Proteoglycan Breakdown Research Articles

Influence of doxorubicin on the extracellular matrix of the liver of rats under conditions of chronic alcoholic hepatitis

The liver has a high regenerative potential that is dependent on many factors, in particular adenosine monophosphate kinase signaling, however, long-term alteration, such as daily alcohol consumption, turns regeneration into a chronic disease such as fibrosis, the end stage of which is cirrhosis. Hepatic extracellular matrix proteins are important triggers of enhanced stellate cell function during the progression of liver fibrosis. The experiments were performed on Wistar rats divided into four groups: control group; doxorubicin injection group (1.25 mg/kg); chronic alcohol hepatitis group; injection of doxorubicin during alcoholic hepatitis group, where we studied the total concentration of glycosaminoglycans, concentration of heparin-heparan, keratan-dermatan and chondroitin fractions of glycosaminoglycans, free oxyproline and sialic acids in the liver tissue homogenate. Cells with Mallory-Denk bodies were present in the liver of rats from the alcohol hepatitis group, which consisted of tangled balls of intermediate filaments and showed eosinophilia in the cytoplasm of degenerative hepatocytes. There were also necrotic changes in cells. Sinusoidal capillaries were locally dilated. In the central sections of the liver lobules of rats injected with doxorubicin against the background of chronic alcoholic hepatitis, the central veins were expanded, their endothelium was thinned. Sinusoidal capillaries were locally expanded, there were no blood cells in their lumens. The nuclei of the vast majority of hepatocytes were in a state of karyopyknosis, karyorrhexis, and karyolysis. Mallory-Denk bodies were present in the cytoplasm of cells. Administration of doxorubicin to animals with chronic alcoholic hepatitis leads to an increase in the total concentration of glycosaminoglycans, the concentration of chondroitin sulfates, a decrease in the heparin-heparan fraction of glycosaminoglycans and no changes in concentration of the keratan-dermatan fraction in the liver of rats compared to the control group. In the group of rats injected with doxorubicin against the background of chronic alcoholic hepatitis, the concentrations of total glycosaminoglycans, heparin-heparan fraction, and chondroitin fraction significantly increased, and the content of the keratan-dermatan fraction of glycosaminoglycans significantly decreased compared to animals with chronic alcoholic hepatitis. In rats injected with doxorubicin against the background of chronic alcoholic hepatitis, the content of free oxyproline decreased by 1.25 times, sialic acids – by 1.36 times, compared to the group of animals with chronic alcoholic hepatitis. Administration of ethanol or doxorubicin in combination with ethanol to rats causes morphological changes in the liver that are characteristic of chronic alcoholic hepatitis. Administration of doxorubicin to rats leads to degenerative morphological changes in the liver lobules. The administration of doxorubicin prevents alcohol-induced collagenolysis and the breakdown of glycoproteins, but increases the breakdown of proteoglycans due to an increase in the content of chondroitin and heparin-heparan fractions.

Role of Serum level of IL-1B and MMP-13 in detection of severity and functional impairment in hand Osteoarthritis patients

Hand Osteoarthritis (HOA) is one of the most common chronic degenerative joint diseases. Pain and decreased function are the leading symptom in HOA. Previously HOA was considered a pure degenerative disease, but this concept has changed and current research has demonstrated that inflammation is one of the key factors leading to the cartilage destruction. Cytokines as Interleukin-1β (IL1- β) induces expression and release of proteolytic enzymes such as matrix metalloproteinases (MMPs) and aggrecanases. Chondrocyte-derived MMPs are the main enzymes involved in the breakdown of cartilage collagens and proteoglycans. Research in the area of Hand OA is important to help early diagnosis, stop the progression of the disease, and preventing disability.The aim was to determine role of serum level of IL-1β and MMP-13 in detection of severity and functional impairment in patients with HOA.The study was carried out on 30 patients diagnosed with HOA, fulfilling the ACR 1990 criteria for diagnosis of HOA. Demographic data and anthropometric measurements were recorded for all participants. Clinical characteristics of the disease including duration of disease, pattern of joint involvement, pain and disease severity as well as functional assessment of HOA were assessed. Radiographic severity of HOA was assessed by Kellgren and Lawrence (K-L) score. Serum IL-1β and MMP-13 were assessed using double-antibody sandwich enzyme-linked immunosorbent assay method in both patients and control.The conclusion was that Interleukin-1B and MMP-13 seem to have an insignificant role in detection of severity and functional impairment in mild to moderate HOA.

A mutation map for human glycoside hydrolase genes.

Glycoside hydrolases (GHs) are found in all domains of life, and at least 87 distinct genes encoding proteins related to GHs are found in the human genome. GHs serve diverse functions from digestion of dietary polysaccharides to breakdown of intracellular oligosaccharides, glycoproteins, proteoglycans and glycolipids. Congenital disorders of GHs (CDGHs) represent more than 30 rare diseases caused by mutations in one of the GH genes. We previously used whole-exome sequencing of a homogenous Danish population of almost 2000 individuals to probe the incidence of deleterious mutations in the human glycosyltransferases (GTs) and developed a mutation map of human GT genes (GlyMAP-I). While deleterious disease-causing mutations in the GT genes were very rare, and in many cases lethal, we predicted deleterious mutations in GH genes to be less rare and less severe given the higher incidence of CDGHs reported worldwide. To probe the incidence of GH mutations, we constructed a mutation map of human GH-related genes (GlyMAP-II) using the Danish WES data, and correlating this with reported disease-causing mutations confirmed the higher prevalence of disease-causing mutations in several GH genes compared to GT genes. We identified 76 novel nonsynonymous single-nucleotide variations (nsSNVs) in 32 GH genes that have not been associated with a CDGH phenotype, and we experimentally validated two novel potentially damaging nsSNVs in the congenital sucrase-isomaltase deficiency gene, SI. Our study provides a global view of human GH genes and disease-causing mutations and serves as a discovery tool for novel damaging nsSNVs in CDGHs.

Induction of Osteoarthritis-like Pathologic Changes by Chronic Alcohol Consumption in an Experimental Mouse Model.

Induction of Osteoarthritis-like Pathologic Changes by Chronic Alcohol Consumption in an Experimental Mouse Model.

PPARdelta promotes the progression of post-traumatic osteoarthritis

Purpose: Osteoarthritis is a chronic degenerative joint disorder, characterized by articular cartilage degeneration, subchondral bone thickening, osteophyte formation, and synovial hyperplasia. The human burden of this disease influences both the independence and quality of life of those afflicted. Currently there are no pharmacological treatments available to delay or reverse disease progression, and the lack of available treatments can be attributed due to our incomplete understanding of the molecular mechanisms underlying disease initiation and progression. Recent findings from our laboratory indicate that activation of the nuclear receptor PPARdelta induces the expression of enzymes involved in proteoglycan breakdown, prompting us to speculate whether inhibition of PPARdelta could be a viable target for OA treatment. We aimed to characterize the role of PPARdelta in OA in order to determine whether activation of PPARdelta would correlate with an OA-like phenotype and whether the inhibition of this nuclear receptor could affect OA progression. We hypothesize that inhibition of PPARdelta will slow the progression of OA in animal models. Methods: Primary mouse chondrocytes and cartilage explants were treated with a pharmacological PPARdelta agonist (GW501516) to evaluate changes in gene expression (qPCR), aggrecan breakdown (Dye-binding assay;Western Blotting) and histology (Safranin-O, immunohistochemistry) consistent with OA development. Microarrays are conducted on primary mouse chondrocytes treated with GW501516 to elucidate information on direct targets of this nuclear receptor. In order to examine the role of PPARdelta in-vivo, cartilage-specific knockout mice and wild-type littermate controls were subjected to destabilization of medial meniscus surgery (DMM) at 20 weeks of age. 8 weeks post-surgery mice were compared through classical histological and biochemical measures of OA progression including Safranin-O staining with OARSI scoring, immunohistochemistry for cartilage matrix breakdown products, and picrosirius red staining for collagen fiber structure and orientation. Results: In vitro, PPARdelta agonism (by GW501516) results in the upregulation of expression of proteases implicated in cartilage degeneration (including MMPs and ADAMTS genes). Activation of PPARdelta also results in proteoglycan breakdown in the cartilage matrix in an explant culture system, as well as increased aggrecan breakdown products in the medium. Western blots of medium and guanidine extracts depict specific aggrecan neoepitopes present in the medium and guanidine extracts. Microarray results have identified targets of PPARdelta, such as those involved in lipid oxidation and transport. Mice with cartilage-specific deletion of PPARdelta do not demonstrate any abnormalities in skeletal growth or development, but show significant chondroprotection in comparison to wild-type littermate controls after surgical induction of OA. OARSI scoring and immunohistochemistry confirm strong protection of mutant mice against cartilage matrix breakdown, and decreased breakdown products in the cartilage of PPARdelta knockout mice. Conclusions: This study provides strong evidence for catabolic roles of endogenous PPARdelta in post-traumatic OA and suggests that pharmacological inhibition of PPARdelta is a promising therapeutic strategy.

PPARdelta promotes the progression of post-traumatic osteoarthritis

s / Osteoarthritis and Cartilage 22 (2014) S57–S489 S370 micro computed tomography (mCT, Skyscan 1172, 4.5 um resolution) of the knee at 3, 7, 14, 28, 56, 84, 112 days (n 8 per group). Changes in subchondral bone at 28 days post DMM (n 1⁄4 6) were measured separating the subchondral plate from the subchondral tabecular structures. Results: Osteophytes were identified in 70% of mice by day 7 post DMM and in all mice by day 14. At day 7 most samples had 1 or 2 osteophytes, with somehavingup to 4 osteophytes by day 14. Fromday 28onwards the osteophytes fused and formed into one or two large osteophytes. Osteophytes increased in size and bone content with time, plateauing by day 112. The trabeculae of the subchondral bone at day 28 showed a significant (p<0.03) decrease in trabecular number on the medial side of both theDMMand shamoperatedmicewhen compared to the respective contralateral leg. In the sham samples, the reduction of trabecular number translated to a significant increase in trabecular space, whereas in the DMM mice an increase in trabecular thickness occurred, eliminating the increase in trabecular space. Although bone changes occurred within a week of DMM induction, cartilage damage scores were not significant until day 56 (p<0.05), compared to earlier time points. Conclusions:We herein charachterised differential bone changes in the DMM murine model of OA: there was accelerated osteophyte development within the first week, whereas subchondral bone changes were not evident until day 28. Interestingly sham operated animals showed subchondral trabecular bone loss in the ipsilateral knee, highlighting the need to use the contralateral knee as an internal control. The early bone changes in this OA model significantly preceded cartilage degradation, consistent with altered bone biology being an important driver of OA pathogenesis.Supported By Arthritis Research UK (20199). 651 PPARDELTA PROMOTES THE PROGRESSION OF POST-TRAUMATIC OSTEOARTHRITIS A. Ratneswaran y, E. LeBlanc y, E. Walser y, I. Welch y, J.S. Mort zx, N. Borradaile y, F. Beier yk. yWestern Univ., London, ON, Canada; zMcGill Univ., Montreal, QC, Canada; x Shriner’s Hosp. for Children, Montreal, ON, Canada; k Children’s Hlth.Res. Inst., London, ON, Canada Background: Osteoarthritis (OA) is a degenerative joint disorder, characterized by the breakdown of articular cartilage, subchondral bone thickening, osteophyte formation and synovial inflammation. The human burden of this disease influences both the independence and quality of life of those afflicted. Currently, there are no pharmaceutical treatments to slow, stop or reverse disease progression, resulting in greatly reduced quality of life for OA patients and the need for joint replacement surgeries in many cases. The lack of available treatments for OA is partially due to our incomplete understanding of themolecular mechanisms promoting disease initiation and progression. Recent findings from our laboratory indicate that activation of the transcription factor PPARdelta induces the expression of enzymes involved in proteoglycan breakdown and can lead to cartilage degeneration in OA, prompting us to speculate whether inhibition of PPARdelta, can protect from cartilage breakdown in OA. Purpose:We aimed to characterize the role of PPARdelta in OA through in-vitro, ex-vivo, and in-vivo studies to determine whether it is a feasible target for OA therapy. Hypothesis: We hypothesize that inhibition of PPARdelta will slow the progression of OA in animal models. Methods: Primary mouse chondrocytes and cartilage explants were treated with a pharmacological PPARdelta agonist (GW501516) to evaluate changes in gene expression (qPCR), and histology (Safranin-O, immunohistochemistry) consistent with OA development. In order to examine the role of PPARdelta in-vivo, cartilage-specific knockout mice and wild-type littermate controls were subjected to destabilization of medial meniscus surgery (DMM) at 20 weeks of age. 8 weeks postsurgery mice were compared through classical histological and biochemical measures of OA progression including Safranin-O staining with OARSI scoring, immunohistochemistry for cartilage matrix breakdown products, and picrosirius red staining for collagen fiber structure and orientation. Results: In vitro, PPARdelta agonism (by GW501516) results in the upregulation of expression of proteases implicated in cartilage degeneration (including MMPs, and ADAMTS genes). Activation of PPARdelta also results in proteoglycan breakdown in the cartilage matrix of an explant culture system. Mice with cartilage-specific deletion of PPARdelta do not demonstrate any abnormalities in skeletal growth or development, but do show significant chondroprotection in comparison to wild-type littermate controls after surgical induction of OA. OARSI scoring and immunohistochemistry confirm strong protection of mutant mice against cartilage matrix breakdown, and decreased breakdown products in the cartilage of PPARdelta knockout mice. Conclusion: This study provides strong evidence for catabolic roles of endogenous PPARdelta in post-traumatic OA and suggests that pharmacological inhibition of PPARdelta is a promising therapeutic strategy. 652 DKK3 PREVENTS MATRIX LOSS INDUCED BY PRO-INFLAMMATORY CYTOKINES IN HUMAN CARTILAGE EXPLANTS S.M. Walzer, D. Bieder, B. Simon, R. Windhager, S. Toegel. Karl Chiari Lab for Orthopaedic Biology, Dept. of Orthopaedics, Med. Univ. of Vienna,

Radikal Bebas pada Gangguan Sendi Rahang

In human, the temporomandibular joint (TMJ) has been proven as a stress-bearing joint, especially during mastication. Overload of shear stress on the joint can cause degenerative diseases of the joint, such as osteoarthrosis or TMJ osteoarthritis (TMJ-OA). Free radicals have been assumed to be associated in the pathogenesis of various degenerative diseases of the joint and oxidative stress is important in the mechanism of TMJ dysfunction. Free radicals are superoxide (O2-), hydrogen peroxide (H2O2) and hydroxyl (OH-), and are called reactive oxygen species (ROS). Hydroxyl is most destructive in patients with TMJ dysfunction. ROS exert their effects on TMJ by reducing the viscosity of synovial fluid, decreasing joint surface lubrication, breakdown of collagen and proteoglycan, and increasing the activity of cartilage degradation enzymes such as metalloproteinases. DOI: 10.14693/jdi.v15i1.88

Cannabinoids: Novel Therapies For Arthritis?

A key feature of osteoarthritis and rheumatoid arthritis is the loss of articular cartilage. Cartilage breakdown is mediated by complex interactions of proinflammatory cytokines, such as IL-1, inflammatory mediators, including nitric oxide and prostaglandin E(2), and proteases, including matrix metalloproteinases and aggrecanases, such as ADAMTS-4 and -5. Cannabinoids have been shown to reduce joint damage in animal models of arthritis. They have also been shown to prevent IL-1-induced matrix breakdown of collagen and proteoglycan, indicating that cannabinoids may mediate chondroprotective effects. Cannabinoids produce their effects via several cannabinoid receptors and it is important to identify the key cannabinoids and their receptors that are involved in chondroprotection. This review aims to outline the current and future prospects of cannabinoids as anti-arthritic therapeutics, in terms of their ability to prevent cartilage breakdown.

Adamts5 (aggrecanase‐2) is widely expressed in the mouse musculoskeletal system and is induced in specific regions of knee joint explants by inflammatory cytokines

ADAMTS5 (aggrecanase-2) is an extracellular matrix-degrading protease implicated in cartilage destruction in arthritis. Our goals were to determine expression sites of Adamts5 in the murine musculoskeletal system and in an ex vivo joint inflammation model. In mice with an intragenic LacZ reporter controlled by the Adamts5 promoter, β-galactosidase staining was used to identify Adamts5 expressing cells. Mice expressing one wild-type Adamts5 allele were used to determine distribution of Adamts5 mRNA, cleaved aggrecan and versican, and the ADAMTS5 activating enzymes furin and PACE4. Quantitative RT-PCR and immunoblotting were used to validate the immunohistochemistry results. Adamts5 was expressed in mouse synovium, tenosynovium, bone marrow sinusoids, tendons, ligaments, ligament insertions, periosteal cells, and bone vasculature. In knee joint explants treated with IL-1α and TNFα, Adamts5 expression was induced in tenocytes, synovium, and in patellar, but not femoral or tibial articular cartilage. In contrast, increased proteoglycan breakdown in tibial and femoral articular cartilage was associated with increased immunohistochemical staining of PACE4 and furin. These studies identify diverse cell types in the musculoskeletal system that express Adamts5. They also suggest that Adamts5 induction in joint components other than cartilage, and its post-translational activation by PACE4 and/or furin may be important in the pathophysiology of arthritis.

Processing of the Matricellular Protein Hevin in Mouse Brain Is Dependent on ADAMTS4

The matricellular SPARC family member hevin (SPARC-like 1/SPARCL-1/SC1/Mast9) contributes to neural development and alters tumor progression in a range of mammalian models. The distribution of hevin in mouse tissues was reexamined with a novel monoclonal antibody that discriminates between hevin and its ortholog SPARC. We now report proteolysis of hevin in many tissues, with the most extensive processing in the brain. We demonstrate a cleavage site within the hevin sequence for the neural tissue proteinase ADAMTS4. Digestion of hevin by ADAMTS4 in vitro produced fragments similar to those present in brain lysates. Monoclonal antibodies revealed a SPARC-like fragment generated from hevin that was co-localized with ADAMTS4 in vivo. We show that proteolysis of hevin by ADAMTS4 in the mouse cerebellum is important for the normal development of this tissue. In conclusion, we have identified the fragmentation of hevin by ADAMTS4 in the mouse brain and propose that this specific proteolysis is integral to cell morphology and extracellular matrix deposition in the developing brain.

Rolipram-induced elevation of cAMP or chondroitinase ABC breakdown of inhibitory proteoglycans in the extracellular matrix promotes peripheral nerve regeneration

The inhibitory growth environment of myelin and extracellular matrix proteoglycans in the central nervous system may be overcome by elevating neuronal cAMP or degrading inhibitory proteoglycans with chondroitinase ABC (ChABC). In this study, we asked whether similar mechanisms operate in peripheral nerve regeneration where effective Wallerian degeneration removes myelin and extracellular proteoglycans slowly. We repaired transected common peroneal (CP) nerve in rats and either elevated cAMP in the axotomized neurons by subcutaneous rolipram, a specific inhibitor of phosphodiesterase IV, and/or promoted degradation of proteoglycans in the distal nerve stump by local ChABC administration. Rolipram treatment significantly increased the number of motoneurons that regenerated axons across the repair site at 1 and 2 weeks, and increased the number of sensory neurons that regenerated axons across the repair site at 2 weeks. Local application of ChABC had a similar effect to rolipram treatment in promoting motor axon regeneration, the effect being no greater when rolipram and ChABC were administered simultaneously. We conclude that blocking inhibitors of axon regeneration by elevating cAMP or degrading proteoglycans in the distal nerve stump promotes peripheral axon regeneration after surgical repair of a transected nerve. It is likely that elevated cAMP is sufficient to encourage axon outgrowth despite the inhibitory growth environment such that simultaneous enzymatic proteoglycan degradation does not promote more axon regeneration than either elevated cAMP or proteoglycan degradation alone.

Influence of Temporary Chondroitinase ABC-Induced Glycosaminoglycan Suppression on Maturation of Tissue-Engineered Cartilage

A fundamental challenge of cartilage tissue engineering has been the inability to promote collagen synthesis up to native levels. In contrast, recent protocols have demonstrated that glycosaminoglycans (GAG) can be synthesized to native levels in 4-6 weeks of in vitro culture. We hypothesize that rapid GAG synthesis may be an impediment to collagen synthesis, possibly by altering transport pathways of nutrients or synthesis products. In this study, this hypothesis is tested by inducing enzymatic GAG loss in the early culture period of cartilage tissue constructs, and monitoring collagen content at various time points after cessation of enzymatic treatment. In Study 1, to induce breakdown of proteoglycans, chondroitinase ABC (CABC, 0.002U/mL) was continuously added into the culture media for the initial 4 weeks of culture or for 2 weeks starting on day 14 of culture. In Study 2, multiple transient CABC treatments (0.15U/mL, for 2 days) were applied to the matured tissue-engineered constructs. Continuous and transient CABC treatments significantly increased the collagen concentration of the constructs, improving their tensile properties. The GAG content of the treated constructs recovered quickly to the pretreatment level after 2-3 weeks. This study demonstrates that tissue-engineered cartilage constructs with improved tensile properties can be achieved by temporarily suppressing the GAG content enzymatically.

I-27 TARGETING NF-KAPPAB SIGNALING IN OA: IS IT ONLY ABOUT INFLAMMATION?

I-27 TARGETING NF-KAPPAB SIGNALING IN OA: IS IT ONLY ABOUT INFLAMMATION?

Benefit of chondroitinase ABC on sensory axon regeneration in a laceration model of spinal cord injury in the rat

Background Chondroitin sulfate proteoglycans are up-regulated in the spinal cord after SCI, creating a molecular barrier inhibitory to axon growth. Chondroitinase ABC degrades CSPGs in vitro and in vivo. Methods We studied whether IT ChABC promotes axonal regeneration in a laceration model of SCI. Three groups of Sprague-Dawley rats were used: control and rats treated with low-dose and high-dose IT ChABC. Chondroitin sulfate proteoglycan breakdown products were measured by 2-B-6 expression, and intact CSPGs by CS-56 expression. Sensory axonal regeneration was traced after CTB injection into the median, ulnar, and sciatic nerves. Results CS-56 expression was down-regulated and 2-B-6 expression was increased in the groups treated with IT ChABC but not in the control. Laminin and GFAP immunoreactivity was unaltered in the ChABC groups. The number of axons growing into the scar was 3.1 times greater ( P < .01) in the high-dose ChABC group and 2.1 times greater ( P < .01) in the low-dose group compared with the controls. The length of axonal growth after high- and low-dose ChABC was 9.9 ( P < .01) and 8.3 ( P < .01) times greater, respectively, than in the control group. Axons extended across the lesion gap and into the distal spinal cord stump in 2 of 8 (low dose) and in 3 of 9 (high dose) rats compared with none in the control group. Conclusions Intrathecal ChABC administration caused a slight decrease in CSPGs in the scar after a laceration SCI with a minimal increase in sensory axonal regeneration into and across the laceration gap.

Proteoglycans and pathophysiology

ventilation-induced lung injury (VILI) is an important clinical entity whose pathophysiology is incompletely understood. The article by Moriondo and coworkers ([14][1]) in the Journal of Applied Physiology offers new insights into potential mechanistic pathways. These authors report that one of the

Proteoglycan Breakdown of Meniscal Explants Following Dynamic Compression Using a Novel Bioreactor

Motivated by our interest in examining meniscal mechanotransduction processes, we report on the validation of a new tissue engineering bioreactor. This paper describes the design and performance capabilities of a tissue engineering bioreactor for cyclic compression of meniscal explants. We showed that the system maintains a tissue culture environment equivalent to that provided by conventional incubators and that its strain output was uniform and reproducible. The system incorporates a linear actuator and load cell aligned together in a frame that is contained within an incubator and allows for large loads and small displacements. A plunger with six Teflon-filled Delrin compression rods is attached to the actuator compressing up to six tissue explants simultaneously and with even pressure. The bioreactor system was used to study proteoglycan (PG) breakdown in porcine meniscal explants following various input loading tests (0–20% strain, 0–0.1 MPa). The greatest PG breakdown was measured following 20% compressive strain. These strain and stress levels have been shown to correspond to partial meniscectomy. Thus, these data suggest that removing 30–60% of meniscal tissue will result in the breakdown of meniscal tissue proteoglycans.

Proinflammatory Cytokine Activities, Matrix Metalloproteinase‐3 Activity, and Sulfated Glycosaminoglycan Content in Synovial Fluid of Dogs with Naturally Acquired Cranial Cruciate Ligament Rupture

To measure and compare activities of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and matrix metalloproteinase-3 (MMP-3); as well as sulfated glycosaminoglycan (S-GAG) content in synovial fluid from dogs with cranial cruciate ligament rupture (CCLR) and dogs with clinically normal stifles. To determine whether correlations exist between demographic and disease-related variables and these synovial markers. Prospective clinical study. Dogs with CCLR (n=23) and Beagles with normal stifle joints (n=21). Synovial fluid activities of proinflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) were determined by bioassay. MMP-3 activity was measured using fluorogenic substrate. S-GAG contents were determined by dimethylmethylene blue dye-binding assay. Mann-Whitney U-test was used to compare results from CCLR joints with normal controls. Spearman's rank correlation test was used to evaluate associations between demographic and disease-related markers and synovial markers. Mean values for synovial markers were significantly higher in CCLR joints compared with controls. IL-1beta and MMP-3 were positively correlated with lameness duration. Activities of proinflammatory cytokines, MMP-3 activity and S-GAG contents were significantly elevated in synovial fluid from canine stifle joints with naturally acquired CCLR. These results indicate that there is joint inflammation and increased release of GAGs into synovial fluid, suggesting that these inflammatory changes are associated with depletion of proteoglycan from articular cartilage. Medical and surgical treatments designed to decrease joint inflammation and breakdown of proteoglycans may be of value in the management of CCLR in the dog.

Arthritis and cannabinoids: HU-210 and Win-55,212–2 prevent IL-1 α-induced matrix degradation in bovine articular chondrocytes in-vitro

Cannabinoids have analgesic, immunomodulatory and anti-inflammatory properties and attenuate joint damage in animal models of arthritis. In this study the mechanisms of action of the synthetic cannabinoid agonists, HU-210 and Win-55,212-2, were studied to determine if they affected interleukin-1 alpha (IL-1alpha)-induced proteoglycan and collagen degradation in bovine nasal cartilage explant cultures and prostaglandin E2 (PGE2) production in primary cultures of bovine articular chondrocytes. The effects of the inactive enantiomer, Win-55,212-3, were compared with those of the active enantiomer, Win-55,212-2, to determine if the effects were cannabinoid (CB)-receptor mediated. The chondrocytes and explants were stimulated by IL-1alpha (100 U mL(-1) identical with 0.06 nM and 500 U mL(-1) identical with 0.3 nM, respectively). Proteoglycan breakdown was determined as sulfated glycosaminoglycan (sGAG) release using the dimethylmethylene blue assay. Collagen degradation was determined as hydroxyproline in the conditioned culture media and cartilage digests. PGE2 was determined by ELISA. Expression of cannabinoid receptors, CB1 and CB2; cyclooxygenase-1 and -2 (COX-1 and COX-2); inducible nitric oxide synthase (iNOS); as well as activation of nuclear factor-kappa B (NF-kappaB) in chondrocytes were studied using immunoblotting techniques and immunofluorescence. The results showed that HU-210 and Win-55,212-2 (5-15 microM) significantly inhibited IL-1-alpha stimulated proteoglycan (P < 0.001) and collagen degradation (P < 0.001). Win-55,212-2 (5-10 microM) also significantly inhibited PGE2 production (P < 0.01). At 5 microM, Win-55,212-2 inhibited the expression of iNOS and COX-2 and activation of NF-kappaB. Chondrocytes appeared to constitutively express cannabinoid receptors CB1 and CB2. It is concluded that biologically stable synthetic cannabinoids protect cartilage matrix from degradation induced by cytokines and this effect is possibly CB-receptor mediated and involves effects on prostaglandin and nitric oxide metabolism. Cannabinoids could also be producing these effects via inhibition of NF-kappaB activation.

Degradation of small leucine-rich repeat proteoglycans by matrix metalloprotease-13: identification of a new biglycan cleavage site

A major and early feature of cartilage degeneration is proteoglycan breakdown. Matrix metalloprotease (MMP)-13 plays an important role in cartilage degradation in osteoarthritis (OA). This MMP, in addition to initiating collagen fibre cleavage, acts on several proteoglycans. One of the proteoglycan families, termed small leucine-rich proteoglycans (SLRPs), was found to be involved in collagen fibril formation/interaction, with some members playing a role in the OA process. We investigated the ability of MMP-13 to cleave members of two classes of SLRPs: biglycan and decorin; and fibromodulin and lumican. SLRPs were isolated from human normal and OA cartilage using guanidinium chloride (4 mol/l) extraction. Digestion products were examined using Western blotting. The identities of the MMP-13 degradation products of biglycan and decorin (using specific substrates) were determined following electrophoresis and microsequencing. We found that the SLRPs studied were cleaved to differing extents by human MMP-13. Although only minimal cleavage of decorin and lumican was observed, cleavage of fibromodulin and biglycan was extensive, suggesting that both molecules are preferential substrates. In contrast to biglycan, decorin and lumican, which yielded a degradation pattern similar for both normal and OA cartilage, fibromodulin had a higher level of degradation with increased cartilage damage. Microsequencing revealed a novel major cleavage site (... G177/V178) for biglycan and a potential cleavage site for decorin upon exposure to MMP-13. We showed, for the first time, that MMP-13 can degrade members from two classes of the SLRP family, and identified the site at which biglycan is cleaved by MMP-13. MMP-13 induced SLRP degradation may represent an early critical event, which may in turn affect the collagen network by exposing the MMP-13 cleavage site in this macromolecule. Awareness of SLRP degradation products, especially those of biglycan and fibromodulin, may assist in early detection of OA cartilage degradation.

Treatment of patients with venous insufficiency with fresh plant horse chestnut seed extract: A review of 5 clinical studies

Extracts from the seed of the horse chestnut (Aesculus hippocastanum L.) have traditionally been used to treat patients with chronic venous insufficiency and to alleviate its associated symptoms, including lower leg swelling. The efficacy of preparations that contain horse chestnut seed extract (HCSE) is believed to be due largely to an inhibitory effect on the catalytic breakdown of capillary wall proteoglycans. Aesculaforce is a fresh plant HCSE that is available as an oral tincture, as tablets (20 mg or 50 mg), and as topical gel. Four clinical trials in patients with chronic venous insufficiency and 1 study in patients with varicose veins demonstrated the effectiveness of these preparations through the objective measure of reduction in lower leg edema and the subjective alleviation of leg pain, heaviness, and itching. Safe, well tolerated, and acceptable to patients, the fresh plant HCSE preparation Aesculaforce offers a real alternative in the treatment of patients with mild to moderate venous insufficiency.