Allan R. McPhaden

Inhibition by leukocyte depletion of neointima formation after balloon angioplasty in a rabbit model of restenosis

OBJECTIVEnThe aim of the current study was to examine neointima formation in balloon injured left subclavian artery of rabbits subjected to two different methods of leukocyte depletion at the time of injury.nnnMETHODSnAngioplasty of the left subclavian artery was performed in leukopenic male New Zealand White rabbits. Depletion of circulating leukocytes was induced by either mustine hydrochloride or an antibody against leukocyte common antigen (anti-LCA) before angioplasty. Left and right subclavian arteries were removed 28 days after injury for morphological analysis and measurement of neointimal size. At the same time, leukocytes were isolated from autologous rabbit blood for 51Cr-labelling for assessment of leukocyte adhesion to injured and non-injured artery segments.nnnRESULTSnLeukopenia decreased neointima formation in injured arteries (neointimal area was 0.09+/-0.03 mm(2) in mustine-treated arteries, n=8, vs. 0.56+/-0.07 mm(2) in control arteries, n=7; P<0.001 and 0.07+/-0.01 mm(2) in anti-LCA treated arteries, n=9, vs. 0.22+/-0.04 mm(2) in non immune serum-treated arteries, n=9; P<0.001). Adventitial fibrosis was also significantly (P<0.05) decreased by both leukopenic interventions. Neither medial nor adventitial area was modified in any of the groups. No differences in leukocyte adhesion were observed between injured and non-injured arteries in any of the experimental groups at the 28 day time point.nnnCONCLUSIONnThese results suggest that leukocytes play a major role in the development of two of the major characteristics of the response to balloon injury, namely formation of neointima and adventitial fibrosis, that currently limit the success of clinical angioplasty. Elucidation of the fine mechanisms involved in leukocyte-mediated injury may lead to the discovery of novel therapeutic targets for the prevention of restenosis.

Adrenomedullin acts via nitric oxide and peroxynitrite to protect against myocardial ischaemia-induced arrhythmias in anaesthetized rats

1 The overall aim of this study was to determine if adrenomedullin (AM) protects against myocardial ischaemia (MI)‐induced arrhythmias via nitric oxide (NO) and peroxynitrite. 2 In sham‐operated rats, the effects of in vivo administration of a bolus dose of AM (1u2003nmolu2003kg−1) was assessed on arterial blood pressure (BP), ex vivo leukocyte reactive oxygen species generation and nitrotyrosine deposition (a marker for peroxynitrite formation) in the coronary endothelium. 3 In pentobarbitone‐anaesthetized rats subjected to ligation of the left main coronary artery for 30u2003min, the effects of a bolus dose of AM (1u2003nmolu2003kg−1, i.v.; n=19) or saline (n=18) given 5u2003min pre‐occlusion were assessed on the number and incidence of cardiac arrhythmias. In a further series of experiments, some animals received infusions of the NO synthase inhibitor N(G)‐nitro‐L‐arginine (LNNA) (0.5u2003mgu2003kg−1u2003min−1) or the peroxynitrite scavenger N‐mercaptopropionyl‐glycine (MPG) (20u2003mgu2003kg−1u2003h−1) before AM. 4 AM treatment significantly reduced mean arterial blood pressure (MABP) and increased ex vivo chemiluminescence (CL) generation from leukocytes in sham‐operated animals. AM also enhanced the staining for nitrotyrosine in the endothelium of coronary arteries. 5 AM significantly reduced the number of total ventricular ectopic beats that occurred during ischaemia (from 1185±101 to 520±74; P<0.05) and the incidences of ventricular fibrillation (from 61 to 26%; P<0.05). AM also induced a significant fall in MABP prior to occlusion. AM‐induced cardioprotection was abrogated in animals treated with the NO synthase inhibitor LNNA and the peroxynitrite scavenger MPG. 6 This study has shown that AM exhibits an antiarrhythmic effect through a mechanism that may involve generation of NO and peroxynitrite.

Correlation of leukocyte adhesiveness, adhesion molecule expression and leukocyte‐induced contraction following balloon angioplasty

The aim of this study was to examine the changes in leukocyte adhesion and leukocyte‐induced contraction in balloon‐injured rabbit subclavian artery and to correlate these changes with vessel morphology and expression of adhesion molecules on the injured arteries. Rabbits were anaesthetized and their left subclavian arteries were injured by balloon inflation and withdrawal followed by sacrifice at 2, 24, 48u2003h or 8 days after injury. The left and right subclavian arteries were removed and leukocytes were isolated from autologous rabbit blood. Leukocyte‐induced contraction was measured in 5‐HT precontracted artery rings and leukocyte adhesion was measured using 51Cr‐labelled leukocytes. Immunocytochemistry using paraffin‐embedded tissue was employed to detect changes in the expression of adhesion molecules on injured arteries. Autologous leukocytes caused a contraction of rabbit subclavian artery rings, which was prevented by L‐NAME (10−3u2003M). Balloon‐induced injury abolished the contractile response to leukocytes, which correlated with loss of carbachol‐induced relaxation Balloon injury markedly enhanced the adhesiveness of the subclavian artery for leukocytes, most notably at 24 and 48u2003h after injury (1.7 and 1.8 fold respectively). Increased leukocyte adhesion at these two time points correlated with an upregulation of E‐selectin, P‐selectin and VCAM‐1 expression on the remaining endothelium of the injured artery. Vessel morphology revealed that balloon inflation had induced an infiltration of inflammatory cells into the vessel wall, the greatest increase being seen at 24u2003h after injury. It is concluded that an increase in the expression of E‐selectin, P‐selectin and VCAM‐1 following balloon‐induced injury leads to enhanced leukocyte adhesion and migration into the injured vessel.

Endothelial nitric oxide synthase (NOS) is upregulated in rapid progressive pulmonary hypertension of the newborn

ObjectiveTo provide evidence for the upregulation of endothelial nitric oxide synthase (eNOS) or inducible nitric oxide synthase (iNOS) in the assumed imbalance in the pathophysiology of rapid progressive pulmonary hypertension of the newborn (RPPHN), which is characterized by abnormal hypertrophy of the pulmonary arterioles and arteries leading to increased pulmonary vascular resistance. Furthermore, to determine the cellular source and topographic distribution of eNOS and iNOS.Material and MethodsLung biopsies were taken from two term neonates with clinical and echocardiographic evidence of RPPH and of three controls. Biopsies were obtained at an early stage of the disease as well as at post mortem and examined immunohistochemically for the presence of eNOS, iNOS and nitrotyrosine.ResultsThe endothelial cells of pulmonary arterioles stained significantly for eNOS protein in RPPHN patients. This was not the case in the control infants. There were no differences for nitrotyrosine or iNOS between RPPHN patients and controls.ConclusionRapid progressive pulmonary hypertension of the newborn leads to compensatory induction of eNOS synthesis specifically in endothelial cells of the pulmonary arterioles. This mechanism of compensation can lead to delayed presentation of RPPHN during the late neonatal period. Exogenous inhaled nitric oxide therapy does not lead to suppression of the endogenous synthesis of nitric oxide.

Endothelial, inducible and neuronal nitric oxide synthase in congenital pulmonary lymphangiectasis

Abnormal growth and development of lymphatic pulmonary structures leads to severe hypoxia in congenital pulmonary lymphangiectasis (CPL). This case study aims to determine the cellular source and topographical distribution of the nitric oxide synthases in CPL. It studies the post mortem tissue of a term newborn with the clinical course and histological findings of CPL and three controls without pulmonary pathology. It was found that endothelial cells of pulmonary arteries and lymphatic structures stained significantly more for endothelial nitric oxide synthase protein in the CPL patient compared to the controls. The authors conclude that synthesis of endothelial nitric oxide synthase is upregulated in vascular and lymphatic endothelial cells in congenital pulmonary lymphangiectasis.

Prevalence and Prognostic Significance of Lipomatous Metaplasia in Patients With Prior Myocardial Infarction.

It has been noted recently, initially in pathological specimens, that infarcted myocardium can be infiltrated by adipose cells, known as lipomatous metaplasia (LM). It has been suggested that the presence of myocardial fat may interfere with the conduction system of the heart, perhaps providing a

Succinobucol-eluting stents increase neointimal thickening and peri-strut inflammation in a porcine coronary model.

The aim of this study was to assess the efficacy of stent‐based delivery of succinobucol alone and in combination with rapamycin in a porcine coronary model. Background: Current drugs and polymers used to coat coronary stents remain suboptimal in terms of long term efficacy and safety. Succinobucol is a novel derivative of probucol with improved antioxidant and anti‐inflammatory properties.

Correlation of changes in nitric oxide synthase, superoxide dismutase and nitrotyrosine with endothelial regeneration and neointimal hyperplasia in the balloon-injured rabbit subclavian artery

ObjectivesAlterations in nitric oxide (NO) and superoxide production within the wall of injured vessels may modulate the development and eventual extent of neointima after balloon injury. MethodsIn this study we have characterized a rabbit model of subclavian artery injury and have used immunocytochemistry to detect NO synthase (NOS) isoforms, Cu–Zn superoxide dismutase (SOD) and nitrotyrosine in the injured vessel from 2u2009h to 28 days after injury. ResultsAt 48u2009h after injury, when cellular proliferation that will ultimately form the neointima is commencing, there was upregulation of inducible NOS, Cu–Zn SOD and nitrotyrosine. Recovery of endothelial NOS occurred at 28 days after injury, when the neointima is stabilizing and cellular proliferation has slowed down. There was no increase in neuronal NOS at any time point studied. ConclusionsNO may serve to limit the development of neointima while superoxide may attenuate the effect of NO by formation of peroxynitrite, detected as increased nitrotyrosine staining. Upregulation of Cu–Zn SOD would limit superoxide both at sites of inflammation in the vessel wall from 48u2009h and in the adventitia up to 28 days after injury. Very early intervention to protect NO may reduce neointimal size.

A rapid, quantitative method for measuring leukocyte adhesion to normal and balloon-injured arteries in vitro.

Many of the currently available techniques for quantifying leukocyte adhesion require monolayers of cells and are therefore unsuitable for use in ex vivo arterial tissue. Here we describe a rapid method to measure adhesion of leukocytes to intact artery strips and to determine the effect of artery injury on adhesiveness of leukocytes with and without activation. Leukocytes were isolated from rabbit blood, labelled with 51Cr, and added to the luminal face of the left and right subclavian arteries derived from the same animal. In some experiments the endothelium was removed before addition of leukocytes and in another series of experiments the artery was injured by inflating a balloon catheter within the lumen in vitro before leukocyte addition. After washing, the adhesion of labelled leukocytes was quantified by gamma counting. To determine localization of the leukocytes, some arteries were fixed in situ and examined microscopically, with confirmation of leukocyte identification by enzyme cytochemistry. The adhesion of leukocytes increased progressively during 60 min and was inhibited by reducing the temperature to 4 degrees C. Adhesion was increased by the nitric oxide synthase inhibitor L-NAME. Stretching the artery wall in vitro using a balloon catheter increased leukocyte adhesion within 1 h after injury. In contrast, this did not occur following simple arterial denudation. Histological examination of stained en face preparations and transverse sections of the subclavian arteries revealed loosely adherent granulocytic leukocytes on the endothelial surface. This technique is straightforward and allows accurate and rapid measurement of autologous leukocyte adhesion to normal and pathologically altered arteries ex vivo.

The induction and detection in vitro of iNOS in the porcine basilar artery.

The expression of iNOS in vascular tissues has an adverse effect on vascular responses to vasoconstrictors and NO-mediated vasodilators. The development of a simple method for detecting the iNOS expression by functional means would be extremely useful. Here we describe a method for inducing iNOS in the porcine basilar artery followed by the detection of iNOS protein by immunocytochemical means and the characterisation of functional responses to U46619 and L-arginine. Porcine basilar arteries were treated with LPS (1, 10 and 100 microg/ml) for between 5 and 18 h at 37 degrees C. Inducible NOS protein was expressed in a concentration-dependent manner in the endothelial and smooth muscle cells after 5 h and persisted for 18 h. Vessels treated with LPS showed a time-dependent reduction in contractile function in response to U46619 (10 nM) reaching significance at the 18-h time point. Moreover, a similar time-dependent increase in the vasodilator response to exogenously applied L-arginine (30 microM) was observed at both 5- and 18-h time points. These effects of LPS at the 18-h time point were prevented by the incubation of vessels with dexamethasone (100 microM) in addition to LPS. The vasodilator response to L-arginine was prevented with the incubation with and in the presence of the inhibitor of inducible NOS, 1400W (10 microM) in addition to LPS. These results show that iNOS protein can be expressed in porcine cerebral arteries and that the iNOS is functional. The assessment of contractile function and responses to L-arginine using single concentrations is a rapid and effective method for establishing whether functional iNOS is present in porcine cerebral arteries.