The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The rat mesenteric angiogenesis assay was used as previously described. In brief, Male Wistar rats (~350 g) were anaesthetized by inhalation of halothane and body temperature maintained at 37°C. A laparotomy was performed and part of the intestine exposed. A mesenteric panel was exposed under an intravital microscope (Leica DMIL). The panel was imaged using a Leica DC350F (Leica, Bucks, UK) digital camera and 25 μL of virus (108
plaque forming units) injected into the nearby fat pad with a Hamilton syringe fitted with a 30-gauge needle. Ad-VEGF-C18
or adenovirus expressing enhanced green fluorescent protein (EGFP), Ad-EGFP (a kind gift of J Uney, University of Bristol), was used. Ten microlitres of Monastral blue (0.6%, diluted in saline) was injected into the fat pads on either side of the virus-injected panel. The intestine was gently put back into the cavity and the animal sutured and recovered. After 14 days (day 14), the animal was re-anaesthetized with halothane, and a laparotomy performed. The mesentery was exposed and the virus injected panel located from Monastral blue injection sites. The mesenteric panel was imaged as described above.
Mesenteric panels with a clear lymphatic vessel as identified by intravital microscopy were chosen for the lymphangiogenesis model. If these panels were injected with Ad-VEGF-C they were termed VEGF-C (L+). To determine any diverse effects of VEGF-C on angiogenesis in the absence of lymphatic vessels, panels not containing pre-existing lymphatics were injected and these were termed VEGF-C (L−). EGFP was used as a control. Animals were injected on day 1, and the mesentery excised and the animal sacrificed on day 14. Five random images were taken of lymphatic vessels, and of regions of the mesentery that had no lymphatic drainage. To calculate lymphatic density low power immunofluorescent images were taken.
The panels were washed with 0.5% Triton-X100 in PBS (0.5% PBS-T), for 1 h, changing solutions every 10 min at room temperature. The panels were blocked in 1% BSA-0.5% PBS-T 1 h. The mesentery was incubated overnight at 4°C, with biotinylated Griffonia simplicifolia isolectin B4 (IB4, Molecular Probes, Cambridge, UK) 10 μg/ml and mouse monoclonal antibodies to Ki-67 (Novocastra Lab, Newcastle upon Tyne, UK, NCL-L-Ki67-MM1), rabbit anti-mouse lymphatic vessel endothelial hyaluronic acid receptor (LYVE-1) (10 μg/μl, a kind gift from Dr David Jackson, University of Oxford), or goat-anti-mouse VEGFR-3 (1 μg/mL, R&D systems).
Panels were washed as before, and TRITC labelled streptavidin (1 μg/mL, S-870, Molecular Probes) and Alexa Fluor 488, goat-anti-mouse IgG (2 μg/mL, Molecular Probes) were used as secondary detection antibodies to Lectin and Ki67, respectively. A donkey-anti-goat Alexa Fluor 555 (2 μg/ml, Molecular Probes) was used to detect VEGFR-3, and sheep anti-rabbit IgG FITC conjugate (product F7515, 1:160, Sigma) was used to detect LYVE-1. Primary antibodies and lectin were incubated overnight, on a rocker at 4°C. The panels were washed for 1 h, changing the PBS-T solution six times and secondary incubation lasted 2 h, at room temperature on a rocker. The panels were washed a further six times and if appropriate Hoechst 33324 (1 μM, Molecular Probes) was added to stain mesenteric nuclei.
The panels were carefully manipulated using fine forceps and flattened on a glass slide before being mounted in Vectashield (Vector Lab, Peterborough) and a coverslip was carefully placed on the tissue. The tissue was imaged using Leica Confocal Microscope (Leica Confocal TCS-NT DMIRBE). Five random sections of each panel were imaged and analysed off line at ×40 magnification with oil immersion. Overlaying the images permitted the quantification of vessel phenotype and the presence of proliferating cells in either vessel type. Intravital microscopy was used to image the increase in functional blood vessel area (FVA). ELISA using antibodies raised against human VEGF-C (R&D Systems, DVEC00) was used to determine effective gene transfer by Ad-VEGF-C.
Vessel parameters were measured as previously described.19,20
Briefly, for each mesentery, 8-12 views were selected randomly using a ×40 objective and Openlab software (Improvision, Coventry, UK) used to measure vessel parameters. The total blood (isolectin positive, LYVE-1 negative) or lymphatic vessels (Lyve-1 positive) were counted and labelled, and branch points, proliferating endothelial cells, and sprouts in each image counted. The diameter and length of each vessel were measured. Branch point density, sprout density, and proliferating endothelial cell density were calculated as the number per unit area within five randomly selected fields of view (×40 objective) containing vessels as previously described.19,20
Blood vessels were classified into two groups: <16 μm (exchange vessels) and >16 μm (conduit or resistance vessels-generally arterioles and venules).
All data are presented as mean ± SEM. EGFP blood vessel data n = 12, VEGF-C (L−) n = 11 and VEGF-C (L+) n = 7 except Ki67 data where EGFP n = 8, VEGF-C n = 4. All lymphatic EGFP n = 9, VEGF-C n = 9 except for Ki67 data where EGFP n = 6. All data were analysed by either Student’s t-test or student Newman-Keuls where appropriate. P < 0.05 was considered statistically significant.