Hepatocyte growth factor as potential cardiovascular therapy

Hepatocyte growth factor is a mesenchyme-derived pleiotropic factor that regulates the growth, motility and morphogenesis of various types of cells, and is also a member of the angiogenic growth factors. Hepatocyte growth factor is secreted by vascular endothelial cells and smooth muscle cells, and the hepatocyte growth factor receptor, c-met, was also observed in these vascular cells. Treatment of human aortic endothelial cells with recombinant hepatocyte growth factor resulted in a significant increase in cell proliferation, accompanied by mitogen-activated protein kinase and Akt/protein kinase B phosphorylation. Recently, a novel therapeutic strategy for ischemic diseases using angiogenic growth factors to augment collateral artery development has been proposed. As preclinical study of gene therapy using hepatocyte growth factor to treat peripheral arterial disease, naked hepatocyte growth factor plasmid was intramuscularly injected into the ischemic hind limb of rabbits in order to evaluate its angiogenic activity. Intramuscular injection of hepatocyte growth factor plasmid once on day 10 following surgery, produced significant augmentation of collateral vessel development in the ischemic limb on day 30. In the clinical setting, the authors further investigated the safety and efficacy of hepatocyte growth factor plasmid DNA in patients with critical limb ischemia, in a prospective open-labeled trial. Intramuscular injection of naked plasmid DNA was performed in the ischemic limbs of six patients with critical limb ischemia with arteriosclerosis obliterans (n = 3) or Buerger disease (n = 3) graded as Fontaine III or IV. In the efficacy evaluation, a reduction of pain scale of more than 1 cm on a visual analog pain scale was observed in five out of six patients. An increase in ankle pressure index of more than 0.1 was observed in five out of five patients. The long diameter of eight out of 11 ischemic ulcers in four patients was reduced by more than 25%. Intramuscular injection of naked hepatocyte growth factor plasmid is safe, feasible and can achieve successful improvement of ischemic limbs. Although the present data were obtained to demonstrate safety in a Phase I/early Phase II trial, the initial clinical outcome with hepatocyte growth factor gene transfer seems to indicate its usefulness as sole therapy for critical limb ischemia. Randomized placebo-controlled clinical trials of alternative dosing regimens of gene therapy will be required to define the efficiency of this therapy.     

Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat and rabbit hindlimb ischemia models: preclinical study for treatment of peripheral arterial disease

Hepatocyte growth factor (HGF) exclusively stimulates the growth of endothelial cells without replication of vascular smooth muscle cells, and acts as a survival factor against endothelial cell death. Recently, a novel therapeutic strategy for ischemic diseases using angiogenic growth factors to expedite and/or augment collateral artery development has been proposed. We have previously reported that intra-arterial administration of recombinant HGF induced angiogenesis in a rabbit hindlimb ischemia model. In this study, we examined the feasibility of gene therapy using HGF to treat peripheral arterial disease rather than recombinant therapy, due to its disadvantages. Initially, we examined the transfection of 'naked' human HGF plasmid into a rat hindlimb ischemia model. Intramuscular injection of human HGF plasmid resulted in a significant increase in blood flow as assessed by laser Doppler imaging, accompanied by the detection of human HGF protein. A significant increase in capillary density was found in rats transfected with human HGF as compared with control vector, in a dose-dependent manner (P < 0.01). Importantly, at 5 weeks after transfection, the degree of angiogenesis induced by transfection of HGF plasmid was significantly greater than that caused by a single injection of recombinant HGF. As an approach to human gene therapy, we also employed a rabbit hindlimb ischemia model as a preclinical study. Naked HGF plasmid was intramuscularly injected in the ischemic hindlimb of rabbits, to evaluate its angiogenic activity. Intramuscular injection of HGF plasmid once on day 10 after surgery produced significant augmentation of collateral vessel development on day 30 in the ischemia model, as assessed by angiography (P < 0.01). Serial angiograms revealed progressive linear extension of collateral arteries from the origin stem artery to the distal point of the reconstituted parent vessel in HGF-transfected animals. In addition, a significant increase in blood flow was assessed by a Doppler flow wire and the ratio in blood pressure of the ischemic limb to the normal limb was observed in rabbits transfected with HGF plasmid as compared with rabbits transfected with control vector (P < 0.01). Overall, intramuscular injection of naked human HGF plasmid induced therapeutic angiogenesis in rat and rabbit ischemic hindlimb models, as potential therapy for peripheral arterial disease.     

Therapeutic angiogenesis for ischemic diseases

The clinical consequences of peripheral arterial disease include pain on walking, pain at rest and loss of tissue integrity in the distal ischemic limbs. Although development of beneficial drugs and intervention devices do contribute to the treatment of this disease, critical limb ischemia is estimated to develop in 500 to 1,000 individuals per million per year. As angiogenic growth factors can stimulate the development of collateral arteries, a concept called "therapeutic angiogenesis" is now evaluated in the clinical fields. Recent progress in molecular biology has led to the development of gene therapy as a new strategy to treat a variety of cardiovascular diseases using angiogenic growth factors such as vascular endothelial growth factor (VEGF). Therapeutic angiogenesis using angiogenic growth factors is expected to be a new treatment for patients with severe ischemic heart or peripheral arterial disease.     

Beperminogene perplasmid for the treatment of critical limb ischemia

Therapeutic angiogenesis for the treatment of ischemic disease can be attained through the delivery of recombinant growth factor proteins, through gene transfer or cell transplantation. Gene transfer associated with adenovirus or naked plasmid DNAs has been extensively studied in clinical trials. An investigational product, beperminogene perplasmid, is the naked plasmid DNA encoding the cDNA of human HGF, which has potent angiogenic activity. In several clinical trials, beperminogene perplasmid showed favorable safety and efficacy profile in the treatment of critical limb ischemia. This article reviews the results of pre-clinical and clinical studies of beperminogene perplasmid in the treatment of critical limb ischemia caused by peripheral arterial disease and Buerger’s disease.     

Glucocorticoid-Regulated VEGF Expression in Ischemic Skeletal Muscle

Vascular endothelial growth factor (VEGF) is a potent neovascular inducer. Gene therapeutic delivery of a plasmid DNA encoding VEGF has been shown to impart collateral vessel development in animal models of hindlimb ischemia. Constitutive, long-lived expression of VEGF through gene transfer, however, may result in hypervascularization and/or leaky blood vessels. To that end, the introduction of regulated VEGF gene transfer technology may provide a safer and more controlled therapy for ischemic tissues. We developed a glucocorticoid-regulated plasmid vector (pNGVL-hAP/GRE₅-vegf-pA) for modulating VEGF gene expression. This plasmid possessed five tandem repeats of the glucocorticoid-responsive element and adenovirus major-late promoter driving the expression of the VEGF₁₆₅ cDNA. Intramuscular delivery of this plasmid to mice, and subsequent treatment with the synthetic glucocorticoid dexamethasone (DEX), led to greatly enhanced VEGF expression. Similar delivery to the gracillis muscle of New Zealand white rabbits that had undergone ligation of their femoral artery to induce ischemia exhibited increased VEGF expression and collateral vessel development only in the presence of DEX. Additionally, reintroduction of DEX at a time point during which initial VEGF transgene levels had subsided resulted in a vigorous reinduction of VEGF transgene expression. This new iteration of VEGF gene delivery provides for finetuned angiogenic factor-based therapy for tissues requiring neovascularization.     

Gene therapy for peripheral arterial disease

Introduction: Gene therapy has emerged as a novel therapy to promote angiogenesis in patients with critical limb ischemia (CLI) caused by peripheral artery disease. Researchers working in this area have focused on pro-angiogenic factors, such as VEGF, fibroblast growth factor (FGF) and hepatocyte growth factor (HGF). Based on the elaborate studies and favorable results of basic research using naked plasmid DNA (pDNA) encoding these growth factors, some clinical Phase I and Phase II trials have been performed. The results of these studies demonstrate the safety of these approaches and their potential for symptomatic improvement in CLI patients. However, the Phase III clinical trials have so far been limited to HGF gene therapy. Because one pitfall of the Phase III trials has been the limited transgene expression achieved using naked pDNA alone, the development of more efficient gene transfer systems, such as ultrasound microbubbles and the needleless injector, as well as the addition of other genes will make these novel therapies more effective and ease the symptoms of CLI.

Areas covered: This study reviews the previously published basic research and clinical trials that have studied VEGF, FGF and HGF gene therapies for the treatment of CLI. Adjunctive therapies, such as the addition of prostacyclin synthase genes and the development of more efficient gene transfer techniques for pDNA, are also reviewed.

Expert opinion: To date, clinical studies have demonstrated the safety of gene therapy in limb ischemia but the effectiveness of this treatment has not been determined. Larger clinical studies, as well as the development of more effective gene therapy, are needed to achieve and confirm beneficial effects.     

Gene therapy for peripheral arterial diseases

Therapeutic angiogenesis using angiogenic growth factor is expected to be a new treatment for with patients with critical limb ischemia. The first human clinical trial treating peripheral vascular disease was started in 1994 using vascular endothelial growth factor (VEGF). To date, other potent angiogenic growth factors, such as fibroblast growth factor(FGF) or hepatocyte growth factor(HGF), have been also estimated in clinical trials for peripheral arterial disease. Several results from phase 1 or 2 trials using VEGF, FGF and HGF gene were encouraging. Phase 3 trials are now ongoing and their results are expected.     

Mesenchymal stem/stromal cells genetically engineered to produce vascular endothelial growth factor for revascularization in wound healing and ischemic conditions

Mesenchymal stem/stromal cells (MSCs) may be able to improve ischemic conditions as they can actively seek out areas of low oxygen and secrete proangiogenic factors. In more severe trauma and chronic cases, however, cells alone may not be enough. Therefore, we have combined the stem cell and angiogenic factor approaches to make a more potent therapy. We developed an engineered stem cell therapy product designed to treat critical limb ischemia that could also be used in trauma‐induced scarring and fibrosis where additional collateral blood flow is needed following damage to and blockage of the primary vessels. We used MSCs from normal human donor marrow and engineered them to produce high levels of the angiogenic factor vascular endothelial growth factor (VEGF). The MSC/VEGF product has been successfully developed and characterized using good manufacturing practice (GMP)‐compliant methods, and we have completed experiments showing that MSC/VEGF significantly increased blood flow in the ischemic limb of immune deficient mice, compared to the saline controls in each study. We also performed safety studies demonstrating that the injected product does not cause harm and that the cells remain around the injection site for more than 1 month after hypoxic preconditioning. An on‐demand formulation system for delivery of the product to clinical sites that lack cell processing facilities is in development.     

Gene therapy in heart disease.

Application of gene therapy to the field of cardiovascular disorders has been the subject of intensive work over the recent period. Gene therapy for cardiovascular disorders is now fast developing with most therapies being devoted to the consequences (ischemia) rather than the causes of atherosclerotic diseases. Recent human clinical trials have shown that injection of naked DNA encoding vascular endothelial growth factor promotes collateral vessel development in patients with critical limb ischemia or chronic myocardial ischemia. Promising studies in animals have also fueled enthusiasm for treatment of human restenosis by gene therapy, but clinical applications are warranted. Application of gene transfer to other cardiovascular diseases will require the coordinated development of a variety of new technologies, as well as a better definition of cellular and gene targets.     

Overview on diagnosis, treatment and therapeutic angiogenesis for arteriosclerosis obliterans

Arteriosclerosis of the extremities is a disease of the blood vessels characterized by hardening and/or narrowing of the arteries that supply the legs and feet. This causes a decrease in blood flow that can injure nerves and other tissues. Therapeutic angiogenesis using angiogenic growth factor is expected to be a new treatment for patients with critical limb ischemia. The first human clinical trial treating peripheral vascular disease was started in 1994 using vascular endothelial growth factor. To date, other potent angiogenic growth factors, such as hepatocyte growth factor(HGF), have been also estimated in clinical trials for peripheral arterial disease. Several results from phase 1 or 2 trials using HGF gene were encouraging. Phase 3 trials are now ongoing and their results are expected.     

Efficacious and Clinically Relevant Conditioned Medium of Human Adipose-derived Stem Cells for Therapeutic Angiogenesis

Using stem cell–conditioned medium (CM) might be a viable alternative to stem cell transplantation, which is often hampered by low grafting efficiency and potential tumorigenesis, but the concentrations of angiogenic growth factors in CM are too low for therapeutic use and some components of the medium are not for human use. We used three-dimensional (3D) spheroid culture of human adipose-derived stem cells (ADSCs) with clinically relevant medium composed of amino acids, vitamins, glucose, and human serum to produce clinically relevant CM containing angiogenic and/or antiapoptotic factors such as vascular endothelial cell growth factor, fibroblast growth factor 2, hepatocyte growth factor, and chemokine (C-X-C motif) ligand 12. The concentrations of these factors were 23- to 27-fold higher than that in CM produced by conventional monolayer culture. Compared with injection of either monolayer culture CM or human ADSC, injection of spheroid culture CM to an ischemic region in mice significantly enhanced endothelial cell growth, CD34⁺/PTPRC⁻ (endothelial progenitor) cell mobilization from bone marrow, and bone marrow cell homing to the ischemic region, resulting in improved blood vessel density, limb salvage, and blood perfusion in a mouse hindlimb ischemia model. The stem cell CM developed in this study will likely be an effective alternative to conventional stem cell transplantation therapy.     

Effective treatment of vascular endothelial growth factor refractory hindlimb ischemia by a mutant endothelial nitric oxide synthase gene

Gene delivery of angiogenic growth factors is a promising approach for the treatment of ischemic cardiovascular diseases. However, success of this new therapeutic principle is hindered by the lack of critical understanding as to how disease pathology affects the efficiency of gene delivery and/or the downstream signaling pathways of angiogenesis. Critical limb ischemia occurs in patients with advanced atherosclerosis often exhibiting deficiency in endothelial nitric oxide production. Similar to these patients, segmental femoral artery resection progresses into severe ischemic necrosis in mice deficient in endothelial nitric oxide synthase (ecNOS-KO) as well as in balb/c mice. We used these models to evaluate the influence of severe ischemia on transfection efficiency and duration of transgene expression in the skeletal muscle following plasmid injection in combination with electroporation. Subsequently, we also explored the potential therapeutic effect of the phosphomimetic mutant of ecNOS gene (NOS1177D) using optimized delivery parameters, and found significant benefit both in ecNOS-KO and balb/c mice. Our results indicate that NOS1177D gene delivery to the ischemic skeletal muscle can be efficient to reverse critical limb ischemia in pathological settings, which are refractory to treatments with a single growth factor, such as vascular endothelial growth factor.     

超声微泡介导VEGF基因治疗下肢血管闭塞

目的探讨用超声微泡造影剂促进VEGF基因治疗兔下肢缺血的可行性.方法将30只大白兔左股动脉结扎后随机分为3组: A组采用局部注射VEGF质粒DNA; B组采用局部注射VEGF质粒与超声微泡造影剂的混合物;C组作为对照.于治疗后4周进行数字减影血管造影(DSA)和免疫组织化学方法检测侧支循环建立和血管新生.结果 DSA可见用超声破坏微泡后促进侧支循环建立较好,新生血管较多;单纯局部注射质粒可促进部分血管新生,而对照组的侧支循环和新生血管较少.各组免疫组化所测血管计数均有显著性差异.结论用超声微泡介导的VEGF基因转染,可促进缺血骨骼肌的血管新生和侧支循环建立,为下肢缺血性疾病的基因治疗提供了一种新途径.     

Angiogenic and antifibrotic actions of hepatocyte growth factor improve cardiac dysfunction in porcine ischemic cardiomyopathy

Impairment of cardiac function in ischemic cardiomyopathy has been postulated to be due to the decrease in blood flow and increase in collagen synthesis. Therefore, an approach to alter them directly by means of a growth factor may open up a new therapeutic concept in ischemic cardiomyopathy. From this viewpoint, hepatocyte growth factor (HGF) is a unique growth factor with angiogenic and antifibrotic effects. Thus, we examined the feasibility of gene therapy using HGF plasmid DNA for ischemic cardiomyopathy. Human HGF plasmid DNA at a dose of 0.4 or 4 mg was injected into ischemic myocardium of pigs induced by ameroid constrictor with the NOGA system. At 1 month after injection, the ischemic area was significantly reduced in the HGF group, accompanied by a significant increase in capillary density and regional myocardial perfusion in the ischemic area (P<0.01). In contrast, a significant decrease in fibrotic area was observed in the HGF group, associated with a significant decrease in collagen I, III and TGF-beta synthesis as compared to the control group (P<0.01). Consistently, cardiac function was significantly improved in the 4 mg HGF group as compared to the control group (P<0.05). Overall, the present in vivo experiments demonstrated that intramyocardial injection of human HGF plasmid DNA in ischemic cardiomyopathy resulted in a significant improvement in cardiac function through an increase in blood flow and decrease in fibrosis. These favorable outcomes suggest potential utility to treat patients with ischemic heart disease using HGF gene transfer. Currently, a phase I study using human HGF plasmid DNA is ongoing to test the validity of this concept.     

Adeno-associated viral vector-mediated gene transfer of VEGF normalizes skeletal muscle oxygen tension and induces arteriogenesis in ischemic rat hindlimb

Critical limb ischemia is an important clinical problem that often leads to disability and limb loss. Vascular endothelial growth factor (VEGF), delivered either as recombinant protein or as gene therapy, has been shown to promote both collateral artery formation (arteriogenesis) and capillary angiogenesis in animal models of hindlimb ischemia. However, none of the previous studies has demonstrated an improvement in tissue hypoxia, the condition that drives the molecular response to ischemia. Furthermore, the optimal vector and route of gene delivery have not been determined. Recently, adeno-associated viral (AAV) vectors, which efficiently transduce skeletal muscle and produce sustained transgene expression, have been used as gene therapy vectors. We asked whether an intra-arterial injection of AAV-VEGF₁₆₅ normalizes muscle oxygen tension by increasing skeletal muscle oxygen tension, and promotes arteriogenesis and angiogenesis in a rat model of severe hindlimb ischemia. We found that AAV-VEGF treatment normalized muscle oxygen tension in the ischemic limb. In contrast, vehicle and AAV-lacZ-treated limbs remained ischemic. Collateral arteries were more numerous in AAV-VEGF-treated rats, but, surprisingly, capillaries were not. We conclude that intra-arterial AAV-mediated gene transfer of AAV-VEGF₁₆₅ normalizes muscle oxygen tension and leads to arteriogenesis in rats with severe hindlimb ischemia.     

Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle

Although clinical trials of stimulation of angiogenesis by transfection of angiogenic growth factors using naked plasmid DNA or adenoviral vector have been successful, there are still unresolved problems for human gene therapy such as low transfection efficiency and safety. From this viewpoint, it is necessary to develop safe and efficient novel nonviral gene transfer methods. As therapeutic ultrasound induces cell membrane permeabilization, ultrasound irradiation might increase the transfection efficiency of naked plasmid DNA into skeletal muscle. Thus, we examined the transfection efficiency of naked plasmid DNA using ultrasound irradiation with echo contrast microbubble (Optison) in vitro and in vivo experiments. First, we examined the feasibility of ultrasound-mediated transfection of naked plasmid DNA into skeletal muscle cells. Luciferase plasmid mixed with or without Optison was transfected into cultured human skeletal muscle cells using ultrasound (1 MHz; 0.4 W(2)) for 30 s. Interestingly, luciferase activity was markedly increased in cells treated with Optison, while little luciferase activity could be detected without Optison (P < 0.01). Electron microscopy demonstrated the transient formation of holes (less than 5 microM) in the cell surface, which could possibly explain the rapid migration of the transgene into the cells. Next, we studied the in vivo transfection efficiency of naked plasmid DNA using ultrasound with Optison into skeletal muscle. Two days after transfection, luciferase activity in skeletal muscle transfected with Optison using ultrasound was significantly increased about 10-fold as compared with plasmid alone. Successful transfection was also confirmed by beta-galactosidase staining. Finally, we examined the feasibility of therapeutic angiogenesis using naked hepatocyte growth factor (HGF) plasmid in a rabbit ischemia model using the ultrasound-Optison method. Five weeks after transfection, the angiographic score and the number of capillary density in rabbits transfected with Optison using ultrasound was significantly increased as compared with HGF plasmid alone (P < 0.01), accompanied by a significant increase in blood flow and blood pressure ratio (P < 0.01). Overall, the ultrasound transfection method with Optison enhanced the transfection efficiency of naked plasmid DNA in vivo as well as in vitro. Transfection of HGF plasmid by the ultrasound-Optison method could be useful for safe clinical gene therapy to treat peripheral arterial disease without a viral vector system.     

大鼠缺血下肢微血管生成与运动训练

背景:研究表明缺血可通过侧支循环的建立和血管的新生得到代偿,运动训练可以改善患者缺血下肢血液供应,但运动是否能够促进侧支循环的建立至今少有报道.目的:探讨运动训练促进大鼠缺血下肢微血管生成的机制.方法:将SD大鼠随机分为运动训练组、模型组和假手术组.除假手术组外,其余组均建立大鼠左下肢缺血模型.建模1周后运动训练组大鼠跑步训练30 min/d.模型组和假手术组均为日常活动.运动训练4周后取各组大鼠大腿内收肌组织块免疫组化检测微血管密度、血管内皮生长因子和碱性成纤维细胞生长因子的表达,同时取骨髓内皮祖细胞,检测其成血管生成能力.结果与结论:运动训练组内皮祖细胞、大鼠肌组织血管内皮生长因子、碱性成纤维细胞生长因子的表达和微血管密度均高于模型组和假手术组(P<0.01).运动组骨髓内皮祖细胞的体外血管生成能力比假手术组及模型组增加(P<0.05).结果提示下肢缺血刺激可以促进微血管新生,而运动训练可以增强该效应.     

Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model.

Vascular endothelial growth factor (VEGF) is a heparin-binding, endothelial cell-specific mitogen. Previous studies have suggested that VEGF is a regulator of naturally occurring physiologic and pathologic angiogenesis. In this study we investigated the hypothesis that the angiogenic potential of VEGF is sufficient to constitute a therapeutic effect. The soluble 165-amino acid isoform of VEGF was administered as a single intra-arterial bolus to the internal iliac artery of rabbits in which the ipsilateral femoral artery was excised to induce severe, unilateral hind limb ischemia. Doses of 500-1,000 micrograms of VEGF produced statistically significant augmentation of collateral vessel development by angiography as well as the number of capillaries by histology; consequent amelioration of the hemodynamic deficit in the ischemic limb was significantly greater in animals receiving VEGF than in nontreated controls (calf blood pressure ratio, 0.75 +/- 0.14 vs. 0.48 +/- 0.19, P < 0.05). Serial angiograms disclosed progressive linear extension of the collateral artery of origin (stem artery) to the distal point of parent vessel (reentry artery) reconstitution in seven of nine VEGF-treated animals. These findings establish proof of principle for the concept that the angiogenic activity of VEGF is sufficiently potent to achieve therapeutic benefit. Such a strategy might ultimately be applicable to patients with severe limb ischemia secondary to arterial occlusive disease.     

IRES-based Vector Coexpressing FGF2 and Cyr61 Provides Synergistic and Safe Therapeutics of Lower Limb Ischemia

Due to the lack of an adequate conventional therapy against lower limb ischemia, gene transfer for therapeutic angiogenesis is seen as an attractive alternative. However, the possibility of side effects, due to the expression of large amounts of angiogenic factors, justifies the design of devices that express synergistic molecules in low controlled doses. We have developed an internal ribosome entry site (IRES)–based bicistronic vector expressing two angiogenic molecules, fibroblast growth factor 2 (FGF2), and Cyr61. Through electrotransfer into the ApoE^−/− mice hindlimb ischemic muscle model, we show that the IRES-based vector gives more stable expression than either monocistronic plasmid. Furthermore, laser Doppler analysis, arteriography, and immunochemistry clearly show that the bicistronic vector promotes a more abundant and functional revascularization than the monocistronic vectors, despite the fact that the bicistronic system produces 5–10 times less of each angiogenic molecule. Furthermore, although the monocistronic Cyr61 vector accelerates B16 melanoma growth in mice, the bicistronic vector is devoid of such side effects. Our results show an active cooperation of FGF2 and Cyr61 in therapeutic angiogenesis of hindlimb ischemia, and validate the use of IRES-based bicistronic vectors for the coexpression of controlled low doses of therapeutic molecules, providing perspectives for a safer gene therapy of lower limb ischemia.     

山莨菪碱对大鼠缺血后肢侧支循环建立的作用

目的探讨山莨菪碱对大鼠缺血后肢侧支循环建立的影响。方法建立左后肢缺血模型后,随机分为治疗组和对照组,分别给予山莨菪碱注射液8 mg/（kg.d）和等量生理盐水,于缺血后肢分5点进行肌内注射,连续14 d;观察术后即刻、7、14、28 d健侧后肢与缺血后肢脚垫温差及术后28 d大鼠后肢肌肉组织中的微血管密度（Microvessel density,MVD）。结果治疗组两侧后肢脚垫温差（1.75±0.18）℃,较对照组（2.57±0.13）℃小（P〈0.05）;治疗组缺血后肢肌肉组织中的MVD（4.60±1.50）较对照组（3.33±1.05）高（P〈0.05）。结论山莨菪碱对大鼠缺血后肢侧支循环的建立有促进的作用。