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Shear stress plays a significant role in endothelial cell biology and atherosclerosis development. Previous work by our group has shown that fluid flow stimulates important functional changes in cells through protein expression regulation. Peroxiredoxins (PRX) are a family of antioxidant enzymes but have yet to be investigated in response to shear stress. Studies have shown that oscillatory shear stress (OS) increases reactive oxygen species (ROS) levels in endothelial cells, whereas laminar shear stress (LS) blocks this response. We hypothesized that PRX are responsible for the anti-oxidative effect of LS. To test this hypothesis, bovine aortic endothelial cells (BAEC) were subjected to LS (15 dyn/cm2), OS (±5 dyn/cm2, 1 Hz), or static conditions for 24 h. Using Western blot and immunofluorescence staining, all six isoforms of PRX were identified in BAEC. When compared with OS and static, exposure to chronic LS up-regulated PRX 1 levels intracellularly. LS also increased expression of PRX 5 relative to static controls, but not OS. PRX exhibited broad subcellular localization, with distribution in the cytoplasm, Golgi, mitochondria, and intermediate filaments. In addition, PRX 1 knock down, using specific small interference RNA, attenuated LS-dependent reactive oxygen species reduction in BAEC. However, PRX 5 depletion did not. Together, these results suggest that PRX 1 is a novel mechanosensitive antioxidant, playing an important role in shear-dependent regulation of endothelial biology and atherosclerosis.

Arterial regions exposed to oscillatory shear (OS) in branched arteries are lesion-prone sites of atherosclerosis, whereas those of laminar shear (LS) are relatively well protected. Here, we examined the hypothesis that OS and LS differentially regulate production of O2- from the endothelial NAD(P)H oxidase, which, in turn, is responsible for their opposite effects on a critical atherogenic event, monocyte adhesion. We used aortic endothelial cells obtained from C57BL/6 (MAE-C57) and p47phox-/- (MAE-p47-/-) mice, which lack a component of NAD(P)H oxidase. O2- production was determined by dihydroethidium staining and an electron spin resonance using an electron spin trap methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Chronic exposure (18 h) to an arterial level of OS (+/- 5 dynes/cm2) increased O2- (2-fold) and monocyte adhesion (3-fold) in MAE-C57 cells, whereas chronic LS (15 dynes/cm2, 18 h) significantly decreased both monocyte adhesion and O2- compared with static conditions. In contrast, neither LS nor OS were able to induce O2- production and monocyte adhesion to MAE-p47-/-. Treating MAE-C57 with a cell-permeable superoxide dismutase compound, polyethylene glycol-superoxide dismutase, also inhibited OS-induced monocyte adhesion. In addition, over-expressing p47phox in MAE-p47-/- restored OS-induced O2- production and monocyte adhesion. These results suggest that chronic exposure of endothelial cells to OS stimulates O2- and/or its derivatives produced from p47phox-dependent NAD(P)H oxidase, which, in turn, leads to monocyte adhesion, an early and critical atherogenic event.

We examined the influence of individual serine phosphorylation sites in endothelial nitric-oxide synthase (eNOS) on basal and stimulated NO release, cooperative phosphorylation, and co-association with hsp90 and Akt. Mutation of the serine phosphorylation sites 116, 617, and 1179 to alanines affected the phospho-state of at least one other site, demonstrating cooperation between multiple phosphorylation events, whereas mutation of serine 635 to alanine did not cause compensation. Mutation of serines 116 and 617 to alanine promoted a greater protein-protein interaction with hsp90 and Akt and greater phosphorylation on serine 1179, the major site for Akt phosphorylation. More importantly, using alanine substitutions, Ser-116 is important for agonist, but not basal NO release, Ser-635 is important for basal, but not stimulated, Ser-617 negatively regulates basal and stimulated NO release, and Ser-1179 phosphorylation is stimulatory for both basal and agonist-mediated NO release. Using putative "gain of function" mutants (serine to aspartate) serines 635 and 1179 are important positive regulators of basal and stimulated NO release. S635D eNOS is the most efficacious, yielding 5-fold increases in basal and 2-fold increases in stimulated NO release from cells. However, S617A and S617D eNOS both increased NO release with opposite actions in NOS activity assays. Thus, multiple serine phosphorylation events regulate basal and stimulate NO release with Ser-635 and Ser-1179 being important positive regulatory sites and Ser-116 as a negative regulatory. Ser-617 may not be important for directly regulating NO release but is important as a modulator of phosphorylation at other sites and protein-protein interactions.

Recently, we have shown that shear stress stimulates NO(*) production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574-1581). Akt has been believed to regulate shear-dependent production of NO(*) by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser(1179) residue (eNOS-S(1179)), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt(AA) or Akt(AAA)) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S(1179). Here, we show that shear-dependent phosphorylation of eNOS-S(1179) is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt(AA) or Akt(AAA) in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S(1179) if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt(AA) inhibited shear-dependent NO(*) production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S(1179) and NO(*) production. In contrast, H89 did not inhibit phosphorylation of eNOS-S(1179) induced by expressing a constitutively active Akt mutant (Akt(Myr)) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S(1179) within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S(1179) in a PKA-dependent, but Aktindependent manner, whereas the NO(*) production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction between Akt and PKA may be an important mechanism by which eNOS activity is regulated in response to physiological stimuli such as shear stress.

Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro and in vivo. The molecular mechanisms regulating phenotypic modulation are unknown. Previous studies have suggested that the serine/threonine protein kinase mediator of nitric oxide (NO) and cyclic GMP (cGMP) signaling, the cGMP-dependent protein kinase (PKG) promotes modulation to the contractile phenotype in cultured rat aortic smooth muscle cells (RASMC). Because of the potential importance of the mitogen-activated protein kinase (MAP kinase) pathways in VSMC proliferation and phenotypic modulation, the effects of PKG expression in PKG-deficient and PKG-expressing adult RASMC on MAP kinases were examined. In PKG-expressing adult RASMC, 8-para-chlorophenylthio-cGMP activated extracellular signal- regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK). The major effect of PKG activation was increased activation by MAP kinase kinase (MEK). The cAMP analog, 8-Br-cAMP inhibited ERK1/2 activation in PKG-deficient and PKG-expressing RASMC but had no effect on JNK activity. The effects of PKG on ERK and JNK activity were additive with those of platelet-derived growth factor (PDGF), suggesting that PKG activates MEK through a pathway not used by PDGF. The stimulatory effects of cGMP on ERK and JNK activation were also observed in low-passaged, contractile RASMC still expressing endogenous PKG, suggesting that the effects of PKG expression were not artifacts of cell transfections. These results suggest that in contractile adult RASMC, NO-cGMP signaling increases MAP kinase activity. Increased activation of these MAP kinase pathways may be one mechanism by which cGMP and PKG activation mediate c-fos induction and increased proliferation of contractile adult RASMC.

Shear stress, the dragging force generated by fluid flow, differentially activates extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) in bovine aortic endothelial cells (BAEC) (Jo, H., Sipos, K., Go, Y. M., Law, R., Rong, J., and McDonald, J. M. (1997) J. Biol. Chem. 272, 1395-1401). Here, we examine whether cholesterol-enriched compartments in the plasma membrane are responsible for such differential regulation. Pretreatment of BAEC with a cholesterol-binding antibiotic, filipin, did not inhibit shear-dependent activation of JNK. In contrast, filipin and other membrane-permeable cholesterol-binding agents (digitonin and nystatin), but not the lipid-binding agent xylazine, inhibited shear-dependent activation of ERK. The effect of cholesterol-binding drugs did not appear to be due to membrane permeabilization, since treatment of BAEC with a detergent, Triton X-100 which also permeabilizes membranes, did not inhibit shear-dependent activation of ERK. Furthermore, shear-dependent activation of ERK, but not JNK, was inhibited by cyclodextrin, a membrane-impermeable cholesterol-binding agent, which removes cell-surface cholesterol. Moreover, the effects of cyclodextrin were prevented by adding cholesterol during the incubation. These results indicate that cholesterol or cholesterol-sensitive compartments in the plasma membrane play a selective and essential role in activation of ERK, but not JNK, by shear stress. Although exposure to shear stress (1 h) increased the number of caveolae by 3-fold, treatment with filipin had no effect in either control or shear-exposed cells suggesting that caveolae density per se is not a crucial determinant in shear-dependent ERK activation. In summary, the current study suggests that cholesterol-sensitive microdomains in the plasma membrane, such as caveolae-like domains, play a critical role in differential activation of ERK and JNK by shear stress.

Shear stress differentially regulates production of many vasoactive factors at the level of gene expression in endothelial cells that may be mediated by mitogen-activated protein kinases, including extracellular signal-regulated kinase (ERK) and N-terminal Jun kinase (JNK). Here we show, using bovine aortic endothelial cells (BAEC), that shear stress differentially regulates ERK and JNK by mechanisms involving Gi2 and pertussis toxin (PTx)-insensitive G-protein-dependent pathways, respectively. Shear activated ERK with a rapid, biphasic time course (maximum by 5 min and basal by 30-min shear exposure) and force dependence (minimum and maximum at 1 and 10 dyn/cm2 shear stress, respectively). PTx treatment prevented shear-dependent activation of ERK1/2, consistent with a Gi-dependent mechanism. In contrast, JNK activity was maximally turned on by a threshold level of shear force (0.5 dyn/cm2 or higher) with a much slower and prolonged time course (requiring at least 30 min to 4 h) than that of ERK. Also, PTx had no effect on shear-dependent activation of JNK. To further define the shear-sensitive ERK and JNK pathways, vectors expressing hemagglutinin epitope-tagged ERK (HA-ERK) or HA-JNK were co-transfected with other vectors by using adenovirus-polylysine in BAEC. Expression of the mutant (alpha)i2(G203), antisense G(alpha)i2 and a dominant negative Ras (N17Ras) prevented shear-dependent activation of HA-ERK, while that of (alpha)i2(G204) and antisense (alpha)i3 did not. Expression of a Gbeta/gamma scavenger, the carboxyl terminus of beta-adrenergic receptor kinase (betaARK-ct), and N17Ras inhibited shear-dependent activation of HA-JNK. Treatment of BAEC with genistein prevented shear-dependent activation of ERK and JNK, indicating the essential role of tyrosine kinase(s) in both ERK and JNK pathways. These results provide evidence that 1) Gi2-protein, Ras, and tyrosine kinase(s) are upstream regulators of shear-dependent activation of ERK and 2) that shear-dependent activation of JNK is regulated by mechanisms involving Gbeta/gamma, Ras, and tyrosine kinase(s).