Growth hormone promotes lymphangiogenesis

Abstract

The lymphatic system plays an important role in inflammation and cancer progression, although the molecular mechanisms involved are poorly understood. As determined using comparative transcriptional profiling studies of cultured lymphatic endothelial cells versus blood vascular endothelial cells, growth hormone receptor was expressed at much higher levels in lymphatic endothelial cells than in blood vascular endothelial cells. These findings were confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction and Western blot analyses. Growth hormone induced in vitro proliferation, sprouting, tube formation, and migration of lymphatic endothelial cells, and the mitogenic effect was independent of vascular endothelial growth factor receptor-2 or -3 activation. Growth hormone also inhibited serum starvation-induced lymphatic endothelial cell apoptosis. No major alterations of lymphatic vessels were detected in the normal skin of bovine growth hormone-transgenic mice. However, transgenic delivery of growth hormone accelerated lymphatic vessel ingrowth into the granulation tissue of full-thickness skin wounds, and intradermal delivery of growth hormone resulted in enlargement and enhanced proliferation of cutaneous lymphatic vessels in wild-type mice. These results identify growth hormone as a novel lymphangiogenic factor.

Figure 1

GHR is expressed at higher levels in human LECs as compared to BVECs in vitro. A: Real-time RT-PCR of two matched pairs of LECs (L) and BVECs (B) that were obtained from the same donor each, revealed an up to 30-fold increase in GHR mRNA levels in LECs. Bars represent mean + SD. B: Immunoprecipitation and Western blot analyses of cell lysates confirmed that these differences in GHR mRNA expression also correlated with the protein levels.

Figure 2

GHR is expressed by lymphatic vessels in normal human skin. A–F: Double-immunofluorescence analyses of normal human foreskin revealed a co-expression of the lymphatic-specific markers LYVE-1 (A, green) and podoplanin (D, green) with GH receptor (B and E, red; C and F, merged pictures). G–I: All CD31-positive vessels (G, green) were labeled for GH receptor (H, red; I, merged picture), confirming GH receptor expression by lymphatic as well as blood vessels in vivo. Scale bars = 100 μm.

Figure 3

GH induces LEC proliferation independent of VEGFR-2 or VEGFR-3 activation in vitro. A: Dose-dependent effect of GH on LEC proliferation, as compared to untreated cells, with a minimal effective concentration of 10 ng/ml (P = 0.0073). B: Treatment with 100 ng/ml of GH led to a more than 1.65-fold induction of proliferation in LECs (L, black bars) but only to a 1.2-fold induction in BVECs (B, white bars), as compared with untreated controls. Addition of an anti-GH receptor (aGHR) antibody completely abolished the GH-induced proliferation in both cell types whereas addition of an anti-VEGFR-3 antibody (aVEGFR3) or isotype control antibodies (CIgG_goat and CIgG_human) had no inhibitory effect. C: Addition of an anti-VEGFR-2 antibody (aVEGFR2) or isotype control antibody (CIgG_human) did not inhibit the stimulation of LEC proliferation by GH (100 ng/ml). Addition of the anti-VEGFR-2 antibody completely blocked VEGF-A-induced proliferation (P < 0.001), confirming its blocking activity. Bars represent mean + SD. ns, Not significant. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Treatment of LECs with GH induces tube formation, sprouting, and migration, and inhibits apoptosis in vitro. A: Confluent LEC monolayers were overlaid with a collagen type I gel containing or not 100 ng/ml of GH. GH treatment significantly induced formation of tube-like structures as compared with untreated controls (P < 0.001). A and B: Preincubation of the cells with a blocking antibody against GHR (aGHR), but not with an isotype control antibody (CIgG), inhibited the GH effects. C: Addition of GH promoted chemotactic LEC migration with a minimal effective concentration of 100 ng/ml (P = 0.0091). D: Preincubation with an anti-GHR antibody, but not with a blocking anti-integrin a9b1 antibody, significantly inhibited the migratory response toward GH, as compared to isotype controls (P = 0.0017). Bars represent mean + SD. ns, Not significant. **P < 0.01, ***P < 0.001. E: Spheroids produced by LECs, cultured in collagen type I gels, formed significantly more sprouts on incubation with 100 ng/ml of GH as compared to controls, but less than the treatment with 20 ng/ml of VEGF-A. F: Treatment of LEC monolayer cultures with 100 ng/ml of GH reduced serum starvation-induced apoptosis (gray line) as compared to vehicle-treated cells (black line). Hydrogen peroxide treatment was used as a positive control (dotted line). Scale bars = 100 μm (B).

Figure 5

Accelerated lymphatic vessel ingrowth into the granulation tissue of bGH transgenic mice. A and B: Differential immunostains of skin samples for LYVE-1 (green) and CD31 (red) revealed accelerated ingrowth of LYVE-1-positive/CD31-positive lymphatic vessels into the wound granulation tissue in bGH tg mice (arrows). C: Merged picture of a LYVE-1/CD31-positive lymphatic vessel in the wound granulation tissue (blue, nuclei). D and E: Increased density of CD31-positive vessels within and in close proximity to the granulation tissue of bGH tg mice (E) compared to wild-type littermates (D: GT, granulation tissue; NT, normal tissue). F–H: Massive accumulation of LYVE-1-positive macrophages (arrows) were observed at the lower border of the granulation tissue in both genotypes. In contrast to lymphatic vessels, these macrophages did not express CD31. CD31-positive vessels are indicated (arrowhead). Scale bars = 100 μm.

Figure 6

Intradermal implantation of GH-containing Matrigels promotes lymphatic vessel enlargement in mice. A and B: Differential immunostains for LYVE-1 (green) and CD31 (red) revealed enlargement of lymphatic vessels (arrows) and blood vessels (arrowheads) in the skin surrounding GH-containing Matrigels and to a lesser extent in the skin surrounding control Matrigel implants. C: Double immunostains for LYVE-1 (green) and BrdU (red) revealed the presence of proliferating LECs in lymphatic vessels surrounding GH-containing implants (arrow). D–I: Quantitative image analyses of LYVE-1/CD31-stained sections showed an increase in the size (average size of individual vessels in μm²) and the average vascular area (percent of tissue area covered by vessels) of lymphatic (D and E) and blood vessels (G and H) surrounding GH-containing Matrigels, whereas, the number of lymphatic (F) and blood vessels (I) was unchanged.