Topical Administration of Heat-Killed Enterococcus faecalis Strain KH2 Promotes Re-Epithelialization and Granulation Tissue Formation during Skin Wound-Healing

Lactic acid bacteria (LAB) are known to have beneficial effects on immune responses when they are orally administered as bacterial products. Although the beneficial effects of LAB have been reported for the genera Lactobacillus and Lactococcus, little has been uncovered on the effects of the genus Enterococcus on skin wound-healing. In this study, we aimed to clarify the effect of heat-killed Enterococcus faecalis KH2 (heat-killed KH2) strain on the wound-healing process and to evaluate the therapeutic potential in chronic skin wounds. We analyzed percent wound closure, re-epithelialization, and granulation area, and cytokine and growth factor production. We found that heat-killed KH2 contributed to the acceleration of re-epithelialization and the formation of granulation tissue by inducing tumor necrosis factor-α, interleukin-6, basic fibroblast growth factor, transforming growth factor (TGF)-β1, and vascular endothelial growth factor production. In addition, heat-killed KH2 also improved wound closure, which was accompanied by the increased production of TGF-β1 in diabetic mice. Topical administration of heat-killed KH2 might have therapeutic potential for the treatment of chronic skin wounds in diabetes mellitus. In the present study, we concluded that heat-killed KH2 promoted skin wound-healing through the formation of granulation tissues and the production of inflammatory cytokines and growth factors.

Lymphatic-specific intracellular modulation of receptor tyrosine kinase signaling improves lymphatic growth and function

Exogenous administration of lymphangiogenic growth factors is widely used to study changes in lymphatic function in pathophysiology. However, this approach can result in off-target effects, thereby generating conflicting data. To circumvent this issue, we modulated intracellular VEGF-C signaling by conditionally knocking out the lipid phosphatase PTEN using the Vegfr3 promoter to drive the expression of Cre-lox in lymphatic endothelial cells (LECs). PTEN is an intracellular brake that inhibits the downstream effects of the activation of VEGFR3 by VEGF-C. Activation of Cre-lox recombination in adult mice resulted in an expanded functional lymphatic network due to LEC proliferation that was independent of lymphangiogenic growth factor production. Furthermore, compared with lymphangiogenesis induced by VEGF-C injection, LECPTEN animals had mature, nonleaky lymphatics with intact cell-cell junctions and reduced local tissue inflammation. Last, compared with wild-type or VEGF-C–injected mice, LECPTEN animals had an improved capacity to resolve inflammatory responses. Our findings indicate that intracellular modulation of lymphangiogenesis is effective in inducing functional lymphatic networks and has no off-target inflammatory effects.

Mechanisms of Macrophage Plasticity in the Tumor Environment: Manipulating Activation State to Improve Outcomes

Macrophages are a specialized class of innate immune cells with multifaceted roles in modulation of the inflammatory response, homeostasis, and wound healing. While developmentally derived or originating from circulating monocytes, naïve macrophages can adopt a spectrum of context-dependent activation states ranging from pro-inflammatory (classically activated, M1) to pro-wound healing (alternatively activated, M2). Tumors are known to exploit macrophage polarization states to foster a tumor-permissive milieu, particularly by skewing macrophages toward a pro-tumor (M2) phenotype. These pro-tumoral macrophages can support cancer progression by several mechanisms including immune suppression, growth factor production, promotion of angiogenesis and tissue remodeling. By preventing the adoption of this pro-tumor phenotype or reprogramming these macrophages to a more pro-inflammatory state, it may be possible to inhibit tumor growth. Here, we describe types of tumor-derived signaling that facilitate macrophage reprogramming, including paracrine signaling and activation of innate immune checkpoints. We also describe intervention strategies targeting macrophage plasticity to limit disease progression and address their implications in cancer chemo- and immunotherapy.

Abnormalities in reparative function of lung-derived mesenchymal stromal cells in emphysema

Mesenchymal stromal cells (MSCs) may provide crucial support in the regeneration of destructed alveolar tissue (emphysema) in COPD. We hypothesized that lung-derived MSCs (LMSCs) from emphysema patients are hampered in their repair capacity, either intrinsically or due to their interaction with the damaged micro-environment. LMSCs were isolated from lung tissue of controls and severe emphysema patients, and characterized at baseline. Additionally, LMSCs were seeded onto control and emphysematous decellularized lung tissue scaffolds and assessed for deposition of extracellular matrix (ECM). We observed no differences in surface markers, differentiation/proliferation potential and expression of ECM genes between control- and COPD-derived LMSCs. Notably, COPD-derived LMSCs displayed lower expression of FGF10 and HGF mRNA, and HGF and decorin protein. When seeded on control decellularized lung tissue scaffolds, control and COPD-derived LMSCs showed no differences in engraftment, proliferation or survival within 2 weeks, with similar ability to deposit new matrix on the scaffolds. Moreover, LMSC numbers and ability to deposit new matrix was not compromised on emphysematous scaffolds. Collectively, our data show that LMSCs from COPD patients compared to controls show less expression of FGF10 mRNA, HGF mRNA and protein and decorin protein, while other features including the mRNA expression of various ECM molecules are unaffected. Furthermore, COPD-derived LMSCs are capable of engraftment, proliferation and functioning on native lung tissue scaffolds. The damaged, emphysematous micro-environment as such does not hamper the potential of LMSCs. Thus, specific intrinsic deficiencies in growth factor production by diseased LMSCs may contribute to impaired alveolar repair in emphysema.

Toll-like receptor 5-mediated signaling enhances liver regeneration in mice

Background Toll-like receptor 5 (TLR5)-mediated pathways play critical roles in regulating the hepatic immune response and show hepatoprotective effects in mouse models of hepatic diseases. However, the role of TLR5 in experimental models of liver regeneration has not been reported. This study aimed to investigate the role of TLR5 in partial hepatectomy (PHx)-induced liver regeneration. Methods We performed 2/3 PHx in wild-type (WT) mice, TLR5 knockout mice, or TLR5 agonist CBLB502 treated mice, as a model of liver regeneration. Bacterial flagellin content was measured with ELISA, and hepatic TLR5 expression was determined with quantitative PCR analyses and flow cytometry. To study the effects of TLR5 on hepatocyte proliferation, we analyzed bromodeoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression with immunohistochemistry (IHC) staining. The effects of TLR5 during the priming phase of liver regeneration were examined with quantitative PCR analyses of immediate early gene mRNA levels, and with Western blotting analysis of hepatic NF-κB and STAT3 activation. Cytokine and growth factor production after PHx were detected with real-time PCR and cytometric bead array (CBA) assays. Oil Red O staining and hepatic lipid concentrations were analyzed to examine the effect of TLR5 on hepatic lipid accumulation after PHx. Results The bacterial flagellin content in the serum and liver increased, and the hepatic TLR5 expression was significantly up-regulated in WT mice after PHx. TLR5-deficient mice exhibited diminished numbers of BrdU- and PCNA-positive cells, suppressed immediate early gene expression, and decreased cytokine and growth factor production. Moreover, PHx-induced hepatic NF-κB and STAT3 activation was inhibited in Tlr5−/− mice, as compared with WT mice. Consistently, the administration of CBLB502 significantly promoted PHx-mediated hepatocyte proliferation, which was correlated with enhanced production of proinflammatory cytokines and the recruitment of macrophages and neutrophils in the liver. Furthermore, Tlr5−/− mice displayed significantly lower hepatic lipid concentrations and smaller Oil Red O positive areas than those in control mice after PHx. Conclusion We reveal that TLR5 activation contributes to the initial events of liver regeneration after PHx. Our findings demonstrate that TLR5 signaling positively regulates liver regeneration and suggest the potential of TLR5 agonist to promote liver regeneration.

Development of stabilized dual growth factor-loaded hyaluronate collagen dressing matrix

Patients with diabetes experience impaired growth factor production such as epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), and they are reportedly involved in wound healing processes. Here, we report dual growth factor-loaded hyaluronate collagen dressing (Dual-HCD) matrix, using different ratios of the concentration of stabilized growth factors—stabilized-EGF (S-EGF) and stabilized-bFGF (S-bFGF). At first, the optimal concentration ratio of S-EGF to S-bFGF in the Dual-HCD matrix is determined to be 1:2 in type I diabetic mice. This Dual-HCD matrix does not cause cytotoxicity and can be used in vivo. The wound-healing effect of this matrix is confirmed in type II diabetic mice. Dual HCD enhances angiogenesis which promotes wound healing and thus, it shows a significantly greater synergistic effect than the HCD matrix loaded with a single growth factor. Overall, we conclude that the Dual-HCD matrix represents an effective therapeutic agent for impaired diabetic wound healing.

Multimodal imaging in subclinical best vitelliform macular dystrophy

BackgroundTo analyse multimodal imaging alterations in the subclinical form of best vitelliform macular dystrophy (BVMD).MethodsThe study was designed as an observational, cross-sectional case series. Eleven eyes of 7 subclinical patients with BVMD and 12 age-matched and sex-matched controls were included. Multimodal imaging included fundus blue-light autofluorescence, near-infrared autofluorescence (NIR-AF), structural optical coherence tomography (OCT) and OCT angiography (OCTA). The quantitative analysis included the calculation of the following parameters: vessel density (VD), vessel tortuosity (VT), vessel dispersion (Vdisp), vessel rarefaction (VR), foveal avascular zone (FAZ) area, reflectivity of the outer retinal bands and choriocapillaris porosity (CCP).ResultsMean best-corrected visual acuity was 0.0±0.0 LogMAR in both groups. The round central hypoautofluorescent alteration on NIR-AF corresponded to a significant reflectivity attenuation of the outer retinal bands on structural OCT (0.55±0.18 vs 0.75±0.08; p<0.001). VD, VT, VR and Vdisp were normal compared with controls (all p>0.05). The FAZ area turned out to be significantly restricted at the level of the deep capillary plexus in subclinical BVMD eyes (p<0.001). Furthermore, quantitative OCTA revealed a significant central increase of CCP, compared with controls (18.25±2.43 vs 4.58±1.36; p<0.001).ConclusionsThe subclinical stage of BVMD is characterised by significant alterations of the outer retinal bands and the choriocapillaris. Quantitative multimodal imaging assessment suggests that subclinical BVMD is affected by the functional impairment of the outer retinal structures, leading to an alteration in melanin and growth factor production.

Gemcitabine and Rapamycin Exhibit Additive Effect against Osteosarcoma by Targeting Autophagy and Apoptosis

The overall prognosis for sarcoma-based cancer patients has remained largely unchanged over the past 10 years. Because there is no effective anticancer drug for patients with chemoresistant osteosarcoma (OS), novel approaches are needed to improve the prognosis. Here, we investigated whether rapamycin (Rapa) could enhance the anti-tumor effects of gemcitabine (Gem) in OS. Gem dose-dependently killed the OS cells, but exhibited much lower cytotoxicity on osteoblasts. Treatment with a combination Gem and Rapa was much more effective than that of either single agent with respect to reducing cell viability, cell invasion, cell migration, and vascular endothelial growth factor production in vitro. Moreover, the combination of these agents suppressed tumor growth, angiogenesis, and lung metastasis in allograft and xenograft murine models of OS with minimal adverse effects. Overall, the combination therapy prolonged the overall survival of tumor-bearing mice. Mechanistically, Gem induced apoptosis and increased the levels of cleaved caspases, while Rapa induced autophagy and microtubule-associated protein light chain 3 (LC3)-I/LC3-II expression both in vitro and in vivo. Our findings suggest that chemotherapy using Gem combined with Rapa may be a novel and promising therapeutic approach for the treatment of OS.

Glucose-Regulated Protein 94 (GRP94): A Novel Regulator of Insulin-Like Growth Factor Production

Mammals have two insulin-like growth factors (IGF) that are key mediators of somatic growth, tissue differentiation, and cellular responses to stress. Thus, the mechanisms that regulate the bioavailability of IGFs are important in both normal and aberrant development. IGF-I levels are primarily controlled via the growth hormone-IGF axis, in response to nutritional status, and also reflect metabolic diseases and cancer. One mechanism that controls IGF bioavailablity is the binding of circulating IGF to a number of binding proteins that keep IGF in a stable, but receptor non-binding state. However, even before IGF is released from the cells that produce it, it undergoes an obligatory association with a ubiquitous chaperone protein, GRP94. This binding is required for secretion of a properly folded, mature IGF. This chapter reviews the known aspects of the interaction and highlights the specificity issues yet to be determined. The IGF–GRP94 interaction provides a potential novel mechanism of idiopathic short stature, involving the obligatory chaperone and not just IGF gene expression. It also provides a novel target for cancer treatment, as GRP94 activity can be either inhibited or enhanced.