Main Proteoglycan Research Articles

Schwartz-Jampel Syndrome Type-1: Compound Heterozygosity of Two Novel Variants.

Schwartz-Jampel Syndrome (SJS) type-1 (OMIM; #255800), a rare cause of skeletal dysplasia, is characterized by myotonic myopathy, chondrodystrophy, short stature, facial and eye abnormalities. SJS Type-1 develops due to variations in the HSPG2 gene which produces the "perlecan" molecule, one of the main proteoglycans of the basement membrane. A 6-year-old girl presented with short stature, a mask face, shrunken lips, narrow palpebral opening due to blepharospasm, stiffness of facial muscles, micrognathia, overlapping teeth, a short neck, and a bell-shaped thorax due to myotonic myopathy. She was diagnosed with SJS type-1 due to compound heterozygosity of two novel variations in the HSPG2 gene. In patients with short stature and an accompanying myotonic myopathy SJS should be considered. Compound heterozygosity may cause typical clinical findings of SJS. In case of suspicion creatinine kinase levels can be measured, and the determination of myotonia may require evaluation with electromyography. Once the diagnosis is made, patients should be carefully monitored in terms of growth, neuromuscular disorders, joints problems and bone health.

Dexamethasone effects on the expression and content of glycosylated components of mouse brain tissue

Introduction. Glucocorticoids are actively used in the treatment of various diseases, however their long-term use leads to numerous negative side-effects, the molecular mechanisms of which remain poorly understood.Aim. Study of the short-term (1–10 days) effects of various doses of dexamethasone (Dex) (0,1–10 mg/kg) on the expression of the glucocorticoid receptor (GR, Nr3c1), core proteins of main proteoglycans and heparan sulfate metabolism-involved genes, as well as the content of carbohydrate macromolecules of glycosaminoglycans in the brain tissue of experimental animals.Materials and methods. In the study, C57Bl/6 mice were used. The expression of GR, proteoglycan core proteins and heparan sulfate metabolism-involved genes was determined by real-time polymerase chain reaction with reverse transcription. The content and localization of GR protein molecule were studied by Western blot and immunohistochemical analysis, and the glycosaminoglycan content was determined by dot-blot analysis and Alcian Blue staining.Results. It was shown that a single Dex administration leads to fast (1–3 days) short-term activation of GR expression (+1.5 times, p <0.05), proteoglycan’s genes (syndecan-3, Sdc3; perlecan, Hspg2; phosphacan, Ptprz1; neurocan, Ncan; +2–3-fold; p <0.05) and heparan sulfate-metabolism-involved genes (Ndst1, Glce, Hs2st1, Hs6st1, Sulf1 / 2; +1.5–2-fold; p <0.05) in the mouse brain, with a return to control values by 7–10 days after Dex administration. At the same time, the effect of Dex on carbohydrate macromolecules of glycosaminoglycans was more delayed and stable, increasing the content of low-sulfated glycosaminoglycans in the brain tissue in a dose-dependent manner starting from day 1 after Dex administration. Highly-sulfated glycosaminoglycans showed more delayed response to Dex administration, and an increase in their content was observed only at higher doses (2.5 and 10 mg/kg) and only on 7–10 days after its administration, apparently, mainly due to an increase in heparan sulfate content.Conclusion. In general, the effect of a single injection of Dex on the transcriptional activity of GR, proteoglycan core proteins and heparan sulfate metabolism-involved genes were short-termed, and the genes expression quickly returned to the normal levels. However, even a single use of Dex significantly increased the content of total as well as highly sulfated glycosaminoglycans in the mouse brain tissue, which can lead to the changes in the composition and structure of the brain tissue, as well as its functional characteristics.

A Novel Fibromodulin Antagonist Peptide RP4 Exerts Antitumor Effects on Colorectal Cancer

Colorectal cancer (CRC) is the leading cause of cancer-related deaths worldwide. Fibromodulin (FMOD) is the main proteoglycan that contributes to extracellular matrix (ECM) remodeling by binding to matrix molecules, thereby playing an essential role in tumor growth and metastasis. There are still no useful drugs that target FMOD for CRC treatment in clinics. Here, we first used public whole-genome expression datasets to analyze the expression level of FMOD in CRC and found that FMOD was upregulated in CRC and associated with poor patient prognosis. We then used the Ph.D.-12 phage display peptide library to obtain a novel FMOD antagonist peptide, named RP4, and tested its anti-cancer effects of RP4 in vitro and in vivo. These results showed that RP4 inhibited CRC cell growth and metastasis, and promoted apoptosis both in vitro and in vivo by binding to FMOD. In addition, RP4 treatment affected the CRC-associated immune microenvironment in a tumor model by promoting cytotoxic CD8+ T and NKT (natural killer T) cells and inhibiting CD25+ Foxp3+ Treg cells. Mechanistically, RP4 exerted anti-tumor effects by blocking the Akt and Wnt/β-catenin signaling pathways. This study implies that FMOD is a potential target for CRC treatment, and the novel FMOD antagonist peptide RP4 can be developed as a clinical drug for CRC treatment.

A High Proportion of Novel ACAN Mutations and Their Prevalence in a Large Cohort of Chinese Short Stature Children.

ContextAggrecan, encoded by the ACAN gene, is the main proteoglycan component in the extracellular cartilage matrix. Heterozygous mutations in ACAN have been reported to cause idiopathic short stature. However, the prevalence of ACAN pathogenic variants in Chinese short stature patients and clinical phenotypes remain to be evaluated.ObjectiveWe sought to determine the prevalence of ACAN pathogenic variants among Chinese short stature children and characterize the phenotypic spectrum and their responses to growth hormone therapies.Patients and MethodsOver 1000 unrelated short stature patients ascertained across China were genetically evaluated by next-generation sequencing–based test.ResultWe identified 10 novel likely pathogenic variants and 2 recurrent pathogenic variants in this cohort. None of ACAN mutation carriers exhibited significant dysmorphic features or skeletal abnormities. The prevalence of ACAN defect is estimated to be 1.2% in the whole cohort; it increased to 14.3% among those with advanced bone age and to 35.7% among those with both advanced bone age and family history of short stature. Nonetheless, 5 of 11 ACAN mutation carries had no advanced bone age. Two individuals received growth hormone therapy with variable levels of height SD score improvement.ConclusionOur data suggest that ACAN mutation is 1 of the common causes of Chinese pediatric short stature. Although it has a higher detection rate among short stature patients with advanced bone age and family history, part of affected probands presented with delayed bone age in Chinese short stature population. The growth hormone treatment was moderately effective for both individuals.

Serum Decorin and Biglycan as Potential Biomarkers to Predict PPROM in Early Gestation

Preterm birth is a leading cause of neonatal mortality in the US and globally, with preterm premature rupture of fetal membranes (PPROM) accounting for one third of preterm births. Currently no predictive diagnostics are available to precisely assess risk and potentially reduce the incidence of PPROM. Bigycan and decorin, the main proteoglycans present in human fetal membranes, are involved in the physiological maturation of fetal membranes as well as in the pathophysiology of preterm birth. The serum protein sex hormone-binding globulin (SHBG) has recently been identified as a predictor of spontaneous preterm birth. We hypothesize that the balance between serum decorin and biglycan on one hand and SHBG on the other hand may provide insight into the status of the fetal membranes in early pregnancy, thereby predicting PPROM prior to symptoms. Using chart review, 18 patients with confirmed cases of PPROM were identified from 2013-2016. Second trimester residual serum was retreived from freezer storage for these cases along with 5 matched controls for each case. The biomarkers biglycan, decorin and SHBG were analyzed first separately, then in combination to determine their ability to predict PPROM. The predictive score for the combined model displays an AUC = 0.774. The ROC curve of the predicted score has an optimal threshold of 0.238 and a sensitivity and specificity of 0.72 and 0.84 respectively. This prenatal serum panel is a promising serum screening-based biochemical model to predict PPROM in asymptomatic women.

Study design of a phase 2 clinical trial with a disease-modifying Osteoarthritis drug candidate GLPG1972/S201086: The roccella trial

Purpose: Osteoarthritis (OA) is characterized by structural changes of the joint, of which loss of articular cartilage is one of the major signs. The main proteoglycan component of the extracellular matrix of articular cartilage is aggrecan. ADAMTS-5, A Disintegrin And Metalloproteinase with ThromboSpondin-motif-5, is a key aggrecan-cleaving enzyme involved in cartilage degradation, resulting in ARGS-aggrecan neo-epitope levels measurable in synovial fluid and serum. GLPG1972/S201086 is a highly specific and potent ADAMTS-5 inhibitor in development as an orally administered DMOAD candidate.

Development and characterization of a novel non-surgical mouse model of intervertebral disc degeneration

Purpose: In the spinal column, the purpose of the intervertebral discs (IVD) is to provide the spine with flexibility and load-bearing capacity. Each IVD is comprised of a highly hydrated nucleus pulposus (NP) core that is enclosed by the concentric collagenous fibers of the annulus fibrosus (AF). Maintenance of this composite structure and its biomechanical function requires proper activity of disc-resident cells, with balanced synthesis and degradation of macromolecular components. Despite that, our understanding of the role of senescence and cell death on the progression of disc degeneration is limited. IVD degeneration is one of the main contributors to low back pain and is known to precede several prevalent and debilitating spinal diseases, such as disc herniation and spondylosis. Although the causes of IVD degeneration are poorly understood, dysfunction and progressive decline in the number of resident cells have been found with aging and disease. Most animal models currently used to investigate the pathophysiology of IVD degeneration rely on surgical intervention, where invasiveness coupled with an inherent inflammatory response, limits our ability to mimic the pathogenesis of non-traumatic human disc degeneration. Hence, we sought to develop a non-surgical mouse model of IVD degeneration using genetically-induced targeted ablation of disc-resident cells that express aggrecan (Acan), the main proteoglycan component found in the extracellular matrix of the IVDs. Methods: We crossed heterozygous mice that express Cre recombinase under the control of the aggrecan gene promoter (AcanCreERT2) with mice that express diphtheria toxin (DTA) following Cre recombination (Gt(ROSA)DTA). The Acan+/Cre ;DTA+/- offspring received tamoxifen (4-OHT) interperitoneally for 5 (five) consecutive days to promote the targeted ablation of Acan-expressing cells. Animals were euthanized, and spines were harvested at 1-, 5-, 9- and 12-weeks post-DTA induction for histology. Sagittal sections were stained with Safranin-O/Fast-green and compared to littermate controls lacking the Cre allele (Acan+/+;DTA+/-) who received the same treatment, to allow a detailed spatiotemporal analysis of changes in the IVDs structure and composition. Long-term histological assessment was also performed at 7-months post-DTA induction, to investigate IVD degenerative changes with aging. Results: Histological analysis revealed mild degenerative changes of the IVDs by 5-weeks post-DTA induction in the Acan+/Cre:DTA mice, with the cells in the NP demonstrating a hypertrophic phenotype by 12-weeks, with few to no notochordal cells identified. Lumbar IVDs exhibited profound histological changes by 7-months post-DTA induction when compared to controls (Figure 1). These changes included decreased cellularity, loss of NP/AF borders, altered AF lamellar fiber structure, and disruption of NP matrix with loss of proteoglycan staining, and are typical characteristics of severe disc degeneration. Conclusions: Targeted cell depletion within the IVD resulted in progressive histological changes in the structure and composition of the NP, with clear degeneration of the AF compared to controls, reaching severe IVD degeneration by 7-months post-DTA induction. This novel mouse model of IVD degeneration does not require direct injury to the IVD; thus, it may contribute to further understanding the mechanisms underlying spontaneous and/or age-related degeneration of IVD, which more closely mimic the progression of human disease. Considering the structure-function relationship of this tissue, future studies should explore the mechanical behavior of IVDs to assess how the overall structural degeneration seen histologically translates into the biomechanical properties of the tissue.

Serum Decorin and Biglycan as Potential Biomarkers to Predict PPROM in Early Gestation.

Preterm birth is a leading cause of neonatal mortality in the US and globally, with preterm premature rupture of fetal membranes (PPROM) accounting for one third of preterm births. Currently no predictive diagnostics are available to precisely assess risk and potentially reduce the incidence of PPROM. Bigycan and decorin, the main proteoglycans present in human fetal membranes, are involved in the physiological maturation of fetal membranes as well as in the pathophysiology of preterm birth. The serum protein sex hormone-binding globulin (SHBG) has recently been identified as a predictor of spontaneous preterm birth. We hypothesize that the balance between serum decorin and biglycan on one hand and SHBG on the other hand may provide insight into the status of the fetal membranes in early pregnancy, thereby predicting PPROM prior to symptoms. Using chart review, 18 patients with confirmed cases of PPROM were identified from 2013-2016. Second trimester residual serum was retreived from freezer storage for these cases along with 5 matched controls for each case. The biomarkers biglycan, decorin and SHBG were analyzed first separately, then in combination to determine their ability to predict PPROM. The predictive score for the combined model displays an AUC = 0.774. The ROC curve of the predicted score has an optimal threshold of 0.238 and a sensitivity and specificity of 0.72 and 0.84 respectively. This prenatal serum panel is a promising serum screening-based biochemical model to predict PPROM in asymptomatic women.

The corneal wound healing and the extracellular matrix

The cornea is the first refractive element of the eye. The transparency of the cornea results from the regularly arranged collagen fibrils, forming lamellar structure and the leucin rich proteoglycans, which make interactions between the fibrils. The adult cornea consists mainly of fibril-forming collagens. The cornea has less amount of fibril associated and non-fibrillar collagens. The main proteoglycans of the cornea are keratan-sulfate proteoglycans and it also contains dermatan-sulfate proteoglycans. Disorders of the proteoglycan synthesis lead to the disruption of the unique pattern and result in thicker collagen fibrils. The abnormal structure of the extracellular matrix can generate corneal disorders and the loss of corneal transparency. Furthermore, proteoglycans and collagens have an important role in wound healing. In injury the keratocytes produce higher amounts of collagens and proteoglycans mediated by growth factors. Depending on the ratio of the cells and growth factors the extracellular matrix returns to normal or corneal scar tissue develops.

Tissue-specificity of proteoglycans expression in different cancers

Background. Proteoglycans (PGs) are complex glycosylated molecules playing an important role in cell-cell and cell-matrix interactions and signaling. Expression of PGs and their expression pattern change considerably during malignant transformation of mammalian cells and tissues. Objective. The aim of our work was to investigate tissue-specificity of main PGs expression (glypican-1, perlecan, syndecan-1, aggrecan, versican, CSPG4/NG2, brevican, decorin, lumican) in normal cells (fibroblasts and normal epithelial prostate cells PNT2) and in different human cancer cell lines (prostate, breast, lung, brain, kidney). Expression patterns of main PGs were determined in these cells using reverse transcription polymerase chain reaction analysis and immunocytochemical staining. Results. It was shown that fibroblasts actively expressed PGs, and PNT2 cells had lower (5–6-fold) expression levels of a limited set of PG. In different cancer cell lines, overall transcriptional activities of PGs varied up to 10-fold, although their expression patterns had tissue-specific properties (for example, expression of syndecan-1 is more specific for prostate cancer cells, while perlecan is typical for lung cancer cell lines). Conclusions. Along with this, variability of the PG expression patterns in cell lines of the same tissue of origin was shown, suggesting a possible contribution of the variable PGs expression to intratumoural heterogeneity of cancer cells and their potential as perspective biomarker (s) for personalised cancer diagnostics.

P11: Proteoglycans expression correlates with the phenotype of malignant and non-malignant EBV-positive B-cell lines

In vitro Epstein-Barr virus (EBV) infects B-cells resulting in immortalization, In vivo the virus resides latent in resting B-cells. Rarely the EBV-host cell interaction may contribute to development of malignant lymphoma. It is well known that both c-myc translocation and the viral infection are observed in patients with EBV+ Burkitt’s lymphoma (BL). Proteoglycans (PGs) are complex glycosylated proteins. They are key components of ECM and play a critical role in cell–cell and cell–matrix interactions. Disruptions of such interactions will affect B cell interaction with surrounding stroma and may thus perturb the cell phenotypes. The purpose of this study was to investigate expressinon of proteoglycans in EBV+ cell lines with different origins and phenotypes. We analysed the expression of 12 of the main proteoglycans in primary B cells, lymphoblastoid cell lines (LCLs) generated by EBV infection of normal human B cells in vitro and EBV-positive BL cell lines. An EBV-negative BL cell line was used for reference. According to RT-PCR analysis, primary B-lymphocytes expressed different PGs, mainly serglycin, CD44, perlecan and syndecan-1. The high expression of PGs in normal B cells probably reflects interactions of these cells with the neighbouring cells and microenvironment. B cell lines which carry EBV, in general, showed lower levels of PGs. The PGs expression pattern was similar in LCLs and in primary B cells, however, distinguished by high levels of perlecan and serglycin and low expression of CD44 in LCLs. BL cells showed the most significant down-regulation of PGs compared to primary B cells. There was a correlation between the type of EBV latency program, and PGs expression. Serglycin was expressed at a low levels in BL-cells with EBV latency III-program compared to LCLs, while in EBV latency I BL cells both serglycin and perlecan were down-regulated. Cells with latency I-program show general hypermethylation of the cellular genome in contrast to cells with latency III-program. Thus we explored the possibility of epigenetic regulation of the PG-coding genes by treating cells with 5′-deoxyazacytidine (5-AzaC, a demethylating agent) and Trichostatin A (TSA, a chromatin structure modulator). There was no significant change in PGs expression upon this treatment in LCLs or in EBV latency III BL cells, while EBV latency I BL cells showed up-regulation of several PGs. This suggests that PGs expression is at least partly regulated by epigenetic mechanisms. Interestingly EBV latency is also partly regulated at the epigenetic level. Similar trends were observed for the key ECM components (collagen 1A1, fibronectin and elastin). Normal B lymphocytes expressed collagen, fibronectin and elastin, whereas LCLs and BL cells showed no expression of these. Treatment of these cells with 5-AzaC or TSA resulted in similar changes in PGs expression patterns. Up-regulation of ECM components was detected only in EBV latency I BL cells. Taken together, our data show that proteoglycans are expressed in primary B lymphocytes whereas they are not or only partly expressed in EBV-carrying cell lines, depending on their latency program. Expression of PGs in latency I BL cells is silenced due to hypermethylation, but by another mechanim in latency III BL cells. These results show that PGs expression patterns follow the EBV latency programs . It will be highly interesting to further investigate if EBV and its transformation associated genes are directly involved in control of PGs, as well as how PGs may contribute to major phenotypic properties of EBV-carrying cell lines, such as adhesion, migration and growth in soft agarose – a property associated with the malignant phenotype of BLs.

T58: An important role of proteoglycans in the interactions of normal and cancer prostate cells with fibroblasts

Over time, study of malignant tumours was concentrated on cancer cells only. Now, however, an important role of cancer cell microenvironment and interactions cell with extracellular matrix (ECM) is becoming evident. Tumour cells modify normal ECM to cancer-associated matrix (CAM) thereby creating favorable conditions for the growth and development of tumors. One of the main cellular components of ECM are fibroblasts that synthesise extracellular matrix components including proteoglycans (PG) – complex glycosylated molecules consisting of core protein and one or more carbohydrate chains of glycosaminoglycans. PGs are expressed on the cell surface and in extracellular matrix for all mammalian cells and tissues, playing an important role in cell–cell and cell–matrix interactions and signaling. We have hypothesised that proteoglycans are related to transformation of normal ECM to cancer-associated matrix under the influence of prostate cancer cells, and understanding of the mechanism this transformation is very important issue. Thus, the aim of our work was to investigate a possible involvement of proteoglycans in the transformation normal fibroblasts to cancer-associated fibroblasts (CAFs). To study this problem, we co-cultivated fibroblasts with normal prostate cells (PNT2) and prostate cancer cells with different physiological characteristics (LNCaP, PC3, DU145) with subsequent separation of the cells and proteoglycans expression analysis. Firstly, we co-cultivated human fibroblasts with normal prostate epithelial cell PNT2. Expression patterns of main proteoglycans (syndecan-1, glypican-1, perlecan, aggrecan, versican, decorin, lumican, CSPG4/NG2, brevican) were determined in fibroblasts before and after co-culture with PNT2 cells using RT-PCR analysis. It was shown that after co-culture the total level of proteoglycans expression in fibroblasts was reduced by 2-fold, although the fibroblasts expressed a similar set of proteoglycans with the exception of CSPG4/NG2 (which was completely disappeared). The results suggest that normal prostate cells PNT2 affect composition and transcriptional activity of proteoglycans in PNT2-exposed fibroblasts compared with fibroblast monoculture, and this is a normal process of mutual adaptation of these cells to each other. At the next step, fibroblasts were co- cultivated with prostate cancer cells of various rate of aggressiveness (hormone-dependent non-metastatic LNCaP cells, hormone-independent metastatic PC3 and DU145 cells). Co-culture fibroblasts with LNCaP cells resulted in significant down-regulation of expression of glypican-1 and up-regulation of CSPG4/NG2 expression in the fibroblasts, changing overall proteoglycans expression pattern in the cells. Interestingly, CSPG4/NG2 expression is more characteristic for nerve cells and cancer stem cells, and up-regulation of the gene in the cancer cell-exposed fibroblasts may argue for their dedifferentiation. Co-culture fibroblasts with more aggressive DU145 and PC3 resulted in similar results with an additional down-regulation of versican expression. This data show, that prostate cancer cells affect normal fibroblasts by the other way than normal prostate cells and stimulate them to express another set of proteoglycans, which usually are expressed in poor-differentiated or morphologically different cells. Data obtained by immunocytochemical analysis confirmed RT-PCR data and showed specific activation of some proteoglycans at the region of contact between fibroblasts and normal but not cancer prostate cells in co-culture. Taken together, obtained results show that normal prostate epithelial cells affect surrounding fibroblasts and modify transcriptional activities of proteoglycan-coding genes in the cells, while cancer cells change the proteoglycans expression in cancer cells-exposed fibroblasts seems contributing to reprogramming of normal fibroblasts to CAFs. Understanding the mechanisms of this reprogramming may help to develop a new approach for cancer therapy based on the return reprogramming of CAFs to normal fibroblasts and correction of tumour microenvironment. The work was supported by the research Grant from Ministry of Education and Science of the Russian Federation, Scholarship of Russian Federation President for young scientists (Suhovskih A.V.), FEBS Short Term Fellowships (AVS) and UICC International Cancer Technology Transfer Fellowships (EVG, ICR/2015/353372).

Fibroblasts Specifically Affect Proteoglycan Expression in Different Prostate Cancer Cells

ABSTRACT Aim: Over time, the study of malignant tumours has concentrated on cancer cells only. However, the important role of the cancer cell microenvironment and cell-extracellular matrix (ECM) interactions is becoming evident. One of the main components of ECM is proteoglycans (PGs) - complex glycosylated molecules, which are expressed on cell surface and in extracellular matrix, and play an important role in cell-cell and cell-matrix interactions and signaling. The aim of our work was to investigate an effect of fibroblasts on proteoglycans expression in morphologically different prostate cancer cells. Methods: Normal human fibroblasts were co-cultured with normal (PNT2) and prostate cancer cells (PC3, LNCaP, DU145) in cells co-culture model in vitro. Expression and localisation of main proteoglycans (Synd-1, Gly-1, Perl, Vers, Brev, NG2, CD44, Sergl, Dec, Agg, Bigl, Lum) were determined in the cell lines before and after magnetic separation of the cells using real-time RT-PCR analysis and immunocytochemical staining. Results: It was shown that co-cultivation with stromal fibroblasts downregulates PGs expression in normal PNT2 cells at total expression level and change their expression pattern. Androgen-dependent non-metastatic LNCaP cells had similar PGs expression level but their response to the fibroblast influence was distorted. Androgen-independent metastatic PC3 and DU145 cells were characterized by significant decrease of PGs expression and the cells were not able to react to the fibroblast influence. However, in spite of the shown similar effects of the fibroblasts to PC3 and DU145 prostate cancer cells at mRNA level, the cell lines revealed differential reaction to fibroblasts at the protein level. Conclusions: Collectively, obtained data show a different ability of aggressive and non-aggresive prostate cancer cells to respond to the stromal fibroblasts, supporting an important role of ECM in prostate carcinogenesis and suggesting proteoglycans as possible microenviromental biomarkers for prostate tumour diagnosis and treatment. Disclosure: All authors have declared no conflicts of interest.

Lysophosphatidylcholine and Carotid Intima-Media Thickness in Young Smokers: A Role for Oxidized LDL-Induced Expression of PBMC Lipoprotein-Associated Phospholipase A2?

BackgroundAlthough cigarette smoking has been associated with carotid intima-media thickness (CIMT) the mechanisms are yet not completely known. Lysophosphatidylcholine (lysoPC), a main product of lipoprotein-associated phospholipase A2 (Lp-PLA2) activity, appears to be a major determinant of the pro-atherogenic properties of oxidized LDL (oxLDL) and to induce proteoglycan synthesis, a main player in intimal thickening. In this study we assessed whether cigarette smoking-induced oxidative stress may influence plasma Lp-PLA2 and lysoPC and Lp-PLA2 expression in peripheral blood mononuclear cells (PBMC), as well as the relationship between lysoPC and CIMT.Methods/Results45 healthy smokers and 45 age and sex-matched subjects participated in this study. Smokers, compared to non-smokers, showed increased plasma concentrations of oxLDL, Lp-PLA2 and lysoPC together with up-regulation of Lp-PLA2 (mRNA and protein) expression in PBMC (P<0.001). Plasma Lp-PLA2 positively correlated with both lysoPC (r=0.639, P<0.001) and PBMC mRNA Lp-PLA2 (r=0.484, P<0.001) in all subjects. Moreover CIMT that was higher in smokers (P<0.001), positively correlated with lysoPC (r=0.55, P<0.001). Then in in vitro study we demonstrated that both oxLDL (at concentrations similar to those found in smoker’s serum) and oxidized phospholipids contained in oxLDL, were able to up-regulate mRNA Lp-PLA2 in PBMC. This effect was likely due, at least in part, to the enrichment in oxidized phospholipids found in PBMC after exposure to oxLDL. Our results also showed that in human aortic smooth muscle cells lysoPC, at concentrations similar to those found in smokers, increased the expression of biglycan and versican, two main proteoglycans.ConclusionsIn smokers a further effect of raised oxidative stress is the up-regulation of Lp-PLA2 expression in PBMC with subsequent increase of plasma Lp-PLA2 and lysoPC. Moreover the correlation between lysoPC and CIMT together with the finding that lysoPC up-regulates proteoglycan synthesis suggests that lysoPC may be a link between smoking and intimal thickening.

A novel fluorimetric assay for detection of aggrecanase‐1 (ADAMTS‐4) activity using a long wavelength FRET peptide substrate

Aggrecanase‐1, also called ADAMTS‐4, is a member of the ADAMTS (A disintegrin and metalloprotease with thrombospondin motif) family of proteases. Aggrecanase cleaves specific peptide bonds in aggrecan, the main proteoglycan of articular cartilage. The high activity of aggrecanase results in cartilage degradation in diseases such as arthritis. To develop homogeneous high throughput screening of ADAMTS‐4 inhibitors, we synthesized a novel internally quenched peptide substrate for aggrecanase‐1 consisting of 5‐FAM/TAMRA as the FRET pair. Active ADAMTS‐4 cleaves this FRET substrate into two separate fragments resulting in an increase of 5‐FAM fluorescence, monitored at excitation/emission = 490 nm/520 nm. The new FRET substrate is more sensitive to aggrecanase‐1 cleavage than the previously described Abz/Dnp substrate and can detect subnanogram amounts of ADAMTS‐4. Its cleavage by other members of aggrecanase family, such as ADAMTS‐1 and ADAMTS‐5, is negligible. Inhibitor screening assays with several known metalloprotease inhibitors were validated using this substrate.This novel FRET substrate provides a convenient homogeneous format for aggrecanase‐1 activity assay where signal is less interfered by the autofluorescence of test compounds due to the long wavelength excitation and emission of 5‐FAM, resulting in an overall increase of signal to background ratio.

Effect of acute resistance exercise and sex on human patellar tendon structural and regulatory mRNA expression

Tendon is mainly composed of collagen and an aqueous matrix of proteoglycans that are regulated by enzymes called matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Although it is known that resistance exercise (RE) and sex influence tendon metabolism and mechanical properties, it is uncertain what structural and regulatory components contribute to these responses. We measured the mRNA expression of tendon's main fibrillar collagens (type I and type III) and the main proteoglycans (decorin, biglycan, fibromodulin, and versican) and the regulatory enzymes MMP-2, MMP-9, MMP-3, and TIMP-1 at rest and after RE. Patellar tendon biopsy samples were taken from six individuals (3 men and 3 women) before and 4 h after a bout of RE and from a another six individuals (3 men and 3 women) before and 24 h after RE. Resting mRNA expression was used for sex comparisons (6 men and 6 women). Collagen type I, collagen type III, and MMP-2 were downregulated (P < 0.05) 4 h after RE but were unchanged (P > 0.05) 24 h after RE. All other genes remained unchanged (P > 0.05) after RE. Women had higher resting mRNA expression (P < 0.05) of collagen type III and a trend (P = 0.08) toward lower resting expression of MMP-3 than men. All other genes were not influenced (P > 0.05) by sex. Acute RE appears to stimulate a change in collagen type I, collagen type III, and MMP-2 gene regulation in the human patellar tendon. Sex influences the structural and regulatory mRNA expression of tendon.

Proteoglycan-collagen interactions.

Among the most important events in connective tissue physiology are the nucleation, growth and calcification of collagen fibrils. It has been speculated that all are associated with, or even controlled by collagen-proteoglycan interactions. We therefore developed methods for investigating these associations in tissues, particularly for understanding their significance for type I collagen, the commonest form of collagen in the body, especially predominant in bone. Using an electron-dense dye, Cupromeronic blue, in the 'critical electrolyte concentration' mode, and digestion by hyaluronidase, chondroitinase ABC or keratanase, supported by biochemical analyses, we found that dermatan sulphate proteoglycan of soft connective tissue (skin, tendon, cornea) was regularly and orthogonally arrayed at the fibril surface, at the d or e band. Keratan sulphate proteoglycan in the cornea associates orthogonally at the a and c bands. Bone, demineralized by a non-aqueous technique which retains proteoglycans in the tissue, does not contain orthogonal arrays; the interfibrillar proteoglycan filaments are oriented parallel to the fibril axis. The main proteoglycan in bone is chondroitin sulphate-rich. There are thus four separate specific binding sites on type I collagen fibrils, each one associating with one particular proteoglycan, and apparently no other. This implies that there are two corresponding binding sites in each proteoglycan. Available evidence shows that there are two species of small dermatan sulphate and keratan sulphate proteoglycans. It is suggested that each species is specific for its own band (a, c, d or e). Hyaluronate and chondroitin sulphate are probably mainly interfibrillar, acting in a space-filling capacity.

Pre-eclampsia-associated alterations in decorin, biglycan and versican of the umbilical cord vein wall

Objective The role of proteoglycans in the rearrangement of the extracellular matrix of the umbilical cord vein wall in pre-eclampsia is not known. Decorin, biglycan and versican are the main proteoglycans of the umbilical cord vein wall. We decided to test whether the amounts of these proteoglycans alter in pre-eclampsia. Study design Study was performed on the umbilical cord veins taken from 10 newborns delivered by healthy mothers (control group) and from 10 newborns delivered by mothers with pre-eclampsia. Proteoglycans were extracted in dissociative conditions, purified by Q-Sepharose anion exchange chromatography and lyophilised. Decorin, biglycan and versican were analysed by SDS-PAGE followed by Western blotting before and after treatment with chondroitinase ABC. The amounts of decorin, biglycan and versican core proteins were assessed by ELISA method. Results We found that both control and pre-eclamptic umbilical cord vein wall contained all the three proteoglycans. ELISA assay showed the amounts of the core proteins of decorin, biglycan and versican were distinctly higher in pre-eclamptic material in comparison to control vessel. Western blotting confirmed that the expression of all these proteoglycan core proteins increased in pre-eclampsia. They featured in the same electrophoretic mobility—45 and 47 kDa for decorin, 45 kDa for biglycan, and 300 and 320 kDa for versican. Conclusion The content of decorin, biglycan and versican in the umbilical cord vein wall is elevated in pre-eclampsia in comparison to the corresponding control vessel. These alterations may affect the mechanical properties of this vessel and disturb foetal blood circulation.

Proteolytic enzymes in skeletal development: histochemical methods adapted to the study of matrix lysis during the transformation of a "cartilage model" into bone

The replacement of a "cartilage model" by definitive bone is characterized by a series of localized excavations of the cartilage which are eventually followed by bone deposition. Each excavation requires lysis of cartilage components (defined here as the breakdown of a peptide bond) and their eventual resorption (defined here as microscopical visible cartilage loss). More precisely we have proposed that the lysis is affected by proteases capable of breaking down the main proteoglycan "aggrecan" and the main fibril element, "type II collagen". Four approaches combining biochemical, immunologic and microscopic techniques have been adapted to test this hypothesis. Each is applied to the rat tibial head's "cartilage model" where proteases have been shown to be major contributors to secondary ossification center formation. The approaches have been found both effective and distinct as cartilage resorbing enzymes have not only been identified but also detected in situ before and after activation. Achieved overall is an understanding of when, where and how specified proteases contribute to tissue component lyses. While the focus resides on the in situ proteolysis of cartilage, three of the approaches could be translated without change to other tissues, whereas one may require tissue specific adjustments before use.

Structural Change in Decorin with Skin Aging

Decorin, the main proteoglycan in skin, has a small size with a core protein of ∼ 40kDa and one chondroitin sulfate/dermatan sulfate glycosaminoglycan (GAG) chain. The main function of decorin is to regulate the collagen matrix assembly. Decorin is distributed along collagen fibrils with the core protein and the decorin GAG chain controls the distance between the collagen fibrils. Reducing the length of the decorin GAG chain reduces the distance between the collagen fibrils. Age-related changes in decorin are apparent in the GAG chain in respect to the molecular size and sulfate position but not in the core protein. Structural changes in the decorin GAG chain may be involved in changes in collagen matrix assembly during the aging process.