Concomitant induction of SLIT3 and microRNA-218-2 in macrophages by toll-like receptor 4 activation limits osteoclast commitment.

BACKGROUND: Toll-like receptor 4 (TLR4) conducts a highly regulated inflammatory process by limiting the extent of inflammation to avoid toxicity and tissue damage, even in bone tissues. Thus, it is plausible that strategies for the maintenance of normal bone-immunity to prevent undesirable bone damage by TLR4 activation can exist, but direct evidence is still lacking. METHODS: Osteoclast precursors (OCPs) obtained from WT or Slit3-deficient mice were differentiated into osteoclast (OC) with macrophage colony-stimulating factor (M-CSF), RANK ligand (RANKL) and lipopolysaccharide (LPS) by determining the number of TRAP-positive multinuclear cells (TRAP+ MNCs). To determine the alteration of OCPs population, fluorescence-activated cell sorting (FACS) was conducted in bone marrow cells in mice after LPS injection. The severity of bone loss in LPS injected WT or Slit3-deficient mice was evaluated by micro-CT analysis. RESULT: We demonstrate that TLR4 activation by LPS inhibits OC commitment by inducing the concomitant expression of miR-218-2-3p and its host gene, Slit3, in mouse OCPs. TLR4 activation by LPS induced SLIT3 and its receptor ROBO1 in BMMs, and this SLIT3-ROBO1 axis hinders RANKL-induced OC differentiation by switching the protein levels of C/EBP-ß isoforms. A deficiency of SLIT3 resulted in increased RANKL-induced OC differentiation, and the elevated expression of OC marker genes including Pu.1, Nfatc1, and Ctsk. Notably, Slit3-deficient mice showed expanded OCP populations in the bone marrow. We also found that miR-218-2 was concomitantly induced with SLIT3 expression after LPS treatment, and that this miRNA directly suppressed Tnfrsf11a (RANK) expression at both gene and protein levels, linking it to a decrease in OC differentiation. An endogenous miR-218-2 block rescued the expression of RANK and subsequent OC formation in LPS-stimulated OCPs. Aligned with these results, SLIT3-deficient mice displayed increased OC formation and reduced bone density after LPS challenge. CONCLUSION: Our findings suggest that the TLR4-dependent concomitant induction of Slit3 and miR-218-2 targets RANK in OCPs to restrain OC commitment, thereby avoiding an uncoordinated loss of bone through inflammatory processes. These observations provide a mechanistic explanation for the role of TLR4 in controlling the commitment phase of OC differentiation. Video Abstract.

MicroRNA expression profiling of adult hippocampal neural stem cells upon cell death reveals an autophagic cell death-like pattern.

Adult hippocampal neural (HCN) stem cells promptly undergo irreversible autophagic cell death (ACD) if deprived of insulin in culture. Small, non-coding microRNAs (miRNA) play an important role in regulating biological processes, including proliferation and cell death. However, there have been no reports thus far regarding miRNA involvement in the induction of adult HCN stem cell death under insulin-deprived conditions, for which we performed a microarray-based analysis to examine the expression signature of miRNAs in adult rat HCN stem cells. Three independent specimens per culture condition either with or without insulin were prepared and a miRNA microarray analysis carried out. A total of 12 exhibited significantly altered expression levels upon cell death due to the absence of insulin when compared to HCN stem cells cultured with insulin present (cut-off limit; p < 0.05 and fold-change >1.3) The resulting volcano plot showed that, among these miRNAs, seven were upregulated and five were downregulated. The upregulated miRNAs were capable of modulating HCN stem cell death. Caspase-3 activity analysis, LC3 conversion, and TEM of autophagosome formation consistently suggested that ACD, not apoptosis, was most likely the mechanism affecting HCN cell death. As such, we have come to term these miRNAs, "HCN stem cell-specific autophagic cell death regulators." Taken together, our data suggest that the miRNA expression profile of HCN stem cells is altered during ACD occurring due to insulin deprivation and that differentially expressed miRNAs are involved in HCN stem cell viability. Detailed explorations of the underlying mechanisms regarding HCN stem cell viability modulation by these miRNAs would be beneficial in further understanding the physiological features of adult HCN stem cells and are currently being investigated.

Human umbilical cord blood mesenchymal stem cells expansion via human fibroblast-derived matrix and their potentials toward regenerative application.

Large expansion of human mesenchymal stem cells (MSCs) is of great interest for clinical applications. In this study, we examine the feasibility of human fibroblast-derived extracellular matrix (hFDM) as an alternative cell expansion setting. hFDM is obtained from decellularized extracellular matrix (ECM) derived from in vitro cultured human lung fibroblasts. Our study directly compares conventional platforms (tissue culture plastic (TCP), fibronectin (FN)-coated TCP) with hFDM using umbilical cord blood-derived MSCs (UCB-MSCs). Early cell morphology shows a rather rounded shape on TCP but highly elongated morphology on hFDM. Cell proliferation demonstrates that MSCs on hFDM were significantly better compared to the others in both 10 and 2% serum condition. Cell migration assay suggests that cell motility was improved and a cell migration marker CXCR4 was notably up-regulated on hFDM. MSCs differentiation into osteogenic lineage on hFDM was also very effective as examined via gene expression, von Kossa staining and alkaline phosphatase activity. In addition, as the MSCs were expanded on each substrate, transferred to 3D polymer mesh scaffolds and then cultivated for a while, the data found better cell proliferation and more CXCR4 expression with MSCs pre-conditioned on hFDM. Moreover, higher gene expression of stemness and engraftment-related markers was noticed with the hFDM group. Furthermore when UCB-MSCs expanded on TCP or hFDM were injected into emphysema (a lung disease) animal model, the results indicate that MSCs pre-conditioned on hFDM (with 2% serum) retain more advanced therapeutic efficacy on the improvement of emphysema than those on TCP. Current works demonstrate that compared to the conventional platforms, hFDM can be a promising source of cell expansion with a naturally derived biomimetic ECM microenvironment and may find some practical applications in regenerative medicine.

Phosphorylation of p62 by AMP-activated protein kinase mediates autophagic cell death in adult hippocampal neural stem cells.

In the adult brain, programmed death of neural stem cells is considered to be critical for tissue homeostasis and cognitive function and is dysregulated in neurodegeneration. Previously, we have reported that adult rat hippocampal neural (HCN) stem cells undergo autophagic cell death (ACD) following insulin withdrawal. Because the apoptotic capability of the HCN cells was intact, our findings suggested activation of unique molecular mechanisms linking insulin withdrawal to ACD rather than apoptosis. Here, we report that phosphorylation of autophagy-associated protein p62 by AMP-activated protein kinase (AMPK) drives ACD and mitophagy in HCN cells. Pharmacological inhibition of AMPK or genetic ablation of the AMPK α2 subunit by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing suppressed ACD, whereas AMPK activation promoted ACD in insulin-deprived HCN cells. We found that following insulin withdrawal AMPK phosphorylated p62 at a novel site, Ser-293/Ser-294 (in rat and human p62, respectively). Phosphorylated p62 translocated to mitochondria and induced mitophagy and ACD. Interestingly, p62 phosphorylation at Ser-293 was not required for staurosporine-induced apoptosis in HCN cells. To the best of our knowledge, this is the first report on the direct phosphorylation of p62 by AMPK. Our data suggest that AMPK-mediated p62 phosphorylation is an ACD-specific signaling event and provide novel mechanistic insight into the molecular mechanisms in ACD.

Radiotherapy-assisted tumor selective metronomic oral chemotherapy.

Chemotherapy have commonly been used in maximum tolerated dose to completely eradicate the cancer. However, such treatments often failed due to the complex and dynamic nature of cancer. Therefore, it has been suggested that cancer should be treated as a chronic disease, controlling its growth by providing continuous therapeutic pressure for long-term. Such an approach, however, requires a therapy that is non-toxic and orally available with sufficient potency. Herein, we propose a radiotherapy-assisted orally available metronomic apoptosis-targeted chemotherapy, which delivers doxorubicin continuously to the irradiated tumor with high selectivity while causing minimal toxicities to the normal tissues. DEVD-S-DOX/DCK complex is the anticancer prodrug for our strategy that could selectively release doxorubicin in the irradiated tumor tissue with sufficient oral bioavailability. The prodrug was completely inactive by itself, but displayed potent anticancer activity when coupled with radiotherapy. Consequently, the daily oral administration of DEVD-S-DOX/DCK in combination with the low-dose radiotherapy effectively suppressed the growth of tumor in vivo with no significant systemic toxicities despite that the accumulated dose of doxorubicin exceeded 150 mg/kg. Therefore, the our novel therapy using DEVD-S-DOX/DCK complex is considered as an outstanding treatment option for treating cancer for long-term attributed to its oral availability and low-toxicity profile as well as the potent anticancer effect.

The Effects of Losartan in Preserving the Structural Integrity of Decellularized Small Diameter Vascular Allograft Conduit Implants In Vivo.

Decellularization is a proposed method of preparing nonautologous biological arterial vascular scaffolding; however, the fate of the supporting medial elastic fiber, which is important in preserving the vascular structural integrity, is uncertain. The influence of losartan on preserving the medial elastic fiber integrity in decellularized small diameter vascular conduits (SDVC) was investigated. Decellularized infrarenal abdominal aortic allografts were implanted in Sprague-Dawley rats treated either with (study rats, n = 6) or without oral losartan (control rats, n = 6) and graded 8 weeks later according to a remodeling scoring system (1-mild, 2-moderate, 3-severe) which we devised based on the intimal hyperplasia degree, morphologic changes, and elastic fiber fragmentation of the conduits. DAPI immunohistochemistry analysis was performed in 47 (25 decellularization only and 22 losartan treatment) cross-sectional slide specimens. The losartan versus decellularization only SDVC showed a significantly lower medial elastic fragmentation score (1.32 vs. 2.24, P < 0.001), superior medial layer preservation, and relatively more normal appearing intimal cellular morphology. The results suggested rats receiving decellularized SDVCs treated with losartan may yield superior medial layer elastic fiber preservation.

Enhanced axonal regeneration by transplanted Wnt3a-secreting human mesenchymal stem cells in a rat model of spinal cord injury.

BACKGROUND: While pure mesenchymal stem cell (MSC) treatment for spinal cord injury (SCI) is known to be safe, its efficacy is insufficient. Therefore, gene-modified stem cells are being developed to enhance the effect of pure MSCs. We investigated the effect of stem cell therapy through the transfection of a Wnt3a-producing gene that stimulates axonal regeneration. METHOD: MSCs obtained from the human umbilical cord blood (hMSCs) were multiplied, cultivated, and transfected with the pLenti-Wnt3a-GFP viral vector to produce Wnt3a-secreting hMSCs. A total of 50 rats were injured with an Infinite Horizon impactor at the level of the T7-8 vertebrae. Rats were divided into five groups according to the transplanted material: (1) phosphate-buffered saline injection group (sham group, n = 10); (Pertz et al. Proc Natl Acad Sci USA 105:1931-1936, 39) Wnt3a protein injection group (Wnt3a protein group, n = 10); (3) hMSC transplantation group (MSC group, n = 10); (4) hMSCs transfected with the pLenti vector transplantation group (pLenti-MSC group, n = 10); (5) hMSCs transfected with the pLenti+Wnt3a vector transplantation group (Wnt3a-MSC group, n = 10). Behavioral tests were performed daily for the first 3 days after injury and then weekly for 8 weeks. The injured spinal cords were extracted, and axonal regeneration markers including choline acetyltransferase (ChAT), growth-associated protein 43 (GAP43), and microtubule-associated protein 2 (MAP2) were investigated by immunofluorescence, RT-PCR, and western blotting. RESULTS: Seven weeks after the transplantation (8 weeks after SCI), rats in the Wnt3a-MSC group achieved significantly higher average scores in the motor behavior tests than those in the other groups (p < 0.05). Immunofluorescent stains showed greater immunoreactivity of ChAT, GAP43, and MAP2 in the Wnt3a-MSC group than in the other groups. RT-PCR and western blots revealed greater expression of these proteins in the Wnt3a-MSC group than in the other groups (p < 0.05). CONCLUSIONS: Wnt3a-secreting hMSC transplantation considerably improved neurological recovery and axonal regeneration in a rat SCI model.

Calpain Determines the Propensity of Adult Hippocampal Neural Stem Cells to Autophagic Cell Death Following Insulin Withdrawal.

Programmed cell death (PCD) has significant effects on the function of neural stem cells (NSCs) during brain development and degeneration. We have previously reported that adult rat hippocampal neural stem (HCN) cells underwent autophagic cell death (ACD) rather than apoptosis following insulin withdrawal despite their intact apoptotic capabilities. Here, we report a switch in the mode of cell death in HCN cells with calpain as a critical determinant. In HCN cells, calpain 1 expression was barely detectable while calpain 2 was predominant. Inhibition of calpain in insulin-deprived HCN cells further augmented ACD. In contrast, expression of calpain 1 switched ACD to apoptosis. The proteasome inhibitor lactacystin blocked calpain 2 degradation and elevated the intracellular Ca(2+) concentration. In combination, these effects potentiated calpain activity and converted the mode of cell death to apoptosis. Our results indicate that low calpain activity, due to absence of calpain 1 and degradation of calpain 2, results in a preference for ACD over apoptosis in insulin-deprived HCN cells. On the other hand, conditions leading to high calpain activity completely switch the mode of cell death to apoptosis. This is the first report on the PCD mode switching mechanism in NSCs. The dynamic change in calpain activity through the proteasome-mediated modulation of the calpain and intracellular Ca(2+) levels may be the critical contributor to the demise of NSCs. Our findings provide a novel insight into the complex mechanisms interconnecting autophagy and apoptosis and their roles in the regulation of NSC death.

Phagocytic function of Kupffer cells in mouse nonalcoholic fatty liver disease models: Evaluation with superparamagnetic iron oxide.

PURPOSE: To evaluate the Kupffer cell (KC) phagocytic function using superparamagnetic iron oxide-enhanced magnetic resonance imaging (SPIO-MRI) in animal models with nonalcoholic fatty liver disease (NAFLD). MATERIALS AND METHODS: Mouse NAFLD models with varying severity were created by feeding high-fat, high-cholesterol (HFHC) diets to ob/ob mice for 3, 6, or 12 weeks. SPIO-MRI was performed on a 4.7-T animal scanner in the mouse NAFLD models, in wildtype control mouse, and in the NAFLD mice (NAFLD treatment group) that received 6 weeks of pioglitazone treatment. The relative signal loss (RSL) of the liver was measured in each animal to represent the magnitude of SPIO-induced signal loss of the liver. Liver samples were analyzed for steatosis, inflammation, fibrosis, and the number of SPIO particles and KCs. RESULTS: RSL values of the NAFLD mice (range of RSL value, 26.3%-53.8%) seen on SPIO-MRI were significantly lower than those of the control mice (67.7%-74.8%, P ≤ 0.008) and decreased in proportion to the duration of their HFHC diet (mean ± SD, 53.7% ± 10.9, 44.7% ± 8.2, and 26.3% ± 12.6, after 3-, 6-, and 12-week HFHC diet, respectively, on 20-minute delayed images). For the NAFLD treatment group, the RSL values increased after 6 weeks of pioglitazone treatment, compared with the values before treatment (P ≤ 0.039). The RSL values had significant independent correlation with both hepatic steatosis (P = 0.007) and inflammation (P = 0.023). CONCLUSION: KC phagocytic dysfunction is aggravated in the progression of NAFLD and may be reversible with therapeutic intervention. SPIO-MRI may be useful for classifying the severity of NAFLD and monitoring the treatment response of NAFLD.

Activation of autophagic pathways is related to growth inhibition and senescence in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (SCC) is a very common resectable cancer; however, cutaneous SCC is highly resistant to chemotherapy if metastasis develops. Activating transcription factor 3 (ATF3) has been suggested as a marker of advanced or metastatic cutaneous SCC. Autophagy is one of the most important mechanisms in cancer biology and commonly induced by in vitro serum starvation. To investigate the role of autophagy activation in cutaneous SCC, we activated autophagic pathways by serum starvation in SCC13 and ATF3-overexpressing SCC13 (ATF3-SCC13) cell lines. ATF3-SCC13 cells demonstrated high proliferative capacity and low p53 and autophagy levels in comparison with control SCC13 cells under basal conditions. Intriguingly, autophagic stimulation via serum starvation resulted in growth inhibition and senescence in both cells, while ATF3-SCC13 cells further demonstrated growth inhibition and senescence. Apoptosis was not significantly induced by autophagy activation. Taken together, autophagy activation may be a promising antitumor approach for advanced cutaneous SCC.

CEACAM1-engineered MSCs have a broad spectrum of immunomodulatory functions and therapeutic potential via cell-to-cell interaction.

Mesenchymal stem cells (MSCs) have garnered attention for their regenerative and immunomodulatory capabilities in clinical trials for various diseases. However, the effectiveness of MSC-based therapies, especially for conditions like graft-versus-host disease (GvHD), remains uncertain. The cytokine interferon (IFN)-γ has been known to enhance the immunosuppressive properties of MSCs through cell-to-cell interactions and soluble factors. In this study, we observed that IFN-γ-treated MSCs upregulated the expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), associated with immune evasion through the inhibition of natural killer (NK) cell cytotoxicity. To co-opt this immunomodulatory function, we generated MSCs overexpressing CEACAM1 and found that CEACAM1-engineered MSCs significantly reduced NK cell activation and cytotoxicity via cell-to-cell interaction, independent of NKG2D ligand regulation. Furthermore, CEACAM1-engineered MSCs effectively inhibited the proliferation and activation of T cells along with the inflammatory responses of monocytes. In a humanized GvHD mouse model, CEACAM1-MSCs, particularly CEACAM1-4S-MSCs, demonstrated therapeutic potential by improving survival and alleviating symptoms. These findings suggest that CEACAM1 expression on MSCs contributes to MSC-mediated regulation of immune responses and that CEACAM1-engineered MSC could have therapeutic potential in conditions involving immune dysregulation.

Highly efficient genome editing via CRISPR-Cas9 ribonucleoprotein (RNP) delivery in mesenchymal stem cells.

The CRISPR-Cas9 system has significantly advanced regenerative medicine research by enabling genome editing in stem cells. Due to their desirable properties, mesenchymal stem cells (MSCs) have recently emerged as highly promising therapeutic agents, which properties include differentiation ability and cytokine production. While CRISPR-Cas9 technology is applied to develop MSC-based therapeutics, MSCs exhibit inefficient genome editing, and susceptibility to plasmid DNA. In this study, we compared and optimized plasmid DNA and RNP approaches for efficient genome engineering in MSCs. The RNP-mediated approach enabled genome editing with high indel frequency and low cytotoxicity in MSCs. By utilizing Cas9 RNPs, we successfully generated B2M-knockout MSCs, which reduced T-cell differentiation, and improved MSC survival. Furthermore, this approach enhanced the immunomodulatory effect of IFN-r priming. These findings indicate that the RNP-mediated engineering of MSC genomes can achieve high efficiency, and engineered MSCs offer potential as a promising therapeutic strategy. [BMB Reports 2024; 57(1): 60-65].

Enhancer of zeste homolog 2 (EZH2)-dependent sirtuin-3 determines sensitivity to glucose starvation in radioresistant head and neck cancer cells.

Sirtuin 3 (SIRT3) regulates mitochondrial function as a mitochondrial deacetylase during oxidative stress. However, the specific regulatory mechanism and function of SIRT3 in radioresistant cancer cells are unclear. In this study, we aim to investigate how SIRT3 determines the susceptibility to glucose deprivation and its regulation in p53-based radioresistant head and neck cancer cells. We observed mitochondrial function using two established isogenic radioresistant subclones (HN3R-A [p53 null] and HN3R-B [p53 R282W]) with intratumoral p53 heterogeneity. Cell counting analysis was performed to evaluate cell proliferation and cell death. The correlation between the regulation of SIRT3 and enhancer of zeste homolog 2 (EZH2) was confirmed by immunoblotting and chromatin immunoprecipitation assay. p53-deficient radioresistant cells (HN3R-A) expression reduced SIRT3 levels and increased sensitivity to glucose deprivation due to mitochondrial dysfunction compared to other cells. In these cells, activation of SIRT3 significantly prevented glucose deprivation-induced cell death, whereas the loss of SIRT3 increased the susceptibility to glucose deficiency. We discovered that radiation-induced EZH2 directly binds to the SIRT3 promoter and represses the expression. Conversely, inhibiting EZH2 increased the expression of SIRT3 through epigenetic changes. Our findings indicate that p53-deficient radioresistant cells with enhanced EZH2 exhibit increased sensitivity to glucose deprivation due to SIRT3 suppression. The regulation of SIRT3 by EZH2 plays a critical role in determining the cell response to glucose deficiency in radioresistant cancer cells. Therefore, EZH2-dependent SIRT3 could be used as a predictive biomarker to select treatment options for patients with radiation-resistance.

Promoter methylation of WNT inhibitory factor-1 and expression pattern of WNT/ß-catenin pathway in human astrocytoma: pathologic and prognostic correlations.

WNT inhibitory factor-1 (WIF1) is an antagonist of the WNT signaling pathway. We investigated the relationship between WIF1 promoter methylation and regulation of the WNT/ß-catenin signaling pathway, tumor grade, and survival in patients with astrocytoma. This study included 86 cases of astrocytoma, comprising 20 diffuse astrocytomas and 66 glioblastomas. In addition, 17 temporal lobectomy specimens from patients with epilepsy were included as controls. The ratio of methylated DNA to total methylated and unmethylated DNA (% methylation) was measured by methylation- and unmethylation-specific PCR. Representative tumor tissue was immunostained for WIF1, ß-catenin, cyclin D1, c-myc, and isocitrate dehydrogenase 1. Levels of WIF1 promoter methylation, mRNA expression, and protein expression in a glioblastoma cell line were compared before and after demethylation treatment. The mean percent methylation of the WIF1 promoter in astrocytomas was higher than that in control brain tissue. WIF1 protein expression was lower in the tumor group with >5% methylation than in the group with <5% methylation. Cytoplasmic ß-catenin staining was more frequently observed in tumors with a low WIF1 protein expression level. Demethylation treatment of a glioblastoma cell line increased WIF1 mRNA and protein expression. Increased WIF1 promoter methylation and decreased WIF1 protein expression were not related to patient survival. In conclusion, WIF1 expression is downregulated by promoter methylation and is an important mechanism of aberrant WNT/ß-catenin pathway activation in astrocytoma pathogenesis.

Multispecies-compatible antitumor effects of a cross-species small-interfering RNA against mammalian target of rapamycin.

Successful development of sequence-specific siRNA (small interfering RNA)-based drugs requires an siRNA design that functions consistently in different organisms. Utilizing the CAPSID program previously developed by our group, we here designed siRNAs against mammalian target of rapamycin (mTOR) that are entirely complementary among various species and investigated their multispecies-compatible gene-silencing properties. The mTOR siRNAs markedly reduced mTOR expression at both the mRNA and protein levels in human, mouse, and monkey cell lines. The reduction in mTOR expression resulted in inactivation of both mTOR complex I and II signaling pathways, as confirmed by reduced phosphorylation of p70S6K (70-kDa ribosomal protein S6 kinase), 4EBP1 (eIF4E-binding protein 1), and AKT, and nuclear accumulation of FOXO1 (forkhead box O1), with consequent cell-cycle arrest, proliferation inhibition, and autophagy activation. Moreover, interfering with mTOR activity in vivo using mTOR small-hairpin RNA-expressing recombinant adeno-associated virus led to significant antitumor effects in xenograft and allograft models. Thus, the present study demonstrates that cross-species siRNA successfully silences its target and readily produces multispecies-compatible phenotypic alterations-antitumor effects in the case of mTOR siRNA. Application of cross-species siRNA should greatly facilitate the development of siRNA-based therapeutic agents.

Granulocyte colony-stimulating factor minimizes negative remodeling of decellularized small diameter vascular graft conduits but not medial degeneration.

BACKGROUND: Poor endothelialization and intimal hyperplasia are major causes of small diameter vascular conduit (SDVC) failure. The present study was aimed to investigate the influence of granulocyte colony-stimulating factor (G-CSF) on inhibiting adverse remodeling of decellularized SDVCs. METHODS: Sprague-Dawley rats implanted with allograft infra renal abdominal aortic conduits were divided into 2 groups according to whether they were treated with G-CSF (+G-CSF group; n=6) or without (Decell group; n=6). The conduits were harvested at 8 weeks after surgery and examined for intimal hyperplasia, collagen deposition, and -actin-staining cells. The medial layer was also examined for signs of cellular repopulation and changes in the elastic fiber morphology. RESULTS: Intergroup comparison of the intimal composition showed relatively sparse collagen content and predominance of -actin-staining cells in the +G-CSF group. The medial layer in the 2 groups showed similar degrees of elastic fiber degeneration and wall thinning relative to the normal aortic wall. However, the enhanced staining for von Willebrand factor and CD31, along with transmission electron microscopy findings of superior cellular and ultrastructural preservation, suggested that the remodeling and endothelialization in the +G-CSF conduits were superior to those in the Decell conduits. CONCLUSIONS: This study suggests that G-CSF exerts a positive influence on inhibiting adverse vascular remodeling of decellularized vascular conduit implants. However, whether G-CSF administration may also effectuate an improved ability to preserve the medial structural integrity is unclear.

Epidermal growth factor and epidermal growth factor signaling attenuate laser-induced melanogenesis.

BACKGROUND: Epidermal growth factor (EGF) is a peptide that promotes cell growth by binding to its receptor (EGFR) on the cell surface. EGF has been used in cosmetics to whiten the skin and for the prevention of postinflammatory hyperpigmentation (PIH), presumably by accelerating wound healing, but the effects of EGF on melanogenesis are not known, and the presence of EGFR on melanocytes has not been confirmed. METHODS: To establish a role of EGF in melanogenesis, we first investigated expression of EGFR on melanocytes. Second, in the search for an effect of EGF on PIH, we investigated the effect of EGF on melanin production by melanocytes with or without laser-treated keratinocyte-conditioned culture media (LCM). RESULTS: Treatment with EGF did not affect proliferation of melan-A, mouse-derived immortalized melanocytes. Melanocytes treated with LCM had greater prostaglandin-E2 (PGE2) expression and tyrosinase enzyme activity than melanocytes treated with control media. Treatment with EGF lowered melanin production of LCM-treated melanocytes but not of melanocytes treated with control media. CONCLUSION: Our results support EGF as a candidate target for development of antimelanogenic agents in PIH.

Enhanced neuroregenerative effects by scaffold for the treatment of a rat spinal cord injury with Wnt3a-secreting fibroblasts.

BACKGROUND: Wnt proteins are bifunctional axon guidance molecules, several of which appear to mediate guidance of corticospinal tract axons along the spinal cord. Here, we studied increasing effect on regeneration by Wnt-containing alginate scaffolds on spinal cord injury (SCI). METHODS: A total of 32 rats were injured at the T7-8 level with an NYU impactor. According to transplantation materials, rats were classified into four groups: a Wnt3a-secreting fibroblast transplantation group (Wnt group, n = 8), a Wnt3a-secreting fibroblast with alginate transplantation group (Wnt + alginate group, n = 8), an alginate transplantation group (alginate group, n = 8), and a contusion-only group (sham group, n = 8). Behavioral tests were performed on the first, second, and third days after injury, and then weekly for 8 weeks. Five of the eight rats from each group were selected for manganese-enhanced magnetic resonance imaging (ME-MRI). Two rats from each group were examined for GAP43 and MAP2 expression using monoclonal and polyclonal primary antibodies, respectively. RESULTS: Seven weeks after transplantation (8 weeks after SCI), Wnt + alginate group rats achieved an average Basso-Beattie-Bresnahan locomotor score of 19.0, which was significantly higher than that of other groups. ME-MRI at 8 weeks after SCI revealed significantly higher relative signal intensities in the Wnt + alginate group. Gap43 and Map2 immunostaining, showed strong positive in the Wnt + alginate group. CONCLUSION: The Wnt + alginate complex exerted significantly enhanced recovery in a rat SCI model compared to alginate or Wnt3a alone. These results suggest that alginate scaffolds facilitate the regeneration of axon working with Wnt3a protein that promotes regeneration of the injured spinal cord.

Comparative analysis of human mesenchymal stem cells from bone marrow, adipose tissue, and umbilical cord blood as sources of cell therapy.

Various source-derived mesenchymal stem cells (MSCs) have been considered for cell therapeutics in incurable diseases. To characterize MSCs from different sources, we compared human bone marrow (BM), adipose tissue (AT), and umbilical cord blood-derived MSCs (UCB-MSCs) for surface antigen expression, differentiation ability, proliferation capacity, clonality, tolerance for aging, and paracrine activity. Although MSCs from different tissues have similar levels of surface antigen expression, immunosuppressive activity, and differentiation ability, UCB-MSCs had the highest rate of cell proliferation and clonality, and significantly lower expression of p53, p21, and p16, well known markers of senescence. Since paracrine action is the main action of MSCs, we examined the anti-inflammatory activity of each MSC under lipopolysaccharide (LPS)-induced inflammation. Co-culture of UCB-MSCs with LPS-treated rat alveolar macrophage, reduced expression of inflammatory cytokines including interleukin-1α (IL-1α), IL-6, and IL-8 via angiopoietin-1 (Ang-1). Using recombinant Ang-1 as potential soluble paracrine factor or its small interference RNA (siRNA), we found that Ang-1 secretion was responsible for this beneficial effect in part by preventing inflammation. Our results demonstrate that primitive UCB-MSCs have biological advantages in comparison to adult sources, making UCB-MSCs a useful model for clinical applications of cell therapy.

ZBTB7A suppresses glioblastoma tumorigenesis through the transcriptional repression of EPB41L5.

Glioblastoma multiforme (GBM), the most aggressive and malignant glioma, has a poor prognosis. Although patients with GBM are treated with surgery, chemotherapy, and radiation therapy, GBM is highly resistant to treatment, making it difficult and expensive to treat. In this study, we analyzed the Gene Expression Profiling Interactive Analysis dataset, the Cancer Genome Atlas dataset, and Gene Expression Omnibus array data. ZBTB7A (also called FBI1/POKEMON/LRF) was found to be highly expressed in low-grade glioma but significantly downregulated in patients with GBM. ZBTB7A is a transcription factor that plays an important role in many developmental stages, including cell proliferation. The activation of epithelial-mesenchymal transition (EMT) is a key process in cancer progression and metastasis. Erythrocyte membrane protein band 4.1 like 5 (EPB41L5) is an essential protein for EMT progression and metastasis in various types of cancer. We found that ZBTB7A depletion in U87 cells induced GBM progression and metastasis. Based on RNA sequencing data, ZBTB7A directly binds to the promoter of the EPB41L5 gene, reducing its expression and inhibiting GBM progression. We demonstrated that ZBTB7A dramatically inhibits GBM tumor growth through transcriptional repression of EPB41L5. Thus, both ZBTB7A and EPB41L5 may be potential biomarkers and novel therapeutic targets for GBM treatment. Overall, we discovered the role of a novel tumor suppressor that directly inhibits GBM progression (ZBTB7A) and identified EPB41L5 as a therapeutic target protein for patients with GBM.