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(Circulation Research. 2004;94:462.)
© 2004 American Heart Association, Inc.

Molecular Medicine

₄ß₁ Integrin Mediates Selective Endothelial Cell Responses to Thrombospondins 1 and 2 In Vitro and Modulates Angiogenesis In Vivo

Maria J. Calzada, Longen Zhou, John M. Sipes, Jane Zhang, Henry C. Krutzsch, M. Luisa Iruela-Arispe, Douglas S. Annis, Deane F. Mosher, David D. Roberts

From the Laboratory of Pathology (M.J.C., L.Z., J.M.S., J.Z., H.C.K., D.D.R.), National Cancer Institute, National Institutes of Health, Bethesda, Md; Department of Molecular, Cell and Developmental Biology (M.L.I.-A.), UCLA, Los Angeles, Calif; and the Department of Medicine (D.S.A., D.F.M.), University of Wisconsin–Madison, Madison, Wis.

Correspondence to David D. Roberts, PhD, NIH, Building 10 Room 2A33, 10 Center Dr, Bethesda, MD 20892-1500. E-mail droberts{at}helix.nih.gov

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We examined the function of ₄ß₁ integrin in angiogenesis and in mediating endothelial cell responses to the angiogenesis modulators, thrombospondin-1 and thrombospondin-2. ₄ß₁ supports adhesion of venous endothelial cells but not of microvascular endothelial cells on immobilized thrombospondin-1, vascular cell adhesion molecule-1, or recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. Chemotactic activities of this region of thrombospondin-1 and thrombospondin-2 are also mediated by ₄ß₁, whereas antagonism of fibroblast growth factor-2–stimulated chemotaxis is not mediated by this region. Immobilized N-terminal regions of thrombospondin-1 and thrombospondin-2 promote endothelial cell survival and proliferation in an ₄ß₁-dependent manner. Soluble ₄ß₁ antagonists inhibit angiogenesis in the chick chorioallantoic membrane and neovascularization of mouse muscle explants. The latter inhibition is thrombospondin-1–dependent and not observed in explants from thrombospondin-1^-/- mice. Antagonizing ₄ß₁ may in part block proangiogenic activities of thrombospondin-1 and thrombospondin-2, because N-terminal regions of thrombospondin-1 and thrombospondin-2 containing the ₄ß₁ binding sequence stimulate angiogenesis in vivo. Therefore, ₄ß₁ is an important endothelial cell receptor for mediating motility and proliferative responses to thrombospondins and for modulation of angiogenesis.

Key Words: adhesion • proliferation • migration • angiogenesis • peptides

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Angiogenesis, the outgrowth of new blood vessels from existing vasculature, is an important event in normal development and pathological conditions such as cancer. Angiogenesis is regulated by interactions of endothelial cells with growth factors and components of the extracellular matrix that either promote or inhibit angiogenesis.¹ Thrombospondins (TSPs) have diverse effects on cell behavior.² Two members of this family, TSP1 and TSP2, are known to modulate angiogenic responses. TSP1 and TSP2 have antiangiogenic as well as antitumor activities in several xenograft models.^3–6 TSP1 and TSP2 presumably regulate angiogenesis through their modulation of endothelial cell proliferation,^7–9 motility,^7,10 and apoptosis.^11,12

Based on in vitro and in vivo angiogenesis assays, antiangiogenic activities of TSP1 and TSP2 have been mapped to the procollagen-homology domain and the type 1 repeats.^13–15 Inhibition is mediated through interactions with CD36 and proteoglycan receptors.^14–16 However, the N-terminal domain of TSP1 stimulates angiogenesis,^17,18 in part through its interaction with ₃ß₁ integrin.¹⁸ TSP1 may also influence their behavior through additional TSP receptors expressed on endothelial cells, including LDL receptor–related protein, _vß₃ and ₆ß₁ integrins, and CD47 or by activating latent TGFß.^2,19

The role of TSP1 in angiogenesis is therefore complex, and includes both proangiogenic and antiangiogenic activities. Acquisition of an angiogenic phenotype is frequently associated with decreased expression of TSP1 or TSP2,^20–22 whereas restoration of TSP1 levels in tumor cells reduces angiogenesis and tumor progression.³ Correlating levels of TSP1 with tumor progression and clinical prognosis in cancer patients, however, has yielded conflicting results.^23–26 To understand its role in cancer and to define a molecular basis for the complex endothelial cell responses to TSP1, we must understand how the simultaneous signals from multiple TSP1 receptors are integrated. The complete identification of endothelial cell TSP1 receptors is the critical first step toward this goal.

In addition to the known endothelial cell receptors, TSP1 interacts with ₄ß₁ integrin on T cells²⁷ and some cancer cells.^28,29 A sequence in TSP1 recognized by ₄ß₁ was mapped to the N-terminal domain and is also conserved in TSP2.²⁷ Leukocyte ₄ß₁ is a well-characterized counter receptor for endothelial cell vascular cell adhesion molecule-1 (VCAM-1),³⁰ but its function in endothelial cells is poorly understood. The ₄ integrin subunit is expressed in endothelial cells in vitro^31,32 and in vivo,³³ suggesting that ₄ß₁ could be an additional receptor for TSPs in these cells. We therefore examined the role of ₄ß₁ in venous and microvascular endothelial cell responses to TSP1 and TSP2 in vitro. Our data show that ₄ß₁ is an endothelial TSP receptor that regulates cell proliferation, adhesion, and migration. In vivo and ex vivo angiogenesis assays indicate that ₄ß₁ plays important roles in angiogenesis and its regulation by TSP1.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Culture
Human umbilical vein endothelial cells (HUVECs) were maintained in M199 containing 20% FBS, 2 mmol/L glutamine, 80 µg/mL endothelial cell mitogen, and 10 µg/mL heparin. Human dermal microvascular endothelial cells (HDMVECs) and human lung microvascular endothelial (HMVE-L) cells were maintained as specified by the manufacturer (Clonetics). Adult iliac vein endothelial cells (AG10773A) were grown on flasks coated with 0.1% gelatin in medium M199 containing 10% FBS, 2 mmol/L glutamine, 30 µg/mL endothelial cell mitogen, and 30 µg/mL heparin.

Proteins, Peptides, and Antibodies
TSP1 was purified from human platelets. Recombinant trimeric proteins consisting of the N-terminal module (N), oligomeration sequence (o), and procollagen module (C) of TSP1 (NoC1) residues 1 to 356, or TSP2 (NoC2) residues 1 to 359³⁴ and monomeric N-terminal module of TSP1 (residues 1 to 175)¹⁴ were prepared as described previously. Synthetic peptides containing TSP1 sequences were prepared as described.³⁵ Recombinant soluble 7-domain VCAM-1 (residues 1 to 674) was prepared as described.²⁷ VEGF was from R&D Systems, FGF2 from Prizm Pharmaceuticals, and Vitrogen type I collagen from Cohesion Technologies.

The ß₁ integrin–activating antibody TS2/16 was purified from hybridoma supernatant.³⁶ Anti-₄ and anti-₃ antibodies, P4C2 and Asc-1, respectively, were from Chemicon. The anti-ß₁ blocking antibody mAb13 was provided by Dr Ken Yamada, National Institutes of Dental and Craniofacial Research, Bethesda, Md. The ₄ß₁ antagonist (4-((2-methylphenyl)aminocarbonyl) aminophenyl)acetyl-LDVP (phLDVP)³⁷ was obtained from Bachem.

Endothelial Cell Functional Assays
Adhesion of endothelial cells was assessed as described.²⁸ Chemotaxis was assessed using modified Boyden chambers and 8-µm pore membranes. Endothelial cell proliferation was quantified using a tetrazolium proliferation assay (Promega).

Integrin Expression Analyses
Cells were washed and incubated with TS2/16 or P4C2 for 1 hour. Cells were washed, incubated with FITC-conjugated anti-mouse antibody, washed, and fixed with 1% formaldehyde. Flow cytometry data were acquired using a Becton Dickinson flow cytometer. Cell lysates labeled with EZ-Link Sulfo-NHS-LC-Biotin (Pierce) were immunoprecipitated using 2 µg of P4C2 or Asc-1. The immune complexes were analyzed by Western blotting.

Angiogenesis Assays
Effects of the ₄ß₁-binding peptide from TSP1 and other ₄ß₁ antagonists on angiogenesis were evaluated using the chick chorioallantoic membrane (CAM) angiogenesis assay as described previously.¹⁵ Muscle explant vascularization in a 3-dimensional collagen matrix was assessed essentially as described.³⁸

siRNA-Mediated Knockdown of ₄ß₁ Integrin
Selection of the target sequence AACUAUCAACGAAAGAACUGG from human ₄ mRNA was done according to Elbashir et al.³⁹ siRNA duplexes were synthesized using the Silencer siRNA kit (Ambion). HUVECs were transfected using a nuclear-localized ß-Gal expression vector alone, in combination with ₄ siRNA or with ₄ siRNA alone. Transfected cells were maintained in complete medium for 48 to 72 hours before use. Three siRNA sequences were screened initially, and the most active was selected. Adhesion assays were stained using 5-bromo-4-chloro-3-indoyl-ß-D-Gal to identify transfected cells.

An expanded Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
₄ß₁ Integrin Mediates Adhesion of Large-Vessel Endothelial Cells on TSP1 and TSP2
₄ß₁ and ₆ß₁ recognize N-terminal regions of both TSP1 and TSP2,^19,27 whereas ₃ß₁ recognizes a sequence in this region of TSP1 that is not conserved in TSP2³⁵ (Figure 1A). We recently showed that ₆ß₁ is not a functional TSP receptor in HUVECs.¹⁹ However, the ß₁ integrin–activating antibody TS2/16 increased adhesion of HUVECs similarly on recombinant portions of both TSP1 (NoC1) and TSP2 (NoC2) (Figure 1B). The cells spread on NoC1 and NoC2 forming prominent lamellipodia (Figure 1C). These results are consistent with the similar activities of NoC1 and NoC2 for interacting with ₄ß₁ on T cells.²⁷

View larger version (59K): [in this window] [in a new window]   Figure 1. 4ß1 integrin mediates large-vessel endothelial cell adhesion on TSP1 and TSP2. A, ß1 Integrin binding sites of TSP1 and recombinant regions of TSP1 and TSP2. 4ß1 Mediates activation-dependent HUVEC (B) and AG10773A iliac vein endothelial cell (D) adhesion on N-terminal regions of TSP1 (NoC1) and TSP2 (NoC2) 30 µg/mL. Unstimulated cells (2 to 2.5x105 cells/mL, C) or cells treated with the ß1-activating antibody TS2/16 (5 µg/mL, S) were incubated in the absence or presence of the 4ß1 inhibitor, phLDVP (1 µmol/L). C, Morphologies of HUVECs activated using TS2/16 attaching on TSP1, NoC1, or NoC2. Bar=50 µm. E, Cell attachment (solid bars) and spreading (striped bars) on TSP1(1–175) (30 µg/mL) was determined using cells activated with TS2/16 and quantified after 1 hour. Results are expressed as the number of cells/mm2 ±SD, from 3 experiments. *P<0.05 relative to control.

Recognition of N-terminal regions of TSP1 and TSP2 is at least partially dependent on ₄ß₁ because an ₄ß₁-specific antagonist, phLDVP, partially inhibited adhesion of HUVECs and AG10773A venous endothelial cells on TSP1, NoC1, and NoC2 (P0.01) (Figures 1B and 1D). Recombinant TSP1(1–175), which contains the ₄ß₁ binding site²⁷ but lacks the recognition site for ₃ß₁,³⁵ also mediated HUVEC adhesion and was inhibited by phLDVP (P0.007) (Figure 1E). Therefore, this region of TSP1 is sufficient for promoting ₄ß₁-dependent endothelial cell adhesion.

To confirm a direct role of ₄ß₁ as an endothelial cell TSP receptor, we suppressed the expression of ₄ integrin in HUVECs using RNA interference (siRNA). In cells cotransfected with a ß-galactosidase expression plasmid and ₄ siRNA, attachment on TSP1, TSP1 (1–175), and NoC2 was significantly decreased relative to control cultures transfected with the ß-galactosidase plasmid alone (Figure 2A). The effect of the siRNA on adhesion to TSPs was specific, in that ₂/₁ß₁-mediated adhesion on type I collagen was not affected (Figure 2A). Loss of ₄ expression in siRNA-transfected cells was confirmed by Western blotting (Figure 2B). Densitometric analysis showed a 5.7-fold decrease in ₄ expression, whereas ₃ expression did not show a significant change (Figure 2B). Therefore, ₄ß₁ is required for venous endothelial cell adhesion to TSP1 and an N-terminal region of TSP2.

View larger version (22K): [in this window] [in a new window]   Figure 2. Suppression of 4ß1 expression inhibits HUVEC adhesion on TSP1 and TSP2. A, HUVECs cotransfected with ßGal plasmid and 4 siRNA or transfected with ßGal plasmid alone were incubated in dishes coated with TSP1 (40 µg/mL), TSP1(1–175), NoC2 (30 µg/mL), or type I collagen (5 µg/mL) in the presence of TS2/16 antibody. Cell adhesion was quantified after 1 hour. Results are expressed as percentage of siRNA-transfected cell adhesion with respect to cells transfected with ßGal alone as a control ±SD. B, Cell lysates from 4 siRNA-transfected cells or control were immunoprecipitated with anti-3 (Asc-1) or anti-4 (P4C2) antibodies. 4 Immunoprecipitates were also immunoblotted with P4C2.

₄ß₁ Does Not Mediate Adhesion of Microvascular Endothelial Cells
Adhesion of microvascular endothelial cells on NoC1 and NoC2 is also ß₁ integrin–dependent, based on complete inhibition by a ß₁-blocking antibody¹⁹ and stimulation by a ß₁-activating antibody (Figure 3A). However, the ₄ß₁ inhibitor phLDVP did not significantly inhibit adhesion of either HDMVECs or HME-L cells on NoC1 or NoC2 (Figures 3A and 3B). Adhesion of microvascular cells, therefore, is mediated exclusively by ₃ß₁ and ₆ß₁.^18,19

View larger version (26K): [in this window] [in a new window]   Figure 3. 4ß1 Integrin does not contribute to microvascular endothelial cell adhesion on TSP1 or TSP2. A, Unstimulated (C) or TS2/16-activated HDMVECs (S, 2 to 2.5x105 cells/mL) were incubated on dishes coated using TSP1 (40 µg/mL), NoC1 or NoC2 (30 µg/mL) in triplicate. B, 4ß1-dependence was tested using phLDVP (1 µmol/L). TS2/16-activated HMVE-L cells were incubated on the indicated substrates alone or in the presence of phLDVP. Cell attachment and spreading after 1 hour are expressed as cells/mm2 ±SD, n=3.

Although microvascular cells exhibited no ₄ß₁-dependent adhesion on TSPs, they express ₄ß₁ (Figures 4A and 4B). The modest difference in ₄ subunit surface expression between HUVECs and HDMVECs measured by flow cytometry (Figure 4A) was not sufficient to account for the differences in adhesion. To exclude the possibility that ₄ pairs only with ß₇ subunits in microvascular cells, cell lysates were immunoprecipitated using an ₄ antibody and immunoblotted with a ß₁ antibody. The amount of ₄ complexed with ß₁ was only slightly lower in HDMVECs than in HUVECs (Figure 4B). Thus, ₄ß₁ is present but may be maintained in an inactive state in microvascular cells. Consistent with this hypothesis, adhesion of HDMVECs to the ₄ß₁ ligand VCAM-1 was minimal relative to HUVECs and was not significantly stimulated by TS2/16 (Figure 4C).

View larger version (24K): [in this window] [in a new window]   Figure 4. 4 Integrin expression in HUVECs and HDMVECs. A, HUVECs and HDMVECs were stained using antibodies specific for 4 (P4C2) and ß1 (TS2/16) subunits (2 µg/106 cells, thick lines) or isotype control (thin lines) and for surface expression analyzed by flow cytometry. B, Cell lysates from both cell types were immunoprecipitated using P4C2, resolved on SDS gels, and immunoblotted with TS2/16 (0.5 µg/mL). C, Adhesion on immobilized S7D-VCAM-1 (5 µg/mL) or TSP1 (40 µg/mL) was measured using unstimulated (C) or TS2/16-activated HUVECs and HDMVECs (S). Attached (solid bars) and spread cells (striped bars) after 1 hour and are expressed as cells/mm2 ±SD, n=3.

TSP1 and TSP2 Promote Endothelial Cell Survival and Proliferation Through ₄ß₁ Integrin
TSP1 has context-dependent effects on endothelial cell proliferation. In solution, it inhibits proliferation,^7,40 whereas immobilized TSP1 stimulates proliferation at least in part through ₃ß₁.¹⁸ We examined whether ₄ß₁ also contributes to the proliferative activity of TSP1 and whether TSP2 shares this activity. Plating HUVECs on immobilized NoC1 and NoC2 at 0.1 µg/mL supported initial adhesion and cell survival, and higher concentrations dose-dependently increased cell numbers after 72 hours (Figure 5A). The cells did not attach or survive after 72 hour on wells without the proteins, indicating that immobilized N-terminal regions of TSP1 and TSP2 have both growth- and survival-promoting activities. Consistent with their differential ₄ß₁ activities, proliferation of HUVECs on NoC1 and NoC2 was greater than for HDMVECs (Figure 5B).

View larger version (38K): [in this window] [in a new window]   Figure 5. Immobilized 4ß1 ligands promote endothelial cell survival and proliferation. A, Immobilized NoC1 and NoC2 stimulate HUVEC proliferation in a concentration-dependent manner. B, NoC1 and NoC2 differentially promote proliferation of HUVECs (circles) and HDMVECs (triangles). Proliferation was assessed after 72 hours at 37°C in the presence of TS2/16 (5 µg/mL). C, HUVEC survival and proliferation on immobilized NoC2 were assessed in the absence or presence of 4ß1-blocking antibody P4C2 (5 µg/mL) or phLDVP (1 µmol/L). D, Effect of immobilized S7D-VCAM-1 on HUVEC survival and proliferation was estimated at the indicated times in the absence or presence of P4C2 (5 µg/mL) or phLDVP (1 µmol/L). Duplicate plates developed at 1 hour were used to determine the starting cell number. Data were corrected for reagent background and are expressed as mean±SD, n=3. *P0.05.

Using NoC2 to avoid contributions of ₃ß₁ to the observed proliferative response,¹⁸ we examined the activities of two ₄ß₁ antagonists (Figure 5C). NoC2 at >0.5 µg/mL stimulated net proliferation, which was significantly inhibited by phLDVP and the ₄ß₁-blocking antibody (P0.04; Figure 5C). Cell adhesion, survival, and proliferation were also promoted by the immobilized ₄ß₁ ligand VCAM-1 at >0.7 µg/mL. Net proliferation stimulated by immobilized VCAM-1 was also inhibited by the ₄ß₁ antibody and phLDVP (P0.02; Figure 5D). Based on these data, ₄ß₁ mediates proliferative responses of venous endothelial cells to immobilized NoC2 and VCAM-1.

₄ß₁ Mediates Endothelial Cell Chemotaxis
TSP1-stimulated chemotaxis of murine lung capillary and bovine aortic endothelial cells was inhibited by an antibody recognizing the N-module of TSP1.⁷ ₃ß₁ and ₆ß₁ contribute to this response for microvascular cells,¹⁹ but the receptor mediating venous endothelial cell motility to TSPs has not been identified. A report that ₄ß₁ mediates HUVEC chemotaxis stimulated by soluble VCAM-1⁴¹ suggested ₄ß₁ may be involved. TSP1, NoC1, and NoC2 stimulated chemotaxis of HUVECs with similar dose dependencies (Figure 6A). The ₄ß₁ antagonist phLDVP strongly inhibited chemotactic responses to both TSP1 and NoC2 (Figures 6B and 6C). This inhibition was specific, because phLDVP did not inhibit the chemotactic activity of a recombinant region of fibronectin containing only its ₅ß₁ binding site (online Figure 1, available in the online data supplement at http://circres.ahajournals.org). Therefore, ₄ß₁ plays a role in endothelial cell chemotaxis to TSP1 and TSP2.

View larger version (22K): [in this window] [in a new window]   Figure 6. 4ß1 Mediates chemotaxis to TSP1, NoC1, and NoC2. TSP1, NoC1, or NoC2 was added in the bottom wells of Boyden chambers at the indicated concentrations (A). HUVECs (0.5 to 1x105/well) added to the top wells were allowed to migrate 6 hours at 37°C in 5% CO2. HUVEC migration induced by the indicated concentrations of TSP1 (B) or NoC2 (C) was assayed with or without phLDVP (1 µmol/L). HUVEC migration induced by FGF2 (5 ng/mL, D) or VEGF (30 ng/mL, E) was assessed with the indicated concentrations of TSP1, NoC1, or NoC2 added with the cells in the upper chamber. Medium with BSA in the bottom chamber was used as a negative control (). Results are presented as cells migrated/field ±SD, n=3.

In addition to its direct chemotactic activity, TSP1 inhibits endothelial cell motility induced by FGF2.^7,10 In microvascular cells, this inhibition is mediated by CD36,¹⁶ although TSP1(1–175), which lacks a CD36-binding site, also inhibits FGF2-stimulated chemotaxis of BAE cells.¹⁴ The inhibitory activity of TSP1(1–175) was originally attributed to HSPG binding,¹⁴ but the present data suggested that ₄ß₁ should also be considered. Using HUVECs, which do not express CD36, TSP1 inhibited FGF2-stimulated chemotaxis, but NoC1 and NoC2 did not (Figure 6D).

When VEGF was used in place of FGF2 to stimulate migration of HUVECs, NoC1 and NoC2 weakly inhibited chemotaxis but were approximately 5-fold less active than native TSP1 (Figure 6E). Therefore, the N-terminal region of TSP1 does not contain its inhibitory activity for FGF2-mediated motility, and receptors recognizing this domain, including ₄ß₁, are not involved. However, these regions of TSP1 and TSP2 do contain inhibitory activities for VEGF-stimulated chemotaxis that are independent of CD36 expression. The mechanism of this inhibitory activity of TSP1 and TSP2 remains to be defined.

₄ß₁ Antagonists Inhibit Angiogenesis
The activities of ₄ß₁ to mediate endothelial adhesion, chemotaxis, and survival responses to TSP1 and TSP2 in vitro suggested that ₄ß₁ may mediate proangiogenic activities of both proteins. Consistent with this hypothesis, a synthetic TSP1 peptide containing its ₄ß₁ binding site inhibited angiogenesis in the CAM assay (Figure 7A). AELDVP (753) inhibited angiogenesis in CAMs overlaid with either a pure type I collagen matrix or with a collagen plus fibronectin matrix to provide an exogenous ₄ß₁ ligand. In both cases, the peptide was specific in that the control VALAEP (755) was inactive. Furthermore, neither peptide altered the angiogenic response in the absence of growth factors (unpublished data, 2003).

View larger version (23K): [in this window] [in a new window]   Figure 7. Role of 4ß1 integrin in angiogenesis. A, 4ß1-binding TSP1 peptide 753 (acetyl-AELDVP) and control peptide 755 (acetyl-VALAEP) were evaluated in a CAM assay. Polymerized matrix of type I collagen alone (circles) or in combination with fibronectin (triangles) containing either 250 ng/mesh VEGF+25 ng FGF2 or vehicle in the presence or absence of peptide was placed onto the CAM of day 11 chicken embryos. Angiogenesis responses ±SEM (n=9 to 12) were normalized to growth factor (defined as 100%) and vehicle controls (0%). Responses were quantified 24 hours after application of matrix to the CAM surface. B, CAM angiogenesis was assessed on the indicated matrices containing either SD7-VCAM-1 or phLDVP, mean±SEM, n=21. C, TSP1-/- and +/+ mouse muscle explants were cultured in Vitrogen matrices in the absence or presence of 1 or 5 µmol/L phLDVP. Vascular outgrowth responses were evaluated by measuring maximal cell migration distances in 4 quadrants from each explant. Data represent the outgrowth migration normalized to controls for 6 explants at each time point, mean±SEM (*P<0.05). These results are representative of 3 independent experiments. D, RNAs from explants were amplified using GAPDH and TSP2 primers±reverse transcriptase (RT) and analyzed after 35 cycles using a 2% agarose gel.

In the presence of growth factors, native TSP1 had equivalent antiangiogenic activities in matrices of type I collagen or collagen plus fibronectin, whereas no response was observed in the absence of growth factors (Table). In contrast to intact TSP1, NoC1 and NoC2 stimulated angiogenesis above the control both in the presence and absence of growth factors. The greater activity of NoC1 compared with NoC2 may result from engaging ₃ß₁,¹⁸ but the activity of NoC2 suggested that ₄ß₁ also contributes to the proangiogenic activities. Engaging ₄ß₁ may be sufficient to stimulate angiogenesis in the CAM, because VCAM-1 had a weak proangiogenic activity, similar to NoC2, in the absence of growth factors, and a significant stimulation in the presence of growth factors was observed on the collagen matrix (Table; P<0.005, n=9).

View this table: [in this window] [in a new window]   Table 1. CAM Angiogenesis in the Presence or Absence of Growth Factors (FGF and VEGF)

This stimulatory activity of VCAM-1 is specific for collagen matrices, however, because addition of soluble VCAM-1 inhibited CAM angiogenesis in a Hydron gel and in a fibronectin matrix (Figure 7B). Under these conditions, soluble VCAM-1 appears to be an ₄ß₁ antagonist. Consistent with this, the specific ₄ß₁ antagonist phLDVP inhibited angiogenesis in CAM assays using collagen matrices with or without fibronectin (Figure 7B). VCAM-1 in collagen and the N-terminal regions of TSP1 and TSP2, in contrast, may be proangiogenic because they become immobilized in the matrix.

Inhibition by three specific antagonists indicated that ₄ß₁ is required for angiogenesis in the CAM. Although exogenous NoC1 stimulated CAM angiogenesis, we could not determine whether TSP1 is an important endogenous ligand for ₄ß₁ in this response. To address this question, we used a muscle explant angiogenesis assay³⁸ to compare the sensitivity of neovascularization to an ₄ß₁ antagonist in wild-type and TSP1^-/- mice (Figure 7C). The ₄ß₁ antagonist dose-dependently inhibited vascular outgrowth in explants from wild-type mice at days 3 to 9 (P0.05), but not in explants from TSP1^-/- mice. Therefore, sensitivity of angiogenesis to ₄ß₁ antagonists in this context is clearly TSP1-dependent. Loss of inhibition in the -/- mice was not due to compensatory upregulation of TSP2, based on comparable TSP2 mRNA levels in the wild-type and null explants (Figure 7D).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Leukocyte ₄ß₁ plays critical roles in adhesion and migration by binding to VCAM-1 on activated endothelium.³⁰ Although VCAM-1 can modulate angiogenesis,^41,42 little is known about ₄ß₁ function in endothelial cells. We show in this study that inhibiting ₄ß₁ disrupts angiogenic responses in vivo and in vitro. In vitro, ₄ß₁ mediates proliferative, adhesive, and chemotactic responses to N-terminal regions of TSP1 and TSP2. Based on differential responses of wild-type and TSP1 null explants to an ₄ß₁ antagonist, the ability of endogenous TSP1 to influence muscle explant vascularization depends on this integrin. However, TSP1 is probably not the only ligand for ₄ß₁ that modulates vascularization. A soluble form of VCAM-1 induces chemotaxis and proliferation of HUVECs and modulates angiogenesis in both cornea⁴¹ and CAM assays. Fibronectin, TSP2,²⁷ and osteopontin⁴³ are also ₄ß₁ ligands. Because angiogenesis involves smooth muscle cells and pericytes as well as endothelial cells, the functions of ₄ß₁ that are disrupted by ₄ß₁ antagonists may not be limited to endothelium.

Endothelial cells now have three identified ß₁ integrins that recognize TSP1, and these integrins have some overlapping functions. ₃ß₁ mediates adhesion and chemotaxis but has both stimulatory and inhibitory effects on proliferation.^18,19 ₆ß₁ mediates adhesion and chemotaxis, but only in microvascular endothelial cells.¹⁹ Our data show that immobilized ₄ß₁ ligands selectively mediate venous endothelial adhesion and stimulate cell survival and proliferation. In addition, ₄ß₁ mediates chemotactic responses of venous endothelial cells to soluble TSP1 and TSP2. Both ₄ß₁ and ₃ß₁ contribute to angiogenesis in the CAM. Stimulation of angiogenesis by NoC2 demonstrates a role for ₄ß₁, but the stronger proangiogenic activity of NoC1 suggests that ₃ß₁ and ₄ß₁ are both proangiogenic TSP1 receptors.

Differential regulation may be key to understanding how functions of the eight endothelial cell TSP receptors identified to date are coordinated. ₄ß₁ and ₃ß₁1 are widely expressed in endothelium,^33,44 but their functional regulation differs. ₃ß₁ is inactivated by cell-cell contact and, therefore, may function as a TSP1 receptor only during vascular remodeling.¹⁸ ₆ß₁ is active only on microvascular cells,¹⁹ whereas ₄ß₁ functions are largely restricted to large-vessel endothelium. ₄ß₁-dependent adhesion is not regulated by cell contact (unpublished data, 2003). CD36 is expressed and functions selectively on microvascular endothelium.^45,46 CD36 is required for inhibition of FGF2-stimulated endothelial cell chemotaxis in vitro¹⁶ and for inhibition of angiogenesis in the cornea.¹² Differential effects of TSP1 on HUVEC and HDMVEC proliferation at concentrations lower than 10 nmol/L⁹ are also consistent with an inhibitory activity mediated by CD36.

Although native TSP1 has no proangiogenic activity in the CAM assay, NoC1, NoC2, and other contructs containing the N-domain^17,18 are proangiogenic. Therefore, proteolytic processing that removes the inhibitory type 1 repeats could generate proangiogenic fragments of TSP1 and TSP2. Previous studies have established that native TSP1 displays proangiogenic activities under certain conditions.^47,48 Whether these activities are mediated by intact TSP1 or by proteolytic fragments similar to NoC1 remains to be determined.

The inhibitory activities of ₄ß₁ antagonists in the CAM assay suggest that ₄ß₁ becomes activated on microvascular endothelium in vivo. Alternatively, ₄ß₁ may be essential for a perivascular cell contribution to angiogenesis. Although we do not know which of these cells express functional ₄ß₁ in vivo, the explant data show that TSP1 is essential for sensitivity of neovascularization to an ₄ß₁ antagonist.

Integrins play complex regulatory roles in angiogenesis. ß₁ Integrins are essential for angiogenesis,⁴⁹ but the roles of specific ß₁ integrins in this process are unclear. The ₄ subunit is widely expressed in endothelial cells during vascular development,³³ but the specific role of ₄ß₁ in angiogenesis remains unclear. As is known for _vß₃ antagonists, our results using ₄ß₁ antagonists do not prove an essential role for ₄ß₁ in angiogenesis. However, like _vß₃,¹ ₄ß₁ may be a useful pharmacological target to control pathological angiogenesis.

	Acknowledgments

 
This work was supported in part by NIH grants 2RO1NIH-NCI-CA-6562-06 (M.L.I.-A.), HL54462, and HL56396 (D.F.M). We thank Dr Jack Lawler for providing TSP1 null mice.

	Footnotes

 
Original received July 18, 2003; resubmission received November 12, 2003; revised resubmission received December 11, 2003; accepted December 17, 2003.

	References

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