Cell proliferation along vascular islands during microvascular network growth

ABSTRACT: BACKGROUND: Observations in our laboratory provide evidence of vascular islands, defined as disconnected endothelial cell segments, in the adult microcirculation. The objective of this study was to determine if vascular islands are involved in angiogenesis during microvascular network growth. RESULTS: Mesenteric tissues, which allow visualization of entire microvascular networks at a single cell level, were harvested from unstimulated adult male Wistar rats and Wistar rats 3 and 10 days post angiogenesis stimulation by mast cell degranulation with compound 48/80. Tissues were immunolabeled for PECAM and BRDU. Identification of vessel lumens via injection of FITC-dextran confirmed that endothelial cell segments were disconnected from nearby patent networks. Stimulated networks displayed increases in vascular area, length density, and capillary sprouting. On day 3, the percentage of islands with at least one BRDU-positive cell increased compared to the unstimulated level and was equal to the percentage of capillary sprouts with at least one BRDU-positive cell. At day 10, the number of vascular islands per vascular area dramatically decreased compared to unstimulated and day 3 levels. CONCLUSIONS: These results show that vascular islands have the ability to proliferate and suggest that they are able to incorporate into the microcirculation during the initial stages of microvascular network growth.     

The quantitative estimation of microvessels in microvascular networks

A novel method is presented that greatly facilitates the determination of vessel segment number and density in both simple and complex microvascular networks. This approach was applied to microvascular networks represented by the Bra-Ket operator technique and accurately predicted the number of vessel segments in both tree-branched and loop-branched (arcade) networks. The method was then applied to the complex hexagonal array network described by Engelson et al. for gastrointestinal mucosa and accurately yielded an average vessel segment number of three around each hexagonal loop. This new method may be used for conveniently estimating tissue microvascular densities, such as vessel rarefaction or proliferation, and for the modelling of microvascular networks.     

An Ex Vivo Model for Anti-Angiogenic Drug Testing on Intact Microvascular Networks

New models of angiogenesis that mimic the complexity of real microvascular networks are needed. Recently, our laboratory demonstrated that cultured rat mesentery tissues contain viable microvascular networks and could be used to probe pericyte-endothelial cell interactions. The objective of this study was to demonstrate the efficacy of the rat mesentery culture model for anti-angiogenic drug testing by time-lapse quantification of network growth. Mesenteric windows were harvested from adult rats, secured in place with an insert, and cultured for 3 days according to 3 experimental groups: 1) 10% serum (angiogenesis control), 2) 10% serum + sunitinib (SU11248), and 3) 10% serum + bevacizumab. Labeling with FITC conjugated BSI-lectin on Day 0 and 3 identified endothelial cells along blood and lymphatic microvascular networks. Comparison between day 0 (before) and 3 (after) in networks stimulated by 10% serum demonstrated a dramatic increase in vascular density and capillary sprouting. Growing networks contained proliferating endothelial cells and NG2+ vascular pericytes. Media supplementation with sunitinib (SU11248) or bevacizumab both inhibited the network angiogenic responses. The comparison of the same networks before and after treatment enabled the identification of tissue specific responses. Our results establish, for the first time, the ability to evaluate an anti-angiogenic drug based on time-lapse imaging on an intact microvascular network in an ex vivo scenario.     

Carotid geometry effects on blood flow and on risk for vascular disease

It has been widely observed that atherosclerotic diseases occur at sites with complex hemodynamics, such as artery bifurcations, junctions, and regions of high curvature. These regions usually have very low or highly oscillatory wall shear stress (WSS). In the present work, 3D pulsatile blood flow through a model of the carotid artery bifurcation was simulated using a finite volume numerical method. The goal was to quantify the risk of atherogenesis associated with different carotid artery geometries. A risk scale based on the average WSS on the sinus wall of the internal carotid artery was proposed-a scale that can be used to quantify the effect of the carotid geometry on the relative risk for developing vascular disease. It was found that the bifurcation angle and the out-of-plane angle of the internal carotid artery affect the formation of low stress regions on the carotid walls. The main conclusions are: (a) larger internal carotid artery angles (theta(IC)) generally increase the frequency and the area of blood recirculation and lower the WSS on the sinus wall, hence increasing the risk of plaque build-up; (b) off-plane angles were found to lower the WSS on the sinus for geometries with theta(IC)25 degrees . Larger off-plane angles generally increase the danger of plague build-up; (c) for theta(IC) < 25 degrees , the off-plane angle does not have an obvious effect on the hemodynamic WSS; (d) symmetric bifurcations were found to increase the WSS on the sinus wall and ease the risk of vascular disease.     

Computer simulation of hydration networks around amino acids

The networks of solvent hydrogen bonds around polar and apolar amino acids have been studied by computer simulation techniques using a non-pair additive model for the water molecules interactions. Analysis of the simulated aqueous solutions has shown the presence of water molecules which (a) form a bridge around individual polar solute atoms (self-bridging loops) and (b) form chains between different polar solute atoms (polar bridging chains). Some of these networks associated with polar solute atoms from pentagons but 4, 6 and 7 sided polygons are also seen. The water molecule close to apolar solute atoms (<4.0 Å) also form irregular networks with polygons of 4, 5, 6 and 7 sides. These networks are compared with those found experimentally in ice, clathrates and crystal hydrates of macromolecules.     

Equal graph partitioning on estimated infection network as an effective epidemic mitigation measure

Controlling severe outbreaks remains the most important problem in infectious disease area. With time, this problem will only become more severe as population density in urban centers grows. Social interactions play a very important role in determining how infectious diseases spread, and organization of people along social lines gives rise to non-spatial networks in which the infections spread. Infection networks are different for diseases with different transmission modes, but are likely to be identical or highly similar for diseases that spread the same way. Hence, infection networks estimated from common infections can be useful to contain epidemics of a more severe disease with the same transmission mode. Here we present a proof-of-concept study demonstrating the effectiveness of epidemic mitigation based on such estimated infection networks. We first generate artificial social networks of different sizes and average degrees, but with roughly the same clustering characteristic. We then start SIR epidemics on these networks, censor the simulated incidences, and use them to reconstruct the infection network. We then efficiently fragment the estimated network by removing the smallest number of nodes identified by a graph partitioning algorithm. Finally, we demonstrate the effectiveness of this targeted strategy, by comparing it against traditional untargeted strategies, in slowing down and reducing the size of advancing epidemics.     

Mapping 3-D functional capillary geometry in rat skeletal muscle in vivo

We have developed a novel mapping software package to reconstruct microvascular networks in three dimensions (3-D) from in vivo video images for use in blood flow and O2 transport modeling. An intravital optical imaging system was used to collect video sequences of blood flow in microvessels at different depths in the tissue. Functional images of vessels were produced from the video sequences and were processed using automated edge tracking software to yield location and geometry data for construction of the 3-D network. The same video sequences were analyzed for hemodynamic and O2 saturation data from individual capillaries in the network. Simple user-driven commands allowed the connection of vessel segments at bifurcations, and semiautomated registration enabled the tracking of vessels across multiple focal planes and fields of view. The reconstructed networks can be rotated and manipulated in 3-D to verify vessel connections and continuity. Hemodynamic and O2 saturation measurements made in vivo can be indexed to corresponding vessels and visualized using colorized maps of the vascular geometry. Vessels in each reconstruction are saved as text-based files that can be easily imported into flow or O2 transport models with complete geometry, hemodynamic, and O2 transport conditions. The results of digital morphometric analysis of seven microvascular networks showed mean capillary diameters and overall capillary density consistent with previous findings using histology and corrosion cast techniques. The described mapping software is a valuable tool for the quantification of in vivo microvascular geometry, hemodynamics, and oxygenation, thus providing rich data sets for experiment-based computational models.     

An Efficient Immunization Strategy for Community Networks

An efficient algorithm that can properly identify the targets to immunize or quarantine for preventing an epidemic in a population without knowing the global structural information is of obvious importance. Typically, a population is characterized by its community structure and the heterogeneity in the weak ties among nodes bridging over communities. We propose and study an effective algorithm that searches for bridge hubs, which are bridge nodes with a larger number of weak ties, as immunizing targets based on the idea of referencing to an expanding friendship circle as a self-avoiding walk proceeds. Applying the algorithm to simulated networks and empirical networks constructed from social network data of five US universities, we show that the algorithm is more effective than other existing local algorithms for a given immunization coverage, with a reduced final epidemic ratio, lower peak prevalence and fewer nodes that need to be visited before identifying the target nodes. The effectiveness stems from the breaking up of community networks by successful searches on target nodes with more weak ties. The effectiveness remains robust even when errors exist in the structure of the networks.     

Rapid analysis of vessel elements (RAVE): a tool for studying physiologic, pathologic and tumor angiogenesis

Quantification of microvascular network structure is important in a myriad of emerging research fields including microvessel remodeling in response to ischemia and drug therapy, tumor angiogenesis, and retinopathy. To mitigate analyst-specific variation in measurements and to ensure that measurements represent actual changes in vessel network structure and morphology, a reliable and automatic tool for quantifying microvascular network architecture is needed. Moreover, an analysis tool capable of acquiring and processing large data sets will facilitate advanced computational analysis and simulation of microvascular growth and remodeling processes and enable more high throughput discovery. To this end, we have produced an automatic and rapid vessel detection and quantification system using a MATLAB graphical user interface (GUI) that vastly reduces time spent on analysis and greatly increases repeatability. Analysis yields numerical measures of vessel volume fraction, vessel length density, fractal dimension (a measure of tortuosity), and radii of murine vascular networks. Because our GUI is open sourced to all, it can be easily modified to measure parameters such as percent coverage of non-endothelial cells, number of loops in a vascular bed, amount of perfusion and two-dimensional branch angle. Importantly, the GUI is compatible with standard fluorescent staining and imaging protocols, but also has utility analyzing brightfield vascular images, obtained, for example, in dorsal skinfold chambers. A manually measured image can be typically completed in 20 minutes to 1 hour. In stark comparison, using our GUI, image analysis time is reduced to around 1 minute. This drastic reduction in analysis time coupled with increased repeatability makes this tool valuable for all vessel research especially those requiring rapid and reproducible results, such as anti-angiogenic drug screening.     

Regulation of endothelial connexin40 expression by shear stress

Endothelial connexin (Cx)40 plays an important role in signal propagation along blood vessel walls, modulating vessel diameter and thereby blood flow. Blood flow, in turn, has been shown to alter endothelial Cx40 expression. However, the timing and shear stress dependence of this relationship have remained unclear, as have the signal transduction pathways involved and the functional implications. Therefore, the aim of this study was to quantify the effects of shear stress on endothelial Cx40 expression, to analyze the role of phosphoinositide 3-kinase (PI3K)/Akt signaling involved, and to assess the possible functional consequences for the adaptation of microvascular networks. First-passage human umbilical vein endothelial cells were exposed to defined shear stress conditions and analyzed for Cx40 using real-time RT-PCR and immunoblot analysis. Shear stress caused long-term induction of Cx40 protein expression, with two short-term mRNA peaks at 4 and 16 h, indicating the dynamic nature of the adaptation process. Maximum shear stress-dependent induction was observed at shear levels between 6 and 10 dyn/cm(2). Simulation of this pattern of shear-dependent Cx expression in a vascular adaptation model of a microvascular network led to an improved fit for the simulated results to experimental measurements. Cx40 expression was greatly reduced by inhibiting PI3K or Akt, with PI3K activity being required for basal Cx40 expression and Akt activity taking part in its shear stress-dependent induction.     

Circulatory responses to vasoconstrictor agents during passive heating in the rat

The purpose of this study was to determine the actions of several pharmacological agents on the circulatory system, and more specifically on the superior mesenteric vascular bed, in response to environmental heat stress in chloralose-anesthetized rats. Animals were instrumented with Doppler flow probes on the mesenteric and renal arteries and exposed to an ambient temperature of 40 degrees C. Heart rate, mean arterial blood pressure (MAP), and core (Tc) and tail skin temperatures were also monitored. As Tc progressively increased from 37 degrees C during heat exposure, MAP rose to a plateau and then fell precipitously as Tc exceeded 41.5 degrees C. Mesenteric resistance increased throughout the early stages of heating before sharply declining prior to the reduction in MAP. The pressor and mesenteric resistance responses to constant infusions of several adrenergic agonists after MAP began falling (Tc = 41.3 degrees C) were significantly (P less than 0.05) attenuated compared with infusions into normothermic animals. In a second set of experiments, injections of both norepinephrine and angiotensin II were made 30 min before and approximately 10, 30, 50, 70, and 90 min after initiation of heating. These injections increased both MAP and mesenteric resistance; however, at TcS greater than 40 degrees C, the responses to both agonists were progressively and significantly attenuated. In a final group of animals, barium chloride infusions produced similar pressor and regional resistance changes during both normothermia and severe hyperthermia (Tc greater than 42 degrees C). These results indicate that, in the chloralose-anesthetized rat, hyperthermia disrupts adrenoceptor function but does not alter the intrinsic ability of vascular smooth muscle to contract.     

利用微血管环技术检测肠系膜动脉三级分支张力的方法

目的：以肠系膜动脉三级分支为样本,观察利用微血管技术检测血管张力功能的整个过程,以便研究各种微血管相关疾病中的微血管功能状态。方法：利用DMT张力测定仪和PowerLab数据采集系统检测微血管收缩、舒张功能,将肠系膜三级动脉血管游离,固定,标准化和激活后,通过加入血管收缩药物及舒张药物,完成对微血管张力功能的检测。结果：本文制备的肠系膜动脉三级分支血管环对血管活性药物出现良好的收缩、舒张反应,加入10^-5mol／L的去甲肾上腺素（NE）后收缩张力达19mN,之后依次加入10^-9～10^-5mol／L的乙酰胆碱（ACh）或硝普钠（SNP）,血管张力呈梯度降低,ACh和SNP引起的最大舒张率分别为80％和95％。结论：利用该微血管环技术成功检测出肠系膜动脉三级分支的收缩舒张功能。     

Network thermodynamic analysis of vasomotion in a microvascular network.

The modulation of microvascular blood flow by vasomotion in the individual vessels of a simple vascular network was simulated by means of a network thermodynamic model. The flow is driven under a pulsating pressure through two arcades of branching vasoactive arterioles into a passive resistance representing the capillary and venular beds. Each vessel was assumed to have the capability of decreasing rhythmically the local diameter over a short section by a specified fraction of the maximum value and to change the average diameter along its total length in response to alterations in intraluminal pressure. Blood was assumed to exhibit a simple linear viscous flow resistance. Alterations in flow rate and distribution through the network were determined as a function of the magnitude and frequency of vasomotion within the individual arterioles supplying blood to the microvascular bed. Specific cases are shown to illustrate how blood flow can be influenced by the patterns of vasomotion within the network.     

Three-dimensional geometry of the human carotid artery

Accurate characterization of carotid artery geometry is vital to our understanding of the pathogenesis of atherosclerosis. Three-dimensional computer reconstructions based on medical imaging are now ubiquitous; however, mean carotid artery geometry has not yet been comprehensively characterized. The goal of this work was to build and study such geometry based on data from 16 male patients with severe carotid artery disease. Results of computerized tomography angiography were used to analyze the cross-sectional images implementing a semiautomated segmentation algorithm. Extracted data were used to reconstruct the mean three-dimensional geometry and to determine average values and variability of bifurcation and planarity angles, diameters and cross-sectional areas. Contrary to simplified carotid geometry typically depicted and used, our mean artery was tortuous exhibiting nonplanarity and complex curvature and torsion variations. The bifurcation angle was 36 deg?±?11 deg if measured using arterial centerlines and 15 deg?±?14 deg if measured between the walls of the carotid bifurcation branches. The average planarity angle was 11 deg?±?10 deg. Both bifurcation and planarity angles were substantially smaller than values reported in most studies. Cross sections were elliptical, with an average ratio of semimajor to semiminor axes of 1.2. The cross-sectional area increased twofold in the bulb compared to the proximal common, but then decreased 1.5-fold for the combined area of distal internal and external carotid artery. Inter-patient variability was substantial, especially in the bulb region; however, some common geometrical features were observed in most patients. Obtained quantitative data on the mean carotid artery geometry and its variability among patients with severe carotid artery disease can be used by biomedical engineers and biomechanics vascular modelers in their studies of carotid pathophysiology, and by endovascular device and materials manufacturers interested in the mean geometrical features of the artery to target the broad patient population.     

A mathematical model for the distribution of hemodynamic parameters in the human retinal microvascular network

To quantitatively assess the arteriovenous distribution of hemodynamic parameters throughout the microvascular network of the human retina, we constructed a retinal microcirculatory model consisting of a dichotomous symmetric branching system. This system is characterized by a diameter exponent of 2.85, instead of 3 as dictated by Murray’s law, except for the capillary networks. The value of 2.85 was the sum of a fractal dimension (1.70) and a branch exponent (1.15) of the retinal vasculature. Following the feeding artery (central retinal artery), each bifurcation was recursively developed at a distance of an individual branch length [L(r) = 7.4r ^1.15] by a centrifugal scheme. The venular tree was formed in the same way. Using this model, we evaluated hemodynamic parameters, including blood pressure, blood flow, blood velocity, shear rate, and shear stress, within the retinal microcirculatory network as a function of vessel diameter. The arteriovenous distributions of blood pressure and velocity in the simulation were consistent with in vivo measurements in the human retina and other vascular beds of small animals. We therefore conclude that the current theoretical model was useful for quantifying hemodynamics as a function of vessel diameter within the retinal microvascular network.     

Early effects of tobacco smoke exposure on vascular dynamics in the microcirculation.

The purpose of these studies is to examine the early effects of chronic tobacco smoke exposure on vascular dynamics in the mesenteric microcirculation. Female rats were exposed daily to tobacco smoke from five reference cigarettes for a period of 2 mo. At the end of this period the smoke-treated rats had gained 12 g less than sham-treated controls, and arterial blood pressure in the smoke-treated animals was slightly less than pressure in the sham-treated animals. These are characteristic effects of tobacco smoke exposure on rats. Following the treatment period, red blood cell (RBC) velocity in single mesenteric capillaries and microvascular pressures in arterioles and venules were measured in accordance to established methods. There was no significant difference in pressure distribution on the arterial side of the mesenteric vascular network, but pressure in the venules of the smoke-treated animals was significantly higher than that of the sham-treated group. In association with the higher venular pressure in the smoke-treated animals, capillary RBC velocity (an index of capillary flow) was significantly lower. The reduction in velocity was in proportion to the decrease in pressure drop (arteriole-venule) across the capillary network.     

Structural adaptation of microvascular networks: functional roles of adaptive responses

Terminal vascular beds continually adapt to changing demands. A theoretical model is used to simulate structural diameter changes in response to hemodynamic and metabolic stimuli in microvascular networks. Increased wall shear stress and decreased intravascular pressure are assumed to stimulate diameter increase. Intravascular partial pressure of oxygen (PO(2)) is estimated for each segment. Decreasing PO(2) is assumed to generate a metabolic stimulus for diameter increase, which acts locally, upstream via conduction along vessel walls, and downstream via metabolite convection. By adjusting the sensitivities to these stimuli, good agreement is achieved between predicted network characteristics and experimental data from microvascular networks in rat mesentery. Reduced pressure sensitivity leads to increased capillary pressure with reduced viscous energy dissipation and little change in tissue oxygenation. Dissipation decreases strongly with decreased metabolic response. Below a threshold level of metabolic response flow shifts to shorter pathways through the network, and oxygen supply efficiency decreases sharply. In summary, the distribution of vessel diameters generated by the simulated adaptive process allows the network to meet the functional demands of tissue while avoiding excessive viscous energy dissipation.     

Effects of hyperoxia on local and remote microcirculatory inflammatory response after splanchnic ischemia and reperfusion

Splanchnic ischemia-reperfusion (I/R) causes tissue hypoxia that triggers local and systemic microcirculatory inflammatory responses. We evaluated the effects of hyperoxia in I/R induced by 40-min superior mesenteric artery (SMA) occlusion and 120-min reperfusion in four groups of rats: 1) control (anesthesia only), 2) sham operated (all surgical procedures without vascular occlusion; air ventilation), 3) SMA I/R and air, 4) SMA I/R and 100% oxygen ventilation started 10 min before reperfusion. Leukocyte rolling and adhesion in mesenteric microvessels, pulmonary microvascular blood flow velocity (BFV), and macromolecular (FITC-albumin) flux into lungs were monitored by intravital videomicroscopy. We also determined pulmonary leukocyte infiltration. SMA I/R caused marked decreases in mean arterial blood pressure (MABP) and blood flow to the splanchnic and hindquarters vascular beds and pulmonary BFV and shear rates, followed by extensive increase in leukocyte rolling and adhesion and plugging of >50% of the mesenteric microvasculature. SMA I/R also caused marked increase in pulmonary sequestration of leukocytes and macromolecular leak with concomitant decrease in circulating leukocytes. Inhalation of 100% oxygen maintained MABP at significantly higher values (P < 0.001) but did not change regional blood flows. Oxygen therapy attenuated the increase in mesenteric leukocyte rolling and adherence (P < 0.0001) and maintained microvascular patency at values not significantly different from sham-operated animals. Hyperoxia also attenuated the decrease in pulmonary capillary BFV and shear rates, reduced leukocyte infiltration in the lungs (P < 0.001), and prevented the increase in pulmonary macromolecular leak (P < 0.001), maintaining it at values not different from sham-operated animals. The data suggest that beneficial effects of normobaric hyperoxia in splanchnic I/R are mediated by attenuation of both local and remote inflammatory microvascular responses.     

The influence of depolarization block on seizure-like activity in networks of excitatory and inhibitory neurons

The inhibitory restraint necessary to suppress aberrant activity can fail when inhibitory neurons cease to generate action potentials as they enter depolarization block. We investigate possible bifurcation structures that arise at the onset of seizure-like activity resulting from depolarization block in inhibitory neurons. Networks of conductance-based excitatory and inhibitory neurons are simulated to characterize different types of transitions to the seizure state, and a mean field model is developed to verify the generality of the observed phenomena of excitatory-inhibitory dynamics. Specifically, the inhibitory population’s activation function in the Wilson-Cowan model is modified to be non-monotonic to reflect that inhibitory neurons enter depolarization block given strong input. We find that a physiological state and a seizure state can coexist, where the seizure state is characterized by high excitatory and low inhibitory firing rate. Bifurcation analysis of the mean field model reveals that a transition to the seizure state may occur via a saddle-node bifurcation or a homoclinic bifurcation. We explain the hysteresis observed in network simulations using these two bifurcation types. We also demonstrate that extracellular potassium concentration affects the depolarization block threshold; the consequent changes in bifurcation structure enable the network to produce the tonic to clonic phase transition observed in biological epileptic networks.