Marko Määttä

EXPRESSION OF THE LAMININ γ2 CHAIN IN PANCREATIC ADENOCARCINOMA

Forty‐two pancreatic adenocarcinomas were investigated immunohistochemically and by in situ hybridization for the expression of the laminin γ2 chain. In 41 cases, intracytoplasmic immunoreactivity for the γ2 chain was seen. Positive tumour cells were located especially at the epithelial–stromal interface of the tumour cell islands. In 22 cases, diffuse laminin γ2 chain immunoreactivity could also be seen in stroma and in seven cases, occasional positivity was detected in the neoplastic basement membranes. Signals for laminin γ2 chain mRNA in tumour cells displayed a distribution similar to that observed on immunohistochemistry. There were significantly more cases with less than 20 per cent of laminin γ2 chain‐positive tumour cells in tumours extending to peripancreatic tissues and/or tumours with regional or distant metastases (P=0·029). A corresponding statistical significance could also be noted in the mRNA level (P=0·025). The results show that pancreatic adenocarcinomas display a high activity of laminin γ2 chain synthesis. Tumours with a strong laminin γ2 chain synthesis show a lower invasive and metastatic potential than tumours with a weak or moderate laminin γ2 chain expression.

Comparative Analysis of the Distribution of Laminin Chains in the Basement Membranes in Some Malignant Epithelial Tumors: The α1 Chain of Laminin Shows a Selected Expression Pattern in Human Carcinomas

Laminins (Ln), together with Type IV collagen and nidogen-1, form the structural integrity of the basement membranes (BM). In this study we used immunohistochemistry to show the distribution of laminin chains α1, α3, α5, β1, β2, β3, γ1, γ2, as well as Type IV collagen, in various types of carcinomas and in normal tissues. Except for diffuse gastric carcinomas and infiltrative breast carcinomas, the malignant epithelial tumor clusters were surrounded by quite a continuous BM in most tumors. These BMs comprised most abundantly Ln α5, β1, and γ1 chains. Conversely, the Ln α1 chain, a component of laminins-1 and -3, showed the most restricted distribution in BMs of both normal tissues and malignancies, being moderately present in carcinomas of thyroid gland and ovary and in intraductal carcinomas of breast. In other types of carcinomas, immunoreactivity for Ln α1 chain was found more randomly and was practically negative in carcinomas of tongue, stomach, and colon. These findings were comparable to those observed by in situ hybridization, which showed that carcinomas of thyroid gland and intraductal carcinomas of breast constitutively expressed Ln α1 mRNA and that the epithelial tumor cells were the main producers of it. The results suggest that epithelial malignancies, except for infiltrative breast and diffuse gastric carcinomas, produce more notable amounts of BM macromolecules in their growth substratum than has previously been anticipated. Corroborating their widespread distribution in normal epithelial tissues, the chains of Lns-5 and -10 are the most abundant Ln molecules in the corresponding carcinomas.

Lack of type XVIII collagen results in anterior ocular defects

Mice lacking type XVIII collagen have defects in the posterior part of the eye, including delayed regression of the hyaloid vasculature and poor outgrowth of the retinal vessels. We report here that these mice also have a fragile iris and develop atrophy of the ciliary body. The irises of Col18a1−;/−; mice can be seen to adhere to the lens and cornea. After the pupils begin to function, the double layer of epithelial cells separates at the apical cell contacts, leading to defoliation of its posterior pigment epithelial cell layer, and extracellular material begins to accumulate in the basement membrane zones of the iris. In contrast to the iris epithelia, where no clear signs of cellular atrophy were detected, the lack of type XVIII collagen resulted in atrophy of the pigmented epithelial cells of the ciliary body, and there were also ultrastructural abnormalities in the basement membrane zones. These changes did not lead to chronically elevated intraocular pressures, however. Our results indicate that type XVIII collagen is needed for the integrity of the epithelial basement membranes of the iris and the ciliary body and that its gene should therefore be taken into account as a new potential cause of anterior segment disorders in the eye.

Collagenolytic proteinases in keratoconus.

PURPOSE To study the proteolytic phenomena contributing to the pathogenesis of keratoconus, corneal enzymes with potential to cleave fibrillar collagen were studied. METHODS Immunohistochemical labeling was undertaken of conventional and novel mammalian collagenases (MMP-1, -2, -8, -13, and -14) of the matrix metalloproteinase (MMP) family and other collagenolytic proteinases of the serine (human trypsin-2) and cysteine (cathepsin K) endoproteinase families. The results were analyzed using a semiquantitative scoring system. RESULTS Labeling of MMP-8 was moderate in healthy controls, but weak in keratoconus. Moderate MMP-2 and weak MMP-14 expressions were similar in controls and keratoconus. MMP-1 was slightly overexpressed in keratoconus. In contrast, MMP-13 was weak in controls compared to moderate in keratoconus and human trypsin-2 and cathepsin K were moderate in controls and strong in keratoconus. CONCLUSIONS The collagenolytic milieu of human cornea is more complex than expected. Mesenchymal isoform of MMP-8 (ie, collagenase-2) participates in normal tissue remodeling, which may be impaired in keratoconus. MMP-2 (gelatinase A with interstitial collagenase activity) and MMP-14 (membrane-type MMP type I with collagenolytic potential) seem to be constitutively expressed and probably play a role in normal corneal remodeling. The most prominent changes in keratoconic cornea were observed in collagenase MMP-13 (ie, collagenase-3), and particularly, in cathepsin K and human trypsin-2, which were strongly expressed in keratoconus suggesting a role in intra- and extracellular pathological collagen destruction, respectively. This may contribute to stromal thinning characteristic for keratoconus.

Expression of the laminin γ2 chain in different histological types of lung carcinoma. A study by immunohistochemistry and in situ hybridization

Sixty‐four malignant lung tumours and 12 of their regional lymph node metastases were analysed for expression of the laminin γ2 chain by immunohistochemistry and in situ hybridization. Expression of the laminin γ2 chain was strongest in squamous cell carcinomas, followed by adenocarcinomas and large cell carcinomas. Positive cells, except for large cell carcinomas, were located at the epithelial–stromal interface of tumour clusters. An important exception was small cell lung carcinoma, with only a low level of laminin γ2 chain expression. Apart from tumour type, this may reflect the relatively scanty fibrous stroma in these tumours and supports previous observations that small cell lung carcinoma cells, contrary to other types, lack surface expression of α6β4 integrin, the specific laminin‐5 binding receptor. In frozen sections, immunohistochemistry showed linear basement membranes around tumour clusters in squamous cell carcinomas and adenocarcinomas. This shows that carcinoma cells are capable of heavy deposition of the laminin γ2 chain around tumour clusters and suggests that a laminin γ2 chain‐containing substrate may be of significance for the spread and growth of malignant tumours. Copyright

Altered expression of type XIII collagen in keratoconus and scarred human cornea: Increased expression in scarred cornea is associated with myofibroblast transformation.

Purpose: Type XIII collagen (ColXIII) is a transmembrane protein thought to be involved in cell-cell and cell-matrix interactions. We report here on its presence in the normal human cornea and compare the results for keratoconus and scarred corneas. Methods: Immunohistochemistry and in situ hybridization were applied to human corneal samples obtained by penetrating keratoplasty. Results: In the normal human cornea, ColXIII was immunolocalized to the corneal epithelial cells, and to a lesser degree to the stromal keratocytes. The keratoconus cases showed otherwise similar results, but in areas containing Bowman membrane disruptions showed thinned epithelial cells reduced immunostaining for ColXIII, whereas occasionally pronounced immunoreactivity was seen in the stromal keratocytes. The corneal scar samples contained highly increased ColXIII immunostaining by stromal cells in the fibrotic foci, whereas the peripheral areas showed less intense immunostaining. In situ hybridization confirmed that the corneal epithelium and keratocytes actively synthesize the transcript. Immunostaining with &agr;SMA revealed that a substantial proportion of the ColXIII mRNA-expressing cells in the stromal scar tissues was myofibroblasts and that these areas lack CD34 immunoreactivity. Conclusions: The results indicate that ColXIII, which is predominantly confined to the basal corneal cells in the normal cornea, may have a role in the adhesion of corneal epithelial cells to each other and to the underlying basement membrane. Additionally, highly increased expression in scarred corneas suggests that it participates in the corneal wound healing process.

Differential expression of basement membrane components in lymphatic tissues

Peripheral lymphoid tissues act as important organs of immunological defense. Characteristic of their architecture is the rich reticular fiber meshwork composed of various extracellular matrix (ECM) molecules with which the stationary non-lymphatic cells stay in intimate contact and form channels through which the lymphatic cells travel. Here we studied the distribution of various laminin (Ln) chains and different types of collagens in human spleen, lymph node, and tonsil to clarify their chain-specific distribution. The most widely distributed proteins in all these organs were Ln chains α5, β1, γ1 and collagen types IV and XVIII, which were present in practically all compartments. Conversely, Ln α1, α2, α4, and type VII collagen showed a more restricted expression pattern. A unique feature was that Ln α3-, β3-, and γ2-chains, which normally are not localized to the vascular wall in non-lymphatic tissues, were present also in capillary basement membranes (BMs) of the follicular structures of lymph node and tonsil and in Ln α1-chain and type VII collagen also in the splenic white pulp. We also found that collagen XVII was exclusively present in the ring fibers of the spleen. The results indicate that BMs of lymphatic tissues contain a variety of macromolecules that probably contribute strongly to immunological events. In addition, capillaries of the lymphoid tissue exhibit a specified BM composition resembling that in epithelial BMs of non-lymphoid tissues.

Differential expression of collagen types XVIII/endostatin and XV in normal, keratoconus, and scarred human corneas.

Purpose: This study was designed to clarify the expression of 2 closely related collagen (Col) types XVIII and XV, and the proteolytically derived endostatin fragment of ColXVIII in normal, keratoconus, and scarred human corneas. Methods: Immunohistochemistry, in situ hybridization, immunoelectron microscopy, and Western immunoblotting were used for human corneal samples obtained from penetrating keratoplasty. Results: In the normal cornea, ColXVIII was immunolocalized to the corneal and conjunctival epithelial basement membrane (EBM), Descemet s membrane, and the limbal and conjunctival capillaries. Immunoreaction for endostatin was otherwise similar, but it also was present in corneal epithelial cells. Western immunoblotting showed that normal cornea contains several endostatin fragments ranging from 20 to 100 kDa. ColXV was present in the EBM of the limbus and conjunctiva, but not in EBM of the clear cornea. In situ hybridization revealed that corneal basal epithelial cells were responsible for the synthesis of ColXVIII mRNA. Keratoconus cases were characterized by an irregular EBM immunoreactivity for ColXVIII and endostatin and patchy immunoreactivity beneath EBM. In scarred corneas, highly increased immunoreactivity for ColXVIII, endostatin, and ColXV was present within stroma. Conclusions: The results indicate that ColXVIII and ColXV are differentially expressed in normal human corneas. Constant expression of ColXVIII by corneal EBM suggests that it is an important structural molecule. Aberrant expression of ColXVIII, endostatin, and ColXV in keratoconus and scarred corneas emphasizes the active role these molecules in the wound healing process.

Collagen XVIII/endostatin shows a ubiquitous distribution in human ocular tissues and endostatin-containing fragments accumulate in ocular fluid samples.

BackgroundThe endostatin domain of type XVIII collagen (ColXVIII) inhibits neovascularization and regulates cell migration and matrix turnover. This study was designed to demonstrate the protein and gene expression patterns of ColXVIII/endostatin in the human eye and to ascertain whether endostatin is detectable in ocular fluid samples.MethodsTwenty human eyes enucleated on account of choroidal melanoma were used for immunohistochemical stainings with antibodies against ColXVIII and endostatin. In situ hybridization was used to localize cells responsible for the production of mRNA for ColXVIII. Tear fluid, aqueous humor, and vitreous gel samples were used for Western immunoblotting to detect endostatin fragments in these samples.ResultsColXVIII was immunolocalized to almost all ocular structures, namely the basement membranes (BMs) of the corneal and conjunctival epithelia, Descement’s membrane, the anterior border layer and posterior pigmented epithelium of the iris, the BMs of the pigmented and non-pigmented ciliary epithelia, the internal wall of Schlemm’s canal and trabeculae, the ciliary and iris muscle cells, the BMs of the pigment epithelium of the retina, and the internal limiting membrane. Universal expression was seen in the BMs of vascular endothelial cells, and in fibroblasts located in the conjunctiva, the iris, and the ciliary body. Endostatin showed a corresponding pattern, but additional immunostaining was present in the corneal and conjunctival epithelial cells. Most epithelial and mesenchymal cells expressed the mRNA for ColXVIII. Endostatin-containing fragments varying in size were detected in tear fluid, aqueous humor and vitreous gel samples.ConclusionsPractically all structures of the human eye contain ColXVIII/endostatin, emphasizing its possible important structural and functional role in the human eye. Furthermore, ocular fluid samples contain endostatin fragments, which may contribute to the antiangiogenic properties of the eye.

Differential Expression of Laminin Isoforms in Ovarian Epithelial Carcinomas Suggesting Different Origin and Providing Tools for Differential Diagnosis

Immunohistochemistry was used to study the distribution of laminin (Ln) chains, collagen types IV (α 1/2), VII, and XVIII and Lutheran antigen (Lu) in 36 frozen ovarian carcinoma samples. Surface epithelial basement membrane (BM) of the normal ovary showed immunoreactivity for Ln α1, α3-α5, β1-3, γ1, and γ2 chains and type IV and XVIII collagens. Chains of Ln-5 (α3β3γ2) and Ln-10 (α5β1γ1) as well as type IV and XVIII collagens were found in most tumor BMs, but Ln α2 chain and type VII collagen were detected only in few tumors. Contrary to serous tumors, BMs of mucinous carcinomas showed Ln α4 chain, but not Ln α1 and β2 chains. Ln α1 chain was found in most endometrioid carcinomas, whereas chains of Ln-5 were only moderately detectable in comparison with serous and mucinous carcinomas. In the normal ovary, Lu immunoreactivity was confined to basal aspect in the ovarian epithelial cells, but in tumor specimens Lu immunostainings showed variable polarized and nonpolarized patterns. The results suggest that the three types of ovarian carcinoma have distinct differences in their Ln distribution and can be grouped based on their expression pattern. This suggests that they may have histogenetically different precursors and may help to distinguish these tumors from each other.