Preclinical and clinical experience in vascular gene therapy: advantages over conservative/standard therapy.

No systemic pharmacological treatment has been shown to convincingly reduce the incidence of restenosis after angioplasty or increase the formation of collaterals in ischemic tissue in patients. The lack of success of many pharmaceutical agents in reducing restenosis rates or in inducing angiogenesis post-angioplasty and following stent implantation has encouraged the development of new technological treatment approaches. Gene therapy is a novel strategy with the potential to prevent some of the sequelae after arterial injury, particularly cell proliferation, and to induce growth of new vessels or remodeling of pre-existing vessel branches, which may help patients with critical ischemia. Gene therapy strategies have the advantage of minimizing systemic side effects and may have a long-term effect as the encoded protein is released. Most clinical trials investigating gene therapy for vascular disease have been uncontrolled phase I and IIa trials. Gene therapy into vessels with the genes for growth factors has been demonstrated to be feasible and efficient. Local drug delivery devices have been used in combination with gene therapy in several trials to maximize safety and efficiency. Data from experimental animal work indicates that gene therapy may modify intimal hyperplasia after arterial injury, but there are few clinical trials on restenosis in patients. Preliminary clinical results show only limited success in altering restenosis rates. In vitro and experimental in vivo investigations into gene therapy for angiogenesis demonstrate increased formation of collaterals and functional improvement of limb ischemia. There is some evidence of increased collateral formation and clinical improvement in patients with critical limb ischemia. Results of placebo-controlled and double-blind trials of gene therapy for vascular disease are awaited.

Elevation of plasma high-density lipoprotein concentration reduces interleukin-1-induced expression of E-selectin in an in vivo model of acute inflammation.

BACKGROUND: Although there is strong evidence that plasma HDL levels correlate inversely with the incidence of coronary artery disease, the precise mechanism(s) for the protective effect of HDLs remains unclear. We recently showed that HDLs inhibit endothelial cell expression of cytokine-induced leukocyte adhesion molecules in vitro. Our study therefore sought to test the hypothesis that elevating the level of circulating HDLs would inhibit endothelial cell activation in vivo. METHODS AND RESULTS: We used a porcine model of inflammation previously established in our laboratory, in which the level of vascular endothelial cell expression of E-selectin in interleukin (IL)-1alpha-induced skin lesions was measured by the uptake of a radiolabeled anti-E-selectin antibody (1.2B6). Porcine plasma HDL levels were elevated by use of a bolus injection of reconstituted discoidal HDL (recHDL). These particles resemble nascent HDL particles in shape and contain apolipoprotein A-I as the sole protein and soybean phosphatidylcholine as the sole phospholipid. We found that recHDLs inhibited the expression of IL-1alpha-induced E-selectin by porcine aortic endothelial cells in vitro, confirming that the inhibitory effect is conserved with synthetic HDLs and demonstrating that the phenomenon is not restricted to human endothelial cells. In vivo, elevating the circulating level of HDLs approximately 2-fold led to significant inhibition of basal and IL-1alpha-induced E-selectin expression by porcine microvascular endothelial cells. CONCLUSIONS: These observations demonstrate the potential anti-inflammatory action of HDLs and provide support for the further investigation of the mechanisms underlying the inhibitory effects of HDLs on endothelial cell activation.

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.

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.

Targeting an adenoviral gene vector to cytokine-activated vascular endothelium via E-selectin.

We have aimed at selective gene delivery to vascular endothelial cells (EC) at sites of inflammation, by targeting E-selectin, a surface adhesion molecule that is only expressed by activated EC. An anti-E-selectin mAb, 1.2B6, was complexed with the adenovirus vector AdZ.FLAG (expressing the FLAG peptide) by conjugating it to an anti-FLAG mAb. Gene transduction of cultured EC was increased 20-fold compared with AdZ.FLAG complexed with a control bsAb providing EC were activated by cytokines. The anti-E-selectin-complexed vector transduced 29 +/- 9% of intimal EC in segments of pig aorta cultured with cytokines ex vivo, compared with less than 0.1% transduced with the control construct (P < 0.05). This strategy could be developed to target endothelium in inflammation with genes capable of modifying the inflammatory response.

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.

Repetitive myocardial stunning in pigs is associated with the increased expression of inducible and constitutive nitric oxide synthases.

OBJECTIVES: Nitric oxide (NO) has complex effects on myocardial function particularly following ischaemia-reperfusion. The goal of this study was to examine the result of repetitive myocardial stunning on myocardial NO release and expression of inducible (iNOS) and constitutive (eNOS) NO synthases. METHODS AND RESULTS: Propofol anaesthetised pigs underwent ten, 2-min episodes of circumflex artery occlusion (n = 6) or acted as sham operated controls (n = 4). Measurements of segment shortening demonstrated a fall in function in the ischaemic territory to 52.5 +/- 7.3% (mean +/- S.E.M.) of baseline shortening 30 min after the stunning stimulus, recovering to 92 +/- 8.7% 5.5 h later. Function remained stable in sham controls. The change in venous-arterial [NO] between baseline and 6 h reperfusion was found to be significantly different between the two groups (0.2 +/- 0.7 in stunned vs. -4.3 +/- 1.6 microM in shams; P < 0.02). Western blotting and band optical density used to compare tissue from stunned territory (S), non-stunned territory (IC) and sham control animals (SC) demonstrated this was associated with an increase in the expression of both iNOS (S: 93 +/- 13.4, IC: 37 +/- 2.4 and SC: 25 +/- 4 [arbitrary units], P < 0.01 and P = 0.031) and eNOS (S: 104 +/- 7.4, IC; 62.5 +/- 7.4 and SC; 75.7 +/- 0.6, P < 0.03 and P < 0.01) in stunned myocardium. Immunocytochemistry localised iNOS reactivity to vascular smooth muscle cells and cardiomyocytes in stunned tissue and eNOS reactivity to endothelial cells. CONCLUSION: Recovery from repetitive myocardial stunning is associated with the increased expression of both iNOS and eNOS and would be compatible with a protective role for both these enzymes. This finding has possible relevance for both the late window of ischaemic preconditioning and myocardial hibernation.

Restenosis in human vein bypass grafts.

The restenosis rate in vein bypass grafts is higher than in native coronary arteries, and both the cascade of regulatory factors and the vessel reaction may be altered. In this study, vein bypass atherectomy specimens were classified as primary (n = 10) or restenotic (n = 12). Immunohistochemistry with 11 primary antibodies showed low levels of proliferation in both tissues and similar amounts of extracellular matrix components in both primary and restenotic specimens at the time points at which tissue was removed for clinical reasons. Inflammation appeared increased in restenotic specimens. Using in situ hybridization, transforming growth factor-beta1 messenger RNA was detected in both primary and restenotic tissue, with a trend to higher expression in restenosis (8.4 +/- 5.3 vs. 9.4 +/- 7.4 grains/nucleus) and further increased expression in multiple compared with single restenoses (15.1 +/- 6.1 vs. 5.6 +/- 5.1 grains/nucleus, P < 0.05). Hence, there were no great differences in cell proliferation or extracellular matrix formation between primary and restenosis vein graft tissue, in contrast to previously described findings in arterial tissue. This suggests that primary vein graft tissue is already in a chronic 'restenosis-like' state and subsequent injury creates minimal additional upregulation.

Use of nuclear magnetic resonance imaging angiography to follow-up arterial remodeling in an animal model.

Appropriately sized arteries in small animals may be possible models for studying the remodeling process as occurs after arterial balloon injury in humans. Magnetic resonance imaging (MRI) is able to noninvasively image tissue in vivo. To date, small animal angiography models have mostly used research-dedicated instruments and resolution, which are not universally available. Experiments were carried out on a rat aorta model of remodeling in vivo (n=40). Arteries were injured by oversized balloon dilation; control arteries were uninjured. Angiography imaging was performed immediately before sacrifice with an unmodified clinical MRI unit, a 1.5 Tesla MR tomograph with a 20-cm-diameter coil. Longitudinal MRI pictures of the aorta and morphometry of tissue sections to measure luminal and arterial wall areas were analyzed with use of computer-assisted techniques. Comparison of dimensions demonstrated correlation between MRI and histology measurements of the lumen. MRI and morphometry showed a gradual increase in mean luminal area over 6 weeks following injury. The lumen increase correlated with total arterial area and thickness. In this rat aorta model, remodeling documented at histology was followed-up in vivo. The use of such clinical MRI scanners has potential to reduce animal numbers needed to follow-up the remodeling process after therapeutic intervention.

Conservative treatment of a paracardial abscess following post-infarct patch grafting.

A patient with previous patch grafts at the site of a ventricular aneurysm and over an ischaemic septal defect presented with an oval hypodense mediastinal mass consistent with a mediastinal abscess with blood cultures positive for Staphylococcus aureus. Surgical re-operation in this region was considered to be too risky and conservative treatment was pursued. Antibiotics were continued for a total of nearly 5 months of treatment. A computed tomographic scan prior to discharge indicated that the abscess was completely resolved.

Current biotechnological approaches to the prevention of restenosis.

No systemic pharmacological treatment has been convincingly shown to reduce the incidence of restenosis after angioplasty in patients. The lack of success of many pharmaceutical agents in reducing restenosis rates post-angioplasty and following stent implantation, as documented in dozens of clinical trials, has encouraged the development of new biotechnological approaches to the treatment of restenosis. Gene therapy and other agents, including antibodies, fusion toxins and ribozymes, have the potential to prevent some of the sequelae after arterial injury, particularly cell proliferation. Mechanical methods of preventing restenosis, for example sophisticated local drug delivery strategies and biodegradable stents using new materials, in combination with novel therapeutic agents or radiation, may also be of use.

Expression of vascular cell adhesion molecule-1 by vascular endothelial cells in immune and nonimmune inflammatory reactions in the skin.

Expression of VCAM-1 was compared with that of E-selectin in cytokine-induced lesions and in delayed-type hypersensitivity reactions to tuberculin purified protein derivative (PPD) in pig skin. Lumenally expressed Ags were quantified by measuring localization in skin of i.v. injected (111)In-mAb 10.2C7 (anti-vascular cell adhesion molecule-1 (anti-VCAM-1), (125)I-mAb 1.2B6 (anti-E-selectin), and (99m)Tc-MOPC21 (control IgG1). Anti-VCAM-1 mAb uptake was greater following intradermal (i.d.) injection of TNF-alpha than following injection of IL-1, while the two cytokines induced similar uptake of anti-E-selectin. In immunologically naive pigs there was no detectable increase in anti-VCAM-1 after i.d. injection of PPD, although anti-E-selectin uptake was increased at 3 and 6 h. In contrast, i.d. injection of PPD in sensitized pigs led to increased uptake of both anti-VCAM-1 and anti-E-selectin at 6, 8, 24, and 48 h, each of which was significantly greater than the uptake of control IgG1 into the same lesions (each p < 0.01). Anti-TNF-alpha mAb abolished the increased uptake of anti-VCAM-1 3 and 8 h following i.d. injection of PPD in sensitized pigs and significantly inhibited uptake at 24 h (p = 0.0025), but did not significantly reduce uptake of anti-E-selectin. We conclude that in this delayed-type hypersensitivity model 1) E-selectin expression by endothelial cells follows sequential Ag nonspecific and immune-specific phases, 2) increased VCAM-1 expression by endothelial cells is only seen in sensitized animals, and 3) expression of VCAM-1 appears to be relatively more dependent on TNF-alpha than E-selectin. Differential expression of E-selectin and VCAM-1 may influence the leukocytic infiltrate during the course of nonspecific and immune-specific inflammatory reactions.

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.

Excessive arterial thrombus in spherocytosis. A case report.

Spherocytosis is the most common of the hereditary hemolytic anemias, with characteristically shaped erythrocytes. An unusually large amount of arterial thrombus was documented in a dissected artery after angioplasty in a patient with spherocytosis. It is hypothesized that the excessive arterial thrombus may have been linked to the spherocytosis.

Significance of the Thrombolysis in Myocardial Infarction scoring system in assessing infarct-related artery reperfusion and mortality rates after acute myocardial infarction.

Thrombolysis in Myocardial Infarction (TIMI) flow scores were originally devised as semiquantitative angiographic measures of coronary artery perfusion. Several studies have indicated an important relation between different TIMI flow grades at 90 minutes after thrombolysis and clinical outcome. To further evaluate this relation we conducted a metaanalysis of all angiographic, postinfarction trials that studied the relation between individual 90-minute TIMI flow grades and mortality rates. In 4687 pooled patients, the mortality rate was lowest in patients with TIMI grade 3 flow (3.7%) and significantly lower than those with TIMI 2 (6.6%, p = 0.0003; odds ratio 0.55; 95% confidence interval [CI] 0.4% to 0.76%) or TIMI 0/1 flow (9.2%, p < 0.0001; odds ratio 0.38; 95% CI 0.29% to 0.5%). The mortality rate difference between TIMI grade 2 and TIMI grade 0/1 patients was also significant (p = 0.02; odds ratio 0.7; 95% CI 0.51% to 0.94%). This study confirms the importance of achieving rapid and complete reperfusion after acute myocardial infarction with the best outcome associated with 90-minute TIMI 3 flow. Furthermore, it shows that although TIMI 2 flow (partial perfusion) is not equivalent to TIMI 3 flow, it nevertheless still confers a significant survival benefit compared with TIMI flow 0/1.

Medical education in Turkey 1996.

Medical education in Turkey has certain similarities with systems in the West, and is struggling to train its doctors to the highest standard. However, as a country it remains very much in between the cultures of East and West. Although the overall organisation of medical training is continually changing, and individual medical schools have varying ideals, the aim of this article is to provide an overview of the current system for training student doctors in Turkey.

Novel uses and potential for calcium antagonists in revascularization.

Calcium antagonists affect the arterial wall and blood components in many ways: these include their classical vasomotor functions, as well as newly-documented effects on platelet aggregation, rheology, the platelet membrane receptor glycoprotein IIb/IIIa complex and on tissue factor, a glycoprotein that initiates the clotting cascade. Calcium antagonists slow atherogenesis in animals, perhaps through inhibiting calcium incorporation, lowering heart rate or reducing thrombus formation, although no benefits were shown in prospective clinical studies of stenosis progression. However, it has been possible to attenuate proliferation in in vitro and in vivo experimental models. These discoveries are leading to novel calcium antagonist applications in revascularization. They have the potential to act synergistically in thrombolysis, but so far there has been very little evaluation of this. During coronary intervention, the myocardial protective action of calcium antagonists could be of benefit against stunning and in the no-reflow phenomenon. Their action on vasomotor tone and thrombus formation might affect acute closure or restenosis, although clinical studies have not yet shown this, perhaps because systemic administration of calcium antagonists does not achieve a high enough local concentration. Local drug delivery into the arterial wall may have potential. Calcium antagonists could be of use in cardiac surgery by preventing spasm or providing myocardial protection.

Endovascular catheter-delivered photodynamic therapy in an experimental response to injury model.

BACKGROUND: The effectiveness of local endovascular photodynamic therapy (PDT) in preventing tissue hyperplasia was evaluated in a vascular injury model. METHODS: Standardized unidirectional arterial injury with a directional atherectomy catheter was performed in porcine arteries (n = 180). Animals (n = 72) were randomly allocated to unidirectional injury only (Group 1), injury followed by drug delivery of photosensitizer with a porous balloon (Group 2), or by local exposure to monochromatic light (Group 3). In Group 4, injury was followed by local drug delivery of photosensitizer and subsequent exposure to light (PDT). Up to 21 days after treatment, all experimental vessels were excised, fixed and processed for histology, immunohistochemistry and transmission electron microscopy. RESULTS: After vascular injury an inflammatory and myoproliferative response was observed in Groups 1, 2 and 3 (mean tissue hyperplasia/media ratio 1.0 +/- 0.5 at 21 days; area tissue hyperplasia: 1.57 +/- 0.9 mm2). Proliferation in injured vascular segments (Group 1-3) reached a maximum at 7 days, with 6%. Only in Group 4, after injury followed by photodynamic therapy, was there no significant vascular response (mean tissue hyperplasia/media ratio 0.3 +/- 0.2: area tissue hyperplasia: 0.1 +/- 0.05 mm2 p < 0.001, proliferating cells 0.3%). CONCLUSION: Vascular response after unidirectional injury was suppressed only by endovascular photodynamic therapy.

Local photodynamic therapy reduces tissue hyperplasia in an experimental restenosis model.

Local photodynamic therapy may have potential in preventing myointimal hyperplasia after angioplasty. In this study, the effect of photodynamic therapy was evaluated in an experimental model of restenosis. Standardized unidirectional arterial injury with a directional atherectomy catheter was performed in porcine arteries. Animals were randomly allocated to four groups: group 1, unidirectional injury only; group 2, injury followed by local delivery of photosensitizer; group 3, injury followed by local exposure to monochromatic light; and group 4, where injury was followed by local drug delivery of photosensitizer and subsequent exposure to light (photodynamic therapy). Seven, 14 or 21 days after treatment, all experimental vessels were excised, fixed and processed for histology. An inflammatory and myoproliferative response was observed after injury in vessels from groups 1, 2 and 3. In group 4, after injury followed by photodynamic therapy, the myoproliferative response was significantly reduced. Thus, in this study, tissue hyperplasia after unidirectional injury was effectively suppressed by photodynamic therapy.

Immune-complex allergic vasculitis in association with the development of transverse myelitis. A case report.

A severe vasculitis, probably therapy related, in a sixty-four-year-old man being treated for possible subacute bacterial endocarditis, was associated with the development of transverse myelitis. It is hypothesized that the vasculitis affected the small vessels to the spinal cord in the same way that systemic vasculitis can also cause a transverse myelitis.