Substance P (SP) is a neuropeptide, predominantly released from sensory nerve fibers, with a potentially protective role in diabetic corneal epithelial wound healing. However, the molecular mechanism remains unclear. We investigated the protective mechanism of SP against hyperglycemia-induced corneal epithelial wound healing defects, using type 1 diabetic mice and high glucose–treated corneal epithelial cells. Hyperglycemia induced delayed corneal epithelial wound healing, accompanied by attenuated corneal sensation, mitochondrial dysfunction, and impairments of Akt, epidermal growth factor receptor (EGFR), and Sirt1 activation, as well as decreased reactive oxygen species (ROS) scavenging capacity. However, SP application promoted epithelial wound healing, recovery of corneal sensation, improvement of mitochondrial function, and reactivation of Akt, EGFR, and Sirt1, as well as increased ROS scavenging capacity, in both diabetic mouse corneal epithelium and high glucose–treated corneal epithelial cells. The promotion of SP on diabetic corneal epithelial healing was completely abolished by a neurokinin-1 (NK-1) receptor antagonist. Moreover, the subconjunctival injection of NK-1 receptor antagonist also caused diabetic corneal pathological changes in normal mice. In conclusion, the results suggest that SP-NK-1 receptor signaling plays a critical role in the maintenance of corneal epithelium homeostasis, and that SP signaling through the NK-1 receptor contributes to the promotion of diabetic corneal epithelial wound healing by rescued activation of Akt, EGFR, and Sirt1, improvement of mitochondrial function, and increased ROS scavenging capacity.

This study provided the first evidence of TSG-6 secreted by MSCs promoting corneal epithelial wound healing in diabetic mice through activating corneal epithelial stem/progenitor cells and accelerating M2 macrophage polarization.

Human mesenchymal stem cells (hMSCs) are currently investigated for a variety of therapeutic applications. However, MSCs isolated from primary tissue cannot meet clinical grade needs and should be expanded in vitro for several passages. Although hMSCs show low possibility for undergoing oncogenic transformation, they do, similar to other somatic cells, undergo cellular senescence and their therapeutic potential is diminished when cultured in vitro. However, the role of senescent MSCs in tumor progression remains largely elusive. In the current study, by establishing senescent human umbilical cord mesenchymal stem cells (s-UCMSCs) through the replicative senescence model and genotoxic stress induced premature senescence model, we show that s-UCMSCs significantly stimulate proliferation and migration of breast cancer cells in vitro and tumor progression in a co-transplant xenograft mouse model compared with ‘young’ counterparts (defined as MSCs at passage 5, in contrast to senescent MSCs at passage 45). In addition, we identified IL-6, a known pleiotropic cytokine, as a principal mediator for the tumor-promoting activity of s-UCMSCs by induction of STAT3 phosphorylation. Depletion of IL-6 from s-UCMSCs conditioned medium partially abrogated the stimulatory effect of s-UCMSCs on the proliferation and migration of breast tumor cells.

Ciliary neurotrophic factor (CNTF), a well-known neuroprotective cytokine, has been found to play an important role in neurogenesis and functional regulations of neural stem cells. As one of the most innervated tissue, however, the role of CNTF in cornea epithelium remains unclear. This study was to explore the roles and mechanisms of CNTF in the activation of corneal epithelial stem/progenitor cells and wound healing of both normal and diabetic mouse corneal epithelium. In mice subjecting to mechanical removal of corneal epithelium, the corneal epithelial stem/progenitor cell activation and wound healing were promoted by exogenous CNTF application, while delayed by CNTF neutralizing antibody. In cultured corneal epithelial stem/progenitor cells, CNTF enhanced the colony-forming efficiency, stimulated the mitogenic proliferation, and upregulated the expression levels of corneal epithelial stem/progenitor cell-associated transcription factors. Furthermore, the promotion of CNTF on the corneal epithelial stem/progenitor cell activation and wound healing was mediated by the activation of STAT3. Moreover, in diabetic mice, the content of CNTF in corneal epithelium decreased significantly when compared with that of normal mice, and the supplement of CNTF promoted the diabetic corneal epithelial wound healing, accompanied with the advanced activation of corneal epithelial stem/progenitor cells and the regeneration of corneal nerve fibers. Thus, the capability of expanding corneal epithelial stem/progenitor cells and promoting corneal epithelial wound healing and nerve regeneration indicates the potential application of CNTF in ameliorating limbal stem cell deficiency and treating diabetic keratopathy. STEM CELLS 2015;33:1566-1576

Corneal epithelium expresses multiple neurotrophic factors, among which NGF and GDNF may play an important role in the corneal nerve regeneration.

Netrins are secreted chemoattractants with the roles in axon guidance, cell migration and epithelial plasticity. In the present study, we investigated the roles of netrin-1 in the regulation of corneal epithelial wound healing, inflammation response and nerve fiber regeneration in diabetic mice and cultured corneal epithelial cells. In diabetic mice, the expression of netrin-1 was decreased when compared with that of normal mice. Furthermore, high glucose blocked the wounding-induced up-regulation of netrin-1 expression in corneal epithelial cells. Exogenous netrin-1 promoted the corneal epithelial wound healing in diabetic mice, and facilitated the proliferation and migration by reactivating the phosphorylation of ERK and EGFR in high-glucose treated corneal epithelial cells. Moreover, netrin-1 decreased the neutrophil infiltration and promoted M2 macrophage transition, accompanied with the attenuated expression of pro-inflammatory factors in diabetic mouse corneal epithelium. The promotions of netrin-1 on corneal epithelial wound healing and inflammation resolution were mediated at least through the adenosine 2B receptor. In addition, netrin-1 promoted the regeneration of corneal nerve fibers that was impaired in diabetic mice. Taken together, netrin-1 regulates corneal epithelial wound healing, inflammation response and nerve fiber regeneration in diabetic mice, indicating the potential application for the therapy of diabetic keratopathy.

The present study was set out to address the therapeutic efficacy of human adipose tissue stem cells derived extracellular vesicles (hADSC-Evs) in a mouse model of dry eye disease and to investigate the underlying mechanisms involved. hADSC-Evs eye drops were topically administered to mice that subjected to desiccating stress (DS). Clinical parameters of ocular surface damage were assessed with fluorescein staining, tear production and PAS staining. For in vitro studies, cell viability assay and TUNEL staining were performed in human corneal epithelial cells (HCECs) treated with hADSC-Evs under hyperosmotic media. In addition, immunofluorescent staining, Real-time PCR (qRT-PCR) and Western blots were used to evaluated NLRP3, ASC, caspase-1, and IL-1β expression levels. Compared with vehicle control mice, topical hADSC-Evs treated mice showed decreased corneal epithelial defects, increased tear production, decreased goblet cell loss, as well as reduced inflammatory cytokines production. In vitro, hADSC-Evs could protect HCECs against hyperosmotic stress-induced cell apoptosis. Mechanistically, hADSC-Evs treatment suppressed the DS induced rises in NLRP3 inflammasome formation, caspase-1 activation and IL-1β maturation. In conclusion, hADSC-Evs eye drops effectively suppress NLRP3 inflammatory response and alleviate ocular surface damage in dry eye disease. Dry eye disease (DED) is a highly prevalent ocular surface disorder in the world 1. It is estimated that more than 16 million adults are diagnosed DED in US, and the prevalence in Asia is even higher than in western countries 2,3. According to the reports of Dry Eye Workshop (DEWS II), DED is a multifactorial disease that characterized by loss of homeostasis of the tear film 4. The tear film instability causes symptoms of discomfort, itching, eye irritation, glare and blurry vision, leading to a reduction in quality of life. Although the pathogenesis of DED is not yet fully understood, mounting evidence showed that the "vicious cycle of inflammation", including tear film instability, tear hyperosmolarity, apoptosis of cornea/conjunctiva and elevated levels of pro-inflammatory cytokines, play a core driver in its initiation and progression 5,6. Accordingly, most of the treatments to date are focused on reducing inflammation and restoring normal tear film 7. Mesenchymal stem cells (MSCs) are self-renewing multipotent stromal cells that can be isolated from mesenchymal tissues such as bone marrow, adipose, umbilical cord, as well as other tissues 8. Due to their immunomodulatory and trophic characteristics, MSC-based therapeutic intervention has been explored in a variety of immune-mediated disorders, including dry eye disease 9-12. Although most of the results were promising, safety issues regarding MSC-based therapy are still a matter of concern. Extracellular vesicles, with nanosized diameter of 30-150 nm, are known as intercellular communication mediators by transferring various bioactive molecules (proteins, lipids and RNAs) 13. Recent studies revealed that MS...

Mesenchymal stem/stromal cells (MSCs) possess some characteristics of immune cells, including a pro-inflammatory phenotype, an immunosuppressive phenotype, antibacterial properties and the expression of Toll-like receptor proteins. Here we show that, similar to immune cells, MSCs retain information from danger signals or environmental stimuli for a period of time. When treated with the pro-inflammatory factors lipopolysaccharide (LPS) or tumor necrosis factor-a (TNF-a), MSCs display increased expression of IL-6, IL-8 and MCP-1. Following re-plating and several rounds of cell division in the absence of stimulating factors, the expression of IL-6, IL-8 and MCP-1 remained higher than in untreated cells for over 7 days. A spike in cytokine secretion occurred when cells were exposed to a second round of stimulation. We primed MSCs with LPS and LPS-primed MSCs had better therapeutic efficacy at promoting skin flap survival in a diabetic rat model than did unprimed MSCs. Finally, we found that several microRNAs, including miR146a, miR150 and miR155, along with the modification of DNA by 5-hydroxymethylcytosine (5hmC), mediate the MSC response to LPS and TNF-a stimulation. Collectively, our data suggest that MSCs have a short-term memory of environmental signals, which may impact their therapeutic potential. Cellular & Molecular Immunology