Basement membrane collagen: degradation by migrating endothelial cells.

One of the first steps in neovascularization is dissolution of the basement membrane at the point of endothelial outgrowth. An assay was developed to determine whether basement membrane collagens (types IV and V) are degraded by endothelial cells migrating toward a chemotactic stimulus. Fetal bovine endothelial cells were placed on one side of a filter containing the collagen substrate, and a chemoattractant derived from retinal extracts was placed on the opposite side. Degradation of both type IV and type V collagens was observed when the retinal factor was placed on the side of the filter opposite the endothelial cells. Metalloproteinases that cleaved type IV and type V collagens could be extracted from the endothelial cells with detergents. Such endothelial cell-associated (possibly membrane-bound) proteinases may locally disrupt the basement membrane and facilitate the outgrowth of capillary sprouts toward the angiogenic stimulus.

Pulmonary alveolar macrophages from patients with active sarcoidosis express type IV collagenolytic proteinase. An enzymatic mechanism for influx of mononuclear phagocytes at sites of disease activity.

Alveolar macrophages (AMs) recovered from the bronchoalveolar lavage (BAL) of 44 patients with sarcoidosis were evaluated for their ability to release type IV collagenolytic metalloproteinase (IV-Case). This enzyme, which is produced by peripheral blood monocytes (PBMs) but not by tissue macrophages, degrades type IV collagen, the major structural component of vessel wall basement membranes, and helps to promote the migration of PBMs from the blood compartment to peripheral tissues. Our results demonstrated that AMs from patients with active sarcoidosis released significantly increased levels of IV-Case with respect to patients with inactive disease and control subjects. After in vitro culture, sarcoid AMs secreted IV-Case during the first 24 h of collection; after that time, AMs progressively lost their ability to release IV-Case. Exposition of both sarcoid and normal AMs to recombinant IL 2 or gamma IFN did not influence their property to release IV-Case. The immunoblot analysis of IV-Case demonstrated complete identity between IV-Case released by AMs and the degradative enzyme obtained from PBMs. The increased property to release IV-Case was significantly related to the increase of the absolute number of AMs and, in particular, of AMs bearing two determinants that are usually expressed by most PBMs (CD11b and CD14). Selective depletion of CD11b+/CD14+ AMs from the entire macrophagic population was associated with the recovery of the IV-Case activity to normal values. A positive correlation was also found between the increase in the absolute number of lung T cells and the enhanced CD4/CD8 pulmonary ratio. A 6-mo follow-up study indicated a significant association between the positivity for the 67Gallium scan and the increased property of AMs to release IV-Case. Our data are consistent with the hypothesis that a IV-Case mediated influx of peripheral monocytes takes place in the lung of sarcoid patients. Furthermore, the correlation found between the IV-Case release and disease activity suggests that this assay could represent a useful tool in sarcoidosis disease staging.

Increased expression of the laminin receptor in human colon cancer.

It has been proposed that among the various cell-surface proteins capable of interacting with laminin, the 67-kd high-affinity laminin receptor plays a crucial role during tumor invasion and metastasis. In this study, the expression of laminin-receptor-precursor messenger RNA (mRNA) and 67-kd protein was analyzed in human colon adenocarcinoma. In 22 of 23 patients with colon cancer, we found a 2- to 23-fold increase in levels of laminin-receptor-precursor mRNA in the cancer tissues compared with those in matched normal adjacent colonic mucosa. In 10 of 11 cases studied, the level of 67-kd laminin receptor, detected by affinity-purified anti-laminin-receptor synthetic peptide antibodies on immunoblots of matched tumor and normal tissue extracts, was higher in the colon carcinoma tissue. Immunodetection of laminin receptor in tissue sections using anti-laminin-receptor-peptide antibodies confirmed that the increased expression of laminin receptor was specifically associated with the cancer cells. In a series of 72 paraffin sections of colon lesions, we observed a correlation between the expression of the laminin receptor and the Dukes' classification. Our observations indicate that increased expression of laminin-receptor-precursor mRNA is associated with enhanced levels of the 67-kd laminin receptor as well as with the invasive phenotype of colon carcinoma. Detection of this metastasis-associated gene product may be a valuable adjunct in the evaluation of human colon cancer.

Effect of plasminogen activator (urokinase), plasmin, and thrombin on glycoprotein and collagenous components of basement membrane.

Tumor cells traverse basement membranes (BM) during the stages of the metastatic process. Penetration of the BM may involve proteolysis by enzymes directly or indirectly associated with tumor cells. This study evaluated the role of the serine proteases urokinase (plasminogen activator), plasmin, and another regulatory protease, alpha-thrombin, in the degradation of the BM. Homogeneously pure enzyme preparations were incubated with isolated components of BM and with whole human amnion BM. The BM components consisted of acid-extracted type IV collagen, pepsin fragments of collagen type IV, laminin, and fibronectin. Collagen type V (alpha A alpha B) associated with the peri-BM zone was also studied. The purity of the enzymes was verified by gel electrophoresis and inhibitor studies. Digestion of the BM components was performed at 25 degrees using matched activity for the different enzymes. Urokinase failed to significantly degrade fibronectin or any of the other BM components. Under the same 25 degrees (native) conditions, plasmin and thrombin cleaved fibronectin and laminin into multiple specific fragments but did not produce a major cleavage of acid-extracted type IV collagen, pepsinized type IV collagen, or alpha A alpha B (type V) collagade fibronectin or any of the other BM components. Under the same 25 degrees (native) conditions, plasmin and thrombin cleaved fibronectin and laminin into multiple specific fragments but did not produce a major cleavage of acid-extracted type IV collagen, pepsinized type IV collagen, or alpha A alpha B (type V) collagade fibronectin or any of the other BM components. Under the same 25 degrees (native) conditions, plasmin and thrombin cleaved fibronectin and laminin into multiple specific fragments but did not produce a major cleavage of acid-extracted type IV collagen, pepsinized type IV collagen, or alpha A alpha B (type V) collagen. alpha-Thrombn selectively degraded only the m.w. 400,000 chain of laminin, whereas plasmin degraded both the laminin chains. Digestion of laminin by the serine proteases was time and concentration dependent, as verified by a new degradation assay using [14C]laminin. A variety of normal and neoplastic cells were tested for the presence of laminin-degrading proteases. macrophages, polymorphonuclear leukocytes, and metastatic tumor cells contained a significant laminin-degarding activity. The activity was enhanced by the addition of plasminogen. Type V collagen was cleaved by thrombin and plasmin at 35 degrees but not at temperatures below 33 degrees. Following treatment of whole-amnion BM with any of these enzymes, electron microscopy demonstrated preservation of the lamina densa. Immunohistology studies indicated that laminin, but not type IV collagen, was removed from the whole BM by plasmin treatment. The results suggest that these BM components are poor substrates for plasminogen activators and that plasmin alone is not sufficient to completely degrade the whole BM...

Photosensitization with zinc (II) phthalocyanine as a switch in the decision between apoptosis and necrosis.

Photodynamic therapy (PDT) of tumors and other diseases is based on the uptake of a photosensitizing dye in target cells, which are damaged by reactive oxygen intermediates generated on irradiation with light in which the wavelengths match the dye absorption spectrum. PDT can induce cell death by necrosis and apoptosis both in vivo and in vitro, but the factors determining the contribution of either mechanism to the overall process are not completely defined. Our studies on the photosensitization of 4R transformed fibroblasts with the second-generation photosensitizer zinc (II) phthalocyanine (ZnPc) aim at determining the effect of important experimental parameters such as time of cell incubation (2 or 24 h) with ZnPc before irradiation and ZnPc concentration in the incubation medium on cell death. Furthermore, we propose possible correlations between the cell death mechanism and primary photo-damage sites; these are mainly determined by the intracellular localization of the photosensitizer. The mechanism of cell death was determined by both electron microscopy analysis of the morphological alterations induced by photosensitization and measurement of caspase 3 activation. The initial photodamage sites were determined by measuring the activities of several functions typical of mitochondria, lysosomes, Golgi apparatus, cytosol, and plasma membrane. The intracellular localization of ZnPc after 2- or 24-h incubation was determined by fluorescence microscopy. Necrosis, associated with early loss of plasma membrane integrity and complete depletion of intracellular ATP, represents the prevailing mode of death for 4R cells dark-incubated for 2 h with ZnPc and irradiated with light doses reducing viability by 99.9%. In contrast, irradiation performed 24 h after ZnPc incubation causes only partial inhibition of plasma membrane activities, and cell death occurs largely by apoptosis. ZnPc is mainly localized in the Golgi apparatus after 2- and 24-h incubation, and in all of the cases this compartment represents a primary target of photodamage. Only after prolonged incubation is mitochondrial localization of ZnPc clearly detected by fluorescence microscopy; this could be a determining factor for promotion of apoptosis. Our data demonstrate that it is possible to modulate the mechanism of cell death by appropriate protocols; this may be relevant for enhancing the therapeutic efficacy of PDT.

Correlation of serum metalloproteinase levels with lung cancer metastasis and response to therapy.

Cancer cells elaborate metalloproteinases which may play a role in invasion and metastasis. The serum level of the M(r) 72,000 type IV collagenase (MMP-2) was measured in 87 lung cancer patients. Stage IV cancer levels were significantly elevated (P less than 0.0001) compared to normal sera. A significant difference (P less than 0.01) was found between enzyme levels in the presence versus the absence of distant metastasis. For 29 patients treated with combination chemotherapy, a positive relationship was noted between response failure and elevated enzyme levels. Serum metalloproteinase levels may provide information relevant to prognosis as well as treatment decisions.

Secretion of type IV collagenolytic protease and metastatic phenotype: induction by transfection with c-Ha-ras but not c-Ha-ras plus Ad2-E1a.

Activated ras oncogene transfection into suitable recipient cells has been shown to induce the metastatic phenotype (Thorgeirsson, et al., Mol. Cell. Biol., 5: 259-262, 1985). We have used this model system to study the correlation of basement membrane collagenolysis with metastatic propensity. The c-Ha-ras oncogene alone, or combined with v-myc, transfected into early passage rat embryo fibroblasts, induce these cells to secrete high levels of type IV collagenolytic metalloproteinase and to concomitantly exhibit a high incidence of spontaneous metastases in nude mice. Cotransfection of c-Ha-ras plus the adenovirus type 2 E1a gene yields cells which are highly tumorigenic but nonmetastatic and fail to produce type IV collagenase. This effect is due to a suppression of collagenase elaboration, not increased production of a collagenase inhibitor, and not decreased production of a collagenase activator. The characteristics of the collagenase are identical to tumor type IV collagenase described previously. The nonmetastatic cells which failed to produce type IV collagenase retain the ability to secrete high levels of plasminogen activator. Transfection with the protooncogenic forms of Ha-ras or mos, or spontaneous transformation of NIH 3T3 cells or chemical transformation of BALB 3T3 cells yields cells which fail to produce collagenase, are tumorigenic, but totally nonmetastatic. These data support a biochemical linkage of type IV collagenase expression with the metastatic phenotype in this rodent system.

Increased expression of the Mr 72,000 type IV collagenase in human colonic adenocarcinoma.

Proteolytic enzymes, such as type IV collagenase, play an important role in tumor invasion and metastasis. To examine Mr 72,000 type IV collagenase expression in human colon carcinoma, blot hybridization of total RNA from 19 primary colon tumors were performed. These filters were probed with complementary DNA probes encoding the Mr 72,000 type IV collagenase metalloenzyme. The results were expressed as the ratio of the messenger RNA (mRNA) levels in the tumor tissue to that in the adjacent normal mucosa (R). The level of the 3.1-kilobase type IV collagenase mRNA was higher in the primary tumor than in the normal adjacent colonic mucosa in 13 of 18 (72%) cases with a diagnosis of adenocarcinoma. These cases were divided into high expression (R, 4.50 to 29.34) and intermediate expression (R, 2.54 to 3.31) subgroups. Both groups showed statistically significant (P less than 0.05) elevations when compared with the five cases showing the lowest levels of Mr 72,000 type IV collagenase mRNA expression (low expression subgroup; R, 0.96 to 1.48). With this demonstrated elevation of Mr 72,000 type IV collagenase mRNA in colorectal adenocarcinoma we examined concomitant expression at the protein level using immunohistochemical techniques. Immunohistochemical examination of 70 cases of colon tumors, including 30 benign adenomas, using anti-Mr 72,000 type IV collagenase antibodies demonstrated a significant correlation with Duke's classification (P less than 0.001). Our results suggest that enhanced expression of the Mr 72,000 type IV collagenase enzyme may be a marker of human colorectal tumor invasiveness.

Lorenzo Gotte (1926-1991): a pioneer of elastin.

Lorenzo Gotte (1926-1991) was an outstanding histologist at the School of Medicine of Padua. This year marks the 25th anniversary of his passing away - commemorated during the recent congress of the Italian Society for the Connective Tissue (SISC), held in Padua (September 30 - October 1, 2016). This brief note recalls this outstanding figure: indeed, forthose who knew him, Lorenzo Gotte was an exceptional scientist and at the same time, an unparalleled teacher - and, for many, a great friend. It is still difficult to separate these aspects of his personality, so intertwined in his life: studying elastin and elastic tissue was a passion central to Gotte's life.

Augmented membrane type 1 matrix metalloproteinase (MT1-MMP):MMP-2 messenger RNA ratio in gastric carcinomas with poor prognosis.

The activation of zymogen and the amount of proteinase and its inhibition are important in determining the eventual activity of matrix-degrading enzymes involved in tumor aggressiveness. To evaluate a gene complement leading to matrix metalloproteinase 2 (MMP-2; Mr 72,000 gelatinase) activity, membrane type 1 MMP (MT1-MMP), urokinase-type plasminogen activator, MMP-2, and tissue inhibitor of metalloproteinase 2 transcriptional levels were measured in gastric carcinoma biopsies. Comparative tumor:normal tissue reverse transcription-PCR in a cohort of 25 patients revealed up to a 10-fold difference in the expression of MT1-MMP, a metalloproteinase that has been proposed as a membrane receptor activator of MMP-2; a 1-unit increment resulted in a 30% risk to survival. A 20% risk also resulted from a 1-unit increment in the MT1-MMP: MMP-2 ratio, which showed differences of up to 15-fold. Instead, the expression of urokinase-type plasminogen activator, which trips off a cascade ending in the activation of MMP-2, as well as the expression of MMP-2 itself and its inhibitor, tissue inhibitor of metalloproteinase 2, lacked correlation with patient follow-up. Zymography revealed MMP-2 activities that were often in conflict with the transcription results and also with follow-up. The results suggest the evaluation of MT1-MMP and/or MT1-MMP:MMP-2 transcription as a new preoperative molecular-level prognostic factor for gastric carcinoma.

Synthesis and refolding of human TIMP-2 from E. coli, with specific activity for MMP-2.

Tissue inhibitors of metalloproteinase (TIMPs) are inhibitory counterparts of collagenases, containing 12 cysteine residues paired to six internal disulphide bridges. TIMP-2, an inhibitory protein of 72 kDa gelatinase/type IV collagenase (MMP-2), was expressed in Escherichia coli as a fusion protein with a 34 amino acid NH2-linked tail containing six consecutive histidine residues. The protein was purified in a single-step using an ion metal affinity column (IMAC) in denaturing conditions. The immobilized fusion TIMP-2 protein was refolded at a high concentration in the column, producing about 5 mg of protein per litre of bacterial cells. It shows specific binding and inhibitory activity against MMP-2, but has no effect against 92 and 45 kDa gelatinases.

Quantitation of basement membrane collagen degradation by living tumor cells in vitro.

In order to study the mechanism of tumor cell invasion of basement membranes, a new method has been developed to quantify basement membrane (type IV) collagen degradation by living tumor cells. Tumor cells are inoculated into 16-mm tissue culture wells coatd with biosynthetically [14C]proline-labeled type IV collagen. Soluble degradation products are detected by measuring the radioactivity present in the medium. Using this method, we find that both highly metastatic and non-metastatic tumor cells and normal cells attach, but only the metastatic cells degrade the type IV collagen layer. The present data suggest that the mechanism of degradation is local activation of a latent type IV collagen specific enzyme at the cell substrate interface with no significant cell phagocytosis of substrate.

Interaction of hydro- or lipophilic phthalocyanines with cells of different metastatic potential.

A highly metastatic (4R) and a nonmetastatic (RE4) transformed rat embryo fibroblast cell line were incubated with lipid-soluble Zn(II)-phthalocyanine (ZnPc) and its water-soluble tetrasulphonated derivative (ZnPcTS) and compared for phthalocyanine uptake. The hydrophobic liposome-delivered ZnPc showed a significantly greater uptake by both cell lines than did ZnPcTS. Moreover, the two phthalocyanines appear to interact with cells according to different pathways, as suggested by the different temperature-dependence of the binding process and the different inhibitory action exerted by selected serum proteins, such as lipoproteins and heavy proteins. Under all experimental conditions, the two cell lines exhibited similar interactions with ZnPc and ZnPcTS, suggesting that heterogeneity of the tumor cell population has a minor influence on the accumulation of photosensitizers.

Gelatinase-a/mmp-2 serum levels and neoplastic progression in breast-cancer patients.

The metastatic potential of cancer cells has been associated to their ability to elaborate and secrete basement membrane degradative enzymes. In this process a major role appears to be played by a protease known as gelatinase A (72 kDa type IV collagenase, MMP-2). In an effort to assess the significance of these findings to breast cancer progression, we have evaluated the gelatinase A/MMP-2 serum levels in a cohort of 80 breast cancer patients, 27 subjects affected by benign breast disease and 27 healthy controls. Although differences between the three groups were observed, with the highest values monitored in benign breast disease, they were not statistically significant. On the contrary, within the breast cancer cohort, the patients presenting clinical evidence of distant metastases (M+, n=40) had statistically elevated enzyme serum levels (p<0.03) compared to those without nodal involvement and distant metastases (N-M-, n=20). The statistical significance was still evident when considering the overall M- cohort (including N+ and N- patients, n=40) compared to the M+. Although indicating that, in general, gelatinase A/MMP-2 is not a useful serum marker for breast cancer screening and diagnosis, the findings point toward its involvement in the breast cancer metastatic process and suggest a possible value in monitoring the outcome of the disease.

CNTF up-regulation of TIMP-2 in neuroblastoma cells.

The ciliary neurotrophic factor (CNTF) can regulate survival and differentiation of many types of developing and adult neurons; in metastatic SK-N-BE neuroblastoma cells, it promotes differentiation and neurite outgrowth. The expression of Gelatinase A (MMP-2) and its specific tissue inhibitor (TIMP-2), a degradative system whose balance is involved in matrix invasion and metastasis, was investigated in SK-N-BE cells cultured with and without CNTF or NGF. Zymographic analysis of conditioned media revealed that the cells constitutively secrete two gelatinases, mainly pro-MMP-2 but also traces of pro-MMP-9. In a time-course experiment in the presence of 25 ng/ml of CNTF, the MMP-2 mRNA expression showed no significant modulation, while TIMP-2 mRNA up-regulated to > 2-fold after 48 h and then fell dramatically. At the same concentrations, NGF showed no effect. TIMP-2 mRNA expression showed a dose-dependent increase of up to 8-fold from 1 to 250 ng/ml of CNTF and increased secretion of TIMP-2 was confirmed by Western blotting. MMP-2 was only slightly over-expressed under the same conditions, at either mRNA or protein level, with no correlation with neurocytokine concentration. These results suggest that boosting the expression of TIMP-2 by CNTF could restrain both matrix degradation following nervous system injury and neuroblastoma aggressiveness.

Gelatinase A (MMP-2) and its mRNA detected in both neoplastic and stromal cells of tumors with different invasive and metastatic properties.

Simultaneous presence of gelatinase A (MMP-2) and MMP-2 messenger RNA (mRNA) in 30 malignant tumors with various degrees of differentiation and biological behavior was evaluated by immunohistochemistry and in situ hybridization. The series consisted of 10 gastric carcinomas, 10 colorectal carcinomas, five squamous skin carcinomas, and five basal cell skin tumors. MMP-2 was detected in all cases. MMP-2 mRNA was expressed in the stromal cells in all cases and was more marked in the less-differentiated gastric and colonic carcinomas; it was also detected in the neoplastic cells of poorly differentiated tumors, particularly in those of the signet-ring cell type, both in the colon and stomach. The study confirmed that stromal cells have a specific role in tumor invasion and suggests a direct relationship between neoplastic epithelium and stromal cells in the most aggressive varieties.

Reverse transcription-polymerase chain reaction phenotyping of metalloproteinases and inhibitors involved in tumor matrix invasion.

The matrix metalloproteinase enzymes have been implicated in tumor invasion and metastasis by a series of correlative immunohistochemical studies. In addition, direct evidence for the role of these enzymes in this pathologic process comes from studies using specific metalloproteinase inhibitors to block tumor invasion and metastasis formation, both in vitro and in vivo. Synthetic oligonucleotide primers for four metalloproteinases (MMP-1, MMP-2, MMP-9, MMP-10) and their tissue inhibitors (TIMP-1, TIMP-2) were selected, synthesized, and optimized in the reverse transcriptase-polymerase chain reaction (RT-PCR) to study the qualitative profile of these enzymes and inhibitors in cultured human tumor cells and tumor tissues. These primers are specific and generate unique amplification products for each appropriate enzyme and inhibitor. Slight enhancement in the amplification of cDNA products was achieved by adding dimethylsulfoxide to the reaction mixture, but commercial enhancement reagents were ineffective. Using this RT-PCR method, cDNA amplification was successful with RNA from as few as 20 cultured tumor cells. The RT-PCR analysis was done on three invasive human colon adenocarcinomas and their paired adjacent normal mucosa. The results show MMP-1 and MMP-2 products in all three tumors, and MMP-2 detected in one of the three normal mucosa samples; TIMP-2 expression was present in two of three patients and awaits quantitative assessment of RT-PCR products.

Microvascular changes in progressive systemic sclerosis: immunohistochemical and ultrastructural study.

Fifteen patients affected by Progressive Systemic Sclerosis have been studied. With immunofluorescence, specific antibodies against collagen type IV and laminin clearly outlined the microvessels, while endothelial cells showed a brilliant heavy fluorescence for vimentin antibodies. In the deep dermis, fibronectin proved to have increased. At electron microscopy, microvessels appeared with occluded lumina due to the presence of swollen endothelial cells. Endothelial cytoplasm was filled with intermediate filaments (vimentin type), generally condensed into peripherally located bundles or in perinuclear rings. The perivascular basal lamina was thickened and laminated. Although these changes do not demonstrate a specific pattern, representing a common step in several connective tissue disorders, the data tend to confirm a clear involvement of the microvasculature in Progressive Systemic Sclerosis.

Tissue and serum metalloproteinase (MMP-2) expression in advanced ovarian serous cystoadenocarcinomas: clinical and prognostic implications.

OBJECTIVE: The object of this study was to analyse the tissue and serum metalloproteinase (MMP-2), an enzyme which degrades the basement membrane collagen type IV, as a potential marker useful in prognostic evaluation and clinical monitoring of the follow-up, in patients with advanced ovarian serous cystadenocarcinoma. MATERIALS AND METHODS: Tissue MMP-2 expression was determined in 21 FIGO stage III ovarian serous cystadenocarcinomas treated with primary surgery and adjuvant chemotherapy, and compared to 10 cystadenomas used as controls. Retrospective analysis of clinical data allowed the comparison of accepted prognostic factors to tissue MMP-2 expression for impact on disease-free survival. In fourteen out of 21 patients, serum MMP-2 levels were also analysed. RESULTS: Compared to cystadenomas, the tissue MMP-2 expression was significantly (P < 0.001) higher in serous cystadenocarcinomas. A significant relationship was observed between tissue MMP-2 and disease-free survival (P = 0.0003), independently of tumor architectural grade, lymph nodal status and residual disease after debulking surgery. Recurrence risk in patients whose carcinomas had a tissue MMP-2 > or = 29% was significantly higher than that in patients whose carcinomas demonstrated lower tissue MMP-2 expression (P = 0.004). Serum MMP-2 levels correlated with tissue staining, and also expressed a significant relationship with disease-free survival (P = 0.002). CONCLUSIONS: Tissue MMP-2 seems to be a prognostic indicator in patients with FIGO stage III ovarian serous cystadenocarcinoma, significantly correlated with recurrence risk and apparently independent of tumor architectural grade, lymph nodal status, and residual disease after debulking surgery. An interesting relationship was observed between tissue staining and MMP-2 serum levels.