Biomedical subjects

M G Kinsella

Publications and source records attributed to M G Kinsella.

At least 19 recordsLinked to original sources

PubMed1998

Heparan sulfate proteoglycans mediate a potent inhibitory signal for migration of vascular smooth muscle cells.

Migration of vascular smooth muscle cells (SMCs) is a key step in vascular remodeling and formation of pathological lesions in diseased arteries and may be controlled by extracellular matrix (ECM) and by factors that regulate ECM composition, such as platelet-derived growth factor (PDGF). In culture, PDGF-AB and -BB enhance but PDGF-AA (although having no effect alone) suppresses SMC migration stimulated by other PDGF isoforms. To determine whether the migration-inhibitory mechanism of PDGF-AA was mediated by ECM composition, we examined baboon SMC migration in a Boyden chamber assay using filters coated with different ECM proteins. PDGF-AA suppressed the PDGF-BB-induced migration of baboon SMCs on a filter coated with basement membrane proteins (Matrigel) and fibronectin but failed to inhibit cell migration on a type I collagen (Vitrogen)-coated filter. Fibronectin and fibronectin fragments that contain heparin-binding domains permitted PDGF-AA inhibition of cell migration, but a fragment lacking heparin-binding domains did not. Treatment of SMCs with heparin lyases II and III, but not with chondroitin ABC lyase, diminished the PDGF-AA-mediated inhibition of migration. PDGF-AA stimulated accumulation of proteoglycan (PG) in the cell layer more potently than did PDGF-BB, whereas the turnover of cell layer PG was unaffected by either PDGF-AA or -BB. Northern blot analysis revealed that PDGF-AA increased syndecan-1 mRNA expression more than did PDGF-BB, whereas both PDGF isoforms decreased perlecan expression. The changes in cell migration and PG synthesis induced by PDGF-AA were accompanied by changes in the morphology of SMCs. PDGF-AA dramatically induced the spreading of SMCs, whereas the heparin lyase treatment of PDGF-AA-stimulated cultures diminished cell spreading. The data suggest that PDGF-AA selectively modifies heparan sulfate PG accumulation on SMCs and thereby influences the interactions of SMCs with heparin-binding ECM proteins. These interactions, in turn, generate signals that suppress SMC migration.

Animals↗

PubMed1997

Genistein selectively inhibits platelet-derived growth factor-stimulated versican biosynthesis in monkey arterial smooth muscle cells.

Platelet-derived growth factor (PDGF) stimulates not only the proliferation and migration of arterial smooth muscle cells (ASMCs), but also the transcription, translation, and posttranslational processing of versican, a large chondroitin sulfate proteoglycan present in the extracellular matrix of blood vessels. PDGF receptor tyrosine kinase activity is required for signaling events associated with mitogenic and motogenic stimulation of cells by PDGF. Therefore, we have asked if inhibiton of tyrosine kinase activity by genistein also blocks the stimulation of both versican core protein synthesis and glycosaminoglycan (GAG) chain modifications induced by PDGF in ASMCs. The tyrosine kinase inhibitor, genistein, in a dose-dependent manner, reversibly inhibits PDGF-stimulated ASMC cell proliferation and RNA and core protein expression of versican, without affecting the expression of decorin and biglycan. In contrast, genistein does not affect the increase in GAG chain elongation that is induced by PDGF. This suggests that different aspects of the biosynthesis of versican are differentially regulated. To determine if such differential regulation involves downstream activation of protein kinase C, ASMCs were treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to directly activate this kinase. In comparison to PDGF stimulation, TPA has little effect on expression of versican mRNA expression, nor does TPA stimulate ASMC cell proliferation. However, like PDGF, TPA increases [35S]sulfate incorporation into proteoglycans and GAG chain elongation. These results indicate that PDGF-induced GAG chain elongation, which is not inhibited by genistein treatment and is stimulated by protein kinase C activation, involves signaling pathways different from those that regulate PDGF-stimulated versican mRNA and protein expression.

Animals↗

PubMed1997

Selective expression and processing of biglycan during migration of bovine aortic endothelial cells. The role of endogenous basic fibroblast growth factor.

Repair of the vascular lumenal surface after injury requires a controlled endothelial cell response that includes cell migration, proliferation, and remodeling of the extracellular matrix. These cellular processes are modulated by growth factors that are released or activated following cell injury. When endothelial cell migration is stimulated in response to monolayer wounding in vitro, cells increase synthesis of small leucine-rich dermatan sulfate proteoglycans (PGs) (Kinsella, M. G., and Wight, T. N. (1986) J. Cell Biol. 102, 679-687). However, the identity of the PGs that are increased during cell migration and the factors that affect this modulation have not been identified. We now report that basic fibroblast growth factor (bFGF) is responsible for the transient increase of [35S]sulfate incorporation into PGs following monolayer wounding. SDS-polyacrylamide gel electrophoresis analysis revealed that bFGF-treated and wounded cultures increase both biglycan core protein synthesis and biglycan proteolytic processing, which results in the accumulation of a approximately 20-kDa N-terminal biglycan fragment in the culture media. Biglycan RNA steady-state levels also selectively increase 2- to 3-fold after wounding or bFGF treatment. Finally, immunocytochemical staining localizes biglycan to the tips and edges of lamellopodia on migrating cells, indicating that biglycan is found at loci at which the formation and dissolution of adhesion plaques occurs, consistent with hypotheses that predict involvement of biglycan in the control of cell migration. Taken together, these results suggest that release of endogenous bFGF is primarily responsible for altered biglycan expression, synthesis, and proteolytic processing as endothelial cells migrate after wounding.

Animals↗

PubMed1997

Localization of perlecan (or a perlecan-related macromolecule) to isolated microglia in vitro and to microglia/macrophages following infusion of beta-amyloid protein into rodent hippocampus.

The origin of the heparan sulfate proteoglycan (PG), perlecan, in beta-amyloid protein (A beta)-containing amyloid deposits in Alzheimer's disease (AD) brain is not known. In the present investigation we used indirect immunofluorescence, SDS-PAGE, and Western blotting with a specific perlecan core protein antibody to identify possible cell candidates of perlecan production in both primary cell cultures and in a rat infusion model. Double and triple-labeled indirect immunofluorescence was performed on dissociated primary rat septal cultures using antibodies for specific identification of cell types and for perlecan core protein. In mixed cultures of both embryonic day 18 (containing neurons and glia) and postnatal day 2-3 (devoid of neurons), microglia identified by labeling with OX-42 or anti-ED1 were the only cell type also double labeled with an affinity-purified polyclonal antibody against perlecan core protein. Similar immunolabeling of microglia with the anti-perlecan antibody was also observed in purified cultures of post-natal rat microglia. Analyses of PGs from cultured postnatal rat microglia by Western blotting using a polyclonal antibody against perlecan core protein revealed an approximately 400 kDa band in cell layer, which was intensified following heparitinase/heparinase digestion, suggestive of perlecan core protein. Other lower Mr bands were also found implicating either degradation of the 400 kDa core protein or the presence of separate and distinct gene products immunologically related to perlecan. Reverse transcription followed by polymerase chain reaction using human perlecan domain I specific primers demonstrated perlecan mRNA in cultured human microglia derived from postmortem normal aged and AD brain. Following a 1-week continuous infusion of A beta (1-40) into rodent hippocampus, immunoperoxidase immunocytochemistry and double-labeled immunofluorescent studies revealed perlecan accumulation primarily localized to microglia/macrophages within the A beta infusion site. These studies have identified microglia/macrophages as one potential source of perlecan (or a perlecan-related macromolecule) which may be important for the ongoing accumulation of both perlecan and A beta in the amyloid deposits of AD.

Amyloid beta-Peptides↗

PubMed1996

Developmental regulation of perlecan gene expression in aortic smooth muscle cells.

Heparan sulfate proteoglycans (HSPGs) are believed to act as potent endogenous regulators of vascular smooth muscle cell (SMC) replication, migration, gene expression and differentiation. Here we describe the pattern of expression of perlecan, the predominant basement membrane HSPG, during aortic development in the rat. Expression of perlecan mRNA and protein in the aortic SMC was first significantly observed at day e19 (day 19 of embryonic development), a time which marks a dramatic switch in SMC replication rate and growth phenotype. Expression of perlecan message and protein was high throughout fetal and early neonatal life, and it remained readily detectable in the adult aorta. Using a double-labeling technique (in situ hybridization for perlecan message coupled with bromodeoxyuridine immunohistochemistry), we determined the relationship between DNA synthesis and perlecan mRNA expression in individual SMC at days e17-e21; we found that perlecan gene expression was largely limited to non-replicating cells. Consistent with the in vivo data, perlecan mRNA was undetectable in cultured e17 SMC by Northern or RT-PCR analysis, while in cultured adult SMC, perlecan mRNA was significantly higher in non-replicating (serum-starved) cultures compared to replicating cultures. Treatment of growth-arrested adult SMC cultures with heparin caused a further accumulation in perlecan mRNA levels. The data suggest that the expression of perlecan by vascular SMC is regulated by apparent developmental age as well as by cellular growth state. The developmentally times expression of perlecan in the aortic wall may contribute to the establishment and/or maintenance of vascular SMC differentiation and quiescence.

Animals↗

PubMed1996

Lung hyaluronan decreases during group B streptococcal pneumonia in neonatal piglets.

Neonatal Group B streptococcus (GBS) sepsis and pneumonia result in lung injury and remain a major cause of morbidity and mortality in the newborn. Increased lung hyaluronan (HA) content is an important component of the lung's early response to damage in diseases such as adult respiratory distress syndrome (ARDS), infant respiratory distress syndrome (IRDS), and bleomycin-induced fibrosis. It is known, however, that GBS virulence factors include specific secretory enzymes such as hyaluronidase, an enzyme which breaks down HA. We therefore hypothesized that in lobar GBS pneumonia, lung HA would be decreased compared with normal values, and that in lobar pneumonia with atelectasis, lung HA would be further decreased because of increased substrate availability. The right lower lobes (RLL) and left lower lobes (LLL) of anesthetized piglets 16 +/- 2 d old were each selectively inoculated with 1 x 10(9) colony-forming units (CFU) GBS via an endobronchial catheter (n = 7). The LLL was subsequently collapsed by endobronchial occlusion following 10 min of 100% O2. Control animals (n = 6) was anesthetized, instrumented, and ventilated without exposure to GBS. At 4 h, lungs were removed and HA extracted and assayed using a competitive inhibition assay. HA extracted from areas of lobar pneumonia was significantly decreased (27 +/- 6.6 micrograms/g wet lung, p < 0.005) when compared with control values of control piglets (51 +/- 19.6 micrograms/g wet lung). Atelectasis plus lobar pneumonia further decreased lung HA to 10 +/- 13.3 micrograms/g wet lung, p < 0.0001. We conclude that lobar GBS decreases lung HA and that this process is augmented by collapsed lung regions, and speculate that this departure from the usual early lung response to injury contributes to GBS invasion of lung parenchyma.

Animals↗

PubMed1995

Differential binding of vascular cell-derived proteoglycans (perlecan, biglycan, decorin, and versican) to the beta-amyloid protein of Alzheimer's disease.

Previous studies have demonstrated the immunolocalization of perlecan, a specific heparan sulfate proteoglycan, to the beta-amyloid protein (A beta)-containing amyloid deposits within the walls of blood vessels (i.e., congophilic angiopathy) in Alzheimer's disease (AD) brain. In the present investigation, the differential binding of previously characterized endothelial cell (EC)- and smooth muscle cell (SMC)-derived PGs to A beta was examined to determine whether the accumulation of A beta in cerebrovascular amyloid deposits may be due to its interactions with perlecan. Pretreatment of AA amyloidotic splenic and liver tissue sections with synthetic A beta (1-28) produced strong immunoreactivity with A beta antibodies at tissue sites enriched in perlecan which was partially removed by pretreatment with heparitinase, but not by chondroitin ABC lyase. [35S]-Sulfate labeled proteoglycans (PGs) derived from cultured ECs and SMCs bound to affinity columns containing A beta (1-28) or (1-40), with virtually no binding to A beta (40-1) (reverse peptide), beta-amyloid precursor protein (410-429), or bovine serum albumin. Characterization of EC and SMC PGs bound to A beta (1-28) revealed strong binding by perlecan, weak binding by decorin and biglycan, two dermatan sulfate proteoglycans, and lack of binding by versican/PG-M, a large chondroitin sulfate proteoglycan. Binding of 125I-labeled perlecan to A beta (1-28) was strongly inhibited by isolated perlecan and to a lesser extent by heparin, but not by chondroitin-6-sulfate or unsulfated dextran sulfate. Heparitinase treatment decreased, but did not eliminate the binding of 125I-labeled perlecan to A beta (1-28). Scatchard analysis of the interaction of A beta (1-28)- and EC-derived perlecan in solid-phase assays indicated high-affinity (Kd = 8.3 x 10(-11) M) and lower-affinity (Kd = 4.2 x 10(-8) M) binding sites, with approximately 1 mol of perlecan binding 1.8 mol of A beta. A significant decrease in binding of EC-derived perlecan to A beta (1-28) was observed when a sequence within the putative heparin-binding motif of A beta (His13His14Gln15Lys16) was replaced by the uncharged peptide sequence, Gly13Gly14Gln15Gly16, indicating a perlecan binding site on A beta near the postulated alpha-secretase site (at Lys-16). Overall, the results indicate that specific vascular cell-derived PGs differentially interact with A beta, and that the interactions of highest affinity occur between A beta and binding sites on both the core protein and glycosaminoglycan chains of perlecan.(ABSTRACT TRUNCATED AT 400 WORDS)

Alzheimer Disease↗

PubMed1995

Migration of bovine aortic smooth muscle cells after wounding injury. The role of hyaluronan and RHAMM.

The migration of smooth muscle cells is a critical event in the pathogenesis of vascular diseases. We have investigated the role of hyaluronan (HA) and the hyaluronan receptor RHAMM in the migration of adult bovine aortic smooth muscle cells (BASMC). Cultured BASMC migrated from the leading edge of a single scratch wound with increased velocity between 1 and 24 h. Polyclonal anti-RHAMM antisera that block HA binding with this receptor abolished smooth muscle cell migration following injury. HA stimulated the random locomotion of BASMC and its association with the cell monolayer increased following wounding injury. Immunoblot analysis of wounded monolayers demonstrated a novel RHAMM protein isoform that appeared within one hour after injury. At the time of increased cell motility after wounding, FACS analysis demonstrated an increase in the membrane localization in approximately 25% of the cell population. Confocal microscopy of injured monolayers confirmed that membrane expression of this receptor was limited to cells at the wound edge. Collectively, these data demonstrate that RHAMM is necessary for the migration of smooth muscle cells and that expression and distribution of this receptor is tightly regulated following wounding of BASMC monolayers.

Animals↗

PubMed1994

Changes in hyaluronan deposition during early respiratory distress syndrome in premature monkeys.

Increased deposition of hyaluronan (HA) is part of the early response to fibrogenic stimulus in the lung exposed to bleomycin injury and has been associated with increased lung water in adult animals. Early respiratory distress syndrome (RDS) in premature infants is characterized by increased lung water, and late sequelae include fibrosis or bronchopulmonary dysplasia. We hypothesized that increased HA in the alveolar interstitium would be associated with increasingly severe RDS in prematurely delivered monkeys and that modes of therapy that affect severity of disease such as treatment with high-frequency oscillatory ventilation or exogenous surfactant would decrease this response. Thirty-four Macaca nemestrina monkeys were delivered at 134 +/- 1 d (term = 168 d) and randomized to high-frequency oscillatory ventilation or conventional mechanical ventilation from birth. Sixteen of these animals received surfactant. At 6 h of age, the right lower lung was frozen in situ during inflation to 30 cm H2O (approximately 2940 Pa) and then dehydrated and processed for microscopy. The presence and severity of RDS were evaluated by clinical and morphologic criteria. HA concentrations in lung extracts increased with progressively severe RDS (p = 0.0003). Treatment with high-frequency oscillatory ventilation decreased the lung injury score (1.69 +/- 0.7 compared with 2.5 +/- 0.9, p = 0.05), but changes in lung HA concentration did not reach significance (37.9 +/- 22.7 compared with 44.8 +/- 22.6). Surfactant treatment decreased lung HA concentration (29.6 +/- 19.0 micrograms/wet lung) compared with non-surfactant-treated animals (54.7 +/- 20.2 micrograms/g wet lung, p = 0.0009).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Interleukin-1 beta regulation of fibroblast proteoglycan synthesis involves a decrease in versican steady-state mRNA levels.

This study investigates the effects of interleukin (IL)-1 beta on proteoglycan metabolism by fibroblasts surrounded by endogenous extracellular matrix. In both three-dimensional matrix cultures and long-term monolayer cultures IL-1 beta caused a significant decrease in synthesis and deposition of sulphated proteoglycans, but had no effect on release of deposited material. The decrease in synthesis became successively more pronounced, and corresponded to 40-60% of the control after 72 h incubation. The reduction was almost totally accounted for by an effect on the chondroitin ABC-lyase-sensitive proteoglycans. Gel electrophoresis showed a significant decrease in a high-molecular-mass chondroitin ABC-lyase-sensitive proteoglycan after incubation with IL-1 beta. Northern-blot analyses of total RNA revealed a pronounced decrease in the steady-state mRNA levels of versican, the large chondroitin sulphate, with levels corresponding to 10-30% of controls. In comparison, the steady-state mRNA level for decorin, the major sulphated proteoglycan synthesized by the cells, was only slightly affected. The prominent decrease in synthesis of sulphated proteoglycans induced in long-term fibroblast cultures, including the pronounced decrease in versican steady-state mRNA levels, is likely to have a significant effect on the structure of the extracellular matrix. Induction of this type of change may constitute a significant mechanism whereby IL-1 beta can affect the properties of connective tissue during inflammation and wound healing.

Blotting, Northern↗

PubMed1993

Platelet-derived growth factor and transforming growth factor-beta 1 differentially affect the synthesis of biglycan and decorin by monkey arterial smooth muscle cells.

Platelet-derived growth factor (PDGF) and transforming growth factor-beta 1 (TGF-beta 1), two growth-regulatory peptides with opposite effects on arterial smooth muscle cell (ASMC) proliferation, were examined for their influence on the synthesis of two small chondroitin sulfate/dermatan sulfate proteoglycans (CS/DS PGs) called biglycan and decorin. Quiescent ASMCs treated with either PDGF or TGF-beta 1 for 24 hours increased [35S]sulfate incorporation into biglycan 3.3- and 2.9-fold, respectively, whereas the incorporation of [35S]sulfate into decorin was not significantly affected. Treatment with TGF-beta 1 but not PDGF more than doubled the steady-state level of messenger RNA (mRNA) transcripts hybridizing to a complementary DNA (cDNA) encoding biglycan. Both growth factors had little or no effect on steady-state levels of mRNA transcripts hybridizing to a decorin cDNA. Incorporation of [35S]sulfate into biglycan glycosaminoglycan (GAG) was maximal by 12 to 18 hours after either PDGF or TGF-beta 1 addition. Both PDGF and TGF-beta 1 increased the molecular sizes of biglycan and decorin. This increase was a result of the synthesis of longer GAG chains substituted on the core proteins of both PGs. PDGF but not TGF-beta 1 led to an increase of more than twofold in the ratio of 6'- to 4'-sulfated disaccharides in these newly synthesized GAG chains. These results indicate that PDGF and TGF-beta 1 have specific but different effects on the synthesis of small CS/DS PGs by monkey ASMCs in culture.

Animals↗

PubMed1993

Alterations in nonhuman primate (M. nemestrina) lung proteoglycans during normal development and acute hyaline membrane disease.

Proteoglycans (PGs) and lung hyaluronan (HA) are important components of the lung matrix both during normal development and in response to injury. We combined morphologic and biochemical techniques to study changes in PG and HA in a developmental series of Macaca nemestrina lungs ranging from 62% gestation to 3 mo post-term (n = 16), in adult lungs (n = 6), and from prematurely delivered, mechanically ventilated monkeys with hyaline membrane disease (HMD) (n = 7). Three groups of cuprolinic blue-positive (CuB) precipitates, identified by size, location, and susceptibility to enzyme digestion were found in lungs from all animals. Immature alveolar interstitium is characterized by loosely woven collagen bundles and an abundance of large (100 to 200 nm) stained filaments representing chondroitin sulfate proteoglycans (CSPGs). As maturation proceeds, the interstitial matrix appears increasingly organized, with large collagen bundles associated with 20 nm CuB-stained deposits (dermatan sulfate proteoglycans, DSPGs), and fewer large CSPGs. Fetal alveolar basement membrane contains CuB-stained heparin sulfate proteoglycans (HSPGs) (10 nm) scattered throughout. Lung matrix from animals with HMD appeared to have a disruption of the collagen-DSPG relationship, in addition to an enrichment in large CSPG. Complementary biochemical analysis of lung PGs and HA was done. Minced lung parenchyma was cultured with [3H]-glucosamine and [35S]-sulfate for 24 h; PGs and HA were extracted and analyzed. While PG synthesis during development tended to be highest at 80% gestation, animals with HMD showed greatly increased synthesis, approximately 2.5-fold higher than comparable fetal animals. In the developmental series, [3H]-glucosamine incorporation into HA was maximal at term, falling abruptly thereafter. HMD animals, however, showed a 2.3-fold increase over controls in net HA synthesis. Extracted PGs were separated according to buoyant density by dissociative cesium chloride density gradient ultracentrifugation. Two peaks of 35S-labeled PGs were separated from each density gradient fraction by chromatography on Sepharose CL-4B. A large CSPG was the principal PG eluting in the voiding volume, while the second broad peak (K(av) = 0.42) contained a mixed population of CSPG, DSPG, and HSPGs, the proportions of which varied with age. Both ultrastructural and biochemical analyses indicate that production of a large, high buoyant density CSPG predominates in fetal lung tissue, and diminishes with developmental age. Synthesis of large CSPG is greatly increased in lung explants from prematurely delivered animals with HMD.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗

PubMed1993

Degradation of endothelial cell matrix heparan sulfate proteoglycan by elastase and the myeloperoxidase-H2O2-chloride system.

The degradation of the heparan sulfate proteoglycans of subendothelial matrix by neutrophil elastase and the myeloperoxidase-H2O2-chloride system added separately, sequentially, or together at pH 4.5 to 7.5 was determined by the release of lower molecular weight 35S-labeled material. Elastase alone and the myeloperoxidase system alone caused degradation, and when 4-hour exposure to elastase was followed by 15 minutes of exposure to the myeloperoxidase system, the effect was greater than additive. A greater than additive effect was not observed when elastase followed the myeloperoxidase system or the two were added together. Chloride (or sulfate) alone increased the release of 35S-labeled material from elastase-treated matrix, although the effect of 0.1 M chloride was not as great as that observed when an equivalent concentration of chloride was combined with myeloperoxidase and H2O2. The release of these systems at sites of adherence of neutrophils to glomerular basement membrane may contribute to neutrophil-associated proteinuria.

Animals↗

PubMed1992

Expression of decorin by sprouting bovine aortic endothelial cells exhibiting angiogenesis in vitro.

In our recent studies, we have demonstrated that monolayer cultures of bovine aortic endothelial (BAE) cells that do not express type I collagen also fail to express and synthesize decorin, a small chondroitin/dermatan sulfate proteoglycan that interacts with type I collagen and regulates collagen fibrillogenesis in vitro. However, BAE cells exhibiting a spontaneous sprouting phenotype and a predisposition toward the formation of cords and tube-like structures (an in vitro model for angiogenesis) initiate the synthesis of type I collagen during their morphological transition from a polygonal monolayer to an angiogenic phenotype. In the present study, we examined whether BAE cells also initiate the synthesis of the proteoglycan decorin during this morphological transition. We show by Northern blot analysis and by immunochemical methods that BAE cell cultures containing sprouting cells and cords, but not monolayer cultures of these cells, express and synthesize decorin (M(r) approximately 100,000). We also show that type I collagen expression by BAE cell cultures is initiated concomitantly. However, the localization of decorin and type I collagen in cord and tube-forming BAE cell cultures is not completely identical. Type I collagen is detected only in sprouting BAE cells and in endothelial cords, whereas decorin is also apparent in BAE cells surrounding the cords and tubes. Our results indicate that the synthesis of decorin as well as type I collagen is associated with endothelial cord and tube formation in vitro.

Animals↗

PubMed1992

The role of proteoglycans in cell adhesion, migration and proliferation.

Proteoglycans comprise a part of the extracellular matrix that participates in the molecular events that regulate cell adhesion, migration and proliferation. Their structural diversity and tissue distribution suggest a functional versatility not generally encountered for other extracellular matrix components. This versatility is mainly dictated by their molecular interactions and their ability to regulate the activity of key molecules involved in several biological events. This molecular cooperativity either promotes or inhibits cell adhesion, migration and proliferation. A growing number of studies indicate that proteoglycans can play a direct role in these cellular events by functioning either as receptors or as ligands for molecules that are required for these events to occur. Such studies support a role for proteoglycans as important effectors of cellular processes that constitute the basis of development and disease.

Animals↗

PubMed1992

Modulation of proteoglycan metabolism by human fibroblasts maintained in an endogenous three-dimensional matrix.

This report describes synthesis and degradation of proteoglycans by human gingival fibroblasts growing in an endogenous three-dimensional matrix. Cells grown in the matrix cultures demonstrated a high rate of proteoglycan synthesis, varying between 2 and 4 times that of cells maintained in monolayer cultures. In addition, the relative amount deposited into the cell layer was increased in the matrix cultures, constituting 70% to 90% of the synthesized material during the first 24 h. Comparable levels for the monolayer cultures were 30% to 60%. The majority of the 35S-sulfate-labeled material in both matrix (80%) and monolayer (62%) cultures was susceptible to chondroitin ABC-lyase digestion. The major product was a low Mr (120,000) proteoglycan which could be immunoprecipitated by an antibody against PGII (decorin). In addition, the cells synthesized two chondroitin ABC-lyase-sensitive proteoglycans, one with Mr greater than 400,000, one with an apparent Mr of 250,000, as well as two heparan sulfate proteoglycans with Mr greater than 250,000. The low Mr dermatan sulfate, decorin, was also the major component deposited in the three-dimensional matrix, constituting about 60% of the total sulfate incorporation. In contrast, fibroblasts in monolayer cultures deposited only a small amount (13%) of decorin (PGII) in the cell layer, and the major proteoglycan in this compartment was heparin sulfate. The rate of release of the newly deposited proteoglycans was the same in the two culture conditions, although material released from the three-dimensional matrix cultures contained small Mr components indicating a higher degree of degradation. These studies show differences in proteoglycan metabolism by gingival fibroblasts grown in an endogenous matrix and in monolayer cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗

PubMed1991

Differential expression of small chondroitin/dermatan sulfate proteoglycans, PG-I/biglycan and PG-II/decorin, by vascular smooth muscle and endothelial cells in culture.

Cultured bovine aortic smooth muscle (BASM) and endothelial (BAE) cells, like articular chondrocytes, synthesize two populations of small chondroitin/dermatan sulfate (CS/DS) proteoglycans (PGs) of similar size as PG-I/biglycan and PG-II/decorin. However, Northern blot analyses demonstrated that BAE cells express detectable amounts of mRNA transcripts only for PG-I/biglycan, whereas BASM cells and articular chondrocytes express mRNA transcripts for both PG-I/biglycan and PG-II/decorin. Endothelial cells from human umbilical vein also expressed detectable amounts of mRNA transcripts only for PG-I/biglycan, and not PG-II/decorin. Antiserum raised against bovine PG-II/decorin immunoprecipitated an apparent single PG species with relative molecular mass (Mr) of approximately 120,000-180,000 from BASM cell and articular chondrocyte cultures but failed to immunoprecipitate an equivalent PG species from BAE cell cultures, consistent with the results from Northern blot analysis. In contrast, immunoprecipitations by antisera to PG-I/biglycan indicated that cultured endothelial cells synthesize two forms of PG-I/biglycan with Mr values slightly larger than 200,000 and 120,000-140,000. It is likely, based on the magnitude of the size difference, that these two forms of PG-I/biglycan differ in the number of glycosaminoglycan chains. Additionally, BASM but not BAE cells were found to express detectable amounts of mRNA transcripts for type I collagen. The above results indicate that the two main cell types of the vascular wall, endothelial and smooth muscle cells, express different sets of small interstitial CS/DS PGs and that the synthesis of PG-II/decorin by these cells correlates with the expression of type I collagen, a collagen known to interact specifically with this PG. These differences in the expression of extracellular matrix molecules may be important in regulating the cell type-specific functions of endothelial and smooth muscle cells within the vascular tissue.

Animals↗

PubMed1990

Formation of high molecular weight dermatan sulfate proteoglycan in bovine aortic endothelial cell cultures. Evidence for transglutaminase-catalyzed cross-linking to fibronectin.

Three glucuronate-rich dermatan sulfate proteoglycan (DS-PG) subclasses were isolated and previously characterized from bovine aortic endothelial cell cultures (Kinsella, M. G., and Wight, T. N. (1988) J. Biol. Chem. 263, 19222-19231). In the present study, pulse-chase experiments indicate that the DS-PG of highest apparent Mr (approximately 1 x 10(6)), denoted previously as HMW-DS, is a relatively stable component of the endothelial extracellular matrix and is formed at the expense of lower Mr DS-PG species. The formation of HMW-DS is reduced in a dose-dependent manner in the presence of dansylcadaverine, an inhibitor of transglutaminase-catalyzed protein cross-linking, but not when the activity of other cross-linking enzymes such as lysyl oxidase is inhibited. The putative DS-PG precursor to HMW-DS accumulates during inhibition of cross-linking only when lysosomal degradation is also inhibited by ammonium chloride, suggesting that the precursor is degraded rapidly in the absence of cross-linking. HMW-DS is precipitable from endothelial cell monolayer extracts with antibodies against fibronectin, a known transglutaminase substrate. Thus, we conclude that the stability of HMW-DS in the subendothelial matrix in culture depends upon the cross-linking of a low Mr DS-PG precursor to matrical protein(s), including fibronectin, resulting in the formation of a DS-PG subclass of high apparent molecular mass.

Animals↗