Effects of pioglitazone on fasting and postprandial levels of lipid and hemostatic variables in overweight non-diabetic patients with coronary artery disease.

OBJECTIVES: To evaluate the effects of pioglitazone on insulin sensitivity and levels of biomarkers associated with thrombotic risk in overweight and obese, non-diabetic subjects with coronary artery disease. BACKGROUND: Little information is available regarding the effects of thiazolidinediones in the absence of diabetes. Further, although postprandial hyperlipemia is a risk factor for cardiovascular diseases, there is limited information about the postprandial effects. METHODS: Twenty overweight and obese, non-diabetic patients with coronary artery disease were enrolled in a randomized, placebo-controlled, double-blind study. Subjects were on atorvastatin for the duration of the study and received either placebo or pioglitazone (45 mg day(-1)) for 12 weeks and then crossed over to the alternative therapy for an additional 12 weeks. Insulin sensitivity, fasting and postprandial levels of lipid, hemostatic, and inflammatory variables were measured, and endothelial function was assessed. RESULTS: Insulin sensitivity improved from 0.03 micromol kg(-1) x min pM(-1) on placebo to 0.04 on pioglitazone (P = 0.0002), and there were decreases in fasting levels of factor (F) VII:C (102 +/- 17% to 92 +/- 18%, P = 0.001), FVII:Ag (68 +/- 12% to 60 +/- 14%, P = 0.01) and in von Willebrand factor (VWF) (174 +/- 94% to 142 +/- 69%, P = 0.01). Pioglitazone lowered postprandial levels of FVII:Ag, FVII:C, plasminogen activator inhibitor-1, VWF, and triglycerides, and increased high-density lipoproteins (+9%, P = 0.02). CONCLUSIONS: Pioglitazone improves insulin sensitivity and favorably modifies fasting and postprandial lipid, hemostatic and inflammatory markers of the metabolic syndrome in overweight and obese non-diabetic patients with coronary artery disease.

Effects of lipids and lipid-lowering therapy on hemostatic factors in patients with myocardial infarction.

BACKGROUND: The risk of cardiovascular disease (CVD) is associated with specific hemostatic markers and lipid profiles, and evidence indicates that there are associations between lipid profiles and the levels of certain hemostatic factors. The disturbances in hemostasis and the risk of CVD can be ameliorated by lipid-lowering therapy. OBJECTIVE: We investigated the associations of lipid profiles with factor (F)VIIa, von Willebrand factor (VWF), D-dimer and plasminogen activator inhibitor-1 (PAI-1), and examined whether lipid-lowering statin therapy would affect the levels of these hemostatic markers. PATIENTS AND METHODS: This cross-sectional study analyzed 1045 postmyocardial infarction patients. RESULTS: In multivariate regression analyses (without adjusting for clinical covariates) HDL-cholesterol (HDL-C) and HDL size were independent and significant predictors of FVIIa; HDL size was a predictor of VWF; HDL size, HDL-C and LDL size were predictors of D-dimer; and triglyceride and HDL size were predictors of PAI-1. After adjusting for clinical covariates, HDL-C, lipoprotein (Lp)(a), apolipoprotein B (apoB) and warfarin were independent and significant predictors of FVIIa; HDL size, age, diabetes mellitus, insulin, race and warfarin were predictors of VWF; HDL-C, HDL size, LDL size, age, warfarin, hypertension and gender were predictors of D-dimer; and triglyceride, HDL size, body mass index, insulin and hypertension were predictors of PAI-1. Patients on statin therapy had significantly lower levels of D-dimer than those who were not on this therapy. CONCLUSION: There are significant associations of lipid profiles with hemostatic factors, the directions of which suggest novel pathways by which dyslipidemia may contribute to coronary heart disease.

Protein S declines during winter respiratory infections.

There is an increase in cardiovascular and cerebrovascular morbidity and mortality in the older adult population during the winter that could be related to prothrombotic changes caused by seasonal effects or acute respiratory tract infections. Therefore, a prospective cohort study was conducted to assess the effect of acute winter respiratory infection on hemostatic parameters including complement 4b-binding protein (C4-BP), functional protein S, total protein S, free protein S, and the inflammatory marker, interleukin-6 (IL-6), in younger and older adults. The changes in the levels of hemostatic and inflammatory markers during winter respiratory infections in the younger and older adults were compared with matched, non-infected controls. In younger and older adults (combined), total protein S increased from 83% [95% confidence interval (CI); 77-88] to 98% (95% CI; 91-106, P < 0.001) while free protein S decreased from 100% (95% CI; 95-105) to 70% (95% CI; 66-75, P < 0.001). There were no significant changes in C4-BP (P = 0.622), functional protein S (P = 0.061) or IL-6 (P = 0.651) from baseline. In a multivariate analysis, only total protein S and free protein S showed significant association with seasonal change after adjusting for the effect of infection. The estimated effect of season on total protein S was 15 +/- 4%, P < 0.001 and on free protein S was -27 +/- 3%, P < 0.001. After adjusting for seasonal effect, only functional protein S showed a significant association with infection, with the estimated effect of -17 +/- 5%, P < 0.001. The results in the younger and older adults were similar to those in the combined groups. Seasonal and infection-related changes in hemostatic parameters including an increase in fibrinogen and a decrease in free protein S, observed in this study, may contribute to thrombotic risk and excess vascular disease morbidity and mortality in older populations in the winter season.

Hypotonicity induces L-selectin shedding in human neutrophils.

Expression levels of adhesion molecules on neutrophils are affected under various conditions, including ischemia, possibly because of associated increases in cell volume. We examined the effects of cell swelling in hypotonic media on the level of L-selectin (CD62L) and beta(2)-integrin (CD18) on human neutrophils. In hypotonic media, neutrophils shed L-selectin. The shedding was greatly reduced by 30 microM RO31-9790, the metalloprotease (sheddase) inhibitor. Hypotonicity-induced L-selectin shedding was also time and tonicity dependent. Decreasing tonicity caused increased shedding. In 0.6x medium (0.6x the normal tonicity of 300 mosmol/kgH(2)O), shedding increased over a 2-h period, after which >70% of the neutrophils had lost L-selectin. In contrast to L-selectin, the level of beta(2)-integrin on the neutrophil surface was not significantly affected. Thus L-selectin shedding, which occurs on neutrophil activation and is usually accompanied by beta(2)-integrin upregulation, was selectively induced by hypotonicity without a corresponding effect on beta(2)-integrin.

Effect of urea and osmotic cell shrinkage on Ca2+ entry and contraction of vascular smooth muscle cells.

The present study was performed to elucidate the effects of urea on vascular smooth muscle cells (SMC). Addition of urea (20, 50, 100 mM) to physiological salt solution blunted the vasoconstrictory effect of phenylephrine (by 17, 25 and 30%, respectively) and of an increased extracellular K+ concentration (by 7, 14 and 19%, respectively) without affecting the basal tone of rabbit arterial rings. According to Fura-2 fluorescence in cultured SMC (A7r5), urea had no effect on basal intracellular calcium activity ([Ca2+]i), but significantly blunted the increase of [Ca2+]i following an increase of extracellular K+. Whole-cell patch-clamp studies revealed that the Ca2+ current through voltage-sensitive Ca2+ channels is significantly inhibited in the presence of urea. As evident from calcein fluorescence, addition of urea leads to sustained cell shrinkage. The effects of urea on vascular tone, [Ca2+]i activity, voltage-gated Ca2+ channels and cell volume are mimicked by addition of raffinose or NaCl. However, the cell shrinkage induced by urea is sustained, whereas the addition of equiosmolar NaCl is only transient and followed by a regulatory cell volume increase. Moreover, hypertonic NaCl increases, whereas urea decreases, the transcription of cell-volume-regulated kinase hsgk. In conclusion, urea leads to sustained shrinkage of vascular smooth muscle cells, which is followed by inhibition of voltage-gated Ca2+ channels, a decrease of [Ca2+]i and thus blunts the vasoconstrictory action of phenylephrine and increased extracellular K+ concentration.

Ion channels involved in insulin release are activated by osmotic swelling of pancreatic B-cells.

Measurements of the membrane potential showed that osmotic swelling (-80 mosmol/l) of pancreatic B-cells led to a transient hyperpolarization followed by a more sustained depolarization of the cell membrane. Cell swelling triggers a transient activation of the K+ATP current and of an inward current, carried by Cl-. This current was inhibited by DIDS, D600, and by omission of extracellular Ca2+. The depolarization opens voltage dependent L-type Ca2+ channels, thereby increasing the intracellular Ca2+ activity ([Ca2+]i). This effect was blunted by D600 or abolished by omission of Ca2+. Moreover, osmotic swelling transiently increased the amplitude of the Ca2+ currents. Replacement of NaCl by d-mannitol proved that the observed effects are due to an increase in cell volume and not to a reduction of extracellular Na+ or Cl-. Our results suggest that regulatory volume decrease is achieved by activation of K+ and Cl- currents. The Cl- current is responsible for the previously described depolarization and increase in insulin release induced by osmotic cell swelling.

The tyrosine kinase p56lck mediates activation of swelling-induced chloride channels in lymphocytes.

Osmotic cell swelling activates Cl- channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56lck, a src-like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (ICl-swell) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of ICl-swell is defective in p56lck-deficient cells. Retransfection of p56lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of ICl-swell. Addition of purified p56lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56lck washed into the cytoplasm activates ICl-swell in native and p56lck-deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56lck, slow single-channel gating events can be observed revealing a unitary conductance of 25-28 pS. In accordance with our patch-clamp data, osmotic swelling increases activity of immunoprecipitated p56lck. We conclude that osmotic swelling activates ICl-swell in lymphocytes via the tyrosine kinase p56lck.

Effect of osmolarity on LDL binding and internalization in hepatocytes.

The present study has been performed to elucidate a possible role of cell volume in low-density lipoprotein (LDL) binding and internalization (LDL(b+i)). As shown previously, increase of extracellular osmolarity (OSMe) and K+ depletion, both known to shrink cells, interfere with the formation of clathrin-coated pits and thus with LDL(b+i). On the other hand, alterations of cell volume have been shown to modify lysosomal pH, which is a determinant of LDL(b+i). LDL(b+i) have been estimated from heparin-releasable (binding) or heparin-insensitive (internalization) uptake of 125I-labeled LDL. OSMe was modified by alterations of extracellular concentrations of ions, glucose, urea, or raffinose. When OSMe was altered by varying NaCl concentrations, LDL(b+i) decreased (by 0.5 +/- 0.1%/mM) with increasing OSMe and LDL(b+i) increased (by 1.2 +/- 0.1%/mM) with decreasing OSMe, an effect mainly due to altered affinity; the estimated dissociation constant amounted to 20.6, 48.6, and 131.6 micro/ml at 219, 293, and 435 mosM, respectively. A 25% increase of OSMe increased cytosolic (by 0.46 +/- 0.03) and decreased lysosomal (by 0.14 +/- 0.02) pH. Conversely, a 25% decrease of OSMe decreased cytosolic (by 0.28 +/- 0.02) and increased lysosomal (by 0.17 +/- 0.02) pH. Partial replacement of extracellular Na+ with K+ had little effect on LDL(b+i), although it swelled hepatocytes and increased lysosomal and cytosolic pH. Hypertonic glucose, urea, or raffinose did not exert similar effects despite a shrinking effect of hypertonic raffinose. Monensin, which completely dissipates lysosomal acidity, virtually abolished LDL(b+i). In conclusion, the observations reveal a significant effect of ionic strength on LDL(b+i). The effect is, however, not likely to be mediated by alterations of cell volume or alterations of lysosomal pH.

Effect of cellular hydration on protein metabolism.

In the past few years, the paramount importance of cell volume for the regulation of cell function, including protein metabolism, has been recognized. Among many other effects, cell swelling inhibits proteolysis and stimulates protein synthesis, whereas cell shrinkage stimulates proteolysis and inhibits protein synthesis. Moreover, cell swelling and cell shrinkage influence the expression of a number of genes, including carriers, enzymes, and signaling molecules. Hormones exploit the influence of cell volume on metabolism to exert their effects. Insulin swells hepatocytes by activation of Na-/H+ exchange and Na+,K+,2Cl- cotransport, while glucagon shrinks hepatocytes by activation of ion channels. The effects of these hormones on hepatic proteolysis completely depend on their influence on cell volume. The effects of cell volume are mediated in part by alterations of lysosomal pH, which modifies the activity of acidic lysosomal proteases. Transforming growth factor-beta 1, as other growth factors, activates the Na+/H+ exchanger, swells cells, leads to lysosomal alkalinization, inhibits proteolysis and may thus contribute to renal hypertrophy in chronic renal disease. Moreover, a decrease in cell volume correlates with catabolic states in a variety of diseases.