The high reactivity of peroxiredoxin 2 with H(2)O(2) is not reflected in its reaction with other oxidants and thiol reagents

Peroxiredoxin 2 is a member of the mammalian peroxiredoxin family of thiol proteins that is important in antioxidant defense and redox signaling. We have examined its reactivity with various biological oxidants, in order to assess its ability to act as a direct physiological target for these species. Human erythrocyte peroxiredoxin 2 was oxidized stoichiometrically to its disulfide-bonded homodimer by hydrogen peroxide, as monitored electrophoretically under nonreducing conditions. The protein was highly susceptible to oxidation by adventitious peroxide, which could be prevented by treating buffers with low concentrations of catalase. However, this did not protect peroxiredoxin 2 against oxidation by added H(2)O(2). Experiments measuring inhibition of dimerization indicated that at pH 7.4 catalase and peroxiredoxin 2 react with hydrogen peroxide at comparable rates. A rate constant of 1.3 x 10(7) M(-1) s(-1) for the peroxiredoxin reaction was obtained from competition kinetic studies with horseradish peroxidase. This is 100-fold faster than is generally assumed. It is sufficiently high for peroxiredoxin to be a favored cellular target for hydrogen peroxide, even in competition with catalase or glutathione peroxidase. Reactions of t-butyl and cumene hydroperoxides with peroxiredoxin were also fast, but amino acid chloramines reacted much more slowly. This contrasts with other thiol compounds that react many times faster with chloramines than with hydrogen peroxide. The alkylating agent iodoacetamide also reacted extremely slowly with peroxiredoxin 2. These results demonstrate that peroxiredoxin 2 has a tertiary structure that facilitates reaction of the active site thiol with hydrogen peroxide while restricting its reactivity with other thiol reagents.     

A hybrid one-dimensional/Womersley model of pulsatile blood flow in the entire coronary arterial tree

Using a frequency-domain Womersley-type model, we previously simulated pulsatile blood flow throughout the coronary arterial tree. Although this model represents a good approximation for the smaller vessels, it does not take into account the nonlinear convective energy losses in larger vessels. Here, using Womersley's theory, we present a hybrid model that considers the nonlinear effects for the larger epicardial arteries while simulating the distal vessels (down to the 1st capillary segments) with the use of Womersley's Theory. The main trunk and primary branches were discretized and modeled with one-dimensional Navier-Stokes equations, while the smaller-diameter vessels were treated as Womersley-type vessels. Energy losses associated with vessel bifurcations were incorporated in the present analysis. The formulation enables prediction of impedance and pressure and pulsatile flow distribution throughout the entire coronary arterial tree down to the first capillary segments in the arrested, vasodilated state. We found that the nonlinear convective term is negligible and the loss of energy at a bifurcation is small in the larger epicardial vessels of an arrested heart. Furthermore, we found that the flow waves along the trunk or at the primary branches tend to scale (normalized with respect to their mean values) to a single curve, except for a small phase angle difference. Finally, the model predictions for the inlet pressure and flow waves are in excellent agreement with previously published experimental results. This hybrid one-dimensional/Womersley model is an efficient approach that captures the essence of the hemodynamics of a complex large-scale vascular network. The present model has numerous applications to understanding the dynamics of coronary circulation.     

Validation of a novel noninvasive cardiac index of left ventricular contractility in patients

Although there are several excellent indexes of myocardial contractility, they require accurate measurement of pressure via left ventricular (LV) catheterization. Here we validate a novel noninvasive contractility index that is dependent only on lumen and wall volume of the LV chamber in patients with normal and compromised LV ejection fraction (LVEF). By analysis of the myocardial chamber as a thick-walled sphere, LV contractility index can be expressed as maximum rate of change of pressure-normalized stress (d sigma*/dt(max), where sigma* = sigma/P and sigma and P are circumferential stress and pressure, respectively). To validate this parameter, d sigma*/dt(max) was determined from contrast cine-ventriculography-assessed LV cavity and myocardial volumes and compared with LVEF, dP/dt(max), maximum active elastance (E(a,max)), and single-beat end-systolic elastance [E(es(SB))] in 30 patients undergoing clinically indicated LV catheterization. Patients with different tertiles of LVEF exhibit statistically significant differences in d sigma*/dt(max). There was a significant correlation between d sigma*/dt(max) and dP/dt(max) (d sigma*/dt(max) = 0.0075 dP/dt(max) - 4.70, r=0.88, P<0.01), E(a,max) (d sigma*/dt(max) = 1.20E(a,max) + 1.40, r=0.89, P<0.01), and E(es(SB)) [d sigma*/dt(max)=1.60 E(es(SB)) + 1.20, r=0.88, P<0.01]. In 30 additional individuals, we determined sensitivity of the parameter to changes in preload (intravenous saline infusion, n = 10 subjects), afterload (sublingual glyceryl trinitrate, n = 10 subjects), and increased contractility (intravenous dobutamine, n=10 patients). We confirmed that the index is not dependent on load but is sensitive to changes in contractility. In conclusion, d sigma*/dt(max) is equivalent to dP/dt(max), E(a,max), and E(es(SB)) as an index of myocardial contractility and appears to be load independent. In contrast to other measures of contractility, d sigma*/dt(max) can be assessed with noninvasive cardiac imaging and, thereby, should have more routine clinical applicability.     

New Concept in Stroke Diagnosis

Stroke is a life-threatening event that is expected to more than double over the next 40 years. Approximately 85% of strokes are ischemic in nature and result from thromboembolic occlusion of a major cerebral artery or its branches. One of the diagnostic methods for detection of the cerebral ischemia is the gadolinium-enhanced MRI imaging. It is mainly used in patients to detect brain tissue damaged by an ischemic stroke and brain hemorrhage. These techniques are expensive, require sophisticated machines and are time consuming. A recent study in acute stroke patients showed gadolinium leakage into ocular structures (GLOS) during MRI imaging with gadolinium administration. The results indicate that at 2 hours after administration of the contrast agent, GLOS was more common in the aqueous chamber alone, compared to the vitreous chamber with increasing amount in 24 hours after the administration of the contrast agent. This could be due to disruption of blood-ocular barrier similar to the disruption of blood-brain barrier in acute stroke. A new approach to diagnosis of acute stroke and transient ischemic attack (TIA) is through the detection of sodium fluorescein contrast agent in the eye by i.v. injection. The agent is safe and is used routinely in eye fluorescein angiography. Fluorescein fluorescence occurs at the visible wavelengths and can be detected by fluorescein angiography camera. The fluorescein angiography camera prices are affordable for any medical clinic. The innovation of this method is to leverage the eye as the window to the brain. The method can potentially detect acute stroke and TIA without MRI. This can have a far-reaching impact on the healthcare system. The eventual feature of the device will be portability and simplicity of operation that can be used by a medical technician in medical office, emergency outfits and even in ambulances given the portability.     

Global Ablation of the Mouse Rab11a Gene Impairs Early Embryogenesis and Matrix Metalloproteinase Secretion

Rab11a has been conceived as a prominent regulatory component of the recycling endosome, which acts as a nexus in the endo- and exocytotic networks. The precise in vivo role of Rab11a in mouse embryonic development is unknown. We globally ablated Rab11a and examined the phenotypic and molecular outcomes in Rab11a^null blastocysts and mouse embryonic fibroblasts. Using multiple trafficking assays and complementation analyses, we determined, among multiple important membrane-associated and soluble cargos, the critical contribution of Rab11a vesicular traffic to the secretion of multiple soluble MMPs. Rab11a^null embryos were able to properly form normal blastocysts but died at peri-implantation stages. Our data suggest that Rab11a critically controls mouse blastocyst development and soluble matrix metalloproteinase secretion.     

Constructal Law of Vascular Trees for Facilitation of Flow

Diverse tree structures such as blood vessels, branches of a tree and river basins exist in nature. The constructal law states that the evolution of flow structures in nature has a tendency to facilitate flow. This study suggests a theoretical basis for evaluation of flow facilitation within vascular structure from the perspective of evolution. A novel evolution parameter (Ev) is proposed to quantify the flow capacity of vascular structures. Ev is defined as the ratio of the flow conductance of an evolving structure (configuration with imperfection) to the flow conductance of structure with least imperfection. Attaining higher Ev enables the structure to expedite flow circulation with less energy dissipation. For both Newtonian and non-Newtonian fluids, the evolution parameter was developed as a function of geometrical shape factors in laminar and turbulent fully developed flows. It was found that the non-Newtonian or Newtonian behavior of fluid as well as flow behavior such as laminar or turbulent behavior affects the evolution parameter. Using measured vascular morphometric data of various organs and species, the evolution parameter was calculated. The evolution parameter of the tree structures in biological systems was found to be in the range of 0.95 to 1. The conclusion is that various organs in various species have high capacity to facilitate flow within their respective vascular structures.     

Cytochrome b5, not superoxide anion radical, is a major reductant of indoleamine 2,3-dioxygenase in human cells

The heme protein indoleamine 2,3-dioxygenase (IDO) initiates oxidative metabolism of tryptophan along the kynurenine pathway, and this requires reductive activation of Fe(3+)-IDO. The current dogma is that superoxide anion radical (O(2)(*-)) is responsible for this activation, based largely on previous work employing purified rabbit IDO and rabbit enterocytes. We have re-investigated this role of O(2)(*-) using purified recombinant human IDO (rhIDO), rabbit enterocytes that constitutively express IDO, human endothelial cells, and monocyte-derived macrophages treated with interferon-gamma to induce IDO expression, and two cell lines transfected with the human IDO gene. Both potassium superoxide and O(2)(*-) generated by xanthine oxidase modestly activated rhIDO, in reactions that were prevented completely by superoxide dismutase (SOD). In contrast, SOD mimetics had no effect on IDO activity in enterocytes and interferon-gamma-treated human cells, despite significantly decreasing cellular O(2)(*-) Similarly, cellular IDO activity was unaffected by increasing SOD activity via co-expression of Cu,Zn-SOD or by increasing cellular O(2)(*-) via treatment of cells with menadione. Other reductants, such as tetrahydrobiopterin, ascorbate, and cytochrome P450 reductase, were ineffective in activating cellular IDO. However, recombinant human cytochrome b(5) plus cytochrome P450 reductase and NADPH reduced Fe(3+)-IDO to Fe(2+)-IDO and activated rhIDO in a reconstituted system, a reaction inhibited marginally by SOD. Additionally, short interfering RNA-mediated knockdown of microsomal cytochrome b(5) significantly decreased IDO activity in IDO-transfected cells. Together, our data show that cytochrome b(5) rather than O(2)(*-) plays a major role in the activation of IDO in human cells.     

The validation of a generalized Hooke's law for coronary arteries

The exponential form of constitutive model is widely used in biomechanical studies of blood vessels. There are two main issues, however, with this model: 1) the curve fits of experimental data are not always satisfactory, and 2) the material parameters may be oversensitive. A new type of strain measure in a generalized Hooke's law for blood vessels was recently proposed by our group to address these issues. The new model has one nonlinear parameter and six linear parameters. In this study, the stress-strain equation is validated by fitting the model to experimental data of porcine coronary arteries. Material constants of left anterior descending artery and right coronary artery for the Hooke's law were computed with a separable nonlinear least-squares method with an excellent goodness of fit. A parameter sensitivity analysis shows that the stability of material constants is improved compared with the exponential model and a biphasic model. A boundary value problem was solved to demonstrate that the model prediction can match the measured arterial deformation under experimental loading conditions. The validated constitutive relation will serve as a basis for the solution of various boundary value problems of cardiovascular biomechanics.     

Scaling of myocardial mass to flow and morphometry of coronary arteries.

There is no doubt that scaling relations exist between myocardial mass and morphometry of coronary vasculature. The purpose of this study is to quantify several morphological (diameter, length, and volume) and functional (flow) parameters of the coronary arterial tree in relation to myocardial mass. Eight normal porcine hearts of 117-244 g (mean of 177.5 +/- 32.7) were used in this study. Various coronary subtrees of the left anterior descending, right coronary, and left circumflex arteries were perfused at pressure of 100 mmHg with different colors of a polymer (Microfil) to obtain rubber casts of arterial trees corresponding to different regions of myocardial mass. Volume, diameter, and cumulative length of coronary arteries were reconstructed from casts to analyze their relationship to the perfused myocardial mass. Volumetric flow was measured in relationship with perfused myocardial mass. Our results show that arterial volume is linearly related to regional myocardial mass, whereas the sum of coronary arterial branch lengths, vessel diameters, and volumetric flow show an approximately 3/4, 3/8, and 3/4 power-law relationship, respectively, in relation to myocardial mass. These scaling laws suggest fundamental design principles underlying the structure-function relationship of the coronary arterial tree that may facilitate diagnosis and management of diffuse coronary artery disease.     

Nuclear prostaglandin signaling system: biogenesis and actions via heptahelical receptors

Prostaglandins are ubiquitous lipid mediators that play pivotal roles in cardiovascular homeostasis, reproduction, and inflammation, as well as in many important cellular processes including gene expression and cell proliferation. The mechanism of action of these lipid messengers is thought to be primarily dependent on their interaction with specific cell surface receptors that belong to the heptahelical transmembrane spanning G protein-coupled receptor superfamily. Accumulating evidence suggests that these receptors may co-localize at the cell nucleus where they can modulate gene expression through a series of biochemical events. In this context, we have recently demonstrated that prostaglandin E2-EP3 receptors display an atypical nuclear compartmentalization in cerebral microvascular endothelial cells. Stimulation of these nuclear EP3 receptors leads to an increase of eNOS RNA in a cell-free isolated nuclear system. This review will emphasize these findings and describe how nuclear prostaglandin receptors, notably EP3 receptors, may affect gene expression, specifically of eNOS, by identifying putative transducing elements located within this organelle. The potential sources of lipid ligand activators for these intracellular sites will also be addressed. The expressional control of G-protein-coupled receptors located at the perinuclear envelope constitutes a novel and distinctive mode of gene regulation.