Increased Hepatocyte Growth Factor Secretion by Placenta-Derived Mesenchymal Stem Cells Improves Ovarian Function in an Ovariectomized Rat Model via Vascular Remodeling by Wnt Signaling Activation.

The vascular network contributes to the development of follicles. However, the therapeutic mechanism between vascular remodeling and ovarian functions is still unclear. Therefore, we demonstrated whether increased HGF by placenta-derived mesenchymal stem cells (PD-MSCs) improves ovarian function in an ovariectomized rat model via vascular remodeling by Wnt signaling activation. We established a half-ovariectomized rat model in which damaged ovaries were induced by ovariectomy of half of each ovary, and PD-MSCs (5 × 105 cells) were transplanted by intravenous injection. Three weeks after transplantation, rats in all groups were sacrificed. We examined the secretion of HGF by PD-MSCs through culture medium. The vascular structure in injured ovarian tissues was restored to a greater extent in the PD-MSC transplantation (Tx) group than in the nontransplantation (NTx) group (* p < 0.05). The expression of genes related to Wnt signaling (e.g., LRP6, GSK3ß, ß-catenin) was significantly increased in the Tx group compared to the NTx group (* p < 0.05). However, the expression of genes related to vascular permeability (e.g., Asef, ERG3) was significantly decreased in the Tx group compared to the NTx group (* p < 0.05). Follicular development was improved in the Tx group compared to the NTx group (* p < 0.05). Furthermore, to evaluate vascular function, we cocultivated PD-MSCs after human umbilical vein endothelial cells (HUVECs) with lipopolysaccharide (LPS), and we analyzed the vascular formation assay and dextran assay in HUVECs. Cocultivation of PD-MSCs with injured HUVECs enhanced vascular formation and decreased endothelial cell permeability (* p < 0.05). Also, cocultivation of PD-MSCs with explanted ovarian tissues improved follicular maturation compared to cocultivation of the Wnt inhibitor-treated PD-MSCs with explanted ovarian tissues. Therefore, HGF secreted by PD-MSCs improved ovarian function in rats with ovarian dysfunction by decreasing vascular permeability via Wnt signaling.

Increased IGFBP2 Levels by Placenta-Derived Mesenchymal Stem Cells Enhance Glucose Metabolism in a TAA-Injured Rat Model via AMPK Signaling Pathway.

The insulin resistance caused by impaired glucose metabolism induces ovarian dysfunction due to the central importance of glucose as a source of energy. However, the research on glucose metabolism in the ovaries is still lacking. The objectives of this study were to analyze the effect of PD-MSCs on glucose metabolism through IGFBP2-AMPK signaling and to investigate the correlation between glucose metabolism and ovarian function. Thioacetamide (TAA) was used to construct a rat injury model. PD-MSCs were transplanted into the tail vein (2 × 106) 8 weeks after the experiment started. The expression of the IGFBP2 gene and glucose metabolism factors (e.g., AMPK, GLUT4) was significantly increased in the PD-MSC group compared to the nontransplantation (NTx) group (* p < 0.05). The levels of follicular development markers and the sex hormones AMH, FSH, and E2 were also higher than those in the TAA group. Using ex vivo cocultivation, the mRNA and protein expression of IGFBP2, AMPK, and GLUT4 were significantly increased in the cocultivation with the PD-MSCs group and the recombinant protein-treated group (* p < 0.05). These findings suggest that the increased IGFBP2 levels by PD-MSCs play an important role in glucose metabolism and ovarian function through the IGFBP2-AMPK signaling pathway.

The Dose-Related Efficacy of Human Placenta-Derived Mesenchymal Stem Cell Transplantation on Antioxidant Effects in a Rat Model with Ovariectomy.

Oxidative stress initiates various degenerative diseases, and it is caused by excessive reactive oxygen species (ROS) production. Oxidative stress is a key factor that causes infertility by inducing ovarian dysfunction, characterized by irregular hormone levels, lower quality of mature follicles, and loss of follicles. Hence, stem cell therapy has been actively studied as an approach to overcome the side effects of hormone replacement therapy (HRT) on ovarian dysfunction. However, there is a lack of evidence about the appropriate number of cells required for stem cell therapy. Therefore, based on the antioxidant effects investigated in this study, we focused on determining the appropriate dose of stem cells for transplantation in an animal model with ovarian dysfunction. One week after half-ovariectomy, placenta-derived mesenchymal stem cells (PD-MSCs, 1 × 105 cells, 5 × 105 cells, or 2.5 × 106 cells) were injected intravenously into the Tx groups through the tail vein. As a result, the mRNA expression of hAlu gradually increased as the transplanted cell concentration increased. Compared with no transplantation (NTx), the transplantation of PD-MSCs improved folliculogenesis, including the levels of secreted hormones and numbers of follicles, by exerting antioxidant effects. Also, the levels of oxidized glutathione in the serum of animal models after transplantation were significantly increased (* p < 0.05). These results indicated that PD-MSC transplantation improved ovarian function in half-ovariectomized rats by exerting antioxidant effects. According to our data, increasing the number of transplanted cells did not proportionally increase the effectiveness of the treatment. We suggest that low-dose PD-MSC transplantation has the same therapeutic effect as described in previous studies. These findings provide new insights for further understanding reproductive systems and provide evidence for related clinical trials.

Deficiency of HtrA4 in BeWo cells downregulates angiogenesis through IL-6/JAK/STAT3 signaling.

In a previous study, we investigated the effects of high-temperature requirement factor A4 (HtrA4) deficiency on trophoblasts using the BeWo KO cell line. However, the effects of this deficiency on angiogenesis remain unclear. To explore the role of HtrA4 in angiogenesis, HUVECs were co-cultured with wild-type BeWo cells (BeWo WT), BeWo KO, and HtrA4-rescued BeWo KO (BeWo KO-HtrA4 rescue) cells. Dil staining and dextran analysis revealed that HUVECs co-cultured with BeWo KO formed tubes, but they were often disjointed compared to those co-cultured with BeWo WT, BeWo KO-HtrA4 rescue, and HUVECs controls. RT-PCR, ELISA, and western blot analysis were performed to assess angiogenesis-related factors at the mRNA and protein levels. HtrA4 deficiency inhibited IL-6 expression in trophoblasts, and the reduced secretion of IL-6 decreases VEGFA expression in HUVECs by modulating the JAK2/STAT3 signaling pathway to prevent tube formation. Moreover, rescuing HtrA4 expression restored the HUVEC tube formation ability. Interestingly, IL-6 expression was lower in supernatants with only cultured HUVECs than in co-cultured HUVECs with BeWo WT cells, but the HUVEC tube formation ability was similar. These findings suggest that the promoting angiogenesis-related signaling pathway differs between only HUVECs and co-cultured HUVECs, and that the deficiency of HtrA4 weakens the activation of the IL-6/JAK/STAT3/VEGFA signaling pathway, reducing the ability of tube formation in HUVECs. HtrA4 deficiency in trophoblasts hinders angiogenesis and may contribute to placental dysfunction.

Hypoxia disrupt tight junctions and promote metastasis of oral squamous cell carcinoma via loss of par3.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a highly malignant tumor that is frequently associated with lymph node metastasis, resulting in poor prognosis and survival in patients. In the tumor microenvironment, hypoxia plays an important role in regulating cellular responses such as progressive and rapid growth and metastasis. In these processes, tumor cells autonomously undergo diverse transitions and acquire functions. However, hypoxia-induced transition of OSCC and the involvement of hypoxia in OSCC metastasis remain unclear. Therefore, in this study, we aimed to elucidate the mechanism of hypoxia-induced OSCC metastasis and particularly, its impact on tight junctions (TJs). METHODS: The expression of hypoxia-inducible factor 1-alpha (HIF-1α) was detected in tumor tissues and adjacent normal tissues from 29 patients with OSCC using reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry (IHC). The migration and invasion abilities of OSCC cell lines treated with small interfering (si)RNA targeting HIF-1α or cultured in hypoxic conditions were analyzed using Transwell assays. The effect of HIF-1α expression on in vivo tumor metastasis of OSCC cells was evaluated using lung metastasis model. RESULTS: HIF-1α was overexpressed in patients with OSCC. OSCC metastasis was correlated with HIF-1α expression in OSCC tissues. Hypoxia increased the migration and invasion abilities of OSCC cell lines by regulating the expression and localization of partitioning-defective protein 3 (Par3) and TJs. Furthermore, HIF-1α silencing effectively decreased the invasion and migration abilities of OSCC cell lines and restored TJ expression and localization via Par3. The expression of HIF-1α was positively regulated the OSCC metastasis in vivo. CONCLUSIONS: Hypoxia promotes OSCC metastasis by regulating the expression and localization of Par3 and TJ proteins. HIF-1α positively correlates to OSCC metastasis. Lastly, HIF-1α expression could regulate the expression of Par3 and TJs in OSCC. This finding may aid in elucidating the molecular mechanisms of OSCC metastasis and progression and developing new diagnostic and therapeutic approaches for OSCC metastasis.

Increased PRL-1 in BM-derived MSCs triggers anaerobic metabolism via mitochondria in a cholestatic rat model.

Mesenchymal stem cell (MSC) therapy in chronic liver disease is associated with mitochondrial anaerobic metabolism. Phosphatase of regenerating liver-1 (PRL-1), known as protein tyrosine phosphatase type 4A, member 1 (PTP4A1), plays a critical role in liver regeneration. However, its therapeutic mechanism remains obscure. The aim of this study was to establish genetically modified bone marrow (BM)-MSCs overexpressing PRL-1 (BM-MSCsPRL-1) and to investigate their therapeutic effects on mitochondrial anaerobic metabolism in a bile duct ligation (BDL)-injured cholestatic rat model. BM-MSCsPRL-1 were generated with lentiviral and nonviral gene delivery systems and characterized. Compared with naive cells, BM-MSCsPRL-1 showed an improved antioxidant capacity and mitochondrial dynamics and decreased cellular senescence. In particular, mitochondrial respiration in BM-MSCsPRL-1 generated using the nonviral system was significantly increased as well as mtDNA copy number and total ATP production. Moreover, transplantation of BM-MSCsPRL-1 generated using the nonviral system had predominantly antifibrotic effects and restored hepatic function in a BDL rat model. Decreased cytoplasmic lactate and increased mitochondrial lactate upon the administration of BM-MSCsPRL-1 indicated significant alterations in mtDNA copy number and ATP production, activating anaerobic metabolism. In conclusion, BM-MSCsPRL-1 generated by a nonviral gene delivery system enhanced anaerobic mitochondrial metabolism in a cholestatic rat model, improving hepatic function.

Can a Large Number of Transplanted Mesenchymal Stem Cells Have an Optimal Therapeutic Effect on Improving Ovarian Function?

Mesenchymal stem cells (MSCs) are next-generation treatment in degenerative diseases. For the application of mesenchymal stem cell therapy to degenerative disease, transplantation conditions (e.g., optimized dose, delivery route and regenerating efficacy) should be considered. Recently, researchers have studied the mode of action of MSC in the treatment of ovarian degenerative disease. However, the evidence for the optimal number of cells for the developing stem cell therapeutics is insufficient. The objective of this study was to evaluate the efficacy in ovarian dysfunction, depends on cell dose. By intraovarian transplantation of low (1 × 105) and high (5 × 105) doses of placenta-derived mesenchymal stem cells (PD-MSCs) into thioacetamide (TAA)-injured rats, we compared the levels of apoptosis and oxidative stress that depend on different cell doses. Apoptosis and oxidative stress were significantly decreased in the transplanted (Tx) group compared to the non-transplanted (NTx) group in ovarian tissues from TAA-injured rats (* p < 0.05). In addition, we confirmed that follicular development was significantly increased in the Tx groups compared to the NTx group (* p < 0.05). However, there were no significant differences in the apoptosis, antioxidant or follicular development of injured ovarian tissues between the low and high doses PD-MSCs group. These findings provide new insights into the understanding and evidence obtained from clinical trials for stem cell therapy in reproductive systems.

Increased phosphatase regenerating liver-1 trigger vascular remodeling in injured ovary via platelet-derived growth factor signaling pathway.

BACKGROUND: Vascular abnormalities in the ovary cause infertility accompanied by ovarian insufficiency due to a microenvironment of barren ovarian tissues. Placenta-derived mesenchymal stem cells (PD-MSCs, Naïve) treatment in ovarian dysfunction shows angiogenic effect, however, the therapeutic mechanism between ovarian function and vascular remodeling still unclear. Therefore, we examined whether by phosphatase regenerating liver-1 (PRL-1), which is correlated with angiogenesis in reproductive systems, overexpressed PD-MSCs could maximize the angiogenic effects in an ovarian tissues injured of rat model with partial ovariectomy and their therapeutic mechanism by enhanced vascular function via PDGF signaling. METHODS: PD-MSCsPRL-1 (PRL-1) were generated by nonviral AMAXA gene delivery system and analyzed the vascular remodeling and follicular development in ovary. One week after Sprague-Dawley (SD) rats ovariectomy, Naïve and PRL-1 was transplanted. The animals were sacrificed at 1, 3 and 5 weeks after transplantation and vascular remodeling and follicular development were analyzed. Also, human umbilical vein endothelial cells (HUVECs) and ovarian explantation culture were performed to prove the specific effects and mechanism of PRL-1. RESULTS: Vascular structures in ovarian tissues (e.g., number of vessels, thickness and lumen area) showed changes in the Naïve and PRL-1-overexpressed PD-MSC (PRL-1) transplantation (Tx) groups compared to the nontransplantation (NTx) group. Especially, PRL-1 induce to increase the expression of platelet-derived growth factor (PDGF), which plays a role in vascular remodeling as well as follicular development, compared to the NTx. Also, the expression of genes related to pericyte and vascular permeability in arteries was significantly enhanced in the PRL-1 compared to the NTx (p < 0.05). PRL-1 enhanced the vascular formation and permeability of human umbilical vein endothelial cells (HUVECs) via activated the PDGF signaling pathway. CONCLUSIONS: Our results show that PRL-1 restored ovarian function by enhanced vascular function via PDGF signaling pathway. These findings offer new insight into the effects of functionally enhanced stem cell therapy for reproductive systems and should provide new avenues to develop more efficient therapies in degenerative medicine.

Combination Therapy of Placenta-Derived Mesenchymal Stem Cells with WKYMVm Promotes Hepatic Function in a Rat Model with Hepatic Disease via Vascular Remodeling.

Changes in the structure and function of blood vessels are important factors that play a primary role in regeneration of injured organs. WKYMVm has been reported as a therapeutic factor that promotes the migration and proliferation of angiogenic cells. Additionally, we previously demonstrated that placenta-derived mesenchymal stem cells (PD-MSCs) induce hepatic regeneration in hepatic failure via antifibrotic effects. Therefore, our objectives were to analyze the combination effect of PD-MSCs and WKYMVm in a rat model with bile duct ligation (BDL) and evaluate their therapeutic mechanism. To analyze the anti-fibrotic and angiogenic effects on liver regeneration, it was analyzed using ELISA, qRT-PCR, Western blot, immunofluorescence, and immunohistochemistry. Collagen accumulation was significantly decreased in PD-MSCs with the WKYMVm combination (Tx+WK) group compared with the nontransplantation (NTx) and PD-MSC-transplanted (Tx) group (p < 0.05). Furthermore, the combination of PD-MSCs with WKYMVm significantly promoted hepatic function by increasing hepatocyte proliferation and albumin as well as angiogenesis by activated FPR2 signaling (p < 0.05). The combination therapy of PD-MSCs with WKYMVm could be an efficient treatment in hepatic diseases via vascular remodeling. Therefore, the combination therapy of PD-MSCs with WKYMVm could be a new therapeutic strategy in degenerative medicine.

Phosphatase of Regenerating Liver-1 (PRL-1)-Overexpressing Placenta-Derived Mesenchymal Stem Cells Enhance Antioxidant Effects via Peroxiredoxin 3 in TAA-Injured Rat Livers.

DNA damage repair is induced by several factors and is critical for cell survival, and many cellular DNA damage repair mechanisms are closely linked. Antioxidant enzymes that control cytokine-induced peroxide levels, such as peroxiredoxins (Prxs) and catalase (CAT), are involved in DNA repair systems. We previously demonstrated that placenta-derived mesenchymal stem cells (PD-MSCs) that overexpress PRL-1 (PRL-1(+)) promote liver regeneration via antioxidant effects in TAA-injured livers. However, the efficacy of these cells in regeneration and the role of Prxs in their DNA repair system have not been reported. Therefore, our objective was to analyze the Prx-based DNA repair mechanism in naïve or PRL-1(+)-transplanted TAA-injured rat livers. Apoptotic cell numbers were significantly decreased in the PRL-1(+) transplantation group versus the nontransplantation (NTx) group (p < 0.05). The expression of antioxidant markers was significantly increased in PRL-1(+) cells compared to NTx cells (p < 0.05). MitoSOX and Prx3 demonstrated a significant negative correlation coefficient (R2 = −0.8123). Furthermore, DNA damage marker levels were significantly decreased in PRL-1(+) cells compared to NTx cells (p < 0.05). In conclusion, increased Prx3 levels in PRL-1(+) cells result in an effective antioxidant effect in TAA-injured liver disease, and Prx3 is also involved in repairing damaged DNA.

Activation of the EGFR-PI3K-CaM pathway by PRL-1-overexpressing placenta-derived mesenchymal stem cells ameliorates liver cirrhosis via ER stress-dependent calcium.

BACKGROUND: Cholesterol accumulation and calcium depletion induce hepatic injury via the endoplasmic reticulum (ER) stress response. ER stress regulates the calcium imbalance between the ER and mitochondria. We previously reported that phosphatase of regenerating liver-1 (PRL-1)-overexpressing placenta-derived mesenchymal stem cells (PD-MSCsPRL-1) promoted liver regeneration via mitochondrial dynamics in a cirrhotic rat model. However, the role of PRL-1 in ER stress-dependent calcium is not clear. Therefore, we demonstrated that PD-MSCsPRL-1 improved hepatic functions by regulating ER stress and calcium channels in a rat model of bile duct ligation (BDL). METHODS: Liver cirrhosis was induced in Sprague-Dawley (SD) rats using surgically induced BDL for 10 days. PD-MSCs and PD-MSCsPRL-1 (2 × 106 cells) were intravenously administered to animals, and their therapeutic effects were analyzed. WB-F344 cells exposed to thapsigargin (TG) were cocultured with PD-MSCs or PD-MSCsPRL-1. RESULTS: ER stress markers, e.g., eukaryotic translation initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP), were increased in the nontransplantation group (NTx) compared to the control group. PD-MSCsPRL-1 significantly decreased ER stress markers compared to NTx and induced dynamic changes in calcium channel markers, e.g., sarco/endoplasmic reticulum Ca2+ -ATPase 2b (SERCA2b), inositol 1,4,5-trisphosphate receptor (IP3R), mitochondrial calcium uniporter (MCU), and voltage-dependent anion channel 1 (VDAC1) (*p < 0.05). Cocultivation of TG-treated WB-F344 cells with PD-MSCsPRL-1 decreased cytosolic calmodulin (CaM) expression and cytosolic and mitochondrial Ca2+ concentrations. However, the ER Ca2+ concentration was increased compared to PD-MSCs (*p < 0.05). PRL-1 activated phosphatidylinositol-3-kinase (PI3K) signaling via epidermal growth factor receptor (EGFR), which resulted in calcium increase via CaM expression. CONCLUSIONS: These findings suggest that PD-MSCsPRL-1 improved hepatic functions via calcium changes and attenuated ER stress in a BDL-injured rat model. Therefore, these results provide useful data for the development of next-generation MSC-based stem cell therapy for regenerative medicine in chronic liver disease.

Increased Phosphatase of Regenerating Liver-1 by Placental Stem Cells Promotes Hepatic Regeneration in a Bile-Duct-Ligated Rat Model.

Phosphatase of regenerating liver-1 (PRL-1) controls various cellular processes and liver regeneration. However, the roles of PRL-1 in liver regeneration induced by chorionic-plate-derived mesenchymal stem cells (CP-MSCs) transplantation remain unknown. Here, we found that increased PRL-1 expression by CP-MSC transplantation enhanced liver regeneration in a bile duct ligation (BDL) rat model by promoting the migration and proliferation of hepatocytes. Engrafted CP-MSCs promoted liver function via enhanced hepatocyte proliferation through increased PRL-1 expression in vivo and in vitro. Moreover, higher increased expression of PRL-1 regulated CP-MSC migration into BDL-injured rat liver through enhancement of migration-related signals by increasing Rho family proteins. The dual effects of PRL-1 on proliferation of hepatocytes and migration of CP-MSCs were substantially reduced when PRL-1 was silenced with siRNA-PRL-1 treatment. These findings suggest that PRL-1 may serve as a multifunctional enhancer for therapeutic applications of CP-MSC transplantation.

Expression of miRNAs Targeting ATP Binding Cassette Transporter 1 (ABCA1) among Patients with Significant Carotid Artery Stenosis.

Background: Carotid artery stenosis is a dynamic process associated with an increased risk of cardiovascular events. However, knowledge of biomarkers useful for identifying and quantifying high-risk carotid plaques associated with the increased incidence of cerebrovascular events is insufficient. Therefore, the objectives of this study were to evaluate the expression of ATP binding cassette transporter 1 (ABCA1) and validate its target microRNA (miRNA) candidates in human carotid stenosis arteries to identify its potential as a biomarker. Methods: In human carotid stenosis arterial tissues and plasma, the expression of ABCA1 and its target miRNAs (miRNA-33a-5p, 33b-5p, and 148a-3p) were evaluated by quantitative real time-polymerase chain reaction (qRT-PCR), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA). Results: The expression of ABCA1 was significantly decreased in the plasma of stenosis patients, but its expression was not different in arterial tissues (p < 0.05). However, significantly more target miRNAs were secreted by stenosis patients than normal patients (p < 0.05). Interestingly, lipotoxicity induced by the oleic and palmitic acid (OAPA) or lipopolysaccharide (LPS) treatment of human umbilical vein endothelial cells (HUVECs) dramatically enhanced the gene expression of adipogenic and inflammatory factors, whereas ABCA1 expression was significantly decreased. Conclusions: Therefore, miRNA-33a-5p, 33b-5p, and 148a-3p represent possible biomarkers of carotid artery stenosis by directly targeting ABCA1.

miR-373-3p Regulates Invasion and Migration Abilities of Trophoblast Cells via Targeted CD44 and Radixin.

Preterm labor (PTL) is one of the obstetric complications, and is known to be associated with abnormal maternal inflammatory response and intrauterine inflammation and/or infection. However, the expression of specific miRNAs associated with PTL is not clear. In this study, we performed combination analysis of miRNA array and gene array, and then selected one miRNA (miR-373-3p) and its putative target genes (CD44 and RDX) that exhibited large expression differences in term and PTL placentas with or without inflammation. Using qRT-PCR and luciferase assays, we confirmed that miR-373-3p directly targeted CD44 and RDX. Overexpression of miR-373-3p reduced the migration and invasion of trophoblast cells, while inhibition of miR-373-3p restored the migration and invasion abilities of trophoblast cells. Finally, we validated the expression of miR-373-3p and its target genes in clinical patients' blood. miR-373-3p was increased in PTL patients' blood, and was the most expressed in PTL patients' blood with inflammation. In addition, by targeting the miR-373-3p, CD44 and RDX was decreased in PTL patients' blood, and their expression were the lowest in PTL patients' blood with inflammation. Taken together, these findings suggest that miR-373-3p and its target genes can be potential biomarkers for diagnosis of PTL.

PEDF-Mediated Mitophagy Triggers the Visual Cycle by Enhancing Mitochondrial Functions in a H2O2-Injured Rat Model.

Retinal degenerative diseases result from oxidative stress and mitochondrial dysfunction, leading to the loss of visual acuity. Damaged retinal pigment epithelial (RPE) and photoreceptor cells undergo mitophagy. Pigment epithelium-derived factor (PEDF) protects from oxidative stress in RPE and improves mitochondrial functions. Overexpression of PEDF in placenta-derived mesenchymal stem cells (PD-MSCs; PD-MSCsPEDF) provides therapeutic effects in retinal degenerative diseases. Here, we investigated whether PD-MSCsPEDF restored the visual cycle through a mitophagic mechanism in RPE cells in hydrogen peroxide (H2O2)-injured rat retinas. Compared with naïve PD-MSCs, PD-MSCsPEDF augmented mitochondrial biogenesis and translation markers as well as mitochondrial respiratory states. In the H2O2-injured rat model, intravitreal administration of PD-MSCsPEDF restored total retinal layer thickness compared to that of naïve PD-MSCs. In particular, PTEN-induced kinase 1 (PINK1), which is the major mitophagy marker, exhibited increased expression in retinal layers and RPE cells after PD-MSCPEDF transplantation. Similarly, expression of the visual cycle enzyme retinol dehydrogenase 11 (RDH11) showed the same patterns as PINK1 levels, resulting in improved visual activity. Taken together, these findings suggest that PD-MSCsPEDF facilitate mitophagy and restore the loss of visual cycles in H2O2-injured rat retinas and RPE cells. These data indicate a new strategy for next-generation MSC-based treatment of retinal degenerative diseases.

MIT-001 Restores Human Placenta-Derived Mesenchymal Stem Cells by Enhancing Mitochondrial Quiescence and Cytoskeletal Organization.

Inflammation is a major cause of several chronic diseases and is reported to be recovered by the immuno-modulation of mesenchymal stem cells (MSCs). While most studies have focussed on the anti-inflammatory roles of MSCs in stem cell therapy, the impaired features of MSCs, such as the loss of homeostasis by systemic aging or pathologic conditions, remain incompletely understood. In this study, we investigated whether the altered phenotypes of human placenta-derived MSCs (hPD-MSCs) exposed to inflammatory cytokines, including TNF-α and IFN-γ, could be protected by MIT-001, a small anti-inflammatory and anti-necrotic molecule. MIT-001 promoted the spindle-like shape and cytoskeletal organization extending across the long cell axis, whereas hPD-MSCs exposed to TNF-α/IFN-γ exhibited increased morphological heterogeneity with an abnormal cell shape and cytoskeletal disorganization. Importantly, MIT-001 improved mitochondrial distribution across the cytoplasm. MIT-001 significantly reduced basal respiration, ATP production, and cellular ROS levels and augmented the spare respiratory capacity compared to TNF-α/IFN-γ-exposed hPD-MSCs, indicating enhanced mitochondrial quiescence and homeostasis. In conclusion, while TNF-α/IFN-γ-exposed MSCs lost homeostasis and mitochondrial quiescence by becoming over-activated in response to inflammatory cytokines, MIT-001 was able to rescue mitochondrial features and cellular phenotypes. Therefore, MIT-001 has therapeutic potential for clinical applications to treat mitochondrion-related inflammatory diseases.

PRL-1 overexpressed placenta-derived mesenchymal stem cells suppress adipogenesis in Graves' ophthalmopathy through SREBP2/HMGCR pathway.

BACKGROUND: Graves' ophthalmopathy (GO) is a disorder, in which orbital connective tissues get in inflammation and increase in volume. Stimulants such as thyroid-stimulating hormone (TSH), insulin-like growth factor 1(IGF-1), IL-1, interferon γ, and platelet-derived growth factor cause differentiation into adipocytes of orbital fibroblasts (OFs) in the orbital fat and extraocular muscles. Human placental mesenchymal stem cells (hPMSCs) are known to have immune modulation effects on disease pathogenesis. Some reports suggest that hPMSCs can elicit therapeutic effects, but to date, research on this has been insufficient. In this study, we constructed PRL-1 overexpressed hPMSCs (hPMSCsPRL-1) in an attempt to enhance the suppressive function of adipogenesis in GO animal models. METHODS: In order to investigate the anti-adipogenic effects, primary OFs were incubated with differentiation medium for 10 days. After co-culturing with hPMSCsPRL-1, the characteristics of the OFs were analyzed using Nile red stain and quantitative real-time polymerase chain reaction. We then examined the in vivo regulatory effectiveness of hPMSCsPRL-1 in a GO mouse model that immunized by leg muscle electroporation of pTriEx1.1Neo-hTSHR A-subunit plasmid. Human PMSCsPRL-1 injection was performed in left orbit. We also analyzed the anti-adipogenic effects of hPMSCsPRL-1 in the GO model. RESULTS: We found that hPMSCsPRL-1 inhibited adipogenic activation factors, specifically PPARγ, C/EBPα, FABP4, SREBP2, and HMGCR, by 75.1%, 50%, 79.6%, 81.8%, and 87%, respectively, compared with naïve hPMSCs in adipogenesis-induced primary OFs from GO. Moreover, hPMSCsPRL-1 more effectively inhibited adipogenic factors ADIPONECTIN and HMGCR by 53.2% and 31.7%, respectively, than hPMSCs, compared with 15.8% and 29.8% using steroids in the orbital fat of the GO animal model. CONCLUSION: Our findings suggest that hPMSCsPRL-1 would restore inflammation and adipogenesis of GO model and demonstrate that they could be applied as a novel treatment for GO patients.

Formyl Peptide Receptor 2 Alleviates Hepatic Fibrosis in Liver Cirrhosis by Vascular Remodeling.

Hexapeptide WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met), a ligand of formyl peptide receptor 2, exhibits anti-inflammatory and angiogenic properties in disease models. However, the therapeutic effects of WKYMVm on hepatic fibrosis have not been evaluated to date. Therefore, we investigated whether WKYMVm exerts antifibrotic effects and induces vascular regeneration in a rat model of bile duct ligation (BDL). The antifibrotic and angiogenic effects of WKYMVm on liver regeneration in the BDL rat model were analyzed using biochemical assays, qRT-PCR, western blotting, immunofluorescence, and immunohistochemistry. To determine the effects of WKYMVm on hepatic fibrosis and angiogenesis in vitro, we measured the expression levels of fibrotic factors in hepatic stellate cells (HSCs) and angiogenic factors in human umbilical vein endothelial cells (HUVECs). WKYMVm attenuated the expression of collagen type I (Col I) and α-smooth muscle actin (α-SMA) and significantly increased the levels of angiogenetic factors in the BDL model (p < 0.05). WKYMVm reduced fibrotic marker expression in transforming growth factor (TGF)-ß-induced HSCs and promoted angiogenic activity through tube formation in 5-Fluorouracil (FU)-treated HUVECs (p < 0.05). Also, WKYMVm administration enhanced hepatocyte proliferation in BDL rats (p < 0.05). The WKYMVm alleviates hepatic fibrosis by inhibiting HSC activation and promotes hepatic regeneration via vascular remodeling. These data suggest that the WKYMVm may be a new therapeutic agent for liver fibrosis.

The mitochondrial-derived peptide MOTS-c promotes homeostasis in aged human placenta-derived mesenchymal stem cells in vitro.

Mesenchymal stem cells (MSCs) are multipotent cells with critical roles in homeostasis and regeneration. MSCs undergo aging in response to various stresses, and this causes many diseases including degenerative disorders. Thus, regulation of aging factors is crucial for healthy aging. Mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) was recently reported to regulate metabolic homeostasis. Here, we investigated the restorative effects of MOTS-c on aged human placenta-derived MSCs (hPD-MSCs). MOTS-c promoted the morphology of old hPD-MSCs. MOTS-c significantly activated AMP-activated protein kinase, which is the main target pathway of MOTS-c, and inhibited its antagonistic effector mTORC1. MOTS-c considerably enhanced mitochondrial homeostasis by decreasing oxygen consumption and reactive oxygen species production. The mitochondrial state of MOTS-c-treated old hPD-MSCs was more similar to that of young hPD-MSCs than the mitochondrial state of non-treated old hPD-MSCs. MOTS-c also decreased lipid synthesis. In conclusion, we demonstrated that MOTS-c promotes homeostasis in aged hPD-MSCs.

Human placenta-derived mesenchymal stem cells induce trophoblast invasion via dynamic effects on mitochondrial function.

The trophoblast is a critical cell for placental development and embryo implantation in the placenta. We previously reported that placenta-derived mesenchymal stem cells (PD-MSCs) increase trophoblast invasion through several signaling pathways. However, the paracrine effects of PD-MSCs on mitochondrial function in trophoblasts are still unclear. Therefore, the objective of the study was to analyze the mitochondrial function of trophoblasts in response to cocultivation with PD-MSCs. The results showed that PD-MSCs regulate the balance between cell survival and death and protect damaged mitochondria in trophoblasts from oxidative stress. Moreover, PD-MSCs upregulate factors involved in mitochondrial autophagy in trophoblast cells. Finally, PD-MSCs improve trophoblast invasion. Taken together, the data indicate that PD-MSCs can regulate trophoblast invasion through dynamic effects on mitochondrial energy metabolism. These results support the fundamental role of mitochondrial energy mechanism in trophoblast invasion and suggest a new therapeutic strategy for infertility.