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Results for “Extracellular Matrix”

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PubMed1995

Extracellular matrix proteins modulate cytochrome P450 2D6 expression in human hepatocytes.

The role of extracellular matrix proteins on the expression of human liver-specific genes has been investigated. Cytochrome P450 2D6 and albumin are two liver-specific genes which are constitutively expressed in hepatocytes. Primary human hepatocytes were cultured on different extracellular matrix proteins, namely collagen, fibronectin, a combination of collagen/fibronectin and EHS-laminin rich gel, and under different culture conditions. After 48 h in culture, primary human hepatocytes showed a peak in DNA-synthesis associated with a downregulation of liver-specific expression of albumin and P450 2D6, indicating the dedifferentiation of the hepatocytes. Differentiation and expression of liver-specific genes started to increase subsequently. Redifferentiation as determined by albumin and P450 2D6 levels was more pronounced if hepatocytes were seeded on fibronectin, compared to cells seeded on EHS-laminin rich gel, collagen or a combination of collagen and fibronectin.

Blotting, Northern↗

PubMed1995

Differential effects of aqueous and vitreous on fiber differentiation and extracellular matrix accumulation in lens epithelial explants.

PURPOSE: Results from this and other laboratories strongly suggest that differences in the properties of the ocular media that bathe cells in the anterior and posterior regions of the lens contribute to its normal growth patterns and polarity. The aim of this study was to compare the effects of aqueous and vitreous on the morphology of lens epithelial explants, with particular attention to changes associated with fiber differentiation. METHODS: Light and electron microscopy were used to assess rat lens epithelial explants cultured with bovine aqueous or vitreous. Immunohistochemistry was used to detect fiber-specific crystallins and extracellular matrix components, and synthesis of extracellular matrix was investigated by autoradiography. RESULTS: Vitreous, but not aqueous, induced morphologic changes characteristic of fiber differentiation, which included cell elongation, organelle loss, and the appearance of ball and socket junctions, as well as the accumulation of beta-crystallin. In addition, vitreous stimulated the synthesis and organization of a distinct basement membrane on explants that resembled the lens capsule, both structurally (regular layers of basal laminae) and immunologically (reactive for laminin and heparan sulphate proteoglycan). CONCLUSIONS: Only one of the ocular media, the posteriorly located vitreous, induced lens epithelial explants to undergo morphologic events characteristic of fiber differentiation. This provides further support for the hypothesis that anteroposterior patterns of cellular responses in the lens are caused by differences in the ocular media. The observation that vitreous also stimulated the synthesis and assembly of capsule-like extracellular matrix suggests that vitreous contains factors that may influence lens capsule formation in situ.

Animals↗

PubMed1997

[Study of electron microscopy histochemistry and immunohistochemistry of extracellular matrix in adenoid cystic carcinoma].

In order to determine the character of the extracellular matrix in adenoid cystic carcinoma (ACC), 22 cases of ACC of salivary gland were examined by immunohistochemical methods and 4 cases were examined by electron microscopy histochemically. Immunohistochemistry, type IV collagen and fibronectin were more intensively localized in pseudocyst space and in extracellular matrix between tumor cells. Ultrastructural study showed that the pseudocystic cells had abundant rough endoplasmic reticulum and a well-developed Golgi apparatus, some cells had muscle microfilament. There were laminin, proteoglycan and collagen fibril in the pseudocyst space. The results indicated that type IV collagen and fibronectin were the important stromal component in ACC, which may be secreted by neoplasmic myoepithelial cells.

Carcinoma, Adenoid Cystic↗

PubMed2006

[Extracellular matrix metalloproteinases. Part II].

Remodeling of extracellular matrix is possible due to activity of specific, skilled proteolytic enzymes present in connective tissue. Metalloproteinases (MMPs) are involved most of physiological and pathological processes in human body. To date 28 enzymes have been identified and 23 in humans. This paper is a systemic review of metalloproteinases family. Identification of enzymes in ethiopathogenesis of various diseases is useful in modern therapy planning.

Extracellular Space↗

PubMed2003

Extracellular matrix metabolism in diabetic nephropathy.

Diabetic nephropathy is characterized by excessive deposition of extracellular matrix proteins in the mesangium and basement membrane of the glomerulus and in the renal tubulointerstitium. This review summarizes the main changes in protein composition of the glomerular mesangium and basement membrane and the evidence that, in the mesangium, these are initiated by changes in glucose metabolism and the formation of advanced glycation end products. Both processes generate reactive oxygen species (ROS). The review includes discussion of how ROS may activate intracellular signaling pathways leading to the activation of redox-sensitive transcription factors. This in turn leads to change in the expression of genes encoding extracellular matrix proteins and the protease systems responsible for their turnover.

Animals↗

PubMed1995

Treatment with chemopreventive agents, difluoromethylornithine and retinyl acetate, results in altered mammary extracellular matrix.

The effect of treatment with D-L-2-difluoromethylornithine (DFMO) plus retinyl acetate (RA) on the promotion stage of 1-methyl-1-nitrosourea (MNU)-induced mammary carcinogenesis was evaluated in female Sprague-Dawley rats. Combined treatment was more effective than single agent treatment in decreasing cancer incidence and multiplicity and in prolonging cancer latency. Increased efficacy was associated with reduced morphological complexity of the gland and increased mammary extracellular matrix. Using ovarian hormones to model mitogen stimulation in the mammary gland, DFMO plus RA treatment reduced mammary gland complexity in the absence of an effect on bromodeoxyuridine (BrDU) labeling index measured immunohistochemically. Morphological and biochemical evaluation of these glands revealed increased levels of extracellular matrix in rats treated with chemopreventive agents. Tenascin expression and fibronectin levels were elevated and laminin levels were decreased. The fact that matrix degrading proteinase activity was also increased indicated that tissue remodeling was modulated by these chemopreventive agents. These data provide evidence of alterations in epithelial cell-extracellular matrix interactions that could account in part for the chemopreventive effects of DFMO plus RA.

Animals↗

PubMed1990

[Effect of extracellular matrix and serum components on cellular adhesion and growth in vitro and in vivo].

The recent cell-biological research have been reported the usefulness of extracellular matrix, such as collagen and fibronectin, in cell culture techniques and in vitro experiments. These matrix began to applied in medical treatments and brought many success. Fibroblast and osteoblast have characteristics to promote stabilization of implant materials in vivo. These cells revealed the additional effects to control mending and healing of surgical invasion. The role of extracellular matrix and serum components for cellular adhesion and growth have been discussed extensively. The recent studies reported that collagen and other materials promoted the cellular behavior. The author discussed the effect of extracellular matrix and serum components or the established cell lines on cellular adhesion and growth, which were performed in vitro and in vivo. The following cell lines were employed in this study, fibroblastic cell lines from mouse (3T12-3) and human (SF-TY), osteogenic cell line from mouse (MC3T3-E1) and osteogenic cell lines from human (SAOS-2, NY), osteoblastic cell line (HuO-3N1) and rabbit cell line. Different types of collagen were used as the coating material on glass surface by the method of collagen-gel culture method. Fetal calf serum, bovine serum albumin, fibronectin and proteoglycan were also employed in this study. After the cultivation, adherent cells were stained by Gimsa's method and counted microscopically. In cellular-adhesion test, the most cell number was found on the collagen type I coated glass surface to compare with the other materials, such as fetal calf serum, bovine serum albumin, proteoglycan, employed in all the tested cell lines. Growth of the defined cell-number on glass surface was compared among used materials, and all the osteogenic cell lines and fibroblastic cell lines were found to grow well on collagen type I-fibronectin, collagen type I, and fibronectin coated glass surface to compare with other materials employed in this study. Collagen-fibronectin and collagen-coated alumina ceramic tubes were embedded in the mandibular bones of rabbits, and the aspect was observed with the passage of time. These treatments promoted the osseous area in the tubes. These findings strongly indicated that surface-coated biomaterial by collagen and collagen-fibronectin enhanced cellular adhesion, and promoted the growth of fibroblast and osteoblast in vitro and in vivo.

Animals↗

PubMed1990

Follow-up of the serum levels of extracellular matrix components in acute and chronic pancreatitis.

Time-dependent serum concentrations of extracellular matrix proteins were studied in 32 patients with pancreatitis in order to find potential markers of the reparative response during the disease. Patients were subdivided by clinical and biochemical criteria: severe acute pancreatitis (n = 10), moderate acute pancreatitis (n = 17), and acute attack of chronic pancreatitis (n = 5). Serum and plasma samples were collected on days 1-7, 10, 14, and 21 for measurements of the aminoterminal propeptide of type III procollagen (PIIINP), hyaluronic acid, laminin, fibronectin, and routine clinical-chemical parameters. During an acute attack of chronic pancreatitis all parameters were within the reference range. In moderate acute pancreatitis concentrations of PIIINP, laminin, and hyaluronic acid fluctuated around the upper reference limit, but declined to mid-normal levels at day 21. In severe acute pancreatitis all three parameters increased. In patients who died as a consequence of sepsis and multi-organ failure the increase in PIIINP, laminin and hyaluronic acid was much more pronounced and paralleled by a decrease in plasma concentrations of fibronectin. In conclusion, this study revealed a relation between the severity of acute pancreatitis and the increase in serum concentrations of extracellular matrix components, especially PIIINP.

Acute Disease↗

PubMed2000

Immunohistochemistry of the extracellular matrix of the normal equine lamina cribrosa.

Purpose To use immunohistochemical techniques to identify and localize the structural macromolecules of the extracellular matrix (ECM) of the normal adult equine lamina cribrosa in order to make comparisons to the extracellular matrix of the lamina cribrosa of horses with glaucoma. METHODS: Normal eyes of five adult horses between 5 and 10 years of age were fixed in 10% neutral buffered formalin and embedded in paraffin. Polyclonal rabbit-derived antibodies against human elastin, laminin, fibrillin-1, and collagen types I, III and IV, and polyclonal goat-derived antibodies against collagen type VI were used as primary antibodies. Transverse and longitudinal histologic sections of the optic nerve head and lamina cribrosa were stained using several dilutions of the primary antibodies, biotinylated link antibody, horseradish peroxidase-labeled streptavidin, and 3,3'-diaminobenzidine as a chromogen. The immunohistochemical staining patterns were qualitatively interpreted. RESULTS: The normal adult horse lamina cribrosa labeled positively for collagen types I, III and VI, laminin, elastin and fibrillin. Collagen type VI staining of the laminar ECM was most intense, followed by labeling for collagen types III and I, respectively. Laminar blood vessels were weakly positive for laminin and slightly positive for type IV collagen. The scleral ECM of the laminar insertion zone had more intense labeling for collagen types I and VI than did the laminar plates. CONCLUSIONS: The extracellular matrix of the laminar plates of the adult equine lamina cribrosa is similar to the dog as it consists of elastic and collagen fibers (with collagen types VI, III and I). Both the normal dog and horse lamina display more intense staining of collagen type VI than is found in the ECM of the normal human lamina cribrosa. The macromolecular structure of the equine lamina cribrosa suggests that it is a very resilient structure that may provide some protection to the optic nerve axons during episodes of elevated intraocular pressure.

Journal Article↗

PubMedDate not supplied

Puromycin aminonucleoside and adriamycin disturb cytoskeletal and extracellular matrix protein organization, but not protein synthesis of cultured glomerular epithelial cells.

Puromycin aminonucleoside (PA) and Adriamycin (ADR) cause glomerular proteinuria associated with degenerative alterations of glomerular visceral epithelial cells (GVEC) and detachment from the glomerular basement membrane when administered to rats. This in vitro study was performed to define, in detail, the quantitative and qualitative changes of a number of adhesion-associated proteins (cytoskeletal, extracellular matrix and integrin proteins) upon exposure to PA and ADR. By immunofluorescence we observed: (1) dose- and incubation-time-dependent filament pattern changes and decreased staining of the cytoskeletal proteins actin, vimentin, keratin, and beta-tubulin; (2) an altered distribution, and decreased expression of the extracellular matrix proteins laminin and heparan sulfate and (3) a loss of the beta 1-integrin focal adhesions upon exposure to PA and ADR. Using an ELISA, a concentration-dependent decrease was found (a 50% reduction with 50 micrograms/ml PA for 48 h and with 2 micrograms/ml ADR for 24 h) in the production of cytoskeletal and extracellular matrix proteins per cell. These general effects were suggestive of a disturbance of protein synthesis but, by metabolic labelling studies, no reduction in overall protein synthesis was found. Using two-dimensional PAGE on 35S-methionine steady-state labeled cells, no changes were found in intracellular protein patterns of PA- and ADR-treated cells (pH 5-7.5, MW 110-20 kD). We hypothesize that exposure of GVEC in vitro to PA and ADR might result, directly or indirectly, in perturbation of the macromolecular organization of cytoskeletal and extracellular matrix proteins with loss of GVEC adhesion.

Animals↗

PubMed1992

Isolation, characterization, and localization of cardiac collagen type VI. Associations with other extracellular matrix components.

We have isolated and characterized collagen type VI from murine, canine, and nonhuman primate hearts. In the three species studied, collagen type I was the major collagenous component of the cardiac interstitium (80% of total collagen), whereas collagen type VI represented approximately 5% of total collagen. To define the exact distribution of collagen type VI and its possible interactions with other components of the cardiac extracellular matrix, collagen types I, III, IV, and VI, laminin, and fibronectin were localized in the rat myocardium by immunohistochemistry, using monospecific antibodies. In the rat myocardium, collagen type VI was prevalent in the media and adventitia of muscular arteries, in fine connective tissue septa, in the area surrounding capillaries, and in the delicate endomysium in proximity to myocardial cells. When compared with the immunohistochemical localization of collagen types I, III, and IV, laminin, and fibronectin, the continuity and hierarchical organization of the cardiac extracellular matrix became apparent. The matrix forms a continuous network extending from the pericardium to the endocardium. Furthermore, there is an arborescent hierarchy in the system such that collagen type I is more prevalent in the wider septa, collagen type III being more obvious in medium-sized branches, and fibronectin and collagen type VI prevailing in the terminal (pericellular) aspects of the network. In this pericellular location, fibronectin and collagen type VI, by means of specific interactions, may act as anchor components linking the myocardial cell basement membranes not only to the extracellular matrix but also to the cardiac interstitial cells. This continuity, organization, and coupling of the cardiac extracellular matrix appears well suited to integrate and distribute the physical stress generated by the continuous contraction and relaxation of the myocardium.

Animals↗

PubMed2001

Expression of three extracellular matrix degradative enzymes in bladder cancer.

The relationship between expression of extracellular matrix degradative enzymes, angiogenesis and survival of multistage bladder cancer was determined. Expression of 3 extracellular matrix degradative enzymes (metalloproteinase-2, -9 and heparanase) and microvessel formation were examined in 40 resected bladder cancer specimens by immunohistostochemic staining, and then the association of the enzyme expression with angiogenesis and various stages of cancer was investigated. Heparanase protein expression in muscular invasive or lymph-node metastatic cancer was significantly higher than in superficial or nonmetastatic cancer, respectively (69% vs. 8%, p < 0.001, and 80% vs. 40%, p = 0.028, respectively). Interestingly, heparanase was expressed at much higher levels than matrix metalloproteinase-2 and -9. The mean microvessel count in cancers with heparanase expression was significantly higher than that in cancers without heparanase expression (32.3 +/- 18.2 vs. 5.5 +/- 6.1, p = 0.0008). The microvessel formation was not associated with the expression of matrix metalloproteinase-2 and -9. The cancer-specific and overall survival rates of patients with heparanase expression were significantly lower than those of patients without it (p = 0.0001 and p = 0.0008, respectively). Multivariate analysis showed that heparanase expression was a significantly independent prognostic factor for both cancer-specific (p = 0.0047) and overall survival (p = 0.0200). Our study suggested that heparanase plays important roles in invasion, angiogenesis and metastasis of bladder cancer, and thus, this molecule could be a new molecule to inhibit invasion, angiogenesis and metastasis of bladder cancer. Moreover, our results indicate that expression of heparanase could be a new prognostic factor of this disease.

Adult↗

PubMed2001

Immunoelectron microscopic localization of the neural recognition molecules L1, NCAM, and its isoform NCAM180, the NCAM-associated polysialic acid, beta1 integrin and the extracellular matrix molecule tenascin-R in synapses of the adult rat hippocampus.

We have investigated the possibility that morphologically different excitatory glutamatergic synapses of the "trisynaptic circuit" in the adult rodent hippocampus, which display different types of long-term potentiation (LTP), may express the immunoglobulin superfamily recognition molecules L1 and NCAM, the extracellular matrix molecule tenascin-R, and the extracellular matrix receptor constituent beta1 integrin in a differential manner. The neural cell adhesion molecules L1, NCAM (all three major isoforms), NCAM180 (the largest major isoform with the longest cytoplasmic domain), beta1 integrin, polysialic acid (PSA) associated with NCAM, and tenascin-R were localized by pre-embedding immunostaining procedures in the CA3/CA4 region (mossy fiber synapses) and in the dentate gyrus (spine synapses) of the adult rat hippocampus. Synaptic membranes of mossy fiber synapses where LTP is expressed presynaptically did not show detectable levels of immunoreactivity for any of the molecules/epitopes studied. L1, NCAM, and PSA, but not NCAM180 or beta1 integrin, were detectable on axonal membranes of fasciculating mossy fibers. In contrast to mossy fiber synapses, spine synapses in the outer third of the molecular layer of the dentate gyrus, which display postsynaptic expression mechanisms of LTP, were both immunopositive and immunonegative for NCAM, NCAM180, beta1 integrin, and PSA. Those spine synapses postsynaptically immunoreactive for NCAM or PSA also showed immunoreactivity on their presynaptic membranes. NCAM180 was not detectable presynaptically in spine synapses. L1 could not be found in spine synapses either pre- or postsynaptically. Also, the extracellular matrix molecule tenascin-R was not detectable in synaptic clefts of all synapses tested, but was amply present between fasciculating axons, axon-astrocyte contact areas, and astrocytic gap junctions. Differences in expression of the membrane-bound adhesion molecules at both types of synapses may reflect the different mechanisms for induction and/or maintenance of synaptic plasticity.

Animals↗

PubMed1995

Increased central nervous system production of extracellular matrix components and development of hydrocephalus in transgenic mice overexpressing transforming growth factor-beta 1.

A number of important neurological diseases, including HIV-1 encephalitis, Alzheimer's disease, and brain trauma, are associated with increased cerebral expression of the multifunctional cytokine transforming growth factor-beta 1 (TGF-beta 1). To determine whether overexpression of TGF-beta 1 within the central nervous system (CNS) can contribute to the development of neuropathological alterations, a bioactive form of TGF-beta 1 was expressed in astrocytes of transgenic mice. Transgenic mice with high levels of cerebral TGF-beta 1 expression developed a severe communicating hydrocephalus, seizures, motor incoordination, and early runting. While unmanipulated heterozygous transgenic mice from a low expressor line showed no such alterations, increasing TGF-beta 1 expression in this line by injury-induced astroglial activation or generation of homozygous offspring did result in the abnormal phenotype. Notably, astroglial overexpression of TGF-beta 1 consistently induced a strong upmodulation of the extracellular matrix proteins laminin and fibronectin in the CNS, particularly in the vicinity of TGF-beta 1-expressing perivascular astrocytes, but was not associated with obvious CNS infiltration by hematogenous cells. While low levels of extracellular matrix protein expression may assist in CNS wound repair and regeneration, excessive extracellular matrix deposition could result in the development of hydrocephalus. As an effective inducer of extracellular matrix components, TGF-beta 1 may also contribute to the development of other neuropathological alterations, eg, the formation of amyloid plaques in Alzheimer's disease.

Animals↗

PubMed1994

Proliferation and extracellular matrix synthesis of smooth muscle cells cultured from human coronary atherosclerotic and restenotic lesions.

OBJECTIVES: The purpose of this study was to examine the proliferative capacity and extracellular matrix synthesis of human coronary plaque cells in vitro. BACKGROUND: Common to both primary atherosclerosis and restenosis are vascular smooth muscle cell proliferation and production of extracellular matrix proteins. The applicability to humans of experimental animal models of these processes has been questioned. METHODS: Primary atherosclerotic and restenotic lesions were excised by percutaneous directional coronary atherectomy in 93 patients. Smooth muscle cells were cultivated by an explant technique and identified by their morphology in culture, ultrastructural features under electron microscopy and immunostaining using monoclonal antibodies to smooth muscle cell alpha-actin. Proliferation in secondary culture was assessed with growth curves and the synthesis of collagen and sulfated glycosaminoglycans by the incorporation of 3H-proline and 35S-sulfate, respectively. These studies were also performed in cells derived from human umbilical artery media. RESULTS: Success rates for primary (45%) and secondary (12%) culture of coronary cells were not influenced by clinical variables or lesion category. Primary culture success was improved by the presence of organized thrombus in the plaque and in relation to increased maximal cell density of the atherectomy specimen. Restenotic cells displayed more rapid growth than did cells of primary atherosclerotic origin, which grew in a manner similar to that of umbilical artery cells. Mean calculated population-doubling times for the three cell groups were 52 h (95% confidence interval [CI] 48 to 58 h), 71 h (95% CI 62 to 83 h) and 74 h (95% CI 65 to 84 h), respectively. Restenotic and primary atherosclerotic cells did not differ in the synthesis of collagen ([mean +/- SEM] 0.034 +/- 0.004 vs. 0.033 +/- 0.004 nmol isotope.microgram protein-1, p = NS) or sulfated glycosaminoglycans (11.47 +/- 1.07 vs. 15.37 +/- 3.10 nmol isotope.microgram protein-1, p = NS), but the coronary cells synthesized significantly more collagen and sulfated glycosaminoglycans than did umbilical artery cells (0.019 +/- 0.004 and 5.43 +/- 1.00 nmol isotope.microgram protein-1, respectively, both p < 0.05). CONCLUSIONS: These data indicate that increased smooth muscle cell proliferation contributes to coronary restenosis in humans and support the concept that the extracellular matrix synthesis of adult smooth muscle cells is important to lesion formation.

Cell Division↗

PubMed2000

Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones.

The changes in tissue architecture that accompany the development of breast cancer have been the focus of investigations aimed at developing new cancer therapeutics. As we learn more about the normal mammary gland, we have begun to understand the complex signaling pathways underlying the dramatic shifts in the structure and function of breast tissue. Integrin-, growth factor-, and steroid hormone-signaling pathways all play an important part in maintaining tissue architecture; disruption of the delicate balance of signaling results in dramatic changes in the way cells interact with each other and with the extracellular matrix, leading to breast cancer. The extracellular matrix itself plays a central role in coordinating these signaling processes. In this review, we consider the interrelationships between the extracellular matrix, integrins, growth factors, and steroid hormones in mammary gland development and function.

Animals↗

PubMed1983

Homology of bone-inductive proteins from human, monkey, bovine, and rat extracellular matrix.

Allogeneic implantation of rat extracellular demineralized diaphyseal bone matrix in subcutaneous sites induces a sequence of events resulting in the local differentiation of endochondral bone. However, xenogenic subcutaneous implantation of human, monkey, and bovine extracellular bone matrix into rat showed that bovine matrix had only a weak capacity to induce bone, whereas human and monkey matrix had none at all. This suggested that extracellular matrix-induced bone differentiation is apparently species-specific. We recently reported that the extraction of matrix with 4 M guanidine X HCl resulted in complete removal of the ability to induce endochondral bone differentiation, with the biological activity of the matrix being again restored when the extracted active matrix components (less than 50,000 daltons) were reconstituted with the inactive residue. To define the possible biochemical basis of species specificity, human, monkey, and bovine extracellular bone matrices were extracted with 4 M guanidine X HCl and the extracts were reconstituted with biologically inactive rat residue and bioassayed. The results were similar to those obtained with intact matrices and showed that total extracts of bovine matrix had a weak capacity to induce bone, whereas corresponding extracts of human and monkey matrix did not induce bone. However, partial purification by gel filtration of 4 M guanidine X HCl extracts from each species followed by reconstitution of the different fractions with inactive rat residue resulted in bone induction by all species from fractions containing proteins of less than 50,000 daltons. These observations demonstrate that species specificity of xenogenic extracellular bone matrix is due to immunogenic or inhibitory components (or both) in the guanidine X HCl residue and solubilized extracellular matrix components of greater than 50,000 daltons. These results imply that there is homology in the bone inductive proteins from human, monkey, bovine, and rat extracellular bone matrices.

Animals↗

PubMed1996

Increased expression of extracellular matrix proteins and decreased expression of matrix proteases after serial passage of glomerular mesangial cells.

The cellular events causing pathological extracellular matrix (ECM) accumulation in vivo are not well understood. Prolonged serial passage of several cell types in culture leads to increased production of extracellular matrix (ECM) proteins, but the mechanism for these putative fibrotic changes is not known. Here, human fetal glomerular mesangial cells were subjected to serial passage (P) in culture and the expression of ECM proteins, proteases and protease inhibitors was comprehensively evaluated. From P11 through P14, a series of phenotypic changes occurred. Steady-state expression of mRNA for alpha 1 chains of type III and type IV (but not type I) collagen, and for laminin beta 1 and gamma 1, increased 2- to 8-fold, while expression of mRNA for interstitial collagenase (MMP-1) and gelatinase A (MMP-2) virtually ceased. Expression of tissue-type plasminogen activator (tPA) mRNA also decreased markedly. Expression of mRNA for the tissue inhibitor of metalloproteinases (TIMP)-1, and of the smaller of two mRNA species for the PA inhibitor PAI-1, ceased by P14. There was a switch in expression of the two species of TIMP-2 mRNA: whereas the ratio of signal intensity comparing the 3.5 kb mRNA species to the 1.0 kb species was 5:1 up to P11, it was reversed (1:5) at P14 and later. Serial passage also led to changes in protein expression, with increased type IV collagen and laminin, but decreased interstitial collagenase and gelatinase A. The cells showed a progressive increase in staining for type IV collagen. These findings define the appearance of a matrix-accumulating phenotype in later-passage mesangial cells. Matrix expansion in vivo has been associated with increased transforming growth factor (TGF)-beta synthesis; the cells were found to show at least 5-fold increased expression of TGF-beta 1 mRNA from P8 to P16. However, treatment of P9 or P10 cells with graded doses of TGF-beta 1 increased expression of both collagen IV and gelatinase A mRNA and did not alter the ratio of signal intensity for TIMP-2 mRNA species. Thus, assumption of a matrix-accumulating phenotype by these cultured fetal glomerular mesangial cells is not accelerated by exogenous TGF-beta. These data describe an in vitro model of mesangial cell matrix turnover in which matrix accumulation could result from a concerted increase in ECM synthesis and decrease in ECM degradation.

Cell Culture Techniques↗