Multi-walled carbon nanotube carpets as scaffolds for U87MG glioblastoma multiforme cell growth.

Carbon Nanotubes (CNTs) are known for effective adhesion, growth, and differentiation of bone, muscle, and cardiac cells. CNTs can provide excellent mechanical and electrical properties for cell scaffolding; however, loose CNTs can cause in-vivo toxicity. To suppress this risk, our team has developed biomimetic scaffolds with multiscale hierarchy where carpet-like CNT arrays are covalently bonded to larger biocompatible substrates. In this study, we investigated the interaction between glioblastoma multiforme (GBM) cells (U87MG) and our unique hierarchical CNT-coated scaffolds upon brain tumor cell proliferation. U87MG cells grown on un-modified carbon scaffolds grew in a bi-phasic fashion. Initially, the scaffolds prevented GBM cell growth; however, prolonged growth on such scaffolds significantly increased GBM cell proliferation. We further defined the importance of the hydrophobicity/hydrophilicity of the CNT-coated scaffolds in this cellular response by utilizing sodium-hypochlorite based bleach treatment prior to cellular exposure. This surface modification increased the hydrophilicity of the CNT-coated scaffolds and ameliorated the biphasic response of U87MG cells allowing for a normal growth curve. Findings highlight the importance of surface modification and wettability of the CNT-coated scaffolds for cell growth applications. The focus for this study was to determine whether scaffold surface features could modulate tumor-scaffold interactions, and thus to improve our understanding of and optimize successful development of future scaffold-based chemotherapy applications. Overall, it appears that the wettability of carbon scaffolds coated with CNTs is an important regulator of U87MG cellular growth. These findings will be important to consider when developing a potential chemotherapy-attached implant to be used post-surgical resection for GBM patient treatment.

Gender- and region-specific changes in estrogen signaling in aging rat brain mitochondria.

Recently epidemiological studies suggest females lose neuroprotection from neurodegenerative diseases as they go through menopause. It has been hypothesized that this neuroprotection is hormone-dependent. The current study characterized cell signaling molecules downstream of estrogen receptor beta that are known to play a role in memory, PKC, ERK, and connexin-43, in regions of the brain associated with memory decline in an attempt to elucidate significant changes that occur post-estrus. Total whole cell lysates were compared to isolated mitochondrial protein because mitochondrial function is known to be altered during aging. As hypothesized, protein concentrations differed depending on age, gender, and brain region. Additionally, many of these changes occurred within mitochondria but not within whole cell lysates indicating that these are epigenetic alterations. These findings accentuate the complexity of aging and provide insight into the gender-specific cellular processes that occur throughout this process.

AMPK Knockdown in Placental Labyrinthine Progenitor Cells Results in Restriction of Critical Energy Resources and Terminal Differentiation Failure.

Placental abnormalities can cause Pregnancy-Associated Disorders, including preeclampsia, intrauterine growth restriction, and placental insufficiency, resulting in complications for both the mother and fetus. Trophoblast cells within the labyrinthine layer of the placenta facilitate the exchange of nutrients, gases, and waste between mother and fetus; therefore, the development of this cell layer is critical for fetal development. As trophoblast cells differentiate, it is assumed their metabolism changes with their energy requirements. We hypothesize that proper regulation of trophoblast metabolism is a key component of normal placental development; therefore, we examined the role of AMP-activated kinase (AMPK, PRKAA1/2), a sensor of cellular energy status. Our previous studies have shown that AMPK knockdown alters both trophoblast differentiation and nutrient transport. In this study, AMPKα1/2 shRNA was used to investigate the metabolic effects of AMPK knockdown on SM10 placental labyrinthine progenitor cells before and after differentiation. Extracellular flux analysis confirmed that AMPK knockdown was sufficient to reduce trophoblast glycolysis, mitochondrial respiration, and ATP coupling efficiency. A reduction in AMPK in differentiated trophoblasts also resulted in increased mitochondrial volume. These data indicate that a reduction in AMPK disrupts cellular metabolism in both progenitors and differentiated placental trophoblasts. This disruption correlates to abortive trophoblast differentiation that may contribute to the development of Pregnancy-Associated Disorders.

Nf1 loss and Ras hyperactivation in oligodendrocytes induce NOS-driven defects in myelin and vasculature.

Patients with neurofibromatosis type 1 (NF1) and Costello syndrome Rasopathy have behavioral deficits. In NF1 patients, these may correlate with white matter enlargement and aberrant myelin. To model these features, we induced Nf1 loss or HRas hyperactivation in mouse oligodendrocytes. Enlarged brain white matter tracts correlated with myelin decompaction, downregulation of claudin-11, and mislocalization of connexin-32. Surprisingly, non-cell-autonomous defects in perivascular astrocytes and the blood-brain barrier (BBB) developed, implicating a soluble mediator. Nitric oxide (NO) can disrupt tight junctions and gap junctions, and NO and NO synthases (NOS1-NOS3) were upregulated in mutant white matter. Treating mice with the NOS inhibitor NG-nitro-L-arginine methyl ester or the antioxidant N-acetyl cysteine corrected cellular phenotypes. CNP-HRasG12V mice also displayed locomotor hyperactivity, which could be rescued by antioxidant treatment. We conclude that Nf1/Ras regulates oligodendrocyte NOS and that dysregulated NO signaling in oligodendrocytes can alter the surrounding vasculature. The data suggest that antioxidants may improve some behavioral deficits in Rasopathy patients.

Neurofibroma-associated macrophages play roles in tumor growth and response to pharmacological inhibition.

Neurofibromatosis type 1 (NF1) is a common genetic disease that predisposes 30-50 % of affected individuals to develop plexiform neurofibromas. We found that macrophage infiltration of both mouse and human neurofibromas correlates with disease progression. Macrophages accounted for almost half of neurofibroma cells, leading us to hypothesize that nerve macrophages are inflammatory effectors in neurofibroma development and/or growth. We tested the effects of PLX3397, a dual kit/fms kinase inhibitor that blocks macrophage infiltration, in the Dhh-Cre; Nf1(flox/flox) mouse model of GEM grade I neurofibroma. In mice aged 1-4 months, prior to development of nerve pathology and neurofibroma formation, PLX3397 did not impair tumor initiation and increased tumor volume compared to controls. However, in mice aged 7-9 months, after tumor establishment, a subset of mice demonstrating the largest reductions in macrophages after PLX3397 exhibited cell death and tumor volume regression. Macrophages are likely to provide an initial line of defense against developing tumors. Once tumors are established, they become tumor permissive. Macrophage depletion may result in impaired tumor maintenance and represent a therapeutic strategy for neurofibroma therapy.

Neurofibromatosis 2011: a report of the Children's Tumor Foundation annual meeting.

The 2011 annual meeting of the Children's Tumor Foundation, the annual gathering of the neurofibromatosis (NF) research and clinical communities, was attended by 330 participants who discussed integration of new signaling pathways into NF research, the appreciation for NF mutations in sporadic cancers, and an expanding pre-clinical and clinical agenda. NF1, NF2, and schwannomatosis collectively affect approximately 100,000 persons in US, and result from mutations in different genes. Benign tumors of NF1 (neurofibroma and optic pathway glioma) and NF2 (schwannoma, ependymoma, and meningioma) and schwannomatosis (schwannoma) can cause significant morbidity, and there are no proven drug treatments for any form of NF. Each disorder is associated with additional manifestations causing morbidity. The research presentations described in this review covered basic science, preclinical testing, and results from clinical trials, and demonstrate the remarkable strides being taken toward understanding of and progress toward treatments for these disorders based on the close interaction among scientists and clinicians.

Perinatal or adult Nf1 inactivation using tamoxifen-inducible PlpCre each cause neurofibroma formation.

Plexiform neurofibromas are peripheral nerve sheath tumors initiated by biallelic mutation of the NF1 tumor suppressor gene in the Schwann cell lineage. To understand whether neurofibroma formation is possible after birth, we induced Nf1 loss of function with an inducible proteolipid protein Cre allele. Perinatal loss of Nf1 resulted in the development of small plexiform neurofibromas late in life, whereas loss in adulthood caused large plexiform neurofibromas and morbidity beginning 4 months after onset of Nf1 loss. A conditional EGFP reporter allele identified cells showing recombination, including peripheral ganglia satellite cells, peripheral nerve S100ß+ myelinating Schwann cells, and peripheral nerve p75+ cells. Neurofibromas contained cells with Remak bundle disruption but no recombination within GFAP+ nonmyelinating Schwann cells. Extramedullary lympho-hematopoietic expansion was also observed in PlpCre;Nf1fl/fl mice. These tumors contained EGFP+/Sca-1+ stromal cells among EGFP-negative lympho-hematopoietic cells indicating a noncell autonomous effect and unveiling a role of Nf1-deleted microenvironment on lympho-hematopoietic proliferation in vivo. Together these findings define a tumor suppressor role for Nf1 in the adult and narrow the range of potential neurofibroma-initiating cell populations.

PAX6 suppression of glioma angiogenesis and the expression of vascular endothelial growth factor A.

We reported that PAX6 suppresses glioblastoma cell growth in vivo and anchorage-independent growth without significant alteration of cell proliferation in vitro, suggesting that PAX6 may alter the tumor microenvironment. Because we found that PAX6 downregulates expression of the gene encoding vascular endothelial growth factor A (VEGFA) in glioma cells, we used a subcutaneous xenograft model to verify PAX6 suppression of VEGFA-induced angiogenesis based on CD31-immunostaining of endothelial cells. The results showed a significant reduction of VEGFA at the transcription level in PAX6-transfected cells in xenografts and PAX6 has a suppressive effect on the microvascular amplification typically seen in glioblastoma. We showed that PAX6 suppression of VEGFA expression requires its DNA binding-domain. The C-terminal truncation mutant of PAX6, however, did not show the dominant negative function in regulating VEGFA expression that it showed previously in regulating MMP2 expression. In the glioma cell line U251HF, we further determined that blocking the PI3K/Akt signaling pathway with either adenoviral-mediated PTEN expression or LY294002 enhanced PAX6-mediated suppression of VEGFA in an additive manner; thus, PAX6-mediated suppression of VEGFA is not via the canonical pathway through HIF1A. These two VEGFA-regulatory pathways can also be similarly modulated in another malignant glioma cell line, U87, but not in LN229 where the basal VEGFA level is low and PTEN is wild-type. PAX6 suppression of VEGFA appears to be blocked in LN229. In conclusion, our data showed that PAX6 can initiate in glioma cells a new signaling pathway independent of PI3K/Akt-HIF1A signaling to suppress VEGFA expression.

PAX6 suppresses the invasiveness of glioblastoma cells and the expression of the matrix metalloproteinase-2 gene.

Glioblastoma multiforme (GBM) is the most invasive brain tumor. We have previously reported that the transcription factor PAX6 suppresses the tumorigenecity of GBM cells. By an in vitro Matrigel invasion assay on two GBM cell lines stably transfected with wild-type and/or two mutant forms of PAX6, this study displays the first evidence that PAX6 inhibits the invasiveness of GBM cells and that the DNA-binding domain of PAX6 is required for this function. Using real-time quantitative reverse transcription-PCR (RT-PCR), gelatin zymography, and immunohistochemistry assays, the expression of the gene encoding matrix metalloproteinase-2 (MMP2) in GBM cell lines grown in vitro or in intracranial xenografts in nude mice was shown to be repressed by either stable or adenoviral-mediated overexpression of PAX6. Luciferase promoter assays revealed PAX6-mediated suppression of MMP2 promoter activity. Electrophoretic mobility shift assays showed direct binding of PAX6 to the MMP2 promoter. A significant reverse correlation (P < 0.05) occurred between PAX6 and MMP2 expression quantified by real-time quantitative RT-PCR in 41 GBMs, 43 anaplastic astrocytomas, and 7 adjacent normal tissues. Interestingly, the degree and significance of the reverse correlation increased after excluding astrocytomas, whereas it became insignificant after excluding GBMs. In GBM cells stably transfected with a dominant negative mutant PAX6 showing increased MMP2 expression and invasiveness, knock-down of MMP2 revealed that MMP2 is one of the PAX6 target genes mediating its suppression of invasion. Overall data delineated a mechanism for the suppressive function of PAX6 in GBM: suppression of cell invasion by repressing the expression of proinvasive genes such as MMP2.

Combining an autologous peripheral nervous system "bridge" and matrix modification by chondroitinase allows robust, functional regeneration beyond a hemisection lesion of the adult rat spinal cord.

Chondroitinase-ABC (ChABC) was applied to a cervical level 5 (C5) dorsal quadrant aspiration cavity of the adult rat spinal cord to degrade the local accumulation of inhibitory chondroitin sulfate proteoglycans. The intent was to enhance the extension of regenerated axons from the distal end of a peripheral nerve (PN) graft back into the C5 spinal cord, having bypassed a hemisection lesion at C3. ChABC-treated rats showed (1) gradual improvement in the range of forelimb swing during locomotion, with some animals progressing to the point of raising their forelimb above the nose, (2) an enhanced ability to use the forelimb in a cylinder test, and (3) improvements in balance and weight bearing on a horizontal rope. Transection of the PN graft, which cuts through regenerated axons, greatly diminished these functional improvements. Axonal regrowth from the PN graft correlated well with the behavioral assessments. Thus, many more axons extended for much longer distances into the cord after ChABC treatment and bridge insertion compared with the control groups, in which axons regenerated into the PN graft but growth back into the spinal cord was extremely limited. These results demonstrate, for the first time, that modulation of extracellular matrix components after spinal cord injury promotes significant axonal regeneration beyond the distal end of a PN bridge back into the spinal cord and that regenerating axons can mediate the return of useful function of the affected limb.