The expression of keratan sulfate reveals a unique subset of microglia in the mouse hippocampus after pilocarpine-induced status epileptics.

Induction of keratan sulfate in microglia has been found in several animal models of neurological disorders. However, the significance of keratan sulfate-expressing microglia is not fully understood. To address this issue, we analyzed the characteristics of microglia labeled by the 5D4 epitope, a marker of high-sulfated keratan sulfate, in the mouse hippocampus during the latent period after pilocarpine-induced status epilepticus (SE). Only 5D4-negative (5D4- ) microglia were found in the CA1 region of vehicle-treated controls and pilocarpine-treated mice at 1 day after SE onset. A few 5D4+ microglia appeared in the strata oriens and radiatum at 5 days post-SE, and they were distributed into the stratum pyramidale at 14 days post-SE. The expressions of genes related to both anti- and pro-inflammatory cytokines were higher in 5D4+ cells than in 5D4- cells at 5 but not 14 days post-SE. The expressions of genes related to phagocytosis were higher in 5D4+ cells than in 5D4- cells throughout the latent period. The phagocytic activity of microglia, as measured by engulfment of the zymosan bioparticles, was higher in 5D4+ cells than in 5D4- cells. The contact ratios between excitatory synaptic boutons and microglia were also higher in 5D4+ cells than in 5D4- cells at 5 and 14 days post-SE. The excitatory/inhibitory ratios of synaptic boutons within the microglial domain were lower in 5D4+ cells than in 5D4- cells at 14 days post-SE. Our findings indicate that 5D4+ microglia may play some role in epileptogenesis via pruning of excitatory synapses during the latent period after SE.

Keratan Sulfate Phenotype in the ß-1,3-N-Acetylglucosaminyltransferase-7-Null Mouse Cornea.

Purpose: Synthesis of keratan sulfate (KS) relies on coordinated action of multiple enzymes, including the N-acetylglucosamine-transferring enzyme, ß-1,3-N-acetylglucosaminyltransferase-7 (ß3GnT7). A mouse model deficient in ß3GnT7 was developed to explore structural changes in KS and the extracellular matrix (ECM; i.e., the corneal stroma), elucidate the KS biosynthesis mechanism, and understand its role in corneal organization. Methods: A knockout vector for the ß3GnT7-encoding gene, B3gnt7, was created to develop heterozygous- (htz) and homozygous-null (null) knockouts. Epithelial, stromal, and whole cornea thicknesses were measured from each group. Proteoglycans were stained with cupromeronic blue for visualization by electron microscopy, and Western blot analyses were conducted on the KS core protein, lumican. Corneal sections were labelled fluorescently for KS and chondroitin sulfate/dermatan sulfate (CS/DS) using monoclonal antibodies 1B4 or 2B6, respectively. Results: Wild-type (WT) and htz corneas were of similar stromal thickness, whereas null specimens measured relatively thin. Electron micrographs revealed that WT and htz samples contained comparable levels of KS- and CS/DS-PGs. Null corneas, however, lacked detectable KS and featured uncharacteristically elongated electron dense PG filaments, which were susceptible to chondroitinase ABC digestion. Western blotting revealed lumican in the null corneas was substituted with low-molecular-weight KS, relative to WT or htz tissue. KS was not immunohistochemically detectable in the null cornea, whereas CS/DS content appeared increased. Conclusions: Addition of N-acetylglucosamine via ß3GnT7 to KS glycosaminoglycans is necessary for their biosynthesis. Without ß3GnT7, murine corneal stromas lack KS and appear to compensate for this loss with upregulation of chondroitinase ABC-sensitive PGs.

Keratan sulfate: biosynthesis, structures, and biological functions.

Keratan sulfate is a glycosaminoglycan that has been investigated in the cornea and skeletal tissues for decades. Endoglycosidases and monoclonal antibodies specific for keratan sulfate have been developed. These materials have facilitated the analysis of keratan sulfate biosynthesis and structures. Likewise, they have expedited study of the biological roles of keratan sulfate in vitro and in vivo. It has been shown that keratan sulfate is also expressed in the central nervous system and functions as a regulator of neuronal regeneration/sprouting. Here, we describe methods to determine the enzymatic activity of GlcNAc6ST, which is involved in keratan sulfate biosynthesis, and to extract and prepare ocular keratan sulfate for a disaccharide composition analysis. Immunohistochemistry for an anti-keratan sulfate epitope in the brain is also described.

Gene expression profiling of giant cell tumor of bone reveals downregulation of extracellular matrix components decorin and lumican associated with lung metastasis.

Giant cell tumor of bone (GCTB) displays worrisome clinical features such as local recurrence and occasionally metastatic disease which are unpredictable by morphology. Additional routinely usable biomarkers do not exist. Gene expression profiles of six clinically defined groups of GCTB and one group of aneurysmal bone cyst (ABC) were determined by microarray (n = 33). The most promising differentially expressed genes were validated by Q-PCR as potential biomarkers in a larger patient group (n = 41). Corresponding protein expression was confirmed by immunohistochemistry. Unsupervised hierarchical clustering reveals a metastatic GCTB cluster, a heterogeneous, non-metastatic GCTB cluster, and a primary ABC cluster. Balanced score testing indicates that lumican (LUM) and decorin (DCN) are the most promising biomarkers as they have lower level of expression in the metastatic group. Expression of dermatopontin (DPT) was significantly lower in recurrent tumors. Validation of the results was performed by paired and unpaired t test in primary GCTB and corresponding metastases, which proved that the differential expression of LUM and DCN is tumor specific rather than location specific. Our findings show that several genes related to extracellular matrix integrity (LUM, DCN, and DPT) are differentially expressed and may serve as biomarkers for metastatic and recurrent GCTB.

Keratan sulfate expression is associated with activation of a subpopulation of microglia/macrophages in Wallerian degeneration.

Wallerian degeneration is a fundamental process of axonal degeneration distal to the injury site. Although axonal degeneration itself is accomplished in a few days, the subsequent process of removing debris, including myelin debris, in the central nervous system takes more time. Since this debris is a potent inhibitor of axonal regeneration, the removal process is critical for functional recovery after neuronal injuries. Although it is known that microglia/macrophages are involved in this process, the underlying mechanisms are not fully understood. Here, we found that keratan sulfate (KS) expression was induced far from the injury site after spinal cord injury. A hemilateral section of the spinal cord at the third cervical level induced KS expression in a restricted area of the ipsilateral column at the first lumbar level 1 week after injury. This localized KS expression lasted for at least 1 month after injury. The KS signal was merged with a portion of Iba1-positive cells, suggesting that a subpopulation of microglia/macrophages expressed KS. KS-positive cells expressed CD68 and CD86, but not CD206 or arginase 1, suggesting that these microglia/macrophages were in an activated state probably polarized to M1. Our study has explored for the first time the relation between KS expression and activation of microglia/macrophages in Wallerian degeneration.

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.

Analysis of protein expression regulated by lumican in PANC­1 cells using shotgun proteomics.

Lumican, a member of the class II small leucine-rich proteoglycan family, regulates the assembly and diameter of collagen fibers in the extracellular matrix of various tissues. We previously reported that lumican expression in the stromal tissues of pancreatic ductal adenocarcinoma (PDAC) correlates with tumor invasion, and tends to correlate with poor prognosis. Lumican stimulates growth and inhibits the invasion of a PDAC cell line. In the present study, we performed a global shotgun proteomic analysis using lumican-overexpressing PANC­1 cells and lumican downregulated PANC­1 cells to identify candidate proteins that are regulated by lumican and related to cell growth and invasion in PDAC cells. A total of 448 proteins were identified from lumican-overexpressing PANC­1 and control cells. Additionally, 451 proteins were identified from lumican-downregulated PANC­1 cells and control cells. As a result of semi-quantification based on spectral counting, 174 differentially expressed proteins were identified by lumican upregulation, and 143 differentially expressed proteins were identified by lumican downregulation. The expression levels of 24 proteins, including apoptosis- and invasion-related proteins correlated with lumican expression levels. It is likely that the expression of these proteins is regulated by lumican, and that they are involved in apoptosis and invasion in PDAC. These findings suggest that lumican may be involved in cell growth and invasion through the regulation of these 24 proteins expressed in PDAC.

UDP-N-acetylglucosamine transporter (SLC35A3) regulates biosynthesis of highly branched N-glycans and keratan sulfate.

SLC35A3 is considered the main UDP-N-acetylglucosamine transporter (NGT) in mammals. Detailed analysis of NGT is restricted because mammalian mutant cells defective in this activity have not been isolated. Therefore, using the siRNA approach, we developed and characterized several NGT-deficient mammalian cell lines. CHO, CHO-Lec8, and HeLa cells deficient in NGT activity displayed a decrease in the amount of highly branched tri- and tetraantennary N-glycans, whereas monoantennary and diantennary ones remained unchanged or even were accumulated. Silencing the expression of NGT in Madin-Darby canine kidney II cells resulted in a dramatic decrease in the keratan sulfate content, whereas no changes in biosynthesis of heparan sulfate were observed. We also demonstrated for the first time close proximity between NGT and mannosyl (α-1,6-)-glycoprotein ß-1,6-N-acetylglucosaminyltransferase (Mgat5) in the Golgi membrane. We conclude that NGT may be important for the biosynthesis of highly branched, multiantennary complex N-glycans and keratan sulfate. We hypothesize that NGT may specifically supply ß-1,3-N-acetylglucosaminyl-transferase 7 (ß3GnT7), Mgat5, and possibly mannosyl (α-1,3-)-glycoprotein ß-1,4-N-acetylglucosaminyltransferase (Mgat4) with UDP-GlcNAc.

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.

Characterizing the effects of VPA, VC and RCCS on rabbit keratocytes onto decellularized bovine cornea.

To investigate the morphological and growth characteristics of rabbit keratocytes when cultured on decellularized cornea under simulate microgravity (SMG) rotary cell culture system (RCCS) and static culture or in plastic culture supplemented with small molecules of valproic acid (VPA) and vitamin C (VC). Bovine corneas were firstly decellularized with Triton X-100 and NH(4)OH and through short-term freezing process. Then cell count kit-8 (CCK-8) and flow cytometry were used to test the effects of VPA and VC on the proliferation, cell cycle and apoptosis of rabbit keratocytes. Hematoxylin-eosin (H&E) staining and scanning electron microscopy (SEM) imaging showed that cells were eliminated in the decellularized bovine corneas. The proliferation of cultured keratocytes was promoted by VPA and VC in the cell proliferation assay. VPA and VC moderately decreased the number of apoptotic cells and obviously promoted cell-cycle entrance of keratocytes. Rabbit keratocytes in plastic displayed spindle shape and rare interconnected with or without VPA and VC. Cells revealed dendritic morphology and reticular cellular connections when cultured on the carriers of decellularized corneas supplemented with VPA and VC even in the presence of 10% fetal bovine serum (FBS). When cultured in RCCS supplemented with VPA, VC and 10% FBS, keratocytes displayed round shape with many prominences and were more prone to grow into the pores of carriers with aggregation. Reverse transcription-polymerase chain reaction (RT-PCR) analysis proved that the keratocytes cultured on decellularized bovine cornea under SMG with VPA and VC expressed keratocan and lumican. Keratocytes cultured on plastic expressed lumican but not keratocan. Immunofluorescence identification revealed that cells in all groups were positively immunostained for vimentin. Keratocytes on decellularized bovine cornea under SMG or in static culture were positively immunostained for keratocan and lumican. Thus, we reasonably made a conclusion that the combination of VPA, VC, RCCS and decellularized corneal carriers provide a good condition for keratocytes to well grow. Keratocytes can be manipulated to be aggregates or physiological morphological growth in vitro, which are important for the research of corneal stem cells and corneal tissue engineering.

Assessment of gene expression profiles in peripheral occlusive arterial disease.

BACKGROUND: Molecular events responsible for the onset and progression of peripheral occlusive arterial disease (POAD) are incompletely understood. Gene expression profiling may point out relevant features of the disease. METHODS: Tissue samples were collected as operatory waste from a total of 36 patients with (n = 18) and without (n = 18) POAD. The tissues were histologically evaluated, and the patients with POAD were classified according to Leriche-Fontaine (LF) classification: 11% with stage IIB, 22% with stage III, and 67% with stage IV. Total RNA was isolated from all samples and hybridized onto Agilent 4×44K Oligo microarray slides. The bioinformatic analysis identified genes differentially expressed between control and pathologic tissues. Ten genes with a fold change ≥ 2 (1 with a fold change ≥ 1.8) were selected for quantitative polymerase chain reaction validation (GPC3, CFD, GDF10, ITLN1, TSPAN8, MMP28, NNMT, SERPINA5, LUM, and FDXR). C-reactive protein (CRP) was assessed with a specific assay, while nicotinamide N-methyltransferase (NNMT) was evaluated in the patient serum by enzyme-linked immunosorbent assay. RESULTS: A multiple regression analysis showed that the level of CRP in the serum is correlated with the POAD LF stages (r(2) = 0.22, P = 0.046) and that serum NNMT is higher in IV LF POAD patients (P = 0.005). The mRNA gene expression of LUM is correlated with the LF stage (r(2) = 0.45, P = 0.009), and the mRNA level of ITLN1 is correlated with the ankle-brachial index (r(2) = 0.42, P = 0.008). CONCLUSIONS: Our analysis shows that NNMT, ITLN1, LUM, CFD, and TSPAN8 in combination with other known markers, such as CRP, could be evaluated as a panel of biomarkers of POAD.

Rhodamine B and 2-acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-D-glucopyranose (F-GlcNAc) inhibit chondroitin/dermatan and keratan sulphate synthesis by different mechanisms in bovine chondrocytes.

MPS disorders result from a deficiency or absence of glycosaminoglycan (GAG) degrading enzymes leading to an imbalance between the synthesis and degradation of GAGs and their subsequent accumulation in a range of cells. The inhibition of GAG synthesis using small chemical inhibitors has been proposed as a novel therapeutic approach to treatment. Several inhibitors have been shown to decrease heparan sulphate GAG synthesis and in this study we evaluated a novel fluorinated analog of N-acetylglucosamine (2-acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-D-glucopyranose (F-GlcNAc)) and rhodamine B for their ability to also inhibit the synthesis of chondroitin/dermatan and keratan sulphate GAGs present in bovine cartilage. Both inhibitors decreased GAG synthesis in chondrocyte monolayer culture and in cartilage chip explant culture in a dose dependent manner. Both inhibitors decreased the size of newly synthesised proteoglycans and in the case of F-GlcNAc this was due to a decrease in newly synthesised GAG chain size. Rhodamine B, however, did not affect GAG chain size, while both inhibitors decreased the amount of chondroitin/dermatan and keratan sulphate GAG equally. The expression of genes responsible for the initiation and elongation of chondroitin/dermatan sulphate and keratan sulphate GAGs were downregulated in the presence of rhodamine B but not in the presence of F-GlcNAc. Thus the 2 inhibitors appear to have differing effects on GAG synthesis, with F-GlcNAc inhibiting the epimerisation of UDP-GlcNAc to UDP-GalNAc thus decreasing the availability of monosaccharides for addition to the growing GAG chain, whereas rhodamine B is more likely to reduce the number of GAG chains. Together with previous data these 2 inhibitors are capable of non-specific inhibition of GAG synthesis, reducing the production of chondroitin/dermatan sulphate, keratan sulphate and heparan sulphate GAGs. As such they would be applicable to therapy in a range of MPS disorders.

Secreted 70kDa lumican stimulates growth and inhibits invasion of human pancreatic cancer.

Lumican expression in the stromal tissues of pancreatic ductal adenocarcinoma (PDAC) correlates with tumor invasion, and tends to correlate with poor prognosis. We used gene transfection techniques to examine the biological roles of lumican secreted from PDAC cells. Lumican-transfected PANC-1 cells secreted a 70-kDa lumican protein and had an active ERK pathway. Transfection stimulated PANC-1 cell growth, increased cell adhesion to laminin, inhibited cell invasion, and decreased active matrix metalloproteinase-9. Down-regulation of lumican using siRNA resulted in opposite cell behavior. Thus, the 70-kDa lumican secreted by PDAC cells plays important roles in cell growth and invasion.

Expression of lumican and fibromodulin following interleukin-1 beta stimulation of disc cells of the human temporomandibular joint.

Small leucine-rich repeat proteoglycans (SLRP) are present in the extracellular matrix of the temporomandibular joint (TMJ) disc. Lumican and fibromodulin, classified as class 2 SLRPs, play important roles in TMJ assembly, proliferation and inflammation. Degenerative change in the TMJ disc gives rise to the process of internal derangement (ID). In this study, we immunohistochemically examined the expression of lumican and fibromodulin in nine human TMJ specimens and examined the gene expression of both proteoglycans in cultured human TMJ disc cells under interleukin-1 beta (IL-1 ß)-stimulated conditions. An articular disc cell line was established by collagenase treatment of a TMJ disc. The subcultured cells were then incubated for 1, 3, 6, 12, 24 or 48 h under both normal and IL-1 ß (1 ng/mL) conditions. The gene expression of lumican and fibromodulin was examined using the reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR. We demonstrated that the expression of lumican significantly differs from that of fibromodulin in the deformed disc and that IL-1 ß induces a significant increase in lumican mRNA, but not in fibromodulin mRNA, after 24∼48 h culture compared to cells cultured in the absence of IL-1 ß (P<0.05). These results indicate that lumican and fibromodulin display different behaviors and that lumican may promote regeneration of the TMJ after degeneration and deformation induced by IL-1 ß.

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.

HMGA2 overexpression-induced ovarian surface epithelial transformation is mediated through regulation of EMT genes.

The AT-hook transcription factor HMGA2 is an oncogene involved in the tumorigenesis of many malignant neoplasms. HMGA2 overexpression is common in both early and late-stage high-grade ovarian serous papillary carcinoma. To test whether HMGA2 participates in the initiation of ovarian cancer and promotion of aggressive tumor growth, we examined the oncogenic properties of HMGA2 in ovarian surface epithelial (OSE) cell lines. We found that introduction of HMGA2 overexpression was sufficient to induce OSE transformation in vitro. HMGA2-mediated OSE transformation resulted in tumor formation in the xenografts of nude mice. By silencing HMGA2 in HMGA2-overexpressing OSE and ovarian cancer cell lines, the aggressiveness of tumor cell growth behaviors was partially suppressed. Global gene profiling analyses revealed that HMGA2-mediated tumorigenesis was associated with expression changes of target genes and microRNAs that are involved in epithelial-to-mesenchymal transition (EMT). Lumican, a tumor suppressor that inhibits EMT, was found to be transcriptionally repressed by HMGA2 and was frequently lost in human high-grade serous papillary carcinoma. Our findings show that HMGA2 overexpression confers a powerful oncogenic signal in ovarian cancers through the modulation of EMT genes.

Aberrant expression of TFF1, TFF2, and PDX1 and their diagnostic value in lobular endocervical glandular hyperplasia.

Lobular endocervical glandular hyperplasia (LEGH) is a distinct benign glandular lesion expressing gastric gland mucous cell-type mucin (N-acetylglucosaminα1 → 4galactose → R [GlcNAcα1 → 4Gal → R]). To investigate histogenesis and diagnostic markers of LEGH, we examined the immunohistochemical expression profile of gastric surface mucous cell (MUC5AC and TFF1), gastric gland mucous cell (MUC6, TFF2, and GlcNAcα1 → 4Gal → R), gastric pyloric epithelial cell (PDX1), and endocervical cell (keratan sulfate) markers in normal endocervix samples and benign glandular lesions (nabothian cysts, tunnel clusters, and LEGHs). MUC5AC and MUC6 were expressed in normal endocervical mucosa and benign glandular lesions. TFF1, TFF2, GlcNAcα1 → 4Gal → R, and PDX1 were expressed only in LEGH. Keratan sulfate was expressed in normal endocervical mucosa and benign glandular lesions. In LEGH, gastric surface mucous cell and gastric gland mucous cell differentiation were demonstrated, and transdifferentiation from endocervical mucosa into gastric pyloric mucosa was suggested. In addition to GlcNAcα1 → 4Gal → R, TFF1, TFF2, and PDX1 are additional useful markers for LEGH.

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.

Transforming growth factor-beta1 upregulates keratan sulfate and chondroitin sulfate biosynthesis in microglias after brain injury.

After injury to the adult central nervous system, levels of extracellular matrix molecules increase at the injury site and may inhibit the repair of injured axons. Among these molecules, the importance of proteoglycans, particularly their chondroitin sulfate chains, has been highlighted. We have recently reported that keratan sulfate-deficient mice show better axonal regeneration after injury. Here, we investigated the regulation of keratan sulfate and chondroitin sulfate biosynthesis after neuronal injuries. Several key enzymes required for glycosaminoglycan biosynthesis (beta3GlcNAcT-7 and GlcNAc6ST-1 for keratan sulfate; CS synthase-1 and C6ST-1 for chondroitin sulfate) were expressed at significantly higher levels in the lesion 7 days after a knife-cut injury was made to the cerebral cortex in adult mice. These increases were accompanied by increased expression of TGF-beta(1) and bFGF. Since microglias at the injury sites expressed both keratan sulfate and chondroitin sulfate, the effects of these cytokines were examined in microglias. TGF-beta(1) induced the expression of the above-named enzymes in microglias, and consequently induced keratan sulfate and chondroitin sulfate biosynthesis as well as the expression of the chondroitin/keratan sulfate proteoglycan aggrecan in these cells. TGF-beta(1) also induced bFGF expression in microglias. bFGF in turn induced TGF-beta(1) expression in astrocytes. Astrocyte-conditioned medium following bFGF stimulation indeed induced keratan sulfate and chondroitin sulfate production in microglias. This production was blocked by TGF-beta(1)-neutralizing antibody. Taken together, our data indicate that the biosyntheses of keratan sulfate and chondroitin sulfate are upregulated in common by TGF-beta(1) in microglias after neuronal injuries.

Localization of small leucine-rich proteoglycans and transforming growth factor-beta in human oral mucosal wound healing.

Wound healing in oral mucosa is fast and results in little scar formation as compared with skin. The biological mechanisms underlying this property are poorly understood but may provide valuable information about the factors that promote wound regeneration. Small leucine-rich proteoglycans (SLRPs) decorin, biglycan, fibromodulin and lumican are extracellular matrix molecules that regulate collagen fibrillogenesis, inhibit transforming growth factor-beta (TGF-beta) activity and reduce scarring. In the present study, we analyzed accumulation of SLRPs and TGF-beta during non-scarring human oral mucosal wound healing. Biopsies were collected from healthy volunteers from unwounded tissue and from standardized experimental wounds 3-60 days postwounding. Localization of SLRPs, TGF-beta1 and TGF-beta3 was analyzed by immunohistochemical staining and quantitated by image analysis. Double immunostaining was used to study localization of SLRPs or active TGF-beta in distinct cells. Decorin, biglycan, fibromodulin, and TGF-beta isoforms showed significantly increased accumulation in the wound extracellular matrix and distinct wound cells while the abundance of lumican in the extracellular matrix was strongly reduced during wound healing. Localization and abundance of fibromodulin, lumican, and TGF-beta isoforms was also spatiotemporally regulated in the wound epithelium. The findings suggest that SLRPs regulate wound reepithelialization and connective tissue regeneration during oral mucosal wound healing.