A microfluidic device for assessment of E-selectin-mediated neutrophil recruitment to inflamed endothelium and prediction of therapeutic response in sickle cell disease.

Neutrophil recruitment to the inflamed endothelium is a multistep process and is of utmost importance in the development of the hallmark vaso-occlusive crisis in sickle cell disease (SCD). However, there lacks a standardized, clinically feasible approach for assessing neutrophil recruitment to the inflamed endothelium for individualized risk stratification and therapeutic response prediction in SCD. Here, we describe a microfluidic device functionalized with E-selectin, a critical endothelial receptor for the neutrophil recruitment process, as a strategy to assess neutrophil binding under physiologic flow in normoxia and clinically relevant hypoxia in SCD. We show that hypoxia significantly enhances neutrophil binding to E-selectin and promotes the formation of neutrophil-platelet aggregates. Moreover, we identified two distinct patient populations: a more severe clinical phenotype with elevated lactate dehydrogenase levels and absolute reticulocyte counts but lowered fetal hemoglobin levels associated with constitutively less neutrophil binding to E-selectin. Mechanistically, we demonstrate that the extent of neutrophil activation correlates with membrane L-selectin shedding, resulting in the loss of ligand interaction sites with E-selectin. We also show that inhibition of E-selectin significantly reduces leukocyte recruitment to activated endothelial cells. Our findings add mechanistic insight into neutrophil-endothelial interactions under hypoxia and provide a clinically feasible means for assessing neutrophil binding to E-selectin using clinical whole blood samples, which can help guide therapeutic decisions for SCD patients.

Global endothelial function assessment using pulse wave analysis in hypercholesterolemia

To compare global endothelial function assessed by pulse wave analysis (PWA) using the ratio of endothelium dependent vasodilatation (EDV) to endothelium independent vasodilatation (EIV) in patients with hypercholesterolemia and controls. 92 subjects [46 hypercholesterolemics, 46 controls] were studied at standardized conditions. Baseline augmentation index (AIx) was assessed followed by the administration of 0.5 mg sublingual nitroglycerine, an endothelium independent vasodilator. AIx was assessed and the maximum change in AIx after nitroglycerine was recorded as EIV. After a washout period of 30 minutes, 400 µg of inhaled salbutamol, an endothelium dependent vasodilator was administered. AIx was assessed again and the maximum change in AIx after salbutamol was recorded as EDV. Global endothelial function was calculated as EDV:EIV ratio. EDV and EIV in patients with hypercholesterolemia compared to controls were 2.97 ± 3.95 and 6.65 ± 3.80 (p<0.001); and 13.41 ± 4.57 and 15.88 ± 4.78 (p=0.01) respectively. EDV:EIV ratio was significantly reduced in patients with hypercholesterolemia compared to controls; 0.21 ± 0.38 and 0.44 ± 0.24 (p<0.001) respectively. EDV:EIV ratio was significantly reduced in patients with hypercholesterolemia compared to controls. PWA is a potential clinical tool to assess global endothelial function in patients with hypercholesterole

Functional assessment of two variants of unknown significance in TEK by endothelium-specific expression in zebrafish embryos.

Vascular malformations are most often caused by somatic mutations of the PI3K/mTOR and the RAS signaling pathways, which can be identified in the affected tissue. Venous malformations (VMs) commonly harbor PIK3CA and TEK mutations, whereas arteriovenous malformations (AVMs) are usually caused by BRAF, RAS or MAP2K1 mutations. Correct identification of the underlying mutation is of increasing importance, since targeted treatments are becoming more and more relevant, especially in patients with extensive vascular malformations. However, variants of unknown significance (VUSs) are often identified and their pathogenicity and response to targeted therapy cannot be precisely predicted. Here, we show that zebrafish embryos can be used to rapidly assess the pathogenicity of novel VUSs in TEK, encoding for the receptor TIE2, present on endothelial cells of VMs. Endothelium-specific overexpression of TEK mutations leads to robust induction of VMs, whereas MAP2K1 mutations cause AVMs in our zebrafish model. TEK mutations are often found as double mutations in cis; using our model, we show that double mutations have an additive effect in inducing VMs compared with the respective single variants. The clinically established mTOR-inhibitor sirolimus (rapamycin) efficiently abrogates the development of VMs in this zebrafish model. In summary, endothelium-specific overexpression of patient-derived TEK variants in the zebrafish model allows assessment of their pathogenic significance as well as testing of candidate drugs in a personalized and mutation-specific approach.

Assessment of tissue perfusion and vascular function in mice by scanning laser Doppler perfusion imaging.

Post-occlusive reactive hyperemia (PORH) is a key feature of physiological vasomotion to appropriately match the supply/demand ratio of tissues. This adaptive mechanism is severely disturbed in endothelial dysfunction with a reduced flow-mediated dilation (FMD). Reduced PORH and FMD are powerful prognostic risk factors in cardiovascular diseases. While these parameters are frequently determined in human beings, comparable methods applicable to mouse models are sparse. We aimed to evaluate the applicability and accuracy of scanning laser Doppler perfusion imaging (LDPI) to measure PORH in the mouse hindlimb. Changes in mean perfusion in response to vasoactive drugs and PORH (assessed by scanning LDPI) were compared with changes in diameter and blood flow in the femoral artery, as assessed by high-resolution ultrasound. We found that the measured LDPI signal significantly correlated with changes of inflow into the femoral artery. Vasodilation induced by administration of nitroglycerine and acetylcholine increased vessel diameter, blood flow and mean perfusion, while vasoconstriction following administration of epinephrine decreased all three parameters. PORH was induced by temporal occlusion of the femoral artery with an external cuff. During occlusion, mean perfusion decreased to a condition of zero-perfusion and release of the cuff induced an immediate increase in blood flow that was followed by femoral artery dilation driving PORH/perfusion. Surgical removal of the femoral artery decreased mean perfusion to a zero-perfusion level and fully abolished PORH. Importantly, the measurement of the PORH response by scanning LDPI is highly reproducible as determined by repeated measurements and intra/interobserver variation analysis. Last, we found that the PORH response was dependent on nitric oxide synthase and cyclooxygenase and declined with age. Thus, we here provide novel and robust non-invasive methods to serially measure tissue perfusion at baseline and during physiological and pharmacological modulation of vasomotor tone in the hindlimb of mice. The application of these LDPI scanning and ultrasound-based methods may be useful for testing the effects of drugs affecting vasomotor function or future elucidation of mechanisms leading to vasomotor dysfunction in mice in vivo.

Assessment of Basilar Artery Reactivity in Stroke and Subarachnoid Hemorrhage Using Wire Myograph.

Blood flow regulation of normal cerebral arteries is a critical and important factor to supply the brain tissue with nutrients and oxygen. Stroke insult results in a disruption or reduction in cerebral arteries' blood flow with subsequent brain tissue damage. Hemorrhagic stroke is one type of stroke and accounts for about 13 % of all of stroke insults. In this type of stroke, the cerebral artery breaks open and causes bleeding in or surrounding the brain. Subsequently, this bleeding causes blood vessels to constrict in a process called vasospasm, in which the vessels narrow and impede the blood flow to brain tissue. Hemorrhagic stroke is the major cause of prolonged constriction of cerebral arteries. This leads to partial brain damage and sometimes death in patients with aneurysmal subarachnoid hemorrhage. Among the key delicate techniques to assess small blood vessel functionality is the wire myograph, which can be utilized in several cerebral injury models including stroke. The wire myograph is a device that provides information about the reactivity, stiffness, and elasticity of small blood vessels under isometric conditions. In this book chapter, we describe the techniques involved in wire myography assessment and the different measures and parameters recorded; we describe the utility of this technique in evaluating the effects of subarachnoid hemorrhage on basilar artery sensitivity to different agonists.

Invasive assessment of the coronary microcirculation in the catheter laboratory.

Historically much of our understanding of the coronary circulation has been centered towards the epicardial vessels. However, recent work has highlighted the importance of the coronary microcirculation across a broad spectrum of clinical conditions in influencing patient outcomes. Therefore an ability to measure microvascular function is most valuable. While evaluation of the epicardial coronary circulation is widely understood, interrogation of the coronary microcirculation is more complex. Many methods are available to assess the integrity of the microcirculation in the catheter laboratory. This review will discuss the physiology of the coronary microcirculation and evaluate the utility of available invasive techniques.

Assessment of endothelial function in the patient with erectile dysfunction: an opportunity for the urologist.

Although there is now ample evidence that men with manifest coronary, cerebral and peripheral vascular disease and their risk factors are at highest risk for suffering from erectile dysfunction, recent findings demonstrate a strong connection between erectile dysfunction and endothelial dysfunction. This review explores how urologists and other providers may utilize the link between these conditions in clinical practice, compares methods of assessing endothelial dysfunction and finally speculates on how this additional information might impact treatment plans.

[Effect of enalapril on endotheliun-dependent contractile reactions and oxygen cost of work of the smooth muscles in experimental diabetes mellitus].

The influence of angiotensin-converting enzyme on endotheliun-dependent contractile vascular reactions was investigated on the rat model of streptozotocin-induced diabetes mellitus. It is shown, that the long-term administration of enalapril results in partial restoration of disturbed at diabetes mellitus reactions and also to reduction of oxygen cost of smooth muscles and myocardial work. Thus, after 28-day's of oral administration of this drug the restoration of endotheliun-dependent dilatation of aorta and coronary vessels, increase of stretch-induced contractile responses of vascular smooth muscles, reduction of stiffness of isolated portal vein strips are observed. Possible mechanisms of such changes are following: increase of nitric oxide synthesis (at the expense of constitutive NOS activity) and reduction of oxidative stress, to what the decrease of diene conjugates contents in tissues of animals with diabetes mellitus after long introduction of enalapril testifies.

Effect of microvillus deformability on leukocyte adhesion explored using adhesive dynamics simulations.

Leukocyte rolling on the endothelium via selectin molecules is an important step in the adhesion cascade, which allows leukocytes in the bloodstream to reach sites of infection. We improve upon Adhesive Dynamics simulations by incorporating deformable microvilli on which adhesion molecules are clustered. As determined in micropipette experiments, microvilli deform like an elastic spring at small forces and a combination of yield and viscous dissipation at high forces. First, we create a modified version of the state diagram for adhesion which includes microvillus deformation, and find four adhesion states-firmly bound; landing; rolling; and no-adhesion. Then, we simulate the effects of receptor clustering on the tips of microvilli, number of adhesion molecules on the cell, and the spring constant of the bonds, within the context of deformable microvilli. We also explore how the microvillus rheology itself controls the dynamics of adhesion. A minimum in rolling velocity occurs at an intermediate value of the microvillus membrane viscosity, remarkably close to the reported physiological value, suggesting that the mechanics of microvilli have evolved ideally for rolling and adhesion of leukocytes. We find that a larger degree of association between the membrane and cytoskeleton leads to slower rolling, and stiffer microvilli result in faster rolling. Decreasing the overall deformability of the microvilli greatly reduces a simulated cell's ability to roll. A comparison to experimental results of in vitro cell rolling agrees with the simulation at low shear rates. Furthermore, simulated rolling trajectories of cells with deformable microvilli display periods of rolling interdispersed with pauses, consistent with that seen in experiments where microvilli were observed to stretch.

Assessment of prostatic protein secretion in tissue recombinants made of urogenital sinus mesenchyme and urothelium from normal or androgen-insensitive mice.

Urogenital sinus mesenchyme (UGM) from normal, androgen receptor-positive mice appears to instructively induce prostatic morphology in urinary tract epithelium from both normal and androgen-insensitive (Tfm) mice. This indicates that epithelial androgen receptors are not necessary for prostatic development. However, the secretory function of these tissue recombinants has never been assessed. In this study antisera to androgen-dependent dorsolateral prostate (DLP) secretion were used in immunocytochemistry, Western blotting, and immunoprecipitation techniques to detect the presence of these prostatic proteins in tissue recombinants made with either normal or Tfm epithelium. In a majority of cases, UGM plus normal bladder or urethral epithelium developed into prostatic tissue that produced androgen-dependent DLP proteins. Hence UGM appeared to be capable of instructively inducing a functional prostatic phenotype in these normal heterotypic epithelia. With rare exceptions, tissue recombinants of UGM plus Tfm bladder or urethral epithelium did not produce full prostatic cytodifferentiation or mouse DLP proteins, indicating that epithelial androgen receptors are important for the final stages of morphogenesis and in the initiation of secretory function in the prostate.

Fractal model of ion-channel kinetics.

Markov models with discrete states, such as closed in equilibrium with closed in equilibrium with open have been widely used to model the kinetics of ion channels in the cell membrane. In these models the transition probabilities per unit time (the kinetic rate constants) are independent of the time scale on which they are measured. However, in many physical systems, a property, L, depends on the scale, epsilon, at which it is measured such that L(epsilon) alpha epsilon 1-D where D is the fractal dimension. Such systems are said to be 'fractal'. Based on the assumption that the kinetic rates are given by k(t) alpha t1-D we derive a fractal model of ion-channel kinetics. This fractal model has fewer adjustable parameters, is more consistent with the dynamics of protein conformations, and fits the single-channel recordings from the corneal endothelium better than the discrete-state Markov model.

Corneal penetration behavior of beta-blocking agents II: Assessment of barrier contributions.

Rabbit corneas were excised and mounted in a chamber to determine the permeability characteristics of a group of beta-blocking agents. By measuring the permeability rate of each drug across intact cornea, stroma alone, epithelium-stroma, and stroma-endothelium, it was possible to determine the resistance to penetration for each corneal layer. The reciprocal of the sum of resistances for the epithelium, stroma, and endothelium equaled the experimentally determined permeability coefficient for the intact cornea (104 +/- 6.0%). Thus, the penetration of beta-blocking agents through the excised rabbit cornea could be treated as three barriers in series. For hydrophilic compounds, the epithelium was the rate-determining barrier. The endothelium offered less resistance, whereas the stroma offered only very minimal resistance. The lipophilic compounds penetrated the excised cornea more rapidly. However, the stroma became rate-determining for the most lipophilic compounds (penbutolol, bufuralol, bevantolol, and propranolol). Although the octanol-buffer (pH 7.65) distribution coefficient of these compounds varied over a fourfold logarithmic range, the permeability coefficient was considered nearly constant [3.4 X 10(-5) (+/- 0.34) cm/sec] for stroma. Also, the ratios of tortuosity to porosity for the stromal layer were 1.58 +/- 0.15. These results suggest that drug diffuses through an aqueous media of gel-like mucopolysaccharide interspersed by a matrix of collagen fibrils. From further analyses intra- and intercellular pathways for epithelium and endothelium were added to the model resulting in a sigmoidal representation of permeability coefficient versus distribution coefficient. However, the intercellular (pore) pathway could not be adequately quantified because of the variation in the data for very hydrophilic compounds.