Collagen Release Research Articles

Imbalance in the Synthesis of Collagen Type I and Collagen Type III in Smooth Muscle Cells Derived from Human Varicose Veins

Varicose veins have a thickening wall. Their smooth muscle cells are disorganized as regards proliferation and production of extracellular matrix protein. An imbalance between the synthesis of collagen type I protein (collagen I) and collagen type III protein (collagen III) could explain the lack of elasticity of varicose veins. Therefore, collagen synthesis was compared in the media and in cultured smooth muscle cells derived from human control and varicose saphenous veins. An increase in total collagen synthesis was observed in the media and in smooth muscle cells derived from varicose veins. This augmentation was due to an overproduction of collagen I in cultured cells from varicose veins consistent with an increase in the release of collagen I metabolites in the media. A concomitant decrease in collagen III was observed in cultures of smooth muscle cells from varicose veins. The increase in the synthesis of collagen I in cells from varicose veins was correlated with an overexpression of the gene since mRNAs for collagen I were augmented without change in mRNA-half-life. This augmentation in the synthesis of collagen I was reduced by the addition of exogenous collagen III in cultures from varicose veins. These findings suggest a dysregulation of the synthesis of collagen I and III in smooth muscle cells derived from varicose veins.

Synergistic effects of glycoprotein 130 binding cytokines in combination with interleukin-1 on cartilage collagen breakdown.

To determine whether other glycoprotein 130 (gp130) binding cytokines can mimic the effects of oncostatin M (OSM) in acting synergistically with interleukin-1alpha (IL-1alpha) to induce cartilage collagen breakdown and collagenase expression, and to determine which receptors mediate these effects. The release of collagen and proteoglycan was assessed in bovine and human cartilage explant cultures. Messenger RNA (mRNA) and protein production from immortalized human chondrocytes (T/C28a4) was analyzed by Northern blotting and specific enzyme-linked immunosorbent assays. Collagenase activity was measured by bioassay. Cell surface receptors were detected by flow cytometry. OSM in combination with IL-1alpha caused a rapid synergistic induction of matrix metalloproteinase 1 mRNA, which was sustained over a 72-hour period. Flow cytometric analyses detected both the OSM-specific receptor and the gp130 receptor at the chondrocyte cell surface, but failed to detect the leukemia inhibitory factor receptor (LIFR). Cartilage degradation assays revealed that, of the gp130 binding cytokines, only OSM and IL-6, in the presence of its soluble receptor (sIL-6R), were able to act synergistically with IL-1alpha to promote collagen release. This study demonstrates that IL-6 can mimic OSM in synergizing with IL-1alpha to induce chondrocyte-mediated cartilage collagen breakdown and collagenase production. In order to have this effect, IL-6 requires the presence of its soluble receptor. The apparent absence of LIFR explains why other gp130 binding cytokines do not act in synergy with IL-1alpha. Since OSM, IL-6, and sIL-6R levels have all been shown to be elevated in the rheumatoid joint, our findings suggest that these cytokines may be key mediators of cartilage collagen catabolism in the inflammatory arthritides.

Activation of procollagenases is a key control point in cartilage collagen degradation: interaction of serine and metalloproteinase pathways.

Bovine and human cartilages in explant culture respond to proinflammatory cytokines with the up-regulation of procollagenases. In stimulated bovine nasal cartilage (BNC), >90% of collagen is released by day 14 of culture, but collagen release is rarely seen before day 7. The aim of this study was to investigate if activation of procollagenases is a rate-limiting step in cartilage collagen breakdown. BNC and human articular cartilage explants were cultured with interleukin-1alpha (IL-1alpha) and/or oncostatin M (OSM) with or without test reagents. Collagen levels were determined by assay of hydroxyproline. Collagenase activity was measured using the diffuse fibril assay. The addition of procollagenase activators, matrix metalloproteinase 3 (MMP-3), and APMA to IL-1alpha/OSM-stimulated BNC resulted in early release of collagen. The release with APMA was completely blocked by the addition of tissue inhibitor of metalloproteinases 1. This shows that procollagenases are present early in the culture period, but cartilage collagen breakdown does not happen until activation occurs. The addition of plasminogen to IL-1alpha/OSM-stimulated cartilage produced early collagen release in bovine and a significant increase in human cartilage. Thus, plasminogen activators (PAs) are present and convert plasminogen to plasmin, a known activator of several MMPs, including collagenases. Addition of alpha1-proteinase inhibitor or a urokinase-type PA inhibitor, 7-amino-4-chloro-3-(3-isothiureidopropoxy) isocoumarin, partially blocked the breakdown of collagen from IL-1alpha/OSM-treated bovine cartilage. This suggests that serine proteinases are involved in the activation cascades of procollagenases that result in cartilage collagen breakdown. The activation of procollagenases is a key control point in cartilage collagen breakdown, and serine proteinase pathways activate MMPs.

TRANSFORMING GROWTH FACTOR-β1 BLOCKS THE RELEASE OF COLLAGEN FRAGMENTS FROM BOVINE NASAL CARTILAGE STIMULATED BY ONCOSTATIN M IN COMBINATION WITH IL-1α

Oncostatin M in combination with interleukin-1 (IL-1) induced a rapid and reproducible release of collagen from bovine nasal cartilage in culture. This release was accompanied by a high collagenolytic activity and low or absent tissue inhibitor of metalloproteinase-1 activity in the culture medium. Transforming growth factor-β1 was able to block this release of collagen from the tissue, and reduce the expression and secretion of collagenases whilst maintaining TIMP-1 levels from bovine nasal chondrocytes. This study shows for the first time that TGF-β1 can protect cartilage collagen from destruction.

Ultrastructural characteristic of collagen resorption in cirrhotic liver

Partial hepatectomy of mouse cirrhotic liver leads to rapid degeneration of hypertrophied connective tissue. Enlarged cisterns of the smooth endoplasmic reticulum lie close to the plasma membrane on the sinusoidal surface of hepatocyte. Golgi apparatus of Kupffer cells is hypertrophied, swollen collagen fibrils lose their specific striation. Microvilli of hepatocyte plasmalemma separate swollen collagen fibrils and draw them into the cytoplasm. Lysosomes, peroxisomes, and hypertrophied Golgi apparatus are spread from the sinusoidal surface to bile capillaries, which indicates that these structures are involved in collagen lysis and release of metabolites into the bile capillary. After partial hepatectomy of cirrhotic liver, hepatocytes, Kupffer cells, and neutrophils play the major role in collagen lysis. Kupffer cells and neutrophils are involved in the lysis of collagen fibrils during CCl4-induced liver cirrhosis.

The regulation of MMPs and TIMPs in cartilage turnover.

The treatment of cartilage with mediators initiates the breakdown of proteoglycan followed by collagen. This is accompanied by the modulation of different proteinases and inhibitors that include members of the MMP family and TIMPs. We have evidence that a chondrocyte membrane-associated metalloproteinase cleaves aggrecan. This activity is rapidly induced after stimulation with IL-1 and OSM and is not inhibited by TIMPs-1 and -2 but is inhibited by synthetic MMP inhibitors. This same combination of cytokines also upregulates the collagenases with the subsequent release of collagen fragments, and there is a close correlation between the amount of collagen released and collagenase activity produced. Collagen release can be prevented after treatment with specific inhibitors of MAP kinases, inhibitors of MMP transcription, synthetic metalloproteinase inhibitors, TIMPs and treatment of cartilage with agents that upregulate TIMPs. The results from bovine cartilage culture models show that collagen release occurs when TIMP levels are low, collagenases are upregulated and then subsequently activated.

Whole Blood Platelet Aggregation in Humans and Animals: A Comparative Study

Background.Many animal species are used to evaluate the performance and blood compatibility of cardiovascular devices, but interspecies differences in platelet activity have not been well characterized. This study measures platelet response to six agonists in human, dog, and calf blood.Materials and methods.We used whole blood impedance lumi-aggregometry to measure platelet aggregation and ATP release in blood samples from adult humans (n= 19), mongrel dogs (n= 19), and Holstein calves (n= 7). The agonists were collagen, ristocetin, arachidonic acid, thrombin, and three concentrations of both ADP and epinephrine.Results.Only collagen (1 μg/ml) and ADP (5, 10, and 20 μM) caused aggregation and ATP release in all samples. Canine platelets responded to all six agonists at all doses. Human platelets responded to everything except epinephrine at 2 and 100 μM. Bovine platelets responded only to collagen, ADP, and thrombin. In bovine platelets, aggregation from collagen and ATP release from thrombin were significantly lower than the corresponding responses in human and canine blood. The aggregation induced by 10 μM ADP was significantly higher in canine than in human platelets.Conclusion.Human, canine, and bovine platelets have very different responses to agonists. In these models, collagen (1 μg/ml) and ADP (10 μM) are the agonists of choice for investigating whole blood platelet aggregation because they provide the most consistent results between species. For ATP release, 1 U/ml thrombin is the recommended agonist and the dose for all three species.

Effect of heparin on mesangial cell growth and gene expression of matrix proteins

Mesangial cell (MC) proliferation and matrix expansion are characteristics of many glomerulopathies. Heparin has been shown to inhibit MC proliferation in vitro and mitigate cell proliferation, matrix expansion, proteinuria, renal insufficiency, and hypertension in experimental glomerulonephritis and subtotal renal ablation. We examined the effect of standard heparin on MC proliferation and matrix protein expression in vitro which necessarily excludes the confounding influences of haemodynamic, inflammatory, haemostatic, and various other processes that are present in vivo. Gene expression and release of fibronectin (FN), collagen IV and laminin by cultured rat MC were tested in the presence and absence of heparin. In addition the effect of transforming growth factor-beta1 (TGF-beta1) on the gene expression of those matrix proteins was assessed. Within a 3-1000 microg/ml concentration range, heparin inhibited gene expression and release of FN by 10% fetal calf serum (FCS)-stimulated MC in a concentration-dependent manner. At concentrations of 300 and 1000 microg/ml, heparin inhibited fibronectin mRNA levels in TGF-beta1 (6 ng/ml) stimulated cells. However, heparin had no effect on gene expression or release of collagen IV or laminin under these conditions. Heparin markedly inhibited 10% FCS-stimulated MC proliferation in a concentration-dependent manner. Heparin inhibited MC growth and fibronectin production. These effects may, in part, account for the reported beneficial effects of heparin on the course of renal disease in experimental animals.

Stromelysin 1, neutrophil collagenase, and collagenase 3 do not play major roles in a model of chondrocyte mediated cartilage breakdown.

AIMS: To determine the collective roles of stromelysin 1, neutrophil collagenase, and collagenase 3 in chondrocyte mediated cartilage proteoglycan and type II collagen degradation in tissue culture model systems. METHODS:...

Matrix degradation by chondrocytes cultured in alginate: IL-1β induces proteoglycan degradation and proMMP synthesis but does not result in collagen degradation

Objective: To determine the role of interleukin-1β (IL-1β) in the degradation of proteoglycans and collagen by articular chondrocytes.Design: Chondrocytes were cultured in alginate beads for 2 weeks to produce extracellular matrix, followed by the addition of IL-1β for 1 or 2 days. Breakdown of extracellular matrix (with and without activation of pro-matrix metalloproteinases (MMPs) by APMA) was monitored by release of glycosaminoglycans (GAG, proteoglycans) and hydroxyproline (collagen) from the beads into the medium, and by the amount of damaged collagen in the bead. Levels of (pro)MMPs in the beads were assayed by zymography and their activity was quantified fluorometrically.Results: IL-1β induced a profound GAG release (∽80% after 2 days at 20 ng/ml IL-1β) that was both time and IL-1β concentration dependent. Under these conditions no increase in collagen release or damaged collagen in the bead was detected. Zymography demonstrated that the synthesis of a variety of proMMPs was induced by IL-1β, without a detectable increase of MMP-activity as measured in the activity assay. After activation of the proMMPs by APMA, a time and IL-1β concentration-dependent increase in MMP-activity was found, which resulted in almost complete deterioration of collagen already after 18 h of incubation. In the presence of APMA, GAG release from IL-1β treated beads was significantly increased from 24 to 31%.Conclusions: Our data suggest that proteoglycan and collagen degradation are regulated through different mechanisms: IL-1β induces the synthesis of active enzymes that degrade proteoglycans, such as ‘aggrecanase’, and inactive proMMPs. Thus, IL-1β alone is not sufficient to result in collagen-degrading MMPs. Once activated, MMPs may account for up to a quarter of the aggrecan degradation in this model.

Phenotypic Changes in Vascular Smooth Muscle Cells during Aging: Insulin Effect on Migration

We examined the mechanisms by which insulin may be atherogenic during aging. We postulated that an increase in insulin secretion during aging produces growth factor effects on vascular smooth muscle cells (VSMCs), promoting these cells to synthesize collagen and to migrate. We have previously demonstrated that insulin stimulates collagen synthesis and release in senescent VSMCs that were obtained from a human organism with high levels of insulin secretion. Using the same experimental model, we now study the effects of insulin on VSMC migration. We demonstrate that insulin has a chemoattractant effect on VSMCs which occurs through insulin binding to its own specific receptors as opposed to its effect on collagen production. Blocking the insulin receptor significantly eliminates the insulin effect on cell migration. At the same molarity, the chemotactic effect of insulin is less pronounced than that of insulin-like growth factor-1. In spite of different mechanisms, there is a remarkable correlation between the insulin effects on collagen secretion and cell migration (r² = 97%, p < 0.0005). Our results indicate that distinct but closely related mechanisms may exist by which insulin becomes atherogenic. Our results also suggest the importance of normal aging processes in the development of atherosclerosis.

Collagen degradation induced by the combination of IL-1alpha and plasminogen in rabbit articular cartilage explant culture.

To investigate the effect of plasminogen on cartilage catabolism, we assessed collagen degradation in rabbit articular cartilage explants treated with or without plasminogen and interleukin-1alpha (IL-1alpha). The combination of IL-1 alpha and plasminogen induced rapid collagen degradation, amounting to more than 60% of the total collagen by day 7, while neither IL-1alpha nor plasminogen alone had any effect. To examine the mechanism of collagen degradation induced by IL-1alpha and plasminogen, the matrix metalloproteinases (MMPs) in the culture supernatants were examined by ELISA, Western blotting and gelatin zymography. We found that the treatment with IL-1alpha induced MMP-1, MMP-3, and MMP-9. In addition, plasminogen converted the pro form of MMPs into the active form. Both a tissue inhibitor of metalloproteinases-1 (TIMP-1) and a synthetic hydroxamate MMP inhibitor prevented this collagen release. These results suggest that plasminogen causes collagen degradation via activation of MMPs induced by IL-1alpha.

Tetracycline derivatives inhibit cartilage degradation in cultured embryonic chick tibiae

Tetracyclines have been used extensively as antibiotics and growth promoters in the poultry industry. However, they can inhibit angiogenesis and matrix degradation, both of which are essential for normal growth plate cartilage development. The purpose of this research was to test the ability of several tetracyclines to inhibit cartilage degradation in cultured embryonic chick tibiae. Based on gross observations and biochemical quantitation of collagen release into the media, minocycline, doxycycline, oxytetracycline, and tetracycline inhibited cartilage degradation at 20, 40, 60, and 80 μg ml −1 respectively. Chlortetracycline did not inhibit cartilage degradation at concentrations tested. The ability of the tetracycline derivative to inhibit cartilage degradation was in general related to its hydrophobicity. Since a majority of the cartilage in the embryonic chick tibia will develop into the post hatched growth plate, it may be important to determine if any of the tetracyclines used as antibiotics could cause problems in in vivo growth plate cartilage development.

Quantitation of collagen types I, III and V in tissue slices by capillary electrophoresis after cyanogen bromide solubilization.

A method for the determination of the proportions of major fiber-forming collagens (types I, III and V) in soft connective tissue was elaborated. The method is based on the release of insoluble collagen by CNBr with subsequent separation of the arising peptides. For routine application the peptides are separated by capillary electrophoresis (50 mM phosphate pH 2.5, 15 kV, 50 degrees C, 70/60 cm x 70 microns I.D. capillary with UV detection at 200 nm). Quantitation of collagen type I can be done either on the basis of spiking the sample with a peptide mixture obtained from a known amount of collagen type I, or by spiking the sample with an equimolar mixture of the two peptides [alpha 1(I)CB2 and alpha 1(I)CB4] (constituting a fused peak) along with alpha 1(III)CB2 and alpha 1(V)CB1. Compared to the previously published methods the procedure is faster and does not require isolation of marker peptides by tedious chromatographic procedures in a preceding preparatory step. Good results are obtained within a wide range of run buffer concentrations and applied voltages; conversely, intensive cleaning of the capillary after every three runs is recommended with a new capillary after 20-30 runs.

Immunocytochemical detection of growth factors (PDGF and TGF β) in equine chronic pneumonia

The roles played by platelet-derived growth factor ( pdgf) and transforming growth factor β ( tgf β) in the development of pulmonary fibrosis in equine chronic pneumonia varied greatly. Macrophages, epithelial cells and fibroblasts were identified as a source of PDGF, the principal role of which was related to its mitotic effect on epithelial cells, and particularly on fibroblasts in the final phase of the disease. tgf β was detected in epithelial cells in all three phases of the disease and in fibroblasts in the last phase. However, the role of tgf β in this pulmonary disease is not clear because its expression in the cytoplasm of fibroblasts in areas of strong collagenation during the last phase was weak. Nonetheless, it was responsible for the production and release of collagen during the stage of total fibrosis.

Quantitative analysis of pyridinium crosslinks of collagen in the synovial fluid of patients with rheumatoid arthritis using high-performance liquid chromatography.

The pyridinium crosslinks are important and definite biomarkers of mature hard tissue collagen degradation. A gradient ion-paired reversed-phase high-performance liquid chromatographic method was used for the simultaneous determination of both crosslinks in synovial fluid (SF) samples of 25 patients with rheumatoid arthritis (RA). The mean +/- SD levels of pyridinoline (Pyd) and deoxypyridinoline (Dpyd) in SF were 107.7 +/- 182.3 nmol/l and 4.8 +/- 8.3 nmol/l, respectively. The Pyd/Dpyd ratio, which indicates the amount of Pyd released from cartilage rather than bone, amounted to 30.8 +/- 29.5. This value is significantly higher than in urine or serum of the same patients. These data suggest increased destruction of joint cartilage in patients with RA and the release of collagen II fragments in SF. In addition, the levels of the crosslinks in SF reflect considerable interindividual variation, indicating substantial individual differences in the amount of collagenous material that is degraded.

Airways remodelling in asthma: no doubt, no more?

Asthma is a chronic inflammatory disease of the airways invariably associated with healing. Remodelling of the airways can be demonstrated by the activation of airways macrophages, the release of growth factors and fibrogenic cytokines, the activation of fibroblasts and myofibroblast, elastolysis and elastosynthesis, collagen deposition, increased synthesis and release of extracellular matrix components, and an increased mass of smooth muscle and mucous glands. However, the control of remodelling is still poorly understood.

Fish Oil Supplementation Inhibits Platelet Aggregation and ATP Release Induced by Platelet-activating Factor and Other Agonists

The anti-inflammatory effects of fish oil may partly be due to the inhibition of platelet activation induced by platelet-activating factor (PAF) and other agonists. To investigate this hypothesis, the diets of 12 healthy volunteers were supplemented with 12 fish oil capsules or 12 olive oil capsules daily for 4 weeks in a double blind crossover study. Aggregation induced by PAF (18 and 12.5 nM) and collagen (20 μg/ml)tended to be reduced after fish oil but the effect was statistically significant only in subjects receiving fish oil in the 6rst 4 weeks of the study (P 0.05, n=6). The effect of fish oil supplementation on platelet ATP release was more marked with significant inhibition of ATP release induced by PAF (1200 and 36 nM, P 0.01, n = 12), collagen (20 μg/ml, P 0.005, n = 12) and ADP (15,10 and 5 μM, P 0.05, n = 12). Olive oil supplementation appeared to inhibit ATP release induced by collagen (45 and 30 μg/ml, P> 0.025, n = 12), while aggregation and ATP release induced by arachidonic acid and adrenaline were unaffected by the supplements. Plasma fibrinogen was significantly reduced after olive oil (P 0.01, n = 12) while prothrombin time was reduced after fish oil (P 0.001, n = 12) and olive oil (P 0.0025). Reduced platelet aggregation and more importantly, inhibition of platelet release induced by PAF and other agonists may contribute to the anti-inflammatory effects of fish oil supplementation in a number of disease states but olive oil may also independently affect platelet function and influence the effect offish oil.

Valproate-mediated disturbances of hemostasis: relationship to dose and plasma concentration.

Valproic acid (VPA) may cause impaired platelet function, thrombocytopenia and, occasionally, severe bleeding. Controversy exists both as to the mechanism of this alteration in hemostasis and as to whether these adverse effects are either dose-related or idiosyncratic. Previous investigations have focused primarily on pediatric patients who commonly were receiving multiple anticonvulsant medications. We evaluated a cohort of 27 adult patients with epilepsy who were receiving VPA monotherapy and compared them with age-matched controls to determine whether a correlation exists between platelet count, function, bleeding time, levels of von Willebrand's factor antigen, and VPA dose or plasma concentration. VPA patients had significantly lower platelet counts and longer bleeding times than did controls (p < 0.05). Platelet count was inversely correlated to VPA dose and both free and total VPA concentration (p < 0.01). There was no significant difference in levels of von Willebrand's factor antigen between patients and controls. We assessed platelet aggregation by measurement of whole-blood platelet aggregation and release with agonists including adenosine diphosphate, thrombin, collagen, arachidonic acid, and ristocetin. VPA patients had significant decreases in platelet aggregation values compared with controls. There were significant differences in collagen, arachidonic acid, and adenosine diphosphate release and aggregation between groups that correlated to both VPA dose (p < 0.01) and concentration (p < 0.05). Prolongation of bleeding time was significantly correlated to both VPA dose and concentration. Our data suggest a significant relationship between impaired platelet function and both VPA dose and plasma concentration.

Circulating Tumor Gangliosides Enhance Platelet Activation

AbstractGangliosides enhance tumor formation in experimental animals, and high circulating concentrations of gangliosides shed by tumor cells are associated with rapid progression of human neuroblastoma. We studied these shed molecules for effects on platelet function, because platelet activation may play a role in the metastatic process. Preincubation of normal platelets in patient (tumor ganglioside-containing) serum resulted in their aggregation upon exposure to a subthreshold concentration (1 µg/mL) of collagen (up to 34% v <10% in normal serum) and ATP release (up to 1.1 nmol/2.5 × 107 platelets v <0.2 in normal serum). Because circulating shed tumor gangliosides are lipoprotein-associated, we next assessed the effects of the serum lipoprotein fraction on platelet ATP release. The patient serum lipoprotein fraction (d > 1.210) enhanced ATP release (up to 3.1 nmol ATP), whereas the same fraction of normal serum, and both patient and control lipoprotein-depleted serum fractions (d > 1.210), were inactive (<0.2 nmol ATP released). Finally, as little as 0.5 µmol/L patient serum gangliosides (purified from the lipoprotein fraction) caused significantly greater ATP release than did normal serum gangliosides (P < .01) and caused maximal release at 50 µmol/L (up to 3.0 nmol ATP released v ≤0.3 nmol released by platelets exposed to normal serum gangliosides). Purified total human neuroblastoma tumor gangliosides, detected in the patient serum and isolated from LA-N5 cells, were highly active; preincubation of platelets with only 5 µmol/L of these gangliosides resulted in release of 2.5 ± 0.1 nmol ATP. Thus, neuroblastoma patient serum, the lipoprotein fraction, and, specifically, the serum gangliosides enhance platelet activation. This activity appears to reside particularly in the tumor cell gangliosides, which are shed in vivo.