Evidence for the importance of angiotensin II type 1 receptor in ischemia-induced angiogenesis (original) (raw)

Animals. To obtain AT1a-deficient heterozygous (AT1a+/–) mice that have C57BL/6 background, a germline chimera derived from TT2 embryonic stem cells with a targeted mutation of the AT1a gene as described previously (16) was backcrossed for five generations with C57BL/6 mice (18). The resulting AT1a+/– F5 mice were then intercrossed to generate the homozygous (_AT1a_–/–) mice (16, 18). The _AT1a_–/– mice were then inbred to obtain an appropriate number of animals for the present study. As wild-type (WT; AT1a+/+) mice, C57BL/6-strain mice were obtained from Clea Japan Inc. (Tokyo, Japan). Male mice at the age of 8–10 weeks were used.

Mouse model of angiogenesis. The study protocols were approved by the Institutional Animal Care and Use Committee of Kurume University School of Medicine. We used a mouse model of angiogenesis, in which the entire left femoral artery and vein were excised surgically (10, 19). When hindlimb ischemia was induced, new blood vessels grew into the ischemic limb (10, 19). We prepared this model in _AT1a_–/– mice and WT mice and employed various pharmacological agents to determine whether ischemia-induced angiogenesis was affected by the deficiency of the AT1a receptor. In brief, mice were subjected to unilateral hindlimb ischemia under anesthesia with sodium pentobarbital (50 mg/kg intraperitoneally) (10). Before surgery and on postoperative days 3, 7, 14, 21, 28, and 35, body weight and SBP were determined. SBP was determined using a tail-cuff pressure analysis system (TK370C; UNICOM, Tokyo, Japan) in the conscious state. Capillary angiogenesis, collateral vessel formation (arteriogenesis), and limb blood flow were examined by the following methods.

Laser Doppler blood flow analysis. We measured hindlimb blood flow using a laser Doppler blood flow (LDBF) analyzer (Moor LDI; Moor Instruments, Devon, United Kingdom) as described previously (10). Before and on postoperative days 3, 7, 14, 21, 28, and 35, we performed LDBF analysis over the legs and feet. Blood flow (i.e., blood cell movement) was displayed as changes in the laser frequency using different color pixels. After scanning, the stored images were analyzed to quantify blood flow, and mean LDBF values of the ischemic and nonischemic legs were calculated. To avoid data variations due to ambient light and temperature, hindlimb blood flow was expressed as the ratio of left (ischemic) to right (nonischemic) LDBF.

Angiographic score. On postoperative day 14, under pentobarbital anesthesia (50 mg/kg intraperitoneally), the peritoneal cavity was opened, and a 26-gauge soft-tip catheter was inserted through the abdominal aorta. The lower limbs were gently perfused with 0.5 ml of warm saline containing heparin (10 U/ml). Postmortem angiography was then performed by injecting 0.3 ml of contrast media through the catheter at a perfusion pressure of 80–90 mmHg. X-ray angiograms were taken using a mammography system (Senographe 500T; GE Medical Systems, Paris, France), and the extent of collateral vessel formation was quantified by angiographic score as described previously (20).

Capillary density. Capillary density within the ischemic thigh adductor skeletal muscles was analyzed to obtain specific evidence of vascularity at the level of microcirculation. Three pieces of ischemic muscles were harvested from each animal, sliced, and fixed in methanol. Tissues were embedded in paraffin, and multiple tissue slices, 5 μm in thickness, were prepared. Capillary endothelial cells (ECs) were identified by immunohistochemical staining with a rat anti-mouse CD31 mAb (PharMingen, San Diego, California, USA). Fifteen random microscopic fields from three different sections in each tissue block were examined for the presence of capillary ECs, and capillary density was expressed as the number of capillaries per high power field (×400).

Effects of hydralazine on ischemia-induced angiogenesis in WT mice. _AT1a_–/– mice had lower SBP than WT mice (16, 17). We thus examined whether the lower SBP itself influenced ischemia-induced angiogenesis. We examined the effects of the reduction of SBP by hydralazine (Novartis Pharmaceuticals Corp., Tokyo, Japan) on angiogenesis in nine WT mice. SBP was reduced by oral hydralazine (50 mg/kg/d) in WT mice; our preliminary experiments showed that the SBP in WT mice treated with this dose of hydralazine was similar to that of _AT1a_–/– mice. On day 10 of hydralazine treatment, hindlimb ischemia was induced, and the ischemic/normal LDBF ratio was examined up to 35 days after surgery.

Effects of TCV-116 on ischemia-induced angiogenesis in WT mice. We examined whether selective pharmacological blockade of the AT1 receptor by candesartan cilexetil (TCV-116; Takeda Chemical Industries, Osaka, Japan) (12, 13, 21) in WT mice mimicked the altered angiogenic response observed in _AT1a_–/– mice. TCV-116 is a specific AT1 receptor antagonist, and its AT1-binding affinity is 80 times greater than that of losartan and 10 times greater than that of EXP 3174, the active form of losartan (12, 13). TCV-116 has been used to block the major vascular effects of ATII in previous in vivo studies (21–23). In preliminary experiments, we examined the dose-response effects of oral TCV-116 on SBP in WT mice. We used 5 mg/kg/d of TCV-116, because this dose reduced SBP in WT mice to a level similar to that of _AT1a_–/– mice. TCV-116 was administered via drinking water to 15 WT mice. On day 10 of the administration, mice were subjected to hindlimb ischemia, and ischemic/normal LDBF ratios were examined up to 35 days after surgery.

Effects of PD123319 on ischemia-induced angiogenesis in AT1a–/– mice. To further test the biological role of ATII in the ischemia-induced angiogenesis, we examined the effects of PD123319, a selective AT2 receptor antagonist, on angiogenesis in _AT1a_–/– mice with hindlimb ischemia. PD123319 has been used to block the biological effects of the ATII–AT2 receptor pathway in various in vivo experiments. We chose the dose of PD123319 (30 mg/kg/d) that effectively suppressed AT2 receptor function in previous studies (24). PD123319 was administered using an osmotic pump (ALZA Corp., Palo Alto, California, USA) to five _AT1a_–/– mice, which were then subjected to surgical hindlimb ischemia. Ischemic/normal LDBF ratios were examined up to 35 days after surgery.

Histological analysis of the inflammatory responses. ATII has been shown to play a proinflammatory role (25–27). We thus examined whether the extent of inflammatory reactions in the ischemic tissues differed between WT and _AT1a_–/– mice. Five-micrometer-thick multiple sections prepared from paraffin-embedded tissues of the ischemic limbs were used for histological analysis. Leukocyte infiltration was examined by hematoxylin and eosin (H&E) staining as well as immunohistochemical staining using an anti-mouse CD45 mAb (PharMingen), a common leukocyte antigen.

VEGF expression in inflammatory mononuclear cells. Inflammatory mononuclear cells (MNCs), mainly macrophages and T lymphocytes, release various angiogenic cytokines including VEGF (19, 28). Therefore, we also examined VEGF expression in MNCs in ischemic tissues using a double immunofluorescence staining technique. Cryostat sections, 5 μm in thickness, from ischemic tissues were mounted on silicone-coated slides. They were then incubated overnight at 4°C with an anti-mouse VEGF mAb (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA), and with either rat anti-mouse F4/80 mAb or rat anti-mouse CD3 mAb (Serotec Inc., Raleigh, North Carolina, USA) in a moist chamber. The slides were then incubated for 30 minutes at 37°C with an FITC-conjugated anti-goat IgG secondary antibody (Zymed Laboratories Inc., South San Francisco, California, USA) to detect VEGF. Then they were further incubated for 30 minutes at 37°C with TRITC-conjugated anti-rat IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, Pennsylvania, USA) to detect macrophages or T lymphocytes. The slides were examined and photographed using a laser confocal microscope (Noran Instruments Inc., Middleton, Wisconsin, USA). The infiltrated macrophages, T lymphocytes, and VEGF-positive leukocytes per high power field (×400) were then counted.

Western blot analysis. Protein extracts were obtained from homogenized ischemic skeletal muscles. One hundred micrograms of protein per sample was separated on a 12.5% polyacrylamide gel and electroblotted onto PVDF membranes (Trans-blot; Bio-Rad Laboratories Inc., Hercules, California, USA). The membrane was blocked with 10% nonfat dry milk in PBS with 0.2% Tween (T-PBS) and then probed with 1:100 of a goat polyclonal anti-mouse VEGF antibody (clone P-20) or with 1:100 of a goat polyclonal anti-mouse monocyte chemoattractant protein-1 (MCP-1) (clone M-18; Santa Cruz Biotechnology Inc.) for 3 hours at room temperature. After incubation with the primary antibody, the membrane was washed three times in T-PBS and then incubated for 1 hour with 1:5000 of anti-goat IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology Inc.). The membrane was then washed in T-PBS, and antigen-antibody complexes were visualized using an enhanced chemiluminescence kit (ECL; Amersham Biosciences, Buckinghamshire, United Kingdom) at room temperature, followed by exposure to x-ray films (Hyperfilm; Amersham Biosciences).

Isolation of peripheral blood MNCs from WT or AT1a–/– mice and implantation into the ischemic hindlimb of AT1a–/– mice. Peripheral blood was obtained from WT and _AT1a_–/– mice. MNCs were isolated by centrifugation through a Histopaque-1083 density gradient (Sigma Chemical Co., St. Louis, Missouri, USA). In 14 _AT1a_–/– mice, left hindlimb ischemia was induced, and WT mouse–derived MNCs (4 × 105 cells per animal, n = 8) or _AT1a_–/– mouse–derived MNCs (4 × 105 cells per animal, n = 6) were implanted into the thigh adductor muscles in the ischemic limb on postoperative day 3. In five additional mice, neutralizing anti-mouse VEGF mAb (R&D Systems Inc., Minneapolis, Minnesota, USA) was continuously administered by a subcutaneously implanted osmotic pump (ALZA Corp.), and ischemia-induced angiogenesis was examined in _AT1a_–/– mice that had been subjected to WT-derived MNC transplantation. The ischemic/normal LDBF ratio was examined up to 35 days after surgery.

Peritonitis model for in vivo leukocyte transendothelial migration assay. To examine the effects of the AT1a receptor deficiency on in vivo leukocyte transendothelial migration, we employed an additional inflammation model: a murine peritonitis model induced by injecting oyster glycogen (intraperitoneally). This is an established model used to examine the degree of leukocyte transendothelial migration in response to inflammation in vivo (29, 30). _AT1a_–/– (n = 10) and WT mice (n = 10) were lightly anesthetized with pentobarbital (30 mg/kg intramuscularly), and 2 ml of 0.1 vol% oyster glycogen type II (Sigma Chemical Co.) in sterile saline was injected into the peritoneal cavity. Four hours after the injection, the abdominal wall was cut and the peritoneal cavity was opened with special care to avoid blood contamination. The peritoneal cavity was washed with 2 ml of warm heparinized (10 U/ml) saline; then peritoneal fluid was collected and centrifuged at 400 g for 10 minutes. The pellets were resuspended in PBS (1 ml), and the total number of transmigrated leukocytes was calculated using a hemocytometer (K-800; Sysmex, Kobe, Japan).

Statistics. All values are presented as the mean ± SE. All data were subjected to ANOVA followed by Fisher’s analysis for comparison between any two means. Probabilities of less than 0.05 were considered to be statistically significant.