Vascular endothelial cell growth factor and fibroblast growth factor 2 expression in patients with critical limb ischemia

BACKGROUND: For patients with critical limb ischemia (CLI), there is a great need for alternative treatment strategies. One option is therapeutic angiogenesis by administration of vascular growth factors. The lack of convincing clinical data supporting this strategy may be due to the ignorance of endogenous angiogenic processes in CLI. To evaluate the importance of vascular growth factors in the pathogenesis in CLI and provide information for clinical growth factor treatment trials, we determined the levels of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF-2) in the ischemic legs of patients with this disease. METHODS: Skin and muscle biopsies from the calf and groin were gathered from 25 patients with CLI. Control samples came from 10 orthopedic patients and from 5 patients who were undergoing coronary artery bypass. The concentration of VEGF and FGF-2 in the biopsies was measured by enzyme-linked immunoassay, and to localize growth factor production, biopsied sections were immunostained. RESULTS: Patients with CLI had lower levels of VEGF in distal skin samples than in proximal ones (mean difference: 16.7 pg/mg total protein, 95% confidence interval: -1.0 to -32.3, P =.038), but these levels were similar to those in distal samples from control subjects (8.0, -4.6 to 20.5, P =.65). In muscle, VEGF concentrations were similar in calf and groin (5.4, -12.4 to 23.1, P =.55), but distal levels were higher than in distal samples from control subjects (23.7, 1.2 to 56.7, P =.028). Skin FGF levels tended to be higher in distal samples (45.3, 26.5 to 117.5, P =.090) and were higher than in skin from control subjects (106.2, -11.4 to 223.8, P =.050). Also in muscle, distal samples had higher levels of FGF-2 (35.6, -1.6 to 59.7, P =.006), but these levels were similar to what was found in control subjects (29.4., -16.3 to 81.2, P =.39). Growth factors were located in connective tissue between muscle fibers. In skin, the predominant FGF-2 staining was just below the epidermal layer, whereas VEGF appeared in the dermal layer. CONCLUSIONS: The results indicate that there are elevated concentrations of FGF-2 in calf muscle, whereas VEGF concentrations do not appear to be higher in the ischemic part of the leg in patients with CLI. These findings suggest that VEGF supplementation may be a more appropriate strategy for therapeutic angiogenesis to the calf area for CLI than FGF-2.     

腺相关病毒介导的血管内皮生长因子基因与内皮祖细胞联合治疗肢体缺血

目的探讨腺相关病毒(AAV)介导的人血管内皮生长因子165(hVEGF165)基因转染的兔骨髓内皮祖细胞(EPCs)自体移植对于移植细胞存活率和缺血肢体血管再生的影响。方法构建、制备携带hVEGF165基因或β-半乳糖苷酶(β-gal)基因的AAV载体AAV-hVEGF165和AAV- LacZ;用AAV—hVEGF165和AAV-LacZ分别转染体外培养的EPCs,得到AAV/VEGF-EPC和AAV/LacZ-EPC,用RT-PCR、免疫细胞化学、流式细胞术检测外源基因的表达。分别用AAV/VEGF-EPC、AAV/LacZ-EPC和PBS自体移植于兔右下肢缺血的肌组织,28 d后行双侧肢体血流和免疫组织化学检测。结果AAV/VEGF—EPC和AAV/LacZ-EPC内可检测到外源基因的表达。AAV/VEGF—EPC、AAV/LacZ-EPC和PBS组患/健侧血流比值、毛细血管密度分别为0.73±0.21、0.64±0.13、0.45±0.10;(962±291)、(557±132)、(361±69)/mm2。AAV/VEGF-EPC和AAV/LacZ-EPC组移植成活的EPCs密度分别为(330±81)/mm2和(204±55)/mm2。结论AAV能够高效将外源基因转入EPCs,对其进行基因修饰。自体移植AAV-hVEGF165转染的EPCs可提高其移植存活率和增强其促进缺血肢体血管再生能力。     

Vascular endothelial growth factor gene delivery by magnetic DNA nanospheres ameliorates limb ischemia in rabbits

BACKGROUND: Critical limb ischemia 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 arteriogenesis and angiogenesis in animal models of limb ischemia. However, most of the studies used a nonspecific targeting system. MATERIALS AND METHODS: Magnetic DNA nanospheres containing expression plasmids encoding VEGF were synthesized, and their morphology, magnetropism, and stability were analyzed. The magnetic DNA nanospheres were administrated via an artery into a rabbit limb ischemia model. The expression of VEGF and vascularization were examined by immunohistochemistry. The angiography was taken to evaluate arteriogenesis. RESULTS: Magnetic DNA nanospheres were very stable and showed a high magnetropism. Gene delivery of such nanospheres via artery under a magnetic field led to the overexpression of VEGF in situ. The capillary density and capillary to muscle fiber ratio were doubled compared with those of the control animals. The arteriogenesis also was promoted in VEGF gene therapy group compared with controls but at later interval than capillary angiogenesis. CONCLUSIONS: Our results suggest that intra-arterial VEGF gene delivery by magnetic DNA nanosphere promotes angiogenesis and arteriogenesis and presents a potent therapeutic strategy for critical limb ischemia.     

严重肢体缺血的干细胞治疗研究进展

大量动物实验和临床试验揭示干细胞移植可望成为一种非常有前途的治疗严重肢体缺血的手段.笔者对干细胞移植治疗严重肢体缺血的适应证、干细胞来源及分离方法、移植途径、安全性及有效性等方面研究进展进行综述.     

血管内皮生长因子-碱性成纤维细胞生长因子基因治疗家兔肢体缺血的研究

目的　了解血管内皮生长因子 (VEGF)和碱性成纤维细胞生长因子 (bFGF)联合克隆基因 (VEGF bFGF)治疗兔下肢动脉缺血模型后新生血管和侧支形成状况。方法　应用 40只家兔制成下肢缺血模型 ,其中VEGF bFGF组 10只 ,VEGF组 12只 ,空载体组 8只 ,生理盐水 (NS)组 10只。构建pcDNA3 /VEGF和pcDNA3 /VEGF bFGF真核表达载体。转染缺血部位肌组织 ,行下肢血管造影。结果　血管造影计数显示 ,VEGF bFGF组在转染后 14d(1.98± 0 .2 2 ) ,2 8d (1.81± 0 .5 2 ) ,5 6d(2 .2 1± 0 .44 )和 3个月 (2 .10± 0 .2 2 ) ;VEGF组在 2 8d(1.3 8± 0 .2 9) ,5 6d(1.94± 0 .2 5 )和 3个月 (2 .2 4± 0 .3 1) ,新生血管形成较对照组显著增加 (P <0 .0 5 )。结论　VEGF bFGF真核表达载体可以获得局部高效表达 ,刺激新生血管生成 ,建立侧枝循环 ,改善肢体缺血。     

Platelet-derived growth factor and vascular endothelial growth factor expression in disc herniation tissue: an immunohistochemical study

Angiogenesis is essential in tissue growth and regeneration. There are several factors that are able to stimulate vascular endothelial cell growth, including platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). Disc herniation tissue (DHT) contains vascular ingrowth, which promotes granulation tissue formation. In this study we observed 50 disc herniations for PDGF and VEGF immunoreactivity. PDGF immunopositivity was detected in 38 samples (78%). In 28 samples (56%) there were PDGF immunopositive capillaries, PDGF immunopositive disc cells were detected in 19 samples (38%) and PDGF immunopositive fibroblasts in 6 DHT samples (12%). VEGF immunopositive capillaries were identified in 44 DHT samples (88%). For neither growth factor was immunopositivity dependent on preoperative radicular pain duration. In extrusions (n = 25) VEGF immunopositive capillaries were detected in 23 samples (92%) and PDGF immunopositivity in 21 samples (84%). PDGF immunopositivity was more commonly associated with capillaries than with nuclei of disc cells. In sequesters (n = 20) VEGF immunopositive capillaries were identified in all samples and PDGF immunopositivity in 16 (80%). As in extrusions, PDGF immunoreaction was more prevalent in capillaries than in disc cells. Patient age did not relate to VEGF expression. In all age groups it was higher than 80%. Thus capillaries in disc herniation tissue are evidently newly formed and our results demonstrate that PDGF and VEGF participate in the neovascularization process. The presence of PDGF in fibroblasts and in disc cells suggests that this growth factor regulates the function of these cells, possibly the proliferation of the cells and the production of extracellular matrix components.     

Endovascular therapy for critical limb ischemia

Bypass surgery is still considered the "gold standard" therapy for the treatment of critical limb ischemia (CLI). However, due to recent advances in endovascular technologies, catheter-based intervention has become a viable option, and percutaneous treatment is becoming more widely used. This paper provides a brief overview of the percutaneous therapies available today and new technologies on the horizon as well as highlights some advantages and limitations of each treatment option in CLI.     

Platelet-derived growth factor production by canine aortic grafts seeded with endothelial cells.

The advantages of an endothelial cell surface on a prosthetic graft may be compromised by endothelial cell production of mitogens for smooth muscle cells. To study platelet-derived growth factor (PDGF) production by cells lining prosthetic grafts, 15 beagles underwent placement of 20 to 22 cm long, 8 mm inner diameter, expanded polytetrafluoroethylene thoracoabdominal aortic grafts, of which seven were seeded with autologous jugular vein endothelial cells, and eight were unseeded control grafts. Grafts and adjacent arteries were explanted after 4 weeks, divided into segments, and studied in organ culture. Platelet-derived growth factor production during a 72-hour incubation period was measured by radioreceptor assay. Midgraft segments of seeded grafts produced significantly more PDGF than control grafts, 43 +/- 8 pg/cm2 and 22 +/- 5 pg/cm2, respectively (mean +/- SEM), p less than 0.05. Platelet-derived growth factor production correlated directly with endothelial cell coverage on graft segments as measured by scanning electron microscopy (r = 0.63, p = 0.01), and inversely with platelet deposition (r = -0.48, p = 0.07). For all dogs, PDGF production by the distal aorta was significantly greater than that by the proximal aorta, 89 +/- 6 and 17 +/- 4 pg/cm2, respectively (p less than 0.0001). These results suggest that endothelial cells on prosthetic vascular grafts are a significant source of PDGF. Furthermore, the higher PDGF production by the distal arteries may offer an explanation for the clinical finding of more severe intimal hyperplasia adjacent to the distal anastomosis.     

Vascular endothelial growth factor gene therapy in ischaemic rat skin flaps.

Gene therapy with the complementary DNA (cDNA) of the angiogenic cytokine vascular endothelial growth factor (VEGF) has emerged as a promising strategy in the treatment of myocardial and lower-limb ischaemia. The objective of this study was to determine whether these principles could be applied to a recognised model of skin-flap ischaemia. Plasmid vectors including the cDNA of green fluorescent protein (GFP) and one of three VEGF isoforms (A165, B167 or B186) were constructed, and their base sequences confirmed. GFP expression was used as a marker of successful in vitro transfection of human endothelial cells with each plasmid. The plasmids were then administered subcutaneously to rat abdominal skin flaps surgically rendered ischaemic, and the percentage of viable tissue was assessed at 1 week. Angiograms of the flaps and histological preparations of flap tissue were assessed for evidence of angiogenesis. The survival of flaps treated with VEGF A165 or B167 cDNA was significantly greater than that of controls (P < 0.05). The survival of flaps treated with VEGF B186 cDNA was greater than that of controls, but statistical significance was not reached. Angiograms and microvessel density counts failed to produce evidence of angiogenesis. With improved delivery strategies, VEGF may have a role in the management of surgical ischaemia.     

Platelet-derived growth factor in experimental glomerulonephritis

Growth factors and in particular platelet-derived growth factor(PDGF) have been implicated in the pathogenesis of glomerulonephritisand glomerulosclerosis. We have studied the distribution ofimmunoreactive PDGF (iPDGF) within serial kidney biopsies (days7, 15, 30, 90 and 120) of eight rats with an accelerated formof nephrotoxic serum nephritis (NTN). The course of NTN wasmild in five rats and severe in three. Two patterns of immunostainfor PDGF were noted. The first consisted of iPDGF-B chain ina glomerular segmental distribution similar to that of infiltratingmonocytes (OX6+cells). At all stages of NTN the distributionof iPDGF-B chain correlated closely with the immunostain formonocytes. The second pattern of immunostain showed iPDGF-Achain in a diffuse distribution along the glomerular capillarylining and to a lesser extent in some mesangial cells. In severelyaffected rats the magnitude of the iPDGF-A increased along withglomerulosclerosis but disappeared later from areas of segmentaland global glomerular obsolescence. By contrast, in rats withmilder NTN glomerular iPDGF-A chain peaked early and decreasedsubsequently. During the acute phase of NTN, on day 7, iPDGFA chain correlated with the severity of proteinuria (r=0.848,P<0.05) and that of glomerular cellular proliferation (r=0.91,P<0.02). On day 30 iPDGF-B correlated positively with proteinuria(r=0.998, P<0.01, n=5) and with serum creatinine (r=0.949,P<0.02, n=5). During the late stages of NTN, both iPDGF-A(day 90, r=0.884, P<0.05) and iPDGF-B (day 120, r=0.941,P<0.01) correlated closely with the extent of glomerulosclerosis.Both PDGF chains (A and B) are detectable through out the courseof nephrotoxic nephritis in rats. PDGF may play a role in theearly proliferative and late sclerotic changes of experimentalglomerulonephritis.     

慢性下肢动脉缺血的腔内治疗

动脉粥样硬化是慢性下肢缺血的主要原因,约有65％的患者为多平面动脉狭窄或闭塞。目前传统的旁路手术仍然是外周动脉阻塞性疾病的主要治疗措施。但腔内治疗技术的发展,使许多原来手术治疗的疾病可以采用更加微创的治疗方法。为挽救肢体,腔内治疗通常应同时包括髂股和胫腓动脉闭塞性疾病。重建胫腓动脉血流不仅可以使髂股动脉腔内治疗效果更加长久,而且可以使缺血症状明显缓解。但小腿血管的腔内技术有一定局限,多采用手术联合腔内技术治疗多节段动脉闭塞。达到了较好的临床效果。     

Amelioration of microvascular myocardial ischemia by gene transfer of vascular endothelial growth factor in rabbits

OBJECTIVES: Restoration of coronary blood flow by angiogenesis may offer a new approach to intractable ischemic heart disease. In the present study, we investigated the angiogenic effects of gene transfer of vascular endothelial growth factor 165 on microvascular myocardial ischemia. METHODS: A rabbit model of microvascular myocardial ischemia was created by plugging coronary microvessels with microspheres (15 microm in diameter, 2.8 x 10(5)/kg, n = 29). Gene transfer was performed by semi-selective intracoronary injection of recombinant adenovirus expressing vascular endothelial growth factor 165 forty minutes after microsphere injection (n = 9). RESULTS: Microsphere injection reduced myocardial perfusion (78% +/- 9% of baseline tissue flow) and diminished myocardial contraction (61% +/- 12% of the baseline ejection fraction) and cardiac performance (elevated left ventricular end-diastolic pressure and decreased systemic flow) in the acute phase. At 17 +/- 3 days, gene transfer of vascular endothelial growth factor 165 had had the following effects: (1) promoted coronary angiogenesis as evidenced by myocardial flow above the baseline (121% +/- 24%), (2) increased vascular density revealed by synchrotron radiation microangiography and histologic analysis, (3) ameliorated the degree of myocardial ischemia as evidenced by myocardial lactate content and the extent of histologic necrosis, and (4) restored heart function as evidenced by increased ejection fraction (95% +/- 10%), reduced left ventricular end-diastolic pressure, and restored body weight. CONCLUSIONS: In vivo vascular endothelial growth factor 165 gene transfer promoted angiogenesis and was an effective approach to treating microvascular myocardial ischemia.     

Combined strategy of mesenchymal stem cell injection with vascular endothelial growth factor gene therapy for the treatment of diabetes-associated erectile dysfunction

This study was designed to investigate the effect of vascular endothelial growth factor 164 adenovirus (Ad-VEGF(164))-transfected mesenchymal stem cells (MSC) on improving erectile function in diabetic rats. Forty-five male Sprague-Dawley rats were injected with streptozotocin to develop type 1 diabetes, whereas 10 served as normal controls. Diabetic rats were randomly divided into 3 groups: rats that underwent intracavernous injection with phosphate-buffered saline (DM+PBS), unmodified MSCs (DM+MSC), and Ad-VEGF(164)-transfected MSCs (DM+VMSC). Normal controls received intracavernous injection of PBS. Four weeks after injection, erectile function was measured by cavernous nerve electrostimulation. Penile tissue was harvested for histology and enzyme-linked immunoassay. Prior to injection, high expression of VEGF was confirmed in Ad-VEGF(164)-transfected MSCs by enzyme-linked immunoassay. Four weeks after injection, the erectile function, as well as the content of smooth muscle and endothelium in corpus cavernosum increased significantly in the MSC-injected groups compared with the DM+PBS group. There was a significant improvement of erectile function, the content of smooth muscle and endothelium, and the VEGF concentration in the corpus cavernosum in the DM+VMSC group compared with the DM+MSC group. Our study validates the effect of intracavernous injection of MSCs for diabetes-associated erectile dysfunction in an animal model. The combined strategy of MSC injection with VEGF gene therapy-enhanced therapy of MSCs for the treatment of diabetes-associated erectile dysfunction.