Inhibition of neointimal hyperplasia in a rabbit vein graft model following non-viral transfection with human iNOS cDNA.

Vein graft failure caused by neointimal hyperplasia (IH) after coronary artery bypass grafting with saphenous veins is a major clinical problem. The lack of safe and efficient vectors for vascular gene transfer has significantly hindered progress in this field. We have developed a Receptor-Targeted Nanocomplex (RTN) vector system for this purpose and assessed its therapeutic efficacy in a rabbit vein graft model of bypass grafting. Adventitial delivery of ß-Galactosidase showed widespread transfection throughout the vein wall on day 7, estimated at about 10% of cells in the adventitia and media. Vein grafts were then transfected with a plasmid encoding inducible nitric oxide synthase (iNOS) and engrafted into the carotid artery. Fluorescent immunohistochemistry analysis of samples from rabbits killed at 7 days after surgery showed that mostly endothelial cells and macrophages were transfected. Morphometric analysis of vein graft samples from the 28-day groups showed approximately a 50% reduction of neointimal thickness and 64% reduction of neointimal area in the iNOS-treated group compared with the surgery control groups. This study demonstrates efficacy of iNOS gene delivery by the RTN formulation in reducing IH in the rabbit model of vein graft disease.

Engaging women in academic medicine in the UK: report of a workshop at the Association of Physicians Annual Meeting, 2 April 2009.

In April 2009, at the Association of Physicians of Great Britain and Ireland (APGBI) Annual Meeting in Birmingham, a workshop was held to consider the changing demographics of the medical profession, its potential effects on the Association's stated aims of promoting academic excellence and ways of ensuring that medical academia is attractive to everyone with the ability and drive it needs. This paper reports the discussions of the workshop participants and also summarises recommendations for actions by both the Association and its membership, which will encourage interest, equal opportunities and personal development for all in academic medicine.

Cardiac involvement in acute thrombotic thrombocytopenic purpura: association with troponin T and IgG antibodies to ADAMTS 13.

INTRODUCTION: Evidence for cardiac involvement in thrombotic thrombocytopenic purpura (TTP) is uncommonly described. METHODOLOGY: We retrospectively reviewed 41 patients assessing troponin T as a marker for cardiac involvement in acute TTP with clinical symptoms, electrocardiograms (ECG) and echocardiograms. A histopathological review of five patients who died of acute TTP was also undertaken. RESULTS: In 54% (22/41) of patients, troponin T was >or=0.05microg L(-1) (normal range 0-0.01 microg L(-1)). Half (12/22) had cardiac symptoms and 8/22 with a raised troponin T reported chest pain. ECG changes were present in 62% of patients with a raised troponin T. Median anti-ADAMTS 13 IgG antibody was significantly higher (P=0.018) in patients with troponin T>or=0.05 microg L(-1) (58.5% (range 17-162%), compared with patients with troponin T<0.05 microg L(-1) (35%, range 9-134%). Patients who died had higher troponin T levels (median 0.305 microg L(-1)) and raised anti-ADAMTS 13 IgG (median 66.5%). On admission, there were no deaths in those with troponin Tor=0.05 microg L(-1)) signify myocardial necrosis associated with microvascular thrombi. Mortality and acute morbidity was associated with higher admission troponin T and raised IgG antibody (>67%) to ADAMTS 13.

Raised blood pressure, not renin-angiotensin systems, causes cardiac fibrosis in TGR m(Ren2)27 rats.

OBJECTIVES: Elevated systemic arterial blood pressure is associated with left ventricular hypertrophy and fibrosis. It has been suggested that both circulating and local myocardial renin-angiotensin systems play a role in mediating these responses. Here we describe the natural history of ventricular hypertrophy and fibrosis in the transgenic (mRen2)27 rat--a monogenetic model--which has a high tissue expression of the murine renin transgene, and suffers severe hypertension. We further explored the relative contribution of both hypertensive burden and circulating and tissue renin-angiotensin systems to the fibrotic process. METHODS: The transgenic rats were treated from 28 days old with (1) a hypotensive dose of the ACE inhibitor ramipril which inhibited both tissue and circulating ACE activity, (2) the calcium antagonist amlodipine, or (3) a non-hypotensive dose of ramipril which inhibited about 60% of tissue ACE activity with little effect on circulating ACE. Normotensive Sprague-Dawley rats were used as controls. RESULTS: The transgenics developed left ventricular hypertrophy along with perivascular and interstitial fibrosis which became progressively worse up to 24 weeks of age. Both the high dose of ramipril and amlodipine prevented the hypertrophy and fibrosis, whereas tissue ACE inhibition without lowering blood pressure had no effect, and actually led to a worsening of the fibrosis by 24 weeks. CONCLUSIONS: These results suggest that the development of left ventricular hypertrophy and fibrosis in the transgenic (mRen2)27 rat are regulated by blood pressure and not activity of the renin-angiotensin systems and that progression of fibrosis at 24 weeks involves a mechanism unrelated to local renin-angiotensin activity.

Reduction in the response to coronary and iliac artery injury with photodynamic therapy using 5-aminolaevulinic acid.

OBJECTIVE: Photodynamic therapy (PDT) uses red light (non-thermal, non-ionising) to activate a previously administered photosensitizing drug. This inhibits neointimal hyperplasia in injured arteries in small animals where it appears safe and well tolerated. Our aim was to develop a method for percutaneous application of PDT to iliac and coronary arteries in a large animal model and investigate its influence on the remodeling and intimal hyperplastic response to balloon injury. METHODS: Studies were undertaken on 13 juvenile Large White-Landrace crossbred pigs (15-20 kg). After intravenous administration of the photosensitizing agent 5-amino laevulinic acid (ALA), the arterial tree was accessed via the left common carotid artery and balloon injuries made by over-distension in both common iliacs (thirteen animals) and one or two main coronary arteries (eight animals). Half the injured sites were then illuminated with red laser light transmitted via the catheter. Animals were culled 28 days later and tissue harvested for histomorphometry. RESULTS: Compared with control injured vessels, PDT treated, balloon injured coronary arteries had a larger lumen (1.4 vs. 0.8 mm2, P = 0.002), larger area within the external elastic lamina (2.8 vs. 2.2 mm2, P = 0.006) and smaller area of neointimal hyperplasia (0.4 vs. 0.7 mm2, P = 0.06), 28 days after intervention. Less neointimal hyperplasia and the absence of negative remodeling resulted in the lumen of PDT-treated, injured segments being the same as that of adjacent reference segments (1.5 vs. 1.6 mm2). Similar trends, but with smaller differences, were seen in the iliac vessels. CONCLUSIONS: Intra-arterial, trans-catheter PDT favourably influences the arterial response to balloon injury in both the coronary and peripheral circulations. This technique offers a promising new approach to restenosis after endovascular procedures.

Clinical study of adjuvant photodynamic therapy to reduce restenosis following femoral angioplasty.

BACKGROUND: Photodynamic therapy (PDT) reduces neointimal hyperplasia and negative remodelling following balloon injury in small and large animal models. This clinical study investigated the role of adjuvant PDT following femoral percutaneous transluminal angioplasty (PTA). METHODS: Eight PTAs in seven patients (two women) with a median age of 70 (range 59-86) years were performed with adjuvant PDT. All patients had previously undergone conventional angioplasty at the same site which resulted in symptomatic restenosis or occlusion between 2 and 6 months. Each was sensitized with oral 5-aminolaevulinic acid 60 mg/kg, 5-7 h before the procedure. Following a second femoral angioplasty, up to 50 J/cm2 red light (635 nm) was delivered to the angioplasty site via a laser fibre within the angioplasty balloon. Patients were kept in subdued light overnight and discharged the following day. Outcome was assessed by duplex imaging at 24 h, 1, 3 and 6 months and by intravenous digital subtraction angiography at 6 months. A peak systolic velocity ratio (PSVR) of more than 2.0 at the angioplasty site was taken to represent restenosis. RESULTS: All patients tolerated the procedure well without adverse complications or death. All were rendered asymptomatic which was sustained throughout the study interval. All vessels remained patent and no lesion attained the duplex definition of restenosis. Median (interquartile range) PSVR across stenotic segments was 4.7 (3.7-5.7) before angioplasty, 1.1 (0.9-1.3) at 24 h and 1.4 (1.0-1.8) at 6 months after intervention (P = 0.04 compared with preoperative value). CONCLUSION: This pilot study suggests that endovascular PDT is safe and may reduce restenosis follow- ing angioplasty. The data justify a randomized controlled trial.

Expression of tissue inhibitor of matrix metalloproteinases 1 by use of an adenoviral vector inhibits smooth muscle cell migration and reduces neointimal hyperplasia in the rat model of vascular balloon injury.

BACKGROUND: Cell migration is a major contributor to injury-induced neointimal hyperplasia and depends on alteration of the proteolytic balance within the arterial wall toward matrix breakdown. This is partly mediated by the matrix metalloproteinases (MMPs) and their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). METHODS AND RESULTS: An increase in expression of biologically active and immunoreactive TIMP-1 was seen in vitro after infection of rat smooth muscle cells (SMCs) with Av1.TIMP1 (an adenoviral vector containing the human TIMP1 cDNA). Infection of rat SMCs with Av1.TIMP1 reduced migration in vitro by 27% compared with control virus-infected cells (37.6+/-4.34 versus 51+/-5.01 cells per high-power field, P<0.05). The adenoviral vector was delivered to the injured rat carotid artery, and 4 days later, immunoreactive protein was identified and migration of SMCs reduced by 60% (5.2+/-0. 5 versus 12.8+/-1.5 cells per section, P<0.05, n=5). Neointimal area 14 days after injury showed a 30% reduction in the animals receiving the Av1.TIMP1 virus compared with controls (0.09+/-0.01 versus 0. 14+/-0.01 mm2, P=0.02, n=14). CONCLUSIONS: The response to arterial balloon injury involves MMP-dependent SMC migration and can be attenuated in vivo by the transmural expression of TIMP-1 by adenoviral gene transfer.

TIMP-4 is regulated by vascular injury in rats.

The role of basement membrane-degrading matrix metalloproteinases (MMPs) in enabling vascular smooth muscle cell migration after vascular injury has been established in several animal models. In contrast, the role of their native inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs), has remained unproven despite frequent coregulation of MMPs and TIMPs in other disease states. We have investigated the time course of expression and localization of TIMP-4 in rat carotid arteries 6 hours, 24 hours, 3 days, 7 days, and 14 days after balloon injury by in situ hybridization, immunohistochemistry, and Western blot analysis. TIMP-4 protein was present in the adventitia of injured carotid arteries from 24 hours after injury. At 7 and 14 days after injury, widespread immunostaining for TIMP-4 was observed throughout the neointima, media, and adventitia of injured arteries. Western blot analysis confirmed the quantitative increase in TIMP-4 protein at 7 and 14 days. In situ hybridization detected increased expression of TIMP-4 as early as 24 hours after injury and a marked induction in neointimal cells 7 days after injury. We then studied the effect of TIMP-4 protein on the migration of smooth muscle cells through a matrix-coated membrane in vitro and demonstrated a 53% reduction in invasion of rat vascular smooth muscle cells. These data and the temporal relationship between the upregulation of TIMP-4, its accumulation, and the onset of collagen deposition suggest an important role for TIMP-4 in the proteolytic balance of the vasculature controlling both smooth muscle migration and collagen accumulation in the injured arterial wall.

Local gene delivery of recombinant adenoviruses to the rat carotid artery in vivo.

A number of animal models are available to investigators wishing to study the use of gene transfer to prevent neointimal formation after vascular injury. The majority are models of primary vascular injury rather than the human situation, where recurrence of a stenosis occurs in an abnormal blood vessel treated by angioplasty. The main animal models used to study vascular balloon injury or angioplasty are the rat carotid, the rabbit iliac, the pig coronary and carotid models, and, less frequently, the dog coronary and primate saphenous or iliac models. A healthy level of skepticism exists about many of these animal models following the success of angiotensin-converting enzyme inhibitors in preventing neointima formation in the rat model but their failure to prevent human restenosis (1,2). Each model, however, has a particular suitability to investigate different aspects of the balloon injury process and all are suitable for gene transfer studies.

Intra-arterial photodynamic therapy using 5-ALA in a swine model.

OBJECTIVES: To test the hypothesis that intravascular light could be delivered via a balloon catheter for arterial photodynamic therapy (PDT). DESIGN: Pig non-injury model. MATERIALS: Clinical catheter equipment. METHODS: Large White pigs (15-20 micrograms) were photosensitised with 5-aminolaevulinic acid (5-ALA) induced protoporphyrin IX (PpIX) at a concentration of 120 mg/kg. Arterial biopsies were taken at intervals between 30 mins and 24 h and frozen sections analysed using a CCD camera to give a temporal profile of fluorescence in each arterial layer. PDT was given to normal arterial segments via a 4 mm transparent PTA balloon inflated so as to occlude flow, but not distend the artery. Animals were culled at 3 and 14 days and the above segments harvested. RESULTS: Fluorescence peaked in the adventitia, intima and medial layers at 1.5, 4 and 6 h respectively. PDT at all time points produced VSMC depletion compared with controls. The degree of depletion mirrored the fluorescence profile of PpIX. CONCLUSIONS: PDT can be delivered via a standard PTA balloon with a transparent channel. This depletes the VSMC population within the arterial wall without complications. Intra-arterial PDT is therefore a potential therapy to reduce the incidence of restenosis post-angioplasty.

Inhibition of tissue angiotensin converting enzyme activity prevents malignant hypertension in TGR(mREN2)27.

BACKGROUND: Activation of the renin-angiotensin system has been implicated strongly in the transition from benign to malignant hypertension. However, the concomitant rise in blood pressure might also have a direct effect on the vascular wall by initiating fibrinoid necrosis and myointimal proliferation. Ascertaining the relative importance of these two factors in this process has proved difficult. TGR(mREN2)27 heterozygotes (HanRen2/Edin- -) have previously been shown to develop malignant hypertension spontaneously and exhibit the characteristic features of human malignant hypertension. OBJECTIVE: Tissue renin-angtiotensin systems have been implicated in the pathogenesis of malignant hypertension. We set out to determine whether inhibition of this system might protect against development of the disease in a rat model. METHOD: Male TGR(mREN2)27 heterozygotes (n = 24) were given a non-hypotensive dose of the angiotensin converting enzyme inhibitor ramipril (5 microg/kg per day) from 28 to 120 days of age, untreated rats acting as controls (n = 40). The incidences of malignant hypertension were compared. Systolic blood pressure was measured by tail-cuff plethysmography during treatment; tissue and plasma angiotensin converting enzyme levels and renal histological changes were assessed at the end of the treatment period or upon development of malignant hypertension. RESULTS: Sixty-three per cent of control rats and 4% of angiotensin converting enzyme inhibitor-treated rats had developed malignant hypertension by 120 days despite there having been no significant difference in systolic blood pressure throughout the course of treatment. Angiotensin converting enzyme activities in kidney, heart and resistance vessels, though not that in plasma, were significantly lower in the treated rats. The degree of medial wall thickening did not differ between the two groups whereas evidence of tissue injury (e.g. intimal fibrosis, fibrinoid necrosis and nephron injury) was significantly less common among rats in the angiotensin converting enzyme inhibitor-treated group. CONCLUSIONS: Tissue angiotensin converting enzyme inhibition at a non-hypotensive dose almost completely prevented mortality from malignant hypertension and significantly reduced tissue injury in this model, implicating angiotensin II rather than high blood pressure as the principal 'vasculotoxic' agent in malignant hypertension.

Expression of matrix metalloproteinases and their inhibitor TIMP-1 in the rat carotid artery after balloon injury.

The temporal relationship of matrix metalloproteinases (MMPs) and a specific tissue inhibitor (TIMP-1) has been examined by reverse transcription-polymerase chain reaction and substrate zymography, after balloon catheter angioplasty of the rat carotid artery. The contralateral uninjured carotid artery was used as a comparative control. Of the MMPs examined, only MMP-2 (72-kDa gelatinase) was produced constitutively by normal uninjured arteries. After injury, MMP-2 mRNA levels fell compared with the uninjured arteries; by 24 hours, levels had increased 2-fold. Zymography showed that the inactive form of MMP-2 predominated in uninjured vessels, but after injury, the level of the active form was increased. MMP-9 (92-kDa gelatinase) mRNA levels and activity peaked at 6 hours after injury and were still detectable at 7 days. MMP-3 (stromelysin) expression was detectable at low levels as early as 2 hours after injury and showed an approximate 2-fold increase of expression at 7 days. The presence of the active protein paralleled the mRNA expression. The inhibitor TIMP-1 mRNA was first detected 6 hours after injury and showed a marked peak of expression at 24 hours; however, no expression was detected by 7 days. The presence of a constitutively expressed, low molecular weight caseinolytic enzyme (27 kDa) was observed, and the induction of a caseinolytic enzyme (30 kDa) was noted that was induced as early as 2 hours after injury, peaked at 6 hours, and was barely detectable by 7 days. These results demonstrate that the process of extracellular matrix breakdown by MMPs after balloon catheter-induced injury is controlled by a tightly regulated temporal response by the genes responsible for the production of these enzymes and their inhibitor and by post-translational activation of the proenzymes.

Association of angiotensin-converting enzyme gene I/D polymorphism with change in left ventricular mass in response to physical training.

BACKGROUND: The absence (deletion allele [D]) of a 287-base pair marker in the ACE gene is associated with higher ACE levels than its presence (insertion allele [I]). If renin-angiotensin systems regulate left ventricular (LV) growth, then individuals of DD genotype might show a greater hypertrophic response than those of II genotype. We tested this hypothesis by studying exercise-induced LV hypertrophy. METHODS AND RESULTS: Echocardiographically determined LV dimensions and mass (n=140), electrocardiographically determined LV mass and frequency of LV hypertrophy (LVH) (n=121), and plasma brain natriuretic peptide (BNP) levels (n=49) were compared at the start and end of a 10-week physical training period in male Caucasian military recruits. Septal and posterior wall thicknesses increased with training, and LV mass increased by 18% (all P<.0001). Response magnitude was strongly associated with ACE genotype: mean LV mass altered by +2.0, +38.5, and +42.3 g in II, ID and DD, respectively (P<.0001). The prevalence of electrocardiographically defined LVH rose significantly only among those of DD genotype (from 6 of 24 before training to 11 of 24 after training, P<.01). Plasma brain natriuretic peptide levels rose by 56.0 and 11.5 pg/mL for DD and II, respectively (P<.001). CONCLUSIONS: Exercise-induced LV growth in young males is strongly associated with the ACE I/D polymorphism.

Evidence for both in vivo and in vitro interaction between vigabatrin and alanine transaminase.

AIMS: Decreases in plasma alanine transaminase (ALA-T) activity of 20-100% have been reported following the use of vigabatrin (Sabril) in patients with uncontrolled epilepsy. This effect has a potential clinical significance as it may mask signs of early, underlying hepatic disease. It is particularly significant in a patient population known to have a higher than average risk of hepatotoxicity due to treatment with other anti-epilepsy drugs or to an independent, but concomitant, disease process. Vigabatrin is a highly specific enzyme antagonist. There is an almost 1000-fold difference between its activity against gamma-amino butyric acid aminotransaminase and ALA-T. However, some activity against other transaminases is not unexpected, and it, is important to determine the degree of vigabatrin's effect against ALA-T in man. METHODS: Two in vitro experiments, using serum samples spiked with vigabatrin, confirmed the presence of an interaction between vigabatrin and ALA-T, and formed the basis for the design of a study in five healthy male volunteers, in whom serum ALA-T activity was measured before and after a single dose of 1.5 g of vigabatrin. RESULTS: Serial sampling confirmed the presence of an in vivo interaction between vigabatrin and ALA-T, causing an inhibition of enzyme activity of 30-40%. A further 20% reduction was found in vitro in samples taken at the time of the peak plasma vigabatrin concentration after they had been stored for 6 h. CONCLUSIONS: The clinical significance of these findings is that the levels of ALA-T activity determined in patients receiving vigabatrin may be inaccurate. The "real' values must be assumed to be higher than those reported after routine testing. To obtain the most relistic measurement of ALA-T activity in patiets, samples should be taken at the times of trough plasma concentration and processed as soon as possible afterwards. Samples stored for any length of time at or above room temperature may also give even more false results.

Ultrastructural localisation of nitric oxide synthase, endothelin and binding sites of lectin (from Bandeirea simplicifolia) in the rat carotid artery after balloon catheter injury.

An immunocytochemical and cytochemical study has been made on the ultrastructural localisation of type III (endothelial) nitric oxide synthase, endothelin-1 and the binding sites of lectin from Bandeirea simplicifolia to the endothelium surface-associated glycoproteins in the rat left common carotid artery at 1 and 28 d after Fogarty embolectomy balloon catheter-induced injury. Controls were carotid arteries from sham operated rats. In the controls, the immunoreactivity to nitric oxide synthase-III and endothelin-1 was localised in different proportions in vascular endothelial cells (36.9% +/- 4.3 and 7.6% +/- 2.7, respectively); immunoreactivity was confined to the cytoplasm and the membranes of intracellular organelles and structures. In contrast, staining with lectin was localised on the luminal surface of all endothelial cells. 1 d after injury, platelets were adherent to the endothelium-denuded intima. Some of the platelets displayed, immunoreactivity to nitric oxide synthase-III and endothelin-1 and were stained with lectin. 28 d after injury, a neointimal thickening of substantial size was present. Subpopulations of the regrown endothelial cells covering the luminal surface of the neointima showed positive immunoreactivity to nitric oxide synthase-III and endothelin-1 but there was a significant decrease in the proportion of nitric oxide synthase-III-containing endothelial cells (17.2% +/- 1.9; P < 0.001) and a significant increase in the proportion of endothelin-1-containing endothelial cells (36.9% +/- 4.7; P < 0.001) compared with the controls. Staining with lectin was associated with the cell membrane of all endothelial cells and in addition with cells located 'deeper' in the neointima which showed lectin-positive plasmalemma, Golgi complex and multivesicular bodies/lysosomes. In conclusion, regenerated endothelial cells of the neointima showed reduced population (2-fold) of nitric oxide synthase-III-and increased population (5-fold) endothelin-1-positive cells. The subendothelial location of some lectin-stained cells after balloon catheter injury indicates the heterogeneity of the neointima and suggests that some of these cells are involved in early angiogenesis. 24 h and 28 d after injury some platelets showed positive immunoreactivity for nitric oxide synthase-III and endothelin-1.

Neurocompensatory responses to balloon-catheter-induced injury of the rat carotid artery.

Percutaneous transluminal angioplasty relieves discrete arterial stenosis but causes extensive vascular injury. There is denudation of the endothelium and variable medial disruption, but the effect on adventitial structures has not been studied in detail. We have investigated the innervation of the left and right carotid arteries after unilateral balloon-catheter-induced injury. Immunohistochemical examination of the arteries 1 day after Fogarty-catheter-induced injury of the left common carotid artery revealed a reduction in the density of protein gene product 9.5 (PGP)-, substance P (SP)- and calcitonin-gene-related peptide (CGRP)-containing nerves close to the medial smooth muscle of the injured vessel. At the same time, on the side contralateral to the injury, there was a substantial increase in the density of PGP-, SP- and CGRP-containing nerves innervating the carotid artery and vasa vasorum compared to controls. Immunoassay data from these vessels showed a selective increase in SP and CGRP contents of the contralateral carotid artery (SP, controls 0.02 +/- 0.01, operated 0.59 +/- 0.32 pmol/cm; CGRP, controls 0.03 +/- 0.01, operated 0.14 +/- 0.03 pmol/cm, n = 6, p < 0.05). Neuropeptide Y levels were unchanged. Twenty-eight days after surgery, at which time a neointima was present, peptide levels were no different from controls, and the innervation of both the left and right carotid arteries and vasa vasorum was indistinguishable from the controls. In conclusion, balloon-catheter-induced injury includes damage to the perivascular nerves and induces a transient increase in the density of sensory neuropeptide-containing nerves innervating the contralateral, uninjured side. This novel observation may reflect neurocompensatory responses to vascular injury.