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Growing evidence suggests that versican is important in the innate immune response to lung infection. Our goal was to understand the regulation of macrophage-derived versican and the role it plays in innate immunity. We first defined the signaling events that regulate versican expression, using bone marrow-derived macrophages (BMDMs) from mice lacking specific Toll-like receptors (TLRs), TLR adaptor molecules, or the type I interferon receptor (IFNAR1). We show that LPS and polyinosinic-polycytidylic acid [poly(I:C)] trigger a signaling cascade involving TLR3 or TLR4, the Trif adaptor, type I interferons, and IFNAR1, leading to increased expression of versican by macrophages and implicating versican as an interferon-stimulated gene. The signaling events regulating versican are distinct from those for hyaluronan synthase 1 (HAS1) and syndecan-4 in macrophages. HAS1 expression requires TLR2 and MyD88. Syndecan-4 requires TLR2, TLR3, or TLR4 and both MyD88 and Trif. Neither HAS1 nor syndecan-4 is dependent on type I interferons. The importance of macrophage-derived versican in lungs was determined with LysM/ Vcan−/−mice. These studies show increased recovery of inflammatory cells in the bronchoalveolar lavage fluid of poly(I:C)-treated LysM/ Vcan−/−mice compared with control mice. IFN-β and IL-10, two important anti-inflammatory molecules, are significantly decreased in both poly(I:C)-treated BMDMs from LysM/ Vcan−/−mice and bronchoalveolar lavage fluid from poly(I:C)-treated LysM/ Vcan−/−mice compared with control mice. In short, type I interferon signaling regulates versican expression, and versican is necessary for type I interferon production. These findings suggest that macrophage-derived versican is an immunomodulatory molecule with anti-inflammatory properties in acute pulmonary inflammation.

Smooth muscle cell (SMC) proliferation is a key process in stabilization of atherosclerotic plaques, and during restenosis after interventions. A clearer understanding of SMC growth regulation is therefore needed to design specific anti-proliferative therapies. Retinoic acid has been shown to inhibit proliferation of SMCs both in vitro and in vivo and to affect the expression of extracellular matrix molecules. To explore the mechanisms behind the growth inhibitory activity of retinoic acid, we hypothesized that retinoids may induce the expression of perlecan, a large heparan sulfate proteoglycan with anti-proliferative properties. Perlecan expression and accumulation was induced in murine SMC cultures by all-trans-retinoic acid (AtRA). Moreover, the growth inhibitory effect of AtRA on wild-type cells was greatly diminished in SMCs from transgenic mice expressing heparan sulfate-deficient perlecan, indicating that the inhibition is perlecan heparan sulfate-dependent. In addition, AtRA influenced activation and phosphorylation of PTEN and Akt differently in wild-type and mutant SMCs, consistent with previous studies of perlecan-dependent SMC growth inhibition. We demonstrate that AtRA regulates perlecan expression in SMCs and that the inhibition of SMC proliferation by AtRA is, at least in part, secondary to an increased expression of perlecan and dependent upon its heparan sulfate-chains.

Viral infection is an exacerbating factor contributing to chronic airway diseases, such as asthma, via mechanisms that are still unclear. Polyinosine-polycytidylic acid (poly(I:C)), a Toll-like receptor 3 (TLR3) agonist used as a mimetic to study viral infection, has been shown to elicit inflammatory responses in lungs and to exacerbate pulmonary allergic reactions in animal models. Previously, we have shown that poly(I:C) stimulates lung fibroblasts to accumulate an extracellular matrix (ECM), enriched in hyaluronan (HA) and its binding partner versican, which promotes monocyte adhesion. In the current study, we aimed to determine the in vivo role of versican in mediating inflammatory responses in poly(I:C)-induced lung inflammation using a tamoxifen-inducible versican-deficient mouse model (Vcan−/− mice). In C57Bl/6 mice, poly(I:C) instillation significantly increased accumulation of versican and HA, especially in the perivascular and peribronchial regions, which were enriched in infiltrating leukocytes. In contrast, versican-deficient (Vcan−/−) lungs did not exhibit increases in versican or HA in these regions and had strikingly reduced numbers of leukocytes in the bronchoalveolar lavage fluid and lower expression of inflammatory chemokines and cytokines. Poly(I:C) stimulation of lung fibroblasts isolated from control mice generated HA-enriched cable structures in the ECM, providing a substrate for monocytic cells in vitro, whereas lung fibroblasts from Vcan−/− mice did not. Moreover, increases in proinflammatory cytokine expression were also greatly attenuated in the Vcan−/− lung fibroblasts. These findings provide strong evidence that versican is a critical inflammatory mediator during poly(I:C)-induced acute lung injury and, in association with HA, generates an ECM that promotes leukocyte infiltration and adhesion.

Objective— Overexpression of decorin reduces neointimal thickening in balloon-injured carotid arteries of rats by decreasing the volume of neointimal extracellular matrix (ECM). We examined the hypothesis that decorin regulates ECM volume by stimulating cell-mediated contraction of collagen-rich ECMs. Methods and Results— Rat arterial smooth muscle cells (ASMCs) transduced with bovine decorin cDNA by retroviral transfection (LDSN) exhibited enhanced contraction of collagen gels in vitro when compared with vector-only transduced (LXSN) cells. Addition of recombinant decorin to LXSN or LDSN cells did not stimulate contraction of collagen gels. Enhanced contraction of collagen by LDSN cells was unaffected by the metalloproteinase inhibitor GM6001. LDSN cells exhibited increased expression of type I collagen mRNA when compared with that of LXSN cells. Correspondingly, collagen gel contraction by LDSN cells was reduced by inhibition of collagen synthesis by 3,4- l -dehydroproline (L-DHP). Antibodies to α1β1-integrin, but not to α2β1-integrin, blocked collagen contraction by both LXSN and LDSN cells. However, LXSN and LDSN cells expressed similar levels of α1- and β1-integrin mRNAs. Conclusions— Decorin synthesized de novo by ASMCs increases type I collagen synthesis and enhances contraction of collagen gels. Regulated synthesis of decorin may be a useful therapeutic approach to reduce ECM volume in vascular disease.

Objective— Vascular smooth muscle cells (SMCs), activated by growth factors after arterial injury, migrate and proliferate to expand the intima of the blood vessel. During intimal expansion, proliferation is suppressed and an increasingly large proportion of the neointimal mass is composed of newly synthesized extracellular matrix (ECM). We sough to determine whether the ECM heparan sulfate proteoglycan (HSPG) perlecan, which inhibits SMC proliferation in vitro, also accumulates and limits SMC proliferation during neointimal expansion. Methods and Results— Perlecan expression and accumulation were analyzed by immunohistochemistry and in situ hybridization during neointima formation after balloon catheter injury to the rat carotid artery. Perlecan expression was low in uninjured vessels and up to 7 days after injury, during maximal SMC proliferation. By 14 days after injury, perlecan was dramatically increased, and immunostaining remained heavy throughout the advanced lesion, 35 to 42 days after injury. Finally, explants of intimal tissue from 35- to 42-day neointimal lesions were digested with glycosaminoglycanases to determine whether endogenous HSPGs inhibit intimal SMC proliferation. SMCs within HS-depleted, but not chondroitinase ABC–treated or mock-incubated, explants were found to proliferate in response to platelet-derived growth factor BB. Conclusions— HSPGs, such as perlecan, may inhibit the proliferative response of SMCs after vascular injury.

Chemokines are a group of structurally related peptides that promote the directed migration of leukocytes in tissue. Mechanisms controlling the retention of chemokines in tissue are not well understood. In this study we present evidence that two different mechanisms control the persistence of the CXC chemokine, IL-8, in lungs and skin. 125I-labeled IL-8 was injected into the airspaces of the lungs and the dermis of the skin and the amount of 125I-labeled IL-8 that remained at specified times was measured by scintillation counting. The 125I-labeled IL-8 was cleared much more rapidly from skin than lungs, as only 2% of the 125I-labeled IL-8 remained in skin at 4 h whereas 50% of the 125I-labeled IL-8 remained in lungs at 4 h. Studies in neutropenic rabbits showed that neutrophils shortened the retention of 125I-labeled IL-8 in skin but not lungs. A monomeric form of IL-8, N-methyl-leucine 25 IL-8, was not retained as long in lungs as recombinant human IL-8, indicating that dimerization of IL-8 is a mechanism that increases the local concentration and prolongs the retention of 125I-labeled IL-8 in lungs. These observations show that the mechanisms that control the retention of IL-8 in tissue include neutrophil migration and dimerization, and that the importance of these varies in different tissues.

Abstract —Decorin is a member of the family of small leucine-rich proteoglycans that are present in blood vessels and synthesized by arterial smooth muscle cells (ASMCs). This proteoglycan accumulates in topographically defined regions of atherosclerotic lesions and may play a role in the development of this disease. However, little is known about whether decorin has specific effects on the cellular events that contribute to atherosclerotic lesion formation. In the present study, rat ASMCs were transduced with a retroviral vector (LDSN) that carries the bovine decorin gene. Compared with vector control cells (LXSN), these cells constitutively overexpress decorin, as verified by Northern and Western analysis and by metabolic labeling. Experiments were performed to examine the responsiveness of decorin-overexpressing rat ASMCs to platelet-derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1), 2 growth factors that affect cell proliferation and extracellular matrix production in atherosclerosis. Decorin-overexpressing cells had decreased [ 3 H]thymidine incorporation into DNA and increased the levels of the cyclin-dependent kinase inhibitors p21 and p27 in the first 24 hours of response to serum and PDGF-BB. However, these effects of decorin were not apparent at 48 or 72 hours after plating and did not result in reduced growth of decorin-overexpressing cells in response to serum and PDGF-BB. In contrast, the growth response of decorin-overexpressing ASMCs to TGF-β1, as well as the expression of TGF-β1–responsive genes, such as plasminogen activator inhibitor-1 and versican (an extracellular matrix proteoglycan), was diminished. These results indicate that decorin selectively inhibits the responsiveness of rat ASMCs to TGF-β1 and suggests that the induction of constitutive decorin overexpression by ASMCs in vivo may have therapeutic value in the inhibition of TGF-β1–mediated effects on the development of atherosclerotic lesions.

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.

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.

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.

Chronic inflammation in vascular disease affects the organization of the extracellular matrix (ECM) in such a way as to promote the events associated with atherosclerosis and restenosis. Versican and hyaluronan are two ECM components that are affected by inflammation and, in turn, affect the inflammatory response. Inflammatory growth factors such as TGF-β1 and PDGF stimulate the production of versican and hyaluronan by arterial smooth muscle cells (ASMCs). These ECM components accumulate in vascular disease and promote the proliferation and migration of ASMCs, contributing to intimal hyperplasia. Hyaluronan also serves as attachment ligand for inflammatory cells and may be partly responsible for leukocyte retention as part of the early inflammatory response. Inflammation also leads to destruction of the ECM and failure of the ECM to remodel. Elastic fibers are absent in atherosclerotic and restenotic lesions and controlled expression of a versican variant, V3, in ASMCs restores the ability of these cells to form elastic fibers. ECM assembly is critical to vascular healing and the prevention of vascular disease.

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.

Syndecans are receptors for soluble ligands, including heparin-binding growth factors, and matrix proteins. However, intracellular targets of syndecan-1 (Sdc-1)-mediated signaling are not fully understood. A yeast two-hybrid protein interaction screening of a mouse embryo library identified the ubiquitin and SUMO-1 E3 ligase, Topors, as a novel ligand of the Sdc-1 cytoplasmic domain (S1CD), a finding confirmed by ligand blotting and co-precipitation with Sdc-1 from cell lysates. Deletion mutagenesis identified an 18-amino acid sequence of Topors required for the interaction with the S1CD. By immunohistochemistry, Topors and Sdc-1 co-localized near the cell periphery in normal murine mammary gland (NMuMG) cells in vitro and in mouse embryonic epithelia in vivo. Finally, siRNA-mediated knockdown of Topors demonstrated that Topors is a growth promoter for murine arterial smooth muscle cells and is required for the inhibitory effect of Sdc-1 on cell growth and platelet-derived growth factor-B induction. These data suggest a novel mechanism for the inhibitory effects of Sdc-1 on cell growth that involves the interaction between the cytoplasmic domain of Sdc-1 and the SUMO-1 E3 ligase, Topors.

The extracellular matrix molecule hyaluronan (HA) accumulates in human atherosclerotic lesions. Yet the reasons for this accumulation have not been adequately addressed. Because abnormalities in lipid metabolism promote atherosclerosis, we have asked whether disrupted cholesterol homeostasis alters HA accumulation in low density lipoprotein receptor-deficient cell cultures. Cultured aortic smooth muscle cells (ASMC) from Watanabe heritable hyperlipidemic (WHHL) rabbits and skin fibroblasts from homozygous patients with familial hypercholesterolemia accumulated 2–4-fold more HA than corresponding cells from age- and sex-matched normolipidemic rabbits and individuals. This occurred in both cell-associated and secreted HA fractions and was independent of cell density or medium serum concentration. WHHL ASMC cultures synthesized twice the proportion of high molecular mass HA (>2 × 106 Da) as normal rabbit ASMC but showed a lower capacity to degrade exogenous [3H]HA. Most importantly, cholesterol depletion or blocking cholesterol synthesis markedly reduced HA accumulation in WHHL ASMC cultures, whereas cholesterol replenishment or stimulation of cholesterol synthesis restored elevated HA levels. We conclude the following: 1) maintaining normal HA levels in cell cultures requires normal cell cholesterol homeostasis; 2) HA degradation may contribute to but is not the predominant mechanism to increase high molecular mass HA accumulation in low density lipoprotein receptor-deficient WHHL ASMC cultures; and 3) elevated accumulation of HA depends on cellular or membrane cholesterol content and, potentially, intact cholesterol-rich microdomains.

Heparan sulfate (HS) has been proposed to be antiatherogenic through inhibition of lipoprotein retention, inflammation, and smooth muscle cell proliferation. Perlecan is the predominant HS proteoglycan in the artery wall. Here, we investigated the role of perlecan HS chains using apoE null (ApoE0) mice that were cross-bred with mice expressing HS-deficient perlecan ( Hspg2 Δ3/Δ3 ). Morphometry of cross-sections from aortic roots and en face preparations of whole aortas revealed a significant decrease in lesion formation in ApoE0/ Hspg2 Δ3/Δ3 mice at both 15 and 33 weeks. In vitro, binding of labeled mouse triglyceride-rich lipoproteins and human LDL to total extracellular matrix, as well as to purified proteoglycans, prepared from ApoE0/ Hspg2 Δ3/Δ3 smooth muscle cells was reduced. In vivo, at 20 minutes influx of human 125 I-LDL or mouse triglyceride-rich lipoproteins into the aortic wall was increased in ApoE0/ Hspg2 Δ3/Δ3 mice compared to ApoE0 mice. However, at 72 hours accumulation of 125 I-LDL was similar in ApoE0/ Hspg2 Δ3/Δ3 and ApoE0 mice. Immunohistochemistry of lesions from ApoE0/ Hspg2 Δ3/Δ3 mice showed decreased staining for apoB and increased smooth muscle α-actin content, whereas accumulation of CD68-positive inflammatory cells was unchanged. We conclude that the perlecan HS chains are proatherogenic in mice, possibly through increased lipoprotein retention, altered vascular permeability, or other mechanisms. The ability of HS to inhibit smooth muscle cell growth may also influence development as well as instability of lesions.

Proteoglycans accumulate in the intimal layer of blood vessels during the early stages of atherosclerosis and predispose the vessel wall to further complications of this disease. Arterial endothelial and smooth muscle cell cultures have been used to study the metabolism of vessel wall proteoglycans in an attempt to determine whether cellular events associated with the genesis of this disease, such as cellular proliferation, ageing, migration and interaction with components of the extracellular matrix, influence the metabolism of arterial proteoglycans. Proteoglycan analyses of vascular cells reveal that endothelial cells synthesize multiple species of heparan sulphate proteoglycan while smooth muscle cells synthesize little heparan sulphate proteoglycan but significant quantities of chondroitin and dermatan sulphate proteoglycan. Each family of proteoglycans synthesized by each cell type differs with regard to charge density, hydrodynamic size, glycosaminoglycan type and size, oligosaccharide content and ability to form high molecular weight aggregates. A monoclonal antibody has been generated against the chondroitin sulphate proteoglycan and used to immunolocalize this antigen to the interstitial matrix of normal and diseased blood vessels. Experiments are presented to indicate that proteoglycan metabolism is modulated when cultured arterial cells are stimulated to proliferate and migrate. Other factors shown to influence proteoglycan metabolism include the age of the cell and the nature of the substratum upon which the cells are grown. These culture systems provide useful models with which to study the factors involved in the regulation of proteoglycan synthesis by vascular cells.

Publisher Summary Perlecan is the product of a single gene and has no close relatives. Perlecan, because of its interaction with many of the other principal structural proteins of basal and external laminae, is a reasonable candidate for a central role in basement membrane structure. Perlecan has been found to interact with components of these extracellular structures, including laminin, type IV collagen, and nidogen. In addition to contributing to the integrity of basement membranes, perlecan binds cell surface proteins and receptors. These interactions both organize cell surface proteins and mediate cellular adhesion to extracellular matrix proteins. A key question continues to be the mechanism by which perlecan downregulates the proliferative response of vascular smooth muscle cells (SMCs). An obvious possibility for perlecan-dependent inhibition of vascular smooth muscle growth is suggested by the binding of heparan binding growth factors, such as bFGF, by perlecan. Heparin (HS) infusion inhibits SMC proliferation and neo-intimal formation in the rat carotid injury model.

Lipoprotein retention on extracellular matrix (ECM) may play a central role in atherogenesis, and a specific extracellular matrix proteoglycan, biglycan, has been implicated in lipoprotein retention in human atherosclerosis. To test whether increased cellular biglycan expression results in increased retention of lipoproteins on ECM, rat aortic smooth muscle cells (SMCs) were transduced with a human biglycan cDNA-containing retroviral vector (LBSN) or with an empty retroviral vector (LXSN). To assess the importance of biglycan's glycosaminoglycan side chains in lipoprotein retention, ECM binding studies were also performed using RASMCs transduced with a retroviral vector encoding for a mutant, glycosaminoglycan-deficient biglycan (LBmutSN). Human biglycan mRNA and protein were confirmed in LBSN and LBmutSN RASMCs by Northern and Western blot analyses. HDL3+E binding to SMC ECM was increased significantly (as determined by 95% confidence intervals for binding curves) for LBSN as compared to either LXSN or LBmutSN cells; the increases for LBSN cell ECM were due primarily to an approximately 50% increase in binding sites (increased Bmax) versus LXSN cell ECM and of approximately 25% versus LBmutSN cell ECM. These results are consistent with the hypothesis that biglycan, through its glycosaminoglycan side chains, may mediate lipoprotein retention on atherosclerotic plaque ECM.

Smooth muscle cells (SMC) of the rat carotid arterial media proliferate and migrate in response to injury during the formation of a neointima. The interaction of fibroblast growth factor (FGF-2), which is released at the site of injury, with heparan sulfate proteoglycans (HSPGs) is necessary to induce signaling, which elicits an FGF-dependent mitogenic response by arterial smooth muscle cells, and also serves as a mechanism for storage of the growth factor within the extracellular matrix. However, whether these interactions are critical during neointimal formation has not been directly tested. In this study, a model of FGF-2-dependent medial SMC mitogenic response in balloon-injured rat carotid artery was used to test the effect of degradation of vessel wall heparan sulfate on subsequent SMC proliferation. Treatment of balloon-catheterized rat carotid arteries with chondroitin ABC lyase and/or heparin lyases eliminated heparan sulfates in the vessel wall, as determined by immunoperoxidase staining. In contrast, the distribution in the carotid vessel wall of the large core protein of perlecan, a major vessel wall HSPG that binds FGF-2, is not decreased. The effect of glycosaminoglycan digestion in situ on medial SMC proliferation in response to a bolus injection of FGF-2 after injury was determined by measuring the percentage of SMC nuclei that incorporated 5-bromo-2'-deoxyuridine (BrdU) 48 h after injury. Enzymatic removal of heparan sulfate reduced BrdU incorporation into medial SMC by 60-70% (P < 0.001) at 48 h after injury. Moreover, pre-incubation of FGF-2 with heparin prior to injection restored SMC replication to the levels present in injured vessels treated with buffer alone (P < 0.01). These experiments indicate that endogenous HSPGs are essential to promote FGF-2-driven medial SMC proliferation following injury, and that heparinase treatment can abrogate FGF-2-dependent responses in vivo.

Versican is an extracellular matrix (ECM) proteoglycan that is synthesized as multiple splice variants. In a recent study, we demonstrated that retroviral-mediated overexpression of the variant V3, which lacks chondroitin sulfate (CS) chains, altered arterial smooth muscle cell (ASMC) phenotype in short-term cell culture. We now report that V3-overexpressing ASMCs exhibit significantly increased expression of tropoelastin and increased formation of elastic fibers in long-term cell cultures. In addition, V3-overexpressing ASMCs seeded into ballooned rat carotid arteries continued to overexpress V3 and, at 4 weeks after seeding, produced a highly structured neointima significantly enriched in elastic fiber lamellae. In contrast to the hydrated, myxoid neointima produced by rounded or stellate vector-alone–transduced cells, V3-expressing cells produced a compact and highly ordered neointima, which contained elongated ASMCs that were arranged in parallel arrays and separated by densely packed collagen bundles and elastic fibers. These results indicate that a variant of versican is involved in elastic fiber assembly and may represent a novel therapeutic approach to facilitate the formation of elastic fibers.

Abstract —Decorin is an extracellular matrix (ECM) proteoglycan that may modify vascular smooth muscle cell (SMC) function by altering the response to growth factors and the accumulation of ECM proteins during vascular injury. To investigate these possibilities in vivo, decorin was overexpressed at the site of arterial injury by cell-mediated gene transfer. Fischer rat SMCs were transduced in vitro with a retroviral construct that contained the bovine decorin gene and were subsequently seeded into injured rat carotid arteries. A species-specific antibody to bovine decorin and polymerase chain reaction primers were used to detect bovine decorin and distinguish it from endogenous rat decorin. Immunohistochemical and Northern analyses of rat carotid arteries revealed only low levels of rat decorin expression up to 8 weeks after balloon injury. However, after cell-mediated transfer of bovine decorin, strong expression of bovine decorin was verified by immunohistochemistry and reverse transcriptase–polymerase chain reaction. Four weeks after injury, the intimal area in vessels seeded with bovine decorin–overexpressing SMCs was significantly reduced by 35±4% (mean±SEM, n=9; P

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.

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.

The formation of the valvular and septal primordia of the embryonic heart depends upon the migration of endocardial cushion tissue mesenchyme (CT) to populate the cardiac jelly (CJ) in specific heart regions (e.g., atrioventricular (AV) pads). It has been proposed that the migration of CT may be directed by macromolecules of the CJ. In this study, [ 3 H]thymidine-labeled endocardial (EC) and CT cells were transplanted onto intact pre- and postmigratory AV pads in vitro to test whether the compositional or structural changes known to occur in the cardiac jelly during development influence the migration of cushion tissue cells. After transplantation of labeled donor cells, host AV pads were fixed, embedded, and sectioned, and autoradiography was performed to determine the distribution of labeled donor cells within the host CJ. The experiments indicate that transplanted mural EC cells remain primarily at the AV pad surface, while grafted CT cells of all developmental ages rapidly invade both developmentally young and older AV pads. Furthermore, CT cells readily migrate in a direction opposite to that of cells in vivo when transplanted to inverted AV pads from which the myocardium has been removed. It is concluded that the CJ matrix, which is clearly a suitable framework for CT cell migration, provides no direct cues to determining the polarity or extent of migration.

Differential interference microscopy and time-lapse cinematography were used to determine unequivocally the origin of cushion tissue cells migrating in situ in the atrioventricular region of the embryonic chick heart. These studies have verified the presumed endocardial origin of cushion tissue mesenchyme.