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(Journal of Leukocyte Biology. 2002;71:47-53.)
© 2002 by Society for Leukocyte Biology

Laminin binding to the calreticulin fragment vasostatin regulates endothelial cell function

Lei Yao, Sandra E. Pike and Giovanna Tosato

Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Correspondence: Lei Yao, Experimental Transplantation and Immunology Branch, National Cancer Institute, Building 10, Room 12N226, MSC 1907, Bethesda, Maryland 20892. E-mail: yaol{at}mail.nih.gov


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ABSTRACT
 
Vasostatin, the 1–180 amino acids NH2 domain of calreticulin, inhibits endothelial cell proliferation, angiogenesis, and tumor growth, but the mechanisms underlying these effects are unclear. We show that endothelial cells express the extracellular matrix protein laminin, including chains {alpha}5 and {gamma}1 and that vasostatin specifically binds to laminin. When added to endothelial cell cultures, vasostatin specifically inhibits endothelial cell attachment to laminin and by this mechanism, can reduce subsequent endothelial cell growth induced by basic fibroblast growth factor. As an angiogenesis inhibitor that specifically disrupts endothelial cell attachment to components of the extracellular matrix, vasostatin has a unique potential as a cancer therapeutic.

Key Words: angiogenesis • extracellular matrix • laminin


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INTRODUCTION
 
An endothelial cell inhibitory factor present in culture supernatants of Epstein-Barr virus-immortalized cells was identified recently as the NH2-terminal domain of calreticulin [1 ]. The purified recombinant NH2 domain of calreticulin (amino acids 1–180) was found to inhibit the proliferation of primary cultures of endothelial cells stimulated with basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) and to suppress neovascularization in vivo. It also significantly reduced tumor growth in mice. We have named this N-terminal domain of calreticulin (amino acids 1–180) vasostatin [1 ]. The full-length calreticulin molecule and an internal 61 amino acids fragment of vasostatin were also found to inhibit endothelial cell proliferation, angiogenesis, and tumor growth in a manner comparable with vasostatin [2 ].

Calreticulin is a highly conserved and ubiquitous protein that is a calcium storage depot in the endoplasmic reticulum and participates in calcium signaling [3 ]. Structure-function analyses have distinguished the protein in three domains, N, P, and C. The proline-rich P domain contains the high-affinity, calcium-binding site of calreticulin, whereas the highly acidic C domain of calreticulin binds the clotting factors IX, X, and prothrombin [4 5 6 ]. Vasostatin, the N domain of calreticulin, can regulate glucocorticoid, androgen, and retinoic acid binding to DNA [5 , 7 ]. It can also bind the cytoplasmic domain of alpha subunits of integrins and the C1q recognition subunit of the first component of the classical complement pathway [8 , 9 ]. However, the mechanisms by which vasostatin inhibits endothelial cell proliferation, angiogenesis, and tumor growth are unknown. Earlier studies aimed at evaluating the potential use of calreticulin as an antithrombotic agent based on its binding to the coagulation factors IX and X showed that calreticulin localized to the vessel wall of most vascular organs when infused into mice [6 ]. Recently, cell-surface calreticulin was found to mediate focal adhesion disassembly to thrombospondin-1 [10 ]. In vitro studies showed that radiolabeled calreticulin can bind to endothelial cells with high affinity, raising the possibility that endothelial cells may express a calreticulin surface receptor [6 ]. Other experiments have characterized a highly conserved amino acid sequence in the cytoplasmic domain of alpha integrins as a specific calreticulin binding site within cells, suggesting that calreticulin may regulate the functional status of intergrins and thereby modulate cell attachment and spread [8 ]. Focusing on structural similarities between calreticulin and the chaperon protein calnexin that recognizes glycosyl structures, other studies have described lectin-like properties of calreticulin and characterized its affinity binding to glycosylated laminin [11 ]. Laminins constitute a family of high-molecular-weight glycoproteins composed of the assembly of three different classes of polypeptides, the {alpha}, ß, and {gamma} chains, held together by disulfide bonds [12 , 13 ]. At least five {alpha}, three ß, and three {gamma} chains have been described to date, which assemble into 12 identified different laminin isoforms: laminin-1 ({alpha}1, ß1, and {gamma}1), laminin-2 ({alpha}2, ß2, and {gamma}2), etc. Endothelial cells are known to produce various forms of laminin, but the structural characteristics and biological features of endothelial cell-derived laminin are incompletely known [14 , 15 ]. Here, we show that vasostatin can bind to laminin and by this mechanism, can inhibit endothelial cell attachment and proliferation.


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MATERIALS AND METHODS
 
Reagents
Laminin from basement membrane of Engelbreth-Holm-Swarm (EHS) mouse sarcoma, native collagen Type IV from basement membrane of EHS murine sarcoma, and bovine fibronectin were from Sigma Chemical Co. (St. Louis, MO); purified laminin {alpha}5 was from Gibco-BRL (Gaithersburg, MD). Human vitronectin was from Becton Dickinson (Bedford, MA); human tenascin-C and human thrombospondin-1 were purchased from Chemicon International (Temecula, CA); bovine bFGF, human VEGF, and murine VEGF were from R&D Systems (Minneapolis, MN). Anti-EHS-laminin (laminin-1) antibodies included an affinity-purified rabbit antilaminin antibody (Sigma Chemical Co.), a mouse monoclonal antibody (mAb) against laminin {alpha}5 (4C7; Gibco-BRL), a goat antiserum against laminin ß1 (Santa Cruz Biotechnology, Santa Cruz, CA), and goat antiserum to laminin {gamma}1 (Santa Cruz Biotechnology). Affinity-purified rabbit antihuman tenascin antibody, polyclonal rabbit antibovine fibronectin antibody, and polyclonal rabbit antimurine collagen Type IV antiserum were from Chemicon International. Murine mAbs to human vitronectin and human thrombospondin, and rabbit polyclonal antiserum against bovine bFGF were from Sigma Chemical Co. Rabbit antihuman VEGF and a goat antimurine VEGF were from R&D Systems. Polyclonal rabbit anticalreticulin antiserum was purchased from Applied BioReagents (ABR; Golden, CO), rabbit antimaltose-binding protein (MBP)-vasostatin antibody was previously described [2 ], and murine mAb against MBP conjugated with horseradish peroxidase (HRP) was from Amersham Pharmacia Biotech (Piscataway, NJ). Purified recombinant MBP, MBP-calreticulin, MBP-vasostatin, and biotin-labeled MBP-vasostatin fragment were previously described [2 ]. Streptavidin-HRP was from Gibco-BRL.

Endothelial cells and membranes
Fetal bovine heart endothelial cells [FBHE; American Type Culture Collection (ATCC), Manassas, VA] and primary cultures of human umbilical vein endothelial cells (HUVEC) were propagated as previously described [1 ]. Cell membranes were prepared essentially as described [16 ]. Briefly, endothelial cells scraped from culture plates and suspended in cold buffer (10 mM Tris, pH 7.6, with 10 mM KCl) were homogenized by a Dounce homogenizer (50–60 times). Homogenates were equilibrated to 150 mM NaCl and spun at 1780 g for 10 min. Supernatants were centrifuged for 45 min at 100,000 g, and the pellets were suspended in cold Triton buffer (50 mM Tris, 300 mM NaCl, pH 7.6, 0.5% Triton X-100 protease inhibitors). After another centrifugation at 14,000 g for 30 min, supernatants were collected and used as a source of cell membranes.

Immunoprecipitation, Western blotting, and protein binding assays
Test samples were subjected to immunoprecipitation as described [17 ]. For Western blotting, proteins separated by electrophoresis were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA) and incubated overnight with the appropriate antibody in phosphate-buffered saline (PBS) containing 5% milk. Antibody bound to the membranes was detected by incubation (1 h) with affinity-purified, HRP-conjugated donkey antirabbit immunoglobulin G (IgG; Amersham Pharmacia Biotech) or antigoat antibodies (Santa Cruz Biotechnology) followed by enhanced chemiluminescence (ECL) detection (ECL kit, Amersham Pharmacia Biotech).

The binding of calreticulin and vasostatin to laminin and to other proteins was evaluated by enzyme-linked immunosorbent assay (ELISA). Briefly, polystyrene microtiter plates (Immulon-1B, Dynex Technologies, Chantilly, VA) were coated overnight at 4°C with murine laminin, bovine fibronectin, human vibronectin, murine collagen type IV, human tenascin-C, human thrombospondin-1, bovine bFGF, and human or murine VEGF in carbonate buffer, pH 9. Coating, optimized for each protein, used concentrations ranging from 0.2–20 µg/ml. Optimal concentration for laminin coating was determined to be 20 µg/ml. After washing the plates with PBS containing 0.05% Tween 20 (PBS-T) and blocking with PBS-T containing 1% fetal bovine serum (FBS; BioWhittaker, Walkersville, MD), recombinant, purified MBP-calreticulin, MBP-vasostatin, or MBP was added to the wells in PBS-T containing 1% FBS. After 2 h incubation and washing, anti-MBP-vasostatin rabbit antibody was added at the appropriate dilution. The plates were incubated at room temperature for 2 h and washed in PBS-T, and affinity-purified, alkaline-phosphatase-conjugated goat antirabbit antiserum (Sigma Chemical Co.; dilution 1:350) was added to the wells. After 2 h incubation and washing in PBS-T, the substrate (p-nitrophenyl-phosphate, substrate 104, Sigma Chemical Co.) was added to the plates. After development, the plates were read at 405 nm. The binding of matrix proteins to calreticulin or calreticulin fragments was assessed by coating the wells with recombinant-purified MBP-calreticulin, MBP-vasostatin, or MBP (0.32–1.0 µM), as described above. After washing, laminin or other extracellular matrix proteins were added to the wells at varying concentrations. The binding of laminin or other protein to MBP-calreticulin, MBP-vasostatin, or MBP was evaluated by addition of appropriately diluted rabbit or murine antibody directed at laminin or another protein. The plates were incubated at room temperature for 2 h and washed in PBS-T, and affinity-purified, alkaline-phosphatase-conjugated goat antirabbit antiserum (Sigma Chemical Co.; dilution 1:350) or goat antimouse antiserum (Sigma Chemical Co.; dilution 1:300) was added. The following steps were done as described above.

Endothelial cell attachment and proliferation
Cell adhesion to extracellular matrix proteins was evaluated as previously described [18 ] with modifications. Briefly, 96-well neutral polystyrene plates (Immulon I, Dynex Technologies) were coated at 4°C overnight with 5 µg/ml murine EHS-laminin, bovine fibronectin, human vitronectin, murine collagen type IV, and 3% bovine serum albumin (BSA) in Dulbecco PBS (DPBS; Gibco-BRL) or 0.2% gelatin, 100 µl/well. Wells were washed in DPBS and blocked with PBS containing 3% BSA at 37°C for 1 h. After washing, endothelial cells (HUVEC or FBHE, 3x104 cells/well) were added with or without MBP-vasostatin and allowed to adhere in a humidified incubator (37°C, 20 h). Nonadherent cells were removed by washing with DPBS. Adherent cells were stained with 0.05% crystal violet in 20% ethanol. After rinsing, plates were allowed to dry. After addition of methanol (100 µl/well), absorbance was measured at 595 nm. Endothelial cell proliferation assays were performed as described previously [1 , 2 ]. Where indicated, 96-well neutral polystyrene plates (Immulon I, Dynex Technologies) were coated overnight with 5 µg/ml murine EHS-laminin, bovine fibronectin, and 0.2% gelatin or 3% BSA in DPBS (Gibco-BRL), 100 µl/well. Wells were washed and blocked (PBS containing 3% BSA at 37°C for 1 h), and endothelial cells [FBHE, 3x104 cells/well in DMEM culture medium supplemented with 10% heat-inactivated fetal calf serum (FCS) and 5 µg/ml gentamicin] were added with or without MBP-vasostatin or antilaminin antibody (Sigma Chemical Co.). After incubation for 72 h, proliferation was measured by [3H]thymidine deoxyribose uptake (0.6 mCi/well, 6.7 Ci/mmol; New England Nuclear, Boston, MA).

Statistical analysis
The means, SD, and SE were derived using conventional formulas. The statistical significance of group differences was determined by Student’s t-test.


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RESULTS
 
Laminin expression by endothelial cells
Endothelial cells have been shown previously to express various forms of laminin [14 , 15 ], but the precise constitution of endothelial cell-derived laminin is not known. As expected, a rabbit antiserum raised against EHS-laminin (laminin {alpha}1, ß1, and {gamma}1), identified by Western blotting two laminin chains migrating at approximately 400 kD (tentatively identified as {alpha}1 based on size) and 200 kD (tentatively identified as ß1 or {gamma}1 based on size) in control EHS-laminin (Fig. 1 A , lane 1). When reacted with endothelial cell lysates (HUVEC and FBHE) and endothelial cell membranes (HUVEC) run under nonreducing conditions (Fig. 1A , lanes 2–4, respectively), this antiserum identified two laminin-related components migrating at approximately 400 kD and 200 kD. No higher molecular-weight bands were visualized. When the endothelial cell lysates (HUVEC and FBHE) and endothelial cell membranes (HUVEC) were run under reducing conditions (Fig. 1A , lanes 5–7, respectively), the antibody against EHS-laminin identified only one band migrating at approximately 200 kD. This suggested that the 400 kD laminin identified in endothelial cell lysates and membranes under nonreducing conditions represented a complex of two laminin chains of approximately 200 kD. Parallel Western blots reacted with a control rabbit antiserum were negative (not shown). To identify the laminin chains in endothelial cells, we used antibodies that specifically recognize the ~200-kD laminin {alpha}5, ß1, and {gamma}1. A mouse mAb to laminin chains {alpha}5 (4C7, not active in Western blot assays) was used in immunoprecipitation assays, and the immunoprecipitates were then immunoblotted with rabbit antibody directed at EHS-laminin. As shown in Fig. 1B , the mAb against laminin {alpha}5 immunoprecipitated material from lysates of HUVEC (lane 2) and FBHE cells (lane 3) that appeared as a ~200-kD band identified by the rabbit antibody against EHS-laminin. In addition, this rabbit anti-EHS-laminin antibody visualized a 200-kD band from purified preparations of laminin {alpha}5 (Fig. 1B , lane 1). A goat antiserum against laminin ß1 identified a ~200-kD band from preparations of EHS-derived laminin (Fig. 1C , lane 1). However, it failed to identify reactive material from HUVEC lysates (Fig. 1C , lane 2) and identified only a very faint band from FBHE cell lysates (Fig. 1C , lane 3). A goat antiserum directed at laminin {gamma}1 (2E8) identified a ~200-kD band from preparations of EHS-derived laminin (Fig. 1D , lane 1). In addition, it identified a ~200-kD band from lysates of HUVEC and FBHE cells (Fig. 1D , lanes 2 and 3). These results confirm that endothelial cells express laminin and identify laminin {alpha}5 and {gamma}1 chains as components of endothelial cell-derived laminin.



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  		Figure 1. Laminin expression in primary cultures of endothelial cells. Cell lysates from HUVEC and FBHE and cell membranes from HUVEC were tested for the presence of laminin by immunoblotting. (A) Immunoblotting with a rabbit antibody against EHS-laminin. Lane 1, murine EHS-laminin; lanes 2 and 5, HUVEC lysate; lanes 3 and 6, FBHE lysate; lanes 4 and 7, HUVEC cell membranes. Samples in lanes 2–4 were run in nonreducing conditions; samples in lanes 5–7 were run under reducing conditions. (B) Immunoprecipitation with a murine mAb against laminin {alpha}5 (4C7) followed by immunoblotting with a rabbit anti-EHS-laminin antibody. Lane 1, purified laminin {alpha}5; lane 2, HUVEC lysate; lane 3, FBHE lysate. (C) Immunoblotting with a goat antilaminin ß1 antiserum. Lane 1, EHS-laminin; lane 2, HUVEC lysate; lane 3, FBHE lysate. (D) Immunoblotting with a goat anti-laminin {gamma}1 antiserum. Lane 1, EHS-laminin; lane 2, HUVEC lysate; lane 3, FBHE lysate.

Binding of vasostatin to laminin
Previously, calreticulin purified from bovine liver was found to bind laminin [11 ]. We examined whether the angiogenesis inhibitor vasostatin representing the NH2 domain of calreticulin encompassing amino acids 1–180 can also bind laminin. Because endothelial cells can express laminin, we first examined whether vasostatin can bind to endothelial cell-associated laminin. To this end, equal amounts of cell lysates from FBHE and HUVEC were run under nonreducing conditions and blotted onto nitrocellulose paper. Western blotting of control lanes with a rabbit antibody against EHS-laminin confirmed the presence of laminin-related bands migrating at approximately 400 and 200 kD in cell lysates of FBHE and HUVEC (Fig. 2 , lanes 1 and 2). Parallel lanes blotted with FBHE (lanes 3, 5, and 7) and HUVEC (lanes 4, 6, and 8) cell lysates (5 µg/lane) were incubated individually with MBP-vasostatin alone (5 µg, lanes 3 and 4) or together with vasostatin (25 and 50 µg). After incubation and washing, MBP-vasostatin binding to the endothelial cell lysates was revealed by HRP-labeled mAb directed at MBP. As shown (Fig. 2A , lanes 3 and 4), two bands at approximately 400 and 200 kD were identified when FBHE and HUVEC cell lysates were incubated with MBP-vasostatin alone. Excess vasostatin (25 and 50 µg/lane) incubated together with MBP-vasostatin resulted in the almost-complete disappearance of the ~400- and ~200-kD bands (Fig. 2A , lanes 5–8). These results suggested that MBP-vasostatin can bind specifically to a component on endothelial cell membranes corresponding in size to ~400- and ~200-kD laminin present in endothelial cells.



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  		Figure 2. Binding of vasostatin to endothelial cell lysates. Cell lysates from FBHE (lanes 1, 3, 5, and 7) and HUVEC (lanes 2, 4, 6, and 8) were run under nonreducing conditions and then blotted onto nitrocellulose paper. Two lanes were immunoblotted with a rabbit anti-EHS-laminin antiserum (lanes 1 and 2). MBP-vasostatin (5 µg) was added to nitrocellulose paper blotted with FBHE or HUVEC, alone (lanes 3 and 4) or together with 25 µg (lanes 5 and 6) or 50 µg (lanes 7 and 8) competing vasostatin. MBP-vasostatin bound to nitrocellulose was revealed by an HRP-conjugated mouse mAb against MBP.

To examine further whether vasostatin can bind to laminin, we used solid-phase assays with purified EHS-laminin. Microtiter wells were coated with EHS-laminin (20 µg/ml), and MBP-calreticulin, MBP-vasostatin, and MBP were tested for binding to immobilized laminin. The binding of test proteins to laminin was revealed by a rabbit antibody directed at MBP-vasostatin. We found that recombinant MBP-vasostatin and MBP-calreticulin, but not MBP, can bind to immobilized laminin and that this binding is specific and concentration-dependent (Fig. 3 A). On a molar basis, MBP-calreticulin and MBP-vasostatin displayed similar binding to laminin-coated plates. In parallel experiments (not shown), vasostatin bound minimally to fibronectin, vitronectin, collagen type IV, tenascin-C, thrombospondin, bFGF, and human VEGF immobilized onto microtiter wells. In each case, we confirmed the presence of these proteins on the wells by use of specific antibodies. We also looked for laminin-binding to ELISA plates coated with MBP, MBP-calreticulin, or MBP-vasostatin (0.5 µg/ml). Similar coating was confirmed by use of an antibody directed at MBP-vasostatin and a mAb directed at MBP. As shown (Fig. 3B ), laminin specifically bound to MBP-calreticulin and MBP-vasostatin, but not MBP-coated plates, and displayed similar dose-response curves. These experiments demonstrate that vasostatin can bind specifically to laminin.



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  		Figure 3. Evaluation of vasostatin binding to laminin. (A) Recombinant-purified MBP-calreticulin (MBP-Cal), MBP-vasostatin (MBP-Vaso), and MBP were tested (0.16–1000 nM) for their ability to bind to murine laminin in solid-phase assays. Laminin (25 µg/ml) was immobilized onto microtiter wells. MBP-calreticulin, MBP-vasostatin, and MBP were tested for binding to murine EHS-laminin by use of a rabbit anti-MBP-vasostatin antiserum, and a secondary affinity-purified goat antirabbit antiserum conjugated to alkaline phosphatase. The results reflect the mean (SE) absorbance (405 nM) of three experiments performed in triplicate. (B) Murine laminin (0.25–60 µg/ml) was tested for binding to recombinant-purified MBP, MBP-calreticulin, or MBP-vasostatin in solid-phase assays. MBP-calreticulin, MBP-vasostatin, or MBP (0.5 µM) was immobilized onto microtiter wells, and comparable coating was established by use of a rabbit anti-MBP-vasostatin antibody and a mouse anti-MBP mAb. Laminin (EHS-laminin) binding to the wells coated with MBP (MBP-coated), MBP-calreticulin (MBP-Cal-coated), or MBP-vasostatin (MBP-Vaso-coated) was detected by use of a rabbit anti-murine laminin serum and a secondary affinity-purified goat anti-rabbit serum conjugated to alkaline phosphatase. The results reflect the mean (SE) absorbance (405 nM) of three experiments performed in triplicate.

Vasostatin inhibits endothelial cell attachment to laminin-coated wells
Several extracellular matrix components are known to favor the attachment, survival, and growth of endothelial cells and other cells in culture [16 , 19 20 21 ]. We found that endothelial cells fail to attach to noncharged polystyrene surfaces, unless these surfaces are appropriately coated. Wells coated with BSA, fibronectin, collagen type IV, and EHS-laminin provided a surface to which FBHE cells attached to varying degrees (Fig. 4 A ). Because vasostatin can bind to laminin, we examined the effects of vasostatin on endothelial cell attachment to polystyrene wells coated with laminin or other proteins. As shown (Fig. 4A) , vasostatin (2.0 µg/ml) inhibited endothelial cell binding to EHS-laminin-coated plates (P=0.04), whereas it had minimal effect on endothelial cell binding to fibronectin, collagen, or BSA-coated plates (P>0.05 all comparisons). This selective inhibition of endothelial cell binding to laminin-coated wells by vasostatin was dose-dependent (Fig. 4B) . We tested whether laminin could reverse this inhibition of cell attachment by vasostatin. As shown (Fig. 4C) , laminin (7–62 µg/ml), added in conjunction with vasostatin (1 µg/ml), dose-dependently reversed inhibition of endothelial cell attachment to laminin-coated plates. When laminin was added to culture at the concentration of 62 µg/ml, the reversal was significant (P<0.01). We also examined whether inhibition of endothelial cell attachment to laminin-coated wells by vasostatin was associated with reduced endothelial cell proliferation compared with control cultures established without vasostatin. As shown, vasostatin added at the start of endothelial cell cultures on laminin-coated wells inhibited endothelial cell proliferation after 3 days of culture (Fig. 4D) . This inhibitory effect of vasostatin (43.2% inhibition) was comparable in magnitude with the effect of an antilaminin antibody (56.8% inhibition) added to laminin-coated plates prior to the addition of endothelial cells (Fig. 4D) . Thus, by interfering with endothelial cell attachment to laminin-coated wells, vasostatin can reduce endothelial cell proliferation.



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  		Figure 4. Effects of vasostatin on endothelial cell attachment to laminin-coated wells and proliferation on laminin-coated wells. FBHE cells (3x104/well) were tested for their ability to bind to polystyrene wells that had been coated with 5 µg/ml BSA, bovine fibronectin, murine collagen Type IV, or murine EHS-derived laminin. Cell binding to the plates was measured by absorbance at 595 nM after staining the cells bound to the wells. Endothelial cell proliferation on laminin-coated wells was measured by 3[H]thymidine uptake after 48-h culture in bFGF (20 ng/ml)-supplemented medium alone, with MBP-vasostatin (1 µg/ml), or with rabbit anti-EHS-laminin antibody (10 µg/ml). (A) The results reflect the mean (SE) endothelial cell binding of five independent experiments performed in triplicate. MBP-vasostatin (MBP-Vaso) was used at 2.0 µg/ml. (B) Endothelial cell attachment to laminin-coated plates was measured as a function of the amount of MBP-vasostatin (MBP-Vaso; 0.06–16 µg/ml) added to the wells. Results reflect means (SD) of triplicate cultures. Three representative experiments were performed. (C) Effects of EHS-laminin (7–62 µg/ml) on endothelial cell attachment to laminin-coated plates in the presence of MBP-vasostatin (1.0 µg/ml). Results reflect means (SD) of triplicate cultures. Three representative experiments were performed. (D) Endothelial cell proliferation on laminin-coated polystyrene wells in bFGF-supplemented medium alone, with MBP-vasostatin (2.0 µg/ml), or with anti-laminin antibody (10 µg/ml). Results reflect means (SD) of triplicate cultures. Three experiments were performed.

In additional experiments, we evaluated the effects of vasostatin on endothelial cells already attached to the culture vessel. As shown in Figure 5 , vasostatin significantly inhibited endothelial cell proliferation induced by bFGF once the cells were attached to laminin (Fig. 5A ; 81.5% inhibition; P<0.01)- or gelatin (Fig. 5B ; 71.9% inhibition; P<0.01)-coated plates. Thus, vasostatin selectively inhibited endothelial cell attachment to laminin-coated plates but nonselectively inhibited endothelial cell proliferation once the cells were attached to laminin or gelatin. To test whether growth inhibition of attached endothelial cells could be attributed to a selective effect of vasostatin on endogenous laminin, we compared the antiproliferative effects of vasostatin with those of antilaminin antibody. In addition, we tested whether exogenous laminin could reverse vasostatin-induced growth inhibition of attached endothelial cells. As shown in Figure 5C , unlike vasostatin, antilaminin antibody had a minimal effect (P>0.05) on the proliferation of endothelial cells that had been allowed to adhere to gelatin-coated plates. Furthermore, laminin added at concentrations as high as 50 µg/ml failed to reverse vasostatin-induced growth inhibition of adherent endothelial cells. Together, these experiments suggest that in addition to its ability to bind laminin, thereby inhibiting endothelial cell attachment, vasostatin displays other laminin-independent activities that contribute to endothelial cell growth inhibition.



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  		Figure 5. Effects of vasostatin, anti-laminin antibody (anti-Lam), and laminin on the proliferation of endothelial cells that have been allowed to attach to the wells. FBHE cells were cultured (650–800 cells/well; polystyrene microtiter wells precoated with laminin or gelatin) with or without bFGF (20 ng/ml) for 4 days. Vasostatin (recombinant MBP-vasostatin) was added at the indicated concentrations (A and B) or at 2 µg/ml (C and D). Anti-laminin antibody (rabbit antilaminin antibody) was used at 5 µg/ml. Laminin was added to the cultures at 3 and 50 µg/ml. 3[H]Thymidine was added during the last 20 h of culture. The results presented in all panels represent the means (SE) of three experiments performed in triplicate cultures.


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DISCUSSION
 
Formation of new vessels in the embryo and in the adult through vasculogenesis and angiogenesis is believed to involve many cell types, soluble mediators, cell-surface molecules, and components of the extracellular matrix participating in a series of complex and orderly processes [22 23 24 ]. Thus far, much of the attention has focused on endothelial cells that line the vessels, and candidate molecules and molecular pathways have been described that regulate endothelial cell growth, migration, differentiation, survival, and interaction with smooth muscle cells [24 , 25 ]. However, there is considerable evidence that the extracellular matrix plays various critical roles in vasculogenesis and angiogenesis [19 , 26 , 27 ]. Mice that lack fibronectin die in utero on the 9th and 10th day of gestation, and the embryos display defects in somitogenesis and vascular development [28 ]. Mice that lack type I collagen die of circulatory collapse just before birth [29 ]. Mice deficient in laminin ß2 display vascular abnormalities primarily in the kidney [30 ]. Knockout of the laminin {alpha}2-chain gene in mice by homologous recombination caused growth retardation, severe muscular dystrophy, and death by 5 weeks of age [31 ].

Recently, a number of candidate drugs have been identified that inhibit angiogenesis and reduce tumor growth in experimental models. The mechanisms of action of some of these inhibitors are understood, at least in part. For example, neutralizing antibodies against VEGF, VEGF receptor 2, or soluble VEGF receptors may reduce neovascularization or promote endothelial cell death because they limit the availability of VEGF, a critical endothelial cell growth and survival factor [25 , 32 , 33 ]. However, for many other angiogenesis inhibitors, the mechanisms of action are not fully understood.

In this study, we explored the mechanisms by which vasostatin can inhibit endothelial cell growth and angiogenesis. We show that endothelial cells express laminin, including the laminin {alpha}5 and {gamma}1 chains and that vasostatin can bind to laminin. We also show that through this binding to laminin, vasostatin can specifically interfere with endothelial cell attachment to laminin-coated wells and inhibit endothelial cell proliferation, in part, because of reduced cell attachment. Laminin can provide the extracellular scaffolding upon which the endothelial cells can attach, migrate, proliferate, and sprout. By binding to laminin, vasostatin interferes with endothelial cell attachment to this component of the extracellular matrix and in doing so, can derail the angiogenic process. Although other laminin-independent mechanisms may contribute to endothelial cell growth inhibition by vasostatin, results here point to a critical role played by the binding of vasostatin to laminin.

Laminins constitute the major component of vascular endothelial basement membranes [34 ]. Endothelial cells are known to produce laminins and to express several laminin-binding sites, including integrins [35 ], a high-affinity 67-kDa receptor [36 ] derived by posttranslational modifications of a 37-kDa laminin receptor precursor [37 ], and dystroglycan [38 ]. It will be important to characterize the effects of vasostatin on these distinct endothelial cell binding sites for laminin. It will also be important to define the vasostatin sequences that mediate laminin binding. We do not know whether the inhibitory effects of vasostatin are general or selective for certain endothelial cells at certain sites. In vitro, vasostatin can inhibit the proliferation of endothelial cells from bovine aorta and from human umbilical cord. In vivo, vasostatin inhibits bFGF and tumor-induced neovascularization in the murine subcutaneous tissues. Given the high degree of conservation among laminins, it is not surprising that vasostatin is active across different species. However, there is evidence that the composition of basal laminae differs somewhat in different tissues, and this could provide selectivity for vasostatin as a potential antiangiogenic agent. For example, laminin and collagen IV isoforms contribute differently to the structure of basal membranes in different tissues [34 , 39 ]. In addition, endothelial cells can shift the relative levels of variant laminin forms they produce depending on exogenous stimuli [15 ]. There is evidence that endothelial cells from various sites are different [40 ]. In particular, tumor vessels appear to differ from normal vessels morphologically and functionally, as well as in the antigens they express [41 ]. It will be important to establish whether production of extracellular matrix proteins, particularly laminins, defines another distinguishing feature of the tumor vasculature that could be exploited therapeutically.


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ACKNOWLEDGEMENTS
 
The authors thank Drs. H. Kleinman, R. Burgeson, J. Miner, K. Jones, G. Wang, and R. Gress for their help on various aspects of this work.

Received May 1, 2001; revised September 12, 2001; accepted September 13, 2001.


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