Effect of MG on platelet [Ca²⁺]_i and degranulation.

<p>(a) Increase in [Ca²⁺]_i measured in washed human platelets treated with either solvent (CTL) or methylglyoxal (MG, 1 mmol/L, 15 minutes) prior to the stimulation with thrombin. (b) Effect of MG pre-treatment on the thrombin (0.03U/ml)-induced release of ATP and (c) on the TRAP-induced surface expression of P-selectin. The graphs summarise the data from at least 6 different individuals; *P<0.05, **P<0.01 versus CTL.</p

Effect of MG on the phosphorylation of β3 integrin and Akt.

<p>(a) Effect of MG (MG, 1 mmol/L, 15 minutes) on fibronectin (Fn) and collagen (coll)-induced tyrosine phosphorylation of β3 integrin (Tyr747). (b) Effect of MG on thrombin -induced tyrosine phosphorylation (Tyr747) of β3 integrin in washed human platelets. (c) Effect of MG on fibronectin (Fn) and collagen (coll)-induced phosphorylation of Akt (Ser 473). (d) Effect of wortmannin (Wt, 20 nmol/L, 30 minutes) on fibronectin (Fn) and collagen (coll)-induced phosphorylation of β3 integrin (Tyr747) and Akt (Ser 473). The graphs summarise the data from 6 different experiments; *P<0.05, ***P<0.001 versus sol or CTL and ^# P<0.05, ^{# # #} P<0.001 versus agonists.</p

Effect of MG on PKC activation.

<p>(a) Membrane translocation of PKCα and β in washed human platelets stimulated with either methylglyoxal (MG, 1 mmol/L, 15 minutes) or thrombin (0.03U/ml) alone or in combination. (b) Effect of methylglyoxal (MG, 1 mmol/L, 15 and 30 minutes) on the phosphorylation of MLC20 in the absence or in the presence of the PKC inhibitor Ro-318820 (Ro, 300 nM, 30 minutes). (c) Effect of MG on thrombin-induced phosphorylation of MLC20. (d) Effect of Ro-318220 on the thrombin-induced aggregation of washed human platelets treated or not with MG. The graphs summarise the data from 6-8 different experiments; *P<0.05, **P<0.01 versus CTL and ^# P<0.05, ^{# #} P<0.01 versus agonists.</p

Effect of MG on platelet adhesion, spreading and <i>in vivo</i> thrombus formation.

<p>(a) Representative pictures and (b) quantification of adherent and spread washed human platelets (to fibronectin (Fn)- or collagen (coll)-coated slides) pre-treated with either solvent (CTL) or methylglyoxal (MG, 1 mmol/L, 15 minutes). (c) Representative pictures (upper panel) and quantification (lower graphs) of the effect of <i>in </i><i>vivo</i> treatment of healthy mice with MG (1 mmol/L, 15 minutes) on thrombus size and time to peak after FeCl₃-induced injury of carotid artery. The graphs summarize data obtained in platelets from 12 subjects or 6 animals per group; *P<0.05, ***P<0.001, versus CTL.</p

Effect of 11,12-EET on the mitochondrial membrane potential.

<p>(A) Representative tracing of the 11,12-EET (3 µmol/L)-induced changes in JC-1 fluorescence in pulmonary artery endothelial cells from wild-type (WT) cells in the absence or presence of iberiotoxin (IbTx, 300 nmol/L) and from BKβ₁^−/− cells. (B) Mitochondrial membrane depolarization by 11,12-EET in WT and BKβ₁^−/− pulmonary artery smooth muscle cells in the presence of solvent (Sol) or IbTx. (C) Mitochondrial membrane depolarization by 11,12-EET in WT cells in the presence of Sol, 14,15-EEZE (10 µmol/L), or Rp-cAMPS (10 µmol/L). All experiments were performed in the presence of diclofenac (10 µmol/L) and L-NA (300 µmol/L). The bar graphs summarize data obtained in 4–10 independent experiments; *P<0.05, ***P<0.001 versus WT+EET.</p

Effect of sEH inhibition on hypoxic pulmonary vasoconstriction in wild-type and BKβ₁^−/− mice.

<p>Hypoxia-induced increases in pulmonary arterial pressure (ΔPAP) were assessed in the presence of solvent (Sol) or ACU (3 µmol/L) in isolated lungs from wild-type (WT) or BKβ₁^−/− mice. Experiments were performed in (A) the absence and (B) the presence of diclofenac (10 µmol/L) and L-NA (300 µmol/L). The graphs summarize data obtained in 4–15 independent experiments; *P<0.05, **P<0.01 versus Sol; §§§P<0.001 versus WT+ACU.</p

11,12-EET-induced association of the BKα and β₁ subunits.

<p>Representative blot and densitometric analysis showing the co-precipitation of BKα with BKβ1 from HEK293 cells overexpressing either one or both BK subunits and stimulated with 11,12-EET (10 µmol/L) for 2–10 minutes. The graph summarizes data from 6 independent experiments; *P<0.05 versus the unstimulated control (CTL).</p

Effect of 11,12-EET on the membrane potential of wild-type and BKβ₁^−/− pulmonary artery smooth muscle cells.

<p>(A) Representative blots showing the expression of the α and β₁ subunits of the BK in cultured pulmonary artery smooth muscle cells. (B) Original tracing showing the effect of 11,12-EET (10 µmol/L) on the fluorescence emission ratio of Di-8-ANEPPS-loaded pulmonary artery smooth muscle cells. (C) Effect of 11,12-EET (10 µmol/L) on the membrane potential of wild-type (WT) and BKβ₁^−/− pulmonary artery smooth muscle cells in the presence of solvent (Sol) or iberiotoxin (IbTx, 300 nmol/L). Experiments were performed in the presence of diclofenac (10 µmol/L) and L-NA (300 µmol/L). The bar graph summarizes data obtained in 4–8 independent experiments; **P<0.01, ***P<0.001 versus WT+Sol.</p

Effect of iberiotoxin on the sensitivity of the acute hypoxic vasoconstriction to sEH inhibition.

<p>Hypoxia-induced increases in pulmonary arterial pressure (ΔPAP) were assessed in the presence of solvent (Sol), ACU (3 µmol/L) and iberiotoxin (IbTx, 300 nmol/L) in isolated lungs from (A) wild-type (WT) or (B) BKβ₁^−/− mice. All experiments were performed in the presence of diclofenac and L-NA. The graphs summarize data obtained in 7–13 independent experiments; **P<0.01 versus Sol; §P<0.05, §§P<0.001 versus WT+ACU.</p

Cardiovascular functions of Ena/VASP proteins: past, present and beyond

Actin binding proteins are of crucial importance for the spatiotemporal regulation of actin cytoskeletal dynamics, thereby mediating a tremendous range of cellular processes. Since their initial discovery more than 30 years ago, the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family has evolved as one of the most fascinating and versatile family of actin regulating proteins. The proteins directly enhance actin filament assembly, but they also organize higher order actin networks and link kinase signaling pathways to actin filament assembly. Thereby, Ena/VASP proteins regulate dynamic cellular processes ranging from membrane protrusions and trafficking, and cell-cell and cell-matrix adhesions, to the generation of mechanical tension and contractile force. Important insights have been gained into the physiological functions of Ena/VASP proteins in platelets, leukocytes, endothelial cells, smooth muscle cells and cardiomyocytes. In this review, we summarize the unique and redundant functions of Ena/VASP proteins in cardiovascular cells and discuss the underlying molecular mechanisms. </p