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 Ser¹¹⁷⁹ residue (eNOS-S¹¹⁷⁹), 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-S¹¹⁷⁹. Here, we show that shear-dependent phosphorylation of eNOS-S¹¹⁷⁹ is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt^AAor Akt^AAA in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S¹¹⁷⁹ if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt^AAinhibited 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-S¹¹⁷⁹and NO^⋅ production. In contrast, H89 did not inhibit phosphorylation of eNOS-S¹¹⁷⁹ 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-S¹¹⁷⁹ within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S¹¹⁷⁹ in a PKA-dependent, but Aktindependent manner, whereas the NO^⋅ production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction between Akt and PKA may be an important mechanism by which eNOS activity is regulated in response to physiological stimuli such as shear stress.

Vascular 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.

Controlling unwanted hyperpigmentation is a major challenge in dermatology and cosmetology, and safe and efficacious antimelanogenic agents are deemed useful for this purpose. p-Coumaric acid is a natural metabolite contained in many edible plants, and its antioxidant activities in reducing oxidative stress and inflammatory reactions have been demonstrated in various experimental models. p-Coumaric acid has the optimal structure to be a competitive inhibitor of tyrosinase that catalyzes key reactions in the melanin biosynthetic pathway. Experimental evidence supports this notion as it was found to be a more potent inhibitor of tyrosinase, especially toward human enzymes, than other well-known tyrosinase inhibitors such as arbutin and kojic acid. p-Coumaric acid inhibited melanin synthesis in murine melanoma cells, human epidermal melanocytes, and reconstituted three-dimensional human skin models. Ex-vivo skin permeation experiments and in-vivo efficacy tests for p-coumaric acid confirmed its efficient transdermal delivery and functional efficacy in reducing erythema development and skin pigmentation due to ultraviolet radiation exposure. Human studies further supported its effectiveness in hypopigmentation and depigmentation. These findings suggest that p-coumaric acid has good potential to be used as a skin-lightening active ingredient in cosmetics. Future studies are needed to extensively examine its safety and efficacy and to develop an optimized cosmetic formulation for the best performance in skin lightening.

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 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 p47^phox-/- (MAE-p47^-/-) mice, which lack a component of NAD(P)H oxidase. 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 (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 compared with static conditions. In contrast, neither LS nor OS were able to induce 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 p47^phox in MAE-p47^-/- restored OS-induced production and monocyte adhesion. These results suggest that chronic exposure of endothelial cells to OS stimulates and/or its derivatives produced from p47^phox-dependent NAD(P)H oxidase, which, in turn, leads to monocyte adhesion, an early and critical atherogenic event.

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 NFκB-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.

Arbutin is a compound of hydroquinone and D-glucose, and it has been over 30 years since there have been serious studies on the skin lightening action of this substance. In the meantime, there have been debates and validation studies about the mechanism of action of this substance as well as its skin lightening efficacy and safety. Several analogs or derivatives of arbutin have been developed and studied for their melanin synthesis inhibitory action. Formulations have been developed to improve the stability, transdermal delivery, and release of arbutin, and device usage to promote skin absorption has been developed. Substances that inhibit melanin synthesis synergistically with arbutin have been explored. The skin lightening efficacy of arbutin alone or in combination with other active ingredients has been clinically evaluated. Combined therapy with arbutin and laser could give enhanced depigmenting efficacy. The use of arbutin causes dermatitis rarely, and caution is recommended for the use of arbutin-containing products, especially from the viewpoint that hydroquinone may be generated during product use. Studies on the antioxidant properties of arbutin are emerging, and these antioxidant properties are proposed to contribute to the skin depigmenting action of arbutin. It is hoped that this review will help to understand the pros and cons of arbutin as a cosmetic ingredient, and will lead to future research directions for developing advanced skin lightening and protecting cosmetic products.