Biomedical subjects

N F Paoni

Publications and source records attributed to N F Paoni.

At least 19 recordsLinked to original sources

PubMed2001

Effects of early angiotensin-converting enzyme inhibition on cardiac gene expression after acute myocardial infarction.

BACKGROUND: ACE inhibition after myocardial infarction (MI) has been shown to have beneficial effects on cardiac anatomy and function. The purpose of this study was to examine the effects of ACE inhibition on cardiac gene expression after MI. METHODS AND RESULTS: Rats were randomized to receive captopril or no treatment 1 day after MI. Eight weeks later, cardiac function and hemodynamics were measured by use of indwelling catheters and perivascular flow probes. Myocardial gene expression was assessed with DNA microarrays and real-time reverse transcription-polymerase chain reaction. The ratios of heart and left ventricular weights to body weight were significantly increased by MI and normalized by captopril. Cardiac index and stroke volume index were lower in the untreated MI group than in sham controls but were normal in the MI+captopril group. Thirty-seven genes were found to be differentially expressed between the untreated MI group and sham controls; 31 were induced and 6 repressed. Captopril partially or completely inhibited changes in 10 of the genes. The 37 genes clustered into 11 functional groups, and 6 had >/=1 genes whose expression was modified by ACE inhibition. CONCLUSIONS: ACE inhibition after MI inhibits cardiac hypertrophy, preserves cardiac function, and attenuates changes in myocardial gene expression. Gene expression profiling reveals, however, that some elements of the pathophysiology may be unaffected by the treatment and be targets for new therapies.

Acute Disease↗

PubMed2001

Expression profiling techniques for cardiac molecular phenotyping.

Evidence from clinical and experimental heart failure studies indicates that cardiac growth and morphogenesis are important determinants for morbidity and mortality. Characterization of the myocardial gene expression patterns that are associated with these processes may be useful in the search for novel therapeutic strategies. Changes in tissue mRNA abundance have traditionally been monitored by a candidate gene approach, in which transcripts of interest have been analyzed one or several at a time. New methodologies for detecting differentially expressed genes, such as DNA microarrays, and restriction fragment display, are now enabling molecular phenotyping to be performed on a much larger scale. Here we describe our work on the application of these methods and the insights gained into the biology and pathophysiology of the myocardium.

Animals↗

PubMed2000

Effects of exercise training on cardiac function, gene expression, and apoptosis in rats.

This study determined the effects of exercise training on cardiac function, gene expression, and apoptosis. Rats exposed to a regimen of treadmill exercise for 13 wk had a significant increase in cardiac index and stroke volume index and a concomitant decrease in systemic vascular resistance compared with both age-matched and body weight-matched sedentary controls in the conscious state at rest. In exercise-trained animals, there was no change in the expression of several marker genes known to be associated with pathological cardiac adaptation, including atrial natriuretic factor, beta-myosin heavy chain, alpha-skeletal and smooth muscle actins, and collagens I and III. Exercise training, however, produced a significant induction of alpha-myosin heavy chain, which was not observed in rats with myocardial infarction. No histological features of cardiac apoptosis were observed in the treadmill-trained rats. In contrast, apoptotic myocytes were detected in animals with myocardial infarction. In summary, exercise training improves cardiac function without evidence of cardiac apoptosis and produces a pattern of cardiac gene expression distinct from pathological cardiac adaptation.

Actins↗

PubMed1998

Effects of cardiotrophin-1 on haemodynamics and cardiac function in conscious rats.

Cardiotrophin-1 (CT-1), a newly discovered cytokine, has been shown to induce cardiac hypertrophy in vitro and in vivo. The present study examined the effects of CT-1 on haemodynamics and cardiac function. The measurements of haemodynamic parameters were made using in-dwelling catheters and flow probes in conscious, unrestrained rats. Intravenous administration of CT-1 caused a dose-dependent decrease in mean arterial pressure (MAP), and an increase in heart rate (HR). CT-1 (100 micrograms/kg) significantly elevated cardiac output and HR, and decreased MAP and systemic vascular resistance. Stroke volume was unaltered, suggesting that the CT-1 induced increase in cardiac output was secondary to increased HR. There was no significant difference in left ventricular maximal dP/dt between the CT-1-treated and vehicle-treated groups, suggesting that CT-1 might not induce a meaningful change in ventricular contractility. Pretreatment with intravenous N omega-nitro-L-arginine methyl ester, a specific inhibitor of nitric oxide synthase, significantly attenuated the depressor and tachycardic responses to CT-1. These results indicate that nitric oxide plays an important role in mediating the haemodynamic effects of CT-1.

Animals↗

PubMed1996

In vivo effects of cardiotrophin-1.

Cardiotrophin-1 (CT-1) is a recently discovered cytokine that was isolated based on its ability to induce cardiac myocyte hypertrophy in vitro. In this study, the effects of chronic administration of CT-1 to mice (0.5 or 2 microg by intraperitoneal injection, twice a day for 14 days) were determined. A dose-dependent increase in both the heart weight and ventricular weight to body ratios was observed in the treated groups. The body weights of the animals were unaffected. These results indicate that CT-1 can induce cardiac hypertrophy in vivo. CT-1 was not specific for the heart, however. It stimulated the growth of the liver, kidney, and spleen, and caused atrophy of the thymus. CT-1 administration also increased the platelet counts by 70%, with no change in mean platelet volume. Red blood cell counts were increased in the treated animals, and there was a concomitant increase in haemoglobin concentration. Thus, CT-1 has a broad spectrum of biological activities in vivo. This observation is consistent with previous in-vitro findings showing that the mRNA for CT-1 is expressed in several tissues, and that CT-1 can function through binding to the leukaemia inhibitory factor (LIF) receptor and signalling through the gp130 pathway.

Animals↗

PubMed1996

Prostaglandin F2 alpha induces cardiac myocyte hypertrophy in vitro and cardiac growth in vivo.

Several prostaglandins [prostaglandin (PG) A2, -B2, -D2, -E2, -F2 alpha, and -I2 and carbaprostacyclin] and the thromboxane analogue U-46619 were analyzed for the ability to induce hypertrophy of rat neonatal cardiac ventricular myocytes. Myocyte hypertrophy was induced specifically by PGF2 alpha. Myocytes exposed to this prostanoid in culture increased in size and protein content. The contractile fibrils within the cells became organized into parallel arrays, and the cells tended to cluster and beat spontaneously. PGF2 alpha also induced the expression of c-fos, atrial natriuretic factor (ANF), and alpha-skeletal actin in these cells. The effects of PGF2 alpha were compared with several known cardiac myocyte hypertrophy factors (phenylephrine, endothelin-1, leukemia inhibitory factor, cardiotrophin-1, and angiotensin II). PGF2 alpha was found to be intermediate in potency among the factors but induced a level of ANF production that was approximately 10-fold higher than any of the other effectors. Responsiveness to PGF2 alpha was not limited to neonatal cardiocytes. Ventricular myocytes isolated from adult rats also responded specifically to PGF2 alpha with a morphological change similar to that observed with phenylephrine and by producing ANF. In rats, chronic administration of fluprostenol, a potent agonist analogue of PGF2 alpha, resulted in a dose-dependent increase in heart weight- and ventricular weight-to-body weight ratios. The amount of PGF2 alpha extractable from the hearts of rats with cardiac hypertrophy induced by myocardial infarction was also found to be greater than that in sham-operated control rats. These results indicate that PGF2 alpha may play an important role in inducing cardiac hypertrophy.

Aging↗

PubMed1996

Substitution of serine or threonine at position 473 of tissue-type plasminogen activator increases its stability in plasma.

Inactivation by slow acting inhibitors in plasma is of little consequence for thrombolysis with wild type t-PA, since it is rapidly cleared from the blood stream and constantly replenished through infusion. However, it becomes increasingly important as the clearance rate of t-PA is reduced, through mutagenesis, to enable the molecule to be long acting and administered by a single bolus injection. The substitution of serine for alanine at position 473 substantially reduced the slow inactivation that occurs at pharmacological levels of t-PA in plasma. Approximately 70% of the activity of A473S remained after 4 h incubation in human plasma compared to approximately 25% for wild type t-PA. Wild type t-PA and A473S showed the same stability in alpha-2-antiplasmin depleted plasma, indicating that the resistance of A473S to inactivation is a result of reduced reactivity towards alpha-2-antiplasmin, the primary slow acting inhibitor of t-PA. The second order rate constant for the inactivation of A473S by purified alpha-2-antiplasmin was approximately 4 fold less than that of wild type t-PA, which is consistent with the results obtained in plasma. Substitution of threonine at position 473 also produced inhibitor resistance, but glycine did not. The substitution of charged or bulky residues at position 473 destroyed enzymatic activity. The mechanism of inhibitor resistance for A473S and A473T appears to be a reduced reactivity towards substrates with arginine at the P1 position. The A473S mutation adds well to T103N, a mutation that causes an approximate 9 fold reduction in the clearance rate of t-PA. The double mutation variant, T103N, A473S had normal plasma clot lysis activity, and was stable in plasma over a 4 h incubation period at 37 degrees C in vitro.

Amino Acid Sequence↗

PubMed1996

An experimental model of intracranial hemorrhage during thrombolytic therapy with t-PA.

Multiple clinical trials have proven that thrombolytic therapy is an effective treatment for acute myocardial infarction. Spontaneous intracranial hemorrhage (ICH) occurs in a small percentage of patients as a result of the treatment. The etiology of the ICH is unknown and there is currently no established experimental model for this side effect. A model of ICH during thrombolytic therapy has been developed using spontaneously hypertensive rats (SHR). The SHR were made susceptible to ICH during thrombolytic therapy by bilateral ligation of the external jugular veins. This procedure produced asymptomatic hemorrhagic lesions in the brains of the animals in the hours preceding the administration of t-PA/heparin. The incidence of ICH following the administration of test substances was assessed by histological examination and by measuring the red blood cell count in a sample of cerebrospinal fluid taken from the atlanto-occipital space. t-PA administration produced a low frequency of ICH in this model. The incidence and severity of ICH were dramatically increased, and significant mortality at 24h was observed, by combining heparin were administered sequentially rather than simultaneously. Furthermore, ICHs were observed whether the t-PA dose was administered over 4 h, 1 h, or as a double bolus 30 min apart. The potentiation of ICH by heparin was dose dependent and proportional to the prolongation of the aPTT. Although the precise mechanism of ICH during thrombolytic therapy is unknown, many similarities exist between the observations made in this model and in the human clinical experience.

Animals↗

PubMed1995

New variant of human tissue plasminogen activator (TPA) with enhanced efficacy and lower incidence of bleeding compared with recombinant human TPA.

BACKGROUND: The thrombolytic properties of a new variant of tissue plasminogen activator (TPA) (T103N, N117Q, KHRR 296-299 AAAA, or TNK-TPA) with longer plasma half-life, greater fibrin specificity, and increased resistance to inhibition by plasminogen activator inhibitor (PAI-1) were investigated in a rabbit thrombosed carotid artery model. METHODS AND RESULTS: After 60 minutes of arterial occlusion, TPA (1.5, 3.0, 6.0, or 9.0 mg/kg as a front-loaded IV infusion for 90 minutes; n = 22) or TNK-TPA (0.38, 0.75, or 1.5 mg/kg as IV bolus; n = 16) was administered. Blood flow through the artery was monitored for an additional 120 minutes. Bleeding was assessed by weighing the amount of blood absorbed in a gauze pad placed in a subcutaneous muscular incision. Recanalization rates and duration of recanalization were dose dependent. The doses that produced > 80% recanalization rates with the longest duration of recanalization were 9.0 mg/kg for TPA and 1.5 mg/kg for TNK-TPA. At these doses, time to reperfusion (mean +/- SEM) was significantly faster (11 +/- 2 versus 23 +/- 7 minutes) and duration of recanalization longer (77 +/- 9 versus 51 +/- 18 minutes) for TNK-TPA compared with TPA (P < .025). Weights of the residual thrombi of the TPA group were greater than those of the TNK-TPA group (P = .004). Concentrations of fibrinogen, plasminogen, and alpha 2-antiplasmin at 120 minutes were significantly higher for TNK-TPA-treated animals compared with TPA-treated animals (P < .001). ANOVA of the blood loss data determined that there were significant differences between thrombolytic agents but not between doses. After correction for saline controls, total blood loss for pooled doses of TPA and TNK-TPA was 82 +/- 6 mg and 40 +/- 4 mg, respectively (P < .01). CONCLUSIONS: From these data, we conclude that TNK-TPA, given as a bolus, produces faster and more complete recanalization of occluded arteries in a rabbit experimental model compared with TPA, without increasing systemic plasmin generation or peripheral bleeding. In addition, we observed that TNK-TPA, unlike TPA, did not potentiate collagen-induced aggregation of platelets obtained from human plasma. This lack of effect on platelet aggregation by TNK-TPA potentially could be associated with a decreased risk of reocclusion after successful thrombolysis.

Animals↗

PubMed1995

Cardiotrophin-1. Biological activities and binding to the leukemia inhibitory factor receptor/gp130 signaling complex.

Cardiotrophin-1 (CT-1) is a newly isolated cytokine that was identified based on its ability to induce cardiac myocyte hypertrophy. It is a member of the family of cytokines that includes interleukins-6 and -11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor, and oncostatin M. These cytokines induce a pleiotropic set of growth and differentiation activities via receptors that use a common signaling subunit, gp130. In this work we determine the activity of CT-1 in six in vitro biological assays and examine the composition of its cell surface receptor. We find that CT-1 is inactive in stimulating the growth of the hybridoma cell line, B9 and inhibits the growth of the mouse myeloid leukemia cell line, M1. CT-1 induces a phenotypic switch in rat sympathetic neurons and promotes the survival of rat dopaminergic and chick ciliary neurons. CT-1 also inhibits the differentiation of mouse embryonic stem cells. CT-1 and LIF cross-compete for binding to M1 cells, Kd [CT-1] approximately 0.7 nM, and this binding is inhibited by an anti-gp130 monoclonal antibody. Both ligands can be specifically cross-linked to a protein on M1 cells with the mobility of the LIF receptor (approximately 200 kDa). In addition, CT-1 binds directly to a purified, soluble form of the LIF receptor in solution (Kd approximately 2 nM). These data show that CT-1 has a wide range of hematopoietic, neuronal, and developmental activities and that it can act via the LIF receptor and the gp130 signaling subunit.

Amino Acid Sequence↗

PubMed1995

Beneficial effects of growth hormone and insulin-like growth factor-1 in experimental heart failure in rats treated with chronic ACE inhibition.

The effects of growth hormone (GH) plus insulin-like growth factor-1 (IGF-1) were tested in an experimental model of cardiac failure treated with chronic angiotensin-converting enzyme (ACE) inhibition. Myocardial infarction was induced in rats by left coronary artery ligation. Two weeks after ligation, the animals received either captopril (2 g/L in drinking water) or water for 3 months. The rats were then given either GH (2 mg/kg/day) plus IGF-1 (2 mg/kg/day) or vehicle for 14 days. Captopril treatment decreased mean arterial pressure (MAP), left ventricular end-diastolic pressure (LVEDP) and systemic vascular resistance (SVR) (p < 0.05), and increased cardiac index (CI) and stroke volume index (SVI) (p < 0.05). GH/IGF-1 or captopril+GH/IGF-1 treatment decreased MAP, LVEDP, and SVR (p < 0.05), and increased left ventricular maximum dP/dt, CI, and SVI (p < 0.05). The increases in CI and SVI were significantly greater in the captopril+GH/IGF-1-treated animals than in those treated with captopril alone (p < 0.05). The beneficial effect of captopril in reducing cardiac hypertrophy was preserved in the captopril+GH/IGF-1 group. The results indicate that GH/IGF-1 and captopril can improve cardiac performance in congestive heart failure by independent and complementary mechanisms.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1994

A faster-acting and more potent form of tissue plasminogen activator.

Current treatment with tissue plasminogen activator (tPA) requires an intravenous infusion (1.5-3 h) because the clearance of tPA from the circulation is rapid (t 1/2 approximately 6 min). We have developed a tPA variant, T103N,N117Q, KHRR(296-299)AAAA (TNK-tPA) that has substantially slower in vivo clearance (1.9 vs. 16.1 ml per min per kg for tPA in rabbits) and near-normal fibrin binding and plasma clot lysis activity (87% and 82% compared with wild-type tPA). TNK-tPA exhibits 80-fold higher resistance to plasminogen activator inhibitor 1 than tPA and 14-fold enhanced relative fibrin specificity. In vitro, TNK-tPA is 10-fold more effective at conserving fibrinogen in plasma compared to tPA. Arterial venous shunt models of fibrinolysis in rabbits indicate that TNK-tPA (by bolus) induces 50% lysis in one-third the time required by tPA (by infusion). TNK-tPA is 8- and 13-fold more potent in rabbits than tPA toward whole blood clots and platelet-enriched clots, respectively. TNK-tPA conserves fibrinogen and, because of its slower clearance and normal clot lysis activity, is effective as a thrombolytic agent when given as a bolus at a relatively low dose.

Animals↗

PubMed1993

A slow clearing, fibrin-specific, PAI-1 resistant variant of t-PA (T103N, KHRR 296-299 AAAA).

Site directed mutagenesis was used to construct a t-PA variant that contains an additional glycosylation site in the first kringle domain (T103N) combined with a tetra-alanine substitution in the protease domain (KHRR 296-299 AAAA). This combination variant has a plasma clearance rate that is 4.5-fold slower in rats and 5.4-fold slower in rabbits than t-PA. It is also less than one tenth as active as t-PA towards plasminogen in the presence of fibrinogen, and has approximately twice the normal activity in the presence of fibrin. It shows substantial resistance to the fast acting inhibitor, plasminogen activator inhibitor-1 (PAI-1), requiring a 10-fold greater molar excess of PAI-1 to reduce its activity by 50%, compared to t-PA. This is the result of a reduction of nearly 100-fold in the second order rate constant for PAI-1 inactivation. These results show that it is possible to combine mutations in different domains of t-PA to construct a variant which is simultaneously slower clearing, less reactive towards plasminogen in the absence of a fibrin clot, and resistant to inactivation by PAI-1.

Animals↗

PubMed1993

A variant of t-PA (T103N, KHRR 296-299 AAAA) that, by bolus, has increased potency and decreased systemic activation of plasminogen.

In the accompanying paper, we reported that the properties of decreased plasma clearance rate, increased fibrin specificity, and resistance to inactivation by PAI-1 could be effectively combined in the t-PA variant T103N, KHRR 296-299 AAAA. In the current study we evaluated the in vivo efficacy of this variant as well as variants containing the individual mutations T103N and KHRR 296-299 AAAA. Plasma clearance and in vivo lysis of whole blood and platelet-rich clots were determined in a rabbit arterio-venous shunt model. The T103N containing variants were administered as an intravenous (i.v.) bolus. KHRR 296-299 AAAA and t-PA were infused i.v. over 90 min. The clearance rate of the KHRR 296-299 AAAA variant was similar to t-PA. However, the clearance of the T103N and T103N, KHRR 296-299 AAAA variants were 8 and 6-fold reduced, respectively. Potency of the variants relative to t-PA on whole blood clots ranged from 0.9 (T103N, KHRR 296-299 AAAA) to 1.7 (T103N). Relative potency on platelet-rich clots ranged from 2.4 (T103N) to 4.2 (T103N, KHRR 296-299 AAAA). Fibrinogen concentrations in rabbits 120 min after dosing with a 2.5 mg/kg bolus were: 24, 16, 82, and 77% of initial for t-PA; T103N; KHRR 296-299 AAAA; and T103N, KHRR 296-299 AAAA treatment groups, respectively. These results suggest that the T103N, KHRR 296-299 AAAA variant of t-PA, given as a bolus, could result in greater efficacy, particularly on refractory platelet-rich clots, without inducing the severe systemic lytic state produced by a bolus of a less fibrin specific variant.

Animals↗

PubMed1993

Making tissue-type plasminogen activator more fibrin specific.

The fibrin specificity of tissue-type plasminogen activator can be increased by mutagenesis within at least four sites in the protease domain. These sites include residue I276, the new N-terminus formed by conversion to a two-chain structure, residues on either side of the active site cleft, KHRR 296-299 or DDD 364-366, a charged surface involved in fibrin interactions, which includes residues H432, R434, D460, R462 and a loop structure, PQANL 466-470, near the fibrin-binding patch. Variants with mutations at any of these sites have low fibrinogen-stimulated activity, whereas fibrin-stimulated activity is at least normal. Kinetic analysis reveals that mutations at these positions reduce the kcat in the presence of fibrinogen, but leave the molecules with normal kinetic constants in the presence of fibrin. A significant exception is found at positions 296-299, where the presence of fibrin manifests significant increases in both kcat and Km. Combinations of mutations at these sites appear to be additive with respect to fibrin specificity.

Amino Acid Sequence↗

PubMed1992

Involvement of residues 296-299 in the enzymatic activity of tissue-type plasminogen activator.

The tetra-alanine substitution variant KHRR 296-299 AAAA of tissue-type plasminogen activator (t-PA) was previously shown to have enhanced fibrin specificity and enhanced activity in the presence of fibrin compared with the wild-type form of the molecule. The structural requirements for these alterations in enzymatic activity were investigated by constructing several amino acid substitution variants at each of the positions from 296 to 299 and evaluating their activities under a variety of conditions. Effects on plasminogen activator activity were common among the point mutants at positions 296-299; nearly all had a phenotype similar to the KHRR 296-299 AAAA variant. The greatest effects on enzymatic function were found with multiple substitution variants, but some single charge reversals and proline substitutions had substantial effects. The enhanced fibrin specificity of KHRR 296-299 AAAA t-PA results in less fibrinogenolysis than seen with wild-type t-PA. Approximately four times greater concentration of KHRR 296-299 AAAA compared with wild-type t-PA was required to consume 50% of the fibrinogen in human plasma.

Alanine↗

PubMed1991

High resolution analysis of functional determinants on human tissue-type plasminogen activator.

Sixty-four variants of human tissue-type plasminogen activator (tPA) were produced using recombinant DNA techniques. Charged residues were converted to alanine in clusters of from one to four changes per variant; these clusters spanned all the domains of the molecule. The variants were expressed by mammalian cells and were analyzed for a variety of properties. Variants of tPA were found that had reduced activity with respect to each tested property; in a few cases increased activity was observed. Analysis of these effects prompted the following conclusions: 1) charged residues in the nonprotease domains are less involved in fibrin stimulation of tPA activity than those in the protease domain, and it is possible to increase the fibrin specificity (i.e. the stimulation of tPA activity by fibrin compared to fibrinogen) by mutations at several sites in the protease domain; 2) the difference in enzymatic activity between the one- and two-chain forms of tPA can be increased by mutations at several sites on the protease domain; 3) binding of tPA to lysine-Sepharose was affected only by mutations to kringle-2, whereas binding to fibrin was affected most by mutations in the other domains; 4) clot lysis was influenced by mutations in all domains except kringle-2; 5) sensitivity to plasminogen activator inhibitor-1 seems to reside exclusively in the region surrounding residue 300. A model of the tPA protease domain has been used to map some of the critical residues and regions.

Amino Acid Sequence↗

PubMed1984

Improved method for detection of glycosidases in bacterial colonies.

An assay has been developed to detect bacterial glycosidases in colonies grown on the surface of agar plates. Advantages of this technique over previously described methods include elimination of the need for replica plating, better visualization of chromagenic reaction products, and a simple permeabilization step to enable better penetration by chromagenic substrates.

Animals↗