Applicability of avidin protein coated mesoporous silica nanoparticles as drug carriers in the lung.

Mesoporous silica nanoparticles (MSNs) exhibit unique drug delivery properties and are thus considered as promising candidates for next generation nano-medicines. In particular, inhalation into the lungs represents a direct, non-invasive delivery route for treating lung disease. To assess MSN biocompatibility in the lung, we investigated the bioresponse of avidin-coated MSNs (MSN-AVI), as well as aminated (uncoated) MSNs, after direct application into the lungs of mice. We quantified MSN distribution, clearance rate, cell-specific uptake, and inflammatory responses to MSNs within one week after instillation. We show that amine-functionalized (MSN-NH2) particles are not taken up by lung epithelial cells, but induced a prolonged inflammatory response in the lung and macrophage cell death. In contrast, MSN-AVI co-localized with alveolar epithelial type 1 and type 2 cells in the lung in the absence of sustained inflammatory responses or cell death, and showed preferential epithelial cell uptake in in vitro co-cultures. Further, MSN-AVI particles demonstrated uniform particle distribution in mouse lungs and slow clearance rates. Thus, we provide evidence that avidin functionalized MSNs (MSN-AVI) have the potential to serve as versatile biocompatible drug carriers for lung-specific drug delivery.

Immunoproteasome dysfunction augments alternative polarization of alveolar macrophages.

The proteasome is a central regulatory hub for intracellular signaling by degrading numerous signaling mediators. Immunoproteasomes are specialized types of proteasomes involved in shaping adaptive immune responses, but their role in innate immune signaling is still elusive. Here, we analyzed immunoproteasome function for polarization of alveolar macrophages, highly specialized tissue macrophages of the alveolar lung surface. Classical activation (M1 polarization) of primary alveolar macrophages by LPS/IFNγ transcriptionally induced all three immunoproteasome subunits, low molecular mass protein 2 (LMP2), LMP7 and multicatalytic endopeptidase complex-like 1, which was accompanied by increased immunoproteasome activity in M1 cells. Deficiency of LMP7 had no effect on the LPS/IFNγ-triggered M1 profile indicating that immunoproteasome function is dispensable for classical alveolar macrophage activation. In contrast, IL-4 triggered alternative (M2) activation of primary alveolar macrophages was accompanied by a transcriptionally independent amplified expression of LMP2 and LMP7 and an increase in immunoproteasome activity. Alveolar macrophages from LMP7 knockout mice disclosed a distorted M2 profile upon IL-4 stimulation as characterized by increased M2 marker gene expression and CCL17 cytokine release. Comparative transcriptome analysis revealed enrichment of IL-4-responsive genes and of genes involved in cellular response to defense, wounding and inflammation in LMP7-deficient alveolar macrophages indicating a distinct M2 inflammation resolving phenotype. Moreover, augmented M2 polarization was accompanied by amplified AKT/STAT6 activation and increased RNA and protein expression of the M2 master transcription factor interferon regulatory factor 4 in LMP7(-/-) alveolar macrophages. IL-13 stimulation of LMP7-deficient macrophages induced a similar M2-skewed profile indicative for augmented signaling via the IL-4 receptor α (IL4Rα). IL4Rα expression was generally elevated only on protein but not RNA level in LMP7(-/-) alveolar macrophages. Importantly, specific catalytic inhibition with an LMP7-specific proteasome inhibitor confirmed augmented IL-4-mediated M2 polarization of alveolar macrophages. Our results thus suggest a novel role of immunoproteasome function for regulating alternative activation of macrophages by limiting IL4Rα expression and signaling.

Identification of a neurite outgrowth-promoting motif within the alternatively spliced region of human tenascin-C.

Our work centers on understanding how the extracellular matrix molecule tenascin-C regulates neuronal growth. We have found that the region of tenascin-C containing only alternately spliced fibronectin type-III repeat D, called fnD, when used by itself, dramatically increases neurite outgrowth in culture. We used overlapping synthetic peptides to localize the neurite outgrowth-promoting site within fnD to a 15 amino acid sequence, called D5. An antibody against D5 blocked promotion of neurite outgrowth by fnD as well as tenascin-C, indicating that this peptide sequence is functional in the context of the native molecule. Further testing of shorter synthetic peptides restricted the neurite outgrowth-promoting site to eight amino acids, VFDNFVLK. Of these, "FD" and "FV" are conserved in tenascin-C sequences derived from all the species available in the GenBank. To investigate the hypothesis that FD and FV are critical for the interaction with neurons, we tested a recombinant fnD protein and synthetic peptides with alterations in FD and/or FV. These molecules did not facilitate process extension, suggesting that the conserved amino acids are required for formation of the active site in fnD. We next investigated whether VFDNFVLK could be used as a reagent to overcome the neurite outgrowth inhibitory properties of chondroitin sulfate proteoglycans, the major inhibitory molecules in the glial scar. The peptide significantly enhanced outgrowth on proteoglycans and was more effective than laminin-1, L1-Fc, or intact tenascin-C, thus demonstrating the potential applicability of tenascin-C regions as therapeutic reagents.

Additive and nonadditive effects of herbivory and competition on tree seedling mortality, growth, and allocation.

The interaction between simulated cotyledon herbivory and interspecific competition was studied in a greenhouse experiment using two species of trees, Acer rubrum and Quercus palustris, which commonly invade abandoned agricultural fields. Herbivory treatments were applied as a gradient of cotyledon removal for A. rubrum with 0, 25, 50, 75, and 100% of cotyledon tissue removed. Cotyledons from Q. palustris were clipped and removed (control, early, and late removal) to create a gradient of seed reserve availability. The competition treatment consisted of plugs of old-field vegetation that filled the pots with perennial cover. Mortality of seedlings was higher with competition. There was a significant interaction between herbivory and competition with the highest mortality occurring with competition at the highest intensity of herbivory in both species. Herbivory reduced biomass for Q. palustris only, while competition reduced biomass in both species. Neither species showed an interaction between herbivory and competition for growth. There was a significant interaction between herbivory and competition on allocation patterns for both species, with greater allocation to roots with competition at the highest intensity of herbivory. This study demonstrates the potential for cotyledon herbivory and competition to interact, altering the invasion of tree seedlings into abandoned agricultural land.

Hepatocyte growth factor effects on motility of stone-diseased and stone-free human gallbladders.

Hepatocyte growth factor (HGF) has unique morphogenic activity for several cell types. Besides its major effect upon liver regeneration, its motogenic activity to enhance motility has not been verified for smooth muscles. Therefore we evaluated the impact of HGF in an in-vitro model of human gallbladder motility. Twelve stone-diseased and eight stone-free muscle strips were preincubated with HGF (100 ng/ml, 200 ng/ml). For the analysis of motility, cholecystokinin (CCK) was added (0.1 nM, 0.5 nM, 2 nM, 10 nM, and 100 nM). Twelve stone-diseased and eight stone-free strips without HGF incubation served as the control group. The tone of healthy (tone/100 nM CCK: control group, 12.4 +/- 3.6 mN; HGF group, 19.5 +/- 4.5 mN) and stone-diseased (tone/100 nM CCK: control group, 10.8 +/- 3.8 mN; HGF group, 17.3 +/- 4.8 mN) muscle strips, preincubated with HGF, was increased, with a higher sensitivity to CCK. Our results suggest that there is a clear motogenic response of stone-diseased human gallbladders to HGF.

Tenascin-C contains domains that independently regulate neurite outgrowth and neurite guidance.

Tenascin-C has been implicated in regulation of both neurite outgrowth and neurite guidance. We have shown previously that a particular region of tenascin-C has powerful neurite outgrowth-promoting actions in vitro. This region consists of the alternatively spliced fibronectin type-III (FN-III) repeats A-D and is abbreviated fnA-D. The purpose of this study was to investigate whether fnA-D also provides neurite guidance cues and whether the same or different sequences mediate outgrowth and guidance. We developed an assay to quantify neurite behavior at sharp substrate boundaries and found that neurites demonstrated a strong preference for fnA-D when given a choice at a poly-L-lysine-fnA-D interface, even when fnA-D was intermingled with otherwise repellant molecules. Furthermore, neurites preferred cells that overexpressed the largest but not the smallest tenascin-C splice variant when given a choice between control cells and cells transfected with tenascin-C. The permissive guidance cues of large tenascin-C expressed by cells were mapped to fnA-D. Using a combination of recombinant proteins corresponding to specific alternatively spliced FN-III domains and monoclonal antibodies against neurite outgrowth-promoting sites, we demonstrated that neurite outgrowth and guidance were facilitated by distinct sequences within fnA-D. Hence, neurite outgrowth and neurite guidance mediated by the alternatively spliced region of tenascin-C are separable events that can be independently regulated.

Neurite outgrowth promotion by the alternatively spliced region of tenascin-C is influenced by cell-type specific binding.

We have investigated the impact of cellular environment on the neurite outgrowth promoting properties of the alternatively spliced fibronectin type-III region (fnA-D) of tenascin-C. FnA-D promoted neurite outgrowth in vitro when bound to the surface of BHK cells or cerebral cortical astrocytes, but the absolute increase was greater on astrocytes. In addition, different neurite outgrowth promoting sites were revealed within fnA-D bound to the two cellular substrates. FnA-D also promoted neurite outgrowth as a soluble ligand; however, the actions of soluble fnA-D were not affected by cell type. Therefore, we hypothesized that different mechanisms of cellular binding can alter the growth promoting actions of bound fnA-D. We found that fnA-D utilizes two distinct sequences to bind to the BHK cell surface as opposed to the BHK extracellular matrix. In contrast, only one of these sequences is utilized to bind to the astrocyte matrix as opposed to the astrocyte surface. Furthermore, Scatchard analysis indicated two types of receptors for fnA-D on BHK cells and only one type on astrocytes. These results suggest that active sites for neurite outgrowth within fnA-D are differentially revealed depending on cell-specific fnA-D binding sites. Therefore, the function of tenascin-C and its various domains must be considered in terms of cellular context.

Cytokine-induced changes in the ability of astrocytes to support migration of oligodendrocyte precursors and axon growth.

Repair of demyelination in the CNS requires that oligodendrocyte precursors (OPs) migrate, divide and then myelinate. Repair of axon damage requires axonal regeneration. Limited remyelination and axon regeneration occurs soon after injury, but usually ceases in a few days. In vivo and in vitro experiments have shown that astrocytic environments are not very permissive for migration of OPs or for axonal re-growth. Yet remyelination and axon sprouting early after injury occurs in association with astrocytes, while later astrocytes can exclude remyelination and prevent axon regeneration. A large and changing cast of cytokines are released following CNS injury, so we investigated whether some of these alone or in combination can affect the ability of astrocytes to support migration of OPs and neuritic outgrowth. Interleukin (IL) 1alpha, tumour necrosis factor alpha, transforming growth factor (TGF) beta, basic fibroblast growth factor (bFGF), platelet-derived growth factor and epidermal growth factor alone exerted little or no effect on migration of OPs on astrocytes, whereas interferon (IFN) gamma was inhibitory. The combination of IL-1alpha + bFGF was found to be pro-migratory, and this effect could be neutralized by TGFbeta. We also examined neuritic outgrowth from dorsal root ganglion explants in three-dimensional astrocyte cultures treated with cytokines and found that IL-1alpha + bFGF greatly increased axon outgrowth and that this effect could be blocked by TGFbeta and IFNgamma. All these effects were absent or much smaller when OP migration or axon growth was tested on laminin, so the main effect of the cytokines was via astrocytes. The cytokine effects did not correlate with expression on astrocytes of laminin, fibronectin, tenascin, chondroitin sulphate proteoglycan, N-cadherin, polysialyated NCAM (PSA-NCAM), tissue plasminogen activator (tPA) or urokinase (uPA).

A truncated SV40 large T antigen lacking the p53 binding domain overcomes p53-induced growth arrest and immortalizes primary mesencephalic cells.

As an alternative to primary fetal tissue, immortalized central nervous system (CNS)-derived cell lines are useful for in vitro CNS model systems and for gene manipulation with potential clinical use in neural transplantation. However, obtaining immortalized cells with a desired phenotype is unpredictable, because the molecular mechanisms of growth and differentiation of CNS cells are poorly understood. The SV40 large T antigen is commonly used to immortalize mammalian cells, but it interferes with multiple cell-cycle components, including p53, p300, and retinoblastoma protein, and usually produces cells with undifferentiated phenotypes. In order to increase the phenotypic repertoire of immortalized CNS cells and to address the molecular mechanisms underlying immortalization and differentiation, we constructed an expression vector containing a truncated SV40 large T gene that encodes only the amino-terminal 155 amino acids (T155), which lacks the p53-binding domain. Constructs were first transfected into a p53-temperature-sensitive cell line, T64-7B. Colonies expressing T155 proliferated at the growth-restrictive temperature. T155 was then transfected into primary cultures from embryonic day-14 rat mesencephalon. Two clonal cell lines were derived, AF-5 and AC-10, which co-expressed T155 and mature neuronal and astrocytic markers. Thus, the amino-terminal portion of SV40 large T is sufficient to: (1) overcome p53-mediated growth arrest despite the absence of a p53-binding region, and (2) immortalize primary CNS cells expressing mature markers while actively dividing. T155 and T155-transfectants may be useful for further studies of cell-cycle mechanisms and phenotyic expression in CNS cells or for further gene manipulation to produce cells with specific properties.

Role of morphogenetic factors in metastasis of mammary carcinoma cells.

We have analysed the role of the morphogenetic factors hepatocyte growth factor/scatter factor (HGF), neuregulin and E-cadherin in the process of metastasis and morphogenesis of mammary carcinoma cells. The cDNAs for HGF, neuregulin and E-cadherin were stably expressed in anaplastic human MDA MB 435 carcinoma cells. The altered cells were then injected into the mammary fat pads of nude mice, where they form tumors which can spontaneously metastasize to the lungs. We found that expression of HGF or neuregulin promoted metastasis whereas expression of the cell adhesion molecule E-cadherin was inhibitory. Moreover, expression of E-cadherin reconstituted the ability of the cells to form morphogenetic structures in matrigel cultures in response to HGF. These data demonstrate that HGF and neuregulin, which control branching or lobulo-alveolar morphogenesis of normal breast epithelium, do have metastasis-promoting effects on breast carcinoma cells. Moreover, our results suggest that the differential activities of the two factors can be explained by the degree of epithelial differentiation: induction of morphogenesis requires an intact epithelial adhesion and differentiation system, whereas induction of metastasis is observed when the cells have lost their epithelial characteristics.

Suramin disrupts the gliotic response following a stab wound injury to the adult rat brain.

Reactive gliosis, observed in numerous pathological states, leads to the formation of a glial scar that is believed to impede axonal regeneration. Astrocyte reactivity can be initiated both in vitro and in vivo by various cytokines. Thus, the aim of this study was to investigate if suramin, a polysulfonated napthylurea that has been shown to inhibit the binding of many different cytokines to their cell surface receptors, could attenuate the glial response after brain injury. A single dose of suramin (5 microl, 75 microM) or saline vehicle was injected intracerebrally through the same needle used to make the stab wound at the time of lesioning. Suramin-treated animals showed an obvious reduction in several parameters of CNS inflammation: cellular proliferation, GFAP levels, and tenascin-C immunoreactivity were reduced in suramin-treated as compared to control animals at early time points. GFAP immunoreactivity was strikingly reduced at 3 days after injury, as confirmed by Western blot analysis. This reduction was transient, however, in that the difference in GFAP expression between suramin-treated and control animals was less apparent at 7 days and had disappeared by 30 days after injury. Likewise, fewer BrdU-positive cells were noted in treated versus control tissue at 1 and 3 days, but this difference was not significant by 7 days. Moreover, tenascin immunoreactivity was significantly diminished at 24 h as confirmed by Western blot analysis in suramin-treated lesion areas, which is analogous to our observations that suramin can antagonize tenascin expression by cultured astrocytes treated with bFGF. In addition, examination of the corpus callosum of saline-treated animals 30 days post-trauma revealed a disruption of the fiber tract within the lesion site, while suramin-treated animals displayed numerous fibers spanning the lesion. These results demonstrate that a single injection of suramin transiently inhibits the gliotic response, which may be sufficient to ameliorate subsequent tissue damage.

Mechanisms of astrocyte-directed neurite guidance.

Astrocytes have recently become better recognized as playing vital roles in regulating the patterning of central nervous system neurites during development and following injury. In general, astrocytes have been shown to be supportive of neurite extension, but alterations in the biochemical properties of astrocytes in particular areas during development and in gliotic tissue may act to confine neurite outgrowth and thus provide guidance cues. In vivo studies indicate that restrictive astrocytes function through their altered expression of specific extracellular matrix molecules, including tenascin, chondroitin, and keratan sulfate proteoglycans. In addition, several in vitro models suggest that other cell surface molecules are utilized by restrictive astrocytes to direct neurite trajectories.

Long and short splice variants of human tenascin differentially regulate neurite outgrowth.

Tenascin-C has been implicated in regulation of neurite outgrowth both during development and after injury; however, its role as permissive vs inhibitory remains controversial. We report that different tenascin splice variants may have dramatically different impacts on neuronal growth. In a cell culture model, the largest and smallest splice variants (TN.L and TN.S) of human tenascin both promoted process extension when surface-bound. In contrast, soluble TN.S inhibited outgrowth, whereas soluble TN.L had no inhibitory effect. Perturbation experiments with antibodies, and outgrowth experiments with recombinant tenascin fragments, indicate that the differential properties of these molecules can be attributed to their distinctive array of FN-III repeats. Monoclonal antibodies were used to demonstrate at least two distinct neurite outgrowth promoting domains within the alternatively spliced region. These results suggest that the effect of tenascin on axon growth is a function of splice variants, as well as the form or conformation of those variants.

Role of HGF/SF and c-Met in morphogenesis and metastasis of epithelial cells.

We have analysed the role of hepatocyte growth factor/scatter factor (HGF/SF) in the process of morphogenesis and metastasis of epithelial (carcinoma) cells. HGF/SF induces various morphogenic responses in epithelial cells that derive from different tissues when these are grown in three-dimensional gels, e.g. branching tubules in kidney, breast, and prostate epithelial cells, crypt-like structures with brush border in colon epithelial cells, and alveolar-like aggregates in lung and pancreas cells. Epithelial cells are thus able to form complex structures in vitro which resemble the structures formed in the organ they originate from. We also examined the response of human breast carcinoma cells to HGF/SF in vivo. MDA MB 435 cells transfected with HGF/SF were injected into the mammary fat pad of nude mice, where they form tumours which spontaneously metastasize to the lungs. We found that expression of HGF/SF promoted metastasis whereas expression of the cell adhesion molecule E-cadherin was inhibitory. Moreover, expression of E-cadherin reconstituted the ability of the cells to form complex structures in response to HGF/SF in vitro. These data demonstrate that the different responses to HGF/SF depend on the state of the epithelial cells: morphogenesis requires epithelial differentiation and cell polarity, whereas metastasis is observed when the cells have lost their epithelial characteristics. Moreover, we have recently identified Gab-1 as a direct-binding substrate of the c-Met receptor. Gab-1 binds to c-Met phosphorylated on tyrosine residues, but not to a number of other tyrosine kinases from different subfamilies. A newly identified proline-rich domain of Gab-1 is responsible for the binding to the bidentate docking site in c-Met. Expression of Gab-1 in epithelial cells is sufficient to induce c-Met-specific cellular responses which include the formation of branching tubules. Thus, Gab-1 seem to correspond to the substrate of the c-Met receptor tyrosine kinase that mediates the epithelial morphogenesis.

Inflammatory cytokines interact to modulate extracellular matrix and astrocytic support of neurite outgrowth.

Following injury to the central nervous system, an astroglial scar forms that is thought to impede neuronal regeneration and recovery of function. It is our hypothesis that inflammatory cytokines act upon astrocytes to alter their biochemical and physical properties, which may in turn be responsible for failed neuronal regeneration. We have therefore examined the interactions of two cytokines with prominent actions following injury, interferon-gamma (IFN-gamma) and basic fibroblast growth factor (FGF2), in modulating the extracellular matrix and proliferation of astrocytes in culture. We also evaluated the effects of these cytokines on the ability of astrocytes to support the growth of neurites. IFN-gamma significantly inhibited the proliferation of rat cortical astrocytes both in serum-free and serum-containing media as measured by [3H]thymidine incorporation. Furthermore, IFN-gamma also antagonized FGF2-induced proliferation. In parallel, IFN-gamma reduced the levels of the ECM molecules tenascin, laminin, and fibronectin as evaluated by Western blot analysis and immunocytochemistry. Similarly, IFN-gamma also antagonized FGF2-induced tenascin formation. While IFN-gamma-pretreated astrocyte monolayers did not differ from control in their ability to support neurite outgrowth of cortical neurons, it antagonized the enhancement of neurite outgrowth on FGF2-treated monolayers. We demonstrate that IFN-gamma did not alter signal transduction through the FGF2 receptor down to the phosphorylation of mitogen-activated protein kinase, suggesting that the interaction is at the level of transcriptional regulation or that an alternate pathway is involved. These results support the hypothesis that inflammatory cytokines interact to modulate several facets of the gliotic response and such interactions may be important in creating the biochemical and physical properties of the glial scar.

A distinct subset of tenascin/CS-6-PG-rich astrocytes restricts neuronal growth in vitro.

Astrocytes provide an optimal surface for attachment, migration, and growth of CNS neurons. Nonetheless, not all astrocytes are alike: our previous work demonstrated a heterogeneity in the ability of cultured astrocyte monolayers to support neuronal growth. Areas displaying a fibrous, uneven surface ("rocky" astrocytes) were shown to be restrictive substrates, whereas surrounding, flat areas were permissive substrates. However, whether these cell types are in fact different cannot be addressed using mixed cultures. Therefore, in the current study we used morphological criteria to isolate the two subpopulations from mixed astrocyte cultures established from the cerebral cortex of neonatal rats. Following isolation, the purified populations only produced progeny with the same phenotype as the parent cells. We then measured production of several extracellular matrix molecules putatively involved in neuronal guidance during development and quantitatively assessed neuronal behavior on the purified populations. Immunocytochemistry and immunoblotting showed that rocky astrocytes were enriched in tenascin and chondroitin-6- sulfate-containing proteoglycans, but not in laminin or fibronectin. In addition, these astrocytes, as well as their isolated matrix, were a less permissive substrate for neuronal growth than flat astrocytes/matrix. Neurite outgrowth was significantly increased on rocky astrocytes following treatment with chondroitinase ABC or AC, but not heparitinase or hyaluronidase. These data support a critical role for matrix-bound chondroitin-6-sulfate-containing proteoglycans. We hypothesize that rocky astrocytes represent a subtype of cells which form barriers to neuronal growth during cortical development.

An analysis of astrocytic cell lines with different abilities to promote axon growth.

The adult mammalian central nervous system (CNS) lacks the capacity to support axonal regeneration. There is increasing evidence to suggest that astrocytes, the major glial population in the CNS, may possess both axon-growth promoting and axon-growth inhibitory properties and the latter may contribute to the poor regenerative capacity of the CNS. In order to examine the molecular differences between axon-growth permissive and axon-growth inhibitory astrocytes, a panel of astrocyte cell lines exhibiting a range of axon-growth promoting properties was generated and analysed. No clear correlation was found between the axon-growth promoting properties of these astrocyte cell lines with: (i) the expression of known neurite-outgrowth promoting molecules such as laminin, fibronectin and N-cadherin; (ii) the expression of known inhibitory molecules such tenascin and chondroitin sulphate proteoglycan; (iii) plasminogen activator and plasminogen activator inhibitor activity; and (iv) growth cone collapsing activity. EM studies on aggregates formed from astrocyte cell lines, however, revealed the presence of an abundance of extracellular matrix material associated with the more inhibitory astrocyte cell lines. When matrix deposited by astrocyte cell lines was assessed for axon-growth promoting activity, matrix from permissive lines was found to be a good substrate, whereas matrix from the inhibitory astrocyte lines was a poor substrate for neuritic growth. Our findings, taken together, suggest that the functional differences between the permissive and the inhibitory astrocyte cell lines reside largely with the ECM.

An immortalized mouse neuroepithelial cell line with neuronal and glial phenotypes.

Evidence from retroviral marking techniques and immortalized cell lines indicates that multipotential stem cells exist in many areas of the developing central nervous system. However, the factors that influence the commitment of these stem cells into distinct neuronal or glial lineages are not known. We have created an immortalized hypothalamic cell line derived from embryonic day 14 hypothalamic cells with a replication-defective retroviral construct containing a temperature-sensitive allele (tsA58) of the large T antigen of the simian virus 40. The clonality of this cell line, which we have named V1, was established by single cell cloning and by Southern blot analysis. V1 cells exhibit two different morphologies: the vast majority of cells are flat and stellate, and a smaller number are phase-bright round cells with processes. V1 cells express nestin and neural-cell adhesion molecule, typical of proliferating neuroepithelial cells. They also express glial fibrillary acidic protein and S100 as well as the low molecular weight neurofilament protein. In addition, the phase-bright, process-bearing V1 cells stain intensely for many typical neuronal proteins, such as low, medium and high molecular weight neurofilament proteins, tau protein, microtubule-associated protein-2, and neuron-specific enolase. The phase-bright cells also have condensed chromatin and display mitotic spindles, indicating that they are in mitosis. When V1 cells are transferred from the permissive temperature (33 degrees C) to the restrictive temperature (39 degrees C), there is a decrease in expression of NF-L and an increase in expression of NF-H and glial fibrillary acidic protein in the flat V1 cells. The enhanced expression of neuronal antigens in mitotically active V1 cells is novel and may represent a more general property of the differentiation process. We suggest that V1 cells arise from a mixed neural/glial neuroepithelial progenitor cell that expresses both neuronal- and glial-specific proteins in the developing hypothalamus.

Regulation of astrocytic tenascin by basic fibroblast growth factor.

Extracellular matrix (ECM) molecules have been implicated in the regulation of neuronal adhesion and neurite outgrowth both during development and after injury. It has been demonstrated in our laboratory that astrocytes are heterogeneous in expression of the ECM molecule tenascin. High-tenascin astrocytes have a reduced ability to support neurite outgrowth. In addition, astrocytes treated with exogenous basic fibroblast growth factor (bFGF) supported reduced neuronal growth and adhesion. In the current study, the hypothesis was tested that bFGF could increase the expression of tenascin by these cells. Basic FGF was added to cultures of rat cerebral cortical astrocytes at concentrations of up to 30 ng/ml, concentrations shown to have a significant effect on neuronal adhesion. Tenascin levels were evaluated by Western blot analysis of both cell extracts and conditioned media and also by immunocytochemistry techniques. Tenascin levels began to increase after 24-48 hr and continued to increase throughout 8 days in culture. The increase in tenascin was concentration-dependent, with the largest increase seen at 5 ng/ml bFGF. Tenascin production was increased approximately 5.5-fold in serum-containing medium but only about 2-fold in serum-free medium. When heparin (10 micrograms/ml) was included along with bFGF in serum-free medium, tenascin production was further enhanced. The bFGF treatment was discontinued after 8 days, and the cells were maintained for an additional 8 days in culture. Tenascin levels returned to control values, demonstrating that the bFGF effect is transient. It is our hypothesis that the action of bFGF during injury may evoke the induction of tenascin on astrocytes, thereby reducing regeneration in the central nervous system.