Basement Membrane Constituents Research Articles

Basement membrane proteoglycans: from cellar to ceiling

The biology of basement membrane proteoglycans extends far beyond the original notion of anionic filters. These complex molecules have dual roles as structural constituents of basement membranes and functional regulators of several growth-factor signalling pathways. As such, they are involved in angiogenesis and, consequently, in tumour progression and their partial or total absence causes several congenital defects that affect the musculoskeletal, cardiovascular and nervous systems. New findings indicate a potential functional coupling between the intricate make-up of basement membrane proteoglycans and their ability to control important biological processes.

Expression pattern of matrilins and other extracellular matrix proteins characterize distinct stages of cell differentiation during antler development

Deer antler regeneration is a uniquely intense and complex process, which involves chondrogenic and intramembranous ossification. Cell differentiation in the developing antler of red deer, Cervus elaphus, was characterized with extracellular matrix markers. Expression of the four matrilin genes was monitored by immunohistochemistry and in situ hybridization and compared to cartilage markers collagen II and cartilage link protein, the bone component collagen I, and the endothelial basement membrane constituent laminin. The mesenchyme layer at the very tip of the velvet antler was enriched in link protein, indicative of the role of hyaluronan in apical morphogenesis. Matrilin-2, formerly described as a component of hard and soft connective tissue matrices, was identified here also as a marker of cells with high differentiation potential: it is expressed predominantly by mesenchyme cells, prechondrocytes and preosteoblasts. In addition to matrilin-3, documented as a component of the bony extracellular matrix, expression of the other three matrilin genes was observed in osteoprogenitor cells and osteoblasts. A layer of presumed osteoprogenitor cells, which surrounded the perivascular channels, expressed all four matrilins and collagen I. As a consequence, all four matrilins, including matrilin-1, previously detected in the skeleton only in cartilage, were found associated to collagen I-rich structures in a thin layer bordering the columns of hypertrophic chondrocytes. Cells with similar morphology and expression pattern were identified in the periosteum. Altogether all cell types of the chondrogenic and osteogenic lineage that expressed the four matrilins were in a separate study [Faucheux, C., Nicholls, B.M., Allen, S., Danks, J.A, Horton, M.A., Price, J.S., 2004. Recapitulation of the parathyroid hormone-related peptide-Indian hedgehog pathway in the regenerating deer antler. Dev. Dyn. 231, 88-97] positive for parathyroid hormone-related peptide and its receptor.

Immunological identification of types I and III collagen in bovine lens epithelium and its anterior lens capsule

To define the molecular structure of bovine lens epithelium and its anterior lens capsule, we investigated the composition of lens capsule basement membrane proteins. Immunofluorescence and immunogold techniques were used to demonstrate the presence of type I and type III collagen in the lens capsule and in primary explant epithelial cultures grown on protein-binding membranes. Immunofluorescence staining with specific antibodies indicated that type I and type III collagen were constituents of lens basement membrane. We observed that deposition of type III collagen was more than type I collagen. The synthesis of fibrillar collagen by lens epithelium and its deposition in the lens capsule was established by localization of fibrillar collagen by transmission immunoelectron microscopy. These results demonstrate for the first time that normal lens epithelium synthesize fibrillar collagen which is an intrinsic component of the anterior lens capsule basement membrane.

Laminin‐5 Expression in Melanocytic Lesions of the Skin

Background: Laminin‐5 is a basement membrane constituent that functions as an anchoring filament to integrin‐derived hemidesmosomes, and has been detected at the invasive front (tumor‐stromal interface) in many types of carcinomas. Our goal was to analyze laminin‐5 expression in melanocytic lesions and evaluate its role in tumor progression. Design: Immunohistochemical staining for laminin‐5 was performed on 101 cutaneous melanocytic lesions including 41 nevi, 31 primary melanomas, and 29 metastatic melanomas. A standard immunoperoxidase technique (ABC) was used with DAB chromagen and a primary monoclonal antibody to the laminin‐5 gamma2 chain (clone D4B5, 1:50, Chemicon International, Temecula, CA). Any degree of staining in melanoma cells or at the tumor‐stromal interface was considered positive. Results: Positive staining for laminin‐5 was observed in three of the primary melanomas (10.7%), three of the metastatic melanomas (10.3%), and none of the nevi. In primary melanomas, staining was observed predominately at the tumor‐stromal interface. In metastatic lesions, staining was observed around individual melanoma cells and melanoma cell nests.Conclusion: Expression of laminin‐5 was detected in a small percentage of primary and metastatic melanomas and in none of the nevi studied. Laminin‐5 expression does not appear to be essential for the development of melanomas or their metastases.

Localization of Type IV Collagen Alpha Chains in the Basement Membrane of Ameloblastoma, Tooth Germ and Oral Mucosa by Using Indirect Immunofluorescence.

Introduction Type IV collagen is a major structural component of basement membrane (BM) and acts as a scaffold for other BM constituents. It is a heterotrimeric molecule that exists in six genetically distinct forms, α1(IV) α6(IV). The ameloblastoma is the most frequently encountered odontogenic epithelial tumor. The BM zone of the ameloblastoma remains a subject of research interest primarily because of increasing evidence of its mediatory role during oncogenesis. However, the expression pattern of specific collagen α (IV) chains in the ameloblastoma BM has not been previously reported. In this preliminary study, indirect immunofluorescence was utilized to localize α1(IV) α6(IV) chains in the BM of two

A single nucleotide polymorphism in the matrix metalloproteinase-2 promoter is associated with colorectal cancer

Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity against matrix proteins, particularly basement membrane constituents. A single nucleotide polymorphism C → T transition at −1306, which disrupts an Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with T allele. Our study investigated whether the MMP-2 −1306 C → T polymorphism contributed to the development and progression of colorectal cancer in the Chinese population. One hundred twenty-six colorectal cancer patients and 126 age- and sex-matched controls were included in this study. PCR-based denaturing high performance liquid chromatography analysis and sequencing were used to determine the MMP-2 genotypes. MMP-2 expression of each genotype was analyzed in four colorectal cancer cell lines by semi-quantitative RT-PCR. The correlation between the genotypes and clinicopathological parameters among colorectal cancer cases was investigated. The results showed that the levels of MMP-2 mRNA expression in cell lines containing CC genotype were much higher compared with cell with CT genotype. The frequency of MMP-2 CC genotype was significantly higher in colorectal cancer patients when compared with controls (OR, 1.959; 95% CI, 1.055–3.637). Colorectal cancers with CC genotype were more common with serosa/adventitia layer involvement compared with CT + TT genotypes. Our data suggest that MMP-2 −1306 C → T polymorphism may be associated with colorectal cancer development and invasion in the Chinese population.

Structurally altered basement membranes and hydrocephalus in a type XVIII collagen deficient mouse line.

Type XVIII collagen/endostatin is known to be crucial for the eye, as witnessed by severe eye defects in Knobloch syndrome patients with mutations in this collagen and in Col18a1(-/-) mice. We show here that in a specific C57BL background, 20% of the Col18a1(-/-) mice developed hydrocephalus, and dilation of the brain ventricles was observed by MRI in all of the mutant mice. Significant broadening was observed in the epithelial basement membrane (BM) of the choroid plexuses (CP), its width being 86.4+/-10.52 nm, compared with 61.4+/-6.05 nm in wild-type mice. The CP epithelial cell morphology was balloon-shaped rather than cuboidal, and the microvilli of the apical surface of the CP epithelium contained more vacuoles in the null mice than in the wild-type, as also did the CP epithelial cells, which is suggestive of alterations in cerebrospinal fluid production. Analysis of BMs elsewhere in the body revealed a broadened epidermal BM in the Col18a1(-/-) mice, but this did not result in any apparent functional deficiencies. Moreover, markedly broadened BMs were found in the atrioventricular valves of the heart and in the kidney tubules, whereas the glomerular mesangial matrix of the kidneys was expanded in the mutant mice and serum creatinine levels were elevated, indicating alterations in kidney filtration capacity. We thus suggest that type XVIII collagen is a structurally important constituent of BMs, and that its absence can result in a variety of phenotypic alterations.

The role of matrix metalloproteinases -9 and -2 in development of neonatal chronic lung disease.

Matrix metalloproteinases (MMPs) -9 and -2 degrade type-IV collagen, a major constituent of lung basement membrane, and may have a role in the pathogenesis of neonatal chronic lung disease (CLD). We determined factors influencing MMP levels in neonatal bronchoalveolar lavage (BAL) fluid to establish whether an imbalance between MMP and its inhibitor could be implicated in CLD. We measured MMP-9 and -2 and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels in 316 BAL fluid samples from 121 babies of gestational ages 23 to 42 wk over the first 14 d of life to determine effects of gestation and postnatal age. Median MMP-9, -2, TIMP-1 and MMP-9/TIMP-1 ratio in BAL were further studied in a subgroup of 85 babies <33 wk gestation to determine their ability to predict CLD and to establish effects of antenatal corticosteroid therapy (ANCS). MMP-9, -2 and TIMP levels did not vary with postnatal age over the first week. Median MMP-9 levels and MMP-9/TIMP-1 ratio increased with decreasing gestation in preterm babies. The MMP-9/TIMP-1 ratio was higher in babies who developed CLD, implying a proteinase/antiproteinase imbalance, but this association disappeared when controlled for gestational age. ANCS had no effect on BAL fluid MMP or TIMP levels. MMPs may have a role in the development of lung injury and fibrosis, but estimating their levels in the first week of life does not help with prediction of CLD.

Kruppel-like factors regulate the Lama1 gene encoding the laminin alpha1 chain.

Laminin-1 (alpha1beta1gamma1), a basement membrane (BM) constituent, has been associated with differentiation processes and also with malignant progression. In the intestinal tissue, the alpha1 chain is expressed and secreted in the subepithelial BM during the developmental period; in the adult rodent tissue, it is restricted to the BM of the dividing cells. To understand how laminin alpha1 chain expression is regulated, we cloned and characterized a 2-kb promoter region of the Lama1 mouse gene. Analysis of the promoter was conducted in the Caco2-TC7 intestinal epithelial cells by transient transfection of serially deleted and site-directed mutated promoter constructs, by electrophoretic mobility shift assays, and expression of selected transcription factors. We determined that a proximal region, which includes an Sp1-binding GC box and a Krüppel-like element, was important for the promoter activity. This region is conserved between the human and mouse genes. Interestingly, two Krüppel-like factors KLF4 and KLF5 exhibit opposing effects on the Lama1 promoter activity that are decreased and increased, respectively, in the intestinal epithelial cells. These data corroborate the complementary expression of KLF4 and KLF5 along the intestinal crypt-villus axis and the parallel expression of KLF5 and laminin alpha1 chain in the crypt region. Finally, we showed that glucocorticoids stimulate the promoter activity. This study is the first characterization of the Lama1 promoter; we identified regulatory elements that may account for the expression pattern of the endogenous protein in the mouse intestine.

Chorioamnionitis and increased neonatal lung lavage fluid matrix metalloproteinase-9 levels: Implications for antenatal origins of chronic lung disease

Objective: Matrix metalloproteinase-9 (MMP-9) degrades type IV collagen, the major constituent of lung basement membrane. We studied the effects of chorioamnionitis and antenatal corticosteroids on bronchoalveolar lavage (BAL) fluid levels of MMP-9, and its inhibitor, TIMP-1 in preterm infants. Study Design: A prospective study was performed on serial BAL samples from 79 ventilated preterm infants at less than 33 weeks' gestation, 18 of whom were from pregnancies complicated by chorioamnionitis. MMP-9 levels were measured by gelatin zymography and TIMP-1 by enzyme-linked immunosorbent assay, and the median value for each infant was calculated. The presence and severity of chorioamnionitis were defined histologically. Results: BAL fluid MMP-9 levels were higher in preterm infants in the chorioamnionitis group (86 [29-518] vs 13 [3-43] ng/mL, P =.001), and levels increased stepwise with the increasing severity of chorioamnionitis. Antenatal corticosteroids had no effect on median MMP-9 levels. Infants in the chorioamnionitis group were more likely to have chronic lung disease (CLD) develop (55% vs 28%, P <.05). TIMP-1 levels were no different between groups. Conclusion: Chorioamnionitis is associated with increased lung type IV collagenase levels in the ventilated preterm infant. Antenatal lung inflammation with up-regulation of MMP-9 may be important in the pathogenesis of CLD. (Am J Obstet Gynecol 2003;188:871-5.)

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.

Determinants of Vascular Permeability in the Kidney Glomerulus

The human kidneys filter 70 liters of blood plasma every day. The hallmark of almost all kidney diseases, whether acquired or genetic, is the leakage of plasma proteins into the urine because of alterations in the glomerular filtration unit of the kidney. In this regard, the human mutations in nephrin, podocin, alpha-actinin-4, COL4A3, and COL4A5 genes expressed in the glomeruli have been implicated to cause alterations in glomerular filtration apparatus. Nevertheless, the expression of these proteins in relation to each other in mouse models for glomerular vascular leak is unknown. Additionally, within the glomerulus, the central question of whether the primary filtration barrier is the basement membrane or the epithelial slit diaphragm remains ambiguous. Therefore, in this study, we examined the localization and expression of glomerular epithelial slit diaphragm and glomerular basement membrane proteins implicated in glomerular vascular leak using mice deficient in either the alpha3 chain of type IV collagen, the major constituent of glomerular basement membrane, or LMX1B transcription factor, which regulates the expression of key glomerular type IV collagen genes COL4A3 and COL4A4 or nephrin, a glomerular epithelial slit diaphragm-associated protein. This study demonstrates that decreased expression of slit diaphragm protein, nephrin, correlates with a loss of glomerular filter integrity. Additionally, we demonstrate that defects induced by proteins of glomerular basement membrane lead to an insidious plasma protein leak, whereas the defects induced by proteins in the glomerular epithelial slit diaphragms lead to a precipitous plasma protein leak.

Impact of Hemodialysis Membrane and Permeability on Neutrophil Transmigration in vitro

Background/Aim: The impact of dialysis membrane permeability on neutrophil transmigration properties in vitro was examined in the present study. This issue has not been fully scrutinized before. Methods: We studied the capacity of neutrophils collected from a group of dialysis patients randomly treated with cuprophan, low- or high-flux polysulfone, to transmigrate in vitro through a membrane covered with fibronectin (a main constituent of the endothelial basement membrane). The hemodialysis-induced quantitative changes in expression of adhesion molecules were examined in parallel. Results: At the end of dialysis, neutrophils collected from patients treated with high-flux polysulfone dialyzers had a significantly higher transmigration index than neutrophils from patients treated with low-flux polysulfone membrane (p < 0.01) or cuprophan membrane (p < 0.01), and approached the level of transmigration observed in neutrophils collected from healthy controls. In the groups treated with low-flux polysulfone and cuprophan dialyzers, the transmigration capacity was significantly lower (p < 0.02) compared to neutrophils from healthy subjects. We also noted that differences between low- and high-flux polysulfone dialysis, in the context of transmigration properties, were not mirrored by changes in adhesion phenotype, which strengthens the view that there is no strict relationship between these two features. Conclusion: The study demonstrates that high-flux polysulfone dialysis, as opposed to low-flux polysulfone and cuprophan treatment, improves the transmigration properties of circulating neutrophils, despite similar effects on adhesion molecule phenotypes. A plausible mechanism is that potentially toxic middle range molecules that inhibit neutrophil migration are more efficiently eliminated during high-flux polysulfone dialysis, but this explanation requires further support.

Laminin alpha-chain expression and basement membrane formation by MLE-15 respiratory epithelial cells.

Basement membranes have a critical role in alveolar structure and function. Alveolar type II cells make basement membrane constituents, including laminin, but relatively little is known about the production of basement membrane proteins by murine alveolar type II cells and a convenient system is not available to study basement membrane production by murine alveolar type II cells. To facilitate study of basement membrane production, with particular focus on laminin chains, we examined transformed murine distal respiratory epithelial cells (MLE-15), which have many structural and biochemical features of alveolar type II cells. We found that MLE-15 cells produce laminin-alpha5, a trace amount of laminin-alpha3, laminins-beta1 and -gamma1, type IV collagen, and perlecan. Transforming growth factor-beta1 significantly induces expression of laminin-alpha1. When grown on a fibroblast-embedded collagen gel, MLE-15 cells assemble a basement membrane-like layer containing laminin-alpha5. These findings indicate that MLE-15 cells will be useful in modeling basement membrane production and assembly by alveolar type II cells.

Synthesis of heterotrimeric collagen peptides containing the alpha1beta1 integrin recognition site of collagen type IV.

Collagen type IV provides a biomechanically stable scaffold into which the other constituents of basement membranes are incorporated, but it also plays an important role in cell adhesion. This occurs with collagen type IV mainly via the alpha1beta1 integrin, and the proposed epitope involved in this type of collagen/integrin interaction corresponds to a non-sequential R/Xaa/D motif, where the arginine and aspartate residues are provided by the alpha2 and alpha1 chains of the collagen molecule, respectively. Since the stagger of the three alpha chains in native collagen type IV is still unknown and different alignments of the chains lead to different spatial epitopes, two heterotrimeric collagen peptides containing the natural 457-469 sequences of the cell adhesion site were synthesized in which the single chains were assembled via disulfide bonds into the two most plausible alpha1alpha2alpha1' and alpha2alpha1alpha1' registers. The differentiated triple-helical stabilities of the two heterotrimers suggest a significant structural role of the chain register in collagen, although the binding to alpha1beta1 integrin is apparently less affected as indicated by preliminary experiments.

Sphingosine-1-phosphate stimulates human Caco-2 intestinal epithelial proliferation via p38 activation and activates ERK by an independent mechanism.

Sphingosine-1-phosphate (S-1-P) has been identified as an extracellular mediator and an intracellular second messenger that may modulate cell motility, adhesion, proliferation, and differentiation and cancer cell invasion. Widely distributed, S-1-P is most abundant in the intestine. Although S-1-P is likely to modulate various intracellular pathways, activation of the mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase 1 (ERK1), ERK2, and p38 is among the best-characterized S-1-P effects. Because the MAPKs regulate proliferation, we hypothesized that S-1-P might stimulate intestinal epithelial cell proliferation by MAPK activation. Human Caco-2 intestinal epithelial cells were cultured on a fibronectin matrix because fibronectin is an important constituent of the gut mucosal basement membrane. We assessed ERK1, ERK2, and p38 activation by Western blotting with antibodies specific for their active forms and proliferation by Coulter counting at 24 h. Specific MAP kinase kinase (MEK) and p38 inhibitors PD98059 (20 microM) and SB202190 and SB203580 (10 and 20 microM) were used to probe the role of ERK and p38 in S-1-P-mediated proliferation. Three or more similar studies were pooled for the analysis. S-1-P stimulated Caco-2 proliferation and dose-responsively activated ERK1, ERK2, and p38. Proliferation peaked at 5 microM, yielding a cell number 166.3 +/- 2.7% of the vehicle control (n = 6, P < 0.05). S-1-P also maximally stimulated ERK1, ERK2, and p38 at 5 microM, to 164.4 +/- 19.9%, 232.2 +/- 38.5%, and 169.2 +/- 20.5% of the control, respectively. Although MEK inhibition prevented S-1-P activation of ERK1 and ERK2 and slightly but significantly inhibited basal Caco-2 proliferation, MEK inhibition did not block the S-1-P mitogenic effect. However, pretreatment with 10 microM SB202190 or SB203580 (putative p38 inhibitors) attenuated the stimulation of proliferation by S-1-P. Twenty micromolars of SB202190 or SB203580 completely blocked the mitogenic effect of S-1-P. Ten to twenty micromolars of SB202190 and SB203580 also dose-dependently ablated the effects of 5 microM S-1-P on heat shock protein 27 accumulation, a downstream consequence of p38 MAPK activation. Consistent with the reports in some other cell types, S-1-P appears to activate ERK1, ERK2, and p38 and to stimulate proliferation. However, in contrast to the mediation of the S-1-P effects in some other cell types, S-1-P appears to stimulate human intestinal epithelial proliferation by activating p38. ERK activation by S-1-P is not required for its mitogenic effect.

Heparanase expression in human leukemias is restricted to acute myeloid leukemias.

Matrix metalloproteinases and an endo-beta-D-glucuronidase (heparanase) are enzymes that degrade the protein and carbohydrate constituents of basement membranes, thereby facilitating transendothelial migration of blood-borne cells. Heparanase activity was found to correlate with the metastatic potential of solid tumors. We evaluated heparanase expression, at the levels of gene and protein expression and activity in a variety of leukemias, and compared it with normal hematopoietic cells. Heparanase expression was evaluated in leukocytes isolated from peripheral blood of 71 patients with myeloid and lymphoid leukemias, or non-Hodgkin's lymphoma. Analysis was performed at two levels: heparanase RNA was determined by reverse transcriptase polymerase chain reaction, and heparanase protein was evaluated by immunocytochemistry and flow cytometry. In eight peripheral blood samples from normal donors, heparanase RNA was detected, and protein was found within the cytoplasm of granulocytes. In mononuclear cells derived from various leukemias, heparanase RNA was expressed in 14 of 15 acute myeloid leukemia (AML) samples. In contrast, cells derived from all 33 chronic lymphoblastic leukemia, all 7 non-Hodgkin's lymphoma, 7 of 8 chronic myeloid leukemia, and 6 of 8 acute lymphoblastic leukemia patients showed no detectable expression of the heparanase RNA. Heparanase protein was detected primarily within the cytoplasm of AML cells, indicating that the enzyme is produced and stored within the cytoplasm of myeloid cells, with limited expression on the cell surface. We propose that heparanase expression is associated with the myeloid lineage and may serve as an independent marker to support the identification of AMLs.

Interaction of disease-related antigen-reactive T-cell lines from multiple sclerosis patients with type IV collagen: role of integrin VLA-1 and effects of irradiation.

Multiple sclerosis (MS), a chronic demyelinating disease, is thought to be initiated by pathogenic T cells that transmigrate the vascular endothelium and enter the brain through vascular and parenchymal basement membranes (BM). Vaccination with T-cell lines reactive with myelin basic protein (MBP) and myelin oligodendrocytic glycoprotein (MOG) epitopes, expanded with interleukin-2 (IL-2), and attenuated by ionizing radiation is currently being evaluated as a therapeutic modality for this disease. We examined mechanisms potentially involved in pathogenic cell migration into the central nervous system (CNS) and the influence of irradiation on these processes. Seven of 7 autoantigen-responsive T-cell lines from MS patients adhered to collagen IV, the major collagenous constituent of BMs. This adhesion was inhibited almost completely by monoclonal antibody (MAb) to very late antigen (VLA)-1 and partially by anti-VLA-2. T-cell lines from healthy donors adhered more variably to collagen IV. Furthermore, patient derived T cells actively transmigrated through a collagen IV gel toward medium containing TNF-a, in a process that was inhibited by MAbs to VLA-1. Ionizing radiation at the dose used in vaccine preparation, inhibited morphological polarization associated with migratory capability, induced integrin clustering on the cell membrane, and abrogated adhesion to collagen IV. These findings may have important implications for understanding the pathogenesis of MS and how irradiation of potentially pathogenic T cells produces a reagent with possible therapeutic effects in T-cell vaccination (TCV).

Tumor deposition of laminin-5 and the relationship with perineural invasion.

Perineural invasion (PNI) is increasingly being recognized as an important indicator of aggressiveness in head and neck squamous cell carcinoma. The mechanisms of PNI are poorly understood. Laminin-5, an important basement membrane constituent, has been shown to be essential in head and neck squamous cell carcinoma invasion and motility. We hypothesized that tumors exhibiting increased expression of laminin-5 are more likely to be neurotropic. Analysis of archived surgical specimens with and without PNI for presence and intensity of laminin-5 tumor staining. Immunohistochemistry of archived head and neck squamous cell carcinoma specimens with known PNI was performed with anti-laminin-5 antibodies and appropriate positive and negative control specimens. The staining patterns were characterized as follows: A, few to no tumor cells positive; B, some peripheral cells positive; C, all peripheral cells positive; and D, almost all tumor cells positive. Statistical analysis was by chi2 analysis. Forty-six PNI-positive and 18 PNI-negative specimens were analyzed. The staining distribution for the PNI-positive specimens was as follows: 2% for A, 41% for B, 46% for C, and 11% for D. For tumors without PNI, the distribution was 28% for A, 50% for B, 22% for C, and 0% for D (P = .005). In PNI-positive tumors, no significant difference in staining was seen between areas with and without PNI. We found a significant correlation between laminin-5 staining and the presence of PNI in head and neck squamous cell carcinoma. Expression of laminin-5 by tumors is, possibly, an important step in the process of PNI. These preliminary findings support the concept that deposition of basement membrane constituents are required in the multistep process of nerve invasion.

Synthesis and degradation of basement membranes and extracellular matrix and their regulation by TGF-beta in invasive carcinomas (Review).

The proper structure of the extracellular matrix, in particular of the basement membrane and the adjacent interstitial matrix, are essential prerequisites for a proper function of tissues. Invasive growth in malignant tumors is associated with a destruction of various matrix structures. Due to extensive recent analyses significant advances have been made in the knowledge of the structure of the extracellular matrix, the composition of its most important constituents, their metabolism and that of matrix degrading enzymes. This information provides insight into the pathophysiology of malignant growth. Thereby, it has been shown that malignant tumor growth is associated with a loss of basement membrane (BM) material which, however, disappears not homogeneously, but affects various BM components to different degree. The loss of an intact BM as the first barrier is therefore the initial step of tumor invasion. Despite this loss there is evidence that the de novo synthesis of BM constituents in tumor and adjacent stromal cells is enhanced. Thus, it is obvious that BM material is degraded during the invasion process to significant degree. In addition, since there is a positive correlation between the amount of retained peritumoral BM and a higher degree of tumor cell differentiation the amount of retained BM material seems to represent a marker for the biological behaviour of the tumor cells. The loss of BM material is well explained by a significant expression of major matrix degrading enzymes, the matrix metalloproteinases (MMPs) both on the mRNA and protein level. Here again, there is considerable data indicating that both tumor and stroma cells are involved in the MMP synthesis. In addition to the loss of BM substances, the interstitial extracellular matrix (ECM) is disarranged. This disarrangement may comprise enhanced de novo synthesis ("desmoplasia") or dissolution by distinct MMPs (collagenases, such as MMP-1) reflecting obviously different reaction statuses of the stromal cells. Finally, significant work has been done on the elucidation of the role of regulating cytokine systems. To this regard, particular attention has been paid to the TGF-beta system and it has been shown that the major three isoforms of TGF-betas are upregulated both in tumor and stroma cells. Since the TGF-beta-effect is mainly mediated by a particular signalling system via the TGF-beta-receptors (TBRs), the investigation of this system has provided considerable insight into the role of TBRs which are now known to represent the most potent tumor suppressor genes. Thus frequent mutations in the TBR-II gene, one of the three TBRs, in various carcinomas suggest that these molecular alterations are responsible for both the loss of the control of cellular proliferation (in tumor cells) and altered matrix metabolism (in tumor and stroma cells). The further analysis of this major cytokine system therefore will provide us with major insights into the molecular abnormalities of invasive tumor growth.