Effects of Training and Overtraining on Intervertebral Disc Proteoglycans.

STUDY DESIGN: Animal experimental study. OBJECTIVE: Evaluate the effect of physical activity and overtraining condition on glycosaminoglycan concentration on the intervertebral disc (IVD) using a rat running model. SUMMARY OF BACKGROUND DATA: Some guidelines recommend the implementation of a physical exercise program as treatment for low back pain; however, cyclic loading impact on the health of the IVD and whether there is a dose-response relationship is still incompletely understood. METHODS: Thirty-two rats ages 8 weeks were divided into four groups with eight animals each. The first 8 weeks were the adaptive phase, the overtraining phase was from the ninth to the eleventh week, which consisted of increasing the number of daily training sessions from 1 to 4 and the recovery phase was represented by the 12th and 13th weeks without training. Control group 1 (CG1) did not undergo any kind of training. Control group 2 (CG2) completed just the adaptive phase. Overtraining group 1 (OT1) completed the overtraining phase. Overtraining group 2 (OT2) completed the recovery phase. Running performance tests were used to assess the "overtraining" status of the animals. IVD glycosaminoglycans were extracted and quantified, and identified by electrophoresis. RESULTS: Glycosaminoglycans showed a distribution between chondroitin sulfate and dermatan sulfate. Glycosaminoglycans quantification showed decreasing concentration at the following order: OT1 > CG2 > OT2 > CG1. Increased expression of dermatan sulfate was verified at the groups submitted to any training. CONCLUSION: Overtraining condition, as assessed by muscle and cardiovascular endurance did not lessen glycosaminoglycan concentration in the IVD. In fact, physical exercise increased glycosaminoglycan concentration in the IVD in proportion to the training load, even at overtraining condition, returning to normal levels after the recovery phase and glycosaminoglycan production is a reversible acute positive response for mechanical stimulation of the IVD. LEVEL OF EVIDENCE: N/A.

Sulfated fucans extracted from algae Padina gymnospora have anti-inflammatory effect

Sulfated polysaccharides were extracted with acetone from brown algae Padina gymnospora. The fraction precipitated with 1.5 volumes of acetone (F1.5) purified in Sephadex G-75 was characterized by infrared and nuclear magnetic resonance of 13C and ¹H, through which the presence of sulfate groups on the C4 of α-L-fucose could be observed. This polysaccharide showed that an MW of 25,000 Da was effective in reducing leukocyte influx into the peritoneal cavity in mice at 10 mg/kg and 25 mg/kg body weight, causing a decrease of 60 and 39 percent, respectively. In the present study, it was observed that this fucan has anti-inflammatory properties but no cytotoxic action, indicating its potential use in the pharmaceutical industry.

Heparin-integrin interaction in endothelial cells: downstream signaling and heparan sulfate expression.

Endothelial cells (ECs) are a source of physiologically important molecules that are synthesized and released to the blood and/or to the subendothelial extracellular matrix such as a heparan sulfate proteoglycan (HSPG) with antithrombotic properties. Previously, we have shown that heparin stimulates the synthesis and modifies the sulfation pattern of this HSPG. Here the molecular mechanisms involved in the up-regulation of HSPG synthesis by heparin in endothelial cells were decoded. The cells were stimulated with heparin and the expression of HSPG and intracellular pathways were evaluated by a combination of methods involving confocal microscopy, flow cytometry, Western blotting analyses, and [(35) S]-sulfate metabolically labeling of the cells. We observed that the up-regulation of HSPG synthesis evoked by heparin is dependent on the interaction of heparin with integrin since RGD peptide abolishes the effect. The activation of integrin leads to tyrosine-phosphorylation of focal adhesion-associated proteins such as FAK, Src, and paxillin. In addition, heparin induces ERK1/2 phosphorylation and inhibitors of Ras and MEK decreased heparin-dependent HSPG synthesis. Moreover, heparin also induced intracellular Ca(2+) release, PLCγ1 (phospholipase Cγ1) and CaMKII (calcium calmodulin kinase II) activation, as well as an increase in nitric oxide (NO) production. Finally, an intracellular Ca(2+) chelator, Ca(2+) signaling inhibitors, and an endothelial NO synthase inhibitor were all able to abolish the effect in heparan sulfate synthesis. In conclusion, the heparin-induced up-regulation of HSPG expression is associated with the phosphorylation of focal adhesion proteins and Ras/Raf/MEK/ERK MAP and Ca(2+) /NO pathways.

Chemical reduction of carboxyl groups in heparin abolishes its vasodilatory activity.

Previous studies have shown that heparin induces vascular relaxation via integrin-dependent nitric oxide (NO)-mediated activation of the muscarinic receptor. The aim of this study was to identify the structural features of heparin that are necessary for the induction of vasodilatation. To address this issue, we tested heparin from various sources for their vasodilatation activities in the rat aorta ring. Structural and chemical characteristics of heparin, such as its molecular weight and substitution pattern, did not show a direct correlation with the vasodilation activity. Principal component analysis (PCA) of circular dichroism (CD), (1)H-nuclear magnetic resonance (NMR) and vasodilation activity measurements confirmed that there is no direct relationship between the physico-chemical nature and vasodilation activity of the tested heparin samples. To further understand these observations, unfractionated heparin (UFH) from bovine intestinal mucosa, which showed the highest relaxation effect, was chemically modified. Interestingly, non-specific O- and N-desulfation of heparin reduced its anticoagulant, antithrombotic, and antihemostatic activities, but had no effect on its ability to induce vasodilation. On the other hand, chemical reduction of the carboxyl groups abolished heparin-induced vasodilation and reduced the affinity of heparin toward the extracellular matrix (ECM). In addition, dextran and dextran sulfate (linear non-sulfated and highly sulfated polysaccharides, respectively) did not induce significant relaxation, showing that the vasodilation activity of polysaccharides is neither charge-dependent nor backbone unspecific. Our results suggest that desulfated heparin molecules may be used as vasoactive agents due to their low side effects.

Glycosaminoglycans of abdominal skin after massive weight loss in post-bariatric female patients.

BACKGROUND: The number of post-bariatric patients had a significant increase over the last years, and a better understanding of the consequences of massive weight loss on skin is imperative. Despite weight-loss-related changes in collagen and elastin have been reported, less is known about changes in another of the matrix components of the skin, the glycosaminoglycans. The objective of this study is to evaluate abdominal skin glycosaminoglycans concentrations and perlecan and collagen III expression in post-bariatric female patients. METHODS: Skin tissue samples from the abdomen of lean (n = 19) and post-bariatric (n = 24) female patients were compared. Sulfated glycosaminoglycans and hyaluronic acid were extracted, characterized and quantified. Perlecan and collagen III expression was assessed by immunofluorescence. RESULTS: The major glycosaminoglycans found were dermatan sultafe and hyaluronic acid; the others were found in smaller amounts. The skin of the post-bariatric patients had lower concentrations of heparan sulfate (p = 0.002) while hyaluronic acid, dermatan sulfate, and chondroitin sulfate concentrations were similar to the lean women's skin. Post-bariatric skin showed decreased expression of perlecan and increased expression of collagen III. No correlation was found among glycosaminoglycans concentrations and age, body mass index, frequency of pregnancies, or skin types, but it was observed in higher skin heparan sulfate concentrations in post-bariatric patients who had their weights stabilized for over than 24 months (p = 0.000). CONCLUSION: Abdominal skin of post-bariatric women presented decreased heparan sulfate concentrations and perlecan expression and increased expression of collagen III.

Heparin induces rat aorta relaxation via integrin-dependent activation of muscarinic M3 receptors.

Previous reports have shown that heparin may promote human hypotension and vascular relaxation by elevation of NO levels through unclear mechanisms. We hypothesized that endothelial muscarinic M(3) receptor activation mediates the heparin-induced vasodilation of rat aortic rings. The experiments were carried out using unfractionated heparin extracted from bovine intestinal mucosa, which elicited an endothelium and NO-dependent relaxation of aortic segments with maximal potency and efficacy (EC(50): 100±10 µmol/L; E(max): 41±3%). Atropine and 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide inhibitors reduced the heparin-dependent relaxation, indicating that M(3) muscarinic receptor is involved in this phenomenon. However, no direct binding of heparin to muscarinic receptors was observed. More importantly, studies performed using the arginine-glycine-aspartic acid peptide and 1-(1,1-dimethylethyl)-3-(1-naphthalenyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine, an Src family inhibitor, reduced by 51% and 73% the heparin-dependent relaxation, respectively, suggesting the coupling of heparin and M(3) receptor through extracellular matrix molecules and integrin. Furthermore, unfractionated heparin induced activation of focal adhesion protein kinase, Src, and paxillin. Finally, fluorescence resonance energy transfer approach confirmed the interaction of the M(3) receptor to integrin. Taken together, these data demonstrate the participation of M(3) receptor and integrin in heparin-dependent relaxation of vascular smooth muscle. These results provide new insights into the molecular mechanism and potential pharmacological action of heparin in vascular physiology.

Anti-inflammatory properties of a heparin-like glycosaminoglycan with reduced anti-coagulant activity isolated from a marine shrimp.

The anti-inflammatory properties of a heparin-like compound from the shrimp Litopenaeus vannamei are related. Besides reducing significantly (p<0.001) the influx of inflammatory cells to injury site in a model of acute inflammation, shrimp heparin-like compound was able to reduce the matrix metalloproteinase (MMPs) activity in the peritoneal lavage of inflamed animals. Moreover, this compound also reduced almost 90% the activity of MMP-9 secreted by human activated leukocytes. Negligible anti-coagulant activities in aPPT assay and a poor bleeding potential make this compound a better alternative than mammalian heparin as a possible anti-inflammatory drug.

A non-anticoagulant heterofucan has antithrombotic activity in vivo.

Fucan is a term used to denominate a family of sulfated L-fucose-rich polysaccharides. The brown alga Spatoglossum schröederi (Dictyotaceae) has three heterofucans namely fucan A, B and C. The 21 kDa fucan A is composed of a core of a beta (1-3) glucuronic acid-containing oligosaccharide of 4.5 kDa with branches at C4 of the fucose chains alpha (1-3) linked. The fucose is mostly substituted at C4 with a sulfate group and at C2 with chains of beta (1-4) xylose. This fucan has neither anticoagulant (from from 0.1 to 100 microg) nor hemorrhagic activities (from 50 to 800 microg/mL). The antithrombotic test in vivo showed that fucan A has no activity in any of the concentrations (from 0.2 to 20 microg/g/day) tested 1 h after polysaccharide administration. However, when fucan A was injected endovenously 24 h before the ligature of the venae cavae, we observed a dose-dependent effect, reaching saturation at around 20 microg/g of rat weight. In addition, this effect is also time-dependent, reaching saturation around 16 h after fucan administration. In addition, regardless of the administration route, fucan A displayed antithrombotic activity. The exception was the oral pathway. Of particular importance was the finding that fucan A stimulates the synthesis of an antithrombotic heparan sulfate from endothelial cells like heparin. The hypothesis has been raised that the in vivo antithrombotic activity of fucan A is related to the increased production of this heparan. Taken together with the fact that the compound is practically devoid of anticoagulant and hemorrhagic activity, the data suggest that it may be an ideal antithrombotic agent in vivo.