Vascular endothelial growth factor receptor-3 (VEGFR-3), phospho-ERK (E4) and ERK 1 (K-23) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Phospho-Akt (Thr308) and total Akt antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) antibody was obtained from Abcam (Cambridge, UK) and β-actin antibody was from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Ceramide C6 was purchased from Sigma-Aldrich and SC79 from Selleck Chemicals (Houston, TX, USA). Green fluorescent protein-adenovirus (Ad-GFP), Ad-kallistatin (Ad-KAL) and kallistatin knock-in transgenic mice (KAL-TG mice) were provided by Professor Jianxing Ma, Department of Physiology, The University of Oklahoma Health Sciences Center (Oklahoma, OK, USA). The transgenic mice were generated through a contracted service at Transgenic Animal Facility at Stanford University and confirmed by genotyping with PCR using a forward primer (5′-AGG GAA GAT TGT GGA TTT GG-3′) and a reverse primer (5′-ATG AAG ATA CCA GTG ATG CTC-3′) specific for the human kallistatin cDNA.

Human LECs (hLECs; purchased from ProCell, Wuhan, China) were cultured in ECM medium (ScienCell, San Diego, CA, USA) with supplements according to the manufacturer's instructions and incubated at 37°C in a humidified incubator with 5% CO₂. To maintain uniform conditions, all experiments were performed using cells between passages 2 and 6.

SGC7901 gastric cancer cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in 10% FBS-supplemented DMEM medium (Hyclone; GE Healthcare Life Sciences, Chalfont, UK) and incubated at 37°C in a humidified incubator at 5% CO₂.

The recombinant kallistatin (rKAL) cDNA containing a sequence encoding the full-length mature peptide was amplified from the total RNA of rat liver by reverse transcription-PCR as described previously (54). The PCR product was cloned into the pET28 vector (Novagen) at the BamHI and SacI sites in frame with the sequence encoding the His tag at the 3′-end. The kallistatin/pET28 construct was introduced into Escherichia coli strain BL-21/DE3 (Novagen). The expression and purification of rKAL protein were carried out as described previously (54). Briefly, expression of kallistatin was induced by the addition of iso propylthio-β-galactoside (IPTG) and carried out for 10 h at 25°C. Periplasmic proteins were released by breaking down bacteria with ultrasonification and separated from cells by centrifugation. Kallistatin purification and LPS deletion were accomplished by dialysis with 1K MWCO (molecular weight cutoff) dialysis membranes and LPS level was detected in allowed scope. Identity of recombinant kallistatin was examined by SDS-PAGE and western blot analysis using antibody specific to His-tag. Then concentration of recombinant kallistatin was measured by BCA assay and bacteria were eliminated with a 0.22-µm filter. An average of 20 mg of purified kallistatin in soluble form was obtained from 1 l of culture.

Male BALB/c mice (18–22 g) were obtained from Center of Experimental Animals, Sun Yat-Sen University (Guangzhou, China). The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University (IACUC SYSU, no. 20061211005). Human gastric cancer cells SGC-7901 (1×10⁷ cells/0.1 ml) were inoculated subcutaneously in the middle dorsum of each animal. When tumors reached a volume of 50 mm³, mice were randomized into two groups. rKAL-treated group received intraperitoneal injection of recombinant kallistatin with 48-h intervals, and the total amount of rKAL was 640 nM/ml blood volume (2.88 mg/kg body mass). Control group was treated with the same volume of PBS. Tumor growth was monitored by external measurement in 2 dimensions. Tumor volume was calculated by the following formula: volume (mm³) = (length x width²)/2, 30 days after the first injection, the mice were sacrificed and tumors were dissected, and weighed.

hLECs were seeded in 96-well plates at a cell density of 5,000 cells per well and were allowed to attach overnight. The cells were treated with Ad-KAL/Ad-GFP or rKAL/PBS for 12–48 h. Cell viability was measured using CCK8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). The absorbance value at 450 nm was read using a Sunrise Microplate Reader (Tecan Group Ltd., Männedorf, Switzerland).

EdU staining was according to the protocol of the EdU staining kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), and the images were captured with ZEISS Axio Imager Z1 (Zeiss GmbH, Jena, Germany).

Apoptosis was determined using flow cytometry with a commercial Annexin V-FITC Apoptosis kit (Vazyme Biotech, Nanjing, China) according to the manufacturer's protocol. Briefly, following treatment with different concentrations of rKAL, the cells were washed in ice-cold PBS and trypsinized gently using a trypsin solution. After centrifugation to remove the trypsin, the cells were re-suspended in binding buffer containing Annexin V-FITC and propidium iodide (PI), then incubated for 15 min at room temperature in the dark and subsequently analyzed on a Beckman CytoFLEX flow cytometer (Beckman Coulter, Inc., Brea, CA, USA).

Following incubation with rKAL and Ad-GFP/Ad-KAL, 5×10⁴ hLECs were seeded into the upper chamber of the inserts in 200 µl basal medium, and the bottom chamber was filled with 500 µl complete ECM as the chemoattractant. After 12 h, the inserts were washed with PBS, then fixed in 5% glutaraldehyde for 15 min, and the non-migrated cells in the upper chamber were removed using cotton swabs. The migrated cells were stained with crystal violet and imaged using a microscope. Finally, cell counting was performed using ImageJ software (NIH, Bethesda, MA, USA).

Cells were seeded in 6-well plates and cultured to 100% confluence. The cell monolayer was scratched using a 10-µl pipette tip to produce scratches of a constant width. Cells were then incubated with the indicated treatments, and cells invading the wound line were imaged with a ZEISS Axio Observer Z1. A video was produced using Axiovison 4.7 software (Zeiss GmbH).

A tube formation assay was performed by pipetting 200 µl Matrigel (BD Biosciences, Bedford, MA, USA) into each well of a 24-well plate, which was then polymerized for 30 min at 37°C. hLECs (2×10⁴) in 200 µl Gibco ECM medium (Thermo Fisher Scientific Inc., Gaithersburg, MD, USA) with rKAL or Ad-GFP/KAL were added to each well and incubated at 37°C, 5% CO₂ for 12 h. Images were captured using a bright-field microscope ZEISS Axio Observer Z1 (Zeiss GmbH).

Histological sections, 5-µm-thick, prepared from frozen tissue samples, were used for immunofluorescence analysis. Identification of tissue lymphatic vessels and hLECs was performed using immunofluorescence with an antibody against mouse LYVE-1 (Abcam) and VEGFR-3 (Santa Cruz Biotechnology, Inc.).

Histological sections, 5-µm-thick, prepared from paraffin-fixed tissue samples, were used for immunohistochemical analysis. Identification of tissue lymphatic vessels and hLECs was performed by immunohistochemistry using antibodies to detect VEGFR-3 (Santa Cruz Biotechnology, Inc.) and LYVE-1 (Abcam).

hLECs transfected with Ad-GFP/Ad-KAL for 48 h were washed with cold PBS and lysed. The cell lysates were resolved using SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked in 10% skimmed milk for 1 h at room temperature and probed with antibodies: VEGFR-3, ERK (Santa Cruz Biotechnology, Inc.), p-ERK, AKT, p-AKT (CST Biotechnology) and β-actin (Sigma). Membranes were incubated with ECL solution (Applygen Technologies, Beijing, China) and bound antibodies were detected using ImageQuant Las 4000mini (GE Healthcare Life Sciences). Western blot quantification was performed using ImageJ (NIH).

Data are expressed as the mean ± standard deviation. Multiple comparisons were assessed by one-way analysis of variance or Student's t-test using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA) and differences with P<0.05 were considered statistically significant. All experiments were performed at least three times.

We aimed to determine whether kallistatin had a direct anti-lymphangiogenic effect on LECs. As the results demonstrate, kallistatin inhibited the proliferation of hLECs in a dose- and time-dependent manner. At a dose of 160 nM, kallistatin had little effect on cell proliferation after a 48-h treatment in complete medium, whereas it exhibited a significant inhibitory effect at 320–1,280-nM concentrations (Fig. 1A, C and D). Overexpression of kallistatin via transfection with Ad-KAL produced a similar effect on cell proliferation (Fig. 1B). To validate the effect of kallistatin on apoptosis, flow cytometry was performed to analyze AnnexinV/PI-stained hLECs. Kallistatin marginally promoted apoptosis of hLECs at doses of 640 and 1,280 nM (Fig. 2). Taken together, the results indicate that kallistatin inhibits the survival of hLECs and directly influences lymphangiogenesis.

As hLEC migration is involved in the process of lymphangiogenesis, a Boyden chamber cell migration assay and wound-healing assay were performed to determine the influence of kallistatin on the migration of hLECs. Following a 12-h incubation, kallistatin inhibited the migration of hLECs (Fig. 3A and B). Overexpression of kallistatin by transfection with Ad-KAL exhibited a similar effect on cell migration (Fig. 3C). These results also demonstrate a direct inhibitory effect of kallistatin on lymphangiogenesis.

To gain more direct evidence of the inhibitory effect of kallistatin on lymphangiogenesis, the action of kallistatin on lymphangiogenic responses was characterized further. After treatment with 640 nM rKAL or Ad-KAL transfection, the number of lymphatic tubes formed was significantly reduced, which suggests that kallistatin is a potent inhibitor of lymphangiogenesis (Fig. 4). These results showed that kallistatin exhibited potent inhibitory effect on lymphangiogenesis ex vivo, we further checked whether this effect also exists in vivo.

To investigate the effect of kallistatin on lymphangiogenesis in vivo, the lymphatic vascular density (LVD) was assessed in 5 adult wild-type and 5 KAL-TG knock-in C57BL/6 mice (6–8 weeks). The lymphatic vasculature was stained with LEC-specific markers, VEGFR-3 and LYVE-1. As the results demonstrate (Fig. 5), LVD in the KAL-TG mice was significantly lower than in wild-type mice. Additionally, recombinant kallistatin was used to treat nude mice with gastric cancer xenografts, and the LVD in the primary tumors was subsequently analyzed by staining for lymphatics. Similarly, we observed that kallistatin reduced the LVD in the gastric tumors (Fig. 6). Taken together, these results indicate that kallistatin also exerts anti-lymphan-giogenic effects in vivo.

As a VEGF-C-specific receptor, VEGFR-3 has critical roles in lymphangiogenesis. Therefore, we investigated the actions of kallistatin on VEGFR-3 expression. After 48 h of Ad-KAL transfection, expression of VEGFR-3 in hLECs was reduced (Fig. 7A). Additionally, the phosphorylation of the downstream signaling proteins ERK and Akt was decreased by Ad-KAL transfection, whereas there was no observable effect on total ERK and Akt expression (Fig. 7B and C). To understand the effect of kallistatin further, hLECs were treated with rKAL, and simultaneously, ERK and Akt signaling were activated using ceramide C6 or SC79, respectively. The proliferation and migration of hLECs was subsequently analyzed. The inhibition of proliferation induced by rKAL was rescued by ERK activation using ceramide C6, and inhibition of migration induced by rKAL was counteracted by Akt activation using SC79 (Fig. 8). These outcomes suggest that kallistatin inhibits proliferation and migration of hLECs by reducing the activation of ERK and Akt signaling, respectively.