Rabbit Model Of Limb Ischemia Research Articles

Adequate Selection of a Therapeutic Site Enables Efficient Development of Collateral Vessels in Angiogenic Treatment With Bone Marrow Mononuclear Cells.

BackgroundInduction of angiogenic mechanisms to promote development of collateral vessels is considered promising for the treatment of peripheral arterial diseases. Collateral vessels generally develop from preexisting arteriolar connections, bypassing the diseased artery. We speculated that induction of angiogenic mechanisms should be directed to such arteriolar connections to achieve efficient collateral development. The aim of this study was to verify this hypothesis using autologous transplantation of bone marrow mononuclear cells in the rabbit model of chronic limb ischemia.Methods and ResultsThe left femoral artery was excised to induce limb ischemia in male rabbits. In this model, arteriolar connections in the left coccygeofemoral muscle tend to develop into collateral vessels, although this transformation is insufficient to alleviate the limb ischemia. In contrast, arteriolar connections in the closely located adductor muscle do not readily develop into collateral vessels. At 21 days after ischemia initiation, a sufficient number of automononuclear cells were selectively injected in the left coccygeofemoral muscle (coccygeo group) or left adductor muscle (adductor group). Evaluation of calf blood pressure ratios, blood flow in the left internal iliac artery, and angiographic scores at day 28 after injection revealed that collateral development and improvement of limb ischemia were significantly more efficient in the coccygeo group than in the adductor group. Morphometric analysis of the coccygeofemoral muscle at day 14 showed similar results.ConclusionsSpecific delivery of mononuclear cells to the coccygeofemoral but not the adductor muscle effectively improves collateral circulation in the rabbit model of limb ischemia and suggests that adequate site selection can facilitate therapeutic angiogenesis.

Therapeutic site selection is important for the successful development of collateral vessels

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. 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 μg) 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. 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 ± 0.05 vs 0.69 ± 0.05, respectively; P < .01). Similar results were observed in blood flow through the internal iliac artery (resting: 24.6 ± 6.1 vs 17.4 ± 8.0 mL/min, P < .01; maximum: 47.4 ± 12.3 vs 33.2 ± 10.7 mL/min, P < .01) and in the angiographic score (0.67 ± 0.13 vs 0.39 ± 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. 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.

Naked DNA expressing two isoforms of hepatocyte growth factor induces collateral artery augmentation in a rabbit model of limb ischemia

Hepatocyte growth factor (HGF) has been shown to induce angiogenesis in vivo and has potential as a candidate gene for 'therapeutic angiogenesis'. In vivo, two isoforms of HGF, HGF₇₂₃ and HGF₇₂₈, consisting of 723 and 728 amino acids, are generated through alternative splicing between exons 4 and 5, but the biological effects of their coexpression have not yet been elucidated. In this study, we generated a series of genomic-complementary DNA (cDNA) hybrids of the HGF gene by inserting various truncated intron 4 into the junction of exons 4 and 5 of HGF cDNA and analyzed the biological activities of these hybrid constructs. We showed that: (1) the hybrid called HGF-X7, which contained 1502 base pairs of intron 4, could drive a higher level of HGF expression than other hybrid constructs and cDNAs of each isoform alone; (2) the pCK vector was most efficient for the gene expression of HGF-X7; (3) coexpression of both isoforms of HGF could more efficiently induce the migration of human umbilical vein endothelial cell (HUVEC) and of the mouse myoblast cell line C₂C₁₂ myoblasts than a single isoform of HGF and human vascular endothelial growth factor (VEGF)₁₆₅ at a given concentration; (4) intramuscular administration of pCK-HGF-X7 resulted in transient and localized HGF expression in the injected muscle without an increase in the HGF protein levels in other tissues including serum; and (5) intramuscular injection of pCK-HGF-X7 could more efficiently increase the number of angiographically recognizable collateral vessels, as well as improve an intra-arterial Doppler wire-measured blood flow in the rabbit model of hindlimb ischemia when compared with the identical vector encoding VEGF₁₆₅ gene. These results showed that transfer of the genomic-cDNA hybrid of the HGF gene could be used as a potential therapeutic approach to human vascular diseases.

Autologous skeletal myoblasts transduced with a new adenoviral bicistronic vector for treatment of hind limb ischemia

We aimed to achieve angiogenic synergism between human vascular endothelial growth factor 165 (VEGF 165 ) and angiopoietin-1 (Ang-1) using a new adenoviral bicistronic vector concurrently with cell therapy to repair an ischemically damaged hind limb in a rabbit model. Rabbit autologous primary skeletal myoblasts were isolated and labeled with retrovirally transduced LacZreporter gene, 4,6-diamidino-2-phenylindole (DAPI), and 5-bromo-2'-deoxyuridine (BrdU). Hind limb ischemia was created in 48 female New Zealand White rabbits by means of femoral artery ligation at 8 different places, and was assessed at angiography. Animals were randomized to receive intramuscular injection of either Dulbeco's Modified Eagle Medium (DMEM;group 1, n = 8), nontransduced myoblasts (group 2, n = 10), or myoblasts transduced with Ad-Null (group 3, n = 10), Ad-VEGF (group 4, n = 10), or Ad-Bicis (group 5, n = 8). Six weeks after treatment neovascularization in the limb was assessed at angiography. The animals were euthanized, and tissue was harvested for histologic study. Extensive transplanted myoblast survival was observed in all cell-transplanted groups, as visualized with DAPI, BrdU, and LacZ staining. Angiographic blood vessel count revealed enhanced neovascularization in group 5 (25.14 +/- 5.14) compared with group 4 (13.62 +/- 4.52), group 3 (6.09 +/- 0.09), group 2 (4.67 +/- 3.49), and group 1 (3.18 +/- 7.76). Immunostaining for von Willebrand factor confirmed significantly increased capillary density ( P < .01) at high-power microscopic field in group 5 (19.04 +/- 1.59) compared with group 4 (15.31 +/- 1.55), group 3 (6.53 +/- 0.97), group 2 (5.69 +/- 0.51), and group 1 (3.03 +/- 0.20). Simultaneous expression of VEGF and Ang-1 from bicistronic vector transduced skeletal myoblasts potently stimulated enhanced functional neovascularization in a rabbit model of limb ischemia.

PO2 and regional blood flow in a rabbit model of limb ischemia

Oxygen tension (pO2) in muscles and regional blood flow were measured in a rabbit model of limb ischemia. pO2 was measured repetitively by EPR oximetry with EMS char in four different muscle groups in the same animals. Blood flow in the same muscles at several time points was measured using microspheres. A linear mixed effects model was developed to analyze the data on pO2 and blood flow. The results suggest that while under normal conditions pO2 in muscles does not depend significantly on blood flow, immediately after arterial occlusion pO2 correlates linearly with blood flow. Within two weeks of occlusion the pO2 is recovered to 45% of baseline. This study demonstrates, for the first time, the applicability of EPR oximetry in animals larger than rodents.