Is the expression of the components of the carotid matrix of rats influenced by estrogen, progestin and tibolone? / A expressão dos componentes da matriz carotídea de ratos é influenciada pelo estrogênio, progestagênio e tibolona?

Abstract Objective To analyze the effects of estrogen alone or in combination with progestogens and tibolone (TIB) on the expression of the extracellular matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), of perlecan, and of heparanase (HPSE) of the vascular walls of the carotid arteries. Methods A total of 30 250-day-old ovariectomized Wistar rats were orally treated for 5 weeks with: a) 1 mg/kg of estradiol benzoate (EB); b) EB + 0.2 mg/kg of medroxyprogesterone acetate (MPA); c) EB + 0.2mg/kg of norethisterone acetate (NETA); d) EB + 2 mg/kg of dydrogesterone (DI); e) 1 mg/kg of TIB; f) placebo (CTR). Following treatment, the expression of mRNA for MMP-2, MMP-9, and HPSE was analyzed by realtime polymerase chain-reaction (PCR), and the expression of MMP-2, of MMP-9, of tissue inhibitor of metalloproteinase 2 (TIMP-2), and of perlecan was quantified by immunohistochemistry in the carotid arteries. Results The groups showed significant differences on mRNA HPSE expression (p = 0.048), which was higher in the EB, EB + MPA, and TIB groups. There was no statistically significant difference in mRNA MMP-2 or MMP-9 expression. The immunohistochemical expression of MMP-2, of TIMP-2, of MMP-9, of HPSE, and of perlecan showed no differences between groups. Conclusion Estradiol alone or associated with MPA and TIB treatment can increase mRNA HSPE expression of the walls of the carotid arteries in ovariectomized rats.

Resumo Objetivo Analisar os efeitos do estrogênio isolado ou em combinação com progestogênios e tibolona (TIB) na expressão das metaloproteinases 2 e 9 da matriz extracelular (MMP-2 e MMP-9), da perlecan e da heparanase (HPSE) das paredes vasculares das artérias carótidas. Métodos Trinta ratas Wistar ovariectomizadas com 250 dias de idade foram tratadas oralmente por 5 semanas com: a) 1 mg/kg de benzoato de estradiol (EB); b) EB + 0,2 mg/kg de acetato de medroxiprogesterona (MPA); c) EB + 0,2mg/kg de acetato de noretisterona (NETA); d) EB + 2 mg/kg de didrogesterona (DI); e) 1 mg/kg de TIB; f) placebo (CTR). Após o tratamento, a expressão de mRNA para MMP-2, MMP- 9, e HPSE foi analisada por reação em cadeia da polimerase (RCP) em tempo real, e a expressão de MMP-2, MMP-9, inibidor tecidual de metaloproteinase 2 (TIMP-2), e de perlecan foi quantificado por imunohistoquímica em artérias carótidas. Resultados Os grupos apresentaram diferenças significativas na expressão do mRNA HPSE (p = 0,048), sendo maiores nos grupos EB, EB + MPA e TIB. Não houve diferença estatisticamente significativa nas expressões de mRNA MMP-2 ou MMP-9. A expressão imunohistoquímica de MMP-2, TIMP-2, MMP-9, HPSE e perlecan não mostrou diferenças entre os grupos. Conclusão O estradiol isolado ou associado ao tratamento com MPA e TIB pode aumentar a expressão de mRNA HSPE nas paredes das artérias carótidas em ratas ovariectomizadas.

Sulfatase 1 mediates the inhibitory effect of angiotensin II type 2 receptor inhibitor on angiotensin II-induced hypertensive mediator expression and proliferation in vascular smooth muscle cells from spontaneously hypertensive rats / 영남의대학술지

BACKGROUND: Extracellular sulfatases (Sulfs), sulfatase 1 (Sulf1) and sulfatase 2 (Sulf2), play a pivotal role in cell signaling by remodeling the 6-O-sulfation of heparan sulfate proteoglycans on the cell surface. The present study examined the effects of Sulfs on angiotensin II (Ang II)-induced hypertensive mediator expression and vascular smooth muscle cells (VSMCs) proliferation in spontaneously hypertensive rats (SHR).METHODS: Ang II receptors, 12-lipoxygenase (12-LO), and endothelin-1 (ET-1) messenger RNA (mRNA) expressions in SHR VSMCs were analyzed by real-time polymerase chain reaction and Western blotting. VSMCs proliferation was determined by [³ H]-thymidine incorporation.RESULTS: Basal Sulfs mRNAs expression and enzyme activity were elevated in SHR VSMCs. However, Sulfs had no effect on the basal or Ang II-induced 12-LO and ET-1 mRNA expression in SHR VSMCs. The inhibition of Ang II-induced 12-LO and ET-1 expression by blockade of the Ang II type 2 receptor (AT₂ R) pathway was not observed in Sulf1 siRNA-transfected SHR VSMCs. However, Sulf2 did not affect the action of AT₂ R inhibitor on Ang II-induced 12-LO and ET-1 expression in SHR VSMCs. The down-regulation of Sulf1 induced a reduction of AT₂ R mRNA expression in SHR VSMCs. In addition, the inhibition of Ang II-induced VSMCs proliferation by blockade of the AT₂ R pathway was mediated by Sulf1 in SHR VSMCs.CONCLUSION: These findings suggest that extracellular sulfatase Sulf1 plays a modulatory role in the AT₂ R pathway that leads to an Ang II-induced hypertensive effects in SHR VSMCs.

Sulfatase 1 mediates the inhibitory effect of angiotensin II type 2 receptor inhibitor on angiotensin II-induced hypertensive mediator expression and proliferation in vascular smooth muscle cells from spontaneously hypertensive rats / 영남의대학술지

BACKGROUND: Extracellular sulfatases (Sulfs), sulfatase 1 (Sulf1) and sulfatase 2 (Sulf2), play a pivotal role in cell signaling by remodeling the 6-O-sulfation of heparan sulfate proteoglycans on the cell surface. The present study examined the effects of Sulfs on angiotensin II (Ang II)-induced hypertensive mediator expression and vascular smooth muscle cells (VSMCs) proliferation in spontaneously hypertensive rats (SHR). METHODS: Ang II receptors, 12-lipoxygenase (12-LO), and endothelin-1 (ET-1) messenger RNA (mRNA) expressions in SHR VSMCs were analyzed by real-time polymerase chain reaction and Western blotting. VSMCs proliferation was determined by [³ H]-thymidine incorporation. RESULTS: Basal Sulfs mRNAs expression and enzyme activity were elevated in SHR VSMCs. However, Sulfs had no effect on the basal or Ang II-induced 12-LO and ET-1 mRNA expression in SHR VSMCs. The inhibition of Ang II-induced 12-LO and ET-1 expression by blockade of the Ang II type 2 receptor (AT₂ R) pathway was not observed in Sulf1 siRNA-transfected SHR VSMCs. However, Sulf2 did not affect the action of AT₂ R inhibitor on Ang II-induced 12-LO and ET-1 expression in SHR VSMCs. The down-regulation of Sulf1 induced a reduction of AT₂ R mRNA expression in SHR VSMCs. In addition, the inhibition of Ang II-induced VSMCs proliferation by blockade of the AT₂ R pathway was mediated by Sulf1 in SHR VSMCs. CONCLUSION: These findings suggest that extracellular sulfatase Sulf1 plays a modulatory role in the AT₂ R pathway that leads to an Ang II-induced hypertensive effects in SHR VSMCs.

Adipose stem cell-derived nanovesicles inhibit emphysema primarily via an FGF2-dependent pathway

Cell therapy using stem cells has produced therapeutic benefits in animal models of COPD. Secretory mediators are proposed as one mechanism for stem cell effects because very few stem cells engraft after injection into recipient animals. Recently, nanovesicles that overcome the disadvantages of natural exosomes have been generated artificially from cells. We generated artificial nanovesicles from adipose-derived stem cells (ASCs) using sequential penetration through polycarbonate membranes. ASC-derived artificial nanovesicles displayed a 100 nm-sized spherical shape similar to ASC-derived natural exosomes and expressed both exosomal and stem cell markers. The proliferation rate of lung epithelial cells was increased in cells treated with ASC-derived artificial nanovesicles compared with cells treated with ASC-derived natural exosomes. The lower dose of ASC-derived artificial nanovesicles had similar regenerative capacity compared with a higher dose of ASCs and ASC-derived natural exosomes. In addition, FGF2 levels in the lungs of mice treated with ASC-derived artificial nanovesicles were increased. The uptake of ASC-derived artificial nanovesicles was inhibited by heparin, which is a competitive inhibitor of heparan sulfate proteoglycan that is associated with FGF2 signaling. Taken together, the data indicate that lower doses of ASC-derived artificial nanovesicles may have beneficial effects similar to higher doses of ASCs or ASC-derived natural exosomes in an animal model with emphysema, suggesting that artificial nanovesicles may have economic advantages that warrant future clinical studies.

From DCPD to NTCP: The long journey towards identifying a functional hepatitis B virus receptor

Hepatitis B virus (HBV) is the prototype of hepatotropic DNA viruses (hepadnaviruses) infecting a wide range of human and non-human hosts. Previous studies with duck hepatitis B virus (DHBV) identified duck carboxypeptidase D (dCPD) as a host specific binding partner for full-length large envelope protein, and p120 as a binding partner for several truncated versions of the large envelope protein. p120 is the P protein of duck glycine decarboxylase (dGLDC) with restricted expression in DHBV infectible tissues. Several lines of evidence suggest the importance of dCPD, and especially p120, in productive DHBV infection, although neither dCPD nor p120 cDNA could confer susceptibility to DHBV infection in any cell line. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a binding partner for the N-terminus of HBV large envelope protein. Importantly, knock down and reconstitution experiments unequivocally demonstrated that NTCP is both necessary and sufficient for in vitro infection by HBV and hepatitis delta virus (HDV), an RNA virus using HBV envelope proteins for its transmission. What remains unclear is whether NTCP is the major HBV receptor in vivo. The fact that some HBV patients are homozygous with an NTCP mutation known to abolish its receptor function suggests the existence of NTCP-independent pathways of HBV entry. Also, NTCP very likely mediates just one step of the HBV entry process, with additional co-factors for productive HBV infection still to be discovered. NTCP offers a novel therapeutic target for the control of chronic HBV infection.

Growth inhibition effection of perlecan anti-sense cDNA on human laryngeal carcinoma xnograft in nude mice / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To observe growth inhibition effect of perlecan anti-sense cDNA (pAP) on human laryngeal carcinoma xnografted in nude mice. To vertify its antitumor effect and mechanism in vivo, and it may be useful as a biomarker in carcinoma of larynx cancer.@*METHOD@#Created the model of human laryngeal carcinoma xnograft in nude mice. To observe growth of those xnografts in nude mice and draw growth curve of xnografted. The expression of perlecan mRNA and portein in xnografts were examined by RT-PCR and immunohistochemistry.@*RESULT@#Volume of xnografts in the group transfected by the plasmids of pAP were significant small as compared with other two groups made by the wild type cells and phpApr-neol cells (P < 0.05). It was showed that the expression of perlecan mRNA and protein were significantly reduced in the tumor of pAP transfected Hep-2 cells as compared with the tumors transfected by the wild type cells and phβApr-neol cells (P < 0.01).@*CONCLUSION@#These data raise the possibility that pAP many play key roles in the growth of those xnografts in nude mice.

Association of single nucleotide polymorphisms of CSPG2 and HSPG2 genes with intracranial aneurysm in ethnic Han Chinese population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To assess the association between CSPG2 and HSPG2 gene polymorphisms and intracranial aneurysm (IA) in ethnic Han Chinese population.</p><p><b>METHODS</b>A case-control study was carried out. A total of 537 IA patients and 1071 normal controls with matched age and gender were recruited. Peripheral blood samples were obtained from all subjects. Following extraction, target DNA was amplified with PCR and genotyped with a SNaPshot method. The association between 2 tag SNPs (rs251124 and rs3767137) of CSPG2 and HSPG2 genes and IA was assessed.</p><p><b>RESULTS</b>The genotype frequencies of rs251124 and rs3767137 were both in Hardy-Weinberg equilibrium. No significant difference has been found in the frequencies of rs251124 of CSPG2 between the two groups. Similarly, the frequency of rs3767137 (HSPG2) did not differ between the IA and control groups (P=0.22), albeit with an OR value of greater than 1 (OR=1.12, 95%CI=0.92-1.37). There were no significant difference in genotypic frequencies of the two SNPs between the two groups (P=0.46, 0.53).</p><p><b>CONCLUSION</b>No association has been found between polymorphisms of rs251124 and rs3767137 loci of CSPG2 and HSPG2 genes and IA in the selected population.</p>

Acute UV Irradiation Increases Heparan Sulfate Proteoglycan Levels in Human Skin

Glycosaminoglycans are important structural components in the skin and exist as various proteoglycan forms, except hyaluronic acid. Heparan sulfate (HS), one of the glycosaminoglycans, is composed of repeated disaccharide units, which are glucuronic acids linked to an N-acetyl-glucosamine or its sulfated forms. To investigate acute ultraviolet (UV)-induced changes of HS and HS proteoglycans (HSPGs), changes in levels of HS and several HSPGs in male human buttock skin were examined by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR) after 2 minimal erythema doses (MED) of UV irradiation (each n = 4-7). HS staining revealed that 2 MED of UV irradiation increased its expression, and staining for perlecan, syndecan-1, syndecan-4, CD44v3, and CD44 showed that UV irradiation increased their protein levels. However, analysis by real-time qPCR showed that UV irradiation did not change mRNA levels of CD44 and agrin, and decreased perlecan and syndecan-4 mRNA levels, while increased syndecan-1 mRNA level. As HS-synthesizing or -degrading enzymes, exostosin-1 and heparanase mRNA levels were increased, but exostosin-2 was decreased by UV irradiation. UV-induced matrix metalloproteinase-1 expression was confirmed for proper experimental conditions. Acute UV irradiation increases HS and HSPG levels in human skin, but their increase may not be mediated through their transcriptional regulation.

SHh activity and localization is regulated by perlecan

Proliferation and cell fate determination in the developing embryo are extrinsically regulated by multiple interactions among diverse secreted factors, such as Sonic Hedgehog (SHh), which act in a concentration-dependent manner. The fact that SHh is secreted as a lipid-modified protein suggests the existence of a mechanism to regulate its movement across embryonic fields. We have previously shown that heparan sulfate proteoglycans (HSPGs) are required for SHh binding and signalling. However, it was not determined which specific HSPG was responsible for these functions. Here we evaluated the contribution of perlecan on SHh localization and activity. To understand the mechanism of action of perlecan at the cellular level, we studied the role of perlecan-SHh interaction in SHh activity using both cell culture and biochemical assays. Our findings show that perlecan is a crucial anchor and modulator of SHh activity acting as an extracellular positive regulator of SHh.

Quantitative evaluation of experimental choroidal neovascularization by confocal scanning laser ophthalmoscopy: fluorescein angiogram parallels heparan sulfate proteoglycan expression

The objective of the present study was to develop a quantitative method to evaluate laser-induced choroidal neovascularization (CNV) in a rat model using Heidelberg Retina Angiograph 2 (HRA2) imaging. The expression of two heparan sulfate proteoglycans (HSPG) related to inflammation and angiogenesis was also investigated. CNV lesions were induced with argon laser in 21 heterozygous Zucker rats and after three weeks a fluorescein angiogram and autofluorescence exams were performed using HRA2. The area and greatest linear dimension were measured by two observers not aware of the protocol. Bland-Altman plots showed agreement between the observers, suggesting that the technique was reproducible. After fluorescein angiogram, HSPG (perlecan and syndecan-4) were analyzed by real-time RT-PCR and immunohistochemistry. There was a significant increase in the expression of perlecan and syndecan-4 (P < 0.0001) in retinas bearing CNV lesions compared to control retinas. The expression of these two HSPG increased with increasing CNV area. Immunohistochemistry demonstrated that the rat retina damaged with laser shots presented increased expression of perlecan and syndecan-4. Moreover, we observed that the overexpression occurred in the outer layer of the retina, which is related to choroidal damage. It was possible to develop a standardized quantitative method to evaluate CNV in a rat model using HRA2. In addition, we presented data indicating that the expression of HSPG parallels the area of CNV lesion. The understanding of these events offers opportunities for studies of new therapeutic interventions targeting these HSPG.

Soluble syndecan-1 at diagnosis and during follow up of multiple myeloma: a single institution study / 대한혈액학회지

BACKGROUND: Syndecan-1 is a heparan sulfate proteoglycan expressed on plasma cells, especially myeloma cells, and can exist in serum as soluble syndecan-1 after shedding from the cell surface. Soluble syndecan-1 has been suggested to promote myeloma cell growth and to be an independent prognostic factor for multiple myeloma. We aimed to evaluate the effect of soluble syndecan-1 levels at the time of diagnosis and during therapy on therapeutic response and prognosis for patients with multiple myeloma. METHODS: We analyzed soluble syndecan-1 levels in 28 patients with multiple myeloma and 50 normal controls, and compared its levels with Durie-Salmon stage and other markers of myeloma. In addition, we evaluated the therapeutic response and determined the 3-year survival rates of these patients. RESULTS: We observed that the median soluble syndecan-1 level in myeloma patients was higher than that in the normal controls (P <0.0001), and the soluble syndecan-1 levels in 21 (75%) patients were higher than the cut-off level (162 ng/mL). Soluble syndecan-1 levels correlated with disease stage, percentage of plasma cells in the bone marrow, beta2 microglobulin level, serum M-component concentration, and creatinine level. The baseline levels of soluble syndecan-1 at the time of diagnosis in the patients who responded to chemotherapy were lower than those in the non-responders (P=0.04); however, the baseline level was not a significant predictor of therapeutic response. The 3-year overall survival rate of the patients with high soluble syndecan-1 levels at the time of diagnosis and 6 months after chemotherapy was lower than the corresponding survival rates of the patients with low levels of soluble syndecan-1; however, the overall survival rate was not statistically significant. CONCLUSION: The use of soluble syndecan-1 has limitations in the diagnosis of multiple myeloma. Soluble syndecan-1 levels correlate with known prognostic factors; however, we could not assess the prognostic value of high levels of soluble syndecan-1 at the time of diagnosis and after chemotherapy.

Heparan sulfate proteoglycans: structure, protein interactions and cell signaling

Heparan sulfate proteoglycans are ubiquitously found at the cell surface and extracellular matrix in all the animal species. This review will focus on the structural characteristics of the heparan sulfate proteoglycans related to protein interactions leading to cell signaling. The heparan sulfate chains due to their vast structural diversity are able to bind and interact with a wide variety of proteins, such as growth factors, chemokines, morphogens, extracellular matrix components, enzymes, among others. There is a specificity directing the interactions of heparan sulfates and target proteins, regarding both the fine structure of the polysaccharide chain as well precise protein motifs. Heparan sulfates play a role in cellular signaling either as receptor or co-receptor for different ligands, and the activation of downstream pathways is related to phosphorylation of different cytosolic proteins either directly or involving cytoskeleton interactions leading to gene regulation. The role of the heparan sulfate proteoglycans in cellular signaling and endocytic uptake pathways is also discussed.

Proteoglicanos de heparam sulfato são encontrados tanto superfície celular quanto na matriz extracelular em todas as espécies animais. Esta revisão tem enfoque nas características estruturais dos proteoglicanos de heparam sulfato e nas interações destes proteoglicanos com proteínas que levam à sinalização celular. As cadeias de heparam sulfato, devido a sua variedade estrutural, são capazes de se ligar e interagir com ampla gama de proteínas, como fatores de crescimento, quimiocinas, morfógenos, componentes da matriz extracelular, enzimas, entreoutros. Existe uma especificidade estrutural que direciona as interações dos heparam sulfatos e proteínas alvo. Esta especificidade está relacionada com a estrutura da cadeia do polissacarídeo e os motivos conservados da cadeia polipeptídica das proteínas envolvidas nesta interação. Os heparam sulfatos possuem papel na sinalização celular como receptores ou coreceptores para diferentes ligantes. Esta ligação dispara vias de sinalização celular levam à fosforilação de diversas proteínas citosólicas ou com ou sem interações diretas com o citoesqueleto, culminando na regulação gênica. O papel dos proteoglicanos de heparam sulfato na sinalização celular e vias de captação endocítica também são discutidas nesta revisão.

Down-regulation of perlecan expression contributes to the inhibition of rat cardiac microvascular endothelial cell proliferation induced by hypoxia / 生理学报

Exposure of endothelial cells (ECs) to hypoxia leads to a decrease in EC proliferation. However, the mechanism by which hypoxia inhibits EC proliferation is unclear. Perlecan has been reported to play an important role in regulating EC proliferation. We hypothesized that perlecan was involved in the hypoxia-induced inhibition of EC proliferation. To test this hypothesis, rat cardiac microvascular ECs were cultured under normoxic or hypoxic conditions for 12 h and harvested for determination of perlecan mRNA expression using real-time reverse transcription-polymerase chain reaction (RT-PCR). The results showed that exposure of ECs to hypoxia for 12 h induced a decrease in perlecan mRNA expression (61.72%, P<0.05). Concomitantly, the down-regulation of endogenous perlecan induced by hypoxia or the neutralization of endogenous perlecan with anti-perlecan antibody significantly inhibited EC proliferation and responsiveness to basic fibroblast growth factor (bFGF), and decreased focal adhesion kinase (FAK) expression and extracellular signal-regulated kinase 1/2 (ERK1/2) activation. These data indicate that down-regulation of perlecan expression contributes to hypoxia-induced inhibition of rat cardiac microvascular EC proliferation by suppressing FAK-mediated and ERK1/2-dependent growth signals.

Effects of sulfated polysaccharides on tumour biology / Efectos de los polisacáridos sulfatados en la biología tumoral

Sulfated polysaccharides can act not only as anticoagulants but also as tumour inhibitors. Recent studies suggest that sulfated polysaccharides could affect tumour cells directly. Sulfated polysaccharides could inhibit the metastasis and proliferation of tumour cells by binding to growth factors and cell adhesion molecules. Moreover, sulfated polysaccharides could inhibit heparanase, which cleaves heparan sulfate chains of heparan sulfate proteoglycans and cause release of growth factors sequestered by heparan sulfate chains. Some sulfated polysaccharides can induce apoptosis and differentiation of tumour cells, but the mechanism is uncertain. In addition, sulfated polysaccharides can enhance the innate and adaptive immune response for tumour cells. Thus, the anti-tumour mechanism of sulfated polysaccharides can be explained, at least partly, through the effects on tumour biology directly.

Los polisacáridos sulfatados podrían actuar no solamente como anticoagulantes, sino también como inhibidores del tumor. Estudios recientes sugieren que los polisacáridos sulfatados podrían afectar directamente las células tumorales. Los polisacáridos tumorales podrían inhibir la metástasis y la proliferación de las células tumorales por medio de la unión con los factores de crecimiento y las moléculas de adhesión celular. Además, los polisacáridos sulfatados podrían inhibir la heparanasa, que rompe las cadenas de heparán-sulfato del proteoglicano de heparán-sulfato, dando lugar a la liberación de los factores de crecimiento secuestrados por las cadenas de heparán-sulfato. Algunos polisacáridos sulfatados podrían inducir la apoptosis y diferenciación de las células tumorales, pero el mecanismo es incierto. Además, los polisacáridos sulfatados podrían mejorar la respuesta inmunológica innata y adaptativa frente a las células tumorales. De este modo, el mecanismo antitumoral de los polisacáridos sulfatados pudiera explicarse – al menos parcialmente – a partir de los efectos sobre la biología tumoral directamente.

Specific structural features of syndecans and heparan sulfate chains are needed for cell signaling

The syndecans, heparan sulfate proteoglycans, are abundant molecules associated with the cell surface and extracellular matrix and consist of a protein core to which heparan sulfate chains are covalently attached. Each of the syndecan core proteins has a short cytoplasmic domain that binds cytosolic regulatory factors. The syndecans also contain highly conserved transmembrane domains and extracellular domains for which important activities are becoming known. These protein domains locate the syndecan on cell surface sites during development and tumor formation where they interact with other receptors to regulate signaling and cytoskeletal organization. The functions of cell surface heparan sulfate proteoglycan have been centered on the role of heparan sulfate chains, located on the outer side of the cell surface, in the binding of a wide array of ligands, including extracellular matrix proteins and soluble growth factors. More recently, the core proteins of the syndecan family transmembrane proteoglycans have also been shown to be involved in cell signaling through interaction with integrins and tyrosine kinase receptors.

The functional links between prion protein and copper

Prion diseases are fatal neurodegenerative disorders associated with the conversion of the cellular prion protein (PrPC) into a pathologic isoform. Although the physiological function of PrPC remains unknown, evidence relates PrPC to copper metabolism and oxidative stress as suggested by its copper-binding properties in the N-terminal octapeptide repeat region. This region also reduces copper ions in vitro, and this reduction ability is associated with the neuroprotection exerted by the octarepeat region against copper in vivo. In addition, the promoter region of the PrPC gene contains putative metal response elements suggesting it may be regulated by heavy metals. Here we address some of the evidence that support a physiological link between PrPC and copper. Also, in vivo experiments suggesting the physiological relevance of PrPC interaction with heparan sulfate proteoglycans are discussed.

Regulation of cell adhesion by heparan sulfate proteoglycan in adenomatoid odontogenic tumour

During recent years, attention was drawn to the role of cell adhesion in tumour development and progression. Cell-cell and cell-extracellular matrix interaction is crucial with regard to tumorous transformation and tumour spreading. There are numerous data indicating that the expression of syndecan-1 an important transmembrane proteoglycan undergoes changes during the development of several tumours. CD 138 antibody reacts with the core protein of syndecan-1, cell surface integral membrane heparin sulfate and chondroitin sulfate containing proteoglycan that binds to interstitial extracellular matrix molecules, thereby regulating cell adhesion. The predominant location of syndecan-1 is on epithelial cells where its expression correlates with normal epithelial organization. Previous studies have demonstrated that decreased expression of CD 138 is linked to malignant progression. Hitherto, few studies on the expression of CD138 in odontogenic tumours have been published and no studies have been found regarding the expression of this marker in adenomatoid odontogenic tumour [AOT]. Therefore, this study was carried out to highlight the recent concepts of cell adhesion in tumour development and progression in AOT using CD 138 monoclonal antibody. In respect to this, formalin-fixed, paraffinembedded tissue sections of 7 cases of AOT were selected in an attempt to clarify the peculiar histopathological features of this lesion. All studied cases showed positive reaction to the marker used, however differences were observed among the studied cases regarding areas of immunoreactivity and optical density of the positive areas. Overexpressions of CD138 were observed as cytoplasmic immunoreactivity especially in the spindle cells bordering the sheets and masses of the polyhedral cells and in the scanty stromal cells. The duct like structures and masses of polyhedral cells showed negative reactions. The results of the present study explain why AOT is a benign non-aggressive lesion

Effects of High Glucose and Advanced Glycosylation Endproducts(AGE) on the in vitro Permeability Model

PURPOSE: We describe the changes of rat glomerular epithelial cells when exposed to high levels of glucose and advanced glycosylation endproducts(AGE) in the in vitro diabetic condition. We expect morphological alteration of glomerular epithelial cells and permeability changes experimentally and we may correlate the results with a mechanism of proteinuria in DM. METHODS: We made 0.2 M glucose-6-phsphate solution mixed with PBS(pH 7.4) containing 50 mg/mL BSA and protease inhibitor for preparation of AGE. As control, we used BSA. We manufactured and symbolized five culture dishes as follows; B5 - normal glucose(5 mM) + BSA, B30 - high glucose(30 mM) + BSA, A5 - normal glucose(5 mM) + AGE, A30 - high glucose(30 mM) + AGE, A/B 25 - normal glucose(5 mM) + 25 mM of mannitol(osmotic control). After the incubation period of both two days and seven days, we measured the amount of heparan sulfate proteoglycan(HSPG) in each dish by ELISA and compared them with the B5 dish at 2nd and 7th incubation days. We observed the morphological changes of epithelial cells in each culture dish using scanning electron microscopy(SEM). We tried the permeability assay of glomerular epithelial cells using cellulose semi-permeable membrane measuring the amount of filtered BSA through the apical chamber for 2 hours by sandwich ELISA. RESULTS: On the 2nd incubation day, there was no significant difference in the amount of HSPG between the 5 culture dishes. But on the 7th incubation day, the amount of HSPG increased by 10% compared with the B5 dish on the 2nd day except the A30 dish(P0.05). In the osmotic control group (A/B 25) no significant correlation was observed. On the SEM, we could see the separated intercellular junction and fused microvilli of glomerular epithelial cells in the culture dishes where AGE was added. The permeability of BSA increased by 19% only in the A30 dish on the 7th day compared with B5 dish on the 7th day in the permeability assay(P<0.05). CONCLUSION: We observed not only the role of a high level of glucose and AGE in decreasing the production of HSPG of glomerular epithelial cells in vitro, but also their additive effect. However, the role of AGE is greater than that of glucose. These results seems to correlate with the defects in charge selective barrier. Morphological changes of the disruption of intercellular junction and fused microvilli of glomerular epithelial cells seem to correlate with the defects in size-selective barrier. Therefore, we can explain the increased permeability of glomerular epithelial units in the in vitro diabetic condition.

Effect of Tumor Necrosis Factor-Alpha on Glomerular Epithelial Cells in Glomerular Permeability

PURPOSE: Minimal Change Disease (MCD) is the most common primary nephrotic syndrome in children. Some suggested that tumor necrosis factor-alpha (TNF-alpha) are involved in the pathogenesis of MCD. METHODS: This study was done to see the changes of plasma and urinary TNF-alpha, and its effect on the determination of permeability of the glomerular basement membrane (BM) contributed by heparan sulfate proteoglycan (HSPG). Study patients consisted of 19 biopsy-proven MCD children aged 2-15 years old. Both plasma and urinary TNF-alpha were measured. Employing the Millicell system, TNF-alpha was screened for the permeability factors. We examined whether TNF-alpha regulated BM HSPG gene expression and HS synthesis in the glomerular epithelial cells (GECs). RESULTS: Urinary TNF-alpha during relapse was significantly increased when compared with that of during remission or controls (364.4+/-51.2 vs 155.3+/-20.8, 36.0+/-4.5 ng/mg cr) (P< 0.05). However, negative results were obtained in the permeability assay using the Millicell system. No difference was seen in the BM HSPG gene expression and HS synthesis in the GECs. CONCLUSION: It seems that TNF-alpha may not play a disease-specific role in the pathogenesis of MCD.

Regulation of Glomerular Endothelial Cell Proteoglycans by Glucose

The presence of heparan sulfate proteoglycan (HSPG) in anionic sites in the lamina rara interna of glomerular basement membrane suggests that the proteoglycan may be deposited by the glomerular endothelial cells (GEndo). We have previously demonstrated that bovine GEndo in vitro synthesize perlecan, a species of glomerular basement membrane HSPG. In this study we examined whether high glucose medium regulates the GEndo metabolism of glycopeptides including perlecan. Metabolic labeling of glycoconjugates with 35S-SO4, sequential ion exchange and Sepharose CL-4B chromatography of labeled glycoconjugates, and northern analysis were performed. Incubation of GEndo for 8 to 14 weeks (but not for 1-2 weeks) in medium containing 30 mM glucose resulted in nearly 50% reduction in the synthesis of cell layer and medium 35SO4-labeled low anionic glycoproteins and proteoglycans, including that of basement membrane HSPG (Kav 0.42) compared to GEndo grown in 5 mM glucose medium; no changes in anionic charge density or hydrodynamic size of proteoglycans were noted. Northern analysis demonstrated that the mRNA abundance of perlecan was reduced by 47% in cells incubated with 30 mM glucose. Our data suggest that high glucose medium reduces the GEndo synthesis of perlecan by regulating its gene expression. Reduced synthesis of perlecan by GEndo may contribute to proteinuria seen in diabetic nephropathy.