Rate Of Proteoglycan Synthesis Research Articles

Cyanidin attenuates the high hydrostatic pressure-induced degradation of cellular matrix of nucleus pulposus cell via blocking the Wnt/β-catenin signaling

PurposeThe treatment of intervertebral disc degeneration is limited, cyanidin can protect chondrocytes from degeneration. This research investigated the effect of cyanidin on human nucleus pulposus cells (HNPC) metabolism and its mechanism. MethodsHNPC were treated with cyanidin, XAV-939 (inhibitor of Wnt/β-catenin signaling) and SKL2001 (activator of Wnt/β-catenin signaling), and pressurized at 1, 3, and 30 atm. Quantitative real-time PCR and Western blot were used to assess the expressions of matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), Collagen-II, Aggrecan, Wnt-3a and β-catenin in treated HNPC. Cell counting kit-8 was used to detect the HNPC cell viability. Radioisotope incorporation method was used to assess the proteoglycan synthesis rate of HNPC. The level of cell matrix was detected by toluidine blue staining. ResultsProper hydrostatic pressure of 3 atm could elevate cell viability, proteoglycan synthesis and the level of cell matrix of HNPC, while high hydrostatic pressure of 30 atm reduced the above effects. Cyanidin and XAV-939 reversed the promoting effect of 30 atm pressure on Wnt/β-catenin signaling pathway and the inhibiting effect on cell viability, proteoglycan synthesis and the level of cell matrix. Subsequently, SKL2001 further reversed the function of cyanidin on HNPC. ConclusionCyanidin attenuated the high hydrostatic pressure-induced degradation of cellular matrix of HNPC via blocking the Wnt/β-catenin signaling pathway. Our findings in this research provided a basis for in vitro experiment of cyanidin and a theoretical fundament on this disease.

Proteoglycan synthesis rate as a novel method to measure blood-induced cartilage degeneration in non-haemophilic and haemophilic rats.

IntroductionHaemophilic animal models are used to study blood‐induced cartilage damage, but quantitative and sensitive outcome measures are needed.AimTo develop a novel quantitative method for detecting early cartilage degeneration in a haemophilic rat model of blood‐induced joint damage.MethodsThe 35Sulphate incorporation (35SO4 2− assay) was applied to tibial and patellar cartilage of wild‐type rats to quantify baseline proteoglycan synthesis and to evaluate the effect of 4‐day blood exposure in vitro. Next, haemarthrosis was induced in 39 FVIII‐deficient rats and characterized by changes in knee joint diameter and development of bone pathology (using micro‐CT). Four‐ and 16‐day posthaemarthrosis proteoglycan synthesis rate (PSR) was assessed using the 35SO4 2− assay, with the contralateral knee as control.ResultsIn vitro, a decrease in PSR in tibial and patellar cartilage was demonstrated following blood exposure. In vivo, joint diameter and development of bone pathology confirmed successful induction of haemarthrosis. In the blood‐exposed knee, tibial and patellar PSR was inhibited 4 and 16 days after induced haemarthrosis. Interestingly, at day 16 the proteoglycan synthesis in the contralateral knee was also inhibited to an extent correlating with that of the blood‐exposed knee.ConclusionFor the first time, early changes in cartilage matrix synthesis upon blood exposure were quantified with the 35SO4 2− assay in a haemophilic rat model, establishing this assay as a novel method to study blood‐induced cartilage damage.

Pathobiochemistry of arthrofibrotic remodeling

Aims and Objectives:Arthrofibrosis is defined as painful impairment of joint flexibility due to fibrotic tissue remodeling after joint trauma or surgery. The incidence of arthrofibrosis after knee replacement surgery is 5 to 10%. Although conventional therapeutic approaches as for instance mobilization and physiotherapy are applied, an effective and causative therapeutic regimen is not known.Materials and Methods:To characterize arthrofibrotic remodeling of the extracellular matrix, to develop new therapeutic approaches and to define diagnostic biomarkers and therapeutic targets, understanding of biochemical principles is urgently required. Fibrotic remodeling was described in several tissues, whereas synovial fibrosis is one of the least investigated fibrotic disorders. Nevertheless, molecular key events in fibrosis seem to be the same and are initiated by exogenic or endogenic tissue damage and differentiation of resident fibroblasts of the connective tissue to myofibroblasts. Known inductors of myofibroblast differentiation are fibrotic growth factors, which are secreted by platelets, damaged tissue and inflammatory cells, as well as mechanical strain. Research studies concerning cardiac fibrosis in tako-tsubo cardiomyopathy also define emotional stress and sympathicotonic destabilization as profibrotic stressors. Myofibroblasts generate contractile forces and synthesize extracellular matrix components, so that scar tissue accumulates. While myofibroblasts disappear by apoptosis in physiological wound healing, they persist in fibrosis.Results:Recently, we could demonstrate that increased expression of human xylosyltransferase (XT)-I, an enzyme which catalyzes the rate limiting step in proteoglycan glycosylation, is linked to abnormal extracellular matrix remodeling. Serum XT activity reflects proteoglycan synthesis rate and is known as fibrosis biomarker in liver fibrosis or scleroderma. Our data also indicate that XT-I is a cellular key mediator of arthrofibrosis. However, we suggest that molecular changes based on arthrofibrosis are, due to local restriction of the affected joint by the blood-synovial-barrier, not detectable in human serum. Currently, we study synovial XT activity of arthrofibrosis patients and controls in a multicenter study.Conclusion:In summary, we give insights into the complex pathobiochemistry of arthrofibrosis as well as current research projects. A deeper characterization of the involved mechanisms might not only contribute to control and inhibit fibrotic remodeling by interfering with components of fibrotic signal cascades but also to establish new therapeutic strategies.

Identification and characterization of human xylosyltransferase II promoter single nucleotide variants

The human isoenzymes xylosyltransferase-I and -II (XT-I, XT-II) catalyze the rate-limiting step in proteoglycan biosynthesis. Therefore, serum XT activity, mainly representing XT-II activity, displays a powerful biomarker to quantify the actual proteoglycan synthesis rate. Serum XT activity is increased up to 44% in disorders which are characterized by an altered proteoglycan metabolism, whereby underlying regulatory mechanisms remain unclear. The aim of this study was to investigate new regulatory pathways by identifying and characterizing naturally occurring XYLT2 promoter sequence variants as well as their potential influence on promoter activity and serum XT activity.XYLT2 promoter single nucleotide variants (SNVs) were identified and genotyped in the genomic DNA of 100 healthy blood donors by promoter amplification and sequencing or restriction fragment length polymorphism analysis. The SNVs were characterized by an in silico analysis considering genetic linkage and transcription factor binding sites (TBSs). The influence of SNVs on promoter activity and serum XT activity was determined by dual luciferase reporter assay and HPLC-ESI mass spectrometry.Allele frequencies of seven XYLT2 promoter sequence variants identified were investigated. In silico analyses revealed a strong genetic linkage of SNVs c.-80delG and c.-188G > A, c.-80delG and c.-1443G > A, as well as c.-188G > A and c.-1443G > A. However, despite the generation of several SNV-associated changes in TBSs in silico, XYLT2 promoter SNVs did not significantly affect promoter activity. Serum XT activities of SNV carriers deviated up to 8% from the wild-type, whereby the differences were also not statistically significant.This is the first study which identifies, genotypes and characterizes XYLT2 promoter SNVs. Our results reveal a weak genetic heterogeneity and a strong conservation of the human XYLT2 promoter region. Since the SNVs detected could be excluded as causatives for strong interindividual variabilities in serum XT activity, our data provide increasing evidence that XT-II activity is obviously regulated by hitherto unknown complex genetic pathways, such as cis- or trans-acting enhancers, silencers or miRNAs.

Interleukin-1β Is Essential for Blood-Induced Cartilage Damage In Vitro

Objective: Joint damage due to recurrent joint bleeds remains the most common complication in hemophilia. The combination of cartilage degeneration and synovial inflammation ultimately lead to hemophilic arthropathy. Cartilage destruction is considered to result from both cartilage matrix-degrading proteases as well as cartilage-destructive pro-inflammatory cytokines such as interleukin (IL)-1b. To unravel the role of IL1b in the pathogenesis of blood-induced cartilage damage, we investigated whether direct blocking of IL1b specifically prevents blood-induced cartilage damage in vitro. Moreover, we investigated whether blocking IL1b after the onset of a bleed can still avert cartilage damage.Methods: Full thickness healthy human articular cartilage explants, obtained post-mortem, were cultured for four days in presence or absence of 50% v/v whole blood. A recombinant human IL1b monoclonal antibody (IL1bmAb) was added at the moment of blood exposure in a concentration of 0, 1, 3, 10, 30, or 100ng/mL (n=8). Furthermore, 100ng/mL IL1bmAb was administered directly or after a delay of several hours up to two days (n=7). Cartilage matrix proteoglycan turnover was determined 12 days later to analyse long-term effects.To investigate the direct effects of IL1bmAb on cartilage, explants were cultured for four days in the presence of 10ng/mL IL1bmAb in the absence of blood (n=7).Results: Exposure to blood decreased the proteoglycan synthesis rate and -content, and increased the proteoglycan release (all p<0.05). Adding IL1bmAb resulted in a dose-dependent increase of the proteoglycan synthesis rate leading to normalisation at higher concentrations (see figure A). Proteoglycan release and -content also normalised after addition of IL1bmAb. Its protective effect was most pronounced when IL1bmAb was administered within 4 hours after the bleed, but significant recovery was still achieved by administration within 8-24 hours (see figure B for proteoglycan synthesis). In the absence of blood, IL1bmAb did not have direct effects on cartilage proteoglycan turnover (for all three parameters p>0.50).Conclusions: This study demonstrates that IL1b is a crucial factor in the development of blood-induced cartilage damage in vitro. Blocking this pro-inflammatory cytokine with a monoclonal antibody protects cartilage from the damaging effects of blood exposure in a dose-dependent way. Early administration after blood-exposure is most beneficial. Further research is warranted to investigate the in vivo capacity of IL1bmAb in prevention and its position as a treatment to limit joint damage upon joint bleeding. [Display omitted] Figure -Effect ofIL1bmAbon cartilage proteoglycan synthesis rate is dose-dependent (A) and time-dependent (B). Median values ± IQR of at least 7 individual cartilage and blood donors are shown. Hash tags indicate a statistically significant difference from control values, whereas asterisks indicate a statistically significant difference from blood-exposed cartilage withoutIL1bmAb addition(p<0.05). DisclosuresSchutgens:CSL Behring: Research Funding.

Long non-coding rnas in osteoarthritis and chondrogenesis

Long non-coding rnas in osteoarthritis and chondrogenesis

Interleukin-1B IS essential for blood-induced cartilage damage in vitro

Purpose: Exposure of joint cartilage to blood can occur after joint trauma, during or after major joint surgery, or due to hemophilia. This ultimately leads to joint damage, both by direct effects of blood on cartilage and via synovial inflammation. Cartilage destruction is considered to result from both cartilage matrix-degrading proteases as well as cartilage-destructive pro-inflammatory cytokines such as IL1β. To unravel the role of IL1β in the pathogenesis of blood-induced cartilage damage, we investigated whether direct blocking of IL1β specifically prevents blood-induced cartilage damage in vitro. Methods: Full thickness healthy human articular cartilage explants, obtained post-mortem, were cultured for 4 days in presence or absence of 50% v/v whole blood. A recombinant human IL1β monoclonal antibody (IL1β mAb) was only added during blood exposure in a concentration of 0, 1, 2, 10, 30, or 100 ng/mL. Cartilage matrix proteoglycan turnover was determined 12 days later to analyse long-term effects. Moreover, to investigate the direct effects of IL1β mAb on cartilage, explants were cultured for 4 days in the presence of 10 ng/mL IL1β mAb in the absence of blood. Results: Exposure to blood decreased the proteoglycan-synthesis rate (-62%) and proteoglycan content (-19%), and increased the proteoglycan release (+191%, all p < 0.05). Adding IL1βmAb resulted in a dose-dependent increase of the proteoglycan synthesis rate leading to normalisation at higher concentrations (see figure A). Moreover, proteoglycan release was statistically significantly decreased from a concentration of 3ng/mL and above (see figure B). Similar, the proteoglycan content increased statistically significantly upon addition of the IL1βmAb (3 ng/mL and above; all p < 0.05). In the absence of blood, IL1βmAb did not have direct effects on cartilage proteoglycan turnover (p = 0.51). Conclusions: This study demonstrates that IL1β is a crucial factor in the development of blood-induced cartilage damage in vitro. Blocking this pro-inflammatory cytokine with a monoclonal antibody protects cartilage from the damaging effects of blood exposure. Further research is warranted to investigate the in vivo capacity of IL1β mAb in prevention and its position as a treatment of hemophilic arthropathy.

A fusion protein of IL4 and IL10 (IL4-10 synerkine), is very effective in protecting cartilage from blood-induced damage

Purpose: Interleukin 4 (IL4), IL10 or the combination of both protects cartilage from blood-induced damage in vitro. Because IL4 and IL10 use different signaling pathways they are able to exert different, but also potentially additive effects. It has been postulated that they can act in synergy enhancing their beneficial effects and controlling their individual adverse effects. Therefore a combination of IL4 and IL10 might be the best option to administer in vivo. To overcome the low bioavailability of IL4 and IL10 in vivo, a fusion protein has been developed: IL4-10 synerkine. It has been demonstrated that this novel drug inhibits numerous pro-inflammatory cytokines secreted by monocytes and T-cells. This study investigates whether this IL4-10 synerkine protects against blood-induced cartilage damage similarly as the combination of the individual components. Methods: Human cartilage explants were exposed to 50% v/v whole blood for 4 days and simultaneously to a broad concentration range (0-100 ng/mL) of the IL4-10 synerkine. Effects of 10 ng/mL IL4-10 synerkine were compared to the same concentrations of the individual cytokines and the combination. Cartilage matrix proteoglycan turnover was assessed after a recovery period of 12 days. Moreover, the influence of IL4-10 synerkine (10 ng/mL) and its individual components on levels of IL1β and IL6 were investigated in a 4 days 50% v/v whole blood culture. Results: Exposure to 50% v/v whole blood resulted in a clear statistical significant decrease of proteoglycan (PG) synthesis rate (-74%; p = 0.012) and an increase of PG release (+92%, p = 0.017) compared to control. Adding of IL4-10 synerkine, resulted in a clear dose-response curve, leading to full normalisation of PG synthesis rate and -release at higher concentrations (>10 ng/mL). These results were similar to the effect of the two individual cytokines combined (see figure). The individual cytokines had a more distinct profile where IL4 was superior over IL10 (see figure). Moreover, addition of IL4-10 synerkine reduced IL1β and IL6 production in whole blood cultures to control levels (both p < 0.05), similar to the combination of IL4 plus IL10 (p = 0.180 and p = 0.173, respectively). Conclusions: IL4-10 synerkine strongly protects against blood-induced cartilage damage in vitro, presumably at least in part by reduction of pro-inflammatory cytokines including IL1β and IL6. Considering better bioavailability and more easy application of this synerkine compared to the individual cytokines, testing the IL4-10 synerkine in an in vivo model of blood-induced cartilage damage is warranted.

AB0135 Four-day continuous blood exposure leads to prolonged joint damage in a canine in vivo model, not the cumulative blood load of intermittent exposures

BackgroundIn a canine model, intra-articular blood injections twice a week during 4 weeks resulted in transient damage only (1). Human in vitro studies have shown that there is a threshold...

AB0134 Blood coagulation aggravates joint damage after an experimental haemorrhage in the canine knee

BackgroundJoint bleeding due to trauma, major joint surgery, or haemophilia leads to joint damage. However, it is unclear if there are differences between coagulating blood and anticoagulated blood with respect...

A short time window to profit from protection of blood-induced cartilage damage by IL-4 plus IL-10

IL-4 plus IL-10 prevents blood-induced cartilage damage. The aim of the present study was to evaluate whether cartilage damage can still be averted by addition of IL-4 plus IL-10 when added after the onset of a bleed and whether aspiration of blood prior to addition of IL-4 plus IL-10 is of additive protective value. Healthy canine hip and human shoulder cartilage was exposed to whole blood for 4 days. IL-4 plus IL-10 was administered directly or after a delay of several hours up to 2 days. Furthermore, blood was aspirated after 1 or 2 days and subsequently IL-4 plus IL-10 was added. IL-1β concentration and cartilage matrix proteoglycan turnover were determined. Exposure of canine and human cartilage to blood decreased the proteoglycan synthesis rate and content and increased proteoglycan release. IL-4 plus IL-10 only prevented blood-induced damage of canine cartilage when added directly, not after 4 h or later. For human cartilage, IL-4 plus IL-10 limited blood-induced damage as well as IL-1β production when administered within 4-8 h after the onset of a bleed, but not thereafter. Aspiration of blood within 24 h fully prevented cartilage damage. Subsequent addition of IL-4 plus IL-10 was not of additive value. For humans, there is a short time window after onset of a joint bleed in which IL-4 plus IL-10 can limit blood-induced cartilage damage. Furthermore, aspiration of a joint to shorten blood exposure fully prevents cartilage damage. Both options can be considered in the treatment of a joint haemorrhage.

Nonoperative management of ankle arthritis

Although hip and knee osteoarthritis are mostly of primary origin, ankle osteoarthritis is of posttraumatic origin. In sports injuries, the ankle is the second most commonly injured body site after the knee. In addition, compared to the cartilage in the knee joint, ankle cartilage has a higher content of proteoglycans and water, and an increased rate of proteoglycan turnover and synthesis, all of which are responsible for its increased incidence of posttraumatic origin osteoarthritis. Nonoperative management of ankle arthritis typically starts with weight reduction, activity modification, and oral non-steroidal anti-inflammatory drugs, physical therapy, and can progress to gait-aids including a cane, shoe-wear modification, patellar tendon weight-bearing ankle-foot orthosis, and intra-articular injections with corticosteroids or hyaluronic acid.

Homing of Mesenchymal Stem Cells in Induced Degenerative Intervertebral Discs in a Whole Organ Culture System

Homing of human bone marrow-derived mesenchymal stem cells (BMSCs) was studied using ex vivo cultured bovine caudal intervertebral discs (IVDs). To investigate in a whole organ culture whether metabolic and mechanical challenges can induce BMSC recruitment into the IVD. Cells from injured tissues release cytokines and mediators that enable the recruitment of progenitor cells. BMSCs have the ability to survive within the IVD. Bovine IVDs with or without endplates were cultured for 1 week under simulated physiological or degenerative conditions; disc cells were analyzed for cell viability and gene expression, whereas media was analyzed for nitric oxide production and chemotaxis. Homing of BMSCs was investigated by supplying PKH-labeled human BMSCs onto cultured IVDs (1 × 10(6) cells/disc on d 8, 10, and 12 of culture); on day 14, the number of homed BMSCs was microscopically assessed. Moreover, a comparative study was performed between transduced BMSCs (transduced with an adenovirus encoding for insulin-like growth factor 1 [IGF-1]) and nontransduced BMSCs. Disc proteoglycan synthesis rate was quantified via (35)S incorporation. The secretion of IGF-1 was evaluated by enzyme-linked immunosorbent assay on both simulated physiological and degenerative discs. Discs cultured under degenerative conditions showed reduced cell viability, upregulation of matrix degrading enzymes, and increased nitric oxide production compared with simulated physiological discs. Greater homing occurred under degenerative compared with physiological conditions with or without endplate. Media of degenerative discs demonstrated a chemoattractive activity toward BMSCs. Finally, discs homed with IGF-1-transduced BMSCs showed increased IGF-1 secretion and significantly higher proteoglycan synthesis rate than discs supplied with nontransduced BMSCs. We have demonstrated for the first time that degenerative conditions induce the release of factors promoting BMSC recruitment in an ex vivo organ culture. Moreover, IGF-1 transduction of BMSCs strongly increases the rate of proteoglycan synthesis within degenerative discs. This finding offers a new delivery system for BMSCs and treatment strategy for IVD regeneration.

Removal of Mitochondria-Generated Reactive Oxygen Species Delays Age-Associated Disk Degeneration

Introduction Reactive oxygen species (ROS) and free radicals are byproducts of oxidative metabolism that cause cellular damage and are implicated in the pathogenesis of age-related diseases. Mitochondria are the main cellular sites of ROS production. Recent studies reported a positive correlation between mitochondria dysfunction and age-associated disk cellular senescence and apoptosis. Hence, we hypothesize that reducing mitochondria-generated ROS would slow the onset of age-related intervertebral disk degeneration (IDD). We tested this hypothesis by chronically treating the Errc1-/D mouse model of accelerated aging with XJB-5-131, a mitochondria-targeted free radical scavenger previously demonstrated to be therapeutic in rodent models of hemorrhagic shock and sepsis. Materials and Methods Pairs of 5-week-old siblings of Ercc1-/▵ mice were treated with XJB-5-131 or oil carrier control (intraperitoneal injections, 2 mg/Kg, 3×/week, 15 weeks). Total proteoglycan content (safranin-O histology, DMMB assay) and synthesis (35S-sulfate incorporation) were measured. Transmission electron microscopy (TEM) was performed to assess disk mitochondria number and morphology in Ercc1-/▵ mice and wild-type littermates. Results TEM showed greater mitochondrial death in discs of Ercc1-/▵ mice compared to their wild-type littermates. Compared to oil-treated Ercc1-/▵ controls, disks from XJB-5-131 treated Ercc1-/▵mice showed greater (1.3×) proteoglycan matrix content and higher (1.9×) rate of proteoglycan synthesis. Conclusion Errc1-/Dmouse model of a human progeria had previously established as an accurate model of spine aging. Treatment of Errc1-/Dmice with a mitochondria-targeted ROS scavenger decreased disk proteoglycan loss. This result is consistent with our in vitro studies which showed XJB-5-131 is able to rescue reduced proteoglycan synthesis mediated by oxidative stress in human disk cells grown at high oxygen (20 vs. 5% oxygen). The findings in this study suggest that oxidative damage originating from mitochondria dysfunction plays an important role in disk health. The mitochondrial-targeted ROS free radical scavenger XJB-5-131 is potentially therapeutic in delaying the onset of age-related IDD. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Removal of Mitochondria-Generated Reactive Oxygen Species Delays Age-Associated Disk Degeneration

IntroductionReactive oxygen species (ROS) and free radicals are byproducts of oxidative metabolism that cause cellular damage and are implicated in the pathogenesis of age-related diseases. Mitochondria are the main cellular sites of ROS production. Recent studies reported a positive correlation between mitochondria dysfunction and age-associated disk cellular senescence and apoptosis. Hence, we hypothesize that reducing mitochondria-generated ROS would slow the onset of age-related intervertebral disk degeneration (IDD). We tested this hypothesis by chronically treating the Errc1-/D mouse model of accelerated aging with XJB-5-131, a mitochondria-targeted free radical scavenger previously demonstrated to be therapeutic in rodent models of hemorrhagic shock and sepsis. Materials and MethodsPairs of 5-week-old siblings of Ercc1-/▵ mice were treated with XJB-5-131 or oil carrier control (intraperitoneal injections, 2 mg/Kg, 3×/week, 15 weeks). Total proteoglycan content (safranin-O histology, DMMB assay) and synthesis (35S-sulfate incorporation) were measured. Transmission electron microscopy (TEM) was performed to assess disk mitochondria number and morphology in Ercc1-/▵ mice and wild-type littermates. ResultsTEM showed greater mitochondrial death in discs of Ercc1-/▵ mice compared to their wild-type littermates. Compared to oil-treated Ercc1-/▵ controls, disks from XJB-5-131 treated Ercc1-/▵mice showed greater (1.3×) proteoglycan matrix content and higher (1.9×) rate of proteoglycan synthesis. ConclusionErrc1-/Dmouse model of a human progeria had previously established as an accurate model of spine aging. Treatment of Errc1-/Dmice with a mitochondria-targeted ROS scavenger decreased disk proteoglycan loss. This result is consistent with our in vitro studies which showed XJB-5-131 is able to rescue reduced proteoglycan synthesis mediated by oxidative stress in human disk cells grown at high oxygen (20 vs. 5% oxygen). The findings in this study suggest that oxidative damage originating from mitochondria dysfunction plays an important role in disk health. The mitochondrial-targeted ROS free radical scavenger XJB-5-131 is potentially therapeutic in delaying the onset of age-related IDD.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declared

Changes in glycosaminoglycans and proteoglycans of normal breast and fibroadenoma during the menstrual cycle

BackgroundFibroadenoma is the most common breast tumor in young women, and its growth and metabolism may be under hormonal control. In the present paper we described the proteoglycan (PG) composition and synthesis rate of normal breast and fibroadenoma during the menstrual cycle. MethodsSamples of fibroadenoma and adjacent normal breast tissue were obtained at surgery. PGs were characterized by agarose gel electrophoresis and enzymatic degradation with glycosaminoglycan (GAG) lyases, and immunolocalized by confocal microscopy. To assess the synthesis rate, PGs were metabolic labeled by 35S-sulfate. ResultsThe concentration of PGs in normal breast was higher during the secretory phase. Fibroadenoma contained and synthesized more PGs than their paired controls, but the PG concentrations varied less with the menstrual cycle and, in contrast to normal tissue, peaked in the proliferative phase. The main mammary GAGs are heparan sulfate (HS, 71%–74%) and dermatan sulfate (DS, 26%–29%). The concentrations of both increased in fibroadenoma, but DS increased more, becoming 35%–37% of total. The DS chains contained more β‐d-glucuronic acid (IdoUA/GlcUA ratios were >10 in normal breast and 2–7 in fibroadenoma). The 35S-sulfate incorporation rate revealed that the in vitro synthesis rate of DS was higher than HS. Decorin was present in both tissues, while versican was found only in fibroadenoma. ConclusionsIn normal breast, the PG concentration varied with the menstrual cycle. It was increased in fibroadenoma, especially DS.General significancePGs are increased in fibroadenoma, but their concentrations may be less sensitive to hormonal control.

Intra-articular blood coagulation aggravates joint damage after a bleed in a canine in vivo model

Intra-articular blood coagulation aggravates joint damage after a bleed in a canine in vivo model

Regulation of the biphasic decline in scleral proteoglycan synthesis during the recovery from induced myopia

During the recovery from form deprivation myopia (myopic defocus), the rate of proteoglycan synthesis in the posterior sclera decreases co-incident with a deceleration of axial elongation. The choroid has been implicated in the regulation of scleral proteoglycan synthesis, possibly through the synthesis and secretion of scleral growth inhibitors. Therefore these studies were carried out to attempt to establish a causal relationship between choroidal secretion and the inhibition of scleral proteoglycan synthesis during the recovery from induced myopia. Chicks were form vision deprived for 10 days followed by a recovery period (3 h–20 days) of unrestricted vision. Sclera and choroids (5 mm punches) were isolated from control and treated eyes. The rate of proteoglycan synthesis was estimated by the incorporation of 35c in cetylpyridinium chloride-precipitable glycosaminoglycans by isolated sclera of control and treated eyes. Additionally, choroids from control and treated eyes were placed in co-culture with untreated age-matched normal chick sclera for 20–24 h, after which time sclera were removed and scleral proteoglycan synthesis rates were determined. Following removal of occluders, a biphasic decline was observed in scleral proteoglycan synthesis: A rapid decline in proteoglycan synthesis (−7.6% per hr; r2 = 0.923) was observed over the first 12 h of recovery, followed by a slow decline extending from 12 to 96 h (−0.3% per hr; r2 = 0.735). Proteoglycan synthesis rates gradually increased to control levels over the next 96 h at a rate of +0.3% per hr. No relative proteoglycan inhibition was observed when untreated sclera were co-cultured with choroids from eyes recovering for 0–4 days, whereas co-culture of untreated sclera with choroids from eyes recovering for 5 and 8 days resulted in significant inhibition of sclera proteoglycan synthesis, relative to that of sclera co-cultured with choroids from control eyes (≈−24%, P < 0.05, paired t-test). In conclusion, recovery from induced myopia is characterized by a rapid decline in proteoglycan synthesis which occurs within the first 12 h of unrestricted vision as a well as a slower more gradual decline that occurs over the next four days. Choroidal inhibition of scleral proteoglycan synthesis in vitro occurs during the second phase of decline and is most likely related to increased choroidal permeability; whereas the rapid decline in proteoglycan synthesis that occurs during the first 12 h of recovery is regulated by an independent, yet to be identified mechanism.

220 BRADYKININ, THROUGH B 2 RECEPTORS, ACTIVATES THE RELEASE OF THE CYTOKINE INTERLEUKIN 6, THE CHEMOKINE INTERLEUKIN 8, AND THE METALLOPROTEINASE 3 IN HUMAN KNEE CHONDROCYTES

Results: Cartilage cultured in the presence of blood showed a decrease of proteoglycan synthesis rate of 70%, an increase of proteoglycan release of 100%, and a decrease of proteoglycan content of 15% after 16 days of culture (all p<0.05). This blood-induced damage of the cartilage matrix was limited by IL-4 in a clear dose-dependent way. Addition of 100 ng/ml IL-4 during blood-exposure reduced the proteoglycan synthesis rate with only 45%, and decreased the proteoglycan release with 30% compared to control (all p<0.05). Moreover, proteoglycan content was normalized. The combination of IL-4 and IL-10 was clearly more protective against damage caused by blood. This was especially evident for the proteoglycan synthesis which was completely normalized. Furthermore, treatment with a combination of the two cytokines was significantly better than the effect of IL-4 and IL-10 alone (p<0.05). Conclusions: Besides IL-10, as shown previously, also IL-4 protects against blood-induced cartilage damage. The combination of these two cytokines is clearly the most protective. In addition to the direct effects on cartilage, both cytokines are synergistic in inhibition of inflammation. As such, this justifies further evaluation of the combination of IL-4 and IL-10 in prevention and treatment of blood-induced joint damage.

Homeomorphic Matlab Model of Myopia Development

A homeomorphic biomechanical model of the eye based on the Incremental Retinal-Defocus Theory (IRDT) was developed to simulate myopic and hyperopic growth under different optical defocus conditions. The IRDT states that the time-integrated reduction in retinal-image defocus magnitude decreases the rate of retinal neuromodulator release, which in turn decreases the rate of proteoglycan synthesis and reduces scleral structural integrity, thus resulting in axial elongation and myopia development. A homeomorphic biomechanical model of the eye was constructed to test the IRDT based only on optical defocus and its resultant contribution to mechanical forces acting on the components of the eye. The Matlab simulation results showed a more fl attened oblate shape for myopic defocus, and a more elongated prolate shape with hyperopic defocus. Thus, the homeomorphic biomechanical model of the eye was able to simulate the emmetropization mechanism using only the forces acting on the masses at the nodes. In addition, neurochemicals injected at local retinal regions modifi ed the spring constants to result in predicted changes in ocular deformation. The fi ndings have clinical implications with respect to basic theories of myopia development, as well as future therapeutic use of direct ocular drug delivery to control human myopia.