Mechanism of Heparin Acceleration of Tissue Inhibitor of Metalloproteases-1 (TIMP-1) Degradation by the Human Neutrophil Elastase

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k2 = 21±1 s−1) was much higher than the HNE deacylation step (k3 = 0.57±0.05 s−1). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k1 2.4-fold and reducing k−1 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k2 value, whereas the k3 value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs

A New Approach for Heparin Standardization: Combination of Scanning UV Spectroscopy, Nuclear Magnetic Resonance and Principal Component Analysis

The year 2007 was marked by widespread adverse clinical responses to heparin use, leading to a global recall of potentially affected heparin batches in 2008. Several analytical methods have since been developed to detect impurities in heparin preparations; however, many are costly and dependent on instrumentation with only limited accessibility. A method based on a simple UV-scanning assay, combined with principal component analysis (PCA), was developed to detect impurities, such as glycosaminoglycans, other complex polysaccharides and aromatic compounds, in heparin preparations. Results were confirmed by NMR spectroscopy. This approach provides an additional, sensitive tool to determine heparin purity and safety, even when NMR spectroscopy failed, requiring only standard laboratory equipment and computing facilities

Response to Letter to the Editor, Tooth Bleaching Increases Dentinal Protease Activity

Universidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilUniv Mogi das Cruzes, Ctr Interdisciplinar Invest Bioquim, Mogi das Cruzes, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilWeb of Scienc

PAR-1 and PAR-2 Expression Is Enhanced in Inflamed Odontoblast Cells

Protease-activated receptors (PARs) are G protein-coupled receptors, which are activated by proteolytical cleavage of the amino-terminus and act as sensors for extracellular proteases. We hypothesized that PAR-1 and PAR-2 can be modulated by inflammatory stimulus in human dental pulp cells. PAR-1 and PAR-2 gene expression in human pulp tissue and MDPC-23 cells were analyzed by quantitative polymerase chain reaction. Monoclonal PAR-1 and PAR-2 antibodies were used to investigate the cellular expression of these receptors using Western blot, flow cytometry, and confocal microscopy in MDPC-23 cells. Immunofluorescence assays of human intact and carious teeth were performed to assess the presence of PAR-1 and PAR-2 in the dentin-pulp complex. The results show for the first time that human odontoblasts and MDPC-23 cells constitutively express PAR-1 and PAR-2. PAR-2 activation increased significantly the messenger RNA expression of matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, and MMP-14 in MDPC-23 cells (P <0.05), while the expression of these enzymes decreased significantly in the PAR-1 agonist group (P <0.05). The high-performance liquid chromatography and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis showed the presence of MMP-13 activity cleaving PAR-1 at specific, noncanonical site TLDPRS42 down arrow(FLL)-L-43 in human dental pulp tissues. Also, we detected a presence of a trypsin-like activity cleaving PAR-2 at canonical site SKGR(20)down arrow S(21)LIGRL in pulp tissues. Confocal microscopy analysis of human dentin-pulp complex showed intense positive staining of PAR-1 and PAR-2 in the odontoblast processes in dentinal tubules of carious teeth compared to intact ones. The present results support the hypothesis of activation of the upregulated PAR-1 and PAR-2 by endogenous proteases abundant during the inflammatory response in dentin-pulp complex.Peer reviewe

The self-assembling peptide P-11-4 prevents collagen proteolysis in dentin

The major goal in restorative dentistry is to develop a true regenerative approach that fully recovers hydroxyapatite crystals within the caries lesion. Recently, a rationally designed self-assembling peptide P-11-4 (Ace-QQRFEWEFEQQ-NH2) has been developed to enhance remineralization on initial caries lesions, yet its applicability on dentin tissues remains unclear. Thus, the present study investigated the interaction of P-11-4 with the organic dentin components as well as the effect of P-11-4 on the proteolytic activity, mechanical properties of the bonding interface, and nanoleakage evaluation to artificial caries-affected dentin. Surface plasmon resonance and atomic force microscopy indicated that P-11-4 binds to collagen type I fibers, increasing their width from 214 +/- 4 nm to 308 +/- 5 nm (P < 0.0001). P-11-4 also increased the resistance of collagen type I fibers against the proteolytic activity of collagenases. The immediate treatment of artificial caries-affected dentin with P-11-4 enhanced the microtensile bonding strength of the bonding interface (P < 0.0001), reaching values close to sound dentin and decreasing the proteolytic activity at the hybrid layer; however, such effects decreased after 6 mo of water storage (P < 0.05). In conclusion, P-11-4 interacts with collagen type I, increasing the resistance of collagen fibers to proteolysis, and improves stability of the hybrid layer formed by artificial caries-affected dentin983347354CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçãosem informação2013/05822-9; 2014/22899-8; 2015/12660-0; 2013/05822-9; 2014//22899-8; 2015/03964-

Protective Effects of Heparin on Hepatic Ischemia and Reperfusion Lesions in Rabbits

Because the role of heparin (HEP) in hepatic ischemia/reperfusion (I/R) injury is still not fully understood, we investigated the effects of treatment with HEP on hepatic I/R injury in rabbits. for I/R procedures, the portal vein and hepatic artery were occluded by a metallic clamp to promote ischemia. the clamp was removed after 30 minutes to allow reperfusion. Rabbits undergoing the I/R procedure were treated with HEP (100 U/kg) or saline solution 0.9% (SS). When compared with levels before I/R, the serum aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, levels were increased by the hepatic I/R procedure, among rabbits treated with SS or HER However, the increase in these enzymes was lower among rabbits treated with HEP. Histologic analysis of hepatic tissue of rabbits undergoing I/R and treated with SS showed marked lesions in the central lobule with significant inflammatory infiltration. in contrast, a significant reduction in lesions caused by I/R was observed in the livers of rabbits treated with HER After starting reperfusion, we visualized apoptotic cells with nuclear staining among rabbits submitted to I/R and treated with SS, but not those treated with HER These results suggested that HEP was able to attenuate hepatic lesions caused by I/R in the livers of rabbits.Universidade Federal de São Paulo, Escola Paulista Med, Dept Surg, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Morphol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biochem, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Pharmacol, BR-04023900 São Paulo, BrazilFed Univ Grande Dourados, Dept Med, Grande Dourados, MS, BrazilUniv Mogi Cruzws, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Surg, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Morphol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biochem, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Pharmacol, BR-04023900 São Paulo, BrazilWeb of Scienc

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), 1H-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. J. Cell. Biochem. 113: 13591367, 2012. (c) 2011 Wiley Periodicals, Inc.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2006/61006-2]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Financiadora de Estudos e Projetos (FINEP)Financiadora de Estudos e Projetos (FINEP)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES