Trabecular Cells Research Articles

Effect of heparin II domain of fibronectin on aqueous outflow in cultured anterior segments of human eyes.

To determine whether an integrin/syndecan-binding domain of fibronectin, called the heparin II (Hep II) domain, affects outflow facility in the human eye. Anterior segments of human eyes were placed in perfusion organ culture. One eye of each pair received the Hep II domain, and the fellow eye received DMEM or a heat-denatured Hep II domain. The Hep II domain was produced as a recombinant glutathione S-transferase (GST)-fusion protein. Microscopic changes were assessed. Outflow facility in anterior segments treated with Hep II domain increased by 93% compared with that in anterior segments treated with DMEM. In contrast, facility in anterior segments treated with the heat-denatured Hep II domain showed very little change. Outflow facility remained high during Hep II domain perfusion and returned to baseline after removal of the protein. Electron microscopy revealed disruptions in the endothelial lining of Schlemm's canal in anterior segments fixed during maximum effect and in anterior segments after facility had returned to baseline. Scattered disruptions of canal cells were noted in control anterior segments. Trabecular cells in other regions looked normal. Major changes in the extracellular matrix of the juxtacanalicular tissue were not observed. Repeated doses of the Hep II domain administered after facility returned to baseline increased facility in two of three anterior segments. The Hep II domain of fibronectin increases outflow facility in the human anterior segment. This suggests that fibronectin-mediated interactions may have a role in modulating aqueous hydrodynamics. Such interactions may represent avenues of novel therapeutic interventions for glaucoma.

Dominant localization of adenosine deaminase in leptomeninges and involvement of the enzyme in sleep

Adenosine is an endogenous hypnotic molecule. However, the mechanism by which the level of extracellular adenosine is regulated remains to be elucidated. We found by Northern hybridization and enzyme assay that ecto-5 ′-nucleotidase and adenosine deaminase (ADA), major enzymes responsible for the production and degradation of adenosine, respectively, were localized most abundantly in the leptomeninges within the rat brain. Immunohistochemical study showed that ADA was dominantly localized in arachnoid barrier and trabecular cells of the leptomeninges. In vivo microdialysis demonstrated that externally applied adenosine was rapidly metabolized by ADA to inosine in the subarachnoid space. Perfusion of an ADA inhibitor, coformycin, increased the extracellular adenosine level in the subarachnoid space under the rostral basal forebrain. When coformycin was continuously infused into the subarachnoid space, non-rapid eye movement sleep was increased with prolonged duration of the sleep episode. These results demonstrate that the leptomeninges control the extracellular level of adenosine in the subarachnoid space by their high 5 ′-nucleotidase and ADA activities and regulate non-rapid eye movement sleep.

The effect of TGF-β2 on human trabecular meshwork extracellular proteolytic system

Our study aimed to investigate whether transforming growth factor-β2 (TGF-β2), increased in the aqueous humor of eyes with primary open angle glaucoma (POAG), can affect factors responsible for the activity of matrix metalloproteinases (MMPs) in human trabecular cell cultures. With this goal in mind cultures of human trabecular meshwork (hTM) cells derived from 8 donors were treated with TGF-β2 for 24, 36 and 48 hr. Influence of TGF-β2 on expression of MMP-2, MMP-9, membrane type 1-MMP (MT1-MMP) and plasminogen activator inhibitor-1 (PAI-1) was examined using RT-PCR, Northern Blot, Western Blot and zymography. The influence of TGF-β2 treatment on PAI-1 expression was also investigated using immunohistochemistry. It appeared that treatment with TGF-β2 significantly increased expression of the proform of MMP-2, whereas the active form was not detectable. MMP-9 and MT1-MMP expression were not influenced by TGF-β2 treatment. There was, however, a significant increase in PAI-1 expression. To investigate whether transformation of the proform of MMP-2 to the active form was inhibited by PAI-1, the influence of treatment with TGF-β2 and a PAI-1 neutralizing antibody on MMP-2 was investigated using zymogram method. With this treatment protocol the active form of MMP-2 was clearly visible, indicating that TGF-β2 enhancement of the PAI-1-expression decreases MMP activity. Inhibition of MMP activity through elevated levels of TGF-β2 might contribute to the increase in ECM in the trabecular meshwork of glaucomatous eyes.

289 In vitro effects of preserved or unpreserved antiglaucoma drugs on apoptotic marker expression by human trabecular cells

This study compares the effects of orbitofrontal and temporal neocortical lesions on the integration and expression of grooming, huddling, and spatial relationships in vervet monkeys (Cercopithecus aethiops sabaeus). Two groups of socially living vervet monkeys were observed in a large outdoor enclosure. Nine members (four male and five female) of one group received orbitofrontal lesions and seven members of the other group (four male and three female) were subjected to temporal neocortical lesions. Unoperated control subjects, matched for age, gender, and rank, received sham operations. Animals with orbitofrontal lesions groomed less, huddled less, and were less likely to be within 1 m of their nearest neighbor. Animals with temporal neocortical lesions also groomed less, but their huddling behavior and spatial relationships were not altered. These data suggest that when a single species of Old World monkey is observed in a similar setting using comparable behavioral units, the effects of orbitofrontal and temporal neocortical lesions on affiliative behaviors can be dissociated.

Activation of p38 and Smads mediates BMP-2 effects on human trabecular bone-derived osteoblasts

The bone morphogenetic proteins (BMPs) are potent osteoinductive factors that accelerate osteoblast maturation, accompanied by increased cell-substrate adhesion. BMP-2 treatment of osteoblastic cells increases phosphorylation of the cytoplasmic BMP-2 signaling molecules, Smad1 and Smad5. We have previously reported that BMP-2 treatment increase cytoskeletal organization of human trabecular bone-derived osteoblast-like cells (osteoblasts), which is also accompanied by an activation of the focal adhesion kinase p125 FAK. We report here that activation of p125 FAK occurs with the same kinetics as the phosphorylation of Smad1, suggesting that BMP-2 initiates cross-talk between Smad signaling and the adhesion-mediated signaling pathway. As an adjunct to these effects, we examined activation of mitogen-activated protein (MAP) kinase family members in response to focal adhesion contact formation. Although phosphorylated forms of all three kinases were apparent, only SAPK2α/p38 (p38) was activated in response to BMP-2 treatment. Inhibition of p38 kinase activity suppressed BMP-2 induced Smad1 phosphorylation, as well as its translocation to the nucleus, suggesting the integration of p38 activation with Smad1 signaling. Finally, inhibition of p38 in osteoblasts also led to the complete abrogation of BMP-2 induced osteocalcin gene expression and matrix mineralization. These findings suggest that BMP-2 must activate p38 in order to mediate osteogenic differentiation and maturation.

Hepatocellular carcinoma with sarcomatous change arising after radiofrequency ablation for well-differentiated hepatocellular carcinoma

We report a case involving well-differentiated hepatocellular carcinoma (HCC) developing to HCC with sarcomatous changes after radiofrequency ablation (RFA). In a cirrhotic patient with both hepatitis B surface antigen and hepatitis C virus RNA, a well-differentiated HCC with a diameter of 2 cm was detected in segment IV of the liver. A combination of transcatheter arterial embolization and percutaneous ethanol injection (PEI) was performed and, after 8 months, PEI was performed for recurrent tumors. Fifteen months after the first treatment, a recurrent tumor measuring 3.5 cm in diameter was detected in segment IV, which was demonstrated as well-differentiated HCC by tumor biopsy, and treated by RFA. Although the treated lesion was reduced to 2.5 cm in diameter 6 months after RFA, the tumor rapidly enlarged to 6 cm in diameter 2 months later and progressed to lymph node metastasis. Aspiration biopsy showed spindle-shaped sarcomatoid cells with positive staining for both vimentin and keratin. The patient died of HCC progression 10 months after RFA. Autopsy findings showed both sarcomatoid cells and trabecular HCC cells. The sarcomatoid cells had metastasized to the lymph nodes and distant organs. This is the first case illustrating a sarcomatous HCC after RFA. Of interest, RFA may be related to the development of sarcomatous HCC.

Why is glaucoma associated with exfoliation syndrome?

Exfoliation syndrome (XFS) is an age-related, generalized disorder of the extracellular matrix characterized by production and progressive accumulation of a fibrillar material in tissues throughout the anterior segment and also in connective tissue portions of various visceral organs. Mature exfoliation fibrils are composed of 8-10 nm microfibrils resembling elastic microfibrils. The exact chemical composition of exfoliation material (XFM) remains unknown. It appears to consist of a complex glycoprotein/ proteoglycan structure composed of a protein core surrounded by abundant glycoconjugates. The protein components include both non-collagenous basement membrane components and epitopes of the elastic fiber system, particularly components of elastic microfibrils. Overall, XFS is the most common identifiable cause of glaucoma, accounting for the majority of cases in some countries, and causing both open-angle glaucoma and angle-closure glaucoma. Iridolenticular friction leads to loss of XFM from the anterior lens surface and disruption of the iris pigment epithelium, resulting in pigment deposition in the trabecular meshwork, which also produces XFM locally. The primary cause of chronic pressure elevation appears to be the active involvement of trabecular cells and Schlemm's canal cells in particular, in the generalized pathologic matrix process with subsequent degenerative changes of Schlemm's canal and adjacent tissues. Narrow angles and angle-closure are common in XFS. Pupillary block may be caused by a combination of posterior synechiae, increased iris thickness or rigidity, or anterior lens movement secondary to zonular weakness or dialysis. Enlargement of the lens due to cataract formation and relative pupillary constriction are additional factors.

Protein expression, genomic structure, and polymorphisms of oculomedin

Purpose: To elucidate protein expression, genomic structure, and genomic polymorphisms of a novel gene, ‘oculomedin’, that has been cloned as a mechanical stretch-response gene from human trabecular cells in culture. Methods: Polyclonal antibody was prepared by immunizing rabbits with a chemically synthesized 15-mer peptide of oculomedin. Protein expression was revealed by Western blot analysis after polyacrylamide gel electrophoresis of extracts of mechanically stretched trabecular cells and control trabecular cells in culture as well as retinal tissue. Protein localization was studied immunohistochemically in the human eye section. Genomic structure was determined by searching the GenBank database. Genomic polymorphisms of the coding region in 163 glaucoma patients and 50 normal subjects were detected by PCR amplification and direct sequencing. Results: Western blot analysis showed that oculomedin protein was expressed only in stretched trabecular cells, not in control trabecular cells in culture. Immunohistochemically, oculomedin protein was localized to the trabecular meshwork, Schlemm’s canal endothelium, retinal photoreceptor cells, and corneal and conjunctival epithelium. The oculomedin gene (OCLM) consists of two exons which are located inside an intron of a different gene of unknown function (C1orf27) on chromosome 1q25, near and telomeric to myocilin (MYOC). Two types of heterozygous nucleotide substitutions resulting in amino acid changes were found in two of 75 patients with primary open-angle glaucoma, but not at all in patients with other types of glaucoma or in normal subjects. Conclusions: Oculomedin may play a role in the function of the trabecular meshwork and also in the development of primary open-angle glaucoma.

In vitro effects of preserved or preservative-free antiglaucoma medications on human complement system

Purpose. Antiglaucoma drugs have been associated with conjunctival and trabecular inflammatory cell infiltrates. However, the underlying mechanisms are still poorly understood. The aim of this study was to assess the effects of antiglaucoma medications on the complement system, an early mediator of the inflammatory response. Methods. Human serum was first treated with a classical or alternative pathway activator (aggregated human IgG or zymosan, respectively) in the presence or the absence of preservative-free or benzalkonium (BAK)-preserved antiglaucoma drugs. CH50 assay was then performed to assess the functional activity of residual complement in treated serum. Results. In the absence of complement activator, the antiglaucoma drugs tested in this study were all devoid of intrinsic complement-activating potency. Preserved and preservative-free carteolol as well as preserved latanoprost did not worsen or prevent complement activation induced by zymosan or aggregated IgG. Unexpectedly, both preserved and unpreserved timolol and betaxolol significantly counteracted the effects of complement activators. Timolol prevented activation triggered by both IgG and zymosan to the same extent (24% to 29%), despite the presence of BAK in the preserved formulation. Betaxolol was twice as effective at preventing the effect of IgG (34% to 37%) than that of zymosan (14%), regardless of the presence of BAK. However, BAK itself strongly aggravated complement activation by both activators. Conclusions. Carteolol, timolol, betaxolol and latanoprost did not activate complement system. On the contrary, the beta-blockers timolol and betaxolol exerted an anti-inflammatory effect by preventing complement activation. The deleterious effect of benzalkonium seems to have been neutralized within the preserved eyedrops through a mechanism that remains to be elucidated. Our study suggests that inflammatory signs in glaucoma patients should not be attributed to complement activation by antiglaucoma drugs.

Molecular mechanism of sleep induced by prostaglandin D 2

Prostaglandin (PG) D 2 is recognized as the most potent endogenous sleep-promoting substance whose mechanism of action is the best characterized among the various potential sleep substances thus far reported. Lipocalin-type PGD synthase is dominantly produced in the arachnoid membrane and choroid plexus of the brain, and is secreted into the cerebrospinal fluid to become β-trace, a major protein in human cerebrospinal fluid. PGD synthase and its resulting product, PGD 2, circulate in the ventricular system, subarachnoidal space and extracellular space of the brain system. PGD 2 then interacts with D type of prostanoid (DP) receptors on arachnoid trabecular cells in the chemosensory region of the ventromedial surface of the rostral basal forebrain to initiate the signal to promote sleep, probably via the activation of adenosine A 2A receptive neurons. The activation of the DP receptors results in activation of a cluster of neurons within the ventrolateral preoptic area, a recently identified sleep center, which may promote sleep by inhibiting the tuberomammillary nucleus, the source of the ascending histaminergic arousal system.

Platelets stimulate proliferation of bone cells: involvement of platelet-derived growth factor, microparticles and membranes.

Platelets have been implicated in accelerated bone regeneration in grafting applications. The beneficial effects of platelets may involve their ability to stimulate the proliferation of osteoblasts. We therefore determined the mitogenic response of human trabecular bone-derived cells to human platelets and supernatants of thrombin-activated platelets. We can show a approximately 50-fold increase in DNA-synthesis of bone cells (BC) cultured in the presence of platelets as determined by [3H]-thymidine incorporation. Preventing cell-to-cell contact by a membrane filter did not abrogate the stimulatory effect, indicating the release of soluble factor(s) that are mitogenic for BC. The lipid fraction of the platelets had no effect on [3H]-thymidine uptake into the DNA of BC. Platelet-released supernatant (PRS) increased the rate of [3H]-thymidine incorporation to approximately 20-fold and retained 56% of their activity after incubation at 56 degrees C, and 27% at 100 degrees C, respectively. Neutralizing antibodies raised against platelet-derived growth factor (PDGF) partially suppressed the mitogenic potential of PRS. Gel exclusion chromatography analysis showed that molecules ranging from 25 kDa to more than 70 kDa within the PRS can stimulate BC proliferation. The highest amount of PDGF was detected in fractions corresponding to a molecular weight of 28-37 kDa as determined by immunoassay. The mitogenic activity was not restricted to soluble growth factors because microparticles in the PRS and platelet membranes also increased BC proliferation. Our data indicate that native platelets, the respective PRS, microparticles, and platelet membranes can stimulate the mitogenic activity of BC, thereby contributing to the regeneration of mineralized tissue.

Spaceflight affects bone formation in rhesus monkeys: a histological and cell culture study.

Using analyses of iliac crest cell and tissue, back-scattered electron imaging, and biochemical techniques, we characterized the effects of a 14-day spaceflight (Bion 11) on bone structure and bone formation in two 3- to 4-yr-old male rhesus monkeys compared with eight age-matched Earth-control monkeys. We found that postflight bone volume was 35% lower than preflight values in flight monkeys. This was associated with reduced osteoid (-40%) and mineralizing (-32%) surfaces and decreased bone formation rate (-53%). Moreover, flight monkeys exhibited trends to lower values of mineralization profile in iliac bone (back-scattered electron imaging) and to decreased osteocalcin serum levels (P = 0.08). The initial number of trabecular bone cells yielded in cultures did not differ in flight and control animals before or after the flight. However, osteoblastic cell proliferation was markedly lower in postflight vs. preflight at 9 and 14 days of culture in one flight monkey. This study suggests that a 14-day spaceflight reduces iliac bone formation, osteoblastic activity, and/or recruitment in young rhesus monkeys, resulting in decreased trabecular bone volume.

P38 MAP Kinase Regulation of AP‐2 Binding in TGF‐β1‐Stimulated Chondrogenesis of Human Trabecular Bone‐Derived Cells

Collagenase-treated, explanted human trabecular-bone chips are an excellent source of osteoblast-like cells. We have recently shown the multiple differentiation potential of these cells; in addition to osteogenesis and adipogenesis, these cells also undergo chondrogenesis when maintained as high-density pellet cultures (250,000 cells/pellet) in a serum-free, chemically defined medium stimulated with TGF-beta1 (10 ng/mL). In this investigation, we have analyzed how transactivating nuclear transcription factors, specifically AP-2 and SP-1, may interact with common cis-acting elements found in the regulatory region of cartilage-specific genes as part of the signal transduction mechanism of TGF-beta1 and p38 during chondrogenesis of human trabecular bone-derived multipotential cells. Both TGF-beta1 stimulation and p38 MAP kinase activation affect the binding of AP-2 as well as SP-1 to oligonucleotides with sequence similarity to the overlapping AP-2/SP-1 sites found in the putative 52-bp immediate upstream regulatory region and the 5'-untranslated region of the human aggrecan gene. Electrophoretic mobility shift assays show that TGF-beta1 treatment of the bone-derived cells inhibits AP-2 DNA binding but enhances the DNA binding ability of SP-1. Additionally, treatment of these TGF-beta1-stimulated cells with p38 MAP kinase inhibitor, SB203580, rescued the AP-2 DNA binding but did not affect SP-1 DNA binding. These findings indicate that AP-2 DNA binding is the target of both TGF-beta1 and p38 MAP kinase signaling pathways and suggest a possible signal transduction cascade whereby TGF-beta1 induction of chondrogenesis involves the activation of p38 MAP kinase and the subsequent inhibition of DNA binding by AP-2, thereby preventing the transcriptional repression of the aggrecan gene.

Different osteochondral potential of clonal cell lines derived from adult human trabecular bone.

Cells derived from human trabecular bones have been shown to have multipotential differentiation ability along osteogenic, chondrogenic, and adipogenic lineages. In this study, we have derived two clonal sublines of human trabecular bone cells by means of stable transduction with human papilloma virus E6/E7 genes. Our results showed that these clonal sublines differ in their osteochondral potential, but are equally adipogenic, indicative of the heterogeneous nature of the parental cell population. The availability of these cell lines should be useful for the analysis of the mechanisms regulating the differentiation of adult mesenchymal progenitor cells.

Bisphosphonates influence the proliferation and the maturation of normal human osteoblasts.

The key pharmacological action for the clinical use of bisphosphonates lies in the inhibition of osteoclast-mediated bone resorption. Osteoblasts could be other target cells for bisphosphonates. We studied the effects of bisphosphonates on the proliferation and the differentiation of normal human bone trabecular osteoblastic cells (hOB). We tested 4 different compounds: clodronate, pamidronate and 2 newer compounds: ibandronate, a nitrogen-containing bisphosphonate and zoledronate, which is a heterocyclic imidazole compound. Ibandronate and zoledronate stimulated hOB cell proliferation by up to 30% (p<0.05) after 72 h for concentrations ranging from 10(-8) M to 10(-5) M. Clodronate transiently enhanced hOB cell survival after only 24 h (+60%, p<0.001) whereas pamidronate had no effect. Longer time course studies, in presence of fetal calf serum, revealed that cell growth was finally reduced by all 4 bisphosphonates (40% after 7 days). Type I collagen synthesis was transiently increased by all 4 bisphosphonates after only 48 h incubation (+17% to +67%, p<0.05). Clodronate increased ALP activity by up to 1.7-fold after 4 days of culture (p<0.05) whereas ibandronate or zoledronate exhibited lesser stimulatory effects (+17 to +30%), and pamidronate had no significant effect. In conclusion, we found that different bisphosphonates, currently used or tested in various clinical conditions, transiently stimulated the growth of preosteoblastic cells and thereafter increased their differentiation according to sequential events (type I collagen synthesis first, then ALP activity to a lesser extent). Our data suggest that the beneficial effects of bisphosphonate treatment on bone mass and integrity could be partly mediated through a direct action on osteoblasts.

Stem cell characteristics of human trabecular bone-derived cells

Human trabecular bone-derived cells (HTBs) have been used for many years as osteoblast progenitors. In this study we tested whether HTBs have stem cell characteristics; that is, whether they are pluripotent and able to self-renew. We show that HTBs readily differentiate into osteoblasts, chondrocytes, and adipocytes if subjected to the appropriate differentiating conditions. Importantly, differentiation into these three lineages is maintained in single cell clones derived by limiting dilution, following expansion over more than 20 cumulative population doublings. We conclude that cultures of HTBs are equivalent to cultures of “mesenchymal stem cells” (MSCs) isolated from bone marrow.

Multilineage mesenchymal differentiation potential of human trabecular bone-derived cells

Explant cultures of adult human trabecular bone fragments give rise to osteoblastic cells, that are known to express osteoblast-related genes and mineralize extracellular matrix. These osteoblastic cells have also been shown to undergo adipogenesis in vitro and chondrogenesis in vivo. Here we report the in vitro developmental potential of adult human osteoblastic cells (hOB) derived from explant cultures of collagenase-pretreated trabecular bone fragments. In addition to osteogenic and adipogenic differentiation, these cells are capable of chondrogenic differentiation in vitro in a manner similar to adult human bone marrow-derived mesenchymal progenitor cells. High-density pellet cultures of hOB maintained in chemically defined serum-free medium, supplemented with transforming growth factor-β1, were composed of morphologically distinct, chondrocyte-like cells expressing mRNA transcripts of collagen types II, IX and X, and aggrecan. The cells within the high-density pellet cultures were surrounded by a sulfated proteoglycan-rich extracellular matrix that immunostained for collagen type II and proteoglycan link protein. Osteogenic differentiation of hOB was verified by an increased number of alkaline phosphatase-positive cells, that expressed osteoblast-related transcripts such as alkaline phosphatase, collagen type I, osteopontin and osteocalcin, and formed mineralized matrix in monolayer cultures treated with ascorbate, β-glycerophosphate, and bone morphogenetic protein-2. Adipogenic differentiation of hOB was determined by the appearance of intracellular lipid droplets, and expression of adipocyte-specific genes, such as lipoprotein lipase and peroxisome proliferator-activated receptor γ2, in monolayer cultures treated with dexamethasone, indomethacin, insulin and 3-isobutyl-1-methylxanthine. Taken together, these results show that cells derived from collagenase-treated adult human trabecular bone fragments have the potential to differentiate into multiple mesenchymal lineages in vitro, indicating their developmental plasticity and suggesting their mesenchymal progenitor nature.

Targeted Gene Transfer to Corneal Stroma in vivo by Electric Pulses

This study was conducted to develop a method of targeted gene transfer by electric pulses to a selected area of corneal stroma in vivo. Plasmid DNA with a green fluorescent protein (GFP) gene under a cytomegalovirus promoter was injected through the corneal pocket into the corneal stroma of the adult Brown Norway rat, and various intensities of electric pulses ranging from 10 to 30V were delivered to the corneal epithelial side with an electric probe. Direct stereomicroscopy of the fluorescent using real-time imaging was used to determine in vivo gene expression on days 1, 2, 4, 6, 8, 10, 15, and 20 after gene transfer. Transgene expression was detected in the corneal stroma as early as day 1 and until day 15. The most intense expression was noted on days 4 and 6. Gene transfer was most effective using eight electric pulses of 20V for 50msec. Histologic study disclosed GFP expression in keratocytes within the targeted area. There was no apparent cell damage in the gene transferred cells. No apparent inflammation was found in the anterior chamber or trabecular cells when electric pulses less than 30V were used. In summary, the present technique transferred the gene of interest to a highly selected area of corneal stroma with no apparent damage. This method will likely be useful not only for developing gene therapy for corneal diseases but also for corneal research in general.

Cellular and molecular properties associated with osteosarcoma cells.

Osteosarcoma cells are recognized by abnormal function that causes a primary bone tumor. Osteosarcoma cells U(2)OS and SAOS-2 were analyzed for the expression of cell surface markers. High expression was quantified for hyaloronidase receptor (CD-44) > moderate for integrins (CD-51 and -61), > and lower for selectins (CD-62). High mitotic capacity were demonstrated by gene expression (measured by RT-PCR) and the protein level (measured by FACS) for cFOS, cMYC, and cJUN. The basic definition of osteosarcoma is excessive production of pathological osteoid. Expression of mRNA for matrix genes osteocalcin, osteonectin, and biglycan was studied. Osteocalcin and osteonectin were detected in RNA from primary cultured marrow stromal, trabecular bone cells, and osteosarcoma cell lines (U(2)OS, SAOS-2). mRNA for biglycan was detected only in primary cells and MG-63 cell line and was undetectable in RNA from U(2)OS, SAOS-2 osteosarcoma cell lines and by RNA extracted from bone biopsies of osteosarcoma patients. The absence of biglycan message observed in osteosarcoma samples provides evidence for the alterations in the extra cellular matrix which result with non-mineralized osteoid produced by the osteosarcoma cells.

Dominant localization of prostaglandin D receptors on arachnoid trabecular cells in mouse basal forebrain and their involvement in the regulation of non-rapid eye movement sleep.

Infusion of prostaglandin (PG) D(2) into the lateral ventricle of the brain induced an increase in the amount of non-rapid eye movement sleep in wild-type (WT) mice but not in mice deficient in the PGD receptor (DP). Immunofluorescence staining of WT mouse brain revealed that DP immunoreactivity was dominantly localized in the leptomeninges (LM) of the basal forebrain but that PGD synthase immunoreactivity was widely distributed in the LM of the entire brain. Electron microscopic observation indicated that DP-immunoreactive particles were predominantly located on the plasma membranes of arachnoid trabecular cells of the LM. The region with the highest DP immunoreactivity was clearly defined as bilateral wings in the LM of the basal forebrain located lateral to the optic chiasm in the proximity of the ventrolateral preoptic area, one of the putative sleep centers, and the tuberomammillary nucleus, one of the putative wake centers. The LM of this region contained DP mRNA 70-fold higher than that in the cortex as judged from the results of quantitative reverse transcription-PCR. PGD(2) infusion into the subarachnoid space of this region increased the extracellular adenosine level more than 2-fold in WT mice but not in the DP-deficient mice. These results indicate that DPs in the arachnoid trabecular cells of the basal forebrain mediate an increase in the extracellular adenosine level and sleep induction by PGD(2).