Nikolaos M. Tsoukias

Endothelial Ca2+ wavelets and the induction of myoendothelial feedback

When arteries constrict to agonists, the endothelium inversely responds, attenuating the initial vasomotor response. The basis of this feedback mechanism remains uncertain, although past studies suggest a key role for myoendothelial communication in the signaling process. The present study examined whether second messenger flux through myoendothelial gap junctions initiates a negative-feedback response in hamster retractor muscle feed arteries. We specifically hypothesized that when agonists elicit depolarization and a rise in second messenger concentration, inositol trisphosphate (IP(3)) flux activates a discrete pool of IP(3) receptors (IP(3)Rs), elicits localized endothelial Ca(2+) transients, and activates downstream effectors to moderate constriction. With use of integrated experimental techniques, this study provided three sets of supporting observations. Beginning at the functional level, we showed that blocking intermediate-conductance Ca(2+)-activated K(+) channels (IK) and Ca(2+) mobilization from the endoplasmic reticulum (ER) enhanced the contractile/electrical responsiveness of feed arteries to phenylephrine. Next, structural analysis confirmed that endothelial projections make contact with the overlying smooth muscle. These projections retained membranous ER networks, and IP(3)Rs and IK channels localized in or near this structure. Finally, Ca(2+) imaging revealed that phenylephrine induced discrete endothelial Ca(2+) events through IP(3)R activation. These events were termed recruitable Ca(2+) wavelets on the basis of their spatiotemporal characteristics. From these findings, we conclude that IP(3) flux across myoendothelial gap junctions is sufficient to induce focal Ca(2+) release from IP(3)Rs and activate a discrete pool of IK channels within or near endothelial projections. The resulting hyperpolarization feeds back on smooth muscle to moderate agonist-induced depolarization and constriction.

Carbon Nanotube Reinforced Polylactide−Caprolactone Copolymer: Mechanical Strengthening and Interaction with Human Osteoblasts in Vitro

This study proposes the use of carbon nanotubes (CNTs) as reinforcement to enhance the mechanical properties of a polylactide-caprolactone copolymer (PLC) matrix. Biological interaction of PLC-CNT composites with human osteoblast cells is also investigated. Addition of 2 wt % CNT shows very uniform dispersion in the copolymer matrix, whereas 5 wt % CNT shows severe agglomeration and high porosity. PLC-2 wt % CNT composite shows an improvement in the mechanical properties with an increase in the elastic modulus by 100% and tensile strength by 160%, without any adverse effect on the ductility up to 240% elongation. An in vitro biocompatibility study on the composites shows an increase in the viability of human osteoblast cells compared to the PLC matrix, which is attributed to the combined effect of CNT content and surface roughness of the composite films.

A mathematical model of Ca2+ dynamics in rat mesenteric smooth muscle cell: agonist and NO stimulation.

A mathematical model of calcium dynamics in vascular smooth muscle cell (SMC) was developed based on data mostly from rat mesenteric arterioles. The model focuses on (a) the plasma membrane electrophysiology; (b) Ca2+ uptake and release from the sarcoplasmic reticulum (SR); (c) cytosolic balance of Ca2+, Na+, K+, and Cl ions; and (d) IP3 and cGMP formation in response to norepinephrine(NE) and nitric oxide (NO) stimulation. Stimulation with NE induced membrane depolarization and an intracellular Ca2+ ([Ca2+]i) transient followed by a plateau. The plateau concentrations were mostly determined by the activation of voltage-operated Ca2+ channels. NE causes a greater increase in [Ca2+]i than stimulation with KCl to equivalent depolarization. Model simulations suggest that the effect of[Na+]i accumulation on the Na+/Ca2+ exchanger (NCX) can potentially account for this difference.Elevation of [Ca2+]i within a concentration window (150-300 nM) by NE or KCl initiated [Ca2+]i oscillations with a concentration-dependent period. The oscillations were generated by the nonlinear dynamics of Ca2+ release and refilling in the SR. NO repolarized the NE-stimulated SMC and restored low [Ca2+]i mainly through its effect on Ca2+-activated K+ channels. Under certain conditions, Na+-K+-ATPase inhibition can result in the elevation of [Na+]i and the reversal of NCX, increasing resting cytosolic and SR Ca2+ content, as well as reactivity to NE. Blockade of the NCXs reverse mode could eliminate these effects. We conclude that the integration of the selected cellular components yields a mathematical model that reproduces, satisfactorily, some of the established features of SMC physiology. Simulations suggest a potential role of intracellular Na+ in modulating Ca2+ dynamics and provide insights into the mechanisms of SMC constriction, relaxation, and the phenomenon of vasomotion. The model will provide the basis for the development of multi-cellular mathematical models that will investigate microcirculatory function in health and disease.

Nitric Oxide Bioavailability in the Microcirculation: Insights from Mathematical Models

Over the last 30 years nitric oxide (NO) has emerged as a key signaling molecule involved in a number of physiological functions, including in the regulation of microcirculatory tone. Despite significant scientific contributions, fundamental questions about NOs role in the microcirculation remain unanswered. Mathematical modeling can assist in investigations of microcirculatory NO physiology and address experimental limitations in quantifying vascular NO concentrations. The number of mathematical models investigating the fate of NO in the vasculature has increased over the last few years, and new models are continuously emerging, incorporating an increasing level of complexity and detail. Models investigate mechanisms that affect NO availability in health and disease. They examine the significance of NO release from nonendothelial sources, the effect of transient release, and the complex interaction of NO with other substances, such as heme‐containing proteins and reactive oxygen species. Models are utilized to test and generate hypotheses for the mechanisms that regulate NO‐dependent signaling in the microcirculation.

A model of nitric oxide capillary exchange

Objective: Our aim was to develop a mathematical model that describes the nitric oxide (NO) transport in and around capillaries. The model is used to make quantitative predictions for (1) the contribution of capillary endothelium to the nitric oxide flux into the parenchymal tissue cells; (2) the scavenging of arteriolar endothelium‐derived NO by capillaries in the surrounding tissue; and (3) the role of myoglobin in tissue cells and plasma‐based hemoglobin on NO diffusion in and around capillaries.

A mathematical model of vasoreactivity in rat mesenteric arterioles. II. Conducted vasoreactivity

This study presents a multicellular computational model of a rat mesenteric arteriole to investigate the signal transduction mechanisms involved in the generation of conducted vasoreactivity. The model comprises detailed descriptions of endothelial (ECs) and smooth muscle (SM) cells (SMCs), coupled by nonselective gap junctions. With strong myoendothelial coupling, local agonist stimulation of the EC or SM layer causes local changes in membrane potential (V(m)) that are conducted electrotonically, primarily through the endothelium. When myoendothelial coupling is weak, signals initiated in the SM conduct poorly, but the sensitivity of the SMCs to current injection and agonist stimulation increases. Thus physiological transmembrane currents can induce different levels of local V(m) change, depending on cells gap junction connectivity. The physiological relevance of current and voltage clamp stimulations in intact vessels is discussed. Focal agonist stimulation of the endothelium reduces cytosolic calcium (intracellular Ca(2+) concentration) in the prestimulated SM layer. This SMC Ca(2+) reduction is attributed to a spread of EC hyperpolarization via gap junctions. Inositol (1,4,5)-trisphosphate, but not Ca(2+), diffusion through homocellular gap junctions can increase intracellular Ca(2+) concentration in neighboring ECs. The small endothelial Ca(2+) spread can amplify the total current generated at the local site by the ECs and through the nitric oxide pathway, by the SMCs, and thus reduces the number of stimulated cells required to induce distant responses. The distance of the electrotonic and Ca(2+) spread depends on the magnitude of SM prestimulation and the number of SM layers. Model results are consistent with experimental data for vasoreactivity in rat mesenteric resistance arteries.

A mathematical model of vasoreactivity in rat mesenteric arterioles: I. Myoendothelial communication

To study the effect of myoendothelial communication on vascular reactivity, we integrated detailed mathematical models of Ca2+ dynamics and membrane electrophysiology in arteriolar smooth muscle (SMC) and endothelial (EC) cells. Cells are coupled through the exchange of Ca2+, Cl−, K+, and Na+ ions, inositol 1,4,5‐triphosphate (IP3), and the paracrine diffusion of nitric oxide (NO). EC stimulation reduces intracellular Ca2+ ([Ca2+ in the SMC by transmitting a hyperpolarizing current carried primarily by K+. The NO‐independent endothelium‐derived hyperpolarization was abolished in a synergistic‐like manner by inhibition of EC SKCa and IKCa channels. During NE stimulation, IP3diffusing from the SMC induces EC Ca2+ release, which, in turn, moderates SMC depolarization and [Ca2+]i elevation. On the contrary, SMC [Ca2+]i was not affected by EC‐derived IP3. Myoendothelial Ca2+ fluxes had no effect in either cell. The EC exerts a stabilizing effect on calcium‐induced calcium release‐dependent SMC Ca2+ oscillations by increasing the norepinephrine concentration window for oscillations. We conclude that a model based on independent data for subcellular components can capture major features of the integrated vessel behavior. This study provides a tissue‐specific approach for analyzing complex signaling mechanisms in the vasculature.

Effect of alveolar volume and sequential filling on the diffusing capacity of the lungs: II. Experiment

The diffusing capacity of the lung, DL, is a critical physiological parameter, yet the currently accepted clinical model (Jones‐Meade) assumes a well-mixed alveolar region, and a constant DL independent of alveolar volume, VA, despite experimental evidence to the contrary. We have formulated a new mathematical model [Tsoukias, N.M, Wilson, A.F., George, S.C., 2000. Respir. Physiol 120, 231‐249] that considers variable alveolar mixing through a single parameter, k (0BkB1), and a DL that is a positive function of VA (DL a bVA or DLaVA b ). The goal of this study is to determine the suitability of this model to determine the unknown parameters a, b, a, b, and k from experimental data in normal subjects. The model predicts that the normal lung fills, in part, sequentially (k 0.519 0.35). The following average values in all seven subjects were obtained: DLNO 48·VA 2:3 ml:min:mmHg and DLCO200.7·VA ml:min:mmHg (STPD) where VA is expressed in L (STPD). We conclude that the mathematical model is suitable for identifying the unknown parameters and thus can be used to characterize the degree of alveolar mixing (or sequential filling) as well as the volume dependence of DL.

Impact of volume-dependent alveolar diffusing capacity on exhaled nitric oxide concentration.

AbstractExhaled endogenous nitric oxide (NO) holds promise as a potential biomarker of pulmonary inflammation. Previous experimental and theoretical work has concluded that the alveolar concentration approaches a constant steady state value at end exhalation due to both a constant maximum flux or release of NO (Jmax,alv) and a constant diffusing capacity (DNO,alv) in the alveolar region. We have recently demonstrated that DNO,alv is not constant, but increases with alveolar volume (VA) given by the following average relationship: DNO,alv=48*VA2/3 ml/min/mmHg (where VA is expressed in liters, STPD). We investigated the potential impact of a variable DNO,alv on exhaled concentration by incorporating the volume dependence into the currently accepted two-compartment model for NO exchange dynamics. Our results suggest that the mechanism underlying the plateau in exhaled concentration is a constant ratio Jmax,alv/DNO,alv.This constant ratio requires a volume dependence of Jmax,alv similar to DNO,alv, and is likely due to a decreasing alveolar surface area during exhalation.

Role of microprojections in myoendothelial feedback – a theoretical study

•  Endothelial microprojections (MPs) are cellular extensions of endothelial cells (ECs) that may be involved in regulation of smooth muscle cell (SMC) constriction in blood vessels. •  We developed computational models to investigate the role of MPs in generating EC feedback during SMC stimulation. The models account for the geometry of MPs and heterogeneous distribution of membrane channels and receptors in an EC. •  Simulations show that SMC stimulation causes calcium release in and around EC MPs that activates hyperpolarizing currents in ECs and moderates SMC constriction. •  The results help us better understand the mechanisms that regulate blood flow and pressure.