Local perivascular application of low amounts of a plasmid encoding for vascular endothelial growth factor (VEGF165) is efficient for therapeutic angiogenesis in pigs.

Clinical trials have demonstrated therapeutic benefit in inducing angiogenesis in chronic occlusive arterial disease. The route of application mostly used was the intramuscular injection of high dosages of plasmid. Therefore, a local perivascular application of low amounts of vascular endothelial growth factor (VEGF) plasmid was used in an interventional occlusion model, and the effect of VEGF on coronary and peripheral occlusions compared in the same animal model. Coronary and peripheral arteries were chronically occluded in Pietrain pigs using a non-surgical, interventional approach. Adventitial delivery of the DNA for VEGF was performed with a needle injection catheter. The DNA was applied as lipoplexes using the novel cationic liposomes DOCSPER. Optimized transfer conditions were used. Angiography, polymerase chain reaction (PCR), reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry were undertaken within a follow-up period of 6 months. Expression of the transfected VEGF gene was observed at 1 and 3 weeks following application. The DNA was detected up to 5 months following application. Around occluded coronary arteries, there was formation of new collaterals and arterial prolongation, whereas surrounding occluded peripheral arteries there was no collateralization but development of new arterial branches was seen. Results demonstrate that the response to VEGF is also sufficient, when minimal amounts of plasmid encoding for VEGF are applied locally into the perivasculature allowing for more safety of this therapy. Comparison of treatment of chronic coronary and peripheral arterial disease revealed differences in angiogenesis following VEGF application during a total follow-up period of almost 6 months which may be related to their different developmental origins. This may have important implications for developing future therapeutic strategies using VEGF in different vessels.

Evaluation of endovascular techniques for creating a porcine femoral artery occlusion model.

PURPOSE: To determine the optimal endovascular approach to achieve long-term occlusion of large arteries, while preserving the integrity of periarterial tissue, in an animal model of ischemia. METHODS: Femoral artery occlusions were created in 16 pigs using detachable balloons, coils, or blinded stent-grafts. Feasibility, safety, primary and long-term success, and the degree of neovascularization were determined over a 6-month period by serial angiography and histological analyses. Four animals served as untreated controls. RESULTS: Overall primary success for all occlusion devices was 100%. The 6-month occlusion rate using detachable balloons or coils was 33% and 0%, respectively; however, all arteries occluded with blinded stent-grafts remained obstructed to the end of the study. There was no significant difference in capillary densities and collateralization of periarterial areas when occluded arteries were compared with nonoccluded controls in the same animal. No increase in collateralization was observed following endovascular arterial occlusion. CONCLUSIONS: Percutaneous insertion of blinded stent-grafts easily, safely, and reliably creates long-term arterial occlusion in pigs, which may make this a more appropriate model for studying the effects of angiogenic factors in vivo.

In vivo perivascular implantation of encapsulated packaging cells for prolonged retroviral gene transfer.

Long-term benefits of coronary angioplasty remain limited by the treatment-induced renarrowing of arteries, termed restenosis. One of the mechanisms leading to restenosis is the proliferation of smooth muscle cells. Therefore, proliferating cells of the injured arterial wall, which can be selectively transduced by retroviruses, are potential targets for gene therapy strategies. A direct single-dose therapeutic application of retroviral vectors for inhibition of cell proliferation is normally limited by too low transduction efficiencies. Encapsulated retrovirus-producing cells release viral vectors from microcapsules, and may enhance the transduction efficiency by prolonged infection. Primary and immortal murine and porcine cells and murine retrovirus-producing cells were encapsulated in cellulose sulphate. Cell viability was monitored by analysing cell metabolism. Safety, stability, transfer efficiency and extent of restenosis using capsules were determined in a porcine restenosis model for local gene therapy using morphometry, histology, in situ beta-galactosidase assay and PCR. Encapsulation of cells did not impair cell viability. Capsules containing retrovirus-producing cells expressing the beta-galactosidase reporter gene were implanted into periarterial tissue or a pig model of restenosis. Three weeks following implantation, beta-galactosidase activity was detected in the pericapsular tissue with a transduction efficiency of approximately 1 in 500 cells. Adventitial implantation of vector-producing encapsulated cells for gene therapy may, therefore, facilitate successful targeting of proliferating vascular smooth muscle cells, and allow stable integration of therapeutic genes into surrounding cells. The encapsulation of vector-producing cells could represent a novel and feasible way to optimize local retroviral gene therapy.

Non-invasive coronary bypass graft imaging after multivessel revascularisation.

Non-invasive imaging techniques for the detection of graft patency after multivessel coronary revascularisation may be useful for follow-up after surgery. Forty consecutive asymptomatic patients (38 men, age 59.9+/-1.3 years) who had undergone coronary bypass surgery with at least three grafts were examined by spiral computed tomography or magnetic resonance angiography 24.9+/-0.3 months after surgery, using conventional angiography as reference. In total, 133 grafts (37 internal mammary artery, 96 venous grafts) were analysed. Spiral computed tomography studies were performed with a subsecond scanner; for magnetic resonance angiography, a three-dimensional contrast-enhanced gradient echo technique with ultrashort echo time during breath holding was used. For spiral computed tomography, sensitivities were 76% (internal mammary artery) and 100% (venous graft). This was compared with 100% (internal mammary artery) and 92% (venous graft) assessed by magnetic resonance angiography (P=ns). The positive predictive values were 100% for internal mammary artery and venous graft (spiral computed tomography) and 100% (internal mammary artery), 92% for venous grafts studied by magnetic resonance angiography (P=ns). Both subsecond spiral computed tomography and contrast-enhanced magnetic resonance angiography are highly accurate and relatively non-invasive approaches of assessing coronary graft patency after multivessel revascularisation and have potential for follow-up assessment in the long term.

[Therapeutic neoangiogenesis of peripheral arteries]. / Therapeutische Neoangiogenese peripherer Arterien.

The identification of angiogenic growth factors was the basis for the development of novel strategies for the treatment of occlusive vascular diseases. Therapeutic angiogenesis resulting in capillary sprouting (angiogenesis) and collateral vessel development (arteriogenesis) may be a potential alternative for patients suffering from critical limb ischemia. Extensive investigations performed in vitro and in vivo demonstrated therapeutic efficacy through the enhancement of collateral growth resulting in augmented blood flow in ischemic tissues following administration of both, recombinant growth factors and genes encoding for such factors. Advantages of gene therapy strategies over the use of proteins include minimal systemic adverse effects and a slow and continuous release of the encoded angiogenic factor which may lead to a long-lasting therapeutic effect. Several uncontrolled phase-I and -IIa trials demonstrated the feasibility and safety of the use of angiogenic growth factors. However, the results of placebo-controlled and double-blind trials are needed to prove their therapeutic potential.

Needle injection catheter delivery of the gene for an antibacterial agent inhibits neointimal formation.

Percutaneous transluminal coronary angioplasty is a routinely used non-surgical revascularization technique for patients with coronary artery disease. Up to 30% of patients undergoing coronary angioplasty develop a renarrowing of treated vessels, called restenosis. Smooth muscle cell proliferation is thought to be an important factor in restenosis; this leads to neointima formation and arterial lumen narrowing. Neointima may be reduced by the transfer of genes encoding proteins with antiproliferative effects. Cecropins are antimicrobial peptides with antiproliferative properties in mammalian cells. Cecropin A is one member of this family of peptides. In this article, a plasmid carrying the gene for the immature form, pre-pro-cecropin A, complexed with liposomes was locally delivered to perivascular tissue in a porcine arterial injury model using a needle injection catheter. Retention of the plasmid in the treated arteries was demonstrated at both 8 and 21 days following application. Transferred plasmid DNA was not detected in any other tissues analyzed. Pre-pro-cecropin A-specific transcripts could also be found in treated arteries. Balloon-injured vessels demonstrated significantly reduced neointima at 21 days in vessels treated with the pre-pro-cecropin A gene compared with neointimal area in those given a control gene (P < 0.05). The needle injection catheter appears to be useful for local intravascular gene delivery. In vivo gene transfer of cecropins may be of therapeutic relevance in restenosis prevention by limiting cell proliferation.

Neointimal growth can be influenced by local adventitial gene manipulation via a needle injection catheter.

Revascularization by percutaneous transluminal coronary angioplasty is limited in the long-term by restenosis, which is luminal renarrowing in the first 6 months after the procedure. Smooth muscle cell proliferation is thought to be an important factor in restenosis; this leads to neointima formation and arterial lumen narrowing. Local therapy delivered perivascularly may have an effect on events in the neointima and reduce restenosis. The effect of delivering expression vector plasmids for senescent cell-derived inhibitor SDI-1, which regulates cell proliferation, and its antisense, into the perivascular tissue of injured arteries was investigated in a porcine arterial injury model using a needle injection catheter. Transfection efficiency, biological effect and plasmid dissemination were evaluated in arterial and organ tissue sections between 2 days and 4 months. A limited number of adventitial, medial and neointimal cells were transfected up to 4 months. sdi gene transfer did not result in a change in neointima. Transfer of antisense sdi resulted in an increase in neointima after 3 weeks. No DNA plasmid was detected in control tissues. Liposomally-mediated adventitial local gene delivery is feasible and safe using the needle injection catheter in a porcine model. A limited number of cells was transfected, with expression of transfected genes up to 4 months after delivery. A transient biological effect with increased neointima was observed after delivery of the antisense sdi gene.

Patency of coronary bypass grafts: assessment with breath-hold contrast-enhanced MR angiography--value of a non-electrocardiographically triggered technique.

PURPOSE: To determine the value of non-electrocardiographically triggered contrast material-enhanced magnetic resonance (MR) angiography in assessing the patency of venous and internal thoracic artery (ITA) grafts after coronary bypass surgery. MATERIALS AND METHODS: Twenty-seven patients with 76 coronary bypass grafts (48 venous, 28 ITA) were examined 26.5 months +/- 5.8 after surgery with MR angiography and conventional angiography. MR angiography was performed with a three-dimensional gradient-echo sequence after automated injection of contrast material; contrast agent administration was based on measurement of the individual transit time of the agent. Results of MR angiography were interpreted by two independent observers and compared with results of conventional angiography. RESULTS: The independent interpretations of the MR angiograms agreed with the results of conventional angiography in 96% and 91% of the grafts. After a final consensus reading, sensitivity was 95% for all grafts, 94% for venous grafts, and 96% for ITA grafts. Specificity was 85% for venous grafts and 67% for ITA grafts. Positive predictive value was 95% for all grafts, 94% for venous grafts, and 96% for ITA grafts. CONCLUSION: Non-electrocardiographically triggered contrast-enhanced MR angiography allows reliable assessment of the patency of venous and arterial coronary bypass grafts.

Accuracy of spiral computed tomography for identifying arterial and venous coronary graft patency.

Late outcome after coronary artery bypass grafting (CABG) mainly depends on the status of graft patency. The recent generation of spiral computed tomography (SCT) scanners may have potential in the long-term follow-up of CABG. In this study, graft patency in patients with internal mammary (IMA) and venous CABG was investigated using SCT and angiography. Forty-nine consecutive patients (age 61 +/- 8 years, 45 men) who had undergone CABG were examined by SCT and angiography 22 +/- 6 months after CABG. In total, 134 bypass grafts (42 IMA and 92 venous grafts) were analyzed. The angiographically determined patency rate of grafts was 86% for IMA (n = 36 of 42) and 74% for venous grafts (n = 68 of 92). By SCT, 32 IMA and 64 venous grafts were diagnosed correctly as patent. Sensitivity was 89% (IMA) and 94% (venous); overall sensitivity was 92%. None of the truly occluded venous grafts was diagnosed falsely patent by SCT (specificity 100%), whereas the specificity of IMA graft visualization was somewhat lower (88%, p = NS [overall 97%]). The accuracy for a patent graft was 88% (IMA) and 96% (venous CABG, p = NS). Compared with previous studies, these data suggest that SCT using one of the recent generation scanners (single scan time 0.75 second) is a highly accurate and relatively noninvasive approach for assessing not only saphenous vein graft patency, but also IMA graft patency. To date, this technique has only limited use in visualizing graft stenosis or distal anastomosis site patency.

[Contrast media enhanced magnetic resonance angiography for determining patency of a coronary bypass. A comparison with coronary angiography]. / Kontrastmittelverstärkte Magnetresonanzangiographie zur Uberprüfung der Durchgängigkeit koronarer Bypasses. Vergleich zur Koronarangiographie.

AIM: Assessment of graft patency with current non-invasive MRA techniques is particularly difficult for evaluating internal mammary artery grafts. Our aim is to determine the accuracy of a contrast enhanced MRA technique is assessing graft patency. METHODS: We examined 19 patients with a total of 53 grafts (32 venous/21 arterial), using an ultrafast contrast enhanced 3D gradient-echo technique and compared this with the results of selective angiography. RESULTS: Sensitivity of the contrast enhanced method was 95.2% for venous grafts, 94.4% for IMA grafts and 94.8% overall. Specificity was 85.7% overall, 90.9% for venous and 66.7% for IMA grafts. Positive predictive value was 94.4%. CONCLUSION: Compared with previous studies, visualisation of IMA grafts was improved by using contrast enhanced MRA. In this preliminary study, contrast enhanced MRA proved promising for the assessment of graft patency.