The α1β1 and α2β1 Integrins Provide Critical Support for Vascular Endothelial Growth Factor Signaling, Endothelial Cell Migration, and Tumor Angiogenesis
2002; Elsevier BV; Volume: 160; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)64363-5
ISSN1525-2191
AutoresDonald R. Senger, Carole Perruzzi, Michael Streit, Victor Koteliansky, Antonin R. de Fougerolles, Michael Detmar,
Tópico(s)Inflammatory mediators and NSAID effects
ResumoAngiogenesis is a complex process, involving functional cooperativity between cytokines and endothelial cell (EC) surface integrins. In this study, we investigated the mechanisms through which the α1β1 and α2β1 integrins support angiogenesis driven by vascular endothelial growth factor (VEGF). Dermal microvascular EC attachment through either α1β1 or α2β1 supported robust VEGF activation of the Erk1/Erk2 (p44/42) mitogen-activated protein kinase signal transduction pathway that drives EC proliferation. Haptotactic EC migration toward collagen I was dependent on α1β1 and α2β1 as was VEGF-stimulated chemotaxis of ECs in a uniform collagen matrix. Consistent with the functions of α1β1 and α2β1 in supporting signal transduction and EC migration, antibody antagonism of either integrin resulted in potent inhibition of VEGF-driven angiogenesis in mouse skin. Moreover, combined antagonism of α1β1 and α2β1 substantially reduced tumor growth and angiogenesis of human squamous cell carcinoma xenografts. Collectively, these studies identify critical collaborative functions for the α1β1 and α2β1 integrins in supporting VEGF signal transduction, EC migration, and tumor angiogenesis. Angiogenesis is a complex process, involving functional cooperativity between cytokines and endothelial cell (EC) surface integrins. In this study, we investigated the mechanisms through which the α1β1 and α2β1 integrins support angiogenesis driven by vascular endothelial growth factor (VEGF). Dermal microvascular EC attachment through either α1β1 or α2β1 supported robust VEGF activation of the Erk1/Erk2 (p44/42) mitogen-activated protein kinase signal transduction pathway that drives EC proliferation. Haptotactic EC migration toward collagen I was dependent on α1β1 and α2β1 as was VEGF-stimulated chemotaxis of ECs in a uniform collagen matrix. Consistent with the functions of α1β1 and α2β1 in supporting signal transduction and EC migration, antibody antagonism of either integrin resulted in potent inhibition of VEGF-driven angiogenesis in mouse skin. Moreover, combined antagonism of α1β1 and α2β1 substantially reduced tumor growth and angiogenesis of human squamous cell carcinoma xenografts. Collectively, these studies identify critical collaborative functions for the α1β1 and α2β1 integrins in supporting VEGF signal transduction, EC migration, and tumor angiogenesis. Vascular endothelial growth factor (VEGF) is a cytokine essential for the vasculogenesis associated with normal embryonic development and for the angiogenesis associated with wound healing, cancers, and a variety of other important pathologies.1Senger DR Van de Water L Brown LF Nagy JA Yeo KT Yeo TK Berse B Jackman RW Dvorak AM Dvorak HF Vascular permeability factor (VPF, VEGF) in tumor biology.Cancer Metastasis Rev. 1993; 12: 303-324Crossref PubMed Scopus (809) Google Scholar, 2Ferrara N The role of vascular endothelial growth factor in pathological angiogenesis.Breast Cancer Res Treat. 1995; 36: 127-137Crossref PubMed Scopus (408) Google Scholar, 3Dvorak HF Brown LF Detmar M Dvorak AM Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis.Am J Pathol. 1995; 146: 1029-1039PubMed Google Scholar Through its receptors, which include two distinct receptor tyrosine kinases,4Mustonen T Alitalo K Endothelial receptor tyrosine kinases involved in angiogenesis.J Cell Biol. 1995; 129: 895-898Crossref PubMed Scopus (474) Google Scholar VEGF exerts multiple effects on vascular endothelium including stimulation of endothelial cell (EC) proliferation,5Ferrara N Henzel WJ Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells.Biochem Biophys Res Commun. 1989; 161: 851-858Crossref PubMed Scopus (1993) Google Scholar rapid induction of microvascular permeability,6Senger DR Galli SJ Dvorak AM Perruzzi CA Harvey VS Dvorak HF Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid.Science. 1983; 219: 983-985Crossref PubMed Scopus (3362) Google Scholar, 7Senger DR Connolly DT Van de Water L Feder J Dvorak HF Purification and NH2-terminal amino acid sequence of guinea pig tumor-secreted vascular permeability factor.Cancer Res. 1990; 50: 1774-1778PubMed Google Scholar promotion of EC survival,8Gerber HP McMurtrey A Kowalski J Yan M Keyt BA Dixit V Ferrara N Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3′-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation.J Biol Chem. 1998; 273: 30336-30343Crossref PubMed Scopus (1722) Google Scholar, 9Alon T Hemo I Itin A Pe'er J Stone J Keshet E Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity.Nat Med. 1995; 1: 1024-1028Crossref PubMed Scopus (1400) Google Scholar, 10Pierce EA Foley ED Smith LE Regulation of vascular endothelial growth factor by oxygen in a model of retinopathy of prematurity.Arch Ophthalmol. 1996; 114: 1219-1228Crossref PubMed Scopus (421) Google Scholar stimulation of EC adhesion and migration,11Byzova TV Goldman CK Pampori N Thomas KA Bett A Shattil SJ Plow EF A mechanism for modulation of cellular responses to VEGF: activation of the integrins.Mol Cell. 2000; 6: 851-860Abstract Full Text Full Text PDF PubMed Google Scholar and induction of EC gene expression.12Senger DR Vascular endothelial growth factor/vascular permeability factor: multiple biological activities for promoting angiogenesis.in: Voest EE D'Amore PA Tumor Angiogenesis and Microcirculation. Marcel Dekker, Inc., New York2001: 167-184Google Scholar Thus, the mechanisms by which VEGF promotes angiogenesis are highly complex and involve the regulation of multiple EC functions. Adhesion to extracellular matrix through cell surface integrins is generally required for cell proliferation, survival, and migration, and for cytokine-stimulation of these processes.13Meredith JE Schwartz MA Integrins, adhesion and apoptosis.Trends Cell Biol. 1997; 7: 146-150Abstract Full Text PDF PubMed Scopus (244) Google Scholar, 14Giancotti FG Ruoslahti E Integrin signaling.Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3757) Google Scholar, 15Aplin AE Short SM Juliano RL Anchorage-dependent regulation of the mitogen-activated protein kinase cascade by growth factors is supported by a variety of integrin alpha chains.J Biol Chem. 1999; 274: 31223-31228Crossref PubMed Scopus (65) Google Scholar The complex integrin family of transmembrane proteins consists of heterodimers, each consisting of one α and one β chain.16Hynes RO Integrins: versatility, modulation, and signaling in cell adhesion.Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8941) Google Scholar, 17Ruoslahti E Noble NA Kagami S Border WA Integrins.Kidney Int. 1994; 44: S17-S22Google Scholar Previously, we reported that VEGF potently induces dermal microvascular ECs to express the α1β1 and α2β1 integrins, two important members of the β1 integrin subfamily.18Senger DR Claffey KP Benes JE Perruzzi CA Sergiou AP Detmar M Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins.Proc Natl Acad Sci USA. 1997; 94: 13612-13617Crossref PubMed Scopus (451) Google Scholar Depending on cell type, α1β1 and α2β1 generally bind collagens and laminins.16Hynes RO Integrins: versatility, modulation, and signaling in cell adhesion.Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8941) Google Scholar, 17Ruoslahti E Noble NA Kagami S Border WA Integrins.Kidney Int. 1994; 44: S17-S22Google Scholar On dermal microvascular ECs, α1β1 and α2β1 are the principal receptors for interstitial collagen type I, a major component of the extracellular matrix; and α1β1 also is a receptor for collagen IV and laminin-1.18Senger DR Claffey KP Benes JE Perruzzi CA Sergiou AP Detmar M Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins.Proc Natl Acad Sci USA. 1997; 94: 13612-13617Crossref PubMed Scopus (451) Google Scholar Our previous findings that VEGF induces α1β1 and α2β1 expression by microvascular ECs suggested that these integrins are important to the mechanism by which VEGF promotes angiogenesis. Consistent with this hypothesis, we found that a combination of α1-blocking and α2-blocking antibodies (Abs) inhibited VEGF-driven angiogenesis in the skin of adult mice.18Senger DR Claffey KP Benes JE Perruzzi CA Sergiou AP Detmar M Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins.Proc Natl Acad Sci USA. 1997; 94: 13612-13617Crossref PubMed Scopus (451) Google Scholar However, the individual functional contributions of these integrins remained undefined. In this study we investigated specific functions of the α1β1 and α2β1 integrins in supporting VEGF-stimulated signal transduction and EC migration. Furthermore, we used a mouse model of VEGF-driven skin neovascularization to test the importance of the α1β1 and α2β1 integrins individually for angiogenesis in vivo. To assess the involvement of α1β1 and α2β1 in tumor angiogenesis, we examined the consequences of combined α1β1 and α2β1 antagonism in a xenograft model of human squamous cell carcinoma. Collectively, findings reported here indicate that the α1β1 and α2β1 integrins each serve important functions in supporting VEGF signaling, EC migration, and tumor angiogenesis within the collagen-rich matrix of skin. Purified recombinant human VEGF165, expressed in Sf21 cells, was obtained from the National Cancer Institute Preclinical Repository, Biological Resources Branch, Frederick, MD. Human dermal microvascular ECs were isolated from neonatal foreskins and cultured as previously described.19Senger DR Ledbetter SR Claffey KP Papadopoulos-Sergiou A Peruzzi CA Detmar M Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the alphavbeta3 integrin, osteopontin, and thrombin.Am J Pathol. 1996; 149: 293-305PubMed Google Scholar All experiments were performed with cells at the fourth to seventh passage. Experiments were performed in Costar 96-well EIA plates coated first overnight with 10 μg/ml Fc-specific goat anti-mouse IgG (Sigma Chemical Co., St. Louis, MO), followed by blocking of remaining nonspecific protein binding sites with 100 mg/ml bovine serum albumin (BSA) (fraction V, no. A9306; Sigma) for 2 hours at 37°C, followed by incubation for 1 hour with either 10 μg/ml or 0.2 μg/ml mouse monoclonal Abs (mAbs), as indicated. mAbs included the following: anti-human integrin α1 [(clone 5E8D9 (Upstate Biotechnology, Lake Placid, NY) and clone FB12 (Chemicon, Temecula, CA)], anti-human integrin α2 (clone A2-IIE10, Upstate Biotechnology), isotype IgG1 control Ab (clone G192-1; PharMingen, La Jolla, CA), and IgG2a isotype control Ab (clone G192-428, PharMingen). After incubation with mAbs, wells were washed three times with phosphate-buffered saline. Cells were gently trypsinized, and washed twice in serum-free medium (EBM-2; Clonetics, San Diego, CA), and 8 × 104 cells in serum-free medium were added to Ab-coated wells. Cells were allowed to attach and spread, and after decay of MAPK activity to baseline, cells were stimulated with 20 ng/ml of VEGF. At harvest, the entire contents (cells and medium) of each well were lysed in standard Laemmli sodium dodecyl sulfate sample buffer without reducing agents but containing protease and phosphatase inhibitors: 1 mmol/L 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), 1 μm aprotinin, 20 μm leupeptin, 35 μm bestatin, 15 μm pepstatin A, and 15 μm E-64 (all from Sigma), and 1 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L EGTA, 2.5 mmol/L sodium pyrophosphate, 5 mmol/L sodium orthovanadate, and 50 mmol/L sodium fluoride. One half the total volume of each sample was electrophoresed under reducing conditions on standard Laemmli gels containing 10% (w/v) polyacrylamide followed by electrophoretic transfer to Transblot membranes (BioRad, Richmond, CA). Blots were blocked for 1 hour with 5% (w/v) nonfat dry milk and stained with phospho-MAPK (Erk1/2) rabbit polyclonal Ab (New England Biolabs, Beverly, MA) and subsequently with total Erk1/2 rabbit polyclonal Ab (K-23; Santa Cruz Biotechnology, Santa Cruz, CA). Bound primary Ab was detected by staining with horseradish peroxidase-conjugated goat anti-rabbit IgG (New England Biolabs) followed by visualization with chemiluminescence (NEN Renaissance). All experiments were repeated at least three times with similar results. Before assay, cells were induced for maximal expression of α1β1 and α2β1 by stimulating with 20 ng/ml of VEGF165 for 3 days as previously described.19Senger DR Ledbetter SR Claffey KP Papadopoulos-Sergiou A Peruzzi CA Detmar M Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the alphavbeta3 integrin, osteopontin, and thrombin.Am J Pathol. 1996; 149: 293-305PubMed Google Scholar Cell migration was assayed with 8-μm-pore size Transwell migration chambers (Costar). For haptotaxis assays, the undersides of membranes were coated at room temperature for 1 hour with 10 μg/ml rat tail collagen (BD Biosciences). For chemotaxis assays, both sides of the membranes were coated with collagen. After 60 minutes, coating solutions were removed and remaining protein-binding sites were blocked by incubation with a solution of 100 mg/ml of BSA at room temperature for 60 minutes. For chemotaxis assays only, 20 ng/ml of VEGF165 was included in the lower chambers as a chemoattractant. Cells (8 × 104) were added to the upper chambers in serum-free EBM-2 containing 10 mg/ml of BSA. Integrin-blocking or control isotype Abs in solution (10 μg/ml) were mixed with cells for 15 minutes before the addition of cells to chambers. The integrin Abs were identical to those used as immobilized ligands to support cell adhesion (see MAPK analyses, above). Cell migration was allowed to proceed for 4 hours at 37°C in a standard tissue culture incubator; cells then were removed from the upper surface of the membranes with a cotton swab, and cells that migrated to the lower surface were stained with 0.2% (w/v) crystal violet in 2% ethanol for 15 minutes and washed with water. Dried membranes were cut out and mounted on glass slides in immersion oil. At least 10 random high-power fields from each of triplicate membranes were counted for each experimental condition. No cell migration was observed when membranes were coated with BSA alone, and in no cases did we observe cells in the lower chamber that had traversed the membranes but did not remain attached. All migration assays were repeated at least twice with similar results. Assays were based on a previously described model20Passaniti A Taylor RM Pili R Guo Y Long PV Haney JA Pauly RR Grant DS Martin GR A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor.Lab Invest. 1992; 67: 519-528PubMed Google Scholar with the following modifications. Athymic NCr nude mice (females, 11 weeks old) were injected subcutaneously midway on the right and left backsides with 0.25 ml of Matrigel (BD Biosciences) at a final concentration of 9 mg/ml together with 1.5 × 106 SK-MEL-2 cells transfected for stable expression of human VEGF165. Soon after injection, the Matrigel implant solidified and persisted without apparent deterioration throughout the 6-day assay interval. Isotype-matched control hamster mAb (150 μg, clone Ha 4/8) or blocking hamster anti-mouse α1 antibody (Ab) (clone Ha 31/8)21Mendrick DL Kelly DM duMont SS Sandstrom DJ Glomerular epithelial and mesangial cells differentially modulate the binding specificities of VLA-1 and VLA-2.Lab Invest. 1995; 72: 367-375PubMed Google Scholar or blocking hamster anti-mouse α2 Ab (clone Ha 1/29)21Mendrick DL Kelly DM duMont SS Sandstrom DJ Glomerular epithelial and mesangial cells differentially modulate the binding specificities of VLA-1 and VLA-2.Lab Invest. 1995; 72: 367-375PubMed Google Scholar were administered to five animals per group by intraperitoneal injection on days 1, 3, and 5. Five additional animals were treated with α1 Ab and α2 Ab in combination (150 μg each), and five animals were treated with the corresponding dose (300 μg) of control isotype Ab. After 6 days, the animals were euthanized and dissected. Implants together with associated skin were fixed for 3 hours in 10% buffered formalin and embedded in paraffin. Sections were cut, deparaffinized, and treated with 0.1% trypsin for 30 minutes at 37°C to enhance antigen availability before staining with 2 μg/ml rat anti-mouse CD31 mAb (clone MEC 13.3, PharMingen). Bound Ab was stained with secondary rabbit anti-rat Ab coupled to horseradish peroxidase (Vectastain Elite Kit; Vector Laboratories, Burlingame, CA) and visualized with liquid DAB-Plus substrate (Zymed, San Francisco, CA). Sections were counterstained with hematoxylin (Vector Laboratories). Cross-sectional diameters of individual new blood vessels within the overlying skin at the Matrigel implant/host interface were measured from representative digitized images (three specimens from each group) with NIH Image Program 1.61 and data were expressed as average diameter ± SE (n = 80 for each group). Combined blood vessel cross-sectional areas within the overlying skin at the Matrigel/host interface, determined as a percentage of the total tissue, were measured from representative digitized images obtained from six specimens of each group, using NIH Image (n = 30 for each group). Statistical analyses were performed with the two-sided unpaired t-test (InStat Program). A431 squamous cell carcinoma cells (2 × 106) (American Type Culture Collection, Rockville, MD) were injected intradermally into both flanks of 8-week-old female BALB/c (nu/nu) mice (two sites per mouse) as described.22Streit M Riccardi L Velasco P Brown LF Hawighorst T Bornstein P Detmar M Thrombospondin-2: a potent endogenous inhibitor of tumor growth and angiogenesis.Proc Natl Acad Sci USA. 1999; 96: 14888-14893Crossref PubMed Scopus (252) Google Scholar Beginning 1 day after implantation, mice (n = 5) received intraperitoneal injections, every third day, of 250 μg of the hamster α1 mAb (clone Ha 31/8) together with 250 μg of the hamster α2 mAb (clone Ha 1/29). The control group (n = 5) received 500 μg of isotype control Ab according to the same schedule. The smallest and largest tumor diameter were measured weekly, using a digital caliper, and tumor volumes were calculated using the following formula: volume = (4/3)(π)(1/2 × smaller diameter)2Ferrara N The role of vascular endothelial growth factor in pathological angiogenesis.Breast Cancer Res Treat. 1995; 36: 127-137Crossref PubMed Scopus (408) Google Scholar (1/2 × larger diameter). Tumor data were analyzed by the two-sided unpaired t-test. Mice were sacrificed after 18 days. Blood vessel size and number within the viable regions of tumors were determined as follows. Six-μm cryostat sections were stained with an anti-mouse CD31 mAb (Pharmingen). Representative sections obtained from five tumors from each cell clone were analyzed using a Nikon E-600 microscope. Images were captured with a Spot digital camera (Diagnostic Instruments, Sterling Heights, MI), and morphometric analyses were performed using the IP LAB software (Scanalytics, Billerica, MA). Three different fields in each section were examined at ×10 magnification, and the number of vessels per mm2, the average vessel size, and the relative area occupied by tumor blood vessels were determined as described.23Detmar M Velasco P Richard L Claffey KP Streit M Riccardi L Skobe M Brown LF Expression of vascular endothelial growth factor induces an invasive phenotype in human squamous cell carcinomas.Am J Pathol. 2000; 156: 159-167Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar The two-sided unpaired t-test was used to analyze differences in microvessel density and vascular size. Angiogenesis requires EC proliferation, and activation of the Erk1/Erk2 (p44/42) MAPK signal transduction pathway is pivotal for cell cycle progression.24Seger R Krebs EG The MAPK signaling cascade.FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3175) Google Scholar, 25Vinals F Pouyssegur J Confluence of vascular endothelial cells induces cell cycle exit by inhibiting p42/p44 mitogen-activated protein kinase activity.Mol Cell Biol. 1999; 19: 2763-2772Crossref PubMed Scopus (89) Google Scholar VEGF potently activates the MAPK pathway and VEGF-stimulated EC proliferation is blocked by inhibitors of MAPK activation.26Parenti A Morbidelli L Cui XL Douglas JG Hood JD Granger HJ Ledda F Ziche M Nitric oxide is an upstream signal of vascular endothelial growth factor-induced extracellular signal-regulated kinase1/2 activation in postcapillary endothelium.J Biol Chem. 1998; 273: 4220-4226Crossref PubMed Scopus (396) Google Scholar, 27Smith LE Shen W Perruzzi C Soker S Kinose F Xu X Robinson G Driver S Bischoff J Zhang B Schaeffer JM Senger DR Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor.Nat Med. 1999; 5: 1390-1395Crossref PubMed Scopus (487) Google Scholar Consequently, VEGF activation of this pathway in ECs is most probably required for VEGF stimulation of angiogenesis. Integrins have been implicated critically in supporting cytokine activation of the MAPK pathway,15Aplin AE Short SM Juliano RL Anchorage-dependent regulation of the mitogen-activated protein kinase cascade by growth factors is supported by a variety of integrin alpha chains.J Biol Chem. 1999; 274: 31223-31228Crossref PubMed Scopus (65) Google Scholar raising the possibility that α1β1 and/or α2β1 collaborate with VEGF in promoting MAPK activation and angiogenesis. To test directly α1β1 and α2β1 integrin function in regulating VEGF activation of the Erk1/Erk2 (p44/42) MAP kinase pathway, dermal microvascular ECs in suspension were added to plastic wells coated with isotype control Abs or functional integrin mAbs directed against either the α1 integrin subunit or the α2 integrin subunit. Because these two α subunits pair exclusively with the β1 integrin subunit, the chosen α1 and α2 Abs selectively probe α1β1 and α2β1 function, respectively. Although these Abs in solution function as integrin antagonists by sterically blocking attachment of α1β1 and α2β1 to collagen I, these same Abs, when immobilized to plastic substratum, serve as α1β1-specific and α2β1-specific ligands that support cell attachment and spreading similar to collagen I. ECs did not attach and spread on plastic coated with control Abs, and VEGF only marginally activated the Erk1/Erk2 MAP kinases in these cells (Figure 1A). In contrast, dermal microvascular ECs adhered and spread efficiently on plastic coated with either α1 Ab or α2 Ab, and attachment of ECs through either α1 Ab or α2 Ab supported marked activation of Erk1/Erk2 by VEGF, as determined with phospho-specific Abs (Figure 1A). Phosphorylation of Erk1/Erk2 after VEGF stimulation in cells attached to either α1 Ab or α2 Ab was rapid (within 10 minutes) and sustained through 30 minutes. Thus, these data demonstrate that dermal microvascular EC adhesion through either the α1β1 integrin or the α2β1 integrin is sufficient to support VEGF activation of the Erk1/Erk2 MAPK pathway. Furthermore, activation was comparable to that observed in cells attached to natural ligands including type I collagen and vitronectin (not shown). At the concentrations of Abs used in Figure 1A (10 μg/ml), we did not observe additive effects of coating substratum with α1 Ab in combination with α2 Ab. However, in related experiments in which substratum was coated with reduced concentrations of Abs (0.2 μg/ml), we observed that both Abs in combination supported MAPK activation by VEGF more potently than either Ab alone, suggesting cooperation between α1β1 and α2β1 (Figure 1B). Finally, we have observed that microvascular EC adhesion through integrins α5β1 and αvβ3 also fulfills the adhesion requirement for MAPK activation by VEGF (data not shown). Regardless, as the most prominent receptors for collagen on dermal microvascular ECs,18Senger DR Claffey KP Benes JE Perruzzi CA Sergiou AP Detmar M Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins.Proc Natl Acad Sci USA. 1997; 94: 13612-13617Crossref PubMed Scopus (451) Google Scholar integrins α1β1 and α2β1 likely serve prominent roles in supporting VEGF activation of MAPK in the collagen-rich matrix of skin. We first examined the functions of the α1β1 and α2β1 integrins in supporting haptotactic migration in a gradient of immobilized collagen I. The activities of the α1β1 and α2β1 integrins in supporting haptotaxis were tested by including soluble α1 Ab and α2 Ab at concentrations sufficient to provide the maximum inhibition of cell attachment to collagen I as determined with cell adhesion assays.18Senger DR Claffey KP Benes JE Perruzzi CA Sergiou AP Detmar M Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins.Proc Natl Acad Sci USA. 1997; 94: 13612-13617Crossref PubMed Scopus (451) Google Scholar As shown in Figure 2, antagonism of each integrin individually resulted in ∼40% inhibition of migration toward collagen type I, in comparison with isotype control Abs. Thus, these data indicate that the α1β1 and α2β1 integrins each function in directed migration toward collagen I. Importantly, both α1 Ab and α2 Ab in combination blocked haptotaxis toward collagen I by nearly 90% (Figure 2). Next, we tested the functions of α1β1 and α2β1 in supporting VEGF-driven chemotaxis, ie, migration in a gradient of soluble VEGF. Filters were coated uniformly with collagen I, soluble Abs were included with the cells in the upper chambers, and VEGF was included in the bottom chambers. As shown in Figure 3, antagonism of α2 alone inhibited chemotaxis by ∼45%, whereas antagonism of α1 resulted in only ∼15% inhibition relative to controls. Combined antagonism of α1 and α2 provided the greatest inhibition (∼60%). Thus, we observed greater inhibition of haptotaxis than chemotaxis with combined antagonism of α1 and α2 and this may relate to the fact that haptotaxis is primarily an adhesion-driven phenomenon. Regardless, experiments described here identify important functions for the α1β1 and α2β1 integrins in supporting directed migration of microvascular ECs. The foregoing observations implicated both the α1β1 and α2β1 integrins in supporting a key VEGF-signaling pathway together with EC migration, raising the possibility that antagonism of either integrin alone might significantly suppress angiogenesis. To test the consequences of individual antagonism of the α1β1 and α2β1 integrins for dermal angiogenesis in vivo, we used blocking Abs in an athymic nude mouse model involving subdermal injection of Matrigel together with immortalized human cells stably transfected for expression of human VEGF165. Neither Matrigel alone nor the untransfected cells in Matrigel provoked angiogenesis in the overlying dermis. In contrast, Matrigel containing VEGF transfectants potently induced neovascularization. Moreover, the hamster monoclonal blocking Abs used in these experiments do not recognize the respective human integrins and therefore did not interact with the transfected cells expressing VEGF. Animals were injected intraperitoneally with integrin-blocking Abs on days 1, 3, and 5. All animals were harvested on day 6, and skin overlying the Matrigel implants was dissected and processed for immunohistochemical analyses. Thus, overlying skin specimens from a total of 10 implants per group were analyzed, and results were highly consistent within each group. Figure 4 illustrates blood vessels in a cross-section stained with Ab to CD 31, indicating that treatment of animals with either α1 Ab or α2 Ab suppressed angiogenesis, and inhibition was greatest with both Abs in combination. Average vessel diameter (±SE) was reduced with Ab treatment from 9.58 ± 0.51 μm (control Ab) to 5.21 ± 0.24 μm (α1 Ab), 5.23 ± 0.23 μm (α2 Ab), and 3.58 ± 0.22 μm (α1 Ab + α2 Ab). Quantitation of total vascular area as a percentage of total tissue area in cross-section (Figure 5) established that cross-sectional area of new blood vessels in the α1 Ab and α2 Ab treatment groups were each reduced ∼45% relative to controls (P < 0.001). Administration of α1 Ab together with α2 Ab resulted in further inhibition of neovascularization, yielding an ∼70% reduction in total vascular area in cross-section (P < 0.001).Figure 5Quantitation of angiogenesis inhibition by α1 Ab and α2 Ab in mouse skin. Vascular cross-sectional area as a percentage of total tissue area was measured at the interface between dermis and the angiogenic stimulus (see Figure 4, above) as described in Materials and Methods. Data are presented as the mean ± SEM. Total cross-sectional area of new blood vessels in the α1 Ab and α2 Ab treatment groups were each reduced ∼45% relative to controls (P < 0.001). Administration of α1 Ab together with α2 Ab resulted in further inhibition of neovascularization, yielding an ∼70% reduction (P < 0.001).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because there is considerable evidence linking VEGF to tumor angiogenesis, we next inves
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