Degradation of basement membrane collagens by metalloproteases released by human,murine and amphibian tumours

In this investigation it has been found that naturally-occurring (i.e. indigenous, not transplanted) tumours of diverse organs in a spectrum of vertebrates from frogs to man can secrete enzymes which degrade basement membrane collagens (types IV and V). The enzymes are inhibited by chelating agents (EDTA) but not by other protease antagonists and are, therefore, specific metalloproteases. Individual tumours do not necessarily secrete collagenases active against all collagen types (I, IV and V) and release of these different enzymes does not, therefore, appear to be coordinated. These biochemical findings support those reported for serially transplanted tumour cell lines and provide a plausible mechanism for the destruction of basement membranes and stromal collagen fibres observed morphologically in tumour spread.     

MT1-MMP correlates with MMP-2 activation potential seen after epithelial to mesenchymal transition in human breast carcinoma cells

We have previously reported that human breast carcinoma (HBC) cell lines expressing the mesenchymal intermediate filament protein vimentin (VIM+) are highly invasive in vitro, and highly metastatic in nude mice when compared to their VIM– counterparts. Since only VIM+ cell lines can be induced to activate matrix metalloproteinase-2 (MMP-2) upon stimulation with Concanavalin A (Con A), we have examined here membrane type 1 MMP (MT1-MMP), a cell surface activator of MMP-2. Northern analysis reveals baseline expression of MT1-MMP in five of the six VIM+ cell lines studied (MDA-MB-231, MDA-MB-435, BT-549, Hs578T, MCF-7ADR), each of which showed variable activation of exogenous MMP-2 after treatment with Con A. In contrast, the four VIM–, poorly invasive HBC cell lines studied (MCF-7, T47D, MDA-MB 468, ZR-75-1) lacked baseline MT1-MMP mRNA expression, and showed no induction of either MT1-MMP expression or MMP-2-activation with Con A. Such differential MT1-MMP expression was confirmed in vivo using in situ hybridization analysis of nude mouse tumor xenografts of representative cell lines. Western analysis of the MDA-MB-231 cells revealed baseline membrane expression of a 60 kDa species, which was strongly induced by Con A treatment along with a weaker band co-migrating with that from MT1-MMP-transfected COS-1 cells (63 kDa), presumably representing latent MT1-MMP. MT1-MMP immunofluorescence strongly decorated Con A-stimulated MDA-MB-231 cells in a manner consistent with membranous staining, but did not decorate the unstimulated MDA-MB-231 cells or MCF-7 cells under either condition. Collectively, the results suggest the constitutive production of active MT1-MMP which is unavailable for either MMP-2 activation or immuno-decoration until Con A treatment. Since VIM expression arises by virtue of the so-called epithelial to mesenchymal transition (EMT) in invasive embryonic epithelia, we propose that this represents a major metastasis mechanism in breast carcinomas. MT1-MMP on the surface of such ÔfibroblastoidÕ carcinoma cells may mediate a paracrine loop for the utilization of stromally produced MMP-2, and contribute to the poorer survival associated with VIM+ breast carcinomas.     

AGE‐modified basement membrane cooperates with Endo180 to promote epithelial cell invasiveness and decrease prostate cancer survival

Biomechanical strain imposed by age‐related thickening of the basal lamina and augmented tissue stiffness in the prostate gland coincides with increased cancer risk. Here we hypothesized that the structural alterations in the basal lamina associated with age can induce mechanotransduction pathways in prostate epithelial cells (PECs) to promote invasiveness and cancer progression. To demonstrate this, we developed a 3D model of PEC acini in which thickening and stiffening of basal lamina matrix was induced by advanced glycation end‐product (AGE)‐dependent non‐enzymatic crosslinking of its major components, collagen IV and laminin. We used this model to demonstrate that antibody targeted blockade of CTLD2, the second of eight C‐type lectin‐like domains in Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) that can recognize glycosylated collagens, reversed actinomyosin‐based contractility [myosin‐light chain‐2 (MLC2) phosphorylation], loss of cell polarity, loss of cell–cell junctions, luminal infiltration and basal invasion induced by AGE‐modified basal lamina matrix in PEC acini. Our in vitro results were concordant with luminal occlusion of acini in the prostate glands of adult Endo180^{ΔEx2–6/ΔEx2–6} mice, with constitutively exposed CTLD2 and decreased survival of men with early (non‐invasive) prostate cancer with high epithelial Endo180 expression and levels of AGE. These findings indicate that AGE‐dependent modification of the basal lamina induces invasive behaviour in non‐transformed PECs via a molecular mechanism linked to cancer progression. This study provides a rationale for targeting CTLD2 in Endo180 in prostate cancer and other pathologies in which increased basal lamina thickness and tissue stiffness are driving factors. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Loss of laminin-5 in the epithelium-stroma interface: an immunohistochemical marker of malignancy in epithelial lesions of the breast

AIMS: To demonstrate immunohistochemically the alpha3 and gamma2 chain of laminin-5 in benign epithelial and malignant lesions of the human breast. METHODS AND RESULTS: The alpha3 chain was identified by the monoclonal antibody BM165 and the gamma2 chain by GB3 in shock frozen samples using APAAP (alkaline phosphatase monoclonal anti-alkaline phosphatase) technique. The pre-existing breast epithelium, the 12 benign ductal and lobular proliferations and the three fibroadenomas showed a continuous immunostaining in the basement membrane region. In contrast to benign epithelial lesions, the 44 cases of invasive breast carcinoma showed a loss of the laminin-5 chains in more than 50% of the carcinoma stroma interface. Twenty-four out of the 44 invasive carcinomas revealed a complete loss of laminin-5 immunostaining. Focal defects of the laminin-5 immunostaining were also found in ductal carcinoma in situ in its pure form. CONCLUSIONS: As recently described, the malignant transformation of breast epithelium with expression of an invasive phenotype is associated with a decrease of hemidesmosomes. The reduced immunostaining of laminin-5 is in line with this finding because laminin-5 represents the major component of the anchoring filaments attaching hemidesmosomes to the basement membrane. We feel that immunohistochemical demonstration of laminin-5 may serve as a marker of benignity in epithelial breast lesions. While other carcinoma types exhibit an increased laminin-5 deposition, which has been suggested as an invasion promoting factor, the loss of laminin-5 in breast cancer supports the view that breast carcinomas do not utilize laminin-5 for invasion.     

Patterns of basement membrane deposition in benign and malignant breast tumours

We have examined epithelial basement membranes in tissue samples of seven normal breasts, 64 benign breast lesions and 63 malignant breast tumours by immunocytochemistry, using polyclonal antisera specific for type IV collagen. In normal breast tissue as well as in all benign tumours a continuous basement membrane was found at the epithelial stromal interface. In benign proliferative lesions, epitheliosis and papillomatosis could be more accurately distinguished with basement membrane staining. This approach also facilitated the differentiation between sclerosing adenosis and tubular carcinoma, since the tubules in sclerosing adenosis are surrounded by a continuous basement membrane whereas in tubular carcinoma basement membranes are almost entirely absent. In radial scar lesions the tubules were always surrounded by intact basement membranes, which underlines the fact that these are benign lesions. In breast carcinoma we could not detect a relationship between histological grade and the extent of basement membrane deposition. However, in different tumour types the basement membrane alterations varied. In infiltrating lobular carcinoma of the alveolar type, fragments of basement membrane were found, whereas in the classic and trabecular type, basement membranes were absent, suggesting that the alveolar type may be an intermediate phase in the progression of lobular carcinoma in situ to infiltrating lobular carcinoma. It is concluded that basement membrane immunocytochemistry, using antibodies to type IV collagen, is useful in the differentiation between benign and malignant breast lesions and in the classification of breast neoplasms.     

The human embryo produces basement membrane collagen (type IV collagen)-degrading protease activity

The biochemical basis and mechanism of embryo implementationis poorly understood. The human embryo has to penetrate theendometrial basement membrane and the thick decidual wall duringimplantation, a process which resembles the active spreadingof invasive tumour cells where the degradation of type IV collagen(basement membrane collagen) plays an important role. This studyreports that human embryos produce type IV collagen-degradingenzyme activity and the secretion of this enzyme increases withtime in culture. The type IV collagen-degrading enzyme activitymight facilitate the penetration of the embryo through the decidua,thus emphasizing an active role of the embryo in implantation.On the other hand, unfertilized oocytes secrete low, stableamounts of type IV collagen-degrading enzyme activity in vitro.It was also found that follicular granulosa cells secrete highamounts of type IV collagenolytic activity in culture. It hasbeen previously shown that there is a pre-ovulatory peak intype IV collagenolytic activity in follicular fluid, and itcan be assumed that the appearance of this enzyme in the follicularfluid is probably connected to follicular rupture and that itis produced by granulosa cells     

Isolation and characterization of the tubular basement membrane antigen associated with human tubulo-interstitial nephritis.

The target antigen, a 54-kD glycoprotein (gp54), reactive with sera from patients with anti-tubular basement membrane (anti-TBM) nephritis, was isolated from collagenase-digested (CD) bovine TBM. The purified gp54 was shown to be non-collagenous by amino acid analysis, and to be a unique basement membrane component by amino-terminal sequencing. The nephritogenicity of gp54 was demonstrated by immunizing strain XIII guineapigs with purified gp54, and producing anti-gp54 antibody and tubulo-interstitial nephritis. Anti-gp54 antibody, affinity-purified from sera of patients with anti-TBM nephritis, bound by immunoblotting to 54-kD and, to a lesser extent, 48-kD components of partially purified human CD-TBM. Indirect immunofluorescence showed that gp54 was present in the basement membrane of proximal tubules of the kidneys of normal human, cow, rabbit, guineapig and Brown-Norway rat but not in Lewis rat. Immunoelectron microscopy revealed localization of gp54 along the interstitial side of the TBM and its association with interstitial collagen fibres. These results indicate that gp54 is the nephritogenic antigen involved in tubulo-interstitial nephritis, and is unique in chemical characteristics and localization in the kidney.     

小儿肾小球薄基膜病的临床病理研究

目的 :探讨小儿肾小球薄基膜病 (TBMN)的临床病理特征。方法 :对 11例TBMN患儿进行了临床、病理及超微结构的系统观察 ,测量了肾小球基膜 (GBM)及致密层的厚度 ,对小儿薄基膜病的临床病理特征进行了分析。结果 :11例小儿TBMN临床主要表现为单纯血尿 ,无其它明显的阳性体征 ,光镜下肾小球无明显改变或轻微异常 ,未见蛋白管型 ,包曼囊内及肾小管腔内可见渗出的红细胞 ,电镜下GBM广泛变薄 ,平均厚度 <2 0 0nm ,致密层厚度 <10 0nm。结论 :小儿肾小球薄基膜病的诊断主要依靠电镜 ,同时必须强调与临床病史、生化检查及病理组织学、免疫组化紧密结合方可确诊。     

A study of glomerular basement membrane anionic sites and glomerular visceral epithelial cell coat in protein overload proteinuria in the rat

The presence and distribution of anionic sites in the glomerular basement membrane and visceral epithelial cell coat has been demonstrated. No definite decrease in intensity or periodicity of staining of basement membrane particulate sites was seen in protein overload proteinuric animals and only one staining technique employed for electron microscopy (alcian blue 8GX) demonstrated a focal decrease in visceral epithelial cell coat staining in severely damaged glomeruli. A decrease in overall glomerular staining was also demonstrated by quantitative analysis of colloidal iron staining by light microscopy. The findings differ from those described in puromycin aminonucleoside nephropathy and nephrotoxic nephritis. Staining was demonstrated also in other basement membranes, in Bowman's capsule and along interstitial collagen fibres.     

Differential distribution of basement membrane type IV collagen alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV) chains in colorectal epithelial tumors

In this study, we examined the relationship between the histopathological grade and immunohistochemical localization of six genetically distinct type IV collagen alpha chains, the major component of basement membrane (BM), in normal and neoplastic colorectal tissues. In the normal colorectal mucosa, alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were stained in all epithelial BM. However, alpha3/alpha4(IV) chains were restrictively immunostained in the BM of the apical surface epithelium. Similar immunostaining profiles for alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were observed in tubular adenomas with mild/moderate atypia. However, in intramucosal carcinomas, both alpha1/alpha2(IV) chains were linearly stained in the BM of cancer cell nests, while the assembly of alpha5/alpha6(IV) chains into the BM was inhibited in a discontinuous or negatively stained pattern. The normal colorectal mucosa forms a second network of BM composed of alpha5/alpha6(IV), partly alpha3/alpha4(IV) chains, in addition to the classic network of alpha1/alpha2(IV) chains. The differential immunohistochemical localization of the type IV collagen alpha5/alpha6 chains could be one diagnostic marker for the invasiveness of colorectal cancer.     

Loss of types XV and XIX collagen precedes basement membrane invasion in ductal carcinoma of the female breast

Ductal and lobular carcinomas comprise most malignancies of the female breast and the morbidity and mortality associated with breast cancer. During the progression from in situ to invasive stages, tumour cells penetrate the epithelial and vascular basement membranes (BM) to realize full metastatic potential. While the definition of these structures has primarily resulted from analysis of laminin and type IV collagen, characterization of newly discovered BM/BM zone (BMZ) proteins will further elucidate the interactions between tumour cells and the host stroma. We have studied the expression of two non-fibrillar BMZ collagens, the type XV proteoglycan and collagen XIX, in breast cancer where a linear, well-formed BM becomes fragmented and even lost in the progression of epithelial malignancy. In the normal breast, types XV and XIX were found in all BMZ: epithelial, muscle, neural, endothelial, and fat. In in situ lesions, these two collagens, and particularly type XV, were often absent from the BM/BMZ displaying a continuous or just focally disrupted type IV/laminin staining pattern. In contrast, infiltrating ductal carcinomas showed only rare traces of laminin and collagen IV reactivity adjacent to the glands and tumour nests, and similarly there was little if any evidence of types XV and XIX collagen. All four molecules were, however, detected in the interstitium associated with some of the invasive carcinomas. The data suggest that types XV and XIX collagen are lost early in the development of invasive tumours, prior to penetration and eventual dissolution of the epithelial BM. Disappearance of these proteins from the BM/BMZ may signal remodelling of the extracellular matrix to promote tumour cell infiltration.     

Objective measurement of basement membrane abnormalities in human neoplasms of colorectum and of breast

The basement membrane (lamina densa) was evaluated as normal, absent, multilayered, or otherwise abnormal at intercepts of an array of parallcl lines superimposed nearly perpendicularly on basement membrane regions. Six to eight consecutive photographs of the basement membrane region of three to four areas of three non-neoplastic lesions and eight neoplastic lesions of colorectum, and two non-neoplastic and six neoplastic lesions of the female breast were thus examined. Statistical analyses showed that there was no significant difference between three regions of three non-neoplastic lesions, nor between the three nonneoplastic lesions of colorectum. The analyses were by χ² tests performed comparing counts of absent basement membrane with those of all other types of basement membrane lumped together. Significant differences were observed between the sum of the non-neoplastic lesions and the individual neoplasms. Differences between regions of the same lesion were observed only in malignant neoplasms. In breast lesions similarly, where the observations were pooled into three groups, i.e. counts of normal, absent, or other type of basement membrane, no difference between regions of the same lesion were observed in a duct hyperplasia and a fibroadenoma, but such differences were seen amongst adenocarcinomas and other lesions. Comparing the sum of observations of the above two lesions with the other lesions individually, there was generally an increasingly high χ² value with increasing malignancy. Thus this approach to evaluation of neoplasms may provide an objective measure which correlates with prognosis. It is adaptable to routine use in hospitals with electron microscopy facilities.     

Specificity of basement membrane thickening in severe asthma

BACKGROUND: Reticular basement membrane (RBM) thickness is considered a hallmark for airway remodeling in airway diseases such as asthma. It is still unclear whether this measurement could be associated with disease severity or apply to chronic obstructive pulmonary disease (COPD). A wide range of results, at baseline or after therapeutic intervention, have been reported using different measurement methods. OBJECTIVE: To determine whether increased RBM thickness could be associated specifically with severe asthma and in COPD in large samples. METHODS: We blindly measured RBM thickness in endobronchial biopsies from 50 patients with severe asthma (mean age, 53 years; FEV(1) 66% predicted, inhaled steroids > or =1500 microg and 20 mg daily dose of oral corticosteroids, lifelong nonsmokers), 50 untreated patients with mild asthma (mean age, 33 years; FEV(1) 93%pred, lifelong nonsmokers), 50 patients with COPD (mean age, 57 years; FEV(1) 53%pred, all current smokers), and 18 control subjects using 2 different validated quantitative and computer-assisted methods (repeated multiple point-to-point vs area by length ratio). RESULTS: Reticular basement membrane thickness was higher in severe asthma compared with mild asthma and COPD (P = .0053). On the basis of receiver operating characteristic curves, RBM thickness was effective in differentiating severe asthma from other groups (sensitivity and specificity, 98% and 95%, respectively, above a threshold of 5 microm vs control, 70% and 75% at 7 microm vs mild, 83% and 68% at 6 microm vs COPD). CONCLUSION: Increased RBM thickness was specifically associated with severe asthma, whereas surprisingly, COPD and mild asthma had similar remodeling features. CLINICAL IMPLICATIONS: Reticular basement membrane thickness can be considered a hallmark of severe asthma.     

Urokinase-type plasminogen activation in three human breast cancer cell lines correlates with their in vitro invasiveness

In order to invade and spread cancer cells must degrade extracellular matrix proteins. This degradation is catalysed by the concerted action of several enzymes, including the serine protease plasmin. Several experimental studies have shown that inhibition of plasmin formation reduces cancer cell invasion and metastasis, indicating a critical role of this proteolytic pathway in these processes. In order to further study the role of plasmin in cancer progression, we have characterized urokinase-type plasminogen activator (uPA) mediated plasmin formation in three human breast cancer cell lines. Using monoclonal antibodies against uPA and its receptor uPAR, we have investigated the contribution of uPA and uPAR to invasive capacity in an in vitro invasion assay. MDA-MB-231 BAG cells were found to express high protein levels of uPA, uPAR and PAI-1. MDA-MB-435 BAG cells produced low amounts of uPA, PAW and moderate amounts of uPAR, whereas MCF-7 BAG cells showed low levels of uPA, uPAR and PAM protein. In a plasmin generation assay MDA-MB-231 BAG cells were highly active in mediating plasmin formation, which could be abolished by adding either an anticatalytic monoclonal antibody to uPA (clone 5) or an anti-uPAR monoclonal antibody (clone R3), which blocks binding of uPA to uPAR. The two other cell lines lacked the capacity to mediate plasmin formation. In the Matrigel invasion assay the cells showed activity in this order: MCF-7 BAG < MDA-MB-435 BAG < MDA-MB-231 BAG. Testing MDA-MB-231 BAG cells in the Matrigel invasion assay revealed that invasion could be inhibited in a dose-dependent manner either by the clone 5 uPA antibody or by the clone R3 uPAR antibody, suggesting that the cell surface uPA system is actively involved in this invasive process. It is concluded that these three cell lines constitute a valuable model system for in vitro studies of the role of cell surface uPA in cancer cell invasion and has application in the search for novel compounds which inhibit mechanisms involved in uPA-mediated plasmin generation on cancer cells.     

The thin glomerular basement membrane in children with haematuria

To determine the specificity and significance of widespread attenuation of the glomerular basement membrane on electron microscopy, 240 renal biopsies from 218 children were studied retrospectively. Twenty-three patients showed diffuse attenuation and three of them are cases of hereditary nephritis. The other 20 patients are characterized by persistent microscopic haematuria, absence of proteinuria, normal blood pressure and renal function, and minimal glomerular changes. In 10 of these 20 children, microscopic haematuria was also present in the family. We conclude that widespread attenuation of the glomerular basement membrane is a characteristic of benign familial and non-familial haematuria. The thin glomerular basement membrane may be responsible for the haematuria and may result from incomplete glomerular maturation.     

粘附分子JAM-A与人宫颈癌的侵袭性

目的观察粘附分子JAM-A在宫颈癌不同肌层侵袭时的表达情况,探讨其与癌细胞侵袭性的关系。方法 35例宫颈癌患者标本,其中癌细胞侵袭浅肌层15例,侵袭深肌层20例,采用免疫组化法观察JAM-A在宫颈癌浅、深两组侵袭深度以及在癌团癌细胞和孤立癌细胞时的表达情况。结果 JAM-A在宫颈癌癌细胞、血管管腔内皮细胞间连接、腺腔内皮细胞连接及血细胞表达,JAM-A在浅肌层癌细胞侵袭组的表达强于深肌层侵袭组(P0.001);JAM-A在癌团的癌细胞表达显著强于孤立癌细胞(P0.0001)。结论宫颈癌JAM-A的表达与癌细胞的侵袭程度呈负相关。     

Antibodies against C1q in anti-glomerular basement membrane nephritis.

The prevalence of antibodies against the collagen-like region of the subcomponent of the first component of complement, C1q, was investigated in 11 patients with anti-glomerular basement membrane (GBM) nephritis. Anti-C1q antibodies (anti-C1qAb) were detected in seven patients. IgG anti-C1qAb were found in four and IgA anti-C1qAb in five patients. During follow up of the patients a relationship was observed between the levels of IgG anti-C1qAb and the levels of anti-GBM antibodies (anti-GBMAb). Gelfiltration experiments indicated that both IgG anti-C1qAb as well as IgG anti-GBMAb were monomeric and that binding also occurred with the F(ab')2 fragments of the antibodies. Although anti-C1qAb and anti-GBMAb are both directed against a collagen-like structure, it was demonstrated by means of inhibition experiments that anti-C1qAb and anti-GBMAb are directed against different antigenic sites. Comparison of patients with anti-GBM nephritis with and without anti-C1qAb revealed that there were no differences in disease activity or disease severity. Therefore, the results of this study suggest that anti-C1qAb do not play a direct pathogenetic role in anti-GBM nephritis.     

Isolation and growth of human mammary epithelial cells

Summary Techniques for the isolation and culture of human mammary epithelial cells are described. The isolation procedure consists of dissection followed by partial enzymatic digestion with collagenase and hyal-uronidase and subsequent filtration to separate the epithelial clusters from the digested stromal elements. Culture procedures utilizing two different growth media are presented. A serum-free medium, MCDB170, permits long-term growth (45 to 60 population doublings) of a pure epithelial population; a less defined medium, MM, yields fewer population doublings but increased expression of some mammary-specific properties.     

A quantitative analysis of perineurial cell basement membrane collagen IV,laminin and fibronectin in diabetic and non-diabetic human sural nerve

The thickness of the perineurial cell basement membrane was examined in diabetic and non-diabetic human sural nerve. A significant increase in thickness was found in the diabetic group. The nature of this thickening was investigated using immunohistochemistry and image analysis in order to semi-quantify three of the major intrinsic components of the perineurial cell basement membrane: collagen IV, laminin and fibronectin. Amounts of all three components were shown to be increased in the diabetic group, but not significantly so. However, significant linear correlations between fascicle size and perineurial collagen IV, laminin and fibronectin were identified in both diabetic and non-diabetic nerve.     

Antimitochondrial autoantibodies in anti-glomerular basement membrane disease.

Anti-glomerular basement membrane (GBM) disease is characterized by the production of an autoantibody with very restricted specificity, with no evidence of polyclonal B cell activation. It was therefore surprising to find that in a solid-phase ELISA a proportion of anti-GBM sera showed significant binding to pyruvate dehydrogenase (PDH), a reactivity usually associated with the antimitochondrial autoantibodies (AMA) found in primary biliary cirrhosis (PBC). The specificity of this reactivity was confirmed by inhibition and competition experiments. The AMA found in anti-GBM sera were of much lower affinity than those found in PBC sera, and recognized a more restricted set of species (mainly the 55-kD and occasionally the 74-kD component of PDH). However, it was possible to block the binding in a Western blot of an anti-GBM serum to both the 55-kD and 74-kD species with F(ab')2 fragments prepared from a PBC serum. Although AMA have been found in diseases other than PBC, such diseases have usually been characterized by polyclonal B cell activation. The stimulus to the production of AMA in anti-GBM disease, and their significance in pathogenesis (if any), are unknown.