Phase I and pharmacodynamic study of high-dose NGR-hTNF in patients with refractory solid tumours.

BACKGROUND: NGR-hTNF exploits the peptide asparagine-glycine-arginine (NGR) for selectively targeting tumour necrosis factor (TNF) to CD13-overexpressing tumour vessels. Maximum-tolerated dose (MTD) of NGR-hTNF was previously established at 45 µg m(-2) as 1-h infusion, with dose-limiting toxicity being grade 3 infusion-related reactions. We explored further dose escalation by slowing infusion rate (2-h) and using premedication (paracetamol). METHODS: Four patients entered each of 12 dose levels (n=48; 60-325 µg m(-2)). Pharmacokinetics, soluble TNF receptors (sTNF-R1/sTNF-R2), and volume transfer constant (K(trans)) by dynamic imaging (dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI)) were assessed pre- and post-treatment. RESULTS: Common related toxicity included grade 1/2 chills (58%). Maximum-tolerated dose was not reached. Both C(max) (P<0.0001) and area under the plasma concentration-time curve (P=0.0001) increased proportionally with dose. Post-treatment levels of sTNF-R2 peaked significantly higher than sTNF-R1 (P<0.0001). Changes in sTNF-Rs, however, did not differ across dose levels, suggesting a plateau effect in shedding kinetics. As best response, 12/41 evaluable patients (29%) had stable disease. By DCE-MRI, 28/37 assessed patients (76%) had reduced post-treatment K(trans) values (P<0.0001), which inversely correlated with NGR-hTNF C(max) (P=0.03) and baseline K(trans) values (P<0.0001). Lower sTNF-R2 levels and greater K(trans) decreases after first cycle were associated with improved survival. CONCLUSION: asparagine-glycine-arginine-hTNF can be safely escalated at doses higher than MTD and induces low receptors shedding and early antivascular effects.

Activity and safety of NGR-hTNF, a selective vascular-targeting agent, in previously treated patients with advanced hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly vascularised and poor-prognosis tumour. NGR-hTNF is a vascular-targeting agent consisting of human tumour necrosis factor-alpha fused to the tumour-homing peptide NGR, which is able to selectively bind an aminopeptidase N overexpressed on tumour blood vessels. METHODS: Twenty-seven patients with advanced-stage disease resistant to either locoregional (59%; range, 1-3), systemic treatments (52%; range, 1-3) or both (33%) received NGR-hTNF 0.8 microg m(-2) once every 3 weeks. The primary aim of the study was progression-free survival (PFS). RESULTS: No grade 3-4 treatment-related toxicities were noted. Common toxicity included mild-to-moderate, short-lived chills (63%). Median PFS was 2.3 months (95% CI: 1.7-2.9). A complete response ongoing after 20 months was observed in a sorafenib-refractory patient and a partial response in a Child-Pugh class-B patient, yielding a response rate of 7%. Six patients (22%) experienced stable disease. The disease control rate (DCR) was 30% and was maintained for a median PFS time of 4.3 months. Median survival was 8.9 months (95% CI: 7.5-10.2). In a subset of 12 sorafenib-resistant patients, the response rate was 8% and the median survival was 9.5 months. CONCLUSION: NGR-hTNF was well tolerated and showed single-agent activity in HCC. Further investigation in HCC is of interest.

Bronchodilator activity of (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino] carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407), a potent, long-acting, and selective muscarinic M3 receptor antagonist.

The novel quaternary ammonium salt (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino]carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407) showed subnanomolar affinities for human muscarinic M1 (hM1), M2 (hM2), and M3 (hM3) receptors and dissociated very slowly from hM3 receptors (t(½) = 166 min) with a large part of the receptorial complex (54%) remaining undissociated at 32 h from radioligand washout. In contrast, [(3)H]CHF5407 dissociated quickly from hM2 receptors (t(½) = 31 min), whereas [(3)H]tiotropium dissociated slowly from both hM3 (t(½) = 163 min) and hM2 receptor (t(½) = 297 min). In the guinea pig isolated trachea and human isolated bronchus, CHF5407 produced a potent (pIC(50) = 9.0-9.6) and long-lasting (up to 24 h) inhibition of M3 receptor-mediated contractile responses to carbachol. In the guinea pig electrically driven left atrium, the M2 receptor-mediated inhibitory response to carbachol was recovered more quickly in CHF5407-pretreated than in tiotropium-pretreated preparations. CHF5407, administered intratracheally to anesthetized guinea pigs, potently inhibited acetylcholine (Ach)-induced bronchoconstriction with an ED(50) value of 0.15 nmol/kg. The effect was sustained over a period of 24 h, with a residual 57% inhibition 48 h after antagonist administration at 1 nmol/kg. In conscious guinea pigs, inhaled CHF5407 inhibited Ach-induced bronchoconstriction for at least 24 h as did tiotropium at similar dosages. Cardiovascular parameters in anesthetized guinea pigs were not significantly changed by CHF5407, up to 100 nmol/kg i.v. and up to 1000 nmol/kg i.t. In conclusion, CHF5407 shows a prolonged antibronchospastic activity both in vitro and in vivo, caused by a very slow dissociation from M3 receptors. In contrast, CHF5407 is markedly short-acting at M2 receptors, a behavior not shared by tiotropium.

The hydrogen sulphide-releasing derivative of diclofenac protects against ischaemia-reperfusion injury in the isolated rabbit heart.

BACKGROUND AND PURPOSE: Hydrogen sulphide (H(2)S) is an endogenous gaseous mediator active in the multilevel regulation of pathophysiological functions in mammalian cardiovascular tissues. EXPERIMENTAL APPROACH: This study investigated the pharmacological activity of a new H(2)S-releasing derivative of diclofenac, S-diclofenac (2-[(2,6-dichlorophenyl)amino]benzeneacetic acid 4-(3H-1,2-dithiole-3-thione-5-yl)-phenyl ester) in the isolated rabbit heart submitted to low-flow ischaemia-reperfusion damage. KEY RESULTS: S-diclofenac (3, 10 and 30 microM), despite inhibiting prostacyclin generation by cardiac tissues, achieved dose-dependent normalization of coronary perfusion pressure, reducing left ventricular contracture during ischaemia and improving left ventricular developed pressure and +/-dP/dt(max) at reperfusion. Creatine kinase and lactate dehydrogenase activities in heart perfusates were significantly reduced during reperfusion. These effects were accompanied by substantial release of reduced glutathione (GSH), indicating that the H(2)S moiety may have up-regulated cysteine transport. The anti-ischaemic activities of S-diclofenac and the H(2)S-donor sodium hydro sulphide (NaHS) were partially prevented by the K(ATP) channel antagonist glibenclamide, suggesting a mechanism similar to H(2)S-induced cardioprotection in metabolic ischaemic preconditioning. Perfusion with the nitric oxide (NO) synthase inhibitor N(G)-monomethyl-L-arginine worsened the myocardial ischaemia-reperfusion damage, but this was dose-dependently prevented by S-diclofenac and NaHS, suggesting that the released H(2)S may have overcome NO deficiency. CONCLUSION AND IMPLICATIONS: These data show that S-diclofenac had marked anti-ischaemic activity in ischaemic-reperfused rabbit hearts despite inhibition of prostaglandin generation. Increased GSH formation leading to activation of K(ATP) channels may have contributed to this beneficial effect. The pharmacological profile of S-diclofenac and its anti-inflammatory activity, with diminished gastrointestinal side effects, offer therapeutic applications in cardiovascular disease.

Sildenafil reduces L-NAME-induced severe hypertension and worsening of myocardial ischaemia-reperfusion damage in the rat.

BACKGROUND AND PURPOSE: Phosphodiesterase-5 inhibitors are beneficial in pulmonary hypertension and congestive heart failure, the two conditions associated with coronary heart disease and ischaemia. We investigated whether sildenafil counteracts the cardiovascular alterations induced by N -nitro-L-arginine methyl ester (L-NAME) in the rat. EXPERIMENTAL APPROACH: Sildenafil was given orally to rats at doses of 0.37, 0.75 or 1.5 mg kg-1day-1 for four weeks, either alone or with L-NAME (35-40 mg kg-1 day-1 in the drinking water). Systolic blood pressure and urinary parameters (6-keto-prostaglandin F1alpha, thromboxane B2, 8-isoprostane-prostaglandin F2 and nitrite/nitrate) were measured in conscious rats. Isolated hearts were subjected to low flow ischaemia-reperfusion, and myocardial levels of guanosine 3', 5'cyclic monophosphate (cGMP) were determined. Endothelial vascular dysfunction was examined in aortic rings. KEY RESULTS: Sildenafil dose-dependently prevented the rise in systolic blood pressure in L-NAME-treated rats. This activity was associated with a normalization of urinary 8-isoprostane-prostaglandin F2alpha and other biochemical parameters. In perfused hearts, the post-ischaemic ventricular dysfunction was worse in preparations from L-NAME-treated rats than in controls. Sildenafil dose-dependently reduced this effect, and creatine kinase and lactate dehydrogenase release were lower too. cGMP levels, which were low in myocardial tissue from L-NAME-treated rats, were restored by sildenafil. In noradrenaline-precontracted aortic rings from L-NAME-treated rats acetylcholine lost its vasorelaxant effect, and sildenafil restored it. CONCLUSION AND IMPLICATIONS: In a rat model of chronic nitric oxide deprivation, where hypertension and aggravation of post-ischaemic ventricular dysfunction are associated with loss of vascular endothelium-relaxant function, sildenafil provided significant cardiovascular protection, primarily by maintaining tissue cGMP levels.

Monoclonal anti-CD18 antibody prevents transcellular biosynthesis of cysteinyl leukotrienes in vitro and in vivo and protects against leukotriene-dependent increase in coronary vascular resistance and myocardial stiffness.

BACKGROUND: Cysteinyl leukotrienes (cys-LT) can constrict small and large vessels and increase vascular permeability. Formation of cys-LT arising from polymorphonuclear leukocytes (PMNL) and endothelial cell cooperation (transcellular synthesis) led to the hypothesis that PMNL-endothelial cell adhesion may represent a key step toward the formation of vasoactive cys-LT. METHODS AND RESULTS: We studied the effect of pretreatment with a monoclonal antibody directed against the CD18 subunit of PMNL beta(2)-integrin on the synthesis of cys-LT in a PMNL-perfused isolated rabbit heart in vitro and in a model of permanent ligature of the left descending coronary artery in the rabbit in vivo. Challenge of PMNL-perfused rabbit hearts with formyl-met-leu-phe (0.3 micromol/L) caused synthesis of cys-LT and increase in coronary perfusion pressure that were prevented by the anti-CD18 antibody. Similar results were obtained with the use of A-23187 (0.5 micromol/L) as a challenge. Persistence of PMNL-associated myeloperoxidase activity in the perfusion buffer was observed in the presence of the anti-CD18 antibody, indicating decreased PMNL infiltration. Coronary artery ligature in vivo increased urinary excretion of leukotriene E(4), supporting the activation of the 5-lipoxygenase pathway during experimental acute myocardial infarction. Pretreatment with the anti-CD18 antibody (1 mg/kg) prevented the increase in leukotriene E(4) excretion. CONCLUSIONS: These data support the importance of adhesion in promoting cys-LT formation, originating from PMNL-endothelial cell cooperation, and contributing to myocardial stiffness and increased coronary resistance.

Growth hormone-independent cardioprotective effects of hexarelin in the rat.

We previously reported that induction of selective GH deficiency in the rat exacerbates cardiac dysfunction induced by experimental ischemia and reperfusion performed on the explanted heart. In the same model, short-term treatment with hexarelin, a GH-releasing peptide, reverted this effect, as did GH. To ascertain whether hexarelin had non-GH-mediated protective effects on the heart, we compared hexarelin and GH treatment in hypophysectomized rats. Hexarelin (80 microg/kg sc), given for 7 days, prevented exacerbation of the ischemia-reperfusion damage induced by hypophysectomy. We also demonstrate that hexarelin prevents increases in left ventricular end diastolic pressure, coronary perfusion pressure, reactivity of the coronary vasculature to angiotensin II, and release of creatine kinase in the heart perfusate. Moreover, hexarelin prevents the fall in prostacyclin release and enhances recovery of contractility. Treatment with GH (400 microg/kg sc) produced similar results, whereas administration of EP 51389 (80 microg/kg sc), another GH-releasing peptide that does not bind to the heart, was ineffective. In conclusion, we demonstrate that hexarelin prevents cardiac damage after ischemia-reperfusion, and that its action is not mediated by GH but likely occurs through activation of specific cardiac receptors.

Formation of sulphidopeptide-leukotrienes by cell-cell interaction causes coronary vasoconstriction in isolated, cell-perfused heart of rabbit.

1. We have studied the transcellular biosynthesis of bioactive leukotrienes (LTs), generated upon blood cell-vascular wall interactions and their functional consequences, in the spontaneously beating, cell-perfused, heart of the rabbit. Rabbit isolated hearts were perfused under recirculating conditions (50 ml) with 5 x 10(6) cells of unpurified (buffy coat) or purified human neutrophils (PMNL), and challenged with 0.5 microM A23187 for 30 min. Coronary perfusion pressure (CPP), heart rate (HR), left ventricular end-diastolic pressure (LVEDP) and left ventricular pressure (LVP) were monitored continuously. Leukotriene formation was measured by specific enzyme-immunoassay and confirmed by reversed phase h.p.l.c. and u.v. spectral analysis. 2. Basal CPP values averaged 44 +/- 1.4 mmHg; A23187 triggered a marked increase in CPP both in the presence of buffy coat cells (+100% above basal) and PMNL (+270% above basal); the latter change in CPP was accompanied by a rise in LVEDP (+138% above basal). 3. The increase in CPP was preceded by a statistically significant rise in iLTC4-D4 concentration in the circulating buffer. Pretreatment with two structurally unrelated LTD4 receptor antagonists, LY171883 and SKF104353 (10 microM), fully prevented the increase in CPP and LVEDP. A similar protection was also observed when the rabbit heart was perfused with PMNL that had been pretreated with MK886 (1 microM), a potent inhibitor of leukotriene biosynthesis. 4. The increased coronary tone was accompanied by a marked release of lactate dehydrogenase (LDH), a marker of ischaemic damage; pretreatment of the heart with the LTD4 receptor antagonists as well as of the PMNL with MK886 resulted in a complete suppression of LDH activity release. 5. Positive identification of LTC4-D4 in the perfusates was obtained and a significant correlation observed between the CPP values and iLTC4-D4 concentrations.6. This study suggests that challenge of PMNL present within the coronary vasculature, causes a LTD4-dependent coronary vasoconstriction, favoured by an efficient uptake of PMNL-derived LTA4 by endothelial cells. The activation of the 5-lipoxygenase pathway in the context of tight interactions between blood cells and coronary vasculature, is suggested to have an important outcome in the alterations of coronary flow and cardiac contractility.

Hyperbaric oxygen increases plasma exudation in rat trachea: involvement of nitric oxide.

This study investigates the microvascular permeability changes in tracheal tissue of rats exposed to hyperbaric oxygen (HBO). Rats, following exposure to HBO or ambient air (control animals) for 1.5, 3 and 6 h, were prepared for recording of nitric oxide exhaled (FENO) in air using a chemiluminescence analyser. The level of FENO was not statistically different in the two groups. Plasma exudation, evaluated by measuring the leakage of Evans blue (EB) dye into the tracheal tissue, was significantly elevated (48, 86 and 105% at 1.5, 3 and 6 h, respectively) in HBO-treated rats. Plasma exudation in the trachea of control rats was significantly increased (42%, P<0.05) by NG-nitro-L-arginine methyl ester (L-NAME), whereas it was significantly reduced (31%, P<0.05) in rats exposed to HBO for 3 h. N-acetylcysteine (NAC) and flunisolide significantly prevented the increase in plasma leakage in HBO-treated rats. In contrast, indomethacin was devoid of anti-exudative activity in these experiments. Western immunoblot showed a significant increase in the level of inducible nitric oxide synthase (iNOS) protein in the tracheal homogenates of HBO-treated rats, as compared to basal levels. These results indicate that nitric oxide (NO) is involved in the maintenance of microvascular permeability in tracheal tissue of rats. The protective effect observed with the steroid seems to support this hypothesis. Furthermore, the beneficial action of NAC underlines that reactive oxygen species participate in the microvascular permeability changes observed in tracheal tissue of rats exposed to HBO.

Nitric oxide modulation of transcellular biosynthesis of cys-leukotrienes in rabbit leukocyte-perfused heart.

1. We have studied the role of nitric oxide (NO) in the regulation of the transcellular biosynthesis of sulphidopeptide leukotrienes (cys-LT) generated upon neutrophil-vascular wall interactions and their functional consequences, in the spontaneously beating, cell-perfused, heart of the rabbit. 2. Hearts were perfused under recirculating conditions (50 ml) with 5 x 10(6) purified human neutrophils (PMNL), and challenged with 0.5 microM A-23187 for 30 min. Coronary perfusion pressure (CPP) and left-ventricular end-diastolic pressure (LVEDP) were monitored. Cys-LT formation was measured by reversed phase high performance liquid chromatography (h.p.l.c.) and u.v. spectral analysis. Myeloperoxidase (MPO) enzyme activity, assayed in aliquots of the recirculating buffer, was used as a marker of PMNL, adhesion to the coronary endothelium. 3. Basal CPP and LVEDP values averaged 45 +/- 1.4 mmHg and 5 +/- 0.1 mmHg, respectively; A-23187 triggered an increase in CPP (134 +/- 9 mmHg, at 30 min) which was significantly attenuated by pretreatment with L-arginine, 100 microM (90 +/- 3 mmHg, at 30 min). Pretreatment with NG-monomethyl-L-arginine, 10 microM (L-NMMA), induced a marked increase in CPP (290 +/- 40 mmHg, at 20 min) and in LVEDP (47 +/- 16 mmHg), so pronounced that it caused cardiac arrest in systole in 5 out of 6 hearts and these were prevented by L-arginine, 100 microM, (CPP 115 +/- 10 mmHg, LVEDP 6 +/- 1.1 mmHg, at 30 min). 4. The increase in CPP was accompanied by the release of cys-LT in the circulating buffer, which was reduced significantly by L-arginine. Pretreatment with L-NMMA, caused a marked rise in cys-LT concentrations which was prevented by L-arginine. 5. Neither L-arginine nor L-NMMA affected directly the A-23187-induced arachidonic acid (AA) metabolism in isolated PMNL alone. 6. Pretreatment with L-NMMA caused a prompt drop in myeloperoxidase (MPO), activity, suggesting rapid adhesion of PMNL to the coronary wall; this effect was significantly blunted by L-arginine. 7. This study suggests that NO provides cardioprotection in an organ model of transcellular metabolism of cys-LT by preventing PMNL adhesion to the coronary intima.

4-Oxystilbene compounds are selective ligands for neuronal nicotinic alphaBungarotoxin receptors.

1. Starting from the structure of an old 4-oxystilbene derivate with ganglioplegic activity (MG624), we synthesized two further derivates (F2 and F3) and two stereoisomers of F3 (F3A and F3B), and studied their selective effect on neuronal nicotinic acetylcholine receptor (AChR) subtypes. 2. MG 624, F3, F3A and F3B inhibited of 125I-alphaBungarotoxin (alphaBgtx) binding to neuronal chick optic lobe (COL) membranes, with nM affinity, but inhibited 125I-alphaBgtx binding to TE671 cell-expressed muscle-type AChR only at much higher concentrations. 3. We immobilized the alpha7, beta2 and beta4 containing chick neuronal nicotinic AChR subtypes using anti-subunit specific antibodies. MG 624, F3, F3A and F3B inhibited 125I-alphaBgtx binding to the alpha7-containing receptors with nM affinity, but inhibited 3H-Epi binding to beta2-containing receptors only at very high concentrations (more than 35 microM); their affinity for the beta4-containing receptors was ten times more than for the beta2-containing subtype. 4. Both MG624 and F3 compounds inhibited the ACh evoked currents in homomeric oocyte-expressed chick alpha7 receptors with an IC50 of respectively 94 and 119 nM. 5. High doses of both MG 624 and F3 depressed the contractile response to vagus nerve stimulation in guinea pig nerve-stomach preparations although at different IC50s (49.4 vs 166.2 microM) The effect of MG624 on rat nerve-hemidiaphragm preparations was 33 times less potent than that of F3 (IC50 486 vs 14.5 microM). 6. In conclusion, MG624 and F3 have a high degree of antagonist selectivity for neuronal nicotinic alphaBgtx receptors containing the alpha7 subunit.

Endocrine, metabolic and cardioprotective effects of hexarelin in obese Zucker rats.

Genetically obese male Zucker rats have an impaired secretion of GH, coupled to hyperinsulinemia, hyperlipidemia and glucose intolerance. The aim of this study was to evaluate whether a chronic treatment with hexarelin, a synthetic enkephalin-derived hexapeptide with a potent GH-releasing activity, might be able to ameliorate the somatotropic function and reverse some metabolic alterations associated with obesity in male obese Zucker rats. Furthermore, as decreased GH secretion and insulin resistance are associated with increased cardiovascular risk, we also tested the capacity of hexarelin to prevent postischemic ventricular dysfunction in hearts of male obese Zucker rats. Obese and lean male rats of the Zucker strain were treated with hexarelin (80 microgram/kg, b.i.d., s.c.) or saline (1 ml/kg, b.i.d., s.c.) for 30 days. An acute hexarelin injection (80 microgram, s.c.) at the 28th day of treatment elicited a rise in plasma GH levels in ! lean but not in obese rats (pretreated or not with hexarelin); lean rats chronically treated with hexarelin showed a greater increase in plasma GH as compared with control counterparts. At the end of the experiment, pituitary GH mRNA levels were significantly reduced in obese rats and hexarelin administration failed to increase pituitary GH mRNA and IGF-I concentrations in plasma and heart. Chronic treatment with hexarelin increased insulinemia and blood glucose levels in obese but not in lean rats, left unaltered the high triglyceride levels but significantly decreased plasma cholesterol concentrations in obese rats. Heart preparations from lean and obese Zucker rats treated with saline, subjected to low flow ischemia and reperfusion, showed at reperfusion: a) a low recovery of postischemic left ventricular developed pressure (LVDP), coupled to a substantial increase in coronary perfusion pressure, and b) a marked increase in creatine kinase released in the perfusates. Hexare! lin administration for 30 days counteracted the heart ischemic damage both in lean and obese Zucker rats. In fact, the recovery of LVDP at reperfusion was significantly higher than in controls and the increase in coronary resistance was minimal. Collectively, these data indicate that a 30-day treatment with hexarelin was unable to improve somatotropic function in male obese Zucker rats but was successful in decreasing plasma cholesterol concentrations. Hexarelin exerted a cardioprotective effect in both lean and obese rats. The heart-protective activity afforded by the peptide was divorced from any stimulation of the GH axis and is probably exerted through activation of specific cardiac receptors.

New data concerning the antianaphylactic and antihistaminic activity of nimesulide.

The time to respiratory crisis in ovalbumin-sensitised guinea-pigs following exposure to aerosol administered antigen was dose dependently delayed by inhalation of nimesulide (0.1% to 1%), whereas indomethacin had no effect. At the same time, nimesulide significantly reduced blood histamine concentrations, in contrast to the slight increase observed with indomethacin. In human bronchial muscle preparations, nimesulide, but not indomethacin, antagonised H1-histamine-receptor activation by histamine and was without effect on acetylcholine-induced responses. Bronchoconstriction was also elicited in anaesthetised guinea-pigs by intravenous acetaldehyde (5% in saline, 1 ml/kg). This effect, which is paralleled by a rise in blood histamine concentrations, was significantly attenuated by inhaled nimesulide (0.1% to 1%), but not by indomethacin (1%). These data, which further support the antihistaminic and antiallergic activity of nimesulide, may have therapeutic relevance in patients who are affected by inflammation of the respiratory tract and who also have a history of allergic bronchoconstriction.

Comparison of endothelium-dependent vasoactivity of internal mammary arteries from hypertensive, hypercholesterolemic, and diabetic patients.

BACKGROUND: Endothelium-dependent relaxation is abnormal in a variety of diseased states. Despite the widespread use of the internal mammary artery (IMA) in coronary artery bypass grafting, there is a lack of comparative studies on IMA endothelial-dependent function in patients with major cardiovascular risk factors. METHODS: An IMA segment from 48 selected patients undergoing coronary artery bypass grafting was harvested intraoperatively and assigned to one of four groups (n = 12): diabetics requiring therapy, hypertensives, hypercholesterolemic, and nondiabetic-normotensive-normocholesterolemic patients. Internal mammary artery specimens were cut into rings and suspended in organ bath chambers, and the isometric tension of vascular tissues was recorded. The IMA rings were (1) precontracted with norepinephrine, and the endothelium-derived relaxation was evaluated by cumulative addition of acetylcholine, (2) contracted with cumulative concentrations of endothelin-1, and (3) contracted with the nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine. Furthermore, the release of prostacyclin by the IMA rings was directly measured during basal tone conditions and at the end of the various pharmacologic interventions. Histology of IMA rings was randomly performed. RESULTS: The results obtained in these experiments showed that IMA rings harvested from hypertensive patients have the greatest impairment of endothelium-dependent response to relaxant and contracting stimuli (p < 0.01 versus nondiabetic-normotensive-normocholesterolemic tissues; p < 0.05 versus hypercholesterolemic and diabetic tissues) and prostacyclin release in normal and stimulated conditions. To a lesser extent, hypercholesterolemic and diabetic tissues show similar depression (diabetic > hypercholesterolemic) both of relaxation and prostacyclin production, with respect to nondiabetic-normotensive-normocholesterolemic specimens (p < 0.05). Histology findings (scanning electron microscopy) did not differ in multiple sections from vessel studies. CONCLUSIONS: Major cardiovascular risk factors affect the endothelium-dependent vasoactive homeostasis of human IMA differently. Depression of relaxation is highest in patients with a history of hypertension. These findings may be pertinent to early and long-term treatment of patients undergoing coronary artery bypass grafting.

Prostacyclin production by different human grafts employed in coronary operations.

Segments of human saphenous vein, internal mammary artery, right gastroepiploic artery, and inferior epigastric artery were incubated in vitro in Krebs-Henseleit solution and compared in terms of their capacity to generate and release into the medium 6-keto-prostaglandin F1 alpha (PGF1 alpha), the stable metabolite of prostacyclin. The four vascular conduits were also challenged with endothelin-1 (40 ng/mL), and accumulation of the lipidic material in the bathing fluid was also studied. The results obtained show clearly that under both normal and endothelin-1-stimulated conditions, the four vascular segments generate a substantial amount of 6-keto-PGF1 alpha. Multiple-comparisons analysis of the results indicates that the rank order in producing 6-keto-PGF1 alpha is as follows: inferior epigastric artery > internal mammary artery > right gastroepiploic artery > saphenous vein (p < 0.01). A similar order of potency was obtained in vascular conduits stimulated with endothelin-1. The rate of formation of immunoreactive 6-keto-PGF1 alpha under both normal and stimulated conditions by the inferior epigastric artery (normal, 301 +/- 8 pg/mg of tissue; stimulated, 519 +/- 15 pg/mg of tissue) was at 10 minutes more than 2 times (p < 0.01) that of the saphenous vein and about 1.5 times (p < 0.01) that of the right gastroepiploic artery.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelial-dependent dynamic and antithrombotic properties of porcine aortic and pulmonary valves.

BACKGROUND: In the present study, the endothelium-dependent antithrombotic and dynamic properties of porcine aortic (AoV) and pulmonary valves (PuV) were investigated. METHODS: Fifteen fresh AoV and 15 fresh PuV were obtained from 25 9-month-old swines. The valves were examined for endothelial function by pharmacologic evaluation (with and without endothelium) of both the endothelial-releasing capacity of prostacyclin and the endothelial-dependent dynamic response to relaxing (acetylcholine from 10[-10] mol/L to 10[-4] mol/L in AoV and PuV segments precontracted with norepinephrine [3 x 10(-6) mol/L]) and contracting (endothelin-1, from 10[-11] mol/L to 10[-5] mol/L; and NG-monomethyl-L-arginine, 10[-4] mol/L) drugs. The ultrastructural integrity of the endothelial valve layer was also examined with transmission electron microscopy. RESULTS: Acetylcholine caused potent relaxation in both AoV and PuV specimens with, but not in those without, endothelium. Endothelin-1 produced a concentration-dependent tension increase in AoV and PuV with and without endothelium. However, the intrinsic activity of the peptide significantly increased in tissues without endothelium. NG-monomethyl-L-arginine evoked a progressive increase in resting tension of the preparations, but the AoV and PuV without endothelium were less sensitive to the inhibition of the nitric oxide generation. Aortic and pulmonary valves with an intact endothelium showed a spontaneous ability to release prostacyclin. The basal release of this lipidic autacoid significantly decreased in cardiac valves without endothelium. This phenomenon was observed in both basal conditions, and under stimulation with the aforementioned drugs. Transmission electron microscopy showed the perfect preservation of endothelial cells in all the preparations examined. CONCLUSIONS: Valvular endothelium of AoV and PuV seems to have similar antithrombotic and dynamic functions of vascular endothelium, actively participating in valvular homeostasis.

Retention of endothelium-dependent properties in human mammary arteries after cryopreservation.

BACKGROUND: We investigated the effects of cryopreservation and antibiotic treatment on endothelium-dependent vasomotor properties of human internal mammary arteries (IMAs). METHODS: Sixty IMA specimens from routine coronary artery bypass grafting procedures were randomly assigned to six groups. Group I (controls) were immediately tested after harvest. Remaining groups were prepared according to a stepwise design: group II, 6 hours of warm ischemia; group III, 6 hours of warm ischemia + 24 hours at 4 degrees C (without antibiotics); group IV, 6 hours of warm ischemia + 24 hours of 4 degrees C antibiotic disinfection; group V, 6 hours of warm ischemia + 24 hours at 4 degrees C (without antibiotics) + cryopreservation; and group VI, 6 hours of warm ischemia + 24 hours of 4 degrees C disinfection+cryopreservation. The IMA specimens were cut into rings and the tension of vascular smooth muscle was recorded. The IMA rings were contracted with norepinephrine (3 x 10(-6) mol/L) and tested with cumulative concentrations of acetylcholine (from 1 x 10(-9) to 1 x 10(-5) mol/L), contracted with endothelin-1 (from 1 x 10(-11) to 1 x 10(-6) mol/L), and contracted with the nitric oxide-synthase inhibitor NG-monomethyl-L-arginine (1 x 10(-4) mol/L). Rings were also tested for their capacity to generate 6-keto-prostaglandin F1 (the stable metabolite of prostacyclin), and endothelial cell viability rate was finally evaluated with the trypan blue dye exclusion method. RESULTS: Our results show that a complete cryopreservation protocol does not significantly modify (p > 0.05) the relaxant activity to acetylcholine in norepinephrine-precontracted IMA rings (controls; 90.2% +/- 4.2% vs group VI, 77.1% +/- 6.2%) or the vasoconstrictor response induced by endothelin-1 (controls, 62.6% +/- 2.8% versus group VI, 73.7% +/- 4.8%) and NG-monomethyl-L-arginine (controls, 22.4% +/- 1.5% versus group VI, 18.9% +/- 1.9%). Furthermore, IMA cryopreservation does not significantly modify (p > 0.05) the endothelial release of prostacyclin either in basal conditions (-20% versus controls) or during pharmacologic intervention with acetylcholine (-18% versus controls), endothelin-1 (-17% versus controls), and NG-monomethyl-L-arginine (-18% versus controls). CONCLUSIONS: We conclude that the IMA endothelial function does not seem significantly injured by any of the current steps of disinfection and cryopreservation.

Effects of warm ischemia on valve endothelium.

BACKGROUND: This study investigates the time-dependent resistance of the endothelium of porcine aortic and pulmonary valves to different periods of warm ischemia (WIT). METHODS: Twenty-five 9-month-old swine were divided after death into five groups of WIT (0, 6, 12, 24, and 36 hours). Aortic and pulmonary valves were removed and a total of 15 aortic and 15 pulmonary valve specimens were obtained for each WIT interval. Valves were then examined for (1) their viability rate by the trypan blue dye exclusion method at light microscopy (percent of viability compared with 0 hours of WIT); (2) ultrastructural signs of irreversible or reversible ischemic damage by transmission electron microscopy (cell disruption, dilation of endoplasmic reticulum, cytoplasmic edema, nuclear and mitochondrial changes); (3) endothelial function by pharmacologic evaluation of both the endothelial-releasing capacity of prostacyclin and the endothelial-dependent dynamic responses to relaxing (acetylcholine from 1 x 10(-10) mol/L to 1 x 10(-4) mol/L) in aortic and pulmonary valve segments precontracted with norepinephrine (1 x 10(-6) mol/L) and contracting (NG-monomethyl-L-arginine, 1 x 10(-4) mol/L) drugs. RESULTS: Our results showed an endothelial progressive time-dependent ischemic injury, which reached significance after 12 hours of exposure. Viability and functional data indicated that 6 hours of WIT only provoked slight endothelial damage (p > 0.05 respect to time 0 hours), with signs at transmission electron microscopy consistent with a reversible injury. At 12 hours of exposure, we observed a significant reduction (p < 0.05) with respect to time 0 of the viability rate of prostacyclin production and of the endothelium-dependent dynamic responses to acetylcholine and NG-monomethyl-L-arginine. These functional impairments, although significant, were not consistent, however, with a complete loss of viability. Transmission electron microscopic observations confirmed the appearance of signs of irreversible injury; nevertheless, some elements were found to be well preserved or presented reversible damage. After 24 hours of WIT, ultrastructural and functional data were consistent with a dramatic decrease compared with controls in endothelial viability and functions (p < 0.01). Finally, after 36 hours of WIT, there was a subtotal loss of viability, of functions (p < 0.001) and, at transmission electron microscopic observations, of the endothelial layer of the valves. CONCLUSIONS: Our data show that the endothelial cells are resistant to short periods of WIT (up to 6 hours), and suggest that these cells can endure longer exposures, up to 12 hours of warm ischemia. Periods of 24 and 36 hours of WIT provoke progressive irreversible damage.

Hexarelin exhibits protective activity against cardiac ischaemia in hearts from growth hormone-deficient rats.

Male rats were treated with growth hormone (GH)-releasing hormone antiserum to induce selective GH deficiency. The chronic administration of hexarelin to these GH-deficient rats had a pronounced protective effect against ischaemic and post-ischaemic ventricular dysfunction. Hexarelin prevented hyper-responsiveness of the coronary vascular bed to angiotensin II and also prevented the reduction in generation of 6-keto-prostaglandin F1alpha in perfused hearts from GH-deficient rats. The most plausible interpretation of these findings is that hexarelin acts via stimulation of specific cardiac and vascular receptors, triggering currently unknown cytoprotective mechanisms that are responsible for resistance to ischaemic insults and for the preservation of the integrity of the endothelial vasodilation function.

Carbamazepine exerts anti-inflammatory effects in the rat.

In a first set of experiments, we evaluated the effects of different doses (5.0, 10, 20 and 40 mg/kg p.o.) of carbamazepine on nociceptive thresholds to thermal and mechanical stimuli, and on paw inflammatory hyperalgesia induced by the injection of brewer's yeast. Moreover, we studied the effect of carbamazepine on paw inflammatory edema by plethysmometry. Carbamazepine did not modify nociceptive latencies, but dose dependently reduced the hyperalgesia and the edema induced by the brewer's yeast injection in the rat hindpaw. In a second set of experiments, we studied the effects induced by the same doses of the drug on subcutaneous carrageenin-induced inflammation. Carbamazepine dose dependently reduced the inflammatory exudate, the prostaglandin E2-like activity in the exudate, and the substance P concentrations in the exudate. Our results demonstrate that carbamazepine is able to inhibit the development of different types of inflammation in the rat.