Role of extracellular matrix and its regulators in human airway smooth muscle biology

Altered extracellular matrix (ECM) deposition contributing to airway wall remodeling is an important feature of asthma and chronic obstructive pulmonary disease (COPD). The molecular mechanisms of this process are poorly understood. One of the key pathological features of these diseases is thickening of airway walls. This thickening is largely to the result of airway smooth muscle (ASM) cell hyperplasia and hypertrophy as well as increased deposition of ECM proteins such as collagens, elastin, laminin, and proteoglycans around the smooth muscle. Many growth factors and cytokines, including fibroblast growth factor (FGF)-1, FGF-2, and transforming growth factor (TGF)-α₁, that are released from the airway wall have the potential to contribute to airway remodeling, revealed by enhanced ASM proliferation and increased ECM protein deposition. TGF-α₁ and FGF-1 stimulate mRNA expression of collagen I and III in ASM cells, suggesting their role in the deposition of extracellular matrix proteins by ASM cells in the airways of patients with chronic lung diseases. Focus is now on the bidirectional relationship between ASM cells and the ECM. In addition to increased synthesis of ECM proteins, ASM cells can be involved in downregulation of matrix metalloproteinases (MMPs) and upregulation of tissue inhibitors of metalloproteinases (TIMPs), thus eventually contributing to the alteration in ECM. In turn, ECM proteins promote the survival, proliferation, cytokine synthesis, migration, and contraction of human airway smooth muscle cells. Thus, the intertwined relationship of ASM and ECM and their response to stimuli such as chronic inflammation in diseases such as asthma and COPD contribute to the remodeling seen in airways of patients with these diseases.     

Reelin: A novel extracellular matrix protein involved in brain lamination

Normal development of the nervous system is achieved through an elaborate program of guided neuronal migration and axonal growth. In the last few years, a flood of research has dissected the molecular bases of these phenomena, and several cell-surface and extracellular matrix molecules, which are implicated in neuronal and axonal targeting processes, have been recognized. Taking this knowledge a step further, a recent paper by Tom Curran's group⁽¹⁾ reports the molecular cloning of the gene deleted in the autosomal recessive mouse mutation reeler, affecting cortical neuronal migration. This gene encodes reelin, a novel extracellular matrix protein.     

Glucosepane is a major protein cross-link of the senescent human extracellular matrix. Relationship with diabetes

The extracellular matrix in most tissues is characterized by progressive age-related stiffening and loss of proteolytic digestibility that are accelerated in diabetes and can be duplicated by the nonenzymatic reaction of reducing sugars and extracellular matrix proteins. However, most cross-links of the Maillard reaction described so far are present in quantities too low to account for these changes. Here we have determined in human skin and glomerular basement membrane (GBM) collagen the levels of the recently discovered lysine-arginine cross-links derived from glucose, methylglyoxal, glyoxal, and 3-deoxyglucosone, i.e. glucosepane, MODIC, GODIC, and DOGDIC, respectively. Insoluble preparations of skin collagen (n = 110) and glomerular basement membrane (GBM, n = 28) were enzymatically digested, and levels were measured by isotope dilution technique using liquid chromatography/mass spectrometry. In skin, all cross-links increased with age (p < 0.0001) except DOGDIC (p = 0.34). In nondiabetic controls, levels at 90 years were 2000, 30, and 15 pmol/mg for glucosepane, MODIC, and GODIC, respectively. Diabetes, but not renal failure, increased glucosepane to 5000 pmol/mg (p < 0.0001), and for all others, increased it to <60 pmol/mg (p < 0.01). In GBMs, glucosepane reached up to 500 pmol/mg of collagen and was increased in diabetes (p < 0.0001) but not old age. In conclusion, glucosepane is the single major cross-link of the senescent extracellular matrix discovered so far, accounting for up to >120 mole% of triple helical collagen modification in diabetes. Its presence in high quantities may contribute to a number of structural and cell matrix dysfunctions observed in aging and diabetes.     

Ovarian surface epithelium: autonomous production of connective tissue-type extracellular matrix.

The ovarian surface epithelium (OSE) is known to contribute to postovulatory repair of the ovarian cortex by proliferation and migration over the site of follicular rupture, and by deposition of a basement membrane. We examined the production of other extracellular matrix components in culture by OSE cells of the rat (ROSE), using immunofluorescence microscopy, electron microscopy, and proline incorporation. We compared recently explanted cells in low passage, the immortal line ROSE 239, whose growth pattern resembles low passage cultures, and the immortal line ROSE 199, which forms ridges and papillae. The epithelial nature of all three cell types was confirmed by the presence of keratin and laminin. All three cell types secreted collagen types I and III and at least one (ROSE 199) produced highly polymerized banded fibrils, which are characteristic for stromal or interstitial extracellular matrix. Simultaneous production of collagen types I and III, keratin, and laminin by cloned subpopulations ruled out an origin of the lines in mixed epithelial/fibroblast populations. The results demonstrate that OSE has the capacity to synthesize major components of connective tissue stroma. They suggest that this epithelium, in addition to its postulated proteolytic role, may also express synthetic activity in the remodelling of the ovarian cortical stroma. A capacity of OSE cells to produce stromal components autonomously might be an important factor in the formation of ovarian surface papillae and in neoplastic progression of OSE-derived ovarian carcinomas.     

Tensin is potentially involved in extracellular matrix production in mesangial cells

Tensin, a focal adhesion protein, is expressed in renal tubular epithelial cells (TECs). Tensin-null mice develop multiple large cysts in the renal proximal tubules. However, the role of tensin in human glomeruli remains unclear. In this study, we assessed tensin localization in human kidney and interaction between tensin and other adhesion components. In human mesangial cells (MCs) and TECs, we confirmed mRNA and protein expressions of tensin by RT-PCR and immunoprecipitation. In normal kidney, immunohistochemistry revealed that tensin was localized in MCs and parietal epithelial cells as well as TECs. In biopsy specimens, the expression of tensin was significantly increased in areas of mesangial expansion in patients with IgA nephropathy and diabetic nephropathy. These results suggest that the expression of tensin is associated with extracellular matrix (ECM) production. In vitro, immunocytochemistry revealed that MCs express tensin mainly at the ends of actin stress fibers and apparently in the focal adhesion areas. Integrin 5, but not 1 and 3, colocalized with tensin. Vinculin and focal adhesion kinase (FAK) were coprecipitated by tensin, suggesting that tensin can mediate signal transduction between cell and ECM through these molecules. Tensin may play important roles in mesangial ECM production through an adhesion complex with integrin 5, FAK, and vinculin.     

Cell adhesion profiling using extracellular matrix protein microarrays

We have developed a microarray-based system for cell adhesion profiling of large panels of cell-adhesive proteins to increase the throughput of in vitro cell adhesion assays, which are currently primarily performed in multiwell plates. Miniaturizing cell adhesion assays to an array format required the development of protocols for the reproducible microspotting of extracellular matrix (ECM) protein solutions and for the handling of cell suspensions during the assay. We generated ECM protein microarrays with high reproducibility in microspot protein content using nitrocellulose-coated glass microslides, combined with piezoelectric microspotting of protein solutions. Protocols were developed that allowed us to use 5000 cells or fewer on an array of 4 x 4 mm consisting of 64 microspots. Using this microarray system, we identified differences of adhesive properties of three cell lines to 14 different ECM proteins. Furthermore, the sensitivity and accuracy of the assays were increased using microarrays with ranges of ECM protein amounts. This microarray system will be particularly useful for extensive comparative cell adhesion profiling studies when only low amounts of adhesive substrate and cells, such as stem cells or cells from biopsies, are available.     

Tenascin-W: An extracellular matrix protein associated with osteogenesis and cancer

Tenascin-W was the last member of a family of four large extracellular matrix glycoproteins to be discovered. The original member of the tenascin family, tenascin-C, has been widely studied due to its association with asthma, fibrosis, infection, inflammation and cancer. Recent studies report multiple common features between tenascin-W and tenascin-C in terms of structure, expression and function, especially in the context of tumorigenesis. Furthermore, specific functions for tenascin-W in osteogenesis have been revealed. This review presents an update on our current knowledge concerning tenascin-W and discusses potential medical applications of this cancer-enriched extracellular matrix protein.     

Stimulation of extracellular protein production in Bacillus brevis 47

Summary Bacillus brevis 47 was cultivated in 2-1 fermentors to study the effect of medium supplementation on extracellular protein production. Additional polypeptone, when supplied initially or at 12 h (late exponential phase), had little stimulatory effect on extracellular protein levels, which reached 6–7 g/l after 48h. A large increase in protein production was observed, however, when polypeptone was added at 21 h (stationary phase). This addition resulted in the accumulation in the medium of 14 g/l protein after 48 h, and a total of 16 g/l when cell-bound protein was included. In all cases, glucose was consumed only very slowly.     

Interaction between cartilage oligomeric matrix protein and extracellular matrix protein 1 mediates endochondral bone growth

In an effort to define the biological functions of COMP, a functional genetic screen was performed. This led to the identification of extracellular matrix protein 1 (ECM1) as a novel COMP-associated partner. COMP directly binds to ECM1 both in vitro and in vivo. The EGF domain of COMP and the C-terminus of ECM1 mediate the interaction between them. COMP and ECM1 colocalize in the growth plates in vivo. ECM1 inhibits chondrocyte hypertrophy, matrix mineralization, and endochondral bone formation, and COMP overcomes the inhibition by ECM1. In addition, COMP-mediated neutralization of ECM1 inhibition depends on their interaction, since COMP largely fails to overcome the ECM1 inhibition in the presence of the EGF domain of COMP, which disturbs the association of COMP and ECM1. These findings provide the first evidence linking the association of COMP and ECM1 and the biological significance underlying the interaction between them in regulating endochondral bone growth.     

Dynamics of extracellular matrix production and turnover in tissue engineered cardiovascular structures

Appropriate matrix formation, turnover and remodeling in tissue-engineered small diameter vascular conduits are crucial requirements for their long-term patency and function. This complex process requires the deposition and accumulation of extracellular matrix molecules as well as the remodeling of this extracellular matrix (ECM) by matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). In this study, we have investigated the dynamics of ECM production and the activity of MMPs and TIMPs in long-term tissue-engineered vascular conduits using quantitative ECM analysis, substrate gel electrophoresis, radiometric enzyme assays and Western blot analyses. Over a time period of 169 days in vivo, levels of elastin and proteoglycans/glycosaminoglycans in tissue-engineered constructs came to approximate those of their native tissue counter parts. The kinetics of collagen deposition and remodeling, however, apparently require a much longer time period. Through the use of substrate gel electrophoresis, proteolytic bands whose molecular weight was consistent with their identification as the active form of MMP-2 (approximately 64--66 kDa) were detected in all native and tissue-engineered samples. Additional proteolytic bands migrating at approximately 72 kDa representing the latent form of MMP-2 were detected in tissue-engineered samples at time points from 5 throughout 55 days. Radiometric assays of MMP-1 activity demonstrated no significant differences between the native and tissue-engineered samples. This study determines the dynamics of ECM production and turnover in a long-term tissue-engineered vascular tissue and highlights the importance of ECM remodeling in the development of successful tissue-engineered vascular structures.     

Adiponectin and leptin up-regulate extracellular matrix production by dermal fibroblasts

Adipocytes were recently shown to secrete adipocytokines, such as adiponectin and leptin, which may have an endocrine role. Subcutaneous adipose tissue lies just beneath the dermis, and dermal condition is correlated with body mass index (BMI). However, it is not clear whether adipocytokines released by adipocytes in subcutaneous adipose tissue influence the adjacent dermis. We found that human dermal fibroblasts express genes encoding receptors for adiponectin and leptin, and that those cytokines both significantly increase production of hyaluronic acid (HA), a major extracellular matrix component (ECM) of dermis, by dermal fibroblasts. This effect is accompanied with up-regulation of HA synthase 2 gene expression. Moreover, adiponectin significantly increases production of collagen, the most abundant component of ECM in dermis, by dermal fibroblasts. These results suggest that subcutaneous adipocytes influence dermal condition by up-regulating collagen and HA production by dermal fibroblasts via secretion of adiponectin and leptin.     

iPSC-derived fibroblasts demonstrate augmented production and assembly of extracellular matrix proteins

Reprogramming of somatic cells to induced pluripotent stem cells (iPSC) provides an important cell source to derive patient-specific cells for potential therapeutic applications. However, it is not yet clear whether reprogramming through pluripotency allows the production of differentiated cells with improved functional properties that may be beneficial in regenerative therapies. To address this, we compared the production and assembly of extracellular matrix (ECM) by iPSC-derived fibroblasts to that of the parental, dermal fibroblasts (BJ), from which these iPSC were initially reprogrammed, and to fibroblasts differentiated from human embryonic stem cells (hESC). iPSC- and hESC-derived fibroblasts demonstrated stable expression of surface markers characteristic of stromal fibroblasts during prolonged culture and showed an elevated growth potential when compared to the parental BJ fibroblasts. We found that in the presence of l-ascorbic acid-2-phosphate, iPSC- and hESC-derived fibroblasts increased their expression of collagen genes, secretion of soluble collagen, and extracellular deposition of type I collagen to a significantly greater degree than that seen in the parental BJ fibroblasts. Under culture conditions that enabled the self-assembly of a 3D stromal tissue, iPSC- and hESC-derived fibroblasts generated a well organized, ECM that was enriched in type III collagen. By characterizing the functional properties of iPSC-derived fibroblasts compared to their parental fibroblasts, we demonstrate that these cells represent a promising, alternative source of fibroblasts to advance future regenerative therapies.     

Fibrillin-1, a calcium binding protein of extracellular matrix

Fibrillin-1 is a large extracellular matrix glycoprotein which assembles to form 10-12 nm microfibrils in extracellular matrix. Mutations in the human fibrillin-1 gene (FBN-1) cause the connective tissue disease Marfan syndrome and related disorders, which are characterised by defects in the skeletal, cardiovascular and ocular systems of the body. Fibrillin-1 has a striking modular organisation which is dominated by multiple tandem repeats of the calcium binding epidermal growth factor-like (cbEGF) domain. This review focuses on recent studies which have investigated the structural and functional role of calcium binding to cbEGF domains in fibrillin-1 and 10-12 nm microfibrils.     

Endothelins stimulate the production of stromelysin-1 in cultured rat astrocytes

The effects of endothelins (ETs) on the production of stromelysins, a sub-family of matrix metalloproteinases, were examined in cultured astrocytes. The treatment of cultured rat astrocytes with ET-1 increased stromelysin-1 mRNA levels, while stromelysin-2 and -3 mRNAs were not affected. Immunocytochemical observations showed that cultured astrocytes produced stromelysin-1 protein. ET-1 and Ala^1,3,11,15-ET-1, an ET_B receptor selective agonist, stimulated the release of stromelysin-1 from cultured astrocytes. Accompanying the increase in protein release, the peptidase activity of stromelysin-1 in the medium was also increased by ET-1. The effects of ET-1 on astrocytic stromelysin-1 expression were inhibited by PD98059, staurosporine, and Ca²⁺ chelation, but not by SB203580 or pyrrolidine dithiocarbamate. These results show that activation of astrocytic ET receptors stimulates the production of stromelysin-1, suggesting a role for ETs in stromelysin production in brain pathologies.     

The role of extracellular matrix metalloproteinase inducer protein in prostate cancer progression

Extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) is a multifunctional membrane glycoprotein overexpressed in many solid tumors, and involved in tumor invasion and angiogenesis. We investigated EMMPRIN expression in human prostate cancer (CaP) tissues and cells, and evaluated whether EMMPRIN expression is related to tumor progression and matrix metalloproteinase (MMPs) expression in human CaP. An immunohistochemical study using tissue microarrays of 120 primary CaPs of different grades and 20 matched lymph node metastases from untreated patients was performed. The association of EMMPRIN expression with clinicopathological parameters was evaluated. Co-immunolocalization for EMMPRIN and MMP-1, MMP-2 or MMP-9 in primary tumors was examined using confocal microscopy. Flow cytometry and immunoblotting were used to examine EMMPRIN expression in 11 metastatic CaP cell lines. Heterogeneous expression of EMMPRIN was found in 78/120 (65%) CaPs, correlated significantly with progression parameters including pre-treatment PSA level (P < 0.05) and increased with progression of CaP (Gleason score, P < 0.05; pathological stage, P < 0.01; nodal involvement, P < 0.05 and surgical margin, P < 0.05). Heterogeneous cytoplasmic MMP-1, MMP-2 and MMP-9 associated with EMMPRIN immunolabeling was observed, particularly in tumors with Gleason scores >3 + 4. Metastatic CaP cell lines, except DuCaP, expressed abundant EMMPRIN protein, indicating highly ( approximately 45 to approximately 65 kDa) and less ( approximately 30 kDa) glycosylated forms, although with no relationship to cells being either androgen responsive or nonresponsive. Our results suggest that EMMPRIN may regulate MMPs and be involved in CaP progression, and as such, could provide a target for treating metastatic CaP disease.     

Immunohistochemical localization of a tenascin-like extracellular matrix protein in sea urchin embryos

Summary We have used polyclonal antisera raised against vertebrate tenascin to identify and localize tenascin-like proteins in the developing sea urchin. These antisera recognize high-molecular weight proteins on immunoblots of sea urchin embryo homogenates that are similar in size and appearance to tenascin from vertebrates. These proteins appear as a doublet with an apparent molecular weight of 150 kDa and a larger, broad band with an apparent molecular weight of 350 kDa. Whole mounts of sea urchin embryos and larvae were stained with one of these antisera. The anti-tenascin stained the surface of primary mesenchyme cells during their phase of active migration. This staining was sensitive to detergent, suggesting that the protein recognized by the antiserum was associated with the cell surface. During later stages of development, the bulk of the antitenascin staining was found dispersed throughout the blastocoel matrix, and was no longer sensitive to detergent. We conclude that sea urchins express tenascin-like proteins during early stages of development, and that these proteins may play a role associated with primary mesenchyme cell morphogenesis.     

The matrix corroded: podosomes and invadopodia in extracellular matrix degradation

Podosomes and invadopodia are unique actin-rich adhesions that establish close contact to the substratum but can also degrade components of the extracellular matrix. Accordingly, matrix degradation localized at podosomes or invadopodia is thought to contribute to cellular invasiveness in physiological and pathological situations. Cell types that form podosomes include monocytic, endothelial and smooth muscle cells, whereas invadopodia have been mostly observed in carcinoma cells. This review highlights important new developments in the field, discusses the common and divergent features of podosomes and invadopodia and summarizes current knowledge about matrix-degrading proteinases at these structures.