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Nitric oxide (NO) produced by vascular endothelial cells is a potent vasodilator and an antiinflammatory mediator. Regulating production of endothelial-derived NO is a complex undertaking, involving multiple signaling and genetic pathways that are activated by diverse humoral and biomechanical stimuli. To gain a thorough understanding of the rich diversity of responses observed experimentally, it is necessary to account for an ensemble of these pathways acting simultaneously. In this article, we have assembled four quantitative molecular pathways previously proposed for shear-stress-induced NO production. In these pathways, endothelial NO synthase is activated 1), via calcium release, 2), via phosphorylation reactions, and 3), via enhanced protein expression. To these activation pathways, we have added a fourth, a pathway describing actual NO production from endothelial NO synthase and its various protein partners. These pathways were combined and simulated using CytoSolve, a computational environment for combining independent pathway calculations. The integrated model is able to describe the experimentally observed change in NO production with time after the application of fluid shear stress. This model can also be used to predict the specific effects on the system after interventional pharmacological or genetic changes. Importantly, this model reflects the up-to-date understanding of the NO system, providing a platform upon which information can be aggregated in an additive way.

Plasmin (PLS) and urokinase-type plasminogen activator (UPA) are ubiquitous proteases that regulate the extracellular environment. Although they are secreted in inactive forms, they can activate each other through proteolytic cleavage. This mutual interplay creates the potential for complex dynamics, which we investigated using mathematical modeling and in vitro experiments. We constructed ordinary differential equations to model the conversion of precursor plasminogen into active PLS, and precursor urokinase (scUPA) into active urokinase (tcUPA). Although neither PLS nor UPA exhibits allosteric cooperativity, modeling showed that cooperativity occurred at the system level because of substrate competition. Computational simulations and bifurcation analysis predicted that the system would be bistable over a range of parameters for cooperativity and positive feedback. Cell-free experiments with recombinant proteins tested key predictions of the model. PLS activation in response to scUPA stimulus was found to be cooperative in vitro. Finally, bistability was demonstrated in vitro by the presence of two significantly different steady-state levels of PLS activation for the same levels of stimulus. We conclude that ultrasensitive, bistable activation of UPA-PLS is possible in the presence of substrate competition. An ultrasensitive threshold for activation of PLS and UPA would have ramifications for normal and disease processes, including angiogenesis, metastasis, wound healing, and fibrosis.

Cytoplasmic actin distributes between monomeric and filamentous phases in cells. As cells crawl, actin polymerizes near the plasma membrane of expanding peripheral cytoplasm and depolymerizes elsewhere. Thus, the finite actin filament lifetime, the diffusivity of actin monomer, and the distribution of actin between the polymer and monomer phases are key parameters in cell motility. The dynamics of cellular actin can be determined by following the evolution of fluorescence in the techniques of photoactivated fluorescence (PAF) or fluorescence recovery after photobleaching (FRAP) of microinjected actin derivatives. A mathematical model is discussed that measures monomer diffusion coefficients, filament turnover rates, and the fraction of actin polymerized from measurements of the evolution of fluorescence from a photoactivated band [Tardy et al. (1995) Biophys. J., 69:1674–1682; McGrath et al. (1998) Biophys. J., in press]. Applying this model to subconfluent endothelial cells shows that ~40% of the actin is polymer and that these filaments turn over on average every 6 minutes. This report dicusses how PAF and FRAP can be combined with more traditional biochemistry to probe actin cytoskeleton remodeling in endothelial cells. Microsc. Res. Tech. 43:385–394 © 1998 Wiley-Liss, Inc.

Cytoplasmic actin distributes between monomeric and filamentous phases in cells. As cells crawl, actin polymerizes near the plasma membrane of expanding peripheral cytoplasm and depolymerizes elsewhere. Thus, the finite actin filament lifetime, the diffusivity of actin monomer, and the distribution of actin between the polymer and monomer phases are key parameters in cell motility. The dynamics of cellular actin can be determined by following the evolution of fluorescence in the techniques of photoactivated fluorescence (PAF) or fluorescence recovery after photobleaching (FRAP) of microinjected actin derivatives. A mathematical model is discussed that measures monomer diffusion coefficients, filament turnover rates, and the fraction of actin polymerized from measurements of the evolution of fluorescence from a photoactivated band [Tardy et al. (1995) Biophys. J., 69:1674–1682; McGrath et al. (1998) Biophys. J., in press]. Applying this model to subconfluent endothelial cells shows that ∼40% of the actin is polymer and that these filaments turn over on average every 6 minutes. This report dicusses how PAF and FRAP can be combined with more traditional biochemistry to probe actin cytoskeleton remodeling in endothelial cells. Microsc. Res. Tech. 43:385–394 © 1998 Wiley‐Liss, Inc.

Objective: The mechanism by which cultured endothelial cells respond to shear stress is controversial. The cell surface and cytoskeleton are involved, but their roles are undefined. In this study, previously unknown changes in the surface detail and actin cytoskeleton of bovine aortic endothelial cells were identified.Methods: Actin filament content and filament number in resting and flow-oriented cells were determined by biochemical assays. The three-dimensional organization of the actin cytoskeleton in cells was defined in the confocal microscope and in the electron microscope after rapid-freezing, freeze-drying, and metal coating of detergent-permeabilized cells.Results: Endothelial cells have smooth apical membranes in situ. However, cultured cells exhibit surface microvilli which increase the apical surface area, exposing the ruffled surface to forces from fluid flow and potentially enhancing cell interactions with blood-borne white cells. Stereoscopic micrographs show that stress fibers are integrated into a complex distributed cytoplasmic structural actin network (DCSA). This lattice is formed by actin filaments that frequently cross and connect to each other, stress fibers, and microfilaments and microtubules. The cytoskeletons of cells cultured in static media lack apparent order when compared to cytoskeletons from cells which have been exposed to 24 hours of laminar flow.Conclusions: The DCSA physically connects the apical and basal cell membranes and fills the volume between nucleus and membrane, providing a mechanism for transmitting mechanical forces across cells and a signaling pathway from membrane to nucleus. Stress fibers increase the mechanical modulus of the DCSA, although this increase is probably unnecessary to withstand the increase in shear stress caused by blood flow in vivo. This implies that actin rearrangements are not required for mechanical integrity, but serve an alternate function.

• We performed an investigation of boundary layer separation in models of side-to-end anastomoses, used clinically in axillofemoral and femorofemoral grafting. Boundary layer separation occurs when momentum causes a fluid to flow against a local pressure gradient. Models of side-to-end anastomoses were constructed from Dacron grafting material and from clear plastic blocks and tubing. Fluid energy loss across the anastomosis was small and physiologically insignificant. Local adverse pressure gradients were demonstrated near the anastomosis. Flow visualization studies demonstrated characteristic areas of boundary layer separation in the region of the adverse pressure gradients. The separation region involved both the main limb and the side arm. The separation forms a shell or ring of slow-moving fluid around the mainstream. Preliminary studies with pulsatile flow and with blood demonstrate that boundary layer separation occurs under clinical flow conditions. Boundary layer separation may play a role in the development of anastomotic hyperplasia and atherosclerotic deposits in the vicinity of surgical anastomoses.(Arch Surg 114:1369-1373, 1979)

Objective: The mechanism by which cultured endothelial cells respond to shear stress is controversial. The cell surface and cytoskeleton are involved, but their roles are undefined. In this study, previously unknown changes in the surface detail and actin cytoskeleton of bovine aortic endothelial cells were identified.

Objective: To determine whether fluid flow influences the action of soluble vasoactive agonists on vascular endothelium.Methods: Confluent monolayers of bovine aortic endothelial cells (BAEC) were cultured on glass coverslips, prelabeled with the Ca2+-sensitive dye fura-2, and placed in a parallel-plate flow chamber designed to generate defined laminar fluid flow. Cytosolic free Ca2+ concentration ([Ca2+];) in individual BAEC was monitored during perfusion with medium containing adenine nucleotide under defined flow conditions.Results: Continuous perfusion with ATP (0.3-3.0 μM) or ADP (0.1-1.0 μM) evoked repetitive oscillations in [Ca2+]; in individual BAEC. The frequency of the [Ca2+]; oscillations was dependent on both nucleotide concentration and levels of applied shear stress; at constant bulk concentration of nucleotide, the frequency increased with shear stress. Stopping flow in the continuous presence of agonists immediately extinguished the oscillatory response. Elimination of extracellular Ca2+ did not inhibit the [Ca2+]; oscillations. In the presence of nonhydrolyzable nucleotide analog, ATPγS or ADPβS, application of flow resulted in similar shear-dependent [Ca2+]; oscillations, suggesting that flow modulation of the [Ca2+]; response was not simply due to depletion of ATP or ADP in the vicinity of BAEC monolayers as a result of hydrolysis of nucleotides by ectonucleotidases.Conclusions: These findings suggest that local hemodynamic conditions may modulate the action of vasoactive agents on the vascular endothelium in vivo.

Vascular endothelial cells subjected to fluid shear stress change their shape from polygonal to ellipsoidal and become uniformly oriented with the flow. In order to study the mechanisms of this response, we have measured the relaxation of bovine aortic endothelial cells that were grown on glass coverslips and exposed to fluid shear stress for 72 hours. An image analysis system was developed to quantify the cell shape relaxation that occurs following the cessation of shear stress. This method provides two different quantitative measures of relaxation: the loss of elongated shape by the cells and the change in cell direction with time. After equilibration to a fluid shear stress level of 8 dynes/cm2, cells immersed in static medium relax their shape in about 20 hours. After 72 hours in this static condition, the cell elongation is comparable to that of unstressed control cells but vestiges remain of the original orientation in the flow direction. This relaxation process contributes to our understanding of the response of vascular endothelium to fluid shear stress.

Phonoangiography, the quantitative analysis of sounds produced by blood flow, has been used to investigate local fluid motion in arteries narrowed by atherosclerosis. The sound spectra measured in vivowere found to be identical to those obtained in laboratory investigations of turbulent pipe flow. A theoretical model is proposed which: (a) quantitatively relates the in vivoand in vitrodata, and (b) allows estimation of the clinically important parameters of arterial diameter, flow velocity, local turbulence intensity, and wall pressure fluctuations. The results of calculations using data from human subjects agree with known arterial diameters and blood flow velocities.