Kallistatin exerts anti-lymphangiogenic effects by inhibiting lymphatic endothelial cell proliferation, migration and tube formation

Abstract

Kallistatin has been recognized as an endogenous angiogenic inhibitor. However, its effects on lymphatic endothelial cells and lymphangiogenesis remain poorly understood. Lymphangiogenesis is involved in tumor metastasis via the lymphatic vasculature in various types of tumors. The aim of this study was to investigate the effects of kallistatin on lymphangiogenesis and the mechanism of action involved. Treatment with kallistatin recombinant protein or overexpression of kallistatin inhibited the proliferation, migration and tube formation of human lymphatic endothelial cells (hLECs), and induced apoptosis of hLECs. Furthermore, our results showed that the lymphatic vessel density (LVD) was reduced in lung and stomach sections from kallistatin-overexpressing transgenic mice. Treatment with kallistatin recombinant protein decreased the LVD in the implanted gastric xenograft tumors of nude mice. To the best of our knowledge, the present study is the first to demonstrate that kallistatin possesses anti-lymphangiogenic activity in vitro and in vivo. Moreover, kallistatin inhibited proliferation and migration of hLECs by reducing the phosphorylation of ERK and Akt, respectively. These findings suggested that kallistatin may be a promising agent that could be used to suppress cancer metastasis by inhibiting both angiogenesis and lymphangiogenesis.

Figure 1

Kallistatin inhibits proliferation of lymphatic endothelial cells. (A) Cells in different groups were treated with various concentrations of rKAL for 24 and 48 h. After treatment with CCK8, changes in the optical density (OD) value at 450 nm in a micro-plate reader were recorded. (B) Cells were transfected with Ad-GFP/Ad-KAL for various periods of time, then the OD values at 450 nm were recorded. (C) Cells in different groups were treated with various concentrations of rKAL for 24 h, during which cells were incubated with EdU, then tested with immunofluorescence; scale bar represents 100 µm. (D) Histogram representing the rate of proliferative hLECs. ^*,#P<0.05, ^**,##P≤0.01, the results are presented as the mean ± standard deviation.

Figure 2

Kallistatin promotes apoptosis of lymphatic endothelial cells. (A) Flow cytometric analysis of cell apoptosis in hLECs treated with various concentrations of rKAL for 48 h. (B) Histogram representing the apoptotic rate of hLECs. ^*P<0.05, ^**P<0.01, the results are presented as the mean ± standard deviation.

Figure 3

Kallistatin inhibits hLEC cell migration. (A) hLECs were treated with 640 nM rKAL or PBS in a Boyden chamber assay for 12 h, then stained with crystal violet; histogram represents the rate of migration; scale bar represents 100 µm. (B) At 100% confluence, a 10-µl pipette was used to create a wound in the layer of hLECs, then cells were treated with rKAL for 12 h and microscopically imaged; migration distances were automatically measured by the software; the histogram represents the migration distance. (C) hLECs were transfected with Ad-GFP/Ad-KAL for 48 h, then distances were measured as described above. ^*P<0.05, ^**P<0.01. Results are presented as the mean ± standard deviation. hLECs, human lymphatic endothelial cells.

Figure 4

Kallistatin inhibits tube formation of hLEC cells. (A) Representative microscope images of hLEC cells. The cells (plated on Matrigel) were treated with 640 nM rKAL or PBS for 12 h; ×100 magnification. The histogram represents the tube numbers per field. (B) hLECs were transfected with Ad-GFP/Ad-KAL for 48 h and then plated on Matrigel for 12 h; tube numbers were counted; scale bar, 50 µm. ^*P<0.05, ^**P<0.01, the results are presented as the mean ± standard deviation. hLECs, human lymphatic endothelial cells.

Figure 5

The lymphatic vessel density (LVD) in different tissues of kallistatin transgenic mice (KAL-TG). (A, C and D) LVD in the lung tissue of wild-type and KAL-TG mice. The lymphatics were stained with VEGFR-3 and LYVE-1. (B) The number of lymphatics per field in lung sections (3–6 fields were counted in each group). (E, G and H) LVD in the stomach of wild-type mice and KAL-TG mice. The lymphatics were stained with VEGFR-3 and LYVE-1. (F) The number of lymphatics per field in stomach sections. n=5, wild-type and KAL-TG mice. Scale bar, 100 µm. ^*P<0.05, ^**P<0.01. Results are presented as the mean ± standard deviation.

Figure 6

The lymphatic vessel density (LVD) in gastric cancer xenografts of nude mice. (A) Xenografts from nude mice. n=6, PBS group and rKAL group. (B) Volume of gastric tumors from each group. (C) Weight of gastric tumors from each group. (D) Lymphatics in the gastric tumors from each group stained with LYVE-1. (E) Histogram representing the LVD in the gastric tumors. (n=6). (F) Double staining of lymphatic tubes with VEGFR-3 and LYVE-1. Scale bar, 200 µm. ^*P<0.05, ^**P<0.01, the results are presented as the mean ± standard deviation.

Figure 7

Kallistatin inhibits the VEGFR-3/ERK and VEGFR-3/Akt signaling pathways in hLECs cells. (A) After transfection with Ad-GFP/Ad-KAL for 24 h, cells were harvested for immunoblotting analysis of VEGFR-3, p-ERK/ERK and p-Akt/Akt expression levels. (B) Histograms representing the expression levels of VEGFR-3/actin, p-ERK/ERK and p-Akt/Akt. (C) Immunofluorescence assay was performed to detect p-ERK in hLECs treated with 640 nM rKAL for 24 h. (D) Immunofluorescence assay was performed to detect p-Akt in hLECs treated with 640 nM rKAL or Ad-KAL for 24 h. Scale bar, 50 µm. ^*P<0.05, ^**P<0.01. Results are presented as the mean ± standard deviation.

Figure 8

Inhibition of proliferation and migration were rescued by ERK and Akt activators respectively. (A) Following incubation with rKAL and the ERK activator ceramide C6 (10 µM) or the Akt activator SC79 (1 µg/ml) for 24 h, hLECs cells were harvested for immunoblotting analysis to detect phosphorylation levels of ERK and Akt. (B) Inhibition of hLEC proliferation was rescued by treatment with an ERK activator. (C) Inhibition of hLECs migration was rescued by the Akt activator. (D) Histogram representing the migrated distance of hLECs. ^*P<0.05, ^**P<0.01. Results are presented as the mean ± standard deviation.