Objective To investigate the feasibility of intramuscular gene therapy for acute arterial ischemic diseases by use of plasmid pcDNA3-VEGF121 and to evaluate therapeutic efficiency of vascular endothelial growth factor(VEGF) by different routes of administration. Methods Fifty New Zealand White rabbits were randomly assigned to either gelation sponge carryingpcDNA3-VEGF121 (n=18), intramuscular injectionpcDNA3-VEGF121 (n=18), or pcDNA3 (as control group,n=14). After ligation of the external iliac artery and complete excision of the femoral artery, 500 μg of the plasmid pcDNA 3-VEGF121 were transfected into the muscles of the ischemic limb by gelation sponge carrying or direct intramuscular-injection. Immediately after gene transfection, blood flow of the internal iliac artery were measured. VEGF121gene expression was detected by RT-PCR after 2 days, 1 week, 2 weeks, 3 weeks and 4 weeks of transfection. After 30 days, blood flow of the internal iliac artery, angiographic score and histologicalvessels of ischemic hindlimbs were measured respectively. Results In the two VEGF-treated groups, VEGF121 mRNA expressed in the transfected ischemic muscles after 2 days and lasted 2 weeks. Immediately after gene transfection, blood flow of the internal iliac artery had no significant difference between three groups. After 30 days, blood flow of the internal iliac artery, angiographicscore and capillary density were significantly greater in both VEGF-treated groups than in control group. Complexity of vascular branching and vessel density of gelation sponge-VEGF treated limbs were significantly greater when comparedwith the intramuscular-injection limbs. Conclusion These findings suggest the feasibility of employing gene therapy of pcDNA3-VEGF121could augmentcollatal development and tissue perfusion in an animal model of hindlimb ischemia, andgelation sponge carrying VEGF gene may respect a potential therapy methods.
Objective To evaluate the effectiveness and safety of sarpogrelate hydrochloride for patients with peripheral arterial disease (PAD). Methods The randomized controlled trials (RCTs) on PAD treated by sarpogrelate hydrochloride were identified from CBM (1978 to September 2011), CNKI (1979 to May 2011), PubMed (1950 to May 2011), EMbase (1970 to May 2011) and The Cochrane Library (Issue 3, 2011). According to the criteria of the Cochrane Handbook, two reviewers independently screened the studies, extracted and cross-checked the data, and assessed the methodological quality. Then meta-analysis was conducted by using RevMan 5.0 software. Results Nine RCTs involving 522 patients and 532 limbs were included, with low methodological quality in most trials. The results of meta-analyses indicated that compared with the conventional treatment, sarpogrelate hydrochloride could reduce the area of ulcers (MD= –3.22, 95%CI –3.99 to –2.45), and it could increase the ankle-brachial index (SMD=0.49, 95%CI 0.07 to 0.91), blood flow of dorsalis pedis artery (MD=0.16, 95%CI 0.09 to 0.23) and pain-free walking distance (MD=200.87, 95%CI 3.39 to 398.36). Five trials reported the adverse effects of sarpogrelate hydrochloride, most of which were mild gastrointestinal symptoms. Conclusion Based on the review, sarpogrelate hydrochloride may have positive effect on patients with PAD. However, the evidence is not b enough due to the general low methodological quality, so the reliable conclusion has to be drawn with more high quality studies in future.
Objective To investigate the application of abdominal aorta and bilateral abdominal perineal artery ligation in a hindlimb ischemia model of rats for research. Methods According to the random digits table, 38 SD rats were divided into 3 groups randomly, sham operation group (SO group, n=12), abdominal aorta ligation group (AAL group, n=12) and abdominal aorta and bilateral abdominal perineal artery ligation group (AAL+BAPA group, n=14). Rats were anesthetized by 6 mg/100 g ketamine via intraperitoneal injection. Blood vessels were ligated via laparotomy according to different procedures. Movement and pale skin color of rat’s hindlimb were observed on 2 and 4 weeks after operation, meanwhile venous blood from unilateral iliac vein was obtained for blood gas analysis and hindlimb skeletal muscle for HE stain. Results Two rats were dead during 3 d after operation in AAL+BAPA group, other rats survived. Rats in SO group had no obviously abnormal appearance. AAL group and AAL+BAPA group immediately presented hindlimbs pale, lower skin temperature, hypofrontality of limb motion after procedure. Symptoms above mentioned had improved gradually after 2 weeks and completely recuperated 4 weeks after operation in AAL group. Ischemia symptoms were still remained obviously such as cold, dried and thin on the 4th weekend in AAL+BAPA group. Each group had no hindlimbs necrosis. Two weeks after operation, pale limbs and muscle strength in AAL+BAPA group were more severe than those of SO group (Plt;0.05); Pale limbs was still worse than that of AAL group on 4 weeks after operation (Plt;0.05). There were no significant differences on different time in each group (Pgt;0.05). Venous blood partial pressure of oxygen of AAL+PABA group was significantly lower than that of the other two groups on 2 and 4 weeks after operation (Plt;0.05). Normal striated muscle structure was presented in SO group pathologically. AAL group revealed coloretur unevenness, swelling and distension, muscle cellular transverse striation elimination, skeletal muscle cell nucleolus deeply stained on the 2nd weekend and no difference with the SO group on the 4th weekend. AAL+BAPA group presented skeletal muscles decoration unevenness, cells swelling and distension, muscle cellular transverse striation elimination, skeletal muscle cell nucleolus stained deeply and intensively, intercellular space widening until the 4th weekend， but no obviously necrocytosis. Conclusion The method of ligating abdominal aorta and bilateral abdominal perineal artery can make a stable SD rat model of hindlimb ischemia
Objective To review the studies on vascular endothelium growth factor (VEGF) gene therapy for patients with chronic critical limb ischemia. Methods Advance in molecular biology of VEGF, mechanism of new vessel formation induced by VEGF and achievement of improving blood flow in patients with critical limb ischemia due to VEGF expressed by gene transfer in recent years has been reviewed in this article. Results Preclinical studies showed that VEGF can stimulate the development of collateral arteries in animals with limb ischemia, a concept called “therapeutic angiogenesis”, clinical results demonstrated that VEGF expressed by gene transfer can promote new vessel formation in patients with critical limb ischemia and improve significantly the prognosis for them.Conclusion VEGF gene transfer provide a novel treatment strategy for patients with critical limb ischemia, who neither had favorable response to phamarcological treatment nor were suitable for surgical reconstruction or revascularization.
ObjectiveTo explore the relationship between the oxygen partial pressure of mice hindlimb muscles with normal blood supply or ischemia and expression of HIF-1αprotein, and to provide a theoretical basis for the study of angiogenesis in vitro hypoxia. MethodsMice hind limb ischemia model were established, tissue oxygen tension of gastrocnemius muscle and bone marrow were measured by micro electrode at different time points of ischemia (24 hours, 1 week, 2 weeks, 3 weeks, and unoperated as control). Protein level of hypoxia inducible factor-1αand histological examination were performed on gastrocnemius muscle as well. ResultsThe oxygen tension baselines of gastrocnemius muscle and femoral bone marrow was (47.78±4.37) mm Hg and (21±3.40) mm Hg, respectively. Muscle oxygen tension decreased significantly at all time points after modeling (P < 0.05), and reached lowest level in 1 week of ischemia. The inflammatory reaction was most serious and HIF-1αprotein reached highest level at the same time point. With the extension of ischemic time, the tissue oxygen tension recovered while HIF-1αlevel was down-regulated, however, There was no statistical correlation(r=-0.86, P > 0.05). Oxygen tension in bone marrow didn't show any significant change at all time points. ConclusionsThe expression level of HIF-1αprotein in ischemic tissue can reflect the degree of ischemic limb. The concept that physiological oxygen level differs in different tissue is highlighted, and may provide basis for ex vivo hypoxic research.
ObjectiveTo evaluate the effects of nerve growth factor (NGF) on angiogenesis and skeletal muscle fiber remodeling in ischemic hindlimbs, and study the relationship of NGF and vascular endothelial growth factor (VEGF) to angiogenesis. MethodsEighteen mice were randomly allocated to normal control group (n=6), blank control group (n=6), and NGF gene transfection group (n=6). The left hindlimb ischemia model was established by ligating the femoral artery. NGF plasmid (125μg) was injected into the mouse ischemic gastrocnemius in the NGF gene transfection group. The same volume of normal saline (200μL) was injected into the mouse ischemic gastrocnemius in the blank control group. The gastrocnemius of left hindlimb was harvested under the condition of peritoneal cavity anesthesia on the 21th day after operation, and then the mice were sacrificed. The gastrocnemius of three groups were tested by hematoxylin-eosin staining, proliferating cell nuclear antigen (PCNA) and CD34 were determined by immunohistochemistry staining. Skeletal muscle fiber type was tested by myosin ATPase staining. NGF and VEGF protein expression were detected by enzyme linked immunosorbent assay. ResultsOn the 21th day after surgery, compared with the blank control group, the skeletal muscle atrophy degree was weaker, the functional assessment score was significantly lower (P < 0.05), the endothelial cell proliferation index, capillary density, the typeⅠskeletal muscle fiber proportion, NGF and VEGF expression were significantly higher (P < 0.05) in the NGF gene transfection group. ConclusionsNGF gene transfection could promote NGF and VEGF expression and angiogenesis in ischemic hindlimbs, and induce typeⅠskeletal muscle fibers formation in ischemic hindlimbs. The molecular regulation mechanism still needs to be further studied.