Microvascular Research 80 (2010) 10–17 Contents lists available at ScienceDirect Microvascular Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y m v r e Regular Article Synergistic effects of FGF-2 and PDGF-BB on angiogenesis and muscle regeneration in rabbit hindlimb ischemia model Jie Li a,b, Yuquan Wei a, Kang Liu a,b, Chuang Yuan a, Yajuan Tang a, Qingli Quan a,b, Ping Chen a, Wei Wang a,b, Huozhen Hu a,b,⁎, Li Yang a,⁎ a b State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Sichuan, China Laboratory of Medic-Biology and Cell-Biology, College of Life Science, Sichuan University, Sichuan, China a r t i c l e i n f o Article history: Received 17 August 2009 Revised 3 December 2009 Accepted 10 December 2009 Available online 4 January 2010 Keywords: Basic fibroblast growth factor Platelet-derived growth factor Naked plasmid DNA Therapeutic angiogenesis Muscle regeneration a b s t r a c t 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. © 2010 Published by Elsevier Inc. Introduction Peripheral arterial disease (PAD) is an increasing health-threatening disorder, which can be caused by hypertension, hyperlipoidemia, hypercholesterolaemia, diabetes, smoking and population aging (Selvin and Erlinger, 2004). It is associated with diminution in quality of life, elevated risk of cardiovascular and cerebrovascular disease and increased mortality (Selvin and Erlinger, 2004; Murabito et al., 2003). However, interventional or surgical procedures are not available in some conditions due to the complexity of the disease. Therapeutic angiogenesis–artificially inducing endogenous angiogenesis and collateralization in ischemic area by delivering therapeutic growth factors, genes or cells–has been a promising approach for the treatment of PAD and other ischemic diseases. However, the optimal therapeutic approach has not been developed. Basic fibroblast growth factor (FGF-2) is a powerful stimulator of angiogenesis in vivo and is known to induce the proliferation, migration and differentiation of many cell types–including endothelial cells, smooth muscle cells (SMC), pericytes and fibroblasts (Galzie et al., 1997). The effects of FGF-2 on therapeutic angiogenesis were confirmed in animal model of myocardium ischemia in early 1990s (Harada et al., 1994; Yanagisawa-Miwa et al., 1992). Platelet⁎ Corresponding authors. L. Yang is to be contacted at fax: +86 28 85164060. H. Hu, fax: +86 28 85503520. E-mail addresses: huhuozhen@163.com (H. Hu), yl_tracy@hotmail.com (L. Yang). 0026-2862/$ – see front matter © 2010 Published by Elsevier Inc. doi:10.1016/j.mvr.2009.12.002 derived growth factor BB (PDGF-BB) is a mitogenic and chemotactic factor for vascular smooth muscle cells (VSMC), monocytes and granulocytes (Risau et al., 1992). A pilot study by Cao et al. (2003) has shown that a combination therapy with recombinant proteins PDGF-BB and FGF-2 successfully induced stable vascular networks in mouse corneal and animal model of hindlimb ischemia. Delivery of a single angiogenic factor is insufficient to induce functionally active blood vessels and even cause serious complications (Carmeliet, 2000; Celletti et al., 2001; Thurston et al., 1999). Thus, exploring a combinatorial strategy has become the major focus of therapeutic angiogenesis for PAD and other ischemic diseases. Positive results have already been reported in many laboratories (Cao et al., 2003; Asahara et al., 1995; Chen et al., 2007; Niagara et al., 2004; Shyu et al., 2003). Several delivery techniques have been used in therapeutic angiogenesis. Direct administration of growth factor protein yielded positive results (Bauters et al., 1994; Unger et al., 1994; Takeshita et al., 1994). However, the short half life of protein is insufficient to achieve effective dosage, repeated and prolonged injection could cause systemic toxicity. The adenoviral vector-based delivery approach, though effective, induces cytotoxic immune reactions. Naked DNA-based delivery system, thus, has become an alternative approach to obviate immunological concerns associated with protein and adenoviral vector. Interestingly, skeletal muscle has been reported to take up and express naked plasmid DNA (Davis et al., 1993). Based on these findings, we investigate whether intramuscular injection of J. Li et al. / Microvascular Research 80 (2010) 10–17 naked plasmid DNA carrying PDGF-BB and FGF-2 genes can establish stable mature vessels and improve collateral blood flow in rabbit hindlimb ischemia model without causing side effects. 11 angiographic score was calculated for each film as the ratio of grid intersections crossed by opacified arteries divided by the total number of grid intersections in the medial thigh. This analysis was performed by a single observer blinded to the treatment regimen. Materials and methods Histologic analysis and immunohistochemical stainings Plasmid vectors The plasmid vectors were constructed containing human FGF-2 (called pFGF2 below) or PDGF-BB (pPDGF) or both FGF-2 and PDGFBB (pPDGF/FGF2) gene, encoding secreted proteins. pNull is the same backbone as the vectors above but contains no gene. Animal model We used a rabbit ischemic hindlimb model that has been described previously (Takeshita et al., 1994). All protocols were approved by Institutional Animal Care and Use Committee. A total of 50 male New Zealand White rabbits weighing 3–3.5 kg were anesthetized with sodium pentobarbital (40 mg/kg). Under sterile surgical conditions, the femoral artery was completely excised from its proximal origin as a branch of the external iliac artery to the point distally where it bifurcates to form the saphenous and popliteal arteries. Consequently, blood flow to the ischemic limb is dependent upon collateral vessels, which may originate from the internal iliac artery. After removal of the femoral artery, five different sites in adductor thigh muscle and semimembranous muscle received direct injections with plasmid (500 μg in 1 ml) with the use of a 1-ml syringe and a 25-gauge needle. After gene delivery, the incision was closed in three layers. All rabbits were closely monitored and received antibiotics. Experimental procedures All the rabbits were randomly divided into five groups (n = 10 each group): PBS, pNull, pPDGF, pFGF2 and pPDGF/FGF2. PBS and pNull groups were referred as control. Gene transfer (GT) was performed immediately after surgery (day 0). Two different time points were chosen to evaluate the effects of growth factors on angiogenesis and arteriogenesis. Rabbits were scheduled for euthanasia at 1 week (n = 5, each group) and 4 weeks (n = 5, each group) after GT, with an overdose of intravenous pentobarbital and potassium chloride. Measurements of popliteal blood flow were performed 0, 7, 14, 21 and 28 days after surgery. Collateral development and capillary density were studied separately with angiography and immunohistochemistry 1 and 4 weeks after GT. Tissue specimens were obtained as transverse sections from the adductor muscle and the semimembranous muscle of the ischemic limb of each animal at the time of death (days 7 and 28). Muscle samples were immersion-fixed in 4% PFA (pH 7.4) for more than 24 h, embedded in paraffin, then cut into 5-μm-thick sections. The staining of endothelial cells was performed using a mouse monoclonal antibody (mAb) against CD31 (DAKO, Glostrup, Denmark; Dilution 1:50). A total of 20 different fields from the two muscles were randomly selected, and capillary density (the capillary/muscle fiber ratio) was measured. The pericytes and VSMCs were immunostained with a mAb against α-smooth muscle actin (SMA; Neomarkers, Fremont, CA, USA). Collaterals with diameter greater than 10 μm were counted manually from 10 different fields (×20) selected from each sample. The results were expressed as the number of collateral arteries per field. Hematoxylin and eosin (H&E) staining was performed for morphological analysis. The areas of normal, regeneration and necrosis were determined as percentages of the total muscle area of the H&E staining sections and quantified using the ImageJ software (NIH Software). Statistical analysis Data were presented as mean ± standard error (m ± SE). Statistical evaluation of the results was performed using unpaired Student's ttest and one-way ANOVA for comparisons between two means. A value of p b 0.05 was considered statistically significant. Results FGF2 and PDGF-BB increase collateral-dependent blood flow At day 0, the Doppler flow signal from the popliteal artery could not be detected immediately after surgery in most rabbits of each group. Therefore, the blood flow in all groups was referred to as zero (Fig. 1), indicating successful inducement of ischemia in hindlimb. The blood flow in the popliteal artery was markedly improved in treated groups (pPDGF/FGF2, pFGF2 and pPDGF group) on day 7 and peaked at the end of follow-up (day 28). However, in the control groups, Arterial blood flow measurement by Doppler ultrasound Collateral vessel-dependent blood flow (ml/min) in the popliteal artery of ischemic limb was measured at rest with a PHILIPS HDI 5000 Sono CT ultrasound instrument (Philips Medical Systems, the Netherlands), using a CL 15-7 transducer. Three separate measurements were done for each rabbit, and the results were averaged. Angiography and quantification of collateral growth One and four weeks after GT, rabbits were reanesthetized. Contrast medium (Omnipaque, 300 mg I/ml, 4 ml; GE, Shanghai, China) was power-injected using an automated injector (Medrad, PA, USA) at a rate of 2 ml/s via a 4F catheter placed above the aortic bifurcation. Serial images were recorded at a rate of three films per second, and the image representing the best arterial filling was chosen for analysis. All of the above-described procedures were completed without the use of heparin. Quantitative angiographic analysis of collateral vessel development was performed as follows. A composite of 5 × 5 mm grids was placed over the medial thigh area of angiogram. The Fig. 1. Collateral artery-dependent blood flow (ml/min) at rest in the popliteal artery of the ischemic limb measured with Doppler ultrasound. Greater blood flow was observed in pPDGF/FGF2-treated limbs than any other groups. ⁎ p b 0.05 and ⁎⁎ p b 0.001 vs. PBS control. 12 J. Li et al. / Microvascular Research 80 (2010) 10–17 the curves were flat and seemed to stop increasing at day 28. Quantitatively, pPDGF/FGF2, pFGF2 and pPDGF caused 2.0-, 1.8- and 1.3-fold increases, respectively, as compared with PBS control. In PDGF/FGF2-treated group, the increase of popliteal blood flow was more pronounced than that in single gene-treated groups. There was no difference between PBS and pNull control. FGF2 and PDGF-BB enhance angiogenesis Angiograms at two time points after GT (days 7 and 28) in all groups were shown in Figs. 2 (a–j). For the entire femoral artery was completely excised, collateral vessels mainly originated from the internal iliac artery as expected. The development of collateral vessels in pNull and PBS groups progressed slightly at day 7 after surgery. In contrast, a remarkable rise could be noted after pPDGF/FGF2, pFGF2 or pPDGF infusion. Up to 28 days following GT, there were much more large arteries formed in the medial thigh of ischemic hindlimb in treated groups compared with control, distributed in a broader area of ischemic muscles. Quantitative analysis of collateral vessel development was performed as described above. As shown in Fig. 2k, the difference in the number of angiographically visible collateral vessels (angiographic score) between treated and control groups was significant 7 days after GT and further intensified at day 28, consistent with the Doppler measurement of collateral-dependent blood flow. Although a progressive increase in angiographic score was observed in all groups from days 7 to 28, the increase calculated for the treated groups was significantly higher than control (p b 0.001 in pPDGF/ FGF2, pFGF2 and pPDGF groups, 0.001 b p b 0.05 in control). In contrast to single angiogenic factor, PDGF-BB and FGF-2 together caused higher angiographic score at both 7 and 28 days after GT. Stimulation of capillary, arteriolar and collateral growth As studied in transversal CD31-immunostained histological sections of the semimembranous and adductor muscle (Figs. 3a–f), both combinatorial and single gene treatments induced remarkable Fig. 2. Angiograms of the experimental limbs in PBS (a, b), pNull (c, d), pPDGF (e, f), pFGF2 (g, h) or pPDFG/FGF2 (i, j) group 7 days (a, c, e, g, i) and 28 days (b, d, f, h, j) after femoral artery excision. (k) The number of angiographically visible collateral vessels (angiographic score) was analyzed as described in Materials and methods. Values are shown as mean ± SEM. ⁎ p b 0.05 and ⁎⁎ p b 0.001 vs. PBS control. J. Li et al. / Microvascular Research 80 (2010) 10–17 13 Fig. 3. Immunohistochemical analysis of capillary in normal (a) and in ischemic muscle tissue treated with PBS (b), pNull (c), pPDGF (d), pFGF2 (e) or pPDFG/FGF2 (f) 7 days after GT. Capillaries were stained positive for CD31 (brown stain) as denoted by arrows. Scale bar, 50 μm. Quantitative analysis of capillaries/myocytes ratio in ischemic hindlimb muscles (g). ⁎p b 0.05 and ⁎⁎p b 0.001 vs. normal. angiogenic effect 7 days after GT. The capillary density (capillary/ muscle fiber ratio) was significantly higher in all treated groups than control and normal muscle tissue from intact limb. Especially in pPDGF/FGF2 group, the ratio was 3.3-fold as compared to normal at day 7, but reduced by 46.7% at day 28. The same phenomenon could be observed in single gene-treated groups that the greatest angiogenic effect appeared on day 7 after GT, but almost fell down to the baseline at the end of follow-up. The ratio also increased slightly in pNull and PBS control at day 7, but with no statistical significance (Fig. 3g). To further validate the effect of these growth factors on stimulation of collateral growth, we carried out immunohistochemical analysis using an antibody against SMA (Figs. 4a–f). Collateral numbers were measured from SMA-positive vessels (Fig. 4g). Although the SMApositive stained vessels were present in muscle tissues treated with growth factors and control, combination of PDGF-BB and FGF-2 delivery resulted in a marked increase in the number of arterioles and relatively large arterial vessels with thickened pericyte coverage parallel to the angiographic analysis. However, in single gene-treated 14 J. Li et al. / Microvascular Research 80 (2010) 10–17 Fig. 4. Anti-SMA staining of histological sections of ischemic hindlimb muscle tissue treated with PBS (b), pNull (c), pPDGF (d), pFGF2 (e) or pPDFG/FGF2 (f) 28 days after GT and in normal tissue (a). Arrows point to positive stained arterial vessels. Scale bar, 50 μm. Quantitative analysis of arterioles and large arterial vessels in ischemic limbs (g). ⁎p b 0.05 and ⁎⁎p b 0.001 vs. normal. groups and especially in control groups, the number of SMA-positive vessels was apparently inferior to that of pPDGF/FGF2-treated group. These results provide compelling evidence that PDGF-BB and FGF-2 synergistically stimulated both capillary sprouting and collateral growth which gave rise to stable arterial vascular networks, but did not further enhance angiogenesis. PDGF-BB and FGF-2 induce muscle regeneration Histological analysis revealed that an enhanced regeneration was detected in pPDGF/FGF2-treated samples after ischemic injury (Fig. 5). The normal muscle was identified by myotubes with uniform caliber size and peripheral nuclei with small and oral structure. The muscle from pNull group 28 days after ischemia was characterized by numerous necrotic fibers, infiltration of inflammatory cells and invasion by connective tissue. Only a few regenerating fibers encircled the residual necrotic myofibers, and a massive fiber loss with adipose substitution was evident. In contrast, the effect on promotion of muscle repair after damage was prominent in pPDGF/FGF2-treated samples. The area of necrosis was significantly decreased, and regenerating area which identified as myofibers with small size and centrally located nuclei was predominant (Fig. 6). It is worth noticing that the regenerating area seemed preferentially adjacent to vessels. In addition, the nuclei in treated muscles appeared to be round and enlarged in comparison of normal and control samples. Discussion Clinical trails based on single gene delivery did not yield satisfactory results, leading to research focused on developing combinatorial strategy in therapeutic angiogenesis, which has already been adopted in treatment of many human diseases on animal models with cancer, leucocythemia and ischemic diseases et al. Previously published data have indicated that single angiogenic agent resulted in deleterious effects. For example, VEGF overexpression led to the formation of angioma-like fragile capillaries in animals and transient edema in human patients (Lee et al., 2000; Schwartz et al., 2000). In the current study, we found that the delivery of naked PDGF-BB and FGF-2 DNA alone induced neovascularization in rabbit model with hindlimb ischemia and the combination of both can give even better results than single gene delivery. It has been demonstrated that PDGF-BB and FGF-2 have stimulatory effects on endothelial cells. Histologic analysis shows that the capillary density was remarkably increased 7 days after GT. In this J. Li et al. / Microvascular Research 80 (2010) 10–17 15 Fig. 5. Hematoxylin and eosin-stained histological section of normal muscle and ischemic muscle treated with pPDGF/FGF2 (pP/F) and pNull 28 days after GT. The two figures in the same row are representative images selected from the same group. An interstitial inflammatory infiltration was observed in both pNull and pPDGF/FGF2-treated muscles but not in normal tissue. Areas of adipose substitution (asterisk) and segmental necrosis (n) were predominantly evident in pNull group. In contrast, pPDGF/FGF2 injection promoted muscle regeneration after ischemia, with the presence of small regenerated fibers with central nucleus (arrow). Intramuscular macrophages (arrowhead) indicated the removal of the necrotic fibers. The inserts show a 400 × original magnification of the PDGF/FGF2-treated muscle. A, artery; F, fibrosis. Scale bar, 50 μm. Fig. 6. Quantification of normal area (a), necrotic area (b) and regenerating area (c) of muscle sections from normal, pNull and pPDGF/FGF2-treated muscles. ⁎p b 0.05 vs. pNulltreated rabbits. 16 J. Li et al. / Microvascular Research 80 (2010) 10–17 process, FGF-2 was assumed to play a critical role on the robust angiogenic response, and up-regulated PDGFR-α and PDGFR-β which subsequently induced potent angiogenic activity of PDGF-BB as well. After that, the capillary density decreased and nearly declined to basal level on day 28 in all groups except PDGF/FGF2-treated group. However, the numbers of collateral arteries and arterioles increased according to the information from angiography and immunostaining. In contrast to angiogenesis, arteriogenesis needs a longer period — several weeks. After angiogenic response, the next task was to stabilize the newly formed vessels and remodel the vascular network. Both blood flow and pericyte coverage are critical determinants of vessel persistence (Benjamin et al., 1998; Sho et al., 2001). It has been shown in the chronic myocardial infarction model that PDGF-BB preferentially stimulates arteriolar growth (Hao et al., 2004). Pericytes recruited by PDGF-BB is important for stabilization and maturation of vascular structure (Darland and D'Amore, 1999). PDGFBB can also up-regulate the promoter activity of FGF receptor 1 (FGFR1) on VSMCs. Our data show that there are much more arterioles with strengthened pericyte coverage and collateral arteries in pPDGF/FGF2-treated muscle than control. Such increases in the number and diameter of collaterals therefore contribute to the tremendous augmentation in perfusion. According to Poeseuille' law, the blood flow is related to fourth power of the vessel's radius, which means the size of vessels is critical for the collateral blood flow. Consequently, arteriogenesis is probably more efficient for therapeutic angiogenesis. Our results are consistent with previous finding in mouse cornea that PDGF-BB and FGF-2 synergistically induced establishment of long-lasting, functional arterial vessels. Unlike repeated injections of protein, in the present study we used one-time administration by intramuscular injection of non-viral DNA. In our previous studies, the treatment of PDGF/FGF2 mediated by adenoviral vector seemed to result in better collateral development in ischemic limb than naked DNA injection. Nonetheless, it should be noticed that adenoviral vectors caused moderate inflammatory reaction (data not shown). Speaking of clinical application, the use of naked DNA is safe and convenient. Moreover, skeletal muscle seems to internalize naked DNA more efficiently than other types of tissues (Davis et al., 1993). We consider that the level of the secreted gene product by direct naked DNA delivery is sufficient to achieve therapeutic angiogenesis. Previous studies have reported that a short period of VEGF expression was unable to support stable vessels but resulted in vessel regression (Gounis et al., 2005). It implicates that vessel development requires continued production of the initiating factors, unless there is a specific combination such as PDGF-BB and FGF-2. The mechanism of how to remodel and stabilize the newly formed vasculature after removal of exogenous growth factors is probably due to aggregation and autophosphorylation of PDGF receptors on endothelial and mural cells independent of ligand (Cao et al., 2003). Therapeutic angiogenesis has a limited time window. According to the observation by Hoefer et al. (2001), there was no difference between treated groups and control when the treatment was performed 3 weeks after femoral occlusion. Once the spontaneous development ended, a late treatment had no significant effect. This finding reminds us that the time of delivery is important for the effect of treatment. We have tried two time schedules of treatment in our studies — one is to treat on day 10 after femoral incision and the other is to treat immediately after surgery. Our results resembled their findings that if there allowed for 10-day interval for postoperative recovery, the results came out to be inferior to that of the treatment performed as soon as after surgery (data not shown). The difference caused by these two time schedules implied us the existence of a narrow time window for responsiveness to the angiogenic actions of PDGF-BB and FGF-2. It should be noted that rabbits have a strong regenerative capacity to recover from ischemic injury, since the blood flow and collateral growth also improved a lot in control groups. When the endogenous collateral development has progressed to an extent, the blood supply to low limb and media thigh seemed to be sufficient, then injection of exogenous growth factors at that time point probably have no further effect on angiogenesis or arteriogenesis. We can assume that, if a 10-day interval was allowed, a moderate angiogenesis could be induced by exogenous growth factors in a short time after GT. Nevertheless, such newly formed vessels were usually small and immature that blood flow was more preferentially to go through the existing collateral vessels developed during the 10-day interval. Blood flow is important to vessel maintenance and growth (Benjamin et al., 1998), the newly formed vessels therefore regressed eventually by a pruning process to the advantage of the existing lager ones that conduct blood more efficiently (Hoefer et al., 2001; Rissanen et al., 2005). We also did not observe visible angiogenesis in normal muscle injected with these growth factors. It was implicated that ischemia is required for optimal effectiveness of angiogenesis and arteriogenesis induced by growth factors. In a word, we conclude that a 10-day interval between surgery and treatment may depress the responsiveness of the angiogenic activity of PDGF-BB and FGF-2. Ischemia can cause ischemic injury to cardiac and skeletal muscle and eventually lead to tissue necrosis. Under normal circumstance, adult skeletal muscle is a stable tissue, and the majority of muscle cells are postmitotic, recognized as terminal differentiated. Muscle satellite cell is a kind of stem cell residing beneath the basal lamina of mature skeletal muscle fibers, quiescent most of the time (Campion, 1984). Upon injury, skeletal muscle has a remarkable ability of regeneration, which is initiated by the proliferation of quiescent satellite cells and differentiation into myocytes to regenerate damaged myofibers (Campion, 1984). However, in some pathological conditions such as PAD patients especially those with critical limb ischemia or in the course of normal aging associated with poor muscle regenerative capacity, is this endogenous regeneration sufficient to prevent the loss of muscle mass? Based on prior reports, PAD has some negative impacts on muscle, causing alterations in fiber type distribution, denervation and apoptosis in skeletal muscle, which will further result in the functional impairment of muscle and exercise intolerance (Askew et al., 2007; Mitchell et al., 2007). Our results presented here suggest that PDGF/FGF2 not only greatly improved the perfusion of ischemic limb, but also took effect on muscle regeneration after ischemic injury. The exact mechanism has remained unclear. On one hand, in vivo experiments have demonstrated that FGF-2 can activate satellite cells (Lefaucheur and Sebille, 1995a) and it is a potent stimulator of myoblasts proliferation and fusion in vitro (Menetrey et al., 2000). FGF-2 plays an important role in muscle regeneration, as delivery of this gene led to an enhancement of skeletal muscle repair and an increase in muscle fiber density and size after injury (Lefaucheur and Sebille, 1995a). Likewise, FGF-2-treated wounds showed an average of 20-fold increase of regenerating myotubes (Doukas et al., 2002). On the contrary, injection of neutralizing antibodies of FGF-2 can inhibit muscle regeneration (Lefaucheur and Sebille, 1995b). On the other hand, reconstruction of vascular networks also has indirect positive impacts on ischemic tissue, supplying abundant nutrients and oxygen for instance. Muscle regeneration depends on oxygen, which is supported by blood perfusion via angiogenesis and collateral formation (Yun et al., 2005). Conversely, regenerated muscle promotes angiogenesis by producing angiogenic factors. Muscle regeneration, angiogenesis and collateral formation are linked and interact with each other. In conclusion, our data imply that a short exposure of ischemic tissue to PDGF-BB and FGF-2 is sufficient to establish stable and functional vessels. It changes the therapeutic strategy from sustained release of angiogenic factors to ‘one-short’ delivery, which is more convenient and safer for clinical application. Moreover, in our laboratory, we confirmed that PDGF-BB and FGF-2 also induce angiogenesis and arteriogenesis in both rat models of cardiac ischemia and cerebral ischemia. Although further studies are required for the J. Li et al. / Microvascular Research 80 (2010) 10–17 safety of clinical trail, our findings provide an alternative strategy for the treatment of PAD and a possible therapeutic guideline for muscular disorder. Acknowledgments This work was supported by National 863 Project of China (2007AA021202 and 2007AA021007). Ms. XR Wen (Department of Ultrasonic diagnosis, West China Medical School) and Mr. CW Zhang (Intervention Operating Room, West China Medical School) are acknowledged for their expert technical help. We also thank the personnel of Experimental Animal Center at Sichuan University for expert care of animals. References Asahara, T., Bauters, C., Zheng, L.P., Takeshita, S., Bunting, S., Ferrara, N., Symes, J.F., Isner, J.M., 1995. Synergistic effect of vascular endothelial growth factor and basic fibroblast growth factor on angiogenesis in vivo. Circulation 92, 365–371. 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