HopX1 in Erwinia amylovora functions as an avirulence protein in apple and is regulated by HrpL.

Fire blight is a devastating disease of rosaceous plants caused by the Gram-negative bacterium Erwinia amylovora. This pathogen delivers virulence proteins into host cells utilizing the type III secretion system (T3SS). Expression of the T3SS and of translocated and secreted substrates is activated by the alternative sigma factor HrpL, which recognizes hrp box promoters upstream of regulated genes. A collection of hidden Markov model (HMM) profiles was used to identify putative hrp boxes in the genome sequence of Ea273, a highly virulent strain of E. amylovora. Among potential virulence factors preceded by putative hrp boxes, two genes previously known as Eop3 and Eop2 were characterized. The presence of functionally active hrp boxes upstream of these two genes was confirmed by ß-glucuronidase (GUS) assays. Deletion mutants of the latter candidate genes, renamed hopX1(Ea) and hopAK1(Ea), respectively, did not differ in virulence from the wild-type strain when assayed in pear fruit and apple shoots. The hopX1(Ea) deletion mutant of Ea273, complemented with a plasmid overexpressing hopX1(E)(a), suppressed the development of the hypersensitivity response (HR) when inoculated into Nicotiana benthamiana; however, it contributed to HR in Nicotiana tabacum and significantly reduced the progress of disease in apple shoots, suggesting that HopX1(Ea) may act as an avirulence protein in apple shoots.

Effect of fatty acid chain length and thioesterification on the augmentation of expression of plasminogen activator inhibitor-1.

BACKGROUND AND AIM: Concentrations in blood of plasminogen activator inhibitor type 1 (PAI-1) and circulating free (non-esterified) fatty acids (FFA) are increased in diabetes and may accelerate atherosclerosis. We have shown that FFA increase expression of PAI-1 by activation of a transcription factor that binds to the repeated sequence 5'-TG(G/C) 1-2CTG-3'. This study was designed to determine whether FFA chain length, saturation, or both affect agonist properties and whether agonist properties are mediated by activated, thioesterified FFA (fatty acyl-CoA). METHODS AND RESULTS: Human hepatoma cells were exposed to selected FFA associated with bovine serum albumin (BSA). Triacsin C (5 microM) was used to inhibit production of fatty acyl-CoA. PAI-1 was assayed by enzyme linked immunosorbent assay. Maximal induction of PAI-1 was similar with medium and long chain FFA (fold induction of PAI-1 accumulated in conditioned media compared with control: C10 = 1.8 +/- 0.1, C12 = 2.0 +/- 0.1, C14 = 2.0 +/- 0.2, C16 = 1.4 +/- 0.1, C18 = 1.6 +/- 0.1, C20 = 1.32 +/- 0.1, p < 0.005 for each compared with control). Increased unsaturation did not alter the agonist properties of FFA (fold induction with C16: 0 = 1.4 +/- 0.1, C16: 1 = 1.4 +/- 0.1; C18: 0 = 1.6 +/- 0.1, C18: 1 = 1.5 +/- 0.1, C18: 2 = 1.6 +/- 0.1, C18: 3 = 1.4 +/- 0.1 and C20: 4 = 1.3 +/- 0.1, C20: 5 = 1.4 +/- 0.1, n = 6). However, maximal effects were seen with lower concentrations of longer chain FFA. Triacsin C consistently attenuated effects of FFA. CONCLUSIONS: Longer chain FFA exhibit maximal effects at lower concentrations. Augmented expression of PAI-1 is mediated by the fatty acyl-CoA derivative. These criteria identify targets for therapy designed to normalize expression of PAI-1 and retard progression of atherosclerosis in subjects with elevated concentrations of FFA in blood including those with insulin resistance.

Platelet reactivity in coronary ostial blood: a reflection of the thrombotic state accompanying plaque rupture and of the adequacy of anti-thrombotic therapy.

BACKGROUND: Optimal anti-thrombotic therapy for acute coronary syndromes (ACS) should suppress pro-thrombotic activity at the site of plaque rupture. We sought to determine whether platelet reactivity is increased in blood in the immediate vicinity of a ruptured plaque and is apparent even when blood is obtained by sampling from a catheter placed proximal to the lesion. METHODS: Blood was obtained from a catheter placed in the aorta and from the same catheter after engaging the culprit coronary artery. Platelet reactivity was determined with the use of flow cytometry by surface expression of P-selectin. RESULTS: In preliminary studies we demonstrated that a marker of thrombin activity, fibrinopeptide A, was similarly increased in blood taken from the coronary sinus and coronary arterial ostium of patients with ACS. Subsequently blood was obtained from the aorta and coronary arterial ostium through a coronary guide catheter for assessment of platelet reactivity in 23 subjects with ACS and 22 subjects with stable angina. The percentage of platelets expressing P-selectin in response to 0.2 microM adenosine diphosphate (ADP) was greater in coronary arterial samples from patients with ACS (aorta=6.1+/-1%, coronary artery=8.8+/-1.6%, p=0.02) compared with that in patients with stable symptoms (aorta=6.9+/-1.2, coronary artery=6.5+/-1.4, p=NS). CONCLUSIONS: Coronary arterial blood obtained from the ostium through a coronary guide catheter can be used to determine whether thrombin activity and platelet reactivity are increased in the immediate vicinity of a ruptured atherosclerotic plaque. The simplicity of the approach developed should facilitate its use in future studies designed to determine the impact of optimal suppression of platelet reactivity and the pro-thrombotic state before coronary interventions on short- and long-term clinical outcomes.

Platelet reactivity characterized prospectively: a determinant of outcome 90 days after percutaneous coronary intervention.

BACKGROUND: Platelet activation is pivotal in the pathogenesis of complications after percutaneous coronary interventions (PCI). We previously reported substantial interindividual variability in activation of glycoprotein (GP) IIb/IIIa in response to a low concentration of ADP. We assessed GP IIb/IIIa activation prospectively to determine whether this could differentiate patients at low risk from those at high risk for complications early and late after PCI. Methods and Results-- A total of 112 patients undergoing PCI were studied. Platelet reactivity was determined with the use of flow cytometry. Patients were classified into high and low platelet reactivity groups on the basis of extent of activation of GP IIb/IIIa in response to 0.2 micromol/L ADP. The median value was used for differentiation. The incidence during 90-day follow-up interval of a composite end point (myocardial infarction, urgent revascularization, or repeat revascularization) was determined in each group. Follow up was completed in all 112 patients. The 2 groups were similar with respect to diverse clinical characteristics. Nevertheless, the incidence of the composite end point occurred in 26.8% of the high and 7.1% in the low platelet reactivity group (P=0.01). The difference in the composite end point was most striking during the 30- to 90-day interval after PCI (16.7% versus 1.9%; P=0.02). Repeat revascularization was more frequent in those with increased platelet reactivity (17.9% versus with 3.6%, P=0.029). CONCLUSIONS: Prospective assessment of platelet GP IIb/IIIa activation permits stratification of patients into low- and high-risk groups with respect to adverse events after PCI.

Identification and localization of a fatty acid response region in the human plasminogen activator inhibitor-1 gene.

The increased expression of plasminogen activator inhibitor type-1 (PAI-1) is associated with increased concentrations of fatty acids in blood and may accelerate atherogenesis in diabetes. The present study was designed to define mechanisms by which nonesterified (free) fatty acids (FFAs) augment the expression of PAI-1. FFAs increased PAI-1 protein and mRNA expression by HepG2 cells. To identify potential regulatory elements, we constructed chimeric genes by fusing 1313, 853, 610, or 328 bp of human PAI-1 5'-flanking DNA to a luciferase reporter (PAI-LUC). A 2-fold increase in luciferase activity was seen when cells were transfected with PAI-LUC 1313, 863, or 610 and exposed to FFAs. No response to FFAs was seen with PAI-LUC 328 and after deletion of a 72-bp (-599 to -528) fragment from PAI-LUC 1313. This 72-bp fragment conferred FFA responsiveness to a different (simian virus 40) promoter. Two footprinted regions were demonstrated by DNase I analysis. Gel mobility shift assays indicated specific binding of extracted proteins to an FFA response element: 5'-TG(G/C)(1-2)CTG-3'. This sequence is repeated 4 times and is similar to an Sp1-binding site. Sp1 consensus oligonucleotides inhibited binding of extracted proteins to the regulatory elements. Accordingly, FFA-induced increased expression of PAI-1 in HepG2 cells is mediated by the binding of a transcription factor or factors to the repeated fatty acid response element, 5'-TG(G/C)(1-2)CTG-3', that is highly homologous to an Sp1-binding site.

The consequences of anterior femoral notching in total knee arthroplasty. A biomechanical study.

BACKGROUND: Notching of the anterior femoral cortex during total knee arthroplasty has been implicated as a cause of subsequent periprosthetic supracondylar femoral fracture. However, other than observational clinical data, no reliable association between these events has been established, to our knowledge. The purpose of the present study was to investigate the biomechanical effects of notching of the anterior femoral cortex. METHODS: The femoral component of a total knee replacement was implanted in twelve matched pairs of human cadaveric femora; one specimen in each pair had preservation of the anterior femoral cortex, and the other had a full-thickness cortical defect created just proximal to the anterior flange of the femoral component. The pairs were then subjected to either bending or torsional loading to failure. Both the fracture pattern and the quantitative load to failure were analyzed. Two matched pairs were excluded from the analysis because of inadvertent fracture during placement of the component. RESULTS: Following the application of a bending load, femora with notching of the anterior femoral cortex sustained a short oblique fracture that originated at the cortical defect proximal to the femoral component and femora without notching had a midshaft fracture. In contrast, notching of the anterior femoral cortex had no effect on the fracture pattern that was observed after the application of a torsional load. The mean load to failure was significantly reduced by notching in both testing modes. Notching decreased bending strength from 11,813 to 9690 newtons (18 percent; p = 0.0034), and it decreased torsional strength from 134.7 to 81.8 newton-meters (39.2 percent; p = 0.01). CONCLUSIONS: Biomechanical testing demonstrated that notching of the anterior femoral cortex significantly lessens the load to failure following total knee arthroplasty and influences the subsequent fracture pattern. These effects are manifested in different ways under the two loading conditions: the fracture pattern is altered under bending load, and there is a greater quantitative decrease in load to failure with torsional loading. CLINICAL RELEVANCE: Weakening of the femur by notching of the anterior cortex after total knee arthroplasty may warrant alteration in the customary postoperative regimen for these patients. Manipulation of a total knee replacement with a notched anterior femoral cortex should probably be avoided.

Chondronecrosis of the hip. The protective role of the ossific nucleus in an animal model.

The presence of the ossific nucleus and its role in reducing the risk of ischemic necrosis in developmental dysplasia of the hip remains a matter of controversy. Ischemic necrosis of the pre-osseous capital femoral epiphysis, defined as chondronecrosis, was evaluated in a rabbit model. Histologic evidence of chondronecrosis after casting in maximum abduction was greater in 1-day-old New Zealand White rabbits before the radiographic appearance of the ossific nucleus, compared with 16-day-old New Zealand White rabbits with an ossific nucleus already present. This preliminary study supports the hypothesis that the ossific nucleus may decrease the risk of intracapsular compressive ischemic injury to the developing capital femoral epiphysis in a rabbit model.

Paradoxical inhibition of fibrinogen binding and potentiation of alpha-granule release by specific types of inhibitors of glycoprotein IIb-IIIa.

OBJECTIVE: To determine whether glycoprotein (GP) IIb-IIIa inhibitors can paradoxically augment activation of platelets, activation of GP IIb-IIIa, alpha-granule degranulation, and lysosome release were induced after exposure of platelets to GP IIb-IIIa inhibitors. METHODS: ADP-induced platelet activation was assessed after exposure of platelets to Abciximab, or to a non-peptide ligand, the free acid of Orbofiban (Orbofiban(a)). Activation of GP IIb-IIIa was detected based on binding of fluorochrome labeled fibrinogen or a labeled monoclonal antibody, PAC-1. alpha-Granule degranulation was detected based on surface expression of P-selectin and lysosome release was detected based on surface expression of CD63. RESULTS: Despite significant inter-individual variability in inhibition of fibrinogen binding in response to each of the GP IIb-IIIa inhibitors used, a concentration dependent decrease in fibrinogen binding was seen with each agent in samples from each subject. Binding of PAC-1 was inhibited in a parallel manner. Abciximab increased ADP-induced P-selectin expression. Orbofiban(a) did not alter ADP-induced P-selectin expression. Neither agent altered ADP-induced CD63 expression. When platelets were exposed to Abciximab and Orbofiban(a), both Abciximab and Orbofiban(a) were found in the alpha-granules (by confocal microscopy), consistent with potentiation of agonist-induced release of alpha-granular products associated with uptake of proteins. CONCLUSIONS: Specific types of GP IIb-IIIa inhibitors can paradoxically augment agonist-induced release of alpha-granules despite inhibiting agonist-induced fibrinogen binding.

Differences between activation thresholds for platelet P-selectin glycoprotein IIb-IIIa expression and their clinical implications.

Increased platelet reactivity is a descriptor of the risk of cardiovascular events in healthy men and in patients with overt coronary artery disease. We sought to determine if differential thresholds exist for activation of platelets with respect to alpha-granule degranulation and fibrinogen binding in healthy volunteers and in patients with acute coronary syndromes. We also sought to characterize the effect of aspirin on activation. Platelet activation was assessed with flow cytometry in whole blood anticoagulated with corn trypsin inhibitor and incubated with fluorescein isothiocyanate conjugated fibrinogen (to define activation of glycoprotein IIb-IIIa), a phycoerythrin conjugated antibody to P-selectin (a marker of alpha-granule degranulation), and selected concentrations of adenosine diphosphate (ADP) or thrombin receptor agonist peptide. ADP-induced fibrinogen binding was found to be a low threshold activation event (40% of platelets bound fibrinogen in response to 0.2 microM ADP). Alpha-granule degranulation was a higher threshold event (33% of platelets expressed P-selectin in response to 1.0 microM ADP). Intra- and interindividual variability were most apparent with low concentrations of agonist (0.2 microM ADP). Patients with acute coronary syndromes (on aspirin) had significantly increased P-selectin expression in response to ADP compared with healthy subjects (on aspirin), but no difference in ADP-induced fibrinogen binding was observed. Daily ingestion of 325 mg of aspirin had no effect on either P-selectin expression or fibrinogen binding in healthy subjects. Analysis of platelet reactivity with flow cytometry characterizes activation with respect to specific components of the process and should facilitate development and optimal titration of antiplatelet therapy.

The contribution of the ossific nucleus to the structural stiffness of the capital femoral epiphysis: a porcine model for DDH.

The preosseous femoral head is thought to be vulnerable to compressive ischemic injury during the treatment of developmental dysplasia of the hip. The ossific nucleus has been proposed to increase the mechanical strength of the capital femoral epiphysis (CFE) and to decrease the risk of avascular necrosis. Sixty mixed-breed fetal and postgestational femoral head specimens were evaluated for structural stiffness in relation to the size of the ossific nucleus within the CFE. The structural stiffness of the CFE in the porcine model was found to increase exponentially with the size of the ossific nucleus. A finite-element model revealed that the presence of an ossific nucleus occupying 40% of the epiphyseal volume reduced the compressive strain in the region of the posterior-superior branch of the medial circumflex artery by an average of 54%. The results of this study support the hypothesis that the presence of the ossific nucleus may protect the CFE from compressive ischemic injury in the treatment of DDH.

Variable responses to inhibition of fibrinogen binding induced by tirofiban and eptifibatide in blood from healthy subjects.

Antiplatelet therapy with glycoprotein IIb-IIIa inhibitors reduces the incidence of cardiac events in patients with acute coronary syndromes. A lack of universal efficacy may result from interindividual variation in the inhibition of fibrinogen binding after exposure to tirofiban and eptifibatide. Accordingly, accurate monitoring of platelet function in individual subjects may be needed. To assess this possibility, blood was drawn from 15 healthy volunteers into syringes containing corn trypsin inhibitor (an anticoagulant that is a specific inhibitor of factor XIIa) and selected concentrations of tirofiban and eptifibatide. Platelets were then activated with adenosine diphosphate (ADP) and thrombin receptor agonist peptide. Flow cytometry was used to assess activation with respect to glycoprotein IIb-IIIa activation as reflected by fibrinogen binding and alpha-granule degranulation as reflected by P-selectin expression. In platelets activated with 1 microM ADP, clinically relevant concentrations of tirofiban caused inhibition of fibrinogen binding ranging from 17% to 88%. Similarly, eptifibatide caused inhibition of fibrinogen binding ranging from 32% to 74%. The highest concentration of eptifibatide tested enhanced agonist-induced degranulation, an effect not seen with tirofiban at concentrations tested. Flow cytometry in minimally altered whole blood can discriminate variation in the response to glycoprotein IIb-IIIa inhibitors with respect to specific components of platelet activation. Thus, the approach developed should facilitate definition of optimal platelet inhibition and individualized tailoring of therapy to induce optimal effects.

The lack of augmentation by aspirin of inhibition of platelet reactivity by ticlopidine.

A decreased threshold for platelet activation apparently contributes to the risk of cardiovascular events, such as acute myocardial infarction. To evaluate the impact of specific agents, we characterized platelet reactivity in 9 healthy subjects before and after 5 days of ingestion of 4 commonly prescribed regimens, 81 mg of aspirin daily, 325 mg of aspirin daily, ticlopidine 250 mg twice daily, and ticlopidine plus 325 mg of aspirin daily. Platelet reactivity was assessed with (1) aggregometry induced by 4 microM adenosine diphosphate (ADP) and collagen (0.19 mg/ml) and performed in platelet-rich plasma; and (2) flow cytometric determination of ADP-induced (0.2, 0.8, and 1.5 microM) P-selectin expression in whole blood. Because anticoagulants alter platelet reactivity, results were obtained with 3 anticoagulants, citrate, enoxaparin, or corn trypsin inhibitor (CTI, a specific inhibitor of factor XIIa without effect on other coagulation factors). Ingestion of aspirin did not alter platelet activation as assessed with flow cytometry. Inhibition of the second phase of aggregation was seen with ADP-induced aggregation in platelet-rich plasma anticoagulated with citrate but not enoxaparin or CTI. Ingestion of ticlopidine led to inhibition of ADP-induced aggregation and P-selectin expression. Inhibition of platelet reactivity after the combination of aspirin and ticlopidine did not differ from ticlopidine alone. Marked interindividual variability in platelet reactivity was seen after ingestion of ticlopidine. The results indicate that assessment of effects of specific pharmacologic regimens with accurate and readily available assays of platelet reactivity may facilitate effective prophylaxis and treatment of high-risk subjects with antiplatelet regimens designed to optimally diminish platelet reactivity.

Increased reactivity of platelets induced by fibrinogen independent of its binding to the IIb-IIIa surface glycoprotein: a potential contributor to cardiovascular risk.

OBJECTIVES: To determine whether augmented activation (degranulation) of platelets might contribute to the association between higher concentrations of fibrinogen and risk of myocardial infarction, we characterized adenosine diphosphate (ADP)-induced expression of P-selectin by platelets in whole blood as a function of this exposure to selected concentrations of fibrinogen. BACKGROUND: An increased risk of myocardial infarction has been associated with increased concentrations of fibrinogen. METHODS: Fibrinogen was added to blood anticoagulated with corn trypsin inhibitor (a specific inhibitor of Factor XIIa without effect on other coagulation factors). Degranulation of platelets was identified by flow cytometry. RESULTS: Addition of fibrinogen to blood did not activate platelets under basal conditions (without ADP). By contrast, a concentration-dependent increase in ADP and thrombin receptor agonist peptide (TRAP)-induced activation occurred with increasing concentrations of fibrinogen. Increased ADP-induced degranulation was apparent with the addition of 100 mg/dl of fibrinogen (p < or = 0.001 for 1.5 micromol/liter ADP, n=10 subjects). Inhibition by abciximab of binding of fibrinogen to the surface glycoprotein IIb-IIIa did not attenuate the observed augmentation of reactivity induced by fibrinogen. Augmented degranulation was associated with uptake of fibrinogen into alpha-granules without surface binding despite pretreatment with abciximab as shown by laser scanning confocal microscopy. CONCLUSIONS: Fibrinogen in blood augments degranulation of platelets in response to ADP and is accompanied by uptake of fibrinogen into alpha-granules. Thus, elevated concentrations of fibrinogen secondary to inflammation implicated in cardiovascular risk may operate, in part, by increasing reactivity of platelets.

Homograft replacement of mitral valve in children.

BACKGROUND: Recent reports have demonstrated successful early outcomes using mitral valve homografts in adults. We report our early results after homograft mitral valve replacement in 4 children with previous atrioventricular septal defects, previous placement of a prosthetic valve, and rheumatic valvular disease. METHODS: Between May 1996 and June 1997, 4 children (ages 5, 11, 13, and 15 years) underwent mitral valve replacement with cryopreserved mitral valve homografts at our institution. Preoperative echocardiography confirmed moderately severe to severe mitral regurgitation, stenosis, or both in all 4 patients. RESULTS: Successful homograft valve replacement was achieved in all 4 patients. Based on symptoms, physical examinations, and echocardiographic follow-up, all four homograft mitral valves are functioning well with normal hemodynamics. None of these patients are receiving warfarin. Follow-up has been limited to 10 months. CONCLUSIONS: In children requiring mitral valve replacement, the use of mitral valve homografts offers advantages over prosthetic valves, such as the avoidance of complications associated with thrombosis and anticoagulation. Homograft mitral valve replacement is technically feasible in children with congenital and rheumatic heart disease and previous prosthetic valves.

Acute coronary syndromes: 2. Antiplatelet agents.

Therapies with novel antiplatelet agents and anticoagulants are the focus of current research. When used separately or in combination, these agents prevent generation of thrombin by activated platelets. The new therapies, in conjunction with judicious use of fibrinolytic drugs and mechanical interventions, are revolutionizing the management of patients with acute coronary syndromes.

The Frank Stinchfield Award. Inhibition of heterotopic ossification with radiation therapy in an animal model.

An animal model for the study of heterotopic ossification was developed and the effects of perioperative radiation were analyzed. In Phase I, New Zealand White rabbits (n = 18) underwent surgery either with or without muscle injury on each hip to establish the most reliable model in which to study heterotopic ossification. In Phase II, rabbits (n = 36) underwent either 400, 800, or 1200 cGy radiation to one hip 24 hours after bilateral hip surgery to establish a dose response relationship for postoperative radiation therapy. In Phase III, rabbits (n = 24) underwent preoperative radiation therapy (800 cGy) at 4, 16, or 24 hours preoperatively to investigate the mechanism of action and efficacy of preoperative radiation therapy. Monthly radiographs were graded by blinded observers for severity of heterotopic ossification. Mean grade, intraobserver and interobserver variability, and statistical significance were evaluated. In Phase II, 17 of 18 rabbits generated heterotopic ossification in both hips, and the mean grade of heterotopic ossification was always greater on the operative side with intentional muscle injury. Variability in the grading was considered excellent. Phase II revealed that 800 cGy was the minimal effective dose. Contrary to hypothesis, Phase III revealed an increasing grade of heterotopic ossification coinciding with a decreasing preoperative time interval, with the difference in heterotopic ossification grade with 24-hour versus 4-hour preoperative radiation being significant. The rabbit model is reliable and reproducible and closely resembles the human clinical situation after hip surgery. Preoperative and postoperative radiation effectively prevented heterotopic ossification formation. The results support the use of preoperative radiation and establish a need for additional investigation regarding the mechanism of action and timing of preoperative radiation therapy.

Differential effects of anticoagulants on the activation of platelets ex vivo.

BACKGROUND: Because activation of platelets and of the coagulation system are interdependent mediators of thrombosis, platelet activation was characterized in whole blood in the presence of anticoagulants used to assess platelet function in vitro or as treatment for patients with occlusive arterial disease. METHODS AND RESULTS: Blood was anticoagulated alone or in combination with citrate, ethylenediaminetetraacetatic acid, corn trypsin inhibitor (CTI, an inhibitor of activated factor XII), heparin, enoxaparin, recombinant tick anticoagulant peptide (rTAP), or recombinant hirudin. Platelet activation in response to adenosine diphosphate (ADP) or collagen was detected by assay of P-selectin on the platelet surface delineated by flow cytometry. Although minimal activation was seen without ADP, the fraction of platelets expressing P-selectin in response to ADP was greatest in blood anticoagulated with citrate compared with CTI and all other anticoagulants. ADP-induced platelet activation was greater in blood anticoagulated with heparin compared with an equipotent anti-Xa concentration of enoxaparin. More variable results were seen with collagen, but platelet activation in the presence of citrate was greater than that with CTI. CONCLUSIONS: Interpretation of assays of inhibition of platelet activation by potentially therapeutic agents in vitro requires consideration of the effects of anticoagulants used. In addition, anticoagulants other than standard heparin may potentiate efficacy of antiplatelet drugs.

Altered expression of troponin T isoforms in mild left ventricular hypertrophy in the rabbit.

Alterations in troponin T (TnT) isoforms have been reported in severe human and experimental heart failure (HF), and may play a role in the depressed myofibrillar ATPase activity observed in this condition. It is unclear whether these alterations reflect very severe hemodynamic derangement or are a component of mild hypertrophic stress. Therefore, we studied the expression of TnT isoforms (SDS-PAGE, Western blots), myosin isoforms, myofibrillar ATPase activity, and left ventricular (LV) mechanoenergetics (rbc perfused, isovolumically contracting isolated heart) in a rabbit model of mild hypertrophy (LVH) due to gradual hypertension caused by 12 weeks of cellophane wrap of the kidneys (n=12). LV/body weight ratio increased by 28% in LVH compared to shams (P<0.001); no animals had evidence of HF. In LVH, the percentage of TnT2 was modestly but significantly increased compared to shams [6.2+/-1.9 (+/-S.D. ) v 3.7+/-1.0%, P<0.05], mainly as a consequence of a parallel decrease in TnT4 (P=0.07). Sham hearts ranged from 75-100% V3 isomyosin, whereas all LVH hearts had 100% of the V3 form. There were no significant differences in myofibrillar ATPase activity or mechanical variables, including contraction and relaxation rates. The slope of the VO2-pressure-volume-area relation (a measure of the energy conversion efficiency of the contractile machinery) was also unchanged. We conclude that in the rabbit, shifts in TnT isoforms toward a more "fetal" pattern occur during mild LVH and, therefore, are likely to be a general feature of the response to hemodynamic stress, rather than a phenomenon confined to end-stage disease. These modest shifts are not associated with major alterations in LV myofibrillar ATPase activity or mechanoenergetics.

Troglitazone improves defects in insulin action, insulin secretion, ovarian steroidogenesis, and fibrinolysis in women with polycystic ovary syndrome.

Women with polycystic ovary syndrome (PCOS) are characterized by defects in insulin action, insulin secretion, ovarian steroidogenesis, and fibrinolysis. We administered the insulin-sensitizing agent troglitazone to 13 obese women with PCOS and impaired glucose tolerance to determine whether attenuation of hyperinsulinemia ameliorates these defects. All subjects had oligomenorrhea, hirsutism, polycystic ovaries, and hyperandrogenemia. Before and after treatment with troglitazone (400 mg daily for 12 weeks), all had 1) a GnRH agonist (leuprolide) test, 2) a 75-g oral glucose tolerance test, 3) a frequently sampled iv glucose tolerance test to determine the insulin sensitivity index and the acute insulin response to glucose, 4) an oscillatory glucose infusion to assess the ability of the beta-cell to entrain to glucose as quantitated by the normalized spectral power for the insulin secretion rate, and 5) measures of fibrinolytic capacity [plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator]. There was no change in body mass index (39.9 +/- 1.4 vs. 40.2 +/- 1.4 kg/m2) or body fat distribution after treatment. Both the fasting (91 +/- 3 vs. 103 +/- 3 mg/dL; P < 0.001) and 2 h (146 +/- 8 vs. 171 +/- 6 mg/dL; P < 0.02) plasma glucose concentrations during the oral glucose tolerance test declined significantly. There was a concordant reduction in glycosylated hemoglobin to 5.7 +/- 0.1 from a pretreatment level of 6.1 +/- 0.1% (P < 0.03). Insulin sensitivity increased from 0.58 +/- 0.14 to 0.95 +/- 0.26 10(-5) min-1/pmol.L (P < 0.01) after treatment as did the disposition index (745 +/- 135 vs. 381 +/- 96; P < 0.05). The ability of the beta-cell to appropriately detect and respond to an oscillatory glucose infusion improved significantly after troglitazone treatment; the normalized spectral power for the insulin secretion rate increased to 5.9 +/- 1.1 from 4.3 +/- 0.8 (P < 0.05). Basal levels of total testosterone (109.3 +/- 15.2 vs. 79.4 +/- 9.8 ng/dL; P < 0.05) and free testosterone (33.3 +/- 4.0 vs. 21.2 +/- 2.6 pg/mL; P < 0.01) declined significantly after troglitazone treatment. Leuprolide-stimulated levels of 17-hydroxyprogesterone, androstenedione, and total testosterone were significantly lower posttreatment compared to pretreatment. The reduction in androgen levels occurred independently of any changes in gonadotropin levels. A decreased functional activity of PAI-1 in blood (from 12.7 +/- 2.8 to 6.3 +/- 1.4 AU/mL P < 0.05) was associated with a decreased concentration of PAI-1 protein (from 64.9 +/- 9.1 to 44.8 +/- 6.1 ng/mL; P < 0.05). No change in the functional activity of tissue plasminogen activator (from 5.3 +/- 0.4 to 5.1 +/- 0.5 IU/mL) was observed despite a decrease in its concentration (from 9.6 +/- 0.9 to 8.2 +/- 0.7 ng/mL; P < 0.05). The marked reduction in PAI-1 could be expected to improve the fibrinolytic response to thrombosis in these subjects. We conclude that administration of troglitazone to women with PCOS and impaired glucose tolerance ameliorates the metabolic and hormonal derangements characteristic of the syndrome. Troglitazone holds potential as a useful primary or adjunctive treatment for women with PCOS.