Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (C-12200; Heidelberg, Germany) and cultured in endothelial cell growth medium (C-22010; PromoCell). Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂. Subcultures were obtained by trypsinization and were used for experiments at passages 3 to 9. Before performing the experiments, the cells were made quiescent by incubating overnight in endothelial cell basal medium (C-22210) containing 0.5% (w/v) fetal bovine serum (FBS). The PCa cell line, PC-3, was purchased from The European Collection of Cell Cultures and grown in F-12K (Gibco-Invitrogen, Carlsbad, CA, USA) containing 10% (w/v) FBS. PC-3 cells were seeded at a concentration of 6×10⁶ cells/T75 flask. On the following day, the medium was replaced with basal medium without FBS, and the supernatants were harvested after a 24-h incubation to serve as conditioned medium (PC3CM). Recombinant human VEGF 165 was purchased from R&D Systems (293-VE; Minneapolis, MN, USA). HUVECs were cultured in endothelial cell basal medium plus 2% (w/v) FBS (control group), in PC3CM plus 2% (w/v) FBS (PC3CM group), or in basal medium plus 2% (w/v) FBS and 30 ng/ml VEGF (VEGF group).

HUVEC proliferation was evaluated using a BrdU incorporation assay kit (Amersham; Cell Proliferation Biotrak ELISA System; RPN250; GE Healthcare, Little Chalfont, UK), according to the manufacturers’ instructions. In brief, HUVECs were plated in 96-well microculture plates (3×10³ cells/well). After a 48-h incubation at 37°C in a 5% CO₂ atmosphere, with or without fasudil (1–100 μM), 10 μl BrdU labeling reagent was added, and the cells were cultured for a further 2 h. Cells were washed twice with Dulbecco’s PBS (D8537; Sigma-Aldrich, St. Louis, MO, USA), fixed with fixative solution and then blocked with blocking buffer. BrdU incorporation was revealed by incubation with 100 μl/well horseradish peroxidase (HRP)-labeled anti-BrdU working solution for ~ 90 min. Tetramethylbenzidine (TMB) substrate at room temperature was added at 100 μl/well for 20 min. Absorbance was measured at 450 nm using a microplate reader. All determinations were performed in octuplicate, and each experiment was repeated three times.

Cell motility was assessed using a wound-healing migration assay. HUVECs were seeded to full confluency in 6-well plates. The following day, a uniform scratch was made down the centre of the well using a 100-μl micropipette tip, and the cells were washed twice with PBS. After incubation for 24 h with or without 30 μM fasudil in the control, PC3CM and VEGF groups, the cells were fixed and photographed. Photographic imaging was performed using a Leica inverted microscope. Cell migration was quantified by measuring the ratio of the migration area to the total area of the wound gap. Each experiment was repeated three times.

Ninety-six-well plates were chilled to 4°C and coated with 50 μl of Matrigel (354234; BD Biosciences, Oxford, UK) per well. Freshly passaged HUVECs were seeded onto the gel. Endothelial tube morphogenesis was carried out in the presence or absence of fasudil (3–30 μM). Endothelial tube formation was observed after 16 h and photographed under phase contrast microscopy using a Leica inverted microscope. Quantification of the digital images was performed by counting the total number of tubes in five 40× fields, and total tube length was quantified using ImageJ™ software (NIH, Bethesda, MD, USA). Tube formation was expressed as fold change or percentage, compared to the controls. All determinations were performed three times, and each experiment was repeated three times.

HUVECs were suspended in culture medium containing 0.2% (w/v) methylcellulose (Sigma-Aldrich) and seeded in non-adherent round-bottom 96-well plates (Greiner, Frickenhausen, Germany). All suspended cells formed a single spheroid in each well of defined size and cell number (~400 cells/spheroid). Spheroids were left to form for 24 h and then embedded in 1.5 mg/ml collagen gel. The spheroid-containing gel was rapidly transferred to pre-warmed 24-well plates and allowed to polymerize for 30 min. Endothelial basal medium or PC3CM with or without fasudil (1–100 μM) was then added to the surface of the gel (500 μl/well). After 16 h, images were captured using a Leica inverted microscope. Sprouting was quantified using NIH ImageJ software by measuring the cumulative sprout length, which consisted of every sprout from 10 spheroids in each group.

Protein was extracted on ice from the cultured HUVECs with cold RIPA lysis buffer (9806; Cell Signaling Technology, Boston, MA, USA) containing Pierce™ Protease and Phosphatase Inhibitor (88669; Thermo Scientific, Rockford, IL, USA). Lysates were centrifuged at 12,000 × g for 20 min at 4°C, and the supernatant was collected. Total protein concentrations were determined using a bicinchoninic acid assay (BCA) protein assay kit (23250; Thermo Scientific). Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then electrically transferred onto nitrocellulose membranes. The membranes were blocked for 1 h with 5% (w/v) non-fat milk in PBS-0.1% (v/v) Tween-20 (PBST) and incubated with primary antibodies against MYPT-1 (1:1,000; sc-25618; Santa Cruz Biotechnology, Dallas, TX, USA), phospho-MYPT-1 (1:500; ABS45; Millipore, Billerica, MA, USA), anti-ROCK1 (1:500; sc-6055), anti-ROCK2 (1:1,000; sc-1851; both from Santa Cruz Biotechnology) and β-actin (1:500; ab8229; Abcam, Cambridge, UK) overnight at 4°C. Finally, the membrane was incubated with HRP-conjugated secondary antibodies as follows: goat anti-mouse IgG-HRP (1:5,000; sc-2005; Santa Cruz Biotechnology), rabbit anti-goat IgG-HRP (1:10,000; sc-2768; Santa Cruz Biotechnology), goat anti-rabbit IgG-HRP (1:10,000; sc-2004; Santa Cruz Biotechnology) for 1 h at room temperature. After washing three times with PBST, proteins were visualized using an ECL Prime Western blotting detection kit (GE Healthcare). Photographs of the protein bands were captured using a digital imaging system (ImageQuant LAS; GE Healthcare), and densitometric measurements of band intensity in the western blotting were performed using NIH ImageJ software. The results shown are representative of three or more independent experiments.

Data are expressed as means ± standard deviation. Significance of differences was determined by the two-tailed Student’s t-test or the analysis of variance least significant difference (ANOVA LSD) test. A P-value <0.05 was considered to indicate a statistically significant difference.

Endothelial cell proliferation is crucial for angiogenesis. PC3CM-treated HUVECs were exposed to fasudil concentrations ranging from 1 to 100 μM, and HUVEC proliferation was examined using a BrdU assay. Fasudil concentrations of ≥ 30 μM had a significant inhibitory effect on PC3CM-induced cell proliferation, while proliferation in the control group was unchanged (Fig. 1).

The inhibitory effects of fasudil on endothelial cell motility were assessed using a wound-healing migration assay. Fasudil (30 μM) significantly decreased the number of cells migrating into the scratched gap in the control, PC3CM and VEGF groups, indicating the potent inhibitory effect of fasudil on HUVEC movement and migration. VEGF increased HUVEC migration significantly more than PC3CM-induced HUVEC migration. After treatment with 30 μM fasudil, all migrations decreased to similar levels (Fig. 2).

The effect of fasudil on capillary-like structure formation in vitro was examined using a 3-dimensional (3D) Matrigel assay. When seeded onto Matrigel, HUVECs form tube structures and connect with each other, mimicking the in vivo process of angiogenesis. Sixteen hours after seeding, untreated HUVECs exhibited a clear capillary-like network formation. However, fasudil treatment dramatically decreased the capillary-like network formation in a dose-dependent manner. As fasudil concentration increased, total tube length gradually decreased (Fig. 3).

In the sprout formation assay, HUVECs seeded in non-adhesive conditions in round bottom 96-well plates contributed to the formation of a single spheroid with a quiescent, non-proliferating surface monolayer within 24 h. The spheroids were then embedded in a 3D collagen matrix. In the untreated control group, baseline sprouting was low (Fig. 4Aa). When cultured with PC3CM (Fig. 4Ab), baseline sprouting increased dramatically, although it was still less than that in the cells cultured with basal medium containing 30 ng/ml VEGF (Fig. 4Ac). Sprouting was almost completely inhibited by treatment with 100 μM fasudil (Fig. 4Ad–f). We then examined the dose-dependent response of fasudil on PCa-induced HUVEC sprouting. As shown in Fig. 4B, fasudil decreased HUVEC sprouting in a dose-dependent manner and 100 μM fasudil again inhibited sprouting almost completely.

Furthermore, when treated with increasing concentrations of fasudil, the sprouts became thinner and the HUVEC nucleus seldom emerged from the spheroids. These sprouts resembled cell protrusions, were markedly thinner compared with the untreated HUVEC sprouts, and were more abundant compared with the ordered architecture of the single HUVEC spheroid sprouts (Fig. 4B).

MYPT-1 is one of the most crucial downstream effectors of ROCK. As fasudil is a ROCK inhibitor, we examined the inhibitory effects of fasudil on ROCK by measuring phospho-MYPT-1 (pMYPT-1), the active form of MYPT-1.

As shown in Fig. 5, when cultured with PC3CM or basal medium containing VEGF, expression of both MYPT-1 and pMYPT-1 was increased in the HUVECs, resulting in a moderate increase in the pMYPT-1/MYPT-1 ratio, indicating ROCK activation. Fasudil treatment lead to a significant decrease in pMYPT-1 and a slight decrease in MYPT-1, resulting in a significant decrease in the pMYPT-1/MYPT-1 ratio (Fig. 5A–D). A moderate increase in ROCK1 and ROCK2 expression was also detected, but ROCK expression was not altered significantly by fasudil treatment (Fig. 5E and F).