Basement Membrane Type Research Articles

Proteomic View of Basement Membranes from Human Retinal Blood Vessels, Inner Limiting Membranes, and Lens Capsules.

Basement membranes (BMs) are extracellular matrix sheets comprising the laminins, type-IV collagens, nidogens, and the heparan sulfate proteoglycans, perlecan, collagen XVIII, and agrin. In intact BMs, BM proteins are physiologically insoluble and partially resistant to proteolytic digestion, making BMs a challenge to study. Here three types of BMs from adult human eyes, the inner limiting membrane (ILM), the retinal vascular BMs, and the lens capsule, were isolated for analysis by 1D-SDS-PAGE and LC-MS/MS. Peptide and protein identifications were done using MaxQuant. 1129 proteins were identified with a 1% false discovery rate. Data showed that BMs are composed of multiple laminins, collagen IVs, nidogens, and proteoglycans. The dominant laminin family member in all BMs was laminin α5β2γ1. The dominant collagen IV trimer in lens capsule (LC) and blood vessel (BV) BMs had a chain composition of α1(IV)2, α2 (IV), whereas the dominant collagen IV in the ILM had the α3(IV), α4(IV), α5(IV) chain composition. The data also showed that the ratio of laminin and collagen IVs varied among different BM types: the ratio of collagen IV to the other BM proteins is highest in LC, followed by BV and lowest for the ILM. The data have been deposited to the ProteomeXchange with identifier PXD001025.

Overexpression of Twist and Matrix Metalloproteinase-9 with Metastasis and Prognosis in Gastric Cancer

Twist, a basic helix-loop-helix transcription factor, plays a key role in the metastatic progression of human cancer. Matrix metalloproteinase (MMP)-9 is an endopeptidase that digests basement membrane type IV collagen, therefore being possibly related to tumor progression. It has been reported that Twist and matrix metalloproteinase-9 (MMP-9) are expressed in gastric cancers. However, the exact roles of Twist and MMP-9 in tumor metastasis and prognosis remain unclear. The aim of this study was to casts light on this question. Twist and MMP-9 expression in tissue sections of 37 gastric carcinomas was evaluated with immunohistochemistry. The staining results were compared with clinicopathologic features and to patients'outcome. Twist positive expression was significantly increased in gastric cancer cases with lymph node metastasis (P=0.023). But no correlations were found between MMP-9 overexpression and clinicopathologic features, such as recurrence, TNM stage, and lymph node metastasis. Overall survival (OS) was significantly correlated with recurrence, serosa invasion, TNM stages, distant metastasis, and MMP-9 (P=0.027, 0.021, 0.000, 0.024 and 0.036, respectively). Disease-free survival (DFS) was prominently related to recurrence location, serosa invasion and TNM stages (P=0.000, 0.038 and 0.003, respectively). In the Cox regression multivariate analysis, TNM stage, distant metastasis and MMP-9 were significantly associated with prognosis of gastric cancer (P=0.002, 0.019, and 0.032, respectively). This study showed Twist positive expression to be significantly correlated with lymph node metastasis in gastric cancer. MMP-9 overexpression is associated with OS, suggesting that MMP-9 is a prognostic indicator for survival in patients with gastric cancer.

NASPGHAN Clinical Report on the Evaluation and Treatment of Pediatric Patients With Internal Penetrating Crohn Disease

Natural History of Pediatric Crohn Disease T he natural history of pediatric Crohn disease (CD) remains unpredictable, although some trends are observed that differentiate children from adults. Pediatric CD often presents with more severe disease and more frequent need for immunosuppressive therapy (1). Growth failure, present in 15% to 20% of patients, is a unique characteristic of pediatric CD not seen in adult-onset CD (2). Colonic disease distribution is common in patients younger than 10 years (1). The need for surgical intervention also varies, with 1 study reporting the actuarial risk of having undergone an extensive intestinal resection being 48.6% 5% in a childhood-onset group versus 14.6% 2% in the adult-onset group (P< 0.001) (1). More recently, long-term follow-up of patients enrolled in pediatric registries shows a cumulative surgical rate of 14% to 17% at 5 years and 28% at 10 years (3,4).

Tissue-Engineered Cartilaginous Constructs for the Treatment of Caprine Cartilage Defects, Including Distribution of Laminin and Type IV Collagen

The purpose of this study was the immunohistochemical evaluation of (1) cartilage tissue-engineered constructs; and (2) the tissue filling cartilage defects in a goat model into which the constructs were implanted, particularly for the presence of the basement membrane molecules, laminin and type IV collagen. Basement membrane molecules are localized to the pericellular matrix in normal adult articular cartilage, but have not been examined in tissue-engineered constructs cultured in vitro or in tissue filling cartilage defects into which the constructs were implanted. Cartilaginous constructs were engineered in vitro using caprine chondrocyte-seeded type II collagen scaffolds. Autologous constructs were implanted into 4-mm-diameter defects created to the tidemark in the trochlear groove in the knee joints of skeletally mature goats. Eight weeks after implantation, the animals were sacrificed. Constructs underwent immunohistochemical and histomorphometric evaluation. Widespread staining for the two basement membrane molecules was observed throughout the extracellular matrix of in vitro and in vivo samples in a distribution unlike that previously reported for cartilage. At sacrifice, 70% of the defect site was filled with reparative tissue, which consisted largely of fibrous tissue and some fibrocartilage, with over 70% of the reparative tissue bonded to the adjacent host tissue. A novel finding of this study was the observation of laminin and type IV collagen in in vitro engineered cartilaginous constructs and in vivo cartilage repair samples from defects into which the constructs were implanted, as well as in normal caprine articular cartilage. Future work is needed to elucidate the role of basement membrane molecules during cartilage repair and regeneration.

Basement membrane molecule expression attendant to chondrogenesis by nucleus pulposus cells and mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) represent an autologous cell source for nucleus pulposus (NP) tissue engineering and regeneration. Although studies have demonstrated the ability of MSCs to differentiate to NP-like chondrocytic cells, few have comparatively studied the matrix synthesis and composition of the cartilaginous tissue formed in vitro from both cell types, particularly with respect to the expression of basement membrane (BM) molecules. The objective of this study was to evaluate chondrogenesis and expression of BM molecules, laminin and type IV collagen, in monolayer and in pellet cultures of caprine NP cells and MSCs. Both cell types demonstrated comparable levels of chondrogenesis, indicated by the percentage of chondrocytic cells, and the amounts of glycosaminoglycan and type II collagen. Laminin and type IV collagen were expressed intracellularly by NP cells and MSCs cultured in monolayer. During chondrogenesis in pellet cultures, the deposition of BM molecules in NP and MSC pellets followed an orderly spatiotemporal shift in pattern from a diffuse territorial and interterritorial distribution to a defined pericellular localization, as seen in normal adult NP. These results inform the use of MSCs for NP regeneration and suggest the possible involvement of certain BM molecules in chondrogenesis and cartilage regeneration.

Kidney diseases caused by glomerular basement membrane typeIVcollagen defects in dogs

To review the pathogenesis, as well as the clinical and pathologic features of canine glomerular diseases caused by genetic type IV collagen defects. Original studies and review articles from human and veterinary medical fields. Presence in glomerular basement membranes (GBM) of a network composed of α3.α4.α5 heterotrimers of type IV collagen is required to maintain structure and function of glomerular capillary walls. Hereditary nephropathy (HN) is the most commonly used name for kidney diseases that occur in dogs due to genetic type IV collagen abnormalities. To date, 4 different collagen IV gene mutations have been identified in dogs with HN; 2 are COL4A5 mutations that cause X-linked HN (XL-HN), and 2 are COL4A4 mutations that cause autosomal recessive HN (AR-HN). Affected males with XL-HN and affected males and females with AR-HN develop juvenile-onset kidney disease manifested by proteinuria typically starting at 3-6 months of age and followed by progressive kidney disease leading to terminal failure usually at 6-24 months of age. Carrier female dogs with XL-HN also develop proteinuria starting at 3-6 months of age, but progressive disease causing kidney failure is uncommon until they are >5 years old. The distinctive pathologic lesions of HN are extensive multilaminar splitting and thickening of the GBM, as demonstrated by electron microscopy, and abnormal type IV collagen α-chain content of basement membranes, as demonstrated by immunolabeling. Identification of the underlying gene mutations has permitted genetic testing and selective breeding practices that currently are minimizing HN in breeds known to be at risk. Canine HN is a rare disease that should be considered whenever a dog exhibits a juvenile-onset kidney disease characterized partly by proteinuria, but highly specialized methods are required to pursue a definitive diagnosis.

Harvesting the Potential of the Human Umbilical Cord: Isolation and Characterisation of Four Cell Types for Tissue Engineering Applications

The human umbilical cord (UC) has attracted interest as a source of cells for many research applications. UC solid tissues contain four cell types: epithelial, stromal, smooth muscle and endothelial cells. We have developed a unique protocol for the sequential extraction of all four cell types from a single UC, allowing tissue reconstruction using multiple cell types from the same source. By combining perfusion, immersion and explant techniques, all four cell types have been successfully expanded in monolayer cultures. We have also characterised epithelial and Wharton’s jelly cells (WJC) by immunolabelling of specific proteins. Epithelial cell yields averaged at 2.3 × 10⁵ cells per centimetre UC, and the cells expressed an unusual combination of keratins typical of simple, mucous and stratified epithelia. Stromal cells in the Wharton’s jelly expressed desmin, α-smooth muscle actin, elastin, keratins (K12, K16, K18 and K19), vimentin and collagens. Expression patterns in cultured cells resembled those found in situ except for basement membrane components and type III collagen. These stromal cells featured a sustained proliferation rate up to passage 12 after thawing. The mesenchymal stem cell (MSC) character of the WJC was confirmed by their expression of typical MSC surface markers and by adipogenic and osteogenic differentiation assays. To emphasise and demonstrate their potential for regenerative medicine, UC cell types were successfully used to produce human tissue-engineered constructs. Both bilayered stromal/epithelial and vascular substitutes were produced, establishing the versatility and importance of these cells for research and therapeutic applications.

Differential expression of basement membrane type IV collagen α2 and α6 chains as a prognostic factor in patients with extrahepatic bile duct carcinoma

The destruction of the basement membrane (BM) is the first step in cancer invasion and metastasis. Type IV collagen is a major component of the BM, and is composed of six genetically distinct α(IV) chains; α1(IV) to α6(IV). The loss of α5(IV) and α6(IV) chains from the epithelial BM at the early stage of cancer invasion has been reported in several types of cancers. However, the expression of α5(IV) and α6(IV) chains in extrahepatic bile duct carcinoma (EBDC) remains unclear. We examined the expression of α(IV) chains by immunohistochemistry using 71 resected EBDC specimens. Prognostic significance of α(IV) chains was examined by Cox regression and Kaplan-Meier analyses. In the invasive cancer, the expression of α6(IV) chain in the BM was lost partially or completely preceded by the loss of α2(IV) chain. The loss of α6(IV) chain in the BM of the invasive cancer was related to the tumor classification, TNM stages, and the expression of α2(IV) chain. The patients with α2(IV)-negative and α6(IV)-negative chains had significantly poorer prognosis than those with α2(IV)-positive and α6(IV)-positive/negative chains (P = 0.04). The loss of α2(IV) and α6(IV) chains might be a useful prognostic factor in patients with EBDC.

Podosomes in migrating microglia: components and matrix degradation

BackgroundTo perform their functions during development and after central nervous system injury, the brain’s immune cells (microglia) must migrate through dense neuropil and extracellular matrix (ECM), but it is not known how they degrade the ECM. In several cancer cell lines and peripheral cells, small multi-molecular complexes (invadopodia in cancer cells, podosomes in nontumor cells) can both adhere to and dissolve the ECM. Podosomes are tiny multi-molecular structures (0.4 to 1 μm) with a core, rich in F-actin and its regulatory molecules, surrounded by a ring containing adhesion and structural proteins.MethodsUsing rat microglia, we performed several functional assays: live cell imaging for chemokinesis, degradation of the ECM component, fibronectin, and chemotactic invasion through Matrigel™, a basement membrane type of ECM. Fluorescent markers were used with high-resolution microscopy to identify podosomes and their components.ResultsThe fan-shaped lamella at the leading edge of migrating microglia contained a large F-actin-rich superstructure composed of many tiny (<1 μm) punctae that were adjacent to the substrate, as expected for cell–matrix contact points. This superstructure (which we call a podonut) was restricted to cells with lamellae, and conversely almost every lamella contained a podonut. Each podonut comprised hundreds of podosomes, which could also be seen individually adjacent to the podonut. Microglial podosomes contained hallmark components of these structures previously seen in several cell types: the plaque protein talin in the ring, and F-actin and actin-related protein (Arp) 2 in the core. In microglia, podosomes were also enriched in phosphotyrosine residues and three tyrosine-kinase-regulated proteins: tyrosine kinase substrate with five Src homology 3 domains (Tks5), phosphorylated caveolin-1, and Nox1 (nicotinamide adenine dinucleotide phosphate oxidase 1). When microglia expressed podonuts, they were able to degrade the ECM components, fibronectin, and Matrigel™.ConclusionThe discovery of functional podosomes in microglia has broad implications, because migration of these innate immune cells is crucial in the developing brain, after damage, and in disease states involving inflammation and matrix remodeling. Based on the roles of invadosomes in peripheral tissues, we propose that microglia use these complex structures to adhere to and degrade the ECM for efficient migration.

Functional Consequences of Cell Type-Restricted Expression of Laminin α5 in Mouse Placental Labyrinth and Kidney Glomerular Capillaries

The labyrinth is the highly vascularized part of the rodent placenta that allows efficient transfer of gases, nutrients, wastes, and other molecules between the maternal and embryonic circulations. These two blood compartments are separated by blastocyst-derived trophoblasts and endothelial cells with an intervening basement membrane that contains laminin and other typical basement membrane components. Previously we reported that the labyrinth of laminin α5 knockout (LMα5−/−) embryos exhibits reduced vascularization and detachment of endothelial cells from the basement membrane, which normally contains LMα5. As very little is known about the origin of this vascular basement membrane, we investigated the cellular requirements for LMα5 expression in the mouse placental labyrinth. By fluorescence-activated cell sorting and RT-PCR we confirmed that both endothelial cells and trophoblasts normally express LMα5. Using Cre-loxP technology and doxycycline-mediated gene expression, we generated genetically mosaic placentas in which either the trophoblasts or the endothelial cells, but not both, expressed LMα5. We found that the overall architecture of the labyrinth was normal as long as one of these two cell types expressed LMα5, even if it was transgene-derived human laminin α5. These results suggest that laminin trimers containing α5 that are synthesized and secreted by endothelium or by trophoblasts are capable of integrating into the basement membrane and promoting normal vascularization of the placenta. Additional studies showed that endothelium-expressed human LMα5 can support vascularization of the kidney glomerulus, consistent with previous studies using a tissue grafting approach.

Hyalinized stroma in clear cell carcinoma of the ovary: how is it formed?

Ovarian clear cell carcinoma often shows stromal hyalinization. The main constituents of hyalinization are basement membrane materials, including laminin and type IV collagen. Although it is known that clear cell carcinoma cells produce these materials, it remains unclear whether they can form hyalinized stroma by themselves or if cooperation with stromal cells is required. In the present study, we first reviewed 35 surgical specimens for the pattern of early hyalinization. It occurred either in a globule-like pattern or in a circumferential pattern. In the former, compact hyaline globules abruptly appeared within tumor cell aggregates. In the latter, hyalinized materials appeared around the preceding spherule-like mucoid spaces among tumor cells. In either pattern, hyalinization is most likely to begin in the intercellular spaces among tumor cells, where stromal cells rarely intervene. To verify this, 2 ovarian clear cell carcinoma cell lines (JHOC-5 and HAC-2) were analyzed in vitro. Each cell line was monocultured in suspension: if any deposition occurred in floating multicellular aggregates, it should be in the intercellular spaces. Deposition of type IV collagen occurred in a globule-like pattern (JHOC-5) or a circumferential pattern (HAC-2) within multicellular aggregates, and it developed into a structure comparable with the hyalinized stroma in surgical specimens. Intercellular deposition of type IV collagen was reproduced by culture in 3-dimensional type I collagen gels. All of these findings showed that clear cell carcinoma cells themselves form hyalinized stroma by depositing self-made basement membrane materials in the intercellular spaces.

Comparative Analysis of the Basement Membrane Composition of the Human Limbus Epithelium and Amniotic Membrane Epithelium

Human amniotic membrane has been widely used as substrate for ex vivo expansion and transplantation of limbal epithelial cells. To further clarify its suitability as a surrogate niche for limbal stem cells and progenitor cells, we analyzed the composition of the amniotic epithelial basement membrane, with special focus on the expression of limbus-specific matrix components. Cryosections of corneoscleral specimens obtained from 10 human donor eyes and of 6 amniotic membrane specimens obtained at cesarean section were stained by indirect immunofluorescence using a broad panel of antibodies against basement membrane components. Both amniotic and limbal epithelial basement membranes showed positive immunoreactivity for collagen type IV α1, α2, α5, and α6 chains; collagens type VII, XV, XVI, XVII, and XVIII; laminin α3, β1, β2, β3, γ1, and γ2 chains; laminin-111 and laminin-332; nidogen-1 and nidogen-2; fibronectin; fibulin-2; fibrillin-2; perlecan; and agrin. Both types of basement membrane were negative for collagen type IV α3 and α4 chains, collagen type V, and laminin α4 chain. Limbal basement membrane components, which were not detected in amniotic membrane, included laminin α1, α2, α5, and γ3 chains; BM40/SPARC; tenascin-C; matrilin-2; endostatin; and collagen type XVIII. Despite extensive similarities in basement membrane composition between amniotic and corneolimbal epithelia, the lack of limbus-specific environmental factors argues against the potential of denuded amniotic membrane as a surrogate niche for limbal stem cells but supports its suitability as a substrate to promote the formation of a well-differentiated stratified corneal epithelial equivalent for tissue engineering strategies.

Goodpasture Antigen-binding Protein/Ceramide Transporter Binds to Human Serum Amyloid P-Component and Is Present in Brain Amyloid Plaques

Serum amyloid P component (SAP) is a non-fibrillar glycoprotein belonging to the pentraxin family of the innate immune system. SAP is present in plasma, basement membranes, and amyloid deposits. This study demonstrates, for the first time, that the Goodpasture antigen-binding protein (GPBP) binds to human SAP. GPBP is a nonconventional Ser/Thr kinase for basement membrane type IV collagen. Also GPBP is found in plasma and in the extracellular matrix. In the present study, we demonstrate that GPBP specifically binds SAP in its physiological conformations, pentamers and decamers. The START domain in GPBP is important for this interaction. SAP and GPBP form complexes in blood and partly colocalize in amyloid plaques from Alzheimer disease patients. These data suggest the existence of complexes of SAP and GPBP under physiological and pathological conditions. These complexes are important for understanding basement membrane, blood physiology, and plaque formation in Alzheimer disease.

Engineering endostatin-expressing cartilaginous constructs using injectable biopolymer hydrogels

The release of an anti-angiogenic agent, such as type XVIII/endostatin, from an implantable scaffold may be of benefit in the repair of articular cartilage. The objectives of this study are to develop an injectable mesenchymal stem cell (MSC)-incorporating collagen-based hydrogel capable of undergoing covalent cross-linking in vivo and overexpressing endostatin using nonviral transfection, and to investigate methods for the retention of the endostatin protein within the scaffolds. The effects of different cross-linking agents (genipin, transglutaminase-2, and microbial transglutaminase) and different binding molecules for endostatin retention (heparin, heparan sulfate, and chondroitin sulfate) are evaluated. Cartilaginous constructs that overexpress endostatin for 3weeks are successfully engineered. Most of the endostatin is released into the surrounding media and is not retained within the constructs. The presence of two common basement membrane molecules, laminin and type IV collagen, which have been reported in developing and mature articular cartilage and are generally associated with type XVIII collagen in vivo, is also observed in the engineered cartilaginous constructs. Endostatin-producing cartilaginous constructs can be formulated by growing nonvirally transfected mesenchymal stem cells in collagen gels covalently cross-linked using genipin, transglutaminase-2, and microbial transglutaminase. These constructs warrant further investigation for cartilage repair procedures. The novel finding of laminin and type IV collagen in the engineered cartilage constructs may be of importance for future work toward understanding the role of basement membrane molecules in chondrogenesis and in the physiology and pathology of articular cartilage.

Folding Delay and Structural Perturbations Caused by Type IV Collagen Natural Interruptions and Nearby Gly Missense Mutations

The standard collagen triple helix requires Gly as every third residue in the amino acid sequence, yet all nonfibrillar collagens contain sites where this repeating pattern is interrupted. To explore the effects of such natural interruptions on the triple helix, a 4- or 15-residue sequence from human basement membrane type IV collagen was introduced between (Gly-Xaa-Yaa)(n) domains within a recombinant bacterial collagen. The interruptions had little effect on melting temperature, consistent with the high thermal stability reported for nonfibrillar collagens. Although the 4-residue interruption cannot be accommodated within a standard triple helix, trypsin and thermolysin resistance indicated a tightly packed structure. Central residues of the 15-residue interruption were protease-susceptible, whereas residues near the (Gly-Xaa-Yaa)(n) boundary were resistant, supporting a transition from an alternate conformation to a well packed triple helix. Both interruptions led to a delay in triple-helix folding, with the 15-residue interruption causing slower folding than the 4-residue interruption. These results suggest that propagation through interruptions represents a slow folding step. To clarify the relation between natural interruptions and pathological mutations, a Gly to Ser missense mutation was placed three triplets away from the 4-residue interruption. As a result of this mutation, the 4-residue interruption and nearby triple helix became susceptible to protease digestion, and an additional folding delay was observed. Because Gly missense mutations that cause disease are often located near natural interruptions, structural and folding perturbations arising from such proximity could be a factor in collagen genetic diseases.

Case report: parotid basal cell adenoma, membranous type

A52-year-old female withbackgroundofnon-Hodgkin's lymphoma and polymyositis presented with a 4 year history of slow growing, left parotid mass. There were no other associated symptoms. Fine needle aspirate of the mass was performed and the diagnosis of an atypical primary salivary gland neoplasm was made. Subsequent left parotidectomy was performed and the diagnosis of parotid basal cell adenoma, membranous type was made. Basal cell adenoma is a benign epithelial neoplasm of the salivary gland which is characterised by uniform proliferation of basaloid cells. Basal cell adenoma often occurs in the parotid gland followed by the submandibular gland and the upper lip region. Basal cell adenoma accounts for 1–3% of all benign parotid tumours, and the peak incidence is in the 6th to 7th decades of life. There are various subtypes based on morphological features which include solid, trabecular, tubular, and membranous types. The membranous subtype has been associated with the dermal cyclindromas which have similar incidence of chromosomal alteration at 16q12– 13. The membranous subtype is characterised by tumour cells arranged in a jig-saw like pattern with prominent PAS+ eosino-philic, basement-membrane type material. The main differential diagnosis is basal cell adenocarcinoma, and it can be difficult to distinguish between the two. Basal cell adeno-carcinoma is characterised by increased number of mitotic figures, increased nuclear pleomorphism, infiltrative growth, and peri-neural and intravascular invasion. In our case, the proliferative index Ki-67 is low, and there is no significant nuclear pleomor-phism, infiltrative growth pattern, or perineural invasion. However, the lesion extends to the diathermied margin, which is associated with a higher rate of recurrence of approximately 25%.

Oral mucosa model based on a collagen–elastin matrix

The collagen-elastin matrix (Matriderm(®)) is used to treat deep and full-thickness burns and was recently described as a suitable scaffold for tissue engineering. The aim of the present study was to investigate the biocompatibility of Matriderm(®) for gingival use through creation of an oral mucosa model ex vivo. Gingival fibroblasts and keratinocytes were cultured. A dermal area on the base of the collagen-elastin matrix was repopulated with fibroblasts. After 14 days, keratinocytes were seeded on this dermal area to engineer a multilayered mucosa. Analysis of the architecture was performed using light and electron microscopy. Immunohistochemical detection of collagen IV and cytokeratin was carried out. Based on this scaffold we generated a multilayered oral mucosa-like structure. Histological, immunohistochemical and electron microscopic analysis of the dermal/epidermal junction showed a typical basement membrane and hemidesmosomal structures. Neighboring keratinocytes formed desmosomes in the epidermal sections. Cytokeratin was detectable in all epidermal layers. These experiments revealed that the collagen-elastin matrix was highly biocompatible with gingival cells under ex vivo conditions. Employing tissue-engineering techniques with dermal and epidermal cells from the gingiva, a multilayered oral mucosa was generated and characterized with respect to biocompatibility for Matriderm(®). The results indicate that Matriderm(®) is suitable for the ex vivo growth of gingival tissue cells and is a useful scaffold with possible applications in periodontal therapy.

Abstract 557: Single-chain type IV collagen (SCCOL-IV), an alternative gene product of COL4A1 as a therapeutic target in tumor tissue expansion as well as a diagnostic marker

Abstract Type IV collagen (COL-IV) is a major component of basement membrane (BM), comprising a polygonal meshwork structure underlying the basal side of polarized epithelia in normal tissues. Modulation of BM due to COL-IV degradation by matrix metalloproteinases is often associated with tumor aggravation and metastasis; Degradation products of BM components, including laminin and type IV collagen e.g., are involved in promoting tumor cell motility. Our previous studies showed that cultured human cells, in particular under ascorbate-deficiency, secrete single-chain COL-IV (SCCOL-IV), which is not derived from the denatured polypeptide of COL-IV triple-helical molecule. SCCOL-IV undergoes posttranslational modifications distinct from triple-helical COL-IV; SCCOL-IV has prolyl- and lysyl-hydroxylation to a lower extent than triple-helical COL-IV. SCCOL-IV contains the O-glycan structure of Galβ1-3GalNAc with a positive reaction to Agaricus bisporus agglutinin. The lectin does not recognize the triple-helical COL-IV-derived polypeptide. The alternative form of COL4A1 gene product is detected in vivo by the antibody that recognizes only SCCOL-IV. The polypeptide is isolated from human placenta as well as cultured media. In the present study, we demonstrate that SCCOL-IV is expressed specifically by cultured tumor cells and in tumor tissues. One of the antibodies against SCCOL-IV repressed tumor growth in an in vivo mouse model. The culture cell production of SCCOL-IV was demonstrated by Western blotting of cultured media, with an anti-SCCOL IV antibody, JK132 (Connective Tissue 31:161-168, 1999) for human cancer cell lines; Lu65A and NCI-H460 (pulmonary cancer), HLF (hepatoma), UO31 and A498 (renal cancer), and Panc-1 (pancreatic cancer). SCCOL-IV expression level was as high in tumor tissues of Lu65A-bearing nude mice as in tumor lesions of pulmonary cancer patients. One of the monoclonal antibodies against purified SCCOL-IV, NK46141, which exhibits a higher affinity compared with JK132, has no cross-reactivity with triple-helical COL-IV. Prophylactic interventions with NK46141 suppressed tumor growth in Lu65A-bearing nude mice. NK46141 had no effect, however, on the proliferation of cultured Lu65A cells. In order to obtain a clue for a mechanism how the antibody suppresses in vivo tumor growth, biological properties of SCCOL-IV in tumor interstitium were examined. SCCOL-IV was localized predominantly in vessel lumens in an in vitro vasculogenesis model, suggesting that SCCOL-IV may play a critical role for vasculogenesis in tumor progression. Prevention tumor growth by the antibody might be due to vasculogenesis inhibition. We propose that SCCOL-IV is a useful target in tumor diagnosis and therapy. Anti-SCCOL-IV antibodies including NK46141 could be potential therapeutics for tumor progression involving vasculogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 557. doi:10.1158/1538-7445.AM2011-557

Update on antiglomerular basement membrane disease

Antiglomerular basement membrane (GBM) disease is a rare form of autoimmune glomerulonephritis often accompanied by lung haemorrhage and characterized by circulating and deposited antibodies that bind basement membrane type IV collagen antigens in the glomerulus and lung alveolus. We review recent findings regarding disease pathogenesis and therapy. The target autoantigens are the noncollagenous (NC1) regions of the α3 and to a lesser extent the α5-chains of type IV collagen, which are exposed following disruption of the α3-α4-α5 collagen heterotrimer allowing autoantibody binding. In addition, antigen-specific T cells are found in the circulation of acute patients at higher frequencies than in healthy controls. These are prevented from inducing damage in healthy individuals or during disease remission, by α3(IV)NC1-specific Treg and possibly through destructive antigen processing of autoreactive peptides. Drugs inducing lymphocyte depletion, such as alemtuzumab, may disrupt these natural lymphocyte regulatory processes and promote disease. With regards to therapy, few advances have been made, with the exception of isolated case reports of the use of rituximab and mycophenolate mofetil in resistant disease. Immunity towards the α3(IV) NC1 is effectively regulated in health but conformational changes in the antigen, alterations in its processing, modifications of B cells and Tregs, following certain environmental events, in susceptible individuals, promote disease induction.

Prognostic Role of MMPs in Colorectal Cancer

See Article on Page 133-139 While most matrix metalloproteinases (MMPs) are produced in stromal cells, MMP-7 is produced in cancer cells and is known to affect the invasion and the metastasis of cancer cells by destroying the basement membrane [1]. MMP-7 is expressed mainly in the epithelial cells of the large intestine [2], and the over-expression of MMP-7 is known to influence early carcinogenesis of colorectal cancer from a normal colorectal mucosa to an adenoma [3]. Due to this mechanism, MMP-7 was reported to be an independent prognostic factor upon which to base a prognosis for colorectal cancer patients [4]. In this study, no correlation between the expression of MMP-7 and the prognosis for colorectal cancer patients was found. However, by using immunohistochemistry, this study confirmed a finding of a previous study by showing that MMP-7 was expressed mainly in cancer cells rather than in interstitial cells. MMP-2 is one of the zinc-dependant matrix metalloproteinases working as main extracellular matrix remodelling enzymes, and it is called gelatinase. MMP-2 is regulated positively or negatively at the gene transcription level by various oncogenes, cytokines or growth factors [5]. The up-regulation of MMP-2 provokes the loss of basement membrane type IV collagen to destroy the extracellular matrix and promote the progression and the local invasion by a tumor [6]. As mentioned in many reports, over-expression of MMP-2 is expected for a higher-stage tumor [7]. However, unlike MMP-7, the down-regulation of MMP-2 was also reported at a higher stage [8], so the role of MMP-2 in colorectal cancer has not been clearly determined. Considering that tissue inhibitor of metalloproteinase (TIMP)-1, (or an inhibitor of MMP-2) is significantly up-regulated at higher stages, such as stage III or IV, than stage I or II [9], the action of MMP-2 is thought to be determined by the interaction with TIMP1. According to the results of this study, no correlations of the expression of MMP-2 with factors related with the tumor's stage, differentiation, lymphovascular invasion, distant metastasis and recurrence were found, so drawing a conclusion in this study about the prognostic role of MMPs was difficult. This study aimed to investigate the expressions of MMP-2 and -7 in patients with colorectal cancer and to determine their meaning as prognostic factors by examining the characteristics of the expressions, the correlations with other pathologic findings and the correlation with prognosis, but it did not fulfill that aim. However, the study did include an in-depth morphologic study on the ways in which MMP-2 and MMP-7 are expressed in colorectal cancer tissues, so it should help in understanding the mechanisms for the expressions of the two MMPs. Future studies to investigate whether the expression of MMP-7 actually has a negative influence on the prognosis for colorectal cancer and how it is related with other substances such as β catenin, survivin or PRL-3 will be meaningful. For MMP-2, continuous studies to examine its correlation with its inhibitor TIMP-1, as mentioned above, at a molecular biological level are recommended.