Introduction

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Angiogenesis is the formation of new capillary blood vessels from existent microvessels and also involves differential recruitment of associated supporting cells to different segments of the vasculature (Folkman and D'Amore, 1996). In the physiological condition, the activity of inducers and inhibitors of angiogenesis maintains it in balance. However, persistent and up-regulated angiogenesis is often found to be a critical causal factor in certain pathological conditions such as cancer, atherosclerosis, and diabetic retinopathy (Fan et al., 1995). These diseases may benefit from therapeutic inhibition of angiogenesis (Folkman, 1995; Hanahan and Folkman, 1996).

A number of angiogenic molecules and angiosuppressors have been reported (Auerbach and Auerbach, 1994; Fan et al., 1995; Griffioen and Molema, 2000). Among them, AII, a vasoconstrictor peptide, has been demonstrated to stimulate angiogenesis in vivo in association with endothelial proliferation and migration in vitro (Fernandez et al., 1985; Bell and Madri, 1990). Intense angiogenic activity of AII has also been demonstrated in rat and mice sponge implant model (Andrade et al., 1996; Hu et al., 1996). AII was demonstrated to stimulate the release of vascular endothelial growth factor (VEGF) that plays a central role in the regulation of vasculogenesis (Neufeld et al., 1999). Thus, AII and VEGF are promising targets for the development of specific angiosuppressive drugs.

Recently, KR31372 [(2R,3R,4S)-N"-cyano-N-(6-nitro-3,4-dihydro-hydroxy-2-methyl-2-dimethoxymethyl-2H-1-benzopyran-4yl)-N'-benzylguanidine] has been synthesized by the Korea Research Institute of Chemical Technology (Daejon, Korea). This agent shows weak vasorelaxant actions, while it has a benzopyran moiety in its structure. In the previous study, KR31372 showed inhibitory action on the oxidized low density lipoprotein-stimulated DNA synthesis and migration of the smooth muscle cells with antioxidative action (Kim et al., 2001). Thus, if KR31372 is demonstrated to have antiangiogenic actions, it might have potential for use in therapeutic approaches to the angiogenic disorders.

In the present study, we used the subcutaneous sponge implant model for investigating the inhibitory action of KR31372 in the new vessel formation by AII and recombinant VEGF₁₆₅. First, we established the roles of the AII and VEGF₁₆₅ in angiogenesis, and then demonstrated that KR31372 inhibits angiogenic responses. Furthermore, the antiangiogenic effects of KR31372 (^99mTc clearance and morphometric responses) were compared with those of levcromakalim, a K⁺ channel opener.

	Materials and Methods

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Reagents. Recombinant human VEGF₁₆₅ was purchased from R&D Systems (Minneapolis, MN). AII was from Sigma-Aldrich Korea (Seoul, Korea). Levcromakalim and KR31372 were donated from The Korea Research Institute of Chemical Technology. ^99mTc-diethylenetriaminepentaacetic acid was obtained from Department of Nuclear Medicine, Pusan National University Hospital, Pusan, Korea. Levcromakalim and KR31372 were dissolved in dimethyl sulfoxide as a 100-mg/ml stock solution and then diluted with phosphate-buffered saline (PBS). Glibenclamide (Sigma-Aldrich Korea was suspended in 0.5% methylcellulose as a 10-mg/ml stock solution, and administered orally with 1 mg/kg in a volume of 1 ml.

Sponge Implantation. The Animal Experimentation Committee of College of Medicine, Pusan National University, approved the experimental design of the study and the guiding principles for research. Sterile circular sponge discs composed of double sheets (5 mm in total thickness, 10 mm in diameter) were prepared from polyester sponge. The sponges for implantation were sterilized for 20 min and ultraviolet-irradiated overnight. The rats were anesthetized with anesthesia cocktail composed of 1 mg/kg acepromazine, 5 mg/kg xylazine, and 50 mg/kg ketamine. Implantation was performed with aseptic techniques. After shaving the hair on the dorsal side, two 12-mm midline dorsal incisions were made 1 cm apart, and two subcutaneous air pockets were prepared using a pair of curved scissors. A sterilized sponge disk was then inserted into each air pocket. A 5-mm polyethylene cannula that was installed inside of each sponge disc was exteriolized through needle puncture in the skin and secured in place by a 5-0 silk suture, and then plugged with a sterile polyethylene stopper. Animals were housed individually in plastic cages and allowed access to a normal diet and water ad libitum. Test substances (50 µl), including PBS, were administered daily through each of installed cannulas from day 1 to the end of experiment. Either AII (100 ng/sponge) or VEGF₁₆₅ (200 ng/sponge) were topically injected (50 µl in volume) daily in the morning.

^99mTc Clearance. Blood flow in the implanted sponge was measured by the ^99mTc (half-life, 6 h) clearance technique. This provided a rapid and convenient estimation of blood flow through the implanted sponge in the dorsal side. Briefly, the anesthetized animals were injected with 50 µl (0.5 mCi) of ^99mTc in diethylenetriaminepentaacetic acid (Techneleit, Technetium Tc 99m generator; DuPont Pharmaceuticals, Boston, MA) through the cannula. The clearance of radioactivity from the implants was monitored with a gamma scintillation detector (Vertex EPIC, ADAC Lab, Milpitas, CA) for 60 min. The ^99mTc clearance values were expressed (Microsystem SUN Computer; ADAC Lab) as the percentages of ^99mTc remaining in the sponge as the half-time for ^99mTc clearance (t_1/2). Percentage clearance value of ^99mTc was calculated at 6 min as [(initial radioactivity  residual radioactivity at 6 min)/initial radioactivity] × 100%.

Vascularity Assay. Vascularity was assessed by measuring the formation of vascular casts incorporating carmine red as described by Colville-Nash et al. (1995). After anesthesia, peripheral vasodilation was raised by placing rats in a heated jacket at 40°C for 10 min. The induction of peripheral vasodilation in the rats is important in overcoming alterations in peripheral vasomotor tone and tissue perfusion related to anesthesia and ambient temperature that may invalidate the results (Orlandi et al., 1988). The cast was formed by intravenous injection (via femoral vein) of 1 ml of 5% carmine red in 10% gelatin at 40°C into the warmed rats. The rats were chilled below 4°C for 2 h to solidify the gelatin in the blood vessels. Thereafter, the sponges were removed and oven dried at 56°C for 24 h and weighed. To overcome dye bleaching by digestion of tissue in sodium hydroxide, the dried sponges were papain digested for 12 h at 56°C in 0.6 ml of digestive buffer (2 mM dithiothreitol, 20 mM disodium hydrogen orthophosphate, 1 mM EDTA, 12 U/ml papain). The dye was then dissolved by addition of 1 ml of 3 M sodium hydroxide. The sponges were solubilized for 30 min at 37°C and 0.5 ml of hydrochloric acid (36%) was then added. After homogenization for 5 min, the mixture was centrifuged at 1100g for 10 min at 4°C and filtered through a 0.2-µm syringe filter. The dye content in the filtrate was assayed spectrophotometrically at 490 nm. The results were expressed either by micrograms of dye content per sponge or by a vascularity index, micrograms of dye per milligram dry weight of tissue.

Morphometric Study. The implanted sponges were removed at day 4, 7, and 14 of the experiment. They were dissected free of adherent connective tissue, fixed in 10% neutral buffered formalin, stained with H&E, and processed for light microscopy. To determine the number of new capillary vessels, the tissue outside the sponge was discarded and the intrasponge tissue was examined. The number of new vessels of each size (every 10 µm in diameter) formed inside the sponges was counted by using LSM 510 laser scanning confocal microscopy (Carl Zeiss, Jena, Germany). The neovascularization was expressed as the number of blood vessels per square millimeter.

Statistics. Results are expressed as means ± S.E.M. Comparisons between groups were performed with two-tailed Student's t test for unpaired data. A value of P < 0.05 was considered statistically significant.

	Results

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Inhibitory Effect of KR31372 on ^99mTc Clearance. Figure 1 shows representative photographs of PBS- and AII-induced angiogenesis in the rat sponge model. The number of new microvessels formed inside the sponge was markedly enhanced by daily topical administration of AII (100 ng/sponge) in comparison to that by PBS (50 µl). In Fig. 2, ^99mTc clearance observed at 6 min in PBS-injected sponge group increased progressively with time (4, 7, and 14 days) after implantation. Daily topical administration of AII (100 ng, for 7 days) significantly enhanced the ^99mTc clearance to 37.7 ± 4.4% (P < 0.05, N = 8) in comparison to PBS group (23.9 ± 3.6%, N = 8). The increased ^99mTc clearance caused by VEGF₁₆₅ (200 ng daily, at day 7) was 48.4 ± 6.9% (N = 4). Administration of 100 ng of DuP753, an AT₁ receptor antagonist, significantly decreased the ^99mTc clearance stimulated by 100 ng AII, but PD123319 (100 ng/sponge), an AT₂ receptor antagonist, was without effect, suggesting AII-induced angiogenic action being mediated via activation of AT₁ receptors (data not shown).

View larger version (69K): [in this window] [in a new window]   Fig. 1.   Representative photographic results of PBS- (A-C) and angiotensin II (AII; D, E, F)-induced angiogenesis at 4, 7, and 14 days in the rat sponge model. The number of newly formed capillaries was largely enhanced by topical administration of angiotensin II (100 ng/sponge) in comparison to that by vehicle (PBS, 50 µl). Image from confocal microscope (100×). All sections were stained with H&E. Arrows indicate the newly formed microvessels. S, sponge.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Graphs showing effects of topical administration of AII (100 ng, daily) and recombinant VEGF165 (200 ng, daily) on 99mTc clearance at 6 min in comparison to those of vehicle (PBS, 50 µl) at 4 (), 7 (), and 14 days (). Data represent means ± S.E.M. from four to eight experiments. *P < 0.05 versus corresponding data of vehicle.

View larger version (105K): [in this window] [in a new window]   Fig. 3.   Representative photographic results showing the inhibitory effect of KR31372 (1 mg/kg p.o., daily) on the angiotensin II- (100 ng, daily; A and B) and recombinant VEGF165 (200 ng, daily; C and D)-stimulated formation of new capillaries. Image from confocal microscope (630×). All sections were stained with H&E. Scale bar, 50 µm. Arrows indicate the newly formed microvessels. S, sponge.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Inhibitory effect of KR31372 on 99mTc clearance stimulated by topical administration of AII (100 ng, daily) and recombinant VEGF165 (200 ng, daily) at day 7 after implantation. Data represent means ± S.E.M. from four to eight experiments. *P < 0.05; **P < 0.01; ***P < 0.001 versus corresponding data of vehicle.

Dye Retention within the Vessels. Administration of AII and VEGF₁₆₅ caused increased carmine red dye retention inside the newly formed vessels as seen by increases in the derived vascularity index (Table 1). Dry mass (13.4 ± 0.3 mg in vehicle) and dye content (92.8 ± 5.9 µg in vehicle) significantly increased to 20.7 ± 0.5 mg (54.5%, P < 0.001) and 164.8 ± 9.3 µg (80.8%, P < 0.001), respectively, in the AII (100 ng)-stimulated group. There was little difference between the effects of AII and VEGF₁₆₅. AII-induced carmine dye content retained inside the vessels in the sponge was concentration dependently suppressed by administration of KR31372 (0.1, 0.3, and 1.0 mg/kg p.o., daily for 7 days). However, dry tissue mass was modestly reduced. Therefore, the derived vascularity index was consequently increased in the AII-stimulated group by 18.8%. In contrast, 1.0 mg/kg KR31372 caused a marked reduction in the basal dye content and vascularity index of the vehicle group by 33.4 and 59.8%, respectively (P < 0.001).

Morphometric Analysis. Figure 5 shows the number of new vessels of each size (every 10 µm in diameter) formed inside the sponge matrix. AII (100 ng, topically for 7 days) significantly increased the number of vessels, especially sizes of 20 to 29, 30 to 39, and 40 to 49 µm in diameter. VEGF₁₆₅ similarly showed increased number of vessels. The number of microvessels newly formed was markedly inhibited by treatment with KR31372 (1.0 mg/kg p.o., for 7 days). KR31372 also suppressed VEGF₁₆₅-induced increased formation of microvessels.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Graphs showing the number of new capillaries of each size formed within the sponge matrix. Both AII (100 ng, topically for 7 days) and VEGF165 (200 ng, topically for 7 days) significantly increased the number of vessels of sizes of 20 to 29, 30 to 39, and 40 to 49 µm in diameter. These were markedly inhibited by treatment with KR31372 (1.0 mg/kg p.o., for 7 days). Data represent means ± S.E.M. Numbers in parentheses are number of experiments. P < 0.05; P < 0.01 versus corresponding values of PBS; *P < 0.05; **P < 0.01 versus corresponding values of angiotensin II; $P < 0.05; $$P < 0.01 versus corresponding values of VEGF165.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Graphs showing effect of levcromakalim (LMK, 100 nmol/sponge, topically for 7 days) on the formation of new capillaries of each size stimulated by AII (100 ng/sponge, topically for 7 days) in comparison with that of KR31372 (100 nmol/sponge, topically for 7 days). Numbers in parentheses are number of experiments. Inset, comparison of the inhibitory effect of KR31372 on 99mTc clearance stimulated by topical administration of angiotensin II with that of levcromakalim at day 7 after implantation. Data represent means ± S.E.M. P < 0.05 versus corresponding values of PBS; *P < 0.05; **P < 0.01 versus corresponding values of angiotensin II; ###P < 0.001 versus vehicle.

Effect of Glibenclamide. Figure 7 shows that treatment with glibenclamide (10-100 mg/kg p.o., daily for 7 days), an ATP-sensitive K⁺ channel blocker, largely reversed the ^99mTc clearance that was suppressed by KR31372 (1.0 mg/kg p.o.).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Reverse by glibenclamide, an ATP-sensitive K+ channel blocker, of KR31372 (1 mg/kg p.o., daily)-induced suppression of 99mTc clearance that was stimulated by AII (100 ng/sponge, topically for 7 days). + and , the presence and absence of drugs. Data represent means ± S.E.M. from four to five experiments. *P < 0.05; **P < 0.01 versus angiotensin II alone.

	Discussion

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The major findings of the present study were that the enhanced neovascularization generated by AII as well as VEGF₁₆₅ was significantly suppressed by oral administration of KR31372. These antiangiogenic effects were assessed by determining the ^99mTc clearance and by the carmine dye retention test followed by direct counting of the number of newly formed microvessels with laser scanning confocal microscopy.

Among the various angiogenic factors identified to date, AII and VEGF have attracted much attention as potent angiogenic factors. AII is a strong vasoconstrictor peptide and stimulator of endothelial proliferation and migration. AII stimulates the angiogenesis via AT₁ receptor (Stoll et al., 1995; Chung et al., 1996), and the intense angiogenic activity of AII was also demonstrated in the rat sponge model (Hu et al., 1996). AII has also been reported to be important in the development of collateral vessels (Fernandez et al., 1985) and in the angiogenesis in chick embryo chorioallantoic membrane (Le Noble et al., 1991). On the other hand, of the various VEGF species, the best characterized is the 165-amino-acid-long form (VEGF₁₆₅, Neufeld et al., 1994). VEGF₁₆₅ specifically acts on endothelial cells and induces angiogenesis in vivo (Neufeld et al., 1994, 1999). The overexpression of VEGF and its receptors has been reported to be associated with chronic inflammation, tumor growth (Kim et al., 1993), and diabetic retinopathy (Williams, 1998).

In the present experiment, the technique with ^99mTc demonstrated a simple method for a continuous monitoring of its clearance for long periods (60 min) with high reproducibility. Thus, a series of experiments was carried out at day 7 after sponge implantation, in which ^99mTc clearance was monitored at fixed time intervals. The clearance of ^99mTc from the sponge increased as a function of time after sponge implantation. Our results clearly show that AII and VEGF₁₆₅ increased the ^99mTc clearance and the number of new capillaries invading the sponge matrix. The clearance increased with time and the isotope began largely to accumulate in the kidney at 15 min and appeared in the bladder at 60 min, indicating an installation of a full circulation in the sponge (data not shown).

Our results showing that KR31372 inhibited the increases in ^99mTc clearance stimulated by AII and VEGF₁₆₅ were further confirmed by a vascular casting method incorporating carmine red dye in gelatin (Colville-Nash et al., 1995). The findings that the intravascular content of carmine red dye was significantly increased by AII as well as VEGF₁₆₅ reflect the intravascular space being fully functional. The results that AII and VEGF₁₆₅-induced accumulation of carmine red dye was significantly diminished by KR31372 were fully consistent with those by ^99mTc clearance. Furthermore, the derived vascularity index was reduced under pretreatment with KR31372 because the dry mass was modestly changed. More interestingly, 1.0 mg/kg KR31372 caused a marked reduction in the dye content and vascularity index by 33.4 and 59.8%, respectively (P < 0.001), suggestive of its inhibition of the sponge-induced basal angiogenesis.

Our morphometric analysis showed that AII and VEGF₁₆₅ significantly increased the number of new vessels that are composed mainly of sizes 20 to 29, 30 to 39, and 40 to 49 µm in diameter. The results showing that the new vessel formation was markedly suppressed by treatment with KR31372 further indicate its antiangiogenic effect. Levcromakalim, an ATP-sensitive K⁺ channel opener, also inhibited AII-induced increased new vessel formation, but to a lesser degree than KR31372. Nevertheless, it showed marginal suppression of the increased clearance of ^99mTc by AII. A further study is required to explain why levcromakalim showed a relatively weak antiagiogenic effect compared with KR31372.

In the present study, treatment with glibenclamide, an ATP-sensitive K⁺ channel blocker, reversed the clearance of ^99mTc that was suppressed by KR31372. A number of studies have reported that K⁺ channel openers exert an increase in K⁺ conductance via activation of K⁺ channels, leading to hyperpolarization of the plasma membrane and consequently inhibiting activation of voltage-sensitive Ca²⁺ channels (Hamilton et al., 1986). Kohn et al. (1995) emphasized a role of Ca²⁺-mediated signal transduction in basic fibroblast growth factor-stimulated proliferation and invasion of the endothelial cells. Thus, one explanation is that KR31372 may inhibit the angiogenesis by inhibition of the Ca²⁺ influx through voltage-sensitive Ca²⁺ channels as reported by Kaneko et al. (1992). They addressed that nicardipine, a Ca²⁺ channel blocker, inhibited the migration of endothelial cells and tube formation. Kumamoto et al. (1999), however, reported the contrasting results, in that the angiogenesis was increased by amlodipine, a Ca²⁺ channel blocker, in the ischemic myocardium in association with increased expression of mRNA for VEGF. However, they did not address the relationship between the expressions of VEGF and amlodipine. At the present time, these relationships remain to be elucidated.

Taken together, oral administration of KR31372 strongly suppressed the AII- and VEGF₁₆₅-induced neovascularization, at least in part, via an activation of glibenclamide-sensitive K⁺ channels. It is suggested that this agent may have a therapeutic potential in the angiogenic disorders such as tumor growth, atherosclerosis, and diabetic retinopathy.