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First published online 23 August 2005
doi: 10.1242/jcs.02541

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Role of PECAM-1 in the shear-stress-induced activation of Akt and the endothelial nitric oxide synthase (eNOS) in endothelial cells

Ingrid Fleming^*,, Beate Fisslthaler^*, Madhulika Dixit and Rudi Busse

Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany

View larger version (48K): [in a new window]   Fig. 1. Effects of shear stress and receptor-dependent agonists on the tyrosine phosphorylation of PECAM-1. Primary cultures of human umbilical vein endothelial cells were either maintained under static conditions, exposed to fluid shear stress (Shear; 12 dynes cm–2, 2-120 minutes) or stimulated with either solvent (CTL), VEGF (100 ng ml–1, 5-30 minutes), histamine (Hist; 1 µmol l–1, 10-30 minutes), or ionomycin (Iono; 0.1 µmol l–1, 10-30 minutes). PECAM-1 was immunoprecipitated and the tyrosine phosphorylation was assessed by western blotting. To verify equal amounts of immunoprecipitated protein the blot was reprobed with an antibody raised against PECAM-1. The western blots shown are representative of data obtained in three independent experiments and the bar graph summarizes data obtained in three independent experiments; **P<0.01, ***P<0.001 versus static conditions.

View larger version (54K): [in a new window]   Fig. 2. Effect of shear stress on the activation of Src and phosphorylation of PECAM-1. (A) Endothelial cells were maintained under static conditions or exposed to fluid shear stress (12 dynes cm–2, 5-60 minutes) and the phosphorylation of Src on Tyr416 as well as total Src levels were detected using specific antibodies. The bar graph summarizes data obtained in four independent experiments; *P<0.05, **P<0.01 versus static conditions. (B) Endothelial cells were exposed to fluid shear stress (12 dynes cm–2, 30 minutes) in the absence (CTL) and presence of the Src kinase inhibitor PP1 (30 µmol l–1). Thereafter, PECAM-1 was immunoprecipitated and the tyrosine phosphorylation of PECAM-1 was assessed by western blotting. The results shown represent data obtained in three independent experiments.

View larger version (32K): [in a new window]   Fig. 3. Effect of shear stress on the association of eNOS with PECAM-1 and the phosphorylation of eNOS, Akt and AMPK and the generation of cyclic GMP. (A) Human endothelial cells were exposed to fluid shear stress (12 dynes cm–2) for the times indicated. Thereafter, PECAM-1 was immunoprecipitated and the co-precipitated eNOS was detected by a specific antibody. The bar graph summarizes data obtained in three independent experiments. (B) Human endothelial cells were exposed to fluid shear stress (12 dynes cm–2, 30 minutes) in the absence (CTL) and presence of PP1 (30 µmol l–1) or its inactive analogue PP3 (30 µmol l–1). Thereafter, the Triton X-100-soluble cell fraction was subjected to SDS-PAGE and the phosphorylation of eNOS (Ser1177), Akt (Ser473) and AMPK (Thr172) was determined using phospho-specific antibodies. The blots shown are representative of data obtained in three additional experiments. (C) Cells were incubated with solvent (CTL), wortmannin (40 nmol l–1) or PP1 (30 µmol l–1) in the presence of IBMX (50 µmol l–1) and either maintained under static conditions or exposed to shear stress (12 dynes cm–2, 30 minutes). Intracellular cyclic GMP levels were determined by radioimmunoassay and the bar graph summarizes data obtained in four independent experiments (each performed in duplicate); *P<0.05, **P<0.01 versus static conditions.

View larger version (64K): [in a new window]   Fig. 4. Down regulation of PECAM-1 using siRNA. The expression of PECAM-1 was assessed by (A) immunofluorescence (PECAM-1=green; TOPRO-3=blue) and (B) western blotting using specific antibodies. Primary cultures of human endothelial cells were either left untreated (a, CTL) or treated with either (b) liposomal transfection agent, (c) scrambled oligonucleotides (Scr) or (d) PECAM-1 siRNA (siRNA) oligonucleotides for 48 hours. The bar graph summarizes the results of six independent experiments; ***P<0.001 versus CTL.

View larger version (32K): [in a new window]   Fig. 5. Effect of PECAM-1 downregulation on the shear-stress-induced phosphorylation of Akt, eNOS and AMPK. Primary cultures of human endothelial cells treated with either scrambled oligonucleotides or siRNA against PECAM-1 were exposed to fluid shear stress (12 dynes cm–2, 30 or 60 minutes) and the phosphorylation and expression of (A) Akt, (B) eNOS and (C) AMPK, were detected using specific antibodies. The western blots were quantified as phospho-protein per total protein and expressed relative to the control (CTL) conditions, i.e. the signal obtained in cells treated with scrambled oligonucleotides and maintained under static conditions. The bar graphs summarize the results of six independent experiments; *P<0.05, **P<0.01 versus CTL.

View larger version (18K): [in a new window]   Fig. 6. Effect of PECAM-1 downregulation on the shear stress- and bradykinin-induced activation of eNOS. Human endothelial cells were left untreated (Control), treated with scrambled oligonucleotides (Scr) or siRNA against PECAM-1 and after 48 hours either (A) exposed to fluid shear stress (12 dynes cm–2) or (B) maintained under static conditions (Static) and stimulated with bradykinin (Bk; 10 nmol l–1, 5 minutes). The graphs presented summarize the results of four independent experiments each performed in triplicate; *P<0.05, **P<0.01 versus static conditions.

View larger version (31K): [in a new window]   Fig. 7. Comparison of the shear-stress-induced phosphorylation of eNOS, Akt and AMPK in endothelial cells from wild-type and PECAM-1–/– mice. Confluent cultures of lung endothelial cells from wild-type (WT) or PECAM-1–/– mice were either maintained under static conditions or subjected to fluid shear stress (Shear; 12 dynes cm–2; 30 minutes). The phosphorylation of eNOS on Ser1177, Akt on Ser473 and AMPK on Thr172 was determined by western blotting with specific antibodies (duplicates are shown). The western blots were quantified as phospho-protein/total protein and expressed relative to the control (CTL) conditions, i.e. the signal obtained under static conditions. The bar graph summarizes data obtained in five independent experiments; *P<0.05, **P<0.01 versus the response recorded in cells from wild-type mice.

View larger version (13K): [in a new window]   Fig. 8. Comparison of the shear-stress-induced increase in cyclic GMP in endothelial cells from wild-type and PECAM-1–/– mice. Endothelial cells were incubated with IBMX (50 µmol l–1) and either maintained under static conditions or exposed to shear stress (12 dynes cm–2) for up to 60 minutes. Intracellular cyclic GMP levels were determined by radioimmunoassay and the bar graph summarizes data obtained in three independent experiments (each performed in duplicate); *P<0.05, **P<0.01, ***P<0.001 versus static conditions.