Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2002 Dec 10;99(25):16075-80.
doi: 10.1073/pnas.252649399. Epub 2002 Nov 26.

Complex interactions between the laminin alpha 4 subunit and integrins regulate endothelial cell behavior in vitro and angiogenesis in vivo

Annette M Gonzalez  1 , Meredith Gonzales, G Scott Herron, Usha Nagavarapu, Susan B Hopkinson, Daisuke Tsuruta, Jonathan C R Jones
Affiliations

Complex interactions between the laminin alpha 4 subunit and integrins regulate endothelial cell behavior in vitro and angiogenesis in vivo

Annette M Gonzalez et al. Proc Natl Acad Sci U S A. .

Abstract

The alpha4 laminin subunit is a component of the basement membrane of blood vessels where it codistributes with the integrins alphavbeta3, alpha3beta1, and alpha6beta1. An antibody against the G domain (residues 919-1207; G(919-1207)) of the alpha4 laminin subunit inhibits angiogenesis in a mouse-human chimeric model, indicating the functional importance of this domain. Additional support for the latter derives from the ability of recombinant G(919-1207) to support endothelial cell adhesion. In particular, endothelial cell adhesion to G(919-1207) is half-maximal at 1.4 nM, whereas residues 919-1018 and 1016-1207 of the G domain are poor cellular ligands. Function blocking antibodies against integrins alphavbeta3 and beta1 and a combination of antibodies against alpha3 and alpha6 integrin subunits inhibit endothelial cell attachment to G(919-1207). Moreover, both alphavbeta3 and alpha3beta1 integrin bind with high affinity to G(919-1207). Together, our studies demonstrate that the G domain of laminin alpha4 chain is a specific, high affinity ligand for the alphavbeta3 and alpha3beta1 integrin heterodimers and that these integrins, together with alpha6beta1, function cooperatively to mediate endothelial cell-alpha4 laminin interaction and hence blood vessel development. We propose a model based on these data that reconcile apparent discrepancies in the recent literature with regard to the role of the alphavbeta3 integrin in angiogenesis.

PubMed Disclaimer

Figures

Fig 1.
Fig 1.
Antibody 2A3 against α4 laminin inhibits angiogenesis in vivo. Human endothelial cells were mixed with 0.5 ml of Matrigel in the presence of 125 μg/ml control IgM (A and C) or 125 μg/ml antibody 2A3 (B and D). The cell-matrix mix was then injected in the ventral midline thoracic tissue of severe combined immunodeficient mice. After 7 days, implants were removed, fixed, and prepared for immunofluorescence microscopy. Samples were stained with either anti-human type IV collagen antibody (A and B) or an antiserum against von Willebrand factor (C and D). Type IV collagen and von Willebrand factor staining appears in an annular and linear organization in A and C (arrows). (E) The number of vascular structures observed in the specimens shown in A–D were quantified. Results represent mean ± SD of five separate fields. (F) Laminin α4 subunit and integrin subunit localization in blood vessels. Cryostat sections of human renal carcinoma tissue were prepared for double-label immunofluorescence and viewed by laser-scanning confocal microscopy. The tissue sections were incubated with a polyclonal rabbit antiserum against the integrin β3 in combination with either antibody 2A3 against the α4 laminin subunit or antibody P1B5 against α3 integrin as indicated. Sections were also processed by using antibody GoH3 against α6 integrin in combination with the α4 laminin subunit as shown. Merged versions of the red and green images are presented (Lower Right). Yellow color indicates overlap in staining. (Bar = 20 μm in D; bar = 50 μm in F.)
Fig 2.
Fig 2.
(A) Gel profiles of the recombinant proteins used in these studies. Proteins purified from bacterial cell extracts were processed for SDS/PAGE. Lanes 1–3 show G919–1207, G919–1018, and G1016–1207, respectively. Molecular weight standards are indicated (Left). (B) Endothelial cells (TrHBMEC or HUVEC as indicated) adhere to the G domain of the α4 laminin subunit. Endothelial cells were added to the wells of a 96-well plate coated with varying concentrations of G919–1207, G919–1018, G1016–1207, or human fibronectin as indicated. Cells were allowed to attach at 37°C for 1 h. Nonadherent cells were washed off the wells, and the remaining cells were fixed and stained with crystal violet. Absorbance was read at 570 nm. The curves are representative of three separate experiments.
Fig 3.
Fig 3.
(A) Cell attachment of TrHBMEC or HUVEC to G919–1207 involves the αvβ3 and α3β1 and α6β1 integrin heterodimers. Cells were pretreated with control IgG or function-blocking antibodies against αvβ3 integrin (LM609), α3 integrin (P1B5), α6 integrin (GoH3), or a combination of P1B5 and GoH3 or β1 integrin (6S6) for 30 min at 37°C before adding cells to wells coated with 100 nM G919–1207 protein. LM609 was used at 25 μg/ml, whereas all other antibodies and control IgG were used at 50 μg/ml. Cell attachment was evaluated as in Fig. 2. (B) TrHBMEC and HUVEC adhesion to laminin 5. Endothelial cells were pretreated with the same function-blocking antibodies as in A for 30 min at 37°C. The attachment of the cells to wells coated with 5 μg/ml laminin 5 was evaluated after 1 h as above. (C) Integrin αvβ3 binds directly to fibronectin but not to laminins 1 and 5. Wells of 96-well plates were coated with equal concentrations of extracellular matrix proteins (5 ng/μl). αvβ3 integrin (5 ng/μl) was then added to the wells and allowed to bind for 1 h at 37°C. Integrin binding was evaluated by ELISA using an antibody against αvβ3, followed by a secondary antibody conjugated to alkaline phosphatase. Absorbance was measured at 405 nm. Values in bar graphs are expressed as means ± SD of three trials.
Fig 4.
Fig 4.
(A and B) Integrin αvβ3 binds directly to the α4 G domain and fibronectin with high affinity. Wells of a 96-well plate were coated with varying concentrations of G919–1207, G919–1018, and G1016–1207 (A) or fibronectin (B). αvβ3 integrin (5 ng/μl) was then added to the wells and allowed to bind for 1 h at 37°C. Integrin binding was evaluated by ELISA using an antibody against αvβ3, followed by a secondary antibody conjugated to alkaline phosphatase. (C) Competition binding curve. Soluble αvβ3 (5 ng/μl) was added to wells coated with G919–1207 in the presence of increasing concentrations of fibronectin at 37°C. Integrin binding was assayed as in A and B after 1 h. (D) α3β1 integrin was added to wells coated with varying concentrations of G919–1207, G919–1018, and G1016–1207 and allowed to bind for 1 h at 37°C. Integrin binding was evaluated by ELISA using MKID2, an antibody against the α3β1 integrin heterodimer, followed by alkaline phosphatase-conjugated secondary antibody. In all studies, absorbance was measured at 405 nm. Each of the graphs is representative of at least three separate experiments.
Fig 5.
Fig 5.
Diagram showing a scheme in which there is crosstalk among integrins in endothelial cell adhesion to the G domain of the α4 laminin subunit. In the model, we show that both the αvβ3 and α3β1 integrins interact directly with the G domain. Endothelial cell adhesion to the G domain can be inhibited by G domain antibodies (2A3), by antibodies against the αvβ3 integrin, by antibodies against the β1 integrin, or by a combination of antibodies against the α3 and α6 integrin subunits. In the model, when αvβ3 integrin function is inhibited, this also perturbs the function of α3β1 and α6β1 integrin heterodimers (bracketed) and vice versa. In the diagram, integrin interplay is indicated by a double-headed arrow. Moreover, angiogenesis is blocked when integrin function is perturbed. In the absence of αvβ3 integrin, the α3β1 and α6β1 integrin heterodimers are capable of supporting angiogenesis but may do so in a relatively unregulated manner.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aumailley M. & Smyth, N. (1998) J. Anat. 193, 1-21. - PMC - PubMed
    1. Timpl R. & Brown, J. C. (1994) Matrix Biol. 14, 275-281. - PubMed
    1. Tunggal P., Smyth, N., Paulsson, M. & Ott, M.-C. (2000) Microsc. Res. Tech. 51, 214-227. - PubMed
    1. Smirnov S., McDearmon, E., Shaohua, L., Ervasti, J., Tryggvason, K. & Yurchenco, P. D. (2002) J. Biol. Chem. 277, 18928-18937. - PubMed
    1. Patton B. L. (2000) Microsc. Res. Tech. 51, 247-261. - PubMed

Publication types

MeSH terms

LinkOut - more resources