Nanoparticles carrying paclitaxel and anti-miR-221 for breast cancer therapy triggered by ultrasound.

Nanomaterials have been well demonstrated to have the potential to be used for tumor cell-targeted drug delivery. Targeted inhibition of miR-221 was proved to promote the sensitivity of triple genitive breast cancer (TNBC) cells to chemo-drugs. In order to improve the chemotherapeutic effect in TNBC, herein, we developed a novel kind of nanoparticles shelled with PLGA and loaded with perfluoropentane (PFP), paclitaxel (PTX), and anti-miR-221 inhibitor, which was named PANP. Ultrasound-triggered vaporization of PFP in PANPs was utilized for real-time imaging track of the nanoparticles in vivo. In addition, macrophages were applied for the internalization of PANPs to form RAW-PANP with strong chemotaxis to accumulate around cancer cells. Nanoparticles with different contents did not cause M2 polarization compared with the control group but caused polarization toward M1. We compared the inherent tumor-homing behavior of macrophages containing different contents with that of normal macrophages and no significant abnormalities were observed. After injection into the tumor-burden mice, RAW-PANPs showed enrichment within tumor tissues. Upon the ultrasound cavitation-triggered burst, PTX was released in the tumor. Meanwhile, the release of anti-miR-221 improved the sensitivity of tumor cells to PTX. As a result, RAW-PANPs showed high efficiency in suppressing TNBC cell proliferation in vitro and inhibiting tumor growth and progression in vivo. The treatments did not induce liver, heart, or kidney injury. In conclusion, the current study not only developed a macrophage-carried, ultrasound-triggered, cancer cell-targeted chemotherapeutic system, but also demonstrated a miRNA-based technique to promote drug sensitivity of cancer cells, which holds strong potential to treat patients with TNBC, especially for those suffering drug-resistance. The innovation of this study is to use macrophages to deliver nanoparticles to the tumors and then use ultrasound locally to burst the nanoparticles to release the miRNA and PTX.

Resveratrol Enhances Temozolomide Efficacy in Glioblastoma Cells through Downregulated MGMT and Negative Regulators-Related STAT3 Inactivation.

Chemoresistance blunts the efficacy of temozolomide (TMZ) in the treatment of glioblastoma (GBM). Elevated levels of O6-methylguanine-DNA methyltransferase (MGMT) and activation of signal transducer and of transcription 3 (STAT3) have been reported to correlate with GBM resistance to alkylator chemotherapy. Resveratrol (Res) inhibits tumor growth and improves drug chemosensitivity by targeting STAT3 signaling. Whether the combined therapy of TMZ and Res could enhance chemosensitivity against GBM cells and the underlying molecular mechanism remains to be determined. In this study, Res was found to effectively improve chemosensitivities of different GBM cells to TMZ, which was evaluated by CCK-8, flow cytometry, and cell migration assay. The combined use of Res and TMZ downregulated STAT3 activity and STAT3-regulated gene products, thus inhibited cell proliferation and migration, as well as induced apoptosis, accompanied by increased levels of its negative regulators: PIAS3, SHP1, SHP2, and SOCS3. More importantly, a combination therapy of Res and TMZ reversed TMZ resistance of LN428 cells, which could be related to decreased MGMT and STAT3 levels. Furthermore, the JAK2-specific inhibitor AG490 was used to demonstrate that a reduced MGMT level was mediated by STAT3 inactivation. Taken together, Res inhibited STAT3 signaling through modulation of PIAS3, SHP1, SHP2, and SOCS3, thereby attenuating tumor growth and increasing sensitivity to TMZ. Therefore, Res is an ideal candidate to be used in TMZ combined chemotherapy for GBM.

Morroniside protects OLN-93 cells against H2O2-induced injury through the PI3K/Akt pathway-mediated antioxidative stress and antiapoptotic activities.

Neurodegenerative disorders, including spinal cord injury (SCI), result in oxidative stress-induced cell damage. Morroniside (MR), a major active ingredient of the Chinese herb Shan Zhu Yu, has been shown to ameliorate oxidative stress and inflammatory response. Our previous study also confirmed that morroniside protects SK-N-SH cell line (human neuroblastoma cells) against oxidative impairment. However, it remains unclear whether MR also plays a protective role for oligodendrocytes that are damaged following SCI. The present study investigated the protective effects of MR against hydrogen peroxide (H2O2)-induced cell death in OLN-93 cells. MR protected OLN-93 cells from H2O2-induced injury, attenuated H2O2-induced increase in reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and blocked the reduction of mitochondrial membrane potential (MMP) induced by H2O2. MR enhanced the activity of the antioxidant enzyme superoxide dismutase (SOD) and suppressed H2O2-induced downregulation of the antiapoptotic protein Bcl-2 and activation of the proapoptotic protein caspase-3. Finally, we found that LY294002, a specific inhibitor of the PI3K/Akt pathway, inhibited the protective effect of MR against H2O2-induced OLN-93 cell injury in the MTT and TUNEL assays. LY294002 also inhibited the expression of SOD and Bcl-2, and increased the expression of iNOS and c-caspase-3 induced by MR treatment. MR exerts protective effects against H2O2-induced OLN-93 cell injury through the PI3K/Akt signaling pathway-mediated antioxidative stress and antiapoptotic activities. MR may provide a potential strategy for SCI treatment or other related neurodegeneration.

Resveratrol Downregulates STAT3 Expression and Astrocyte Activation in Primary Astrocyte Cultures of Rat.

Astrocytes respond to all forms of central nervous system (CNS) insults by a process referred to as reactive astrogliosis. Inhibition of astrocyte growth and activation is an important strategy for promoting injured CNS repair. STAT3 (signal transducer and activator of transcription 3) is reported to be a critical regulator of astrogliosis, and resveratrol (RES, a dietary polyphenol) is considered to be a natural inhibitor of STAT3 expression and phosphorylation. In this study, we investigated the effects of RES on STAT3 expression and phosphorylation, and then on the proliferation and activation of astrocytes, a critical process in reactive astrogliosis, in rat primary cultured astrocytes and an in vitro scratch-wound model. RES downregulated the expression levels of STAT3, P-STAT3 and GFAP (glial fibrillary acidic protein) in cultured astrocytes. The positive index of Ki67 was apparently reduced in cultured astrocytes after RES treatment. Meanwhile, cultured astrocyte proliferation and activation were attenuated by RES. Moreover, in the established in vitro scratch-wound model the increased expression levels of STAT3, P-STAT3 and GFAP induced by scratching injury were also clearly inhibited by RES. In addition, the inhibitory effect of RES on cell proliferation was similar to that of AG490 (a selective inhibitor of STAT3 phosphorylation) and abrogated by Colivelin (a STAT3 activator) stimuli. Taken together, our data suggest that RES is able to inhibit reactive astrocyte proliferation and activation mainly via deactivating STAT3 pathway. So RES may have a therapeutic benefit for the treatment of the injured CNS.

Resveratrol Suppresses the Growth and Enhances Retinoic Acid Sensitivity of Anaplastic Thyroid Cancer Cells.

Anaplastic thyroid cancer (ATC) is a highly lethal undifferentiated malignancy without reliable therapies. Retinoic acid (RA) has been employed to promote redifferentiation of thyroid cancers by increasing their I131 uptake and radio-sensitivity, but its effect(s) on ATCs has not yet been ascertained. Likewise, resveratrol induces cancer redifferentiation but, also in this case, its effects on ATCs remain unknown. These issues have been addresses in the current study using three human ATC cell lines (THJ-11T, THJ-16T, and THJ-21T) through multiple experimental approaches. The results reveal that RA exerts a small inhibitory effect on these cell lines. In comparison with normally cultured cells, the total cell number in resveratrol-treated THJ-16T and THJ-21T cultures significantly decreased (p < 0.05), and this effect was accompanied by reduced Cyclin D1 immuno-labeling, increased apoptotic fractions, and distinct caspase-3 activation. Resveratrol failed to inhibit growth but enhanced RA sensitivity of THJ-11T cells, suppressed peroxisome proliferator-activated receptor-ß/δ (PPAR-ß/δ), and upregulated cellular retinoic acid-binding protein 2 (CRABP2) and retinoic acid receptor beta (RAR-ß) expression. Increased thyroglobulin (Tg) and E-cadherin levels and appearance of membranous E-cadherin were evidenced in resveratrol-treated THJ-11T cells. Our results demonstrate for the first time: (1) the therapeutic value of resveratrol by itself or in combination with RA in the management of ATCs, (2) the capacity of resveratrol to overcome RA resistance in ATC cells by reprogramming CRABP2/RAR- and fatty acid-binding protein 5 (FABP5)/PPAR-ß/δ-mediated RA signaling, and (3) the redifferentiating potential of resveratrol in ATC cells.

Differential CRABP-II and FABP5 expression patterns and implications for medulloblastoma retinoic acid sensitivity.

Medulloblastoma (MB) cells exhibit different responses to retinoid acid (RA) for reasons that are poorly understood. RA signaling can be transduced by two approaches that are mediated by cellular retinoic acid-binding protein 2 (CRABP-II) as a tumor-suppressive pathway, and by fatty acid-binding protein 5 (FABP5) as a tumor-promoting pathway. The biological effects of RA on cancer cells are largely determined by the patterns of CRABP-II and FABP5 expression. This study aims to profile the statuses of CRABP-II and FABP5 expression in MB and to evaluate their correlation with RA sensitivities using RA-sensitive (Med-3) and RA-insensitive (UW228-2, UW228-3) MB cells. Our results show that CRABP-II is distinctly expressed and the level of FABP5 is extremely low in Med-3 cells, while the patterns of CRABP-II and FABP5 expression are reversed in UW228-2 and UW228-3 cells. RA up-regulates CRABP-II expression in Med-3 cells, whereas it up-regulates FABP5 expression in the other two cell lines. The FABP5-specific inhibitor BMS309403 increases the RA sensitivity of UW228-2 cells (p < 0.01). Tissue microarray-based immunohistochemical staining showed CRABP-II/FABP5 expression patterns in MB that were variable (CRABP-II-/FABP5-, CRABP-II-/FABP5+, CRABP-II+/FABP5- and CRABP-II+/FABP5+) and imbalanced (CRABP-II↑/FABP5↓ and CRABP-II↓/FABP5↑). MB cases exhibited patterns ofCRABP-II-/FABP5- (12.24%, 6/49), CRABP-II-/FABP5+ (30.61%, 15/49) or CRABP-II↓/FABP5↑ (12.24%, 6/49), implicating unresponsiveness or insensitivity to RA. In conclusion, the ratios of CRABP-II/FABP5 levels are closely related to the RA sensitivities of MB cells. The differential CRABP-II and FABP5 expression patterns are prospective parameters, and of potential value in personalized RA therapy for MB.

Apolipoprotein E as a novel therapeutic neuroprotection target after traumatic spinal cord injury.

Apolipoprotein E (apoE), a plasma lipoprotein well known for its important role in lipid and cholesterol metabolism, has also been implicated in many neurological diseases. In this study, we examined the effect of apoE on the pathophysiology of traumatic spinal cord injury (SCI). ApoE-deficient mutant (apoE-/-) and wild-type mice received a T9 moderate contusion SCI and were evaluated using histological and behavioral analyses after injury. At 3days after injury, the permeability of spinal cord-blood-barrier, measured by extravasation of Evans blue dye, was significantly increased in apoE-/- mice compared to wild type. The inflammation and spared white matter was also significantly increased and decreased, respectively, in apoE-/- mice compared to the wild type ones. The apoptosis of both neurons and oligodendrocytes was also significantly increased in apoE-/- mice. At 42days after injury, the inflammation was still robust in the injured spinal cord in apoE-/- but not wild type mice. CD45+ leukocytes from peripheral blood persisted in the injured spinal cord of apoE-/- mice. The spared white matter was significantly decreased in apoE-/- mice compared to wild type ones. Locomotor function was significantly decreased in apoE-/- mice compared to wild type ones from week 1 to week 8 after contusion. Treatment of exogenous apoE mimetic peptides partially restored the permeability of spinal cord-blood-barrier in apoE-/- mice after SCI. Importantly, the exogenous apoE peptides decreased inflammation, increased spared white matter and promoted locomotor recovery in apoE-/- mice after SCI. Our results indicate that endogenous apoE plays important roles in maintaining the spinal cord-blood-barrier and decreasing inflammation and spinal cord tissue loss after SCI, suggesting its important neuroprotective function after SCI. Our results further suggest that exogenous apoE mimetic peptides could be a novel and promising neuroprotective reagent for SCI.

Correlative analyses of the expression levels of PIAS3, p-SHP2, SOCS1 and SOCS3 with STAT3 activation in human astrocytomas.

The importance of signal transducer and activator of transcription 3 (STAT3) signaling in the growth and survival of glioblastoma cells has been well documented, while the reasons leading to STAT3 activation remains to be elucidated. Suppressors of cytokine signaling (SOCS) 1 and SOCS3, SH2 domain­containing phosphatase (SHP2) and protein inhibitors of activated STAT3 (PIAS3) are known to inhibit STAT3 signal transduction, while their expression statuses in the four grades of astrocytomas and relevance with STAT3 activation remain to be described. The present study aimed to address these issues by tissue microarray­based immunohistochemical profiling the expression levels of phosphorylated (p)­STAT3, SOCS1, SOCS3, PIAS3 and p­SHP2. The results revealed that p­STAT3 nuclear translocation was rarely observed in non­cancerous brain tissues and its frequencies were increased in a tumor grade­associated manner (65.2, 77.1, 81.8 and 85.7% for grade I­IV, respectively). PIAS3, p­SHP2, SOCS1 and SOCS3 were expressed in higher levels (++ and +++) in 63.6, 90, 87.5 and 81.8% of tumor surrounding brain tissues, which reduced to 13.1, 47.8, 33.3 and 50% in grade I, 11.4, 65.7, 58.3 and 77.1% in grade II, 9.1, 63.6, 38.1 and 31.8% in grade III and 7.1, 66.7, 30.8 and 7.1% in grade IV astrocytomas. The above results revealed that although the expression levels of SOCS1, SOCS3 and, in particular, p­SHP2, tend to decrease in the four types of astrocytomas, PIAS3 downregulation is more negatively correlated with STAT3 activation in the stepwise progress of astrocytomas and would indicate an unfavorable outcome.

Effects of social isolation and re-socialization on cognition and ADAR1 (p110) expression in mice.

It has been reported that social isolation stress could be a key factor that leads to cognitive deficit for both humans and rodent models. However, detailed mechanisms are not yet clear. ADAR1 (Adenosine deaminase acting on RNA) is an enzyme involved in RNA editing that has a close relation to cognitive function. We have hypothesized that social isolation stress may impact the expression of ADAR1 in the brain of mice with cognitive deficit. To test our hypothesis, we evaluated the cognition ability of mice isolated for different durations (2, 4, and 8 weeks) using object recognition and object location tests; we also measured ADAR1 expression in hippocampus and cortex using immunohistochemistry and western blot. Our study showed that social isolation stress induced spatial and non-spatial cognition deficits of the tested mice. In addition, social isolation significantly increased both the immunoreactivity and protein expression of ADAR1 (p110) in the hippocampus and frontal cortex. Furthermore, re-socialization could not only recover the cognition deficits, but also bring ADAR1 (p110) immunoreactivity of hippocampus and frontal cortex, as well as ADAR1 (p110) protein expression of hippocampus back to the normal level for the isolated mice in adolescence. In conclusion, social isolation stress significantly increases ADAR1 (p110) expression in the hippocampus and frontal cortex of the mice with cognitive deficit. This finding may open a window to better understand the reasons (e.g., epigenetic change) that are responsible for social isolation-induced cognitive deficit and help the development of novel therapies for the resulted diseases.

Subclinical Hypothyroidism after 131I-Treatment of Graves' Disease: A Risk Factor for Depression?

OBJECTIVES: Although it is well accepted that there is a close relationship between hypothyroidism and depression, previous studies provided inconsistent or even opposite results in whether subclinical hypothyroidism (SCH) increased the risk of depression. One possible reason is that the etiology of SCH in these studies was not clearly distinguished. We therefore investigated the relationship between SCH resulting from 131I treatment of Graves' disease and depression. DESIGN AND METHODS: The incidence of depression among 95 patients with SCH and 121 euthyroid patients following 131I treatment of Graves' disease was studied. The risk factors of depression were determined with multivariate logistic regression analysis. Thyroid hormone replacement therapy was performed in patients with thyroid-stimulating hormone (TSH) levels exceeding 10 mIU/L. RESULTS: Patients with SCH had significantly higher Hamilton Depression Scale scores, serum TSH and thyroid peroxidase antibody (TPOAb) levels compared with euthyroid patients. Multivariate logistic regression analysis revealed SCH, Graves' eye syndrome and high serum TPO antibody level as risk factors for depression. L-thyroxine treatment is beneficial for SCH patients with serum TSH levels exceeding 10 mIU/L. CONCLUSIONS: The results of the present study demonstrated that SCH is prevalent among 131I treated Graves' patients. SCH might increase the risk of developing depression. L-thyroxine replacement therapy helps to resolve depressive disorders in SCH patients with TSH > 10mIU/L. These data provide insight into the relationship between SCH and depression.

Schwann cells induce Proliferation and Migration of Oligodendrocyte Precursor Cells Through Secretion of PDGF-AA and FGF-2.

Our previous study has showed that co-grafted Schwann cells (SCs) promote proliferation and migration of the grafted oligodendrocyte precursor cells (OPCs). However, how the co-grafted SCs affect OPCs has not been clarified. In the present study, we confirmed that SC-induced proliferation and migration of OPCs were mediated by SC-secreted factors using SC-conditioned medium (SCM). Then, we detected several candidate factors, PDGF-AA, FGF-2, and IGF-1, in SCs and SCM, and their receptors in OPCs. Finally, by using the selective inhibitors, the effects of these candidate factors on proliferation and migration of OPCs were examined. Our results showed that SCM-stimulated proliferation and migration of OPCs could be markedly decreased by both AG1295 (the inhibitor of PDGFR) and PD173074 (the inhibitor of FGFR). Together, our study suggests that SCs affect proliferation and migration of OPCs through secreting PDGF-AA and FGF-2. Identity of these molecules not only contributes to understand the mechanism of SC-induced proliferation and migration of OPCs but also provides possible target for treatment of CNS diseases.

Effect of type-2 astrocytes on the viability of dorsal root ganglion neurons and length of neuronal processes.

The role of type-2 astrocytes in the repair of central nervous system injury remains poorly understood. In this study, using a relatively simple culture condition in vitro, type-2 astrocytes, differentiated from oligodendrocyte precursor cells by induction with bone morphogenetic protein-4, were co-cultured with dorsal root ganglion neurons. We examined the effects of type-2 astrocytes differentiated from oligodendrocyte precursor cells on the survival and growth of dorsal root ganglion neurons. Results demonstrated that the number of dorsal root ganglion neurons was higher following co-culture of oligodendrocyte precursor cells and type-2 astrocytes than when cultured alone, but lower than that of neurons co-cultured with type-1 astrocytes. The length of the longest process and the length of all processes of a single neuron were shortest in neurons cultured alone, followed by neurons co-cultured with type-2 astrocytes, then neurons co-cultured with oligodendrocyte precursor cells, and longest in neurons co-cultured with type-1 astrocytes. These results indicate that co-culture with type-2 astrocytes can increase neuronal survival rate and process length. However, compared with type-1 astrocytes and oligodendrocyte precursor cells, the promotion effects of type-2 astrocytes on the growth of dorsal root ganglion neurons were weaker.

A behavioral and micro positron emission tomography imaging study in a rat model of hypothyroidism.

Hypothyroidism leads to somatic, neuropsychological, and psychiatric changes that are similar to depression. The mechanisms underlying the behavioral abnormalities in adult onset hypothyroidism remain ambiguous. Hypothyroidism was induced in adult male Wistar rats by the maintenance of 0.05% propylthiouracil (PTU) in drinking water for 5 weeks (hypothyroid group; HP group); control rats (CON group) received an equivalent amount of water. The open field and sucrose preference tests were employed, and the link between behavioral changes and brain glucose metabolism was evaluated using micro positron emission tomography imaging. The open field test revealed slightly decreased locomotor activity and significantly reduced rearing and defecation in the hypothyroid group. Hypothyroid rats were also characterized by decreased body weight, sucrose preference, and relative sucrose intake compared to control rats. Hypothyroidism induced reduced brain glucose metabolism in the bilateral motor cortex, the caudate putamen, the cortex cingulate, the nucleus accumbens, and the frontal association cortex. A decreased sucrose preference was positively correlated with metabolic glucose changes in the caudate putamen and the nucleus accumbens. The results indicate that the activity pattern in adult onset hypothyroidism is different from the activity pattern when hypothyroidism is induced in the developmental period of the central nervous system. Decreased sucrose preference in hypothyroid rats may be attributed to anhedonia. Furthermore, these findings suggest there may be a common mechanism underlying adult onset hypothyroidism and depression.

Transplantation of D15A-expressing glial-restricted-precursor-derived astrocytes improves anatomical and locomotor recovery after spinal cord injury.

The transplantation of neural stem/progenitor cells is a promising therapeutic strategy for spinal cord injury (SCI). In this study, we tested whether combination of neurotrophic factors and transplantation of glial-restricted precursor (GRPs)-derived astrocytes (GDAs) could decrease the injury and promote functional recovery after SCI. We developed a protocol to quickly produce a sufficiently large, homogenous population of young astrocytes from GRPs, the earliest arising progenitor cell population restricted to the generation of glia. GDAs expressed the axonal regeneration promoting substrates, laminin and fibronectin, but not the inhibitory chondroitin sulfate proteoglycans (CSPGs). Importantly, GDAs or its conditioned medium promoted the neurite outgrowth of dorsal root ganglion neurons in vitro. GDAs were infected with retroviruses expressing EGFP or multi-neurotrophin D15A and transplanted into the contused adult thoracic spinal cord at 8 days post-injury. Eight weeks after transplantation, the grafted GDAs survived and integrated into the injured spinal cord. Grafted GDAs expressed GFAP, suggesting they remained astrocyte lineage in the injured spinal cord. But it did not express CSPG. Robust axonal regeneration along the grafted GDAs was observed. Furthermore, transplantation of D15A-GDAs significantly increased the spared white matter and decreased the injury size compared to other control groups. More importantly, transplantation of D15A-GDAs significantly improved the locomotion function recovery shown by BBB locomotion scores and Tredscan footprint analyses. However, this combinatorial strategy did not enhance the aberrant synaptic connectivity of pain afferents, nor did it exacerbate posttraumatic neuropathic pain. These results demonstrate that transplantation of D15A-expressing GDAs promotes anatomical and locomotion recovery after SCI, suggesting it may be an effective therapeutic approach for SCI.

Astrocytes from the contused spinal cord inhibit oligodendrocyte differentiation of adult oligodendrocyte precursor cells by increasing the expression of bone morphogenetic proteins.

Promotion of remyelination is an important therapeutic strategy to facilitate functional recovery after traumatic spinal cord injury (SCI). Transplantation of neural stem cells (NSCs) or oligodendrocyte precursor cells (OPCs) has been used to enhance remyelination after SCI. However, the microenvironment in the injured spinal cord is inhibitory for oligodendrocyte (OL) differentiation of NSCs or OPCs. Identifying the signaling pathways that inhibit OL differentiation in the injured spinal cord could lead to new therapeutic strategies to enhance remyelination and functional recovery after SCI. In the present study, we show that reactive astrocytes from the injured rat spinal cord or their conditioned media inhibit OL differentiation of adult OPCs with concurrent promotion of astrocyte differentiation. The expression of bone morphogenetic proteins (BMP) is dramatically increased in the reactive astrocytes and their conditioned media. Importantly, blocking BMP activity by BMP receptor antagonist, noggin, reverse the effects of active astrocytes on OPC differentiation by increasing the differentiation of OL from OPCs while decreasing the generation of astrocytes. These data indicate that the upregulated bone morphogenetic proteins in the reactive astrocytes are major factors to inhibit OL differentiation of OPCs and to promote its astrocyte differentiation. These data suggest that manipulation of BMP signaling in the endogenous or grafted NSCs or OPCs may be a useful therapeutic strategy to increase their OL differentiation and remyelination and enhance functional recovery after SCI.

Transplantation of ciliary neurotrophic factor-expressing adult oligodendrocyte precursor cells promotes remyelination and functional recovery after spinal cord injury.

Demyelination contributes to the dysfunction after traumatic spinal cord injury (SCI). We explored whether the combination of neurotrophic factors and transplantation of adult rat spinal cord oligodendrocyte precursor cells (OPCs) could enhance remyelination and functional recovery after SCI. Ciliary neurotrophic factor (CNTF) was the most effective neurotrophic factor to promote oligodendrocyte (OL) differentiation and survival of OPCs in vitro. OPCs were infected with retroviruses expressing enhanced green fluorescent protein (EGFP) or CNTF and transplanted into the contused adult thoracic spinal cord 9 d after injury. Seven weeks after transplantation, the grafted OPCs survived and integrated into the injured spinal cord. The survival of grafted CNTF-OPCs increased fourfold compared with EGFP-OPCs. The grafted OPCs differentiated into adenomatus polyposis coli (APC(+)) OLs, and CNTF significantly increased the percentage of APC(+) OLs from grafted OPCs. Immunofluorescent and immunoelectron microscopic analyses showed that the grafted OPCs formed central myelin sheaths around the axons in the injured spinal cord. The number of OL-remyelinated axons in ventrolateral funiculus (VLF) or lateral funiculus (LF) at the injured epicenter was significantly increased in animals that received CNTF-OPC grafts compared with all other groups. Importantly, 75% of rats receiving CNTF-OPC grafts recovered transcranial magnetic motor-evoked potential and magnetic interenlargement reflex responses, indicating that conduction through the demyelinated axons in VLF or LF, respectively, was partially restored. More importantly, recovery of hindlimb locomotor function was significantly enhanced in animals receiving grafts of CNTF-OPCs. Thus, combined treatment with OPC grafts expressing CNTF can enhance remyelination and facilitate functional recovery after traumatic SCI.

Differential expression of sPLA2 following spinal cord injury and a functional role for sPLA2-IIA in mediating oligodendrocyte death.

After the initial mechanical insult of spinal cord injury (SCI), secondary mediators propagate a massive loss of oligodendrocytes. We previously showed that following SCI both the total phospholipase activity and cytosolic PLA(2)-IV alpha protein expression increased. However, the expression of secreted isoforms of PLA(2) (sPLA(2)) and their possible roles in oligodendrocyte death following SCI remained unclear. Here we report that mRNAs extracted 15 min, 4 h, 1 day, or 1 month after cervical SCI show marked upregulation of sPLA(2)-IIA and IIE at 4 h after injury. In contrast, SCI induced down regulation of sPLA(2)-X, and no change in sPLA(2)-IB, IIC, V, and XIIA expression. At the lesion site, sPLA(2)-IIA and IIE expression were localized to oligodendrocytes. Recombinant human sPLA(2)-IIA (0.01, 0.1, or 2 microM) induced a dose-dependent cytotoxicity in differentiated adult oligodendrocyte precursor cells but not primary astrocytes or Schwann cells in vitro. Most importantly, pretreatment with S3319, a sPLA(2)-IIA inhibitor, before a 30 min H(2)O(2) injury (1 or 10 mM) significantly reduced oligodendrocyte cell death at 48 h. Similarly, pretreatment with S3319 before injury with IL-1 beta and TNFalpha prevented cell death and loss of oligodendrocyte processes at 72 h. Collectively, these findings suggest that sPLA(2)-IIA and IIE are increased following SCI, that increased sPLA(2)-IIA can be cytotoxic to oligodendrocytes, and that in vitro blockade of sPLA(2) can create sparing of oligodendrocytes in two distinct injury models. Therefore, sPLA(2)-IIA may be an important mediator of oligodendrocyte death and a novel target for therapeutic intervention following SCI.

Bone morphogenetic protein signaling and olig1/2 interact to regulate the differentiation and maturation of adult oligodendrocyte precursor cells.

Promotion of remyelination is an important therapeutic strategy for the treatment of the demyelinating neurological disorders. Adult oligodendrocyte precursor cells (OPCs), which normally reside quiescently in the adult central nervous system (CNS), become activated and proliferative after demyelinating lesions. However, the extent of endogenous remyelination is limited because of the failure of adult OPCs to mature into myelinating oligodendrocytes (OLs) in the demyelinated CNS. Understanding the molecular mechanisms that regulate the differentiation of adult OPCs could lead to new therapeutic strategies to treat these disorders. In this study, we established a stable culture of adult spinal cord OPCs and developed a reliable in vitro protocol to induce their sequential differentiation. Adult OPCs expressed bone morphogenetic protein (BMP) type Ia, Ib, and II receptor subunits, which are required for BMP signal transduction. BMP2 and 4 promoted dose-dependent astrocyte differentiation of adult OPCs with concurrent suppression of OL differentiation. Treatment of OPCs with BMP2 and 4 increased ID4 expression and decreased the expression of olig1 and olig2. Overexpression of olig1 or olig2 blocked the astrocyte differentiation of adult OPCs induced by BMP2 and 4. Furthermore, overexpression of both olig1 and olig2, but not olig1 or olig2 alone, rescued OL differentiation from inhibition by BMP2 and 4. Our results demonstrated that downregulation of olig1 and olig2 is an important mechanism by which BMP2 and 4 inhibit OL differentiation of adult OPCs. These data suggest that blocking BMP signaling combined with olig1/2 overexpression could be a useful therapeutic strategy to enhance endogenous remyelination and facilitate functional recovery in CNS demyelinated disorders. Disclosure of potential conflicts of interest is found at the end of this article.

Differential caspase-3 expression in noncancerous, premalignant and cancer tissues of stomach and its clinical implication.

BACKGROUND: Caspase-3 is a critical apoptosis-promoting element but its status during stepwise gastrocarcinogenesis needs to be further clarified. MATERIALS AND METHODS: By the use of frozen tissue microarrays constructed with the tissue spots cored from defined histological regions in tissue blocks, the pattern of caspase-3 expression in noncancerous, premalignant (atrophic gastritis and intestinal metaplasia) tissue and cancer spots were analyzed under the same experimental conditions by the methods of immunohistochemistry and mRNA-in situ hybridization. RESULTS: Caspase-3 was expressed in all 34 of the noncancerous mucosa (100%), in 16 of the 17 premalignant tissues (94.1%) and in 15 of the 48 gastric cancers (31.3%). The incidences of caspase-3 detection were significantly different (p<0.01) between noncancerous mucosa and intestinal as well as diffuse gastric cancers. CONCLUSION: Down-regulated caspase-3 is closely correlated with gastric cancer formation and would be a potential indicator of tumor formation and progression. Helicobacter pylori (H. pylori; Hp) infection is but not the only one element responsible to the enhanced caspase-3 expression in gastric epithelia.

Nuclear translocations of beta-catenin and TCF4 in gastric cancers correlate with lymph node metastasis but probably not with CD44 expression.

Interaction of nuclear beta-catenin and TCF4 is the end point of canonical Wnt signaling, which is believed to trigger the transcription of multiple cancer-associated genes, including CD44. So far, the combined status of beta-catenin and TCF4 and its relevance for lymph node metastasis and CD44 expression have not been well studied in gastric cancers (GCs). To address these issues, we examined 31 GCs, 17 premalignant tissues, 10 noncancerous gastric mucosae, 17 regional lymph node metastases, and 4 human GC cell lines (MGC803, MGC823, AGS, and HGC-27) using immunohistochemical and immunofluorescence staining, reverse transcriptase polymerase chain reaction, and Western blot analysis. Frequent TCF4 up-regulation and nuclear translocation of beta-catenin were found in both primary and metastatic tumors. Standard CD44 was detected in all gastric tissue samples. The frequency of variant CD44 expression increased in parallel with stepwise gastrocarcinogenesis and tumor spread, but the rates of detection did not match that of nuclear beta-catenin and TCF4, especially in the premalignant and noncancerous samples. The data from the 4 cell lines were in accordance with the in vivo findings in terms of beta-catenin nuclear translocation, TCF4 activation, and CD44 expression. Our results suggest an established Wnt signaling pathway in most GCs, a close correlation of beta-catenin/TCF4-mediated signaling with tumor dissemination, and the unlikelihood of a direct effect of activated Wnt signaling on CD44 expression. The influence of beta-catenin-TCF4 interaction on alternative CD44 splicing was not established. These 3 alterations may be regarded as unfavorable features of GC.