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.

Clinical and pathological features of dilated cardiomyopathy in Holstein-Friesian cattle.

Dilated cardiomyopathy is a primary disease of the heart muscle that has been reported in Holstein-Friesian cattle worldwide in the past 20 years. Nine cases of the condition were compared in terms of their clinical and pathological characteristics with nine unaffected animals matched for age, sex and breed. Their clinical signs included right-sided heart failure with severe subcutaneous oedema, ascites and/or hydrothorax and distended jugular veins. There were no characteristic biochemical or haematological changes. Postmortem, the affected hearts were enlarged with all the chambers dilated and walls of variable thickness. In most cases the kidneys were pale with a pitted surface. Histologically there was marked perimysial and endomysial fibrosis, extensive loss of cardiomyocytes by coagulative or colliquative necrosis, increased variation in the cross-sectional area of the myocardial fibres, and multifocal disarray and vacuolation of myocytes. Scanning electron microscopy showed that in all cases there was a mild myocardial inflammatory infiltrate, either diffuse or multifocal, which was identified by immunohistochemical labelling as T cells.

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.

Validation of a technique to measure leukocyte adhesion to arterial segments: effects of drug treatments.

Adhesion and transmigration of leukocytes into arterial walls occurs after vascular injury and may play a role in the development of atherosclerosis and restenosis. This protocol presents a simple, rapid method for quantifying leukocyte adhesion to artery segments ex vivo. The procedure involves isolating leukocytes from rabbit whole blood and labelling with the gamma-emitting isotope 51Cr. Labelled leukocytes are added to open rings of subclavian artery taken from the same rabbit. After gamma counting, percentage leukocyte adhesion can be calculated with reference to a sample containing a quantity of labelled leukocytes equivalent to that which was added to the artery. Leukocyte adhesion was increased by L-NAME, thrombin and increasing incubation time and decreased by low temperatures. In addition, leukocyte adhesion was found to be increased following a vascular stretch injury performed in vitro. This protocol offers a number of advantages: the rapidity of the leukocyte isolation and labelling; the small quantity of leukocytes required; the ability to use autologous leukocytes; the applicability to whole arteries and arteries injured in vitro or in vivo, allowing the effects of vascular injury on leukocyte adhesion to be studied.

Short-term local delivery of an inhibitor of Ras farnesyltransferase prevents neointima formation in vivo after porcine coronary balloon angioplasty.

BACKGROUND: Mitogenic stimuli present at the site of coronary arterial balloon injury contribute to the progression and development of a restenotic lesion, many signaling through a common pathway involving the small G protein p21(ras). Our aim was to demonstrate in biochemical studies that farnesyl protein transferase inhibitor III (FPTIII) is an inhibitor of p21(ras) processing and that when it is given locally in vivo at the site of coronary balloon injury in a porcine model, it can inhibit neointima formation. METHODS AND RESULTS: FPTIII (1 to 25 micromol/L) concentration-dependently reduced p21(ras) levels in porcine coronary artery smooth muscle cell membranes. FPTIII also prevented p42/p44 MAPK activation and DNA synthesis in response to platelet-derived growth factor in these cells at a concentration of 25 micromol/L. Application of 25 micromol/L FPTIII locally for 15 minutes to balloon-injured porcine coronary arteries in vivo prevented neointima formation assessed at 4 weeks, reduced proteoglycan deposition, and inhibited adventitial hypertrophy. Coronary arteries from FPTIII-treated pigs had no deterioration in contraction or in endothelium-dependent relaxation. CONCLUSIONS: The study demonstrates in the pig that short-term local delivery of inhibitors of p21(ras)-dependent mitogenic signal transduction prevents restenosis after balloon angioplasty.

Is hydrogen peroxide an EDHF in human radial arteries?

In human radial arteries, a nitric oxide/prostanoid-independent mechanism that has the pharmacological characteristics of an EDHF contributes to endothelium-dependent relaxation. H2O2 can act as an EDHF in some vascular beds. We examined the hypothesis that endogenously produced H2O2 mediated the nitric oxide/prostanoid-independent relaxation to carbachol in radial arteries obtained from patients undergoing coronary artery bypass surgery. Superoxide levels, measured by chemiluminescence, were similar in radial and internal mammary arteries, but immunohistochemical staining for Cu/Zn superoxide dismutase (SOD) was lower in endothelium from radial arteries. In organ chamber studies, neither addition of catalase nor addition of SOD to the bathing fluid modified nitric oxide/prostanoid-independent relaxations to carbachol in radial arteries. However, nitric oxide-dependent vasorelaxation was enhanced in the presence of SOD. Thus the nitric oxide/prostanoid-independent relaxation to carbachol is not due to H2O2 and, unlike nitric oxide-mediated vasorelaxation, is not attenuated by superoxide. Blood vessels showing EDHF-mediated relaxations resistant to oxidative stress may provide favorable outcomes in revascularization surgery.

Atypical fibroxanthoma of the scalp.

BACKGROUND: Atypical fibroxanthoma occurs most frequently in the head and neck region of the elderly. Previous reports have identified that the condition usually arises at the following sites: the nose, cheeks, forehead, and the ears; its development at other sites is unusual. METHOD: We report a series of 10 cases with lesions all occurring at an apparently unusual site, the scalp, over a 10-year period. We compared the clinical and histologic appearances and behavior of this series with the existing reports of these lesions elsewhere in the head and neck region to investigate whether there were differences with those occurring at a conventional site. RESULTS: Despite the identification of a range of clinical and histologic findings in our cases, we were unable to find any significant differences with those arising at a conventional site. CONCLUSION: This clustering of cases at an apparently unusual site leads us to propose that this condition occurs more commonly on the scalp than current literature suggests. The possibility of its development at this site should be remembered by head and neck surgeons in their differential diagnosis of exophytic lesions of the scalp.

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

OBJECTIVE: The 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. METHODS: Angioplasty 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. RESULTS: Leukopenia 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. CONCLUSION: These 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.

Ventricular fibrillation threshold and local dispersion of refractoriness in isolated rabbit hearts with left ventricular dysfunction.

Patients with congestive heart failure or left ventricular dysfunction (LVD) have a high incidence of ventricular tachyarrhythmias and sudden cardiac death. In addition to structural, metabolic and neuroendocrine changes, mechanoelectrical feedback may play a role in arrhythmogenesis in heart failure. Three groups of rabbits (n = 10 for each) were studied: chronic coronary ligation with ejection fraction (EF) > or = 0.45 or < 0.45, and sham-operated controls. Ventricular fibrillation (VF) thresholds were measured at LV pressures of 0 and 40 mm Hg during modified Langendorff perfusion. Intervals between local activations during VF (VFI) were used as an index of refractoriness. Global dispersion was expressed as coefficient of variation of VFI; local dispersion by maximum difference in VFI between adjacent sites. Median VF threshold was lower at 0 mm Hg in the lower EF group compared to controls (30 vs. 67.5 mA, P<0.05). VF threshold in control hearts was lower at 40 mm Hg than at 0 mm Hg (P<0.01), but there was no further reduction in threshold in LVD hearts at 40 mm Hg. Global dispersion of VFI did not differ significantly between groups. Local dispersion of VFI in the lower EF group was greater than in controls at 0 mm Hg in the infarct border zone (P<0.05). At 40 mm Hg, local dispersion of VFI in zones bordering and remote from the infarct were greater in both LVD groups than in controls (P<0.05). Local inhomogeneity of refractoriness is more marked in the infarct border zone, but latent abnormalities are evident in normal myocardium of rabbits with left ventricular dysfunction and are revealed by left ventricular distension.

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.

Accumulation of nitrotyrosine correlates with endothelial NO synthase in pulmonary resistance arteries during chronic hypoxia in the rat.

Nitrotyrosine and eNOS were detected immunocytochemically using specific antibodies in paraffin sections of lung from rats subjected to hypoxia for 2, 7, or 14 days. The staining intensity for eNOS was enhanced in the endothelium of both resistance and conduit pulmonary arteries at 2 days. Staining intensity for eNOS remained elevated at 7 and 14 days in conduit arteries, whereas it progressively increased further in resistance arteries. Nitrotyrosine staining was elevated to a similar degree in endothelium and adjacent vascular smooth muscle. In resistance pulmonary arteries, there was a progressive increase in nitrotyrosine, which matched the increase in eNOS. In conduit pulmonary arteries, nitrotyrosine increased only after 14 days of hypoxia. The results suggest that in chronic hypoxia the up-regulation of eNOS leads to the formation of peroxynitrite which has access to both endothelium and vascular smooth muscle.

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

1. 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. 2. Rabbits were anaesthetized and their left subclavian arteries were injured by balloon inflation and withdrawal followed by sacrifice at 2, 24, 48 h 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 (51)Cr-labelled leukocytes. Immunocytochemistry using paraffin-embedded tissue was employed to detect changes in the expression of adhesion molecules on injured arteries. 3. Autologous leukocytes caused a contraction of rabbit subclavian artery rings, which was prevented by L-NAME (10(-3) M). Balloon-induced injury abolished the contractile response to leukocytes, which correlated with loss of carbachol-induced relaxation 4. Balloon injury markedly enhanced the adhesiveness of the subclavian artery for leukocytes, most notably at 24 and 48 h 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. 5. Vessel morphology revealed that balloon inflation had induced an infiltration of inflammatory cells into the vessel wall, the greatest increase being seen at 24 h after injury. 6. 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.

Pharmacological characterisation of endothelium-dependent relaxation in human radial artery: comparison with internal thoracic artery.

OBJECTIVE: The aim of this study was to investigate the contribution of nitric oxide/prostanoid-independent pathways to endothelium-dependent vasorelaxation in human conduit arteries. METHODS: Rings of internal thoracic artery (ITA) and radial artery (RA) taken from patients undergoing coronary artery bypass graft surgery were suspended in 10-ml organ baths and relaxation to carbachol and bradykinin studied in the presence and absence of nitric oxide synthase (NOS) inhibitors and potassium channel blockers. RESULTS: No significant relaxation to carbachol or bradykinin was observed in ITA after NOS inhibition. In contrast, in RA less than 40% attenuation of relaxation to carbachol or bradykinin was achieved with any of the NOS inhibitors. In the presence of 20 mM K+ relaxation to carbachol and bradykinin was inhibited by 28 +/- 9% and 42 +/- 9% while in the presence of L-NAME 200 microM + 20 mM K+ relaxation was inhibited by 66 +/- 6% and 70 +/- 4% respectively in this artery. Tetraethylammonium, glibenclamide, apamin and iberiotoxin had little effect on relaxation to carbachol but charybdotoxin alone and charybdotoxin plus apamin attenuated relaxation to carbachol by 23 +/- 4% and 49 +/- 9% in RA. In the presence of L-NAME 200 microM attenuation of these relaxations were increased to 60 +/- 4% and 78 +/- 4%. CONCLUSION: In ITA relaxations to carbachol and bradykinin were mediated via nitric oxide. In contrast in RA, a conduit vessel of similar diameter, both nitric oxide-dependent and independent pathways appeared to contribute to vascular relaxation. This nitric oxide-independent relaxation involved opening of Ca2+ activated potassium channel(s). The existence of alternative pathways mediating endothelium-independent relaxation could be important under pathological conditions and may contribute to the long term survival of radial artery grafts.

Vascular smooth muscle cell polyploidy and cardiomyocyte hypertrophy due to chronic NOS inhibition in vivo.

To assess the vascular and cardiac response to NO (nitric oxide) synthase (NOS) blockade in vivo, Wistar-Kyoto rats (WKY) were treated for 3 wk with NG-nitro-L-arginine methyl ester (L-NAME; 10 mg.kg-1.day-1). L-NAME treatment induced hypertension that was associated with increased plasma renin activity. Flow cytometry cell cycle DNA analysis showed that aortic vascular smooth muscle cells (VSMC) from L-NAME-treated WKY had a significantly higher polyploid population compared with WKY controls. Using organ bath experiments, we have shown that aortic rings from L-NAME-treated WKY have an increased contractile response to phenylephrine and impaired relaxation to carbachol compared with control rings. NOS blockade in vivo caused a significant increase in cardiac and left ventricular hypertrophy. Northern mRNA analysis of the myocardium showed that L-NAME treatment caused reexpression of the fetal skeletal alpha-actin isoform without alterations in collagen type I expression, a pattern indicating true hypertrophy of the cardiomyocytes. These studies provide further insight to confirm that NO deficiency in vivo results in the development of vascular and cardiac hypertrophy.

Detection of cytomegalovirus in upper gastrointestinal biopsies from heart transplant recipients: comparison of light microscopy, immunocytochemistry, in situ hybridisation, and nested PCR.

AIM: To establish the diagnostic value of in situ hybridisation and the nested polymerase chain reaction (PCR) in detecting clinically relevant cytomegalovirus (CMV) infection in upper gastrointestinal biopsies from heart transplant patients. METHODS: Test sensitivity and specificity for detection of CMV early gene RNA by in situ hybridisation and CMV intermediate early gene by PCR were established and then compared with haematoxylin and eosin (H&E) and immunocytochemical detection of CMV in order to establish the best pathological diagnostic approach. All investigations were carried out on formalin fixed, paraffin embedded tissue. RESULTS: Nested PCR had the highest test sensitivity, followed by in situ hybridisation and immunocytochemistry with the same sensitivity; H&E had the lowest. H&E and immunocytochemistry were the most specific but both had a significant false negative rate which was less of a problem with PCR. However, PCR gave no other diagnostic information, and in situ hybridisation was no better than immunocytochemistry. Both in situ hybridisation and PCR were technically complex and more expensive. CONCLUSIONS: H&E and immunocytochemistry represent the best initial screen for CMV and other diseases in upper gastrointestinal biopsies from heart transplant patients. If H&E and immunocytochemistry were negative, nested PCR could significantly increase the diagnostic yield of clinically relevant CMV infection. In situ hybridisation appeared to have no advantages and some drawbacks compared with immunocytochemistry and PCR.

Effects of nitric oxide and superoxide on relaxation in human artery and vein.

Endothelium-derived relaxing and contracting factors play an important role in atherosclerosis, re-stenosis and graft survival. Internal thoracic artery (ITA) and saphenous vein (SV) are used as conduit vessels in coronary artery bypass graft surgery (CABG). The long-term graft patency rate is higher with ITA than SV. Effects of nitric oxide and superoxide on vascular relaxation in isolated rings of ITA and SV from patients undergoing CABG were investigated. NG-nitro-L-Argenine methylester (L-NAME) was used to block nitric oxide synthesis and superoxide dismutase (SOD) and tiron to scavenge superoxide. Responses to carbachol were taken as a measure of stimulated nitric oxide release and increased responses to phenylephrine after addition of L-NAME as a measure of basal nitric oxide release. Immunocytochemical demonstration of endothelial nitric oxide synthase was performed using anti-endothelial nitric oxide synthetase (anti-eNOS) NOS antibody. Stimulated nitric oxide release was observed in ITA and SV but basal release was reduced or absent in SV. Treatment with SOD and tiron potentiated carbachol stimulated relaxation in ITA and SV. Tiron treatment resulted in a significant increase in basal nitric oxide in veins. eNOS immunoreactivity was more intense in ITA than SV, compatible with reduced nitric oxide production in veins. This may contribute to the reduced patency of venous grafts.

Histologically diagnosed Helicobacter pylori in heart transplant recipients.

BACKGROUND: The role of Helicobacter pylori in the pathogenesis of nonautoimmune gastritis and peptic ulceration is well recognized. H. pylori is widely prevalent in the general population, but the incidence among heart transplant recipients has not been reported. Furthermore, the natural history of this infection may be modified by immunosuppression. METHODS: Gastric and duodenal biopsy specimens from 47 heart transplant recipients were examined over a period of 44 months. RESULTS: Twenty-three (49%) patients had H. pylori infection (15 men, 8 women; mean age 49 [range 35 to 59] years). Eight of the 23 (35%) had symptoms. These eight patients were treated for H. pylori with bismuth, metronidazole, and amoxicillin, followed by maintenance H2-receptor antagonists. Dyspepsia continued in six of these patients, with persistence or recurrence of H. pylori being demonstrated in four. CONCLUSIONS: This study shows that although histologically diagnosed H. pylori infection is widely prevalent among heart transplant recipients, this prevalence is very similar to the general population. Immunosuppression may play a role in the recurrence or persistence of this infection and may diminish the mucosal inflammatory response to the organism.

Vascular remodelling in intramyocardial resistance vessels in hypertensive human cardiac transplant recipients.

OBJECTIVE: Cardiac transplant recipients often develop hypertension as a side effect of immunosuppressive treatment. The aim of this study was to use the serial endomyocardial biopsies taken to monitor rejection to study the early and sequential arterial changes in human myocardial resistance arteries as hypertension develops. METHODS: At least 14 biopsies were studied from each of 23 patients, divided into a normotensive group (12 patients with a diastolic pressure never greater than 90 mm Hg) and a hypertensive group (11 patients with more than 10% of diastolic pressure measurements above 100 mm Hg). Morphometric analysis of between 30 and 50 arteries and arterioles in two widely separated histological levels from each biopsy was undertaken using an Optomax image analyser. RESULTS: There was a correlation between blood pressure, particularly diastolic pressure, and rate of medial thickening of intramyocardial coronary resistance arteries and arterioles (P = 0.0025). There was also a correlation between serum cyclosporin A concentrations and mean artery wall thickness (P = 0.003). CONCLUSIONS: Hypertension and cyclosporin A treatment are associated with significant wall thickening of intramyocardial resistance vessels in cardiac allograft recipients. These changes may be functionally and clinically important.