© 1997 American Heart Association, Inc.
Articles |
Syndecan-4 Is a Primary-Response Gene Induced by Basic Fibroblast Growth Factor and Arterial Injury in Vascular Smooth Muscle Cells
the Sigfried and Janet Weis Center for Research, Geisinger Clinic, 26-13, Danville, Pa.
Correspondence to Gunay Cizmeci-Smith, PhD, Sigfried and Janet Weis Center for Research, Geisinger Clinic, 26-13, Danville, PA 17822-2613.
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Abstract |
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Syndecans are a family of transmembrane proteoglycans that have been implicated in cell–extracellular matrix adhesion and growth factor binding. We reported previously that syndecan-1 expression by cultured rat vascular smooth muscle cells (VSMCs) is induced by serum- or platelet-derived growth factor (PDGF). We now report that syndecan-4 mRNA is rapidly induced in cultured VSMCs in response to basic fibroblast growth factor (bFGF) or serum stimulation. In the presence of cycloheximide, induction of syndecan-4 mRNA was enhanced. These characteristics identified syndecan-4 as a primary-response gene product in VSMCs. In contrast, syndecan-1 mRNA expression in response to serum was completely blocked in the presence of cycloheximide. We also examined the expression of syndecan mRNAs in VSMCs in response to balloon catheter injury in vivo. A reverse transcriptase–polymerase chain reaction technique was developed that enabled us to amplify all four syndecan mRNAs in a single reaction tube and determine relative changes in their expression. All four syndecan mRNAs were detected in uninjured rat carotid arteries. In endothelium-denuded arteries, the medial layer (presumably VSMCs) accounted for 70% to 90% of the syndecan mRNAs in the vessel wall. The levels of syndecan-2 and syndecan-3 mRNAs were not altered significantly after balloon injury. In contrast, syndecan-4 mRNA was increased at early times after injury but then decreased to control level by 7 days. Syndecan-1 mRNA levels showed a slower but prolonged increase that reached a maximum at 7 days after injury. Immunostaining with anti–syndecan-4 antibodies demonstrated a rapid increase in syndecan-4 proteoglycan expression in the injured carotid artery.
Key Words: balloon catheter injury • syndecans • gene expression
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Introduction |
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Vascular smooth muscle cells synthesize a wide variety of ECM- and membrane-associated proteoglycans. In the blood vessel wall, the ECM proteoglycans provide structural support, regulate tissue permeability, and influence viscoelasticity and hemostasis. The composition and the quantity of proteoglycans synthesized are affected by the physiological state of the cells. Factors such as cell density1 and the presence of growth factors2 3 and cytokines4 can modulate proteoglycan synthesis by VSMCs.
We are interested in the regulation and function of a gene family of transmembrane proteoglycans, the syndecans.5 The four known mammalian syndecans are syndecan-1 (also called syndecan),6 syndecan-2 (fibroglycan),7 syndecan-3 (N-syndecan)8 and syndecan-4 (ryudocan).9 The syndecans have been implicated in a number of important functions, including growth factor binding,10 11 12 13 cell adhesion,14 15 16 and hemostasis.17 These functions are dependent on binding interactions with extracellular ligands, mediated by the covalently attached heparan sulfate chains of the syndecans. The syndecan core proteins are characterized by highly conserved membrane-spanning and cytoplasmic domains. The extracellular domains of the syndecans, in contrast, vary in their length and in the number and placement of the glycosaminoglycan chains. This structural diversity, plus their highly regulated patterns of expression,5 strongly suggests that different syndecans carry out distinct functions.
In previous work we showed that syndecan-1 was expressed by cultured VSMCs in response to a variety of agents, including serum, PDGF, and angiotensin II.2 VSMCs also synthesize syndecan-2, but in contrast to syndecan-1, the expression of syndecan-2 is not regulated by growth factors in these cells.
VSMCs within the medial layer of arteries are normally in a quiescent state. In response to an injury to the vessel wall, such as balloon angioplasty, a subpopulation of medial VSMCs proliferates and migrates to the intima. Continued replication of these cells, coupled with enhanced accumulation of ECM, leads to pronounced intimal thickening. This response to injury poses a serious clinical problem. Approximately one third of atherosclerotic coronary arteries treated by angioplasty or bypass surgery become occluded as a result of intimal thickening.18 19 Balloon catheter injury of the rat carotid artery is an established animal model for this wound-healing response.20 21 22 There are several reports describing the biochemical changes that take place in rat carotid arteries after balloon injury. Several growth factors, such as bFGF,23 released from damaged VSMCs, and PDGF,24 25 released from platelets that aggregate at the site of injury, are thought to be involved in this process. With the exception of growth factor receptors,26 27 28 however, other cell-surface molecules involved in this process have not been extensively studied.
We wanted to extend our earlier findings on regulation of syndecan expression in VSMCs. This article reports experiments with cultured VSMCs demonstrating that induction of syndecan-4 mRNA but not syndecan-1 mRNA is a primary response to growth factor stimulation. We also observed an early and transient induction of syndecan-4 mRNA in the rat carotid artery after balloon catheter injury.
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Methods |
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Cell Culture
VSMCs were cultured as described previously.1 The cells were grown to 70% to 80% confluence in DMEM containing 10% FCS. Subconfluent cells were made quiescent by being switched to DMEM containing 0.4% FCS for 48 hours. The cells were then switched to medium containing 10% FCS or 0.4% FCS plus bFGF (20 ng/mL) as indicated in the "Results." Cycloheximide (Sigma Chemical Co) was used at a concentration of 10 µg/mL. Human bFGF was purchased from Upstate Biotechnology Inc. Media and FCS were purchased from Life Technologies, Inc.
Surgery
Arterial injury was performed essentially as described previously.21 Male Sprague-Dawley rats (350 to 400 g) were anesthetized with ketamine (45 mg/kg IP) and xylazine (5 mg/kg IP). The right internal and external carotid as well as the right common carotid arteries were exposed through a midline neck incision. A 2F balloon catheter was advanced through the right external carotid into the right common carotid artery. The catheter was rotated as it was passed three times through the carotid artery with the balloon distended with saline sufficiently to generate slight resistance. After balloon injury, the right external artery was ligated and blood flow was restored through the right internal artery. Uninjured contralateral carotid arteries were used as controls. Tissues were harvested at 4 hours, 8 hours, 7 days, and 12 days after injury. Groups of 6 to 10 injured or control arteries were pooled for each experiment. A small segment of each injured artery was processed for light microscopy to monitor the extent of endothelial damage and neointimal thickening.
Preparation of Anti–Syndecan-4 Antibodies
A 400-bp PCR product corresponding to the rat syndecan-4 cDNA ectodomain was subcloned into the pGEX-4t-1 vector (Promega). A sense orientation clone was identified by PCR and used to transform BL21(pLysS) host cells. Protein expression was induced with 0.1 mmol/L IPTG. The cells were lysed by sonication, and the GST–syndecan-4 fusion protein was purified by glutathione/agarose affinity chromatography. The purified fusion protein was used to immunize rabbits as described previously.1 Antibodies were affinity-purified on a column of immobilized fusion protein. The antibodies recognize both syndecan-4 core protein and processed proteoglycan on immunoblots.
Immunohistology of Rat Carotid Arteries
Paraffin-embedded sections of rat carotid arteries were immunostained with affinity-purified anti–syndecan-4 antibodies according to the protocol described previously.1 Briefly, after dewaxing and rehydration, the sections were incubated in 3% H2O2 to block endogenous peroxidase activity. Tissues were treated with 1 mg/mL hyaluronidase in 0.1 mol/L sodium acetate/0.15 mol/L NaCl, pH 5.5, for 30 minutes and blocked by incubation with 10% goat serum in Tris-buffered saline (0.01 mol/L Tris-HCl/0.15 mol/L NaCl, pH 7.4). The polyclonal primary antibody (1:25 or 1:50 dilution) incubation was performed overnight at 4°C. After two washes, the specimens were incubated for 1 hour with unconjugated goat anti-rabbit IgG (Sigma) followed by a 1-hour peroxidase-antiperoxidase (Sigma) incubation. Specific immunostaining was visualized with 3,3'-diaminobenzidine tetrahydrochloride as substrate (Sigma).
Extraction of RNA and Amplification of mRNA Sequences by RT-PCR
Injured and control carotid arteries were excised, opened longitudinally, and cleaned of the surrounding tissue. Prepared arteries were placed in Ultraspec RNAzol (0.5 mL/carotid). The tissue was homogenized in a polytron for 1 minute at high speed, and total RNA was isolated as described previously.2 One microgram of total RNA obtained from rat carotid arteries was used as a template for reverse transcriptase using random hexamers as primers in a reaction mixture containing recombinant reverse transcriptase from Moloney murine leukemia virus (GIBCO-BRL). After 40 minutes at 40°C, the reaction mixture was heated at 98°C to inactivate the enzyme and then used as a template for PCR amplification. Oligonucleotides for amplification were based on published sequences and are shown in the Table
. Sense and antisense primers were chosen in different exons to eliminate the possibility of amplification of genomic DNA. Primer pairs were chosen with the aid of a computer program (PRIMER) that optimizes the selected pairs for G/C content and annealing temperature and minimizes false priming and primer-dimer formation. The PCR cycles were 1 minute at 94°C, 1 minute at 60°C, and 1.5 minutes at 72°C for up to 30 cycles. Up to five different PCR products, corresponding to the four syndecan and 
-tubulin mRNAs, were amplified in the same reaction tube containing 10 µCi of [-32P]dCTP and Taq polymerase (Promega). Starting from the 14th cycle, 10-µL aliquots were taken from each reaction and analyzed on 8% acrylamide gels in Tris-borate/EDTA buffer. Amplified PCR products were visualized by ethidium bromide staining. Gels were dried and subsequently analyzed with a radioanalytic detector (Ambis). [32P]dCTP incorporated into the amplified products was determined, and the amount of radioactivity for each product was plotted as a function of the number of PCR cycles completed.
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Northern Blot Analysis
Total RNA was extracted from the cultured cells with Ultraspec RNAzol (Cinna Biotech) as described previously.2 29 Twenty micrograms of total RNA from each sample was separated by electrophoresis in agarose/formaldehyde gels, transferred to nylon membranes, and hybridized to random-primed 32P-labeled cDNA probes, as described previously. The preparation of cDNAs for rat syndecan-1,1 syndecan-2,2 and N-syndecan (syndecan-3)8 has been described. cDNAs coding for rat syndecan-4 and rat ß-actin were produced by PCR amplification using sequence-specific primers based on published sequences.9 The resulting PCR products were subcloned into plasmid pCRII (Invitrogen). Their authenticity was verified by DNA sequence analysis. For quantification of the hybridization signals, the exposed x-ray films were scanned with a laser densitometer (Molecular Dynamics). These values were normalized to the quantity of 18S rRNA in the samples, determined by scanning a photograph of the ethidium bromide–stained membranes, as described previously.1 2
Analysis of RT-PCR Data
Data were analyzed by kinetic analysis as described.30 Under ideal conditions, the amount of product doubles during each cycle of a PCR reaction. Experimentally, however, the efficiency of amplification is less than ideal; therefore, the amplification is described by the equation
 | (E1) |
 | (E2) |
If two products are amplified simultaneously in the same reaction tube, the relationship between the quantities of amplified products is described by N0 prod 1/N0 prod 2=Nprod 1 (1+E1)n/N prod 2 (1+E2)n, which can be expressed as
 | (E3) |
 | (E4) |
). Thus, in Equation 4, Ac/Ainj equals 1 and Equation 4 can be simplified to
 | (E5) |
-tubulin mRNA was used as an internal control to compare the relative quantities of each syndecan mRNA in different layers of the vascular wall. This is based on the assumption that the adventitia, medial layer, and endothelium have similar contents of -tubulin mRNA. In other experiments, syndecan-2 mRNA was used as an internal control because it is the most abundant syndecan mRNA in VSMCs and its levels are stable under the conditions we have studied. In our experiments, the rates of amplification of all templates were exponential between cycles 16 and 22. The data reported here were calculated from Equation 5 and the quantities of amplification products measured at cycle 20.
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Results |
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We wanted to determine whether the induction of syndecan-1 mRNA by serum in VSMCs exhibited characteristics of a primary-response gene product. We were also interested in comparing the induction of syndecan-1 mRNA with that of other VSMC products, including syndecan-4, ß-actin, and c-fos. Products of primary-response genes (sometimes called immediate early genes) are thought to be responsible for the functional changes required to initiate cell division. Examples include proto-oncogenes such as c-myb and c-fos.31 32 A characteristic of primary-response gene products is their "superinduction" in the presence of a protein synthesis inhibitor, such as cycloheximide. For these experiments, quiescent VSMCs were exposed to serum in the absence or presence of cycloheximide, and steady-state mRNA levels were determined by Northern blot analysis. Fig 1
shows representative Northern blot signals and quantitative data on the time course of mRNA induction in response to serum stimulation. Serum stimulation produced a large but delayed increase in syndecan-1 mRNA levels in VSMCs. In contrast, syndecan-2 mRNA levels showed a rapid but moderate decrease in response to serum stimulation. Syndecan-4 and ß-actin mRNAs were induced rapidly in response to serum stimulation and continued to increase for at least 5 hours. c-fos mRNA increased rapidly after serum stimulation but then declined. In the presence of cycloheximide serum, induction of syndecan-1 mRNA was totally blocked. In contrast, serum induction of mRNAs coding for syndecan-4, ß-actin, and c-fos was enhanced in the presence of cycloheximide.
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) or presence (
) of cycloheximide (10 µg/mL). Data shown are from a single representative experiment that was repeated three times with identical results.
In our earlier studies, when we tested individual growth factors for their effects on syndecan-1 mRNA induction, we found that PDGF and angiotensin II but not bFGF or transforming growth factor-ß were able to elicit increases in syndecan-1 mRNA.1 2 As observed for serum stimulation, the induction of syndecan-1 mRNA by PDGF was blocked by cycloheximide treatment (not shown). When we tested individual growth factors for their ability to induce syndecan-4 mRNA expression, we found that PDGF produced a weak induction (not shown) and that, in contrast to what was found for syndecan-1, bFGF produced an induction even larger than that produced by serum (Fig 2). The effect of bFGF stimulation on syndecan-4 mRNA was rapid (within 1 hour), and in the presence of cycloheximide, the bFGF induction was enhanced after 2 hours ("superinduction"). The time course of syndecan-4 mRNA induction by bFGF differed from that of c-fos and ß-actin (Figs 3 and 4). The increase in syndecan-4 mRNA levels did not peak for several hours, in contrast to c-fos mRNA, which peaked at 1 hour and then declined. Syndecan-4 mRNA levels continued to increase until 8 hours, but declined by 24 hours (not shown). These results indicated that syndecan-4 induction by bFGF was a primary response to bFGF stimulation that did not require the synthesis of new proteins.
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RT-PCR Amplification of Syndecan Family Proteoglycan Sequences From Carotid Artery mRNA
The finding that synthesis of different members of the syndecan family of transmembrane proteoglycans was regulated by apparently different mechanisms in VSMCs and that syndecan-4 was a primary-response gene product prompted us to investigate the expression of these mRNAs in an in vivo model of VSMC physiological response. Balloon catheter injury of rat carotid arteries is a well-established model that elicits a reproducible vascular smooth muscle response that includes cell proliferation, migration, and synthesis of ECM. The small quantities of mRNA that could be isolated from these vessels precluded the use of Northern blot analysis for detection of low-abundance mRNAs, such as syndecans. For this reason, we adapted RT-PCR techniques for amplification of all four syndecan mRNAs in a single reaction tube in a manner that could be used to provide data on the relative abundance of these mRNAs. The major advantages of the RT-PCR technique are the ability to determine the relative amounts of low-abundance mRNAs with limited amounts of tissue and to analyze several mRNA species in the same reaction.
To demonstrate that specific amplification of syndecan sequences could be achieved with carotid artery mRNA as the template, total RNA isolated from rat carotid arteries was reverse-transcribed with random primers and amplified by PCR with primers specific for syndecan-1, syndecan-2, syndecan-3, or syndecan-4 as described in "Methods." The amplified products were analyzed by electrophoresis on 1% agarose gels (Fig 3
) or 8% polyacrylamide gels (see below). Amplified products of the appropriate size (Table) were observed by ethidium bromide staining for each of the four syndecan types. To verify the authenticity of the products, the amplified products were blotted onto nylon membranes and hybridized to radiolabeled cDNA probes specific for each of the four syndecans. In each case, specific hybridization of the appropriate cDNA probe was observed (Fig 3).
The ability of the RT-PCR method to measure relative changes in syndecan mRNA expression was verified by measuring syndecan-1 mRNA expression in cultured VSMCs and comparing the results with those obtained by Northern blot analysis. Total RNA was extracted from quiescent VSMCs stimulated with 10% FCS. Twenty micrograms of total RNA was used for Northern blot analysis, and 1 µg of the same RNA was used for RT-PCR. As shown in Fig 4, 10% FCS caused a time-dependent induction of syndecan-1 mRNA levels that were easily detected by both methods. Syndecan-1 signals were normalized to syndecan-2 mRNA levels in both methods. In the PCR experiments, the linear amplification range was between 18 and 22 cycles. Yields obtained at the end of cycle 20 were used for calculation of relative mRNA levels. Comparison of the data obtained by the two methods demonstrated that RT-PCR is as reliable as Northern blot analysis in measuring relative changes in mRNA levels.
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Distribution of Syndecan mRNAs Within the Carotid Artery
We next determined the relative distribution of the four syndecan mRNAs among the medial, adventitial, and endothelial layers of the vessel. For these experiments, RNA was isolated from whole carotid arteries (containing the endothelium) or arteries that had been dissected to yield isolated adventitial or medial layers. These were analyzed by RT-PCR as described in "Methods." Since linear amplification was between 20 and 26 cycles, cycle 23 was chosen for comparison. As shown in Fig 5, all four syndecan mRNAs were detected in the medial layer, presumably in VSMCs. The relative abundance of specific syndecans varied within different layers of the artery. Syndecan-1 was enriched in whole artery compared with the medial or adventitial layers, which suggested that it was most abundant in the endothelial layer. Syndecan-2 and syndecan-4 mRNAs were enriched in the medial layer compared with the adventitia or whole vessel. The yield of RNA from the medial layer was approximately three times greater than from the adventitia. From this information plus data on the relative abundance of the syndecan mRNAs (Fig 5), it can be calculated that the medial layer accounts for 80% of the syndecan-1 and 90% of the syndecan-4 mRNAs in the vessel wall, ignoring the contribution of the endothelium. Thus, changes in mRNA levels for these syndecans measured in endothelium-denuded arteries would presumably reflect changes in VSMC expression.
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Syndecan mRNA Expression After Balloon Catheter Injury
To determine whether expression of syndecan mRNAs was regulated in vivo after balloon catheter injury, we measured by RT-PCR syndecan mRNA levels in rat carotid arteries at early (4 and 8 hours) and late (7 and 12 days) times after injury. For these experiments, contralateral uninjured carotid arteries served as controls. Representative data for the 4-hour and 7-day samples are shown in Figs 6 and 7. A summary of the data is shown in Fig 8. Consistent with results obtained with cultured VSMCs, there was no detectable difference in the syndecan-2/tubulin ratio between the injured and control arteries at any of the times examined. For this reason, values for the other syndecan mRNAs were normalized to the syndecan-2 mRNA levels measured in the same sample. As shown in Fig 6, at 4 hours after injury there was an increase in the level of syndecan-4 mRNA. Similar results were obtained for carotid arteries 8 hours after injury (Fig 8). This increase was transient, and by 7 days the syndecan-4 mRNA level returned to a value that was approximately equal to the control value (Figs 7 and 8).
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Syndecan-1 mRNA, which was present at the lowest level among the four syndecans in control vessels, was also increased over control levels at 4 and 8 hours after injury as well as at 7 days after injury. Syndecan-3 mRNA levels dropped slightly in the injured vessels. These results suggested that syndecan-4 mRNA was induced rapidly and transiently in VSMCs after injury. Syndecan-1 mRNA was also induced but to a lesser extent and for a longer period. In contrast, the other syndecan mRNAs were not changed significantly.
Syndecan-4 Proteoglycan Increases After Injury
To determine whether the increase in syndecan-4 mRNA levels resulted in an increase in syndecan-4 proteoglycan levels in the tissues, control and injured carotid arteries were stained with anti–syndecan-4 antibodies as described in "Methods." As shown in Fig 9, injured carotid arteries showed stronger immunostaining in the medial layer than did uninjured control arteries. These results demonstrate that expression of syndecan-4 by VSMCs is induced after injury.
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Discussion |
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Experiments with cultured rat aortic VSMCs have shown that syndecan-4 mRNA was induced in response to serum or bFGF in a manner that was characteristic of a primary-response gene product. This response was unique among syndecan family genes in these cells. We have also examined changes in mRNA levels of syndecan family transmembrane proteoglycans after balloon catheter injury in the rat carotid artery using a quantitative RT-PCR amplification method. The accuracy of the RT-PCR method was verified by comparison of the results with those obtained with Northern blot analysis of syndecan-1 expression in cultured VSMCs. The major advantages of the RT-PCR technique are the ability to determine the relative amounts of low-abundance mRNAs using limited amounts of tissue and to analyze several mRNA species in the same reaction. Because of the latter, differences in sample preparation, conditions of reverse transcription, and changes in amplification efficiency caused by contaminants in the template affect all products equally. Our results showed that medial tissue, which consists almost entirely of VSMCs, contains the mRNAs coding for all four mammalian syndecans. The mRNAs coding for syndecan-1 and syndecan-4 were preferentially induced after balloon catheter injury in the rat carotid artery but with different temporal patterns. Syndecan-4 mRNA induction was rapid and transient, whereas syndecan-1 mRNA induction was slower and more prolonged, reaching a maximum at 7 days after injury. Results of these experiments were consistent with those carried out with cultured VSMCs that indicated distinct mechanisms for regulation of expression of syndecan RNAs.
Previous animal studies have shown that the tissue response to balloon catheter injury progresses in several phases.21 During the early response (0 to 8 hours after injury), platelets adhere to the endothelium-denuded vessel wall and release growth factors, including PDGF, which has potent mitogenic and chemotactic activity for VSMCs. Basic bFGF is also released by damaged cells and from storage sites within the vessel wall ECM. The blood coagulation cascade is also activated by tissue factors, which results in the production of thrombin at the injury site. VSMC proliferation begins 2 to 3 days after injury, with the majority of proliferative activity occurring within 7 days. VSMC migration to the intima also occurs at this time. At 2 weeks, the number of VSMCs in the intima peaks and remains constant for up to 1 year. According to this progression of events, syndecan-4 induction occurs during the initial cellular response and before the initiation of VSMC proliferation. The temporal pattern of syndecan-4 mRNA increase is thus similar to that of several proto-oncogenes. The increase in syndecan-1 mRNA occurs during the period of active VSMC proliferation and migration.
Of interest in the context of this report is the fact that syndecan expression by VSMCs is regulated by exposure to PDGF, bFGF, and thrombin but that the mechanisms regulating expression of individual syndecan subtypes are distinct. Syndecan-2 appears to be constitutively expressed by VSMCs.2 Its expression is not increased by growth factors or serum or in response to balloon injury of the carotid artery. We have obtained similar findings for syndecan-3 expression in VSMCs. In contrast, expression of syndecan-1 and syndecan-4 is highly regulated. Syndecan-1 mRNA is induced by serum, PDGF, thrombin, and angiotensin II but not bFGF.2 In contrast, syndecan-4 is induced by bFGF and less strongly by serum. The mechanism of induction appears to be different as well. Syndecan-4 mRNA is superinduced by these agents in the presence of cycloheximide. Thus, this appears to be a direct effect of growth factor stimulation and does not require the synthesis of new proteins. In this respect, syndecan-4 can be considered a primary-response gene32 in VSMCs. Other examples of primary-response genes include those coding for the cytoskeletal proteins actin, vimentin, and tropomyosin as well as the ECM-adhesive protein fibronectin and its integrin receptor. Syndecan-1 mRNA is also induced in response to serum and mitogens in these cells. In contrast to what we found for syndecan-4, syndecan-1 induction by these agents was abolished in the presence of cycloheximide. Thus, syndecan-1 can be classified as a secondary-response gene product. Data obtained with cultured VSMCs agree well with the findings presented here on the induction of syndecan mRNAs after balloon catheter injury in vivo. Syndecan-4 mRNA is induced rapidly and transiently, syndecan-1 mRNA is induced after a lag period of several days, and syndecan-2 and syndecan-3 mRNAs do not change significantly.
The functional consequences of changes in the syndecan composition of the vessel wall during the cellular responses to injury are not known. Syndecans are thought to act as multifunctional cell-surface receptors for a variety of ligands involved in cell-ECM adhesion, growth factor binding, and hemostasis. For example, bFGF binds to glycosaminoglycan chains present on proteoglycans in the ECM or on cell surfaces. This could provide a reservoir for storage and gradual release of this growth factor, which may be important in wound healing. A similar mechanism has been proposed for PDGF. In addition, the activation of high-affinity bFGF receptors has been shown to be dependent on the presence of cell-surface heparin-like molecules.10 33 Several syndecan types have been shown to bind bFGF.11 13 Syndecan-1 and syndecan-2 have been reported to inhibit bFGF-stimulated proliferation of some cells.34 The mechanism of this inhibition is not known, but it may be related to the well-known inhibitory activity of heparin and heparan sulfate for VSMC proliferation. Another possible ligand for syndecan-4 is heparin-binding epidermal growth factor, which is a component of wound fluid and has been shown to stimulate smooth muscle cell migration. Its biological activity has been shown to be glycosaminoglycan-dependent.35 Syndecan-1 and syndecan-4 isolated from cultured endothelial cells also have been shown to bind antithrombin III,17 36 which may regulate local hemostasis and thrombosis. Finally, syndecan-1 has been shown to bind to fibronectin and other ECM molecules that are present in the vascular wall. Syndecan-1 can mediate cell adhesion to fibronectin,15 and its expression leads to increased cell spreading on fibronectin-coated surfaces.37 Syndecan-1 has been shown to associate with microfilaments, and its expression results in changes in cytoskeletal organization.37 38 Syndecan-4 is enriched in focal adhesion components in fibroblasts.39 Syndecan-4 association with focal contacts correlates with fibronectin deposition, suggesting a possible role in matrix assembly.40 Syndecan-ECM interactions could be relevant to VSMC migration that occurs in response to injury.
A complete understanding of the function of syndecans in the vessel wall and their role in mediating the cellular responses to injury will require additional information on the ligand-binding activity of vascular syndecans.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by NIH grant HL-48740 to Dr Carey and a Grant-in-Aid from the American Heart Association, Pennsylvania Affiliate, to Dr Cizmeci-Smith. We thank John Showalter for excellent technical assistance, Dr Sally Wixson for advice on animal surgery, and Charlotte Gonzales and Linda Dearman for help in preparing the manuscript.
Received February 14, 1995;
revision received September 3, 1996;
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