Katarzyna Winsz-Szczotka

Age- and Gender-Dependent Changes in Connective Tissue Remodeling: Physiological Differences in Circulating MMP-3, MMP-10, TIMP-1 and TIMP-2 Level

Background: The mechanisms which cause age-dependent remodeling of connective tissue are still not fully understood. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) constitute an important proteolytic pathway affecting physiological matrix remodeling. Objective: The way in which changes in the extracellular matrix metabolism during the ageing process influence the level of circulating MMP-3 and MMP-10, as well as their tissue inhibitors TIMP-1 and TIMP-2, in a healthy population was investigated in this study. Methods: Blood samples were taken from 81 healthy individuals aged 6–62 years and measured for MMP-3, MMP-10, TIMP-1 and TIMP-2 levels using enzyme-linked immunosorbent assays. Polyacrylamide gel electrophoresis followed by Western immunoblotting allowed for the detection of pro- and active forms of both MMPs. Results: Serum MMP-3 and TIMP-1 values were positively correlated with age (r = 0.44, p = 0.00001 and r = 0.28, p = 0.012, respectively). A contrary tendency was found for MMP-10 and TIMP-2 serum levels. A strong age-related decrease in MMP-10 (–0.53; p = 0.000) and TIMP-2 (–0.52; p = 0.000) was noticed in our study. Gender was a significant factor modifying MMP/TIMP potential, except for the MMP-10 level. Conclusions: The data presented indicate that changes in MMP/TIMP balance occur in physiological ageing. Moreover, these findings highlight the necessity of utilizing age- and sex-matched values for analysis of MMPs and TIMPs in the pathological conditions.

Propolis Induces Chondroitin/Dermatan Sulphate and Hyaluronic Acid Accumulation in the Skin of Burned Wound

Changes in extracellular matrix glycosaminoglycans during the wound repair allowed us to apply the burn model in which therapeutic efficacy of propolis and silver sulfadiazine was compared. Burns were inflicted on four pigs. Glycosaminoglycans isolated from healthy and burned skin were quantified using a hexuronic acid assay, electrophoretic fractionation, and densitometric analyses. Using the reverse-phase HPLC the profile of sulfated disaccharides released by chondroitinase ABC from chondroitin/dermatan sulfates was estimated. Chondroitin/dermatan sulfates and hyaluronic acid were found in all samples. Propolis stimulated significant changes in the content of particular glycosaminoglycan types during burn healing. Glycosaminoglycans alterations after silver sulfadiazine application were less expressed. Propolis maintained high contribution of 4-O-sulfated disaccharides to chondroitin/dermatan sulfates structure and low level of 6-O-sulfated ones throughout the observed period of healing. Propolis led to preservation of significant contribution of disulfated disaccharides especially 2,4-O-disulfated ones to chondroitin sulfates/dermatan sulfates structure throughout the observed period of healing. Our findings demonstrate that propolis accelerates the burned tissue repair by stimulation of the wound bed glycosaminoglycan accumulation needed for granulation, tissue growth, and wound closure. Moreover, propolis accelerates chondroitin/dermatan sulfates structure modification responsible for binding growth factors playing the crucial role in the tissue repair.

Age-related changes of plasma glycosaminoglycans

Abstract Background: This study was undertaken to reveal how the physiological ageing process influences plasma glycosaminoglycans (GAGs) content and composition. Methods: GAGs isolated from plasma samples obtained from 105 healthy subjects were quantified using a hexuronic acid assay and subjected to electrophoretic fractionation. Results: We found that total GAGs decreased gradually with advancing age. The electrophoretic analysis allowed identifying chondroitin sulfates, dermatan sulfates and heparan sulfates and heparin in plasma of healthy individuals. Chondroitin sulfates, predominant plasma GAGs, showed declining values with age and displayed higher structural diversity in subjects from the first five decades of life compared to older subjects. An age-related increase in plasma dermatan sulfates content during the first four decades of life followed by a decrease in a later period of life was apparent. Structural heterogeneity of these glycans was found in subjects until 10 years of age. Heparan sulfates and heparin plasma levels were inversely correlated with age until 25 years of age, without age-dependent changes thereafter. These macromolecules displayed higher heterogeneity of structure in subjects over 50 years of age than in younger subjects. Conclusions: The obtained results indicate that age should be taken into account in all studies investigating blood GAGs alterations during pathological conditions. Clin Chem Lab Med 2008;46:219–24.

Propolis modulates vitronectin, laminin, and heparan sulfate/heparin expression during experimental burn healing *

ObjectiveThis study was aimed at assessing the dynamics of vitronectin (VN), laminin (LN), and heparan sulfate/heparin (HS/HP) content changes during experimental burn healing.MethodsVN, LN, and HS/HP were isolated and purified from normal and injured skin of domestic pigs, on the 3rd, 5th, 10th, 15th, and 21st days following thermal damage. The wounds were treated with apitherapeutic agent (propolis), silver sulfadiazine (SSD), physiological salt solution, and propolis vehicle. VN and LN were quantified using an immunoenzymatic assay and HS/HP was estimated by densitometric analysis.ResultsPropolis treatment stimulated significant increases in VN, LN, and HS/HP contents during the initial phase of study, followed by a reduction in the estimated extracellular matrix molecules. Similar patterns, although less extreme, were observed after treatment with SSD.ConclusionsThe beneficial effects of propolis on experimental wounds make it a potential apitherapeutic agent in topical burn management.

Alterations of glycosaminoglycan metabolism in the development of diabetic complications in relation to metabolic control.

Abstract Disturbed metabolism of glycosaminoglycans (GAGs) has been proposed to play an important role in the pathogenesis of late diabetic complications. The effect of diabetic complications and metabolic control on both total serum GAGs content and the serum activity of lysosomal glycosidases (N-acetyl-β-D-glucosaminidase, α-L-fucosidase, β-D-galactosidase, and α-D-mannosidase) contributing to GAGs degradation, was investigated in 48 patients with type 2 diabetes mellitus. The activity of β-D-glucosidase and acid phosphatase, the lysosomal enzymes unrelated to GAGs metabolism, was determined for comparison. The elevated serum total GAG concentration in diabetic patients was strongly and positively influenced by poor metabolic compensation of diabetes and the presence of vascular complications. A similar tendency has been shown in regard to the activity of enzymes involved in GAG degradation, especially N-acetyl-β-D-glucosaminidase, α-L-fucosidase and β-D-galactosidase. Furthermore, the total serum GAG concentrations, as well as the activity of lysosomal enzymes involved in the extracellular matrix degradation, closely followed metabolic compensation, regardless of diabetic vascular complications. Thus, we suggest that increased values of the investigated parameters may indicate the degree of endothelial cell dysfunction and may be useful to predict the development of diabetic vascular pathology.

Relationship between adiponectin, leptin, IGF-1 and total lipid peroxides plasma concentrations in patients with systemic sclerosis: possible role in disease development

The relationship between adiponectin, leptin, insulin‐like growth factor‐1 (IGF‐1) and total lipid peroxide (TLP) concentrations, and its possible role in the development of diffuse cutaneous systemic sclerosis (dcSSc), were evaluated in this study.

Age- and gender-related alteration in plasma advanced oxidation protein products (AOPP) and glycosaminoglycan (GAG) concentrations in physiological ageing.

Abstract Background: The authors studied the role of increased oxidative stress in the development of oxidative protein damage and extracellular matrix (ECM) components in ageing. The age- and gender-associated disturbances in connective tissue metabolism were evaluated by the plasma chondroitin sulphated glycosaminoglycans (CS-GAG) and non-sulphated GAG-hyaluronan (HA) measurements. Plasma concentration of advanced oxidation protein products (AOPP) was analysed in order to assess oxidative protein damage and evaluate the possible deleterious role of oxidative phenomenon on tissue proteoglycans’ metabolism during the physiological ageing process. Methods: Sulphated and non-sulphated GAGs as well as AOPP were quantified in plasma samples from 177 healthy volunteers. Results: A linear age-related decline of plasma CS-GAG level was found in this study (r=–0.46; p<0.05). In contrast, HA concentrations rise gradually with age (r=0.44; p<0.05) in plasma samples. For both ECM components, the observed differences were not gender-specific. A strong age-dependent relationship has been shown in regard to AOPP. AOPP levels significantly increased with age (r=0.63; p<0.05), equally strongly in both men (r=0.69; p<0.05) and women (r=0.57; p<0.05) during physiological ageing. A significant correlation was found between the concentrations of AOPP and both CS-GAG (r=–0.31; p<0.05) and HA (r=0.33; p<0.05). Conclusions: Proceeding with age changes in the ECM are reflected by CS-GAG and HA plasma levels. Strong correlations between AOPP and ECM components indicate that oxidative stress targets protein and non-protein components of the connective tissue matrix during human ageing.

Age- and gender-dependent changes in circulating concentrations of tumor necrosis factor-α, soluble tumor necrosis factor receptor-1 and sulfated glycosaminoglycan in healthy people

Abstract Background: In this study, the effect of gender and physio-logical ageing on circulating concentrations of plasma sulfated glycosaminoglycans (sGAG) as well as molecules involved in pro- (tumor necrosis factor-α; TNF-α) and anti-inflammatory responses (soluble tumor necrosis factor receptor-1, sTNF-RI) were assessed. The relationships between sGAG and molecules involved in age-dependent extracellular matrix (ECM) remodeling during physiological ageing were also investigated. Methods: Circulating TNF-α and sTNF-RI were measured in 91 healthy volunteers using enzyme-linked immunosorbent assays. sGAG were quantified using an Alcian blue-binding assay. Results: A linear age-related decline in plasma sGAG was found during the first five decades of life (r=–0.61, p<0.05), followed by an increase occurring only in females (r=0.46, p<0.05). Circulating TNF-α concentrations were inversely correlated with age (r=–0.24, p<0.05) over the lifetime. For TNF-α, the observed changes were gender specific. Serum sTNF-RI concentrations were not affected by age in either men or women. A significant positive correlation was found between the concentrations of TNF-α and both sGAG (r=0.22, p<0.05) and sTNF-RI (r=0.21, p<0.05). Conclusions: Our data demonstrate that physiological ageing is associated with ECM remodeling, reflected by plasma sGAGs concentrations. Changes in the ECM metabolism during the ageing process were influenced by circulating TNF-α. Furthermore, serum concentrations of biomolecules involved in pro- and anti-inflammatory responses are not increased in healthy elderly subjects.

Adiponectin, Leptin, and Leptin Receptor in Obese Patients with Type 2 Diabetes Treated with Insulin Detemir

The aim of the present study is to quantitatively assess the expression of selected regulatory molecules, such as leptin, leptin receptor, and adiponectin in the blood of obese patients with type 2 diabetes both before treatment and after six months of pharmacological therapy with the long-lasting insulin analogue, insulin detemir. A significant decrease in the analysed regulatory molecules, i.e., leptin receptor and adiponectin, was found in blood plasma of the patients with untreated type 2 diabetes. These changes were accompanied by an increase in plasma leptin concentrations. Insulin treatment resulted in the normalization of plasma leptin receptor and adiponectin concentrations. The circulating leptin level did not change following anti-diabetic therapy with insulin detemir. Gender was a significant factor modifying the circulating level of all the analysed regulatory active compounds. Bioinformatic analysis was performed using Matlab with the Signal Processing Toolbox. The conducted discriminant analysis revealed that the leptin receptor, Δw(19), and adiponectin, Δw(21), were the parameters undergoing the most significant quantitative changes during the six-month therapy with insulin detemir. The conducted examinations indicated the contribution of adipocytokines—the biologically-active mediators of systemic metabolism, such as leptin and adiponectin in the pathomechanism of disorders being the basis for obesity which leads to development of insulin resistance, which, in turn, results in the occurrence of type 2 diabetes.

Urinary glycosaminoglycan (uGAG) excretion in healthy pediatric and adolescent population.

OBJECTIVES The influence of age and gender factor on the urinary excretion of total glycosaminoglycans (uGAGs) and their particular types: chondroitin/dermatan sulfates (CS/DSs), heparan sulfates (HSs) and hyaluronan (HA) was analyzed in healthy pediatric and adolescent population. DESIGN AND METHODS Urine samples were collected from 95 healthy children. Sulfated GAGs excreted in the urine were quantitated using standardized dye-binding method, while the concentrations of HA were determined by immunoassay. RESULTS Age-dependent decline in total uGAG excretion (r=-0.686; p<0.001), resulting from a decrease in particular GAG fractions i.e. CS/DS (r=-0.757; p<0.001), HS (r=-0.401; p<0.05) and HA (r=-0.638; p<0.001), was found in healthy subjects. The observed differences were not gender specific with the exception of HS, in which excretion declines with age in males (r=-0.501; p<0.05) and does not change in females. Changes in the distribution pattern of uGAG were also found. CS/DS were the predominant uGAGs fraction, representing from 55% to 76% of the total GAGs. Children up to 3 years excreted more GAGs than older subjects and with a higher proportion of CS/DS and less content of HS. Moreover, the relative contribution of HA was increased twofold in adolescents, aged 15-18, as compared to younger subjects. A negative correlation existed between uGAG excretion and body height, except for HS, for which this relationship was found only in males. CONCLUSIONS Changes in urinary distribution pattern of particular GAG types during physiological human growth and development were found. Evaluation of urinary GAG screening procedures during pathological conditions should be based on the GAG/creatinine ratios with age and gender taken into account.