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T N Wight

Publications and source records attributed to T N Wight.

At least 55 records · Page 3Linked to original sources
PubMed1996

Heparin causes the accumulation of heparan sulfate in cultures of arterial smooth muscle cells.

Heparin infusion in an experimental animal model of arterial injury causes a significant increase in the proteoglycan (PG) component of the extracellular matrix in the injured arteries (Snow, A. D., Bolender, R. P., Wight, T. N., and Clowes, A. W. (1990) Am. J. Pathol. 137, 313-330). The mechanisms responsible for this heparin-induced increase in arterial PGs are not understood. To address this question, we have examined the effect of heparin on PG synthesis and accumulation by aortic smooth muscle cells in culture. Heparin causes a dose-dependent increase in the accumulation of PGs while the greatest percentage change among the different types of PGs is a doubling of the amount of heparan sulfate (HS) accumulated in the cell layer. There is also a selective enrichment of HS in the trypsin-resistant component of the cell layer indicating a specific modification in intracellular PGs. This change is due to the accumulation of large HS glycosaminoglycans (GAGs) which elute at K(av) approximately 0.3 on Sepharose CL-6B (Mr approximately 50,000) under dissociative conditions and which are absent from the controls. These GAGs are located inside the cell as is indicated by their retention after heparitinase or trypsin treatment of the cell layer. Furthermore, the increased accumulation of labeled HS in steady-state heparin-treated cells above controls does not appear for several hours after the addition of [35S]sulfate, indicating that HS accumulation is due to decreased degradation of the HS and not new synthesis. This possibility is further supported by the fact that degradation of the intracellular large HS in trypsinized cells is delayed for 2 h by incubation with heparin. These results suggest that heparin may reduce the intracellular degradation of HS through competitive inhibition of endogenous heparitinase.

Animals↗
PubMed1996

Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme.

Certain matrix metalloproteinases (MMP) are expressed within the fibrous areas surrounding acellular lipid cores of atherosclerotic plaques, suggesting that these proteinases degrade matrix proteins within these areas and weaken the structural integrity of the lesion. We report that matrilysin and macrophage metalloelastase, two broad-acting MMPs, were expressed in human atherosclerotic lesions in carotid endarterectomy samples (n = 18) but were not expressed in normal arteries (n = 7). In situ hybridization and immunohistochemistry revealed prominent expression of matrilysin in cells confined to the border between acellular lipid cores and overlying fibrous areas, a distribution distinct from other MMPs found in similar lesions. Metalloelastase was expressed in these same border areas. Matrilysin was present in lipid-laden macrophages, identified by staining with anti-CD-68 antibody. Furthermore, endarterectomy tissue in organ culture released matrilysin. Staining for versican demonstrated that this vascular proteoglycan was present at sites of matrilysin expression. Biochemical studies showed that matrilysin degraded versican much more efficiently than other MMPs present in atherosclerotic lesions. Our findings suggest that matrilysin, specifically expressed in atherosclerotic lesions, could cleave structural proteoglycans and other matrix components, potentially leading to separation of caps and shoulders from lipid cores.

Antigens, CD↗
PubMed1996

Distribution of hyaluronan during extracellular matrix remodeling in human restenotic arteries and balloon-injured rat carotid arteries.

BACKGROUND: The glycosaminoglycan hyaluronan (HA) is present in developing tissues and healing wounds and forms a loose, hydrated extracellular matrix (ECM) that promotes processes such as cell migration. To investigate the potential contribution of HA to the pathogenesis of restenosis, we studied (1) human lesions obtained by directional atherectomy and (2) experimentally induced neointima formation in balloon-injured rat carotid arteries. METHODS AND RESULTS: A biotinylated proteoglycan fragment that binds specifically to HA was used to stain atherectomy specimens from 29 human restenotic lesions (mean restenosis interval, 6.0+/-4.4 months) and 8 human primary lesions. The loose myxoid ECM typical of human restenotic arteries demonstrated intense, diffuse staining for HA. The intensity was inversely related to the density of immunostaining for collagen types I and III and was lowest in hypocellular primary atherosclerotic plaque. Among 24 rat carotid arteries retrieved 3, 7, 14, 28, 42, or 56 days after balloon injury and immunostained as well for proliferating cell nuclear antigen, staining for HA in the neointima reached a maximum 7 days after balloon injury and was associated with the presence of proliferating, PCNA-positive smooth muscle cells. CONCLUSIONS: Hyaluronan is a characteristic constituent of the loose myxoid ECM in human restenotic arteries and of the neointima in experimentally injured arteries. The presence of hyaluronan may be a marker for an initial phase of the extracellular matrix remodeling that occurs during the development of a fibroproliferative lesion and could facilitate biological processes such as cell migration.

Animals↗
PubMed1996

A chondroitin sulfate/keratan sulfate proteoglycan, PG-1000, forms complexes which are concentrated in the reticular laminae of electric organ basement membranes.

Previously, we identified PG-1000 as part of a disulfide-linked complex of two large proteoglycans (PG-1000 and the beta component) and three smaller proteins purified from the extracellular matrix of elasmobranch electric organ (Iwata and Carlson, 1991, J. Biol. Chem. 266: 323-333). PG-1000 is a chondroitin sulfate/keratan sulfate proteoglycan with a molecular mass of about 1.2 x 16(6) daltons. When visualized in the electron microscope, PG-1000 has the typical "bottle-brush" appearance expected for a proteoglycan with an average total length of about 345 nm and about 20 chains of approximately 110 nm (Carlson and Wight, 1987, J. Cell Biol. 105: 3075-3086). Using immunocytochemical methods, we now demonstrate that PG-1000 is a component of the interstitial extracellular matrix of the electric organ. PG-1000 immunoreactivity is found throughout the interstitial matrix, but it is highly concentrated in that region of the matrix immediately adjacent to the basal lamina, the reticular lamina. The reticular and basal laminae together form the basement membrane. PG-1000 immunoreactivity is especially apparent on basal laminae that surround nerve fibers and nerve terminals. When the disulfide-linked PG-1000 complexes are purified and examined in the electron microscope following rotary shadowing, they appear as bottle-brush structures which are often attached at a central region and radiate like spokes of a wheel. These aggregates contain two to six proteoglycan monomers. We hypothesize that the PG-1000 complexes are disulfide-stabilized parts of an extended network of linked proteoglycans in the reticular lamina.

Animals↗
PubMed1996

Distinct rat aortic smooth muscle cells differ in versican/PG-M expression.

Smooth muscle cells (SMCs) with distinct phenotypes are present in blood vessels, and distinct culture types appear when SMCs are maintained in vitro. For example, cultured SMCs from rat adult media grow as bipolar cells, which differ in gene expression from the predominantly cobblestone-shaped SMCs from rat pup aortas and rat neointimas that we call pi SMCs. Since proteoglycans are present at different concentrations in the normal intima and media and are elevated in atherosclerotic plaque, we sought to determine whether pi and adult medial SMC types synthesize different or unique proteoglycans that are characteristic of each phenotype. [35S]sulfate-labeled proteoglycans were purified by ion-exchange chromatography. An adult medial SMC line synthesized a large proteoglycan (0.2 Kav on Sepharose CL-2B) that was not detectable in a pi SMC line. Digestion of this proteoglycan with chondroitin ABC lyase revealed three core glycoproteins of 330, 370, and 450 kD. By Western blot analysis, the two smallest of these reacted with two antibodies to the human fibroblast proteoglycan versican. RNAs hybridizing to versican probes were found only in adult medial-type SMCs, including an adult medial type clone from pup aorta, by Northern blot analysis. Both SMC types synthesize RNAs that hybridize to probes for other proteoglycans, such as perlecan, biglycan, and decorin. We conclude that rat pi SMC cultures, unlike monkey, human, and rat adult medial SMC cultures, express little or no versican. This difference in expression may be responsible for the different morphologies and growth properties of the two cell types.

Age Factors↗
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

Fibronectin attachment is permissive for IL-1 mediated gene regulation.

This study examines the effect of cell-matrix interaction on IL-1 induced gene regulation. In fibroblasts and smooth muscle cells attached to fibronectin, IL-1 caused a pronounced reduction in proteoglycan synthesis, while no reduction occurred in cells plated on bare plastic. Further, fibronectin attachment was permissive for IL-1 mediated suppression of both versican and collagen mRNA levels, initially noted after 4-6 h of IL-1 incubation. Attachment to vitronectin was less potent in influencing regulation, and collagen had no effect, suggesting specificity of the matrix modulation of the IL-1 induced response. Similar fibronectin induced dependence was demonstrated for IL-1 regulation of IL-6 gene expression, supporting the notion of a general effect of fibronectin receptor engagement on IL-1 induced signal transduction.

Cell Adhesion↗
PubMed1995

Organized type I collagen influences endothelial patterns during "spontaneous angiogenesis in vitro": planar cultures as models of vascular development.

Selected strains of vascular endothelial cells, grown as confluent monolayers on tissue culture plastic, generate flat networks of cellular cords that resemble beds of capillaries--a phenomenon referred to as "spontaneous angiogenesis in vitro". We have studied spontaneous angiogenic activity by a clonal population (clone A) of bovine aortic endothelial cells to identify processes that mediate the development of cellular networks. Confluent cultures of clone A endothelial cells synthesized type I collagen, a portion of which was incorporated into narrow, extracellular cables that formed a planar network beneath the cellular monolayer. The collagenous cables acted as a template for the development of cellular networks: flattened, polygonal cells of the monolayer that were in direct contact with the cables acquired spindle shapes, associated to form cellular cords, and became elevated above the monolayer. Networks of cables and cellular cords did not form in a strain of bovine aortic endothelial cells that did not synthesize type I collagen, or when traction forces generated by clone A endothelial cells were inhibited with cytochalasin D. In a model of cable development, tension applied by a confluent monolayer of endothelial cells reorganized a sheetlike substrate of malleable type I collagen into a network of cables via the formation and radial enlargement of perforations through the collagen sheet. Our results point to a general involvement of extracellular matrix templates in two-dimensional (planar) models of vascular development in vitro. For several reasons, planar models simulate invasive angiogenesis poorly. In contrast, planar models might offer insights into the growth and development of planar vascular systems in vivo.

Animals↗
PubMed1995

The extracellular matrix and atherosclerosis.

The vascular extracellular matrix is a collection of vastly different macromolecules organized by entanglement and cross-linking into a biomechanically active polymer that imparts regional tensile strength, viscoelasticity, and compressibility to the atherosclerotic lesion. Each component of the extracellular matrix possesses unique structural properties that determine its separate roles during atherogenesis. Not only does this matrix provide the architectural framework that influences the structural integrity of the lesion, but it also provides the milieu for vascular cells and participates in the adhesive, proliferative, and migratory events that characterize lesion development. The fact that different components of the extracellular matrix selectively bind plasma proteins, growth factors, cytokines, and enzymes implicate these molecules in the regulation of key metabolic events in the formation of the atherosclerotic plaque. The extracellular matrix can no longer be thought of as simply an inert structural mass, but rather as a collection of molecules that possesses the capacity to 'instruct' and 'transduce the information' that 'drives' events central to the atherogenic process. A more thorough understanding of the nature and properties of the vascular extracellular matrix and the factors that regulate its accumulation would seem to be a reasonable goal if progress is to be made towards alleviating this disease.

Arteriosclerosis↗
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

Characterization of proteoglycans synthesized by murine embryonal carcinoma cells (P19) reveals increased expression of perlecan (heparan sulfate proteoglycan) during neuronal differentiation.

Proteoglycans (PGs) incorporated into cell layer and secreted into media were characterized during retinoic acid-induced neuronal differentiation of cultured P19 murine embryonal carcinoma cells. Heparan sulfate significantly increased (P < 0.01) in cell layer following neuronal differentiation of P19 cells by 3.9-fold. CL-4B gel chromatography revealed the major PGs present in cell layer of stem cells eluted as a broad peak with a Kav = 0.65, and was susceptible to chondroitin ABC lyase. The chondroitin ABC lyase resistant material eluted as a broad peak between Kav = 0.40 and Kav = 0.60, and was only partially digested with heparitinase/heparinase (with resistant material eluting at Kav = 0.70). Therefore, the cell layer of stem cells contained primarily chondroitin sulfate/dermatan sulfate (CS/DS) PGs, with lesser amounts of heparan sulfate proteoglycans (HSPGs). This was confirmed by SDS-PAGE. The CS/DS PGs in the cell layer of stem cells had an apparent M(r) of approximately > 200 kDa, and the HSPGs had an apparent M(r) of approximately 140-230 kDa. In contrast, the major PGs in the cell layer of neurons consisted primarily of HSPGs, with only a minor proportion of CS/DS PGs. Furthermore, both gel filtration chromatography and SDS-PAGE analysis revealed a larger HSPG in the cell layer of neurons (Kav = 0.3-0.6 on CL-4B following chondroitin ABC lyase digestion; M(r) 170 kDa- > 400 kDa on SDS-PAGE) in comparison to stem cells (Kav = 0.4-0.6 on CL-4B following chondroitin ABC lyase digestion; M(r) 140-230 kDa on SDS-PAGE). Likewise, the major PGs secreted into media of stem cells consisted almost exclusively of CS/DS PGs, with lesser amounts of HSPGs, whereas an increase in HSPGs in the media of neurons was apparent. Western, Northern, and immunocytochemical analysis demonstrated that mRNA transcript and protein levels for a specific HSPG (i.e., perlecan) markedly increased in cell layer following P19 neuronal differentiation. Perlecan core protein was identified by Western blot analysis using specific monoclonal and polyclonal antibodies, as a large HSPG with a core protein of apparent M(r) approximately 370-400 kDa, and was observed primarily in extracts from neurons. Northern blot analysis with a cDNA to perlecan revealed a significant (P < 0.01) 12.7-fold increase in expression of perlecan in neurons (day 9) in comparison to stem cells. The increase in perlecan message during P19 neuronal differentiation was concomitant with a significant (P < 0.01) 26.3-fold increase in message for beta-amyloid precursor protein (beta PP).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Identification of the proteoglycan versican in aorta and smooth muscle cells by DNA sequence analysis, in situ hybridization and immunohistochemistry.

Versican is a large chondroitin sulfate proteoglycan (CSPG) initially identified in cultured human fibroblasts. Previous studies have shown that there is a versican-like molecule in cultured monkey smooth muscle cells. In this study, we have cloned and sequenced the large CSPG from cultured monkey smooth muscle cells, fetal and juvenile monkey aorta, and human fetal aorta. The cDNA sequence from human fetal aorta is completely homologous to the human fibroblast versican. We obtained 2.5 kb of cDNA sequence from monkey aortic RNA and cultured monkey smooth muscle cell RNA. This sequence covers three distinct domains of versican (hyaluronic acid binding domain, glycosaminoglycan attachment domain and protein binding domain) and demonstrates over 90% homology to the human versican sequence. In situ hybridization histochemistry indicates that the versican RNA transcript is located in the epithelium throughout the tunica media of the aorta. Western blot analysis and immunohistochemistry also confirm the presence of versican in human and monkey aorta.

Amino Acid Sequence↗
PubMed1994

Regional differences in the distribution of the proteoglycans biglycan and decorin in the extracellular matrix of atherosclerotic and restenotic human coronary arteries.

Proteoglycans are important constituents of blood vessels and accumulate in various forms of vascular disease. Little is known concerning the proteoglycan composition of restenotic lesions formed after angioplasty and whether the proteoglycan composition of these lesions differs from that of primary atherosclerosis. Accordingly, we sought to characterize the distribution of two proteoglycans, biglycan and decorin, in primary atherosclerotic and restenotic lesions of human coronary arteries. Restenosis (n = 37) and primary (n = 11) lesions obtained from 48 patients by directional atherectomy of human coronary arteries were stained with antibodies against biglycan and decorin. To further characterize the extracellular matrix of restenotic tissues, we studied the co-distribution of these proteoglycans with collagen types I, III, and IV. The loose fibroproliferative tissue seen predominantly in restenosis lesions consistently stained positively for biglycan in patterns of deposition ranging from disseminated to homogeneous. The density and intensity of biglycan staining was correlated with the density of collagen type I and III fiber networks, both of which were observed to interweave among the loose fibroproliferative tissue. The compact connective tissue of primary atherosclerotic plaque was characterized by strong biglycan staining which co-localized with intense collagen type I and III staining. Only basement membrane-like structures rich in collagen type IV demonstrated negative biglycan staining. In contrast, loose fibroproliferative tissue exhibited no significant staining for decorin. Strong immunostaining for decorin, however, was found in primary atherosclerotic plaque. There are thus regional differences in the distribution of extracellular matrix proteoglycans of restenotic and primary human atherosclerotic lesions; these observations suggest that differences established for the biological roles of biglycan and decorin in other organ systems may extend as well to pathologically altered human coronary arteries.

Angioplasty, Balloon↗
PubMed1994

Smooth muscle cell expression of extracellular matrix genes after arterial injury.

Accumulation of extracellular matrix (ECM) after arterial injury is an important event in the development of intimal thickening and is modulated by heparin. To investigate the regulation of matrix protein expression, we have analyzed messenger RNA levels by Northern blotting for various ECM proteins in the rat carotid artery balloon injury model. RNA was extracted from normal arteries and from intima-medial preparations at 2 days, 1 week, 2 weeks, and 4 weeks after balloon injury of arteries in animals receiving either saline or heparin infusion. Transcripts for the heparan sulfate proteoglycans perlecan, syndecan, and ryudocan; the chondroitin sulfate proteoglycan versican; the dermatan sulfate proteoglycan biglycan; type I procollagen; and tropoelastin all were increased on Northern blots beginning at 1 week after injury. By in situ hybridization, the transcripts for elastin nd biglycan were primarily localized to smooth muscle cells in the intima and were diminished by heparin in proportion to the decrease in intimal mass. Other matrix genes (perlecan, ryudocan) were expressed in the intima and media and were not affected by heparin. The results support the conclusion that ECM gene expression is a relatively late event in the response of the carotid artery, and that some of the genes are expressed only in the intima whereas others are expressed in both the intima and media.

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

A brain extracellular matrix proteoglycan forms aggregates with hyaluronan.

Unlike many tissues, the adult central nervous system extracellular matrix (ECM) has few known components. Previously, we characterized a large chondroitin sulfate proteoglycan, pgT1, from adult rat brain which has the properties of a general brain ECM component and is immunologically distinct from aggrecan and versican (Iwata, M., and Carlson, S.S. (1993) J. Neurosci. 13, 195-207). In this study we demonstrate that pgT1 binds hyaluronan with relatively high affinity. The pgT1 preparation isolated from rat brain aggregates in non-denaturing conditions. This aggregation is abolished by incubation of pgT1 with Streptomyces hyaluronidase. Examination of these aggregates by electron microscope reveals a structure in which an average of 18 subunits arise laterally from opposite sides of an elongated 350-nm filament. These pgT1 aggregates resemble the proteoglycan aggregates in cartilage which are composed of aggrecan and hyaluronan. Using affinity coelectrophoresis, we measure a dissociation constant (Kd) of 0.9 +/- 0.2 nM for the interaction of pgT1 and hyaluronan. These new findings, combined with the general distribution of pgT1 in brain, suggest that pgT1/hyaluronan aggregates are an extended general structure of the brain extracellular matrix network.

Animals↗
PubMed1993

Partial characterization of proteoglycans synthesized by human gingival epithelial cells in culture.

Proteoglycans (PGs) were extracted from the [35S]-sulfate labelled medium and cell layer of proliferating human gingival epithelial cells and analyzed by ion exchange and molecular sieve chromatography, and by SDS-PAGE. The majority of the incorporated radioactivity secreted into the medium eluted from a DEAE Sephacel ion exchange column as a single peak at 0.44 M NaCl with a small shoulder at 0.52 M NaCl. This material, when chromatographed on Sepharose CL-6B contained two species--a quantitatively major peak at K(av) = 0.30 (M(r) congruent to 235,000 on SDS-PAGE) and a quantitatively minor peak at K(av) = 0.39. The major peak was sensitive to alkaline borohydride, shifting to K(av) = 0.45, and nitrous acid degradation, indicating the presence of heparan sulfate PG with glycosaminoglycan chains with M(r) congruent to 26,000. The minor peak is chondroitin/dermatan sulfate PG with glycosaminoglycan chains of M(r) = 22,200 as indicated by sensitivity to alkaline borohydride (shifting to K(av) = 0.48) and chondroitin ABC lyase digestion. The [35S]-sulfate labelled material from the cell layer eluted in a broad peak between 0-0.50 M NaCl from DEAE Sephacel. Chromatography of this material on Sepharose CL-6B revealed the presence of three peaks at K(av) = 0.20, 0.31, and 0.75. The largest peak (K(av) = 0.20 and M(r) congruent to 245,000 on SDS-PAGE) shifted elution position to K(av) = 0.50 after alkaline borohydride treatment and was completely sensitive to nitrous acid degradation. These results indicate that this peak contains heparan sulfate PG with glycosaminoglycan chains of M(r) congruent to 20,000. Two peaks containing [35S]-sulfate labelled glycosaminoglycan chains were detected by chromatography of the cell layer extract over Sepharose CL-6B with K(av)S = 0.42 (M(r) congruent to 30,500) and 0.75 (M(r) congruent to 5300). The larger peak was predominantly chondroitin/dermatan glycosaminoglycan as indicated by susceptibility to chondroitin ABC lyase while the chains at K(av) = 0.75 were predominantly heparan sulfate with 83% susceptibility to nitrous acid. These results indicate that cultured human gingival epithelial cells synthesize and secrete principally heparan sulfate PGs with small amounts of chondroitin/dermatan sulfate PGs. This work will serve as a basis for future studies designed to examine those factors involved in regulation of PG synthesis by these cells.

Cells, Cultured↗