The serpin protease nexin-1 regulates vascular smooth muscle cell adhesion, spreading, migration and response to thrombin.

BACKGROUND: Protease nexin-1 (PN-1) is an important physiological regulator of thrombin in the brain. PN-1 is also present in aortic smooth muscle cells and may thus participate in vascular biology. However, little is known about its function in the vessel wall. OBJECTIVES: In this study, we investigated the effect of PN-1 overexpression in smooth muscle cells (SMCs), on their sensitivity to thrombin, and their capacity for adhesion, spreading and migration. RESULTS: Two clones exhibiting a two- to threefold increase in PN-1 expression were selected and compared with untransfected and mock-transfected cells. Overexpression of PN-1 was observed to inhibit thrombin-induced cell responses as indicated by a twofold decrease in induction of PAI-1 expression, a decreased calcium mobilization in response to low thrombin concentrations and a twofold increase in the capacity to inhibit thrombin catalytic activity. Overexpression of PN-1 did not modify adhesion, spreading, and migration of SMCs on type I collagen. In contrast, SMCs overexpressing PN-1 exhibited a 40% reduction in adhesion, a 50% reduction in spreading and a complete absence of migration on vitronectin when compared with control SMCs. CONCLUSIONS: Our studies thus reveal that PN-1 is likely to play a critical role in regulating essential cell functions such as (i) thrombin-induced responses, which are dependent on its antiprotease activity, and (ii) adhesion, spreading, and migration, which are independent of its antiprotease activity and may be related to its interaction with other partners, such as vitronectin in the present case.

Platelet-derived growth factor inhibits smooth muscle cell adhesion to fibronectin by ERK-dependent and ERK-independent pathways.

The adhesion of cells to the extracellular matrix plays a major role in cell migration. Pretreatment with platelet-derived growth factor (PDGF) inhibited the adhesion of smooth muscle cells to fibronectin by 80%. This inhibition decreased as concentrations of fibronectin increased. In the presence of 200 microm GRGDS peptide, only 45% of PDGF-treated cells adhered to fibronectin compared with 80% of control cells. This indicates that a decrease in integrin avidity was induced by PDGF. Cell adhesion was partially restored when the activation of the extracellular signal-regulated kinase (ERK) was inhibited with PD98059. The remaining inhibition of adhesion (50%) was independent of the fibronectin concentration, suggesting that the ERK pathway is involved in the decrease in integrin avidity. This was confirmed by depleting ERK protein levels by treatment with ERK antisense oligonucleotide. The adhesion of ERK control oligonucleotide-treated cells decreased by 41% when the concentration of GRGDS peptide was increased from 50 to 200 microm but only decreased by 11% in ERK antisense oligonucleotide-treated cells. Treatment with PDGF also delayed focal complex assembly and inhibited stress fiber formation. Consistent with a delay in tyrosine phosphorylation of paxillin, PDGF treatment caused a lag in focal complex formation, although this was not associated with any change in Src family tyrosine kinase activity. Our results indicate that PDGF inhibits smooth muscle cells adhesion by two pathways. The first involves an ERK-dependent decrease in integrin avidity; the second involves the ERK-independent inhibition of focal complex assembly.

Regulatory role of prostaglandin E2 in induction of cyclo-oxygenase-2 by a thromboxane A2 analogue (U46619) and basic fibroblast growth factor in porcine aortic smooth-muscle cells.

U46619, a thromboxane A2 analogue, and basic fibroblast growth factor (FGF-2) both induced the expression of the inducible cyclo-oxygenase (Cox)-2 in porcine aortic smooth-muscle cells. This induction was dose-dependent (submaximal at 300 nM for U46619 and 1 ng/ml for FGF-2) and time-dependent, with similar intensity and maximal expression at 2 h. Under these conditions, both inducers stimulated rapid activation of extracellular signal-regulated kinase (ERK2) at 5-10 min, a transient and lower intensity being induced by U46619 whereas that induced by FGF-2 was sustained (>1 h). PD98059, an inhibitor of the ERK pathway, inhibited the expression of Cox-2. In contrast, activation of Jun-N-terminal kinase (JNK1) was sustained with U46619 but poorly induced by FGF-2. Cox-2 expression induced by U46619 or FGF-2 was similarly reduced by prostaglandin (PGE2), forskolin or dibutyryl-cAMP, suggesting a regulatory effect of adenylate cyclase on Cox-2 expression. However, activation of ERK2 by FGF-2 was not affected by PGE2 whereas that of JNK1 by U46619 was inhibited, suggesting that inhibition of COX-2 expression by cAMP may be downstream of ERK2. The effects of PGE2 and forskolin on Cox-2 and phosphorylation of JNK1 were reversed with the protein kinase A inhibitor H89. In addition, endogenous PGE2 down-regulated the expression of Cox-2 by the two inducers, as stimulation of the cells in the presence of different Cox inhibitors increased the expression of the protein. Overall, these results suggest that exogenous and endogenous PGE2 exert negative inhibitory effects on Cox-2 expression mediated by stimulation of protein kinase A.

Differential up-regulation of Rap1a and Rap1b proteins during smooth muscle cell cycle.

The relationship between Rap1 proteins and cell proliferation was assessed by investigating the effect of isoforms AA and BB of platelet-derived growth factor (PDGF) on Rap1 protein and mRNA expression throughout the smooth muscle cell cycle. Firstly, PDGF BB-induced cell cycle traverse was studied, thus demonstrating entry into S phase at 18 to 20 h. Western blotting carried out on total Rap1 proteins showed that 5 ng/ml of PDGF BB instigated a biphasic induction of total Rap1 proteins during the cell cycle. This involved a 2.1 +/- 0.4-fold increase at 6 h (early G1) and a 2.8 +/- 0.6-fold increase at 20 to 24 h (G1/S transition). Such an up-regulation was abolished by addition of 1 ng/ml of transforming growth factor-beta 1 (TGF-beta 1), which inhibited up to 80% of the PDGF BB-induced entry into S phase. Comparative RT-PCR of both rap1a and rap1b mRNAs throughout the cell cycle allowed us to differentiate between the two rap1a and rap1b species. PDGF BB induced a 1.9 +/- 0.3-fold increase at 4 h and a 2.4 +/- 0.2-fold relative increase at 16 h for rap1b mRNA, whereas a unique 1.9 +/- 0.5-fold increase in rap1a mRNA was observed at 14 h. Again, this induction of rap1a and rap1b mRNAs by PDGF BB was totally abolished by TGF-beta 1. We conclude that the differential up-regulation of Rap1a and Rap1b proteins during the smooth muscle cell cycle is directly linked to cell proliferation.

Transforming growth factor beta 1 inhibits mitogen-activated protein kinase induced by basic fibroblast growth factor in smooth muscle cells.

Stimulation of smooth muscle cells with basic fibroblast growth factor (bFGF) results in the activation of the mitogen-activated protein kinase (MAP kinase) cascade and leads to cell proliferation. We show that transforming growth factor beta 1 (TGF-beta 1), at concentrations that completely inhibited bFGF-induced mitogenic activity, decreased bFGF-induced MAP kinase activity. Under these conditions, tyrosine and threonine phosphorylations of MAP kinase were differentially affected depending on the time period of TGF-beta 1 pretreatment. After a short (30 min) TGF-beta 1 pretreatment, the bFGF-mediated increase in phosphorylation of p42mapk on threonine was inhibited, with no effect on the level of phosphotyrosine or decrease in the electrophoretic mobility of p42mapk. This suggests that TGF-beta 1 inhibited MAP kinase activity through the action of a serine/threonine phosphatase. In contrast, a longer TGF-beta 1 pretreatment (4 h) partly inhibited the bFGF-induced MAP kinase mobility shift and correlated with the inhibition of phosphorylation on both threonine and tyrosine, suggesting that long-term TGF-beta 1 treatment prevented activation of the MAP kinase cascade or directly blocked MAP kinase. The ability of long-term (4 h) but not short-term (30 min) TGF-beta 1 pretreatment to inhibit MAP kinase activity was completely dependent on protein synthesis and suggests that TGF-beta 1 inhibits MAP kinase activity by two distinct mechanisms. These findings provide a molecular basis for the growth-inhibitory action TGF-beta 1 on bFGF-induced mitogenic activity.

In vitro changes in plasma membrane heparan sulfate proteoglycans and in perlecan expression participate in the regulation of fibroblast growth factor 2 mitogenic activity.

Fibroblast growth factor 1 (FGF1) and 2 (FGF2) bind to two classes of receptors: the high affinity receptors, a family of four known transmembrane tyrosine kinases (FGF R1-R4), and the low affinity receptors, cell surface and basement membrane heparan sulfate proteoglycan (HSPG). During early (first and second) passages of retinal pigmented epithelial (RPE) cells, both FGF1 and FGF2 exhibited low mitogenic activity, while in later (fifth to ninth) passages the activity of FGF1 remained constant but FGF2 activity increased two- to threefold. We have investigated aspects of FGF receptor interactions and the role of heparin/heparan sulfate which modulates FGF activity on RPE cells during in vitro senescence. Northern blot analysis demonstrated that FGF receptor type 1 (FGF R1) is the major high affinity receptor expressed in RPE cells and that its level of expression did not change during serially passage. Both the FGF R1 and the FGF low affinity receptors' binding characteristics (i.e., Kd and number of sites per cell) for FGF1 were unaffected by passage number, whereas the capacity of FGF2 binding to FGF R1 and to the low affinity receptors increased by two- and fivefold, respectively, in late passages, although the affinities were unchanged. This change in the capacity of FGF2 to bind to FGF R1 and to HSPG was not due to a switch of the IIIc splice form of FGF R1 to the IIIb splice form since the exon IIIc was the most predominant splice form of FGF R1 during RPE cell cultures. Furthermore the ratio of the IIIb to the IIIc splice form was not modified during cell subcultures. In parallel in the older RPE cell passages, expression of perlecan, the major FGF low affinity binding site localized on the extracellular matrix of RPE cells, was much elevated compared to early RPE cell passages. Moreover, the cell surface of late passage RPE cells had 79% more HSPG than early passage cells. Therefore, it is suggested that the increase in the number of FGF low affinity receptors present on the cell surface or basement membrane could account for a part of the greater proliferative response of aged RPE cells to FGF2.

Transforming growth factor-beta 1 increases internalization of basic fibroblast growth factor by smooth muscle cells: implication of cell-surface heparan sulphate proteoglycan endocytosis.

Basic fibroblast growth factor (bFGF) was internalized by smooth muscle cells (SMC) from pig aorta. Correlation between heparin inhibition of binding and late internalization (8 h) implicated low-affinity sites in bFGF internalization. Transforming growth factor-beta 1 (TGF-beta 1) induced a 38% increase in bFGF internalized between 4 and 8 h. While bFGF and/or TGF-beta 1 enhanced cell-surface proteoglycan synthesis, 35S-labelled proteoglycans of the extracellular matrix (ECM) were not affected. This might be explained by the different turnover rates displayed by the two populations of proteoglycans. Although bFGF and/or TGF-beta 1 induced a similar stimulation in cell-surface chondroitin sulphate/dermatan sulphate and heparan sulphate (HS) proteoglycan synthesis, only the turnover of HS proteoglycans was increased. Twice as much HS proteoglycan was internalized in the presence of TGF-beta 1 or bFGF. Furthermore, TGF-beta 1 induced a 43 +/- 12% increase in HS proteoglycan internalized in the presence of bFGF with a parallel 38% increase in bFGF internalization. Overall, the results indicated that bFGF bound to two HS proteoglycan populations. bFGF storage (70% of bFGF bound to SMC) was not affected by TGF-beta 1 under our conditions and involved ECM proteoglycans characterized by a low turnover. bFGF internalization up-regulated by TGF-beta 1 involved cell-surface HS proteoglycan characterized by a high turnover.

Increased proliferation of bone marrow-derived fibroblasts in primitive hypertrophic osteoarthropathy with severe myelofibrosis.

Pachydermoperiostosis or primary hypertrophic osteoarthropathy (HOA) is a rare congenital growth disorder of connective tissue. We report a case of severe myelofibrosis in a patient with HOA. When cultured in vitro, patient bone marrow-derived fibroblasts displayed a high proliferative potential with a shortened doubling time (24 hours v 36 to 48 hours for normal fibroblasts). The role of platelet-derived growth factor (PDGF), previously implicated in the pathogenesis of secondary acquired myelofibrosis, was studied. HOA fibroblasts expressed an increased number of PDGF-BB binding sites (300,000 sites/cell v 200,000 sites/cell for normal fibroblasts) without any modification of affinity. The increased expression of PDGF-R beta appeared to result from an accelerated rate of PDGF-R beta resynthesis with normal kinetics of endocytosis. As a consequence, a several-fold increase of PDGF-R beta tyrosine kinase activity was observed. No autocrine mechanism of growth was suspected as neither spontaneous PDGF-R beta autophosphorylation nor mitogenic activity in HOA fibroblast-conditioned medium was detected. Patient serum and platelet lysate were less potent than controls in inducing [3H]thymidine incorporation into HOA fibroblasts. This was inconsistent with a paracrine mechanism of growth. In vitro, human serum or PDGF-BB were not more mitogenic for HOA than normal fibroblasts. High levels of cyclin D1, a putative oncogene, were detected in serum-deprived HOA fibroblasts. Cyclin D1 overexpression could be implicated in the accelerated growth of these cells. Our results suggest that the mechanism of fibroblastic proliferation observed in this case of myelofibrosis might differ from those reported in other acquired myeloproliferative syndromes and could be associated with an intrinsic abnormality of HOA fibroblast growth.

Growth-related production of proteoglycans and hyaluronic acid in synchronous arterial smooth muscle cells.

1. The growth-stimulating effect of serum on the proteoglycan and hyaluronic acid production in arterial smooth muscle cells was investigated, using cells synchronized by serum deprivation. 2. After stimulation, synthesis of [35S]sulfated proteoglycans and [14C]hyaluronic acid increased during G1 and G2 phases (about 2- and 5-fold, respectively, in the culture medium), in comparison with quiescent cells. 3. Neither the size, nor the charge, nor the relative proportions of [35S]glycosaminoglycans of the proteoglycans were modified. 4. However, when the cells were stimulated to divide, increased synthesis of large [14C]hyaluronic acid was observed concomitantly with the production of higher hydrodynamic size [35S]proteoglycans, which aggregated with hyaluronic acid (20%).

Stimulation of large proteoglycan synthesis in cultured smooth muscle cells from pig aorta by endothelial cell-conditioned medium.

We have previously shown (Berrou et al., J. Cell. Phys., 137:430-438, 1988) that porcine endothelial cell-conditioned medium (ECCM) stimulates proteoglycan synthesis by smooth muscle cells from pig aorta. ECCM stimulation requires protein cores for glycosaminoglycan chain initiation and is accompanied by an increase in the hydrodynamic size of proteoglycans secreted into the medium. This work investigates the mechanisms involved in the ECCM effect. 1) Control and ECCM stimulated proteoglycan synthesis (measured by a 20 min [35S]-sulfate labeling assay) was not inhibited by cycloheximide, indicating that the proteoglycans were composed of preexisting protein cores and that ECCM stimulates glycosylation of these protein cores. 2) Whereas ECCM stimulation of [35S]-methionine incorporation into secreted proteins only occurred after a 6 h incubation, the increase in [35S] methionine-labeled proteoglycans was observed after 1 h, and the increase was stable for at least 16 h. 3) As analysed by electrophoresis in SDS, chondroitinase digestion generated from [14C] serine-labeled proteoglycans 7 protein cores of high apparent molecular mass (550-200 kDa) and one of 47 kDa. The two protein cores of highest apparent molecular masses (550 and 460 kDa), but not the 47 kDa protein cores, showed increased [14C]-serine incorporation in response to ECCM (51%, as measured by Sepharose CL-6B chromatography). 4) Finally, incorporation of [35S]-sulfate into chondroitinase-generated glycosaminoglycan linkage stubs on protein cores was determined by Sepharose CL-6B chromatography: ECCM did not modify the ratio [35S]/[14C] in stimulated protein cores, indicating that ECCM did not affect the number of glycosaminoglycan chains. The results of these studies reveal that 1) endothelial cells secrete factor(s) that preferentially stimulate synthesis of the largest smooth muscle cell proteoglycans without structural modifications and 2) the stimulation proceeds via increased glycosylation of protein core through enhancement of xylosylated protein core, followed by enhanced protein synthesis.

Changes in proteoglycans of cultured pig aortic smooth muscle cells during subculture.

Smooth muscle cells were cultured from pig aorta. Changes in both the growth and the properties of sulfated proteoglycans were observed during passage. The population doubling time during log phase growth was 34 h from Passages 3 to 7-8 but 20 h at the Passage 11, and the cell density at the stationary phase, was 86,000 and 136,000 cells/cm2 at Passages 3 and 11, respectively. Structural characteristics of sulfated proteoglycans secreted into the medium were investigated after metabolic labeling with [35S]-sulfate. Significant differences were observed with age in vitro: a) [35S]proteoglycan complexes were in a greater amount at Passage 10 than at Passage 3; b) the hydrodynamic size of at least 45% of subunits and about 90% of monomers decreased with in vitro aging; c) this decrease in the size of proteoglycans was partly due to a decrease in the size of their glycanic chains; d) an increase of 15% in the proportion of dermatan sulfate was observed when cells were subjected to 10 passages.

Effects of Ginkgo biloba extract on glucose transport and glycogen synthesis of cultured smooth muscle cells from pig aorta.

We have examined the effect of an extract of Ginkgo biloba (Gbe) on glucose uptake and on glycogen synthesis in cultured smooth muscle cells (SMC) from pig aorta. Initial rates of glucose transport were determined by measurements of 2-deoxy-D-glucose (2-DG) uptake. From kinetic analyses apparent KM and Vmax values of facilitated glucose transport in cultured SMC were evaluated at 2.2 mM and 9.1 nmol/min/10(6) cells respectively. Gbe stimulated glucose transport in a dose-dependent manner; the maximum effect was reached at a Gbe concentration of 0.25 micrograms/ml and represented an increase of 35 +/- 4% above basal activity. This stimulation mainly occurred on facilitated glucose transport. The passive diffusion measured when cells were treated with cytochalasin B represented 15 +/- 3% of glucose total transport activity either in the absence or the presence of Gbe. The effect of Gbe on glycogen synthesis in cultured SMC was then tested by the incorporation of U14C-glucose into cellular glycogen. This process was enhanced by Gbe, the maximal effect was observed at a Gbe concentration of 0.25 micrograms/ml, and represented a 41 +r4% increase above basal activity. These data argue for a direct effect of Gbe upon glucose transport and glucose utilization in cultured SMC thus allowing a better nutriment disposal in the vascular wall.

Effect of endothelial-cell-conditioned medium on proteoglycan synthesis in cultured smooth muscle cells from pig aorta.

The effect of porcine endothelial-cell-conditioned medium on proteoglycan synthesis by pig aorta smooth muscle cells was studied under serum-free conditions. Maximal stimulation of [35S]-sulfate incorporation (50%) into medium-secreted and cell layer proteoglycans was observed after 20 min and 4 h incubation, respectively. This stimulation can be explained neither by increased secretion nor by oversulfation of medium-secreted [35S]-labeled proteoglycans. Those [35S]-proteoglycans secreted (for 24 h) in the presence of endothelial cell-conditioned medium were characterized by a higher hydrodynamic size than those secreted in the presence of control medium, without modification of glycosaminoglycan chain length. Agreement between the stimulation of incorporation of [35S]-sulfate into glycanic chains (50.1%) and [14C]-serine residues associated with glycosaminoglycans (49.9%) involved an increase in the number of glycanic chains linked to protein cores. The lesser stimulation of [14C]-serine incorporation into secreted proteins (18%) suggested that stimulation of glycosaminoglycan synthesis was not the direct consequence of enhanced protein synthesis. Proteoglycan synthesis was studied in the presence of para-nitrophenyl-beta-D-xyloside. Fractionation of medium-secreted [35S]-proteoglycans and xyloside-initiated glycosaminoglycans revealed that stimulation of [35S]-glycosaminoglycan protein core acceptor for glycanic chain initiation. Our results suggest that the factor(s) secreted by endothelial cells are able to modify smooth muscle cell proteoglycan synthesis by stimulating the first step of protein core glycosylation. This stimulation was accompanied by an increase in proteoglycan hydrodynamic size.

Effect of insulin on sulfated proteoglycan synthesis in cultured smooth muscle cells from pig aorta.

The effect of insulin upon proteoglycan synthesis was studied in cultured smooth muscle cells from pig aorta blocked in the G0 phase by serum deprivation. Insulin enhanced [35S]sulfate incorporation into cell layer and medium-secreted proteoglycans. The increase in incorporation of the precursor was not due to a mitogenic response by smooth muscle cells to the hormone and the specific radioactivity of proteoglycans showed that the stimulation reflected a real increase in sulfated proteoglycan synthesis. Maximal stimulation was observed, for the cell layer as well as for the medium, 40 h after the addition of 1.7 x 10(-7) M insulin and reached respectively 65 and 53%. This stimulation was about 80 and 60% of the level achieved with 10% fetal calf serum for cell layer and medium-secreted proteoglycans, respectively. The half-maximal effect was attained, for both the cell layer and the medium, in the presence of 2.1 x 10(-9) M insulin. Proteoglycans secreted into the medium, in the presence of 1.7 x 10(-8) M insulin for 40 h, showed a higher proportion of complexes (24%) than those synthesized in control medium (11%) and at least 95% of the monomers from culture treated with insulin were characterized by a smaller hydrodynamic size than those synthesized by cells maintained in control medium. This decrease in the size of proteoglycans was partly due to a decrease in the size of their glycanic chains.

Role of proteoglycan in the binding of low-density lipoprotein to cultured arterial smooth muscle cells.

The role of proteoglycans in the binding of 125I-labeled low-density lipoproteins (LDL) to cultured arterial smooth muscle cells was examined. About 60% of cell bound 125I-labeled LDL could be released by unlabeled LDL, heparin, dextran sulfate or proteoglycan. Binding of 125I-labeled LDL decreased by about 50% when incubated in the presence of exogenous arterial proteoglycans. Exposure of cell cultures to rho-nitrophenyl-beta-D xyloside resulted in a 40% decrease in both the amount of 35S-labeled proteoglycan in the cell layer and the 125I-labeled LDL binding, without modifying significantly the cell number and amount of cell layer protein. These data suggest that cell surface and/or cell matrix proteoglycans may influence binding of LDL to either specific receptor or non-receptor sites and thereby play a role in the intracellular deposition of lipid in the arterial wall.

Characterization and macromolecular association of proteoglycans produced by pig arterial smooth muscle cells in culture.

1. Medium and cell-layer proteoglycans from pig aorta smooth muscle cells in culture were compared. In both compartments, the main proteoglycans contained chondroitin sulfate-dermatan sulfate chains of 40 kDalton. 2. However, cell-layer proteoglycans differed from those of the medium by the presence of: (a) some small-size proteoglycans; (b) a greater amount of heparan sulfate; (c) chondroitin sulfate-dermatan sulfate enriched in iduronate and in 4 sulfate- (instead of 6 sulfate-) residues. 3. During dissociation-reassociation assays of arterial proteoglycans with exogenous hyaluronate or "aggregate" proteoglycans, the in vitro formation of complexes appeared to involve inter-associations between proteoglycans molecules, in addition to aggregation with hyaluronate.

Synthesis of sulfated proteoglycans throughout the cell cycle in smooth muscle cells from pig aorta.

Cultured smooth muscle cells from pig aorta arrested in G0 phase by serum deprivation were stimulated to proliferate by replacing the medium with one containing 10% serum. Studies in DNA replication and proliferation of cells showed a relatively good synchrony: 90% of the cells were in G1 phase for 16 h after addition of serum; they entered S phase between 18 and 24 h, completed S phase and traversed G2 phase between 24 and 30-32 h; 75% of these cells multiplied after 30-32 h and the remainder were blocked at the end of G2 phase. The synthesis and secretion of sulfated proteoglycans were examined throughout a full cell cycle using metabolic labelling with [35S]sulfate. Smooth muscle cells in G1 or G2 phase synthesized and secreted sulfated proteoglycans with a possible pause at the end of the G2 phase but at the beginning of the S phase and during mitosis the incorporation of [35S]sulfate into these macromolecules stopped entirely. Structural characteristics of sulfated proteoglycans secreted into the medium during G1 phase and an entire cell cycle were investigated. The proportion of proteoglycan complexes and the relative hydrodynamic size of monomers and of constituent subunits of complexes were determined after chromatography on Sepharose CL-2B and CL-6B columns run under both associative and dissociative conditions. No significant differences were observed for the periods of the cell cycle that were studied: [35S]Proteoglycan complexes represented at the end of G1 phase and of the cell cycle respectively 19 and 16% of the total [35S]proteoglycans secreted into the medium. More than 90% of the subunits, obtained after dissociation of complexes, were characterized by a similar kav after chromatography on Sepharose CL-2B columns eluted under dissociative conditions (kav 0.68 at the end of G1 phase and 0.65 at the end of full cell cycle). About 95% of monomers synthesized at the two stages of the cell cycle were eluted at kav 0.25 after chromatography on Sepharose CL-6B column run under associative conditions and were characterized by a similar glycosaminoglycan distribution. These results suggest that smooth muscle cells in culture liberate similar populations of proteoglycans into the medium during the G1 and G2 phases.

The structural characterization of proteoglycans of cultured aortic smooth muscle cells and arterial wall of the pig.

Aortic proteoglycans, from the growth medium of cultured smooth muscle cells and from sequential associative and dissociative extracts of the tissue of origin, the pig aorta, were isolated and purified by precipitation with cetylpiridinium chloride. After isopycnic CsCl gradient centrifugation under associative conditions 94% of the cell-secreted proteoglycans were recuperated in the bottom one fifth (rho av = 1.62 g/ml) fraction. In contrast 80% of the tissue proteoglycans of both extracts, fractionated into two fractions: the bottom one fifth (rho av = 1.60 g/ml) fraction and three fifths (rho av = 1.42 g/ml) fraction. Fractionated tissue proteoglycans were composed predominantly of chondroitin sulfate-dermatan sulfate (83-90%) with lower proportions of heparan sulfate (5-11%) and hyaluronic acid (3-6%) whilst cell-secreted proteoglycans showed a similar glycosaminoglycan composition but with a higher proportion of hyaluronic acid (11-13%). Sepharose 2B and C1-2B chromatography of tissue proteoglycans of high buoyant density showed the presence of only subunit proteoglycans whilst those of intermediate density contained a complex species, partially dissociable in 4 M guanidinium chloride, along with Kav 0.50 subunit species. The latter was also observed for cell-secreted proteoglycans although obtained at high buoyant density. The cell-secreted subunit proteoglycans became separated into two distinct populations when chromatographed on Sepharose 4B and C1-4B, half of which eluted in the column Vo and the rest at a Kav of 0.34. The majority of subunit macromolecules eluted at the Vo fractions of Sepharose 6B and C1-6B columns. Unlike the major species of cartilage proteoglycans, only approx. 20% of purified arterial proteoglycans formed complexes. This proportion could be increased by only 4-7% by interaction, of a mixture of subunit cell-secreted and tissue-extracted proteoglycans, with hyaluronic acid. These results suggest that proteoglycans secreted by cultured aortic smooth muscle cells and present in the aortic tissue possess certain similar physicochemical properties and are present in the form of complex and several subunit species.

Variations in the distribution of glycosaminoglycans in the uterine cervix of the pregnant woman.

A study was made of the modifications of glycosaminoglycans in the uterine cervix and the relationship to gestation. These substances are essential constituents of connective tissue, and a modification of their concentration could affect the physical and chemical characteristics of the cervix. Glycosaminoglycans were extracted from cervical biopsies obtained from pregnant and non-pregnant women. This study showed dermatan sulfte to be quantitatively the most important glycosaminoglycan in the cervix of both the groups studied, and that a significant decrease in the concentration of both dermatan sulfate and chondroitin sulfates occurred in the biopsies obtained just after delivery. This was related to a decrease of collagen in the cervix at the end of gestation, as the proteoglycans containing dermatan sulfate are principally associated with collagen.