Pulsatile Versus Oscillatory Shear Stress Regulates NADPH Oxidase Subunit Expression

Abstract

Abstract-Shear stress regulates endothelial nitric oxide and superoxide (O 2 Ϫ· ) production, implicating the role of NADPH oxidase activity. It is unknown whether shear stress regulates the sources of reactive species production, consequent low-density lipoprotein (LDL) modification, and initiation of inflammatory events. Bovine aortic endothelial cells (BAECs) in the presence of 50 g/mL of native LDL were exposed to (1) pulsatile flow with a mean shear stress ( ave ) of 25 dyne/cm 2 and (2) oscillating flow at ave of 0. After 4 hours, aliquots of culture medium were collected for high-performance liquid chromatography analyses of electronegative LDL species, described as LDL Ϫ and LDL 2Ϫ . In response to oscillatory shear stress, gp91 phox mRNA expression was upregulated by 2.9Ϯ0.3-fold, and its homologue, Nox4, by 3.9Ϯ0.9-fold (PϽ0.05, nϭ4), with a corresponding increase in O 2 Ϫ· production rate. The proportion of LDL Ϫ and LDL 2Ϫ relative to static conditions increased by 67Ϯ17% and 30Ϯ7%, respectively, with the concomitant upregulation of monocyte chemoattractant protein-1 expression and increase in monocyte/BAEC binding (PϽ0.05, nϭ5). In contrast, pulsatile flow downregulated both gp91 phox and Nox4 mRNA expression (by 1.8Ϯ0.2-fold and 3.0Ϯ0.12-fold, respectively), with an accompanying reduction in O 2 Ϫ· production, reduction in the extent of LDL modification (51Ϯ12% for LDL Ϫ and 30Ϯ7% for LDL 2Ϫ ), and monocyte/BAEC binding. The flow-dependent LDL oxidation is determined in part by the NADPH oxidase activity. The formation of modified LDL via O 2 Ϫ· production may also affect the regulation of monocyte chemoattractant protein-1 expression and monocyte/BAEC binding.

Oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine induces vascular endothelial superoxide production: Implication of NADPH oxidase

“…BAEC remained viable compared to the control as reported previously [25]. 2-DOG has been widely used to study intracellular redox status in pulmonary arterial endothelial cell electron transport [63,64].…”

“…Once the BAEC monolayers became confluent, growth medium was changed to glucose-free medium comprised of glucosefree DMEM, 15% heat-inactivated FBS, 100 U/ml penicillin-streptomycin, and 0.05% amphotericin B, and 10 mM 2-DOG. No significant differences in cell survival were found under these conditions [25]. Confluent BAEC monolayers were incubated overnight (>12 h), followed by treatment with ox-PAPC at 50 μg/ml for 4 h. Measurement of extracellular O 2 −• formation was performed at 1-, 2-, 3-, and 4-h intervals as described below.…”

“…Dihydroethidium (DHE) was used to localize intracellular O 2 −• production as previously described [25,27]. Cells are permeable to DHE in the presence of O 2 −• , and DHE is oxidized to fluorescent ethidium which is trapped by intercalation within the double-stranded nuclear DNA.…”

Nox enzymes, ROS, and chronic disease: An example of antagonistic pleiotropy

“…Oscillatory sheer stress occurs preferentially in branched or curved regions of arteries and is associated with atherogenesis. Oscillatory sheer stress results in several-fold induction of Nox1, Nox2 and Nox4 in vascular endothelium (with opposite effects of the anti-atherogenic laminar flow that occurs in straight portions of vessels) [80,81]. Oscillatory sheer stress is associated with induction of bone morphogenic protein 4 (BMP4) [82], which induces Nox1 and p47phox, resulting in an oxidative stress that leads to ICAM-1 expression and monocyte adhesion [81].…”

Circulating progenitor cells are increased in newly diagnosed untreated hypertensive patients with arterial stiffening but normal carotid intima-media thickness

“…17,18 The NADP + -Noxs, multisubunit membrane-associated enzymes that catalyze the one-electron reduction of oxygen using NADH or NADPH as the electron donor represent an important source of ROS for EPCs. 18,19 The Noxs, including the isoform containing the gp91phox or Nox2 isoform, are sensitively modulated by a wide range of factors, 20,21 including angiotensin-II (AngII), 21 the main effector of the renin-Ang system, which modulates vascular functions and arterial pressure. AngII seems to accelerate the onset of senescence in EPCs isolated from healthy individuals via stimulation of gp91phox expression and ROS production.…”