Astrocyte scar formation aids central nervous system axon regeneration.

Transected axons fail to regrow in the mature central nervous system. Astrocytic scars are widely regarded as causal in this failure. Here, using three genetically targeted loss-of-function manipulations in adult mice, we show that preventing astrocyte scar formation, attenuating scar-forming astrocytes, or ablating chronic astrocytic scars all failed to result in spontaneous regrowth of transected corticospinal, sensory or serotonergic axons through severe spinal cord injury (SCI) lesions. By contrast, sustained local delivery via hydrogel depots of required axon-specific growth factors not present in SCI lesions, plus growth-activating priming injuries, stimulated robust, laminin-dependent sensory axon regrowth past scar-forming astrocytes and inhibitory molecules in SCI lesions. Preventing astrocytic scar formation significantly reduced this stimulated axon regrowth. RNA sequencing revealed that astrocytes and non-astrocyte cells in SCI lesions express multiple axon-growth-supporting molecules. Our findings show that contrary to the prevailing dogma, astrocyte scar formation aids rather than prevents central nervous system axon regeneration.

Reactive astrocytes in multiple sclerosis impair neuronal outgrowth through TRPM7-mediated chondroitin sulfate proteoglycan production.

Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS), characterized by inflammation-mediated demyelination, axonal injury and neurodegeneration. The mechanisms underlying impaired neuronal function are not fully understood, but evidence is accumulating that the presence of the gliotic scar produced by reactive astrocytes play a critical role in these detrimental processes. Here, we identified astrocytic Transient Receptor Potential cation channel, subfamily M, member 7 (TRPM7), a Ca2+ -permeable nonselective cation channel, as a novel player in the formation of a gliotic scar. TRPM7 was found to be highly expressed in reactive astrocytes within well-characterized MS lesions and upregulated in primary astrocytes under chronic inflammatory conditions. TRPM7 overexpressing astrocytes impaired neuronal outgrowth in vitro by increasing the production of chondroitin sulfate proteoglycans, a key component of the gliotic scar. These findings indicate that astrocytic TRPM7 is a critical regulator of the formation of a gliotic scar and provide a novel mechanism by which reactive astrocytes affect neuronal outgrowth.

LAR and PTPσ receptors are negative regulators of oligodendrogenesis and oligodendrocyte integrity in spinal cord injury.

Following spinal cord injury (SCI), the population of mature oligodendrocytes undergoes substantial cell death; promoting their preservation and replacement is a viable strategy for preserving axonal integrity and white matter repair in the injured spinal cord. Dramatic upregulation of matrix chondroitin sulfate proteoglycans (CSPGs) is shown to pose an obstacle to endogenous repair processes, and targeting CSPGs improves functional recovery after SCI. However, the cellular and molecular mechanisms underlying the inhibitory effects of CSPGs remain largely undefined. Modulation of CSPGs specific signaling receptors, leukocyte common antigen-related (LAR), and protein tyrosine phosphatase-sigma (PTPσ) allows us to uncover the role and mechanisms of CSPGs in regulating oligodendrocytes in SCI. Here, utilizing specific functionally blocking peptides in a clinically relevant model of contusive/compressive SCI in the rat, we demonstrate that inhibition of PTPσ and LAR receptors promotes oligodendrogenesis by endogenous precursor cells, attenuates caspase 3-mediated cell death in mature oligodendrocytes, and preserves myelin. In parallel in vitro systems, we have unraveled that CSPGs directly induce apoptosis in populations of neural precursor cells and oligodendrocyte progenitor cells and limit their ability for oligodendrocyte differentiation, maturation, and myelination. These negative effects of CSPGs are mediated through the activation of both LAR and PTPσ receptors and the downstream Rho/ROCK pathway. Thus, we have identified a novel inhibitory role for PTPσ and LAR in regulating oligodendrocyte differentiation and apoptosis in the injured adult spinal cord and a new feasible therapeutic strategy for optimizing endogenous cell replacement following SCI.

Prognostic relevance of NG2/CSPG4, CD44 and Ki-67 in patients with glioblastoma.

Neuron-glial antigen 2 (NG2, also known as CSPG4) and hyaluronic acid receptor CD44 are chondroitin sulphate proteoglycans actively involved in brain development and its malignant transformation. Here, we aimed to compare prognostic significances of NG2, CD44 and Ki-67 expression in glioblastoma multiforme patients. Totally, 45 tissue samples and 83 paraffin-embedded tissues for 75 patients were analysed. The prognostic values of the genes were analysed using Kaplan-Meier survival curves. Grade III gliomas showed 2-fold difference in NG2 expression between anaplastic astrocytoma and oligoastrocytoma (10.1 ± 3.5 and 25.5 ± 14.5, respectively). For grade IV gliomas, upregulated NG2 expression (21.0 ± 6.8) was associated with poor glioblastoma multiforme prognosis (overall survival < 12 months) compared with glioblastoma multiforme patients with good prognosis (4.4 ± 3.2; overall survival > 12 months). Multivariate survival analysis using Cox proportional hazards model confirmed that high NG2 expression was associated with low survival of the patients (hazard ratio: 3.43; 95% confidence interval: 1.18-9.93; p = 0.02), whereas age (hazard ratio: 1.02; 95% confidence interval: 0.96-1.09; p = 0.42), tumour resection (hazard ratio: 1.03; 95% confidence interval: 0.98-1.08; p = 0.25) and sex (hazard ratio: 0.62; 95% confidence interval: 0.21-1.86; p = 0.40) did not show significant association with prognosis. Although the positive correlation was shown for NG2 and CD44 expression in the glioblastomas (Pearson coefficient = 0.954), Kaplan-Meier and multivariate survival analyses did not revealed a significant association of the increased CD44 expression (hazard ratio: 2.18; 95% confidence interval: 0.50-9.43; p = 0.30) or high Ki-67 proliferation index (hazard ratio: 1.10; 95% confidence interval: 1.02-1.20; p = 0.02) with the disease prognosis. The results suggest that upregulation of NG2/CSPG4 rather than changes in CD44 or Ki-67 expression is associated with low overall survival in glioblastoma multiforme patients, supporting NG2/CSPG4 as a potential prognostic marker in glioblastoma.

Effects of sesamin on chondroitin sulfate proteoglycan synthesis induced by interleukin-1beta in human chondrocytes.

BACKGROUND: Numerous studies have reported on the health benefits of sesamin, a major lignin found in sesame (S. indicum) seeds. Recently, sesamin was shown to have the ability to promote chondroitin sulfate proteoglycan synthesis in normal human chondrocytes. This study assesses the anti-inflammatory effect of sesamin on proteoglycans production in 3D chondrocyte cultures. METHODS: To evaluate the effects of sesamin on IL-1ß-treated human articular chondrocytes (HAC) pellets, the pellets were pre-treated with IL-1ß then cultured in the presence of various concentrations of sesamin for 21 days. During that period, the expression of IL-1ß, glycosaminoglycans (GAGs) content and Chondroitin sulfate proteoglycans (CSPGs) synthesis genes (ACAN, XT-1, XT-2, CHSY1 and ChPF) was measured. The GAGs accumulation in the extracellular matrix was determined on day 21 by histological analysis. RESULTS: There was clear evidence that sesamin upregulated expression of all the CSPGs synthesis genes, in contrast to the down-regulation of IL-1ß expression both in genes and in protein levels. The level of release and matrix accumulation of GAGs in IL-1ß pre-treated HAC pellets in the presence of sesamin was recovered. These results correlate with the histological examination which showed that sesamin enhanced matrix CSPGs accumulation. CONCLUSIONS: Sesamin enhances CSPGs synthesis, suppresses IL-1ß expression and ameliorates IL-1ß induced inflammation in human chondrocytes. Sesamin could have therapeutic benefits for treating inflammation in osteoarthritis.

OASIS regulates chondroitin 6-O-sulfotransferase 1 gene transcription in the injured adult mouse cerebral cortex.

Old astrocyte specifically induced substance (OASIS), a basic leucine zipper transcription factor of the cAMP response element binding/Activating transcription factor family, is induced in reactive astrocytes in vivo and has important roles in quality control of protein synthesis at the endoplasmic reticulum. Reactive astrocytes produce a non-permissive environment for regenerating axons by up-regulating chondroitin sulfate proteoglycans (CSPGs). In this study, we focus on the potential role of OASIS in CSPG production in the adult mouse cerebral cortex. CS-C immunoreactivity, which represents chondroitin sulfate moieties, was significantly attenuated in the stab-injured cortices of OASIS knockout mice compared to those of wild-type mice. We next examined expression of the CSPG-synthesizing enzymes and core proteins of CSPGs in the stab-injured cortices of OASIS knockout and wild-type mice. The levels of chondroitin 6-O-sulfotransferase 1 (C6ST1, one of the major enzymes involved in sulfation of CSPGs) mRNA and protein increased after cortical stab injury of wild-type, but not of OASIS knockout, mice. A C-terminal deletion mutant OASIS over-expressed in rat C6 glioma cells increased C6ST1 transcription by interacting with the first intron region. Neurite outgrowth of cultured hippocampal neurons was inhibited on culture dishes coated with membrane fractions of epidermal growth factor-treated astrocytes derived from wild type but not from OASIS knockout mice. These results suggest that OASIS regulates the transcription of C6ST1 and thereby promotes CSPG sulfation in astrocytes. Through these mechanisms, OASIS may modulate axonal regeneration in the injured cerebral cortex. OASIS, an ER stress-responsive CREB/ATF family member, is up-regulated in the reactive astrocytes of the injured brain. We found that the up-regulated OASIS is involved in the transcriptional regulation of C6ST1 gene, which promotes chondroitin sulfate proteoglycan (CSPG) sulfation. We conclude that OASIS functions as an anti-regenerative transcription factor by establishing a non-permissive microenvironment to regenerating axons.

Effects of sesamin on the biosynthesis of chondroitin sulfate proteoglycans in human articular chondrocytes in primary culture.

Osteoarthritis (OA) is a degenerative joint disease that progressively causes a loss of joint functions and the impaired quality of life. The most significant event in OA is a high degree of degradation of articular cartilage accompanied by the loss of chondroitin sulfate-proteoglycans (CS-PGs). Recently, the chondroprotective effects of sesamin, the naturally occurring substance found in sesame seeds, have been proved in a rat model of papain-induced osteoarthritis. We hypothesized that sesamin may be associated with possible promotion of the biosynthesis of CS-PGs in human articular chondrocytes. The aim of the study was to investigate the effects of sesamin on the major CS-PG biosynthesis in primary human chondrocyte. The effects of sesamin on the gene expression of the PG core and the CS biosynthetic enzymes as well as on the secretion of glycosaminoglycans (GAGs) in monolayer and pellet culture systems of articular chondrocytes. Sesamin significantly increased the GAGs content both in culture medium and pellet matrix. Real-time-quantitative PCR showed that sesamin promoted the expression of the genes encoding the core protein (ACAN) of the major CS-PG aggrecan and the biosynthetic enzymes (XYLT1, XYLT2, CHSY1 and CHPF) required for the synthesis of CS-GAG side chains. Safranin-O staining of sesamin treated chondrocyte pellet section confirmed the high degree of GAG accumulation. These results were correlated with an increased level of secreted GAGs in the media of cultured articular chondrocytes in both culture systems. Thus, sesamin would provide a potential therapeutic strategy for treating OA patients.

Lumican expression, localization and antitumor activity in prostate cancer.

The stromal reaction surrounding tumors leads to the formation of a tumor-specific microenvironment, which may play either a restrictive role or a supportive role in the growth and progression of the tumors. Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), regulates collagen fibrillogenesis. Recently, lumican has also been shown to regulate cell behavior during embryonic development, tissue repair and tumor progression. The role of lumican in cancer varies according to the type of tumor. In this study we analyze the role of lumican in the pathogenesis of prostate cancer both in vivo and in vitro. Overall lumican up-regulation was observed in the primary tumors analyzed through both real-time PCR and immunostaining. The increase in lumican expression was observed in the reactive stroma surrounding prostate primary tumors with fibrotic deposition surrounding the acinar glands. In vitro analysis demonstrated that lumican inhibited both the migration and invasion of metastatic prostate cancer cells isolated from lymph node, bone and brain. Moreover, prostate cancer cells seeded on lumican presented a decrease in the formation of cellular projections, lamellipodia detected by a decreased rearrangement in ZO-1, keratin 8/18, integrin ß1 and MT1-MMP, and invadopodia detected by disruption of α-smooth muscle actin, cortactin and N-WASP. Moreover, a significant increase in prostate cancer cell invasion was observed through the peritoneum of lumican knockout mice, further demonstrating the restrictive role lumican present in the ECM has on prostate cancer invasion. In conclusion, lumican present in the reactive stroma surrounding prostate primary tumors plays a restrictive role on cancer progression, and we therefore postulate that lumican could be a valuable marker in prostate cancer staging.

Mutations in fam20b and xylt1 reveal that cartilage matrix controls timing of endochondral ossification by inhibiting chondrocyte maturation.

Differentiating cells interact with their extracellular environment over time. Chondrocytes embed themselves in a proteoglycan (PG)-rich matrix, then undergo a developmental transition, termed "maturation," when they express ihh to induce bone in the overlying tissue, the perichondrium. Here, we ask whether PGs regulate interactions between chondrocytes and perichondrium, using zebrafish mutants to reveal that cartilage PGs inhibit chondrocyte maturation, which ultimately dictates the timing of perichondral bone development. In a mutagenesis screen, we isolated a class of mutants with decreased cartilage matrix and increased perichondral bone. Positional cloning identified lesions in two genes, fam20b and xylosyltransferase1 (xylt1), both of which encode PG synthesis enzymes. Mutants failed to produce wild-type levels of chondroitin sulfate PGs, which are normally abundant in cartilage matrix, and initiated perichondral bone formation earlier than their wild-type siblings. Primary chondrocyte defects might induce the bone phenotype secondarily, because mutant chondrocytes precociously initiated maturation, showing increased and early expression of such markers as runx2b, collagen type 10a1, and ihh co-orthologs, and ihha mutation suppressed early perichondral bone in PG mutants. Ultrastructural analyses demonstrated aberrant matrix organization and also early cellular features of chondrocyte hypertrophy in mutants. Refining previous in vitro reports, which demonstrated that fam20b and xylt1 were involved in PG synthesis, our in vivo analyses reveal that these genes function in cartilage matrix production and ultimately regulate the timing of skeletal development.

Phenotypic characterization of culture expanded rabbit limbal corneal keratocytes.

The in vivo quiescent corneal stroma keratocytes need to be transformed to activated state in order to obtain sufficient number of cells either for monolayer evaluation or corneal stroma reconstruction. This study aimed to investigate the phenotypic characterization of corneal stromal cells during culture expansion from the limbal region of the cornea. Isolated corneal keratocytes from limbal tissue of New Zealand White Strain rabbits' corneas (n = 6) were culture expanded until three passages. Keratocytes morphology was examined daily with viability, growth rate, number of cell doubling and population doubling time were recorded at each passage. The expression of collagen type 1, aldehyde dehydrogenase (ALDH), lumican and alpha smooth muscle actin (α-SMA) were detected by RT-PCR. Immunocytochemistry was also used to detect ALDH, α-SMA, collagen type I and Cytokeratin-3 (CK3). Growth kinetic study revealed that the growth rate was low at the initial passage but increase to about two folds with concomitant reduction in population doubling time in later passages. Freshly isolated and cultured keratocytes expressed collagen type 1, ALDH and lumican but α-SMA expression was absent. However, α-SMA was expressed along with the other genes during culture expansion. Keratocytes at P1 expressed all the proteins except CK3. These results suggest that cultured keratocytes maintained most of the gene expression profile of native keratocytes while the emergence of α-SMA in serial passages showed a mix population of various phenotypes. The phenotypic characterization of monolayer keratocytes provides useful information before reconstruction of bioengineered tissue or in vitro pharmaceutical applications.

High chondroitin sulfate proteoglycan 4 expression correlates with poor outcome in patients with breast cancer.

Chondroitin sulfate proteoglycan 4 (CSPG4), a transmembrane proteoglycan originally identified in melanoma cells, has been reported to be expressed in breast cancer cells. This study was performed to examine the expression and significance of CSPG4 in a cohort of breast cancer patients. Immunohistochemical analysis of CSPG4 was performed on tissue microarrays constructed from tissue specimens from 240 breast cancer patients. CSPG4 staining was correlated with clinical and pathological characteristics, overall survival (OS), and disease recurrence. Contradicting to a previous report, our results showed that high CSPG4 expression was not related to triple-negative status of breast cancer patients. The Kaplan-Meier method showed that high CSPG4 expression was significantly associated with shorter time to recurrence (TTR). Patients with high CSPG4 expression had poorer OS and shorter TTR in a multivariate survival analysis after adjustment for stage, tumor grade, expression of estrogen receptor and progesterone receptor, and HER2 overexpression. This study showed that high CSPG4 expression correlates with disease recurrence and OS in breast cancers.

Dermatopontin: a potential predictor for metastasis of human oral cancer.

Dermatopontin (DPT), a component of the extracellular matrix (ECM), is involved in promotion of cellular adhesion and ECM assembly activities. However, the role of DPT in the pathogenesis of carcinoma is unclear. We evaluated DPT expression in human oral cancer and its possible roles including cellular adhesion and invasiveness. We first investigated the DPT mRNA and protein expression status in human oral squamous cell carcinoma (OSCC)-derived cells. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunoblotting analysis detected frequent downregulation of DPT in OSCC-derived cells compared to human normal oral keratinocytes. To assess the epigenetic regulation of DPT, OSCC-derived cells were treated with a histone deacetylase inhibitor, sodium butyrate (NaB). NaB restored the DPT expression in OSCC-derived cells. DPT-overexpressed cells were examined whether DPT could contribute to cellular adhesion and invasiveness. Markedly, increased adhesion and decreased invasiveness in DPT-overexpressed cells were found compared to mock-transfected cells. Adhesion of DPT-overexpressed cells was inhibited by α3ß1 integrin functional blocking antibody. OSCC-derived cells treated with NaB also decreased invasiveness. The expression status of DPT in primary OSCCs (n = 97) was analyzed and compared to clinicopathological behavior. DPT expression in primary OSCCs was significantly lower (p < 0.05) than in the normal counterparts and was correlated significantly (p < 0.05) with regional lymph node metastasis. Our data provided strong evidence that downregulation of DPT is a characteristic event in OSCCs and that DPT was correlated with cellular adhesion and invasiveness. Therefore, DPT might play an important role in regulating tumor invasion and metastasis.

Excitability and synaptic communication within the oligodendrocyte lineage.

The mammalian CNS contains an abundant, widely distributed population of glial cells that serve as oligodendrocyte progenitors. It has been reported that these NG2-immunoreactive cells (NG2(+) cells) form synapses and generate action potentials, suggesting that neural-evoked excitation of these progenitors may regulate oligodendrogenesis. However, recent studies also suggest that NG2(+) cells are comprised of functionally distinct groups that differ in their ability to respond to neuronal activity, undergo differentiation, and experience injury following ischemia. To better define the physiological properties of NG2(+) cells, we used transgenic mice that allowed an unbiased sampling of this population and unambiguous identification of cells in discrete states of differentiation. Using acute brain slices prepared from developing and mature mice, we found that NG2(+) cells in diverse brain regions share a core set of physiological properties, including expression of voltage-gated Na(+) (NaV) channels and ionotropic glutamate receptors, and formation of synapses with glutamatergic neurons. Although small amplitude Na(+) spikes could be elicited in some NG2(+) cells during the first postnatal week, they were not capable of generating action potentials. Transition of these progenitors to the premyelinating stage was accompanied by the rapid removal of synaptic input, as well as downregulation of AMPA and NMDA receptors and NaV channels. Thus, prior reports of physiological heterogeneity among NG2(+) cells may reflect analysis of cells in later stages of maturation. These results suggest that NG2(+) cells are uniquely positioned within the oligodendrocyte lineage to monitor the firing patterns of surrounding neurons.

Chondroitin sulfate proteoglycans regulate astrocyte-dependent synaptogenesis and modulate synaptic activity in primary embryonic hippocampal neurons.

It has been shown that astrocyte-derived extracellular matrix (ECM) is important for formation and maintenance of CNS synapses. In order to study the effects of glial-derived ECM on synaptogenesis, E18 rat hippocampal neurons and primary astrocytes were co-cultivated using a cell-insert system. Under these conditions, neurons differentiated under low density conditions (3500 cells/cm(2) ) in defined, serum-free medium and in the absence of direct, membrane-mediated neuron-astrocyte interactions. Astrocytes promoted the formation of structurally intact synapses, as documented by the co-localisation of bassoon- and ProSAP1/Shank2-positive puncta, markers of the pre- and postsynapse, respectively. The development of synapses was paralleled by the emergence of perineuronal net (PNN)-like structures that contained various ECM components such as hyaluronic acid, brevican and neurocan. In order to assess potential functions for synaptogenesis, the ECM was removed by treatment with hyaluronidase or chondroitinase ABC. Both enzymes significantly enhanced the number of synaptic puncta. Whole-cell voltage-clamp recordings of control and enzyme-treated hippocampal neurons revealed that chondroitinase ABC treatment led to a significant decrease in amplitude and a reduced charge of miniature excitatory postsynaptic currents, whereas inhibitory postsynaptic currents were not affected. When the response to the application of glutamate was measured, a reduced sensitivity could be detected and resulted in decreased currents in response to the excitatory neurotransmitter. These findings are consistent with the interpretation that the ECM partakes in the regulation of the density of glutamate receptors in subsynaptic sites.

Modulation of small leucine-rich proteoglycans (SLRPs) expression in the mouse uterus by estradiol and progesterone.

BACKGROUND: We have previously demonstrated that four members of the family of small leucine-rich-proteoglycans (SLRPs) of the extracellular matrix (ECM), named decorin, biglycan, lumican and fibromodulin, are deeply remodeled in mouse uterine tissues along the estrous cycle and early pregnancy. It is known that the combined action of estrogen (E2) and progesterone (P4) orchestrates the estrous cycle and prepares the endometrium for pregnancy, modulating synthesis, deposition and degradation of various molecules. Indeed, we showed that versican, another proteoglycan of the ECM, is under hormonal control in the uterine tissues. METHODS: E2 and/or medroxiprogesterone acetate (MPA) were used to demonstrate, by real time PCR and immunoperoxidase staining, respectively, their effects on mRNA expression and protein deposition of these SLRPs, in the uterine tissues. RESULTS: Decorin and lumican were constitutively expressed and deposited in the ECM in the absence of the ovarian hormones, whereas deposition of biglycan and fibromodulin were abolished from the uterine ECM in the non-treated group. Interestingly, ovariectomy promoted an increase in decorin, lumican and fibromodulin mRNA levels, while biglycan mRNA conspicuously decreased. Hormone replacement with E2 and/or MPA differentially modulates their expression and deposition. CONCLUSIONS: The patterns of expression of these SLRPs in the uterine tissues were found to be hormone-dependent and uterine compartment-related. These results reinforce the existence of subpopulations of endometrial fibroblasts, localized into distinct functional uterine compartments, resembling the organization into basal and functional layers of the human endometrium.

Fibromodulin regulates collagen fibrillogenesis during peripheral corneal development.

Fibromodulin regulates collagen fibrillogenesis, but its existence/role(s) in the cornea is controversial. We hypothesize that fibromodulin regulates fibrillogenesis during postnatal development of the anterior eye. Fibromodulin is weakly expressed in the limbus at post-natal day (P) 4, increases and extends into the central cornea at P14, becomes restricted to the limbus at P30, and decreases at P60. This differential spatial and temporal expression of fibromodulin is coordinated with emmetropization; the developmental increase in axial length and globe size. Genetic analysis demonstrated that fibromodulin regulates fibrillogenesis in a region-specific manner. At the limbus, fibromodulin is dominant in regulating fibril growth during postnatal development. In the posterior peripheral cornea, cooperative interactions of fibromodulin and lumican regulate fibrillogenesis. These data indicate that fibromodulin plays important roles in the regulation of region-specific fibrillogenesis required for the integration of the corneal and scleral matrices and sulcus development required for establishment of the visual axis.

Formation of the ovarian follicular antrum and follicular fluid.

The formation of the follicular antrum and follicular fluid has received scant attention from researchers, yet both are important processes in follicular development. The central hypothesis on follicular fluid formation suggests that production by granulosa cells of hyaluronan and the chondroitin sulfate proteoglycan versican generates an osmotic gradient. This gradient draws in fluid derived from the thecal vasculature. Inter-alpha-trypsin inhibitor is also present in follicular fluid at least in species with large follicles, and inter-alpha-trypsin inhibitor and versican could additionally bind or cross-link with hyaluronan, resulting in the retention of these molecules within the follicular antrum. Barriers to the movement of fluid across the membrana granulosa are apparently minimal, as even relatively large serum proteins are present in follicular fluid. Despite the relative permeability of the follicular wall, aquaporins are present in granulosa cells and could be actively involved in the transport of water into the follicle. The formation of an antrum also requires movement of granulosa cells relative to each other to allow the fluid to accumulate. This presumably involves remodeling of cell-cell junctions and in species with small follicles may involve death of centrally located granulosa cells. Remodeling of the stroma and thecal layers also accompanies growth and expansion of the antrum and presumably involves similar processes that accompany growth of other glands.

Cloning, expression and purification of recombinant dermatopontin in Escherichia coli.

Dermatopontin (DPT) is an extracellular matrix (ECM) protein with diversified pharmaceutical applications. It plays important role in cell adhesion/migration, angiogenesis and ECM maintenance. The recombinant production of this protein will enable further exploration of its multifaceted functions. In this study, DPT protein has been expressed in Escherichia coli (E.coli) aiming at cost effective recombinant production. The E.coli GJ1158 expression system was transformed with constructed recombinant vector (pRSETA-DPT) and protein was expressed as inclusion bodies on induction with NaCl. The inclusion bodies were solubilised in urea and renaturation of protein was done by on-column refolding procedure in Nickel activated Sepharose column. The refolded Histidine-tagged DPT protein was purified and eluted from column using imidazole and its purity was confirmed by analytical techniques. The biological activity of the protein was confirmed by collagen fibril assay, wound healing assay and Chorioallantoic Membrane (CAM) angiogenesis assay on comparison with standard DPT. The purified DPT was found to enhance the collagen fibrillogenesis process and improved the migration of human endothelial cells. About 73% enhanced wound closure was observed in purified DPT treated endothelial cells as compared to control. The purified DPT also could induce neovascularisation in the CAM model. At this stage, scaling up the production process for DPT with appropriate purity and reproducibility will have a promising commercial edge.

Traumatic brain injury results in disparate regions of chondroitin sulfate proteoglycan expression that are temporally limited.

Axonal injury is a major hallmark of traumatic brain injury (TBI), and it seems likely that therapies directed toward enhancing axon repair could potentially improve functional outcomes. One potential target is chondroitin sulfate proteoglycans (CSPGs), which are major axon growth inhibitory molecules that are generally, but not always, up-regulated after central nervous system injury. The current study was designed to determine temporal changes in cerebral cortical mRNA or protein expression levels of CSPGs and to determine their regional localization and cellular association by using immunohistochemistry in a controlled cortical impact model of TBI. The results showed significant increases in versican mRNA at 4 and 14 days after TBI but no change in neurocan, aggrecan, or phosphacan. Semiquantitative Western blot (WB) analysis of cortical CSPG protein expression revealed a significant ipsilateral decrease of all CSPGs at 1 day after TBI. Lower CSPG protein levels were sustained until at least 14 days, after which the levels began to normalize. Immunohistochemistry data confirm previous reports of regional increases in CSPG proteins after CNS injury, seen primarily within the developing glial scar after TBI, but also corroborate the WB data by revealing wide areas of pericontusional tissue that are deficient in both extracellular and perineuronal net-associated CSPGs. Given the evidence that CSPGs are largely inhibitory to axonal growth, we interpret these data to indicate a potential for regional spontaneous plasticity after TBI. If this were the case, the gradual normalization of CSPG proteins over time postinjury would suggest that this may be temporally as well as regionally limited.

Chondroitin sulfate proteoglycans and microglia prevent migration and integration of grafted Müller stem cells into degenerating retina.

At present, there are severe limitations to the successful migration and integration of stem cells transplanted into the degenerated retina to restore visual function. This study investigated the potential role of chondroitin sulfate proteoglycans (CSPGs) and microglia in the migration of human Müller glia with neural stem cell characteristics following subretinal injection into the Lister hooded (LH) and Royal College of Surgeons (RCS) rat retinae. Neonate LH rat retina showed minimal baseline microglial accumulation (CD68-positive cells) that increased significantly 2 weeks after transplantation (p < .001), particularly in the ganglion cell layer (GCL) and inner plexiform layer. In contrast, nontransplanted 5-week-old RCS rat retina showed considerable baseline microglial accumulation in the outer nuclear layer (ONL) and photoreceptor outer segment debris zone (DZ) that further increased (p < .05) throughout the retina 2 weeks after transplantation. Marked deposition of the N-terminal fragment of CSPGs, as well as neurocan and versican, was observed in the DZ of 5-week-old RCS rat retinae, which contrasted with the limited expression of these proteins in the GCL of the adult and neonate LH rat retinae. Staining for CSPGs and CD68 revealed colocalization of these two molecules in cells infiltrating the ONL and DZ of the degenerating RCS rat retina. Enhanced immune suppression with oral prednisolone and intraperitoneal injections of indomethacin caused a reduction in the number of microglia but did not facilitate Müller stem cell migration. However, injection of cells with chondroitinase ABC combined with enhanced immune suppression caused a dramatic increase in the migration of Müller stem cells into all the retinal cell layers. These observations suggest that both microglia and CSPGs constitute a barrier for stem cell migration following transplantation into experimental models of retinal degeneration and that control of matrix deposition and the innate microglial response to neural retina degeneration may need to be addressed when translating cell-based therapies to treat human retinal disease.