The effects of VEGF on survival of a random flap in the rat: examination of various routes of administration.

The purpose of the present study was to determine the effects of vascular endothelial growth factor (VEGF) on survival of a full thickness random pattern, McFarlane musculocutaneous flap in the rat. In addition, this study examined a number of different methods of VEGF delivery in an attempt to determine the most effective route of administration. A 2 x 8 cm full thickness dorsal flap with the pedicle remaining attached at the anterior end was elevated in 72 male Sprague-Dawley rats. The rats were randomised into six groups and immediately received the following treatment: Group I (n = 12): systemic VEGF injection into the femoral vein (50 microg/ml); Group II (n = 10): multiple systemic VEGF injections at 0, 24 and 48 h post flap elevation (50 microg/ml); Group III (n = 12): subdermal VEGF injection into the flap (1 microg/ml); Group IV (n = 12): subfascial VEGF injections into the recipient bed (1 microg/ml); Group V (n = 10): topical VEGF onto the recipient bed (1 microg/ml); Group VI (n = 16): control group with no treatment. Following 5 days recovery, the area of flap survival was measured. Mean flap survival ranged from 91% in Group II to 78% in Group V, and was significantly greater in all experimental groups (P< 0.001 for Groups I-IV and P< 0.05 for Group V) as compared to the control group (mean survival of 66%). The only significant difference between the experimental groups was between the mean survival in Group II and Group V (P< 0. 05). Histological analysis demonstrated a qualitatively greater amount of granulation tissue and neovascularisation in the experimental groups. These results support the notion that VEGF rescues tissue at risk of hypoxic damage by inducing angiogenesis, and the use of growth factors such as VEGF holds promise as a method of increasing skin viability.     

Synergistically therapeutic effects of VEGF165 and angiopoietin-1 on ischemic rat myocardium

PURPOSE: The aim of this study was to determine whether the combination of 2 angiogenic growth factor, vascular endothelial growth factor 165(VEGF165) and angiopoietin-1 (Ang1), could increase angiogenesis and cardiomyocyte(CM) proliferation in an infarcted myocardium. METHODS: Myocardial ischemia was induced in rats by ligation of the left anterior descending (LAD) coronary artery. Replication-deficient adenoviruses encoding VEGF165 (Ad-VEGF165), Ang1 (Ad-Ang1) or enhanced green fluorescence protein (Ad-EGFP) was injected into the ischemic myocardium immediately. Bromodexyuridine (BrdU) was administered intraperitoneally 1 week after ligation. One week later, the hearts were harvested and sectioned for hematoxylin-eosin (HE) and immunohistochemistry to evaluate densities of capillary, arteriole and double labelled BrdU(+) CM. M-mode echocardiography was used to evaluate the cardiac function. RESULTS: Ang1 significantly increased collateral vessel formation. Both VEGF165 and Ang1 significantly increased densities of capillary and arteriole, as well as the number of double labelled BrdU(+) CM, and improved cardiac function. CONCLUSION: Our results suggest that the combination of VEGF165 and Ang1 can increase both myocardial angiogenesis and CM proliferation following myocardial ischemia in rats, leading to improved cardiac function.     

The effect of single administration of vascular endothelial growth factor or L-arginine on necrosis and vasculature of the epigastric flap in the rat model.

OBJECTIVES: Vascular endothelial growth factor (VEGF) and nitric oxide (NO) produce vasodilation, induce angiogenesis, and improve survival of surgical flaps. We used the rat epigastric skin flap to study the effect of a single intra-arterial dose of VEGF or L-arginine, a substrate for NO production, on flap regional necrosis and pedicle dependence of flap perfusion. METHODS: In 30 Sprague-Dawley rats an 8 x 8 cm2 skin flap, consisting of four vertical zones marked A through D (right to left), based on the proximal right inferior epigastric vessels was raised. Subsequently, 1 ml of either saline (control, n =10), 5 microg VEGF (VEGF, n = 10), or 50 mg of L-arginine (L-arginine, n = 10) was injected into the arterial pedicle by cannulating the right saphenous artery, and the flap was resutured in place. After 8 days, the animals were perfused systemically with 15 microm coloured fluorescent microspheres before (blue) and after (yellow-green) ligation of the right inferior epigastric vascular pedicle. After sacrifice, the area of flap necrosis was measured in each zone by templates and weight-to-surface ratio, and the flap zones were harvested and processed for determination of fluorescence and blood flow. RESULTS: Administration of VEGF or L-arginine resulted in decreased total and regional (zone D) flap necrosis (ANOVA <0.001). The total and regional flap shrinkage was greater in the experimental groups (ANOVA <0.02). While VEGF and L-arginine decreased the percentage of necrosis in the zone most distal to the pedicle (ANOVA <0.01) only L-arginine diminished percentage of total flap necrosis (p = 0.04). In the VEGF group, total and regional flap perfusion did not change after pedicle ligation, but perfusion decreased significantly in zones B through D in the L-arginine treated rats. CONCLUSION: Single intra-pedicle administration of VEGF or L-arginine decreased necrosis of the epigastric skin flap at 8 days postoperatively, but flap shrinkage also increased in the zone with the greatest degree of necrosis. Perfusion data suggest that beneficial effects of VEGF and L-arginine on flap survival may be based on different mechanisms.     

血管内皮生长因子基因治疗联合外科延迟法改善大鼠腹壁超范围轴型皮瓣成活的研究

目的探讨皮下注射血管内皮生长因子（vascular endothelial growth factor,VEGF）基因、外科延迟两种方法及其联合应用对大鼠腹壁超范围轴型皮瓣成活的影响。方法取雄性Wistar大鼠48只,体重400～450g,制作大鼠以腹壁浅动脉为血管蒂的8cm×8cm超范围轴型皮瓣模型。随机分为六组,每组8只,分别为空白对照组（A组）、术时基因治疗组（B组）、术前基因治疗组（C组）、单纯延迟组（D组）、延迟同时基因治疗组（E组）和延迟后基因治疗组（F组）。术后7d,计算各组的皮瓣成活率;取皮瓣组织标本行HE染色,检测平均微血管密度及内径;取皮瓣组织标本行VEGF免疫组织化学染色检测VEGF165的表达。结果各实验组皮瓣成活率均显著高于A组（P〈0.05）;实验组中,E组皮瓣成活率显著高于其他各组（P〈0.05）,余各实验组间差异无统计学意义（P〉0.05）。微血管密度：B、C、E、F组显著高于A、D组,差异有统计学意义（P〈0.05）;B、C、E、F组间以及A、D组间比较差异无统计学意义（P〉0.05）。微血管内径：D组显著大于E、F组,差异有统计学意义（P〈0.05）;而D组和E、F组均显著大于A、B、C组,差异有统计学意义（P〈0.05）。免疫组织化学染色示A组及D组有少量VEGF165沉积,染色深度明显浅于其他各组。B、C组和E、F组可见毛细血管内皮细胞胞浆内有棕褐色VEGF165抗原抗体复合物的沉积,染色较深,部分沉积物呈带状围绕血管腔。各组实验动物角膜层均未见新生血管。结论皮下注射pcDNA4-VEGF165和外科延迟均能有效改善大鼠皮瓣的成活,但二者作用机制不同,而延迟的同时皮下注射VEGF基因能进一步提高大鼠皮瓣成活率。     

Intraventricular infusion of vascular endothelial growth factor promotes cerebral angiogenesis with minimal brain edema

OBJECTIVE: Therapeutic cerebral angiogenesis, i.e., using angiogenic factors to enhance collateral vessel formation within the central nervous system, is a potential method for cerebral revascularization. Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen that also increases capillary permeability, particularly in ischemic tissue. The purpose of this study was to assess the angiogenic and capillary permeability effects of chronic intraventricular infusion of exogenous VEGF in nonischemic brain tissue, because many patients with impaired cerebrovascular reserve do not exhibit chronic cerebral ischemia. METHODS: Recombinant human VEGF(165) was infused into the right lateral ventricle of rats at a rate of 1 microl/h for 7 days, at concentrations of 1 to 25 microg/ml, with osmotic minipumps. Control animals received vehicle only. Vessels were identified in laminin immunohistochemical analyses. Capillary permeability and brain edema were assessed with Evans blue extravasation, [(3)H]inulin permeability, and brain water content measurements. RESULTS: Vessel density was dose-dependently increased by VEGF(165) infusions, with significant increases occurring in animals treated with 5 or 25 microg/ml, compared with control animals (P h 0.01). Significant enlargement of the lateral ventricles was observed for the highest-dose group but not for animals treated with other doses. Capillary permeability was assessed in animals treated with a dose of 5 microg/ml. An increase in capillary permeability in the diencephalon was identified with Evans blue extravasation and [(3)H]inulin permeability assessments; however, the brain water content was not significantly increased. CONCLUSION: Chronic intraventricular infusions of VEGF(165) increased vascular density in a dose-dependent manner. There seems to be a therapeutic window, because infusion of VEGF(165) at a concentration of 5 microg/ml resulted in a significant increase in vessel density with minimal associated brain edema and no ventriculomegaly.     

Platelet-derived growth factor-AA-mediated functional angiogenesis in the rat epigastric island flap after genetic modification of fibroblasts is ischemia dependent

BACKGROUND: The aim of this study was to induce therapeutic angiogenesis in ischemically challenged flap tissue by means of gene transfer. METHODS: Isogenic rat fibroblasts were retrovirally transfected to produce platelet-derived growth factor (PDGF)-AA. Stable gene expression was monitored by PDGF-AA enzyme-linked immunosorbent assay. Eighty animals were divided into 2 groups (1 and 2), each with 4 subgroups. The angiogenic target was a 7 x 7-cm epigastric island flap used as a necrosis model. Group 1 received flap treatment 1 week before flap elevation: 10(7) genetically modified fibroblasts, expressing PDGF-AA (genetically modified fibroblasts) plus 1 mL of Dulbecco's modified Eagle's medium (DMEM) (1A), 10(7) nonmodified fibroblasts (NMFB) plus 1 mL of DMEM (1B), 1 mL of DMEM (1C), and 1 mL of sodium chloride 0.9% (1D). All substances were injected at evenly distributed spots into the panniculus carnosus of the entire flap. Group 2 had the same flap treatment at the day of flap elevation. All flaps were sutured back. Seven days later, the flaps were harvested and examined both clinically, histologically, and immunohistochemically. RESULTS: In vitro, the GMFB produced up to 117.9 +/- 57.2 ng of PDGF-AA/mL medium during a 4-day period, compared with 0.7 +/- 0.6 ng of PDGF-AA/mL medium produced by NMFB in the same time period. In vivo production of PDGF-AA in flaps amounted to 1.3 +/- 0.7 ng of PDGF-AA/1 microL flap tissue for group 1A and 1.7 +/- 1.1 ng of PDGF-AA/1 microL flap tissue for group 2A seven days after cell transplantation. Fibroblasts persisted in all flaps from groups 1A, 1B, 2A, and 2B without major inflammatory reaction. Clinically, group 2A developed significantly less flap necrosis compared with all other groups, including group 1A. Accordingly, only group 2A gave significant histologic and immunohistochemical evidence for enhanced angiogenesis within the flap tissue. CONCLUSIONS: After retroviral gene transfer, isogenic rat fibroblasts produce high amounts of PDGF-AA in vitro. In vivo, PDGF-AA can be detected in flaps receiving genetically modified fibroblasts, which suggests survival of the implanted fibroblasts in this model. PDGF-AA produced by GMFB can induce flap angiogenesis only under ischemic conditions in this model. Transplantation of PDGF-AA-overexpressing fibroblasts results in higher flap survival in this model.     

Therapeutic angiogenesis in chronically ischemic porcine myocardium: comparative effects of bFGF and VEGF

BACKGROUND: Both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been used in preclinical studies to induce new blood vessel growth in ischemic cardiac muscle with promising results. However, clinical trials have been much less convincing and further work is needed. This study expands on prior work by comparing the long-term proangiogenic effects of direct intramyocardial (IM) injection of bFGF, as well as IM and intravenous (IV) VEGF in a porcine model of chronic hibernating myocardium. METHODS: Mini-swine with proximal 90% left circumflex (LCx) coronary stenosis subtending chronically ischemic, viable (hibernating) myocardium by positron emission tomography (PET) and dobutamine stress echocardiography (DSE) were randomized to IM bFGF (n = 5), IM VEGF(165) (n = 5), IV VEGF(165) (n = 5), IM vehicle (n = 5), or sham redo-thoracotomy (n = 4). The bFGF protein was administered in a total dose of 1.35 microg divided into 30 IM injections. IM VEGF(165) protein was administered in a total dose of 15 microg/kg divided into 30 injections; IV VEGF(165) was given at a dose of 50 ng. kg(-1). min(-1) for 200 minutes at three 72-hour intervals (30 microg/kg total dose). After 3 and 6 months the PET and DSE studies were repeated, and the animals were sacrificed for tissue vascular density and angiogenic protein analysis. RESULTS: Myocardial blood flow (MBF) by PET was significantly improved 3 months posttreatment in the IM bFGF and IM VEGF(165) groups, differences that were sustained at 6 months. There was no significant increase in MBF 3-months posttreatment in the IV VEGF(165) group; however, at 6 months MBF was significantly improved. No change in MBF was seen in the IM vehicle or sham groups. Regional wall motion at rest and peak stress in the LCx region demonstrated small but statistically significant improvements by 6 months in the IM bFGF and IV VEGF(165) groups only; no improvement was seen in the IM VEGF(165), IM vehicle, or sham groups. Quantitative vascular density was significantly increased in the LCx regions of all treatment groups (IM bFGF, IM VEGF(165), IV VEGF(165)) 6-months postoperatively. No significant increase in LCx region myocardial bFGF or VEGF protein levels was seen in the treated animals at 6 months. CONCLUSIONS: The IM bFGF, IM VEGF(165), and IV VEGF(165) all improve regional perfusion and vascular density 6-months posttherapy in the animal model utilized. Functional improvements were less consistent. Both bFGF and VEGF(165) may be useful therapies for improving regional perfusion in chronically ischemic myocardium, although combination therapy with additional growth factors or cellular therapies may be necessary if concomitant improvements in function are to be seen.     

血管内皮生长因子基因治疗与皮瓣延迟术对大鼠腹壁轴型皮瓣成活的影响

目的 探讨血管内皮生长因子基因治疗、皮瓣延迟术两种方法及二者联合应用对大鼠腹壁轴型皮瓣成活的影响.方法 制作大鼠以右侧腹壁浅动脉为血管蒂的超范围轴型皮瓣模型,分别采用皮下注射pcDNA4-VEGF165、皮瓣延迟术及二者联合应用,按处理方法的不同分为6组,①计算各组的皮瓣成活率;②取皮瓣组织标本行常规HE染色,检测平均微血管数目及内径;③取皮瓣组织标本行VEGF免疫组化染色检测VEGF的表达情况.各试验组与空白组相对比,各试验组之间两两对比.结果 各试验组的皮瓣成活率明显高于空白组,延迟同时基因治疗组的皮瓣成活率明显高于其他试验组;基因治疗组及联合应用组的平均微血管数目明壶高于延迟组及空白组;平均微血管内径:延迟组＞联合应用组＞基因治疗组＞空白组;免疫组化染色显示基因治疗组及联合应用组VEGF表达明显高于空白组及单纯延迟组.结论 皮下注射pcDNA4-VEGF165和皮瓣延迟均能有效地改善大鼠皮瓣的成活,但其作用机制不同,而二者的联合应用则能进一步地提高大鼠皮瓣的成活率.     

血管内皮生长因子基因改善大鼠腹壁下动脉皮瓣成活的实验研究

目的　通过血管内皮生长因子 (vascularendothelialgrowthfactor,VEGF)基因对腹壁下动脉皮瓣的转染 ,探讨应用VEGF基因对皮瓣成活的影响。方法　以SD大鼠为实验模型制作腹壁下动脉皮瓣 ,并分别注入脂质体包裹的PCD VEGF1 6 5(目的基因组 ) ,PCD(空白质粒组 )和生理盐水 (生理盐水组 )。术后 ,①通过腹腔注射荧光素钠估计皮瓣的血流量 ;②计算断蒂后各组大鼠皮瓣的成活面积 ;③取大鼠皮肤标本行常规染色检测平均血管数目及内径 ;④皮肤标本行VEGF免疫组化染色检测VEGF表达情况。结果　目的基因组、空白质粒组和生理盐水组的平均荧光染色面积分别为6 0 6 4%、30 15 %、2 9 89%(P <0 0 5 ) ,大鼠断蒂后的平均成活面积分别为 92 3%、30 5 %、31 8%(P<0 0 5 ) ,平均血管数目分别为 10 1 72、91 35、89 85 (P <0 0 5 ) ,平均血管内径分别为 2 6、31 0 9、32 5 1μm(P <0 0 5 ) ,免疫组化染色示目的基因组染色深度明显高于空白质粒组和生理盐水组 (P <0 0 5 )。结论　PCD VEGF1 6 5转染能够改善皮瓣的成活 ,且通过转染表达了丰富的VEGF1 6 5。     

重组腺病毒介导的血管内皮生长因子165基因增强大鼠横形腹直肌肌皮瓣活力的研究

目的探讨重组腺病毒介导的血管内皮生长因子165(adenovirusmediatedvascularendothelialgrowthfactor165,Ad-VEGF165)基因,增强大鼠横形腹直肌肌皮瓣(transverserectusabdominismusculocutaneousflap,TRAM)局部缺血区域活力的基因治疗。方法SD大鼠30只,制备30mm×80mm以下腹部血管作为血管蒂的右侧矩形TRAM皮瓣,随机平均分成5组,分别在大鼠TRAM皮瓣皮下、皮下+腹直肌及腹直肌局部注射Ad-VEGF165(1～3组:治疗组),同时在TRAM皮瓣皮下+腹直肌局部注射重组腺病毒载体介导的绿色荧光蛋白(adenovirusmediatedgreenfluorescentprotein,Ad-GFP,4组:重组腺病毒自身对照组)和DMEM培养液(5组:空白对照组),于注射后第7天手术,术后第7天检测各组大鼠TRAM皮瓣成活面积、CD34微血管密度(microvasculardensity,MVD)、VEGF免疫组织化学染色和原位杂交组织化学(insituhybridizationhistochemistry,ISHH)。结果11～3组TRAM皮瓣成活面积分别为14.19±2.77、15.18±2.18、8.30±1.28cm2,均较4、5组4.12±1.86、3.60±1.95cm2明显增加,且差异有统计学意义(P<0.05);21～3组TRAM皮瓣CD34MVD均高于4、5组,差异有统计学意义(P<0.05);31～3组VEGF免疫组织化学染色和ISHH呈阳性表达。结论重组腺病毒载体介导的VEGF165基因局部注射,能增加大鼠TRAM皮瓣局部缺血区域成活面积。     

联合转染血管内皮生长因子165和组织纤溶酶原激活物基因抑制兔血管损伤后内膜增生的研究

目的观察血管局部联合转染血管内皮生长因子165(VEGF165)和组织纤溶酶原激活物(tPA)基因对损伤动脉内膜增生的影响并探讨可能机制。方法采用显微外科手术方法建立兔动脉损伤模型;用微注射装置将基因转染液注入损伤血管壁,按实验终点(术后2d、1周、2周、4周和8周)分为5个亚组(每个亚组8只兔);术后各实验终点取损伤段血管用于病理学检测、电镜观察、RTPCR和免疫组织化学检查。结果术后各时间点基因转染组血管壁的VEGF165基因的mRNA表达量和tPA阳性细胞数明显高于对照组(P<0.01)。与对照组比较,术后各时间点基因转染组血管内膜面积和狭窄率均显著减小(P<0.01),术后8周时基因转染组管腔狭窄率比对照组降低了59.0%。结论局部联合转染VEGF165和tPA基因可抑制血管新生内膜增生及血管再狭窄。     

Vascular endothelial growth factor (VEGF) expression and the effect of exogenous VEGF on survival of a random flap in the rat.

The induction of endogenous vascular endothelial growth factor (VEGF) production in the skin flap with ischemic injury and the effect of exogenous VEGF on survival of the ischemic skin flap were studied in rats. A dorsal flap model (3x10 cm(2)) was used in this study. In Part I, biopsies were taken from the flap at 2.5, 5.5, and 8.5 cm distances from the distal edge at 0, 6, 12, and 24 h after the flaps were sutured. Malonyldialdehyde (MDA) and VEGF(165) protein level were measured. In Part II, exogenous VEGF (1 microg/ml) was injected subdermally into the flaps in 14 rats before the flaps were replaced. Flaps that received a saline injection were used as the controls. The skin paddle survival was measured on postoperative day five. The results showed that the MDA level in the distal part of the flap significantly increased at 24 h postoperatively when compared to MDA in other parts of the flap. However, VEGF levels in the distal part of the flap significantly decreased when compared to the middle part of the flap. Subdermal injection of exogenous VEGF to the distal area of the flap could significantly improve survival of the distal flap (89% of total skin paddle) when compared to the control, which had a 64% mean percent survival. We conclude that production of endogenous VEGF protein is significantly increased in the skin flap with mild ischemia, but decreased in the flap with severe ischemia. Administration of exogenous VEGF could significantly enhance survival of ischemic flaps.     

Revascularization of rat fasciocutaneous flap using CROSSEAL® with VEGF protein or plasmid DNA expressing VEGF

BACKGROUND: Fasciocutaneous tissue transfer is a common reconstructive procedure. Revascularization of flap tissue is an important component of tissue healing. Gene therapy offers an avenue through which the period of pedicle vascular dependency can be reduced. MATERIALS AND METHODS: Rat fasciocutaneous flaps were elevated and a two-hour ischemic time induced. Polycation complex (jet PEI) and human fibrin sealant CROSSEAL was applied between flap and underlying abdominal tissues. Group 1 (six rats) was the control; Group 2 (seven rats) had vascular endothelial growth factor (VEGF) protein applied; Group 3 (seven rats) had plasmid DNA expressing VEGF applied. Vascular pedicles were ligated on postoperative day 5, percentage flap survival evaluated on day 7. RESULTS: All flaps survived initial ischemia. Mean +/- SD percentage area of the flap that survived was 28.1 +/- 12.4 (Group 1), 71.6 +/- 16.2 (Group 2), and 77.5 +/- 12.7 (Group 3) (P < 0.001, Group 1-3, 2-3). No differences were observed between Groups 2 and 3. CONCLUSIONS: Locally administered VEGF protein or plasmid DNA expressing VEGF enhanced survival of fasciocutaneous flaps.     

基因修饰的骨髓间充质干细胞移植于慢性心肌梗死模型的实验研究

目的 研究血管内皮生长因子（VEGF）165基因修饰的骨髓间充质干细胞（MSCs）移植于慢性心肌梗死模型后的血管新生及对心功能的作用。方法 同源重组法构建含有VEGF。基因的重组腺病毒载体（rAd—VEGF165）;密度梯度离心法分离骨髓单个核细胞,贴壁法培养兔MSCs;rAd—VFGF165转染MSCs,并用4,6-联脒-2-苯基吲哚（DAPI）标记MSCs;结扎前降支法建立兔心肌梗死模型,存活6周的实验动物36只随机分为3组：移植rAd—VFGF165转染的MSCs组（Ⅰ组）12只、单纯移植MSCs组（Ⅱ组）12只和只注射无血清培养基的对照组（Ⅲ组）12只。移植术后4周,超声法检测心脏功能,并同术前比较;荧光显微镜下观察MSCs分布情况;免疫组化法检测梗死区微血管密度。结果 移植4周后Ⅰ 组的MSCs存活率大于其他2组;Ⅰ组的左室射血分数、E／A比值和梗死区微血管密度与其他2组比较差异有统计学意义（P〈0．05）。结论 VEGF165基因和MSCs联合策略是治疗心肌梗死的有效方法,所产生的协同治疗效果大于单纯的MSCs移植。     

Intra-arterial bone marrow cell transplantation induces angiogenesis in rat hindlimb ischemia

BACKGROUND: Bone marrow (BM) cells have been shown to augment local angiogenesis by differentiating vessels themselves and/or secreting paracrinally angiogenic growth factors. Herein, the angiogenic effects of intra-arterial BM mononuclear cell (BM-MNC) transplantation were evaluated in a rat ischemic hindlimb model. METHODS: Unilateral hindlimb ischemia was created by excising the femoral artery and its branch in Lewis rats. BM-MNCs were isolated by centrifugation through a Histopaque density gradient. One week after excision of the unilateral femoral artery, BM-MNCs (5 x 10(6) cells, Group A, n = 6) or PBS (Group B, n = 7) were injected into the ischemic thigh skeletal muscles at the six points with a gauge needle. Another injection of BM-MNCs (3 x 10(7) cells, Group C, n = 6) or PBS (Group D, n = 7) was administered via the indwelling catheter in the right common iliac artery. RESULTS: Four weeks after the BM-MNC transplantation, angiographic examination revealed the development of collateral vessels in both BM-MNC-transplanted groups. The difference in skin temperature between right and left hindlimbs was significantly reduced in both BM-MNC-transplanted groups (0.93 +/- 0.15 vs. 2.84 +/- 0.35 vs. 1.20 +/- 0.26 vs. 2.61 +/- 0.37 degrees C, Group A vs. Group B vs. Group C vs. Group D, p < 0.05). Moreover, immunohistochemical analysis demonstrated that capillary endothelial cells were increased in both BM-MNC-transplanted groups. CONCLUSION: BM-MNC implantation was able to induce functional neovascularization in rat ischemic hindlimb. The intra-arterial administration offered similar levels of angiogenic activity as intramuscular injection.     

Expansion of surviving skin paddle of neurocutaneous island flaps in rats by VEGF

The purpose of this study was to investigate whether vascular endothelial growth factor (VEGF) can enlarge the skin paddles of neurocutaneous flaps in rats. Wistar albino rats were used in four groups: Group 1, (n = 10): neurocutaneous island flap; Group 2, (n = 10): neurocutaneous island flap, surgical delay; Group 3, (n = 10): neurocutaneous island flap, VEGF; Group 4, (n = 10): graft. A 3 x 3-cm, neurocutaneous island flap was elevated on the anterolateral skin of the thigh of the rats. The surviving flap areas were 29.7 +/- 1.43 percent in Group 1, 41.3 +/- 3.24 percent in Group 2, 94.2 +/- 1.46 percent in Group 3. There were no surviving areas in Group 4. The vascular networks of Group 3 animals were more intensive and diffused on microangiography and the histopathologic findings were better in this group. The surviving flap areas in Group 3 animals were enlarged approximately three times over the original size.     

Induction of angiogenesis by cationic lipid-mediated VEGF165 gene transfer in the rabbit ischemic hindlimb model

PURPOSE: The purpose of this study was to test the efficacy of a new cationic lipid formulation coupled with the cDNA encoding for the 165-residue form of vascular endothelial growth factor (VEGF(165)) to induce neovascularization and enhance blood flow in the rabbit ischemic hindlimb model. METHODS: Two days after removal of their right femoral arteries, rabbits received intramuscular injections of different concentrations of VEGF(165) or saline solution in the ischemic thigh. Tissue perfusion and increased neovascularization of the ischemic limb were assessed weekly on the basis of the calf blood pressure ratio for the ischemic/nonischemic limbs, regional blood flow to the skeletal muscles as measured with radioactive microspheres, postmortem angiography, and histology. RESULTS: At weeks 1 and 2 after surgery, animals treated with 1000 microgram of VEGF(165) had a 1.5-fold increase and a 2.5-fold increase, respectively, in the regional blood flow to both the adductor and gastrocnemius muscles of the ischemic limb. The blood pressure ratio was also greater in the treated animals than in the controls at weeks 2 and 3 after surgery. Early neovascularization in the VEGF(165) group was further documented at week 1 after surgery by more angiographically recognizable collateral vessels (angioscores were 64.13 +/- 2.51 and 38.28 +/- 3.82 for VEGF(165) and saline solution, respectively; P <.001) and by a threefold increase in the number of capillaries (vascular density) relative to the controls (P <.005). CONCLUSIONS: Intramuscular administration of a single dose of plasmid-liposomes encoding for VEGF(165) accelerates angiogenesis and increases blood flow in the rabbit hindlimb ischemic model. Therefore, this nonviral vector could be recommended for further testing for use in therapeutic angiogenesis.     

Adenovirus-mediated transforming growth factor-beta ameliorates ischemic necrosis of epigastric skin flaps in a rat model

BACKGROUND: Gene therapy has been recently introduced as a novel approach to treat ischemic tissues by using the angiogenic potential of certain growth factors. We investigated the effect of adenovirus-mediated gene therapy with transforming growth factor-beta (TGF-beta) delivered into the subdermal space to treat ischemically challenged epigastric skin flaps in a rat model. MATERIAL AND METHODS: A pilot study was conducted in a group of 5 animals pretreated with Ad-GFP and expression of green fluorescent protein in the skin flap sections was demonstrated under fluorescence microscopy at 2, 4, and 7 days after the treatment, indicating a successful transfection of the skin flaps following subdermal gene therapy. Next, 30 male Sprague Dawley rats were divided into 3 groups of 10 rats each. An epigastric skin flap model, based solely on the right inferior epigastric vessels, was used as the model in this study. Rats received subdermal injections of adenovirus encoding TGF-beta (Ad-TGF-beta) or green fluorescent protein (Ad-GFP) as treatment control. The third group (n = 10) received saline and served as a control group. A flap measuring 8 x 8 cm was outlined on the abdominal skin extending from the xiphoid process proximally and the pubic region distally, to the anterior axillary lines bilaterally. Just prior to flap elevation, the injections were given subdermally in the left upper corner of the flap. The flap was then sutured back to its bed. Flap viability was evaluated seven days after the initial operation. Digital images of the epigastric flaps were taken and areas of necrotic zones relative to total flap surface area were measured and expressed as percentages by using a software program. RESULTS: There was a significant increase in mean percent surviving area between the Ad-TGF-beta group and the two other control groups (P < 0.05). (Ad-TGF-beta: 90.3 +/- 4.0% versus Ad-GFP: 82.2 +/- 8.7% and saline group: 82.6 +/- 4.3%.) CONCLUSIONS: In this study, the authors were able to demonstrate that adenovirus-mediated gene therapy using TGF-beta ameliorated ischemic necrosis in an epigastric skin flap model, as confirmed by significant reduction in the necrotic zones of the flap. The results of this study raise the possibility of using adenovirus-mediated TGF-beta gene therapy to promote perfusion in random portion of skin flaps, especially in high-risk patients.     

Remote ischemic preconditioning modulates p38 MAP kinase in rat adipocutaneous flaps

Ischemic preconditioning has been shown to improve survival of cutaneous flaps. The authors examined the effect of remote ischemic preconditioning (RIPC) on phosphorylation of p38 MAP kinase and related the results to flap survival. Female Wistar rats had 8 x 12-cm abdominal adipocutaneous flaps raised on the medial branch of the superficial epigastric artery. Controls (Group 1) had the flap elevated and the pedicle clamped for 3 hr, then closed with a sheet of plastic between the flap and abdominal wall. Group 2 animals had RIPC by tourniquet on the contralateral hind limb before the flap was dissected. Group 3 animals mimicked Group 2 and also had an infusion of the nitric oxide blocker, N-nitro-L-arginine methyl ester (L-NAME) 5 min prior to the RIPC. Group 4 had the flap elevated prior to the RIPC. All groups except Group 1 had 10 min of RIPC with 30 min of reperfusion, then 3 hr of ischemia. Tissue samples were taken at the distal margins of the flaps before preconditioning and 30 min after preconditioning for detection of p38 MAP kinase and phosphorylated p38 MAP kinase (pp38 MAP kinase). Group 2 flaps (RIPC before flap elevation) exhibited better flap tissue survival and had well-defined phosphorylation of p38 MAP kinase 30 min post RIPC, when compared to the other groups. Pre-infusion with the nitric oxide blocker (Group 3) before RIPC blocked the survival advantage conferred by preconditioning and diminished the phosphorylation of p38 MAP kinase. Tissue from all groups showed very little phosphorylation of p38 MAP kinase following 3 hr of ischemia. Thus, increased tissue survival is correlated with elevated levels of p38 MAP kinase phosphorylation following RIPC. This effect is inhibited by blockade of nitric oxide. Modulation of the p38 MAP kinase pathway may represent a protection pathway for ischemic preconditioning.