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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:37-43.)
© 1995 American Heart Association, Inc.

Articles

Augmented Urokinase Receptor Expression in Atheroma

Hiroko Noda-Heiny; Alan Daugherty; Burton E. Sobel

From the Cardiovascular Division and the Department of Biochemistry and Molecular Biophysics (A.D.), Washington University School of Medicine, St Louis, Mo.

Correspondence to Hiroko Noda-Heiny, MD, Cardiovascular Division, Box 8086, Washington University School of Medicine, 660 S Euclid Ave, St Louis, MO 63110.

	Abstract

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Abstract Smooth muscle cell proliferation and migration into neointima are hallmarks of atherogenesis. However, mechanisms responsible have not yet been fully elucidated. One potential mediator of both smooth muscle cell proliferation and migration is activation of plasminogen by activators bound to receptors on cells within the vessel wall. To determine whether vascular smooth muscle cells within atheroma express the receptor for urokinase-type plasminogen activator (uPA-R), we characterized atheroma in cholesterol-fed New Zealand White rabbits and human subjects by immunostaining. Intense immunostaining of uPA-R was observed throughout the neointima in both rabbit and human atherosclerotic lesions with the use of a monoclonal antibody to uPA-R. uPA-R was not detectable in normal arterial tissues. uPA-R was localized to macrophages and neointimal smooth muscle cells identified by immunostaining in serial sections. Furthermore, uPA-R protein in extracts from atheroma was present in at least a ninefold greater quantity compared with extracts from normal vessels, as shown by Western blotting. Expression of uPA-R mRNA in migrating vascular smooth muscle cells did not increase significantly. Thus, altered posttranscriptional regulation may be contributing to the increased uPA-R. In vitro, antibodies to uPA-R delayed the migration of cultured vascular smooth muscle cells. Our results suggest that increased cell-surface uPA-R contributes to pericellular proteolysis and consequently increased neointimalization secondary to increased vascular smooth muscle cell migration in atheroma.

Key Words: atherogenesis • restenosis • cell migration • plasminogen activators

	Introduction

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Proliferation and migration of vascular smooth muscle cells are hallmarks of atherogenesis. However, mechanisms responsible have not yet been fully elucidated.¹ One potential mediator is activation of plasminogen by activators bound to cell surface receptors as judged from factors implicated in an analogous process, migration of tumor cells that metastasize.² Urokinase-type plasminogen activator (uPA) is thought to potentiate pericellular proteolysis in tissues^{3 4} in contrast to tissue-type plasminogen activator (TPA), which functions largely in the circulating blood.

The uPA receptor (uPA-R) is a single-chain polypeptide, 313 amino acids long with three homologous repeated sequences comprising three domains.^{5 6 7} It is anchored to cell membranes by glycosyl-phosphatidyl-inositol (GPI) and has five potential N-linked glycosylation sites that influence ligand-binding affinity and intracellular trafficking.⁸

uPA-R can bind either two-chain uPA (enzymatically active) or single-chain pro-uPA (inactive) that can, in turn, be activated by plasmin to yield receptor-bound, active uPA.^{2 3 4 5 6} Unoccupied receptor "floats" within the cell membrane, but receptors cluster in relatively stable loci when bound to uPA.⁹ Unmodified uPA bound to receptor can be inactivated by plasminogen activator inhibitor type-1 (PAI-1) or type-2 (PAI-2).¹⁰ uPA-PAI complexes bound to receptor are internalized quickly and degraded, and the receptor is recycled.^{11 12 13 14}

uPA-R is found on human monocytes, neoplastic cells, and normal cells capable of migration.^{15 16 17} In human tumors, both uPA and uPA-R are present consistently at the invasive front.^{18 19} In nonmalignant cells, such as human trophoblasts, cell surface proteolysis is evident despite secretion of PAI-1 and PAI-2 into conditioned media.²⁰ Metastasis and local invasion of tumor cells have been prevented with antibodies blocking uPA or its receptor.^{21 22 23} Such cells are richly endowed with both uPA and uPA-R.^{24 25 26}

uPA-R is expressed in human umbilical vein endothelial cells.²⁷ Migrating compared with nonmigrating endothelial and vascular smooth muscle cells express increased amounts of cell-associated uPA.²⁸ -Thrombin and other mitogens upregulate uPA-R on vascular smooth muscle cells in vitro.²⁹ Accordingly, the uPA–uPA-R system may modulate cell migration in atheroma.

In the present study, uPA-R was detected in and localized to macrophages and vascular smooth muscle cells in atheroma from New Zealand White rabbits and human subjects. Regulation of uPA-R expression was found to be largely posttranslational. In vitro, vascular smooth muscle cell migration was shown to be attenuated by antibodies against uPA-R.

	Methods

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Vessels
New Zealand White rabbits were obtained from Shady Grove and fed a 2% cholesterol–enriched diet (Purina Test Diets) for 3 months before study. Human carotid, iliac, femoral, and tibial artery sections were obtained at the time of clinically mandated vascular surgery with written consent from each patient. Samples were frozen immediately in liquid nitrogen. Tissues were either treated with paraformaldehyde, embedded in paraffin, and stored at room temperature or frozen in liquid N₂ and stored at -80°C. Frozen arteries were sectioned with a cryostat and placed on glass slides precoated with polylysine for immunostaining.

Antibodies and Chromogens
Monoclonal antibody against uPA-R (MoAb 3936) and rabbit anti-human uPA-R (#399R) were purchased from American Diagnostica, Inc (ADI); chicken anti–uPA-R, uPA-R standard, and biotin–anti-chicken IgG were provided by Dr John Bognacki of ADI. Monoclonal antibodies against -smooth muscle actin were obtained from Boehringer Mannheim. Antibody against factor VIII–related proteins, anti-CD68, HAM-56, and RAM-11 were obtained from Dako Corp. Vectastain avidin/biotinylated enzyme complex (ABC) elite kits, diaminobenzidine tetrahydrochloride (DAB) peroxidase kit, Vectastain ABC–alkaline phosphatase, and Vector blue substrate were purchased from Vector Laboratories. Microprobe slides, 10X automation buffer, Redusol, and peroxidase chromogen kit 3-amino-9-ethyl-carbazole (AEC) were obtained from Biomeda Corp.

Immunohistochemistry
Frozen sections were fixed in acetone cooled with ice for 5 minutes. Paraffin sections were deparaffinized with xylene followed by 100% EtOH. All immunostaining was performed with a Fisher MicroProbe system. Briefly, acetone-fixed sections were incubated in Redusol for 2 minutes followed by four washes with automation buffer (Biomeda). To eliminate endogenous peroxidase, sections were quenched with 1% H₂O₂ in methanol for 2 minutes followed by four washes with automation buffer. Sections were blocked with buffer containing preimmune serum from the donor animal used to raise the secondary antibody for 5 minutes, followed by incubation with primary antibody. A biotinylated secondary antibody against the primary antibody was used. Substrates used for enzyme reactions for visualization were DAB, which yields a brown-black color; AEC, identified as a red precipitate; or a Vector Blue chromogen with an alkaline phosphatase substrate that produces a blue color. Specificity of the monoclonal antibody was verified by preabsorption of the antibody with uPA-R standard before immunostaining. Colocalization of uPA-R protein and cell-specific antigens was determined with serial use of two Vector stain kits: the first, a Vector alkaline phosphatase kit and Vector Blue (blue) chromogen; the second, a peroxidase kit and AEC as chromogen (red).

Western Blotting for Detection of uPA-R in Human Atheroma
Sections from thoracic aorta from an organ donor provided by the Mid-America Eye and Tissue Bank were used as normal controls. Surgical specimens were used as a source of atherosclerotic tissue. Tissues were frozen in liquid nitrogen and crushed with a precooled stainless steel pestle and mortar. The powdered tissue was dissolved in extraction buffer containing 2% sodium dodecyl sulfate (SDS) in phosphate-buffered saline (PBS) and 2 mmol/L phenylmethylsulfonyl fluoride at pH 7.0. Total protein was quantified with the Bradford procedure. Samples (5 ng protein) were resolved by SDS–polyacrylamide gel electrophoresis in duplicate and transferred onto nitrocellulose membranes. After overnight incubation with transfer buffer solution (0.025 mol/L Tris, 0.2 mol/L glycine, and 10% methanol) plus 3% bovine serum albumin, each sample was exposed to either 1:100 or 1:200 dilution of rabbit anti-human uPA-R (ADI 399R). After a 1-hour wash with the transfer buffer solution, the membrane was exposed to [¹²⁵I] goat anti-rabbit IgG, washed for 1 hour, dried briefly, and used for autoradiography with Kodak XAR-5 film and Cronex intensifying screens (New England Nuclear Corp). Bands comprising radiolabeled uPA-R were quantified by densitometry (Ultrascan XL, Pharmacia LKB Biotechnology) and compared with those seen with a uPA-R standard binding supplied by ADI.

Cell Culture
Human vascular smooth muscle cells were cultured from aortic explants of organ donors provided by the Mid-America Eye and Tissue Bank. Bovine aortic smooth muscle cells were cultured from aortas from freshly slaughtered cattle as described previously.³⁰ Initial cultures were exposed to trypsin and plated again on glass coverslips before immunocytochemical analysis. Monoclonal antibody to -smooth muscle actin verified the cells to be vascular smooth muscle cells, which were then maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin/streptomycin/amphotericin (1000 U/mL, 1 mg/mL, 2.5 nL) (Sigma Chemical Co).

Cell Migration
Eight parallel "wounds" were created with a 2.0-mm-wide cell scraper in confluent vascular smooth muscle cell monolayers in 75-cm² flasks (Falcon Labware). Cells were washed twice with PBS and fresh media was added. The cultures were visualized with a microscope to verify consistency of wound size and to delineate the area to be photographed subsequently. Migrating cells were photographed periodically with a Nikon inverted photomicroscope until wound healing was complete (24 hours). These cells were cultured in DMEM with 10% FBS plus 1% PSA, with or without monoclonal antibody to uPA-R (20 ng/mL).

Preparation of cDNA and Northern Blotting
A human uPA-R cDNA probe was generated by EcoRI and Xba I digestion of uPA-R cDNA in a Bluescript M13+ plasmid (365768, American Type Culture Collection). Probe size was confirmed by ethidium bromide staining after electrophoresis on agarose gels. The probe was radiolabeled with deoxycytodine-5'-[a-³²P]triphosphate (Amersham) by the random primer method with kits obtained from Boehringer Mannheim Corp. Smooth muscle cells cultured for Northern blotting were plated on 75-cm² flasks and grown to confluence. Eight parallel wounds were created as described above. Either -thrombin (5 National Institutes of Health [NIH] U/mL) or platelet-derived growth factor (PDGF) (BB form, 5 ng/mL) was added to the fresh media after injury. Total RNA was extracted at 0, 1, 4, 8, and 24 hours in acid guanidinium isothiocyanate.³¹ Agarose gel electrophoresis and transfer of size-fractionated RNA to nylon membranes were performed as described previously.³⁰ Equal loading of RNA was verified by visualization of 28S and 18S RNA by ethidium bromide staining and UV detection. Nylon membranes (Biodyne) were prehybridized in a solution containing 1.0 mol/L NaCl, 1% SDS, 0.05 mol/L Tris-HCl, 10% dextran sulfate, and 200 ng/mL denatured calf thymus DNA (D8661, Sigma) for 24 to 36 hours at 42°C. Hybridization with uPA-R (6.5x10⁶ cpm) was performed at 42°C with agitation for 22 to 24 hours. Membranes were probed again with glyceraldehyde-3-phosphate dehydrogenase to verify of equal loading after stripping the probe by boiling the membrane in stripping buffer (1% SDS, 1 mmol/L EDTA, 10 mmol/L Tris) for 15 minutes and washing it with 1% SDS and 2xSSC at 60°C for 5 minutes. Autoradiography was performed with XAR-5 film (Kodak) and intensifying screens (Cronex Lightening Plus, Dupont-New England Nuclear) at -70°C. The relative intensities of the autoradiographic bands were quantified by laser densitometry.

	Results

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Histological Analyses of Rabbit Aortas
Aortas from New Zealand White rabbits fed 2% cholesterol–enriched diet for 3 months exhibited hyperplastic neointima populated by mostly lipid-ladened macrophages and lipid-rich smooth muscle cells. Vessel wall thickness was 2- to 2.5-fold greater compared with vessels from rabbits fed normal chow. A thin layer of endothelial cells covering the luminal surface (Fig 1) in the abnormal vessels was consistent with previous observations.^{32 33}

View larger version (2K): [in this window] [in a new window]   Figure 1. Urokinase-type plasminogen activator (uPA-R)–expressing cells in serial sections of an aorta from a New Zealand White rabbit fed a 2% cholesterol–enriched diet for 3 months. a, Macrophages identified by RAM-11 (blue). b, uPA-R–positive cells identified by anti-human uPA-R (red). c, Smooth muscle cells identified by HHF-35 (blue). d, Double immunostaining of uPA-R (red) and smooth muscle cells (dark blue). Counterstaining was done with hematoxylin. All sections are x40 magnification.

Localization of uPA-R in Macrophages and Vascular Smooth Muscle Cells in Rabbit Atherosclerotic Lesions
Most cells in the atheroma identified as macrophages (RAM-11 positive) (blue in Fig 1a) expressed uPA-R (red in Fig 1b). However, some were smooth muscle cells (blue in Fig 1c) in the media and in neointima. Double immunostaining of adjacent sections for smooth muscle cells and uPA-R confirmed the localization (Fig 1d).

To identify the type of cell that expressed uPA-R, serial double immunostaining studies were performed. uPA-R was first localized in the neointima with the red stain (Fig 2a and 2b). In Fig 2a, macrophages were subsequently stained dark brown (labeled m). In Fig 2b, smooth muscle cells were subsequently stained dark brown (labeled s). Some small cells clustered at the luminal edge were smooth muscle cells, as shown in adjacent sections immunostained for uPA-R (red) and smooth muscle (dark brown) (Fig 2b). Most cells identified as macrophages (Fig 2a) were positive for uPA-R (Fig 2b). Thus, uPA-R in atheroma in rabbits was present in macrophages and neointimal vascular smooth muscle cells.

View larger version (2K): [in this window] [in a new window]   Figure 2. Localization of uPA-R and cell-specific antigens in serial sections of an aorta from a New Zealand White rabbit fed 2% cholesterol–enriched diet for 3 months. a, Localizations of urokinase-type plasminogen activator (uPA-R) (red) in macrophages (dark brown). b, Localization of uPA-R (red) in smooth muscle cells (dark brown). L indicates lumen; m, macrophage; short arrows, internal elastic lamina; s, smooth muscle cells (x40 magnification).

Histological Analyses of Human Atheroma
A total of 21 pieces from carotid, iliac, femoral, and tibial arteries obtained at the time of clinically mandated surgical excision were sectioned serially and analyzed for the presence of uPA-R. In contrast to rabbit vessels, the histological appearance of human atherosclerotic vessels was quite variable. Virtually all plaques contained macrophages and smooth muscle cells in the neointima and variable uPA-R expression. Approximately 20% of the plaque sections showed uPA-R in the adventitia and media.

Serial sections through an atherosclerotic, human tibial artery are shown in Fig 3 with endothelial cells (factor VIII–related protein-positive cells) immunostained brown in Fig 3a and smooth muscle cells (-actin–positive cells) immunostained brown in Fig 3b. The neointima is populated mostly by -actin–positive smooth muscle cells. In other sections, large amounts of amorphous plaque and thrombus were evident at the luminal surface (Fig 3c). Intense, red-stained uPA-R protein was evident at high magnifications of the same plaque (Fig 3d). The plaque and thrombus were also positive for macrophages. In thrombi, cells expressing uPA-R were present in contrast to cells in the medial layer of a normal vessel. In another section taken from femoral artery, large numbers of lipid-laden macrophage also were present (Fig 3e) that were later found to be positive for uPA-R. All atherosclerotic vessels examined exhibited irregularities of luminal surfaces with variable but substantial neointimal hyperplasia. However, in nonatherosclerotic vessels, an intact endothelial monolayer was seen close to the internal elastic lamina (Fig 3f).

View larger version (2K): [in this window] [in a new window]   Figure 3. Sections from human atherosclerotic vessels. a, Atherosclerotic tibial artery immunostained for endothelial cells with anti-factor VIII–related antibody (brown) (DAB) followed by counterstaining with hematoxylin. b, Adjacent section with smooth muscle cells immunostained brown (DAB). c, Atherosclerotic carotid artery section with plaque formation and urokinase-type plasminogen (uPA-R) immunostained red with AEC (x40 magnification). d, Higher magnification of uPA-R–positive cells in the atheroma. These cells also stained positive for macrophages and smooth muscle cells. e, Higher magnification compared with panel a of a section from an atherosclerotic femoral artery with macrophage (blue) infiltration into neointima. f, Nonatherosclerotic human carotid artery immunostained for the presence of uPA-R (red) and endothelial cells (brown). A section from a fresh frozen human carotid artery with intact endothelial cells immunostained with anti-factor VIII–related proteins and DAB (brown) and counterstained with hematoxylin. No uPA-R was seen (x100 magnification). Short arrows indicate internal elastic lamina; e and long arrows, endothelium.

Localization of uPA-R in Macrophages and Vascular Smooth Muscle Cells in Human Atherosclerotic Lesions
Serial sections of atherosclerotic human femoral arteries exhibited uPA-R expression (Fig 4b) in macrophages identified by CD68 (Fig 3a) and smooth muscle cells identified by -actin (Fig 4c). As in aortic sections from cholesterol-fed rabbit, uPA-R was present in macrophages within atherosclerotic lesions and in neointimal smooth muscle cells, particularly near the fibrous cap.

View larger version (2K): [in this window] [in a new window]   Figure 4. Identification of urokinase-type plasminogen (uPA-R)–expressing cells in serial sections from an atherosclerotic femoral artery. a, Macrophages are immunostained blue. b, uPA-R is immunostained red. c, Smooth muscle cells are immunostained blue. d, Control with preimmune serum without primary antibody and with a hematoxylin counterstain.

uPA-R Protein
Western blotting was performed to compare the amounts of uPA-R protein in normal and atherosclerotic vessel wall extracts. Cell lysates from U937 cells, a human histiocytic cell line known to express large amounts of uPA-R, were used as a positive control (Fig 5). On immunoblots, the monoclonal antibody against uPA-R reacted with more than one protein band. However, the most abundant protein detected by far was identified as a 55-kD protein, corresponding to glycosylated, cell surface uPA-R. Atherosclerotic vessels (labeled iliac) exhibited approximately 10-fold more immunoreactive uPA-R compared with normal aorta. The other bands detected are consistent with nonglycosylated forms of uPA-R of approximately 35 kD and partially deglycosylated or cleaved forms of approximately 45 kD.

View larger version (48K): [in this window] [in a new window]   Figure 5. Autoradiograph of a Western blot of tissue extracts exposed to a monoclonal antibody to urokinase-type plasminogen (uPA-R). U937 cells were used as controls. Protein (5 ng, in duplicate) was loaded on each lane on a 10% polyacrylamide gel. Relative intensities of the 55-kD bands were compared by densitometry. Tissue extracts from atheroma contained approximately ninefold uPA-R than extracts from normal aortas (n=3).

uPA-R in Migrating Vascular Smooth Muscle Cells
Migration of bovine and human smooth muscle cells in a monolayer subjected to trauma was decreased by an antibody to uPA-R in vitro. Serial photographs of the bovine cells migrating into a region of injury implemented with a cell scraper demonstrated 50% closure in 16 hours and complete closure in 24 hours under control conditions. Antibody to uPA-R decreased migration, with closure declining to approximately 20% in 24 hours (Fig 6). Similar results were obtained with cells from both species. Bovine cell monolayers were more compact; therefore, changes were visualized more easily because the bovine cells were smaller than the human cells.

View larger version (2K): [in this window] [in a new window]   Figure 6. Inhibition by antibodies against urokinase-type plasminogen (uPA-R) of migration of wounded smooth muscle cell in a monolayer subjected to injury. Vascular smooth muscle cells were grown to confluence in a 75-cm2 flask. Eight parallel wounds were induced with a cell scraper. Consistency of wound size was verified by visualization with an inverted microscope, and the region involved was delineated for photography. a, Wound 1 hour after injury, a time at which uPA-R was often but not universally increased. b, Wounded monolayer cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin/amphotericin (PSA) 24 hours after injury. c, Monolayer cultured in DMEM with 10% FBS, 1% PSA, and monoclonal antibody against uPA-R (20 ng/mL) 24 hours after injury. Arrowheads indicate wounded edges.

Assessment of Transcription
PDGF and -thrombin induce migration of bovine and human vascular smooth muscle cells in modified Boyden chambers (unpublished observations). We have found by flow cytometry that this phenomenon is associated with upregulation of cell surface uPA-R expression. To determine whether increased expression is regulated at the transcriptional level, we subjected monolayers of confluent bovine vascular smooth muscle cells to injury with a cell scraper and migration stimulated by adding either PDGF (5 ng/mL) or -thrombin (5 NIH U/mL). Total RNA was harvested 0, 1, 4, 8, and 24 hours after injury, and the relative abundance of uPA-R and PAI-1 mRNA was determined by Northern blot analyses. Ethidium bromide staining of gels before transfers is used to verify equal loading of total RNA onto gels. As shown previously, both PDGF and thrombin increased PAI-1 mRNA in a time-dependent manner.³⁰ uPA-R-mRNA was less prominent than PAI-1 mRNA but did increase with PDGF in 4 to 8 hours and with thrombin in 8 hours. uPA-R-mRNA levels remained elevated for 24 hours. However, the increase in uPA-R–mRNA levels appeared to be too modest to account for the large increase in protein (Fig 7).

View larger version (123K): [in this window] [in a new window]   Figure 7. Autoradiograph of a Northern blot showing plasminogen activator inhibitor type 1 (PAI-1) (3.1 kD) and urokinase-type plasminogen (uPA-R) (1.6 kD) in extracts from bovine aortic smooth muscle cells cultured 0, 1, 4, 8, and 24 hours after injury to the monolayer. Migration was stimulated by platelet-derived growth factor (PDGF) (5 ng/mL) or -thrombin (5 National Institutes of Health U/mL). Bottom panel shows the same membrane stained with ethidium bromide depicting 28S and 18S ribosomal RNAs. Use of bovine cells permitted extraction of larger amounts of RNA without degradation compared with human cells. Results with human cells were qualitatively similar, but the amounts of RNA extracted were reduced and degraded components were evident.

	Discussion

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uPA-R has been implicated as a mediator of tumor growth and invasion, tissue remodeling, and migration of cells involved in ovulation, implantation, and metastasis.³⁴ However, the primary focus on mediators of vascular pathology has been on chemoattractants and mitogens such as PDGF and fibroblast growth factor.^{35 36} In the present study, uPA-R was found in abundance in rabbit and human atheroma. None was detected in normal arterial tissue. The uPA-R was localized specifically to macrophages and vascular smooth muscle cells, consistent with the hypothesis that its upregulation predisposes to neointimalization.

Neointimal vascular smooth muscle cells in vitro produce fivefold more PDGF-like growth factors than quiescent counterpart controls, vascular smooth muscle cells from the media.³⁷ Thus, neointimal smooth muscle cells differ functionally from medial cells. Furthermore, cells with high proteolytic activity release excessive amounts of growth factors.³⁸

In 95% of atherosclerotic lesions examined, uPA-R was present in association with macrophages and smooth muscle cells in the neointima. In addition, uPA-R was present in approximately 20% of the adventitia and media samples examined, especially near the internal elastic lamina. The relative lack of uPA-R expression in cells in the media may reflect the lack of cells migrating within the media compared with in neointima.

Atherosclerotic vessels contained more immunoreactive uPA-R than did normal vessels. In extracts from atheroma, several bands were identified with the use of antibody to uPA-R. The 35-kD band was consistent with a control antigen (uPA-R standard). By far the most abundant band was the 55-kD band, followed by a 65-kD band corresponding to the uPA-R described previously.³⁹ The 35-kD protein is consistent with an unmodified translational product found in endoplasmic reticulum⁴⁰ -the larger protein with glycosylated uPA-R that is transported to the cell surface, where it is anchored by GPI.^{41 42}

Cell-associated uPA is upregulated in both migrating endothelial cells and smooth muscle cells in vitro.⁴³ Our results show that uPA-R also is upregulated. In addition, we found that vascular smooth muscle cells in culture migrate in response to a gap induced by injury to monolayers. In control cultures with growth media containing 10% FBS, the gap closed within 24 hours. However, in the presence of antibodies against uPA-R, closure was delayed. Thus, upregulated cell-surface uPA-R appeared to facilitate cell migration. The concept that uPA but not TPA appears to participate in smooth muscle cell migration is supported by recent studies with mutant mice lacking one or both of these enzymes.⁴⁴ Because migration appears to depend on uPA-R, the receptor is an attractive target for prevention or retardation of atherogenesis.

	Acknowledgments

 
This work was supported in part by NIH grant HL-17646, SCOR in Coronary and Vascular Diseases. Dr Daugherty is an Established Investigator of the American Heart Association. We thank John Botz, Jeffrey Labuda, and Debra L. Rateri for technical assistance and Kelly Hall for secretarial assistance.

Received June 8, 1994; accepted October 10, 1994.

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