Discovery of Lactoferrin as a Stimulant for hADSC-Derived EV Secretion and Proof of Enhancement of Resulting EVs through Skin Model.

Extracellular vesicles (EVs) are secreted from hADSCs in low concentrations, which makes it difficult to utilize them for the development of therapeutic products. To overcome the problem associated with low concentration, we proposed human lactoferrin (hLF) as a stimulant for the secretion of hADSC-derived EVs. hLF has been reported to upregulate intracellular Ca2+, which is known to be capable of increasing EV secretion. We cultured hADSCs in hLF-supplemented media and analyzed the changes in intracellular Ca2+ concentration. The characteristics of hADSC-derived EVs secreted by hLF stimulation were analyzed through their number, membrane protein markers, and the presence of hLFs to EVs. The function of hADSC-derived EVs was investigated through their effects on dermal fibroblasts. We found that hLF helped hADSCs effectively uptake Ca2+, resulting in an increase of EVs secretion by more than a factor of 4. The resulting EVs had enhanced proliferation and collagen synthesis effect on dermal fibroblasts when compared to the same number of hADSC-derived EVs secreted without hLF stimulation. The enhanced secretion of hADSC-derived EVs increased collagen synthesis through enhanced epidermal penetration, which resulted from increased EV numbers. In summary, we propose hLF to be a useful stimulant in increasing the secretion rate of hADSC-derived EVs.

In Vivo Electroporation Improves Retinal Delivery of Intravitreally Injected Exosomes.

Purpose: Mesenchymal stem cell (MSC)-derived exosomes are promising therapeutic agents and natural nanoscale delivery platforms for treating degenerative retinal diseases. This study investigated the effect of electroporation on the retinal delivery of intravitreally administered MSC-derived exosomes in a murine model. Methods: Exosomes isolated from adipose tissue-derived MSCs were stained with ExoGlow exosome-specific dye and administered to the right eyes of 40 Sprague-Dawley rats. Electroporation was performed in 20 rats immediately after intravitreal injection (electroporation group); 5 square pulses of 40 V/cm for 50 ms each with 950-ms intervals were administered. The remaining 20 rats were assigned to the no-electroporation group. The eyeballs were harvested 24 h later for evaluation. The total number of fluorescent particles per hyperfield was counted from the retinal flat mounts to quantify the retinal delivery of exosomes. Tissue damage after electroporation was evaluated using retinal histological sections and a terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labeling (TUNEL) assay. Results: A significantly higher number of fluorescent particles per hyperfield were observed in the retinal flat mounts of the electroporation group compared with that in the no-electroporation group (599.0 ± 307.5 vs. 376.9 ± 175.4; P = 0.013). Retinal histological sections and TUNEL assays showed no signs of tissue damage after electroporation. Conclusions: In vivo electroporation can improve the retinal delivery of intravitreally injected exosomes.

Adipose-Derived Stem Cell Exosomes Alleviate Psoriasis Serum Exosomes-Induced Inflammation by Regulating Autophagy and Redox Status in Keratinocytes.

Introduction: Exosomes play a key role in cell communication and are involved in both pathological and physiological processes. Autophagy dysfunction and oxidative stress are linked to immune-mediated inflammatory diseases such as psoriasis. Stem cell-derived exosomes exhibit immunomodulatory and antioxidant efficacy. Methods: We aimed to investigate the impact of psoriasis serum-derived exosomes on inflammation, oxidative stress, and autophagy in keratinocytes. Additionally, we explored the therapeutic potential of adipose-derived stem cell (ADSC) exosomes against inflammation induced by psoriasis serum exosomes. To validate psoriasis patient serum-derived exosomes and ADSC exosomes, we used nanoparticle tracking analysis, Western blotting, flow cytometry, and immunofluorescence. qPCR was used to study changes in the gene expression of proinflammatory cytokines and oxidative stress markers in HaCaT cells treated with psoriasis serum-derived exosomes or ADSC exosomes. The effects of these exosomes on autophagy in HaCaT cells were evaluated by Western blotting and immunofluorescence. Result: The treatment of HaCaT cells with psoriasis serum-derived exosomes increased proinflammatory cytokine production and oxidative stress-related factor (Nox2 and Nox4) expression and decreased Nrf2 expression via P65/NF-κB and P38/MAPK activation. Compared with healthy control serum-derived exosomes, psoriasis serum-derived exosomes decreased ATG5, P62, Beclin1, and LC3 expression and autophagosome production in HaCaT cells. Conversely, ADSC exosomes suppressed proinflammatory cytokine and oxidative stress production, and restored autophagy in HaCaT cells treated with psoriasis serum-derived exosomes. Discussion: These findings suggest that ADSC exosomes exhibit a suppressive effect on psoriasis serum exosome-induced inflammation and oxidative stress by regulating autophagy in keratinocytes.

Exosomes released by environmental pollutant-stimulated Keratinocytes/PBMCs can trigger psoriatic inflammation in recipient cells via the AhR signaling pathway.

Introduction: Exosomes, pivotal in intercellular communication during skin disease pathogenesis, have garnered substantial attention. However, the impact of environmental pollutants, such as benzo[a]pyrene (BaP) and 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), on exosome release amid inflammatory skin diseases remains unexplored. This study addresses this gap by examining the influence of BaP and TCDD on exosome function, specifically focusing on immune-related pathway alterations in normal recipient keratinocytes and peripheral blood mononuclear cells (PBMCs). Methods: HaCaT cells were treated with exosomes from BaP- or TCDD-treated keratinocytes. Proinflammatory cytokines and chemokines, including TNF-α, IL-1ß, IL-6, IL-8, CXCL1, and CXCL5, were assessed. The involvement of the p65NF-κB/p38MAPK/ERK signaling pathway in recipient keratinocytes was investigated. Aryl hydrocarbon receptor (AhR) silencing was employed to elucidate its role in mediating the proinflammatory response induced by exosomes from BaP- or TCDD-treated keratinocytes. Results and discussion: Treatment with exosomes from BaP- or TCDD-treated keratinocytes induced a significant increase in proinflammatory cytokines and chemokines in HaCaT cells. The upregulation implicated the p65NF-κB/p38MAPK/ERK signaling pathway. AhR silencing attenuated this response, suggesting a role for AhR in mediating this response. In PBMCs from healthy controls, exosomes from BaP-stimulated PBMCs of psoriatic patients led to increased expression of proinflammatory cytokines and modulation of Th1/Th17 cell distribution via AhR activation. These findings unveil a novel dimension in the interplay between environmental xenobiotic agents (BaP and TCDD) and exosomal functions. The study establishes their influence on psoriatic inflammatory responses, shedding light on the underlying mechanisms mediated through the AhR signaling pathway in recipient keratinocytes and PBMCs.

Identification of cellular responses to low-dose radiation by the profiling of phosphorylated proteins in human B-lymphoblast IM-9 cells.

PURPOSE: The aim of this study was to explore the potential for radiation-specific signaling of various LDIR-induced effects in human B-lymphoblast IM-9 cells. MATERIALS AND METHODS: Human lymphoblast IM-9 cells were exposed to ionizing radiation at 0.1 and 2 Gy using a 137Cs γ-irradiator at a dose rate of 0.8 Gy/min. Cell viability and DNA fragmentation were determined using MTT assay and TUNEL assay at 24 h after irradiation. Profiling of protein phosphorylation by radiation was identified using a phospho-antibody array at 4 h after irradiation and Dataset of the profiling was analyzed by IPA. RESULTS: Cell survival and apoptotic signaling were not affected by 0.1 Gy of radiation, whereas 2 Gy induced cellular damage. The analysis of low-dose ionizing radiation (LDIR) or high-dose ionizing radiation (HDIR)-specific responses by IPA generated different results. Various cell maintenance functions were only apparent following the analysis of increased protein phosphorylation by LDIR, whereas several cancer formation- and development-related functions were only detected following the analysis of increased protein phosphorylation by HDIR. CONCLUSIONS: The LDIR-induced protein phosphorylation patterns might be involved in various cell survival responses or cellular maintenance functions, which provide important insight into our understanding of the different effects of LDIR and HDIR.

Lipoteichoic acid isolated from Lactobacillus plantarum inhibits melanogenesis in B16F10 mouse melanoma cells.

Lipoteichoic acid (LTA) is a major component of the cell wall of Gram-positive bacteria. Its effects on living organisms are different from those of lipopolysaccharide (LPS) found in Gram-negative bacteria. LTA contributes to immune regulatory effects including anti-aging. In this study, we showed that LTA isolated from Lactobacillus plantarum (pLTA) inhibited melanogenesis in B16F10 mouse melanoma cells. pLTA reduced the cellular activity of tyrosinase and the expression of tyrosinase family members in a dose-dependent manner. The expression of microphthalmia-associated transcription factor (MITF), a key factor in the synthesis of melanin, was also decreased by pLTA. Further, we showed that pLTA activated melanogenesis signaling, such as extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinse (PI3K)/AKT. In addition, the expression of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and HuR, which are important RNA-binding proteins (RBPs), was reduced. pLTA likely degrades MITF via regulation of melanogenic signaling and RNA stability of melanogenic proteins, resulting in the reduction of melanin. Thus, our data suggest that pLTA has therapeutic potential for treating hyperpigmentation disorders and can also be used as a cosmetic whitening agent.

Role of AKT and ERK pathways in controlling sensitivity to ionizing radiation and adaptive response induced by low-dose radiation in human immune cells.

Despite many studies of the effect of ionizing radiation, biological mechanisms of action might differ greatly depend on dose, dose rate, and cell type. This study was performed to explore the effects of low- and high-dose radiation in human immune cell lines. We examined cell sensitivity after irradiation with 0.05, 0.1, or 2Gy in two normal cell lines and three tumor cell lines. Low-dose radiation of 0.05 and 0.1Gy had no effect on cell survival in any tested cell line, with the exception of IM-9 cells, whose viability was transiently increased. However, IM-9 and C1R-sB7 cells were very sensitive to high-dose radiation-induced cell death, whereas Jurkat and JM1 cells showed moderate sensitivity, and THP-1 cells were completely resistant. This radiosensitivity was correlated with basal AKT activation, which is induced by phosphorylation. In radiosensitive IM-9 cells, priming with chronic low-dose irradiation blocked cell death induced by high-dose radiation challenge via inhibition of caspase activation and PARP cleavage. AKT phosphorylation was not altered in IM-9 cells, but ERK phosphorylation was greatly elevated immediately after chronic low-dose irradiation. Taken together, our results suggest that the different responses of normal and tumor cells to low-dose and high-dose radiation depend on AKT activation, which is regulated by protein phosphatase 2 (PP2A). In radiosensitive normal cells lacking basal AKT activity, chronic low-dose radiation increases activation of the ERK pathway, which plays an important role in the adaptive response to radiation, providing a very important insight into understanding the effects of ionizing radiation on health.

Lactobacillus sakei lipoteichoic acid inhibits MMP-1 induced by UVA in normal dermal fibroblasts of human.

Human skin is continuously exposed to ultraviolet (UV)-induced photoaging. UVA increases the activity of MMP-1 in dermal fibroblasts through mitogen-activated protein kinase (MAPK), p38, signaling. The irradiation of keratinocytes by UVA results in the secretion of the inflammatory cytokine, tumor necrosis factor-α (TNF-α), and the stimulation of MMP-1 in normal human dermal fibroblasts (NHDFs). Lipoteichoic acid (LTA) is a component of the cell wall of gram-positive Lactobacillus spp. of bacteria. LTA is well known as an anti-inflammation molecule. LTA of the bacterium Lactobacillus plantarum has an anti-photoaging effect, but the potential anti-photoaging effect of the other bacteria has not been examined to date. The current study showed that L. sakei LTA (sLTA) has an immune modulating effect in human monocyte cells. Our object was whether inhibitory effects of sLTA on MMP-1 are caused from reducing the MAPK signal in NHDFs. It inhibits MMP-1 and MAPK signaling induced by UVA in NHDFs. We also confirmed effects of sLTA suppressing TNF-α inducing MMP-1 in NHDFs.