNFα and PAF sets the stage for reparative events mediated by PAFR signaling.

PEG hydrogel containing calcium-releasing particles and mesenchymal stromal cells promote vessel maturation.

The use of human mesenchymal stromal cells (hMSC) for treating diseased tissues with poor vascularization has received significant attention, but low cell survival has hampered its translation to the clinic. Bioglasses and glass-ceramics have also been suggested as therapeutic agents for stimulating angiogenesis in soft tissues, but these effects need further evaluation in vivo. In this study, calcium-releasing particles and hMSC were combined within a hydrogel to examine their vasculogenic potential in vitro and in vivo. The particles provided sustained calcium release and showed proangiogenic stimulation in a chorioallantoic membrane (CAM) assay. The number of hMSC encapsulated in a degradable RGD-functionalized PEG hydrogel containing particles remained constant over time and IGF-1 release was increased. When implanted in the epidydimal fat pad of immunocompromised mice, this composite material improved cell survival and stimulated vessel formation and maturation. Thus, the combination of hMSC and calcium-releasing glass-ceramics represents a new strategy to achieve vessel stabilization, a key factor in the revascularization of ischemic tissues. STATEMENT OF SIGNIFICANCE: Increasing blood vessel formation in diseased tissues with poor vascularization is a current clinical challenge. Cell therapy using human mesenchymal stem cells has received considerable interest, but low cell survival has hampered its translation to the clinic. Bioglasses and glass-ceramics have been explored as therapeutic agents for stimulating angiogenesis in soft tissues, but these effects need further evaluation in vivo. By incorporating both human mesenchymal stem cells and glass-ceramic particles in an implantable hydrogel, this study provides insights into the vasculogenic potential in soft tissues of the combined strategies. Enhancement of vessel formation and maturation supports further investigation of this strategy.