Shear Stress Stimulates Phosphorylation of Endothelial Nitric-oxide Synthase at Ser1179 by Akt-independent Mechanisms: Role of Protein Kinase A

The Journal of Biological Chemistry. Feb, 2002  |  Pubmed ID: 11729190

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.

Activation of C-Jun N-terminal Kinase and Apoptosis in Endothelial Cells Mediated by Endogenous Generation of Hydrogen Peroxide

Biological Chemistry. Mar-Apr, 2002  |  Pubmed ID: 12033458

Reactive oxygen species have been implicated in the activation of signal transduction pathways. However, extracellular addition of oxidants such as hydrogen peroxide (H2O2) often requires concentrations that cannot be readily achieved under physiological conditions to activate biological responses such as apoptosis. Explanations for this discrepancy have included increased metabolism of H2O2 in the extracellular environment and compartmentalization within the cell. We have addressed this issue experimentally by examining the induction of apoptosis of endothelial cells induced by exogenous addition of H2O2 and by a redox cycling agent, 2,3-dimethoxy-1,4-naphthoquinone, that generates H2O2 in cells. Here we show that low nanomolar steady-state concentrations (0.1-0.5 nmol x min(-1) x 10(6) cells) of H2O2 generated intracellularly activate c-Jun N terminal kinase and initiate apoptosis in endothelial cells. A comparison with bolus hydrogen peroxide suggests that the low rate of intracellular formation of this reactive oxygen species results in a similar profile of activation for both c-Jun N terminal kinase and the initiation of apoptosis. However, a detailed analysis reveals important differences in both the duration and profile for activation of these signaling pathways.

Role of P47(phox) in Vascular Oxidative Stress and Hypertension Caused by Angiotensin II

Hypertension. Oct, 2002  |  Pubmed ID: 12364355

Hypertension caused by angiotensin II is dependent on vascular superoxide (O2*-) production. The nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase is a major source of vascular O2*- and is activated by angiotensin II in vitro. However, its role in angiotensin II-induced hypertension in vivo is less clear. In the present studies, we used mice deficient in p47(phox), a cytosolic subunit of the NADPH oxidase, to study the role of this enzyme system in vivo. In vivo, angiotensin II infusion (0.7 mg/kg per day for 7 days) increased systolic blood pressure from 105+/-2 to 151+/-6 mm Hg and increased vascular O2*- formation 2- to 3-fold in wild-type (WT) mice. In contrast, in p47(phox-/-) mice the hypertensive response to angiotensin II infusion (122+/-4 mm Hg; P<0.05) was markedly blunted, and there was no increase of vascular O2*- production. In situ staining for O2*- using dihydroethidium revealed a marked increase of O2*-production in both endothelial and vascular smooth muscle cells of angiotensin II-treated WT mice, but not in those of p47(phox-/-) mice. To directly examine the role of the NAD(P)H oxidase in endothelial production of O2*-, endothelial cells from WT and p47(phox-/-) mice were cultured. Western blotting confirmed the absence of p47(phox) in p47(phox-/-) mice. Angiotensin II increased O2*- production in endothelial cells from WT mice, but not in those from p47(phox-/-) mice, as determined by electron spin resonance spectroscopy. These results suggest a pivotal role of the NAD(P)H oxidase and its subunit p47(phox) in the vascular oxidant stress and the blood pressure response to angiotensin II in vivo.

NAD(P)H Oxidase-derived Hydrogen Peroxide Mediates Endothelial Nitric Oxide Production in Response to Angiotensin II

The Journal of Biological Chemistry. Dec, 2002  |  Pubmed ID: 12377764

Recently, it has been shown that the exogenous addition of hydrogen peroxide (H(2)O(2)) increases endothelial nitric oxide (NO(.)) production. The current study is designed to determine whether endogenous levels of H(2)O(2) are ever sufficient to stimulate NO(.) production in intact endothelial cells. NO(.) production was detected by a NO(.)-specific microelectrode or by an electron spin resonance spectroscopy using Fe(2+)-(DETC)(2) as a NO(.)-specific spin trap. The addition of H(2)O(2) to bovine aortic endothelial cells caused a potent and dose-dependent increase in NO(.) release. Incubation with angiotensin II (10(-7) mol) elevated intracellular H(2)O(2) levels, which were attenuated with PEG-catalase. Angiotensin II increased NO(.) production by 2-fold, and this was prevented by Losartan and by PEG-catalase, suggesting a critical role of AT1 receptor and H(2)O(2) in this response(.) In contrast, NO(.) production evoked by either bradykinin or calcium ionophore was unaffected by PEG-catalase. As in bovine aortic endothelial cells, angiotensin II doubled NO(.) production in aortic endothelial cells from C57BL/6 mice but had no effect on NO(.) production in endothelial cells from p47(phox-/-) mice. In contrast, stimulated NO(.) production to a similar extent in endothelial cells from wild-type and p47(phox-/-) mice. In summary, the present study provides direct evidence that endogenous H(2)O(2), derived from the NAD(P)H oxidase, mediates endothelial NO(.) production in response to angiotensin II. Under disease conditions associated with elevated levels of angiotensin II, this response may represent a compensatory mechanism. Because angiotensin II also stimulates O(2)() production from the NAD(P)H oxidase, the H(2)O(2) stimulation of NO(.) may facilitate peroxynitrite formation in response to this octapeptide.

Shear Stress Stimulates Phosphorylation of ENOS at Ser(635) by a Protein Kinase A-dependent Mechanism

American Journal of Physiology. Heart and Circulatory Physiology. Nov, 2002  |  Pubmed ID: 12384459

Shear stress stimulates nitric oxide (NO) production by phosphorylating endothelial NO synthase (eNOS) at Ser(1179) in a phosphoinositide-3-kinase (PI3K)- and protein kinase A (PKA)-dependent manner. The eNOS has additional potential phosphorylation sites, including Ser(116), Thr(497), and Ser(635). Here, we studied these potential phosphorylation sites in response to shear, vascular endothelial growth factor (VEGF), and 8-bromocAMP (8-BRcAMP) in bovine aortic endothelial cells (BAEC). All three stimuli induced phosphorylation of eNOS at Ser(635), which was consistently slower than that at Ser(1179). Thr(497) was rapidly dephosphorylated by 8-BRcAMP but not by shear and VEGF. None of the stimuli phosphorylated Ser(116). Whereas shear-stimulated Ser(635) phosphorylation was not affected by phosphoinositide-3-kinase inhibitors wortmannin and LY-294002, it was blocked by either treating the cells with a PKA inhibitor H89 or infecting them with a recombinant adenovirus-expressing PKA inhibitor. These results suggest that shear stress stimulates eNOS by two different mechanisms: 1) PKA- and PI3K-dependent and 2) PKA-dependent but PI3K-independent pathways. Phosphorylation of Ser(635) may play an important role in chronic regulation of eNOS in response to mechanical and humoral stimuli.

Compensatory Phosphorylation and Protein-protein Interactions Revealed by Loss of Function and Gain of Function Mutants of Multiple Serine Phosphorylation Sites in Endothelial Nitric-oxide Synthase

The Journal of Biological Chemistry. Apr, 2003  |  Pubmed ID: 12591925

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.

IkappaBalpha-dependent Regulation of Low-shear Flow-induced NF-kappa B Activity: Role of Nitric Oxide

American Journal of Physiology. Cell Physiology. Apr, 2003  |  Pubmed ID: 12620896

We have investigated the role of inhibitor kappaBalpha (IkappaBalpha) in the activation of nuclear factor kappaB (NF-kappaB) observed in human aortic endothelial cells (HAEC) undergoing a low shear stress of 2 dynes/cm(2). Low shear for 6 h resulted in a reduction of IkappaBalpha levels, an activation of NF-kappaB, and an increase in kappaB-dependent vascular cell adhesion molecule 1 (VCAM-1) mRNA expression and endothelial-monocyte adhesion. Overexpression of IkappaBalpha in HAEC attenuated all of these shear-induced responses. These results suggest that downregulation of IkappaBalpha is the major factor in the low shear-induced activation of NF-kappaB in HAEC. We then investigated the role of nitric oxide (NO) in the regulation of IkappaBalpha/NF-kappaB. Overexpression of endothelial nitric oxide synthase (eNOS) inhibited NF-kappaB activation in HAEC exposed to 6 h of low shear stress. Addition of the structurally unrelated NO donors S-nitrosoglutathione (300 microM) or sodium nitroprusside (1 mM) before low shear stress significantly increased cytoplasmic IkappaBalpha and concomitantly reduced NF-kappaB binding activity and kappaB-dependent VCAM-1 promoter activity. Together, these data suggest that NO may play a major role in the regulation of IkappaBalpha levels in HAEC and that the application of low shear flow increases NF-kappaB activity by attenuating NO generation and thus IkappaBalpha levels.

Chronic Shear Induces Caveolae Formation and Alters ERK and Akt Responses in Endothelial Cells

American Journal of Physiology. Heart and Circulatory Physiology. Sep, 2003  |  Pubmed ID: 12763750

Caveolae are plasmalemmal domains enriched with cholesterol, caveolins, and signaling molecules. Endothelial cells in vivo are continuously exposed to shear conditions, and their caveolae density and location may be different from that of static cultured cells. Here, we show that chronic shear exposure regulates formation and localization of caveolae and caveolin-1 in bovine aortic endothelial cells (BAEC). Chronic exposure (1 or 3 days) of BAEC to laminar shear increased the total number of caveolae by 45-48% above static control. This increase was due to a rise in the luminal caveolae density without changing abluminal caveolae numbers or increasing caveolin-1 mRNA and protein levels. Whereas some caveolin-1 was found in the plasma membrane in static-cultured cells, it was predominantly localized in the Golgi. In contrast, chronic shear-exposed cells showed intense caveolin-1 staining in the luminal plasma membrane with minimum Golgi association. The preferential luminal localization of caveolae may play an important role in endothelial mechanosensing. Indeed, we found that chronic shear exposure (preconditioning) altered activation patterns of two well-known shear-sensitive signaling molecules (ERK and Akt) in response to a step increase in shear stress. ERK activation was blunted in shear preconditioned cells, whereas the Akt response was accelerated. These results suggest that chronic shear stimulates caveolae formation by translocating caveolin-1 from the Golgi to the luminal plasma membrane and alters cell signaling responses.

Bone Morphogenic Protein 4 Produced in Endothelial Cells by Oscillatory Shear Stress Stimulates an Inflammatory Response

The Journal of Biological Chemistry. Aug, 2003  |  Pubmed ID: 12766166

Atherosclerosis is now viewed as an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions, including oscillatory shear stress (OS), in branched arteries. In contrast, the arterial regions exposed to laminar shear (LS) are relatively lesion-free. The mechanisms underlying the opposite effects of OS and LS on the inflammatory and atherogenic processes are not clearly understood. Here, through DNA microarrays, protein expression, and functional studies, we identify bone morphogenic protein 4 (BMP4) as a mechanosensitive and pro-inflammatory gene product. Exposing endothelial cells to OS increased BMP4 protein expression, whereas LS decreased it. In addition, we found BMP4 expression only in the selective patches of endothelial cells overlying foam cell lesions in human coronary arteries. The same endothelial patches also expressed higher levels of intercellular cell adhesion molecule-1 (ICAM-1) protein compared with those of non-diseased areas. Functionally, we show that OS and BMP4 induced ICAM-1 expression and monocyte adhesion by a NFkappaB-dependent mechanism. We suggest that BMP4 is a mechanosensitive, inflammatory factor playing a critical role in early steps of atherogenesis in the lesion-prone areas.

Optimization of Isolation and Functional Characterization of Primary Murine Aortic Endothelial Cells

Endothelium : Journal of Endothelial Cell Research. 2003  |  Pubmed ID: 12791518

In this study the authors sought to improve the technique of isolating and culturing murine aortic endothelial cells (MAECs) for future use in studying endothelium-specific roles in complex pathologies such as atherosclerosis. The authors utilized a transgenic mouse expressing green fluorescent protein (GFP) under control of the endothelial specific Tie-2 promoter, allowing the tracking of endothelial cells throughout the isolation and purification. Briefly, aortas were harvested, the adventitia was removed, and strips were placed lumen side down into Matrigel. After 14 days, endothelial cells were isolated from the total cell population by fluorescence-activated cell sorting (FACS) using the GFP signal. The authors confirmed the endothelial phenotype of sorted cells based upon endothelial-specific Griffonia simplicifolia lectin staining, uptake of acetylated low-density lipoprotein (LDL), and von Willebrand factor (vWF) and VE-cadherin staining. The authors also confirmed the cells' ability to form tubes in Matrigel, and for cellular alignment with flow to occur following monolayer culture under unidirectional laminar shear stress but not following culture under oscillatory flow. Although GFP fluorescence--based vital sorting was used initially to optimize the isolation, the authors have verified that this method is applicable to the isolation of MAECs from other strains and backgrounds of mice through the use of other endothelial markers.

A Role for PYK2 in ANG II-dependent Regulation of the PHAS-1-eIF4E Complex by Multiple Signaling Cascades in Vascular Smooth Muscle

American Journal of Physiology. Cell Physiology. Dec, 2003  |  Pubmed ID: 12890645

Regulation of the PHAS-1-eukaryotic initiation factor-4E (eIF4E) complex is the rate-limiting step in the initiation of protein synthesis. This study characterized the upstream signaling pathways that mediate ANG II-dependent phosphorylation of PHAS-1 and eIF4E in vascular smooth muscle. ANG II-dependent PHAS-1 phosphorylation was maximal at 10 min (2.47 +/- 0.3 fold vs. control). This effect was completely blocked by the specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase, LY-294002), mammalian target of rapamycin, and extracellular signal-regulated kinase 1/2 (ERK1/2, U-0126) or by a recombinant adenovirus encoding dominant-negative Akt. PHAS-1 phosphorylation was followed by dissociation of eIF4E. Increased ANG II-induced eIF4E phosphorylation was observed at 45 min (2.63 +/- 0.5 fold vs. control), was maximal at 90 min (3.38 +/- 0.3 fold vs. control), and was sustained at 2 h. This effect was blocked by inhibitors of the ERK1/2 and p38 mitogen-activated protein (MAP) kinase pathways, but not by PI3-kinase inhibition, and was dependent on PKC, intracellular Ca2+, and tyrosine kinases. Downregulation of proline-rich tyrosine kinase 2 (PYK2) by antisense oligonucleotides led to a near-complete inhibition of PHAS-1 and eIF4E phosphorylation in response to ANG II. Therefore, PYK2 represents a proximal signaling intermediate that regulates ANG II-induced vascular smooth muscle cell protein synthesis via regulation of the PHAS-1-eIF4E complex.

Flow-dependent Regulation of Endothelial Nitric Oxide Synthase: Role of Protein Kinases

American Journal of Physiology. Cell Physiology. Sep, 2003  |  Pubmed ID: 12900384

Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca2+/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases-protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases-are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation.

Role of Xanthine Oxidoreductase and NAD(P)H Oxidase in Endothelial Superoxide Production in Response to Oscillatory Shear Stress

American Journal of Physiology. Heart and Circulatory Physiology. Dec, 2003  |  Pubmed ID: 12958034

Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.

Oscillatory Shear Stress Stimulates Endothelial Production of O2- from P47phox-dependent NAD(P)H Oxidases, Leading to Monocyte Adhesion

The Journal of Biological Chemistry. Nov, 2003  |  Pubmed ID: 12958309

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.

Endothelial NO Synthase Phosphorylated at SER635 Produces NO Without Requiring Intracellular Calcium Increase

Free Radical Biology & Medicine. Oct, 2003  |  Pubmed ID: 14583337

Shear stress stimulates NO production involving the Ca2+-independent mechanisms in endothelial cells. We have shown that exposure of bovine aortic endothelial cells (BAEC) to shear stress stimulates phosphorylation of eNOS at S635 and S1179 by the protein kinase A- (PKA-) dependent mechanisms. We examined whether phosphorylation of S635 of eNOS induced by PKA stimulates NO production in a calcium-independent manner. Expression of a constitutively active catalytic subunit of PKA (Cqr) in BAEC induced phosphorylation of S635 and S1179 residues and dephosphorylation of T497. Additionally, Cqr expression stimulated NO production, which could not be prevented by treating cells with the intracellular calcium chelator BAPTA-AM. To determine the role of each eNOS phosphorylation site in NO production, HEK-293 cells transfected with eNOS point mutants whereby S116, T497, S635, and S1179 were mutated to either A or D. Maximum NO production from S635D-expressing cells was significantly higher than that of either wild type or S635A in both basal and elevated [Ca2+]i conditions. More interestingly, S635D cells produced NO even when [Ca2+]i was nearly depleted by BAPTA-AM. We confirmed these results obtained in HEK-293 cells in BAEC transfected with S635D, S635A, or wild-type eNOS vector. These findings suggest that, once phosphorylated at S635 residue, eNOS produces NO without requiring any changes in [Ca2+]i. PKA-dependent phosphorylation of eNOS S635 and subsequent basal NO production in a Ca2+-independent manner may play an important role in regulating vascular biology and pathophysiology.

High Glucose Induced NF-kappaB DNA-binding Activity in HAEC is Maintained Under Low Shear Stress but Inhibited Under High Shear Stress: Role of Nitric Oxide

Atherosclerosis. Dec, 2003  |  Pubmed ID: 14644391

In the present study, we investigated whether low shear (LS, 2 dyn/cm2) favors high glucose (HG, 30 mM) induced nuclear factor kappa B (NF-kappaB) activity by regulating NO release in human aortic endothelial cells (HAEC). The results show that (i) under LS, the NF-kappaB activity of HAEC exposed to HG was significantly higher than HAEC in normal glucose (NG, 5.5mM) (P < 0.05). In contrast, under HS, the activation of NF-kappaB in HAEC exposed to HG showed no significant difference compared to that of NG. (ii) The NF-kappaB activity induced by HG is suppressed by high shear (HS) in the absence of a NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME) but restored in its presence, while LS + HG induced NF-kappaB activity remains the same in the presence or absence of L-NAME. (iii) Endothelial nitric oxide synthase (eNOS) protein expression and quantitative detection of NO indicated that high shear stress significantly induced higher eNOS expression and NO production compared to low shear stress condition. Collectively, these data suggest that HS exerts a protective effect on HG induced NF-kappaB activity through NO mediated signaling. LS, on the other hand, may down-regulate eNOS expression resulting in reduced NO release, and thereby maintain high glucose induced NF-kappaB DNA-binding activity. These observations explain, in part, the mechanism by means of which hyperglycemia accelerates the focal development of atherosclerotic lesions in low shear (lesion prone) areas of the arterial tree.

Bovine Caveolin-2 Cloning and Effects of Shear Stress on Its Localization in Bovine Aortic Endothelial Cells

Endothelium : Journal of Endothelial Cell Research. May-Aug, 2004  |  Pubmed ID: 15370296

Caveolae are plasmalemmal domains enriched with cholesterol, caveolins, and signaling molecules. Normally, cells that express caveolin-1 also express caveolin-2, but this has not been demonstrated in bovine aortic endothelial cells (BAECs). Here, we show that BAECs express caveolin-2, which localizes in caveolae with caveolin-1. We have cloned the bovine caveolin-2 gene and after comparison with known protein sequences (human, murine, rat, and canine) have found divergent immunogenic regions (amino acid [aa] 21 to aa 50 and aa 79 to 88), which may explain the inability to detect caveolin-2 in different cell types. We developed a bovine caveolin-2-specific antibody to examine this protein's expression and localization in BAECs. We used differential gradient centrifugations and immunoprecipitation to show that bovine caveolin-2 and caveolin-1 form a hetero-oligomer in plasma membrane caveolae. Using immunocytochemistry we show that a pool of caveolin-2 also colocalizes with the cis-Golgi in static culture, but unlike caveolin-1, this Golgi associated pool is maintained after 1 day of shear exposure. Therefore, the interaction of caveolin-2 with caveolin-1 could play an important role in caveolae biogenesis and shear stimulated mechano-signal transduction.

Bone Morphogenic Protein 4 Produced in Endothelial Cells by Oscillatory Shear Stress Induces Monocyte Adhesion by Stimulating Reactive Oxygen Species Production from a Nox1-based NADPH Oxidase

Circulation Research. Oct, 2004  |  Pubmed ID: 15388638

Atherosclerosis is an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions including oscillatory shear stress (OS). OS exposure induces endothelial expression of bone morphogenic protein 4 (BMP4), which in turn may activate intercellular adhesion molecule-1 (ICAM-1) expression and monocyte adhesion. OS is also known to induce monocyte adhesion by producing reactive oxygen species (ROS) from reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, raising the possibility that BMP4 may stimulate the inflammatory response by ROS-dependent mechanisms. Here we show that ROS scavengers blocked ICAM-1 expression and monocyte adhesion induced by BMP4 or OS in endothelial cells (ECs). Similar to OS, BMP4 stimulated H2O2 and O2- production in ECs. Next, we used ECs obtained from p47phox-/- mice (MAE-p47-/-), which do not produce ROS in response to OS, to determine the role of NADPH oxidases. Similar to OS, BMP4 failed to induce monocyte adhesion in MAE-p47-/-, but it was restored when the cells were transfected with p47phox plasmid. Moreover, OS-induced O2- production was blocked by noggin (a BMP antagonist), suggesting a role for BMP. Furthermore, OS increased gp91phox (nox2) and nox1 mRNA levels while decreasing nox4. In contrast, BMP4 induced nox1 mRNA expression, whereas nox2 and nox4 were decreased or not affected, respectively. Also, OS-induced monocyte adhesion was blocked by knocking down nox1 with the small interfering RNA (siRNA). Finally, BMP4 siRNA inhibited OS-induced ROS production and monocyte adhesion. Together, these results suggest that BMP4 produced in ECs by OS stimulates ROS release from the nox1-dependent NADPH oxidase leading to inflammation, a critical early atherogenic step.

Simulated Microgravity Using the Random Positioning Machine Inhibits Differentiation and Alters Gene Expression Profiles of 2T3 Preosteoblasts

American Journal of Physiology. Cell Physiology. Jun, 2005  |  Pubmed ID: 15689415

Exposure to microgravity causes bone loss in humans, and the underlying mechanism is thought to be at least partially due to a decrease in bone formation by osteoblasts. In the present study, we examined the hypothesis that microgravity changes osteoblast gene expression profiles, resulting in bone loss. For this study, we developed an in vitro system that simulates microgravity using the Random Positioning Machine (RPM) to study the effects of microgravity on 2T3 preosteoblast cells grown in gas-permeable culture disks. Exposure of 2T3 cells to simulated microgravity using the RPM for up to 9 days significantly inhibited alkaline phosphatase activity, recapitulating a bone loss response that occurs in real microgravity conditions without altering cell proliferation and shape. Next, we performed DNA microarray analysis to determine the gene expression profile of 2T3 cells exposed to 3 days of simulated microgravity. Among 10,000 genes examined using the microarray, 88 were downregulated and 52 were upregulated significantly more than twofold using simulated microgravity compared with the static 1-g condition. We then verified the microarray data for some of the genes relevant in bone biology using real-time PCR assays and immunoblotting. We confirmed that microgravity downregulated levels of alkaline phosphatase, runt-related transcription factor 2, osteomodulin, and parathyroid hormone receptor 1 mRNA; upregulated cathepsin K mRNA; and did not significantly affect bone morphogenic protein 4 and cystatin C protein levels. The identification of gravisensitive genes provides useful insight that may lead to further hypotheses regarding their roles in not only microgravity-induced bone loss but also the general patient population with similar pathological conditions, such as osteoporosis.

Transcriptional Profiles of Valvular and Vascular Endothelial Cells Reveal Phenotypic Differences: Influence of Shear Stress

Arteriosclerosis, Thrombosis, and Vascular Biology. Jan, 2006  |  Pubmed ID: 16293796

The similarities between valvular and vascular lesions suggest pathological initiation mediated through endothelium, but the role of hemodynamics in valvular endothelial biology is poorly understood.

Mechanical Inhibition of RANKL Expression is Regulated by H-Ras-GTPase

The Journal of Biological Chemistry. Jan, 2006  |  Pubmed ID: 16306046

Mechanical input is known to regulate bone remodeling, yet the molecular events involved in mechanical signal transduction are poorly understood. We here investigate proximal events leading to the ERK1/2 activation that is required for mechanical repression of RANKL (receptor activator of NF-kappaB ligand) expression, the factor that controls local recruitment of osteoclasts. Using primary murine bone stromal cells we show that dynamic mechanical strain via substrate deformation activates Ras-GTPase, in particular the H-Ras isoform. Pharmacological inhibition of H-Ras function prevents strain activation of H-Ras as well as the downstream mechanical repression of RANKL. Furthermore, small interfering RNA silencing of H-Ras, but not K-Ras, abrogates mechanical strain repression of RANKL. H-Ras-specific inhibition of mechanorepression of RANKL was also demonstrated in a murine pre-osteoblast cell line (CIMC-4). The requirement of cholesterol for H-Ras activation was probed; cholesterol depletion of rafts using methyl-betacyclodextrin prevented mechanical H-Ras activation. That the mechanical repression of RANKL requires activation of H-Ras, a specific isoform of Ras-GTP that is known to reside in the lipid raft microdomain, suggests that spatial arrangements are critical for generation of specific downstream events in response to mechanical signals. By partitioning signals this way, cells may be able to generate different downstream responses through seemingly similar signaling cascades.

Plasma Membrane Requirements for 1alpha,25(OH)2D3 Dependent PKC Signaling in Chondrocytes and Osteoblasts

Steroids. Apr, 2006  |  Pubmed ID: 16325216

1,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] acts on chondrocytes and osteoblasts through traditional nuclear Vitamin D receptor (VDR) mechanisms as well as through rapid actions on plasma membranes that initiate intracellular signaling pathways. We have investigated the mechanisms involved in activation of protein kinase C (PKC) and downstream biological responses that depend on the latter pathway. These studies show that PKC activation depends on presence of a membrane receptor ERp60 and rapid increases in phospholipase A(2) (PLA(2)) activity. Cells that are responsive to 1alpha,25(OH)(2)D(3) express PLA(2) activating protein (PLAA), suggesting a link between ERp60 and PLA(2). Increased PLA(2) results in increased arachidonic acid release and formation of lysophospholipid, which then activates phospholipase C beta (PLCbeta), leading to rapid formation of inositol-trisphosphate (IP3) and diacylglycerol (DAG). PLA(2), PLC, and DAG are all associated with lipid rafts including caveolae in many cells, suggesting that the caveolar environment may be an important mediator of PKC activation by 1alpha,25(OH)(2)D(3). Here, we use the VDR(-/-) mouse costochondral cartilage growth plate to examine the expression of ERp60 and PLAA in vivo in 1alpha,25(OH)(2)D(3)-responsive hypertrophic chondrocytes (growth zone cells) and in resting zone cells that do not respond to this Vitamin D metabolite in vitro. In addition, we determined if intact lipid rafts are required for the response of rat costochondral cartilage growth zone cells to 1alpha,25(OH)(2)D(3). The results show that ERp60 and PLAA are localized to 1alpha,25(OH)(2)D(3)-responsive growth zone cells and metaphyseal osteoblasts, even in VDR(-/-) mice. Disruption of lipid rafts using beta-cyclodextrin blocks the activation of PKC by 1alpha,25(OH)(2)D(3) and reduces the ability of 1alpha,25(OH)(2)D(3) to regulate [(35)S]-sulfate incorporation.

Caveolin-1 is Transiently Dephosphorylated by Shear Stress-activated Protein Tyrosine Phosphatase Mu

Biochemical and Biophysical Research Communications. Jan, 2006  |  Pubmed ID: 16325778

Endothelial cells are subjected to hemodynamic shear stress, which regulates multiple vascular functions partially by the caveolin-1-dependent mechanisms. Caveolin-1 is a principal protein in the plasma membrane microdomains called caveolae and interacts with various signaling molecules. Recently, caveolin-1 was elucidated to be phosphorylated on tyrosine 14. However, it is not known how phosphorylation of caveolin-1 is controlled in endothelium. In this study, we found that caveolin-1 is phosphorylated by p38 mitogen-activated protein kinase (MAPK) under a static condition. When endothelial cells were exposed to shear stress, caveolin-1 was transiently dephosphorylated. Since the activity of p38 MAPK was not affected by shear stress, the shear-dependent dephosphorylation of caveolin-1 was not mediated by p38 MAPK. Of interest, sodium orthovanadate, an inhibitor for phosphatases, blocked the shear-dependent dephosphorylation of caveolin-1. We also observed that protein tyrosine phosphatase mu was transiently activated by shear stress, suggesting its role in the dephosphorylation of caveolin-1.

Ascorbic Acid Synthesis Due to L-gulono-1,4-lactone Oxidase Expression Enhances NO Production in Endothelial Cells

Biochemical and Biophysical Research Communications. Jul, 2006  |  Pubmed ID: 16737683

As a primary antioxidant, ascorbic acid (AA) provides beneficial effects for vascular health mitigating oxidative stress and endothelial dysfunction. However, the association of intracellular AA with NO production occurring inside the endothelial cells remains unclear. In the present study, we addressed this issue by increasing intracellular AA directly through de novo synthesis. To restore AA synthesis pathway, bovine aortic endothelial cells were transfected with the plasmid vector encoding L-gulono-1,4-lactone oxidase (GULO, EC 1.1.3.8), the missing enzyme converting L-gulono-1,4-lactone (GUL) to AA. Functional expression of GULO was verified by Western blotting and in vitro enzyme activity assay. GULO expression alone did not lead to AA synthesis but the supply of GUL resulted in a marked increase of intracellular AA. When the cells were stimulated with calcium ionophore, A23187, NO production was more active in the GULO-expressing cells supplied with GUL, in comparison with the cells without GULO expression or without GUL supply, indicating that intracellular AA regulated NO production. Enhancement of NO production by intracellular AA was further verified in aortic endothelial cells obtained from eNOS knockout mice that were cotransfected with eNOS and GULO constructs. GULO-dependent AA synthesis also elevated intracellular tetrahydrobiopterin content, implicating that this essential cofactor of endothelial nitric oxide synthase (eNOS) might mediate the AA effect. The present study strongly suggests that intracellular AA plays critical roles in vascular physiology through enhancing endothelial NO production.

Coordinated Regulation of Endothelial Nitric Oxide Synthase Activity by Phosphorylation and Subcellular Localization

Free Radical Biology & Medicine. Jul, 2006  |  Pubmed ID: 16781462

Endothelial nitric oxide synthase (eNOS) is regulated by multiple mechanisms including Ca(2+)/calmodulin binding, protein-protein interactions, phosphorylation, and subcellular locations. Emerging evidence suggests that these seemingly independent mechanisms may be closely correlated. In the present study, the interplay between membrane targeting and phosphorylation of eNOS was investigated by using various mutants designed to target specific subcellular locations or to mimic different phospho states. Phospho-mimicking mutations of wild-type eNOS at S635 and S1179 synergistically activated the enzyme. The targeted eNOS mutants to plasma membrane and Golgi complex exhibited higher NO production activities than that of a myristoylation-deficient cytosolic mutant. Phospho-mimicking mutations at S635 and S1179 rescued the activity of the cytosolic mutant and increased those of the plasma membrane- and Golgi-targeted mutants. In contrast, phospho-deficient mutations at these sites led to inactivation of eNOS. Unlike the other targeted mutants, the cytosolic eNOS mutant was unresponsive to cAMP, indicating that membrane association and phosphorylation are required for eNOS activation. These findings suggest that the coordinated interplay between phosphorylation and subcellular localization of eNOS plays an important role in regulating NO production in endothelial cells.

Laminar Shear Stress Inhibits Cathepsin L Activity in Endothelial Cells

Arteriosclerosis, Thrombosis, and Vascular Biology. Aug, 2006  |  Pubmed ID: 16709945

The cysteine proteases, cathepsins, have been implicated in vascular remodeling and atherosclerosis, processes known to be regulated by shear stress. It is not known, however, whether shear regulates cathepsins. We examined the hypothesis that shear stress regulates cathepsin activity in endothelial cells.

Bone Morphogenic Protein-4 Induces Hypertension in Mice: Role of Noggin, Vascular NADPH Oxidases, and Impaired Vasorelaxation

Circulation. Jun, 2006  |  Pubmed ID: 16769910

Recent in vitro studies have shown that disturbed flow and oxidative conditions induce the expression of bone morphogenic proteins (BMPs 2 and 4) in cultured endothelial cells. BMPs can stimulate superoxide production and inflammatory responses in endothelial cells, raising the possibility that BMPs may play a role in vascular diseases such as hypertension and atherosclerosis. In this study, we examined the hypothesis that BMP4 would induce hypertension in intact animals by increasing superoxide production from vascular nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and an impairment of vasodilation responses.

Embryonic Stem Cell-derived Endothelial Cells May Lack Complete Functional Maturation in Vitro

Journal of Vascular Research. 2006  |  Pubmed ID: 16877873

Stem cell therapies will only become clinically relevant if the stem cells differentiated in vitro function as their in vivo counterparts. Here, we employed our previously developed techniques for deriving endothelial cells (>96% purity) from mouse embryonic stem cells (ESC) and compared these with mouse aortic endothelial cells (MAEC) obtained from thoracic aortas. Immunocytochemical analysis of ESC-derived endothelial cells (EC) demonstrates that both cell types are positive for the EC markers endothelial nitric oxide synthase (eNOS), Flk-1, Flt-1, vascular endothelial cadherin (VEcad), platelet-endothelial cell adhesion molecule-1 (PECAM-1), and CD34. However, ESC-derived EC express slightly lower levels of PECAM-1 and VE-cadherin, and significantly lower levels of acetylated low-density lipoprotein (LDL) uptake and von Willebrand factor. Although ESC-derived EC do express VE-cadherin, the VE-cadherin in the ESC-derived EC did not localize as well at the cell-cell junctions as in the MAEC. Interestingly, ESC-derived EC express much greater levels of the endothelial and hematopoietic stem cell marker CD34 and vasculogenic and angiogenic sprouting than MAEC. These results indicate that ESC-derived EC share some key characteristics of 'mature' EC, while retaining markers of alternate phenotypes including immature endothelium.

Early Determinants of H2O2-induced Endothelial Dysfunction

Free Radical Biology & Medicine. Sep, 2006  |  Pubmed ID: 16895801

Reactive oxygen species (ROS) can stimulate nitric oxide (NO(*)) production from the endothelium by transient activation of endothelial nitric oxide synthase (eNOS). With continued or repeated exposure, NO(*) production is reduced, however. We investigated the early determinants of this decrease in NO(*) production. Following an initial H(2)O(2) exposure, endothelial cells responded by increasing NO(*) production measured electrochemically. NO(*) concentrations peaked by 10 min with a slow reduction over 30 min. The decrease in NO(*) at 30 min was associated with a 2.7-fold increase in O(2)(*-) production (p < 0.05) and a 14-fold reduction of the eNOS cofactor, tetrahydrobiopterin (BH(4), p < 0.05). Used as a probe for endothelial dysfunction, the integrated NO(*) production over 30 min upon repeated H(2)O(2) exposure was attenuated by 2.1-fold (p = 0.03). Endothelial dysfunction could be prevented by BH(4) cofactor supplementation, by scavenging O(2)(*-) or peroxynitrite (ONOO(-)), or by inhibiting the NADPH oxidase. Hydroxyl radical (()OH) scavenging did not have an effect. In summary, early H(2)O(2)-induced endothelial dysfunction was associated with a decreased BH(4) level and increased O(2)(*-) production. Dysfunction required O(2)(*-), ONOO(-), or a functional NADPH oxidase. Repeated activation of the NADPH oxidase by ROS may act as a feed forward system to promote endothelial dysfunction.

Role of NADPH Oxidases in Disturbed Flow- and BMP4- Induced Inflammation and Atherosclerosis

Antioxidants & Redox Signaling. Sep-Oct, 2006  |  Pubmed ID: 16987015

Atherosclerosis is an inflammatory disease, occurring preferentially in branched or curved arterial regions exposed to disturbed flow conditions including oscillatory shear stress (OS). In contrast, straight portions exposed to undisturbed laminar shear stress (LS) are relatively lesion free. The opposite effects of atheroprotective LS and proatherogenic OS are likely to be determined by differential expression of genes and proteins, including redox regulating factors. OS induces inflammation via mechanisms involving increased reactive oxygen species (ROS) production from the NADPH oxidases. Through a transcript profiling study and subsequent verification and functional studies, the authors discovered that OS induces inflammation by producing bone morphogenic protein 4 (BMP4) in endothelial cells. BMP4 stimulates expression and activity of NADPH oxidase requiring p47phox and Nox-1 in an autocrine-like manner. The NADPH oxidase activation by BMP4 then leads to ROS production, NF-kappaB activation, intercellular adhesion molecule 1 (ICAM-1) expression, and subsequent increased monocyte adhesivity of endothelial cells. It is proposed that endothelial NADPH oxidases play a critical role in disturbed flow- and BMP4-dependent inflammation, which is the critical early atherogenic response occurring in atheroprone areas. This emerging field of shear stress, BMP4, NADPH oxidases, inflammation, and atherosclerosis is reviewed.

Regulation of Growth Plate Chondrocytes by 1,25-dihydroxyvitamin D3 Requires Caveolae and Caveolin-1

Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research. Oct, 2006  |  Pubmed ID: 16995819

We examined the role of caveolae and caveolin-1 in the mechanism of 1alpha,25(OH)(2)D(3) action in growth plate chondrocytes. We found that caveolae are required for rapid 1alpha,25(OH)(2)D(3)-dependent PKC signaling, and caveolin-1 must be present based on studies using chondrocytes from Cav-1(-/-) mice.

An Ex Vivo Study of the Biological Properties of Porcine Aortic Valves in Response to Circumferential Cyclic Stretch

Annals of Biomedical Engineering. Nov, 2006  |  Pubmed ID: 17031600

Normal physiological mechanical forces cause constant tissue renewal in aortic valve leaflets (AVL) while altered mechanical forces incite changes in their structural and biological properties. The current study aims at characterizing the remodeling properties of AVL subjected to cyclic circumferential stretch in a sterile ex vivo bioreactor. The leaflets cultured were stretched at a maximum rate of 300%s(-1) corresponding to a 15% strain for 48 h. Collagen, sulfated glycosaminoglycan (sGAG), and elastin contents of the stretched, fresh, and statically incubated leaflets were measured. Cusp morphology and cell phenotype were also examined. AVLs exposed to cyclic stretch showed a significant increase in collagen content (p < 0.05) when compared to fresh and statically incubated AVLs. sGAG content was significantly reduced in the stretched AVLs (p < 0.05) when compared to the fresh leaflets and was comparable between stretched and statically incubated AVLs. There was no statistically significant change in elastin content in all the three groups of AVLs (p > 0.05). Native aortic valve morphology was well preserved in stretched leaflets. Immunohistochemistry and immunoblotting studies showed an increased expression of alpha-smooth muscle actin (alpha-SMA) in stretched leaflets while alpha-SMA expression was reduced in statically incubated AVLs when compared to the fresh leaflets. To conclude, circumferential cyclic stretch altered the extracellular matrix remodeling activity of valvular cells, and consequently the extracellular matrix composition of the AVLs. Most interestingly, the contractile and fibrotic phenotypic expression of valve interstitial cells was enhanced. These results show that circumferential cyclic stretch is a possible mediator for AVL remodeling activity.

Cyclic Pressure and Shear Stress Regulate Matrix Metalloproteinases and Cathepsin Activity in Porcine Aortic Valves

The Journal of Heart Valve Disease. Sep, 2006  |  Pubmed ID: 17044366

It has been shown previously that the exposure of porcine aortic valve leaflets (PAVL) to elevated pressure or steady shear stress increases extracellular matrix (ECM) synthesis. It was hypothesized that exposure of the aortic valves to pressure or shear stress would regulate valvular expression of proteases such as matrix metalloproteinases (MMPs) and cathepsins.

An Improved Method to Measure Nitrate/nitrite with an NO-selective Electrochemical Sensor

Nitric Oxide : Biology and Chemistry / Official Journal of the Nitric Oxide Society. Mar, 2007  |  Pubmed ID: 17056288

Nitric oxide (NO) produced from NO synthase(s) (NOS) is an important cell signaling molecule in physiology and pathophysiology. It remains challenging, however, to measure NO accurately and reproducibly in many cell types producing relatively low levels of NO from the enzymes such as endothelial NO synthase (eNOS). In the present study, we describe a very sensitive and convenient analytical method that affords measurement of 1 to 2 nM concentration of NO(x) (nitrite plus nitrate) in culture media. In the present study, we used an ultra-sensitive NO-selective electrochemical sensor (AmiNO700) in combination with a highly efficient nitrate conversion method, which coupled the nitrate reductase step with the glucose-6-phosphate dehydrogenase system. An aliquot of conditioned culture media was first treated with nitrate reductase, NADPH, glucose-6-phosphate dehydrogenase and glucose-6-phosphate to convert nitrate to nitrite quantitatively. The nitrite (that is present originally plus the reduced nitrate) was then reduced to equimolar NO in an acidic iodide bath while NO was being detected by the sensor. With this analytical method, we can quantitatively and reliably measure basal and stimulated NO release from cultured endothelial cells. We believe this improved assay should be useful in measuring a wide range of NO levels, especially the low but physiologically relevant levels, in many cell types.

Expression of Cathepsin K is Regulated by Shear Stress in Cultured Endothelial Cells and is Increased in Endothelium in Human Atherosclerosis

American Journal of Physiology. Heart and Circulatory Physiology. Mar, 2007  |  Pubmed ID: 17098827

Cathepsins, the lysosomal cysteine proteases, are involved in vascular remodeling and atherosclerosis. Genetic knockout of cathepsins S and K in mice has shown to reduce atherosclerosis, although the molecular mechanisms remain unclear. Because atherosclerosis preferentially occurs in arteries exposed to disturbed flow conditions, we hypothesized that shear stress would regulate cathepsin K expression and activity in endothelial cells. Mouse aortic endothelial cells (MAEC) exposed to proatherogenic oscillatory shear (OS, +/- 5 dyn/cm(2) for 1 day) showed significantly higher cathepsin K expression and activity than that of atheroprotective, unidirectional laminar shear stress (LS, 15 dyn/cm(2) for 1 day). Western blot and active-site labeling studies showed an active, mature form of cathepsin K in the conditioned medium of MAEC exposed to OS but not in that of LS. Functionally, MAEC exposed to OS significantly increased elastase and gelatinase activity above that of LS. The OS-dependent elastase and gelatinase activities were significantly reduced by knocking down cathepsin K with small-interfering (si) RNA, but not by a nonsilencing siRNA control, suggesting that cathepsin K is a shear-sensitive protease. In addition, immunohistochemical analysis of atherosclerotic human coronary arteries showed a positive correlation between the cathepsin K expression levels in endothelium and elastic lamina integrity. These findings suggest that cathepsin K is a mechanosensitive, extracellular matrix protease that, in turn, may be involved in arterial wall remodeling and atherosclerosis.

Reactive Oxygen Species-selective Regulation of Aortic Inflammatory Gene Expression in Type 2 Diabetes

American Journal of Physiology. Heart and Circulatory Physiology. May, 2007  |  Pubmed ID: 17237245

Vascular diseases are a major complication of diabetes mellitus (DM), although their etiology is poorly understood. NADPH oxidase-derived reactive oxygen species (ROS) production and inflammation are potential mediators of DM-associated vascular diseases. Using db/db mice as a Type 2 diabetes model, we examined the relationship between NADPH oxidase-derived ROS and vascular inflammation. When compared with control m+/+ mice, aortas from 4- and 12-wk-old db/db mice had higher NADPH oxidase activity and increased superoxide levels, leading to NADPH oxidase-dependent impaired vasodilation at 12 wk. Diabetes progression from 4 to 12 wk led to increased Nox1, Nox4, and p22(phox) subunit mRNAs and induced the expression of a group of matrix remodeling-related cytokines: connective tissue growth factor (CTGF), bone morphogenetic protein 4 (BMP-4), and osteopontin (OPN). After 8 wk of treatment with the superoxide scavenger Tempol, 12-wk-old db/db mice had lower superoxide production, reduced plasma glucose and lipids, and lower BMP-4 and OPN protein expression when compared with nontreated mice. No changes were observed with Tempol in CTGF or m+/+ mice. The ability of Tempol to reverse ROS production as well as OPN and BMP-4, but not CTGF, induction suggests that DM-induced vascular inflammation involves both ROS-sensitive and -insensitive pathways.

Identification of Mechanosensitive Genes in Osteoblasts by Comparative Microarray Studies Using the Rotating Wall Vessel and the Random Positioning Machine

Journal of Cellular Biochemistry. Jun, 2007  |  Pubmed ID: 17243119

Weightlessness or microgravity of spaceflight induces bone loss due in part to decreased bone formation by unknown mechanisms. Due to difficulty in performing experiments in space, several ground-based simulators such as the Rotating Wall Vessel (RWV) and Random Positioning Machine (RPM) have become critical venues to continue studying space biology. However, these simulators have not been systematically compared to each other or to mechanical stimulating models. Here, we hypothesized that exposure to RWV inhibits differentiation and alters gene expression profiles of 2T3 cells, and a subset of these mechanosensitive genes behaves in a manner consistent to the RPM and opposite to the trends incurred by mechanical stimulation of mouse tibiae. Exposure of 2T3 preosteoblast cells to the RWV for 3 days inhibited alkaline phosphatase activity, a marker of differentiation, and downregulated 61 and upregulated 45 genes by more than twofold compared to static 1 g controls, as shown by microarray analysis. The microarray results were confirmed by real-time PCR and/or Western blots for seven separate genes and proteins including osteomodulin, runx2, and osteoglycin. Comparison of the RWV data to the RPM microarray study that we previously published showed 14 mechanosensitive genes that changed in the same direction. Further comparison of the RWV and RPM results to microarray data from mechanically loaded mouse tibiae reported by an independent group revealed that three genes including osteoglycin were upregulated by the loading and downregulated by our simulators. These mechanosensitive genes may provide novel insights into understanding the mechanisms regulating bone formation and potential targets for countermeasures against decreased bone formation during space flight and in pathologies associated with lack of bone formation.

Reversible Glutathiolation of Caspase-3 by Glutaredoxin As a Novel Redox Signaling Mechanism in Tumor Necrosis Factor-alpha-induced Cell Death

Circulation Research. Feb, 2007  |  Pubmed ID: 17272816

Emerging Role of IGF-1R in Stretch-induced Neointimal Hyperplasia in Venous Grafts

Arteriosclerosis, Thrombosis, and Vascular Biology. Aug, 2007  |  Pubmed ID: 17634521

NFKB1 Promoter Variation Implicates Shear-induced NOS3 Gene Expression and Endothelial Function in Prehypertensives and Stage I Hypertensives

American Journal of Physiology. Heart and Circulatory Physiology. Oct, 2007  |  Pubmed ID: 17644577

In endothelial cells, NF-kappaB is an important intracellular signaling molecule by which changes in wall shear stress are transduced into the nucleus to initiate downstream endothelial nitric oxide synthase (NOS3) gene expression. We investigated whether NF-kappa light-chain gene enhancer in B cells 1 (NFKB1) promoter polymorphism ((-94)NFKB1 I/D, where I is the insertion allele and D is the deletion allele) was associated with 1) NOS3 gene expression in endothelial cells under physiological levels of unidirectional laminar shear stress (LSS) and 2) endothelial function in prehypertensive and stage I hypertensive individuals before and after a 6-mo supervised endurance exercise intervention. Competitive EMSAs revealed that proteins present in the nuclei of endothelial cells preferentially bound to the I allele NFKB1 promoter compared with the D allele. Reporter gene assays showed that the I allele promoter had significantly higher activity than the D allele. In agreement with these observations, homozygous II genotype cells had higher p50 expression levels than homozygous DD genotype cells. Cells with the homozygous II genotype showed a greater increase in NOS3 protein expression than did homozygous DD genotype cells under LSS. Functional experiments on volunteers confirmed higher baseline reactive hyperemic forearm blood flow, and, furthermore, the subgroup analysis revealed that DD homozygotes were significantly less prevalent in the exercise responder group compared with II and ID genotypes. We conclude that the (-94)NFKB1 I/D promoter variation contributes to the modulation of vascular function and adaptability to exercise-induced flow shear stress, most likely due to differences in NFKB1 gene transactivity.

Laminar Shear Inhibits Tubule Formation and Migration of Endothelial Cells by an Angiopoietin-2 Dependent Mechanism

Arteriosclerosis, Thrombosis, and Vascular Biology. Oct, 2007  |  Pubmed ID: 17673702

Fluid shear stress plays a role in angiogenesis. Laminar shear stress (LS) promotes endothelial cell (EC) quiescence, whereas oscillatory shear stress (OS) promotes EC turnover and dysfunction, which could lead to pathological angiogenesis. We hypothesized that LS inhibits EC migration and tubule formation, 2 functions important in angiogenesis, by inhibiting the secretion of proangiogenic factors.

Bone Morphogenic Protein Antagonists Are Coexpressed with Bone Morphogenic Protein 4 in Endothelial Cells Exposed to Unstable Flow in Vitro in Mouse Aortas and in Human Coronary Arteries: Role of Bone Morphogenic Protein Antagonists in Inflammation and Atherosclerosis

Circulation. Sep, 2007  |  Pubmed ID: 17785623

Exposure to disturbed flow, including oscillatory shear stress, stimulates endothelial cells (ECs) to produce bone morphogenic protein (BMP) 4, which in turn activates inflammation, a critical atherogenic step. BMP activity is regulated by the level of BMP antagonists. Until now it was not known whether shear also regulates the expression of BMP antagonists and whether they play a role in EC pathophysiology.

Downregulation of Bone Morphogenetic Protein 4 Expression in Coronary Arterial Endothelial Cells: Role of Shear Stress and the CAMP/protein Kinase A Pathway

Arteriosclerosis, Thrombosis, and Vascular Biology. Apr, 2007  |  Pubmed ID: 17272757

Bone morphogenetic protein 4 (BMP-4) is a transforming growth factor beta family member cytokine that exerts proinflammatory effects on the endothelium and is likely to play a role in atherogenesis. Recent studies suggested that atheroprotective levels of shear stress control endothelial BMP-4 expression; however, the underlying mechanisms remained unknown.

Caveolin-1 Knockout Mice Have Increased Bone Size and Stiffness

Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research. Sep, 2007  |  Pubmed ID: 17550335

The skeletal phenotype of the cav-1(-/-) mouse, which lacks caveolae, was examined. muCT and histology showed increased trabecular and cortical bone caused by the gene deletion. Structural changes were accompanied by increased mechanical properties. Cell studies showed that cav-1 deficiency leads to increased osteoblast differentiation. These results suggest that cav-1 helps to maintain osteoblast progenitors in a less differentiated state.

Laminar Shear Stress Up-regulates Peroxiredoxins (PRX) in Endothelial Cells: PRX 1 As a Mechanosensitive Antioxidant

The Journal of Biological Chemistry. Jan, 2008  |  Pubmed ID: 18024958

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/cm(2)), OS (+/-5 dyn/cm(2), 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.

Target Accessibility and Signal Specificity in Live-cell Detection of BMP-4 MRNA Using Molecular Beacons

Nucleic Acids Research. Mar, 2008  |  Pubmed ID: 18276638

The ability to visualize mRNA in single living cells and monitor in real-time the changes of mRNA level and localization can provide unprecedented opportunities for biological and disease studies. However, the mRNA detection specificity and sensitivity are critically dependent on the selection of target sequences and their accessibility. We carried out an extensive study of the target accessibility of BMP-4 mRNA using 10 different designs of molecular beacons (MBs), and identified the optimal beacon design. Specifically, for MB design 1 and 8 (MB1 and MB8), the fluorescent intensities from BMP-4 mRNA correlated well with the GFP signal after upregulating BMP-4 and co-expressing GFP using adenovirus, and the knockdown of BMP-4 mRNA using siRNA significantly reduced the beacon signals, demonstrating detection specificity. The beacon specificity was further confirmed using blocking RNA and in situ hybridization. We found that fluorescence signal from MBs depends critically on target sequences; the target sequences corresponding to siRNA sites may not be good sites for beacon-based mRNA detection, and vice versa. Possible beacon design rules are identified and approaches for enhancing target accessibility are discussed. This has significant implications to MB design for live cell mRNA detection.

X-linked Inhibitor of Apoptosis Protein is an Important Regulator of Vascular Endothelial Growth Factor-dependent Bovine Aortic Endothelial Cell Survival

Circulation Research. Apr, 2008  |  Pubmed ID: 18309102

Vascular endothelial growth factor (VEGF) is a critical regulator of endothelial cell biology and vascular function. Chronic VEGF treatment has been shown to inhibit tumor necrosis factor-induced apoptosis in endothelial cells. However, the mechanism for this cell survival is unclear. Interestingly, VEGF also enhances the expression of X-linked inhibitor of apoptosis (XIAP), a well-established antiapoptotic factor. XIAP has been shown to suppress apoptosis by blocking caspase activity in cancer cells, but it remains under studied in the endothelium. Therefore, we hypothesized that VEGF affects important endothelial functions, such as apoptosis and cell migration, by regulating XIAP expression and downstream caspase activity. To test this hypothesis, caspase activity, apoptosis, and cell migration were assessed following XIAP overexpression or depletion in bovine aortic endothelial cells. Much like VEGF treatment, ectopic expression of XIAP blocked tumor necrosis factor-induced apoptosis. Surprisingly, the mechanism was caspase-independent. In addition, XIAP-associated cell survival was the result of enhanced nitric oxide (NO) production, and XIAP was partially localized in caveolae. In these lipid rafts, XIAP interacted with a regulator of NO production, caveolin-1, via a binding motif (FtFgtwiY, where the bold letters represent aromatic amino acids) in the baculoviral IAP repeat-3 domain. Endothelial NO synthase binding to caveolin-1 was competitively inhibited by XIAP, suggesting that XIAP acts as a modulator of NO production by releasing endothelial NO synthase from caveolin-1. Further studies showed that endothelial cell migration was also controlled by XIAP-dependent NO. Taken together, these results suggest that XIAP plays a novel role in endothelial cells, interacting with caveolin-1 and acting as a regulator of vascular antiatherogenic function.

Design of an Ex Vivo Culture System to Investigate the Effects of Shear Stress on Cardiovascular Tissue

Journal of Biomechanical Engineering. Jun, 2008  |  Pubmed ID: 18532871

Mechanical forces are known to affect the biomechanical properties of native and engineered cardiovascular tissue. In particular, shear stress that results from the relative motion of heart valve leaflets with respect to the blood flow is one important component of their mechanical environment in vivo. Although different types of bioreactors have been designed to subject cells to shear stress, devices to expose biological tissue are few. In an effort to address this issue, the aim of this study was to design an ex vivo tissue culture system to characterize the biological response of heart valve leaflets subjected to a well-defined steady or time-varying shear stress environment. The novel apparatus was designed based on a cone-and-plate viscometer. The device characteristics were defined to limit the secondary flow effects inherent to this particular geometry. The determination of the operating conditions producing the desired shear stress profile was streamlined using a computational fluid dynamic (CFD) model validated with laser Doppler velocimetry. The novel ex vivo tissue culture system was validated in terms of its capability to reproduce a desired cone rotation and to maintain sterile conditions. The CFD results demonstrated that a cone angle of 0.5 deg, a cone radius of 40 mm, and a gap of 0.2 mm between the cone apex and the plate could limit radial secondary flow effects. The novel cone-and-plate permits to expose nine tissue specimens to an identical shear stress waveform. The whole setup is capable of accommodating four cone-and-plate systems, thus concomitantly subjecting 36 tissue samples to desired shear stress condition. The innovative design enables the tissue specimens to be flush mounted in the plate in order to limit flow perturbations caused by the tissue thickness. The device is capable of producing shear stress rates of up to 650 dyn cm(-2) s(-1) (i.e., maximum shear stress rate experienced by the ventricular surface of an aortic valve leaflet) and was shown to maintain tissue under sterile conditions for 120 h. The novel ex vivo tissue culture system constitutes a valuable tool toward elucidating heart valve mechanobiology. Ultimately, this knowledge will permit the production of functional tissue engineered heart valves, and a better understanding of heart valve biology and disease progression.

Differential Proinflammatory and Prooxidant Effects of Bone Morphogenetic Protein-4 in Coronary and Pulmonary Arterial Endothelial Cells

American Journal of Physiology. Heart and Circulatory Physiology. Aug, 2008  |  Pubmed ID: 18539760

There is increasing evidence that TGF-beta family member cytokine bone morphogenetic protein (BMP)-4 plays different pathophysiological roles in the pulmonary and systemic circulation. Upregulation of BMP-4 has been linked to atherosclerosis and hypertension in the systemic circulation, whereas disruption of BMP-4 signaling is associated with the development of pulmonary hypertension. To test the hypothesis that BMP-4 elicits differential effects in the pulmonary and systemic circulation, we compared the prooxidant and proinflammatory effects of BMP-4 in cultured human coronary arterial endothelial cells (CAECs) and pulmonary arterial endothelial cells (PAECs). We found that BMP-4 (from 0.3 to 10 ng/ml) in CAECs increased O(2)(*-) and H(2)O(2) generation, induced NF-kappaB activation, upregulated ICAM-1, and induced monocyte adhesiveness to ECs. In contrast, BMP-4 failed to induce oxidative stress or endothelial activation in PAECs. Also, BMP-4 treatment impaired acetylcholine-induced relaxation and increased O(2)(*-) production in cultured rat carotid arteries, whereas cultured rat pulmonary arteries were protected from these adverse effects of BMP-4. Thus, we propose that BMP-4 exerts prooxidant, prohypertensive, and proinflammatory effects only in the systemic circulation, whereas pulmonary arteries are protected from these adverse effects of BMP-4. The vascular bed-specific endothelial effects of BMP-4 are likely to contribute to its differential pathophysiological role in the systemic and pulmonary circulation.

Angiopoietin-2 Stimulates Blood Flow Recovery After Femoral Artery Occlusion by Inducing Inflammation and Arteriogenesis

Arteriosclerosis, Thrombosis, and Vascular Biology. Nov, 2008  |  Pubmed ID: 18772493

Recently, we have shown that shear stress regulates the angiogenic potential of endothelial cells in vitro by an Angiopoietin-2 (Ang2)-dependent mechanism; however its pathophysiological significance in vivo was not clear. We hypothesized that Ang2 plays an important role in blood flow recovery after arterial occlusion in vivo by regulating angiogenesis and arteriogenesis.

Laminar Shear Stress Inhibits Lipid Peroxidation Induced by High Glucose Plus Arachidonic Acid in Endothelial Cells

American Journal of Physiology. Heart and Circulatory Physiology. Nov, 2008  |  Pubmed ID: 18790833

Elevated blood glucose and free fatty acids induce oxidative stress associated with the incidence of cardiovascular disease. In contrast, laminar shear stress (LSS) plays a critical role in maintaining vascular health. The present study examined the mechanism for the antioxidant effect of LSS attenuating the oxidative stress induced by high glucose (HG) and arachidonic acid (AA) in human umbilical vein endothelial cells. HG and AA synergistically decreased cell viability and increased glutathione (GSH) oxidation and lipid peroxidation. The lipid peroxidation was markedly prevented by LSS as well as tetrahydrobiopterin (BH4) and GSH. LSS increased BH4 and GSH contents, and expression of GTP cyclohydrolase-1 and glutamylcysteine ligase (GCL) involved in their biosynthesis. Inhibition of GCL activity by DL-buthionine-(S,R)-sulfoximine and small-interfering RNA-mediated knockdown of GCL lessened the antioxidant effect of LSS. Therefore, it is suggested that LSS enhances antioxidant capacity of endothelial cells and thereby attenuates the oxidative stress caused by cardiovascular risk factors.

Altered Shear Stress Stimulates Upregulation of Endothelial VCAM-1 and ICAM-1 in a BMP-4- and TGF-beta1-dependent Pathway

Arteriosclerosis, Thrombosis, and Vascular Biology. Feb, 2009  |  Pubmed ID: 19023092

Hemodynamics has been associated with aortic valve (AV) inflammation, but the underlying mechanisms are not well understood. Here we tested the hypothesis that altered shear stress conditions stimulate the expression of cytokines and adhesion molecules in AV leaflets via a bone morphogenic protein (BMP)- and transforming growth fact (TGF)-beta1-dependent pathway.

Low Magnitude and High Frequency Mechanical Loading Prevents Decreased Bone Formation Responses of 2T3 Preosteoblasts

Journal of Cellular Biochemistry. Feb, 2009  |  Pubmed ID: 19125415

Bone loss due to osteoporosis or disuse such as in paraplegia or microgravity is a significant health problem. As a treatment for osteoporosis, brief exposure of intact animals or humans to low magnitude and high frequency (LMHF) mechanical loading has been shown to normalize and prevent bone loss. However, the underlying molecular changes and the target cells by which LMHF mechanical loading alleviate bone loss are not known. Here, we hypothesized that direct application of LMHF mechanical loading to osteoblasts alters their cell responses, preventing decreased bone formation induced by disuse or microgravity conditions. To test our hypothesis, preosteoblast 2T3 cells were exposed to a disuse condition using the random positioning machine (RPM) and intervened with an LMHF mechanical load (0.1-0.4 g at 30 Hz for 10-60 min/day). Exposure of 2T3 cells to the RPM decreased bone formation responses as determined by alkaline phosphatase (ALP) activity and mineralization even in the presence of a submaximal dose of BMP4 (20 ng/ml). However, LMHF mechanical loading prevented the RPM-induced decrease in ALP activity and mineralization. Mineralization induced by LMHF mechanical loading was enhanced by treatment with bone morphogenic protein 4 (BMP4) and blocked by the BMP antagonist noggin, suggesting a role for BMPs in this response. In addition, LMHF mechanical loading rescued the RPM-induced decrease in gene expression of ALP, runx2, osteomodulin, parathyroid hormone receptor 1, and osteoglycin. These findings suggest that preosteoblasts may directly respond to LMHF mechanical loading to induce differentiation responses. The mechanosensitive genes identified here provide potential targets for pharmaceutical treatments that may be used in combination with low level mechanical loading to better treat osteoporosis or disuse-induced bone loss.

Expression of CYP1A1 and CYP1B1 in Human Endothelial Cells: Regulation by Fluid Shear Stress

Cardiovascular Research. Mar, 2009  |  Pubmed ID: 19126602

CYP1A1 and CYP1B1, members of the cytochrome P450 protein family, are regulated by fluid shear stress. This study describes the effects of duration, magnitude and pattern of shear stress on CYP1A1 and CYP1B1 expressions in human endothelial cells, towards the goal of understanding the role(s) of these genes in pro-atherogenic or anti-atherogenic endothelial cell functions.

Elevated Cyclic Stretch Alters Matrix Remodeling in Aortic Valve Cusps: Implications for Degenerative Aortic Valve Disease

American Journal of Physiology. Heart and Circulatory Physiology. Mar, 2009  |  Pubmed ID: 19151254

Matrix metalloproteinases (MMPs) and cathepsins are proteolytic enzymes that are upregulated in diseased aortic valve cusps. The objective of this study was to investigate whether elevated cyclic stretch causes an increased expression and activity of these proteolytic enzymes in the valve cusp. Circumferentially oriented fresh porcine aortic valve cusp sections were stretched to 10% (physiological), 15% (pathological), and 20% (hyperpathological) in a tensile stretch bioreactor for 24 and 48 h. The expression and activity of MMP-1, MMP-2, MMP-9, tissue inhibitor of MMP-1, and cathepsin L, S, and K were quantified and compared with fresh controls. Cell proliferation and apoptosis were also analyzed. As a result, at 10% physiological stretch, the expression and activity of remodeling enzymes were comparable with fresh controls. At 15% stretch, the expression of MMP-1, -2, -9 and cathepsin S and K were upregulated, whereas the expression of cathepsin L was downregulated compared with controls. A similar trend was observed at 20% stretch, but the magnitudes of upregulation and downregulation of the expression were less than those observed at 15%. In addition, there were significantly higher cell proliferation and apoptosis at 20% stretch compared with those of other treatment groups. In conclusion, elevated mechanical stretch on aortic valve cusps may detrimentally alter the proteolytic enzyme expression and activity in valve cells. This may trigger a cascade of events leading to an accelerated valve degeneration and disease progression.

Redox-sensitive Akt and Src Regulate Coronary Collateral Growth in Metabolic Syndrome

American Journal of Physiology. Heart and Circulatory Physiology. Jun, 2009  |  Pubmed ID: 19376806

We have recently shown that the inability of repetitive ischemia (RI) to activate p38 MAPK (p38) and Akt in metabolic syndrome [JCR:LA-cp (JCR)] rats was associated with impaired coronary collateral growth (CCG). Furthermore, Akt and p38 activation correlated with optimal O(2)(-). levels and were altered in JCR rats, and redox-sensitive p38 activation was required for CCG. Here, we determined whether the activation of Src, a possible upstream regulator, was altered in JCR rats and whether redox-dependent Src and Akt activation were required for CCG. CCG was assessed by myocardial blood flow (microspheres) and kinase activation was assessed by Western blot analysis in the normal zone and collateral-dependent zone (CZ). RI induced Src activation (approximately 3-fold) in healthy [Wistar-Kyoto (WKY)] animals but not in JCR animals. Akt inhibition decreased (approximately 50%), and Src inhibition blocked RI-induced CCG in WKY rats. Src inhibition decreased p38 and Akt activation. Myocardial oxidative stress (O(2)(-). and oxidized/reduced thiols) was measured quantitatively (X-band electron paramagnetic resonance). An antioxidant, apocynin, reduced RI-induced oxidative stress in JCR rats to levels induced by RI in WKY rats versus the reduction in WKY rats to very low levels. This resulted in a significant restoration of p38 (approximately 80%), Akt (approximately 65%), and Src (approximately 90%) activation in JCR rats but decreased the activation in WKY rats (p38: approximately 45%, Akt: approximately 65%, and Src: approximately 100%), correlating with reduced CZ flow in WKY rats (approximately 70%), but significantly restored CZ flow in JCR rats (approximately 75%). We conclude that 1) Akt and Src are required for CCG, 2) Src is a redox-sensitive upstream regulator of RI-induced p38 and Akt activation, and 3) optimal oxidative stress levels are required for RI-induced p38, Akt, and Src activation and CCG.

Partial Carotid Ligation is a Model of Acutely Induced Disturbed Flow, Leading to Rapid Endothelial Dysfunction and Atherosclerosis

American Journal of Physiology. Heart and Circulatory Physiology. Oct, 2009  |  Pubmed ID: 19684185

Atherosclerosis is closely associated with disturbed flow characterized by low and oscillatory shear stress, but studies directly linking disturbed flow to atherogenesis is lacking. The major reason for this has been a lack of an animal model in which disturbed flow can be acutely induced and cause atherosclerosis. Here, we characterize partial carotid ligation as a model of disturbed flow with characteristics of low and oscillatory wall shear stress. We also describe a method of isolating intimal RNA in sufficient quantity from mouse carotid arteries. Using this model and method, we found that partial ligation causes upregulation of proatherogenic genes, downregulation of antiatherogenic genes, endothelial dysfunction, and rapid atherosclerosis in 2 wk in a p47(phox)-dependent manner and advanced lesions by 4 wk. We found that partial ligation results in endothelial dysfunction, rapid atherosclerosis, and advanced lesion development in a physiologically relevant model of disturbed flow. It also allows for easy and rapid intimal RNA isolation. This novel model and method could be used for genome-wide studies to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases.

GTP Cyclohydrolase I Phosphorylation and Interaction with GTP Cyclohydrolase Feedback Regulatory Protein Provide Novel Regulation of Endothelial Tetrahydrobiopterin and Nitric Oxide

Circulation Research. Feb, 2010  |  Pubmed ID: 19926872

GTP cyclohydrolase I (GTPCH-1) is the rate-limiting enzyme involved in de novo biosynthesis of tetrahydrobiopterin (BH(4)), an essential cofactor for NO synthases and aromatic amino acid hydroxylases. GTPCH-1 undergoes negative feedback regulation by its end-product BH(4) via interaction with the GTP cyclohydrolase feedback regulatory protein (GFRP). Such a negative feedback mechanism should maintain cellular BH(4) levels within a very narrow range; however, we recently identified a phosphorylation site (S81) on human GTPCH-1 that markedly increases BH(4) production in response to laminar shear.

Intimal Cushions and Endothelial Nuclear Elongation Around Mouse Aortic Branches and Their Spatial Correspondence with Patterns of Lipid Deposition

American Journal of Physiology. Heart and Circulatory Physiology. Feb, 2010  |  Pubmed ID: 19933414

Spatial variation in hemodynamic stresses acting on the arterial wall may explain the nonuniform distribution of atherosclerosis. In thoracic aortas of LDL receptor/apolipoprotein E double knockout mice, lesions develop preferentially around the entire circumference of intercostal branch ostia, regardless of age, with the highest prevalence occurring upstream. Additional chevron-shaped lesions occur further upstream of the ostia. This pattern differs from the age-related ones occurring in people and rabbits. In the present study, patterns of near-wall blood flow around intercostal ostia in wild-type mice were estimated from the morphology of endothelial nuclei, which were shown in vitro to elongate in response to elevated shear stress and to align with the flow, and wall structure was assessed from confocal and scanning electron microscopy. A triangular intimal cushion surrounded the upstream part of most ostia. Nuclear length-to-width ratios were lowest over this cushion and highest at the sides of branches, regardless of age. Nuclear orientations were consistent with flow diverging around the branch. The pattern of nuclear morphology differed from the age-related ones observed in rabbits. The intimal cushion and the distribution of shear stress inferred from these observations can partly account for the pattern of lesions observed in knockout mice. Nuclear elongation in nonbranch regions was approximately constant across animals of different size, demonstrating the existence of a mechanism by which endothelial cells compensate for the dependence of mean aortic wall shear stress on body mass.

HuR Regulates the Expression of Stress-sensitive Genes and Mediates Inflammatory Response in Human Umbilical Vein Endothelial Cells

Proceedings of the National Academy of Sciences of the United States of America. Apr, 2010  |  Pubmed ID: 20351266

An important aspect of vascular biology is the identification of regulators of stress-sensitive genes that play critical roles in mediating inflammatory response. Here, we show that expression of HuR in human umbilical vein endothelial cells is regulated by shear stress and statin treatment; HuR, in turn, regulates other stress-sensitive genes such as Kruppel-like factor 2 (Klf2), endothelial nitric oxide synthase (eNOS), and bone morphogenic protein 4 (BMP-4). We found that siRNA knockdown of HuR-inhibited inflammatory responses in endothelial cells, including ICAM-1 and VCAM-1 up-regulation, NFkappaB phosphorylation, and adhesion of monocytes. Tissue staining of the mouse aorta revealed increased HuR expression in the lesser curvature region of the arch that is exposed to disturbed flow, consistent with our in vitro data. Taken together, these results suggest that HuR plays a critical role in inducing inflammatory response of endothelial cells under mechanical and biochemical stresses.

X-linked Inhibitor of Apoptosis Protein Controls Alpha5-integrin-mediated Cell Adhesion and Migration

American Journal of Physiology. Heart and Circulatory Physiology. Aug, 2010  |  Pubmed ID: 20472758

The association of integrins with caveolin-1 regulates cell adhesion. However, the vascular ramifications of this association remain to be clearly determined. We recently reported that the X chromosome-linked inhibitor of apoptosis protein (XIAP)-caveolin-1 interaction is critical to endothelial cell survival. Thus, we hypothesized that XIAP performs a crucial function in integrin/caveolin-1-mediated endothelial cell survival. In this study, we demonstrated that XIAP is recruited into the alpha(5)-integrin complex via caveolin-1 binding and mediates cell adhesion. We also determined that XIAP is critical to shear stress-stimulated ERK activation in an alpha(5)-integrin-dependent manner but is not important to VEGF-induced ERK activation. This differential activation of ERK is partly attributable to unique localizations of the receptors. Furthermore, we confirmed that XIAP is an essential molecule in the efficient recruitment of focal adhesion kinase (FAK) into the alpha(5)-integrin-associated complex. This alpha(5)-integrin-caveolin-1-XIAP-FAK multicomplex regulates endothelial cell migration via a mechanism that involves shear-dependent ERK activation. Together, our results indicate that XIAP stabilizes the alpha(5)-integrin-associated focal adhesion complex, thereby further regulating endothelial cell adhesion and migration. The findings of this study provide us with greater insight into the molecular mechanisms underlying the control of vascular function by integrins.

Elevated Cyclic Stretch Induces Aortic Valve Calcification in a Bone Morphogenic Protein-dependent Manner

The American Journal of Pathology. Jul, 2010  |  Pubmed ID: 20489151

Calcified aortic valve (AV) cusps have increased expression of bone morphogenic proteins (BMPs) and transforming growth factor-beta1 (TGF-beta1). Elevated stretch loading on the AV is known to increase expression of matrix remodeling enzymes and pro-inflammatory proteins. Little, however, is known about the mechanism by which elevated stretch might induce AV calcification. We investigated the hypothesis that elevated stretch may cause valve calcification via a BMP-dependent mechanism. Porcine AV cusps were cultured in a stretch bioreactor, at 10% (physiological) or 15% (pathological) stretch and 70 beats per minute for 3, 7, and 14 days, in osteogenic media supplemented with or without high phosphate (3.8 mmol/L), TGF-beta1 (1 ng/ml), as well as the BMP inhibitor noggin (1, 10, and 100 ng/ml). Fresh cusps served as controls. Alizarin red and von Kossa staining demonstrated that 15% stretch elicited a stronger calcification response compared with 10% stretch in a fully osteogenic medium containing high phosphate and TGF-beta1. BMP-2, -4, and Runx2 expression was observed after 3 days on the fibrosa surface of the valve cusp and was stretch magnitude-dependent. Cellular apoptosis was highest at 15% stretch. Tissue calcium content and alkaline phosphatase activity were similarly stretch-dependent and were significantly reduced by noggin in a dose dependent manner. These results underline the potential role of BMPs in valve calcification due to altered stretch.

Systems Analysis of the Role of Bone Morphogenic Protein 4 in Endothelial Inflammation

Annals of Biomedical Engineering. Feb, 2010  |  Pubmed ID: 19851868

Shear stress is an important factor in the onset and progression of atherosclerosis. High and unidirectional laminar stress is seen as protective, while low and oscillatory shear stress is considered pro-inflammatory and pro-atherogenic. The mechanosensitive response of endothelial cells is governed by a complex system of genes, proteins, and signals that operate at distinctly different time scales. We propose a dynamic mathematical model that quantitatively describes this mechanosensing system and permits novel insights into its functioning. The model, the first of its kind, is constructed within the guidelines of Biochemical Systems Theory and accounts for different time scales by means of approximated delays. Parameter values are obtained directly from biochemical observations in an ad hoc fashion. The model reflects most documented observations well and leads to a number of predictions and novel hypotheses. In particular, it demonstrates the crucial role of Bone Morphogenic Protein 4 and p47(phox)-dependent NADPH oxidases in endothelial inflammation.

Shear Stress and Plaque Development

Expert Review of Cardiovascular Therapy. Apr, 2010  |  Pubmed ID: 20397828

Although traditional cardiovascular risk factors 'prime the soil' for atherogenesis systemically, atherosclerosis primarily occurs in a site-specific manner with a predilection towards the inner wall of curvatures and outer wall of bifurcations with sparing of flow-dividers. Wall shear stress is a frictional force exerted parallel to the vessel wall that leads to alteration of the endothelial phenotype, endothelial cell signaling, gene and protein expression leading to a proinflammatory phenotype, reduced nitric oxide availability and disruption of the extracellular matrix, which in turn leads to plaque development. Clinical and experimental data are emerging that suggest the pathobiology associated with abnormal wall shear stress results in atherosclerotic plaque development and progression.

Discovery of Novel Mechanosensitive Genes in Vivo Using Mouse Carotid Artery Endothelium Exposed to Disturbed Flow

Blood. Oct, 2010  |  Pubmed ID: 20551377

Recently, we showed that disturbed flow caused by a partial ligation of mouse carotid artery rapidly induces atherosclerosis. Here, we identified mechanosensitive genes in vivo through a genome-wide microarray study using mouse endothelial RNAs isolated from the flow-disturbed left and the undisturbed right common carotid artery. We found 62 and 523 genes that changed significantly by 12 hours and 48 hours after ligation, respectively. The results were validated by quantitative polymerase chain reaction for 44 of 46 tested genes. This array study discovered numerous novel mechanosensitive genes, including Lmo4, klk10, and dhh, while confirming well-known ones, such as Klf2, eNOS, and BMP4. Four genes were further validated for protein, including LMO4, which showed higher expression in mouse aortic arch and in human coronary endothelium in an asymmetric pattern. Comparison of in vivo, ex vivo, and in vitro endothelial gene expression profiles indicates that numerous in vivo mechanosensitive genes appear to be lost or dysregulated during culture. Gene ontology analyses show that disturbed flow regulates genes involved in cell proliferation and morphology by 12 hours, followed by inflammatory and immune responses by 48 hours. Determining the functional importance of these novel mechanosensitive genes may provide important insights into understanding vascular biology and atherosclerosis.

Endothelial Metallothionein Expression and Intracellular Free Zinc Levels Are Regulated by Shear Stress

American Journal of Physiology. Cell Physiology. Dec, 2010  |  Pubmed ID: 20861469

We examined the effects of fluid shear stress on metallothionein (MT) gene and protein expression and intracellular free zinc in mouse aorta and in human umbilical vein endothelial cells (HUVECs). Immunostaining of the endothelial surface of mouse aorta revealed increased expression of MT protein in the lesser curvature of the aorta relative to the descending thoracic aorta. HUVECs were exposed to high steady shear stress (15 dyn/cm(2)), low steady shear stress (1 dyn/cm(2)), or reversing shear stress (mean of 1 dyn/cm(2), 1 Hz) for 24 h. Gene expression of three MT-1 isoforms, MT-2A, and zinc transporter-1 was upregulated by low steady shear stress and reversing shear stress. HUVECs exposed to 15 dyn/cm(2) had increased levels of free zinc compared with cells under other shear stress regimes and static conditions. The increase in free zinc was partially blocked with an inhibitor of nitric oxide synthesis, suggesting a role for shear stress-induced endothelial nitric oxide synthase activity. Cells subjected to reversing shear stress in zinc-supplemented media (50 μM ZnSO(4)) had increased intracellular free zinc, reduced surface intercellular adhesion molecule-1 expression, and reduced monocyte adhesion compared with cells exposed to reversing shear stress in normal media. The sensitivity of intracellular free zinc to differences in shear stress suggests that intracellular zinc levels are important in the regulation of the endothelium and in the progression of vascular disease.

Peroxiredoxin 2 Deficiency Exacerbates Atherosclerosis in Apolipoprotein E-deficient Mice

Circulation Research. Sep, 2011  |  Pubmed ID: 21835911

Peroxiredoxin 2 (Prdx2), a thiol-specific peroxidase, has been reported to regulate proinflammatory responses, vascular remodeling, and global oxidative stress.

Detection of Low Levels of Nitric Oxide Using an Electrochemical Sensor

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21161631

Nitric oxide produced from nitric oxide synthases mediates various physiological and pathological events in biological systems. However, quantitative assessment of nitric oxide from biological sources remains a difficult task. Here we describe a procedure for the quantification of low levels of nitric oxide using a nitric oxide - selective electrochemical sensor. Nitric oxide is oxidized to nitrite and/or nitrate and accumulated in the aqueous media. First, nitrate in biological fluids or culture media is converted to nitrite by an enzymatic method. Nitrite is then chemically converted to equimolar NO in an acidic iodide bath, where nitric oxide is detected by the sensor. Using this method, the present study demonstrates siRNA -mediated suppression of nitric oxide synthase 3 leading to a significant decline of basal nitric oxide production in human umbilical vein endothelial cells. Basal nitric oxide production from HUVECs is also shown to be inhibited by N (G)-nitro-L: -arginine methyl ester but not by N (G)-nitro-D: -arginine methyl ester (D-NAME) D-NAME . The analytical method presented here provides a sensitive and convenient tool for measuring basal and stimulated nitric oxide production from biological sources.

Differences in Valvular and Vascular Cell Responses to Strain in Osteogenic Media

Biomaterials. Apr, 2011  |  Pubmed ID: 21284997

Calcification is the primary cause of failure of bioprosthetic and tissue-engineered vascular and valvular grafts. We used tissue-engineered collagen gels containing human aortic smooth muscle cells (HASMC) and human aortic valvular interstitial cells (HAVIC) as a model to investigate cell-mediated differences in early markers of calcification. The HASMCs and HAVICs were isolated from non-sclerotic human tissues. After 21 days of culture in either regular or osteogenic media with or without 10% cyclic strain at 1 Hz, the collagen gels were assessed for DNA content, collagen I, matrix metalloproteinase (MMP)-2 and glycosaminoglycan (GAG) content. The collagen gels containing HASMCs contained significantly greater amounts of collagen I and GAG compared to HAVICs. Although strain increased MMP-2 activity for both cell types, this trend was significant (p ≤ 0.05) only for HAVICs. Cultured gels were also assessed for osteogenic markers calcium content, alkaline phosphatase (ALP), and Runx2 and were present at greater amounts in gels containing HASMCs than HAVICs. Calcium content, Runx2 expression, and ALP activity were also modulated by mechanical strain. The results indicate that cell-mediated differences exist between the vascular and valvular calcification processes. Further investigation is necessary for improved understanding and to detect biomarkers for early detection or prevention of these diseases.

Angiotensin Type I Receptor Blockade in Conjunction with Enhanced Akt Activation Restores Coronary Collateral Growth in the Metabolic Syndrome

American Journal of Physiology. Heart and Circulatory Physiology. May, 2011  |  Pubmed ID: 21335466

We have previously demonstrated that Akt was required for repetitive ischemia (RI)-induced coronary collateral growth (CCG) in healthy rats but was not activated by RI in the metabolic syndrome (JCR:LA-cp rats) where CCG was impaired. Here we hypothesized that failure of angiotensin type I receptor (AT₁R) blockers to restore Akt activation is a key determinant of their inability to completely restore CCG in the metabolic syndrome. Therefore, we investigated whether adenovirus-mediated delivery of constitutively active Akt (MyrAkt-Adv) in conjunction with AT₁R blockade (candesartan) was able to restore RI-induced CCG in JCR:LA-cp rats. Successful myocardial MyrAkt-Adv delivery was confirmed by a >80% transduction efficiency and an approximately fourfold increase in Akt expression and activation. CCG was assessed by myocardial blood flow measurements in the normal and collateral-dependent zones. MyrAkt-Adv alone significantly increased RI-induced CCG in JCR:LA-cp rats (~30%), but it completely restored CCG in conjunction with administration of candesartan. In contrast, dominant negative Akt (DN-Akt-Adv) reversed the beneficial effect of candesartan on CCG in JCR:LA-cp rats. We conclude that optimal restoration of coronary collateral growth in JCR:LA-cp rats requires a combination of AT₁R blockade with constitutive Akt activation. These findings may carry implications for metabolic syndrome patients in need of coronary revascularization.

The Effects of Combined Cyclic Stretch and Pressure on the Aortic Valve Interstitial Cell Phenotype

Annals of Biomedical Engineering. Jun, 2011  |  Pubmed ID: 21347552

Aortic valve interstitial cells (VIC) can exhibit phenotypic characteristics of fibroblasts, myofibroblasts, and smooth muscle cells. Others have proposed that valve cells become activated and exhibit myofibroblast or fibroblast characteristics during disease initiation and progression; however, the cues that modulate this phenotypic change remain unclear. We hypothesize that the mechanical forces experienced by the valve play a role in regulating the native phenotype of the valve and that altered mechanical forces result in an activated phenotype. Using a novel ex vivo cyclic stretch and pressure bioreactor, we subjected porcine aortic valve (AV) leaflets to combinations of normal and pathological stretch and pressure magnitudes. The myofibroblast markers α-SMA and Vimentin, along with the smooth muscle markers Calponin and Caldesmon, were analyzed using immunohistochemistry and immunoblotting. Tissue structure was analyzed using Movat's pentachrome staining. We report that pathological stretch and pressure inhibited the contractile and possibly myofibroblast phenotypes as indicated by downregulation of the proteins α-SMA, Vimentin, and Calponin. In particular, Calponin downregulation implies depolymerization of actin filaments and possible conversion to a more synthetic (non-contractile) phenotype. This agreed well with the increase in spongiosa and fibrosa thickness observed under elevated pressure and stretch that are typically indicative of increased matrix synthesis. Our study therefore demonstrates how cyclic stretch and pressure may possibly act together to modulate the AVIC phenotype.

MicroRNA-663 Upregulated by Oscillatory Shear Stress Plays a Role in Inflammatory Response of Endothelial Cells

American Journal of Physiology. Heart and Circulatory Physiology. May, 2011  |  Pubmed ID: 21378144

The mechanisms by which oscillatory shear stress (OS) induces, while high laminar shear stress (LS) prevents, atherosclerosis are still unclear. Here, we examined the hypothesis that OS induces inflammatory response, a critical atherogenic event, in endothelial cells by a microRNA (miRNA)-dependent mechanism. By miRNA microarray analysis using total RNA from human umbilical vein endothelial cells (HUVECs) that were exposed to OS or LS for 24 h, we identified 21 miRNAs that were differentially expressed. Of the 21 miRNAs, 13 were further examined by quantitative PCR, which validated the result for 10 miRNAs. Treatment of HUVECs with the miR-663 antagonist (miR-663-locked nucleic acids) blocked OS-induced monocyte adhesion, but not apoptosis. In contrast, overexpression of miR-663 increased monocyte adhesion in LS-exposed cells. Subsequent mRNA expression microarray study using HUVECs treated with miR-663-locked nucleic acids and OS revealed 32 up- and 3 downregulated genes, 6 of which are known to be involved in inflammatory response. In summary, we identified 10 OS-sensitive miRNAs, including miR-663, which plays a key role in OS-induced inflammatory responses by mediating the expression of inflammatory gene network in HUVECs. These OS-sensitive miRNAs may mediate atherosclerosis induced by disturbed flow.

Redox Going with Vascular Shear Stress

Antioxidants & Redox Signaling. Sep, 2011  |  Pubmed ID: 21457103

Shear stress plays a critical role in the regulation of vascular biology and diseases, such as atherosclerosis, via modulation of signal transduction and redox balance. Atherosclerosis preferentially occurs in a site-specific manner linked to disturbed flow. In this Forum on Vascular Shear Stress, emerging role of redox-dependent molecular mechanisms by which shear stress regulates pro- and antiatherogenic responses in endothelial cells both in vitro and in vivo are reviewed in depth by experts. This Forum also provides comprehensive reviews regarding experimental apparatus and in vivo, ex vivo, and in vitro systems used for shear stress studies.

Tetrahydrobiopterin Deficiency and Nitric Oxide Synthase Uncoupling Contribute to Atherosclerosis Induced by Disturbed Flow

Arteriosclerosis, Thrombosis, and Vascular Biology. Jul, 2011  |  Pubmed ID: 21512164

Tetrahydrobiopterin (BH(4)) is a critical cofactor for nitric oxide (NO) synthesis by NO synthase (NOS). Recently, we demonstrated that disturbed flow produced by partial carotid ligation decreases BH(4) levels in vivo. We therefore aimed to determine whether atherosclerosis induced by disturbed flow is due to BH(4) deficiency and NOS uncoupling and whether increasing BH(4) would prevent endothelial dysfunction, plaque inflammation, and atherosclerosis.

Disturbed Flow: P53 SUMOylation in the Turnover of Endothelial Cells

The Journal of Cell Biology. May, 2011  |  Pubmed ID: 21624951

Disturbed blood flow induces apoptosis of vascular endothelial cells, which causes atherosclerosis. In this issue, Heo et al. (2011. J. Cell Biol. doi:10.1083/jcb.201010051) sheds light on p53's role in this phenomenon. Disturbed flow induces peroxynitrite production, which activates protein kinase C ζ and it's binding to the E3 SUMO (small ubiquitin-like modifier) ligase PIASy (protein inhibitor of activated STATy). This leads to p53 SUMOylation and its export to the cytosol, where it binds to the antiapoptotic protein Bcl-2 to induce apoptosis.

Preferential Activation of SMAD1/5/8 on the Fibrosa Endothelium in Calcified Human Aortic Valves--association with Low BMP Antagonists and SMAD6

PloS One. 2011  |  Pubmed ID: 21698246

Aortic valve (AV) calcification preferentially occurs on the fibrosa side while the ventricularis side remains relatively unaffected. Here, we tested the hypothesis that side-dependent activation of bone morphogenic protein (BMP) pathway in the endothelium of the ventricularis and fibrosa is associated with human AV calcification.

Discovery of Shear- and Side-specific MRNAs and MiRNAs in Human Aortic Valvular Endothelial Cells

American Journal of Physiology. Heart and Circulatory Physiology. Sep, 2011  |  Pubmed ID: 21705672

The role of endothelial cells (ECs) in aortic valve (AV) disease remains relatively unknown; however, disease preferentially occurs in the fibrosa. We hypothesized oscillatory shear (OS) present on the fibrosa stimulates ECs to modify mRNAs and microRNAs (miRNAs) inducing disease. Our goal was to identify mRNAs and miRNAs differentially regulated by OS and laminar shear (LS) in human AVECs (HAVECs) from the fibrosa (fHAVECs) and ventricularis (vHAVECs). HAVECs expressed EC markers as well as some smooth muscle cell markers and functionally aligned with the flow. HAVECs were exposed to OS and LS for 24 h, and total RNA was analyzed by mRNA and miRNA microarrays. We found over 700 and 300 mRNAs down- and upregulated, respectively, by OS; however, there was no side dependency. mRNA microarray results were validated for 26 of 28 tested genes. Ingenuity Pathway Analysis revealed thrombospondin 1 (Thbs1) and NF-κB inhibitor-α (Nfkbia) as highly connected, shear-sensitive genes. miRNA array analysis yielded 30 shear-sensitive miRNAs and 3 side-specific miRNAs. miRNA validation confirmed 4 of 17 shear-sensitive miRNAs and 1 of 3 side-dependent miRNAs. Using miRWalk and several filtering steps, we identified shear-sensitive mRNAs potentially targeted by shear-sensitive miRNAs. These genes and signaling pathways could act as therapeutic targets of AV disease.

Serum BMP-4 Levels in Relation to Arterial Stiffness and Carotid Atherosclerosis in Patients with Type 2 Diabetes

Biomarkers in Medicine. Dec, 2011  |  Pubmed ID: 22103619

The aim of this study was to evaluate the association between the circulating BMP-4 levels and atherosclerosis in patients with Type 2 diabetes.

Animal, in Vitro, and Ex Vivo Models of Flow-dependent Atherosclerosis: Role of Oxidative Stress

Antioxidants & Redox Signaling. Sep, 2011  |  Pubmed ID: 20712399

Atherosclerosis is an inflammatory disease preferentially occurring in curved or branched arterial regions, whereas straight parts of the arteries are protected, suggesting a close relationship between flow and atherosclerosis. However, evidence directly linking disturbed flow to atherogenesis is just emerging, thanks to the recent development of suitable animal models. In this article, we review the status of various animal, in vitro, and ex vivo models that have been used to study flow-dependent vascular biology and atherosclerosis. For animal models, naturally flow-disturbed regions such as branched or curved arterial regions as well as surgically created models, including arterio-venous fistulas, vascular grafts, perivascular cuffs, and complete, incomplete, or partial ligation of arteries, are used. Although in vivo models provide the environment needed to mimic the complex pathophysiological processes, in vitro models provide simple conditions that allow the study of isolated factors. Typical in vitro models use cultured endothelial cells exposed to various flow conditions, using devices such as cone-and-plate and parallel-plate chambers. Ex vivo models using isolated vessels have been used to bridge the gap between complex in vivo models and simple in vitro systems. Here, we review these flow models in the context of the role of oxidative stress in flow-dependent inflammation, a critical proatherogenic step, and atherosclerosis.

Dynamic Immune Cell Accumulation During Flow-Induced Atherogenesis in Mouse Carotid Artery: An Expanded Flow Cytometry Method

Arteriosclerosis, Thrombosis, and Vascular Biology. Jan, 2012  |  Pubmed ID: 22247254

OBJECTIVE: Inflammation plays a central role in atherosclerosis. However, the detailed changes in the composition and quantity of leukocytes in the arterial wall during atherogenesis are not fully understood in part because of the lack of suitable methods and animal models. METHODS AND RESULTS: We developed a 10-fluorochrome, 13-parameter flow cytometry method to quantitate 7 major leukocyte subsets in a single digested arterial wall sample. Apolipoprotein E-deficient mice underwent left carotid artery (LCA) partial ligation and were fed a high-fat diet for 4 to 28 days. Monocyte/macrophages, dendritic cells, granulocytes, natural killer cells, and CD4 T cells significantly infiltrated the LCA as early as 4 days. Monocyte/macrophages and dendritic cells decreased between 7 and 14 days, whereas T-cell numbers remained steady. Leukocyte numbers peaked at 7 days, preceding atheroma formation at 14 days. B cells entered LCA by 14 days. Control right carotid and sham-ligated LCAs showed no significant infiltrates. Polymerase chain reaction and ELISA arrays showed that expression of proinflammatory cytokines and chemokines peaked at 7 and 14 days postligation, respectively. CONCLUSIONS: This is the first quantitative description of leukocyte number and composition over the life span of murine atherosclerosis. These results show that disturbed flow induces rapid and dynamic leukocyte accumulation in the arterial wall during the initiation and progression of atherosclerosis.