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Research Communications |
1 and ß1 chain peptides active for endothelial cell adhesion, tube formation, and aortic sprouting
a Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental Research, NIH, Bethesda, Maryland 20892–4370, USA
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
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1 and ß1 chains to identify potential sites on laminin-1 active with endothelial cells. Peptides were initially screened by testing both endothelial cell adhesion to peptide-coated wells and tube formation on Matrigel in the presence of soluble peptide. Twenty active peptides were identified in these screens. A secondary screen using the rat aortic ring sprouting assay identified 13 of the 20 peptides that stimulated endothelial sprouting. Several of these active peptides were also found to stimulate human umbilical vein endothelial cell migration in Boyden chamber assays. Differences in the amount of peptide needed for the response and in the resultant morphologies/responses were observed between the peptides in all of the assays. Our results suggest that several active domains on laminin-1 may play important roles in stimulating different steps in angiogenesis.—Malinda, K. M., Nomizu, M., Chung, M., Delgado, M., Kuratomi, Y., Yamada, Y., Kleinman, H. K., Ponce, M. L. Identification of laminin 1 and ß1 chain peptides active for endothelial cell adhesion, tube formation, and aortic sprouting. FASEB J. 13, 53–62 (1999)
Key Words: angiogenesis • basement membrane • synthetic peptides
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
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1, ß1, and 
1 chains. Laminin-1 not only has a structural role in potentially organizing the basement membrane matrix, but also has several well-documented biological activities such as neurite outgrowth, tumor metastasis, cell attachment and spreading, and angiogenesis (2–5).
Using synthetically generated peptides, investigators have identified several active sequences on laminin-1. Laminin-1-derived peptides with the IKVAV sequence from the 1 chain promote neurite outgrowth, tumor metastasis and growth, protease activity, cell adhesion, and angiogenesis (4–8). Other sequences, such as YIGSR on the ß1 chain, have different biological activities, including inhibiting angiogenesis, tumor growth and metastasis (9–13). Furthermore, sequential screening of peptides, from the 1 chain G domain (14), ß1 (M. Nomizu, unpublished results), and 1 (15) chains has identified several sequences that promote adhesion with a variety of tumor cells. One sequence from the 1 G domain, LQVQLSIR, is active for cell adhesion, neurite outgrowth, and tumor metastasis (14). This sequence is active with some but not all laminin-1-responsive neuronal cells. These data suggest that a number of additional active sites exist on laminin that could be cell type-specific.
Several laminin-1 peptides have previously been found to regulate angiogenesis, but the mechanisms are not known. Endothelial cell attachment, migration, and proliferation as well as basement membrane degradation and synthesis are key steps in angiogenesis. Angiogenesis plays an important role in tissue formation, reproduction, wound healing, and pathological conditions such as cancer and autoimmune diseases. In in vitro studies, the laminin-derived peptides IKVAV and YIGSR both alter the formation of capillary-like structures by human umbilical endothelial cells (HUVECs)3 when plated on Matrigel, a basement membrane matrix enriched in laminin-1. Very different morphologies were observed with the two peptides, with IKVAV showing sprouting and spreading of the tubes and cells (4, 16) and YIGSR showing incomplete tubes (9). IKVAV promotes angiogenesis and tumor growth in vivo, whereas YIGSR has the opposite effect.
In the current study, we screened 405 overlapping synthetic peptides derived from the 1 and ß1 chains using in vitro assays to determine sequences that were active with endothelial cells. Using a combination of adhesion assays on peptide-coated plastic dishes and by determining the capillary tube formation potential of HUVECs on Matrigel in the presence of soluble peptide, an initial screen identified 20 active peptides for further assays. Inhibition of HUVEC adhesion to laminin-1 by several of the peptides was also observed, which suggests that these sites may be physiologically active in the intact molecule. Rat aortic ring sprouting assays were then used as a secondary screen to look for potential angiogenic peptides among those peptides active in cell adhesion and in capillary tube formation on Matrigel. Thirteen peptides stimulated endothelial sprouting in the ring assay and several of these peptides were found to stimulate HUVEC migration in Boyden chamber assays. These results suggest that the peptide sequences either in the intact molecule, or existing as fragments when laminin-1 is degraded, could stimulate different steps in angiogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
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Cell culture
HUVECs were isolated from freshly delivered cords, as reported previously (17), and grown on Nunclon dishes (Nunc, Denmark) in RPMI 1640 (Life Technologies, Gaithersburg, Md.) supplemented with 20% bovine calf serum (Hyclone Laboratories, Logan, Utah), 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamycin, 2.5 µg/ml amphotericin B (fungizone) (Life Technologies), 5 U/ml sodium heparin (Fisher Scientific, Pittsburgh, Pa.), and 200 µg/ml endothelial cell growth supplement (ECGS) (Collaborative Research, Bedford, Mass.). Cells between passages 3 and 5 were used for all experiments.
Cell adhesion assay
Wells of a 96-well plate were coated at room temperature overnight with either 2 µg of each peptide or 0.5 µg of laminin-1 in phosphate-buffered saline (PBS) (positive control) in a final volume of 100 µl. The wells were then washed three times with 100 µl PBS and blocked for 2 h with 0.2% bovine serum albumin (BSA) in 100 µl PBS at room temperature. Additional uncoated wells were incubated with BSA to serve as a negative control. The wells were then washed three times with 100 µl PBS. HUVECs were detached from the dish with Versene (Life Technologies), resuspended in RPMI, and added (25,000 cells/100 µl) to each well. The plates were incubated for 1 h at 37°C and then washed twice with PBS. The attached cells were fixed and stained with 0.2% crystal violet in 20% methanol for 10 min and washed three times with PBS. For quantitation, cells were solubilized in 100 µl 2% sodium dodecyl sulfate and the optical density was measured at 560 nm. Inhibition studies were performed by coating wells with 0.5 µg of laminin-1 and then adding cells mixed with various amounts of peptide. Attachment was quantitated after 1 h as described above. Each peptide was tested in triplicate and each experiment was repeated three times.
Tube assay
The tube formation assay was performed as previously described (16) with the following modifications: 96-well plates were coated with 106 µl of Matrigel (10 mg/ml) and incubated at 37°C for 30 min to promote gelling. 13,000 HUVECs were resuspended in growth medium (serum concentration 10%) and added to each well with 200 µg of each peptide in a final volume of 100 µl. The angiogenic laminin-1 peptide IKVAV (100 µg) was added to at least three wells as a positive control. After 18 h, the plates were fixed with Diff-Quik (Baxter Healthcare Corporation, McGraw Park, Ill.) and a blinded observer assessed the morphology of the tubes. Each peptide was tested in triplicate and each experiment was repeated at least three times. Tube formation in the presence of peptides was compared to tube formation in media alone and scored, + meaning altered tube formation compared to media alone and - meaning tubes resemble the media alone control.
Aortic ring assay
Aortas were harvested from 6-wk-old Sprague-Dawley rats and immediately placed in RPMI. Fatty tissues were removed by gentle scraping and the aortas were cut into thin rings (18). Since the ends of the aortas were held by forceps during the cleaning and cutting and may have been damaged, they were discarded. Forty-eight-well plates were coated with 110 µl of Matrigel; after gelling, the rings were placed in the wells and sealed in place with an overlay of 40 µl of Matrigel. Various amounts (0.4, 0.2, or 0.1 mg) of each peptide were added to the wells in a final volume of 200 µl of human endothelial serum free media (Life Technologies). As controls, medium alone and medium containing 200 µg/ml of ECGS were assayed. Additional peptide was added on day 4 and the assay was fixed and stained with Diff-Quik on days 5–7. Each data point was assayed in quadruplicate (triplicates for the dose response) and each experiment was repeated at least three times. A blinded observer scored outgrowth by comparing responses with media alone (background levels) to that observed with the peptides and with ECGS (positive control). Results were scored so that 
meant sprouting comparable to ECGS; meant significant sprouting but lower than positive control; : significant sprouting above background levels; : low levels of sprouting; and 
: some sprouting above negative control levels.
Migration assay
Migration assays were conducted in a 48-well microchemotaxis chamber (Neuro Probe Inc., Cabin John, Md.). PVP-free polycarbonate membranes with 12 µm pores (Neuro Probe Inc.) were coated with a 0.1 mg/ml solution of collagen IV (Trevigen, Gaithersburg, Md.) in dH2O and dried. HUVECs were harvested using Versene (Life Technologies) and resuspended in RPMI 1640 containing 0.1% BSA. The bottom chamber was loaded with RPMI containing 0.1% BSA and various concentrations of laminin-1 peptides. Basic fibroblast growth factor (bFGF) (a gift from Gera Neufeld, Technion, Israel) was added to several wells as a positive control. Medium alone was used as the negative control. 50,000 cells per well were added to the upper chamber. Chambers were incubated at 37°C for 4 h and the filters were then fixed and stained using Diff-Quik. The cells that migrated through the filter were quantitated by counting the center of each well at 10x using an Olympus CK2 microscope. Each condition was assayed in triplicate wells and each experiment was repeated at least three times. In Stat software was used to calculate statistical significance using the Welch's t test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
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Tables 1A–C
show that 13 1 chain and 5 ß1 chain peptides demonstrated significant HUVEC adhesion when compared to the BSA-coated wells. Of these 13, A-208 contained the sequence IKVAV, previously found to be active with endothelial cells (4, 7, 16). Table 1B shows an additional 4 1 chain peptides from the carboxy-terminal globular domain of laminin-1 that significantly stimulate HUVEC adhesion. Three of these peptides (AG10, AG56, and AG73) have previously been identified as promoting the adhesion of several cancer cell lines (14). Three 1 chain peptides—A-55, A-203, and AG86—showed only a slight increase in adhesion over control levels, whereas the other peptides had more activity. These data suggest that endothelial cells have the potential to bind multiple sites on laminin-1.
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Tube formation screen
Although most of the peptides did not promote endothelial cell attachment, none of the 405 peptides were eliminated from consideration until a second screen was performed. HUVECs placed on the basement membrane matrix Matrigel undergo capillary-like tube formation, mimicking certain steps in angiogenesis such as migration and differentiation. The tube-forming activity of HUVECs on Matrigel in the presence of soluble peptides was assessed to determine whether the peptides could influence in vitro angiogenesis. Initially, 0.2 mg of each peptide was tested. Cells plated on Matrigel in the absence of peptides showed typical baseline tube formation ( Fig. 1,
part 1H). In this screen, 8 1 and 5 ß1 chain peptides significantly altered tube formation (
Table 1and Fig. 1, parts 1 and 2). IKVAV, an angiogenic peptide that has been shown to alter tube formation (4, 16), was used as a positive control ( Fig. 1, part 1G). A-208, a peptide containing the IKVAV sequence, served as an internal control and showed activity similar to the IKVAV sequence ( Table 1). With IKVAV-containing peptides, tubes were disrupted and the cells appeared to sprout and spread on the Matrigel. Based on the adhesion and tube assay results, 8 1 chain and 5 ß1 chain peptides were selected for further study. Three of these peptides—A-118, A-119, and A-124—showed no enhancement of HUVEC adhesion in the first screen whereas the other peptides active in the tube forming assay did promote HUVEC adhesion. Some of the peptides (A-29, A-71, A-182, A-185, AG85, and AG95) showed weak activity in the tube formation assay and were not evaluated further. Testing different doses of peptide ( Table 2)
showed that A-10 was active only at the highest concentration tested (0.2 mg/ml), whereas the remaining seven 1 chain peptides were also active at 0.1 mg/ml ( Fig. 1, part 1B, D). None of the ß1 chain peptides were active at the lowest dose tested ( Table 2).
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The morphology of the tubes on Matrigel varied depending on the peptide added. None of the tubes formed in the presence of the active peptides resembled that of the positive control IKVAV ( Fig. 1, part 1G). Cells treated with A-13 ( Fig. 1, part 1A, B) formed small clumps and few intact tubes were observed, whereas cells treated with A-55 (C, D), A-64 (E), and A-119 (F) formed partial tubes. The greatest morphological differences were observed between the ß1 chain peptides. B-49 treatment ( Fig. 1, part 2B) resulted in thick, short tubes with very condensed clusters of cells at the tube junctions. B-7, B-77, and B-160 treatments resulted in discontinuous patches of partial tubes ( Fig. 2,
part 2A, D, E). An inactive peptide, B-76 ( Fig. 1, part 2C), and the control lacking peptide ( Fig. 1, part 2F) are shown as a comparison. The peptides were not toxic as evidenced by trypan blue exclusion. Altered tube formation was not reversible when the peptide-containing media were replaced after an overnight incubation (data not shown). Also, no effect on tube formation was observed when the peptides were added after the tubes had formed on Matrigel (data not shown). Based on the adhesion and tube formation screens of all 405 peptides, 13 peptides (8 1 and 5 ß1 chain peptides) were considered highly active and were selected for further study.
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Inhibition of laminin-1 adhesion by peptides
We also tested for the ability of the active peptides to inhibit HUVEC binding to laminin to determine whether these sites are available in intact laminin-1. As shown in Fig. 2, most active peptides inhibited HUVEC adhesion to laminin-1. Six of the seven 1 chain peptides tested significantly reduced HUVEC adhesion to laminin-1 ( Fig. 2A). A-13, A-55, and A-119 significantly inhibited HUVEC adhesion at both 0.1 and 0.2 mg/ml by as much as 60% for A-13, 84% for A-55, and 83% for A-119. A-10 and A-203 were also very active inhibitors of HUVEC adhesion to laminin-1, showing the most significant reduction in HUVEC adhesion at the 0.2 mg/ml level (63 and 57%, respectively). A-64 was very active at the 0.1 mg/ml dose but showed no statistically significant inhibition at the 0.2 mg/ml dose. This may be due to aggregation of the peptide at this dose. One peptide, A-124, showed no inhibitory activity in the competition assay, which suggests that this site may not be available on intact laminin-1. Figure 2B shows the inhibition of HUVEC adhesion by the ß1 chain peptides. All the peptides significantly inhibited HUVEC adhesion to laminin-1, with B-49 and B-160 being the most active, reducing adhesion as much as 91 and 87%, respectively. B-77 was less active at the higher concentration (77 vs. 42%), perhaps also due to peptide aggregation. B-97 reduced HUVEC adhesion by 77% at the 0.2 mg/ml dose. When peptides B-4, B-5, B-8, B-10, GRDGSP, and an unrelated angiogenic peptide, thymosin ß4, were tested in the adhesion assay, there was no significant inhibition of HUVEC attachment to laminin-1 (
16 ±10.5%). Therefore, most of the active peptide sites are likely to be available on the intact molecule.
Endothelial sprouting
The sprouting of vessels from aortic ring explants was used next to determine whether the 13 peptides active in the tube assay stimulated or inhibited in vitro angiogenesis. Rat aortic rings were placed in Matrigel and incubated with serum-free medium in the presence or absence of peptides. As shown in Table 2, all 13 peptides stimulated vessel sprouting above background levels at all doses tested. None were as active as the positive control ECGS, where dense vessel sprouting was observed ( Fig. 3H).
A-13 and B-160 ( Fig. 3A, F) were considered to be the most active, resulting in dense networks of sprouts. A-13, A-119, B-49, and B-97 consistently stimulated a high level of sprouting. Other peptides were very active at 0.2 mg/ml (A-10, A-64, Fig. 3B, C) but showed less activity at 0.4 mg/ml ( Table 2). Five peptides (A-55, A-118, A-124, A-203, and B-160) showed dose-dependent activity from almost background levels at 0.1 mg/ml to high levels of sprouting at the highest dose of 0.4 mg/ml ( Fig. 3D). Morphological differences in the sprouts in the presence of some peptides were also observed. Figure 3E shows that sprouting induced by B-77 resulted in significant but less dense sprouting, and that the sprouting cells migrated at least twice the distance from the aortic ring as the positive control and did not appear to be connected to the aortic ring. A-119 showed morphology similar to B-77 (data not shown). Most of the other peptides tested showed sprouting and migration patterns similar to the positive control, although the number of sprouts was lower. These data demonstrate that several of the peptides were active for angiogenesis and further suggest that the peptides may act by different pathways.
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Migration activity
Boyden chamber assays were used to determine whether the active peptides stimulated HUVEC migration, an important step in tube formation and vessel sprouting. Peptides were tested at 0.2 mg/ml, the concentration shown to stimulate aortic ring sprouting with all 13 active peptides. Three peptides (A-10, A-55, and A-64) stimulated HUVEC migration ( Table 2) at this concentration. Three (A-119, B-7, and B-49) significantly inhibited migration at 0.2 mg/ml. Five peptides were selected for testing at various doses. As shown in Table 2, A-119 ( Fig. 4c)
, which was inhibitory at 0.2 mg/ml, and B-160 ( Fig. 4D), which was stimulatory at 0.2 mg/ml, significantly stimulated HUVEC migration at all lower doses tested. A-10 was active at all doses tested ( Fig. 4A). However, A-13, one of the most active peptides in stimulating sprouting and altering tube formation, significantly stimulated migration only at the 0.02 mg/ml level ( Fig. 4B). A-55 stimulated migration only at the 0.2 mg/ml level and had background levels of migration at the other levels tested (data not shown). These results suggest that some of the peptides may enhance sprouting by stimulating migration. Large differences in the effective concentration for migration are also observed between the peptides.
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DISCUSSION
Although laminin-1 is the best characterized of the laminins, the location and number of potential active sites have not been fully investigated. In the current study, we systematically screened a series of 405 synthetic peptides from the 1 and ß1 chains to identify sites active for endothelial cell adhesion and differentiation. A total of 22 peptides were identified that stimulated HUVEC adhesion. Of these, 13 were active in the tube formation assay and in stimulating aortic sprouting. Three of these—A-118, A-119, and A-124—were active in the tube and ring assays but did not promote HUVEC adhesion. This may be due to different properties of the peptides in solution and in the solid state (i.e., a lack of 3-dimensional structure when the peptides bind the plastic for the adhesion assay) and/or could be a result of these peptides acting through a different mechanism to stimulate endothelial cell differentiation.
The location of the active peptides suggests that certain areas of laminin-1 are critical for HUVEC binding and differentiation in vitro. The 1 chain peptides identified by our screening were clustered mainly in the first, second, and third globular domains ( Fig. 5A).
This suggests that the globular domains of the 1 chain may play a significant role in angiogenesis and that the active sites are, for the most part, clustered on the globular domains. This is not the case for the ß1 chain, where the active sites are in EGF repeats, globular regions, and the coiled-coil domain ( Fig. 5B). Active sites are not clustered as on the 1 chain. Thus, whereas the 1 chain appears to contain specialized areas of activity for endothelial cells, the ß1 chain has active sites in several different domains, perhaps related to the areas of the intact protein, and in cleavage products that are available to interact with endothelial cells.
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The peptide B-7 showed moderate levels of activity in all the assays used. This was unexpected since this pep~tide is highly hydrophobic and located in the putative signal sequence of ß1. B-7 is the most hydrophobic of the active peptides detected in the screening and in screenings of the chain with other cell types (Y. Kuratomi, unpublished data). Although it is likely to be cleaved from ß1 during posttranslational processing, it may not be degraded and could continue to influence a variety of cell types. Similar hydrophobic peptides in the 1 chain- C-3 (15) and 1 chain- A-4 (M. Nomizu, unpublished results) are very active with a variety of cell types, but not active with endothelial cells. Also, adjacent sequences B-4, B-5, B-8, and B-10 were not active in any of the assays, demonstrating that the effects of B-7 are specific. More studies are needed to determine the role of B-7 in vivo.
The mechanism by which the peptides are promoting tube formation and sprouting from the edge of the aorta is not clear. Tube formation in vitro mimics several of the steps of angiogenesis including adhesion, basement membrane breakdown, cell migration/invasion, differentiation, and synthesis of basement membrane proteins. The tube assay was therefore used as a quick high throughput screen to identify potential angiogenic/antiangiogenic factors. The drawback of this screen is that response to the peptide can appear as the formation of incomplete tubes or clumps or patches of cells, the inhibitory or stimulatory nature of which cannot be determined by this assay alone. IKVAV, a previously identified angiogenic peptide, alters tube formation resulting in patches of cells (16). YIGSR, an antiangiogenic peptide, altered tube formation resulting in isolated, round endothelial cells (7). Other angiogenic factors such as haptoglobin (19) and thymosin ß4 (20) form more tubes than do controls. Therefore, although this assay is an effective method of identifying factors that influence angiogenesis, the results must be confirmed in other angiogenesis assays before definite conclusions can be reached as to their angiogenic or antiangiogenic properties. The aortic ring sprouting assay not only mimics the same steps of angiogenesis as the tube assay, but also the proliferation step, and was used to determine whether the peptides active in the tube assay were angiogenic.
The differing morphology of the processes formed on Matrigel in the tube assay in response to the different peptides does suggest that the peptide sequences either on the intact molecule, which are available as shown by the competition studies, or existing as fragments when laminin is degraded could stimulate different steps in angiogenesis. Most active peptides clearly promote adhesion. All active peptides were also tested using zymograms to determine whether any stimulated matrix metalloproteinase activity. No significant elevation of activity was observed (data not shown) with any of the peptides, suggesting they do not influence the degradation of the basement membrane that occurs in angiogenesis. Migration assays suggest that six of the peptides (A-10, A-13, A-55, A-64, A-119, and B-160) stimulate HUVEC migration. A-119 also seems to inhibit cell migration at 0.2 mg/ml. Since migration dose response curves usually show inhibition at levels above the optimum, it is possible that the inhibition observed with A-119 is due to the high peptide levels tested.
The effects of the peptides appear to be specific. Competition assays were also performed on fibronec~tin, collagen I, and plastic (data not shown). Four of the 12 peptides tested (A-13, A-55, A-64, and A-203) inhibited attachment on all substrates except plastic, suggesting that the competition effects were specific for the proteins tested. Two peptides, B-49 and B-160, inhibited attachment on plastic as well, suggesting that they may have nonspecific competition interactions. Five of the remaining six peptides (A-10, A-119, B-7, B-77, and B-97) inhibited only on laminin-1. This suggests that four of the peptides on laminin-1 may have sequences related to active sites on other matrix molecules. Such promiscuity has been observed with matrix molecules recognizing common integrin receptors (for reviews, see refs 21, 22). The specificity to laminin-1 observed with five of the peptides suggests important functional sites that may be involved in angiogenesis.
The reason for the large number of active peptides for endothelial cells on laminin-1 is not clear. Many steps are involved in the process of angiogenesis; our studies suggest that based on the level and type of responses, the active peptides may function by different mechanisms. There are many other regulators of angiogenesis, each with different mechanisms of action, including multiple growth factors (i.e., bFGF and VEGF), cytokines (i.e., TNF), protein fragments (i.e., angiostatin and endostatin), and proteins (i.e., thrombospondin); for reviews, see refs 23–26. That so many molecules exist points to the complexity of the process of angiogenesis. Understanding factors at the peptide level that affect the process is important in defining key mechanistic events.
Several of the active sites identified in this endothelial cell screen appear to be cell type-specific. Most were not active with B16-F10 melanoma cells (Y. Kuratomi, unpublished data). One peptide, A-13, was active with B16-F10 cells and endothelial cells. A-13 was the most active in promoting endothelial cell adhesion, tube formation, and sprouting and also promoted migration. A-13 was the most active of the 1 chain peptides in stimulating melanoma lung colonization. A-64 and A-119 also were active in all assays (except for A-119 in the adhesion assay) but showed a lower level of activity. These peptides share no homology, suggesting there are at least three potential sites on the 1 and ß1 chains that are active in the processes leading to angiogenesis and could influence metastasis. The other active peptides also are not homologous, indicating that there are at least nine other potential angiogenesis-promoting sites. All of the peptides (except possibly A-124) appear to be physiologically important sites on the intact laminin-1 molecule, as competition studies in vitro revealed they could block attachment to laminin-1. In vivo angiogenesis activities of the peptides are currently under investigation. Preliminary studies with A-13 and B-160 in the chick chorioallantoic membrane assay suggest that both are active in promoting angiogenesis in vivo (unpublished observations). The active sequences we have identified may function in a temporal- and tissue-specific manner in processes such as tissue development, wound repair, and tumor growth.
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FOOTNOTES |
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2 Correspondence: Craniofacial Developmental Biology and Regeneration Branch, NIDR, NIH, Bldg. 30, Room 433, Bethesda, MD 20892–4370, USA. E-mail: kleinman{at}yoda.nidr.nih.gov
3 Abbreviations: bFGF, basic fibroblast growth factor; BSA, bovine serum albumin; ECGS, endothelial cell growth supplement; HUVECs, human umbilical endothelial cells; PBS, phosphate-buffered saline.
Received for publication April 17, 1998.
Revision received August 7, 1998.
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1 chain carboxyl-terminal globular domain by systematic screening of synthetic peptides. J. Biol. Chem. 270, 20583–205901 chain by systematic peptide screening. J. Biol. Chem. 272, 32198–32205This article has been cited by other articles:
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S. Hibino, M. Shibuya, J. A. Engbring, M. Mochizuki, M. Nomizu, and H. K. Kleinman Identification of an Active Site on the Laminin {alpha}5 Chain Globular Domain That Binds to CD44 and Inhibits Malignancy Cancer Res., July 15, 2004; 64(14): 4810 - 4816. [Abstract] [Full Text] [PDF]
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M. L. Ponce, S. Hibino, A. M. Lebioda, M. Mochizuki, M. Nomizu, and H. K. Kleinman Identification of a Potent Peptide Antagonist to an Active Laminin-1 Sequence That Blocks Angiogenesis and Tumor Growth Cancer Res., August 15, 2003; 63(16): 5060 - 5064. [Abstract] [Full Text] [PDF]
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T. S. Udayakumar, M. L. Chen, E. L. Bair, D. C. von Bredow, A. E. Cress, R. B. Nagle, and G. T. Bowden Membrane Type-1-Matrix Metalloproteinase Expressed by Prostate Carcinoma Cells Cleaves Human Laminin-5 {beta}3 Chain and Induces Cell Migration Cancer Res., May 1, 2003; 63(9): 2292 - 2299. [Abstract] [Full Text] [PDF]
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 E. J. Su, C. L. Cioffi, S. Stefansson, N. Mittereder, M. Garay, D. Hreniuk, and G. Liau Gene therapy vector-mediated expression of insulin-like growth factors protects cardiomyocytes from apoptosis and enhances neovascularization Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1429 - H1440. [Abstract] [Full Text] [PDF]
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I. Okazaki, N. Suzuki, N. Nishi, A. Utani, H. Matsuura, H. Shinkai, H. Yamashita, Y. Kitagawa, and M. Nomizu Identification of Biologically Active Sequences in the Laminin alpha 4 Chain G Domain J. Biol. Chem., September 27, 2002; 277(40): 37070 - 37078. [Abstract] [Full Text] [PDF]
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J. A. Engbring, M. P. Hoffman, A. J. Karmand, and H. K. Kleinman The B16F10 Cell Receptor for a Metastasis-promoting Site on Laminin-1 Is a Heparan Sulfate/Chondroitin Sulfate-containing Proteoglycan Cancer Res., June 1, 2002; 62(12): 3549 - 3554. [Abstract] [Full Text] [PDF]
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 B. A. Ruggeri, C. Robinson, T. Angeles, J. Wilkinson IV, and M. L. Clapper The Chemopreventive Agent Oltipraz Possesses Potent Antiangiogenic Activity in Vitro, ex Vivo, and in Vivo and Inhibits Tumor Xenograft Growth Clin. Cancer Res., January 1, 2002; 8(1): 267 - 274. [Abstract] [Full Text] [PDF]
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 D. Gebarowska, A. W. Stitt, T. A. Gardiner, P. Harriott, B. Greer, and J. Nelson Synthetic Peptides Interacting with the 67-kd Laminin Receptor Can Reduce Retinal Ischemia and Inhibit Hypoxia-Induced Retinal Neovascularization Am. J. Pathol., January 1, 2002; 160(1): 307 - 313. [Abstract] [Full Text] [PDF]
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R. E. B. Seftor, E. A. Seftor, N. Koshikawa, P. S. Meltzer, L. M. G. Gardner, M. Bilban, W. G. Stetler-Stevenson, V. Quaranta, and M. J. C. Hendrix Cooperative Interactions of Laminin 5 {gamma}2 Chain, Matrix Metalloproteinase-2, and Membrane Type-1-Matrix/Metalloproteinase Are Required for Mimicry of Embryonic Vasculogenesis by Aggressive Melanoma Cancer Res., September 1, 2001; 61(17): 6322 - 6327. [Abstract] [Full Text] [PDF]
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 M. L. PONCE, M. NOMIZU, and H. K. KLEINMAN An angiogenic laminin site and its antagonist bind through the {alpha}v{beta}3 and {alpha}5{beta}1 integrins FASEB J, June 1, 2001; 15(8): 1389 - 1397. [Abstract] [Full Text] [PDF]
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E.-A. B. Gjerde, K. Woie, E. T. Wei, and R. K. Reed Lowering of interstitial fluid pressure after neurogenic inflammation is inhibited by mystixin-7 peptide Am J Physiol Heart Circ Physiol, September 1, 2000; 279(3): H1377 - H1382. [Abstract] [Full Text] [PDF]
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 A. W. Griffioen and G. Molema Angiogenesis: Potentials for Pharmacologic Intervention in the Treatment of Cancer, Cardiovascular Diseases, and Chronic Inflammation Pharmacol. Rev., June 1, 2000; 52(2): 237 - 268. [Abstract] [Full Text] [PDF]
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