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S Garbisa

Publications and source records attributed to S Garbisa.

88 records · Page 5Linked to original sources
PubMed1981

Partial purification and characterization of a neutral protease which cleaves type IV collagen.

A neutral protease has been extracted from the media of cultured metastatic tumor cells and purified approximately 1000 times after sequential ammonium sulfate fractionization, concanavalin A column chromatography, and molecular sieve chromatography. The protease has an apparent molecular weight of 70 000--80 000, is inactive at acid pH, requires trypsin activation, and is inhibited by ethylene-diaminetetraacetic acid but not by phenylmethanesulfonyl fluoride, N-ethylmaleimide, or soybean trypsin inhibitor. The enzyme produces specific cleavage products for both chains of pro type IV collagen isolated without pepsinization and apparently cleaves at one point in a major pepsin-extracted chain of placenta type IV collagen. The partially purified enzyme fails to significantly degrade other collagens or fibronectin under digestion conditions in which specific reaction products are produced for type IV collagen. The existence of this enzyme is significant since previously described animal collagenases fail to degrade type IV collagen. Such a type IV specific collagenase could play a role in tumor invasion and may be secreted by other cells such as endothelial cells, epithelial cells, and immune cells.

Animals↗
PubMed1981

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...

Amnion↗
PubMed1980

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.

Animals↗
PubMed1979

Hormonal requirements for basement membrane collagen deposition by cultured rat mammary epithelium.

Alveoli and ducts isolated from virgin rat mammary glands synthesize basement membrane collagen (typeIV) in primary culture. Using purified antibodies to type IV collagen, prominent intracellular and extracellular fluorescence is observed in the epithelium. No fluorescence is observed with antibodies to collagen type I and III. From quantitation of the incorporation of [14c]proline-labeled proteins, 1.5 to 2.5 per cent of the newly synthesized proteins are collagen. Type IV collagen from these cultures was biochemically identified on the basis of (1) the high ratio of labeled 3-hydroxyproline to 4-hydroxyproline (1:10), (2) the gel electrophoretic pattern of the collagenase-sensitive proteins precipitated with 1.7 M NaCl, (3)the failure of the collagen to bind to diethylaminoethyl-cellulose, and(4)the immunologic cross-reactivity with mouse tumor type IV is identical with that of type IV collagen from other sources. When the supportive hormones, insulin, prolactin, hydrocortisone, progesterone, and estradiol are removed from the cultures, there is a 90 per cent reduction in the amount of [3H]proline recovered in collagen synthesis coincides with only a 30 percentdrop in the growht rate and a 20 per cent drop in total protein synthesis of the sells over the 24-hour period without hormones. Pulse-chase experimout hormones. Pulse-chase experiments revealed an enhanced turnover of collagen following hormone withdrawal. This system may be an in vitro model of collagen turnover in mammary gland in involution.

Animals↗
PubMed1978

Structural organization of collagen fibrils in media aortic wall.

Small-angle X-ray diffraction patterns of bovine, human and porcine media aortic wall show meridional reflections corresponding to a periodicity which suggest a molecular packing typical of tendon collagen. However the meridional intensity distribution of stretched air dried aortic samples appears different from that of air-dried tendon, probably because of the presence of a large amount of type III collagen with the environment, which are specific for aortic tissue. The stretched wet aortic samples show a marked decrease in intensity, revealing an extensive disorder in the axially-projected structure of the fibrils. When a loading system simulating the effect of blood pressure is applied to a ring of aorta, no evidence of orientation of collagen is seen by X-ray diffraction, as would be expected if collagen fibrils had an isotropic distribution inside the aorta media. Scanning electroni microscopy supports the existence of a network of collagen fibrils surrounding elastic lamellae.

Adult↗
PubMedDate not supplied

Basement membrane production by hepatocytes in chronic liver disease.

The immunohistologic distribution of fibronectin, laminin, type IV collagen and whole basement membrane was evaluated in liver biopsies from patients with chronic active liver disease. Fibronectin was consistently increased in the areas of piecemeal necrosis, portal tracts and fibrous septa. Laminin was not detected in normal liver parenchyma. In contrast, laminin positive linear basement membrane structures were prevalent in portal tracts, fibrous septa and the peripheral sinusoids of cirrhotic nodules. In areas of piecemeal necrosis, the hepatocytes, single or assembled in "rosettes", were frequently underlined by linear deposits of laminin and type IV collagen. This immunoreactivity was often polarized, being confined to the stromal side of liver cells, while the parenchymal side was negative for both proteins. Electron microscopy revealed a typical basement membrane in corresponding areas. Hepatocytes normally do not produce a basement membrane, but do so following chronic injury. We suggest that the polarized basement membrane accumulation by hepatocytes is a hallmark of hepatocyte regeneration following damage.

Basement Membrane↗
PubMedDate not supplied

Age-related changes in composition and mechanical properties of the tunica media of the upper thoracic human aorta.

A cylindrical segment, free of complex atherosclerotic lesions, was resected at autopsy from each of 59 descending human thoracic aortas by cutting just below the level of the first pair of intercostal arteries and 35 mm distal to this incision. Each isolated tunica media was defatted and subjected to successive treatment with EDTA-Tris, 5 M guanidine hydrochloride-Tris, 5 M guanidine hydrochloride-Tris-DTE, collagenase and either trypsin or hot alkali. After each extraction or digestion, the dimensions and weight of the segments were measured and the extracted materials were analyzed and quantitated. This allowed the total content of the various components of the tunica media to be assessed by both gravimetric and analytical means. An age-related rise was observed in the total content of the following components: proteins and glycoproteins soluble in chaotropic solvents (ranging from 24 mg/cm in the youngest samples to 46 mg/cm in the oldest) and collagen (38 mg/cm to 69 mg/cm). In contrast, the total content of elastin remained constant at 70 mg/cm at all ages, but its concentration decreased due to the rise in the concentration of the other tissue components as the tunica media thickened with age. It was also noted that with increasing age there was an accumulation of protein(s) which could not be solubilized by extraction with chaotropic agents or with collagenase, but which could be removed by treatment with either trypsin or hot alkali. Mechanical measurements conducted before and after trypsin digestion on samples previously subjected to purification with the first four agents used suggest that this accumulated protein(s) influenced the elastic response of the tissue to the applied stress by increasing the incremental modulus, the breaking stress, and the hysteresis. After the removal of this additional protein(s), the mechanical behavior of the elastin component was found to be identical in all samples, irrespective of age. It is therefore proposed that the morphological changes and the stiffening observed in the aging aortic wall are not due to degradation of its elastin network but to variations in the supramolecular organization of connective tissue components.

Adult↗
PubMedDate not supplied

Rapid method for cultured trophoblast cells identification.

The addition of human foetal cord serum to the culture medium improves the in vitro growth of human chorionic villi cells. The expression of HCG, laminin, laminin receptor, and type IV collagen has been studied on first passage (4-7 weeks) cultured villi cells by the immunoperoxidase method. No cells were positive for HCG, while various patterns of basement membrane markers were always detectable. The presence of laminin and collagen type IV excludes fibroblast contamination and can be used for a rapid trophoblast cells identification.

Antibodies↗
PubMedDate not supplied

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.

Adult↗
PubMedDate not supplied

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.

Basement Membrane↗