Extracellular Diffusion Research Articles

Real-time MR Imaging With Gadoteridol Predicts Distribution of Transgenes After Convection-enhanced Delivery of AAV2 Vectors

Gene therapies that utilize convention-enhanced delivery (CED) will require close monitoring of vector infusion in real time and accurate prediction of drug distribution. The magnetic resonance imaging (MRI) contrast agent, Gadoteridol (Gd), was used to monitor CED infusion and to predict the expression pattern of glial cell line-derived neurotrophic factor (GDNF) protein after administration of adeno-associated virus type 2 (AAV2) vector encoding human pre-pro-GDNF complementary DNA. The nonhuman primate (NHP) thalamus was utilized for modeling infusion to allow delivery of volumes more relevant to planned human studies. AAV2 encoding human aromatic L-amino acid decarboxylase (AADC) was coinfused with AAV2-GDNF/Gd to confirm regions of AAV2 transduction versus extracellular GDNF diffusion. There was a close correlation between Gd distribution and GDNF or AADC expression, and the ratios of expression areas of GDNF or AADC versus Gd were both close to 1. Our data support the use of Gd and MRI to monitor AAV2 infusion via CED and to predict the distribution of GDNF protein after AAV2-GDNF administration.

TH-D-201B-06: Evaluation of the Intracellular and Extracellular Anomalous Diffusion from MR Diffusion Tensor in Neural Structure

Purpose: To improve the characterization of the distribution and microarchitecture of white matter by comparing Monte Carlo simulations of the diffusion weighted MR signal from both the Gaussian and non‐Gaussian PDF with generalized models of anomalous diffusion α tα α≠1 that allows for deviations from standard Fickian diffusion as a function of axon diameter packing fraction and fiber spatial distribution. Method and Materials: The diffusion weighted MR signal is evaluated from a 3D Monte Carlo simulator that tracks the pulsed gradient spin echo within a voxel size 100×100×100μm3 TE= 80ms Δ =40ms δ=30ms . White matter is modeled as cylindrical fibers with intracellular and extracellular molecules distributed homogeneously under δ correlated and general stochastic force within and between the axons. The semi‐analytic models are taken from a general Fokker‐Planck equation that allows deviations from Gaussian diffusion. The mean square displacement predicted from the models is compared to the results of the Monte Carlo simulations for different fiber configurations.Results: Fickian diffusion assumption in conventional diffusiontensor imaging (DTI) can only sustain in a condition of mean spatial homogeneity that depends on the architecture and measurement time. Non‐Fickian diffusion are predicted and observed in the simulations at early times and weaken at very late times. Even when the diffusion is inferred to be Fickian higher order statistical measures of the molecular distribution such as kurtosis reveal an opposite distribution to apparent diffusion coefficient. The mean diffusivity increases with α while the fractional anisotropy remains yet to indentify relationship between the fiber packing geometry.Conclusion: We develop a novel tool to evaluate the MR diffusion weighted signal from the intracellular and extracellular compartment as a function of fiber diameter fraction and geometry using tunable parameters and find that the observed diffusionanisotropy mainly originates from the extracellular compartment between axons.

Effect of Membrane Permeability and Extracellular Diffusion on NO uptake by Erythrocytes

Quantification of nitric oxide (NO)‐red blood cells (RBC) reaction metabolites is necessary to evaluate the extent of diffusional resistances for nitric oxide transport in vascular lumen. Delivery of physiologically relevant concentration of nitric oxide and RBC‐hemoglobin are a critical factor in quantification of in vitro NO‐RBC reaction. In this study, we developed a flask bioreactor which delivers gaseous NO to the RBCs and studied the effects of hematocrit (hct) and NO concentrations on NO‐RBC interactions. We developed a computational model of NO transport to single RBCs using experimental data to analyze the effect of RBC membrane permeability on steady state NO concentrations in the bulk of liquid surrounding the RBCs and at the RBC membrane. We observed that the steady state NO concentrations in the bulk of liquid and at the RBC membrane 1) decreased with increase in RBC membrane permeability for a given equilibrium NO concentration and hct; 2) increased with increase available NO concentrations at a given RBC membrane permeability; 3) decreased with increase in hct for a given RBC membrane permeability. Our results indicate that the extracellular diffusion and RBC membrane permeability are significant diffusional barriers for NO transport to the RBCs and the RBC NO membrane permeability should be in the range of 0.0415‐ 0.4 cm/s. Supported by: AHA 0530050N, NIH R01 HL084337 & R15 HL087287.

β-Amyloid Fibrillation and/or Hyperhomocysteinemia Modify Striatal Patterns of Hyaluronic Acid and Dermatan Sulfate: Possible Role in the Pathogenesis of Alzheimers Disease

A key event in Alzheimer's disease (AD) pathogenesis is the formation of insoluble peptides beta-amyloid aggregates and this process is favoured by a condition of hyperhomocysteinemia. To date, there is growing evidence that implicates glycosaminoglycans (GAGs) in the pathophysiology of amyloidosis but no data are available on the characterization of brain GAGs involved in the enhancing beta-amyloid fibrillogenesis in relationship to their structure and physico-chemical properties. Furthermore, few studies have been performed on the relationship between hyperhomocysteinemia and extracellular matrix (ECM) modifications. The aim of this study was to evaluate the amount and chemical structure of GAGs in rat striatal areas where beta-amyioid fibrillogenesis was induced, and in conditions of hyperhomocysteinemia. The intrastriatal injection of beta-amyloid produced a significant decrease (-40.8%) in the hyaluronic acid (HA) percentage and an increase (+14.5%) in the dermatan sulfate (DS) with a total charge density increasing of 14.9%. A significant decrease (-19.5%) in the HA percentage and an increase (+6.9%) in the DS % was also observed in striata obtained from the hyperhomocysteinemic animals. The total charge density increased by 6.8%. Quite the same trend was observed in rats after intrastriatal injection of beta-amyloid and in a condition of hyperhomocysteinemia. The observed increase of DS concentration and the correspondent decrease of the nonsulfated polymer HA after in vivo treatment with beta-amyloid and in a condition of hyperhocysteinemia support the hypothesis that an increase in local production of sulfated GAGs may reduce beta-amyloid neurotoxicity. However, the consequent modification of the ECM network might impair the extracellular diffusion pathways of different signal molecules and participate in the progression of AD.

Bral1: Its Role in Diffusion Barrier Formation and Conduction Velocity in the CNS

At the nodes of Ranvier, excitable axon membranes are exposed directly to the extracellular fluid. Cations are accumulated and depleted in the local extracellular nodal region during action potential propagation, but the impact of the extranodal micromilieu on signal propagation still remains unclear. Brain-specific hyaluronan-binding link protein, Bral1, colocalizes and forms complexes with negatively charged extracellular matrix (ECM) proteins, such as versican V2 and brevican, at the nodes of Ranvier in the myelinated white matter. The link protein family, including Bral1, appears to be the linchpin of these hyaluronan-bound ECM complexes. Here we report that the hyaluronan-associated ECM no longer shows a nodal pattern and that CNS nerve conduction is markedly decreased in Bral1-deficient mice even though there were no differences between wild-type and mutant mice in the clustering or transition of ion channels at the nodes or in the tissue morphology around the nodes of Ranvier. However, changes in the extracellular space diffusion parameters, measured by the real-time iontophoretic method and diffusion-weighted magnetic resonance imaging (MRI), suggest a reduction in the diffusion hindrances in the white matter of mutant mice. These findings provide a better understanding of the mechanisms underlying the accumulation of cations due to diffusion barriers around the nodes during saltatory conduction, which further implies the importance of the Bral1-based extramilieu for neuronal conductivity.

Intercellular calcium wave propagation in linear and circuit-like bone cell networks

In the present study, the mechanism of intercellular calcium wave propagation in bone cell networks was identified. By using micro-contact printing and self-assembled monolayer technologies, two types of in vitro bone cell networks were constructed: open-ended linear chains and looped hexagonal networks with precisely controlled intercellular distances. Intracellular calcium responses of the cells were recorded and analysed when a single cell in the network was mechanically stimulated by nano-indentation. The looped cell network was shown to be more efficient than the linear pattern in transferring calcium signals from cell to cell. This phenomenon was further examined by pathway-inhibition studies. Intercellular calcium wave propagation was significantly impeded when extracellular adenosine triphosphate (ATP) in the medium was hydrolysed. Chemical uncoupling of gap junctions, however, did not significantly decrease the transferred distance of the calcium wave in the cell networks. Thus, it is extracellular ATP diffusion, rather than molecular transport through gap junctions, that dominantly mediates the transmission of mechanically elicited intercellular calcium waves in bone cells. The inhibition studies also demonstrated that the mechanical stimulation-induced calcium responses required extracellular calcium influx, whereas the ATP-elicited calcium wave relied on calcium release from the calcium store of the endoplasmic reticulum.

An ATP-dependent mechanism mediates intercellular calcium signaling in bone cell network under single cell nanoindentation

To investigate the roles of intercellular gap junctions and extracellular ATP diffusion in bone cell calcium signaling propagation in bone tissue, in vitro bone cell networks were constructed by using microcontact printing and self-assembled monolayer technologies. In the network, neighboring cells were interconnected through functional gap junctions. A single cell at the center of the network was mechanically stimulated by using an AFM nanoindenter. Intracellular calcium ([Ca 2+] i) responses of the bone cell network were recorded and analyzed. In the untreated groups, calcium propagation from the stimulated cell to neighboring cells was observed in 40% of the tests. No significant difference was observed in this percentage when the intercellular gap junctions were blocked. This number, however, decreased to 10% in the extracellular ATP-pathway-blocked group. When both the gap junction and ATP pathways were blocked, intercellular calcium waves were abolished. When the intracellular calcium store in ER was depleted, the indented cell can generate calcium transients, but no [Ca 2+] i signal can be propagated to the neighboring cells. No [Ca 2+] i response was detected in the cell network when the extracellular calcium source was removed. These findings identified the biochemical pathways involved in the calcium signaling propagation in bone cell networks.

Astrocytic Processes Ensheathing Synaptic Glomeruli cause Anomalous Extracellular Diffusion

Brain extracellular space forms a channel where signaling molecules, growth factors and therapeutics diffuse. Characterization and quantification of the extracellular diffusion is thus important for intercellular signaling and drug delivery. We have recently reported that the extracellular diffusion is anomalous in the granular layer of rat cerebellar slices (anomalous diffusion model quantified an average anomalous exponent dw = 5.0). In this respect, the granular layer significantly differs from most brain regions where the extracellular diffusion is normal (i.e., dw ≈ 2.0). In biological systems, anomalous diffusion may be caused by concave geometrical formations that transiently trap diffusing particles. In rat granular layer, large and abundant synaptic glomeruli formed by concave processes of astrocytes that ensheathe complex synapses may function as the traps. To test this hypothesis, we employed Integrative Optical Imaging method to measure diffusion of small dextran molecules (MW 3000) in the granular layer of isolated turtle cerebellum where complex synapses are present but have no or very limited astrocytic ensheathment. In turtle granular layer, the anomalous diffusion model quantified dw = 2.6, which is significantly less than dw = 5.0 observed in rat granular layer but similar to dw extracted from normal diffusion in a free medium (2.1) and in other brain regions (e.g., 2.3 in rat neocortex). Thus the extracellular diffusion was normal in the granular layer of turtle cerebellum that lacks trap-like astrocytic ensheathment of synaptic glomeruli. In conclusion, diffusion measurements in turtle support our hypothesis that astrocytic processes ensheathing synaptic glomeruli cause anomalous extracellular diffusion. Our approach is potentially useful for quantification of astrocytic ensheathment in the living brain under normal and pathological conditions. Our result also has implications for the interstitial transport of molecules and drugs in brain regions containing synaptic glomeruli. Supported by NIH(NS047557).

Extracellular diffusion and permeability effects on NO–RBCs interactions using an experimental and theoretical model

Nitric oxide (NO) is a potent vasodilator and its homeostasis depends on interaction with RBCs. A key factor in understanding NO–RBC interactions in vascular lumen is a comprehensive analysis of product identification and quantification. In this context, administration of NO during in vitro NO–RBC interactions becomes a crucial variable. In this study, we designed a bioreactor that maintains a precise NO concentration in the headspace that diffuses to RBCs suspension to study the quantitative effect of NO concentration and hematocrit (Hct) on NO–RBC interactions. The products of NO–RBC reaction (nitrite and total nitrogen species (total NOx)) were measured by chemiluminescence assay. A mathematical model simulating NO biotransport to a single RBC was developed to (1) estimate NO–RBC reaction rate constant; (2) predict the NO concentrations in the bulk RBC suspension and at the RBC membrane for RBC membrane NO permeability ( P m) values of 0.0415–40 cm/s. Measured nitrite and total NOx concentrations increased with increase in headspace NO concentration whereas nitrite concentrations decreased with hematocrit and total NOx concentrations increased with hematocrit. This indicates that the extracellular resistance is a controlling factor for RBC uptake of NO. Modeling results showed that the effective reaction rate constant ( k eff) for NO–RBC interactions was 2.32 × 10 4–1.08 × 10 6 M − 1 s − 1 . Results also predict that the membrane permeability in the range of 0.0415–0.4 cm/s is required to maintain physiologically relevant levels of NO at the smooth muscle cell layer. The effective reaction rate constant increased with increase in P m and magnitude of increase was higher at 45% Hct. For all P m values, the k hb/ k eff ratios were lower for 45% Hct as compared to 5% Hct indicating extracellular resistance is important for RBC NO uptake. Our experimental and mathematical analyses of NO–RBC interactions indicate that both unstirred layer and RBC membrane have a significant effect on NO transport to RBCs. In addition, the membrane permeability in the range of 0.0415–0.4 cm/s is required to maintain sufficient NO concentrations at the smooth muscle cell layer.

Aldehyde fixative solutions alter the water relaxation and diffusion properties of nervous tissue.

Chemically-fixed nervous tissues are well-suited for high-resolution, time-intensive MRI acquisitions without motion artifacts, such as those required for brain atlas projects, but the aldehyde fixatives used may significantly alter tissue MRI properties. To test this hypothesis, this study characterized the impact of common aldehyde fixatives on the MRI properties of a rat brain slice model. Rat cortical slices immersion-fixed in 4% formaldehyde demonstrated 21% and 81% reductions in tissue T(1) and T(2), respectively (P < 0.001). The T(2) reduction was reversed by washing slices with phosphate-buffered saline (PBS) for 12 h to remove free formaldehyde solution. Diffusion MRI of cortical slices analyzed with a two-compartment analytical model of water diffusion demonstrated 88% and 30% increases in extracellular apparent diffusion coefficient (ADC(EX)) and apparent restriction size, respectively, when slices were chemically-fixed with 4% formaldehyde (P <or= 0.021). Further, fixation with 4% formaldehyde increased the transmembrane water exchange rate 239% (P < 0.001), indicating increased membrane permeability. Karnovsky's and 4% glutaraldehyde fixative solutions also changed the MRI properties of cortical slices, but significant differences were noted between the different fixative treatments (P < 0.05). The observed water relaxation and diffusion changes help better define the validity and limitations of using chemically-fixed nervous tissue for MRI investigations.

Autocrine signal transmission with extracellular ligand degradation

Traveling waves of cell signaling in epithelial layers orchestrate a number of important processes in developing and adult tissues. These waves can be mediated by positive feedback autocrine loops, a mode of cell signaling where binding of a diffusible extracellular ligand to a cell surface receptor can lead to further ligand release. We formulate and analyze a biophysical model that accounts for ligand-induced ligand release, extracellular ligand diffusion and ligand–receptor interaction. We focus on the case when the main mode for ligand degradation is extracellular and analyze the problem with the sharp threshold positive feedback nonlinearity. We derive expressions that link the speed of propagation and other characteristics of traveling waves to the parameters of the biophysical processes, such as diffusion rates, receptor expression level, etc. Analyzing the derived expressions we found that traveling waves in such systems can exhibit a number of unusual properties, e.g. non-monotonic dependence of the speed of propagation on ligand diffusivity. Our results for the fully developed traveling fronts can be used to analyze wave initiation from localized perturbations, a scenario that frequently arises in the in vitro models of epithelial wound healing, and guide future modeling studies of cell communication in epithelial layers.

FGF9 monomer–dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

The spontaneous dominant mouse mutant, Elbow-knee-synostosis (Eks), exhibits elbow and knee joint synosotsis, and premature fusion of cranial sutures. Here we identify a missense mutation in the Fgf9 gene that is responsible for the Eks mutation. Through investigation of the pathogenic mechanisms of joint and suture synostosis in Eks mice, we identify a key molecular mechanism that regulates FGF9 signaling in developing tissues. We show that the Eks mutation prevents homodimerization of the FGF9 protein and that monomeric FGF9 binds to heparin with a lower affinity than dimeric FGF9. These biochemical defects result in increased diffusion of the mutant FGF9 protein (FGF9Eks) through developing tissues, leading to ectopic FGF9 signaling and repression of joint and suture development. We propose a mechanism in which the range of FGF9 signaling in developing tissues is limited by its ability to homodimerize and its affinity for extracellular matrix heparan sulfate proteoglycans.

Eosinophilia-associated muscle disorders: an immunohistological study with tissue localisation of major basic protein in distinct clinicopathological forms

Aims:(a) To evaluate tissue eosinophil density, location of eosinophil cytotoxic products, histopathological muscle changes and inflammatory cell types in different eosinophilia-associated myopathies that are clinicopathologically heterogeneous. (b) To determine the...

Preventing Extracellular Diffusion of Trigeminal Nitric Oxide Enhances Formalin-induced Orofacial Pain

Nitric oxide (NO), a diffusible gas, is produced in the central nervous system, including the spinal cord dorsal horn and the trigeminal nucleus, the first central areas processing nociceptive information from periphery. In the spinal cord, it has been demonstrated that NO acts as pronociceptive or antinociceptive mediators, apparently in a concentration-dependent manner. However, the central role of NO in the trigeminal nucleus remains uncertain in support of processing the orofacial nociception. Thus, we here investigated the central role of NO in formalin (3%)-induced orofacial pain in rats by administering membrane-permeable or -impermeable inhibitors, relating to the NO signaling pathways, into intracisternal space. The intracisternal pretreatments with the NO synthase inhibitor L-NAME, the NO-sensitive guanylate cyclase inhibitor ODQ, and the protein kinase C inhibitor GF109203X, all of which are permeable to the cell membrane, significantly reduced the formalin-induced pain, whereas the membrane-impermeable NO scavenger PTIO significantly enhanced it, compared to vehicle controls. These data suggest that an overall effect of NO production in the trigeminal nucleus is pronociceptive, but NO extracellularly diffused out of its producing neurons would have an antinociceptive action.

Diffusion Pathways for Heartwood Substances in Acacia Mangium

The cellular distribution of heartwood substances and the structure of the pathways for their diffusion were studied in Acacia mangium Willd. Apart from ray parenchyma cells, axial parenchyma cells also are involved in the formation of heartwood substances. Heartwood substances were unevenly distributed in the heartwood. A closer inspection of interfibre pit pairs revealed that, although many pit membranes were completely covered with encrusting materials, some pit pairs had many small openings on their pit membranes. The openings possibly function as intercellular diffusion pathways for heartwood substances. The sizes of the pits varied considerably, ranging from 0.4 to 2.3 μm in diameter. These structural variations in the interfiber pits might be one of the factors contributing to the uneven distribution of the heartwood substances. A large number of blind pits were present in the ray parenchyma cells and faced the intercellular spaces, into which heartwood substances from the ray parenchyma cells were released via these blind pits. Resin-cast replicas demonstrated that the intercellular spaces and the blind pits formed a three-dimensional network that is considered to serve as an extracellular diffusion pathway for heartwood substances.

Estimation of Cell Membrane Permeability and Intracellular Diffusion Coefficient of Human Gray Matter

The signal intensity of diffusion-weighted imaging (DWI) is sensitive to the intra- and extracellular diffusion coefficient of water and cell membrane permeability. We applied a method we proposed in previous papers to estimate noninvasively the membrane permeability and intracellular diffusion coefficient of normal human brain (gray matter) in 3 normal volunteers. We theoretically compared predicted signals and experiment results using a 1.5-tesla magnetic resonance (MR) imaging system. We acquired images using an echo planar imaging (EPI) sequence, applying motion-probing gradient (MPG) pulses in 3 directions. We periodically performed numerical simulations for various combinations of membrane permeability and intracellular diffusion coefficients using the finite-difference method. By minimizing the difference between signals obtained experimentally and those from numerical simulation, we could estimate membrane permeability (76+/-9 mm2/s mum) and intracellular diffusion coefficient (1.0+/-0.0 mm2/s) for the human brain. The estimated membrane permeability was the criterion value for diagnosing disease in gray matter.

Receptor Actions of Synaptically Released Glutamate: The Role of Transporters on the Scale from Nanometers to Microns

Actions of the excitatory neurotransmitter glutamate inside and outside the synaptic cleft determine the activity of neural circuits in the brain. However, to what degree local glutamate transporters affect these actions on a submicron scale remains poorly understood. Here we focus on hippocampal area CA1, a common subject of synaptic physiology studies. First, we use a two-photon excitation technique to obtain an estimate of the apparent (macroscopic) extracellular diffusion coefficient for glutamate, ∼0.32 μm 2/ms. Second, we incorporate this measurement into a Monte Carlo model of the typical excitatory synapse and examine the influence of distributed glutamate transporter molecules on signal transmission. Combined with the results of whole-cell recordings, such simulations argue that, although glutamate transporters have little effect on the activation of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, this does not rule out the occurrence of up to several dozens of transporters inside the cleft. We further evaluate how the expression pattern of transporter molecules (on the 10–100 nm scale) affects the activation of N-methyl-D-aspartic acid or metabotropic glutamate receptors in the synaptic vicinity. Finally, we extend our simulations to the macroscopic scale, estimating that synaptic activity sufficient to excite principal neurons could intermittently raise extracellular glutamate to ∼1 μM only at sparse (microns apart) hotspots. Greater rises of glutamate occur only when <5% of transporters are available (for instance, when an astrocyte fails). The results provide a quantitative framework for a better understanding of the relationship between glutamate transporters and glutamate receptor signaling.

Tunneling nanotube (TNT)-like structures facilitate a constitutive, actomyosin-dependent exchange of endocytic organelles between normal rat kidney cells

Tunneling nanotube (TNT)-like structures are intercellular membranous bridges that mediate the transfer of various cellular components including endocytic organelles. To gain further insight into the magnitude and mechanism of organelle transfer, we performed quantitative studies on the exchange of fluorescently labeled endocytic structures between normal rat kidney (NRK) cells. This revealed a linear increase in both the number of cells receiving organelles and the amount of transferred organelles per cell over time. The intercellular transfer of organelles was unidirectional, independent of extracellular diffusion, and sensitive to shearing force. In addition, during a block of endocytosis, a significant amount of transfer sustained. Fluorescence microscopy revealed TNT-like bridges between NRK cells containing F-actin but no microtubules. Depolymerization of F-actin led to the disappearance of TNT and a strong inhibition of organelle exchange. Partial ATP depletion did not affect the number of TNT but strongly reduced organelle transfer. Interestingly, the myosin II specific inhibitor S-(−)-blebbistatin strongly induced both organelle transfer and the number of TNT, while the general myosin inhibitor 2,3-butanedione monoxime induced the number of TNT but significantly inhibited transfer. Taken together, our data indicate a frequent and continuous exchange of endocytic organelles between cells via TNT by an actomyosin-dependent mechanism.

Diffusion of Flexible Random-Coil Dextran Polymers Measured in Anisotropic Brain Extracellular Space by Integrative Optical Imaging

There are a limited number of methods available to quantify the extracellular diffusion of macromolecules in an anisotropic brain region, e.g., an area containing numerous aligned fibers where diffusion is faster along the fibers than across. We applied the integrative optical imaging method to measure diffusion of the fluorophore Alexa Fluor 488 (molecular weight (MW) 547) and fluorophore-labeled flexible random-coil dextran polymers (dex3, MW 3000; dex75, MW 75,000; dex282, MW 282,000; dex525, MW 525,000) in the extracellular space (ECS) of the anisotropic molecular layer of the isolated turtle cerebellum. For all molecules, two-dimensional images acquired an elliptical shape with major and minor axes oriented along and across, respectively, the unmyelinated parallel fibers. The effective diffusion coefficients, D major * and D minor * , decreased with molecular size. The diffusion anisotropy ratio ( D A R = D major * / D minor * ) increased for Alexa Fluor 488 through dex75 but then unexpectedly reached a plateau. We argue that dex282 and dex525 approach the ECS width and deform to diffuse. In support of this concept, scaling theory shows the diffusion behavior of dex282 and dex525 to be consistent with transition to a reptation regime, and estimates the average ECS width at ∼31 nm. These findings have implications for the interstitial transport of molecules and drugs, and for modeling neurotransmitter diffusion during ectopic release and spillover.

Phospholipase C‐delta extends intercellular signalling range and responses to injury‐released growth factors in non‐excitable cells

Intercellular communication in non-excitable cells is restricted to a limited range close to the signal source. Here, we have examined whether modification of the intracellular microenvironment could prolong the spatial proposition of signal generation and could increase cell proliferation. Mathematical models and experimental studies of endothelial repair after controlled mechanical injury were used. The models predict the diffusion range of injury-released growth factors and identify important parameters involved in a signalling regenerative mode. Transfected human umbilical vein endothelial cells (HUVECs) were used to validate model results, by examining intercellular calcium signalling range, cell proliferation and wound healing rate. The models predict that growth factors have a limited capacity of extracellular diffusion and that intercellular signals are specially sensitive to cell phospholipase C-delta (PLCdelta) levels. As basal PLCdelta levels are increased by transfection, a significantly increased intercellular calcium range, enhanced cell proliferation, and faster wound healing rate were observed. Our in silico and in vitro studies demonstrated that non-excitable endothelial cells respond to stimuli in a complex manner, in which intercellular communication is controlled by physicochemical properties of the stimulus and by the cell microenvironment. Such findings may have profound implications for our understanding of the tight nature of autocrine cell growth control, compensation to stress states and response to altered microenvironment, under pathological conditions.