Study of normal and pathological blood vessel morphogenesis in Flt1-tdsRed BAC Tg mice

Blood vessel development and network patterning are controlled by several signaling molecules, including VEGF, FGF, TGF‐ß, and Ang‐1,2. Among these, the role of VEGF‐A signaling in vessel morphogenesis is best understood. The biological activity of VEGF‐A depends on its reaction with specific receptors Flt1 and Flk1. Roles of VEGF‐A signaling in endothelial cell proliferation, migration, survival, vascular permeability, and induction of tip cell filopodia have been reported. In this study, we have generated Flt1‐tdsRed BAC transgenic (Tg) mice to monitor Flt1 gene expression during vascular development. We show that tdsRed fluorescence is observed within blood vessels of adult mice and embryos, indicative of retinal angiogenesis and tumor angiogenesis. Flt1 expression recapitulated by Flt1‐tdsRed BAC Tg mice overlapped well with Flk1, while Flt1 was expressed more abundantly in endothelial cells of large blood vessels such as dorsal aorta and presumptive stalk cells in retina, providing a unique model to study blood vessel development. genesis 50:561–571, 2012. © 2012 Wiley Periodicals, Inc.     

Multiscale model for pulmonary oxygen uptake and its application to quantify hypoxemia in hepatopulmonary syndrome

This paper presents a novel multiscale methodology for quantitative analysis of pulmonary gas exchange. The process of oxygen uptake in the lungs is a complex multiscale process, characterized by multiple time and length scales which are coupled nonlinearly through the processes of diffusion, convection and reaction, and the overall oxygen uptake is significantly influenced by the transport and reaction rate processes at the small-scales. Based on the separation of length scales, we characterize these disparate scales by three representative ones, namely micro (red blood cell), meso (capillary and alveolus) and macro (lung). We start with the fundamental convection-diffusion-reaction (CDR) equation that quantifies transport and reaction rates at each scale and apply spatial averaging techniques to reduce the dimensionality of these models. The resultant low-dimensional models embed each scale hierarchically within the other while retaining the important parameters of the small-scales in the averaged equations, and drastically reduce the computational efforts involved in solving them. We use our multiscale model for pulmonary gas exchange to quantify the oxygen uptake abnormalities in patients with hepatopulmonary syndrome (HPS), a disease which is characterized by coupled abnormalities in multiple length scales. Based on our multiscale modeling, we suggest a strategy to stratify patients with HPS into two categories--those who are oxygen-responsive and those who are oxygen non-responsive with intractable hypoxemia.     

Derangement of Erythrocytic AE1 in Beta-Thalassemia by Caspase 3: Pathogenic Mechanisms and Implications in Red Blood Cell Senescence

Considering its complex molecular pathophysiology, beta-thalassemia could be a good in vivo model to study some aspects related to erythrocyte functions with potential therapeutic implications not only within the frame of this particular hemoglobinopathy but also with respect to conditions in which the cellular milieu, altered by a deranged anion exchanger, could display a significant pathogenetic role (i.e., erythrocyte senescence, complications of red cell storage, renal tubular acidosis and some abnormal protein thesaurismosis). This work evaluates the anionic influx across band 3 protein in normal and beta-thalassemic red blood cells (RBCs) and ghosts. Since redox-mediated injury is an important pathway in the destruction of beta-thalassemic RBCs, we studied the anion transport and the activity of caspase 3 in the absence and presence of t-butylhydroperoxide in order to evaluate the effect of an increase of cellular oxidative stress. Interestingly, beta-thalassemic erythrocytes show a faster rate of anion exchange than normal RBCs and absence of any modulation mechanism of anion influx. These findings led us to formulate a hypothesis about the metabolic characteristics of beta-thalassemic erythrocytes, outlining that one of the main targets of caspase 3 in RBCs is the cytoplasmic domain of band 3 protein.     

The extracellular matrix and blood vessel formation: not just a scaffold

The extracellular matrix plays a number of important roles, among them providing structural support and information to cellular structures such as blood vessels imbedded within it. As more complex organisms have evolved, the matrix ability to direct signalling towards the vasculature and remodel in response to signalling from the vasculature has assumed progressively greater importance. This review will focus on the molecules of the extracellular matrix, specifically relating to vessel formation and their ability to signal to the surrounding cells to initiate or terminate processes involved in blood vessel formation.     

Mathematical modeling of the response of a resistive vessel to pressure

The response of small arterial vessels to internal pressure makes an essential contribution to autoregulation in the vascular bed. It is believed that free cytosolic Ca²⁺ concentration plays a pivotal role in the regulation of smooth muscle contractility and hence of the vascular lumen. A simple mathematical model of blood flow in a resistive vessel is suggested. The model is based on the experimental data obtained for cerebral arteries, but may be used for any other resistive vessel. The model not only describes the regulation of the vascular lumen by transmural pressure but also shows realistic behavior of the vessel radius and cytosolic [Ca²⁺] at different rates of pressure change. Possible variations in the radius along the vessel due to the Bayliss effect are considered.     

Development of a capillary electrophoresis method for analyzing adenosine deaminase and purine nucleoside phosphorylase and its application in inhibitor screening

A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237–0.833 U/ml and 9.013–10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis. The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity.     

The kinetics of anion equilibrium exchange across the red blood cell membrane as measured by means of35S thiocyanate

Summary Up to a SCN^– concentration of about 110mm, the concentration dependence of SCN^– equilibrium exchange in human red cell ghosts can be represented by the superimposition of two flux components. One component shows saturation kinetics, the other does not. The saturable component has an activation enthalpy of 105 kJ/mole, exhibits arans acceleration by Cl^– and can be inhibited by H₂DIDS. The nonsaturable component has a much lower activation enthalpy of 33 kJ/mole, is slightly reduced intrans acceleration experiments with Cl^– and insensitive to H₂DIDS but susceptible to inhibition by phloretin. At SCN^– concentrations exceeding 110mm, the saturable component undergoes irreversible self inhibition while the nonsaturable component remains unaltered.The half saturation concentration of the saturable flux component increases with decreasing pH from 3.0mm at pH 7.4 to 13.3mm at pH 6.0. Over this pH range, the maximal flux is only slightly increased from 19×10^–12 to 22×10^–12 moles×cm^–2×sec^–1. The nonsaturable flux component also increases slightly.In accordance with previous observations of Wieth (J. Physiol. (London) 207:563–580, 1970), we find that SCN^– increases K⁺ and Na⁺ permeability. The induced cation-permeability is considerably smaller than the SCN^– exchange and the latter does not show the paradoxical temperature dependence that is known to pertain to the former.     

He-Ne激光照射对血液及其组分荧光光谱影响的实验研究

为研究弱激光照射对人血液携氧能力的影响及机制,我们用荧光仪分别测量了He-Ne激光照射前后正常血液及其组分（血浆、红细胞）的荧光光谱,研究了激光照射导致的光谱变化,并分析了光谱变化与血液携氧能力改变的关系。实验结果显示：全血液标本在490nm及614nm附近有荧光峰值;血浆的荧光则主要分布在420－500nm之间;红细胞在500nm及614nm附近有荧光。He-Ne激光照射后,全血液及红细胞在614nm处的荧光谱都有较明显的变化,且较相似。由此可得出结论,He-Ne激光照射可影响血液的携氧能力。     

顺式CA1P对肿瘤细胞增殖和血管生成的影响

目的:研究顺式考布他汀二磷酸四钠(cis-CA1P)对体外培养肿瘤细胞增殖的作用,以及对鸡胚尿囊膜血管和离体培养的大鼠主动脉环血管生成的影响。方法:通过测定MTT评价药物对体外培养肿瘤细胞的作用;建立培养鸡胚尿囊膜模型、离体培养大鼠动脉环,研究顺式CA1P对血管生成的影响。结果:顺式CA1P对MGC-803人胃癌细胞、U937人急性髓系白血病细胞、A375人黑色素瘤细胞、HCT116人结肠癌细胞、MDA-MB-231人乳腺癌细胞、K562人白血病细胞具有显著的生长抑制作用,且呈明显的浓度依赖性;顺式CA1P呈剂量依赖性抑制鸡胚尿囊膜血管及离体培养的大鼠主动脉环血管生成,并可以抑制血管内皮细胞的运动及血管网络状结构形成。结论:顺式CA1P具有抑制肿瘤细胞增殖和抗血管生成的作用。     

Thermodynamic studies on oxygen binding by human red blood cells

Oxygen equilibrium curves have been measured on human normal red blood cells, at the temperatures of 20, 25, 30, 37 and 41 degrees C, and at pHs ranging from 6.8 to 8.2. The thermodynamical parameters have been determined for the four successive steps of oxygenation and for overall oxygenation, according to the Adair and MWC models [Monod J, Wyman J, Changeux JP. On the nature of allosteric transitions: a plausible model. J Mol Biol 1965;12:88-118]. The heat release appears to be nearly equal for the four steps. At the first three steps, the delta H change is counterbalanced by a nearly equivalent change of delta S, resulting in a rather small delta G value. delta G is greater at the fourth step, because of diminution of this enthalpy-entropy compensation phenomenon. The four steps are both enthalpy and entropy driven. According to the MWC model, the T to R transition is endothermic, and allosteric quaternary transition occurs at binding of the third oxygen. The average heat release increases by 2.8 kcal/mol when pH raises from 7.4 to 8.2, but flattens below pH 7.4. After correction for the heat of solution of oxygen and for the heat of proton release (referred to intracellular pH), an intrinsic heat for oxygenation of the heme of approximately--13 kcal/mol is obtained for the successive steps of oxygenation (at pH 7.4, 37 degrees C). These results are compared with those previously obtained for pigeon and trout red blood cells.     

Absence of adrenergic red cell pH and oxygen content regulation in American eel (Anguilla rostrata) during hypercapnic acidosis in vivo and in vitro

Summary American eels (Anguilla rostrata) were exposed to acute (30 min) external hypercapnia (1% CO₂ or 5% CO₂ in air) in order to assess the involvement of circulating catecholamines in regulating red blood cell (RBC) pH and oxygen content during whole blood acidosis. Plasma adrenaline levels increased approximately 5-fold during severe hypercapnia yet absolute levels remained below 1.0 nM; plasma noradrenaline levels were unchanged. Both RBC pH and oxygen bound to haemoglobin ([O₂]/[Hb]) conformed to in vitro relationships with whole blood pH (pHe) indicating absence of regulation during hypercapnia in vivo. Pre-treatment of eels with - or -adrenoceptor antagonists, phentolamine or propranolol was without effect on RBC pH or [O₂]/[Hb] during hypercapnia. Further, intra-arterial injection of adrenaline (final plasma concentration=134 nM) or noradrenaline (final plasma concentration = 34 nM) into hypercapnic eels 5 min prior to blood sampling did not modify any measured blood variable RBC nucleoside triphosphate (NTP) levels, RBC pH and [O₂]/[Hb]. In vitro, the application of adrenaline or noradrenaline to eel RBC's during graded normoxic hypercapnia or hypoxic hypercapnia (noradrenaline only) did not affect RBC pH significantly. RBC NTP levels were depressed by noradrenaline in vitro but only during hypoxic hypercapnia.The results demonstrate adrenergic insensitivity of eel RBC's in vivo even under conditions (acidosis, hypoxemia) known to enhance catecholamine-mediated RBC responses in other species. We conclude that the American eel has no capacity to regulate RBC pH during hypercapnia and consequently [O₂]/[Hb] is reduced in accordance with the in vitro Root effect.     

红细胞分布宽度与高血压靶器官损害研究进展

高血压是最常见的心血管疾病,血压持续升高可导致左室肥厚、心力衰竭、脑卒中以及慢性肾病等相关靶器官损害。红细胞分布宽度(RDW)是一项测量红细胞变量宽度的指标,近年来研究证实RDW与心脑血管疾病存在一定相关性,RDW升高提示高血压患者预后不良,说明RDW可作为高血压患者预后风险评估的潜在指标。本文对近年来RDW与高血压引起的心脏损害、脑卒中、肾损害及血管损害等相关研究进行综述,进一步探讨RDW对相关疾病预防及治疗的预测价值。     

Antiepileptic carbamazepine drug treatment induces alteration of membrane in red blood cells: Possible positive effects on metabolism and oxidative stress

Carbamazepine (CBZ) is an iminostilbene derivative commonly used for treatment of neuralgic pain and bipolar affective disorders. CBZ blood levels of treated patients are within the range of micromolar concentrations and therefore, significant interactions of this drug with erythrocytes are very likely. Moreover, the lipid domains of the cell membrane are believed to be one of the sites where iminostilbene derivatives exert their effects. The present study aimed to deeply characterize CBZ effects on erythrocytes, in order to identify extra and/or cytosolic cell targets. Our results indicate that erythrocyte morphological changes promoted by the drug, may be triggered by an alteration in band 3 functionality i.e. at the level of anionic flux. In addition, from a metabolic point of view this perturbation could be considered, at least in part, as a beneficial event because it could favour the CO₂ elimination.     

A new erythrocyte-based biochemical approach to predict the antiproliferative effects of heterocyclic scaffolds: The case of indolone

Background

The indole core is a key structural feature of many natural products and biomolecules with broad spectrum chemotherapeutic properties. Some of us have recently synthesized a library of biologically promising indolone-based compounds. The present study focuses on the effects of one of them, namely DPIT, on human erythrocytes.

Methods

We have examined the influence of DPIT on band 3 protein, intracellular ATP concentration and transport, caspase 3 activation, metabolic adaptation and membrane stability.

Results

Our study elucidates that DPIT, intercalated into the phospholipid bilayer, decreases the anion transport, the intracellular ATP concentration and the cytosolic pH, inducing a direct activation of caspase 3.

Conclusions

Starting from the metabolic similarity between erythrocytes and cancer cells, we investigate how the metabolic derangements and membrane alterations induced by selected heterocycles could be related to the antiproliferative effects.

General significance

Our work aims to propose a new model of study to predict the antiproliferative effects of heterocyclic scaffolds, pointing out that only one of the listed conditions would be unfavorable to the life cycle of neoplastic cells.     

A computer model of oxygen dynamics in human colon mucosa: implications in normal physiology and early tumor development

Based on the geometry of the colon mucosa, we built a model to compute the oxygen supply, the oxygen diffusion across the interstitial matrix, and the oxygen consumption by cryptal and stromal cells. By using an iterative algorithm, we have been able to solve a set of discretized (time and space) oxygen balance equations and determine the three-dimensional distribution of pO₂ in the mucosa. Although significant longitudinal and radial pO₂ variations were found, cells appeared to operate at their maximum respiratory capacity, regardless of their location in the tissue. The estimated oxygen extraction fraction was 47%, while the capillary oxygen permeability was 1.57×10⁻⁵ cm m s⁻¹. We concluded that cellular metabolism in normal colon mucosa is not limited by oxygen supply, thus prompting the idea that oxygenation does not determine the characteristic microenvironments occurring along the normal Lieberkhün crypts. In an extended model, simulation of an aberrant crypt focus (ACF)—the earliest stage in the adenomatous polyp-carcinoma sequence—showed instead that respiratory activity decreased when the capillary array symmetry is disrupted due to the ACF growth. A unified explanation about the alternative of a hypoxic-independent and/or a hypoxic-dependent early angiogenic response associated to the development of ACF is proposed.     

Tissue engineering a blood vessel: Regulation of vascular biology by mechanical stresses

Important to the tissue engineeping of a substitute blood vessel is an understanding of those faators which regulat vascular biology. A major factor in the mechanical environment imposed by the hemodynamics of the vascular system. In this the vascular endothelium play a critical role, and mver the past two deaades much has been learned about the influence of hemodynamics on vascular endothelial biology, to a large degree using cell culture to study the effects of flow and cyclic stretch. In our laboratory, such studies ape low being extended through the development of a model of the arterial wall involving the co-culture of endothelial cells and smomth muscle cells. The development of such a model and its use in the study of endothelial cells and smmooth muscle the evolution of approaaheq to tissue engileeping a blood vessel.     

Intracellular mechanisms involved in basement membrane induced blood vessel differentiation in vitro

Summary The extracellular matrix, particularly basement membranes, plays an important role in angiogenesis (blood vessel formation). Previous work has demonstrated that a basement membranelike substrate (Matrigel) induces human umbilical vein endothelial cells to rapidly form vessel-like tubes (Kubota, et al., 1988; Grant et al., 1989b); however, the precise mechanism of tube formation is unclear. Using this in vitro model, we have investigated morphologic changes occurring during tube formation and the cytoskeletal and protein synthesis requirements of this process. Electron microscopy showed that endothelial cells attach to the Matrigel surface, align, and form cylindrical structures that contain a lumen and polarized cytoplasmic organelles. The cytoskeleton is reorganized into bundles of actin filaments oriented along the axis of the tubes and is located at the periphery of the cells. The addition of colchicine or cytochalasin D blocked tube formation, indicating that both microfilaments and microtubules are involved in this process. Cycloheximide blocked tube formation by 100%, indicating that the process also required protein synthesis. In particular, collagen synthesis seems to be required for tube formation because cis-hydroxyproline inhibited tube formation, whereas either the presence of ascorbic acid or the addition of exogenous collagen IV to the Matrigel increased tube formation. Our results indicate that endothelial cell attachment to Matrigel induces the reorganization of the cytoskeleton and elicits the synthesis of specific proteins required for the differentiated phenotype of the cells.     

Effects of antioxidants and haemoglobin status on the t-butyl hydroperoxide-induced oxygen uptake by red blood cells

Oxygen uptake by erythrocytes exposed to t-butyl hydroperoxide (t-BHP) exhibited an induction period. The rate of oxygen consumption can be reduced by antioxidants and blood plasma. The induction time was not appreciably modified by the antioxidants tested, however, plasma increased it by a factor of two. The in vivo pretreatment with diethyl maleate (0.6 g kg-1) produced increased rates of oxygen uptake without changes in the induction period, while vitamin E (12.5 mg kg-1) elicited lower oxygen consumption rates and longer induction times, compared to those observed in cells from control rats upon addition of the hydroperoxide. These results suggest that the antioxidants tested on the t-BHP lipid peroxidation in erythrocyte suspensions act as inhibitors and/or retarders of the process. Furthermore, lipid peroxidation induced in these conditions seems to depend upon the haemoglobin status of the cells as oxygen uptake, malondialdehyde production and chemiluminescence were significantly higher in methaemoglobin-containing cells than in those containing oxyhaemoglobin.