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As a form of therapeutic angiogenesis, we sought to investigate the safety and efficacy of a sustained-release system of basic fibroblast growth factor (bFGF) using biodegradable gelatin hydrogel in patients with critical limb ischemia (CLI). We conducted a phase I-IIa study that analyzed 10 CLI patients following a 200-μg intramuscular injection of bFGF-incorporated gelatin hydrogel microspheres into the ischemic limb. Primary endpoints were safety and transcutaneous oxygen pressure (TcO2) at 4 and 24 weeks after treatment. During the follow-up, there was no death or serious procedure-related adverse event. After 24 weeks, TcO2 (28.4 ± 8.4 vs. 46.2 ± 13.0 mmHg for pretreatment vs after 24 weeks, p < 0.01) showed significant improvement. Regarding secondary endpoints, the distance walked in 6 min (255 ± 105 vs. 318 ± 127 m, p = 0.02), the Rutherford classification (4.4 ± 0.5 vs. 3.1 ± 1.4, p = 0.02), the rest pain scale (1.7 ± 1.0 vs. 1.2 ± 1.3, p = 0.03), and the cyanotic scale (2.0 ± 1.1 vs. 0.9 ± 0.9, p < 0.01) also showed improvement. The blood levels of bFGF were within the normal range in all patients. A subanalysis of patients with arteriosclerosis obliterans (n = 7) or thromboangiitis obliterans (Buerger's disease) (n = 3) revealed that TcO2 had significantly improved in both subgroups. TcO2 did not differ between patients with or without chronic kidney disease. The sustained release of bFGF from biodegradable gelatin hydrogel may offer a safe and effective form of angiogenesis for patients with CLI.

Objective: The objective of this investigation was to establish the effectiveness of sustained-release platelet-rich plasma (PRP) on perfusion and neovascularization in diabetic murine hind limb ischemia., Methods: After surgery in streptozotocin-induced diabetic mice, the mice were randomly assigned to the following 4 experimental groups: control (C), 100 μl of the sustained-release form of platelet-poor plasma (PPP), 100 μl of the solution form of PRP (PRP-sol), and 100 μl of the sustained-release form of PRP (PRP-sr). Endpoint evaluations were: blood perfusion by laser Doppler perfusion imaging (LDPI), vascular density by anti-vWF, and mature vessel density by anti-smooth muscle actin antibody., Results: This study demonstrated that a sustained release of PRP increases the perfusion of ischemic tissue as measured by LDPI (57 ± 12; 56 ± 9; 72 ± 7, and 98 ± 4 for the C, PPP, PRP-sol, and PRP-sr groups, respectively; p < 0.05), capillary density (151 ± 16; 158 ± 12; 189 ± 39, and 276 ± 39 for groups C, PPP, PRP-sol, and PRP-sr, respectively; p < 0.05), and mature vessel density (28 ± 2; 31 ± 3; 52 ± 10, and 85 ± 13 for the C, PPP, PRP-sol, and PRP-sr groups, respectively; p < 0.05)., Conclusion: A sustained release of PRP containing potent angiogenic growth factors restores blood perfusion by stimulating angiogenesis and arteriogenesis., (Copyright © 2010 S. Karger AG, Basel.)

Background: While single growth factor has limitation to induce optimal neovascularization, platelet-rich plasma (PRP) is an autologous reserver of various growth factors. However, little is known about the mechanism of PRP-related neovascularization.The objective of this investigation was to characterize the angiogenic and growth factor content of PRP and to determine, in vitro, its effect on endothelial cell proliferation. Additionally, this experiment sought to determine the effectiveness of different compositions of PRP (solution versus sustained release) on perfusion and neovascularization in a murine model of hind limb ischemia., Methods: Different growth factors were measured by enzyme-linked immunosorbent assay (ELISA). In vivo study, we used gelatin hydrogel as a sustained release carrier for growth factors in PRP. We induced hind limb ischemia by excising right femoral artery in wild type C57BL6 mice. After surgery, mice were randomly assigned to four experimental groups; control (C), 100 muL of sustained release form of platelet-poor plasma (PPP), 100 muL of solution form of PRP (PRP-sol), 100 muL of sustained release form of PRP (PRP-sr); each formulation was administered via an intramuscular injection to the ischemic hind limb. Endpoint evaluations were blood perfusion by laser Doppler perfusion image, vascular density by anti Von Willebrand factor (vWF), and mature vessel density by anti smooth muscle actin (SMA) antibody. Green fluorescent protein (GFP+) transgenic mice were generated by transplantation of bone marrow derived mononuclear cells to wild type C57BL6 mice, and finally CD34+ cell in the ischemic site of transgenic mice was detected by staining with anti-CD34 antibody., Results: In vitro study showed that PRP containing different growth factors induces endothelial cell proliferation and capillary tube formation. In vivo study demonstrated that sustained release of PRP increased perfusion of ischemic tissue as measured by laser Doppler perfusion imaging (LDPI) (57 +/- 12, 56 +/- 9, 72 +/- 7, 98 +/- 4 for groups C, PPP, PRP-sol, and PRP-sr, respectively; P < .05); capillary density (151 +/- 16, 158 +/- 12, 189 +/- 39, 276 +/- 39 for groups C, PPP, PRP-sol, and PRP-sr, respectively, P < .05) and mature vessel density (28 +/- 2, 31 +/- 3, 52 +/- 10, 85 +/- 13 for groups C, PPP, PRP-sol, and PRP-sr, respectively, P < .05) . Sustained release PRP also increases CD34+ cells in the ischemic site of transgenic mice (6 +/- 3 vs 18 +/- 5/mm(2) for groups control and PRP-sr respectively, P < .05)., Conclusion: Sustained release of PRP containing potent angiogenic growth factors restores blood perfusion presumably by stimulating angiogenesis, arteriogenesis, as well as vasculogenesis in the mouse hind limb ischemia., Clinical Relevance: PRP is a natural reserver of various growth factors that can be collected autologously and is costeffective. Thus for clinical use, no special considerations concerning antibody formation and infection risk are needed.Some clinical devices to automatically prepare PRP are available at present. PRP are consistently being used clinically inthe department of orthopedics and plastic surgery (oral, maxillary facial) for a long time. On the basis of researchevidence, some publications have reported positive results in either bone or soft tissue healing. However, some researchconcludes that there is no or little benefit from PRP. This is likely due to faster degradation of growth factors in PRP sincesome authors suggest using sustained release form of PRP to deliver optimal effect of PRP. Gelatin hydrogel is also beingused clinically as a slow, sustained release of carrier for growth factors in our center recently.

Background: Although intravenous administration of prostaglandin E(1) (PGE(1)) is commonly used in the treatment of peripheral arterial disease, it rapidly becomes inactivated in the lung. Whether local administration of sustained-release (SR) PGE(1) enhances neovascularization in murine hindlimb ischemia was investigated., Methods and Results: Poly lactide-co-glycolide (PLGA) microspheres were the 4-week SR carrier of PGE(1). C57BL/6 mice with unilateral hindlimb ischemia were randomly treated as follows: no treatment (Group N); single administration of 100 microg/kg PGE(1) solution (Group L) into the ischemic muscles; daily systemic administration of PGE(1) for 2 weeks at a total dose 100 microg/kg (Group S); and single administration of PGE(1)-100 microg/kg-loaded PLGA (Group P100) into the ischemic muscles. The blood perfusion in Group P100 was higher than in Groups N, L and S (ischemic/nonischemic blood perfusion ratio 88 +/-11% vs 73 +/-11% (P<0.01), 77 +/-9% (P<0.05), 79 +/-11% (P<0.05), respectively). Vascular density and alphaSMA-positive-vessel density in Group P100 were higher than in Groups N, L and S (vascular density (vessels/m(2)): 241 +/-39 vs 169 +/-49 (P<0.01), 169 +/-54 (P<0.01), 201 +/-42 (P<0.05), respectively; alphaSMA-positive-vessel density (vessels/m(2)): 34 +/-10 vs 18 +/-6 (P<0.01), 21 +/-11 (P<0.01), 22 +/-10 (P<0.01), respectively), Conclusions: Local administration of a single dose of SR PGE(1) enhances neovascularization in mice hindlimb ischemia more efficiently than daily systemic administration.

Background: Basic fibroblast growth factor (bFGF) is a potent mitogen; however, diabetes mellitus might impair its angiogenic property. Prostaglandin E1 (PGE1) is a potent vasodilator and improves endothelial function. Thus, PGE1 could potentiate the angiogenic properties of bFGF in patients with diabetes mellitus., Methods and Results: Streptozotocin-induced diabetic mice with unilateral hindlimb ischemia were randomly treated as follows: no treatment, 0.2 microg of PGE1, 10 microg of bFGF, and combined administration of PGE1 and bFGF. Blood perfusion was evaluated by the ratio of ischemic-to normal-limb blood perfusion. Four weeks after the treatment, the combined administration of bFGF and PGE1 increased the blood perfusion ratio as compared with single bFGF or PGE1 (77+/-10% vs 56+/-10% and 58+/-10%; p < 0.05, respectively). A histological evaluation showed that vascular density in the combined therapy was higher than single bFGF or PGE1 (418+/-59 vs 306+/-69 and 283+/-71 vessels/mm2; p < 0.01, respectively); the maturity in combined therapy was also higher than single bFGF or PGE1 (46+/-14 vs 30+/-14 and 28+/-6 vessels/mm2; p < 0.01, respectively)., Conclusions: PGE1 potentiated the impaired angiogenic properties of bFGF in diabetic murine hindlimb ischemia. This new strategy might contribute to more effective therapeutic angiogenesis for ischemic limb in patients with diabetes.

Background: The combined treatment of sustained-release basic fibroblast growth factor (Sr-bFGF) and a 5-hydroxytryptamine(2A) blocker, sarpogrelate, was evaluated to see whether it reversed the impaired collateral circulation in diabetic (DM) mouse hindlimb ischemia., Method and Results: Diabetic and normal mice with ischemic hindlimb were randomly assigned to 1 of 5 experimental groups (no treatment, sarpogrelate 50 mg . kg(-1) . day(-1), 20 microg or 50 microg Sr-bFGF and a combined treatment of 20 microg Sr-bFGF and sarpogrelate), and treated for 4 weeks. Tissue blood perfusion (TBP), vascular density (angiogenesis) and the number of mature vessels (arteriogenesis) were checked by the use of standard methods. Although angiogenesis was comparable (161+/-14 vs 154+/-12 vessels/mm(2)), the laser Doppler perfusion image index (LDPII) (0.43+/-0.11 (SD) vs 0.63+/-0.08, p<0.05) and arteriogenesis (8+/-3 vs 12+/-4 vessels/mm(2), p<0.05) were significantly lower in DM mice than those in normal mice. The dose of Sr-bFGF for the sufficient number of mature vessels (>or=45 vessels/mm(2)) and LDPII (>or=0.9) was 20 microg for the normal mice, and 50 microg for the DM mice, which was reduced with the aid of sarpogrelate. Conclusions A combined therapy of Sr-bFGF and sarpogrelate is effective for neovascularization to reverse the impaired arteriogenesis and TBP in DM mice.

Recently we have developed new sustained release system of basic fibroblast growth factor (bFGF) using gelatin hydrogel as a carrier. Using this system, we examined the effect of topical sustained release of bFGF on angiogenesis and tissue blood perfusion in a rabbit model of hind limb ischemia. Thirty-two rabbits underwent excision of right femoral artery under general anesthesia. Two weeks later the rabbits were randomized into four groups (n = 8 each): no treatment, intramuscular injection of gelatin hydrogel alone, and intramuscular injection of gelatin hydrogel incorporating 30 microg and 100 microg of bFGF. Four weeks after each treatment, selective angiography, tissue blood flowmetry using laser Doppler perfusion imaging, and histological examination of thigh muscle were performed. In groups treated with bFGF incorporating gelatin hydrogel, tissue blood flow, number of arterioles, and vascular density were significantly increased in a dose-dependent manner 4 weeks after the treatment. Serum concentrations of bFGF and vascular endothelial growth factor were not elevated 4 weeks after the treatment. In conclusion, sustained release of bFGF using gelatin hydrogel augmented angiogenesis and improved tissue blood flow after excision of the femoral artery.

Background: Whether the combined treatment with sustained-release basic fibroblast growth factor (bFGF) and heparin enhances neovascularization in hypercholesterolemic mouse hindlimb ischemia was investigated., Methods and Results: Wild-type C57BL/6 and low density lipoprotein receptor-deficient mice were assigned to 1 of the following 4 experimental groups and treated for 2 weeks after femoral artery extraction: group N, no treatment; group H, daily subcutaneous injection of heparin calcium; group F, single intramuscular injection of the sustained-release bFGF microspheres; and group FH, combined treatment with sustained-release bFGF and heparin. Among the wild-type mice at 4 weeks after femoral artery extraction, the laser Doppler perfusion image index (LDPII) in groups H, F, and FH was significantly higher than that in group N. The vascular density in group FH was the highest among the 4 groups. The maturation index in the 3 treated groups was significantly higher than that in group N. Among the hypercholesterolemic mice, the LDPII in group FH was significantly higher than that in the other 3 groups. The vascular density and maturation index in group FH were the highest among the 4 groups., Conclusions: Combined treatment with sustained-release bFGF and heparin enhanced neovascularization in the hypercholesterolemic hindlimb ischemia model.

Recently we have developed new sustained release system of basic fibroblast growth factor (bFGF) using gelatin hydrogel as a carrier biomaterial. We examined the effect of topical sustained release of bFGF on blood flow recovery and tissue regeneration in various animal models of cardiovascular area. These results revealed effectiveness and safety of this therapy. We evaluated the effect of sustained release of bFGF for not only normal but also diabetes model. Slow release system of bFGF from biodegradable gelatin hydrogel prevents bFGF glycation and its combination with 5-hydroxytryptamine blocker has shown sufficient neovascularization in diabetic limb ischemia. The method may provide a more effective therapeutic angiogenesis in patients with diabetes. Clinical trial of therapeutic angiogenesis for severe hindlimb ischemia has already started.

Background: The effectiveness of sustained-release basic fibroblast growth factor (bFGF) in potentiating arteriogenesis and angiogenesis was evaluated, as well as determining whether chronic oral administration of sarpogrelate, a serotonin blocker, would further increase collateral blood flow in the rabbit hindlimb following surgical induction of ischemia by femoral artery extraction., Methods and Results: Two weeks after femoral artery removal, the rabbits were assigned to 1 of 4 experimental groups and treated for 4 weeks: group A, no treatment; group B, supplemented with diet containing sarpogrelate; group C, single intramuscular injection of sustained-release form of bFGF microspheres; group D: combined treatment with sustained-release bFGF and sarpogrelate. Endpoint measurements performed at 6 weeks found that the ischemic hindlimb blood flow was significantly improved in the rabbits that received sustained-release bFGF, with a further significant improvement in those with the additional administration of sarpogrelate. Angiographic assessment revealed augmented density of collateral vessels in the medial thigh region in the rabbits given the combined treatment., Conclusions: The findings demonstrate that sustained-release bFGF stimulated the development of collateral vessels, and additional administration of sarpogrelate produced a further improvement in hindlimb blood flow in the rabbit hindlimb ischemia model.

Objective: The present study investigated whether the simultaneous application of basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) enhances blood vessel formation in murine ischemic hindlimb compared with bFGF or HGF applied alone., Methods: Unilateral hindlimb ischemia was created in C57BL/6 mice. Hindlimb blood flow was evaluated by laser Doppler perfusion image index (LDPII) (ratio (%) of ischemic-to-normal-limb blood flow). The ischemic limbs were treated with bFGF and HGF separately, or bFGF and HGF together, and their therapeutic effects were assessed. Collagen microspheres (CM) were used as a sustained-release carrier for bFGF and HGF., Results: A single intramuscular injection of 5 microg or less of bFGF-incorporated CM (bFGF/CM) into the ischemic limb did not significantly increase the LDPII compared with the control (no treatment) 4 weeks after the treatment. Similarly, 20 microg or less of HGF/CM did not increase LDPII. Based on these results, we compared the dual release of CM incorporating 5 microg of bFGF and 20 microg of HGF with either the single release of 5 mug of bFGF/CM alone or 20 microg of HGF/CM alone. The LDPII of the dual release (94.2% +/- 10.9%) was higher than either single release (51.2% +/- 5.8% or 52.5% +/- 8.0%, P < .01). Furthermore, the LDPII in the dual release (94.2% +/- 10.9%) was equivalent to that with 80 microg of bFGF/CM (95.1% +/- 7.6%) alone or 80 microg of HGF/CM (92.8% +/- 7.6%) alone. A histologic evaluation at 4 weeks showed capillary density in the dual release (868 +/- 173 vessels/mm(2)) was higher than that in either single release (204 +/- 68 vessels/mm(2) or 185 +/- 98 vessels/mm(2) , P < .01). The percentage of mature vessels assessed by alpha-smooth muscle actin staining was also higher in the dual release (43.8% +/- 7.8% vs 9.5% +/- 3.0% or 11.7% +/- 3.8%, respectively; P < .01)., Conclusions: This study demonstrates that the sustained dual release of a lower dose of bFGF and HGF from a carrier matrix can achieve equivalent blood perfusion recovery and more mature vasculature in the ischemic limb than a higher dose of bFGF or HGF alone. This approach may be a highly promising strategy for the future treatment of peripheral vascular disease.

We review our studies on therapeutic angiogenesis using basic fibroblast growth factor (bFGF) released in a controlled manner from biodegradable gelatin hydrogel (GH). The bFGF-GH was intramuscularly injected in rabbits with limb ischemia. The group treated with bFGF showed an increase in tissue blood flow under laser Doppler imaging and histology showed a greater vascular density compared with controls. Also, bFGF-GH was subepicardially injected into old heart infarcts in rats. In the group treated with bFGF, improved left ventricular function was shown by echocardiography and cardiac catheterization, increased regional blood flow in the peri-infarct area was detected by pinhole single-photon emission computed tomography using (201)Tl, and increased vascular density was demonstrated by histology. In rabbits with acute myocardial infarction, the heart was wrapped with the omentum (including the gastroepiploic artery) and a bFGF-GH sheet was applied. Postoperative assessment revealed rich communication from the gastroepiploic artery to the coronary artery and improved cardiac function. The controlled release of bFGF was effective for both limb and heart ischemia and is considered to be suitable for clinical use because its application in animals was feasible and safe with minimal invasiveness.