Connective Tissue Extracellular Matrix Research Articles

Structural changes in cartilage and collagen studied by high temperature Raman spectroscopy.

Understanding the high temperature behavior of collagen and collagenous tissue is important for surgical procedures and biomaterials processing for the food, pharmaceutical, and cosmetics industries. One primary event for proteins is thermal denaturation that involves unfolding the polypeptide chains while maintaining the primary structure intact. Collagen in the extracellular matrix of cartilage and other connective tissue is a hierarchical material containing bundles of triple-helical fibers associated with water and proteoglycan components. Thermal analysis of dehydrated collagen indicates irreversible denaturation at high temperature between 135°C and 200°C, with another reversible event at ∼60-80°C for hydrated samples. We report high temperature Raman spectra for freeze-dried cartilage samples that show an increase in laser-excited fluorescence interpreted as conformational changes associated with denaturation above 140°C. Spectra for separated collagen and proteoglycan fractions extracted from cartilage indicate the changes are associated with collagen. The Raman data also show appearance of new features indicating peptide bond hydrolysis at high temperature implying that molecular H2 O is retained within the freeze-dried tissue. This is confirmed by thermogravimetric analysis that show 5-7 wt% H2 O remaining within freeze-dried cartilage that is released progressively upon heating up to 200°C. Spectra obtained after exposure to high temperature and re-hydration following recovery indicate that the capacity of the denatured collagen to re-absorb water is reduced. Our results are important for revealing the presence of bound H2 O within the collagen component of connective tissue even after freeze-drying and its role in denaturation that is accompanied by or perhaps preceded by breakdown of the primary polypeptide structure.

TGF-β Family Signaling in Connective Tissue and Skeletal Diseases.

The transforming growth factor β (TGF-β) family of signaling molecules, which includes TGF-βs, activins, inhibins, and numerous bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs), has important functions in all cells and tissues, including soft connective tissues and the skeleton. Specific TGF-β family members play different roles in these tissues, and their activities are often balanced with those of other TGF-β family members and by interactions with other signaling pathways. Perturbations in TGF-β family pathways are associated with numerous human diseases with prominent involvement of the skeletal and cardiovascular systems. This review focuses on the role of this family of signaling molecules in the pathologies of connective tissues that manifest in rare genetic syndromes (e.g., syndromic presentations of thoracic aortic aneurysm), as well as in more common disorders (e.g., osteoarthritis and osteoporosis). Many of these diseases are caused by or result in pathological alterations of the complex relationship between the TGF-β family of signaling mediators and the extracellular matrix in connective tissues.

The role of serum hyaluronic acid determination in the diagnosis of liver fibrosis.

The common pathway leading to liver fibrosis and cirrhosis is growing deposition of extracellular matrix (ECM). It results from molecular and histological rearrangement of collagens, glycoproteins and hyaluronans. Hyaluronic acid is a chief component of the extracellular matrix of connective tissues and plays the main structural role in the formation of ECM. The most important organ involved in the synthesis of hyaluronic acid is the liver. In this paper the meaning of hyaluronic acid in the diagnostics of liver diseases is discussed. Here, we focus on the described changes of hyaluronic acid concentration in the pathological processes of the liver, including alcoholic and non-alcoholic liver diseases. The results of published clinical studies have shown its high diagnostic sensitivity, which probably enables its application in laboratory diagnosis.

ROCK2/rasHa co-operation induces malignant conversion via p53 loss, elevated NF-κB and tenascin C-associated rigidity, but p21 inhibits ROCK2/NF-κB-mediated progression.

To study ROCK2 activation in carcinogenesis, mice expressing 4-hydroxytamoxifen (4HT)-activated ROCK2 (K14.ROCKer) were crossed with mice expressing epidermal-activated rasHa (HK1.ras1205). At 8 weeks, 4HT-treated K14.ROCKer/HK1.ras1205 cohorts exhibited papillomas similar to HK1.ras1205 controls; however, K14.ROCKer/HK1.ras1205 histotypes comprised a mixed papilloma/well-differentiated squamous cell carcinoma (wdSCC), exhibiting p53 loss, increased proliferation and novel NF-κB expression. By 12 weeks, K14.ROCKer/HK1.ras1205 wdSCCs exhibited increased NF-κB and novel tenascin C, indicative of elevated rigidity; yet despite continued ROCK2 activities/p-Mypt1 inactivation, progression to SCC required loss of compensatory p21 expression. K14.ROCKer/HK1.ras1205 papillomatogenesis also required a wound promotion stimulus, confirmed by breeding K14.ROCKer into promotion-insensitive HK1.ras1276 mice, suggesting a permissive K14.ROCKer/HK1.ras1205 papilloma context (wound-promoted/NF-κB+/p53-/p21+) preceded K14.ROCKer-mediated (p-Mypt1/tenascin C/rigidity) malignant conversion. Malignancy depended on ROCKer/p-Mypt1 expression, as cessation of 4HT treatment induced disorganized tissue architecture and p21-associated differentiation in wdSCCs; yet tenascin C retention in connective tissue extracellular matrix suggests the rigidity laid down for conversion persists. Novel papilloma outgrowths appeared expressing intense, basal layer p21 that confined endogenous ROCK2/p-Mypt1/NF-κB to supra-basal layers, and was paralleled by restored basal layer p53. In later SCCs, 4HT cessation became irrelevant as endogenous ROCK2 expression increased, driving progression via p21 loss, elevated NF-κB expression and tenascin C-associated rigidity, with p-Mypt1 inactivation/actinomyosin-mediated contractility to facilitate invasion. However, p21-associated inhibition of early-stage malignant progression and the intense expression in papilloma outgrowths, identifies a novel, significant antagonism between p21 and rasHa/ROCK2/NF-κB signalling in skin carcinogenesis. Collectively, these data show that ROCK2 activation induces malignancy in rasHa-initiated/promoted papillomas in the context of p53 loss and novel NF-κB expression, whereas increased tissue rigidity and cell motility/contractility help mediate tumour progression.

SHIP, a novel factor to ameliorate extracellular matrix accumulation via suppressing PI3K/Akt/CTGF signaling in diabetic kidney disease

Tubular interstitial extracellular matrix accumulation, which plays a key role in the pathogenesis and progression of diabetic kidney disease (DKD), is believed to be mediated by activation of PI3K/Akt signal pathway. However, it is still not clear whether SH2 domain-containing inositol 5'-phosphatase (SHIP), known as a negative regulator of PI3K/Akt pathway is also involved in extracellular matrix metabolism of diabetic kidney. In the present study, decreased SHIP and increased phospho-Akt (Ser 473, Thr 308) were found in renal tubular cells of diabetic mice accompanied by overexpression of connective tissue growth factor (CTGF) and extracellular matrix deposition versus normal mice. Again, high glucose attenuated SHIP expression in a time-dependent manner, concomitant with activation of PI3K/Akt signaling and extracellular matrix production in human renal proximal tubular epithelial cells (HK2) cultured in vitro, which was significantly prevented by transfection of M90-SHIP vector. Furthermore, in vivo delivery of rAd-INPP5D vector (SHIP expression vector) via intraperitoneal injection in diabetic mice increased SHIP expression by 3.36 times followed by 65.26%, 70.38% and 46.71% decreases of phospho-Akt (Ser 473), phospho-Akt (Thr 308) and CTGF expression versus diabetic mice receiving rAd-EGFP vector. Meanwhile, increased renal extracellular matrix accumulation of diabetic mice was also inhibited with intraperitoneal injection of rAd-INPP5D vector. These above data suggested that overexpression of SHIP might be a potent method to lessen renal extracellular matrix accumulation via inactivation of PI3K/Akt pathway and suppression of CTGF expression in DKD.

Comparison of Enzymatic Hydrolysis of Polysaccharides from Eggshells Membranes

Abstract AHyaluronic acid (HA) is part of the extracellular matrix of connective, epithelial and neural tissues, as well as the synovial fluid, skin, and cartilage. It is composed of repeating disaccharide units of D-glucuronic acid and N-acetyl glucosamine. Hyaluronic acid is used in abdominal surgery, ophthalmology, dermatology, rhinology; it is usable for the osteoarthritis treatment. The membranes of eggshell are a natural source of hyaluronic acid, collagen, glycosaminoglycan and collagenous proteins. In paper, we tested the possibility of extraction hyaluronic acid from the eggshell membranes by enzymatic hydrolysis. We identified optimal conditions of hydrolysis with trypsin at reaction temperature of 37 °C and pH 8; with pepsin at 40 °C and pH 3, as well as with papain at 60 °C and pH 7.5. The content of hyaluronic acid in samples was determined spectrophotometrically using the carbazole method. The experimental results showed a yield of ~ 4 -4.5 % hyaluronic acid per 1 g of dry eggshell membranes.

Proteomic Analysis of Human Tendon and Ligament: Solubilization and Analysis of Insoluble Extracellular Matrix in Connective Tissues.

Connective tissues such as tendon, ligament and cartilage are mostly composed of extracellular matrix (ECM). These tissues are insoluble, mainly due to the highly cross-linked ECM proteins such as collagens. Difficulties obtaining suitable samples for mass spectrometric analysis render the application of modern proteomic technologies difficult. Complete solubilization of them would not only elucidate protein composition of normal tissues but also reveal pathophysiology of pathological tissues. Here we report complete solubilization of human Achilles tendon and yellow ligament, which is achieved by chemical digestion combined with successive protease treatment including elastase. The digestion mixture was subjected to liquid chromatography-mass spectrometry. The low specificity of elastase was overcome by accurate mass analysis achieved using FT-ICR-MS. In addition to the detailed proteome of both tissues, we also quantitatively determine the major protein composition of samples, by measuring peak area of some characteristic peptides detected in tissue samples and in purified proteins. As a result, differences between human Achilles tendon and yellow ligament were elucidated at molecular level.

Hyaluronic Acid (HA) Scaffolds and Multipotent Stromal Cells (MSCs) in Regenerative Medicine

Traditional methods for tissue regeneration commonly used synthetic scaffolds to regenerate human tissues. However, they had several limitations, such as foreign body reactions and short time duration. In order to overcome these problems, scaffolds made of natural polymers are preferred. One of the most suitable and widely used materials to fabricate these scaffolds is hyaluronic acid. Hyaluronic acid is the primary component of the extracellular matrix of the human connective tissue. It is an ideal material for scaffolds used in tissue regeneration, thanks to its properties of biocompatibility, ease of chemical functionalization and degradability. In the last few years, especially from 2010, scientists have seen that the cell-based engineering of these natural scaffolds allows obtaining even better results in terms of tissue regeneration and the research started to grow in this direction. Multipotent stromal cells, also known as mesenchymal stem cells, plastic-adherent cells isolated from bone marrow and other mesenchymal tissues, with self-renew and multi-potency properties are ideal candidates for this aim. Normally, they are pre-seeded onto these scaffolds before their implantation in vivo. This review discusses the use of hyaluronic acid-based scaffolds together with multipotent stromal cells, as a very promising tool in regenerative medicine.

Abnormal pigment epithelium-derived factor processing in progressive myopia

Pigment Epithelium-Derived Factor (PEDF) is a secreted glycoprotein belonging to the family of non-inhibitory serpins. It is known, that in cases of complicated myopia, the content of PEDF in aqueous humor of the anterior chamber is significantly reduced. Here we examined a bulk of Tenon's capsule samples obtained from various groups of myopes, to examine PEDF processing in progressive myopia. We have analyzed the distribution of full length PEDF50 and its truncated form PEDF45 in the soluble and insoluble fractions extracted from Tenon's capsule of myopic and control (non-myopic) patients using SDS-polyacrylamide gel electrophoresis, as well as monitored the proteolytic degradation of PEDF ex vivo by enzyme-linked immunosorbent assay. These results were complemented by PEDF mRNA analysis in correspondent tissues by using qPCR and immunohistochemistry analysis of PEDF distribution in normal and myopic specimens. We found that in the Tenon's capsule of patients suffering from a high myopia the level of “soluble” 45 kDa PEDF reduced by 2-fold, while the content of “insoluble” 50 kDa form of PEDF was increased by 4-fold compared to controls. Excessive amount of PEDF50 in myopic specimens have been shown to correlate with the abrogated PEDF processing rather than with an increase of its expression. Moreover, immunohistochemical staining of the myopic Tenon's capsule tissue sections revealed the halo of deposited PEDF50 in the fibroblast extracellular space. These findings suggest that in myopia limited proteolysis of PEDF is altered or abrogated. Accumulation of full-length PEDF insoluble aggregates in the fibroblast intercellular space may affect cell survival and consequently causes the destructive changes in the extracellular matrix of the eye connective tissues. As a result, the abrogation of full-length PEDF normal processing can be an important mechanism leading to biomechanical destabilization of the scleral capsule and myopia progression.

TGF-β1, in association with the increased expression of connective tissue growth factor, induce the hypertrophy of the ligamentum flavum through the p38 MAPK pathway.

Hypertrophy of the ligamentum flavum (LF) is one of the key pathomechanisms of lumbar spinal stenosis (LSS). Transforming growth factor (TGF)-β1 is abundantly expressed in hypertrophied degenerative LF tissues from LSS. However, the molecular mechanisms underling the association between TGF-β1 and LF hypertrophy have not yet been fully elucidated. In this study, we investigated the important role of the mitogen-activated protein kinase (MAPK) pathway in the pathogenesis of LSS by analyzing the expression of connective tissue growth factor (CTGF) and extracellular matrix (ECM) components (collagen I and collagen III) in TGF-β1-treated LF cells. Cell growth assay revealed that TGF-β1, in association with CTGF, enhanced the the proliferation of LF cells, and we found that TGF-β1 also elevated CTGF expression and subsequently enhanced the mRNA expression of collagen I and collagen III. The increased mRNA expression levels of CTGF, collagen I and collagen III were abolished by p38 inhibitors. Both immunofluorescence imaging and western blot analysis of p38 and p-p38 revealed the increased expression and phosphorylation of p38. Silencing the expression of p38 by siRNA in LF cells decreased the protein expression of p38, p-p38 and CTGF, as well as the mRNA expression of CTGF, collagen I and collagen III. Taken together, our findings indicate that TGF-β1, in association with the increased expression of CTGF, contribute to the homeostasis of the ECM and to the hypertrophy of LF through the p38 MAPK pathway.

Matrix-bound nanovesicles within ECM bioscaffolds.

Biologic scaffold materials composed of extracellular matrix (ECM) have been used in a variety of surgical and tissue engineering/regenerative medicine applications and are associated with favorable constructive remodeling properties including angiogenesis, stem cell recruitment, and modulation of macrophage phenotype toward an anti-inflammatory effector cell type. However, the mechanisms by which these events are mediated are largely unknown. Matrix-bound nanovesicles (MBVs) are identified as an integral and functional component of ECM bioscaffolds. Extracellular vesicles (EVs) are potent vehicles of intercellular communication due to their ability to transfer RNA, proteins, enzymes, and lipids, thereby affecting physiologic and pathologic processes. Formerly identified exclusively in biologic fluids, the presence of EVs within the ECM of connective tissue has not been reported. In both laboratory-produced and commercially available biologic scaffolds, MBVs can be separated from the matrix only after enzymatic digestion of the ECM scaffold material, a temporal sequence similar to the functional activity attributed to implanted bioscaffolds during and following their degradation when used in clinical applications. The present study shows that MBVs contain microRNA capable of exerting phenotypical and functional effects on macrophage activation and neuroblastoma cell differentiation. The identification of MBVs embedded within the ECM of biologic scaffolds provides mechanistic insights not only into the inductive properties of ECM bioscaffolds but also into the regulation of tissue homeostasis.

Lichen sclerosis of urethra (literature review using PUBMED sources http://www.ncbi.nlm.nih.gov/pubmed)

The review analyses data on possible causes of lichen sclerosis development, lists the histologic reports on changes in skin and urethra in genital lichen sclerosis with urethra strictures. The description of immunological barriers of urethra and the role of cell and humoral immunity disruption in development of pathological protein and extracellular matrix of connective tissue is provided. The review also lists the role of fibroblasts and fibroblast-mediated factors of inflammation in disruption of connective tissue and outlines the changes of extracellular matrix during immune response along with the influence of matrix metalloproteinases and their inhibitors. The data on regulating role of sex hormones, role of folate cycle disorders and related increase of homocysteine blood levels on the protein structure and extracellular marix of skin and urethra connective tissue is provided. A role of androgen deficiency in development of lichen sclerosis is listed. Finally the review describes the natural course of lichen sclerosis development and lists the methods of substitutive urethroplasties along with the materials used for urethra stricture treatment.

Collagens XII and XIV: Two collagen types both associated with bovine muscle and intramuscular lipid metabolism

In continental Europe, intramuscular fat (IMF) content in beef is low but plays a key role in determining flavor. IMF expands inside the extracellular matrix (ECM) of intramuscular connective tissue. This ECM contains among others, collagens XII and XIV, two minor collagens probably involved in muscle metabolism. So, our purpose was to determine if there was an association between the contents in collagens XII and XIV and muscle metabolism. Rectus abdominis (RA, oxidative) and Semitendinosus (ST, oxido-glycolytic) muscles were sampled from steers of two genotypes with high (Angus [Ang], n=10) or low (Limousine [Lim], n=10) intramuscular fat level. Collagens XII and XIV relative abundance was evaluated by Western blot analysis. The characteristic parameters of metabolism were previously measured. Ang muscles contained more collagen XIV than Lim muscles (p<0.001). RA muscle contained more collagen XII than ST muscle (p<0.01). Across the two breeds and muscles, collagen XII was positively correlated with A- and H-fatty acid binding protein (FABP) contents (+0.43 and +0.53, respectively, p<0.05) and activities of three oxidative enzymes (cytochrome-c oxidase: +0.41, isocitrate dehydrogenase [ICDH]: +0.40, citrate synthase [CS]: +0.32) (p<0.05) and collagen XIV was positively correlated with triacylglycerol content (+0.35, p<0.35), A- and H-FABP protein contents (+0.35 and +0.38, respectively, p<0.05) and ICDH (+0.34, p<0.05) and negatively with activities of two enzymes of glycolytic metabolism (phosphofructokinase: −0.31 and lactate dehydrogenase: −0.42, p<0.05). After removing the genotype and muscle effects, only collagen XII remained correlated with A- and H-FABP and CS activity (+0.30, +0.34 and +0.46, respectively, p<0.0505). H-FABP is expressed in various tissues but predominantly in cardiac and oxidative skeletal muscles, whereas A-FABP is exclusively expressed within adipocytes. From these results, we can conclude that collagen XII and XIV are both associated with bovine muscle fiber and intramuscular lipid metabolism, but probably by different mechanisms. As a matter of fact, collagens XII is associated with intramuscular fat differentiation (from its correlation with A-FABP content) and oxidative metabolism (from its correlation with H-FABP content and CS activities) regardless of breed and muscle, whereas type XIV collagen is associated with the same parameters plus enzymes of glycolytic metabolism that discriminate breeds.

Biotechnological production of hyaluronic acid: a mini review.

Hyaluronic acid (HA) is a polysaccharide found in the extracellular matrix of vertebrate epithelial, neural and connective tissues. Due to the high moisture retention, biocompatibility and viscoelasticity properties of this polymer, HA has become an important component of major pharmaceutical, biomedical and cosmetic products with high commercial value worldwide. Currently, large scale production of HA involves extraction from animal tissues as well as the use of bacterial expression systems in Streptococci. However, due to concerns over safety, alternative sources of HA have been pursued which include the use of endotoxin-free microorganisms such as Bacilli and Escherichia coli. In this review, we explore current knowledge of biosynthetic enzymes that produce HA, how these systems have been used commercially to produce HA and how the challenges of producing HA cheaply and safely are being addressed.

PDGF-BB induces PRMT1 expression through ERK1/2 dependent STAT1 activation and regulates remodeling in primary human lung fibroblasts

Tissue remodeling of sub-epithelial mesenchymal cells is a major pathology occurring in chronic obstructive pulmonary disease (COPD) and asthma. Fibroblasts, as a major source of interstitial connective tissue extracellular matrix, contribute to the fibrotic and inflammatory changes in these airways diseases. Previously, we described that protein arginine methyltransferase-1 (PRMT1) participates in airway remodeling in a rat model of pulmonary inflammation. In this study we investigated the mechanism by which PDGF-BB regulates PRMT1 in primary lung fibroblasts, isolated from human lung biopsies. Fibroblasts were stimulated with PDGF-BB for up-to 48h and the regulatory and activation of signaling pathways controlling PRMT1 expression were determined. PRMT1 was localized by immuno-histochemistry in human lung tissue sections and by immunofluorescence in isolated fibroblasts. PRMT1 activity was suppressed by the pan-PRMT inhibitor AMI1. ERK1/2 mitogen activated protein kinase (MAPK) was blocked by PD98059, p38 MAPK by SB203580, and STAT1 by small interference (si) RNA treatment. The results showed that PDGF-BB significantly increased PRMT1 expression after 1h lasting over 48h, through ERK1/2 MAPK and STAT1 signaling. The inhibition of ERK1/2 MAPK or of PRMT1 activity decreased PDGF-BB induced fibroblast proliferation, COX2 production, collagen-1A1 secretion, and fibronectin production. These findings suggest that PRMT1 is a central regulator of tissue remodeling and that the signaling sequence controlling its expression in primary human lung fibroblast is PDGF–ERK–STAT1. Therefore, PRMT1 presents a novel therapeutic and diagnostic target for the control of airway wall remodeling in chronic lung diseases.

쇠무릎 에탄올 추출물의 DPPH, 히알루로니다아제 및 리폭시게나아제 저해 효과

The 1, 1- diphenyl 2-picrylhyorazyl (DPPH) is a well-known radical and a trap (scavenger) for other radicals. Hyaluronidase (HAase) is an enzyme that depolymerizes the polysaccharide hyaluronic acid (HA) in the extracellular matrix of connective tissue. Lipoxygenase (LOX) enzyme was reported to convert the arachidonic, linoleic and other polyunsaturated fatty acid into biologically active metabolites involved in the inflammatory and immune responses. The purpose of the present study is to evaluate plant extracts as sources of natural antioxidants and to examine whether Achyranthes japonica having significant DPPH, HAase and LOX inhibitory activity. The inhibitory effect of HAase by A. japonica was assayed using a Morgan microplate assay. The antioxidant activity of the A. japonica extracts was measured on the basis of the scavenging activity of the stable 1, 1- diphenyl 2-picrylhyorazyl (DPPH) free radical. DPPH scavenging activity of matured roots of A. japonica was evaluated at 4.0 mg/ml was 87.8% and that of young roots was 86.2% at same concentration. The roots of A. japonica showed maximum inhibition of HAase activity (IC 50 = 27.7 μg/ml). The highest LOX inhibition was recorded in the root extract among three vegetative parts. Inhibition of HAase activity of roots may contribute towards the development of herbal medicines. Although percent inhibition of lipoxygenase by Achyranthes japonica for all young and matured groups for leaves, stems, and roots at different concentrations, there were not show a statistically significant difference (p<0.05).

The role of matrix metalloproteinases in glaucoma pathogenesis

Matrix metalloproteinases belong to an enzyme family, which assure a proteolysis of practically all components of the extracellular matrix of connective tissues in normal and pathological conditions. At physiological conditions, there are evidences on the impact of this enzyme group in the embryogenesis, morphogenesis, angiogenesis, and tissue involution. The activity impairment of matrix metalloproteinases and of their specific inhibitors leads to the biosynthesis misbalance and to the degradation of extracellular matrix components; it plays a role in the development of such diseases as diabetes mellitus, rheumatoid arthritis, and arteriosclerosis. Laboratory tests and clinical investigation results confirm the role of these enzymes in tissue remodeling of different eyeball structures in glaucoma (in particular, of the trabecular meshwork and the optic disc); it leads to intraocular fluid outflow impairment and to the glaucomatous optic neuropathy development. In the review, the analysis of clinical and experimental studies is performed that are dedicated to the investigation of matrix metalloproteinases role in the pathogenesis of different glaucoma types, of the possibility to use them as biomarkers, as well as therapeutic action targets in this disease.

Glycomics expression analysis of sulfated glycosaminoglycans of human colorectal cancer tissues and non-neoplastic mucosa by electrospray ionization mass spectrometry.

ABSTRACTObjective To determine the presence of glycosaminoglycans in the extracellular matrix of connective tissue from neoplastic and non-neoplastic colorectal tissues, since it has a central role in tumor development and progression.Methods Tissue samples from neoplastic and non-neoplastic colorectal tissues were obtained from 64 operated patients who had colorectal carcinoma with no distant metastases. Expressions of heparan sulphate, chondroitin sulphate, dermatan sulphate and their fragments were analyzed by electrospray ionization mass spectrometry, with the technique for extraction and quantification of glycosaminoglycans after proteolysis and electrophoresis. The statistical analysis included mean, standard deviation, and Student’s t test.Results The glycosaminoglycans extracted from colorectal tissue showed three electrophoretic bands in agarose gel. Electrospray ionization mass spectrometry showed characteristic disaccharide fragments from glycosaminoglycans, indicating their structural characterization in the tissues analyzed. Some peaks in the electrospray ionization mass spectrometry were not characterized as fragments of sugars, indicating the presence of fragments of the protein structure of proteoglycans generated during the glycosaminoglycan purification. The average amount of chondroitin and dermatan increased in the neoplastic tissue compared to normal tissue (p=0.01). On the other hand, the average amount of heparan decreased in the neoplastic tissue compared to normal tissue (p= 0.03).Conclusion The method allowed the determination of the glycosaminoglycans structural profile in colorectal tissue from neoplastic and non-neoplastic colorectal tissue. Neoplastic tissues showed greater amounts of chondroitin sulphate and dermatan sulphate compared to non-neoplastic tissues, while heparan sulphate was decreased in neoplastic tissues.

Abstract 12878: The Effect of Advanced Glycation End Products (AGE) Breaker, Alagebrium on the Cardiac Structural and Functional Change in Type I Diabetic Rat Model

Background: Chronic hyperglycemia contributes to diabetic complications through the formation of advanced glycation end products (AGEs). The crosslink breaker, 3-phenacyl-4,5-dimethylthiazolium chloride (ALT-711), alagebrium chloride is known to reduce the pathological condition through breaking AGE cross-links. However, there has been little assessment regarding the impact of alagebrium on AGE-dependent signaling or restoring structural molecular physiology as well as the myocardial functional change. Methods: We investigated the effect of alagebrium on myocardial functional change using echocardiography as well as structural change in diabetic rat model based on the cellular mechanism. Results: AGE enhanced reactive oxygen species (ROS) synthesis dose-dependently in cardiomyocytes. Alagebrium induced decrease in enhanced ROS synthesis by AGE and increased expression of receptor for AGE (RAGE). In western blot and RT-PCR experiments, alagebrium also inhibited AGE-induced activation of MAP Kinase and decreased AGE-induced connective tissue growth factor (CTGF) and extracellular matrix. In echocardiography, the left ventricular (LV) dimension was increased (6.33 ±0.58mm at initial vs. 8.33±0.32mm at 16weeks after diabetes, p=0.003), and systolic contractility in radial and circumferential direction was decreased as diabetes was progressed in diabetic rat model. Diabetic progress also modified collagen structure. However, the alagebrium treatment reversed these LV remodeling (8.33±0.32mm at initial vs. 7.03±0.46mm at 4weeks after medication, p=0.049) and systolic dysfunction. The value of radial strain improved from 15.2±5.8% to 38.7±6.9% at 4weeks after medication. (p=0.032) Conclusion: In conclusion, alagebrium treatment in diabetic rat heart improved myocardial systolic dysfunction and LV remodeling by blocking the AGE-induced intracellular ROS synthesis resulting in inhibition of contractile dysfunction induced by AGE. The beneficial effect of alagebrium on diabetic cardiomyopathy was mediated by reduction of extracellular matrix production and this might be mediated by decreased activation of ERK (extracellular signal-regulated kinase) MAP kinase.

Migration in Confined 3D Environments Is Determined by a Combination of Adhesiveness, Nuclear Volume, Contractility, and Cell Stiffness

In cancer metastasis and other physiological processes, cells migrate through the three-dimensional (3D) extracellular matrix of connective tissue and must overcome the steric hindrance posed by pores that are smaller than the cells. It is currently assumed that low cell stiffness promotes cell migration through confined spaces, but other factors such as adhesion and traction forces may be equally important. To study 3D migration under confinement in a stiff (1.77 MPa) environment, we use soft lithography to fabricate polydimethylsiloxane (PDMS) devices consisting of linear channel segments with 20 μm length, 3.7 μm height, and a decreasing width from 11.2 to 1.7 μm. To study 3D migration in a soft (550 Pa) environment, we use self-assembled collagen networks with an average pore size of 3 μm. We then measure the ability of four different cancer cell lines to migrate through these 3D matrices, and correlate the results with cell physical properties including contractility, adhesiveness, cell stiffness, and nuclear volume. Furthermore, we alter cell adhesion by coating the channel walls with different amounts of adhesion proteins, and we increase cell stiffness by overexpression of the nuclear envelope protein lamin A. Although all cell lines are able to migrate through the smallest 1.7 μm channels, we find significant differences in the migration velocity. Cell migration is impeded in cell lines with larger nuclei, lower adhesiveness, and to a lesser degree also in cells with lower contractility and higher stiffness. Our data show that the ability to overcome the steric hindrance of the matrix cannot be attributed to a single cell property but instead arises from a combination of adhesiveness, nuclear volume, contractility, and cell stiffness.