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PubMed1998

Genetic diseases of the extracellular matrix: more than just connective tissue disorders.

The rapidly increasing knowledge about the molecular biology of the extracellular matrix has changed the concepts for the pathomechanisms of heritable connective tissue diseases. The spectrum of genetic matrix disorders is much broader than previously thought and now also includes diseases of organs such as the kidney, eye, and muscles. In addition, evidence is emerging that certain "acquired" diseases may be inherited, and that defects in signal transduction and patterning genes contribute to the pathology of connective tissue disorders. The phenotypes of genetic matrix disorders are determined by basic biological characteristics of the extracellular matrix. (a) The extracellular matrix occurs ubiquitously and is important for organ development and functions. (b) Matrix macromolecules are often large oligomers that polymerize into suprastructures at several hierarchic levels. They form insoluble fibrils or filaments that are further assembled into tissue suprastructures, for example, bundles or networks of fibrils. (c) Matrix suprastructures share characteristics with metal alloys. Tissue-specific mixtures of matrix molecules form specific arrays that differ from those of the pure components. Therefore the phenotypes of matrix diseases reflect a cascade of pathological events disturbing alloy formation, such as abnormal protein synthesis and folding, defective fibrillogenesis, and bundling, all capable of leading to abnormal cell-matrix interactions.

Connective Tissue Diseases↗
PubMed2003

The effect of extracellular pH on matrix turnover by cells of the bovine nucleus pulposus.

It has long been known that very acidic conditions can be found in degenerate discs. The effect of these acid conditions on matrix turnover are, however, unknown. This study aimed to examine the effect of acidity on production of matrix components and on agents which break down the matrix in order to gain insight into the effect of pathological values of pH on matrix turnover. Cells were isolated from the nucleus of bovine discs and from bovine articular cartilage, embedded in alginate beads and cultured at pH levels maintained within the ranges seen in normal and pathological discs: pH 7.4-pH 6.3 for 48 h. Rates of sulphated glycosaminoglycan (GAG) and protein synthesis were measured, as well as rates of production of some agents involved in matrix breakdown, i.e. total and activated matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). The results showed that acid conditions had a profound effect on cell matrix turnover; at pH 6.4, total production of most species measured was inhibited by more than 50% compared to production at pH 7.2; production of sulphated GAGs and of TIMP-1 fell by >90%. However production of active metalloproteinases by disc cells was relatively insensitive to pH, with activity at pH 6.3 not statistically different from that at pH 7.2. These findings indicate that exposure to acid conditions appears particularly deleterious for the disc matrix, as it inhibits the disc cells from synthesising functionally important molecules such as the sulphated GAGs but does not prevent the production of agents able to degrade matrix components. The low values of pH seen in some degenerate discs are thus likely to be involved in breakdown of the disc matrix.

Animals↗
PubMed2004

Matrix metalloproteinase activity in thoracic aortic aneurysms associated with bicuspid and tricuspid aortic valves.

OBJECTIVE: Matrix metalloproteinases are endopeptidases that function in cell matrix turnover. Abnormal matrix metalloproteinase activity has been implicated in the formation of atherosclerotic abdominal aortic aneurysms. Recent studies suggest that abnormal matrix metalloproteinase activity may also be associated with the formation of atherosclerotic and nonatherosclerotic thoracic aortic aneurysms. Bicuspid aortic valves are associated with an intrinsic aortic pathology that predisposes to formation of proximal thoracic aneurysms while tricuspid aortic valves are not. The objective of this study was to compare the activities of matrix metalloproteinases and levels of their inhibitors in thoracic aneurysms of patients with bicuspid and tricuspid aortic valves. METHODS: Endogenous and total activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 were measured in proximal nonatherosclerotic thoracic aortic aneurysms of 16 patients with bicuspid aortic valves and 12 patients with tricuspid aortic valves. Levels of tissue inhibitor metalloproteinase-1 and -2 were also measured. Results were standardized to total protein (mg). RESULTS: Total matrix metalloproteinase-2 activity was greater in aneurysms associated with bicuspid valves when compared with those from tricuspid valves (43 +/- 11 ng/mg vs 14 +/- 2 ng/mg, P =.02). Total matrix metalloproteinase-9 activity was also greater in aneurysms associated with bicuspid aortic valves (4.0 +/- 0.9 vs 1.5 +/- 0.3, P =.02). There was no meaningful difference between groups in levels of tissue inhibitor-1 and -2. CONCLUSION: The increased activity of matrix metalloproteinases in the walls of aneurysms associated with bicuspid aortic valves may partly explain the predilection to aneurysm formation in these patients.

Aorta, Thoracic↗
PubMed2004

Gamma-irradiation modulates vascular smooth muscle cell and extracellular matrix function: Implications for neointimal development.

OBJECTIVE: Migration of vascular smooth muscle cells (SMCs) into the subintimal space, and their proliferation and resultant deposition of extracellular matrix are key processes in the development of intimal hyperplasia, leading to vascular recurrent stenosis. The purpose of this study was to investigate the effects of clinically administered doses of gamma-radiation on SMCs and extracellular matrix proteins in vitro, to better understand how it impinges on cellular and extracellular components of recurrent stenosis. METHODS: The effects of gamma-irradiation (10, 20 Gy) on SMC migration into three-dimensional collagen matrix gels was quantitated by calibrated light microscopy, and the release of metalloproteinases into conditioned media was investigated with an enzyme-linked immunosorbent assay and zymography. Collagen production was assayed with [(3)H]-proline incorporation, and SMC phenotype changes with confocal microscopy with a fluorescent alpha-actin antibody. The effect of gamma-irradiation on extracellular matrix was investigated by quantitating untreated SMC proliferation ((3)H-thymidine incorporation) on irradiated endothelial cell-derived matrix and by assessing structural collagen matrix changes with sodium dodecylsulfate polyacrylamide gel electrophoresis. All groups were compared with nonirradiated control groups. RESULTS: SMC vertical migration was significantly decreased by gamma-irradiation (48% and 55%, respectively; P <.0001). Irradiation did not generate measurable matrix protein crosslinks, nor did it alter the production of metalloproteinases or collagen synthesis. However, gamma-irradiation decreased the ability of extracellular matrix to induce nonirradiated SMC proliferation (15% reduction; P =.0028). Moreover, gamma-irradiation reversed the secretory phenotype of cultured SMCs to a contractile type. CONCLUSIONS: The gamma-irradiation-induced reduction of cellular migration, changes in SMC phenotype, and functional activity of matrix-bound factors, and no measurable effects on the production of extracellular matrix proteins, may in part explain the diverse effects of gamma-irradiation on the restenotic response.

Animals↗
PubMed2001

Extracellular matrix remodeling in the vascular wall.

The extracellular matrix provides a structural framework essential for the functional properties of vessel walls. The three dimensional organization of the extracellular matrix molecules--elastin, collagens, proteoglycans and structural glycoproteins--synthesized during fetal development--is optimal for these functions. Early in life, the vessel wall is subjected to injury: lipid deposition, hypoxia, enzyme secretion and reactive oxygen species production during inflammatory processes, and the extracellular matrix molecules are hydrolyzed by proteases--matrix metalloproteinases, leukocyte elastase, etc. In uninjured arteries and veins, some proteases are constitutively expressed, but through the control of their activation and/or their inhibition by inhibitors, these proteases have a very low activity. During the occurrence of vascular pathologies--atherosclerosis, hypertension, varicosis, restenosis, etc.--the balance between proteases and their inhibitors is temporally destroyed through the induction of matrix metalloproteinase gene expression or the secretion of enzymes by inflammatory cells. Smooth muscle cells, the most numerous cells in vascular walls, have a high ability to respond to injury through their ability to synthesize extracellular matrix molecules and protease inhibitors. However, the three dimensional organization of the newly synthesized extracellular matrix is never functionally optimal. In some other pathologies--aneurysm--the injury overcomes the responsive capacity of smooth muscle cells and the quantity of extracellular matrix decreases. In conclusion, care should be taken to maintain the vascular extracellular matrix reserve and any therapeutic manipulation of the protease/inhibitor balance must be perfectly controlled, because an accumulation of abnormal extracellular matrix may have unforeseen adverse effects.

Aneurysm↗
PubMed2002

Membrane type 1 matrix metalloproteinase regulates cellular invasiveness and survival in cutaneous epidermal cells.

Membrane type 1 matrix metalloproteinase is a member of the membrane-anchored matrix metalloproteinase family and is involved in tissue remodeling events ranging from tumor invasion and angiogenesis to growth and development. We sought to clarify the role of membrane type 1 matrix metalloproteinase in cutaneous epidermal cells using anti-sense cDNA expression in human keratinocytes. Modulation of membrane type 1 matrix metalloproteinase transcript and protein levels was achieved via retroviral expression of a 5' 1.4 kb anti-sense membrane type 1 matrix metalloproteinase construct and a 3.4 kb full-length sense membrane type 1 matrix metalloproteinase construct in primary and immortalized keratinocytes and SCC-25 cells. Maximal reductions were observed 48-72 h after transduction with 1.4 kb anti-sense membrane type 1 matrix metalloproteinase construct that correlated with significant decreased pro-matrix metalloproteinase-2 activation. Functionally, we found decreased cell migration, reduced cellular proliferation, and increased apoptotic nuclear fragmentation after 1.4 kb anti-sense membrane type 1 matrix metalloproteinase construct expression. Our findings suggest a role for membrane type 1 matrix metalloproteinase in human cutaneous epidermal cell invasion and survival mechanisms in vivo.

Antisense Elements (Genetics)↗
PubMed2002

Integration of concepts: cardiac extracellular matrix remodeling after myocardial infarction.

The cardiac extracellular matrix consists of a three-dimensional structural network of interstitial collagens to which other matrix components are attached. The main physiological functions of this network are to retain tissue integrity and cardiac pump function. Collagen deposition is controlled and can be modulated by hormonal factors, growth factors, cytokines, regulatory proteins and/or hemodynamic factors. Increased collagen deposition is a prerequisite to prevent dilatation of the infarcted area. Excessive accumulation of collagen leads to ventricular diastolic and systolic dysfunction and ultimately contributes to heart failure. An appropriate balance of extracellular matrix synthesis and degradation is required for normal morphogenesis and maintenance of tissue architecture. A disbalance in the extracellular matrix turnover either by decreased matrix synthesis and/or increased degradation leads to less than normal extracellular matrix in the myocardium which in its turn may lead to cardiac dilatation or even rupture. Extracellular matrix degrading enzymes expressed after myocardial infarction belong to the families of serine and matrix metalloproteinases (MMPs) and are secreted as latent proenzymes that have to be activated. It is crucial to keep the activity of these enzymes under tight control by either influencing the synthesis, activation or inhibition by tissue inhibitors of MMPs (TIMPs) or alpha2-macroglobulin. First studies using MMP inhibitors in experimental models of myocardial infarction seem to give attenuation of ventricular geometry but not always improvement of cardiac function. A central role in the activation of MMPs plays the plasminogen-plasmin system. Invasion of inflammatory cells and hitherto the rest of the wound healing cascade is inhibited in plasminogen or uPA deficient mice, most likely by the inhibition of MMP activity. Regulating the balance of extracellular matrix remodeling either by extracellular matrix synthesis or degradation might be one of the possible prevention mechanisms for heart failure. But also regeneration of the vascular and cardiomyocyte network might be potential new treatments for people with heart failure.

Animals↗
PubMed2001

Cardiac surgery increases the activity of matrix metalloproteinases and nitric oxide synthase in human hearts.

OBJECTIVES: Heart function is variably impaired after cardiopulmonary bypass. We hypothesized that, similar to other myocardial injury states, cardiopulmonary bypass leads to enhanced activity of nitric oxide synthase and matrix metalloproteinases. METHODS: We obtained right atrial biopsy specimens and plasma samples at the onset and termination of cardiopulmonary bypass in 10 patients. Biopsy specimens were analyzed for nitric oxide synthase activity by using a citrulline assay, whereas plasma and tissue were analyzed for matrix metalloproteinase-9 and matrix metalloproteinase-2 activity by using zymography. Tissue inhibitor of metalloproteinase-4 was analyzed by means of Western blotting. The cellular expression of inducible nitric oxide, endothelial nitric oxide synthase, matrix metalloproteinase-2, and matrix metalloproteinase-9 was determined in right atrial biopsy samples from 3 additional patients by using the appropriate conjugated antibodies. RESULTS: Nitric oxide synthase activity increased from the beginning to the end of bypass (4.46 +/- 1.07 vs 16.77 +/- 4.86 pmol citrulline/mg of protein per minute, respectively; P =.018). Pro-matrix metalloproteinase-9 activity increased in hearts (199 +/- 41 vs 660 +/- 177 density units/mg protein; P =.008) and plasma (14.1 +/- 4.6 vs 52.2 +/- 5.9 density units/mg protein; P =.008). Pro-matrix metalloproteinase-2 activity increased in the heart (201 +/- 23 vs 310 +/- 35 density units/mg protein, P <.05) but not in plasma. Tissue inhibitor of metalloproteinase-4 expression in the heart decreased (1574 +/- 280 vs 864 +/- 153 density units, P =.014). CONCLUSIONS: Cardiopulmonary bypass activates enzymes mediating acute inflammation and organ injury (ie, nitric oxide synthase, matrix metalloproteinase-9, and matrix metalloproteinase-2). Decreased tissue inhibitor of metalloproteinase-4 expression allows relatively unopposed increases in matrix metalloproteinase tissue activity. We postulate that these changes play a role in the pathogenesis of heart dysfunction after bypass surgery.

Aged↗
PubMed2003

Matrix metalloproteinase inhibition modifies left ventricular remodeling after myocardial infarction in pigs.

BACKGROUND: Global and regional shape changes that occur within the left ventricular wall after myocardial infarction have been termed infarct expansion. A potential mechanism for this postinfarction remodeling is activation of the matrix metalloproteinases. Accordingly, the present study examined the effects of matrix metalloproteinase inhibition on left ventricular global geometry after myocardial infarction in pigs. METHODS: Myocardial infarction was created in pigs by means of occlusion of the first and second obtuse marginal branches of the circumflex coronary artery, resulting in a uniform left ventricular free wall infarct size of 21% +/- 2%. At 5 days after infarction, the pigs were randomized to undergo broad-spectrum matrix metalloproteinase inhibition (n = 9; PD166793, 20 mg. kg(-1). d(-1) by mouth) or myocardial infarction alone (n = 8). Ten pigs served as noninfarction control animals. Left ventricular end-diastolic area, determined by means of echocardiography, was measured 8 weeks after infarction. RESULTS: Left ventricular end-diastolic area increased in both the myocardial infarction plus broad-spectrum matrix metalloproteinase inhibition and myocardial infarction only groups compared to reference control animals (3.7 +/- 0.2 cm(2)), but was reduced with broad-spectrum matrix metalloproteinase inhibition compared to myocardial infarction alone (4.5 +/- 0.2 vs 4.9 +/- 0.2 cm(2), respectively; P <.05). Regional radial stress within the infarct region increased in both infarction groups when compared to values obtained from reference control animals (599 +/- 152 g/cm(2)), but was attenuated in the myocardial infarction plus broad-spectrum matrix metalloproteinase inhibition group compared to the myocardial infarction alone group (663 +/- 108 vs 1242 +/- 251 g/cm(2), respectively; P <.05). Similarly, regional myocardial stiffness increased in both the myocardial infarction plus broad-spectrum matrix metalloproteinase inhibition and the myocardial infarction only groups compared with that observed in reference control animals (14 +/- 1 rkm, P <.05) but was lower with broad-spectrum matrix metalloproteinase inhibition than with myocardial infarction alone (42 +/- 6 vs 68 +/- 10 rkm, respectively; P <.05). CONCLUSIONS: Matrix metalloproteinase inhibition reduced postinfarction left ventricular dilation, reduced regional myocardial wall stress, and modified myocardial material properties. These unique findings suggest that increased myocardial matrix metalloproteinase activation after infarction contributes directly to the left ventricular remodeling process.

Analysis of Variance↗
PubMed2005

TGF-beta1 reverses the effects of matrix anchorage on the gene expression of decorin and procollagen type I in tendon fibroblasts.

Transforming growth factor-beta1 is known for its effect on the production of extracellular matrix in tendons. Elevated levels of transforming growth factor-beta1 have been reported in tendon adhesion and tendinosis, which suggests that transforming growth factor-beta1 plays an important role in matrix disturbances. Tendon adhesion involves excessive collagen deposition, whereas tendinosis is associated with increased proteoglycan deposition. It seems that other factors also may affect matrix deposition and modulate the effects of transforming growth factor-beta1. We assessed whether matrix anchorage to Type I collagen or fibronectin could change the gene expression of matrix proteins in tendon fibroblasts, and studied whether the effects of transforming growth factor-beta1 were altered by matrix anchorage. Human patellar tendon fibroblast cultures were prepared in different cell anchorages, and the cellular responses to transforming growth factor-beta1 were measured as gene expression of procollagen Type I, Type III, decorin, and biglycan by real-time reverse transcriptase-polymerase chain reaction. Fibronectin anchorage significantly increased the messenger ribonucleic acid level of decorin, and the messenger ribonucleic acid level of procollagen Type I was decreased by matrix anchorage to either fibronectin or Type I collagen. Transforming growth factor-beta1 increased the messenger ribonucleic acid level of procollagen Type I in Type I collagen-coated plates, but it suppressed the messenger ribonucleic acid level of decorin in fibronectin-coated plates. These findings suggest that interaction of matrix anchorage and transforming growth factor-beta1 is an important determinant of matrix deposition in healing tendons and the development of matrix disturbances in tendons.

Cells, Cultured↗
PubMed2002

Effects of mesangium glycation on matrix metalloproteinase activities: possible role in diabetic nephropathy.

High glucose concentrations can decrease degradation of mesangium by reducing the activities of matrix metalloproteinases (MMPs). The aim of this study was to investigate the effects of glycation of mesangium matrix on MMP-2, the principal MMP secreted by mesangial cells to degrade type IV collagen. Also examined were membrane type 1 MMP (MT1-MMP), tissue inhibitors of MMPs (TIMP)-1 and -2, and transforming growth factor-beta (TGF-beta), which together regulate MMP-2 activities in an interacting manner. Human fetal mesangial cells were grown on mesangium matrix glycated by incubation in 500 mmol/l ribose, with or without aminoguanidine. The activities and gene expression of the abovementioned enzymes/inhibitors were measured by degradation of radiolabeled mesangium matrix, RT-PCR, and zymography. Glycation of mesangium matrix resulted in a threefold increase in advance glycation end products and reduced by 45% the matrix-degrading activity of MMPs secreted by mesangial cells. Analogous to the direct effects of high glucose concentrations, glycation of matrix increased the gene expression of MMP-2 and TIMP-1 (control 100 +/- 16.9 vs. glycated 197.3 +/- 30.6% and control 100 +/- 5.3 vs. glycated 152.1 +/- 20.1%, respectively; P < 0.05) and decreased MT1-MMP (control 100 +/- 1.17 vs. glycated 54.1 +/- 15.2%; P < 0.05). However, unlike high glucose concentrations, glycation was not associated with decreased activation of MMP-2. Similarly, glycation but not high glucose increased expression of TIMP-2 (control 100 +/- 5.9 vs. glycated 168.2 +/- 31.4%; P < 0.05), and the effects of glycation on degradation can be abolished by anti-TIMP-2 antibody. Glycation of matrix decreased TGF-beta mRNA by 38.2% and total and active TGF-beta by 35.5 and 21.5%, respectively, opposite the effects of high glucose concentrations. Our results indicate that glycation of matrix affects the balance between MMP-2 and its activator and inhibitors, but this phenomenon is not due to TGF-beta. The process of glycation may impart to the mesangium matrix a memory effect that contributes to the long-term toxicity of hyperglycemia.

Cells, Cultured↗
PubMed2004

[Expression of matrix metalloproteinases in patients with malignant tumors].

The cancer cells secrete proteolytic enzymes, which are important in the tumor spreading. The cells must cross basement membrane and extracellular matrix barriers in order to spread. The matrix metalloproteinases are a family of endopeptidases, which enzymatic activity depends on the presence of zinc ion in the catalytic domain. Matrix metalloproteinases hydrolyze extracellular matrix components such as collagen, laminin, fibronectin, proteoglycans and contribute to the spreading of tumor cells by eliminating the surrounding extracellular matrix and basement membrane barriers. This review describes matrix metalloproteinases family classification and structure, their role under physiological conditions and induced proteolysis during pathological processes. There is a balance between proteolytic extracellular matrix degradation and proteolysis inhibition, but under pathological state (e. g. tumor development) the proteolysis becomes uncontrolled. We review tissue inhibitors of matrix metalloproteinases and synthetic matrix metalloproteinase inhibitors, their perspective in cancer treatment; as well as different matrix metalloproteinases expression in patients with tumors and its prognostic significance during cancer progression.

Antineoplastic Agents↗
PubMed1997

Reactive oxygen species cause direct damage of Engelbreth-Holm-Swarm matrix.

Reactive oxygen species (ROS) are produced and released into the extracellular spaces in numerous diseases and contribute to development and progression, for example, of inflammatory diseases, proteinuria, and tumor invasion. However, little is known about ROS-induced chemical changes of interstitial matrix proteins and their consequences for the integrity of the matrix meshwork. As basement membranes and other matrices are highly cross-linked and complex, the relatively simple matrix produced by Engelbreth-Holm-Swarm (EHS) sarcoma, and proteins isolated therefrom, were incubated in vitro with defined concentrations of ROS that were generated by the Fenton or xanthine oxidase/xanthine reactions. This resulted in two counter-current effects. Although up to approximately 15% of the EHS matrix proteins were released into the supernatant in a ROS dose-response relationship, the residual insoluble matrix was partially cross-linked by ROS. Matrix proteins released into the supernatants were examined by rotary shadowing, quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, and fluorospectrometry for loss of tryptophans and formation of bityrosine residues. At relatively low ROS concentrations, selective liberation of morphologically intact laminin/entactin was found that, however, failed to reassociate and showed oxidative damage of its tryptophan residues. At higher ROS concentrations, laminin and entactin were progressively disintegrated, partially fragmented, and eventually completely degraded. At this point oligomers of type IV collagen predominated in the supernatant, and proteoglycans were not encountered at any concentration of ROS. Similar gradual molecular changes were also obtained when fractions of isolated soluble EHS matrix proteins were incubated with graded concentrations of ROS. In these experiments, the formation of covalently linked oligomers and aggregates paralleled the ROS-dependent formation of cross-linking bityrosine groups. ROS scavengers pinpointed to the hydroxyl radical as the most damaging radical species. Protease inhibitor experiments suggested that degradation of matrix proteins was caused primarily by the direct action of ROS and not by proteolysis by potentially contaminating proteases. Collectively, these results provide evidence that EHS matrix proteins show differential sensitivity to ROS-induced damage in a reproducible, sequential pattern, in the order entactin > laminin > type IV collagen, and that ROS cause partial dissociation and cross-linking of the EHS matrix.

Animals↗
PubMed1991

Rod and cone specific domains in the interphotoreceptor matrix.

The insoluble matrix domain of the interphotoreceptor matrix (IPM) from normal dog, cat, and mouse retinae were characterized using lectin cytochemistry. The lectins WGA (wheat germ agglutinin) and PNA (peanut agglutinin) were used to label interphotoreceptor matrix microdomains in cryosections of retinal tissue and in extracted insoluble matrix. Retinal cryosections and extracted matrix were examined by epifluorescence microscopy and scanning confocal laser microscopy, the latter allowed for the removal of all background fluorescence and gave increased resolution. The insoluble matrix was extracted as a continuous sheet that was comprised of two photoreceptor-specific matrix domains distinguished both by the size of the domains, and by differential binding of WGA and PNA lectins. Each domain encloses a photoreceptor inner and outer segment. Individual rod-associated domains were connected into a hexagonal lattice and this pattern was regularly interrupted by the larger cone-associated domains which have 8-10 surrounding rod domains. The PNA lectin primarily labeled the cone-associated matrix with faint binding to the rod matrix; the WGA lectin labeled both the rod- and cone-associated matrix.

Animals↗
PubMed1995

Partial circumvention of multi-drug resistance by annamycin is associated with comparable inhibition of DNA synthesis in the nuclear matrix of sensitive and resistant cells.

We studied the subcellular and subnuclear distributions of the partially cross-resistant anthracycline Annamycin (Ann) in KB-3-1 and multi-drug resistant KB-VI cells. Subcellular drug localization was assessed qualitatively by fluorescence microscopy and quantitatively by cell fractionation and fluorescence measurements. Doxorubicin (Dox) localized predominantly in the nucleus in KB-3-1 cells and in the membranes in KB-VI cells. In contrast, the subcellular distribution of Ann was identical in both cell lines, with preferential drug localization in the perinuclear region, Golgi apparatus, endoplasmic reticulum and endosomes. Dox rate of efflux from the nucleus was negligible in KB-3-1 cells but markedly enhanced in KB-VI cells, whereas Ann was lost at a similar rate from the nucleus in both cell lines. In KB-3-1 cells Dox levels in the nuclear non-matrix were about 2-fold higher than those of Ann, while in the matrix the inverse relationship was observed. In spite of these differences, Dox and Ann had a similar inhibitory effect on new DNA synthesis in the nuclear matrix and non-matrix of KB-3-1 cells. Dox levels were reduced by 10-fold in the nuclear non-matrix and 2-fold in the matrix in KB-VI cells compared with KB-3-1 cells, whereas Ann levels were reduced by about 2- to 3-fold in the non-matrix and were unchanged in the matrix. In correlation with these findings, Dox did not cause inhibition of new DNA synthesis in either nuclear fraction in KB-VI cells, whereas inhibition of new DNA synthesis in the matrix by Ann was similar in both cell lines. Our results indicate that Ann's partial circumvention of multi-drug resistance is associated with its ability to cause comparable new DNA synthesis inhibition in the nuclear matrix of sensitive and resistant cells.

Antibiotics, Antineoplastic↗
PubMed1993

Pericyte growth and contractile phenotype: modulation by endothelial-synthesized matrix and comparison with aortic smooth muscle.

We compared the effects of endothelial-synthesized matrix and purified matrix molecules on pericyte (PC) and aortic smooth muscle cell (SMC) growth, heparin sensitivity, and contractile phenotype in vitro. When PC are plated on endothelial-synthesized (EC) matrix, cell number is, on average, 3.1-fold higher than identical populations grown on plastic. Under the same conditions, SMC proliferation is stimulated 1.6-fold. Purified matrix molecules, such as collagen type IV (COLL) or fibronectin (FN), both major components of the EC matrix, stimulate PC/SMC growth 1.2-1.7-fold. Heparin (100 micrograms/ml), which inhibits the growth of early passage SMC by 60%, inhibits PC growth approximately 50%, when cells were plated on plastic. However, PC plated on EC matrix in the presence of heparin (100 micrograms/ml) grow as well as parallel cultures grown on plastic (in the absence of heparin). Concomitant with matrix-stimulated proliferation, we observed a marked reduction in PC containing alpha vascular smooth muscle actin (alpha VSMA), as seen by immunofluorescence using affinity-purified antibodies (173/615 positive pericytes on DOC matrix (28%) vs. 221/285 (77%) positive on glass). SMC respond similarly. Whereas alpha VSMA protein is markedly altered when PC and SMC are cultured on EC matrix, similar reductions in mRNA are not observed. However, Northern blotting does reveal that PC contain 17-30 times the steady-state levels of alpha VSMA mRNA compared to SMC. When SMC and PC cultures on plastic are treated with heparin, the steady-state levels of vascular smooth muscle actin mRNA increase 5 and 1.5 fold, respectively. Similarly, heparin treatment of PC grown on plastic induces a 1.8 fold increase in nonmuscle actin mRNA. These heparin-induced alterations in isoactin mRNA levels are not seen when PC are cultured on EC matrix. We also observed reductions in alpha VSMA and beta actin mRNA levels when PC are plated on FN, where they maintain a ratio of 13:1 (alpha:beta). Similar ratios are found in SMC present in rat and bovine aortae in vivo. These steady-state isoactin mRNA ratios are slightly different from those seen in cultured PC (8-10:1; alpha:beta). These results suggest that selective synthesis and remodelling of the endothelial basal lamina may signal alterations in pericyte growth and contractile phenotype during normal vascular morphogenesis, angiogenesis, or during the microvascular remodelling that accompanies hypertensive onset.

Actins↗
PubMed2000

TGF-beta-induced matrix proteins inhibit p42/44 MAPK and JNK activation and suppress TNF-mediated IkappaBalpha degradation and NF-kappaB nuclear translocation in L929 fibroblasts.

The role of transforming growth factor beta1 (TGF-beta1)-induced extracellular matrix proteins in the modulation of cellular response to the cytotoxic effect of tumor necrosis factor (TNF) or Fas ligand was investigated. Murine L929 fibroblasts were prestimulated with or without TGF-beta1 for 1-24 h and the resulting extracellular protein matrices were prepared. Unstimulated control L929 cells were then cultured on these matrices. Compared to control matrix-stimulated L929 cells, the TGF-beta1 matrix-stimulated cells resisted TNF killing in the presence of actinomycin D (ActD), but became more susceptible to killing by anti-Fas antibodies/ActD. The induced TNF resistance is independent of the NF-kappaB antiapoptotic effect. For example, exposure of TGF-beta1 matrix-stimulated L929 cells to TNF failed to result in IkappaBalpha degradation and NF-kappaB nuclear translocation or activation. Also, control matrix stimulated the activation of p42/44 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) in L929 cells, whereas TGF-beta1 matrix suppressed the activation. Nonetheless, in response to TNF, JNK activation was restored in the TGF-beta1 matrix-stimulated cells. By metabolic labeling, ammonium sulfate precipitation and N-terminal amino acid microsequencing, TGF-beta1 was shown to induce a novel matrix protein of 46 kDa (p46) from L929 cells. Adsorption of p46 by peptide antibodies against its N-terminus removed the TGF-beta1 matrix protein-mediated protection against TNF/ActD cytotoxicity and its enhancement of anti-Fas/ActD killing, indicating that p46 is responsible for these effects. Immunostaining of L929 cells revealed that the antibodies were bound to a membrane protein of 100 kDa (p100). Thus, the matrix p46 is likely derived from the released membrane p100.

Amino Acid Sequence↗
PubMed1996

Extracellular matrix penetration by epithelial cells is influenced by quantitative changes in basement membrane components and growth factors.

We have previously shown that isolated mouse fetal choroid plexus epithelial (CPE) cells penetrate a basement membrane matrix (Matrigel) substrate in vitro to form single-layered epithelial vesicles embedded within the matrix. To determine which properties of the matrix are important for inducing or permitting cells to penetrate the substrate and organize into multicellular vesicles we have made quantitative changes to the basement membrane components and growth factors in cell cultures. Matrigel diluted to 33 or 10% with a collagen I gel was not permissive to cell invasion, and CPE cells formed a polarized epithelial monolayer on the substrate surface which had ultrastructural characteristics similar to those of CPE vesicles. Cells in these monolayers proliferated more rapidly than cells in epithelial vesicles. When deliberately embedded within a 33 or 10% Matrigel matrix, CPE cells were able to form vesicles, indicating that a dilute matrix is nonpermissive to cell invasion but promotes epithelial polarization and organization into vesicles. Cells embedded within a 100% collagen I matrix did not proliferate or form epithelial vesicles and the majority of cells did not remain viable. Addition of laminin to the collagen I gel promoted cell adhesion and cell survival, but did not promote the formation of extensive monolayers on the substrate nor the formation of epithelial vesicles within the matrix. Cell invasion into the 33% Matrigel matrix was induced by addition of laminin, nidogen, or a laminin-nidogen complex to the substrate or by addition of TGFbeta2 to the culture medium, but not TGFbeta1 or PDGF. These studies show that CPE cells are sensitive to quantitative changes in matrix composition, which influences their survival and proliferation and also their ability to penetrate the matrix and organize into multicellular epithelial vesicles.

Animals↗