Figure 1.
Lodamin inhibits angiogenesis and inflammation in Matrigel plugs and inhibits corneal neovascularization after local or oral treatment.
(A) Matrigel containing VEGF and bFGF was injected subcutaneously (s.c., n = 5) to determine the effect of Lodamin on angiogenesis and macrophage infiltration. Upper panel shows representative plugs removed from Lodamin treated or untreated mice (bar = 1 cm). Bottom panel shows vessel staining with anti-CD31 antibody (brown) and nuclei staining with Hematoxilin Gill's (blue), bar = 100µm; arrows point to large vessel with open lumans (B) Quantification of infiltrating endothelial cell or macrophages in Matrigel plugs. Data are presented as percent of specific cell population out of the total cell population (n = 3–4, p<0.05). (C) Antiangiogenic activity of Lodamin following topical or systemic administration was evaluated in the mouse corneal micropocket assay using bFGF-induced neovascularization. Quantification of vessel area (mm2) in different corneal assays performed with Lodamin which were administered either orally (30 mg/kg q.d), by eye drops (30 mg/ml, q.d) or by subconjunctival injections (30 mg/ml, q.d). Graphs indicate the significant inhibition of vessel formation after 5 days of treatment. Vessel area was reduced by 38% after subconjunctival injection (P = 0.0002), by 30% after eye drops treatment (P = 0.003), and by 37% (P = 0.04) after oral administration. In each group n = 10. (D) Representative images taken from the different groups at day 5; bFGF pellet is detected as a white spot in the center of the cornea, blood vessels growing from limbal periphery are reduced in Lodamin treated group compared to controls. Vessel area in mm2 is calculated by the following formula: π×clock hours×vessel length (mm)×0.2 mm.
Figure 2.
Lodamin inhibits angiogenesis, inflammation and edema in the DTH reaction.
(A) Lodamin suppressed ear swelling in DTH reactions elicited by oxazolone, photos of representative ears of Lodamin treated or untreated mice are circled. Bar = 1 cm (B) Ear swelling is represented as change in thickness (in µm) compared to original ear thickness. Mice treated with Lodamin (white squares) showed a significant reduction in ear thickness compared to control mice (black squares). The differences were statistically significant from day 2 to day 12 post challenge, excluding day 5 (P<0.05). Data are presented as an average ±SEM, n = 5. (C) Immunohistological analysis of mouse ears post DTH reaction: H&E staining (a–f) and specific staining of endothelial cell marker CD-31 (g–h). In H&H staining, control ears exhibited an excessive inflammation and edema (a–d), including fibroblast proliferation (marked as F) and spongiosis pointed by arrows compared with Lodamin treated mice (e–f). Bar = 50µm.
Figure 3.
Oral treatment of Lodamin reduces CNV progression.
(A) CNV lesion size of Lodamin-treated mice compared to controls. A dose of 15 mg/kg/day was administered for 7 or 14 days and a dose of 30 mg/kg/day was administered for 7 days. CNV lesions in choroidal flat mounts were evaluated after staining of blood vessels using isolectin-IB4 conjugated with Alexa Fluor 488. Data are presented as percent of blood vessel area in choroidal flat mounts (pixels) of treatment per controls. Data are expressed as mean ± SEM, (U-test, * P<0.05, ** P<0.005, n = 10). (B) Representative images of CNV lesions stained with a lectin-FITC in flat mount of mouse choroids. Bar = 20 µm. (C) H&E stained histological side sections of CNV site in Lodamin treated or untreated mice. Substantial differences in fibrous tissues thickness and choroidal vessel invasion to CNV site are detected, Bars = 50 µm (D) FACS analysis of single-cell suspension originated from retinas after 3 and 7 days of oral Lodamin treatment. Quantification of macrophage infiltration in mouse retinas originated from Lodamin treated (30 mg/kg, daily, oral) or mice which were treated with equivalent dose of vehicle. Macrophage population was detected as a double positive CD45+ and F4/80+ staining.
Figure 4.
Lodamin injected intravitreal suppresses CNV progression.
(A) CNV lesions were induced by a diode pumped solid state laser around the optic nerve through a slit lamp delivery system. Only lesions in which a subretinal bubble or focal serous detachment of the retina developed were used for the experiments. Four burns were performed per eye while leaving a space around the optic disc. At the day of laser induction intravitreal injection of Lodamin was performed. Size of CNV lesions after intravitreal injections of Lodamin: 100 µg Lodamin per eye (12.5 µg TNP-470 equivalent) or 300 µg Lodamin per eye. Data is presented as mean pixel number ± SEM (n = 7–17, *P<0.05, U-test). (B) Representative images of retinal flat-mounts stained with a lectin-FITC, Bars = 10 µm.
Figure 5.
Comparison of the effect of Lodamin or sFlk-1 on choroidal vessel regression.
For CNV regression studies intravitreal injections of Lodamin or sFlk-1, a recombinant mouse VEGF R2/KDR Flk-1 Fc Chimera, were performed on established CNV lesions at day 7 post laser induction. After 14 days of laser induction (7 days post intravitreal injection of Lodamin or sFlk-1) fluorescent images of choroidal flat-mounts were captured and CNV area (presented in µm2) in choroidal flat mount was evaluated using Scion image software. (A) CNV size data of untreated mice bearing laser induced CNV at day 7 and 14, compared to Lodamin or sFlk-1 treated mice 7 days after their injection. Data is presented as mean pixel number ± SEM (n = 15–27 spots, *P<0.05, U-test).
Figure 6.
Retinal ERG and protein analysis post Lodamin treatments.
Effects of intravitreal Lodamin on retinal function. (A) Representative full-field flash ERG responses from the eyes of mice 14 days after intra-ocular injection of vehicle (left) or 300µg /eye Lodamin (right). (B) Representative responses to 8 Hz flicker (C) The 5th to 95th prediction limits for ERG parameters in vehicle treated rats are shown; the dashed line represents the normal mean of all the paramters. Symbols are parameters values of individual Lodamin-treated mice, and represent the same mouse in each column. Data are ΔLogNormal (eq. 3). Only the saturating amplitude of the rod photoresponse (RmP3) differed significantly after Lodamin. (D) Effect of oral Lodamin treatment on protein levels of pro-angiogenic and pro-inflammatory factors. (D) Levels of MCP-1 and VEGF extracted from whole eye tissues from naïve mice, mice treated with Lodamin (30mg/kg, oral every day for 7 d) or vehicle (same equivalent amount of mPEG-PLA micelles). Data of ELISA assay are presented as a mean of pg/ml concentration ± SD (n = 6, * P<0.05, t-test). Bottom: ELISA quantification of secreted MCP-1 from RPE to the medium post 24 h incubation with Lodamin at different doses (0.1–100 nM TNP-470 equivalent). (E) Western blot protein analysis of samples of tissue lysis of retina or choroid of the different treatment groups: N = naïve mice, V = vehicle treated mice, L = Lodamin treated mice. Factors detected are TNFα (26 kDa) (F) Zymogram showing MMP-2 and MMP-9 activity in ocular tissue following oral Lodamin treatment of mice (30 mg/kg/day 7 days).
Figure 7.
Lodamin reduces vessel permeability in the modified Miles assay and in retinal angiography.
(A) Left: quantification of modified Miles assays performed in mice after induction of vessel permeability using MCP-1 (50 pg) and VEGF (50 ng); extracted dye contents were quantified by measuring at 620 nm. Data are expressed as mean ± SEM (n = 10, *P<0.05, t-test). (B) Representative photos of mouse skin showing diminished dye in mice treated with oral Lodamin 1 and 3 h before conducting the assay. MCP-1 induces vessel permeability in a dose dependent manner, already at 10 pg/ml, MCP-1 induced significant vessel leak compared with PBS. (C) Fluorescein angiography of CNV lesions of rats treated with Lodamin or vehicle. Fluorescein angiography was performed at day 7 after laser photocoagulation and Lodamin was administered 1 and 3 h before performing the imaging. (D) The percentage of lesions graded as 0, I, IIA, defined as no leakage to moderate leakage, and IIB, considered clinically relevant leakage, in vehicle-treated (n = 6 eyes). A significant reduction of vessel leak was observed after an acute treatment of Lodamin. (E) Lodamin reduces whole eye vessel permeability as determined by Evan's blue extraction method. Mouse eyes treated with Lodamin (3 h before conducting the assay) were 65% less than the control. Data are presented as mean ± SD (n = 5, *P<0.05, t-test). (F) Accumulation of 6-coumarin labeled Lodamin injected i.v at the site of CNV lesion in mouse compared to an injection of free 6-coumarin. In red: blood vessels stained with rhodamine concanavalin A, in green: 6-coumarin dye. Bar = 20 µm. (G) Bioditribution of 6-coumarin labeled Lodamin (green) in mouse retina side sections 1 day or 7 days post intravitreal injection. 10 µm Section were imaged by confocal microscopy using optical sections with 488-nm argon, diode lasers. Bar = 20 µm.