Evidence for prothrombotic effects of exercise and limited protection by aspirin.
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BACKGROUND: Exercise may activate platelets and leukocytes and promote thrombosis. The effects of aspirin treatment on the prothrombotic effects of exercise have not been established. METHODS AND RESULTS: A total of 15 healthy men performed exhaustive exercise without and with 1 week of pretreatment with aspirin (500 mg/day). Before and immediately after exercise, platelet aggregability ex vivo was measured by filtragometry, and venous blood samples were obtained. Whole-blood flow cytometry was used to determine platelet and leukocyte activation and platelet-leukocyte aggregates. Exercise increased platelet P-selectin expression, CD11b expression in neutrophils and lymphocytes, and platelet and leukocyte responses to thrombin, ADP, platelet activating factor, and N-formyl-methionyl-leucyl-phenylalanine (fMLP) in vitro. Consistent with enhanced platelet and leukocyte activation, more circulating platelet-platelet and platelet-leukocyte aggregates were detected after exercise (P<0.001 for both). Filtragometry readings were shortened, and plasma soluble P-selectin and prothrombin fragment 1+2 were elevated. Aspirin markedly reduced the urinary excretion of 11-dehydrothromboxane B(2), decreased P-selectin expression in single platelets at rest (P<0.05), and inhibited fMLP-induced neutrophil CD11b expression, but it did not attenuate exercise-induced increases in platelet aggregability, platelet P-selectin expression, leukocyte CD11b expression, platelet-leukocyte aggregate formation, soluble P-selectin, or prothrombin fragment 1+2. CONCLUSIONS: Exercise induced platelet and leukocyte activation and platelet-leukocyte aggregation in vivo, and it increased platelet and leukocyte responsiveness to in vitro stimulation. Aspirin treatment attenuated certain signs of platelet activity in vivo at rest and fMLP-induced neutrophil activation in vitro, but it did not attenuate the prothrombotic effects of exercise. (+info)
Microarray analysis of replicative senescence.
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BACKGROUND: Limited replicative capacity is a defining characteristic of most normal human cells and culminates in senescence, an arrested state in which cells remain viable but display an altered pattern of gene and protein expression. To survey widely the alterations in gene expression, we have developed a DNA microarray analysis system that contains genes previously reported to be involved in aging, as well as those involved in many of the major biochemical signaling pathways. RESULTS: Senescence-associated gene expression was assessed in three cell types: dermal fibroblasts, retinal pigment epithelial cells, and vascular endothelial cells. Fibroblasts demonstrated a strong inflammatory-type response, but shared limited overlap in senescent gene expression patterns with the other two cell types. The characteristics of the senescence response were highly cell-type specific. A comparison of early- and late-passage cells stimulated with serum showed specific deficits in the early and mid G1 response of senescent cells. Several genes that are constitutively overexpressed in senescent fibroblasts are regulated during the cell cycle in early-passage cells, suggesting that senescent cells are locked in an activated state that mimics the early remodeling phase of wound repair. CONCLUSIONS: Replicative senescence triggers mRNA expression patterns that vary widely and cell lineage strongly influences these patterns. In fibroblasts, the senescent state mimics inflammatory wound repair processes and, as such, senescent cells may contribute to chronic wound pathologies. (+info)
MAP kinase cascade is required for p27 downregulation and S phase entry in fibroblasts and epithelial cells.
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The present report delineates the critical pathway in the G(1) phase involved in downregulation of p27(Kip1), a cyclin-dependent kinase inhibitor, which plays a pivotal role in controlling entry into the S phase of the cell cycle. In resting CCL39 fibroblasts and IEC-6 intestinal epithelial cells, protein levels of p27(Kip1) were elevated but dramatically decreased on serum stimulation, along with hyperphosphorylation of pRb and increased CDK2 activity. In both cell types, expression of ras resulted in an increase of basal and serum-stimulated E2F-dependent transcriptional activity and a reduction in p27(Kip1) protein levels as well. The role of the mitogen-activated protein (MAP) kinase cascade in p27(Kip1) reduction and S phase reentry was reinforced by the blockades of serum-induced E2F-dependent transcriptional activity and p27(Kip1) downregulation with the MKK-1/2 inhibitor PD-98059. In both cell lines, downregulation of p27(Kip1) was associated with a repression of its synthesis, an event mediated by the p42/p44 MAP kinase pathway. Using an antisense approach, we demonstrated that p27(Kip1) may control cell cycle exit in both cell types. These data indicate that activation of the MAP kinase cascade is required for S phase entry and p27(Kip1) downregulation in fibroblasts and epithelial cells. (+info)
Endothelial cell nitric oxide production in acute chest syndrome.
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Acute chest syndrome (ACS) is the most common form of acute pulmonary disease associated with sickle cell disease. To investigate the possibility that alterations in endothelial cell (EC) production and metabolism of nitric oxide (NO) products might be contributory, we measured NO products from cultured pulmonary EC exposed to red blood cells and/or plasma from sickle cell patients during crisis. Exposure to plasma from patients with ACS caused a 5- to 10-fold increase in S-nitrosothiol (RSNO) and a 7- to 14-fold increase in total nitrogen oxide (NO(x)) production by both pulmonary arterial and microvascular EC. Increases occurred within 2 h of exposure to plasma in a concentration-dependent manner and were associated with increases in endothelial nitric oxide synthase (eNOS) protein and eNOS enzymatic activity, but not with changes in nitric oxide synthase (NOS) III or NOS II transcripts, inducible NOS (iNOS) protein nor iNOS enzymatic activity. RSNO and NO(x) increased whether plasma was obtained from patients with ACS or other forms of vasoocclusive crisis. Furthermore, an oxidative state occurred and oxidative metabolites of NO, particularly peroxynitrite, were produced. These findings suggest that altered NO production and metabolism to damaging oxidative molecules contribute to the pathogenesis of ACS. (+info)
Regulation of CDK4 activity by a novel CDK4-binding protein, p34(SEI-1).
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The p16(INK4a) tumor suppressor inhibits cyclin-dependent kinases (CDK4 and CDK6). Here we report the isolation of a novel gene, SEI-1, whose product (p34(SEI-1)) appears to antagonize the function of p16(INK4a). Addition of p34(SEI-1) to cyclin D1-CDK4 renders the complex resistant to inhibition by p16(INK4a). Expression of SEI-1 is rapidly induced on addition of serum to quiescent fibroblasts, and ectopic expression of p34(SEI-1) enables fibroblasts to proliferate even in low serum concentrations. p34(SEI-1) seems to act as a growth factor sensor and may facilitate the formation and activation of cyclin D-CDK complexes in the face of inhibitory levels of INK4 proteins. (+info)
Protective effect of a thrombin receptor (protease-activated receptor 1) gene polymorphism toward venous thromboembolism.
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The human protease-activated receptor 1 (PAR-1) is activated by thrombin at the surface of platelets and endothelial cells, 2 cells that are implicated in hemostasis and thrombosis. We studied the PAR-1 gene in a large case-control study from the Paris Thrombosis Study (PATHROS), and the possible implication of polymorphisms in venous thromboembolism was evaluated. Two polymorphisms were found in the 5' regulatory region. The first is a C to T transition that is 1426 nucleotides upstream from the translation start site (-1426 C/T), and the second is a 13-bp insertion repeating the preceding -506 5'-CGGCCGCGGGAAG-3' sequence (-506 I/D, where I indicates insertion and D indicates deletion), a putative cis-acting element of the Ets family. The third polymorphism is an A to T transversion in the intervening sequence (IVS) that is 14 nucleotides upstream from the exon 2 start site (IVS-14 A/T). The distribution of the 3 polymorphisms was otherwise similar in the 250 cases and the 1214 controls. A noteworthy sex heterogeneity led us to analyze men and women separately with regard to the -506 I/D polymorphism. We found that allele I was less frequent in male cases than in male controls (0.154 versus 0.247, P<0.01), with an odds ratio at 0.52 (95% CI 0. 32 to 0.82, P<0.01). Furthermore, a reduction of prothrombin fragment 1+2 levels was observed in homozygous carriers of allele -506 I (P=0.04). Altogether, these data suggested a protective effect in men of -506 I/D polymorphism for venous thromboembolism. (+info)
Relation of disease stage to humoral and cellular impairment of spleen reticuloendothelial system depression in a rat leukemia.
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In the rat leukemia studied there is an early reversible depression of reticuloendothelial system (RES) function followed by a late-stage secondary depression, culminating in death, associated with an infiltration of leukemic cells into the bone marrow and spleen. In one set of experiments to assess RES function, isolated spleens of nonleukemic rats were perfused with blood from rats in progressively advanced stages of the leukemia. In a reciprocal set of experiments, isolated spleens of rats in comparable stages of the leukemia were perfused with blood from nonleukemic rats. The clearance of 99mTc-sulfur colloid from the blood perfusate was used as a test of RES function. In the first set, clearance was depressed in nonleukemic spleens perfused with blood collected from rats 1 hr after they had been inoculated with tumor cells, and it was more markedly depressed in a late stage of the leukemia 6 days later. Perfusion of nonleukemic spleens with blood from rats in intermediate stages of the leukemia did not depress clearance. In the reciprocal study, clearance was depressed in spleens of rats that were inoculated with tumor cells 1 hr before perfusion with nonleukemic blood. Clearance values were at the control level in spleens of rats in all other stages of the leukemia when calculated on the basis of total spleen weight. When calculated on a unit weight basis, clearance values were lower in the enlarged spleens of rats in advanced stages of the leukemia. The results indicate that impairment of RES clearance of 99mTc-sulfur colloid is associated with alternations in the humoral content of the blood of this leukemic rat. There is indirect evidence to suggest that in the terminal stage of the leukemia an impairment of splenic RES cells to clear 99mTc-sulfur colloid may develop secondarily. (+info)
Plasma from ESRD patients inhibits nitric oxide synthase activity in cultured human and bovine endothelial cells.
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Our recent observations of reduced total nitric oxide synthesis in renal failure patients on peritoneal dialysis and haemodialysis suggest that hypertension in end-stage renal disease involves lack of vasodilatory endothelial NO. To directly test this, uraemic plasma was obtained from dialysis patients and incubated with cultured vascular endothelial cells, to determine the effect on nitric oxide synthase (NOS) activity in comparison with plasma from subjects with normal renal function. After incubation for 6 h with 20% uraemic plasma from peritoneal dialysis and immediately prehaemodialysis patients, NOS activity was reduced in human dermal microvascular endothelial cells. Haemodialysis did not remove the NOS-inhibitory activity of uraemic plasma nor did it activate inducible NOS, as NOS activity was always similar in control and dexamethasone pretreated cells. Nitric oxide production (accumulation of nitrite and nitrate) was lower in cells incubated with uraemic vs. normal plasma and excess arginine increased nitric oxide production by cells previously exposed to uraemic medium. This inhibitory effect was not associated with co-factor deficiency but did correlate with plasma concentrations of endogenous NOS inhibitors. These in vitro findings suggest that low endothelial NOS activity may contribute to hypertension in end stage renal disease patients. (+info)