Serum endostatin levels are elevated in colorectal cancer and correlate with invasion and systemic inflammatory markers.

BACKGROUND: Endostatin, a fragment of collagen XVIII, is an endogenous angiogenesis inhibitor with anti-tumour functions. However, elevated circulating endostatin concentrations have been found in several human cancers including colorectal cancer (CRC). METHODS: Serum endostatin levels were measured by enzyme-linked immunoassay from a series of 143 patients with CRC and from 84 controls, and correlated with detailed clinicopathological features of CRC, serum leukocyte differential count and C-reactive protein (CRP) levels. RESULTS: Patients with CRC had higher serum endostatin levels than the controls (P=0.005), and high levels associated with age, tumour invasion through the muscularis propria and poor differentiation, but not with metastases. Endostatin levels showed a positive correlation with the markers of systemic inflammatory response and a negative correlation with the densities of tumour-infiltrating mast cells and dendritic cells. Collagen XVIII was expressed in tumour stroma most strikingly in blood vessels and capillaries, and in the muscle layer of the bowel wall. CONCLUSIONS: Elevated endostatin levels in CRC correlate with systemic inflammation and invasion through the muscularis propria. Increased endostatin level may be a result of invasion-related cleavage of collagen XVIII expressed in the bowel wall. The negative correlations between serum endostatin and intratumoural mast cells and immature dendritic cells may reflect angiogenesis inhibition by endostatin.

Tumor tissue inhibitor of metalloproteinases-1 (TIMP-1) in hormone-independent breast cancer might originate in stromal cells, and improves stratification of prognosis together with nodal status.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is shown to be a potential marker for poor prognosis in breast cancer, but the biology of TIMP-1 is only partially understood. In this study, TIMP-1 production was studied in a co-culture model of hormone-independent breast cancer cell lines and mesenchymal stem cells mimicking the stromal components of the tumor. In addition, the prognostic value of TIMP-1 was histologically evaluated in a clinical material of 168 patients with hormone-independent breast tumors. The hormone-independent breast cancer (BC) cell lines MDA-MB-231, M4A4 and NM2C5 did not produce TIMP-1 protein in measureable quantities. Six tested primary mesenchymal stem cell lines all produced TIMP-1. Co-culturing of mesenchymal stem cells and breast cancer cells resulted in positive immunocytochemical diffuse staining for TIMP-1 for both cell types. Culturing breast cancer cells with MSC-conditioned media resulted in a positive cytoplasmic immunoreactivity for TIMP-1, and TIMP-1 protein concentration in cell lysates increased 2.7-fold (range 1.1-4.7). The TIMP-1 mRNA levels remained unaffected in BC cells. This might suggest that breast cancer cells can take up TIMP-1 produced by stromal cells and are thus displaying cellular immunoreactivity. In addition, TIMP-1 was shown to improve stratification of prognosis in clinical material.

Toxic acute hepatitis and hepatic fibrosis after consumption of chaparral tablets.

In this report we describe a young, previously healthy woman who developed severe acute hepatitis after consumption of chaparral tablets, a commonly used herbal product. In this case, the elimination-rechallenge event and the exclusion of other possible aetiologic factors strongly supported true causality between the herbal product and the liver damage. Primary liver biopsy showed severe toxic hepatitis consistent with previous reports of chaparral-induced liver damage. Later, 6 months after the liver function tests had normalized, permanent hepatic fibrosis could still be seen.

Abnormal adherence junctions in the heart and reduced angiogenesis in transgenic mice overexpressing mutant type XIII collagen.

Type XIII collagen is a type II transmembrane protein found at sites of cell adhesion. Transgenic mouse lines were generated by microinjection of a DNA construct directing the synthesis of truncated alpha1(XIII) chains. Shortened alpha 1(XIII) chains were synthesized by fibroblasts from mutant mice, and the lack of intracellular accumulation in immunofluorescent staining of tissues suggested that the mutant molecules were expressed on the cell surface. Transgene expression led to fetal lethality in offspring from heterozygous mating with two distinct phenotypes. The early phenotype fetuses were aborted by day 10.5 of development due to a lack of fusion of the chorionic and allantoic membranes. The late phenotype fetuses were aborted by day 13.5 of development and displayed a weak heartbeat, defects of the adherence junctions in the heart with detachment of myofilaments and abnormal staining for the adherence junction component cadherin. Decreased microvessel formation was observed in certain regions of the fetus and the placenta. These results indicate that type XIII collagen has an important role in certain adhesive interactions that are necessary for normal development.

Prolyl 4-hydroxylase isoenzymes I and II have different expression patterns in several human tissues.

Prolyl 4-hydroxylase plays a central role in the synthesis of all collagens. We have previously reported that the recently identified Type II isoenzyme is its main form in chondrocytes and possibly in capillary endothelial cells, while Type I is the main form in many other cell types. We report here that the Type II isoenzyme is clearly the main form in capillary endothelial cells and also in cultured umbilical vein endothelial cells, whereas no Type I isoenzyme could be detected in these cells by immunostaining or Western blotting. The Type II isoenzyme was also the main form in cells of the developing glomeruli in the fetal kidney and tubular structures of collecting duct caliber in both fetal and adult kidney, in occasional sinusoidal structures and epithelia of the bile ducts in the liver, and in some cells of the decidual membrane that probably represented invasive cytotrophoblasts in the placenta. Osteoblasts in a fetal calvaria, i.e., a bone developing by intramembranous ossification, stained strongly for both types of isoenzyme. The Type I isoenzyme was the main form in undifferentiated interstitial mesenchymal cells of the developing kidney, for example, and in fibroblasts and fibroblastic cells in many tissues. Skeletal myocytes and smooth muscle cells appeared to have the Type I isoenzyme as their only prolyl 4-hydroxylase form. Hepatocytes expressed small amounts of the Type I enzyme and very little if any Type II, the Type I expression being increased in malignant hepatocytes and cultured hepatoblastoma cells. The data suggest that the Type I isoenzyme is expressed especially by cells of mesenchymal origin and in developing and malignant tissues, whereas the Type II isoenzyme is expressed, in addition to chondrocytes and osteoblasts, by more differentiated cells, such as endothelial cells and cells of epithelial structures. (J Histochem Cytochem 49:1143-1153, 2001)

Podocyte-specific expression of a novel trans-Golgi protein Vear in human kidney.

BACKGROUND: Vear is a recently identified Golgi apparatus-associated protein. It has been suggested to be involved in vesicular trafficking between the Golgi and the vacuolar/lysosomal system. Proteins similar to Vear have also been shown to interact with activated ARF proteins (ADP ribosylation factor), and they are probably involved in membrane trafficking from the trans-Golgi network (TGN). We have previously shown that Vear is widely distributed in human tissues, with an especially high level of mRNA in the kidney. This study further characterizes the distribution and subcellular localization of Vear in normal adult kidney and shows its association with glomerulogenesis in fetal kidney. METHODS: Immunofluorescence and immunoelectron microscopy were used to study the expression of Vear in fetal and adult kidney. The expression of Vear in isolated glomeruli was shown by immunoblotting. The distribution of its mRNA was analyzed by using in situ and Northern hybridization. RESULTS: In situ hybridization and immunofluorescence microscopy showed that in the kidney, Vear is present in glomerular structures. By fluorescence microscopy, the immunoreactivity for Vear was found only in podocytes, as judged by its distinct colocalization with podocalyxin and vimentin, well-established marker proteins of podocytes. Its specific expression in the glomeruli versus other compartments of the kidney was also verified by Western blotting. By using immunogold electron microscopy, Vear was seen in the Golgi apparatus, tubulovesicular structures, and membranes adjacent to the Golgi complex. In fetal kidney, expression of Vear coincided with the formation of segmental structures of the glomeruli. It was first seen close to the undifferentiated luminal cells at the vesicular stage and increasingly in the differentiating podocytes at the more advanced stages of glomerulogenesis. CONCLUSIONS: In the kidney, Vear shows a distinct, specific, and developmentally regulated expression in glomerular podocytes. This suggests that Vear has a specific function in podocytes. It could be associated with the known high secretory and synthetic activity of the podocytes, especially the production of the basement membrane components, which are critically involved in the glomerulogenesis and the maintenance of the glomerular function.

Distinct expression of type XIII collagen in neuronal structures and other tissues during mouse development.

Type XIII collagen is a type II transmembrane protein found in adhesive structures of mature tissues. We describe here its expression and spatio-temporal localization during mouse fetal development. Type XIII collagen mRNAs were expressed at a constant rate during development, with an increase of expression towards birth. Strong type XIII collagen expression was detected in the central and peripheral nervous systems of the developing mouse fetus in mid-gestation. Cultured primary neurons also expressed this collagen, and it was found to enhance neurite outgrowth. The results suggest that type XIII collagen is a new member among the proteins involved in nervous system development. Strong expression during early development was also detected in the heart, with localization to cell-cell contacts and accentuation in the intercalated discs perinatally. During late fetal development, type XIII collagen was observed in many tissues, including cartilage, bone, skeletal muscle, lung, intestine and skin. Clear developmental shifts in expression suggest a role in endochondral ossification of bone and the branching morphogenesis in the lung. Notable structures lacking type XIII collagen were the endothelia of most blood vessels and the endocardium. Its initially unique staining pattern began to concentrate in the same adhesive structures where it exists in adult tissues, and started to resemble that of the beta1 integrin subunit and vinculin during late intrauterine development and in the perinatal period.

Expression of MMP2, MMP9, MT1-MMP, TIMP1, and TIMP2 mRNA in valvular lesions of the heart.

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play an important role in several diseases. This study was undertaken to investigate the mRNA synthesis of MMP2, MMP9, membrane-type 1 (MT1)-MMP, and matrix metalloproteinase inhibitors TIMP1 and TIMP2 by in situ hybridization in a set of heart mitral and aortic valves operatively removed due to degenerative or inflammatory valvular diseases. The material consisted of 21 valves, eight with endocarditis and 13 with a degenerative valvular disease. The samples were studied by in situ hybridization with specific probes for MMP2, MMP9, MT1-MMP, TIMP1, and TIMP2. Synthesis of MMP2 mRNA was found in seven valves, five with endocarditis and two with degenerative valvular disease. Signals for MMP9 mRNA were found in two cases with endocarditis and five cases with degenerative valvular disease. No signal for MT1-MMP mRNA was found in the lesions. TIMP1 mRNA, on the other hand, was found in 17 cases, both endocarditis and degenerative valvular disease. TIMP2 mRNA was found in three cases of endocarditis. The signals for MMP2, MMP9, TIMP1, and TIMP2 mRNA were localized in endothelial cells and in fibroblast-like cells expressing alpha-smooth muscle actin, thus showing myofibroblast-type differentiation. The results show that matrix metalloproteinases MMP2 and MMP9, and matrix metalloproteinase inhibitors TIMP1 and TIMP2 mRNAs are synthesized in diseased valves and suggest that they may contribute to matrix remodelling in valvular disease.

Comparative analysis of the distribution of laminin chains in the basement membranes in some malignant epithelial tumors: the alpha1 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 alpha1, alpha3, alpha5, beta1, beta2, beta3, gamma1, gamma2, 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 alpha5, beta1, and gamma1 chains. Conversely, the Ln alpha1 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 alpha1 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 alpha1 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.

Localization of MT1-MMP, TIMP-1, TIMP-2, and TIMP-3 messenger RNA in normal, hyperplastic, and neoplastic endometrium. Enhanced expression by endometrial adenocarcinomas is associated with low differentiation.

We studied membrane-type matrix metalloproteinase-1 (MT1-MMP), tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2, and TIMP-3 messenger RNA (mRNA) using in situ hybridization to elucidate their temporal and spatial expression patterns in normal, hyperplastic, and neoplastic endometrium. All mRNAs studied were expressed weakly in proliferating endometrium but were induced strongly in late secretory endometrium except MT1-MMP. Endometrial hyperplasia samples did not show increased MT1-MMP or TIMP mRNA expression, indicating that the overall expression patterns in hyperplasia are comparable to those in proliferating endometrium under estrogen effect and that synthesis of extracellular matrix proteins, rather than degradation, predominates in this condition. Exceptionally, stromal cells in areas of desquamation were seen to express focally intense MT1-MMP mRNA in hyperplasia samples. All mRNAs investigated were expressed increasingly in endometrial adenocarcinomas, especially in less differentiated carcinomas. Furthermore, gelatin zymography revealed higher functional degradative activities in carcinoma tissues than in normal endometrium. Our results indicate that MT1-MMP expression, together with that of TIMPs, is involved most notably in normal endometrium under progesterone effect and, without being connected to cyclic hormonal levels, has an important role in the invasive growth of endometrial adenocarcinomas.

Differential expression of matrix metalloproteinase (MMP)-2, MMP-9, and membrane type 1-MMP in hepatocellular and pancreatic adenocarcinoma: implications for tumor progression and clinical prognosis.

In the present study, we used in situ hybridization to study 36 primary hepatocellular carcinomas (HCCs) and 35 pancreatic adenocarcinomas to analyze the expressions of membrane-type 1 matrix metalloproteinase (MT1-MMP), MMP-2, and MMP-9 mRNAs. In HCCs, MT1-MMP mRNA was mainly expressed by cancer cells and to a lesser extent by stromal cells. MMP-2 mRNA was expressed predominantly by cells of tumor stroma, whereas MMP-9 mRNA was seen mainly in neoplastic epithelial cells. In pancreatic adenocarcinomas, MT1-MMP and MMP-9 mRNA were seen at moderate levels both in cancer and in stromal cells, whereas MMP-2 mRNA was predominantly expressed by the tumor stroma. Antigens of MMP-2, MMP-9, and MT1-MMP immunolocalized to the neoplastic epithelium and to the stromal cells in both tumor types. In gelatin zymography, increased amounts of latent and active MMP-2 were found in tumor samples of HCC as compared with adjacent nontumorous liver tissue. On the other hand, the latent form of MMP-9 was found in almost equal amounts both in tumor and normal liver samples, but its active form was present only in HCC. Expression of MT1-MMP mRNA had a tendency to be associated with a lower degree of differentiation in HCC, but such association was not noticed in pancreatic tumors. Correlation to the clinical data showed that MT1-MMP expression had a strong statistical association with a poor outcome of patients (P < 0.01). A similar tendency was also observed in pancreatic adenocarcinomas, but the association did not reach statistical significance. MMP-2 and MMP-9 mRNA expression did not have significant correlation with prognosis. The results of this study support the previous suggestions of the importance of MT1-MMP for malignant growth and indicate that increased MT1-MMP mRNA expression by tumor cells in HCCs and pancreatic adenocarcinomas may have prognostic significance.

Expression of the laminin gamma2 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 gamma2 chain by immunohistochemistry and in situ hybridization. Expression of the laminin gamma2 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 gamma2 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 alpha(6)beta(4) 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 gamma2 chain around tumour clusters and suggests that a laminin gamma2 chain-containing substrate may be of significance for the spread and growth of malignant tumours.

The short and long forms of type XVIII collagen show clear tissue specificities in their expression and location in basement membrane zones in humans.

Two N-terminal ends of human type XVIII collagen chains have recently been identified. The two chains have different signal peptides and variant N-terminal noncollagenous NC1 domains of 493 (NC1-493) and 303 (NC1-303) amino acid residues, respectively, but share 301 residues of their NC1 domains as well as the collagenous and C-terminal noncollagenous portions of the molecule. Antibodies were produced against the NC1 region common to both human alpha1(XVIII) chain variants and against NC1 sequences specific to the long variant and were used in combination with in situ hybridization to localize this collagen in a number of human tissues. They were also used for Western blotting, which resulted in detection of overlapping high-molecular weight bands above the 200-kd standard in a kidney extract. Heparin lyase II and heparin lyase III digestions of kidney and placenta extracts indicated that at least in these tissues, type XVIII collagen contains heparin sulfate glycosaminoglycan side chains. Type XVIII collagen was found to be a ubiquitous basement membrane component, occurring prominently at vascular and epithelial basement membranes throughout the body. Comparison of the expression of the NC1-493 and NC1-303 variants revealed marked differences. The short variant was found in most conventional basement membranes, including blood vessels and the various epithelial structures, and around muscular structures. The long variant was expressed very strongly in liver, where it was virtually the only variant in the liver sinusoids, and it occurred only in minor amounts elsewhere. Thus, the 192 N-terminal residues specific to the long variant apparently confer some functional property needed above all in the liver sinusoids, but also at certain other locations.

The novel type II prolyl 4-hydroxylase is the main enzyme form in chondrocytes and capillary endothelial cells, whereas the type I enzyme predominates in most cells.

Procollagen-proline dioxygenase (EC 1.14.11.2), an alpha2beta2 tetramer in vertebrates, plays a central role in the synthesis of all collagens. Recently an isoform of the alpha subunit, the alpha(II) subunit, was characterized in man and mouse and found to form a tetramer with the same beta subunit as the previously known alpha(I) subunit. We report here that the (alpha(I))2beta2 type I tetramer is the main enzyme form in most cell types and tissues and that its contribution to total prolyl 4-hydroxylase activity in cultured cells increases in confluence. Surprisingly, however, the (alpha(II))2beta2 type II enzyme was found to represent at least about 70% of the total prolyl 4-hydroxylase activity in cultured mouse chondrocytes and about 80% in mouse cartilage, the corresponding percentage in mouse bone being about 45% and that in many other mouse tissues about 10% or less. Immunofluorescence studies on samples from a fetal human foot confirmed these data and additionally indicated that the type II enzyme represents the main or only enzyme form in capillary endothelial cells. Thus the type II prolyl 4-hydroxylase is likely to play a major role in the development of cartilages and cartilaginous bones and also of capillaries.

Production of membrane-type matrix metalloproteinase-1 (MT-MMP-1) in early human placenta. A possible role in placental implantation?

The extracellular matrix proteolytic machinery is known to play a major role in trophoblast invasion, a process that shares similar features with the pathology of tumor invasion. In this study we investigated the expression of the recently described membrane-type matrix metalloproteinase-1 (MT-MMP-1; MMP-14) in early human placenta and decidual membrane to determine whether it might play a role in invasion. With in situ hybridization, the cytotrophoblasts of trophoblastic columns and the infiltrating intermediate trophoblasts in the decidual membrane were found to be the main producers of MT-MMP-1 mRNA. Gene expression was also seen in the villous double-layered trophoblastic epithelium and in the decidual cells of the decidual membrane. In endothelial and fibroblastic cells, however, the hybridization signal was either very weak or nonexistent. Immunohistochemical analysis and immunoelectron microscopy correlated well with the in situ hybridization findings. The most significant exception to this consisted of pericytes of spiral arteries, which appeared to lack MT-MMP-1 mRNA but showed intensive intracytoplasmic staining for the antigen. Our results show that MT-MMP-1 mRNA production is highly characteristic of intermediate trophoblasts, and MT-MMP-1 may have general importance in the tissue organization of early human placenta. We propose that MT-MMP-1 could be one of the key enzymes in the process of trophoblast invasion, acting alone or as a cell-surface activator of other proteinases.

Location of type XV collagen in human tissues and its accumulation in the interstitial matrix of the fibrotic kidney.

An antipeptide antibody was produced against the carboxyl-terminal noncollagenous domain of human type XV collagen and used to localize this recently described collagen in a number of human tissues. The most conspicuous findings were powerful staining of most of the capillaries and staining of the basement membrane (BM) zones of muscle cells. Not all of the BM zones were positive, however, as shown by the lack of staining in the developing fetal alveoli and some of the tubules in developing kidney. Nor was type XV collagen staining restricted to the BM zones, as some could be observed in the fibrillar collagen matrix of the papillary dermis and placental villi, for example. Interestingly, differences in the expression of type XV collagen could be observed during kidney development, and staining of fetal lung tissue suggested that changes in its expression may also occur during the formation of vascular structures. Another intriguing finding was pronounced renal interstitial type XV collagen staining in patients with kidney fibrosis due to different pathological processes. This suggests that the accumulation of type XV collagen may accompany fibrotic processes. Full-length human type XV collagen chains with an apparent molecular mass of approximately 200 kd were produced in insect cells using a baculovirus expression system. The fact that these had a markedly higher molecular mass than the 100- to 110-kd type XV collagen chains found in homogenates of heart and kidney tissue suggests either proteolytic processing during the synthesis of type XV collagen or an inability to solubilize complete molecules from tissues.

Altered distribution and synthesis of laminin-5 (kalinin) in oral lichen planus, epithelial dysplasias and squamous cell carcinomas.

Laminin-5 is a glycoprotein which mediates epithelial cell adhesion to the basement membrane. This study describes the distribution and synthesis of laminin-5 in oral lichen planus, epithelial dysplasias, squamous cell carcinomas and a lymph node metastasis using immunohistochemistry and in situ hybridization. In normal oral mucosa and lichen planus, immunoreaction to the laminin-5 was seen as a thin continuous, delicate line in the basement membrane region, although slight irregularities in the thickness and intensity of the immunoreaction could be detected in some cases with lichen planus. In epithelial dysplasias, the laminin-5 staining was discontinuous and more diffuse compared to lichen planus and normal mucosa. The immunoreaction was generally extracellular, although in some cases with lichen planus and epithelial dysplasia there were a few basal epithelial cells showing cytoplasmic staining. The invasive carcinomas and the lymph node metastasis showed a striking, intense cytoplasmic, staining of the carcinoma cells along the invasive border of the neoplastic islands and in individual infiltrating carcinoma cells. Using in situ hybridization, the laminin-5 gamma 2 chain mRNA expression could not be detected in normal oral mucosa whereas, in non-dysplastic lichen planus and, more strongly, in dysplasias, there was a clear increase in the expression of laminin-5 mRNA in the basal epithelial cells. The most intensive signal was detected in the invasive front of the oral squamous cell carcinomas and the lymph node metastasis. We conclude that, in oral squamous cell carcinoma, there is altered synthesis and secretion of laminin-5 mRNA and protein. It is also evident that in dysplastic lesions of oral epithelium the synthesis and distribution of laminin-5 is abnormal.

Expression of messenger RNAs for metalloproteinases 2 and 9, type IV collagen, and laminin in nonneoplastic and neoplastic endometrium.

In this study, we analyzed the messenger RNA (mRNA) synthesis of matrix metalloproteinases (MMPs) 2 and 9, and their main substrates laminin and type IV collagen by in situ hybridization in nonneoplastic, hyperplastic, and neoplastic endometrial tissues. In nonneoplastic endometrium stromal synthesis of MMP2, laminin and type IV collagen messenger RNA (mRNA) could be detected throughout the cycle. Their synthesis was strongest in the late secretory and decidualized endometrium. In epithelial cells, only laminin mRNA synthesis was observed. MMP9 mRNA was expressed in only stromal and epithelial cells of the decidualized endometrium. In hyperplastic and neoplastic endometrium, the expression of MMP2, MMP9, laminin, and type IV collagen mRNA was variably present in stromal cells, whereas epithelial cells expressed only laminin mRNA, except for one case of a grade III carcinoma, which also showed signals for MMP2 and MMP9 mRNAs in the carcinoma cells. The presence of MMP2, laminin, and IV collagen mRNA in normal endometrium reflects their importance in the tissue response and matrix remodeling during the proliferative and secretory phases of the menstrual cycle. Their mRNA synthesis follows a similar pattern, suggesting that it is associated with hormonal changes during the menstrual cycle. According to the results, MMP9 does not participate in tissue remodeling during the secretory or proliferative phases, but like MMP2, it is found in the neoplastic endometrial stroma, suggesting that it contributes to the invasion of malignant endometrial cells.

Tenascin expression in lichen sclerosus.

In this study, the distribution of tenascin immunoreactivity in 44 cases of vulvar lichen sclerosus (LS) was investigated. To assess the epithelial basement membrane structure, immunostaining with an antibody to type IV collagen was performed. Ten selected cases were also analyzed by in situ hybridization with a tenascin RNA probe to study the cellular distribution of tenascin mRNA synthesis in LS. Strong tenascin immunoreactivity could be found in LS, especially in areas with subepithelial edema and marked inflammation. By in situ hybridization, signals for tenascin mRNA could be found in basal keratinocytes, dermal fibroblasts, and endothelial cells. Staining for type IV collagen often revealed attenuation and discontinuity in the basement membrane. The abnormal accumulation of tenascin in LS suggests that it may participate in the pathogenesis of this disease. As shown by in situ hybridization, the cell types responsible for tenascin synthesis are basal keratinocytes, dermal fibroblasts, and endothelial cells. Because tenascin, together with fibronectin, is able to upregulate the expression of 92 kDa collagenase and stromelysin in fibroblasts, the matrix destruction and basement membrane damage in LS may partly be a consequence of an abnormal accumulation and synthesis of tenascin. The upregulation of tenascin synthesis in dermal fibroblasts, endothelial cells, and keratinocytes in LS could be mediated by an abnormal expression of growth factors, most notably TGF-beta, which are able to stimulate tenascin synthesis in many non-neoplastic and neoplastic cell lines.

mRNA expressions of TIMP-1, -2, and -3 and 92-KD type IV collagenase in early human placenta and decidual membrane as studied by in situ hybridization.

Cytotrophoblasts of early placenta invade the decidual membrane, gestational endometrium, and spiral arteries during early pregnancy. Unlike tumor invasion, this physiological invasion is well controlled, although its molecular mechanisms are largely unknown. We have previously shown that cytotrophoblasts synthesize significant mRNAs for 72-KD Type IV collagenase, laminin, and Type IV collagen, proteins implicated in extracellular matrix turnover and migration. In this study we used in situ hybridization and immunohistochemistry to investigate the mRNA expression pattern of 92-KD Type IV collagenase and the matix metalloproteinase inhibitors TIMP-1, TIMP-2, and TIMP-3 in early human placenta and decidual membrane. mRNAs for 92-KD Type IV collagenase, TIMP-1, TIMP-2, and TIMP-3 were found in the cells of cytotrophoblastic columns, the endothelial and fibroblastic stromal cells of villi, and the large decidualized cells of decidual membrane. TIMP-1 expression was notably accentuated in the fibroblasts of fibrotic villi. In the decidual membrane, the signals for 92-KD Type IV collagenase and TIMP-1 mRNA were particularly strong around the glandular structures. The trophoblastic epithelium of villi and the epithelial cells of decidual glands showed a signal for 92-KD Type IV collagenase and TIMP-2, but not for TIMP-1 or TIMP-3. The coincidental expression of the proteolytic 92-KD Type IV collagenase and inhibitors TIMP-1, TIMP-2, and TIMP-3 generally in the same cells suggests that the activity of 92-KD Type IV collagenase, which is regulated by TIMPs, plays an important role in placental tissue organization and in the invasion of trophoblastic cells into the uterine wall.