Syndecan-1 mRNA Expression Research Articles

Neuroprotective Effect of Ganoderic Acid against Focal Ischemic Stroke Induced by Middle Cerebral Artery Occlusion in the Rats via Suppression of Oxidative Stress and Inflammation.

Swiss Wistar rats were used for the current study. The rats were subjected to middle cerebral artery occlusion (MCAO) to simulate transient focal ischemia, followed by reperfusion. Various neurological parameters, including infarct size, neurological deficit score, brain water content, Evans blue leakage, nitric oxide (NO), inducible nitric oxide synthase (iNOS), lactate dehydrogenase (LDH), antioxidant levels, inflammatory cytokines, apoptosis markers, inflammatory parameters, and matrix metalloproteinases (MMP) levels, were estimated. Additionally, mRNA expressions were evaluated in the brain tissue. Dose dependently treatment of GA significantly (P < 0.001) suppressed the infarct size, neurological deflects score, brain water, evans blue leakage, NO, iNOS, LDH, C-X-C chemokine receptor type 4 (CXCR-4), monocyte chemoattractant protein-1 (MCP-1), S100 calcium-binding protein B (S-100β) and K+-Cl- cotransporter 1 (KCC1) positive cells. GA altered the level of oxidative stress parameters like Total antioxidant capacity (T-AOC), 8-hydroxy-2'-deoxyguanosine (8-OhdG), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione (GSH), malonaldehyde (MDA); cytokines viz., tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-1β, IL-6, IL-9, IL-10; inflammatory parameters such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), prostaglandin (PGE2), Nuclear factor kappa B (NF-κB); apoptosis parameters like B-cell leukemia/lymphoma 2 protein (Bcl-2), Bcl-2-associated protein x (Bax), Caspase-3; matrix metallopeptidase (MMP) parameters like MMP-2, MMP-3, and MMP-9, respectively. GA remarkably suppressed the mRNA expression of TRL-4, Syndecan-1, CSF, Aquaporin-1, OCT3, and RFX1. Ganoderic acid exhibited the protection against the cerebral ischemia reperfusion via multiple mechanism.

Extracellular matrix alterations in the skin of patients affected by psoriasis

BackgroundPsoriasis is a chronic inflammatory disease dependent upon a complex interaction between genetic predisposition and immunological factors. It is characterized by skin lesions throughout the body, causing great morbidity and affecting life quality. The present study aimed to evaluate the protein and mRNA expression of heparanase-1 (HPSE), heparanase-2 (HPSE2), syndecan-1 (SYND1), metalloproteinases (MMP2, MMP9), and tissue inhibitor metalloproteinases 2 (TIMP2) in skin samples.MethodsFrom each psoriasis patient, two samples were collected, one sample from a psoriasis plaque (n = 23) and the other sample from non-affected skin (n = 23), as well as tissue collected by blepharoplasty from control individuals (n = 18). Protein expression was investigated by immunohistochemistry, followed by digital quantification. Quantitative RT-PCR obtained mRNA expression. Statistical analyses were done, and p values < 0.05 were considered significant.ResultsA significant increase in protein and mRNA expression was observed in both heparanases (HPSE and HPSE2), and higher protein levels of MMP9 and TIMP2 were observed in the psoriasis plaque compared to the non-affected skin. The data point to a probable activation of MMP2 by TIMP2. Moreover, there was a significant increase in HPSE2, SYND1, MMP9, and TIMP2 in non-affected skin samples from patients with psoriasis than in the control sample (tissue obtained by individuals who do not have psoriasis).ConclusionsThese results show a possible correlation between the characteristic inflammatory process and alterations in the expression of the extracellular matrix in psoriasis. The increased expression of HPSE2, SYND1, MMP9, and TIMP2, even in the absence of psoriatic plaque, indicates that these molecules may be involved with extracellular matrix changes in the initial alterations the psoriatic process and may be candidates for the development of target treatments.

Ultrasensitive Immunoprofiling of Plasma Extracellular Vesicles Identifies Syndecan-1 as a Potential Tool for Minimally Invasive Diagnosis of Glioma.

Liquid biopsy has great potential to improve the management of brain tumor patients at high risk of surgery-associated complications. Here, the aim was to explore plasma extracellular vesicle (plEV) immunoprofiling as a tool for noninvasive diagnosis of glioma. PlEV isolation and analysis were optimized using advanced mass spectrometry, nanoparticle tracking analysis, and electron microscopy. We then established a new procedure that combines size exclusion chromatography isolation and proximity extension assay-based ultrasensitive immunoprofiling of plEV proteins that was applied on a well-defined glioma study cohort (n = 82). Among potential candidates, we for the first time identify syndecan-1 (SDC1) as a plEV constituent that can discriminate between high-grade glioblastoma multiforme (GBM, WHO grade IV) and low-grade glioma [LGG, WHO grade II; area under the ROC curve (AUC): 0.81; sensitivity: 71%; specificity: 91%]. These findings were independently validated by ELISA. Tumor SDC1 mRNA expression similarly discriminated between GBM and LGG in an independent glioma patient population from The Cancer Genome Atlas cohort (AUC: 0.91; sensitivity: 79%; specificity: 91%). In experimental studies with GBM cells, we show that SDC1 is efficiently sorted to secreted EVs. Importantly, we found strong support of plEVSDC1 originating from GBM tumors, as plEVSDC1 correlated with SDC1 protein expression in matched patient tumors, and plEVSDC1 was decreased postoperatively depending on the extent of surgery. Our studies support the concept of circulating plEVs as a tool for noninvasive diagnosis and monitoring of gliomas and should move this field closer to the goal of improving the management of cancer patients.

Impact of Different Tidal Volume Levels at Low Mechanical Power on Ventilator-Induced Lung Injury in Rats.

Tidal volume (VT) has been considered the main determinant of ventilator-induced lung injury (VILI). Recently, experimental studies have suggested that mechanical power transferred from the ventilator to the lungs is the promoter of VILI. We hypothesized that, as long as mechanical power is kept below a safe threshold, high VT should not be injurious. The present study aimed to investigate the impact of different VT levels and respiratory rates (RR) on lung function, diffuse alveolar damage (DAD), alveolar ultrastructure, and expression of genes related to inflammation [interleukin (IL)-6], alveolar stretch (amphiregulin), epithelial [club cell secretory protein (CC)16] and endothelial [intercellular adhesion molecule (ICAM)-1] cell injury, and extracellular matrix damage [syndecan-1, decorin, and metalloproteinase (MMP)-9] in experimental acute respiratory distress syndrome (ARDS) under low-power mechanical ventilation. Twenty-eight Wistar rats received Escherichia coli lipopolysaccharide intratracheally. After 24 h, 21 animals were randomly assigned to ventilation (2 h) with low mechanical power at three different VT levels (n = 7/group): (1) VT = 6 mL/kg and RR adjusted to normocapnia; (2) VT = 13 mL/kg; and 3) VT = 22 mL/kg. In the second and third groups, RR was adjusted to yield low mechanical power comparable to that of the first group. Mechanical power was calculated as [(Δ/Est,L)/2]× RR (ΔP,L = transpulmonary driving pressure, Est,L = static lung elastance). Seven rats were not mechanically ventilated (NV) and were used for molecular biology analysis. Mechanical power was comparable among groups, while VT gradually increased. ΔP,L and mechanical energy were higher in VT = 22 mL/kg than VT = 6 mL/kg and VT = 13 mL/kg (p < 0.001 for both). Accordingly, DAD score increased in VT = 22 mL/kg compared to VT = 6 mL/kg and VT = 13 mL/kg [23(18.5–24.75) vs. 16(12–17.75) and 16(13.25–18), p < 0.05, respectively]. VT = 22 mL/kg was associated with higher IL-6, amphiregulin, CC16, MMP-9, and syndecan-1 mRNA expression and lower decorin expression than VT = 6 mL/kg. Multiple linear regression analyses indicated that VT was able to predict changes in IL-6 and CC16, whereas ΔP,L predicted pHa, oxygenation, amphiregulin, and syndecan-1 expression. In the model of ARDS used herein, even at low mechanical power, high VT resulted in VILI. VT control seems to be more important than RR control to mitigate VILI.

Clinicopathological and prognostic significance of SDC1 overexpression in breast cancer.

BackgroundBreast cancer is the leading cause of cancer death among global women, and its early diagnosis and treatment are very urgent. Syndecan-1 (SDC1) is a heparin sulfate proteoglycan, which has been linked with the prognosis and treatment response in a various tumor type. To investigate whether SDC1 can serve as a prognostic indictor in breast cancer, bioinformatic analyses were performed in the present study.MethodsSDC1 expression was assessed using Oncomine analysis. Kaplan-Meier Plotter and bc-GenExMiner were performed to identify the prognostic roles of SDC1 in breast cancer. COSMIC analysis and cBioPortal database were performed to analysis the mutations of SDC1. The heat map and methylation status of SDC1 were identified by performing the UCSC.ResultsWe found that SDC1 was more frequently overexpressed in breast cancer than their normal tissues and its expression might be negatively related with some CpG sites. Meanwhile, pooled data suggested that SDC1 mRNA expression is associated worse prognosis of breast cancer. Following data mining in multiple big databases confirmed a positive correlation between SDC1 mRNA expression and PLAU mRNA expression in breast cancer tissues. In addition, high SDC1 expression is associated with increased risked of age, nodal, HER2 and higher SBR grade status.ConclusionOur findings suggest that overexpressed SDC1 was identified in breast cancer than in matched normal tissues and is associated with methylation status of SDC1 promoter. Additionally, SDC1 is positively associated with PLAU and might act as a potential prognostic indicator for breast cancer.

Incipient renal transplant dysfunction associates with tubular syndecan-1 expression and shedding.

Syndecan-1 is a transmembrane heparan sulfate proteoglycan involved in regenerative growth and cellular adhesion. We hypothesized that the induction of tubular syndecan-1 is a repair response to incipient renal damage in apparently stable, uncomplicated renal transplant recipients. We quantified tubular syndecan-1 in unselected renal protocol biopsies taken 1 yr after transplantation. Spearman rank correlation analysis revealed an inverse correlation between tubular syndecan-1 expression and creatinine clearance at the time of biopsy (r = -0.483, P < 0.03). In a larger panel of protocol and indication biopsies from renal transplant recipients, tubular syndecan-1 correlated with tubular proliferation marker Ki67 (r = 0.518, P < 0.0001). In a rat renal transplantation model, 2 mo after transplantation, mRNA expression of syndecan-1 and its major sheddase, A disintegrin and metalloproteinase-17, were upregulated (both P < 0.03). Since shed syndecan-1 might end up in the circulation, in a stable cross-sectional human renal transplant population (n = 510), we measured plasma syndecan-1. By multivariate regression analysis, we showed robust independent associations of plasma syndecan-1 with renal (plasma creatinine and plasma urea) and endothelial function parameters (plasma VEGF-A, all P < 0.01). By various approaches, we were not able to localize syndecan-1 in vessel wall or endothelial cells, which makes shedding of syndecan-1 from the endothelial glycocalyx unlikely. Our data suggest that early damage in transplanted kidneys induces repair mechanisms within the graft, namely, tubular syndecan-1 expression for tubular regeneration and VEGF production for endothelial repair. Elevated plasma syndecan-1 levels in renal transplantation patients might be interpreted as repair/survival factor related to loss of tubular and endothelial function in transplanted kidneys.

CD138 mRNA expression from peripheral blood of patients with follicular and diffuse large B cell lymphoma: relation to disease progression and treatment response

Syndecans are among those transmembrane PGs which act via their heparan sulphate chains as receptors for different matrix elements (e.g. collagen I–III, fibronectin, thrombospondin, tenascin) and co-receptors for growth factors (e.g. bFGF, aFGF, GM-CSF, IL-3, IFNg). We hypothesized that there is a positive relationship between the expression of syndecan-1 (CD138) and treatment response in non-Hodgkin’s lymphoma. To identify the expression of syndecan-1 (CD138) in cases of non-Hodgkin’s lymphoma and to correlate its expression with treatment response and disease stage, this study was carried out as a cross-sectional study and included 30 patients with non-Hodgkin’s lymphomas attending Suez Canal University Hospital; diagnosis of patients was made by routine histological and immunohistochemical examination. CD138 mRNA expression was assessed by TaqMan technique using real-time PCR method. There was increased expression of CD138 mRNA in patients with follicular lymphoma than in patients with diffuse large B cell lymphoma (6.25 versus 3.46, respectively) (p ≤ 0.05). Syndecan-1 (CD138) mRNA expression from peripheral blood may be a useful marker for follicular lymphoma and can be used as a marker of treatment response in patients with follicular and diffuse large B cell lymphoma.

S72 RAGE Activation and Endothelial Cell Injury Associated with Cardiopulmonary Bypass

Introduction and objectivesThe systemic inflammatory response syndrome (SIRS) which complicates most cases of surgery necessitating cardiopulmonary bypass (snCPB) is associated with endothelial cell injury and activation of the receptor for...

Trastuzumab resistance in breast cancer cell lines: The role of extracellular matrix.

e11135 Background: Trastuzumab is an antibody that blocks the activity of HER2, a member of epidermal growth factor receptor family that is amplified in approximately 20% of breast cancer patients. Trastuzumab significantly improves disease-free and overall survival in these cases. Nevertheless, many patients become resistant to this antibody. Methods: Different human breast cancer cell lines MCF7, SKBR3 and a stable transfected heparanase-1 cDNA MCF7 cells (HPSE-1-MCF7), as well a non tumoral cell line, MCF10A were used for the experimental assays of cell viability, confocal immunofluorescence microscopy and quantitative RT-PCR analysis. Results: Our findings show the role of some extracellular matrix (ECM) components in trastuzumab resistance using breast cancer cell lines assays. MCF7 cells were transfected with heparanase-1 (HPSE-1) cDNA and became completely resistant to trastuzumab. HPSE-1 transfection changes the glycosaminoglycans profile in the transfected MCF7 cells, suggesting that glycosaminoglycans/proteoglycans could be involved in the resistance process. The results showed that trastuzumab binds to heparan sulfate (HS) and this effect is necessary to antibody activity. It was also demonstrated that trastuzumab decreases heparanases isoforms (HPSE-1, HPSE-2), HER2 and the proteoglycan syndecan-1 (Syn-1) mRNA expression in MCF7 cell line, while in MCF7 HPSE-1 transfected cells trastuzumab increases mRNA expression of these molecules. SKBR3 cells presented the highest expression levels of these molecules. The relative proportion of HS chains in Syn-1 core protein seems to influence MCF7 cells sensibility and resistance to this antibody. In addition, HPSE-1/HER2 ratio seems to define trastuzumab resistance, once the relative increase in HPSE-1 expression decrease breast cancer cells sensitivity to trastuzumab. Conclusions: These new insights show the effect of ECM components for trastuzumab efficacy and could be useful when devising strategies for overcoming drug resistance in HER2+ cancers.

Plasma Restoration of Endothelial Glycocalyx in a Rodent Model of Hemorrhagic Shock

The use of plasma-based resuscitation for trauma patients in hemorrhagic shock has been associated with a decrease in mortality. Although some have proposed a beneficial effect through replacement of coagulation proteins, the putative mechanisms of protection afforded by plasma are unknown. We have previously shown in a cell culture model that plasma decreases endothelial cell permeability in comparison with crystalloid. The endothelial glycocalyx consists of proteoglycans and glycoproteins attached to a syndecan backbone, which together protect the underlying endothelium. We hypothesize that endothelial cell protection by plasma is due, in part, to its restoration of the endothelial glycocalyx and preservation of syndecan-1 after hemorrhagic shock. Rats were subjected to hemorrhagic shock to a mean arterial blood pressure of 30 mm Hg for 90 minutes followed by resuscitation with either lactated Ringer's (LR) solution or fresh plasma to a mean arterial blood pressure of 80 mm Hg and compared with shams or shock alone. After 2 hours, lungs were harvested for syndecan mRNA, immunostained with antisyndecan-1, or stained with hematoxylin and eosin. To specifically examine the effect of plasma on the endothelium, we infused small bowel mesentery with a lanthanum-based solution, identified venules, and visualized the glycocalyx by electron microscopy. All data are presented as mean ± SEM. Results were analyzed by 1-way analysis of variance with Tukey post hoc tests. Electron microscopy revealed degradation of the glycocalyx after hemorrhagic shock, which was partially restored by plasma but not LR. Pulmonary syndecan-1 mRNA expression was higher in animals resuscitated with plasma (2.76 ± 0.03) in comparison with shock alone (1.39 ± 0.22) or LR (0.82 ± 0.03) and correlated with cell surface syndecan-1 immunostaining. Shock also resulted in significant lung injury by histopathology scoring (1.63 ± 0.26), which was mitigated by resuscitation with plasma (0.67 ± 0.17) but not LR (2.0 ± 0.25). The protective effects of plasma may be due in part to its ability to restore the endothelial glycocalyx and preserve syndecan-1 after hemorrhagic shock.

Role of the Heparan Sulfate Proteoglycan Syndecan-1 (CD138) in Delayed-Type Hypersensitivity

The cell surface heparan sulfate proteoglycan syndecan-1 (CD138) modulates the activity of chemokines, cytokines, integrins, and other adhesion molecules which play important roles in the regulation of inflammation. We have previously shown that syndecan-1-deficient murine leukocytes display increased interactions with endothelial cells and increased diapedesis in vivo and in vitro. In this study, we demonstrate that syndecan-1 has an important function as a negative modulator in the murine contact allergy model of oxazolone-mediated delayed-type hypersensitivity (DTH). Following elicitation of the DTH response, syndecan-1-deficient mice showed an increase in leukocyte recruitment, resulting in an increased and prolonged edema formation. Expression of the cytokines TNF-alpha and IL-6 of the chemokines CCL5/RANTES and CCL-3/MIP-1alpha and of the adhesion molecule ICAM-1 were significantly increased in syndecan-1-deficient compared with wild-type mice. In wild-type mice, syndecan-1 mRNA and protein expression was reduced during the DTH response. The differentially increased adhesion of syndecan-1-deficient leukocytes to ICAM-1 was efficiently inhibited in vitro by CD18-blocking Abs, which emerges as one mechanistic explanation for the anti-inflammatory effects of syndecan-1. Collectively, our results show an important role of syndecan-1 in the contact DTH reaction, identifying syndecan-1 as a novel target in anti-inflammatory therapy.

118. CHARACTERISATION OF HEPARAN SULPHATE PROTEOGLYCANS IN THE MATURING CUMULUS OOCYTE COMPLEX

Many growth factors including members of the transforming growth factor beta (TGFβ) superfamily and epidermal growth factor (Egf)-like ligands signal via interactions with heparan sulphate proteoglycans (HSPGs). Cell surface HSPGs can act by sequestering ligands at their site of action, by presenting a ligand to its signalling receptor, or by preventing ligand-receptor interaction. The oocyte secreted factors (OSF) growth differentiation factor 9 and bone morphogenetic protein 15 are members of the TGFβ superfamily that act selectively on cumulus cells. Conversely Egf-like ligands are secreted by mural granulosa cells and transmit LH-induced signals to cumulus cells. We investigated the possibility that HSPGs contribute to the spatially restricted responses these signals exert on cumulus cells. Syndecan-1 and Glypican-1 are cell surface HSPGs that are involved in numerous biological processes, including growth factor regulation, cell proliferation and differentiation. Microarray analysis showed Syndecan-1 and Glypican-1 mRNA expression induced 6-fold (P=10-9) and 3-fold (P=10-7) respectively in Egf+FSH stimulated cumulus oocyte complexes (COCs). Furthermore, Syndecan-1 and Glypican-1 mRNA were induced 27- and 16-fold respectively in COCs after hCG treatment of mice. Syndecan-1 and Glypican-1 protein was localised specifically to the COC through immunohistochemical analysis. In Vitro Maturation (IVM) of oocytes is a valuable alternative to gonadotropin mediated superovulation, but IVM COCs are less competent than those matured in vivo. Several components of the COC have been shown to be altered in IVM, including the chondroitin sulphate proteoglycan Versican. COCs from mice that underwent IVM in the presence of Egf+FSH and cilostamide for 16 hours had &gt;16 fold reduced mRNA for Syndecan-1 when compared with In Vivo matured COCs. The lack of Syndecan-1 in IVM COCs could reduce signalling capacity of growth factors including OSFs. This may contribute to the reduced capacity of IVM oocytes to fertilise and produce a healthy embryo, and ultimately, a healthy offspring.

Distinct Effects of N-Acetylgalactosamine-4-sulfatase and Galactose-6-sulfatase Expression on Chondroitin Sulfates

The sulfatase enzymes, N-acetylgalactosamine-4-sulfatase (arylsulfatase B (ASB)) and galactose-6-sulfatase (GALNS) hydrolyze sulfate groups of CS. Deficiencies of ASB and GALNS are associated with the mucopolysaccharidoses. To determine if expression of ASB and GALNS impacts on glycosaminoglycans (GAGs) and proteoglycans beyond their association with the mucopolysaccharidoses, we modified the expression of ASB and GALNS by overexpression and by silencing with small interference RNA in MCF-7 cells. Content of total sulfated GAG (sGAG), chondroitin 4-sulfate (C4S), and total chondroitin sulfates (CSs) was measured following immunoprecipitation with C4S and CS antibodies and treatment with chondroitinase ABC. Following silencing of ASB or GALNS, total sGAG, C4S, and CS increased significantly. Following overexpression of ASB or GALNS, total sGAG, C4S, and CS declined significantly. Measurements following chondroitinase ABC treatment of the cell lysates demonstrated no change in the content of the other sGAG, including heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Following overexpression of ASB and immunoprecipitation with C4S antibody, virtually no sGAG was detectable. Total sGAG content increased to 23.39 (+/-1.06) microg/mg of protein from baseline of 12.47 (+/-0.68) microg/mg of protein following ASB silencing. mRNA expression of core proteins of the CS-containing proteoglycans, syndecan-1 and decorin, was significantly up-regulated following overexpression of ASB and GALNS. Soluble syndecan-1 protein increased following increases in ASB and GALNS and reduced following silencing, inversely to changes in CS. These findings demonstrate that modification of expression of the lysosomal sulfatases ASB and GALNS regulates the content of CSs.

Increased Expression of Heparan Sulfate Proteoglycan on the Cultured Renal Epithelial Cells during Oxalate Exposure

We have previously reported that heparan sulfate (HS) / heparan sulfate proteoglycan (HSPG, syndecan-1) expression significantly increased in the rat kidney during calcium oxalate (CaOx) nephrolithiasis. Although the exact mechanism of the increased syndecan expression still remains unclear, HS/syndecan is thought to have some important roles in CaOx crystal formation. The present study examined the role of HS during oxalate exposure by using a newly developed cell line (KIC-synd-1) that expresses human heparan sulfate proteoglycan (syndecan-1). Quantitative competitive (QC)-RT-PCR was used to examine change of syndecan-1 mRNA expression in KIC-synd-1 cells. Production of syndecan-1 core protein and glycosaminoglycans (GAGs) were also confirmed by Western blot, immunohistochemistry and HPLC, respectively. Wild type Mardin-Darby canine kidney (MDCK) cells were also examined in the same manner. The stable expression of syndecan-1 gene and production of both core protein and HS chains were confirmed in the newly developed KIC-synd-1 cell line. Increased syndecan-1 mRNA expression and production of core proteins were confirmed in KIC-synd-1 cells during oxalate exposure. MTT assay revealed that the cell viability decreased significantly in the MDCK cells after 1 mM oxalate exposure (p<0.05). On the other hand, there was no significant difference in the oxalate exposed KIC-synd-1 cells. However, the cell viability in KIC-synd-1 cells pretreated with heparitinase digestion decreased significantly before oxalate exposure (p<0.05). The present data suggests that both exogenous and endogenous HS exerts protective effect against oxalate-induced cell injuries. Previous studies in our laboratory have indicated that hyperoxaluria and deposition of CaOx crystals resulted in renal tubular cellular injury inducing the synthesis of HSPG to protect and repair the damaged epithelial cell surface. The present data offers strong support for this hypothesis. Finally, HS could be potent inhibitor of CaOx nephrolithiasis and the absence of this substance on the tubular surface may increase the risk of CaOx crystal formation and retention.

Mechanical strain induces a persistent upregulation of syndecan‐1 expression in smooth muscle cells

Syndecan-1 belongs to a family of transmembrane proteoglycans, acts as a coreceptor for growth factor binding, as well as cell-matrix and cell-cell interactions, and is induced in smooth muscle cells (SMCs) following balloon catheter injury. In this report, we investigated syndecan-1 expression in SMCs in response to several distinct biomechanical force profiles and the related syndecan shedding response. Syndecan-1 mRNA expression increased in response to 5% and 10% cyclic strain (24 h: 206 +/- 40% and 278 +/- 33%, respectively, P < 0.05) when compared to unstrained controls. When subjected to 10% cyclic strain for periods of up to 48 h, syndecan-1 mRNA levels remained elevated at 294 +/- 31%. Notably, the SMC mechanosensor mechanism remained responsive after an initial 24 h "preconditioning" period, as evident by a fivefold increase in syndecan-1 gene expression following a change in cyclic stress from 10% to 20% (48 h: 516 +/- 55%, P < 0.05). Of note, similar behavior was not observed in an analysis of syndecan-2 mRNA levels. Commensurate with mRNA responses, mechanical stress induced an increase in cell-associated syndecan-1 protein levels with an associated increase in protein shedding. Given the varied functions of syndecan-1, stress-induced effects on SMC syndecan-1 expression and shedding may represent an additional component of the pro-inflammatory, growth-stimulating pathways that are activated in response to changes in the mechanical microenvironment of the vascular wall. Syndecan-1 expression is uniquely influenced by changes in the phase and magnitude of the local stress field.

Expression of syndecans, cell–cell interaction regulating heparan sulfate proteoglycans, within the human endometrium and their regulation throughout the menstrual cycle

To evaluate the expression of syndecan-1, -2, -3, and -4 in different phases of eutopic endometrium of normal cycling women. Prospective observational study. University-based research center for reproductive medicine. Twenty-nine healthy ovulatory volunteers. mRNA and protein expression of syndecan-1 to -4 in human endometrium. Real-time polymerase chain reaction of syndecan members and further characterization of mRNA expression of syndecan-1 and -4 with multiprobe RNase protection assays of epithelial and stromal cells after purification with antibody-coated magnetic beads. For confirmation of results, protein expression and localization using immunohistochemistry for syndecan-1 and -4 was performed. All syndecans were expressed within human endometrium. Syndecan-1 and -4 proved to be significantly upregulated in whole endometrium during the secretory phase (2.73-fold and 2.85-fold, respectively). Using multiprobe RNase protection assays, a significant upregulation of mRNA was noted in epithelial cells during the secretory phase for both syndecan-1 and -4 (7.46-fold and 2.52-fold, respectively) and confirmed by immunohistochemistry. Cycle-dependent expression of syndecan-1 and -4 suggests that these adhesion proteins are involved in the regulation of the cycling endometrium.

Heparanase Influences Expression and Shedding of Syndecan-1, and Its Expression by the Bone Marrow Environment Is a Bad Prognostic Factor in Multiple Myeloma.

The heparan sulfate (HS) proteoglycan (PG) syndecan-1 plays a major role in multiple myeloma (MM), in part by concentrating HS-binding growth factors on the surface of MM cells (MMC). The function of HSPG is regulated by extracellular enzymes that modulate the structure of HS-chains. One such enzyme is heparanase (HPSE), an endo-glucuronidase that cleaves HS chains at only a few sites, resulting in fragments of 10–20 sugar units long that are biologically active.Using Affymetrix microarrays and real-time RT-PCR, we showed that the gene encoding HPSE was expressed by 11/19 myeloma cell lines (HMCLs). In HSPE-positive HMCLs, syndecan-1 mRNA expression and production of soluble syndecan-1, unlike expression of membrane syndecan-1, were significantly increased. Downregulation of HPSE by HPSE-siRNA resulted in a decrease of syndecan-1 mRNA expression and soluble syndecan-1 production, without affecting membrane syndecan-1. Contrary to HMCLs, HPSE was expressed in only 4/39 primary MMC samples at a low level, whereas it was highly expressed in 36/39 whole bone marrow (WBM) microenvironment samples. In the latter, HPSE was expressed at a median level in polynuclear cells and T cells, at high level in monocytes and osteoclasts, and it was not expressed in BM stromal cells. In order to investigate the connection of clinical parameters with HPSE gene expression, 30/39 myeloma patients treated with high dose chemotherapy and autologous stem cell transplantation where classified into two groups: 15 patients with the lowest or the highest HPSE expression in the WBM. The HPSEhigh group had an increased percentage of patients with elevated C-reactive protein and β2-microglobulin. According to the international staging system (ISS), the HPSEhigh group included a significant higher frequency of patients with stage III MM and a lower frequency of patients with stage I MM. HPSEhigh patients had a shorter event free survival (EFS) (p=.017) and overall survival (OAS) (p=.023) than HPSElow patients. Classifying these 30 patients into two groups according to HPSE expression in MMC did not yield to any EFS or OAS significant differences between the two groups.Altogether, those data provide evidence that HPSE not only modulate syndecan-1 activity through the cleavage of HS chains but also regulates syndecan-1 mRNA expression and shedding. We report for the first time that the expression of a gene in the BM, i.e. HSPE, is an indicator of poor prognostic for MM patients. Therefore, HPSE inhibitors like PI-88, which is currently in clinical trial, will be of interest for the treatment of patients with MM.

Strategies to enhance transductional efficiency of adenoviral‐based gene transfer to primary human fibroblasts and keratinocytes as a platform in dermal wounds

Genetically modified keratinocytes and fibroblasts are suitable for delivery of therapeutic genes capable of modifying the wound healing process. However, efficient gene delivery is a prerequisite for successful gene therapy of wounds. Whereas adenoviral vectors (Ads) exhibit superior levels of in vivo gene transfer, their transductional efficiency to cells resident within wounds may nonetheless be suboptimal, due to deficiency of the primary adenovirus receptor, coxsackie-adenovirus receptor (CAR). We explored CAR-independent transduction to fibroblasts and keratinocytes using a panel of CAR-independent fiber-modified Ads to determine enhancement of infectivity. These fiber-modified adenoviral vectors included Ad 3 knob (Ad5/3), canine Ad serotype 2 knob (Ad5CAV-2), RGD (Ad5.RGD), polylysine (Ad5.pK7), or both RGD and polylysine (Ad5.RGD.pK7). To evaluate whether transduction efficiencies of the fiber-modified adenoviral vectors correlated with the expression of their putative receptors on keratinocytes and fibroblasts, we analyzed the mRNA levels of CAR, alpha upsilon integrin, syndecan-1, and glypican-1 using quantitative polymerase chain reaction. Analysis of luciferase and green fluorescent protein transgene expression showed superior transduction efficiency of Ad5.pK7 in keratinocytes and Ad5.RGD.pK7 in fibroblasts. mRNA expression of alpha upsilon integrin, syndecan-1 and glypican-1 was significantly higher in primary fibroblasts than CAR. In keratinocytes, syndecan-1 expression was significantly higher than all the other receptors tested. Significant infectivity enhancement was achieved in keratinocytes and fibroblasts using fiber-modified adenoviral vectors. These strategies to enhance infectivity may help to achieve higher clinical efficacy of wound gene therapy.

Syndecan‐1 Expression During the Formation of Junctional Epithelium

Syndecans are cell surface heparan sulfate proteoglycans (PG) which can bind to and modulate the action of growth factors and extracellular matrix components (ECM). Syndecan- 1 has been shown to play important roles during early tooth development and wound healing and repair. Among diverse cells and tissues that comprise the periodontium, the junctional epithelium (JE) constitutes a region of significant anatomic and clinical importance, but the nature of inductive signals and molecules involved in its formation is still unclear. Therefore, this work examines if syndecan-1 is associated with formation of JE, and the distribution of other syndecan family members in the epithelium. In situ hybridization and immunohistochemical techniques were performed using oral tissues from 4-day-old to 10-week-old mice to investigate the expression of syndecan- 1, -2, -3 and -4 mRNAs and their corresponding proteins. Based on in situ hybridization experiments, all syndecan mRNAs were detected in sulcular epithelium (SE), gingival epithelium (GE), and JE with varying intensity and distribution. Syndecan-1 immunostaining was localized on the cell surface while that of syndecan-2 did not show clear membrane localization. Our experiments in the developing tooth demonstrated that syndecan-1 protein followed characteristic patterns of expression during JE formation and that immunoreactivity for syndecan-1 protein decreased with age when JE cells underwent terminal differentiation. Results of syndecan-1 mRNA and protein expression patterns suggested that this proteoglycan might be an important molecule during the formation of JE.

High syndecan-1 expression in breast carcinoma is related to an aggressive phenotype and to poorer prognosis.

Syndecan-1 is a transmembrane heparan sulphate proteoglycan that is involved in cell-cell adhesion, organization of cell-matrix adhesion, and regulation of growth factor signaling. Specimens from 254 consecutive breast carcinoma (BC) cases (110 N0, 144 N1/2) with long-term follow-up (median, 95 months) were immunostained for syndecan-1, estrogen receptor (ER), progesterone receptor (PgR), and p53; in 154 cases, c-erbB-2 status was known. Syndecan-1 mRNA and protein expression also were evaluated in 20 breast tissue samples (10 normal and tumor pairs). Syndecan-1 was expressed at high levels in 106 (42%) BCs; syndecan-1 up-regulation was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) studies. High syndecan-1 expression was associated with high histologic grade, large tumor size, high mitotic count, c-erbB-2 overexpression, and ER and PgR negative status. At univariate survival analysis syndecan overexpression was related to poor prognosis (P < 0.01 for both overall survival (OS) and disease-free survival). Bivariate survival analysis showed an additive adverse effect for syndecan-1 and c-erbB-2 overexpression. At multivariate analysis, syndecan-1 overexpression was independently associated with poor OS (hazard ratio [HR], 1.71; 95% confidence interval [CI], 1.08-2.69). High syndecan-1 expression also was of independent prognostic value for OS in the group of 102 ER-negative patients (HR, 2.42; 95% CI, 1.21-4.82). Stratifying patients on the basis of the type of adjuvant therapy given, high syndecan-1 expression was associated with a higher risk of death only in patients treated with the cyclophosphamide-methotrexate-fluorouracil regimen (HR, 1.9; P = 0.09); at multivariate analysis for OS, this association proved to be of independent statistical significance (P = 0.03; HR, 2.15). Syndecan-1 is expressed at high levels in a significant percentage of breast carcinomas and is related to an aggressive phenotype and poor clinical behavior.