Discovery of LLC355 as an Autophagy-Tethering Compound for the Degradation of Discoidin Domain Receptor 1.

Discoidin domain receptor 1 (DDR1) is a potential target for cancer drug discovery. Although several DDR1 kinase inhibitors have been developed, recent studies have revealed the critical roles of the noncatalytic functions of DDR1 in tumor progression, metastasis, and immune exclusion. Degradation of DDR1 presents an opportunity to block its noncatalytic functions. Here, we report the discovery of the DDR1 degrader LLC355 by employing autophagosome-tethering compound technology. Compound LLC355 efficiently degraded DDR1 protein with a DC50 value of 150.8 nM in non-small cell lung cancer NCI-H23 cells. Mechanistic studies revealed compound LLC355 to induce DDR1 degradation via lysosome-mediated autophagy. Importantly, compound LLC355 potently suppressed cancer cell tumorigenicity, migration, and invasion and significantly outperformed the corresponding inhibitor 1. These results underline the therapeutic advantage of targeting the noncatalytic function of DDR1 over inhibition of its kinase activity.

Promotion of hair growth by a conditioned medium from human umbilical cord mesenchymal stem cells cultivated in a 3D scaffold of gelatin sponge.

BACKGROUND: This study aims to investigate the effects of a conditioned medium (CM) from human umbilical cord mesenchymal stem cells (HuMSCs) cultivated in gelatin sponge (GS-HuMSCs-CM) on hair growth in a mouse model. METHODS: CM was collected from the HuMSCs cultivated in a monolayer or in a gelatin sponge. Vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF) levels in CMs were measured by enzyme-linked immunosorbent assays (ELISAs). A hair loss model by a C57 BL/6J mouse was prepared. The effects of GS-HuMSCs-CM and HuMSCs on hair regrowth in mice were investigated by intradermal injection in the depilated back skin with normal saline (NS) as the control. The time for hair regrowth and full covering in depilated areas was observed, and the hair growth was evaluated histologically and by grossly measuring hair length and diameter. RESULTS: Compared with monolayer cultured cells, the three-dimensional (3D) culture of HuMSCs in gelatin sponge drastically increased VEGF, IGF-1, KGF, and HGF production. GS-HuMSCs-CM and HuMSCs injection both promoted hair regeneration in mice, while GS-HuMSCs-CM presented more enhanced effects in hair length, hair diameter, and growth rate. GS-HuMSCs-CM significantly promoted angiogenesis in injected skin areas, which might also contribute to faster hair regrowth. CONCLUSION: GS-HuMSCs-CM exerted significant effects on inducing hair growth and promoted skin angiogenesis in C57BL/6J mice.

Cryopreserved amniotic membrane in the treatment of limb skin defects of aplasia cutis congenita: a case study.

OBJECTIVE: To report the efficacy and long-term outcomes of treating the skin defects of aplasia cutis congenita (ACC) with cryopreserved amniotic membrane (AM). METHOD: Human amnion was obtained from the caesarean delivery of a full-term healthy pregnancy and processed in a sterile laminar flow hood, and cryopreserved in liquid nitrogen. The structure of the AM was investigated histologically and the viability of the epithelial cells was assessed after cryopreservation and compared with fresh AM and with AM preserved in phosphate-buffered saline (PBS) at 4°C. The cryopreserved AM was applied onto the lower limb skin defects of a one-month old baby with ACC. Timely AM changes were performed as necessary until the wounds healed. RESULTS: The structure of the cryopreserved AM was intact, with little visible difference compared with fresh AM. The viability of the epithelial cells was partially lost but still much better retained than in those preserved in PBS at 4°C. The limb skin defects were gradually re-epithelialised upon application of the AM and were completely healed after one month. The 4-month and 2-year follow-ups presented good skin texture and colour, without hypertrophic scar formation. CONCLUSION: In this case study, cryopreservation of AM presented a well preserved stromal compartment and viable epithelial layer. It also offered features such as pain relief, good attachment and adhesiveness, improved wound healing and suppressed scar formation in the treatment of ACC skin defects.

An investigation of a self-assembled cell-extracellular complex and its potentials in improving wound healing.

BACKGROUND: To maintain and enhance the wound healing effects of mesenchymal stem cells (MSCs), a scaffold for hosting MSCs is needed, which ought to be completely biocompatible, durable, producible, and of human source. OBJECTIVE: To build a cell-extracellular matrix (ECM) complex assembled by human umbilical cord mesenchymal stem cells (HuMSCs) and to investigate its clinical potentials in promoting wound healing. METHOD: HuMSCs were isolated and expanded. When the cells of third passage reached confluency, ascorbic acid was added to stimulate the cells to deposit ECM where the cells grew in. Four weeks later, a cells-loaded ECM sheet was formed. The cell-ECM complex was observed under the scanning electron microscopy (SEM) and subjected to histological studies. The supernatants were collected and the cell-ECM complex was harvested at different time points and processed for enzyme-linked immune sorbent assay (ELISA) and mRNA analysis. The in vivo experiments were performed by means of implanting the cell-ECM complex on the mice back for up to 6 months and the specimens were collected for histological studies. RESULTS: After 4 weeks of cultivation with ascorbic stimulation, a sheet was formed which is mainly composed with HuMSCs, collagen and hyaluronic acid. The cell-ECM complex can sustain to certain tensile force. The mRNA and protein levels of vascular endothelial growth factor-α (VEGF-α), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), and transforming growth factor-ß1 (TGF-ß1) were remarkably increased compared to monolayer-cultured cells. The implanted cell-ECM complex on mice was still noticeable with host cells infiltration and vascularization on 6 months. CONCLUSION: Our studies suggested that HuMSCs can be multi-cultivated through adding ascorbic stimulation and ECM containing collagen and hyaluronic acid were enriched around the cells which self-assembly formed a cell-ECM complex. Cell-ECM complex can improve growth factors secretion remarkably which means it may promote wound healing by paracrine.

Effects of caloric overload before caloric restriction in the murine heart.

The beneficial effects of caloric restriction (CR) against cardiac aging and for prevention of cardiovascular diseases are numerous. However, to our knowledge, there is no scientific evidence about how a high-calorie diet (HCD) background influences the mechanisms underlying CR in whole heart tissue (WHT) in experimental murine models. In the current study, CR-treated mice with different alimentary backgrounds were subjected to transthoracic echocardiographic measurements. WHT was then analyzed to determine cardiac energetics, telomerase activity, the expression of energy-sensing networks, tissue-specific adiponectin, and cardiac precursor/cardiac stem cell markers. Animals with a balanced diet consumption before CR presented marked cardiac remodeling with improved ejection fraction (EF) and fractional shortening (FS), enhanced OXPHOS complex I, III, and IV, and CKMT2 enzymatic activity. Mice fed an HCD before CR presented moderate changes in cardiac geometry with diminished EF and FS values, but improved OXPHOS complex IV and CKMT2 activity. Differences in cardiac remodeling, left ventricular systolic/diastolic performance, and mitochondrial energetics, found in the CR-treated mice with contrasting alimentary backgrounds, were corroborated by inconsistencies in the expression of mitochondrial-biogenesis-related markers and associated regulatory networks. In particular, disruption of eNOS and AMPK -PGC-1α-mTOR-related axes. The impact of a past habit of caloric overload on the effects of CR in the WHT is a scarcely explored subject that requires deeper study in combination with analyses of other tissues and organs at higher levels of organization within the organ system. Such research will eventually lead to the development of preventative and therapeutic strategies to promote health and longevity.

Characteristics and clinical potential of a cellularly modified gelatin sponge.

BACKGROUND: Human umbilical cord mesenchymal stem cells (HuMSCs) injected directly have been proven effective for improving chronic wounds. However, HuMSCs largely die within 14 days. The aim of study is to establish a cellularly modified gelatin sponge and investigate its characteristics and clinical potential. METHODS: HuMSCs were isolated, expanded and seeded in a poly-L-lysine (PLL)-coated gelatin sponge. Fabricated gelatin sponges were estimated through observation of morphological surface and ultrastructure, following confirmed by histology method. Supernatants were collected at different times for enzyme-linked immunosorbent assays (ELISAs) to measure growth factors. The cell embedded gelatin sponges were implanted subcutaneously on the backs of mice and the samples were harvested and studied histologically. RESULTS: HuMSCs gradually modified the gelatin sponge by depositing collagen and hyaluronic acid, and degrading the structure of gelatin, resulting in a dense, and elastic structure. Compared with cells cultured in monolayer, the levels of growth factors increased remarkably when HuMSCs were cultivated in the gelatin sponge. Upon subcutaneous implantation in the backs of mice, the cellularized gelatin sponges persisted for up to 2 months and eventually integrated into the host tissue, while blank gelatin sponges degraded completely by the end of the second month. CONCLUSION: Gelatin sponge is a clinically accessible scaffold for HuMSCs implantation to maintain short-term survival of the cells and high-level production of growth factors, which demonstrates good clinical potential for enhancing wound healing.

Articular chondrocyte-derived extracellular vesicles promote cartilage differentiation of human umbilical cord mesenchymal stem cells by activation of autophagy.

BACKGROUND: Umbilical cord mesenchymal stem cell (HUCMSC)-based therapies were previously utilised for cartilage regeneration because of the chondrogenic potential of MSCs. However, chondrogenic differentiation of HUCMSCs is limited by the administration of growth factors like TGF-ß that may cause cartilage hypertrophy. It has been reported that extracellular vesicles (EVs) could modulate the phenotypic expression of stem cells. However, the role of human chondrogenic-derived EVs (C-EVs) in chondrogenic differentiation of HUCMSCs has not been reported. RESULTS: We successfully isolated C-EVs from human multi-finger cartilage and found that C-EVs efficiently promoted the proliferation and chondrogenic differentiation of HUCMSCs, evidenced by highly expressed aggrecan (ACAN), COL2A, and SOX-9. Moreover, the expression of the fibrotic marker COL1A and hypertrophic marker COL10 was significantly lower than that induced by TGF-ß. In vivo, C-EVs induced HUCMSCs accelerated the repair of the rabbit model of knee cartilage defect. Furthermore, C-EVs led to an increase in autophagosomes during the process of chondrogenic differentiation, indicating that C-EVs promote cartilage regeneration through the activation of autophagy. CONCLUSIONS: C-EVs play an essential role in fostering chondrogenic differentiation and proliferation of HUCMSCs, which may be beneficial for articular cartilage repair.

Plasma membrane vesicles of human umbilical cord mesenchymal stem cells ameliorate acetaminophen-induced damage in HepG2 cells: a novel stem cell therapy.

BACKGROUND: Acetaminophen (APAP) overdose is the common cause of acute liver failure (ALF) due to the oxidative damage of multiple cellular components. This study aimed to investigate whether plasma membrane vesicles (PMVs) from human umbilical cord mesenchymal stem cells (hUCMSCs) could be exploited as a novel stem cell therapy for APAP-induced liver injury. METHODS: PMVs from hUCMSCs were prepared with an improved procedure including a chemical enucleation step followed by a mechanical extrusion. PMVs of hUCMSCs were characterized and supplemented to hepatocyte cultures. Rescue of APAP-induced hepatocyte damage was evaluated. RESULTS: The hUCMSCs displayed typical fibroblastic morphology and multipotency when cultivated under adipogenic, osteogenic, or chondrogenic conditions. PMVs of hUCMSCs maintained the stem cell phenotype, including the presence of CD13, CD29, CD44, CD73, and HLA-ABC, but the absence of CD45, CD117, CD31, CD34, and HLA-DR on the plasma membrane surface. RT-PCR and transcriptomic analyses showed that PMVs were similar to hUCMSCs in terms of mRNA profile, including the expression of stemness genes GATA4/5/6, Nanog, and Oct1/2/4. GO term analysis showed that the most prominent reduced transcripts in PMVs belong to integral membrane components, extracellular vesicular exosome, and extracellular matrix. Immunofluorescence labeling/staining and confocal microscopy assays showed that PMVs enclosed cellular organelles, including mitochondria, lysosomes, proteasomes, and endoplasmic reticula. Incorporation of the fusogenic VSV-G viral membrane glycoprotein stimulated the endosomal release of PMV contents into the cytoplasm. Further, the addition of PMVs and a mitochondrial-targeted antioxidant Mito-Tempo into cultures of APAP-treated HepG2 cells resulted in reduced cell death, enhanced viability, and increased mitochondrial membrane potential. Lastly, this study demonstrated that the redox state and activities of aminotransferases were restored in APAP-treated HepG2 cells. CONCLUSIONS: The results suggest that PMVs from hUCMSCs could be used as a novel stem cell therapy for the treatment of APAP-induced liver injury.

Overexpression of retinoblastoma­binding protein 4 contributes to the radiosensitivity of AGS gastric cancer cells via phosphoinositide3­kinase/protein kinase B pathway suppression.

In the present study, the effects and underlying mechanism of RbAp48 on the radiosensitivity of AGS gastric cancer cells was investigated. Cell proliferation was determined with an MTT assay. Flow cytometry was performed to evaluate the cell cycle and apoptosis. Reverse transcription­quantitative polymerase chain reaction and western blot analysis were performed to detect mRNA and protein expression, respectively, including RbAp48, phosphoinositide 3­kinase (PI3K) and protein kinase B (Akt). The results revealed that radiation enhanced the expression level of RbAp48 in AGS cells, and that RbAp48 combined with radiation reduced AGS cell proliferation. In addition, RbAp48 combined with radiation resulted in G2 phase arrest and induced apoptosis via regulation of the PI3K/Akt pathway. In conclusion, it was demonstrated that overexpression of RbAp48 enhanced the radiosensitivity of AGS gastric cancer cells via suppression of PI3K/Akt pathway activity, suggesting that RbAp48 may hold potential as a gene therapeutic strategy in the future, aiding in the treatment of gastric cancer.

Basic Fibroblast Growth Factor Influences Epidermal Homeostasis of Living Skin Equivalents through Affecting Fibroblast Phenotypes and Functions.

AIMS: To elucidate the possible mechanisms of how basic fibroblast growth factor (bFGF) influences epidermal homeostasis in a living skin equivalent (LSE) model. METHODS: Several wound healing-related growth factors were analyzed at protein and mRNA levels for dermal fibroblasts of induced alpha-smooth muscle actin (α-SMA)-positive or α-SMA-negative phenotypes. During culturing an LSE model by seeding normal human keratinocytes on a fibroblast-populated type I collagen gel, bFGF or neutralizing antibody for keratinocyte growth factor (KGF) was added to investigate its effects on fibroblast phenotypes and, subsequently, epidermal homeostasis by histology and immunohistochemistry. RESULTS: The α-SMA-positive phenotype of fibroblasts induced by transforming growth factor beta-1 (TGF-ß1) markedly suppressed the expression of KGF and hepatocyte growth factor (HGF), and slightly upregulated vascular endothelial growth factor (VEGF) and TGF-ß1 at mRNA and protein levels, compared with α-SMA-negative fibroblasts treated with bFGF. α-SMA expression of fibroblasts at the epidermal-mesenchymal junction of the LSEs was suppressed by the addition of bFGF, and a better-differentiated epidermis was presented. The abrogation of KGF from fibroblasts by the addition of the KGF neutralizing antibody disenabled the LSE culturing system to develop an epidermis. CONCLUSIONS: bFGF, through affecting the phenotypes and functions of fibroblasts, especially KGF expression, influenced epidermal homeostasis in an LSE model.

Genome-Wide DNA Methylation Analysis During Palatal Fusion Reveals the Potential Mechanism of Enhancer Methylation Regulating Epithelial Mesenchyme Transformation.

Epithelial mesenchyme transformation (EMT) of the medial edge epithelium (MEE) is the crucial process during palatal fusion. This work is aimed to elucidate the enhancer regulatory mechanism by genome-wide DNA methylation analysis of EMT during palatal fusion. Over 800 million clean reads, 325 million enzyme reads, and 234 million mapping reads were generated. The mapping rate was 68.85-74.32%, which included differentially methylated 17299 CCGG sites and 2363 CCWGG sites (p < 0.05, log2FC >1). Methylated sites in intron and intergenic regions were more compared to other regions of all DNA elements. GO and KEGG analysis indicated that differential methylation sites related to embryonic palatal fusion genes (HDAC4, TCF7L2, and PDGFRB) at the enhancer were located on CCWGG region of non-CpG islands. In addition, the results showed that the enhancer for HDAC4 was hypermethylated, whereas the enhancers for TCF7L2 and PDGFRB were hypomethylated. The methylation status of enhancer regions of HDAC4, PDGFRB, and TCF7L2, involved in the regulation of the EMT during palatal fusion, may enlighten the development of novel epigenetic biomarkers in the treatment of cleft palate.

Human umbilical cord Wharton jelly cells promote extra-pancreatic insulin formation and repair of renal damage in STZ-induced diabetic mice.

BACKGROUND: We evaluated the therapeutic effect and fate of high doses of human umbilical cord Wharton jelly cells (hUCWJCs) after IP administration to streptozotocin (STZ)-induced diabetic mice. METHODS: Type 1 diabetes (T1D) was induced in Kunming mice via IP injection of STZ. hUCWJCs were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). Diabetic animals with sustained hyperglycemia for at least 2 weeks were administered 1 × 107 Dil-hUCWJCs via intraperitoneal injection. Insulin, glucagon and PDX-1 were detected by immunofluorescence with confocal microscopy. Serum mouse and human C-peptide was assayed in blood collected via intracardiac puncture. Specific ß-cell differentiation markers and human DNA were assessed using qPCR performed with 200 ng of target DNA. RESULTS: hUCWJCs migrated to the STZ-damaged organs and contributed to lower blood glucose levels in 30% of the treated mice. Confocal microscopy revealed the presence of resident insulin-positive cells in the liver and kidneys. hUCWJC-treated mice with restored hyperglycemia also showed increased serum mouse C-peptide levels. The qPCR results, particularly in the liver, revealed that after transplantation hUCWJCs upregulated genes of endocrine precursors but failed to express endocrine stage markers. Mice with restored hyperglycemia had reduced urinary volume and lacked glomerular hypertrophy, exhibiting a morphology resembling that of normal glomeruli. Moreover, we also verified that one of the possible mechanisms by which hUCWJCs exert immunosuppressive effects is through down-regulation of the cell surface receptor HLA-1. CONCLUSIONS: We confirmed the potential of IP administration of hUCWJCs and the capability of these cells to migrate to damaged tissues and promote insulin secretion from non-pancreatic local cells and to improve renal damage. These findings confer unique therapeutic properties to hUCWJCs, suggesting a promising future in the treatment of diabetes mellitus.

Platelet poor plasma gel combined with amnion improves the therapeutic effects of human umbilical cord­derived mesenchymal stem cells on wound healing in rats.

The aim of the present study was to investigate the efficacy of human umbilical cord­derived mesenchymal stem cell (HUMSCs) embedded in platelet poor plasma (PPP) gel combined with amnion (PPPA) in improving wound healing on Sprague­Dawley (SD) rats. HUMSCs were cultured and labeled with chloromethylbenzamido­1,1'­dioctadecyl­3,3,3'3'­tetramethylindocarbocyanine perchlorate (CM­DiI) on their third passage. The expression levels of growth factors of HUMSCs in PPPA were assessed by ELISA. Full­thickness excisional skin wounds were induced in 36 male SD rats, which were treated with PPPA grafted with HUMSCs (PPPAC), PPPA, or HUMSC or PBS injection. The degree of healing and the distribution of labeled HUMSCs in the wound were evaluated by hematoxylin and eosin (H&E) staining and immunofluorescence. On day 14 post­surgery, wound healing in PPPAC­treated rats was significantly higher than the PPPA group, compared with rats treated with HUMSCs alone and control rats (P<0.05 and P<0.01, respectively). H&E staining showed that morphology and thickness of the epidermis in the PPPAC group was similar to that of healthy skin. ELISA revealed that levels of growth factors of HUMSCs in PPPAC were higher than in monolayer cells. In conclusion, PPPA can modify growth factor expression levels of HUMSCs and improve the efficiency of HUMSCs in the healing of full thickness wounds in rats.

[Application of amniotic membrane-living skin equivalent in repairing skin defect after removal of congenital giant nevus].

Objective: To investigate the feasibility of human amniotic membrane-living skin equivalent (AM-LSE) in repairing the skin defect. Methods: A 5-year-old boy with giant nevus at neck, shoulder, and back was admitted in July 2016. Normal skin tissue of the patient was harvested and keratinocytes and dermal fibroblasts were separated and expanded in vitro. Human AM was donated from a normal delivery and de-epithelialized for constructing an LSE as a matrix. Keratinocytes were seeded on the epithelial side of the AM which was previously seeded with fibroblasts on the stromal side and then the complex was lifted for air-liquid surface cultivation for 10 days and observed under naked eyes and sampled for histological study. The nevus was excised to deep fascia and the skin defect in size of 20 cm×15 cm was covered with artificial skin of collagen sponge for 2 weeks to enhance granulation tissue formation, and then the AM-LSE grafts of stamp size were grafted on. The dressing was changed until the wound healed. Results: After 10 days of air-liquid surface cultivation, the AM-LSE developed a multilayered and differentiated epidermis with the fibroblasts-populated amnion as the dermal matrix. The LSE stamps survived and expanded to cover the whole wound. The grafted area showed normal skin color and soft contexture at 6 months after operation, and histological study showed well developed epidermis with compactly aligned basal cells, stratified and well differentiated squamous, granular layers and stratum corneum and well vascularized dermal compartment without inflammatory cells infiltration. Conclusion: The cultivated AM-LSE with autologous cells can repair skin defect and survive for a long term without rejection.

Targeting Notch1 and proteasome as an effective strategy to suppress T-cell lymphoproliferative neoplasms.

The T-cell lymphoproliferative neoplasms (T-LPN) are characterized by a poor clinical outcome. Current therapeutics are mostly non-selective and may induce harmful side effects. It has been reported that NOTCH1 activation mutations frequently associate T-LPN. Because anti-Notch1 based therapies such as γ-secretase inhibitors (GSI) are less efficient and induce considerable side effects, we hypothesized that combining low concentrations of GSI and the proteasome inhibitor bortezomib (BTZ) may provide an effective and tolerable approach to treat T-LPN. Hence, we analyzed the in vitro and in vivo effects of GSI-I and BTZ, alone or in combination, against T-LPN. GSI-I and BTZ synergistically decreased cell viability, proliferation, and colony formation, and induced apoptosis in T-LPN cell lines. Furthermore, combining GSI-I and BTZ decreased the viability of primary T-LPN cells from patients. These effects were accompanied by deregulation of Notch1, AKT, ERK, JNK, p38 MAPK, and NF-κB survival pathways. Moreover, combination treatment inhibited T-LPN tumor growth in nude mice. In all experiments, combining low concentrations of GSI-I and BTZ was superior to using a single agent. Our data support that a synergistic antitumor activity exists between GSI-I and BTZ, and provide a rationale for successful utilization of dual Notch1 and proteasome inhibition to treat T-LPN.

Stromal-epithelial interaction study: The effect of corneal epithelial cells on growth factor expression in stromal cells using organotypic culture model.

Interactions between stromal and epithelial cells play important roles in the development, homeostasis, and pathological conditions of the cornea. Soluble cytokines are critical factors in stromal-epithelial interactions, and growth factors secreted from corneal stromal cells contribute to the regulation of proliferation and differentiation of corneal epithelial cells (CECs). However, the manner in which the expression of growth factors is regulated in stromal cells has not been completely determined. To study stromal-epithelial cell interactions, we used an organotypic culture model. Human or rabbit CECs (HCECs or RCECs) were cultured on amniotic membranes placed on human corneal fibroblasts (HCFs) embedded in a collagen gel. The properties of the organotypic culture were examined by hematoxylin-eosin staining and immunofluorescence. In the organotypic culture, HCECs or RCECs were stratified into two-three layers after five days and five-seven layers after nine days. However, stratification was not observed when the HCECs were seeded on a collagen gel without fibroblasts. K3/K12 were expressed on day 9. The HCF-embedded collagen gels were collected on days 3, 5, or 9 after seeding the RCECs, and mRNA expression of growth factors FGF7, HGF, NGF, EGF, TGF-α, SCF, TGF-ß1, TGF-ß2, and TGF-ß3 were quantified by real-time PCR. mRNA expression of the growth factors in HCFs cultured with RCECs were compared with those cultured without RCECs, as well as in monolayer cultures. mRNA expression of TGF-α was markedly increased in HCFs cultured with RCECs. However, mRNA expression of the TGF-ß family was suppressed in HCFs cultured with RCECs. Principal component analysis revealed that mRNA expression of the growth factors in HCFs were generally similar when they were cultured with RCECs. In organotypic cultures, the morphological changes in the CECs and the expression patterns of the growth factors in the stromal cells clearly demonstrated stromal-epithelial cell interactions, and the results suggest that stromal cells and epithelial cells may act in concert in the cornea.

The Ca(2+) channel inhibitor 2-APB reverses ß-amyloid-induced LTP deficit in hippocampus by blocking BAX and caspase-3 hyperactivation.

BACKGROUND AND PURPOSE: At the early stage of Alzheimer's disease (AD), the accumulation of ß-amyloid (Aß) oligomers disturbs intracellular Ca(2+) homeostasis and disrupts synaptic plasticity of brain neurons. Prevention of Aß-induced synaptic failure remains an unsolved problem for the treatment of AD. Here, the effects of 2-aminoethoxydiphenyl borate (2-APB), a non-specific, but moderately potent Ca(2+) channel inhibitor, on Aß-induced deficit of synaptic long-term potentiation (LTP) and the underlying molecular mechanisms were explored. EXPERIMENTAL APPROACH: We used hippocampal slices and primary cultures of hippocampal neurons from C57BL/6 mice. Methods applied in our study included electrophysiological recording, membrane protein extraction, Western blot assay and Ca(2+) imaging. KEY RESULTS: 2-APB at 10 µM effectively reversed suppression by oligomeric Aß1-42 (500 nM) of LTP in hippocampal slices. 2-APB also restored phosphorylation and trafficking of the glutamate receptor subunit GluA1 in Aß-treated hippocampal slices, supporting its protective action on synaptic function. Aß-mediated abnormal neuronal [Ca(2+) ]i elevation and hyperactivation of the mitochondrial apoptotic proteins BAX, caspase-3, and glycogen synthase kinase-3ß, were blocked by 2-APB pretreatment. Moreover, the defict in long term potentiation deficit in hippocampal slices from APPswe /PS1ΔE 9 gene mutant mice was rescued by 2-APB at 10 µM. CONCLUSIONS AND IMPLICATION: These data demonstrate that 2-APB is a potentially useful chemical to protect synaptic plasticity against neurotoxic effects of Aß in AD.

Transient axonal glycoprotein-1 induces apoptosis-related gene expression without triggering apoptosis in U251 glioma cells.

Previous studies show that transient axonal glycoprotein-1, a ligand of amyloid precursor protein, increases the secretion of amyloid precursor protein intracellular domain and is involved in apoptosis in Alzheimer's disease. In this study, we examined the effects of transient axonal glycoprotein-1 on U251 glioma cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that transient axonal glycoprotein-1 did not inhibit the proliferation of U251 cells, but promoted cell viability. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that transient axonal glycoprotein-1 did not induce U251 cell apoptosis. Real-time PCR revealed that transient axonal glycoprotein-1 substantially upregulated levels of amyloid precursor protein intracellular C-terminal domain, and p53 and epidermal growth factor receptor mRNA expression. Thus, transient axonal glycoprotein-1 increased apoptosis-related gene expression in U251 cells without inducing apoptosis. Instead, transient axonal glycoprotein-1 promoted the proliferation of these glioma cells.

Decreased percentages of regulatory T cells are necessary to activate Th1-Th17-Th22 responses during acute rejection of the peripheral nerve xenotransplantation in mice.

BACKGROUND: T cells have major functions in the initiation and perpetuation of nerve graft rejection. Our study aimed to investigate the function of regulatory T cells (Treg)-Th1-Th17-Th22 cells in the rejection of peripheral nerve xenotransplantation. METHODS: Adult male C57 BL/6 mice were used as the recipient for nerve xenotransplantation, and Sprague-Dawley rats were used as the donor. These nerve xenotransplanted mice were used as the experimental groups, and those that received autograft transplant were chosen as the control group. All of the animals were pretreated with interferon (IFN)-γ, interleukin (IL)-17, and IL-22 before the experiment was conducted. The percentages of spleen Treg-Th1-Th17-Th22 cells were evaluated by flow cytometry 1, 3, 7, 14, and 28 days after transplantation. Serum levels of IFN-γ, IL-17, and IL-22 were assessed by enzyme-linked immunosorbent assay. Statistical analysis was performed by Wilcoxon rank sum and Spearman correlation test. RESULTS: During acute rejection, the percentages of Th1-Th17-Th22 cells in the spleen and serum IFN-γ, IL-17, and IL-22 levels in the experimental group increased compared with those in the control group. By contrast, CD4CD25Foxp3 T cell level decreased. The rejection of xenograft was significantly prevented after the mice were treated with IL-17-neutralizing, IL-22-neutralizing, and IFN-γ-neutralizing antibodies. Moreover, the percentage of CD4CD25Foxp3 Treg was negatively correlated with the percentages of Th1-Th17-Th22 cells and levels of IL-17, IL-22, and IFN-γ. CONCLUSION: These results suggested that the Treg-Th1-Th17-Th22 cells involved in xenotransplant rejection and imbalance between Tregs and Th1-Th17-Th22 cells contribute to the acute rejection of peripheral nerve xenotransplant.

Multiple systemic transplantations of human amniotic mesenchymal stem cells exert therapeutic effects in an ALS mouse model.

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disease involving degeneration of motor neurons in the central nervous system. Stem cell treatment is a potential therapy for this fatal disorder. The human amniotic membrane (HAM), an extremely rich and easily accessible tissue, has been proposed as an attractive material in cellular therapy and regenerative medicine because of its advantageous characteristics. In the present study, we evaluate the long-term effects of a cellular treatment by intravenous administration of human amniotic mesenchymal stem cells (hAMSCs) derived from HAM into a hSOD1(G93A) mouse model. The mice received systemic administration of hAMSCs or phosphate-buffered saline (PBS) at the onset, progression and symptomatic stages of the disease. hAMSCs were detected in the spinal cord at the final stage of the disease, in the form of isolates or clusters and were negative for ß-tubulin III and GFAP. Compared with the treatment with PBS, multiple hAMSC transplantations significantly retarded disease progression, extended survival, improved motor function, prevented motor neuron loss and decreased neuroinflammation in mice. These findings demonstrate that hAMSC transplantation is a promising cellular treatment for ALS.