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Biomedical subjects

E S Barnathan

Publications and source records attributed to E S Barnathan.

At least 37 records · Page 2Linked to original sources
PubMed1996

Inhibition of vein graft intimal and medial thickening by periadventitial application of a sulfated carbohydrate polymer.

PURPOSE: The purpose of this study was to determine whether the wall thickening observed in vein grafts after they were placed into the arterial circulation could be inhibited by periadventitial delivery of an insoluble sulfated polymer of beta-cyclodextrin (P-CDS) capable of tightly binding heparin binding growth factors. METHODS: Thirty-four New Zealand white rabbits underwent implantation of reversed autologous jugular vein interposition grafts in the common carotid artery and were randomized to receive either 20 mg P-CDS (n = 18) topically around the graft or no additional therapy (n = 16). Before being killed at 28 days, animals were given bromodeoxyuridine to assess smooth muscle cell proliferation. Histomorphometric analyses were performed after perfusion fixation. RESULTS: Compared to controls, treatment with P-CDS was associated with reduced mean intimal thickness (24 +/- 3 vs 38 +/- 4 microns; mean SEM, p < 0.01) and intimal area (0.25 +/- 0.03 vs 0.54 +/- 0.09 mm2; p < 0.01). There was also significantly less medial thickness in the P-CDS group (45 +/- 3 vs 63 +/-3, p < 0.001). There was no significant difference in intimal or medial smooth muscle cell proliferation between P-CDS-treated and control vein grafts at 28 days. The polymer persisted in the adventitia with a mild foreign body reaction. CONCLUSION: Periadventitial placement of P-CDS, a novel, insoluble, sulfated carbohydrate polymer, inhibits intimal and medial thickening of vein bypass grafts in this model of vein grafting. The persistence of P-CDS in vivo for prolonged periods, and the ease of topical application of P-CDS during vascular bypasses may have important implications for its future use in vascular surgery.

Administration, Topical↗
PubMed1996

Endothelial cells internalize monoclonal antibody to angiotensin-converting enzyme.

We investigated the fate of MAb 9B9, a monoclonal antibody to angiotensin-converting enzyme (ACE), which binds to endothelium both in vitro and in vivo. Using cultured human umbilical vein endothelial cells (HUVEC) and isolated perfused rat lungs (IPL), we demonstrated specific and saturable binding of 125I-labeled MAb 9B9 at 4 degrees C [affinity constant (Kd) = 20-50 nM, maximal number of binding sites (Bmax) = 1.5-3.0 x 10(5) sites/cell]. When 125I-MAb 9B9 was bound to HUVEC at 37 degrees C, only 40% of cell-associated radioactivity was acid elutable, suggesting antibody internalization. This was confirmed by finding that 1) the amount of MAb 9B9 uptake at 37 degrees C was higher than at 4 degrees C both in HUVEC and IPL; 2) binding of 125I-labeled streptavidin with HUVEC and IPL pretreated with biotinylated MAb 9B9 (b-MAb 9B9) was diminished in a temperature- and time-dependent fashion at 37 degrees C; and 3) b-MAb 9B9 bound to HUVEC at 37 degrees C was found intracellularly by ultrastructural analysis using streptavidin gold. Intracellular 125I-MAb 9B9 was found in microsomal fractions of lung homogenate from IPL and after intravenous (iv) injections in rats. Degradation of internalized MAb 9B9 was minimal, since > 90% of cell-associated 125I label remained precipitable by trichloracetic acid in HUVEC, IPL, and in vivo. Autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of lung homogenates made as late as several days after iv injections of 125I-MAb 9B9 in rats demonstrated a predominant band above 140 kDa. These data indicate that endothelial cells either in vitro or in vivo internalize the ACE ligand MAb 9B9 without significant intracellular degradation. Therefore MAb 9B9 may be useful for selective intracellular delivery of drugs to the pulmonary vascular endothelium after systemic administration.

Animals↗
PubMed1996

Contrasting effects of plasminogen activators, urokinase receptor, and LDL receptor-related protein on smooth muscle cell migration and invasion.

Smooth muscle cell (SMC) migration is an early response to vascular injury and contributes to the development of intimal thickening. Upregulation of several components of the plasminogen activator (PA) system has been documented after vascular injury. Utilizing a Transwell filter assay system and human umbilical vein SMCs, we sought to define the role of four different PA system components on SMC migration and matrix invasion: (1) PAs, (2) plasmin, (3) PA receptors, and (4) PA clearance receptors (ie, low density lipoprotein receptor-related protein [LRP]). Addition of active two-chain urokinase-type PA (UPA) stimulated random migration (192 +/- 30% of control, 0.36 nmol/L, P < .001). The stimulation was inhibited by pretreatment with diisopropylfluorophosphate, PA inhibitor type 1 (PAI-1), or aprotinin, a plasmin inhibitor. Augmented migration was also observed with either low-molecular-weight UPA or the amino terminal fragment of UPA (ATF), with the effects being additive. Stimulation by ATF alone, however, was not inhibited by aprotinin. The stimulatory effect was not specific for UPA, in that tissue-type PA (TPA) also increased migration (169 +/- 9% of control, 10 nmol/L, P < .001); the augmentation was inhibited by pretreatment with DFP, PAI-1, or aprotinin and was additive to the UPA effect. Antibodies to the UPA receptor but not 5'-nucleotidase (another glycosylphosphatidylinositol-anchored cell surface protein) inhibited baseline and UPA-stimulated migration. Similarly, both UPA and TPA stimulated invasion of a collagen gel; this augmentation was inhibited by aprotinin, whereas antibodies to the UPA receptor reduced baseline invasion. Finally, we tested whether inhibition of LRP function, which mediates internalization of PA/inhibitor complexes, affected either process. Both antibodies to LRP and recombinant receptor associated protein, a known inhibitor of ligand binding to the LRP, significantly inhibited migration but did not affect collagen gel invasion. These data demonstrate the ability of several components of the PA system to modulate SMC migration and invasion in vitro via plasmin-dependent and -independent mechanisms.

Aprotinin↗
PubMed1996

Targeting of antibody-conjugated plasminogen activators to the pulmonary vasculature.

Thrombolytic therapy has not been widely used for pulmonary embolism due to less than optimal results with conventional plasminogen activators. We propose a new approach to deliver plasminogen activators to the luminal surface of the pulmonary vasculature to potentially improve dissolution of pulmonary thromboemboli. Our previous studies have documented that a monoclonal antibody (mAb) to angiotensin-converting enzyme (anti-angiotensin-converting enzyme mAb 9B9) accumulates in the lungs of various animal species after systemic administration. We coupled 125I-labeled biotinylated plasminogen activators (single-chain urokinase plasminogen activator, tissue-type plasminogen activator and streptokinase) to biotinylated mAb 9B9, using streptavidin as a cross-linker. The fibrinolytic activity of plasminogen activators was not changed significantly by either biotinylation or by coupling to streptavidin. Antibody-conjugated plasminogen activators bind to the antigen immobilized in plastic wells and provide lysis of fibrin clots formed in these wells. Therefore, antibody-conjugated plasminogen activators bound to their target antigen retain their capacity to activate plasminogen. One hour after i.v. injection of mAb 9B9-conjugated radiolabeled biotinylated single-chain urokinase plasminogen activator, biotinylated tissue-type plasminogen activator or biotinylated-streptokinase in rats, the level of radiolabel was 7.4 +/- 0.8, 5.9 +/- 0.4 and 3.6 +/- 0.4% of injected dose/g (ID/g) of lung tissue vs. 0.5 +/- 0.01, 0.3 +/- 0.01 and 0.6 +/- 0.3% ID/g after injection of the same activators conjugated with control mouse IgG (P < .01 in all cases). Injection of mAb 9B9-conjugated radiolabeled plasminogen activator led to its rapid pulmonary uptake with a peak value 6.2 +/- 1.2% ID/g attained 3 hr after injection. One day later, 2.2 +/- 0.5% of the injected radioactivity was found per gram of lung tissue, although the blood level was 0.13 +/- 0.03% ID/g (lung/blood ratio 16.7 +/- 0.3). Therefore, conjugation of plasminogen activators with anti-angiotensin-converting enzyme mAb 9B9 provides their specific targeting to and prolonged association with the pulmonary vasculature. These results provide a basis for study of the local pulmonary fibrinolysis by mAb 9B9-conjugated plasminogen activators.

Animals↗
PubMed1995

The functional effects of biotinylation of anti-angiotensin-converting enzyme monoclonal antibody in terms of targeting in vivo.

The effect of modification with biotin N-hydroxysuccinimide ester of mouse monoclonal antibody to angiotensin-converting enzyme, anti-ACE Mab 9B9, on its targeting to endothelial cells has been studied in vitro and in vivo. By in vitro assay, Mab 9B9 biotinylated at a biotin/IgG molar ratio in reaction mixture (B/IgG ratio) of 0.7-2.2 bound streptavidin monovalently and retained antigen-binding capacity. Mab 9B9 biotinylated at a B/IgG ratio of 20 and higher bound streptavidin polyvalently. Extensive biotinylation (B/IgG ratio of 60 and higher) led to dramatic reduction of Mab 9B9 Ag-binding capacity and to reduction of Mab 9B9 recognition by goat polyclonal antibody to mouse IgG. Radiolabeled Mab 9B9 biotinylated at a B/IgG ratio of 6 (b6-Mab 9B9) bound effectively to cultured vascular endothelium, with affinity characteristics similar to nonbiotinylated Mab 9B9. Endothelial cells internalized both Mab 9B9 and b6-Mab 9B9 to the same extent (60% internalization at 3 h incubation at 37 degrees C). Degradation of cell surface-associated Mab 9B9 or b6-Mab 9B9 was very low (< 1% as measured by TCA solubility of radiolabel). In contrast, degradation of internalized b6-Mab 9B9 was more profound than that of Mab 9B9 (20 +/- 3% vs 6 +/- 1%, P < 0.01). After injection in rats, radiolabeled b6-Mab 9B9 had a biodistribution pattern similar to that of radiolabeled Mab 9B9. Both preparations effectively accumulated in the lung (15-20% of injected dose/g of tissue vs 2% of injected dose/g of blood).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Stimulatory effect of unsaturated fatty acids on the level of plasminogen activator inhibitor-1 mRNA in cultured human endothelial cells.

To determine whether unsaturated fatty acids induce changes in the mRNA level of plasminogen activator inhibitor type-1 (PAI-1), Northern analyses were performed on human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells that were treated with two common fatty acids. Supplementation of cultured HUVEC with docosahexanoic acid (DHA) or with dihomogamma linolenic acid (DGLA), resulted in a concentration dependent, specific increase of the PAI-1 transcript levels, which was detectable within 2 h. DHA and DGLA treatment of smooth muscle cells did not result in changes in the PAI-1 mRNA levels. Homology search of the upstream regulatory region of the PAI-1 gene sequences identified a consensus nucleotide sequence for a fatty acid-responsive element. Our results indicate that unsaturated fatty acids selectively increase PAI-1 mRNA levels in endothelial cells, the primary source of circulating PAI-1 in vivo.

Amino Acid Sequence↗
PubMed1995

Plasminogen activators augment endothelial cell organization in vitro by two distinct pathways.

Endothelial cell differentiation into capillary structures is a complex process that requires the concerted effects of several extracellular matrix proteases, including plasminogen activators. Here, the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) was evaluated in an in vitro model of endothelial morphogenesis involving organization of human umbilical vein endothelial cells into tubular structures when they are cultured on the basement membrane preparation, Matrigel. Both uPA and tPA were detected in HUVEC cultures on Matrigel, and inhibitors of plasminogen activators or of serine proteases decreased the extent of the tube network formed by the cells. The decrease resulting from serine protease inhibitors was additive to that from matrix metalloproteinase inhibitors which have previously been shown to decrease tube formation in this model, suggesting that the two classes of proteases modulate tube formation by distinct mechanisms. Plasminogen activator inhibitor (PAI)-1 decreased tube formation by 50% when added up to 4.5 h after the initiation of an 18 h assay and caused 25% inhibition when added 9.5 h after culture initiation, indicating that the effects of plasminogen activators are not limited to an early event in the differentiation process. Steady-state expression of mRNA for uPA increased during the first several hours of culture on Matrigel, further supporting a role for PA activity throughout the process of tube formation. These findings suggested that PAs may affect multiple events during tube-forming activity. A fucosylated peptide comprising the amino-terminal domain of uPA that binds to the uPA receptor (uPAR) but lacking proteolytic activity enhanced tube formation. In contrast, a defucosylated form of the same peptide had no effect. Since fucosylation of this fragment has been shown to be essential in other models of cell stimulation by uPA-uPAR interaction, these data support the hypothesis that uPA enhances endothelial morphogenesis both through proteolytic activity and via uPAR occupancy. Plasminogen activators could facilitate angiogenesis in vivo.

Capillaries↗
PubMed1995

Migrating vascular smooth muscle cells polarize cell surface urokinase receptors after injury in vitro.

The localization of proteases to cell surfaces via receptors may facilitate cell migration, invasion, and matrix degradation. Since vascular smooth muscle cell (SMC) migration may be an important event in atherosclerosis and in intimal thickening after vascular injury, we studied the cell surface expression of a receptor for urokinase-type plasminogen activator (u-PAR) in cultured human vascular SMC. Using immunofluorescence microscopy, we demonstrated several staining patterns of SMC u-PAR: at the periphery of the cell membrane, at the leading edge, and at cell-cell contact sites. When migration experiments were performed using a wound assay, one-third of the SMC at the wound edge demonstrated polarization of cell surface u-PAR toward the leading edge of the cell membrane (32 +/- 2%, +/- SEM, n = 7). A similar pattern was seen with an antibody to caveolin, a transmembrane protein found in caveolae, but not with an antibody to 5'-nucleotidase, another cell surface glycophosphatidylinositol-anchored protein, which was homogeneously expressed on the cell surface. Low-density lipoprotein receptor-related protein, which mediates internalization of u-PAR bound ligands, was distributed in a diffuse punctate pattern, not polarized to the leading edge. Double immunofluorescent studies demonstrated codistribution of SMC u-PAR with vinculin and caveolin in migrating SMC at the leading edge in a wound assay. Polarization of cell surface u-PAR was not observed in either nonwounded or subconfluent cultures, despite random migratory behavior. These studies suggest that in response to wounding, human vascular SMC polarize and concentrate cell surface u-PAR to their leading edge, perhaps facilitating directional migration.

5'-Nucleotidase↗
PubMed1995

Plasminogen activator system in human coronary atherosclerosis.

Altered coronary artery expression of plasminogen activator (PA) system components may predispose to thrombosis and modulate the vascular response to injury. By immunohistochemistry, we studied the expression of PAs (tPA and uPA), their major physiological inhibitor (PAI-1), and a receptor for uPA (uPAR) in human coronary arteries with either pure fibrointimal proliferation (n = 15) or developed atherosclerotic plaques (n = 10). Overall, the degree of staining showed the following rank order: PAI-1 > tPA > uPAR > uPA. A similar pattern was seen in two normal coronary arteries. There were no significant differences in the extent of staining in any vascular compartment between atherosclerotic arteries and those with only fibrointimal proliferation. However, the ratio of intimal to medial expression of tPA (P = .001) and uPAR (P = .004) was significantly increased in atherosclerotic arteries, with a similar trend for uPA (P = .069) but not for PAI-1 (P = .73). Four of 10 atherosclerotic arteries had higher uPAR expression in the intima than in the media, whereas none of the 15 arteries with only fibrointimal proliferation had this pattern (P < .01). Dual labeling studies demonstrated colocalization of all four PA system components in endothelial cells, smooth muscle cells, and macrophages, with a predominance of PAI-1. Thus, coronary arteries with a wide range of vascular pathology express an abundance of antifibrinolytic potential with enhanced local expression of profibrinolytic proteins, mainly within atherosclerotic plaques.

Adult↗
PubMed1995

Sustained inhibition of intimal thickening. In vitro and in vivo effects of polymeric beta-cyclodextrin sulfate.

Intimal thickening after vascular injury may be modulated in part by heparin binding growth factors. We hypothesized that placement of a therapeutic polymer in the periadventitial space capable of tightly binding growth factors might alter the vascular response to injury. We first demonstrated that incubation of rat aortic smooth muscle cells with an insoluble, sulfated polymer of beta-cyclodextrin (P-CDS) was associated with a dose-dependent inhibition of proliferation induced by fetal calf serum, fibroblast growth factor-2 (FGF-2), platelet-derived growth factor BB, or epidermal growth factor. Preincubation studies of P-CDS with FGF-2 revealed a very rapid removal of mitogenic activity. Using radiolabeled FGF-2 (0.25 microg/ml), we observed a very rapid association rate (0.34 +/- 0.07 min-1, n=4) and a very slow dissociation rate (3.3 +/- 0.2 X 10(-7) min-1) at 37 degrees C, suggesting a high affinity interaction. Using both Transwell and linear under-agarose assays, we demonstrated a significant inhibition of random migration (chemokinesis) by P-CDS. Unsulfated polymeric beta-cyclodextrin (P-CD) had little if any of these effects, suggesting that the high negative charge density of P-CDS was important for the effects. Finally, rats undergoing carotid artery balloon injury were randomized to treatment with periadventitial P-CDS or no treatment, and were killed at 4 (n=20), 14 (n=59), and 88 d (n=14). Morphometric analysis demonstrated significant and sustained inhibition of intimal thickening in P-CDS-treated rats at 14 (P < 0.01) and 88 d (P < 0.05) using absolute intimal area or intima/media area ratios. No inhibition was seen in a group of rats treated with P-CD. In P-CDS-treated rats, bromodeoxyuridine labeling studies revealed fewer labeled smooth muscle cells in the intima at 14 d (P=0.01), while staining with Evans blue revealed enhanced late endothelial cell regrowth. Thus, periadventitially applied sulfated beta-cyclodextrin polymer, which can tightly bind heparin binding growth factors, inhibits intimal thickening in vivo in a sustained fashion without using an additional delivery system. These studies suggest that cellular processes mediated by heparin binding growth factors may be modulated by P-CDS.

Angioplasty, Balloon↗
PubMed1995

Inhibition of human vascular smooth muscle cell migration and proliferation by beta-cyclodextrin tetradecasulfate.

Smooth muscle cell migration and proliferation are important regulatory processes in the development of intimal thickening after vascular injury. beta-cyclodextrin tetradecasulfate, an orally active synthetic heparin mimic, is effective in inhibiting rabbit aortic smooth muscle cell proliferation in vitro and in limiting restenosis in an experimental angioplasty restenosis model in rabbits (Hermann et al., 1993). However, its effects on migration are unknown, as are its effects on human vascular smooth muscle cell biology in general. Using a Transwell assay system, we demonstrated a dose dependent inhibition of human vascular smooth muscle cell random migration for beta-cyclodextrin tetradecasulfate with half maximal inhibition between 100-500 micrograms/ml and 85 +/- 1% inhibition at 10(3) micrograms/ml (P < .001, n = 4). At this latter concentration, inhibition of migration was also demonstrable using a linear under-agarose assay, where the mean velocity for beta-cyclodextrin tetradecasulfate-treated cells (8 +/- 2 microns/h, +/- S.E.) was significantly less than for control cells (21 +/- 4, P < .01), but equaled that of control cells 48 h after drug withdrawal (21 +/- 1, P = NS). Single cell analysis over 17 h using time lapse video microscopy revealed significant inhibition of total migration pathway distance for beta-cyclodextrin tetradecasulfate-treated smooth muscle cells compared with control smooth muscle cells (0.40 +/- 0.03 mm vs. 0.73 +/- 0.03, P < .01). We also demonstrated a dose-dependent inhibition of serum-induced proliferation by beta-cyclodextrin tetradecasulfate in both cultured human umbilical vein and coronary artery smooth muscle cells. To assess whether beta-cyclodextrin tetradecasulfate had any direct cellular toxicity, we measured the release of intracellular LDH at 6 or 24 h. At 10(3) micrograms/ml or less, there was no increase in LDH release compared with untreated cultures. Thus, beta-cyclodextrin tetradecasulfate may be an effective agent in inhibiting intimal thickening after vascular injury by limiting both smooth muscle cell migration and proliferation.

Cell Division↗
PubMed1994

Angioplasty in multivessel coronary artery disease.

Randomized trials directly comparing angioplasty with bypass surgery for complex multivessel disease are under way. Preliminary results suggest that angioplasty can be effective in some patient groups, so that neither procedure will predominate. The early results also suggest criteria to guide physicians in selecting the most appropriate method for individual patients.

Angioplasty, Balloon, Coronary↗
PubMed1994

Lipofectin-aided cell delivery of ribozyme targeted to human urokinase receptor mRNA.

A 37-mer hammerhead ribozyme has been designed to efficiently cleave the 1.4 kb mRNA of the urokinase plasminogen activator receptor (uPAR). Under in vitro conditions, the chemically synthesized ribozyme cleaved uPAR mRNA and inhibited its translation in a concentration-dependent fashion. The ribozymes were 5'-[35S]thiophosphorylated and used as a model to analyze conditions for RNA delivery in a cultured human osteosarcoma cell system. Ribozymes degraded immediately in cell-conditioned medium but ribozymes complexed with lipofectin were protected from RNases for up to 22 h. Lipofectin rapidly transported ribozyme into the cell, where it accumulated almost exclusively in the cytoplasm. Thus, lipofectin dramatically enhances stability and cytoplasmic delivery of ribozymes, potentially enabling targeting of mRNA in vivo.

Base Sequence↗
PubMed1994

Endocytosis of urokinase-plasminogen activator inhibitor type 1 complexes bound to a chimeric transmembrane urokinase receptor.

The urokinase receptor (uPAR) is linked to plasma membranes through a glycosylphosphatidylinositol (GPI) anchor. It has been posited that the GPI anchor facilitates clearance of uPAR-bound complexes between two chain urokinase (tcuPA) and plasminogen activator inhibitor type 1 (PAI-1) by the alpha 2-macroglobulin receptor (alpha 2MR) which permits re-expression of unoccupied uPA receptors on the cell surface. To test this hypothesis we compared internalization and degradation of 125I-labeled tcuPA-PAI-1 by COS cells expressing either transfected wild-type, GPI-linked uPAR (uPAR/GPI), or a chimeric receptor composed of the extracellular domains of uPAR linked to the transmembrane and cytosolic domains of the alpha chain (p55 subunit) of the interleukin-2 receptor (uPAR/IL-2R alpha). The kinetics of binding, internalization and degradation of tcuPA-PAI-1 by COS cells expressing each form of uPAR were virtually identical. However, internalization of complexes by uPAR/IL-2R alpha was more susceptible to inhibition by recombinant soluble 39-kDa alpha 2MR-associated protein (RAP) which competes for binding of tcuPA-PAI-1 complexes to alpha 2MR (p < 0.001), and the internalization was accompanied by a greater reduction in the number of surface uPAR/IL-2R alpha, than uPAR/GPI (p < 0.05). These studies indicate that the rate of internalization of tcuPA-PAI-1 is governed primarily by the extracellular domains of uPAR, whereas the GPI anchor may facilitate internalization of complexes and re-expression of uPAR when binding sites on alpha 2MR are limiting.

Animals↗
PubMed1993

Overexpression of urokinase receptor increases matrix invasion without altering cell migration in a human osteosarcoma cell line.

Proteolysis triggered by receptor-bound urokinase-type plasminogen activator (uPA) involves a cascade of species-specific molecular interactions. To study the role of the uPA receptor (uPAR) in such interactions, a human osteosarcoma cell line (HOS), which normally expresses low levels of uPAR, was transfected with human uPAR complementary DNA. One of several stably transformed clonal cells lines, designated 2A2, was characterized and compared to the parental HOS, revealing the following: (a) stable incorporation of uPAR complementary DNA into the genome demonstrated by Southern blot analysis; (b) a 10-fold increase in steady state mRNA levels of uPAR assessed by Northern blot analysis; (c) a 2-fold increase in the surface expression of glycosylphosphatidylinositol anchored uPAR protein determined by enzyme-linked immunosorbent assay and by the specific binding of radiolabeled single chain uPA; (d) a 2-fold increase in internalization and degradation of radiolabeled uPA/PAI-1 complexes; and (e) a 2-fold increase in receptor-bound uPA-mediated plasmin generation measured by the cleavage of a chromogenic substrate and degradation of 125I-labeled laminin. The involvement of uPAR in cellular processes was determined by comparing 2A2 and HOS cells in in vitro migration and invasion assays. The migration of 2A2 cells were slower on fibronectin-coated surfaces in a linear under-agarose assay, but both cell lines migrated at the same rate on uncoated polycarbonate filters in Boyden chamber assays. In the invasion experiments, 4 times more 2A2 than HOS cells penetrated through the barrier of reconstituted basement membrane Matrigel. These data suggest that uPAR does not potentiate random cell migration but facilitates matrix degradation and subsequent cell invasion.

Base Sequence↗
PubMed1993

Determinants of binding and internalization of tissue-type plasminogen activator by human vascular smooth muscle and endothelial cells.

Vascular injury induced by angioplasty is often followed by smooth muscle cell (SMC) proliferation, migration, and accumulation of extracellular matrix. Since plasminogen activators and their receptors may be important both in cell migration and the clearance of plasminogen activators, we studied the binding, internalization, and degradation of radiolabeled tissue-type plasminogen activator (t-PA) by explant cultures of human vascular SMC. Binding of t-PA to SMC at 4 degrees C was rapid, specific, saturable, and inhibitable by antibodies to plasminogen activator inhibitor type 1 (PAI-1). At 37 degrees C, labeled t-PA was internalized and degraded by SMC but not by human umbilical vein endothelial cells. Internalization and degradation was mediated by the low density lipoprotein receptor related protein/alpha 2-macroglobulin receptor (LRP) in that these processes were inhibited by an anti-LRP antibody, recombinant LRP-associated protein, urokinase-type plasminogen activator-PAI-1 complexes, and lactoferrin. The portion of t-PA most important for internalization after complexing with PAI-1 is likely to be in the finger and/or epidermal growth factor domains or in the carbohydrate at amino acid 117, in that the internalization of preformed t-PA.PAI-1 complexes or complexes formed on the cell surface was inhibited by an excess of active site-blocked wild type t-PA, but not by an active site blocked t-PA variant missing these domains. These studies are consistent with a model in which t-PA binds initially to SMC-associated PAI-1 with subsequent t-PA.PAI-1 internalization via LRP. SMC may play an important role in clearing t-PA-PAI-1 complexes from within the vessel wall.

Binding Sites↗