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An in vitro whole blood reperfusion model was employed to quantify: (a) initial rates of lysis of mural platelet deposits from flowing blood onto fibrin-coated surfaces and (b) plasmin-mediated consumption of plasma plasminogen and fibrinogen, by recombinant tissue-type plasminogen activator (rt-PA) and two t-PA variants, KHRR 296–299 AAAA (K-tPA) and T103N, N117Q, KHRR 296–299 AAAA (TNK-tPA), at wall shear rates of either 500 or 1000 s−1. K- and TNK-tPA are more fibrin-specific than rt-PA, and are also resistant to inactivation by plasminogen activator inhibitor−1 (PAI−1). At 500 s−1, no agent showed significant lysis of mural platelet deposits on fibrin, even at concentrations as high as 10 μ g/ml of blood. At 1000 s−1, each agent demonstrated a dose-dependent lysis of mural platelet deposits, due to plasmin-mediated lysis of the fibrin substrate (fibrinolysis). The local concentration of thrombolytic agents close to the fibrin-coated surface is probably higher than the concentration of released PAI−1 from the adherent and activated platelets. Hence, the initial rates of lysis achieved by K- and TNK-tPA were not significantly different from that by rt-PA, when each agent was tested at either 1 or 10 μ g/ml of blood. However, TNK-tPA, at 1 μ g/ml,> caused the most extensive lysis at the end of the 50 min reperfusion period (50% vs 29% and 17% by rt-PA and K-tPA, respectively). K- and TNK-tPA, at concentrations as high as 10 μ g/ml of blood, caused plasminogen activation that was controlled by the natural plasmin inhibitors, and, thus, no proteolytic degradation of plasma fibrinogen (fibrinogenolysis). On the contrary, rt-PA at 1 μ g/ml revealed slight fibrinogenolysis that became extensive at 10 μ g/ml. This study demonstrates the potential use of an in vitro model, that mimics the in vivo hemodynamic environment, in evaluating the performance of thrombolytic agents. The data suggest that: (a) adequate flow must accompany fibrinolysis for successful embolization, and (b) the TNK variant may lyse annular thrombi after recanalization, at least as efficiently as rt-PA does, while causing lesser defect of systemic hemostasis. © 1998 Biomedical Engineering Society. PAC98: 8745Hw, 8790+y, 8380Lz, 8715Kg

The mitochondrial network is dynamic with conformations that vary between a tubular continuum and a fragmented state. The equilibrium between mitochondrial fusion/fission, as well as the organelle motility, determine network morphology and ultimately mitochondrial/cell function. Network morphology has been linked with the energy state in different cell types. In this study, we examined how bioenergetic factors affect mitochondrial dynamics/motility in cultured vascular endothelial cells (ECs). ECs were transduced with mitochondria-targeted green fluorescent protein (mito-GFP) and exposed to inhibitors of oxidative phosphorylation (OXPHOS) or ATP synthesis. Time-lapse fluorescence videos were acquired and a mathematical program that calculates size and speed of each mitochondrial object at each time frame was developed. Our data showed that inner mitochondrial membrane potential (ΔΨ(m)), ATP produced by glycolysis, and, to a lesser degree, ATP produced by mitochondria are critical for maintaining the mitochondrial network, and different metabolic stresses induce distinct morphological patterns (e.g., mitochondrial depolarization is necessary for "donut" formation). Mitochondrial movement, characterized by Brownian diffusion with occasional bursts in displacement magnitude, was inhibited under the same conditions that resulted in increased fission. Hence, imaging/mathematical analysis shed light on the relationship between bioenergetics and mitochondrial network morphology; the latter may determine EC survival under metabolic stress.

The effect of flow-induced shear stress on membrane K+ permeability was investigated by measuring86Rb+ efflux in cultured calf pulmonary artery endothelial cells. Cells were subjected to step changes in shear stress from 1 dyn/cm2 to 2.4, 4.8, or 10 dyn/cm2 in a parallel-plate flow chamber. Increasing shear stress produced a graded, transient increase in86Rb+ efflux which peaked within 1 min and subsequently declined rapidly toward pre-stimulus levels. Upon returning shear stress to 1 dyn/cm2,86Rb+ efflux initially decreased, but returned slowly to basal values. In contrast, application of bradykinin at a constant shear stress of 1 dyn/cm2 produced a transient increase in86Rb+ efflux that was followed by a sustained elevated phase during which time efflux gradually returned to pre-stimulus levels. In order to exclude the possibility that the transient increase in86Rb+ efflux with shear stress simply reflects a flow-dependent change in the washout of radiotracer, the transient convection-diffusion equation was solved using finite element simulation. When the flux of86Rb+ from the cell monolayer was assumed to be constant with time, the mathematical model predicted an increase in efflux rate coefficients upon step increases in flow that were only 7–19% of that observed experimentally. The numerical predictions correlated well with the experimentally obtained peaks when the flux of86Rb+ from the cell monolayer was simultaneously increased with flow to a new steady value. These simulations however, could not predict the transient nature of the response to increased shear stress. The results from the computer modeling suggest that the transient increase in86Rb+ efflux does not reflect a washout phenomenon and supports the hypothesis that shear stress produces a graded, transient increase in the K+ permeability of vascular endothelial cells.

Study of the mechanisms of monocyte adhesion initiating atheroslerotic lesions has engaged investigators for decades. Single-phase computational fluid dynamics (CFD) analyses fail to account for particulate migration. Consequently, inconsistencies arise when correlating adhesion with wall shear stress (WSS). The purpose of this paper is to present, to our knowledge, the first computational analysis of in vitro U937 monocyte-like human cell adhesion data using a coupled multiphase CFD-population balance adhesion model. The CFD model incorporates multiphase non-Newtonian hemodynamic models to compute the spatial distributions of freely flowing monocytes and WSSs in control volumes adjacent to the wall. Measurements of monocyte adhesion onto an E-selectin-coated flow model that included an idealized stenosis and an abrupt expansion were available from the literature. In this study, we develop a new monolayer population balance adhesion model, based on the widely accepted mechanism of ligand-receptor binding, coupled to the CFD results. The monolayer population balance model accounts for the interactions of freely flowing, rolling, and adhering monocytes with surfaces via first-order reactions, transport of rolling cells in the monolayer, and the concept of a WSS detachment threshold, clearly evident in the adhesion experiments. The new paradigm of coupling the multiphase hemodynamic CFD model with the proposed adhesion model is illustrated by determining and interpreting the model parameters for experimental datasets having Reynolds numbers of 100 and 140. The coupled multiphase CFD adhesion model is able to simultaneously predict the spatial variations in freely flowing monocytes, their adherent number density, and carrier fluid WSSs adjacent to ligand-coated flow cell surfaces.