Biological Action of Leptin as an Angiogenic Factor
10.1126/science.281.5383.1683
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

Leptin is a hormone that regulates food intake, and its receptor (OB-Rb) is expressed primarily in the hypothalamus. Here, it is shown that OB-Rb is also expressed in human vasculature and in primary cultures of human endothelial cells. In vitro and in vivo assays revealed that leptin has angiogenic activity. In vivo, leptin induced neovascularization in corneas from normal rats but not in corneas from fa/fa Zucker rats, which lack functional leptin receptors. These observations indicate that the vascular endothelium is a target for leptin and suggest a physiological mechanism whereby leptin-induced angiogenesis may facilitate increased energy expenditure.

Bibliography

Sierra-Honigmann, M. R., Nath, A. K., Murakami, C., Garcı́a-Cardeña, G., Papapetropoulos, A., Sessa, W. C., Madge, L. A., Schechner, J. S., Schwabb, M. B., Polverini, P. J., & Flores-Riveros, J. R. (1998). Biological Action of Leptin as an Angiogenic Factor. Science, 281(5383), 1683–1686.

Authors 11
  1. M. Rocı́o Sierra-Honigmann (first)
  2. Anjali K. Nath (additional)
  3. Chiaki Murakami (additional)
  4. Guillermo Garcı́a-Cardeña (additional)
  5. Andreas Papapetropoulos (additional)
  6. William C. Sessa (additional)
  7. Lisa A. Madge (additional)
  8. Jeffrey S. Schechner (additional)
  9. Michael B. Schwabb (additional)
  10. Peter J. Polverini (additional)
  11. Jaime R. Flores-Riveros (additional)
References 37 Referenced 1,031
  1. Flier J. S., Proc. Natl. Acad. Sci. U.S.A. 94, 4242 (1997); (10.1073/pnas.94.9.4242) / Proc. Natl. Acad. Sci. U.S.A. by Flier J. S. (1997)
  2. Levin N., Nelson C., Gurney A., Vandlen R., de Sauvage F., ibid. 93, 1726 (1996); / ibid. by Levin N. (1996)
  3. 10.1126/science.7624776
  4. ; J. L. Halaas et al. ibid. p. 543; L. A. Campfield F. J. Smith Y. Guisez R. Devos P. Burn ibid. p. 546.
  5. Shimabukuro M., et al., Proc. Natl. Acad. Sci. U.S.A. 94, 4637 (1997); (10.1073/pnas.94.9.4637) / Proc. Natl. Acad. Sci. U.S.A. by Shimabukuro M. (1997)
  6. Gainsford T., et al., ibid. 93, 14564 (1996); / ibid. by Gainsford T. (1996)
  7. Kieffer T. J., Heller R. S., Leech C. A., Holz G. G., Habener J. F., Diabetes 46, 1087 (1997); (10.2337/diab.46.6.1087) / Diabetes by Kieffer T. J. (1997)
  8. Zachow R. J., Magoffin D. A., Endocrinology 138, 847 (1997); (10.1210/endo.138.2.5035) / Endocrinology by Zachow R. J. (1997)
  9. Tuominen J. A., Ebeling P., Heiman M. L., Stephens T., Koivisto V. A., Acta Physiol. Scand. 160, 83 (1997). (10.1046/j.1365-201X.1997.00102.x) / Acta Physiol. Scand. by Tuominen J. A. (1997)
  10. 10.1016/0014-5793(96)00473-5
  11. Vaisse C., et al., Nature Genet. 14, 95 (1996). (10.1038/ng0996-95) / Nature Genet. by Vaisse C. (1996)
  12. Lee G. H., et al., Nature 379, 632 (1996). (10.1038/379632a0) / Nature by Lee G. H. (1996)
  13. White D. W., Tartaglia L. A., Cytokine Growth Factor Rev. 7, 303 (1996). (10.1016/S1359-6101(96)00040-8) / Cytokine Growth Factor Rev. by White D. W. (1996)
  14. Baumann H., et al., Proc. Natl. Acad. Sci. U.S.A. 93, 8374 (1996); (10.1073/pnas.93.16.8374) / Proc. Natl. Acad. Sci. U.S.A. by Baumann H. (1996)
  15. ; N. Ghilardi et al. ibid. p. 6231; C. Bjøbæk et al. J. Biol. Chem. 272 32686 (1997).
  16. Frederich R. C., et al., J. Clin. Invest. 96, 1658 (1995). (10.1172/JCI118206) / J. Clin. Invest. by Frederich R. C. (1995)
  17. Crandall D. L., Hausman G. J., Kral J. G., Microcirculation 4, 211 (1997). (10.3109/10739689709146786) / Microcirculation by Crandall D. L. (1997)
  18. Peptides based on the sequence of the human leptin receptor (4) corresponding to regions within the intracellular or the extracellular domain were synthesized and coupled to keyhole limpet hemocyanin (KLH). The intracellular region peptides were (i) IC-1 for residues 1148 to 1165 at the COOH-terminal end of the receptor (CSTQTHKIMENKMCDLTV) and (ii) IC-2 for residues 1062 to 1078 (KLEGNFPEENNDKKSIY) (24). The extracellular region peptides were (i) EC-1 for residues 247 to 263 (ITDDGNLKISWSSPPLV) (ii) EC-2 for residues 473 to 487 (CSDIPSIHPISEPKD) and (iii) EC-3 for residues 753 to 767 (CVIVSWILSPSDYKL) (24). The KLH-peptide conjugates were used to generate polyclonal antibodies in rabbits and immunoglobulin (IgG) fractions prepared from bleeds with the highest enzyme-linked immunosorbent assay titers. Unless otherwise indicated antibodies to IC-1 and IC-2 were combined in equal amounts producing OB-R int antibodies to intracellular epitopes of the leptin receptor's OB-Rb form. Likewise equal amounts of antibodies to EC-1 EC-2 and EC-3 were mixed producing OB-R ext antibodies to extracellular epitopes of the leptin receptor.
  19. M. R. Sierra-Honigmann et al. data not shown.
  20. Tissue specimens of human abdominal skin and subcutaneous fat were collected during cosmetic surgery. Subcutaneous adipose tissue specimens were also obtained from adult female leptin receptor–deficient db/db mice or normal db/ + littermates. After delipidation the tissue was flash-frozen and cut into 5-μm-thick sections. Sections were stained with anti–OB-R int Ulex europaeus agglutinin I or normal rabbit serum. After incubation with primary antibodies tissue sections were developed with secondary horseradish peroxidase–conjugated goat antibody to rabbit IgG with a Vectastain Elite ABC kit (Vector Labs). The slides were counterstained with hematoxylin.
  21. Freshly isolated HUVECs (subculture 1) were treated as indicated in Fig. 2A and washed with tris-buffered saline and homogenized on ice with radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors and insoluble material was collected by centrifugation at 12 000 g for 10 min at 4°C. Equal amounts of protein were precleared by incubation with Affigel-15 (Bio-Rad) beads for 2 hours at 4°C and the resin was removed by centrifugation. Lysates were incubated for 6 hours at 4°C with 40 μl of leptin-Affigel beads [containing 30 μg of covalently coupled human recombinant leptin per milliliter of packed Affigel-15 resin; coupling was conducted following the manufacturer's instructions (Bio-Rad)]. Adsorbed complexes were washed with RIPA buffer and boiled with SDS–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer for 10 min. Proteins were separated by SDS-PAGE with 4 to 20% gradient gels followed by overnight transfer to nitrocellulose membranes. Immunoblots were developed with anti–OB-R int (9) or with RC20 recombinant anti-phosphotyrosine (Transduction Laboratories Lexington KY).
  22. Confluent monolayers of HUVECs grown in T75 flasks were washed with PBS twice and total cell extracts were prepared by adding 1 ml of SDS-PAGE sample buffer supplemented with 100 μM dithiothreitol (DTT). Lysed cells were briefly sonicated and insoluble material was removed by centrifugation. Supernates were collected boiled for 3 min and applied to a 4 to 20% gradient gel. Gels were processed for immunoblotting as described (25). The PhosphoPlus Stat3 (Tyr 705 ) Antibody kit (New England Biolabs) containing a phospho-specific Stat3 (Tyr 705 ) rabbit polyclonal antibody was used for detecting the presence of Stat3 and phospho-Stat3 in the cell lysates.
  23. HUVECs were washed with PBS pelleted and resuspended into a minimal volume of low-salt buffer [20 mM Hepes (pH 7.9) 25% glycerol 1.5 mM MgCl 2 20 mM KCl 0.2 mM EDTA 0.5 mM DTT 0.2 mM phenylmethylsulfonyl fluoride and a mixture of protease inhibitors]. After a 15-min incubation on ice cells were homogenized and nuclei were collected by centrifugation. The pellet was resuspended in low-salt buffer adjusted to 0.5 M KCl extracted for 30 min at 4°C and centrifuged at 25 000 g for 30 min. Supernates were collected dialyzed against buffer made 0.1 M with KCl and divided into small portions kept at −80°C until used.
  24. The double-stranded oligonucleotide Stat3 (Santa Cruz Biotechnology Santa Cruz CA) was first end-labeled with T4 polynucleotide kinase and [γ- 32 P]adenosine triphosphate. After labeling 10 μg of nuclear extract was incubated with 100 000 cpm (<1 pmol) of 32 P-labeled probe for 20 min at room temperature in binding buffer [20 mM tris-HCl (pH 7.9) 50 mM NaCl 1 mM EDTA 10% glycerol 0.1% NP-40 1 mM DTT bovine serum albumin (50 μg/ml) and polydeoxyinosine/polydeoxycytosine (2 μg/ml)]. Electrophoresis sample buffer was added to each sample before separation in a nondenaturing gel (89 mM tris 89 mM boric acid 10 mM EDTA and 4.95% acrylamide). Gels were dried and exposed to autoradiography.
  25. G. Garcı́a-Cardeña P. Oh J. Liu J. E. Schnitzer W. C. Sessa Proc. Natl. Acad. Sci. U.S.A. 93 6448 (1996); (10.1073/pnas.93.13.6448)
  26. Kundra V., Anand-Apte B., Feig L. A., Zetter B. R., J. Cell Biol. 130, 725 (1995). (10.1083/jcb.130.3.725) / J. Cell Biol. by Kundra V. (1995)
  27. Ferrara N., Henzel W. J., Biochem. Biophys. Res. Commun. 161, 851 (1989). (10.1016/0006-291X(89)92678-8) / Biochem. Biophys. Res. Commun. by Ferrara N. (1989)
  28. Jain R. K., Schlenger K., Hockel M., Yuan F., Nature Med. 3, 1203 (1997); (10.1038/nm1197-1203) / Nature Med. by Jain R. K. (1997)
  29. Auerbach R., Auerbach W., Polakowski I., Pharmacol. Ther. 51, 1 (1991). (10.1016/0163-7258(91)90038-N) / Pharmacol. Ther. by Auerbach R. (1991)
  30. 3D cultures of HUVECs were established in gel matrices with rat tail Type I collagen at a final concentration of 1.75 mg/ml. The collagen solution was prepared in M199 culture medium and adjusted to fibronectin (90 mg/ml) 150 mM Hepes and sodium bicarbonate neutralizing the pH by the addition of 1 M NaOH. HUVECs were added immediately to a final concentration of 2 × 10 6 cells/ml. Drops (0.2 ml each) of the cell-collagen mixture were added to Petri dishes and placed in a humidified CO 2 incubator at 37°C for 2 to 5 min allowing them to solidify. Growth medium supplemented with endothelial cell growth supplement with or without human recombinant leptin was then added to each dish. Leptin was replenished every 24 hours. HUVECs were allowed to form tubelike structures for 2 days in culture and were then frozen and sectioned to a thickness of 40 μm. For immunofluorescence analysis the sections were fixed with acetone (−20°C) and stained with tetramethylrhodamine isothiocyanate–labeled Ulex europaeus agglutinin I (0.5 μg/ml) and 4′ 6′-diamidino-2-phenylindole (DAPI) (0.0001%).
  31. Castle V. P., Dixit V. M., Polverini P. J., Lab. Invest. 77, 51 (1997). / Lab. Invest. by Castle V. P. (1997)
  32. Koyama K., et al., Diabetes 48, 1276 (1997). (10.2337/diab.46.8.1276) / Diabetes by Koyama K. (1997)
  33. Zhou Y. T., et al., Proc. Natl. Acad. Sci. U.S.A. 94, 6386 (1997). (10.1073/pnas.94.12.6386) / Proc. Natl. Acad. Sci. U.S.A. by Zhou Y. T. (1997)
  34. Sarmiento U., et al., Lab. Invest. 77, 243 (1997). / Lab. Invest. by Sarmiento U. (1997)
  35. Abbreviations for the amino acid residues are as follows: C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; S Ser; T Thr; V Val; W Trp; and Y Tyr.
  36. Sierra-Honigmann M. R., Bradley J. R., Pober J. S., Lab. Invest. 74, 684 (1996). / Lab. Invest. by Sierra-Honigmann M. R. (1996)
  37. We thank T. Buckholz and J. Kupcho for the synthetic peptides and conjugates L. Friedman for the anti–leptin receptor L. Benson and G. Davis for HUVEC cultures D. O'Connor for help in migration studies and J. Pober for laboratory resources.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:43 a.m.)
Deposited 1 year, 7 months ago (Jan. 12, 2024, 10:24 p.m.)
Indexed 3 days, 2 hours ago (Aug. 29, 2025, 5:46 a.m.)
Issued 26 years, 11 months ago (Sept. 11, 1998)
Published 26 years, 11 months ago (Sept. 11, 1998)
Published Print 26 years, 11 months ago (Sept. 11, 1998)
Funders 0

None

@article{Sierra_Honigmann_1998, title={Biological Action of Leptin as an Angiogenic Factor}, volume={281}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.281.5383.1683}, DOI={10.1126/science.281.5383.1683}, number={5383}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Sierra-Honigmann, M. Rocı́o and Nath, Anjali K. and Murakami, Chiaki and Garcı́a-Cardeña, Guillermo and Papapetropoulos, Andreas and Sessa, William C. and Madge, Lisa A. and Schechner, Jeffrey S. and Schwabb, Michael B. and Polverini, Peter J. and Flores-Riveros, Jaime R.}, year={1998}, month=sep, pages={1683–1686} }