Rabbit Model Of Hindlimb Ischemia Research Articles

Cord Lining Mesenchymal Stem Cells Have a Modest Positive Effect on Angiogenesis in Hindlimb Ischemia

Purpose: We investigated the use of human Cord Lining Mesenchymal Stem Cells (CL-MSCs) (US Patent number 9,737,568), in a rabbit hindlimb ischemia model, and evaluated their potential in stimulating neovascularization. Allogenic human CL- MSCs could potentially be used to treat patients with lower limb ischemia and non-healing wounds.Methods: Twenty rabbits were divided into two separate groups. We created a hindlimb ischemia model surgically. At 21 and 49 days post-operatively, animals in the treatment group were injected with CL-MSCs (500,000 cells per 0.2 ml on each site) at 10 different sites (Quadriceps- 4 sites, Hamstrings- 4 sites and Calf-−2 sites) in the hindlimb muscles. The control group received only saline injection to the corresponding sites at the same time point as the treatment group. We then evaluated the effects of treatment on neovascularization by angiography, laser doppler perfusion imaging, as well as by histology. We evaluated the tissue samples for any signs of local immune reaction to the cell implantation. We also observed the rabbit clinically for any adverse effects after treatment.Results: We found a higher number of CD31 positive cells in the treatment group, with a greater number of capillaries found in the treated muscles. The Rectus Femoris demonstrated a median vessel count/muscle fiber of 0.121 for the treatment group, compared to 0.076 in the control group (median difference 0.04; 95% CI 0.001–0.11; p = 0.041). The Gastrocnemius demonstrated a median vessel count/muscle fiber of 0.175 for the treatment group, compared to 0.089 in the control group (median difference 0.087; 95% CI −0.006 to 0.234; p = 0.07). Blood perfusion quantification through Laser Doppler Perfusion Imaging (LDPI) also demonstrated a non-statistically significant increase in perfusion in favor of the treatment group. CL-MSCs demonstrated no toxicity associated morbidity and minimal local immune reaction to implantation.Conclusion: CL-MSCs have a positive effect on angiogenesis in a rabbit hindlimb ischemia model. This preliminary data is encouraging and paves the way for future large animal studies or for clinical trials.

Invited commentary

A major limitation in developing molecular therapies for critical limb ischemia has been the lack of a clinically relevant model to test efficacy. The existing animal models primarily depend on acute limb ischemia generated by femoral artery ligation or excision. These models have provided insights into cellular and molecular mechanisms that underlie collateral artery growth, primarily because young rodents collateralize quickly in response to ischemia. This trait is less helpful when trying to test potential treatments that will be effective in older, atherosclerotic, diabetic human patients. Many single growth factor therapies have showed efficacy in these rodent-based models, only to fail in human clinical trials. Furthermore, these surgical models require invasive isolation of the femoral artery, creating an inflammatory milieu for arteriogenesis different from that caused by atherosclerosis. They also lack the tibial level occlusions seen in diabetic patients. The current article addresses the last two concerns. The authors have modified the model developed by Liddell et al,1Liddell R.P. Patel T.H. Weiss C.R. Lee D.S. Matsuhashi T. Browh P.R. et al.Endovascular model of rabbit hindlimb ischemia: a platforms to evaluate therapeutic angiogenesis.J Vasc Interv Radiol JVIR. 2005; 16: 991-998Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar where the authors used a percutaneous transauricular approach to coil embolize the rabbit superficial femoral artery, by instead using 300- to 500-μm microspheres to embolize more distal targets: the saphenous (posterior tibial equivalent) and anterior tibial arteries and their outflow beds. The resulting acute ischemia resulted in significant hindlimb ulcerations, similar to the pedal gangrene seen in patients with critical limb ischemia. The ulcerations and decreased saphenous artery blood flow were persistent up to 14 days at the time of humane killing. It is likely that this model will need further refinement to be made practical for use in testing arteriogenic therapies. The embolization resulted in a significant acute ischemic injury to the muscle, with a large inflammatory cell infiltrate and muscle necrosis. The footpad necrosis in most of the rabbits was severe, with high pain scores, resulting in ethical concerns for maintaining the model for longer than 14 days. Staged embolizations of the anterior tibial and saphenous arteries with particles sized for the tibial arteries rather than their outflow beds might better reflect the tibial level occlusions seen in diabetic humans and provide a less severe and more chronic ischemia that would better support therapeutic testing. This method would also facilitate use of the model in hypercholesterolemic or diabetic rabbits, who would presumably be less tolerant of the severe arterial ischemic insult generated by the current protocol. Limitations aside, this percutaneous embolization-based model of hindlimb ischemia represents a step forward in the creation of a more relevant animal model of critical limb ischemia. It remains to be seen whether it is enough of a step forward to support robust preclinical testing of arteriogenic therapies. Evaluation of a new model of hind limb ischemia in rabbitsJournal of Vascular SurgeryVol. 68Issue 3PreviewVarious animal models of critical limb ischemia have been developed in the past. However, there is no animal model that can undergo endovascular treatment, while providing reproducible true critical limb ischemia with arterial ulcers and rest pain. We evaluated the efficacy of a new model of rabbit hindlimb ischemia created through a percutaneous approach using embolization with calibrated particles. Full-Text PDF Open Archive

Evaluation of a new model of hind limb ischemia in rabbits

ObjectiveVarious animal models of critical limb ischemia have been developed in the past. However, there is no animal model that can undergo endovascular treatment, while providing reproducible true critical limb ischemia with arterial ulcers and rest pain. We evaluated the efficacy of a new model of rabbit hindlimb ischemia created through a percutaneous approach using embolization with calibrated particles. MethodsThrough a percutaneous transauricular artery approach and selective catheterization of the superficial femoral artery, embolization of distal limb vessels was performed using a mixture of 300- to 500-μm calibrated microparticles (Embosphere, Merit Medical, Salt Lake City, Utah), saline solution, and iodine contrast. Clinical and ultrasound imaging-based blood flow evaluation was performed before embolization and during follow-up. Histologic evaluation was performed at humane killing 14 days after the procedure. ResultsThe model was successfully created in 10 rabbits (10 limbs). One rabbit died of sudden death at 8 days after the procedure. The nine surviving rabbits developed hind ulcers. All rabbits had a higher pain score in the follow-up compared to baseline value (P < .0001). Blood flow in the saphenous artery decreased significantly after the procedure and later at 14 days follow-up (baseline value 63.4 ± 31.3 μL per cardiac cycle vs 32.0 ± 28.4 μL per cardiac cycle postprocedure [P = .0013] and 32.0 ± 28.4 μL per cardiac cycle at 14 days [P = .0015]). Pathology showed signs of severe limb ischemia in all rabbits with subacute and chronic injury patterns. ConclusionsA rabbit hind limb ischemia model created by percutaneous transauricular distal femoral artery embolization with calibrated particles may overcome some of the limitations of existing animal models. As such, this model could prove useful for assessing therapies designed to improve arterial perfusion and collateral growth.

Steerable Induction of the Thymosin β4/MRTF-A Pathway via AAV-Based Overexpression Induces Therapeutic Neovascularization.

Viral vectors have been frequently used in a variety of preclinical animal models to deliver genetic constructs into tissues. Among the vectors used, adeno-associated viral vectors (AAVs) may be targeted to specific tissues, depending on the serotype used. Moreover, they show robust expression for prolonged periods of time and have a low immunogenic potential. Furthermore, AAVs, unlike other vector systems, only display a low rate of genomic integration. However, to ensure efficient transgene production, expression is typically driven by constitutively active promoters, such as the cytomegalovirus (CMV) promoter. Tetracyclin responsive promoters represent a promising alternative to unregulated promoters. The present study compares AAVs encoding either constitutively active CMV or tet-off promoter regions in the preclinical models of hindlimb and chronic myocardial ischemia. Therapeutically, mediators regulating vessel maturation, specifically thymosin beta 4 (Tβ4) and the downstream signaling molecule myocardin-related transcription factor A (MRTF-A) as well as the endothelial activator angiopoietin-2 (Ang2) were overexpressed via AAVs using both promotors. In the model of rabbit hindlimb ischemia, temporary (tet-off) expression of Tβ4 improved capillary density, collateralization, and perfusion in the ischemic hindlimb, with no detectable difference to constitutive Tβ4 overexpression. Similarly, constitutive overexpression of MRTF-A alone was able to improve capillarization, collateralization and perfusion. Temporary expression of Ang2 for 7 days further increased capillary density and pericyte coverage compared with MRTF-A alone, without further improving collateralization or perfusion. In the pig model of chronic myocardial ischemia constitutive expression of Tβ4 for 4 weeks induced capillary and collateral growth similarly to a pulsed expression (2 day expression per week for 3 weeks). Taken together these findings demonstrate for two models of preclinical interventions that temporary gene expression may lead to similar results as constitutive expression, highlighting the potential of controlled temporary gene expression for induction of vascular growth as a therapeutic approach.

Hypoxic preconditioning reinforces cellular functions of autologous peripheral blood-derived cells in rabbit hindlimb ischemia model

Peripheral blood mononuclear cell (PBMNC) is one of powerful tools for therapeutic angiogenesis in hindlimb ischemia. However, traditional approaches with transplanted PBMNCs show poor therapeutic effects in severe ischemia patients. In this study, we used autograft models to determine whether hypoxic pretreatment effectively enhances the cellular functions of PBMNCs and improves hindlimb ischemia. Rabbit PBMNCs were cultured in the hypoxic condition. After pretreatment, cell adhesion, stress resistance, and expression of angiogenic factor were evaluated in vitro. To examine in vivo effects, we autografted preconditioned PBMNCs into a rabbit hindlimb ischemia model on postoperative day (POD) 7. Preconditioned PBMNCs displayed significantly enhanced functional capacities in resistance to oxidative stress, cell viability, and production of vascular endothelial growth factor. In addition, autologous transplantation of preconditioned PBMNCs significantly induced new vessels and improved limb blood flow. Importantly, preconditioned PBMNCs can accelerate vessel formation despite transplantation on POD 7, whereas untreated PBMNCs showed poor vascularization. Our study demonstrated that hypoxic preconditioning of PBMNCs is a feasible approach for increasing the retention of transplanted cells and enhancing therapeutic angiogenesis in ischemic tissue.

Abstract 9452: Comparative Study of Adipose Derived Regenerative Cells versus Bone Marrow-Mononuclear Cells for Therapeutic Angiogenesis

Objective: Due to the poor prognosis of therapeutic angiogenesis by bone marrow-mononuclear cells (BM-MNCs) to severe atherosclerotic patients, we focused on adipose-derived regenerative cells (ADRCs) as an alternative choice. Here, we compared the therapeutic efficacy between these two cells in two animal models of hindlimb ischemia (HLI) and further tested the hypothesis that ADRC augments angiogenesis by regulating local hemostasis through macrophage polarization. Methods and Results: We observed that fluorescence labeled ADRCs incorporated into vasculature in vitro and vivo. Then, we compared the paracrine manner between ADRCs and BM-MNCs using conditioned media (CM). Accelerated migratory ability and prevented apoptosis of human umbilical vein endothelial cells (HUVECs) were observed treated by both CM compared with control. In vivo study, implantation of either ADRCs or BM-MNCs revealed more angiographically detectable collateral vessels formation, higher capillary density, greater cuff blood pressure ratio, and stronger laser Doppler blood perfusion signals than control in rabbit HLI model. In mice study, we proved ADRCs changed the polarity of infiltrated macrophage into M2 phenotype through prostaglandin E2 (PGE2) and EP2/4 receptors. Next, the mRNA expression of several angiogenic cytokines were up-regulated in the macrophages cultured in ADRC-CM compared with BM-MNC-CM. ADRCs implantation also muted local inflammation and apoptosis via down-regulating the gene expression of pro-inflammatory cytokines, as well as increasing interleukin (IL)-10. Finally, blockade of IL-10 by neutralizing monoclonal antibody resulted in harsher microenvironment and attenuated ischemic-induced angiogenesis in mouse HLI model. Conclusions: In conclusion, the safety and efficacy of therapeutic angiogenesis by ADRCs implantation was comparable with BM-MNCs in rabbit HLI model. Moreover, slanted M2 polarity of infiltrated macrophages was observed after ADRCs implantation through PGE2 - EP2/4 - IL-10 axis, leading to facilitation of postnatal neovascularization via their angiogenic, anti-inflammatory and anti-apoptotic manners.

Induction of Angiogenesis by Controlled Delivery of Vascular Endothelial Growth Factor Using Nanoparticles

The study reports the feasibility and efficiency of vascular endothelial growth factor (VEGF) delivery using nanoparticles synthesized from glycidyl methacrylated dextran (Dex-GMA) and gelatin for therapeutic angiogenesis. The nanoparticles were prepared using phase separation method, and the drug release profile was determined by ELISA study. The bioactivity of VEGF-incorporated nanoparticles (VEGF-NPs) were determined using tube formation assay. A rabbit hind limb ischemia model was employed to evaluate the in vivo therapeutic effect. Blood perfusion was measured by single-photon emission computed tomography (SPECT). Vessel formation was evaluated by contrast angiography and immunohistochemistry. The nanoparticles synthesized were spherical in shape with evenly distributed size of about 130 ± 3.5 nm. The VEGF encapsulated was released in a biphase manner, with the majority of 69% released over 1-12 days. Tube formation assays showed increased tubular structures by VEGF-NP compared with empty nanoparticles and no treatment. Both free VEGF and VEGF-NP significantly increased blood perfusion compared with empty nanoparticles (both P < 0.001), but it was much higher in VEGF-NP-treated limbs (P < 0.001). Contrast angiography and immunohistological analysis also revealed more significant collateral artery formation and higher capillary density in VEGF-NP-treated limbs. Dex-GMA and gelatin-based nanoparticles could provide sustained release of VEGF and may serve as a new way for angiogenesis.

Effects of platelet‐rich plasma‐containing fragmin/protamine microparticles in enhancing endothelial and smooth muscle cell growth and inducing collateral vessels in a rabbit model of hindlimb ischemia

The purpose of the study was to evaluate the effects of isogenous platelet-rich plasma (PRP)-containing fragmin/protamine microparticles (F/P MPs) as a delivery system for proteins in PRP on growth of endothelial and smooth muscle cells (SMCs) in vitro and as an alternative treatment for peripheral arterial disease (PAD) and critical limb ischemia. Frozen and thawed PRP contains high concentrations of growth factors that are adsorbed by F/P MPs. Human aorta endothelial cells (AECs) and SMCs were grown in a medium with PRP. Addition of F/P MPs significantly enhanced the proliferative effects of PRP on AECs and SMCs at 37 °C for >10 days. Intramuscular administration of phosphate-buffered saline (PBS; 2 mL, control), F/P MPs (12 mg in 2 mL PBS), PRP (2 mL), or PRP (2 mL) containing F/P MPs (12 mg) was then performed in a rabbit model of hindlimb ischemia prepared by resection of the left femoral artery. Blood flow and pressure were measured on days 0, 14, and 28, and angiography to assess arteriogenesis was performed on day 28. PRP-containing F/P MPs strongly induced functional collateral vessels in the rabbit model of hindlimb ischemia, indicating possible use of these microparticles in therapy for PAD.

Vascular regeneration by pinpoint delivery of growth factors using a microcatheter reservoir system in a rabbit hind-limb ischemia model

The purpose of this study was to compare the results of delivering low doses of growth factor iteratively (20 μg x5) via a reservoir system with results obtained following a single administration of 100 μg of growth factor. The delivery systems using gelatin microspheres (GMS) facilitate the controlled release of drugs. The controlled release of growth factors at specific sites is essential for vascular regeneration. An ischemic hind-limb model was established in nine rabbits. A reservoir system was implanted in each rabbit. GMS impregnated with basic fibroblast growth factor (bFGF) through an indwelling 2-Fr catheter was infused in the reservoir system. The rabbits were divided into three equal groups: group 1 received 20 μg iteratively (x5) via the reservoir, a single dose of 100 μg growth factor was administered to group 2 and group 3 was the saline control. The therapeutic effects were evaluated by measuring the thigh temperature, blood pressure and blood flow. An immunohistological analysis was also performed for CD31. No significant difference was observed between preand post-treatment (4 weeks following bFGF infusion) in the thigh temperature, blood pressure and blood flow results from each group. Pathological analysis revealed that the number of regenerated vessels was significantly higher in the group treated iteratively with low-dose bFGF.

Therapeutic Angiogenesis Using Basic Fibroblast Growth Factor in Combination with a Collagen Matrix in Chronic Hindlimb Ischemia

Although therapeutic angiogenesis by angiogenic cytokines is a feasible strategy to improve regional blood flow in ischemic regions, the optimal delivery mode needs to be established. Here we designed a complex of collagen matrix (CM) and basic fibroblast growth factor (bFGF) and evaluated its proangiogenic effect in ischemic hindlimbs. The bFGF-CM was prepared using lyophilization. The morphology, porosity and toxicity of CM were examined. The bFGF releasing profile and bioactivity of released bFGF were assessed. bFGF-CM was intramuscularly implanted into the rabbit ischemic hindlimb model. Oxygen saturation parameters (OSP) of ischemic hindlimbs was measured to evaluate the extremity perfusion at intervals. Histological examination was performed to evaluate the level of angiogenesis. The CM and bFGF-CM were of identical multiporous structure lacking cytotoxicity. The releasing profile lasted 10 days and the released bFGF remained bioactive. OSP in bFGF-CM group was significantly higher than that in CM, bFGF and ischemic groups at 2 and 4 weeks. The number of capillaries and mature vessels in bFGF-CM group were significantly greater than that in untreated control, CM and bFGF groups. Therefore, bFGF-CM enables the safe and effective long-term release of bFGF with improved angiogenesis in ischemic hindlimbs compared with CM devoid of bFGF.

Efficacy of fragmin/protamine microparticles containing fibroblast growth factor-2 (F/P MPs/FGF-2) to induce collateral vessels in a rabbit model of hindlimb ischemia

The localized delivery of exogenous, angiogenic growth factors such as fibroblast growth factor (FGF)-2 has become a promising alternative treatment of peripheral artery disease (PAD) and critical limb ischemia (CLI). The present study describes the efficacy of fragmin/protamine microparticles containing FGF-2 (F/P-MPs/FGF-2) to promote vessel growth in a rabbit model of hindlimb ischemia. A total of 24 rabbits were used to construct a model of hindlimb ischemia by resection of the left femoral artery. The rabbits were randomly divided into four groups 10 days after surgery (day 0); group A: control (non-treated; 1 mL of phosphate-buffered saline [PBS]); group B: FGF-2 (100 μg FGF-2 in 1 mL PBS)-treated; group C: F/P-MPs (12 mg dried F/P MPs in 1 mL PBS)-treated; and group D; F/P MPs/FGF-2 (100 μg FGF-2 and 12 mg dried F/P MPs in 1 mL PBS)-treated (n = 6 each). The drugs were administered intramuscularly to each group. Blood flow and blood pressure were measured in each group on days 0, 14, and 28. Angiography was performed to assess arteriogenesis on day 28. The number of capillaries on day 28 was determined by direct counting CD31(-) and α-smooth muscle antibody (α-SMA)-positive vessels. Neither death nor wound infection was observed throughout the experiment. The F/P MPs/FGF-2-treated group showed marked improvement in the blood flow ratio, blood pressure ratio, and capillary number in comparison to the control group, FGF-2-treated group, and F/P MPs-treated group. The F/P MPs-treated group showed intermediate improvement in blood flow ratio and capillary number in comparison to the control group and FGF-2-treated group. The F/P MPs/FGF-2-treated group strongly induced functional collateral vessels in the rabbit model of hindlimb ischemia, indicating a possible therapy for PAD.

MR Angiography of Collateral Arteries in a Hind Limb Ischemia Model: Comparison between Blood Pool Agent Gadomer and Small Contrast Agent Gd-DTPA

The objective of this study was to compare the blood pool agent Gadomer with a small contrast agent for the visualization of ultra-small, collateral arteries (diameter<1 mm) with high resolution steady-state MR angiography (SS-MRA) in a rabbit hind limb ischemia model. Ten rabbits underwent unilateral femoral artery ligation. On days 14 and 21, high resolution SS-MRA (voxel size 0.49×0.49×0.50 mm3) was performed on a 3 Tesla clinical system after administration of either Gadomer (dose: 0.10 mmol/kg) or a small contrast agent (gadopentetate dimeglumine (Gd-DTPA), dose: 0.20 mmol/kg). All animals received both contrast agents on separate days. Selective intra-arterial x-ray angiograms (XRAs) were obtained in the ligated limb as a reference. The number of collaterals was counted by two independent observers. Image quality was evaluated with the contrast-to-noise ratio (CNR) in the femoral artery and collateral arteries. CNR for Gadomer was higher in both the femoral artery (Gadomer: 73±5 (mean ± SE); Gd-DTPA: 40±3; p<0.01) and collateral arteries (Gadomer: 18±4; Gd-DTPA: 9±1; p = 0.04). Neither day of acquisition nor contrast agent used influenced the number of identified collateral arteries (p = 0.30 and p = 0.14, respectively). An average of 4.5±1.0 (day 14, mean ± SD) and 5.3±1.2 (day 21) collaterals was found, which was comparable to XRA (5.6±1.7, averaged over days 14 and 21; p>0.10). Inter-observer variation was 24% and 18% for Gadomer and Gd-DTPA, respectively. In conclusion, blood pool agent Gadomer improved vessel conspicuity compared to Gd-DTPA. Steady-state MRA can be considered as an excellent non-invasive alternative to intra-arterial XRA for the visualization of ultra-small collateral arteries.

Transfection of Endothelial Nitric Oxide Synthase Gene Improves Angiogenic Efficacy of Endothelial Progenitor Cells in Rabbits with Hindlimb Ischemia

Background: The present study explored the effect of endothelial nitric oxide synthase (eNOS) gene transfer on the angiogenic potential of ex vivo expanded endothelial progenitor cells (EPCs) in a rabbit model of hindlimb ischemia. Methods: Rabbit peripheral blood EPCs were cultured and transfected with mammalian expression vector pcDNA3.1-eNOS containing full-length human eNOS gene. Ischemia was induced in the right hind limb of three groups of rabbits by ligation of the distal external iliac artery and excision of the common and superficial femoral arteries. In one group of animals, ten days after the surgery, autologous eNOS-EPCs were transplanted intramuscularly in the ischemic limb. Two other groups received an equivalent number of unmodified EPCs or phosphate buffered saline (PBS) respectively. Results: Two weeks after cell transplantation, the in vivo expression of eNOS was detected in limb tissue sections of eNOS-EPCs treated animals. Animals treated with eNOS-EPCs had a significant reduction in ischemic muscle necrosis and inflammation, augmentation in the capillary density (P< 0.05) and angiographic scores demonstrating distal arterial reconstitution and enhanced angiogenesis in comparison to animals transplanted with EPCs or PBS (P< 0.05). Conclusion: We conclude that modification of EPCs by eNOS constitutes an effective strategy to improve the efficacy of EPCs for therapeutic angiogenesis.

Cotransfection of Vascular Endothelial Growth Factor-A and Platelet-Derived Growth Factor-B Via Recombinant Adeno-Associated Virus Resolves Chronic Ischemic Malperfusion: Role of Vessel Maturation

We set out to investigate the ability of cardiotropic adeno-associated viral vector (AAV2.9 = recombinant adeno-associated virus [rAAV]) to induce prolonged expression of vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF)-B in a rabbit hindlimb ischemia model and a pig model of hibernating myocardium. Gene therapy to induce angiogenesis and arteriogenesis has produced mixed results. However, long-acting viruses, such as rAAV, as well as combined induction of angiogenesis and vessel maturation might extend the therapeutic potential. In rabbits, 0.5 x 10(11) particles rAAV.VEGF-A with or without 1 x 10(12) particles rAAV.PDGF-B were retroinfused at day 7 after femoral artery excision. At days 7 and 35, collateral counts and perfusion were determined, each value given as the day 35/day 7 ratio. Capillary-to-muscle fiber ratio was determined at day 35. In pigs, implantation of a reduction stent graft into the circumflex artery led to complete occlusion at day 28. At this time point, retroinfusion of rAAV.VEGF-A (1 x 10(13) particles), rAAV.VEGF-A/PDGF-B (2 x 10(12) and 4 x 10(12) particles, respectively) or mock transfection was performed. Ejection fraction and left ventricular end-diastolic pressure were assessed at days 28 and 56. In rabbits, rAAV.VEGF-A strongly induced angiogenesis (capillary-to-muscle fiber ratio; 1.67 +/- 0.09 vs. 1.32 +/- 0.11 in rAAV.LacZ-treated limbs, p < 0.05), but not collateral growth (125 +/- 7% vs. 106 +/- 7%, p = NS) or perfusion (136 +/- 12% vs. 107 +/- 9%, p = NS). With VEGF-A/PDGF-B cotransfection, collateral growth increased to 146 +/- 9%, perfusion to 163 +/- 8% of the respective day 7 value (p < 0.05). In the pig model, retroinfusion of rAAV.VEGF-A/PDGF-B increased regional myocardial blood flow reserve from 101 +/- 4% (rAAV.Mock) to 129 +/- 8% (p < 0.05), based on collateral growth (3.2 +/- 0.3 in rAAV.Mock vs. 9.0 +/- 0.4 in rAAV.VEGF-A/PDGF-B, p < 0.05), whereas rAAV.VEGF-A did not alter flow reserve (112 +/- 7%) or collateral count (5.2 +/- 0.7). rAAV.VEGF-A/PDGF-B improved ejection fraction (55 +/- 5% vs. 34 +/- 3% in rAAV.Mock, p < 0.05) unlike rAAV.VEGF-A (37 +/- 2%). Retroinfusion of rAAV.VEGF-A alone induces angiogenesis, but fails to enhance collateralization and perfusion, unless PDGF-B is cotransfected. In addition to neovascularization, rAAV.VEGF-A/PDGF-B improves regional and global myocardial function in hibernating myocardium.

Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

To investigate the therapeutic potential of adeno-associated virus (AAV)-mediated expression of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP). Four experimental groups were administered the following AAV vector constructs: rAAV-hVEGF(165)-internal ribosome entry site (IRES)-hBMP-7 (AAV-VEGF/BMP), rAAV-hVEGF(165)-GFP (AAV-VEGF), rAAV-hBMP-7-GFP (AAV-BMP), and rAAV-IRES-GFP (AAV-GFP). VEGF(165) and BMP-7 gene expression was detected using RT-PCR. The VEGF(165) and BMP-7 protein expression was determined by Western blotting and ELISA. The rabbit ischemic hind limb model was adopted and rAAV was administered intramuscularly into the ischemic limb. Rabbit bone marrow-derived mesenchymal stem cells (BMSCs) were cultured and infected with the four viral vectors. The expression of GFP increased from the 7th day of infection and could be detected on the 28th day post-infection. In the AAV-VEGF/BMP group, the levels of VEGF165 and BMP-7 increased with prolonged infection time. The VEGF(165) and BMP-7 secreted from BMSCs in the AAV-VEGF/BMP group enhanced HUVEC tube formation and resulted in a stronger osteogenic ability, respectively. In rabbit ischemic hind limb model, GFP expression increased from the 4th week and could be detected at 8 weeks post-injection. The rAAV vector had superior gene expressing activity. Eight weeks after gene transfer, the mean blood flow was significantly higher in the AAV-VEGF/BMP group. Orthotopic ossification was radiographically evident, and capillary growth and calcium deposits were obvious in this group. AAV-mediated VEGF and BMP gene transfer stimulates angiogenesis and bone regeneration and may be a new therapeutic technique for the treatment of avascular necrosis of the femoral head (ANFH).

SU‐GG‐I‐180: Application of Molecular Imaging in the Monitoring of Angiogenesis of a Hindlimb Ischemia Model

Purpose: Avβ3 integrin expression is a characteristic feature of the angiogenic phenotype of endothelial cells. Targeted molecular imaging of integrins may be a further step forward in the evaluation of specific and early aspects of angiogenesis. The purpose of our study was to investigate the potential of molecular imaging with the aid of a dedicated high resolution γ camera in the rabbit hindlimb ischemia model. Method and Materials: A 99mTc labeled cyclic RGD peptide ([c RGDfk‐His]‐99mTc) was employed for angiogenesis imaging in the New Zealand white rabbits hindlimb ischemia model. Imaging of αvβ3 expression was performed in two animal groups. In group A imaging was employed 3 days and in group B 9 days post femoral artery occlusion (2 rabbits in each group). Each rabbit was injected intravenously with 200 μl of [c RGDfk‐His]‐99mTc (0.5 mCi). Consequently dynamic planar imaging was performed with a high resolution gamma camera with FOV 5×10 cm, equipped with a parallel hole collimator. Static 8 minute images at 80 minutes post injection of the radiotracer were acquired. Results: We have imaged 4 rabbits. An increase of 12.2% was observed in group A in relative radiotracer retention at the ischemic hindlimb compared to control. In group B this relative radiotracer retention was increased to 25.8%. The control limbs showed an increase in the radiotracer retention at day 9 but still much lower compared to ischemic limbs. Moreover, radiotracer accumulation was observed to a region distal to the original area of occlusion, where the newly formed proximal collaterals are located. Conclusion: Our study demonstrates that the phenomenon of angiogenesis proceeds and is maximized within on day 9 following induction of ischemia in a mammalian experimental model. Further studies are deemed necessary in order to establish this conclusion and provide quantitative results.

Abstract No. 46: Computed tomography perfusion imaging in an endovascular model of rabbit hindlimb ischemia: A functional quantification of arteriogenesis

Abstract No. 46: Computed tomography perfusion imaging in an endovascular model of rabbit hindlimb ischemia: A functional quantification of arteriogenesis

Efficient revascularization by VEGF administration via heparin-functionalized nanoparticle–fibrin complex

We investigated the angiogenic bioactivity and therapeutic angiogenic effect of vascular endothelial growth factor (VEGF) administration by the heparin-functionalized nanoparticle–fibrin gel complex. The markedly increased bioactivity was observed by the VEGF-loaded nanoparticle–fibrin gel complex, compared to the VEGF-loaded fibrin gel, the nanoparticle–fibrin gel complex without VEGF, or fibrin gel (control) in terms of the capillary density in a mouse subcutaneous implantation model. Furthermore, the VEGF-loaded nanoparticle–fibrin gel complex significantly enhanced the therapeutic angiogenic effect in a rabbit ischemic hind limb model: the noticeable increase in the recovered calf blood pressure, the angiographic score, and the density of collaterals, as well as the stable maintenance of the organized collaterals, compared to the VEGF-loaded fibrin gel. These results show the enhanced angiogenic potential of VEGF administration by the proposed heparin-functionalized nanoparticle–fibrin gel complex.

Synergistic effects of FGF-2 and PDGF-BB on angiogenesis and muscle regeneration in rabbit hindlimb ischemia model

Combinatorial strategy has been used in therapeutic angiogenesis in animal models of peripheral arterial disease (PAD) and coronary artery disease for decades. Previous studies have shown that basic fibroblast growth factor (FGF-2) and platelet-derived growth factor BB (PDGF-BB) proteins together establish functional and stable vascular networks on mouse corneal and also in animal model of hindlimb ischemia. However, the short half life of protein by single injection is not sufficient to achieve effective dosage, repeated and prolonged injection causes systemic toxicity. Here we study the synergistic effects of FGF-2 and PDGF-BB by intramuscular injection of naked plasmid DNA on therapeutic angiogenesis in rabbit model of hindlimb ischemia. We found that transient delivery of FGF-2 and PDGF-BB naked DNA together resulted in greater increases in capillary growth, collateral formation and popliteal blood flow compared with control and single gene delivery. Our data provided novel evidence of beneficial effects of DNA-based FGF-2 and PDFG-BB on muscle repair after ischemic injury. These findings reveal an alternative therapeutic approach in the treatment of ischemic diseases and even in muscular disorders.

Vascular Regeneration by Repeated Infusions of Basic Fibroblast Growth Factor in a Rabbit Model of Hind-Limb Ischemia

Regenerative therapy is a new treatment of vascular occlusive diseases. The purpose of this study was to examine the advantages of repeated low-dose growth factor infusions compared with a single high-dose infusion in an ischemic hind-limb rabbit model. Thirty-two rabbits were used to construct an ischemic hind-limb model by resection of the left femoral artery. For the vascular regenerative method, basic fibroblast growth factor (bFGF) was impregnated into 3 mg of gelatin microspheres 30 microm in diameter and a reservoir system was implanted in the left femoral artery for infusion. The gelatin microspheres were then infused into the left internal iliac artery via the reservoir system. The rabbits were divided into three groups according to different infusion methods: single high-dose infusion, repeated low-dose infusions, and saline (control). Therapeutic effects were evaluated by thigh temperature, blood pressure, blood flow, angiography, and pathology. There was no significant difference between the two infusion methods in thigh temperature, blood pressure, blood flow, angiography, and pathology. In pathologic analyses at 2 and 4 weeks, both the repeated low-dose infusion and the single high-dose infusion groups showed significant differences in the number of vessels when compared with the control group. The efficacy of repeated bFGF infusions for neovascularization via the reservoir method was investigated. Despite the pathologic confirmation of neovascularization, there was no significant difference in treatment effect by the two administration methods.