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Biological Action of Leptin as an Angiogenic Factor

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Science  11 Sep 1998:
Vol. 281, Issue 5383, pp. 1683-1686
DOI: 10.1126/science.281.5383.1683

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 fromfa/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.

Leptin, a circulating hormone secreted by adipocytes, influences body weight homeostasis through effects on food intake and energy expenditure (1). It also modulates other physiological actions, including lipid metabolism, hematopoiesis, pancreatic β cell function, ovarian function, and thermogenesis (2). Despite this multiplicity of biological effects in extraneural tissues, the leptin receptor is expressed predominantly in the hypothalamus (3). Alternative splicing of a single transcript encoded by the db gene produces several variants of the leptin receptor, including a transmembrane full-length, long form (OB-Rb) expressed at high levels in discrete hypothalamic regions (4). The OB-Rb form has a cytoplasmic domain that transduces the leptin signal through the Jak-STAT pathway (5, 6).

The discovery of leptin and its receptor strongly supports the hypothesis that adipose tissue mass is regulated by a hormone that is produced by adipocytes and released into the bloodstream (7). This endocrine concept of white adipose tissue implies a plastic microvascular bed that not only provides adequate blood supply for this endocrine function or for proper lipid utilization and storage but also undergoes the requisite adaptive changes that occur during physiological or pathological fluctuations in adiposity (8).

We hypothesized that leptin might play an important physiological role in the microvasculature. To test this hypothesis, we first determined whether the leptin receptor is expressed in human umbilical vein endothelial cells (HUVECs) using confocal immunofluorescence microscopy and rabbit polyclonal antibodies to synthetic peptides based on the sequence of the human leptin receptor (9). With antibodies specific for the intracellular domain (or region) of the OB-Rb form of the receptor (anti–OB-Rint) (9), a strong signal was detected (Fig. 1A, panel 1). This signal is characterized by a scattered, punctuate, intracellular staining, suggesting that the bulk of OB-Rb resides in a vesicular compartment. In contrast, an intense perinuclear staining was seen (Fig. 1A, panels 2 and 3) when antibodies to extracellular epitopes of the receptor were used (anti–OB-Rext) (9). The same pattern was found in endothelial cells (ECs) from other sources, including microvascular and aortic bovine ECs and human adipose or dermal microvascular ECs (10). In immunoblots prepared with total HUVEC extracts, a single >200-kD protein band was detected with anti–OB-Rint (Fig. 1B, lane 1). This species was also seen with anti–OB-Rext, but in this case an additional 170-kD band was observed (Fig. 1B, lane 2). To demonstrate the immunological specificity of the antibodies used, we studied frozen sections of adipose tissue from db/db leptin receptor–deficient mice ordb/+ heterozygous normal littermates. A strong immunostaining was seen in the vascular endothelial lining ofdb/+ mice but not in that of db/db mice (Fig. 1C). Finally, an intense immunostaining was observed in vascular structures of human dermis and adipose tissue when anti–OB-Rint was used (Fig. 1D) (11). Thus, the OB-Rb long form of the receptor is expressed in ECs. This expression was confirmed by reverse transcriptase polymerase chain reaction analysis of HUVEC RNA with OB-Rb–specific primers (10). The presence of the leptin receptor in ECs suggests that the endothelium may be a target for leptin action.

Figure 1

Expression of the leptin receptor in endothelial cells. (A) Confocal immunofluorescence microscopy of HUVECs permeabilized (panels 1, 2, and 4) or not (panel 3) with 0.1% Triton X-100. Immunostaining was performed (25) with normal rabbit IgG (panel 4), anti–OB-Rext (9) (panels 2 and 3), or anti–OB-Rint (9) (panel 1). Scale bar, 5 μm. (B) Immunoblotting of total HUVEC cell lysates (25) with anti–OB-Rint (lane 1), anti–OB-Rext (lane 2), or normal rabbit IgG (lane 3). Arrows point to the two bands detected. Molecular mass markers are given at right in kilodaltons. (C) Immunostaining pattern of frozen sections (11) of adipose tissue from leptin receptor–deficient db/db mice (panel 1) or db/+ normal littermates (panel 2) with anti–OB-Rint. Arrows indicate blood vessels. Scale bar, 12.5 μm. (D) Histochemical analysis of frozen sections (11) from human dermis (panels 1 to 3; scale bar, 25 μm ) or human adipose tissue (panels 4 to 6; scale bar, 12.5 μm) immunostained with normal rabbit IgG (panels 1 and 4), the endothelial marker Ulex europaeusagglutinin I (panels 2 and 5), or anti–OB-Rint (panels 3 and 6). Arrows indicate blood vessels.

To determine whether the leptin receptor in ECs is functional, we evaluated its signaling properties. First, we found that treatment of HUVECs with leptin markedly stimulates tyrosine phosphorylation of OB-Rb (Fig. 2A) (12). Second, we examined the ability of leptin to induce tyrosine phosphorylation of the transcription factor Stat3 (5, 6). Treatment of HUVECs with leptin rapidly stimulated Stat3 phosphorylation, as demonstrated by immunoblotting of cellular extracts with antibodies specific for the tyrosine-phosphorylated form of Stat3 (Fig. 2B) (13). Finally, we determined whether leptin-induced Stat3 tyrosine phosphorylation enhances its DNA-binding activity. Electrophoretic mobility gel shift assays performed with nuclear extracts from HUVECs treated with leptin [or interferon-γ (IFN-γ) as a positive control] revealed increased formation of a DNA-protein complex with the use of a Stat3-binding probe (Fig. 2C) (14,15). Thus, the endothelial leptin receptor is functionally competent with respect to ligand-induced tyrosine phosphorylation and activation of Stat3.

Figure 2

Leptin signaling in endothelial cells. (A) Tyrosine phosphorylation of leptin receptor in HUVECs treated with 120 nM leptin for 5 min. Total soluble extracts were prepared and adsorbed to a leptin-affinity matrix (12). Equivalent amounts of adsorbed proteins (12) were then fractionated by SDS-PAGE and immunoblotted with either anti–OB-Rint (αOB-R) or anti-phosphotyrosine (αPY). Molecular mass markers are given at left in kilodaltons. (B) Tyrosine phosphorylation of Stat3 in HUVECs treated with 50 nM leptin for 15 min. Total cell lysates were fractionated by SDS-PAGE followed by immunoblotting with anti-Stat3 (left) or anti–phospho-Stat3 (right) (13). (C) Stimulation of Stat3 DNA-binding activity in untreated HUVECs (lane 1) or HUVECs treated with interferon-γ (IFN-γ) (30 U/ml) (lane 2) or 5 nM leptin (lane 3) for 10 min. Nuclear extracts (14) were incubated with a 32P-labeled Stat3-binding oligonucleotide probe (Santa Cruz Biotechnology) (15). The resulting DNA-protein complexes were then separated by nondenaturing gel electrophoresis and detected by autoradiography (15). In competition reactions, leptin-treated cell extracts were incubated with unlabeled Stat3 probe (lanes 4 to 6) or a Stat3mut probe (lanes 7 to 9) that cannot bind DNA (15).

We next investigated whether leptin has angiogenic activity. First, in vitro experiments were performed. In a modified Boyden chamber assay (16), cultured HUVECs exhibited a robust directional migration in response to leptin treatment with an apparent half-maximal concentration of about 4 nM (Fig. 3A). Vascular endothelial growth factor (VEGF) (17) was used as a positive control in this assay. Leptin also promoted the formation of capillary-like tubes in three-dimensional (3D) collagen gels containing HUVECs (18, 19) (Fig. 3B). In contrast to the control (Fig. 3B, panels 1 and 2), exposure of HUVECs to VEGF (Fig. 3B, panels 3 and 4) or to leptin (Fig. 3B, panels 5 and 6) induced formation of elongated, bifurcating tubules that pervaded the gel matrix. The tubes formed in the presence of leptin displayed a reticular array reminiscent of tissue microvasculature (Fig. 3B, panels 5 and 6). Finally, proliferation assays with several types of human and bovine ECs (from microvascular and large vessel origin) exhibited variable mitogenic activity in response to leptin (10).

Figure 3

In vitro angiogenic activity of leptin. (A) Directional migration of HUVECs as determined by the number of cells moving across a porous membrane in a Boyden chamber in response to leptin (16). Under these conditions, addition of 1 nM VEGF caused a migration response of 118 ± 6 cells per field. Shown are representative data from four independent experiments. (B) Tube formation in Type I collagen gel cultures of HUVECs (19) after 2 days of treatment with vehicle (panels 1 and 2), 2 nM VEGF (panels 3 and 4 ), or 10 nM leptin (panels 5 and 6), as visualized by immunofluorescence microscopy. The red stain (TRITC-labeledUlex europaeus agglutinin I) is specific for human endothelial cell membranes and shows the complex arrangement of the 3D tubes in the collagen gel matrix. The blue stain (DAPI) is specific for DNA and depicts the nuclei of the same microscopic field for each corresponding panel above, demonstrating the multicellular organization of the tubes. Scale bar, 25 μm.

To test leptin's angiogenic activity in vivo, we surgically implanted Hydron polymer pellets containing phosphate-buffered saline (PBS), VEGF, or leptin into the corneas of normal or leptin receptor–deficient fa/fa Zucker fatty rats and monitored neovascularization (20). In normal rats, leptin caused a vigorous angiogenic response (Fig. 4A), whereas in fa/fa rats angiogenesis was seen only with VEGF; leptin had no effect in these animals (Fig. 4B).

Figure 4

In vivo angiogenic activity of leptin. (A) Corneal response (20) 7 days after implantation of a Hydron pellet containing PBS (panel 1) or 50 ng of leptin (panel 2). (B) Lack of corneal response in leptin receptor– deficient fa/fa Zucker fatty rats 7 days after implantation of a Hydron pellet containing 50 ng of leptin (panel 1). A strong positive response with 25 ng of VEGF in fa/fa rats is shown for comparison (panel 2).

The direct angiogenic action of leptin suggests a peripheral mechanism whereby the increase in energy expenditure produced by leptin (which together with the hypothalamus-mediated satiety effect contributes to body weight loss) may be facilitated. By providing a local angiogenic signal, leptin might improve the efficiency of lipid release from fat stores to maintain energy homeostasis. An artificially induced hyperleptinemic state in normal rats causes increased lipolysis and lipid oxidation (21), accompanied by augmented expression of genes encoding enzymes that regulate fatty acid metabolism and thermogenesis, including UCP2 (22). Similar observations have been made in lean C57BL/6J mice after systemic administration of leptin (23). In this case, increased lipid oxidation seems to coincide with an increase in adipose tissue vascularity (23). Thus, leptin produced in adipocytes is not only secreted into the bloodstream, but it may also act locally upon ECs in a paracrine fashion, causing increased fatty acid oxidation and an angiogenic response that maintains an appropriate balance between blood supply and fat depot size. In addition, leptin-induced angiogenesis may assist in heat dissipation at sites of active thermogenesis in the body, including adipose tissue. Our observations suggest that leptin, acting as a functional link between adipocytes and the vasculature, might also play an important extrahypothalamic role in the modulation of adipose tissue mass.

  • * To whom correspondence should be addressed. E-mail: rocio_sierra-honigmann{at}qm.yale.edu

  • Present address: Harvard Medical School, Vascular Research Division, Brigham and Women's Hospital, Boston, MA 02115, USA.

  • Present address: Institute for Diagnostic Research, Branford, CT 06405, USA.

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