Composition Of Extracellular Matrices Research Articles

Adipose tissue: A valuable resource of biomaterials for soft tissue engineering

Extracellular matrices (ECMs), isolated through decellularization of mammalian tissues, have been successfully used in a variety of tissue engineering and regenerative medicine applications. The composition and spatial structure of ECMs provide not only specific instructive cues for the growth, migration, and differentiation of various cells in vitro, but afford ideal substrates for in vivo tissue reconstruction. Adipose tissue, which is the most prevalent and expendable tissue in the body and can be harvested in large quantities with minimal morbidity, has received much attention as a rich source of ECMs. Recent studies have designed different processes to isolate intact ECMs from adipose tissue and have fabricated various three-dimensional (3-D) tissue engineering scaffolds such as microparticles, powders, sponges, sheets, and hydrogels for use in regenerative medicine, particularly for patients requiring soft tissue regeneration. Indeed, because of the abundance of ECM components within adipose tissue, combined with the relative ease of large tissue harvesting, adipose tissue is a valuable resource in tissue regeneration therapy, encompassing autotransplantation, allotransplantation, and xenotransplantion. We briefly review extraction and decellularization techniques of ECMs from adipose tissue, biological characterization and fabrication of ECM-based tissue engineering scaffolds, and their use in soft tissue engineering.

Targeting the Tumor Proteasome as a Mechanism to Control the Synthesis and Bioactivity of Matrix Macromolecules

Extracellular matrices (ECMs) are dynamic structures that provide cells not only with a structural support but, importantly, exhibit significant functional roles in the control of key cellular events such as adhesion, migration, proliferation, differentiation, and survival. In tumors, matrix effectors such as proteoglycans (PGs) and matrix metalloproteinases (MMPs) constitute major regulators of the interactions between tumor cells and their microenvironment and, therefore, they have been identified as potential molecular targets that are expected to advance the pharmacological treatment of cancer. ECMs composition is highly affected by cells through intrinsic regulatory mechanisms, such as the ubiquitin-proteasome system (UPS). Proteasome is a major cellular protease complex that controls the concentration and turnover of molecules in ECMs, including certain types of PGs, MMPs and collagens, and consequently, in the tumor microenvironment. Furthermore, proteasome activity is regulated by PG-derived intracellular glycosaminoglycan moieties revealing a critical inter-dependence of these compounds. Since ECMs renewal and degradation can be tightly regulated by proteasome activities, its modulation may be considered as a novel strategy to control the properties of tumor microenvironment. Currently, there are several proteasome inhibitors targeting distinct molecular pathways either approved or in clinical trials for the treatment of multiple cancers. In this review, the novel approach of targeting the proteasome to selectively regulate the synthesis and the bioactivity of certain matrix PGs and MMPs is presented and discussed.

Loss of Cell-Surface Laminin Anchoring Promotes Tumor Growth and Is Associated with Poor Clinical Outcomes

Perturbations in the composition and assembly of extracellular matrices (ECM) contribute to progression of numerous diseases, including cancers. Anchoring of laminins at the cell surface enables assembly and signaling of many ECMs, but the possible contributions of altered laminin anchoring to cancer progression remain undetermined. In this study, we investigated the prominence and origins of defective laminin anchoring in cancer cells and its association with cancer subtypes and clinical outcomes. We found loss of laminin anchoring to be widespread in cancer cells. Perturbation of laminin anchoring originated from several distinct defects, which all led to dysfunctional glycosylation of the ECM receptor dystroglycan. In aggressive breast and brain cancers, defective laminin anchoring was often due to suppressed expression of the glycosyltransferase LARGE. Reduced expression of LARGE characterized a broad array of human tumors in which it was associated with aggressive cancer subtypes and poor clinical outcomes. Notably, this defect robustly predicted poor survival in patients with brain cancers. Restoring LARGE expression repaired anchoring of exogenous and endogenous laminin and modulated cell proliferation and tumor growth. Together, our findings suggest that defects in laminin anchoring occur commonly in cancer cells, are characteristic of aggressive cancer subtypes, and are important drivers of disease progression.

Abstract 1333: Organ specific biomatrix scaffolds for ex vivo characterization of colorectal cancer metastases

Abstract Colorectal cancer is the second leading cause of cancer-related deaths in the United States with more than 140,000 new cases being diagnosed this year. However, morbidity and mortality is not generally caused by the primary tumor, but by metastases to other organs. There are few models in which to study colorectal cancer metastasis, either in two-dimensional (2D) monolayer tissue culture or in animal xenografts. Both of these models have limitations. Standard 2D tissue culture, though inexpensive and accessible, poorly mimics the metastatic process and xenograft models are costly and technically challenging. Two of the most common sites of colorectal metastasis are the liver and lungs, however, there are few ex vivo models in which to study metastases to these sites. Advances in materials science have led to the development of ex vivo tissue specific models of extracellular matrices (ECM). We have utilized a novel material, biomatrix scaffolds, to engineer a colorectal cancer metastasis ex vivo model. We hypothesized that through the use of liver and lung specific biomatrix, (1) we can better characterize the colorectal cancer metastatic process by mimicking the organ specific microenvironment and (2) more accurately determine the response to therapeutic agents to treat metastases. In this study, we characterized biomatrix derived from rat liver and lung as a growth substrate for human colon cancer cell lines. We used the human colon cancer derived cell lines: HT29, SW480, CaCo2 and Lovo. Chemotherapeutic response to oxaliplatin, irinotecan and 5-fluorouracil for these cell lines grown on biomatrix was examined. We also used primary cultures of colorectal cancer metastases derived from patient samples. The biomatrix scaffolds were generated through decellularization of rat liver or lungs. We preformed mass spectrometry and determined the ECM composition and the associated growth factors found in lysates of liver and lung remain bound to the biomatrix in concentrations similar to those found in vivo. Colorectal cancer cell lines demonstrated different growth rates and morphologies when grown on biomatrix, resulting in organized, 3-dimenasional growth of non-hollow spheroids several hundred microns in diameter. We demonstrated differential responses to chemotherapeutics and observed differential gene expression via microarray analysis when colorectal cancer cell lines were grown on biomatrix compared to plastic, collagen, or matrigel. We also demonstrated increased tumor cell colony formation in primary cultures of patient derived colorectal metastases on biomatrix compared to plastic or collagen. In conclusion, we have developed a decellularized tissue material, biomatrix, that can provide an ex vivo model of colorectal cancer metastasis to aid in therapeutic drug development and genetic characterization of metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1333. doi:1538-7445.AM2012-1333

Inhibition of tumor growth and angiogenesis by a lysophosphatidic acid antagonist in an engineered three‐dimensional lung cancer xenograft model

: We developed an engineered three-dimensional (3D) tumor xenograft model of nonsmall cell lung cancer (NSCLC) in nude mice, and we used this model to evaluate a dual-activity inhibitor of lysophosphatidic acid (LPA) biosynthesis and receptor activation. : First, BrP-LPA, a pan-antagonist for 4 LPA receptors and inhibitor of the lyosphospholipase D activity of autotaxin, was examined for inhibition of cell migration and cell invasion by human NSCLC A549 cells. Second, A549 cells were encapsulated in 3D in 3 semisynthetic extracellular matrices (ECMs) based on chemically modified glycosaminoglycans, and injected subcutaneously in nude mice. Tumor volume and vascularity were determined as a function of semisynthetic ECMs composition. Third, engineered NSCLC xenografts were formed from A549 cells in either Extracel-HP or Matrigel, and mice were treated with 4 intraperitoneal injections of 3 mg/kg of BrP-LPA. : First, BrP-LPA inhibited cell migration and invasiveness of A549 cells in vitro. Second, tumor growth and microvessel formation for 3D encapsulated A549 cells in vivo in nude mice increased in the following order: buffer only < Extracel < Extracel-HP < Extracel-HP containing growth factorss plus laminin. Third, tumor volumes increased rapidly in both Matrigel and Extracel-HP encapsulated A549 cells, and tumor growth was markedly inhibited by BrP-LPA treatment. Finally, tumor vascularization was dramatically reduced in the A549 tumors treated with BrP-LPA. : Engineered A549 lung tumors can be created by 3D encapsulation in an ECM substitute with user controlled composition. The engineered tumors regress and lose vascularity in response to a dual activity inhibitor of the LPA signaling pathway. Cancer 2010. (c) 2010 American Cancer Society.

Acellularization of embryoid bodies via physical disruption methods

Embryonic stem cells (ESCs) are capable of differentiating into all somatic cell types and have therefore attracted significant interest for use in tissue repair and regeneration therapies. Transplanted ESCs can not only integrate into compromised tissues, but can also stimulate endogenous regeneration via secreted factors. In this study, several acellularization protocols were applied to spheroids of differentiating ESCs, termed embryoid bodies (EBs), to develop a potential route to deliver ESC-derived molecules, independent of cells, to damaged tissues. The objective of this study was to physically disrupt EBs via lyophilization or freeze–thaw cycling, and in combination with DNase treatment, determine the efficacy of acellularization based upon cell viability, DNA removal, and protein retention. Mechanical disruption and DNase treatment of EBs efficiently inhibited viability and removed DNA while retaining protein content to produce an acellular EB matrix. The EB-derived acellular matrices permitted attachment and repopulation of the constructs by 3T3 fibroblasts in vitro. Overall, these studies demonstrate that effective mechanical means to acellularize EBs may be used in order to further elucidate the composition and function of embryonic extracellular matrices and serve as novel naturally-derived scaffolds for tissue repair and regeneration.

Commercial Extracellular Matrix Scaffolds for Rotator Cuff Tendon Repair

We are not aware of any in vitro study comparing the biomechanical, biochemical, and cellular properties of commercial extracellular matrix materials marketed for rotator cuff tendon repair. In this study, the properties of GraftJacket, TissueMend, Restore, and CuffPatch were quantified and compared with each other. The elastic moduli were also compared with that of normal canine infraspinatus tendon. Samples were tested from different manufacturing lots of four materials: GraftJacket (ten lots), TissueMend (six), Restore (ten), and CuffPatch (six). The Kruskal-Wallis test was used to compare thickness, stiffness, and modulus as well as hydroxyproline, chondroitin/dermatan sulfate glycosaminoglycan, hyaluronan, and DNA contents among these matrices. The moduli of the extracellular matrices were also compared with those of normal canine infraspinatus tendon. All four extracellular matrices required 10% to 30% stretch before they began to carry substantial load. Their maximum moduli were realized in their linear region at 30% to 80% strain. The elastic moduli of all four commercial matrices were an order of magnitude lower than that of canine infraspinatus tendon. TissueMend had significantly higher DNA content than the other three matrices (p<0.0001), although both Restore and GraftJacket also had measurable amounts of DNA. Our data demonstrate chemical and mechanical differences among the four commercial extracellular matrices that we evaluated. Probably, the source (dermis or small intestine submucosa), species (human, porcine, or bovine), age of the donor (fetal or adult), and processing of these matrices all contribute to the unique biophysical properties of the delivered product. The biochemical composition of commercial extracellular matrices is similar to that of tendon. However, the elastic moduli of these materials are an order of magnitude lower than that of tendon, suggesting a limited mechanical role in augmentation of tendon repair.

Changes in tendon extracellular matrix composition with age

Introduction A major aspect of the normal ageing process is the loss of suppleness of tissues; skin becomes wrinkled and there is a decline in joint flexibility. These gradual changes predominantly result due to long‐term alterations in the extracellular matrices (ECMs) of structures including skin, tendons/ligaments, bones, cartilage and blood vessels. ECMs are generally remodelled during an individual's life, but for tendons/ligaments this occurs at a slow rate. Parameters such as slow turnover, long‐term post‐translational modifications and extensive cell–matrix interactions are three aspects of ECM biology, which influence the mechanisms of ageing. Ageing of tendon/ligament is a major problem affecting the mobility of an increasingly ageing population, and age‐related changes lead to numerous musculoskeletal pathologies in old age. Therefore, the initial objective of these studies was to compare the biochemical composition of young vs. old tendon, with an aim of elucidating the mechanisms controlling the ageing process in tendon ECM.Materials and methods Tail tendons were dissected from 2‐, 9‐, 12‐ or 22‐month‐old C57/Blacks, and both protein and RNA were analysed for differences in ECM tendon composition with age (n = 4). Proteoglycans were extracted in 4 m guanidine hydrochloride and sulphated glycosaminoglycans measured using the DMMB assay. Collagens were extracted by acid hydrolysis and total collagen measured using the hydroxyproline assay; additionally, the ratio of collagen types in the ageing tail tendons was analysed by Western blotting with antibodies raised against collagen types I, III and V. The degree of tissue hydration was determined by measuring the water content of the tendons. Matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 expression and activation were detected by gelatin substrate zymography, and the presence of their inhibitors was assessed by reverse zymography. To assess age changes at the transcriptional level, RNA was extracted from tendons, labelled with biotin and cDNA arrays (Affymetrix) performed.Results The results indicate that there is a change in tendon matrix composition with increasing age (Table 1). There is a significant loss in tissue hydration and a reduction in the amount of sulphated GAGs present in the 22‐month tendons compared with young tissue, whilst the amount of collagen present does not significantly alter. 2 months 9 months 12 months 22 months sGAG (µg/mg)* 49.6 ± 8.7 — 40.1 ± 4.6 28.9 ± 0.9 Collagen (µg/mg)† 48.4 ± 5.3 39.9 ± 3.4 40.2 ± 4.7 47.2 ± 4.3 Water content (%) 80.4 ± 3.9 74.6 ± 2.4 74.5 ± 3.8 69.3 ± 2.8 µg/mg wet weight tissue. µg/mg dry weight tissue. Analysis of MMP expression by gelatin zymography demonstrated that there was a significant loss of MMP‐9 expression in the tendons of 9‐ and 12‐month‐old animals. MMP‐9 was only evident in the 2‐ and 22‐month‐old tendons indicative of developmental turnover and a remodelling response, respectively. MMP‐2 expression and activation was evident in all tendon ages analysed, as were the MMP inhibitors – TIMP‐1 and TIMP‐2.Discussion The data demonstrate that during the ageing process, the composition of tendon ECM is modified. This may compromise the response of the tissue to application of mechanical loads hence the increased incidences of musculoskeletal injuries, e.g. tendon/ligament sprains in elderly people. The reduction in both water and sGAGs, and the increased expression of MMP‐9 may be responsible for changes observed in biomechanical properties where the tendons become more brittle and less able to withstand load. Understanding changes in the biochemical composition may allow us to manipulate cell activity and ultimately ECM structure and function to combat these age‐related effects and the musculoskeletal pathologies incurred.

Comparison of bone marrow extracellular matrices

We have compared the structure and composition of adult and fetal bovine bone marrow extracellular matrices. In contrast to fetal bone marrow, adult bone marrow has more oval fenestration and accumulation of adipocytes as well as lower protein content. These differences could be due to remodeling of bone marrow tissue as it develops. Zymogram analysis of matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) activities showed that fetal, but not adult bone marrow extract contained a 96-kDa MMP and TIMP-1 and -2. These activities may contribute to the structural differences between adult and fetal bone marrow tissues.

Differential expression of laminin α chain in human hepatocellular carcinoma: a switch from α2 to α3 chain

The expression of laminin α chains including α1, α2 and α3, were examined in 12 cases of human liver and hepatocellular carcinoma (HCC) by using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Differential expression pattern of these three laminin α chains were shown. Laminin α1 was detectable in five out of the 12 cases but expressed only weakly, and showed no difference of expression between noncancerous liver and HCC. Laminin α2 was expressed in all cases predominantly in noncancerous liver tissue and down-regulated in HCC tissue, while laminin α3 was only weakly detected in noncancerous liver and up-regulated in HCC. Therefore, the changes in the laminin α chain expression in HCC may be related to the altered composition of extracellular matrices in HCC and the behavior of HCC cells.

Composition and organisation of extracellular matrices around germ tubes and appressoria ofColletotrichum lindemuthianum

The ultrastructure and composition of the extracellular matrices (ECMs) associated with germ tubes and appressoria ofColletotrichum lindemuthianum have been examined. Flexuous fibres (fimbriae), up to 6 μm long and 4–30 nm in diameter, protruded from the surface of germ tubes and appressoria. Anionic colloidal gold and lectin cytochemistry showed that ECMs of germ tubes and appressoria contain basic proteins, α-D-mannose and α-D-galactose residues. A monoclonal antibody, UB26, was raised to infection structures isolated from leaves ofPhaseolus vulgaris infected withC. lindemuthianum. UB26 recognised a protein epitope on two glycoproteins (Mr 133,000 and 146,000). Reductions in the Mr of these proteins after treatment with peptide-N-glycosidase and trifluoromethane sulphonic acid suggest that they carry N- and O-linked side-chains. Immunofluorescence and EM-immunogold labelling showed that glycoproteins recognised by UB26 were restricted to the ECMs around germ tubes and appressoria but fimbriae were not labelled. Unlike appressorial germ tubes formed in vitro, intracellular infection hyphae were not labelled, suggesting that the glycoproteins recognised by UB26 are not present on fungal structures formed within host cells. In liquid culture, these glycoproteins were not released into the medium, suggesting they are physically linked to the cell wall. Also, the glycoproteins were not removed from glass surfaces by ultrasonication. These results suggest that glycoproteins recognised by UB26 may be involved in the adhesion of germ tubes and appressoria to substrata. Our results show that the ECMs of germ tubes and appressoria differ markedly in structure and composition from those of conidia and intracellular hyphae, and that extracellular glycoproteins are associated with specific regions of the fungal cell surface.

Inhibition of Elastin Synthesis by High Potassium Salt Is Mediated by Ca2+ Influx in Cultured Smooth Muscle Cells In Vitro: Reciprocal Effects of K+ on Elastin and Collagen Synthesis

Elastin synthesis in cultured smooth muscle cells was inhibited by one fourth in the presence of 0.1 M K+ in the medium. The degree of inhibition paralleled the decrease in the steady-state levels of elastin mRNA. The inhibition of elastin synthesis was blocked by addition of 1 microM nifedipine, a Ca2+ antagonist. Comparable inhibition of elastin synthesis was observed by addition of A23187, a Ca2+ ionophore. In contrast, collagen synthesis and thymidine uptake were stimulated threefold and twofold respectively in the presence of 0.1 M K+ with a concomitant increase in collagen mRNA. The stimulation of collagen synthesis was also blocked by nifedipine. These results indicate that K+ modulates elastin and collagen synthesis and their gene expression reciprocally, and these effects are mediated by Ca2+ influx. Thus K+ exerts profound effects on the composition of extracellular matrices in aorta.

Alpha 1-VIII collagen is expressed in the rat glomerulus and in resident glomerular cells

Current knowledge regarding the molecular composition of extracellular matrices in the glomerulus does not explain how these components interact to form stable three-dimensional structures. The recent recognition that short-chain collagens such as type VIII collagen function as molecular bridges in some nonrenal tissues has raised the possibility that such molecules may serve a similar function in the glomerulus. We have recently shown that cultured rat mesangial cells synthesize and secrete several short-chain collagenous proteins, one of which has properties similar to alpha 1-VIII collagen. In the present study we have isolated a rat mesangial cell alpha 1-VIII collagen cDNA clone, the sequence of which is 81% homologous to mouse alpha 1-VIII collagen. We used this cDNA to determine that alpha 1-VIII collagen mRNA is expressed in rat renal cortex and in cultured glomerular mesangial, epithelial, and endothelial cells. Additionally, we demonstrated that alpha 1-VIII collagen is secreted by cultured mesangial cells as an 80-kDa translation product. By immunocytochemistry, alpha 1-VIII collagen localized to the media of large intrarenal arteries and to the capillary loops and the mesangium of normal rat kidney. These results indicate that type VIII collagen is a normal constituent of the rat glomerulus as well as large intrarenal arteries. We speculate that type VIII collagen may function in part to determine the three-dimensional organization of the subendothelial and mesangial matrices.

Basement membrane matrices in mouse embryogenesis, teratocarcinoma differentiation and in neuromuscular maturation.

In this paper we discuss studies on basement membrane and interstitial matrix molecules in early development and teratocarcinoma differentiation. In the early embryo a compartmentalization of newly formed cell types takes place immediately by formation of basement membranes. The stage-specific developmental appearance of extracellular matrix molecules such as type IV collagen, laminin, entactin, fibronectin and proteoglycans seems to reflect a diversified role of extracellular matrices already in the earliest stages of development. In teratocarcinoma cultures the appearance and composition of extracellular matrices during the differentiation of endoderm cells closely resembles that found in the early embryo. Also in this respect the teratocarcinoma system can be used as a model for studies on early development. In later developmental phenomena other matrix molecules can also be of importance. Merosin, a novel tissue-specific basement membrane-associated protein that appears during muscle and nerve maturation is an example of such molecules.

Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-beta.

Human platelet-derived transforming growth factor-beta (TGF-beta 1) increases the accumulation of the extracellular matrix proteins, fibronectin and type I collagen, in mesenchymal and epithelial cells. To determine the basis for this effect, we have examined the levels of mRNAs corresponding to fibronectin and alpha 2(I) procollagen in NRK-49 rat fibroblasts and L6E9 rat myoblasts treated with TGF-beta 1. TGF-beta 1 increased severalfold the levels of mRNAs for both proteins. The kinetics of this effect were similar for both mRNA species. The increase in fibronectin and alpha 2(I) procollagen mRNAs was detectable 2 h after addition of TGF-beta 1 to the cells and their maximal levels remained constant for several days. Actinomycin D, but not cycloheximide, inhibited the increase in fibronectin and alpha 2(I) procollagen mRNA levels induced by TGF-beta 1. The results indicate that TGF-beta 1 controls the composition and abundance of extracellular matrices at least in part by inducing a coordinate increase in the levels of fibronectin and type I collagen mRNAs.

Chapter 2 The Extracellular Matrix: A Dynamic Component of the Developing Embryo

This chapter discusses the studies of some molecular characteristics of extracellular matrix and temporal correlations with other developmental events. In this manner, some aspects of the cellular environments during embryonic development are explained in this chapter. By concentrating on the early stages of embryonic development, an attempt is made to elucidate the matrix synthesis at the time the embryo is still histologically simple, yet when major interactions are occurring. Mature tissues and organs are functionally integrated units that consist of both cellular and extracellular elements. Extracellular regions contain a variety of substances, including macromolecules, water, and ions. Collectively these substituents form the extracellular matrix. Extracellular matrices exhibit a great deal of quantitative variation among different tissues. The macromolecules of the extracellular matrix, for example, glycosaminoglycans, collagen, and glycoproteins, are all cell products. Macromolecular composition to a large extent determines the degree of hydration, ionic composition, permeability, and mechanical properties of different extracellular matrices. Chronological changes in macromolecules differ among different matrices, but there is no evidence that each different developing tissue synthesizes with unique embryonic variants of collagen or glycosaminoglycan polysaccharide.