Biglycan expression and its function in human ligamentum flavum.

Hypertrophy of the ligamentum flavum (LF) is a major cause of lumbar spinal stenosis (LSS), and the pathology involves disruption of elastic fibers, fibrosis with increased cellularity and collagens, and/or calcification. Previous studies have implicated the increased expression of the proteoglycan family in hypertrophied LF. Furthermore, the gene expression profile in a rabbit experimental model of LF hypertrophy revealed that biglycan (BGN) is upregulated in hypertrophied LF by mechanical stress. However, the expression and function of BGN in human LF has not been well elucidated. To investigate the involvement of BGN in the pathomechanism of human ligamentum hypertrophy, first we confirmed increased expression of BGN by immunohistochemistry in the extracellular matrix of hypertrophied LF of LSS patients compared to LF without hypertrophy. Experiments using primary cell cultures revealed that BGN promoted cell proliferation. Furthermore, BGN induces changes in cell morphology and promotes myofibroblastic differentiation and cell migration. These effects are observed for both cells from hypertrophied and non-hypertrophied LF. The present study revealed hyper-expression of BGN in hypertrophied LF and function of increased proteoglycan in LF cells. BGN may play a crucial role in the pathophysiology of LF hypertrophy through cell proliferation, myofibroblastic differentiation, and cell migration.

Expression of proteoglycans in two types of ameloblastoma: novel Immunohistochemical findings.

Alterations in cellular and extracellular matrix components play an important role during tumorigenesis; proteoglycans are included among these components. Ameloblastomas are odontogenic tumors distinguished as invasive and infiltrative neoplasms and are divided into different histological types, the most common of which are the unicystic ameloblastoma and the conventional ameloblastoma. The aim of this study was to identify the presence of two proteoglycans, perlecan and biglycan, in different types of ameloblastoma. Using immunohistochemistry, we determined the presence of both proteins in 28 unicystic ameloblastomas and 23 conventional ameloblastomas. We identified the cytoplasmic and nuclear presence of perlecan and the cytoplasmic presence of biglycan in both types of ameloblastoma. The mean values of immunoexpression were higher in the conventional type compared to the unicystic type. Neither the presence of biglycan in ameloblastomas nor the nuclear presence of perlecan in any odontogenic tumor has previously been reported. The differential immunoexpression of perlecan and biglycan in these types of ameloblastomas suggests their participation in the developmental process of these tumors.

Flavopiridol Inhibits TGF-ß-Stimulated Biglycan Synthesis by Blocking Linker Region Phosphorylation and Nuclear Translocation of Smad2.

Transforming growth factor-ß (TGF-ß) is a pleiotropic growth factor implicated in the development of atherosclerosis for its role in mediating glycosaminoglycan (GAG) chain hyperelongation on the proteoglycan biglycan, a phenomenon that increases the binding of atherogenic lipoproteins in the vessel wall. Phosphorylation of the transcription factor Smad has emerged as a critical step in the signaling pathways that control the synthesis of biglycan, both the core protein and the GAG chains. We have used flavopiridol, a well-known cyclin-dependent kinase inhibitor, to study the role of linker region phosphorylation in the TGF-ß-stimulated synthesis of biglycan. We used radiosulfate incorporation and SDS-PAGE to assess proteoglycan synthesis, real-time polymerase chain reaction to assess gene expression, and chromatin immunoprecipitation to assess the binding of Smads to the promoter region of GAG Synthesizing genes. Flavopiridol blocked TGF-ß-stimulated synthesis of mRNA for the GAG synthesizing enzymes, and chondroitin 4-sulfotransferase (C4ST-1), chondroitin sulfate synthase-1 (ChSy-1) and TGF-ß-mediated proteoglycans synthesis as well as GAG hyperelongation. Flavopiridol blocked TGF-ß-stimulated Smad2 phosphorylation at both the serine triplet and the isolated threonine residue in the linker region. The binding of Smad to the promoter region of the C4ST-1 and ChSy-1 genes was stimulated by TGF-ß, and this response was blocked by flavopiridol, demonstrating that linker region phosphorylated Smad can pass to the nucleus and positively regulate transcription. These results demonstrate the validity of the kinases, which phosphorylate the Smad linker region as potential therapeutic target(s) for the development of an agent to prevent atherosclerosis.

VEGF synthesis is induced by prostacyclin and TGF-ß in distal lung fibroblasts from COPD patients and control subjects: Implications for pulmonary vascular remodelling.

BACKGROUND AND OBJECTIVE: Involvement of pulmonary vascular remodelling is a characteristic sign in COPD. Vascular mediators such as vascular endothelial growth factor (VEGF) and prostacyclin may regulate fibroblast activity. The objective was to study the synthesis of VEGF and interactions with prostacyclin and transforming growth factor (TGF)-ß1 in lung fibroblasts from patients with COPD and healthy control subjects. To further explore the autocrine role of synthesized VEGF on fibroblast activity, studies were performed in human lung fibroblasts (HFL-1). METHODS: Primary distal lung fibroblast cultures were established from healthy individuals and from COPD patients (GOLD stage IV). Lung fibroblasts were stimulated with the prostacyclin analogue iloprost and the profibrotic stimuli TGF-ß1 . VEGF synthesis was measured in the cell culture medium. Changes in proliferation rate, migration and synthesis of the extracellular matrix (ECM) proteins proteoglycans were analysed after stimulations with VEGF-A isoform 165 (VEGF165 ; 1-10 000 pg/mL) in HFL-1. RESULTS: Iloprost and TGF-ß1 significantly increased VEGF synthesis in both fibroblasts from COPD patients and control subjects. TGF-ß1 -induced VEGF synthesis was significantly reduced by the cyclooxygenase inhibitor indomethacin in fibroblasts from COPD patients. VEGF significantly increased proliferation rate and migration capacity in HFL-1. VEGF also significantly increased synthesis of the ECM proteins biglycan and perlecan. The VEGF receptors (VEGFR), VEGFR1, VEGFR2 and VEGFR3, were all expressed in primary lung fibroblasts and HFL-1. CONCLUSION: VEGF is synthesized in high amounts by distal lung fibroblasts and may have a crucial role in ongoing vascular remodelling processes in the distal lung compartments.

Altered expression of small leucine-rich proteoglycans (Decorin, Biglycan and Lumican): Plausible diagnostic marker in urothelial carcinoma of bladder.

Small leucine-rich proteoglycans are components of extracellular matrix that regulates neoplastic transformation. Among small leucine rich proteoglycans, Decorin, Biglycan and Lumican are most commonly implicated markers, and their expression is well studied in various malignancies. In this novel study, we have collectively evaluated expression of these three molecules in urothelial carcinoma of bladder. Thirty patients of confirmed untreated bladder cancer, 30 healthy controls for blood and 30 controls for adjacent non-tumour tissue were enrolled. Blood was collected from all subjects and tumour/adjacent normal tissue was obtained from the patients. Circulatory levels were estimated by enzyme-linked immunosorbent assay, relative messenger RNA expression by quantitative polymerase chain reaction and protein expression by immunohistochemistry and western-blotting. Circulatory levels of Biglycan (p = 0.0038) and Lumican (p < 0.0001) were significantly elevated, and that of Decorin (p < 0.0001) was significantly reduced in patients as compared with controls. Protein expression by immunohistochemistry and western-blotting showed elevated expression of Lumican and Biglycan and lower expression of Decorin in urothelial carcinoma of bladder. Quantitative polymerase chain reaction for messenger RNA expression from tissue specimens revealed significantly higher expression of Biglycan (p = 0.0008) and Lumican (p = 0.01) and lower expression of Decorin (p < 0.0001) in urothelial carcinoma of bladder. Out of all molecules receiver operating characteristic curve showed that the 0.207 ng/ml cut-off of serum Lumican provided optimum sensitivity (90.0%) and specificity (90.0%). Significant alteration of matrix small leucine-rich proteoglycans in urothelial carcinoma of bladder was observed. Higher expression of Lumican in Bladder cancer patients with the cut-off value of highest optimum sensitivity and specificity shows its importance as a potential non-invasive marker for early detection of UBC following further validation in large patient cohort.

UV irradiation-induced production of monoglycosylated biglycan through downregulation of xylosyltransferase 1 in cultured human dermal fibroblasts.

BACKGROUND: Biglycan (BGN) is a proteoglycan composed of a 42-kDa core protein and two glycosaminoglycan (GAG) chains, and known to be involved in structural, space-filling functions and many physiological regulations in the skin. OBJECTIVE: To investigate ultraviolet (UV) irradiation-induced changes of BGN protein and its GAG chain synthesis in cultured human dermal fibroblasts. METHODS: UV irradiation-induced or xylosyltransferase (XYLT) 1 siRNA-mediated smaller-sized protein bands detected by Western blot using BGN antibodies were identified as monoglycosylated forms of BGN, using BGN siRNA-mediated knockdown and chondroitinase ABC (ChABC). Differential activity of XYLT1 and 2 on BGN core protein was investigated by size shift of S42A- and S47A-BGN mutants to core protein size caused by XYLT1 siRNA transfection or UV irradiation. RESULTS: After UV irradiation, intact form of BGN protein (I-BGN) and core protein form were reduced in cultured fibroblasts, but other smaller-sized bands were observed to be increased. These smaller-sized ones were reduced by transfection of BGN siRNA, and shifted to the core protein size by treatment with ChABC, suggesting that they are defectively-glycosylated forms of BGN (D-BGN) protein. UV irradiation also decreased mRNA expression levels of XYLT1 and 2, which are responsible for initiation of GAG chain synthesis. UV-mediated reduction of XYLT1 expression was much stronger than that of XYLT2. Furthermore, siRNA-mediated down-regulation of XYLT1 resulted in the increase of D-BGN and the decrease of I-BGN, while down-regulation of XYLT2 resulted in no change of D-BGN and I-BGN, suggesting that the XYLT1 may react with both GAG-attaching serine sites of BGN; however, XYLT2 may prefer to react one of them. Another dermatan sulfate (DS) proteoglycan, decorin, showed no or a little change of its molecular weight by UV irradiation or XYLT1 siRNA transfection, suggesting that DS synthesis may not be a critical factor in formation of D-BGN. Co-transfection with XYLT1, 2 siRNAs and wild-type or mutant forms of BGN overexpression vectors revealed that S42A-BGN showed size reduction to core protein size by XYLT1 downregulation, but S47A-BGN did not, suggesting that XYLT2 can react only with S42 on BGN core protein. With UV irradiation, both S42A-BGN and S47A-BGN showed size reduction, which is probably because UV-caused downregulation of both XYLTs and overexpression condition resulted in incomplete glycosylation and secretion. CONCLUSIONS: UV irradiation-induced increase of BGN monoglycosylated forms in cultured human dermal fibroblasts is resulted from dominance of XYLT2 activity, which acts only at S42 on BGN core protein, caused by UV-mediated stronger reduction of XYLT1.

Potential use of human adipose mesenchymal stromal cells for intervertebral disc regeneration: a preliminary study on biglycan-deficient murine model of chronic disc degeneration.

INTRODUCTION: Biglycan is an important proteoglycan of the extracellular matrix of intervertebral disc (IVD), and its decrease with aging has been correlated with IVD degeneration. Biglycan deficient (Bgn-/0) mice lack this protein and undergo spontaneous IVD degeneration with aging, thus representing a valuable in vivo model for preliminary studies on therapies for human progressive IVD degeneration. The purpose of the present study was to assess the possible beneficial effects of adipose-derived stromal cells (ADSCs) implants in the Bgn-/0 mouse model. METHODS: To evaluate ADSC implant efficacy, Bgn-/0 mice were intradiscally (L1-L2) injected with 8x104 ADSCs at 16 months old, when mice exhibit severe and complete IVD degeneration, evident on both 7Tesla Magnetic Resonance Imaging (7TMRI) and histology. Placebo and ADSCs treated Bgn-/0 mice were assessed by 7TMRI analysis up to 12 weeks post-transplantation. Mice were then sacrificed and implanted discs were analyzed by histology and immunohistochemistry for the presence of human cells and for the expression of biglycan and aggrecan in the IVD area. RESULTS: After in vivo treatment, 7TMRI revealed evident increase in signal intensity within the discs of mice that received ADSCs, while placebo treatment did not show any variation. Ultrastructural analyses demonstrated that human ADSC survival occurred in the injected discs up to 12 weeks after implant. These cells acquired a positive expression for biglycan, and this proteoglycan was specifically localized in human cells. Moreover, ADSC treatment resulted in a significant increase of aggrecan tissue levels. CONCLUSION: Overall, this work demonstrates that ADSC implant into degenerated disc of Bgn-/0 mice ameliorates disc damage, promotes new expression of biglycan and increased levels of aggrecan. This suggests a potential benefit of ADSC implant in the treatment of chronic degenerative disc disease and prompts further studies in this field.

De novo expression of circulating biglycan evokes an innate inflammatory tissue response via MyD88/TRIF pathways.

Matrix-bound constituents, such as the small leucine-rich proteoglycan biglycan, can act as powerful signaling molecules when released by limited proteolysis of the extracellular matrix or de novo synthesized by macrophages in the circulation and body fluids. Specifically, biglycan acts as an endogenous ligand of innate immunity by directly engaging the Toll-like receptor (TLR)-2 and -4. In this study, we generated a transient transgenic mouse model where biglycan was de novo overproduced by hepatocytes driven by the albumin promoter. Transgenic biglycan was rapidly and abundantly synthesized by hepatocytes and released into the bloodstream. Notably, we found that circulating biglycan accumulated in the kidneys where it caused recruitment of leukocytes infiltrating the renal parenchyma concurrent with abnormal renal levels of chemoattractants CXCL1, CXCL2, CCL2 and CCL5. Using mice deficient in either TLR adapter proteins MyD88 or TRIF we discovered that MyD88 deficiency drastically reduced neutrophil and macrophage infiltration in the kidney, whereas TRIF deficiency decreased T cell infiltrates. Production of CXCL1, CXCL2 and CCL2 required MyD88, whereas the levels of T cell and macrophage attractant CCL5 required TRIF. Thus, we provide robust genetic evidence for circulating biglycan as a powerful pro-inflammatory mediator targeting the renal parenchyma. Furthermore, our results provide the first evidence that biglycan differentially triggers chemoattraction of leukocytes via two independent pathways, both under the control of TLR2/4, utilizing either MyD88 or TRIF adaptor proteins. As aberrant expression of biglycan occurs in several inflammatory diseases, this transient transgenic mouse model could serve as a valuable research tool in investigating the effects of increased biglycan expression in vivo and for the development of therapeutic strategies in the treatment of inflammatory diseases.

Differential effects of fluticasone on extracellular matrix production by airway and parenchymal fibroblasts in severe COPD.

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal repair in the lung resulting in airway obstruction associated with emphysema and peripheral airway fibrosis. Because the presence and degree of airways disease and emphysema varies between COPD patients, this may explain the heterogeneity in the response to treatment. It is currently unknown whether and to what extent inhaled steroids can affect the abnormal repair process in the airways and lung parenchyma in COPD. We investigated the effects of fluticasone on transforming growth factor (TGF)-ß- and cigarette smoke-induced changes in mothers against decapentaplegic homolog (Smad) signaling and extracellular matrix (ECM) production in airway and parenchymal lung fibroblasts from patients with severe COPD. We showed that TGF-ß-induced ECM production by pulmonary fibroblasts, but not activation of the Smad pathway, was sensitive to the effects of fluticasone. Fluticasone induced decorin production by airway fibroblasts and partly reversed the negative effects of TGF-ß treatment. Fluticasone inhibited biglycan production in both airway and parenchymal fibroblasts and procollagen 1 production only in parenchymal fibroblasts, thereby restoring the basal difference in procollagen 1 production between airway and parenchymal fibroblasts. Our findings suggest that the effects of steroids on the airway compartment may be beneficial for patients with severe COPD, i.e., restoration of decorin loss around the airways, whereas the effects of steroids on the parenchyma may be detrimental, since the tissue repair response, i.e., biglycan and procollagen production, is inhibited. More research is needed to further disentangle these differential effects of steroid treatment on the different lung compartments and its impact on tissue repair and remodeling in COPD.

Prevention of TGFß induction attenuates angII-stimulated vascular biglycan and atherosclerosis in Ldlr-/- mice.

Angiotensin II (angII) accelerates atherosclerosis, but the mechanisms are not fully understood. The aim of this study was to determine whether TGFß is required for angII-induced atherosclerosis. Ldlr-null mice fed a normal chow diet were infused with angII or saline for 28 days. A single injection of TGFß neutralizing antibody 1D11 (2 mg/kg) prevented angII-induction of TGFß1 levels, and strikingly attenuated angII-induced accumulation of aortic biglycan content. To study atherosclerosis, mice were infused with angII or saline for 4 weeks, and then fed Western diet for a further 6 weeks. 1D11 had no effect on systolic blood pressure or plasma cholesterol; however, angII-infused mice that received 1D11 had reduced atherosclerotic lesion area by 30% (P < 0.05). Immunohistochemical analyses demonstrated that angII induced both lipid retention and accumulation of biglycan and perlecan which colocalized with apoB. 1D11 strikingly reduced the effect of angII on biglycan but not perlecan. 1D11 decreased total collagen content (P < 0.05) in the lesion area without changing plaque inflammation markers (CD68 and CD45). Thus, this study demonstrates that neutralization of TGFß attenuated angII stimulation of biglycan accumulation and atherogenesis in mice, suggesting that TGFß-mediated biglycan induction is one of the mechanisms underlying angII-promoted atherosclerosis.

Identification of differentially expressed proteins in atherosclerotic aorta and effect of vitamin E.

Atherosclerosis is a chronic inflammatory disorder that occurs as a result of mononuclear lymphocyte infiltration to the arterial wall, smooth muscle cell proliferation and damage in the arterial wall caused by extracellular matrix accumulation. Besides several genetic and environmental factors, increased serum cholesterol and oxidized low density lipoproteins are considered to be major inducing factors of atherosclerosis. Several protective agents have been used to prevent the progression of atherosclerosis and recently vitamin E has been focused because of its significant role in signaling mechanisms. Since many different cell types are involved in the development of hypercholesterolemia induced atherosclerosis, it is important to investigate wide range of proteins to highlight the pathologic and diagnostic mechanisms. In this study, by using proteomic technique, we identified differentially expressed proteins following cholesterol and also vitamin E treatments. The expressions of apolipoprotein A I and apolipoprotein E involved in lipid metabolism, peroxiredoxin 1, peroxiredoxin 2 and thioredoxin involved in antioxidant system, 14-3-3 protein zeta delta and 14-3-3 protein beta alpha in cell signaling, biglycan, vimentin, tropomyosin and smooth muscle α-actin as structural and contractile proteins have been discussed. BIOLOGICAL SIGNIFICANCE: We observed several protein alterations in aorta of cholesterol fed and vitamin E treated rabbits.These differentially expressed proteins associated with key mechanisms involved in atherosclerosis and signaling mechanisms related with vitamin E. These findings for different proteins might be helpful for deciphering the pathogenesis in atherosclerosis. In addition it provides a new perspective to understand mechanisms of beneficial effect of vitamin E on the signaling pathways in atherogenesis. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

(S)-[6]-Gingerol inhibits TGF-ß-stimulated biglycan synthesis but not glycosaminoglycan hyperelongation in human vascular smooth muscle cells.

OBJECTIVES: (S)-[6]-Gingerol is under investigation for a variety of therapeutic uses. Transforming growth factor (TGF)-ß stimulates proteoglycan synthesis, leading to increased binding of low-density lipoproteins, which is the initiating step in atherosclerosis. We evaluated the effects of (S)-[6]-gingerol on these TGF-ß-mediated proteoglycan changes to explore its potential as an anti-atherosclerotic agent. METHODS: Purified (S)-[6]-gingerol was assessed for its effects on proteoglycan synthesis by [(35) S]-sulfate incorporation into glycosaminoglycan chains and [(35) S]-Met/Cys incorporation into proteoglycans and total proteins in human vascular smooth muscle cells. Biglycan level was assessed by real-time quantitative polymerase chain reactions and the effects of (S)-[6]-gingerol on TGF-ß signalling by assessment of the phosphorylation of Smads and Akt by western blotting. KEY FINDINGS: (S)-[6]-Gingerol concentration-dependently inhibited TGF-ß-stimulated proteoglycan core protein synthesis, and this was not secondary to inhibition of total protein synthesis. (S)-[6]-Gingerol inhibited biglycan mRNA expression. (S)-[6]-Gingerol did not inhibit TGF-ß-stimulated glycosaminoglycan hyperelongation or phosphorylation of Smad 2, in either the carboxy terminal or linker region, or Akt phosphorylation. CONCLUSIONS: The activity of (S)-[6]-gingerol to inhibit TGF-ß-stimulated biglycan synthesis suggests a potential role for ginger in the prevention of atherosclerosis or other lipid-binding diseases. The signalling studies indicate a novel site of action of (S)-[6]-gingerol in inhibiting TGF-ß responses.

Intervertebral disc degeneration and ectopic bone formation in apolipoprotein E knockout mice.

Cardiovascular risk factors are known to be associated with intervertebral disc degeneration, but the underlying mechanism is still unclear. The ApoE knockout (KO) mouse is a well-established model for atheroscelorosis. We hypothesized that ApoE is involved in maintaining disc health and that ApoE KO mice will develop early disc degeneration. Discs of ApoE KO and wild-type (WT) mice were characterized with histological/immunological, biochemical, and real-time RT-PCR assays. A comparison of the extracellular matrix production was also performed in disc cells. We demonstrated that ApoE was highly expressed in the endplates of WT discs, and ectopic bone formed in the endplates of ApoE KO discs. Glycosaminoglycan content was decreased in both ApoE KO annulus fibrosus (AF) and nucleus pulposus (NP) cells. Collagen levels were increased in AF and decreased in NP cells. Matrix metalloproteinase-3, -9, and -13 expressions were increased, which may partially explain the impaired matrix production. We also found collagen I, II, aggrecan, and biglycan mRNA expressions were increased in AF cells but decreased in NP cells. Apoptosis was increased in the ApoE KO NP tissue. These results suggest early disc degeneration changes in the ApoE KO mice. ApoE may play a critical role in disc integrity and function.

Glycosaminoglycan chains of biglycan promote bone morphogenetic protein-4-induced osteoblast differentiation.

Biglycan (BGN) has been reported to promote bone morphogenetic protein-4 (BMP-4) stimulated osteoblastic differentiation. However, the underlying mechanism has yet to be fully elucidated. The glycosaminoglycan (GAG) chains of BGN have a variety of biological functions. In the present study, we explored the potential role of the GAG chains of BGN in promoting BMP-4-induced osteoblast differentiation. BGN knockout (KO) murine calvarial cells were transfected with adenovirus overexpressing wild-type BGN (Adv-BGN), adenovirus expressing GAG-mutant BGN (Adv-BGNm) and adenovirus without BGN (Adv-Emp). Transfected cells were treated with or without BMP-4. Subsequently, BMP-4 signaling and function were assessed by evaluating the expression of the osteoblast differentiation-related proteins, Smad1/5/8 phosphorylation and alkaline phosphatase (ALP) activity. Furthermore, the binding specificity of the transfected cells to BMP-4 was also investigated using immunofluorescence staining. Our study demonstrated that a mutant BGN lacking GAG chains decreased BGN-assisted BMP-4 signaling and osteoblast differentiation and that the expression of this mutant BGN in biglycan knockout (BGN­KO) calvarial osteoblasts could not rescue its differentiation deficiency as efficiently as wild-type (WT) BGN. These results strongly suggest that the GAG chains of BGN promote BGN-assisted BMP-4 function.

Differential expression of class I small leucine-rich proteoglycans in an animal model of partial bladder outlet obstruction.

PURPOSE: Partial bladder outlet obstruction has been shown in a rat model to progress from inflammation to hypertrophy to fibrosis. Small leucine-rich proteoglycans are extracellular matrix components associated with collagen fibrillogenesis and resultant scar formation. Two such critical small leucine-rich proteoglycans are decorin and biglycan. We hypothesized that in keeping with other scar models, decorin would be down-regulated and biglycan would be up-regulated with the onset of fibrosis compared to sham. MATERIALS AND METHODS: We challenged our hypothesis with female Fisher rats that underwent ligation of the bladder neck or sham surgery. Animals were sacrificed at 4, 8 and 12 weeks, and bladders were harvested. Frozen sections were stained for immunofluorescence for decorin and biglycan. mRNA expression for decorin and biglycan was analyzed using quantitative reverse transcriptase polymerase chain reaction. RESULTS: All rats survived to specified experimental end points in good health. Immunofluorescent stains showed progressive down-regulation of decorin and up-regulation of biglycan during the 12-week course by 0.36 and 1.82-fold, respectively (p = 0.02 and p = 0.02), compared to shams. Quantitative real-time reverse transcriptase polymerase chain reaction confirmed these findings in 12-week specimens, showing a down-regulation of decorin by a factor of 0.45 (p = 0.02) and up-regulation of biglycan by a factor of 2.04-fold (p = 0.08). CONCLUSIONS: We present the first identification to our knowledge of small leucine-rich proteoglycans in normal and abnormal bladder tissue, and their differential expression in the process of bladder fibrosis, consistent with experimental findings in other anatomical sites. Further investigation into small leucine-rich proteoglycan expression and regulation may allow for the development of new antifibrotic therapeutics.

Gene expression analysis of demineralized bone matrix-induced osteogenesis in human periosteal cells using cDNA array technology.

Demineralized bone matrix (DBM) has been widely investigated as a biomaterial to promote new bone formation and is utilized clinically for bone repair and regeneration. We investigated gene expression patterns of osteogenic differentiation in human periosteal (HPO) cells cultured with demineralized bone matrix, using cDNA array technology. Osteogenic differentiation of HPO cells was determined using alkaline phosphatase assay. In order to examine differential gene expression during osteogenic differentiation, total RNA was isolated from HPO cells in the absence or presence of DBM on day seven and analyzed using osteogenesis cDNA gene array. The selected genes were verified using reverse transcriptase (RT)-PCR analysis. Human periosteal cells differentiated along an osteogenic lineage after treatment of DBM. The alkaline phosphatase activity assay showed that HPO cells differentiated into an osteogenic lineage. Gene expression of HPO cells treated with DBM for seven days was analyzed with cDNA array and RT-PCR analyses. Expression of biglycan, TGF-ß1, and TGF-ßR1 was upregulated, whereas collagen14A1 expression was downregulated, as confirmed by RT-PCR. Human periosteal cells expressed osteogenesis genes when treated with DBM. These findings provide new insight into the capability of demineralized bone matrix to modulate the osteogenic differentiation of human periosteal cells.

TGF-ß stimulates biglycan core protein synthesis but not glycosaminoglycan chain elongation via Akt phosphorylation in vascular smooth muscle.

Transforming growth factor-ß (TGF-ß) can mediate proteoglycan synthesis via Smad and non-Smad signalling pathways in vascular smooth muscle (VSM). We investigated whether TGF-ß-mediated proteoglycan synthesis is via PI3K/Akt. TGF-ß induced a rapid phosphorylation of Akt that continued upto 4 h. Akt phosphorylation was blocked by Akt1/2 inhibitor SN30978; however, it did not block Smad2 phosphorylation at either the carboxy or linker regions indicating that TGF-ß-mediated Akt phosphorylation is independent of Smad2 signalling. The role of Akt in TGF-ß-mediated proteoglycan synthesis was investigated. Treatment with SN30978 showed a concentration-dependent decrease in TGF-ß-mediated [(35)S]-sulphate and [(35)S]-Met/Cys incorporation into secreted proteoglycans; however, SDS-PAGE showed no change in biglycan size. In TGF-ß-treated cells, biglycan mRNA levels increased by 40-100% in 24 h and was significantly blocked by SN30978. Our findings demonstrate that Akt is a downstream signalling component of TGF-ß-mediated biglycan core protein synthesis but not glycosaminoglycan chain hyper-elongation in VSM.

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.

Effects of the angiotensin II receptor blocker losartan on the monocyte expression of biglycan in hypertensive patients.

1. Recently, we demonstrated that biglycan (BGN) is increased in circulating monocyte cells from hypertensive patients and that angiotensin (Ang) II is able to increase BGN expression. The present study was designed to investigate the effects of treatment with the angiotensin AT(1) receptor antagonist losartan on monocyte BGN mRNA and protein expression in essential hypertension. 2. One hundred and twenty-six newly diagnosed hypertensive patients without additional risk factors for atherosclerosis and cardiovascular disease were treated with 100 mg losartan once daily for 6 months. Biglycan mRNA and protein expression was determined in monocytes isolated from peripheral blood before (T(0)) and after (T(1)) therapy. Plasma levels of interleukin (IL)-6, tumour necrosis factor (TNF)-alpha and high sensitivity C-reactive protein (hs-CRP) were also determined. In addition, BGN mRNA and protein expression was determined after the ex vivo addition of 1 micromol/L AngII to monocytes isolated from 20 randomly selected hypertensive patients. 3. Biglycan mRNA and protein expression, blood pressure and plasma levels of fibrinogen, IL-6, TNF-alpha and CRP were significantly lower at T(1) than at T(0). Variations in BGN expression were associated with inflammatory markers, but not directly with blood pressure. In AngII-stimulated monocytes, BGN mRNA and protein expression was significantly lower at T(1) that at T(0). Moreover, mean BGN mRNA expression in AngII-stimulated monocytes isolated from losartan-treated patients was similar to baseline expression in unstimulated monocytes from untreated patients. 4. The results of the present study show that losartan can reduce BGN expression in monocytes from hypertensive patients, without any linear association with blood pressure, suggesting that the effects of AngII on BGN expression in monocytes may be modulated, in part, by an AT(1) receptor blocker.