Glycosaminoglycan Levels Research Articles

Cell cycle disturbances in mucopolysaccharidoses: Transcriptomic and experimental studies on cellular models.

Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases caused by defects in genes coding for proteins involved in degradation of glycosaminoglycans (GAGs). These complex carbohydrates accumulate in cells causing their serious dysfunctions. Apart from the physical GAG storage, secondary and tertiary changes may contribute significantly to the pathomechanism of the disease. Among processes which were not systematically investigated in MPS cells to date there is the cell cycle. Here, we studied perturbances in this crucial cellular process in majority of MPS types. Transcriptomic analyses indicated that expression of many genes coding for proteins involved in the cell cycle is dysregulated in all tested MPS cells. Importantly, levels of transcripts of particular genes were changed in the same manner (i.e. either up- or down-regulated) in most or all types of the disease, indicating a common mechanism of the dysregulation. Flow cytometric studies demonstrated that the cell cycle is disturbed in all MPS types, with increased fractions of cells in the G0/G1 phase in most types and decreased fractions of cells in the G2/M phase in all types. We found that increased levels of cyclin D1 and disturbed timing of its appearance during the cell cycle may contribute to the mechanism of dysregulation of this process in MPS. Reduction of GAG levels by either a specific enzyme or genistein-mediated inhibition of synthesis of these compounds improved, but not fully corrected, the cell cycle in MPS fibroblasts. Therefore, it is suggested that combination of the therapeutic approaches devoted to reduction of GAG levels with cyclin D1 inhibitors might be considered in further works on developing effective treatment procedures for MPS.

Fluvastatin promotes chondrogenic differentiation of adipose-derived mesenchymal stem cells by inducing bone morphogenetic protein 2

BackgroundAdipose-derived mesenchymal stem cells (ADMSCs) are a promising source of material source for medical regeneration of cartilage. Growth factors, including transforming growth factor-β (TGFβ) subfamily members and bone morphogenetic proteins (BMPs), play important roles in inducing and promoting chondrogenic differentiation of MSCs. However, these exogenous growth factors have some drawbacks related to their cost, biological half-life, and safety for clinical application. Several studies have reported that statins, the competitive inhibitors of 3-hydroxy-2-methylglutaryl coenzyme A (HMG-CoA) reductase, induce the expression of BMP2 in multiple cell types as the pleotropic effects. The objective of this study was to investigate the effects of fluvastatin during chondrogenic differentiation of human ADMSCs (hADMSCs).MethodsThe effects of fluvastatin were analyzed during chondrogenic differentiation of hADMSCs in the pellet culture without exogenous growth factors by qRT-PCR and histology. For functional studies, Noggin, an antagonist of BMPs, mevalonic acid (MVA) and geranylgeranyl pyrophosphate (GGPP), metabolites of the mevalonate pathway, ROCK inhibitor (Y27632), or RAC1 inhibitor (NSC23766) were applied to cells during chondrogenic differentiation. Furthermore, RhoA activity was measured by RhoA pulldown assay during chondrogenic differentiation with or without fluvastatin. Statistically significant differences between groups were determined by Student’s t-test or the Tukey–Kramer test.ResultsFluvastatin-treated cells expressed higher levels of BMP2, SOX9, ACAN, and COL2A1 than control cells, and accumulated higher levels of glycosaminoglycans (GAGs). Noggin significantly inhibited the fluvastatin-mediated upregulation of ACAN and COL2A1. Both MVA and GGPP suppressed the effects of fluvastatin on the expressions of BMP2, SOX9, ACAN, and COL2A1. Furthermore, fluvastatin suppressed the RhoA activity, and inhibition of RhoA–ROCK signaling by Y27632 increased the expressions of BMP2, SOX9, ACAN, and COL2A1, as well as fluvastatin.ConclusionsOur results suggest that fluvastatin promotes chondrogenic differentiation of hADMSCs by inducing endogenous BMP2, and that one of the mechanisms underlying the effects is inhibition of RhoA–ROCK signaling via suppression of GGPP. Fluvastatin is a safe and low-cost compound that holds promise for use in transplantation of hADMSCs for cartilage regeneration.

Efficacy of Nerve-Derived Hydrogels to Promote Axon Regeneration Is Influenced by the Method of Tissue Decellularization.

Injuries to large peripheral nerves are often associated with tissue defects and require reconstruction using autologous nerve grafts, which have limited availability and result in donor site morbidity. Peripheral nerve-derived hydrogels could potentially supplement or even replace these grafts. In this study, three decellularization protocols based on the ionic detergents sodium dodecyl sulfate (P1) and sodium deoxycholate (P2), or the organic solvent tri-n-butyl phosphate (P3), were used to prepare hydrogels. All protocols resulted in significantly decreased amounts of genomic DNA, but the P2 hydrogel showed the best preservation of extracellular matrix proteins, cytokines, and chemokines, and reduced levels of sulfated glycosaminoglycans. In vitro P1 and P2 hydrogels supported Schwann cell viability, secretion of VEGF, and neurite outgrowth. Surgical repair of a 10 mm-long rat sciatic nerve gap was performed by implantation of tubular polycaprolactone conduits filled with hydrogels followed by analyses using diffusion tensor imaging and immunostaining for neuronal and glial markers. The results demonstrated that the P2 hydrogel considerably increased the number of axons and the distance of regeneration into the distal nerve stump. In summary, the method used to decellularize nerve tissue affects the efficacy of the resulting hydrogels to support regeneration after nerve injury.

Effect of High Pressure/Heating Combination on the Structure and Texture of Chinese Traditional Pig Trotter Stewed with Soy Sauce

In order to clarify the effect of a high pressure/heating combination on the texture of Chinese traditional pig trotter with soy sauce, textural parameters (springiness, chewiness, hardness, and gumminess), the secondary structures, cross-links, decorin (DCN), glycosaminoglycans (GAGs) levels, and the histochemical morphology of collagen fibers under different treatments (0.1 MPa, 150 MPa, 300 MPa, 0.1 MPa + 50 °C, 150 MPa + 50 °C, and 300 MPa + 50 °C) were assessed. At room temperature, the 150 and 300 MPa treatments increased the hardness and chewiness of the pig trotter with weak denaturation of collagen proteins compared with the control group. Textural parameters were improved at 300 MPa + 50 °C, accompanied by an ultrastructural collapse of collagen fibers, the reduction in cross-links, DCN and GAGs levels, and unfolded triple-helix structure. We concluded that the positive effects on the textural parameters of pig trotters by a combination of treatments could be attributed to the collapse of collagen structure.

Facial skin ageing: Key concepts and overview of processes.

IntroductionThe face is a cosmetically sensitive region where the process of ageing is most clearly manifested. With increased focus on anti‐ageing and longevity, more anti‐senescent treatments are being proposed despite limited evidence. This study outlines the pathways and mechanisms underpinning the biological process of ageing in the face.MethodsComprehensive searches of MEDLINE, EMBASE, Cochrane Library and CINAHL from inception to 2020. Inclusion criteria included all empirical human research studies specific to facial ageing features, written in the English language.ResultsA total of 65 papers met inclusion criteria for analysis. Pathways were subdivided into intrinsic and extrinsic senescence mechanisms. Intrinsic pathways included genetics, generation of reactive oxygen species and hormonal changes. Extrinsic pathways included photoageing and damage to skin layers. The combined intrinsic and extrinsic pathway alterations result in wrinkles, higher laxity, slackness and thinning of the skin. Skin functions such as barrier immune function, wound healing, thermoregulation and sensory function are also impaired.ConclusionThe ageing process is unique to the individual and depends on the interplay between an individual's genetics and external environmental factors. Through understanding the molecular and cellular mechanisms, an appreciation of the consequent structural and functional changes can be achieved. Based on this knowledge, further research can focus on how to slow or impede the ageing process and identify specific targets to develop and evolve new treatment strategies.

Peculiarities of the structural and functional state of bone tissue in children during the second growth spurt

Background. Bone health is relevant for patients of all age groups. Special attention is paid to childhood and adolescence, since it is during this period that bone mass is actively accumulated, which depends on the structural and functional state of the bone tissue of an adult. The purpose of this study was to determine the features of the structural and functional state of bone tissue in children during the second growth spurt based on the analysis of markers of bone modeling, the level of 25(OH)D and the indicators of ultrasound densitometry and DXA. Materials and methods. 205 children aged 9–17 years were examined. They divided into three groups depending on the presence and intensity of growth spurts. Examination included: general clinical examination with an assessment of the levels of physical and sexual development, determine of the levels of 25(OH)D, glycosaminoglycans, chondroitin sulfates and alkaline phosphatase, ultrasound densitometry and DXA. Results. 83.9 % of children had vitamin D deficiency, 16.1 % of children had insufficiency. A decrease in BMD according to UD data was diagnosed in 48.0 % (n = 24) of children with growth spurt, in 60.87 % (n = 28) of children with intense growth spurt, and in 39.45% (n = 43) of children without growth spurt. According to DXA data, BMD reduction was confirmed in 56.25 % (n=18) of 32 examined children: children with growth spurt — 38.9 % (n=7), children with intense growth spurt— 50.0% (n = 9), children without growth spurt — 11.1 % (n = 2). The level of chondroitin sulfates in children with growth spurt and intense growth spurt is significantly higher than in children without growth spurt, which indicates the instability of metabolic processes. At the same time, the level of total glycosaminoglycans is significantly lower in the group of children with growth spurt and intense growth spurt than in the group of children without growth spurt, which indicates the intensity of metabolic processes in children of these groups. Conclusions. 100 % of children had insufficiency and deficiency of vitamin D. A decrease in BMD is more often diagnosed in children during an intense growth spurt and does not have a reliable relationship with the level of vitamin D, in contrast to children without a growth spurt. Children with growth spurts and intense growth spurts had an imbalance in the level of chondroitin sulfates and glycosaminoglycans, which indicates the instability and activity of bone tissue growth processes.

The Texture Change of Chinese Traditional Pig Trotter with Soy Sauce during Stewing Processing: Based on a Thermal Degradation Model of Collagen Fibers.

In order to clarify the influence of the thermal degradation of collagen fibers on the texture profile analysis (TPA) parameters of pig trotter stewed with soy sauce (PTSWSS), TPA (springiness, chewiness, hardness, and gumminess), the secondary structures, the cross-linkage, decorin (DCN) and glycosaminoglycan (GAG) levels, and the histochemical morphology of collagen fibers during the stewing process (0, 30, 60, 120 min) were assessed. The springiness and hardness increased after 30 min of stewing, along with the denaturation of collagen proteins. TPA parameters improved with the prolonged stewing times of 60 and 120 min, along with the ultra-structural dissolution of collagen fibers, and a substantial reduction in cross-linkage, DCN, and GAG levels, and the unfolded triple-helix structure. This study concluded that the TPA parameters of PTSWSS were dependent on the stewing time, and that the improvement in TPA parameters with longer stewing time could primarily be attributed to the thermal degradation of collagen fibers.

Застосування методів консервативної терапії в дітей з післяопіковими рубцевими деформаціями та їх вплив на метаболізм сполучної тканини

One of the factors of scarring is changes in the connective tissue (CT). Defeat of CT is accompanied by changes in the content of glycosaminoglycans (GAG), activity of lysosomal enzymes, levels of mineral metabolism. To eliminate the influence of inflammatory phenomena and disorders of CT metabolism on the formation of scar tissue, it is urgent to use conservative rehabilitation measures. Purpose - to determine the effectiveness of measures of conservative therapy of post-burn scars in children for the clinical course and metabolism of connective tissue. Material and methods. 27 children aged 5 to 18 were supervised. The group of healthy children without clinical manifestations of inflammatory reactions consisted of 17 people. Conservative treatment of children with scarring included local, drug, physiotherapy, balneological and efferent therapy methods. The impact of treatment measures was assessed on a modified scar score scale, which consisted of a patient (parent) scale and a researcher scale. Laboratory criteria of effectiveness were the amount of ceruloplasmin (CP), lactic (MC) and sialic (SC) acids, GAG content, lysosomal enzyme activity (bone and alkaline phosphatase), mineral metabolism (total calcium). Laboratory parameters in patients were studied in the period from 150 to 170 and 310 to 340 days after injury. SPSS v was used for statistical analysis. 17.0. Results. Improvements in clinical signs of scar tissue were 1.7-fold according to objective estimates and 1.6-fold according to patient estimates. Laboratory monitoring showed the effectiveness of conservative treatment of convalescents, which was manifested in a reduction in GAG levels 1.6 times after 6 months and 2.0 times after a year. There was a decrease in the activity of acid phosphatase in 1.6 times, alkaline phosphatase and its bone fraction in 1.4 times. In addition, after treatment, there was an increase in ceruloplasmin activity by 1.5 times at all times. A 1.5-fold increase in the number of MCs was determined at all observation times. After treatment, the amount of MC decreased by 1.2 times (P<0.05). Significant confirmation of an increase in the level of SC in 1.4 times (P<0.05) was observed only on the 160th day of the study. At other times, the increase in the IC was unreliable. Conclusions. Long-term studies have shown that in children after thermal trauma in the process of productive scarring there are deviations in the metabolism of CT in the direction of increasing its synthetic function, metabolic disorders and inflammation continue. The use of complex conservative therapy in children with post-burn scars during convalescence allowed to reduce the severity of inflammatory reactions, improve CT metabolism and reduce scarring. The research was carried out in accordance with the principles of the Helsinki declaration. The study protocol was approved by the Local ethics committee of all participating institutions. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors. Key words: post-burn scarring in children, connective tissue, metabolism, inflammation, conservative therapy.

Long-term open-label phase I/II extension study of intrathecal idursulfase-IT in the treatment of neuronopathic mucopolysaccharidosis II

Intrathecal (IT) idursulfase-IT for the treatment of cognitive impairment is being investigated in pediatric patients with neuronopathic mucopolysaccharidosis II (MPS II) in addition to intravenous idursulfase. In this article, we report the findings for 54 months of idursulfase-IT treatment in an ongoing phase I/II extension trial (NCT01506141). A total of 15 male participants with neuronopathic MPS II (aged 3-11 years at enrollment) who were previously treated with intravenous idursulfase entered the extension study. Idursulfase-IT 10 mg or 30 mg was administered monthly via an IT drug delivery device or lumbar puncture, if indicated. The primary endpoint was safety and tolerability; secondary endpoints included pharmacokinetics, cerebrospinal fluid glycosaminoglycan levels, and cognitive function. In total, 15 participants received a median (range) of 50 (18-55) idursulfase-IT doses. Idursulfase-IT was generally well tolerated; there were no life-threatening adverse events (AEs) or deaths. Most serious AEs were related to the IT drug delivery device; only 2 serious AEs were related solely to idursulfase-IT. After treatment with idursulfase-IT, cerebrospinal fluid glycosaminoglycans were decreased in all participants; these decreases were maintained. Cognitive function was stabilized in 3 of 4 testable participants at month55. These long-term results support the clinical development of idursulfase-IT for patients with MPS II with cognitive impairment.

Endothelin‐1 dependent expression of GAG genes involves NOX and p38 mediated Smad linker region phosphorylation

Endothelin‐1 (ET‐1) is implicated in the development of atherosclerosis and mediates glycosaminoglycan (GAG) chain hyperelongation on proteoglycans. Our aim was to identify the ET‐1‐mediated signalling pathway involving NADPH oxidase (NOX), p38 MAP kinsae and Smad2 linker region phosphorylation (phospho‐Smad2L) regulate GAG synthesising enzymes mRNA expression (C4ST‐1 and ChSy1) involved in GAG chains hyperelongation in human vascular smooth muscle cells (VSMCs). Signalling intermediates were detected and quantified by Western blotting and the mRNA levels of GAG synthesising enzymes were assessed by quantitative real‐time polymerase chain reaction (qRT‐PCR). ET‐1 treatment of human VSMCs resulted in an increase in phospho‐Smad2L level. The TGF‐β receptor antagonist, SB431542 and the mixed ETA and ETB receptor antagonist bosentan, inhibited ET‐1‐mediated phospho‐Smad2L level. In the presence of apocynin and diphenyleneiodonium chloride (DPI) (NOX inhibitors) and SB239063 (p38 inhibitor) ET‐1‐mediated phospho‐Smad2L levels were inhibited. The gene expression levels of GAG synthesising enzymes post‐ET‐1 treatment were increased compared to untreated controls (p < 0.01). The ET‐mediated the mRNA levels of these enzymes were blocked by the bosentan, SB431542, SB239063, DPI, apocynin and antioxidant N‐acetyl‐L‐cysteine (NAC). ET‐1‐mediated signalling to GAG synthesising enzymes gene expression occurs via transactivation‐dependent pathway involving NOX, p38 MAP kinsae and Smad2 linker region phosphorylation.

Effect of high glucose on glycosaminoglycans in cultured retinal endothelial cells and rat retina.

The endothelial glycocalyx regulates vascular permeability, inflammation, and coagulation, and acts as a mechanosensor. The loss of glycocalyx can cause endothelial injury and contribute to several microvascular complications and, therefore, may promote diabetic retinopathy. Studies have shown a partial loss of retinal glycocalyx in diabetes, but with few molecular details of the changes in glycosaminoglycan (GAG) composition. Therefore, the purpose of our study was to investigate the effect of hyperglycemia on GAGs of the retinal endothelial glycocalyx. GAGs were isolated from rat retinal microvascular endothelial cells (RRMECs), media, and retinas, followed by liquid chromatography-mass spectrometry assays. Quantitative real-time polymerase chain reaction was used to study mRNA transcripts of the enzymes involved in GAG biosynthesis. Hyperglycemia significantly increased the shedding of heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA). There were no changes to the levels of HS in RRMEC monolayers grown in high-glucose media, but the levels of CS and HA decreased dramatically. Similarly, while HA decreased in the retinas of diabetic rats, the total GAG and CS levels increased. Hyperglycemia in RRMECs caused a significant increase in the mRNA levels of the enzymes involved in GAG biosynthesis (including EXTL-1,2,3, EXT-1,2, ChSY-1,3, and HAS-2,3), with these increases potentially being compensatory responses to overall glycocalyx loss. Both RRMECs and retinas of diabetic rats exhibited glucose-induced alterations in the disaccharide compositions and sulfation of HS and CS, with the changes in sulfation including N,6-O-sulfation on HS and 4-O-sulfation on CS.

Effect of Anti-Iduronate 2-Sulfatase Antibodies in Patients with Mucopolysaccharidosis Type II Treated with Enzyme Replacement Therapy

To assess the relationship between anti-Iduronate 2-sulfatase (IDS) antibodies, IDS genotypes, phenotypes and their impact in patients with enzyme replacement therapy (ERT)-treated Mucopolysaccharidosis type II. Dutch patients treated with ERT were analyzed in this observational cohort study. Antibody titers were determined by enzyme-linked immunosorbent assay. Neutralizing effects were measured in fibroblasts. Pharmacokinetic analysis of ERT was combined with immunoprecipitation. Urinary glycosaminoglycans were measured using mass spectrometry and dimethylmethylene blue. Eight of 17 patients (47%) developed anti-IDS antibodies. Three patients with the severe, neuronopathic phenotype, two of whom did not express IDS protein, showed sustained antibodies for up to 10years of ERT. Titers of 1:5120 or greater inhibited cellular IDS uptake and/or intracellular activity invitro. In 1 patient who was neuronopathic with a titer of 1:20 480, pharmacokinetic analysis showed that all plasma recombinant IDS was antibody bound. This finding was not the case in 2 patients who were not neuronopathic with a titer of 1:1280 or less. Patients with sustained antibody titers showed increased urinary glycosaminoglycan levels compared with patients with nonsustained or no-low titers. Patients with the neuronopathic form and lack of IDS protein expression were most at risk todevelop sustained anti-IDS antibody titers, which inhibited IDS uptake and/or activity invitro, and the efficacy of ERT in patients by lowering urinary glycosaminoglycan levels.

Фенотипическая трансформация гладких миоцитов стенки желчного пузыря при развитии некалькулезного холецистита

The aim of the study was to analyse ultrastructural changes in the smooth muscle tissue of different gallbladder sections during the development of experimental acalculous cholecystitis. Materials and methods. The research was performed on 20 guinea pigs. The duration of the experiment ranged from 4 to 15 days. Each experimental and control group included 5 animals. A standard model of chronic acalculous cholecystitis was used, in which ligation of the proximal part of the common bile duct is accompanied by inflammation and impaired gallbladder motility. Results. In guinea pigs with cholecystitis, on day 15 of the experiment using electron microscopic examination we revealed cells in the smooth muscle tissue that retain a well-structured contractile apparatus, represented by separate thin bundles of myofilaments, and a developed synthetic apparatus. The ultrastructural organization of this type of cells is quite characteristic of myofibroblasts. Studying the modulation of gallbladder smooth muscle cells in an experiment, we need to take into account that in the conditions of inflammation and impaired gallbladder motility, during the restructuring of the intercellular matrix, myofibroblasts can appear, causing the development of sclerotic processes in the organ wall due to their ability to express high levels of collagen and glycosaminoglycans, as well as numerous other extracellular matrix molecules and fibrogenic cytokines. The research showed that adenomyomatous hyperplasia of the gallbladder wall is accompanied by proliferation of myofibroblasts and smooth muscle cells. Thus, impaired stromal-epithelial interactions can be assumed to underlie the pathology in adenomyomatous hyperplasia of the gallbladder.

Mucopolysaccharidosis Type IVA: Extracellular Matrix Biomarkers in Cardiovascular Disease.

Cardiovascular disease (CVD) in Mucopolysaccharidosis Type IVA (Morquio A), signified by valvular disease and cardiac hypertrophy, is the second leading cause of death and remains untouched by current therapies. Enzyme replacement therapy (ERT) is the gold-standard treatment for MPS disorders including Morquio A. Early administration of ERT improves outcomes of patients from childhood to adulthood while posing new challenges including prognosis of CVD and ERT’s negligible effect on cardiovascular health. Thus, having accurate biomarkers for CVD could be critical. Here we show that cathepsin S (CTSS) and elastin (ELN) can be used as biomarkers of extracellular matrix remodeling in Morquio A disease. We found in a cohort of 54 treatment naïve Morquio A patients and 74 normal controls that CTSS shows promising attributes as a biomarker in young Morquio A children. On the other hand, ELN shows promising attributes as a biomarker in adolescent and adult Morquio A. Plasma/urine keratan sulfate (KS), and urinary glycosaminoglycan (GAG) levels were significantly higher in Morquio A patients (p < 0.001) which decreased with age of patients. CTSS levels did not correlate with patients’ phenotypic severity but differed significantly between patients (median range 5.45–8.52 ng/mL) and normal controls (median range 9.61–15.9 ng/mL; p < 0.001). We also studied α -2-macroglobulin (A2M), C-reactive protein (CRP), and circulating vascular cell adhesion molecule-1 (sVCAM-1) in a subset of samples to understand the relation between ECM biomarkers and the severity of CVD in Morquio A patients. Our experiments revealed that CRP and sVCAM-1 levels were lower in Morquio A patients compared to normal controls. We also observed a strong inverse correlation between urine/plasma KS and CRP (p = 0.013 and p = 0.022, respectively) in Morquio A patients as well as a moderate correlation between sVCAM-1 and CTSS in Morquio A patients at all ages (p = 0.03). As the first study to date investigating CTSS and ELN levels in Morquio A patients and in the normal population, our results establish a starting point for more elaborate studies in larger populations to understand how CTSS and ELN levels correlate with Morquio A severity.

In vitroevaluation of anti‐aging and regenerative properties of dermatan sulfate for skin care

IntroductionDermatan sulfate (DS) is a linear polysaccharide classified as a sulfated glycosaminoglycan (GAGs). GAGs form an integral part of the skin and make up 0.1–0.3% of total skin weight. DS is covalently attached to the core proteins to form proteoglycans (PGs). PGs predominates in dermis presenting higher level of DS than other sulfated GAGs.The intrinsic aging processes of the skin entails the reduction of total sulfated GAGs levels causing a thinning of the dermis and modifying the quality of the skin. DS possess strong biological activity that can be modulated to improve skin quality.This study was performed to determine the effects of DS on Human Dermal Fibroblasts (HDF) to evaluate its anti‐aging and regenerative properties.MethodsHuman Dermal Fibroblasts (HDF) were isolated from a sample of the foreskin of a neonatal caucasian donor (Lonza). The effect of the DS on cell proliferation was assessed at 24 h and 48 h in HDF, using the method bromodeoxyuridine (BrdU) incorporation, detected by ELISA. The cell migration was evaluated in HDF at 48 h using the Oris™ Cell Migration Assay (Platypus Technologies). To monitor the migration progress, the cells were labelled with calcein. The wound healing capacity of DS was evaluated in HDF using the scratch test. The inductive capacity on Extracellular matrix proteins (ECMp) synthesis was evaluated by immunolocalization‐ELISA quantifying type I and type III collagen and elastin.ResultsThe results showed that DS induced HDF proliferation after 24 h and 48 h of treatment at all tested concentrations. A dose‐dependent increase in proliferation was observed after 48 h of treatment of these cells, reaching a maximum increase of 5.10 (± 0.82) fold at the highest concentration tested (5 mg/mL) compared to uHDF. Regarding HDF cell migration, no significant differences were observed at 24 h after treatment with DS.The results showed a significant improvement in the in vitro wound healing capacity of HDF treated with 0.25 and 1 mg/mL of DS, with an increase in the healed area of 30.28% (± 4.27%) and 23.76% (± 8.87%) respectively, compared to uHDF.Regarding the ECMp synthesis‐stimulating capacity of DS, the results showed a significant increase of both type III Collagen (50.98 ± 34.48% increase) and Elastin (32.25 ± 2.61%) levels in HDF treated for 72 h with 1 mg/mL of DS compared to uHDF. HDF treated with 0.25 mg/mL also showed higher type I Collagen production than uHDF, although the increase (30.96 ±7.04%) was not statistically significant.ConclusionsThe results showed that DS improves the proliferation and regeneration of HDF present in dermis. In addition, DS increases the synthesis of ECMp as collagen type III and elastin, promoting the maintenance and functionality of the dermis. All these properties support the regenerative and anti‐aging activity of DS acting against intrinsic aging in dermis.

Complex Changes in the Efficiency of the Expression of Many Genes in Monogenic Diseases, Mucopolysaccharidoses, May Arise from Significant Disturbances in the Levels of Factors Involved in the Gene Expression Regulation Processes.

Monogenic diseases are primarily caused by mutations in a single gene; thus, they are commonly recognized as genetic disorders with the simplest mechanisms. However, recent studies have indicated that the molecular mechanisms of monogenic diseases can be unexpectedly complicated, and their understanding requires complex studies at the molecular level. Previously, we have demonstrated that in mucopolysaccharidoses (MPS), a group of monogenic lysosomal storage diseases, several hundreds of genes reveal significant changes in the expression of various genes. Although the secondary effects of the primary biochemical defect and the inefficient degradation of glycosaminoglycans (GAGs) might be considered, the scale of the changes in the expression of a large fraction of genes cannot be explained by a block in one biochemical pathway. Here, we demonstrate that in cellular models of 11 types of MPS, the expression of genes coding for proteins involved in the regulation of the expression of many other genes at various stages (such as signal transduction, transcription, splicing, RNA degradation, translation, and others) is significantly disturbed relative to the control cells. This conclusion was based on transcriptomic studies, supported by biochemical analyses of levels of selected proteins encoded by genes revealing an especially high level of dysregulation in MPS (EXOSC9, SRSF10, RPL23, and NOTCH3 proteins were investigated). Interestingly, the reduction in GAGs levels, through the inhibition of their synthesis normalized the amounts of EXOSC9, RPL23, and NOTCH3 in some (but not all) MPS types, while the levels of SRSF10 could not be corrected in this way. These results indicate that different mechanisms are involved in the dysregulation of the expression of various genes in MPS, pointing to a potential explanation for the inability of some therapies (such as enzyme replacement therapy or substrate reduction therapy) to fully correct the physiology of MPS patients. We suggest that the disturbed expression of some genes, which appears as secondary or tertiary effects of GAG storage, might not be reversible, even after a reduction in the amounts of the storage material.

New approaches suggest term and preterm human fetal membranes may have distinct biomechanical properties

Preterm prelabour rupture of membranes is the leading cause of preterm birth and its associated infant mortality and morbidity. However, its underlying mechanism remains unknown. We utilized two novel biomechanical assessment techniques, ball indentation and Optical Coherence Elastography (OCE), to compare the mechanical properties and behaviours of term (≥ 37 weeks) and preterm (33–36 weeks) human fetal membranes from ruptured and non-ruptured regions. We defined the expression levels of collagen, sulfated glycosaminoglycans (sGAG), matrix metalloproteinase (MMP-9, MMP-13), fibronectin, and interleukin-1β (IL-1β) within membranes by biochemical analysis, immunohistochemical staining and Western blotting, both with and without simulated fetal movement forces on membrane rupture with a new loading system. Preterm membranes showed greater heterogeneity in mechanical properties/behaviours between ruptured and non-ruptured regions compared with their term counterparts (displacement rate: 36% vs. 15%; modulus: 125% vs. 34%; thickness: 93% vs. 30%; collagen content: 98% vs. 29%; sGAG: 85% vs 25%). Furthermore, simulated fetal movement forces triggered higher MMP-9, MMP-13 and IL-1β expression in preterm than term membranes, while nifedipine attenuated the observed increases in expression. In conclusion, the distinct biomechanical profiles of term and preterm membranes and the abnormal biochemical expression and activation by external forces in preterm membranes may provide insights into mechanisms of preterm rupture of membranes.

Time-dependent mechanical behaviour of the aortic chronic dissection flap.

OBJECTIVESThe transition of aortic dissection from acute to chronic is poorly understood. We examined time-dependent mechanical behaviour and biochemical properties of chronic dissection tissues.METHODSAorta samples were obtained from 14 patients with mixed aetiology who were undergoing elective surgery for chronic dissected aneurysms, ranging from 3 months to 15 years post-dissection. The tissue elastic modulus and tissue deformation following application of loading for 5 h were measured for the false lumen (FL), true lumen (TL) and flap (FP) tissues with a custom-indentation technique. Collagen, elastin and glycosaminoglycan levels were determined with established biochemical assays. Elastin fragmentation was graded from histological sections. The number of tissues characterized was as follows: FP (n = 10), TL (n = 5 for biomechanical testing, n = 8 for biochemical analysis, n = 8 for histological assessment) and FL (n = 4).RESULTSTissues stiffness was highest in FP [59.8 (14.8) kPa] as compared with TL [50.7 (6.2) kPa] and FL [40.5 (4.7) kPa] (P = 0.023 and P = 0.006, respectively). FP [0.5 (0.08) mm] also exhibited reduced deformation relative to TL [0.7 (0.02) mm] and FL [0.9 (0.08) mm] (P = 0.003 and P = 0.006, respectively), lowest collagen concentration [FP: 40.1 (19.6) µg/mg, TL: 59.9 (19.5) µg/mg, P = 0.008; FL: 79.1 (32.0) µg/mg, P = 0.006] and the lowest collagen: elastin ratio [0.4 (0.1)] relative to the other tissues [TL; 0.6 (0.3), P = 0.006, FL; 1.5 (0.4); P = 0.003]. Significant elastin loss was evident in the FL-stained tissue sections whereas highly aligned, long fibres were visible in the FP and TL. A linear relationship was found between the stiffness, deformation and the time from the dissection event to surgical intervention for the FP. All data are presented as median (interquartile range).CONCLUSIONSFP exhibited reduced time-dependent deformation and distinct biochemical properties relative to TL and FL irrespective of connective tissue disorder or the anatomical region of the dissection.

Cell-derived and enzyme-based decellularized extracellular matrix exhibit compositional and structural differences that are relevant for its use as a biomaterial.

Due to its availability and minimal invasive harvesting human adipose tissue-derived extracellular matrix (dECM) is often used as a biomaterial in various tissue engineering and healthcare applications. Next to dECM, cell-derived ECM (cdECM) can be generated by and isolated from in vitro cultured cells. So far both types of ECM were investigated extensively toward their application as (bio)material in tissue engineering and healthcare. However, a systematic characterization and comparison of soft tissue dECM and cdECM is still missing. In this study, we characterized dECM from human adipose tissue, as well as cdECM from human adipose-derived stem cells, toward their molecular composition, structural characteristics, and biological purity. The dECM was found to exhibit higher levels of collagens and lower levels of sulfated glycosaminoglycans compared with cdECMs. Structural characteristics revealed an immature state of the fibrous part of cdECM samples. By the identified differences, we aim to support researchers in the selection of a suitable ECM-based biomaterial for their specific application and the interpretation of obtained results.

Exploitation of Sulfated Glycosaminoglycan Status for Precision Medicine of Triplatin in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is a subtype of breast cancer lacking targetable biomarkers. TNBC is known to be most aggressive and when metastatic is often drug-resistant and uncurable. Biomarkers predicting response to therapy improve treatment decisions and allow personalized approaches for patients with TNBC. This study explores sulfated glycosaminoglycan (sGAG) levels as a predictor of TNBC response to platinum therapy. sGAG levels were quantified in three distinct TNBC tumor models, including cell line-derived, patient-derived xenograft (PDX) tumors, and isogenic models deficient in sGAG biosynthesis. The in vivo antitumor efficacy of Triplatin, a sGAG-directed platinum agent, was compared in these models with the clinical platinum agent, carboplatin. We determined that >40% of TNBC PDX tissue microarray samples have high levels of sGAGs. The in vivo accumulation of Triplatin in tumors as well as antitumor efficacy of Triplatin positively correlated with sGAG levels on tumor cells, whereas carboplatin followed the opposite trend. In carboplatin-resistant tumor models expressing high levels of sGAGs, Triplatin decreased primary tumor growth, reduced lung metastases, and inhibited metastatic growth in lungs, liver, and ovaries. sGAG levels served as a predictor of Triplatin sensitivity in TNBC. Triplatin may be particularly beneficial in treating patients with chemotherapy-resistant tumors who have evidence of residual disease after standard neoadjuvant chemotherapy. More effective neoadjuvant and adjuvant treatment will likely improve clinical outcome of TNBC.