- Email: [email protected]
Therapeutic site selection is important for the successful development of collateral vessels
Therapeutic site selection is important for the successful development of collateral vessels Ayako Nishiyama, MD, PhD,a Hiroyuki Koyama, MD, PhD,a,b Tetsuro Miyata, MD, PhD, Prof,a and Toshiaki Watanabe, MD, PhD, Prof,c Tokyo, Japan Background: Induction of collateral development to improve tissue perfusion is a promising approach for the treatment of arterial occlusive diseases. Several growth factors and cells have been reported to increase collateral circulation; however, the appropriate site for the delivery of these factors and cells is unclear. In this study, we identified the delivery site for growth factor in a rabbit model of limb ischemia and evaluated whether specific delivery of basic fibroblast growth factor (bFGF) to this site enhanced collateral augmentation. Methods: The left femoral artery of Japanese white rabbits was excised to induce limb ischemia. Twenty-eight days thereafter, angiograms were obtained to identify the typical pattern of collateral development in this model. Subsequently, bFGF (100 mg) was selectively injected into the left coccygeofemoral muscle (coccygeo group) or adductor muscle (adductor group), major thigh muscles in proximity. Collateral development was evaluated at 28 days after injection, and its mechanism was assessed by immunologic and morphometric analyses of muscle samples. Results: Angiographic evaluation of this model revealed that after femoral artery excision, collateral vessels generally developed in the left coccygeofemoral muscle, whereas few collateral vessels were detected in the left adductor muscle. At 28 days after injection, calf blood pressure ratio, defined as left pressure to right pressure, was significantly higher in the coccygeo group than in the adductor group (0.85 6 0.05 vs 0.69 6 0.05, respectively; P < .01). Similar results were observed in blood flow through the internal iliac artery (resting: 24.6 6 6.1 vs 17.4 6 8.0 mL/min, P < .01; maximum: 47.4 6 12.3 vs 33.2 6 10.7 mL/min, P < .01) and in the angiographic score (0.67 6 0.13 vs 0.39 6 0.11; P < .01). Immunologic analyses of the coccygeofemoral muscle at day 3 showed marked expressions of Ki-67, monocyte chemotactic protein 1, and FGF receptor 1 in the coccygeo group compared with the adductor group. Morphometric analyses of the same muscle at day 14 also revealed that collateral vessel density and wall thickness were significantly increased in the coccygeal group compared with the adductor group. Conclusions: These findings demonstrated that selective bFGF delivery to the coccygeofemoral muscle markedly improved collateral development and limb perfusion compared with delivery to the adductor muscle, suggesting that site selection is important in increasing therapeutic efficacy. (J Vasc Surg 2014;-:1-10.) Clinical Relevance: Several clinical trials of angiogenic therapy for the treatment of peripheral arterial diseases have been conducted. In these trials, angiogenic factors or cells were delivered by intra-arterial or intramuscular injection. However, wide dispersion of the delivered substances to distal areas occurs with intra-arterial injections, and intramuscular injections usually involve broad, multiple injections to the ischemic limb; therefore, both methods might fail to achieve appropriate target site delivery for collateral development. On the basis of the conclusions of this study, accurate delivery of angiogenic substances to the appropriate site could markedly enhance the therapeutic efficacy of human angiogenic therapy.
Induction of angiogenic reactions to improve tissue perfusion is a promising approach for the treatment of arterial occlusive diseases, and the most important mechanism of angiogenic therapy is considered to be the enhancement of collateral circulation to the ischemic lesion. Although a variety of strategies for effective angiogenic therapies have From the Division of Vascular Surgery, Department of Surgery, Graduate School of Medicine, The University of Tokyoa; the Division of Translational Research Center, The University of Tokyo Hospitalb; and the Department of Surgery, Graduate School of Medicine, The University of Tokyo.c Author conflict of interest: none. Reprint requests: Hiroyuki Koyama, MD, PhD, Division of Vascular Surgery, Department of Surgery, Graduate School of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214/$36.00 Copyright Ó 2014 by the Society for Vascular Surgery. http://dx.doi.org/10.1016/j.jvs.2014.02.004
been presented, the basic concept in most of these strategies is the local delivery of bioactive factors or cells that potentially promote angiogenic processes in vivo.1-4 Previous studies have attempted to deliver several bioactive factors or cells in animal models of ischemia and reported favorable therapeutic effects of some growth factors or cells, such as basic fibroblast growth factor (bFGF),5-7 vascular endothelial growth factor,2,8-11 and bone marrow mononuclear cells.12-14 Those studies have successfully identified growth factors or cells that are adequate for delivery substances in angiogenic therapy; however, the appropriate site for such factors or cells is currently unclear. Recently, several clinical trials testing angiogenic therapies have been carried out.15-19 In most of these trials, angiogenic factors or cells were delivered by intra-arterial injection or intramuscular injection. When angiogenic substances are administrated by an intraarterial injection, they disperse widely to areas distal to the injection site and might fail to specifically target areas that are appropriate for collateral vessel formation. When substances are delivered by intramuscular injection, the 1
2 Nishiyama et al
procedure is usually broad, multiple injections to the muscles of the ischemic limb, which might also increase the possibility of irrelevant delivery. Indeed, large-scale clinical trials testing these substances have shown limited therapeutic efficacy.15-19 We hypothesized that one reason for poor outcome might be the potential inefficiency of the delivery. If the delivery site for angiogenic therapy can be clearly identified, it might be possible to notably increase the therapeutic efficacy by specific delivery to this region. The purpose of this study was to identify the therapeutic site for injection of the growth factor for the most effective development of collateral vessels in a rabbit model of chronic limb ischemia. METHODS Animal model of chronic limb ischemia. This study used a rabbit model of chronic limb ischemia to evaluate the development of collateral vessels. Reliable methods to evaluate the perfusion of rabbit limb have been previously reported, and several previous studies testing angiogenic therapies have used the rabbit model of limb ischemia.2,6,20 Male Japanese white rabbits weighing 2.5 to 3.0 kg (Saitama Rabbitry, Saitama, Japan) were anesthetized with an intramuscular injection of a mixture of ketamine (50 mg/kg) and xylazine (2.5 mg/kg). The left femoral artery was completely excised from its proximal origin to the bifurcation formed by the saphenous and popliteal arteries. At 28 days after femoral artery excision, the left hind limb of the rabbits developed chronic ischemia.6,7,10,20 All protocols conformed to the Guide for the Care and Use of Laboratory Animals (NIH publication No. 84-23, revised 1996). Anatomic analyses of collateral development in the rabbit model of limb ischemia. To clarify the anatomic pattern of collateral development in the rabbit model of limb ischemia, we carried out aortic angiography at 28 days after femoral artery removal. Immediately after an overdose injection of pentobarbital, an 18-gauge polyethylene infusion catheter (Terumo, Tokyo, Japan) was introduced into the abdominal aorta in the distal direction, and a mixed solution of lead oxide (20 g; Wako, Tokyo, Japan), gelatin (0.3 g; Wako), and water (10 mL) was injected. Because lead oxide is a radiocontrast reagent and has a vivid orange color, the arteries containing the mixed solution are recognizable under a fluoroscope and also visible from the outside when the arteries are exposed. With the fluoroscopic image as reference, we dissected the collateral vessels by using the orange color as a guide and analyzed the anatomic pattern of collateral development in this model. This experiment was repeated three times. Administration of bFGF. To investigate if the delivery site of bFGF influences the development of collateral vessels in the rabbit model of chronic limb ischemia, we injected bFGF (trafermin, human recombinant bFGF; Kaken Pharmaceutical, Tokyo, Japan) in either the left coccygeofemoral muscle (coccygeo group) or the left adductor muscle (adductor group) at 28 days after femoral artery removal, when a stable, chronic state of limb ischemia has developed.21,22
JOURNAL OF VASCULAR SURGERY --- 2014
The left coccygeofemoral muscle is the region where the collateral vessels are expected to develop in this model, and the location of this muscle could be identified through the buttock skin because the outline of it appears clearly by moistening of the skin. In contrast, few collateral vessels are expected to develop in the left adductor muscle. In the coccygeo group, 100 mg of bFGF in 100 mL of phosphatebuffered saline (PBS) was injected into three different sites in the left coccygeofemoral muscle, and 100 mL of PBS alone was injected into three different sites in the left adductor muscle. In the adductor group, 100 mg of bFGF in PBS was injected into three different sites in the adductor muscle, and 100 mL of PBS alone was injected into three different sites in the coccygeofemoral muscle. The dose of bFGF (100 mg) was chosen on the basis of previous studies in which bFGF protein was administered for the treatment of limb ischemia.6,16 As a control, another set of rabbits with chronic limb ischemia received 100 mL of PBS in both the coccygeofemoral muscle and adductor muscle (vehicle group). Evaluation of collateral development. Collateral vessel development was evaluated at 28 days after the administration of bFGF/PBS (coccygeo group, n ¼ 9; adductor group, n ¼ 9; vehicle group, n ¼ 10), a period evaluated in previous studies with the rabbit model.6,7,20 First, the calf blood pressure was measured in both hind limbs, and the calf blood pressure ratio, defined as the ratio of left systolic pressure to right systolic pressure, was calculated. This measurement was also carried out immediately before the administration of bFGF/PBS. After the measurement of calf blood pressure, a 3F end-hole catheter was introduced into the left iliac artery through the carotid artery, and a 0.014-inch Doppler guidewire (EndoSonics, Rancho Cordova, Calif) was introduced through the 3F catheter to the proximal part of the left internal iliac artery. The average peak velocity was measured at rest, and then the maximum average peak velocity was determined after the injection of 2 mg of papaverine (Dainippon Pharmaceutical, Osaka, Japan). In vivo blood flow was calculated as previously described.6,7,20 Angiograms were then taken, and the angiographic score was determined as described previously.6,7,20,21 Briefly, angiographic score was determined by a grid overlay composed of 2.5-mm-diameter circles arranged in rows spaced 5 mm apart. This overlay was placed over the angiogram, recorded for 4 seconds, at the level of the medial thigh. The number of contrast-opacified arteries crossing over the circles (CA) and the total number of circles encompassing the medial thigh area (CT) were counted. The angiographic score was calculated for each film as CA/CT. Analyses of coccygeofemoral muscle during collateral development. To study the mechanism of collateral development after bFGF administration, we analyzed the histologic findings and protein expression in the coccygeofemoral muscle at sequential time points after bFGF injection. At 28 days after the artery excision, 100 mg of bFGF in PBS or PBS alone was injected into the left coccygeofemoral muscle and adductor muscle according to the
JOURNAL OF VASCULAR SURGERY Volume -, Number -
respective protocol for the coccygeo group, adductor group, or vehicle group. The rabbits in each group were sacrificed at 3, 7, and 14 days after the administration (n ¼ 4 at each time point and in each group). An infusion catheter was introduced into the abdominal aorta, and 200 mL of lactated Ringer solution was infused at 120 mm Hg. In the rabbits sacrificed at 3 and 7 days after bFGF/PBS injection, the left coccygeofemoral muscle was excised and divided transversely into eight parts with equal thickness. The fifth part from the muscle origin was embedded in optimal cutting temperature (OCT) compound (Miles Scientific, Naperville, NJ) for histologic analysis, and the sixth part was snap-frozen in liquid nitrogen for protein lysate extraction. The samples were stored at 80 C. Meanwhile, the animals that were sacrificed at day 14 received an additional perfusion of phosphatebuffered 4% paraformaldehyde at 120 mm Hg, and the left coccygeofemoral muscle was divided into eight parts in the same manner. Then, the fifth part from the muscle origin was embedded in paraffin. Histologic analyses. Transverse histologic sections (4-mm-thick) were cut from OCT-embedded muscle samples excised from rabbits at 3 and 7 days after injection and were immune stained for Ki-67, monocyte chemotactic protein 1 (MCP-1), and FGF receptor 1 (FGFR-1). Ki-67 is a nuclear protein associated with cell proliferation, and MCP-1 and FGFR-1 are considered to play important roles in collateral development.4,7,11,23,24 Previous studies demonstrated that Ki-67, MCP-1, and FGFR-1 were markedly expressed in and around collateral vessels in the early days after induction of collateral development.23-25 After hydrogen peroxide treatment and blocking by 1.5% horse serum, monoclonal antibody against Ki-67 (1:100; Dako, Glostrup, Denmark), goat polyclonal antibody against MCP-1 (1:100; Santa Cruz Biotechnology, Santa Cruz, Calif), or monoclonal antibody against FGFR-1 (1:100; QED Bioscience, San Diego, Calif) was applied, and samples were incubated for 60 minutes at 37 C. Subsequently, biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, Calif) or rabbit anti-goat IgG (Vector Laboratories) was applied, and then analysis was performed with the ABC Elite Kit (Vector Laboratories). Photomicrographs were taken, and the specimen area was measured in each section by the ImageJ software (version 1.42q; National Institutes of Health, Bethesda, Md). In addition, Ki-67-positive cells, MCP-1-positive cells, and FGFR-1positive vessels were counted in each section, and parameters were calculated as follows: Ki 67 or MCP 1 positive cell density =mm2 ¼ Ki 67 or MCP 1 positive cell number=specimen area FGFR 1 positive vessel density =mm2 ¼ FGFR 1 positive vessel number=specimen area In addition, 4-mm-thick sections were cut from the paraffin-embedded muscle samples obtained from animals
Nishiyama et al 3
at 14 days after bFGF/PBS injection and used to analyze the morphometry of developing collateral vessels.22,26 The sections were stained with hematoxylin and eosin, and photomicrographs of each section were taken and assessed with the ImageJ software. The entire specimen area in each section (specimen area) was measured, and the number of arteries with diameters of 50 to 250 mm (arteries50-250) was counted. The diameters were measured from several directions, and the lowest value was used. Arteries50-250 correspond to typical collateral vessels that are imaged by angiography in this model.22,26 Although arteries50-250 are not always developed collateral vessels, most collateral vessels range from 50 to 250 mm in diameter, as newly formed collateral vessels generally develop from arterioles.22,26 The density of arteries50-250 was calculated as follows: Arterie50250 density =mm2 ¼ number of arteries50250 =specimen area in mm2 In addition, the minimum thickness of the arterial walls was measured in all arteries50-250 in each section, and mean values were calculated in the respective sections (arteries50-250 wall thickness). Immunoprecipitation and Western blot analyses. To assess the expression of FGFR-1, frozen muscle samples, excised from animals at 3 and 7 days after injection, were lysed in lysis solution (20 mmol/L Tris [pH 7.5], 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L b-glycerophosphate, and protease inhibitors). The muscle lysate (100 mg total protein) was diluted in 1 mL of lysis solution and treated with monoclonal antibody against FGFR-1 (5 mL; QED Bioscience) at 4 C for 16 hours after precleaning with protein AeSepharose (Invitrogen, Carlsbad, Calif). Immune complexes were recovered with protein GeSepharose (GE Healthcare, Uppsala, Sweden) and washed with lysis solution. Samples were separated on sodium dodecyl sulfateepolyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane. The blot was immunostained with a monoclonal antibody against FGFR-1 (1:500; QED Bioscience) or a monoclonal antibody against phosphorylated tyrosine (1:500, PY99; Santa Cruz Biotechnology) and visualized with an ECL Plus system (GE Healthcare), as described previously.7 We also evaluated the bFGF level in the coccygeofemoral muscles after injection, as described previously.7 Briefly, frozen muscle samples (0.5 g) were lysed in 1 mL of minimum essential medium (Invitrogen), and bFGF in each lysate was concentrated with heparineSepharose CL-6B (GE Healthcare). Samples were then analyzed by Western blot with a monoclonal antibody against bFGF (1:500; Upstate Biotechnology, Lake Placid, NY). Statistical analysis. All measurements were carried out in a blinded fashion. Results are expressed as mean 6 standard deviation. Statistical significance was evaluated by the Tukey-Kramer method for multiple comparisons. All data were considered significant at P < .05.
JOURNAL OF VASCULAR SURGERY --- 2014
4 Nishiyama et al
RESULTS
Fig 1. The anatomic pattern of collateral development in the rabbit model of limb ischemia was analyzed by aortic injection of a lead oxide solution at 28 days after excision of the left femoral artery. a, Aortic angiogram. Collateral vessels (open arrowheads) developed between the left posterior gluteal artery and the left popliteal artery. b, Photograph showing orange collateral vessels (open arrowheads) in the coccygeofemoral muscle. Note that collateral vessels are considerably larger than the right posterior gluteal artery (closed arrowheads). c, Schematic diagram of arterial anatomy in the coccygeofemoral muscle of the rabbit model. d, Schematic diagram of arterial anatomy in the adductor muscle of the rabbit model. Animals in all images are in the prone position. *Developed collateral artery. yTotal removal of the left femoral artery. zNormal size branch of posterior gluteal artery. xNormal size branch of deep femoral artery. **The branch of the
Anatomic pattern of collateral development in the rabbit model of limb ischemia. In all analyzed rabbits (n ¼ 4), the main route of collateral vessels tended to develop between the left posterior gluteal artery and the left popliteal artery, which is located in the coccygeofemoral muscle (Fig 1). Furthermore, the most evident enlargements in collateral images were observed from the middle to the distal part of the muscle, which corresponded to the sampling parts for histologic and protein analyses in the present study. In contrast, little development of collateral vessels was observed in the left adductor muscle. Evaluation of collateral vessel development. The calf blood pressure ratio measured at 28 days after injection of bFGF/PBS showed significant improvement in the coccygeo group compared with the adductor and vehicle groups, whereas there was no significant difference in data before the injection (Fig 2, a). In addition, the calf blood pressure ratio in the adductor group was significantly better than that of the vehicle group. The resting blood flow and the maximum blood flow of the left internal iliac artery demonstrated a significant increase of blood perfusion in the coccygeo group compared with the adductor and vehicle groups (Fig 2, b). Although the resting blood flow in the adductor group was significantly higher than that in the vehicle group, no significant difference was detected between the maximum blood flows of the two groups. Selective internal iliac angiography at 28 days after bFGF/PBS injection demonstrated marked collateral development in the coccygeo group, whereas few collateral vessels were observed in the adductor and vehicle groups (Fig 2, c-e). The angiographic score was significantly higher in the coccygeo group than in the adductor and vehicle groups; however, no significant difference was detected between the scores of the adductor and vehicle groups (Fig 2, f). Immunohistologic analyses. At 3 days after the bFGF/PBS injection, Ki-67-positive cells, MCP-1-positive cells, and FGFR-1-positive vessels were markedly observed in the coccygeofemoral muscle of the coccygeo group compared with the adductor and vehicle groups (Fig 3, a-i), and quantitative analyses showed that the densities of Ki-67-positive cells, MCP-1-positive cells, and FGFR-1positive vessels of the coccygeo group were significantly higher than those of the adductor and vehicle groups (Fig 3, j-l). At 7 days after administration, no significant differences in the expression of Ki-67, MCP-1, and FGFR1 were detected between the coccygeo, adductor, and vehicle groups (Fig 3, j-l). Morphometry of the collateral vessels. The density and wall thickness of arteries50-250 were significantly increased in the coccygeo group compared with the adductor and vehicle groups, but there were no differences in values between the adductor and vehicle groups (Fig 4, a-e). deep femoral artery whose blood flow was lost by femoral artery removal. FA (red asterisk), Femoral artery; PA, popliteal artery; IIA (yellow arrow), internal iliac artery.
JOURNAL OF VASCULAR SURGERY Volume -, Number -
Nishiyama et al 5
Fig 2. a, Calf blood pressure ratio before basic fibroblast growth factor/phosphate-buffered saline (bFGF/PBS) injection and at 28 days after the injection. b, Resting blood flow and maximum blood flow of the left internal iliac artery at 28 days after the injection. a-c, Selective internal iliac angiograms at 28 days after bFGF/PBS injection: c, coccygeo group; d, adductor group; e, vehicle group. f, Angiographic score. *P < .01 (vs other groups). yP < .05.
Immunoprecipitation and Western blot analyses. The expression of FGFR-1 at 3 days after administration of bFGF/PBS was markedly enhanced in the coccygeo group compared with the adductor and vehicle groups, although the enhanced expression in the coccygeo group decreased at 7 days (Fig 5, a). To assess FGFR-1 phosphorylation, the blot was stained with an antibody against phosphorylated tyrosine. Considerable phosphorylation of FGFR-1 was observed in the coccygeo group at day 3 (Fig 5, a). Further, bFGF accumulation in the coccygeofemoral muscle after administration was investigated by Western blot after concentration with heparineSepharose CL-6B.
In the coccygeo group, bFGF accumulation at day 3 was considerably higher than in the adductor and vehicle groups, and the abundant accumulation of bFGF continued until day 7 (Fig 5, b). Also in the adductor group, some accumulation of bFGF was observed at 7 days after administration. DISCUSSION First, in this study, we demonstrated that collateral vessels developed through a specific anatomic route in the rabbit model of limb ischemia.6,7,20 Because the femoral artery was completely excised to induce an ischemic limb, little
6 Nishiyama et al
JOURNAL OF VASCULAR SURGERY --- 2014
Fig 3. Immunohistologic images of coccygeofemoral muscle at 3 days after basic fibroblast growth factor/phosphatebuffered saline (bFGF/PBS) injection. Immunostaining for Ki-67 (a-c), monocyte chemotactic protein 1 (MCP-1) (d-f), and fibroblast growth factor receptor 1 (FGFR-1) (g-i): a, d, and g, coccygeo group; b, e, and h, adductor group; c, f, and i, vehicle group. Antigen-positive parts were stained brown; arrowheads also indicate stained parts. Bar, 100 mm. To quantify the immunostaining results, the densities of Ki-67-positive cells (j), MCP-1-positive cells (k), and FGFR-1-positive vessels (l) were calculated. *P < .01 (vs other groups at 3 days).
JOURNAL OF VASCULAR SURGERY Volume -, Number -
Nishiyama et al 7
Fig 4. a-c, Microphotographs of coccygeofemoral muscle at 14 days after basic fibroblast growth factor/phosphatebuffered saline (bFGF/PBS) injection: a, Coccygeo group; b, adductor group; c, vehicle group. The sections were stained by hematoxylin and eosin. Bar, 200 mm. To quantitatively analyze collateral development, the density (d) and wall thickness (e) of arteries50-250 were calculated. *P < .01. yP < .05 (vs other groups).
perfusion was supplied to the limb through the ipsilateral external iliac artery.26-28 Under this condition, collateral vessels tended to develop between the posterior gluteal artery and the popliteal artery. The posterior gluteal artery is a distal branch of the internal iliac artery and a dominant feeder of the coccygeofemoral muscle, which is located from the buttock to the posterior thigh. The distal part of the coccygeofemoral muscle also receives minor blood supply from branches of the popliteal artery. Therefore, collateral vessels in this model might have developed from a connecting route between the two feeding arteries in the coccygeofemoral muscle. Collateral vessels in the arterial system are an alternative perfusion channel that develops to bypass an occlusive lesion of a native artery, which is a critical mechanism for the recovery from ischemia.10,20 Previous studies have attempted to clarify this mechanism and revealed that natural prototypes of collateral vessels generally preexist in vivo as arteriolar connections between distal branches of different arterial trees.4,29-33 However, when an occlusive lesion occurs upstream from one feeding artery of a certain muscle, the blood supply decreases consequently in the perfusion area of this feeding artery, which induces
blood inflow from the adjoining perfusion area in the same muscle through the arteriolar connections.4,6,25,33 The arteriolar connections subsequently increase their size and undergo maturation to increase the blood supply to the ischemic muscle area. Further, if there are other muscles or tissues in the distal part of the occlusive lesion and they develop ischemia, the enlargement and maturation of the arteriolar connections would be enhanced to conduct blood perfusion to these other muscles or tissues with ischemia. The enlarged and mature arteriolar connections might then function as an acquired perfusion channel, introducing alternative blood inflow to the ischemic muscles or tissues, which indicates a development of collateral vessels. Thus, favorable development of collateral vessels could be promoted by enhancing the enlargement and maturation of the arteriolar connections, a natural prototype of collateral vessels.4,25,30,33 When the femoral artery was removed in the rabbit, the arteriolar connections in the coccygeofemoral muscle developed into collateral vessels, which conducted blood flow from the posterior gluteal artery to the ischemic area through the branches of the popliteal artery. The coccygeofemoral muscle contains preexisting collateral vessels
8 Nishiyama et al
JOURNAL OF VASCULAR SURGERY --- 2014
Fig 5. a, Fibroblast growth factor receptor 1 (FRFR-1) expression and phosphorylation in coccygeofemoral muscle at 3 and 7 days after basic fibroblast growth factor/phosphate-buffered saline (bFGF/PBS) injection. FGFR-1 in each muscle lysate was recovered by immunoprecipitation (IP) and analyzed by Western blotting (WB) with an antibody against FGFR-1 (top) or phosphorylated tyrosine (bottom). b, bFGF expression in coccygeofemoral muscle at 3 and 7 days after injection. bFGF in each lysate was concentrated with heparin-Sepharose and analyzed by Western blotting with an antibody for bFGF. C, Coccygeo group; A, adductor group, V, vehicle group.
in this model. However, under normal conditions, collateral development in the coccygeofemoral muscle is generally insufficient, and chronic ischemia of the limb ensues. Therefore, we considered that the therapeutic site for effective collateral development in the rabbit model might be the arteriolar connections in the coccygeofemoral muscle (Fig 6). Meanwhile, the enlargement and maturation of arterioles are promoted by the process of arteriogenesis. Arteriogenesis is the remodeling process of preexisting vessels that promotes the growth and stabilization of the vasculature. Previous studies showed that bFGF plays an important role in this process.4,6,8,20,25,29,34-36 To estimate whether the therapeutic site for collateral enhancement in this model is the arteriolar connections in the coccygeofemoral muscle, we delivered an effective dose of recombinant bFGF to the coccygeofemoral muscle or the adductor muscle at 28 days after femoral artery removal. The adductor muscle is a major muscle in the thigh like the coccygeofemoral muscle2,10,37; however, the adductor muscle does not contain arteriolar connections that potentially develop into collateral vessels in this model. The reason that bFGF was administrated only once was that bFGF could bind with the extracellular
Fig 6. Therapeutic site for effective collateral development in the rabbit model of limb ischemia. a, The coccygeofemoral muscle receives dominant perfusion through the posterior gluteal artery and minor perfusion through the popliteal artery, and arteriolar connections exist between the distal branches of the two feeding arteries. When the feeding arteries function adequately, the arteriolar connections do not perform an important role. b, Removal of the femoral artery induced limb ischemia, and then the arteriolar connections in the coccygeofemoral muscle developed into collateral vessels. If the maturation of the arteriolar connections is insufficient, the limb develops chronic ischemia. Thus, the immature arteriolar connection might be a therapeutic site in this model. c, The enlargement and maturation of arterioles are promoted by the process of arteriogenesis, and basic fibroblast growth factor (bFGF) is a potent inducer of arteriogenesis. Selective delivery of bFGF (closed squares) to the arteriolar connection markedly enhanced collateral development and limb perfusion. *Arteriolar connections. **Premature collateral arteries. yDeveloped collateral arteries. yyTotal removal of the left femoral artery. IIA, Internal iliac artery; FA, femoral artery; PA, popliteal artery.
matrix and maintain activity in vivo for a certain period. Indeed, the Western blot analysis in this study demonstrated abundant accumulation of bFGF in the muscle even at 7 days after administration. The calf blood pressure
JOURNAL OF VASCULAR SURGERY Volume -, Number -
ratio and the blood flow of the left internal iliac artery are conventional parameters representing the degree of perfusion in the limb.6,20,36 This study showed that 28 days after injection, these parameters were significantly higher in the coccygeo group than in the adductor and vehicle groups, demonstrating that bFGF delivery to the coccygeofemoral muscle improved the limb perfusion compared with delivery to the adductor muscle. Angiograms provide images of vessels of >50 mm in diameter, and functional collateral vessels with a mature wall structure must be included in the imaged vessels.6,20,24,36 In this case, the higher angiographic score after bFGF delivery to the coccygeofemoral muscle represented a higher development of collateral vessels in the ischemic limb compared with that to the adductor muscle. A similar assessment was carried out by morphometric analysis of arteries50-250 in the coccygeofemoral muscle. Because arteries50-250 might contain most of the developed collateral vessels,20,25 the higher arteries50-250 density and wall thickness measurements in the coccygeo group reflected favorable development and maturation of the collateral vessels compared with the adductor and vehicle groups. The results indicate that although the coccygeofemoral and adductor muscles are close, the development of collateral circulation was remarkably dependent on which muscle was selected as the therapeutic site, suggesting that correct selection of the site is critical for successful angiogenic therapy. In addition, the calf blood pressure ratio and the resting blood flow in the adductor group were significantly higher than those in the vehicle group. One possible explanation for this is that bFGF in the adductor muscle was partially distributed to the coccygeofemoral muscle and promoted weak development of collateral vessels. In fact, Western blot for bFGF showed some accumulation of bFGF in the coccygeofemoral muscle at 7 days after the delivery to the adductor muscle. Schaper et al have reported that arteriogenesis could be triggered by an increase in the shear stress to the vascular lumen.14,24,29,32,34 The increase of shear stress induces the expression of MCP-1 and several adhesive molecules on vascular wall cells, promoting an infiltration of inflammatory cells to the vessels. These cells release several growth factors, cytokines, and proteases, and then these bioactive substances cooperatively induce the enlargement and maturation of the vessels, which lead to arteriogenesis.38,39 Among these substances, bFGF is considered a critical growth factor during adaptive arteriogenesis, and this study indeed showed that bFGF delivery to the coccygeofemoral muscle significantly enhanced the collateral development. Because bFGF is a potent mitogen for cells in the vascular wall,34,35,40 the increase of Ki-67-positive cells suggested that the delivery of bFGF directly induced cell proliferation in and around the arteriolar connections in the coccygeofemoral muscle, which potently accelerated the arteriogenesis of these vessels. Meanwhile, increased MCP-1 expression might indicate other effects of bFGF treatment. Fujii et al24 reported that bFGF potently stimulated nonendothelial mesenchymal cells to release MCP-1. Therefore, there is a possibility that bFGF treatment
Nishiyama et al 9
promoted the expression of MCP-1 and then the increased MCP-1 enhanced arteriogenesis of the arteriolar connections. Further, it is also known that a specific receptor for bFGF, FGFR-1, is markedly expressed in vascular wall cells during the process of arteriogenesis.41,42 This study demonstrated abundant expression of FGFR-1 and phosphorylation of FGFR-1 in the vessels of the bFGF-treated coccygeofemoral muscle. These findings suggested that the therapeutic effect of bFGF delivery might be further reinforced by the upregulation of the specific receptor. CONCLUSIONS This study first determined with use of a rabbit model that arteriolar connections in the coccygeofemoral muscle developed into collateral vessels after excision of the femoral artery. On the basis of this finding, we selectively delivered bFGF to the coccygeofemoral muscle in the rabbit with chronic limb ischemia and demonstrated remarkable development of collateral vessels and improvement of limb perfusion compared with bFGF delivery to the other thigh muscle. These findings suggested that selection of the therapeutic site is a key factor in achieving effective augmentation of collateral vessels in chronic peripheral ischemia. Our results suggest that in the clinical setting, substances or cells aimed at promoting angiogenic reactions should be delivered specifically to the appropriate site as determined by angiographic findings in the patient and be based on an understanding of the pathophysiologic mechanism of human collateral vessel development. The pattern of collateral vessel development in the rabbit differs markedly from that of humans, so further study is needed to identify appropriate therapeutic sites for angiogenic therapy in humans. AUTHOR CONTRIBUTIONS Conception and design: AN, HK Analysis and interpretation: AN, HK Data collection: AN, HK Writing the article: AN, HK Critical revision of the article: TM, TW Final approval of the article: TM, TW Statistical analysis: AN, HK Obtained funding: HK Overall responsibility: HK REFERENCES 1. Unger EF, Banani S, Shou M, Lazarous DF, Jaklitsch MT, Scheinowitz M, et al. Basic fibroblast growth factor enhances myocardial collateral flow in a canine model. Am J Physiol 1994;266: H1588-95. 2. Takeshita S, Zheg LP, Brogi E, Kearney M, Pu LQ, Bunting S, et al. Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J Clin Invest 1994;93:662-70. 3. Sellke FW, Li J, Stamler A, Lopez JJ, Thomas KA, Simons M. Angiogenesis induced by acidic fibroblast growth factor as an alternative method of revascularization for chronic myocardial ischemia. Surgery 1996;120:182-8. 4. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med 2000;6:389-95.
10 Nishiyama et al
5. Ninomiya M, Koyama H, Miyata T, Hamada H, Miyatake S, Shigematsu H, et al. Ex vivo gene transfer of basic fibroblast growth factor improves cardiac function and blood flow in a swine chronic myocardial ischemia model. Gene Ther 2003;10:1152-60. 6. Hosaka A, Koyama H, Kushibiki T, Tabata Y, Nishiyama N, Miyata T, et al. Gelatin hydrogel microspheres enable pinpoint delivery of basic fibroblast growth factor for the development of functional collateral vessels. Circulation 2004;110:3322-8. 7. Ohara N, Koyama H, Miyata T, Hamada H, Miyashita SI, Akimoto M, et al. Adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor promotes collateral development in a rabbit model of hind limb ischemia. Gene Ther 2001;8:837-45. 8. Bernotat-Danielowski S, Sharma HS, Schott RJ, Schaper W. Generation and localisation of monoclonal antibodies against fibroblast growth factors in ischemic collateralised porcine myocardium. Cardiovasc Res 1993;27:1220-8. 9. Schwarz ER, Speakman MT, Patterson M, Hale SS, Isner JM, Kedes LH, et al. Evaluation of the effects of intramyocardial injection of DNA expressing vascular endothelial growth factor (VEGF) in a myocardial model in the ratdangiogenesis and angioma formation. J Am Coll Cardiol 2000;35:1323-30. 10. Hershey JC, Baskin EP, Glass JD, Hartman HA, Gilberto DB, Rogers IT, et al. Revascularization in the rabbit hind limb: dissociation between capillary sprouting and arteriogenesis. Cardiovasc Res 2001;49:618-25. 11. Cao R, Brakenhielm E, Pawliuk R, Wariaro D, Post MJ, Wahlberg E, et al. Angiogenic synergism, vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2. Nat Med 2003;9:604-13. 12. Shintani S, Murohara T, Ikeda H, Ueno T, Sasaki K, Duan J, et al. Augmentation of postnatal neovascularization with autologous bone marrow transplantation. Circulation 2001;103:897-903. 13. Fuchs S, Baffour R, Zhou YF, Shou M, Pierre A, Tio FO, et al. Transendocardial delivery of autologous bone marrow enhances collateral perfusion and regional function in pigs with chronic experimental myocardial ischemia. J Am Coll Cardiol 2001;37:1726-32. 14. He Y, Luo Y, Tang S, Rajantie I, Salven P, Heil M, et al. Critical function of Bmx/Etk in ischemia-mediated arteriogenesis and angiogenesis. J Clin Invest 2006;116:2344-55. 15. Haro JD, Acin F, Quintana AL, Florez A, Aguilar EM, Varela C. Metaanalysis of randomized, controlled clinical trials in angiogenesis: gene and cell therapy in peripheral arterial disease. Heart Vessels 2009;24:321-8. 16. Lederman RJ, Mendelsohn FO, Anderson RD, Saucedo JF, Tenaglia AN, Hermiller JB, et al; TRAFFIC Investigators. Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (the TRAFFIC study): a randomized trial. Lancet 2002;359:2053-8. 17. Rajagopalan S, Mohler ER 3rd, Lederman RJ, Mendelsohn FO, Saucedo JF, Goldman CK, et al. Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease: a phase II randomized, double-blind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication. Circulation 2003;108:1933-8. 18. Grossman PM, Mendelsohn F, Henry TD, Hermiller JB, Litt M, Saucedo JF, et al. Results from a phase II multicenter, double-blind placebo-controlled study of Del-1 (VLTS-589) for intermittent claudication in subjects with peripheral arterial disease. Am Heart J 2007;153:874-80. 19. Isner JM, Baumgartner I, Rauh G, Schainfeld R, Blair R, Manor O, et al. Treatment of thromboangiitis obliterans (Buerger’s disease) by intramuscular gene transfer of vascular endothelial growth factor: preliminary clinical results. J Vasc Surg 1998;28:964-73. 20. Kondoh K, Koyama H, Miyata T, Takato T, Hamada H, Shigematsu H. Conduction performance of collateral vessels induced by vascular endothelial growth factor or basic fibroblast growth factor. Cardiovasc Res 2004;61:132-42. 21. Longland CJ. The collateral circulation of the limb: Arris and Gale lecture delivered at the Royal College of Surgeons of England on 4th February, 1953. Ann R Coll Surg Engl 1953;13:161-76.
JOURNAL OF VASCULAR SURGERY --- 2014
22. Pu LQ, Jackson S, Lachapelle KJ, Arekat Z, Graham AM, Lisbona R, et al. A persistent hindlimb ischemia model in the rabbit. J Invest Surg 1992;7:49-60. 23. Troidl C, Nef H, Voss S, Schilp A, Kostin S, Troidl K, et al. Calciumdependent signalling is essential during collateral growth in the pig hind limbeischemia model. J Mol Cell Cardiol 2010;49:142-51. 24. Fujii T, Yonemitsu Y, Onimaru M, Tani M, Nakano T, Egashira K, et al. Nonendothelial mesenchymal cellederived MCP-1 is required for FGF-2 mediated therapeutic neovascularization: critical role of the inflammatory/arteriogenic pathway. Arterioscler Thromb Vasc Biol 2006;26:2483-9. 25. Cai W, Schaper W. Mechanisms of arteriogenesis. Acta Biochim Biophys Sin (Shanghai) 2008;40:681-92. 26. Scholz D, Ito W, Fleming I, Deindl E, Sauer A, Wiesnet M, et al. Ultrastructure and molecular histology of rabbit hind-limb collateral artery growth (arteriogenesis). Virchows Arch 2000;436:257-70. 27. Popesko P, Rajtova V, Horak J. A colour atlas of anatomy of small laboratory animals, vol. 1: rabbit, guinea pig. London: Wolfe Publishing Ltd; 1992. p. 126-33. 28. Guiuriat L, Scatena M, Chiavesgato A, Guidolin D, Pauleto P, Sartore S. Rabbit ductus arteriosus during development: anatomical structure and smooth muscle cell composition. Anat Rec 1993;235: 95-110. 29. Ali M, Gilanpour H. Anatomical study of copulatory organ in male rabbit. Adv Environ Biol 2010;5:3159-63. 30. Heil M, Eitenmuller I, Schmitz-Rixen T, Schaper W. Arteriogenesis versus angiogenesis: similarities and differences. J Cell Mol Med 2006;10:45-55. 31. Van Royen NJ, Piek J, Buschmann I, Hoefer I, Voskuil M, Schaper W. Stimulation of arteriogenesis; a new concept for the treatment of arterial occlusive disease. Cardiovasc Res 2001;49:543-53. 32. Oostrom M, Oostrom O, Quax P, Verhaar M, Hoefer I. Insight into mechanisms behind arteriogenesis: what does the future hold? J Leukoc Biol 2008;84:1379-91. 33. SchaperW. Collateral circulation: past and present. Basic Res Cardiol 2009;104:5-21. 34. Klein S, Roghani M, Rifkin DB. Fibroblast growth factors as angiogenesis factors: new insights into their mechanism of action. EXS 1997;79:159-92. 35. Schaper W. Collateral vessel growth in the human heartdrole of fibroblast growth factor-2. Circulation 1996;94:600-1. 36. Patel TH, Kimura H, Weiss CR, Semenza GL, Hofmann LV. Constitutively active HIF-1a improves perfusion and arterial remodeling. Cardiovas Res 2005;68:144-54. 37. Scholz D, Ziegelhoeffer T, Helisch A, Wangner S, Friedrich C, Podzuweit T, et al. Contribution of arteriogenesis and angiogenesis to postocclusive hind limb perfusion in mice. J Mol Cell Cardiol 2002;34: 775-87. 38. Demicheva E, Hecker M, Kroff T. Stretch-induced activation of the transcription factor activator protein-1 controls monocyte chemoattractant protein-1 expression during arteriogenesis. Circ Res 2008;103:477-84. 39. Ito W, Arras DM, Winkler B, Scholz D, Schaper J, Schaper W. Monocyte chemotactic protein-1 increases collateral and peripheral conductance after femoral artery occlusion. Circ Res 1997;80:829-37. 40. Schierling W, Troidl K, Apfelbeck H, Troidl C, Kasprzak PM, Schaper W, et al. Cerebral arteriogenesis enhanced by pharmacological as well as fluid-shear-stress activation of the Trpv4 calcium channel. Eur J Vasc Endovasc Surg 2011;41:589-96. 41. Banquet Gomez S, Nicol L, Edwards-Lévy F, Henry JP, Cao R, Schapman D, et al. Arteriogenic therapy by intramyocardial sustained delivery of a novel growth factor combination prevents chronic heart failure. Circulation 2011;124:1059-69. 42. Pöling Szibor J, Schimanski S, Ingelmann ME, Rees W, Gajawada P, Kochfar Z, et al. Induction of smooth muscle cell migration during arteriogenesis is mediated by Rap2. Arterioscler Thromb Vasc Biol 2011;31:2297-305. Submitted Oct 29, 2013; accepted Feb 6, 2014.
Induction of angiogenic reactions to improve tissue perfusion is a promising approach for the treatment of arterial occlusive diseases, and the most important mechanism of angiogenic therapy is considered to be the enhancement of collateral circulation to the ischemic lesion. Although a variety of strategies for effective angiogenic therapies have From the Division of Vascular Surgery, Department of Surgery, Graduate School of Medicine, The University of Tokyoa; the Division of Translational Research Center, The University of Tokyo Hospitalb; and the Department of Surgery, Graduate School of Medicine, The University of Tokyo.c Author conflict of interest: none. Reprint requests: Hiroyuki Koyama, MD, PhD, Division of Vascular Surgery, Department of Surgery, Graduate School of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214/$36.00 Copyright Ó 2014 by the Society for Vascular Surgery. http://dx.doi.org/10.1016/j.jvs.2014.02.004
been presented, the basic concept in most of these strategies is the local delivery of bioactive factors or cells that potentially promote angiogenic processes in vivo.1-4 Previous studies have attempted to deliver several bioactive factors or cells in animal models of ischemia and reported favorable therapeutic effects of some growth factors or cells, such as basic fibroblast growth factor (bFGF),5-7 vascular endothelial growth factor,2,8-11 and bone marrow mononuclear cells.12-14 Those studies have successfully identified growth factors or cells that are adequate for delivery substances in angiogenic therapy; however, the appropriate site for such factors or cells is currently unclear. Recently, several clinical trials testing angiogenic therapies have been carried out.15-19 In most of these trials, angiogenic factors or cells were delivered by intra-arterial injection or intramuscular injection. When angiogenic substances are administrated by an intraarterial injection, they disperse widely to areas distal to the injection site and might fail to specifically target areas that are appropriate for collateral vessel formation. When substances are delivered by intramuscular injection, the 1
2 Nishiyama et al
procedure is usually broad, multiple injections to the muscles of the ischemic limb, which might also increase the possibility of irrelevant delivery. Indeed, large-scale clinical trials testing these substances have shown limited therapeutic efficacy.15-19 We hypothesized that one reason for poor outcome might be the potential inefficiency of the delivery. If the delivery site for angiogenic therapy can be clearly identified, it might be possible to notably increase the therapeutic efficacy by specific delivery to this region. The purpose of this study was to identify the therapeutic site for injection of the growth factor for the most effective development of collateral vessels in a rabbit model of chronic limb ischemia. METHODS Animal model of chronic limb ischemia. This study used a rabbit model of chronic limb ischemia to evaluate the development of collateral vessels. Reliable methods to evaluate the perfusion of rabbit limb have been previously reported, and several previous studies testing angiogenic therapies have used the rabbit model of limb ischemia.2,6,20 Male Japanese white rabbits weighing 2.5 to 3.0 kg (Saitama Rabbitry, Saitama, Japan) were anesthetized with an intramuscular injection of a mixture of ketamine (50 mg/kg) and xylazine (2.5 mg/kg). The left femoral artery was completely excised from its proximal origin to the bifurcation formed by the saphenous and popliteal arteries. At 28 days after femoral artery excision, the left hind limb of the rabbits developed chronic ischemia.6,7,10,20 All protocols conformed to the Guide for the Care and Use of Laboratory Animals (NIH publication No. 84-23, revised 1996). Anatomic analyses of collateral development in the rabbit model of limb ischemia. To clarify the anatomic pattern of collateral development in the rabbit model of limb ischemia, we carried out aortic angiography at 28 days after femoral artery removal. Immediately after an overdose injection of pentobarbital, an 18-gauge polyethylene infusion catheter (Terumo, Tokyo, Japan) was introduced into the abdominal aorta in the distal direction, and a mixed solution of lead oxide (20 g; Wako, Tokyo, Japan), gelatin (0.3 g; Wako), and water (10 mL) was injected. Because lead oxide is a radiocontrast reagent and has a vivid orange color, the arteries containing the mixed solution are recognizable under a fluoroscope and also visible from the outside when the arteries are exposed. With the fluoroscopic image as reference, we dissected the collateral vessels by using the orange color as a guide and analyzed the anatomic pattern of collateral development in this model. This experiment was repeated three times. Administration of bFGF. To investigate if the delivery site of bFGF influences the development of collateral vessels in the rabbit model of chronic limb ischemia, we injected bFGF (trafermin, human recombinant bFGF; Kaken Pharmaceutical, Tokyo, Japan) in either the left coccygeofemoral muscle (coccygeo group) or the left adductor muscle (adductor group) at 28 days after femoral artery removal, when a stable, chronic state of limb ischemia has developed.21,22
JOURNAL OF VASCULAR SURGERY --- 2014
The left coccygeofemoral muscle is the region where the collateral vessels are expected to develop in this model, and the location of this muscle could be identified through the buttock skin because the outline of it appears clearly by moistening of the skin. In contrast, few collateral vessels are expected to develop in the left adductor muscle. In the coccygeo group, 100 mg of bFGF in 100 mL of phosphatebuffered saline (PBS) was injected into three different sites in the left coccygeofemoral muscle, and 100 mL of PBS alone was injected into three different sites in the left adductor muscle. In the adductor group, 100 mg of bFGF in PBS was injected into three different sites in the adductor muscle, and 100 mL of PBS alone was injected into three different sites in the coccygeofemoral muscle. The dose of bFGF (100 mg) was chosen on the basis of previous studies in which bFGF protein was administered for the treatment of limb ischemia.6,16 As a control, another set of rabbits with chronic limb ischemia received 100 mL of PBS in both the coccygeofemoral muscle and adductor muscle (vehicle group). Evaluation of collateral development. Collateral vessel development was evaluated at 28 days after the administration of bFGF/PBS (coccygeo group, n ¼ 9; adductor group, n ¼ 9; vehicle group, n ¼ 10), a period evaluated in previous studies with the rabbit model.6,7,20 First, the calf blood pressure was measured in both hind limbs, and the calf blood pressure ratio, defined as the ratio of left systolic pressure to right systolic pressure, was calculated. This measurement was also carried out immediately before the administration of bFGF/PBS. After the measurement of calf blood pressure, a 3F end-hole catheter was introduced into the left iliac artery through the carotid artery, and a 0.014-inch Doppler guidewire (EndoSonics, Rancho Cordova, Calif) was introduced through the 3F catheter to the proximal part of the left internal iliac artery. The average peak velocity was measured at rest, and then the maximum average peak velocity was determined after the injection of 2 mg of papaverine (Dainippon Pharmaceutical, Osaka, Japan). In vivo blood flow was calculated as previously described.6,7,20 Angiograms were then taken, and the angiographic score was determined as described previously.6,7,20,21 Briefly, angiographic score was determined by a grid overlay composed of 2.5-mm-diameter circles arranged in rows spaced 5 mm apart. This overlay was placed over the angiogram, recorded for 4 seconds, at the level of the medial thigh. The number of contrast-opacified arteries crossing over the circles (CA) and the total number of circles encompassing the medial thigh area (CT) were counted. The angiographic score was calculated for each film as CA/CT. Analyses of coccygeofemoral muscle during collateral development. To study the mechanism of collateral development after bFGF administration, we analyzed the histologic findings and protein expression in the coccygeofemoral muscle at sequential time points after bFGF injection. At 28 days after the artery excision, 100 mg of bFGF in PBS or PBS alone was injected into the left coccygeofemoral muscle and adductor muscle according to the
JOURNAL OF VASCULAR SURGERY Volume -, Number -
respective protocol for the coccygeo group, adductor group, or vehicle group. The rabbits in each group were sacrificed at 3, 7, and 14 days after the administration (n ¼ 4 at each time point and in each group). An infusion catheter was introduced into the abdominal aorta, and 200 mL of lactated Ringer solution was infused at 120 mm Hg. In the rabbits sacrificed at 3 and 7 days after bFGF/PBS injection, the left coccygeofemoral muscle was excised and divided transversely into eight parts with equal thickness. The fifth part from the muscle origin was embedded in optimal cutting temperature (OCT) compound (Miles Scientific, Naperville, NJ) for histologic analysis, and the sixth part was snap-frozen in liquid nitrogen for protein lysate extraction. The samples were stored at 80 C. Meanwhile, the animals that were sacrificed at day 14 received an additional perfusion of phosphatebuffered 4% paraformaldehyde at 120 mm Hg, and the left coccygeofemoral muscle was divided into eight parts in the same manner. Then, the fifth part from the muscle origin was embedded in paraffin. Histologic analyses. Transverse histologic sections (4-mm-thick) were cut from OCT-embedded muscle samples excised from rabbits at 3 and 7 days after injection and were immune stained for Ki-67, monocyte chemotactic protein 1 (MCP-1), and FGF receptor 1 (FGFR-1). Ki-67 is a nuclear protein associated with cell proliferation, and MCP-1 and FGFR-1 are considered to play important roles in collateral development.4,7,11,23,24 Previous studies demonstrated that Ki-67, MCP-1, and FGFR-1 were markedly expressed in and around collateral vessels in the early days after induction of collateral development.23-25 After hydrogen peroxide treatment and blocking by 1.5% horse serum, monoclonal antibody against Ki-67 (1:100; Dako, Glostrup, Denmark), goat polyclonal antibody against MCP-1 (1:100; Santa Cruz Biotechnology, Santa Cruz, Calif), or monoclonal antibody against FGFR-1 (1:100; QED Bioscience, San Diego, Calif) was applied, and samples were incubated for 60 minutes at 37 C. Subsequently, biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, Calif) or rabbit anti-goat IgG (Vector Laboratories) was applied, and then analysis was performed with the ABC Elite Kit (Vector Laboratories). Photomicrographs were taken, and the specimen area was measured in each section by the ImageJ software (version 1.42q; National Institutes of Health, Bethesda, Md). In addition, Ki-67-positive cells, MCP-1-positive cells, and FGFR-1positive vessels were counted in each section, and parameters were calculated as follows: Ki 67 or MCP 1 positive cell density =mm2 ¼ Ki 67 or MCP 1 positive cell number=specimen area FGFR 1 positive vessel density =mm2 ¼ FGFR 1 positive vessel number=specimen area In addition, 4-mm-thick sections were cut from the paraffin-embedded muscle samples obtained from animals
Nishiyama et al 3
at 14 days after bFGF/PBS injection and used to analyze the morphometry of developing collateral vessels.22,26 The sections were stained with hematoxylin and eosin, and photomicrographs of each section were taken and assessed with the ImageJ software. The entire specimen area in each section (specimen area) was measured, and the number of arteries with diameters of 50 to 250 mm (arteries50-250) was counted. The diameters were measured from several directions, and the lowest value was used. Arteries50-250 correspond to typical collateral vessels that are imaged by angiography in this model.22,26 Although arteries50-250 are not always developed collateral vessels, most collateral vessels range from 50 to 250 mm in diameter, as newly formed collateral vessels generally develop from arterioles.22,26 The density of arteries50-250 was calculated as follows: Arterie50250 density =mm2 ¼ number of arteries50250 =specimen area in mm2 In addition, the minimum thickness of the arterial walls was measured in all arteries50-250 in each section, and mean values were calculated in the respective sections (arteries50-250 wall thickness). Immunoprecipitation and Western blot analyses. To assess the expression of FGFR-1, frozen muscle samples, excised from animals at 3 and 7 days after injection, were lysed in lysis solution (20 mmol/L Tris [pH 7.5], 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L b-glycerophosphate, and protease inhibitors). The muscle lysate (100 mg total protein) was diluted in 1 mL of lysis solution and treated with monoclonal antibody against FGFR-1 (5 mL; QED Bioscience) at 4 C for 16 hours after precleaning with protein AeSepharose (Invitrogen, Carlsbad, Calif). Immune complexes were recovered with protein GeSepharose (GE Healthcare, Uppsala, Sweden) and washed with lysis solution. Samples were separated on sodium dodecyl sulfateepolyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane. The blot was immunostained with a monoclonal antibody against FGFR-1 (1:500; QED Bioscience) or a monoclonal antibody against phosphorylated tyrosine (1:500, PY99; Santa Cruz Biotechnology) and visualized with an ECL Plus system (GE Healthcare), as described previously.7 We also evaluated the bFGF level in the coccygeofemoral muscles after injection, as described previously.7 Briefly, frozen muscle samples (0.5 g) were lysed in 1 mL of minimum essential medium (Invitrogen), and bFGF in each lysate was concentrated with heparineSepharose CL-6B (GE Healthcare). Samples were then analyzed by Western blot with a monoclonal antibody against bFGF (1:500; Upstate Biotechnology, Lake Placid, NY). Statistical analysis. All measurements were carried out in a blinded fashion. Results are expressed as mean 6 standard deviation. Statistical significance was evaluated by the Tukey-Kramer method for multiple comparisons. All data were considered significant at P < .05.
JOURNAL OF VASCULAR SURGERY --- 2014
4 Nishiyama et al
RESULTS
Fig 1. The anatomic pattern of collateral development in the rabbit model of limb ischemia was analyzed by aortic injection of a lead oxide solution at 28 days after excision of the left femoral artery. a, Aortic angiogram. Collateral vessels (open arrowheads) developed between the left posterior gluteal artery and the left popliteal artery. b, Photograph showing orange collateral vessels (open arrowheads) in the coccygeofemoral muscle. Note that collateral vessels are considerably larger than the right posterior gluteal artery (closed arrowheads). c, Schematic diagram of arterial anatomy in the coccygeofemoral muscle of the rabbit model. d, Schematic diagram of arterial anatomy in the adductor muscle of the rabbit model. Animals in all images are in the prone position. *Developed collateral artery. yTotal removal of the left femoral artery. zNormal size branch of posterior gluteal artery. xNormal size branch of deep femoral artery. **The branch of the
Anatomic pattern of collateral development in the rabbit model of limb ischemia. In all analyzed rabbits (n ¼ 4), the main route of collateral vessels tended to develop between the left posterior gluteal artery and the left popliteal artery, which is located in the coccygeofemoral muscle (Fig 1). Furthermore, the most evident enlargements in collateral images were observed from the middle to the distal part of the muscle, which corresponded to the sampling parts for histologic and protein analyses in the present study. In contrast, little development of collateral vessels was observed in the left adductor muscle. Evaluation of collateral vessel development. The calf blood pressure ratio measured at 28 days after injection of bFGF/PBS showed significant improvement in the coccygeo group compared with the adductor and vehicle groups, whereas there was no significant difference in data before the injection (Fig 2, a). In addition, the calf blood pressure ratio in the adductor group was significantly better than that of the vehicle group. The resting blood flow and the maximum blood flow of the left internal iliac artery demonstrated a significant increase of blood perfusion in the coccygeo group compared with the adductor and vehicle groups (Fig 2, b). Although the resting blood flow in the adductor group was significantly higher than that in the vehicle group, no significant difference was detected between the maximum blood flows of the two groups. Selective internal iliac angiography at 28 days after bFGF/PBS injection demonstrated marked collateral development in the coccygeo group, whereas few collateral vessels were observed in the adductor and vehicle groups (Fig 2, c-e). The angiographic score was significantly higher in the coccygeo group than in the adductor and vehicle groups; however, no significant difference was detected between the scores of the adductor and vehicle groups (Fig 2, f). Immunohistologic analyses. At 3 days after the bFGF/PBS injection, Ki-67-positive cells, MCP-1-positive cells, and FGFR-1-positive vessels were markedly observed in the coccygeofemoral muscle of the coccygeo group compared with the adductor and vehicle groups (Fig 3, a-i), and quantitative analyses showed that the densities of Ki-67-positive cells, MCP-1-positive cells, and FGFR-1positive vessels of the coccygeo group were significantly higher than those of the adductor and vehicle groups (Fig 3, j-l). At 7 days after administration, no significant differences in the expression of Ki-67, MCP-1, and FGFR1 were detected between the coccygeo, adductor, and vehicle groups (Fig 3, j-l). Morphometry of the collateral vessels. The density and wall thickness of arteries50-250 were significantly increased in the coccygeo group compared with the adductor and vehicle groups, but there were no differences in values between the adductor and vehicle groups (Fig 4, a-e). deep femoral artery whose blood flow was lost by femoral artery removal. FA (red asterisk), Femoral artery; PA, popliteal artery; IIA (yellow arrow), internal iliac artery.
JOURNAL OF VASCULAR SURGERY Volume -, Number -
Nishiyama et al 5
Fig 2. a, Calf blood pressure ratio before basic fibroblast growth factor/phosphate-buffered saline (bFGF/PBS) injection and at 28 days after the injection. b, Resting blood flow and maximum blood flow of the left internal iliac artery at 28 days after the injection. a-c, Selective internal iliac angiograms at 28 days after bFGF/PBS injection: c, coccygeo group; d, adductor group; e, vehicle group. f, Angiographic score. *P < .01 (vs other groups). yP < .05.
Immunoprecipitation and Western blot analyses. The expression of FGFR-1 at 3 days after administration of bFGF/PBS was markedly enhanced in the coccygeo group compared with the adductor and vehicle groups, although the enhanced expression in the coccygeo group decreased at 7 days (Fig 5, a). To assess FGFR-1 phosphorylation, the blot was stained with an antibody against phosphorylated tyrosine. Considerable phosphorylation of FGFR-1 was observed in the coccygeo group at day 3 (Fig 5, a). Further, bFGF accumulation in the coccygeofemoral muscle after administration was investigated by Western blot after concentration with heparineSepharose CL-6B.
In the coccygeo group, bFGF accumulation at day 3 was considerably higher than in the adductor and vehicle groups, and the abundant accumulation of bFGF continued until day 7 (Fig 5, b). Also in the adductor group, some accumulation of bFGF was observed at 7 days after administration. DISCUSSION First, in this study, we demonstrated that collateral vessels developed through a specific anatomic route in the rabbit model of limb ischemia.6,7,20 Because the femoral artery was completely excised to induce an ischemic limb, little
6 Nishiyama et al
JOURNAL OF VASCULAR SURGERY --- 2014
Fig 3. Immunohistologic images of coccygeofemoral muscle at 3 days after basic fibroblast growth factor/phosphatebuffered saline (bFGF/PBS) injection. Immunostaining for Ki-67 (a-c), monocyte chemotactic protein 1 (MCP-1) (d-f), and fibroblast growth factor receptor 1 (FGFR-1) (g-i): a, d, and g, coccygeo group; b, e, and h, adductor group; c, f, and i, vehicle group. Antigen-positive parts were stained brown; arrowheads also indicate stained parts. Bar, 100 mm. To quantify the immunostaining results, the densities of Ki-67-positive cells (j), MCP-1-positive cells (k), and FGFR-1-positive vessels (l) were calculated. *P < .01 (vs other groups at 3 days).
JOURNAL OF VASCULAR SURGERY Volume -, Number -
Nishiyama et al 7
Fig 4. a-c, Microphotographs of coccygeofemoral muscle at 14 days after basic fibroblast growth factor/phosphatebuffered saline (bFGF/PBS) injection: a, Coccygeo group; b, adductor group; c, vehicle group. The sections were stained by hematoxylin and eosin. Bar, 200 mm. To quantitatively analyze collateral development, the density (d) and wall thickness (e) of arteries50-250 were calculated. *P < .01. yP < .05 (vs other groups).
perfusion was supplied to the limb through the ipsilateral external iliac artery.26-28 Under this condition, collateral vessels tended to develop between the posterior gluteal artery and the popliteal artery. The posterior gluteal artery is a distal branch of the internal iliac artery and a dominant feeder of the coccygeofemoral muscle, which is located from the buttock to the posterior thigh. The distal part of the coccygeofemoral muscle also receives minor blood supply from branches of the popliteal artery. Therefore, collateral vessels in this model might have developed from a connecting route between the two feeding arteries in the coccygeofemoral muscle. Collateral vessels in the arterial system are an alternative perfusion channel that develops to bypass an occlusive lesion of a native artery, which is a critical mechanism for the recovery from ischemia.10,20 Previous studies have attempted to clarify this mechanism and revealed that natural prototypes of collateral vessels generally preexist in vivo as arteriolar connections between distal branches of different arterial trees.4,29-33 However, when an occlusive lesion occurs upstream from one feeding artery of a certain muscle, the blood supply decreases consequently in the perfusion area of this feeding artery, which induces
blood inflow from the adjoining perfusion area in the same muscle through the arteriolar connections.4,6,25,33 The arteriolar connections subsequently increase their size and undergo maturation to increase the blood supply to the ischemic muscle area. Further, if there are other muscles or tissues in the distal part of the occlusive lesion and they develop ischemia, the enlargement and maturation of the arteriolar connections would be enhanced to conduct blood perfusion to these other muscles or tissues with ischemia. The enlarged and mature arteriolar connections might then function as an acquired perfusion channel, introducing alternative blood inflow to the ischemic muscles or tissues, which indicates a development of collateral vessels. Thus, favorable development of collateral vessels could be promoted by enhancing the enlargement and maturation of the arteriolar connections, a natural prototype of collateral vessels.4,25,30,33 When the femoral artery was removed in the rabbit, the arteriolar connections in the coccygeofemoral muscle developed into collateral vessels, which conducted blood flow from the posterior gluteal artery to the ischemic area through the branches of the popliteal artery. The coccygeofemoral muscle contains preexisting collateral vessels
8 Nishiyama et al
JOURNAL OF VASCULAR SURGERY --- 2014
Fig 5. a, Fibroblast growth factor receptor 1 (FRFR-1) expression and phosphorylation in coccygeofemoral muscle at 3 and 7 days after basic fibroblast growth factor/phosphate-buffered saline (bFGF/PBS) injection. FGFR-1 in each muscle lysate was recovered by immunoprecipitation (IP) and analyzed by Western blotting (WB) with an antibody against FGFR-1 (top) or phosphorylated tyrosine (bottom). b, bFGF expression in coccygeofemoral muscle at 3 and 7 days after injection. bFGF in each lysate was concentrated with heparin-Sepharose and analyzed by Western blotting with an antibody for bFGF. C, Coccygeo group; A, adductor group, V, vehicle group.
in this model. However, under normal conditions, collateral development in the coccygeofemoral muscle is generally insufficient, and chronic ischemia of the limb ensues. Therefore, we considered that the therapeutic site for effective collateral development in the rabbit model might be the arteriolar connections in the coccygeofemoral muscle (Fig 6). Meanwhile, the enlargement and maturation of arterioles are promoted by the process of arteriogenesis. Arteriogenesis is the remodeling process of preexisting vessels that promotes the growth and stabilization of the vasculature. Previous studies showed that bFGF plays an important role in this process.4,6,8,20,25,29,34-36 To estimate whether the therapeutic site for collateral enhancement in this model is the arteriolar connections in the coccygeofemoral muscle, we delivered an effective dose of recombinant bFGF to the coccygeofemoral muscle or the adductor muscle at 28 days after femoral artery removal. The adductor muscle is a major muscle in the thigh like the coccygeofemoral muscle2,10,37; however, the adductor muscle does not contain arteriolar connections that potentially develop into collateral vessels in this model. The reason that bFGF was administrated only once was that bFGF could bind with the extracellular
Fig 6. Therapeutic site for effective collateral development in the rabbit model of limb ischemia. a, The coccygeofemoral muscle receives dominant perfusion through the posterior gluteal artery and minor perfusion through the popliteal artery, and arteriolar connections exist between the distal branches of the two feeding arteries. When the feeding arteries function adequately, the arteriolar connections do not perform an important role. b, Removal of the femoral artery induced limb ischemia, and then the arteriolar connections in the coccygeofemoral muscle developed into collateral vessels. If the maturation of the arteriolar connections is insufficient, the limb develops chronic ischemia. Thus, the immature arteriolar connection might be a therapeutic site in this model. c, The enlargement and maturation of arterioles are promoted by the process of arteriogenesis, and basic fibroblast growth factor (bFGF) is a potent inducer of arteriogenesis. Selective delivery of bFGF (closed squares) to the arteriolar connection markedly enhanced collateral development and limb perfusion. *Arteriolar connections. **Premature collateral arteries. yDeveloped collateral arteries. yyTotal removal of the left femoral artery. IIA, Internal iliac artery; FA, femoral artery; PA, popliteal artery.
matrix and maintain activity in vivo for a certain period. Indeed, the Western blot analysis in this study demonstrated abundant accumulation of bFGF in the muscle even at 7 days after administration. The calf blood pressure
JOURNAL OF VASCULAR SURGERY Volume -, Number -
ratio and the blood flow of the left internal iliac artery are conventional parameters representing the degree of perfusion in the limb.6,20,36 This study showed that 28 days after injection, these parameters were significantly higher in the coccygeo group than in the adductor and vehicle groups, demonstrating that bFGF delivery to the coccygeofemoral muscle improved the limb perfusion compared with delivery to the adductor muscle. Angiograms provide images of vessels of >50 mm in diameter, and functional collateral vessels with a mature wall structure must be included in the imaged vessels.6,20,24,36 In this case, the higher angiographic score after bFGF delivery to the coccygeofemoral muscle represented a higher development of collateral vessels in the ischemic limb compared with that to the adductor muscle. A similar assessment was carried out by morphometric analysis of arteries50-250 in the coccygeofemoral muscle. Because arteries50-250 might contain most of the developed collateral vessels,20,25 the higher arteries50-250 density and wall thickness measurements in the coccygeo group reflected favorable development and maturation of the collateral vessels compared with the adductor and vehicle groups. The results indicate that although the coccygeofemoral and adductor muscles are close, the development of collateral circulation was remarkably dependent on which muscle was selected as the therapeutic site, suggesting that correct selection of the site is critical for successful angiogenic therapy. In addition, the calf blood pressure ratio and the resting blood flow in the adductor group were significantly higher than those in the vehicle group. One possible explanation for this is that bFGF in the adductor muscle was partially distributed to the coccygeofemoral muscle and promoted weak development of collateral vessels. In fact, Western blot for bFGF showed some accumulation of bFGF in the coccygeofemoral muscle at 7 days after the delivery to the adductor muscle. Schaper et al have reported that arteriogenesis could be triggered by an increase in the shear stress to the vascular lumen.14,24,29,32,34 The increase of shear stress induces the expression of MCP-1 and several adhesive molecules on vascular wall cells, promoting an infiltration of inflammatory cells to the vessels. These cells release several growth factors, cytokines, and proteases, and then these bioactive substances cooperatively induce the enlargement and maturation of the vessels, which lead to arteriogenesis.38,39 Among these substances, bFGF is considered a critical growth factor during adaptive arteriogenesis, and this study indeed showed that bFGF delivery to the coccygeofemoral muscle significantly enhanced the collateral development. Because bFGF is a potent mitogen for cells in the vascular wall,34,35,40 the increase of Ki-67-positive cells suggested that the delivery of bFGF directly induced cell proliferation in and around the arteriolar connections in the coccygeofemoral muscle, which potently accelerated the arteriogenesis of these vessels. Meanwhile, increased MCP-1 expression might indicate other effects of bFGF treatment. Fujii et al24 reported that bFGF potently stimulated nonendothelial mesenchymal cells to release MCP-1. Therefore, there is a possibility that bFGF treatment
Nishiyama et al 9
promoted the expression of MCP-1 and then the increased MCP-1 enhanced arteriogenesis of the arteriolar connections. Further, it is also known that a specific receptor for bFGF, FGFR-1, is markedly expressed in vascular wall cells during the process of arteriogenesis.41,42 This study demonstrated abundant expression of FGFR-1 and phosphorylation of FGFR-1 in the vessels of the bFGF-treated coccygeofemoral muscle. These findings suggested that the therapeutic effect of bFGF delivery might be further reinforced by the upregulation of the specific receptor. CONCLUSIONS This study first determined with use of a rabbit model that arteriolar connections in the coccygeofemoral muscle developed into collateral vessels after excision of the femoral artery. On the basis of this finding, we selectively delivered bFGF to the coccygeofemoral muscle in the rabbit with chronic limb ischemia and demonstrated remarkable development of collateral vessels and improvement of limb perfusion compared with bFGF delivery to the other thigh muscle. These findings suggested that selection of the therapeutic site is a key factor in achieving effective augmentation of collateral vessels in chronic peripheral ischemia. Our results suggest that in the clinical setting, substances or cells aimed at promoting angiogenic reactions should be delivered specifically to the appropriate site as determined by angiographic findings in the patient and be based on an understanding of the pathophysiologic mechanism of human collateral vessel development. The pattern of collateral vessel development in the rabbit differs markedly from that of humans, so further study is needed to identify appropriate therapeutic sites for angiogenic therapy in humans. AUTHOR CONTRIBUTIONS Conception and design: AN, HK Analysis and interpretation: AN, HK Data collection: AN, HK Writing the article: AN, HK Critical revision of the article: TM, TW Final approval of the article: TM, TW Statistical analysis: AN, HK Obtained funding: HK Overall responsibility: HK REFERENCES 1. Unger EF, Banani S, Shou M, Lazarous DF, Jaklitsch MT, Scheinowitz M, et al. Basic fibroblast growth factor enhances myocardial collateral flow in a canine model. Am J Physiol 1994;266: H1588-95. 2. Takeshita S, Zheg LP, Brogi E, Kearney M, Pu LQ, Bunting S, et al. Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J Clin Invest 1994;93:662-70. 3. Sellke FW, Li J, Stamler A, Lopez JJ, Thomas KA, Simons M. Angiogenesis induced by acidic fibroblast growth factor as an alternative method of revascularization for chronic myocardial ischemia. Surgery 1996;120:182-8. 4. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med 2000;6:389-95.
10 Nishiyama et al
5. Ninomiya M, Koyama H, Miyata T, Hamada H, Miyatake S, Shigematsu H, et al. Ex vivo gene transfer of basic fibroblast growth factor improves cardiac function and blood flow in a swine chronic myocardial ischemia model. Gene Ther 2003;10:1152-60. 6. Hosaka A, Koyama H, Kushibiki T, Tabata Y, Nishiyama N, Miyata T, et al. Gelatin hydrogel microspheres enable pinpoint delivery of basic fibroblast growth factor for the development of functional collateral vessels. Circulation 2004;110:3322-8. 7. Ohara N, Koyama H, Miyata T, Hamada H, Miyashita SI, Akimoto M, et al. Adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor promotes collateral development in a rabbit model of hind limb ischemia. Gene Ther 2001;8:837-45. 8. Bernotat-Danielowski S, Sharma HS, Schott RJ, Schaper W. Generation and localisation of monoclonal antibodies against fibroblast growth factors in ischemic collateralised porcine myocardium. Cardiovasc Res 1993;27:1220-8. 9. Schwarz ER, Speakman MT, Patterson M, Hale SS, Isner JM, Kedes LH, et al. Evaluation of the effects of intramyocardial injection of DNA expressing vascular endothelial growth factor (VEGF) in a myocardial model in the ratdangiogenesis and angioma formation. J Am Coll Cardiol 2000;35:1323-30. 10. Hershey JC, Baskin EP, Glass JD, Hartman HA, Gilberto DB, Rogers IT, et al. Revascularization in the rabbit hind limb: dissociation between capillary sprouting and arteriogenesis. Cardiovasc Res 2001;49:618-25. 11. Cao R, Brakenhielm E, Pawliuk R, Wariaro D, Post MJ, Wahlberg E, et al. Angiogenic synergism, vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2. Nat Med 2003;9:604-13. 12. Shintani S, Murohara T, Ikeda H, Ueno T, Sasaki K, Duan J, et al. Augmentation of postnatal neovascularization with autologous bone marrow transplantation. Circulation 2001;103:897-903. 13. Fuchs S, Baffour R, Zhou YF, Shou M, Pierre A, Tio FO, et al. Transendocardial delivery of autologous bone marrow enhances collateral perfusion and regional function in pigs with chronic experimental myocardial ischemia. J Am Coll Cardiol 2001;37:1726-32. 14. He Y, Luo Y, Tang S, Rajantie I, Salven P, Heil M, et al. Critical function of Bmx/Etk in ischemia-mediated arteriogenesis and angiogenesis. J Clin Invest 2006;116:2344-55. 15. Haro JD, Acin F, Quintana AL, Florez A, Aguilar EM, Varela C. Metaanalysis of randomized, controlled clinical trials in angiogenesis: gene and cell therapy in peripheral arterial disease. Heart Vessels 2009;24:321-8. 16. Lederman RJ, Mendelsohn FO, Anderson RD, Saucedo JF, Tenaglia AN, Hermiller JB, et al; TRAFFIC Investigators. Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (the TRAFFIC study): a randomized trial. Lancet 2002;359:2053-8. 17. Rajagopalan S, Mohler ER 3rd, Lederman RJ, Mendelsohn FO, Saucedo JF, Goldman CK, et al. Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease: a phase II randomized, double-blind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication. Circulation 2003;108:1933-8. 18. Grossman PM, Mendelsohn F, Henry TD, Hermiller JB, Litt M, Saucedo JF, et al. Results from a phase II multicenter, double-blind placebo-controlled study of Del-1 (VLTS-589) for intermittent claudication in subjects with peripheral arterial disease. Am Heart J 2007;153:874-80. 19. Isner JM, Baumgartner I, Rauh G, Schainfeld R, Blair R, Manor O, et al. Treatment of thromboangiitis obliterans (Buerger’s disease) by intramuscular gene transfer of vascular endothelial growth factor: preliminary clinical results. J Vasc Surg 1998;28:964-73. 20. Kondoh K, Koyama H, Miyata T, Takato T, Hamada H, Shigematsu H. Conduction performance of collateral vessels induced by vascular endothelial growth factor or basic fibroblast growth factor. Cardiovasc Res 2004;61:132-42. 21. Longland CJ. The collateral circulation of the limb: Arris and Gale lecture delivered at the Royal College of Surgeons of England on 4th February, 1953. Ann R Coll Surg Engl 1953;13:161-76.
JOURNAL OF VASCULAR SURGERY --- 2014
22. Pu LQ, Jackson S, Lachapelle KJ, Arekat Z, Graham AM, Lisbona R, et al. A persistent hindlimb ischemia model in the rabbit. J Invest Surg 1992;7:49-60. 23. Troidl C, Nef H, Voss S, Schilp A, Kostin S, Troidl K, et al. Calciumdependent signalling is essential during collateral growth in the pig hind limbeischemia model. J Mol Cell Cardiol 2010;49:142-51. 24. Fujii T, Yonemitsu Y, Onimaru M, Tani M, Nakano T, Egashira K, et al. Nonendothelial mesenchymal cellederived MCP-1 is required for FGF-2 mediated therapeutic neovascularization: critical role of the inflammatory/arteriogenic pathway. Arterioscler Thromb Vasc Biol 2006;26:2483-9. 25. Cai W, Schaper W. Mechanisms of arteriogenesis. Acta Biochim Biophys Sin (Shanghai) 2008;40:681-92. 26. Scholz D, Ito W, Fleming I, Deindl E, Sauer A, Wiesnet M, et al. Ultrastructure and molecular histology of rabbit hind-limb collateral artery growth (arteriogenesis). Virchows Arch 2000;436:257-70. 27. Popesko P, Rajtova V, Horak J. A colour atlas of anatomy of small laboratory animals, vol. 1: rabbit, guinea pig. London: Wolfe Publishing Ltd; 1992. p. 126-33. 28. Guiuriat L, Scatena M, Chiavesgato A, Guidolin D, Pauleto P, Sartore S. Rabbit ductus arteriosus during development: anatomical structure and smooth muscle cell composition. Anat Rec 1993;235: 95-110. 29. Ali M, Gilanpour H. Anatomical study of copulatory organ in male rabbit. Adv Environ Biol 2010;5:3159-63. 30. Heil M, Eitenmuller I, Schmitz-Rixen T, Schaper W. Arteriogenesis versus angiogenesis: similarities and differences. J Cell Mol Med 2006;10:45-55. 31. Van Royen NJ, Piek J, Buschmann I, Hoefer I, Voskuil M, Schaper W. Stimulation of arteriogenesis; a new concept for the treatment of arterial occlusive disease. Cardiovasc Res 2001;49:543-53. 32. Oostrom M, Oostrom O, Quax P, Verhaar M, Hoefer I. Insight into mechanisms behind arteriogenesis: what does the future hold? J Leukoc Biol 2008;84:1379-91. 33. SchaperW. Collateral circulation: past and present. Basic Res Cardiol 2009;104:5-21. 34. Klein S, Roghani M, Rifkin DB. Fibroblast growth factors as angiogenesis factors: new insights into their mechanism of action. EXS 1997;79:159-92. 35. Schaper W. Collateral vessel growth in the human heartdrole of fibroblast growth factor-2. Circulation 1996;94:600-1. 36. Patel TH, Kimura H, Weiss CR, Semenza GL, Hofmann LV. Constitutively active HIF-1a improves perfusion and arterial remodeling. Cardiovas Res 2005;68:144-54. 37. Scholz D, Ziegelhoeffer T, Helisch A, Wangner S, Friedrich C, Podzuweit T, et al. Contribution of arteriogenesis and angiogenesis to postocclusive hind limb perfusion in mice. J Mol Cell Cardiol 2002;34: 775-87. 38. Demicheva E, Hecker M, Kroff T. Stretch-induced activation of the transcription factor activator protein-1 controls monocyte chemoattractant protein-1 expression during arteriogenesis. Circ Res 2008;103:477-84. 39. Ito W, Arras DM, Winkler B, Scholz D, Schaper J, Schaper W. Monocyte chemotactic protein-1 increases collateral and peripheral conductance after femoral artery occlusion. Circ Res 1997;80:829-37. 40. Schierling W, Troidl K, Apfelbeck H, Troidl C, Kasprzak PM, Schaper W, et al. Cerebral arteriogenesis enhanced by pharmacological as well as fluid-shear-stress activation of the Trpv4 calcium channel. Eur J Vasc Endovasc Surg 2011;41:589-96. 41. Banquet Gomez S, Nicol L, Edwards-Lévy F, Henry JP, Cao R, Schapman D, et al. Arteriogenic therapy by intramyocardial sustained delivery of a novel growth factor combination prevents chronic heart failure. Circulation 2011;124:1059-69. 42. Pöling Szibor J, Schimanski S, Ingelmann ME, Rees W, Gajawada P, Kochfar Z, et al. Induction of smooth muscle cell migration during arteriogenesis is mediated by Rap2. Arterioscler Thromb Vasc Biol 2011;31:2297-305. Submitted Oct 29, 2013; accepted Feb 6, 2014.