Many findings suggest that steady laminar flow in blood vessels activates signal transduction events that lead to expression of atheroprotective genes (1, 2). The nature and magnitude of shear stress plays an important role in long-term maintenance of the structure and function of the blood vessel. In ''linear'' areas of the vasculature blood flows in ordered laminar patterns in a pulsatile fashion and endothelial cells (ECs) experience pulsatile shear stress with fluctuations in magnitude that yield a mean positive shear stress. At areas of abrupt curvatures in the vasculature, as in the carotid bifurcation, the laminar flow of blood is disrupted and separate flow patterns result (3-8). The significance of these flow patterns is demonstrated by studies that correlate development of atherosclerotic lesions (fatty streaks and small plaques) with areas of the carotid that experience these flow reversals with low time-averaged shear stress (3, 4). Regions of the carotid bifurcation that experience steady nonoscillatory shear stress as the result of laminar blood flow patterns are relatively protected from atherosclerosis. Examples of the atheroprotective nature of steady laminar flow are inhibition of E-selectin expression and suppression of vascular cell adhesion molecule 1 (VCAM-1) induction by cytokines such as IL-1 and tumor necrosis factor ␣

SUMMARYHuman pluripotent stem cells (hPSCs) represent a novel source of hepatocytes for drug metabolism studies and cell-based therapy for the treatment of liver diseases. These applications are, however, dependent on the ability to generate mature metabolically functional cells from the hPSCs. Reproducible and efficient generation of such cells has been challenging to date, owing to the fact that the regulatory pathways that control hepatocyte maturation are poorly understood. Here, we show that the combination of three-dimensional cell aggregation and cAMP signaling enhance the maturation of hPSC-derived hepatoblasts to a hepatocyte-like population that displays expression profiles and metabolic enzyme levels comparable to those of primary human hepatocytes. Importantly, we also demonstrate that generation of the hepatoblast population capable of responding to cAMP is dependent on appropriate activin/nodal signaling in the definitive endoderm at early stages of differentiation. Together, these findings provide new insights into the pathways that regulate maturation of hPSC-derived hepatocytes and in doing so provide a simple and reproducible approach for generating metabolically functional cell populations.

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Atherosclerosis preferentially occurs in areas of turbulent flow and low fluid shear stress, whereas laminar flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor‐α (TNF), have been shown to stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent data suggest that steady laminar flow decreases EC apoptosis and blocks TNF‐mediated EC activation. EC apoptosis is likely important in the process termed “plaque erosion” that leads to platelet aggregation. Steady laminar flow inhibits EC apoptosis by preventing cell cycle entry, by increasing antioxidant mechanisms (e.g., superoxide dismutase), and by stimulating nitric oxide‐dependent protective pathways that involve enzymes PI3‐kinase and Akt. Conversely, our laboratory has identified nitric oxide‐independent mechanisms that limit TNF signal transduction. TNF regulates gene expression in EC, in part, by stimulating mitogen‐activated protein kinases (MAPK) which phosphorylate transcription factors. We hypothesized that fluid shear stress modulates TNF effects on EC by inhibiting TNF‐mediated activation of MAP kinases. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm2) on TNF‐stimulated activity of two MAP kinases: extracellular signal regulated kinase (ERK1/2) and c‐Jun N‐terminal kinase (JNK). Flow alone stimulated ERK1/2 activity, but decreased JNK activity compared to static controls. TNF (10 ng/ml) alone activated both ERK1/2 and JNK maximally at 15 minutes in human umbilical vein EC (HUVEC). Pre‐exposing HUVEC for 10 minutes to flow inhibited TNF activation of JNK by 46%, but it had no significant effect on ERK1/2 activation. Incubation of EC with PD98059, a specific mitogen‐activated protein kinase kinase inhibitor, blocked the flow‐mediated inhibition of TNF activation of JNK. Flow‐mediated inhibition of JNK was unaffected by 0.1 mM L‐nitroarginine, 100 μM 8‐bromo‐cyclic GMP, or 100 μM 8‐bromo‐cyclic AMP. Transfection studies with dominant negative constructs of the protein kinase MEK1 and MEK5 suggested an important role for BMK1 in flow‐mediated regulation of TNF signals. In summary, the atheroprotective effects of steady laminar flow on the endothelium involve multiple synergistic mechanisms.

Abstract-Endothelial barrier dysfunction leading to increased permeability and vascular leakage is an underlying cause of several pathological conditions, including edema and sepsis. Whereas cAMP has been shown to decrease endothelial permeability, the role of cGMP is controversial. Endothelial cells express cGMP-inhibited phosphodiesterase (PDE)3A and cGMP-stimulated PDE2A. Thus we hypothesized that the effect of cGMP on endothelial permeability is dependent on the concentration of cGMP present and on the relative expression levels of PDE2A and PDE3A. When cAMP synthesis was slightly elevated with a submaximal concentration of 7-deacetyl-7-(O-[N-methylpiperazino]-␥-butyryl)-dihydrochloride-forskolin (MPB-forskolin), we found that low doses of either atrial natriuretic peptide (ANP) or NO donors potentiated the inhibitory effects of MPB-forskolin on thrombininduced permeability. However, this inhibitory effect of forskolin was reversed at higher doses of ANP or NO. These data suggest that cGMP at lower concentrations inhibits PDE3A and thereby increases a local pool of cAMP, whereas higher concentrations cGMP activates PDE2A, reversing the effect. Inhibitors of PDE3A mimicked the effect of low-dose ANP on thrombin-induced permeability, and inhibition of PDE2A reversed the stimulation of permeability seen with higher doses of ANP. Finally, increasing PDE2A expression with tumor necrosis factor-␣ reversed the inhibition of permeability caused by low doses of ANP. As predicted, the effect of tumor necrosis factor-␣ on permeability was reversed by a PDE2A inhibitor. These findings suggest that the effect of increasing concentrations of cGMP on endothelial permeability is biphasic, which, in large part, is attributable to the relative amounts of PDE2A and PDE3A in endothelial cells. Key Words: phosphodiesterase Ⅲ cGMP Ⅲ endothelial permeability Ⅲ TNF-␣ T he endothelium plays an important role in maintaining normal vascular function. In addition to maintaining a nonthrombogenic surface, secreting anticoagulation factors, responding to and participating in inflammatory signaling and regulating vascular tone, the endothelium also acts as an active barrier between the blood and the underlying tissue. Endothelial dysfunction caused by injury or inflammatory signals has been shown to lead to numerous pathological conditions. For example, inflammatory cytokines can induce adhesion molecule expression in endothelial cells, thereby promoting the adherence and migration of immune cells into the vessel wall, leading to atherosclerosis. 1 Dysfunctional endothelium also leads to decreased barrier function or increased permeability. Increased endothelial permeability is characteristic of many diseases and pathological conditions, including atherosclerosis, asthma, tumor growth, edema, and sepsis. 2,3 Several inflammatory mediators, including vascular endothelial growth factor, tumor necrosis factor (TNF)-␣, histamine, and thrombin, are known to increase endothelial permeability. 2-4 TNF-␣ increases permeability, at least in part...