Nitric oxide influences red blood cell velocity independently of changes in the vascular tone

Abstract

Nitric oxide (NO) plays a key role in regulation of vascular tone and blood flow. In the microcirculation blood flow is strongly dependent on red blood cells (RBC) deformability. In vitro NO increases RBC deformability. This study hypothesized that NO increases RBC velocity in vivo not only by regulating vascular tone, but also by modifying RBC deformability. The effects of NO on RBC velocity were analysed by intra-vital microscopy in the microcirculation of the chorioallantoic membrane (CAM) of the avian embryo at day 7 post-fertilization, when all vessels lack smooth muscle cells and vascular tone is not affected by NO. It was found that inhibition of enzymatic NO synthesis and NO scavenging decreased intracellular NO levels and avian RBC deformability in vitro. Injection of a NO synthase-inhibitor or a NO scavenger into the microcirculation of the CAM decreased capillary RBC velocity and deformation, while the diameter of the vessels remained constant. The results indicate that scavenging of NO and inhibition of NO synthesis decrease RBC velocity not only by regulating vascular tone but also by decreasing RBC deformability.     

Study of tumor blood perfusion and its variation due to vascular normalization by anti-angiogenic therapy based on 3D angiogenic microvasculature

The coupling of intravascular and interstitial flow is a distinct feature of tumor microcirculation, due to high vessel permeability, low osmotic pressure gradient and absence of functional lymphatic system inside tumors. We have previously studied the tumor microcirculation by using a 2D coupled model. In this paper, we extend it to a 3D case with some new considerations, to investigate tumor blood perfusion on a more realist microvasculature, and the effects of vascular normalization by anti-angiogenic therapy on tumor microenvironment.The model predict the abnormal tumor microcirculation and the resultant hostile microenvironment: (1) in the intra-tumoral vessels, blood flows slowly with almost constant pressure values, haematocrit is much lower which contributes to hypoxia and necrosis formation of the tumor centre; (2) the total transvascular flux is at the same order of magnitude as intravascular flux, the intravasation appears inside of the tumor, the ratio of the total amount of intravasation flux to extravasation flux is about 16% for the present model; (3) the interstitial pressure is uniformly high throughout the tumor and drops precipitously at the periphery, which leads to an extremely slow interstitial flow inside the tumor, and a rapidly rising convective flow oozing out from the tumor margin into the surrounding normal tissue. The investigation of the sensitivity of flows to changes in transport properties of vessels and interstitium as well as the vascular density of the vasculature, gains an insight into how normalization of tumor microenvironment by anti-angiogenic therapies influences the blood perfusion.     

The effect of geometry on tumor thermal profile and its use in tumor functional state estimation

Thermal differences between transplanted tumors and tumors in humans prevent the implementation of thermographic methods developed in mice models to human models and vise‐versa. Transplantable tumors tend to have an extruding shape, which may affect the thermal patterns. This hypothesis was studied in phantom experiments and simulations. A correlation between tumor dimensions and relative temperature was found and used to estimate tumor functional state from previously published in vivo experiments. A correlation was found between temperature differences and tumor growth rates (tumor aggressiveness) and the effect of tumor treatment was demonstrated, showing the potential for in vivo, non‐invasive tumor monitoring. (© 2015 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

A simple approach for non‐invasive transcranial optical vascular imaging (nTOVI)

In vivo imaging of cerebral vasculature is highly vital for clinicians and medical researchers alike. For a number of years non‐invasive optical‐based imaging of brain vascular network by using standard fluorescence probes has been considered as impossible. In the current paper controverting this paradigm, we present a robust non‐invasive optical‐based imaging approach that allows visualize major cerebral vessels at the high temporal and spatial resolution. The developed technique is simple to use, utilizes standard fluorescent dyes, inexpensive micro‐imaging and computation procedures. The ability to clearly visualize middle cerebral artery and other major vessels of brain vascular network, as well as the measurements of dynamics of blood flow are presented. The developed imaging approach has a great potential in neuroimaging and can significantly expand the capabilities of preclinical functional studies of brain and notably contribute for analysis of cerebral blood circulation in disorder models.

An example of 1 × 1.5 cm color‐coded image of brain blood vessels of mouse obtained in vivo by transcranial optical vascular imaging (TOVI) approach through the intact cranium.     

Automatic and label‐free identification of blood vessels in gliomas using the combination of multiphoton microscopy and image analysis

Currently, the targeted treatment of tumor based on the tumor microenvironment is newly developed. Blood vessels are the key parts in the tumor microenvironment, which is taken as a new visible target for tumor therapy. Multiphoton microscopy (MPM), based on the second harmonic generation and two‐photon excited fluorescence, is available to make the label‐free analysis on the blood vessels in human gliomas. MPM can reveal the vascular morphological characteristics in gliomas, including vascular malformation, intense vascular proliferation, perivascular collagen deposition, perivascular lymphocytes aggregation and microvascular proliferation. In addition, the image analysis algorithms were developed to automatically calculate the perivascular collagen content, vascular cavity area, lumen area, wall area and vessel number. Thus, the vascular morphology, the perivascular collagen deposition and intense vascular proliferation degree can be further quantitatively characterized. Compared with the pathological analysis, the combination of MPM and image analysis has potential advantages in making a quantitative and qualitative analyzing on vascular morphology in glioma microenvironment. As micro‐endoscope and two‐photon fiberscope are technologically improved, this combined method will be a useful imaging way to make the real‐time research on the targeting tumor microenvironment in gliomas.     

A Novel In Vivo Vascular Imaging Approach for Hierarchical Quantification of Vasculature Using Contrast Enhanced Micro-Computed Tomography

The vasculature of body tissues is continuously subject to remodeling processes originating at the micro-vascular level. The formation of new blood vessels (angiogenesis) is essential for a number of physiological and pathophysiological processes such as tissue regeneration, tumor development and the integration of artificial tissues. There are currently no time-lapsed in vivo imaging techniques providing information on the vascular network at the capillary level in a non-destructive, three-dimensional and high-resolution fashion. This paper presents a novel imaging framework based on contrast enhanced micro-computed tomography (micro-CT) for hierarchical in vivo quantification of blood vessels in mice, ranging from largest to smallest structures. The framework combines for the first time a standard morphometric approach with densitometric analysis. Validation tests showed that the method is precise and robust. Furthermore, the framework is sensitive in detecting different perfusion levels after the implementation of a murine ischemia-reperfusion model. Correlation with both histological data and micro-CT analysis of vascular corrosion casts confirmed accuracy of the method. The newly developed time-lapsed imaging approach shows high potential for in vivo monitoring of a number of different physiological and pathological conditions in angiogenesis and vascular development.     

In vivo detection of tumor boundary using ultrahigh‐resolution optical coherence angiography and fluorescence imaging

Accurate detection of early tumor margin is of great preclinical and clinical implications for predicting the survival rate of subjects and assessing the response of tumor microenvironment to chemotherapy or radiation therapy. Here, we report a multimodality optical imaging study on in vivo detection of tumor boundary by analyzing neoangiogenesis of tumor microenvironment (microangiography), microcirculatory blood flow (optical Doppler tomography) and tumor proliferation (green fluorescent protein [GFP] fluorescence). Microangiography demonstrates superior sensitivity (77.7 ± 6.4%) and specificity (98.2 ± 1.7%) over other imaging technologies (eg, optical coherence tomography) for tumor margin detection. Additionally, we report longitudinal in vivo imaging of tumor progression and show that the abrupt tumor cell proliferation did not occur until local capillary density and cerebral blood flow reached their peak approximately 2 weeks after tumor implantation. The unique capability of longitudinal multimodality imaging of tumor angiogenesis may provide new insights in tumor biology and in vivo assessment of the treatment effects on anti‐angiogenesis therapy for brain cancer.     

Perfectly registered multiphoton and reflectance confocal video rate imaging of in vivo human skin

We present a multimodal in vivo skin imaging instrument that is capable of simultaneously acquiring multiphoton and reflectance confocal images at up to 27 frames per second with 256 × 256 pixel resolution without the use of exogenous contrast agents. A single femtosecond laser excitation source is used for all channels ensuring perfect image registration between the two‐photon fluorescence (TPF), second harmonic generation (SHG), and reflectance confocal microscopy (RCM) images. Images and videos acquired with the system show that the three imaging channels provide complementary information in in vivo human skin measurements. In the epidermis, cell boundaries are clearly seen in the RCM channel, while cytoplasm is better seen in the TPF imaging channel, whereas in the dermis, SHG and TPF channels show collagen bundles and elastin fibers, respectively. The demonstrated fast imaging speed and multimodal imaging capabilities of this MPM/RCM instrument are essential features for future clinical application of this technique. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Multispectral optoacoustic and MRI coregistration for molecular imaging of orthotopic model of human glioblastoma

Multi‐modality imaging methods are of great importance in oncologic studies for acquiring complementary information, enhancing the efficacy in tumor detection and characterization. We hereby demonstrate a hybrid non‐invasive in vivo imaging approach of utilizing magnetic resonance imaging (MRI) and Multispectral Optoacoustic Tomography (MSOT) for molecular imaging of glucose uptake in an orthotopic glioblastoma in mouse. The molecular and functional information from MSOT can be overlaid on MRI anatomy via image coregistration to provide insights into probe uptake in the brain, which is verified by ex vivo fluorescence imaging and histological validation.

In vivo MSOT and MRI imaging of an orthotopic glioma mouse model injected with IRDye800‐2DG. Image coregistration between MSOT and MRI enables multifaceted (anatomical, functional, molecular) information from MSOT to be overlaid on MRI anatomy images to derive tumor physiological parameters such as perfusion, haemoglobin and oxygenation.     

A biopsy‐needle compatible varifocal multiphoton rigid probe for depth‐resolved optical biopsy

In this work, we report a biopsy‐needle compatible rigid probe, capable of performing three‐dimensional (3D) two‐photon optical biopsy. The probe has a small outer diameter of 1.75 mm and fits inside a gauge‐14 biopsy needle to reach internal organs. A carefully designed focus scanning mechanism has been implemented in the rigid probe, which, along with a rapid two‐dimensional MEMS scanner, enables 3D imaging. Fast image acquisition up to 10 frames per second is possible, dramatically reducing motion artifacts during in vivo imaging. Equipped with a high‐numerical aperture micro‐objective, the miniature rigid probe offers a high two‐photon resolution (0.833 × 6.11 μm, lateral × axial), a lateral field of view of 120 μm, and an axial focus tuning range of 200 μm. In addition to imaging of mouse internal organs and subcutaneous tumor in vivo, first‐of‐its‐kind depth‐resolved two‐photon optical biopsy of an internal organ has been successfully demonstrated on mouse kidney in vivo and in situ.      

Synthesis,radiolabeling, and preclinical evaluation of a new octreotide analog for somatostatin receptor‐positive tumor scintigraphy

Radiolabeled somatostatin analogs have become powerful tools in the diagnosis and staging of neuroendocrine tumors, which express somatostatin receptors. The aim of this study was to evaluate a new somatostatin analog, 6‐hydrazinopyridine‐3‐carboxylic acid‐Ser³‐octreotate (HYNIC‐SATE) radiolabeled with ^99mTc, using ethylenediamine‐N,N′‐diacetic acid and tricine as coligands, to be used as a radiopharmaceutical for the in vivo imaging of somatostatin receptor subtype 2 (SSTR2)‐positive tumor. Synthesis of the peptide was carried out on a solid phase using a standard Fmoc strategy. Peptide conjugate affinities for SSTR2 were determined by receptor binding affinity on rat brain cortex and C6 cell membranes. Internalization rate of ^99mTc‐HYNIC‐SATE was studied in SSTR2‐expressing C6 cells that were used for intracranial tumor studies in rat brain. A reproducible in vivo C6 glioma model was developed in Sprague–Dawley rat and confirmed by histopathology and immunohistochemical analysis. Biodistribution and imaging properties of this new radiopeptide were also studied in C6 tumor‐bearing rats. Radiolabeling was performed at high specific activities, with a radiochemical purity of >96%. Peptide conjugate showed high affinity binding for SSTR2 (HYNIC‐SATE IC₅₀ = 1.60 ± 0.05 n m ) and specific internalization into rat C6 cells. After administration of ^99mTc‐HYNIC‐SATE in C6 glioma‐bearing rats, a receptor specific uptake of radioactivity was observed in SSTR‐positive organs and in the implanted intracranial tumor and rapid excretion from nontarget tissues via kidneys. ^99mTc‐HYNIC‐SATE is a new receptor‐specific radiopeptide for targeting SSTR2‐positive brain tumor and might be of great promise in the scintigraphy of SSTR2‐positive tumors. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Structural Adaptation and Heterogeneity of Normal and Tumor Microvascular Networks

Relative to normal tissues, tumor microcirculation exhibits high structural and functional heterogeneity leading to hypoxic regions and impairing treatment efficacy. Here, computational simulations of blood vessel structural adaptation are used to explore the hypothesis that abnormal adaptive responses to local hemodynamic and metabolic stimuli contribute to aberrant morphological and hemodynamic characteristics of tumor microcirculation. Topology, vascular diameter, length, and red blood cell velocity of normal mesenteric and tumor vascular networks were recorded by intravital microscopy. Computational models were used to estimate hemodynamics and oxygen distribution and to simulate vascular diameter adaptation in response to hemodynamic, metabolic and conducted stimuli. The assumed sensitivity to hemodynamic and conducted signals, the vascular growth tendency, and the random variability of vascular responses were altered to simulate ‘normal’ and ‘tumor’ adaptation modes. The heterogeneous properties of vascular networks were characterized by diameter mismatch at vascular branch points (d³_var) and deficit of oxygen delivery relative to demand (O_2def). In the tumor, d³_var and O_2def were higher (0.404 and 0.182) than in normal networks (0.278 and 0.099). Simulated remodeling of the tumor network with ‘normal’ parameters gave low values (0.288 and 0.099). Conversely, normal networks attained tumor-like characteristics (0.41 and 0.179) upon adaptation with ‘tumor’ parameters, including low conducted sensitivity, increased growth tendency, and elevated random biological variability. It is concluded that the deviant properties of tumor microcirculation may result largely from defective structural adaptation, including strongly reduced responses to conducted stimuli.     

NADPH oxidase subunit 4-mediated reactive oxygen species contribute to cycling hypoxia-promoted tumor progression in glioblastoma multiforme

Background

Cycling and chronic tumor hypoxia are involved in tumor development and growth. However, the impact of cycling hypoxia and its molecular mechanism on glioblastoma multiforme (GBM) progression remain unclear.

Methodology

Glioblastoma cell lines, GBM8401 and U87, and their xenografts were exposed to cycling hypoxic stress in vitro and in vivo. Reactive oxygen species (ROS) production in glioblastoma cells and xenografts was assayed by in vitro ROS analysis and in vivo molecular imaging studies. NADPH oxidase subunit 4 (Nox4) RNAi-knockdown technology was utilized to study the role of Nox4 in cycling hypoxia-mediated ROS production and tumor progression. Furthermore, glioblastoma cells were stably transfected with a retroviral vector bearing a dual reporter gene cassette that allowed for dynamic monitoring of HIF-1 signal transduction and tumor cell growth in vitro and in vivo, using optical and nuclear imaging. Tempol, an antioxidant compound, was used to investigate the impact of ROS on cycling hypoxia-mediated HIF-1 activation and tumor progression.

Principal Findings

Glioblastoma cells and xenografts were compared under cycling hypoxic and normoxic conditions; upregulation of NOX4 expression and ROS levels were observed under cycling hypoxia in glioblastoma cells and xenografts, concomitant with increased tumor cell growth in vitro and in vivo. However, knockdown of Nox4 inhibited these effects. Moreover, in vivo molecular imaging studies demonstrated that Tempol is a good antioxidant compound for inhibiting cycling hypoxia-mediated ROS production, HIF-1 activation, and tumor growth. Immunofluorescence imaging and flow cytometric analysis for NOX4, HIF-1 activation, and Hoechst 3342 in glioblastoma also revealed high localized NOX4 expression predominantly in potentially cycling hypoxic areas with HIF-1 activation and blood perfusion within the endogenous solid tumor microenvironment.

Conclusions

Cycling hypoxia-induced ROS via Nox4 is a critical aspect of cancer biology to consider for therapeutic targeting of cycling hypoxia-promoted HIF-1 activation and tumor progression in GBM.     

A transgenic Tie2-GFP athymic mouse model; a tool for vascular biology in xenograft tumors

We report the generation of a transgenic Tie2-GFP athymic nude mouse, carrying green fluorescent blood vessels throughout the body. This transgenic mouse is a tool for studies in vascular biology, and is especially of interest for imaging of tumor angiogenesis and the study of anti-angiogenesis strategies in (human) xenografts. Intravital microscopy identified the presence of blood conducting structures that are not lined by endothelial cells. Dedifferentiation of aggressive tumor cells can lead to acquisition of endothelial characteristics. This process of tumor cell plasticity, also referred to as vasculogenic mimicry, has been suggested to contribute to the circulatory system in a tumor. In plastic EW7 Ewing sarcoma tumors in these Tie2-GFP mice, we observed blood flow in both regular blood vessels and non-fluorescent tumor cell-lined channels, visualizing in vivo hemodynamics in vasculogenic channels. These results demonstrate that the transgenic Tie2-GFP athymic mouse model is a valuable tool for vascular biology research.     

Subdiffraction‐resolution live‐cell imaging for visualizing thylakoid membranes

The chloroplast is the chlorophyll‐containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live‐cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three‐dimensional structured illumination microscopy (3D‐SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D‐SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild‐type and mutant strains. Using 3D‐SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D‐SIM. This study demonstrates that 3D‐SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.     

A bright blue fluorescent dextran for two-photon in vivo imaging of blood vessels

Fluorescence-based in vivo imaging is one of the most important tools for monitoring of biological processes in cells and tissues of live animal models. Fluorescence imaging agents have also been used to monitor the microcirculation. Tracking microcirculation of the blood is vital to gain further insight into various vascular disease-related anomalies within the human body. As monitoring of vascular circulation is performed with visualization of both immune cells and pathogens, which are mainly labelled with red and green, the favorable color option for blood vessels could be blue. However, currently available blueish color-labeled agents for vascular monitoring is generally confronted with quick bleaching, because of its short excitation and emission wavelengths. Hereby, what we propose in this report is a newly generated bright blue fluorescent dextran, named HCD-70K that monitors the blood vessels using blue and inter-compatible typical fluorescent materials. DBCO-functionalized dextran-70K was fabricated with hydroxy-coumarin dye via metal-free bioorthogonal click chemistry, and generated HCD-70K, which can flow within the blood vessel and decipher the whole structure of the blood vessel successfully. The synthesis, spectroscopic analysis, and quantum chemical calculations were conducted. Using two-photon microscopy, efficient deep in vivo blood vessel imaging of a mouse model revealed exceptional bio-imaging capabilities of the HCD-70K and consequently it provided a promising opportunity for efficient vascular visualization in various research areas.     

Coupled modeling of blood perfusion in intravascular, interstitial spaces in tumor microvasculature

The coupling of intravascular and interstitial flow is a distinct feature of tumor microcirculation, due to the high vessel permeability, the low osmotic pressure gradient as well as the absence of functional lymphatic system inside tumors. In this paper, a coupled mathematical model of tumor microcirculation is developed, which provides the link between microvasculature and interstitial space perfusion through the matrices determining a neighbor point belonging to either connected vessel (matrix B) or interstitial space (matrix A), and combines the intravascular and interstitial flow by vascular leaky terms. In addition, the compliance of tumor vessels, blood rheology with hematocritic distribution at branches is also considered. The microvascular network, on which the microcirculation calculation is carried out, is generated from our two-dimensional 9-point (2D9P) model of tumor angiogenesis, improved from the previous 2D5P one. A specific coupling procedure is developed in the study to couple the intravascular and interstitial flow. It is based on the iteratively numerical simulation techniques, including local iterations at individual parameter level and one global loop to provide coupling and simulation convergence. The simulation results not only present the basic features and characteristics of tumor microcirculation, which agree with the corresponding experimental observations reported, but also predict an intimate relationship between the tumor intravascular and interstitial flow quantitatively. Among the parameters, the vascular leakiness is a key to govern the systemic flowing pattern, influence the tumor internal environment and contribute to the metastasis of tumor cells, which could not be presented by the previous uncoupled models.     

综述——基于纳米材料的生物检测与医学诊断技术：功能化量子点在肿瘤活体成像中的应用

量子点表面经生物分子或药物分子修饰而具有生物功能．功能化量子点具有独特的光学性质和生物相容性,在生物医学光学诊断和治疗领域具有广泛的应用．本文简要介绍了功能化量子点制备及修饰方法,综合评述了量子点在肿瘤活体诊断和治疗中的应用,包括活体淋巴结成像、血管动态成像、肿瘤成像和抗肿瘤药物示踪等,讨论了功能化量子点在肿瘤活体诊断和治疗中的应用前景以及面临的挑战．