Additive effect of red blood cell rigidity and adherence to endothelial cells in inducing vascular resistance

To explore the contribution of red blood cell (RBC) deformability and interaction with endothelial cells (ECs) to circulatory disorders, these RBC properties were modified by treatment with hydrogen peroxide (H(2)O(2)), and their effects on vascular resistance were monitored following their infusion into rat mesocecum vasculature. Treatment with 0.5 mM H(2)O(2) increased RBC/EC adherence without significant alteration of RBC deformability. At 5.0 mM H(2)O(2), RBC deformability was considerably reduced, inducing a threefold increase in the number of undeformable cells, whereas RBC/EC adherence was not further affected by the increased H(2)O(2) concentration. This enabled the selective manipulation of RBC adherence and deformability and the testing of their differential effect on vascular resistance. Perfusion of RBCs with enhanced adherence and unchanged deformability (treatment with 0.5 mM H(2)O(2)) increased vascular resistance by about 35% compared with untreated control RBCs. Perfusion of 5.0 mM H(2)O(2)-treated RBCs, with reduced deformability (without additional increase of adherence), further increased vascular resistance by about 60% compared with untreated control RBCs. These results demonstrate the specific effects of elevated adherence and reduced deformability of oxidized RBCs on vascular resistance. These effects can be additive, depending on the oxidation conditions. The oxidation-induced changes applied in this study are moderate compared with those observed in RBCs in pathological states. Yet, they caused a considerable increase in vascular resistance, thus demonstrating the potency of RBC/EC adherence and RBC deformability in determining resistance to blood flow in vivo.     

内皮细胞粘附分子与血管壁通透性

内皮细胞是血管壁的主要通透屏障,对血管壁通透性有多种调节作用。细胞粘附分参与细胞间连接形成以及细胞－基底膜间的粘附,与内皮层连续完整性以及通透性密切相关。参与内皮细胞中间连接的粘附分子有三大家庭,即整合素家庭、钙依赖性粘附素家族和免疫球蛋白家族,对这些粘附分子家族的研究有助于阐明内皮细胞损伤修复、新生血管形成、血管通透性调节以及循环血细胞游出的机制。     

Dependence of red blood cell dynamics in microvessel bifurcations on the endothelial surface layer’s resistance to flow and compression

Red blood cells (RBCs) make up 40–45% of blood and play an important role in oxygen transport. That transport depends on the RBC distribution throughout the body, which is highly heterogeneous. That distribution, in turn, depends on how RBCs are distributed or partitioned at diverging vessel bifurcations where blood flows from one vessel into two. Several studies have used mathematical modeling to consider RBC partitioning at such bifurcations in order to produce useful insights. These studies, however, assume that the vessel wall is a flat impenetrable homogeneous surface. While this is a good first approximation, especially for larger vessels, the vessel wall is typically coated by a flexible, porous endothelial glycocalyx or endothelial surface layer (ESL) that is on the order of 0.5–1 µm thick. To better understand the possible effects of this layer on RBC partitioning, a diverging capillary bifurcation is analyzed using a flexible, two-dimensional model. In addition, the model is also used to investigate RBC deformation and RBC penetration of the ESL region when ESL properties are varied. The RBC is represented using interconnected viscoelastic elements. Stokes flow equations (viscous flow) model the surrounding fluid. The flow in the ESL is modeled using the Brinkman approximation for porous media with a corresponding hydraulic resistivity. The ESL’s resistance to compression is modeled using an osmotic pressure difference. One cell passes through the bifurcation at a time, so there are no cell–cell interactions. A range of physiologically relevant hydraulic resistivities and osmotic pressure differences are explored. Decreasing hydraulic resistivity and/or decreasing osmotic pressure differences (ESL resistance to compression) produced four behaviors: (1) RBC partitioning nonuniformity increased slightly; (2) RBC deformation decreased; (3) RBC velocity decreased relative to blood flow velocity; and (4) RBCs penetrated more deeply into the ESL. Decreasing the ESL’s resistance to flow and/or compression to pathological levels could lead to more frequent cell adhesion and clotting as well as impaired vascular regulation due to weaker ATP and nitric oxide release. Potential mechanisms that can contribute to these behaviors are also discussed.

     

Cell-cell interactions mediate the response of vascular smooth muscle cells to substrate stiffness

The vessel wall experiences progressive stiffening with age and the development of cardiovascular disease, which alters the micromechanical environment experienced by resident vascular smooth muscle cells (VSMCs). In vitro studies have shown that VSMCs are sensitive to substrate stiffness, but the exact molecular mechanisms of their response to stiffness remains unknown. Studies have also shown that cell-cell interactions can affect mechanotransduction at the cell-substrate interface. Using flexible substrates, we show that the expression of proteins associated with cell-matrix adhesion and cytoskeletal tension is regulated by substrate stiffness, and that an increase in cell density selectively attenuates some of these effects. We also show that cell-cell interactions exert a strong effect on cell morphology in a substrate-stiffness dependent manner. Collectively, the data suggest that as VSMCs form cell-cell contacts, substrate stiffness becomes a less potent regulator of focal adhesion signaling. This study provides insight into the mechanisms by which VSMCs respond to the mechanical environment of the blood vessel wall, and point to cell-cell interactions as critical mediators of VSMC response to vascular injury.     

Mechanisms of uremic erythrocyte-induced adhesion of human monocytes to cultured endothelial cells

In end-stage renal disease (ESRD) endothelium may represent a key target for the action of circulating elements, such as modified erythrocytes (RBC) and/or plasmatic factors, that may facilitate inflammation and the vasculopathy associated with uremia. We have previously demonstrated that phosphatidylserine (PS) exposure on the surface of RBC from ESRD patients increases RBC-human umbilical vein endothelial cell (HUVEC) interactions and causes decreased nitric oxide (NO) production. We postulated that, besides the pro-inflammatory effects due to decreased NO bio-availability, enhanced ESRD-RBC-HUVEC interactions might directly stimulate pro-inflammatory pathways leading to increased vascular adhesion molecule expression. ESRD-RBC-endothelial cell interactions induced a time-dependent up-regulation of VCAM-1 and ICAM-1 (measured by Western blot (WB) and real-time PCR), associated with mitogen-activated protein kinase (MAPK) activation and impairment of the Akt/endothelial nitric oxide synthase (eNOS) signaling cascade, measured by WB. In reconstitution experiments, normal RBC incubated with uremic plasma showed increased PS exposure and significantly increased VCAM-1 and ICAM-1 mRNA levels when incubated on HUVEC. Interestingly, ESRD-RBC induced increased expression of adhesion molecules was prevented by Annexin-V (AnV, able to mask PS on RBC surface), anti-integrin-alpha(v)beta3, anti-thrombospondin-1 (TSP-1), and PD98059 (a selective inhibitor of MAPK phosphorylation). Moreover, AnV reversed the ESRD-RBC effects on MAPK and Akt/eNOS signaling pathways. Our data demonstrate that, possibly via a direct interaction with the endothelial thrombospondin-(alpha(v)beta3) integrin complex, ESRD-RBC-HUVEC adhesion induces a vascular inflammatory phenotype. Thus, intervention targeting ESRD-RBC increased adhesion to endothelium and/or MAPK and Akt/eNOS pathways may have the potential to prevent vascular lesions under uremic conditions.     

Effects of fibulin-5 on attachment, adhesion, and proliferation of primary human endothelial cells

BACKGROUND: Fibulin-5 is a novel extracellular protein that is thought to act as a bridging peptide between elastin fibers and cell surface integrins in blood vessel wall. Fibulin-5 binding to endothelial cell (EC) surface integrins may effect cell proliferation and cell attachment to extracellular matrix (ECM) or to artificial surfaces. In this paper, we describe the effects of fibulin-5 on attachment, adhesion, and proliferation of primary human EC. After demonstrating that fibulin-5 over-expression inhibited EC proliferation, we tested the hypothesis that co-expression of fibulin-5 and VEGF165 will lead to unique EC phenotype that will exhibit increased adherence properties and retain its proliferation capacity. METHODS AND RESULTS: Fibulin-5 and VEGF165 gene transfer to primary human saphenous vein endothelial cells was accomplished using retroviral vectors encoding the two genes. Transgene expression was verified using immunohistochemistry, Western blotting, and ELISA. Fibulin 5 over-expression tended to improve immediate EC attachment (30 min after seeding) and improved significantly adhesion (>40%) under shear stress tested 24h after EC seeding. The effects of fibulin-5 and VEGF165 on EC proliferation in the presence or absence of basic FGF were also tested. EC expressing fibulin-5 had reduced proliferation while VEGF165 co-expression ameliorated this effect. CONCLUSION: Fibulin-5 improved EC attachment to artificial surfaces. Dual transfer of fibulin-5 and VEGF165 resulted in EC phenotype with increased adhesion and improved proliferation. This unique EC phenotype can be useful for tissue engineering on endovascular prostheses.     

Red blood cells initiate leukocyte rolling in postcapillary expansions: a lattice Boltzmann analysis

Leukocyte rolling on the vascular endothelium requires initial contact between leukocytes circulating in the blood and the vessel wall. Although specific adhesion mechanisms are involved in leukocyte-endothelium interactions, adhesion patterns in vivo suggest other rheological mechanisms also play a role. Previous studies have proposed that the abundance of leukocyte rolling in postcapillary venules is due to interactions between red blood cells (RBCs) and leukocytes as they enter postcapillary expansions, but the details of the fluid dynamics have not been elucidated. We have analyzed the interactions of red and white blood cells as they flow from a capillary into a postcapillary venule using a lattice Boltzmann approach. This technique provides the complete solution of the flow field and quantification of the particle-particle forces in a relevant geometry. Our results show that capillary-postcapillary venule diameter ratio, RBC configuration, and RBC shape are critical determinants of the initiation of cell rolling in postcapillary venules. The model predicts that an optimal configuration of the trailing red blood cells is required to drive the white blood cell to the wall.     

Plasma Factor in Red Blood Cells Adhesion to Endothelial Cells: Humans and Rats

Erythrocyte adhesion to the vascular endothelium is one of the key determinants of microcirculatory blood flow. Adhesion is a complex process determined by the intricate interaction among red blood cells (RBC), plasma factors, and the vascular endothelium. Rats are commonly used as disease models to investigate the pathophysiology of various hematological disease processes occurring in humans and their response to prospective treatments. The aim of our study was to characterize the adhesion of RBC in adult blood from rat and human subjects, in order to test the validity of rat models for adhesion-related disease processes. We demonstrated that adhesion of RBC from rats (rRBC), to endothelial cells (EC) in plasma-free buffer, is stronger than from human subjects (hRBC). In addition, plasma proteins induced elevation of hRBC (eightfold) but depression of rRBC (threefold) adhesion to EC. It is thus suggested to be aware of the difference in RBC/EC interaction for human and rat subjects, when studying models of blood flow.     

Endothelial Cell E- and P-Selectin and Vascular Cell Adhesion Molecule-1 Function as Signaling Receptors

Previous studies have shown that polymorphonuclear leukocyte (PMN) adherence to endothelial cells (EC) induces transient increases in EC cytosolic free calcium concentration ([Ca²⁺]_i) that are required for PMN transit across the EC barrier (Huang, A.J., J.E. Manning, T.M. Bandak, M.C. Ratau, K.R. Hanser, and S.C. Silverstein. 1993. J. Cell Biol. 120:1371–1380). To determine whether stimulation of [Ca²⁺]_i changes in EC by leukocytes was induced by the same molecules that mediate leukocyte adherence to EC, [Ca²⁺]_i was measured in Fura2-loaded human EC monolayers. Expression of adhesion molecules by EC was induced by a pretreatment of the cells with histamine or with Escherichia coli lipopolysaccharide (LPS), and [Ca²⁺]_i was measured in single EC after the addition of mAbs directed against the EC adhesion proteins P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or platelet/endothelial cell adhesion molecule-1 (PECAM-1). Both anti–P- and anti–E-selectin mAb, as well as anti–VCAM-1 mAb, induced transient increases in EC [Ca²⁺]_i that were comparable to those induced by 200 μM histamine. In contrast, no effect was obtained by mAbs directed against the endothelial ICAM-1 or PECAM-1. PMN adherence directly stimulated increases in [Ca²⁺]_i in histamine- or LPS-treated EC. mAbs directed against leukocyte CD18 or PECAM-1, the leukocyte counter-receptors for endothelial ICAM-1 and PECAM-1, respectively, did not inhibit PMN-induced EC activation. In contrast, mAb directed against sialyl Lewis x (sLe^x), a PMN ligand for endothelial P- and E-selectin, completely inhibited EC stimulation by adherent PMN. Changes in EC [Ca²⁺]_i were also observed after adherence of peripheral blood monocytes to EC treated with LPS for 5 or 24 h. In these experiments, the combined addition of mAbs to sLe^x and VLA-4, the leukocyte counter-receptor for endothelial VCAM-1, inhibited [Ca²⁺]_i changes in the 5 h–treated EC, whereas the anti–VLA-4 mAb alone was sufficient to inhibit [Ca²⁺]_i changes in the 24 h-treated EC. Again, no inhibitory effect was observed with an anti-CD18 or anti–PECAM-1 mAb. Of note, the conditions that induced changes in EC [Ca²⁺]_i, i.e., mAbs directed against endothelial selectins or VCAM-1, and PMN or monocyte adhesion to EC via selectins or VCAM-1, but not via ICAM-1 or PECAM-1, also induced a rearrangement of EC cytoskeletal microfilaments from a circumferential ring to stress fibers. We conclude that, in addition to their role as adhesion receptors, endothelial selectins and VCAM-1 mediate endothelial stimulation by adhering leukocytes.     

Acridine orange induces translocation of phosphatidylserine to red blood cell surface

Clustering of band-3 on red blood cell (RBC) surface has been assumed to catalyze RBC phagocytosis. In studying this subject, acridine orange (AO) has commonly been employed on the assumption that it specifically induces band-3 clustering. In the present study, we show that AO strongly induces translocation of phosphatidylserine (PS) to RBC surface. Because surface PS is well known to induce RBC intercellular interaction, these findings suggest that the use of AO as a specific inducer of band-3 clustering is questionable. It is possible that band-3 clustering and PS translocation are interdependent, and this interrelationship has yet to be explored. erythrocytes; adherence; acridine orange; band-3; phosphatidylserine     

The art of arteriogenesis

The identification of collateral artery growth (arteriogenesis) as the only mechanism to compensate for the loss of an occluded artery forced us to define the mechanisms responsible for this type of vessel growth. To achieve this, a variety of coronary as well as peripheral models of arteriogenesis have been developed. Based on these studies it is obvious that arteriogenesis obeys different mechanisms than angiogenesis, the sprouting of capillaries. Upon occlusion of an artery, the blood flow is redirected into preexisting arteriolar anastomoses that experience increased mechanical forces such as shear stress and circum ferential wall stress. The endothelium of the arteriolar connections is then activated, resulting in an increased release of monocyte-attracting proteins as well as an upregulation of adhesion molecules. Upon adherence and extravasation, monocytes promote arteriogenesis by supplying growth factors and cytokines that bind to receptors that are expressed on vascular cells within a limited time frame. Animal studies evidenced that factors, such as monocyte chemoattractant protein-1, granulocyte-monocyte colony-stimulating factor, or transforming growth factor-β₁, that either attract or prolong the lifetime of monocytes efficiently enhance collateral artery growth, an effect that was seen only to a minor degree after application of a single growth factor. Bone marrow-derived stems cells and endothelial progenitor cells do not incorporate in growing arteries but, rather, function as supporting cells. Complete elucidation of the mechanisms of arteriogenesis may lead to efficacious therapies counteracting the devastating consequences of vascular occlusive diseases.     

VE-cadherin links tRNA synthetase cytokine to anti-angiogenic function

A natural fragment of an enzyme that catalyzes the first step of protein synthesis-human tryptophanyl-tRNA synthetase (T2-TrpRS) has potent anti-angiogenic activity. A cellular receptor through which T2-TrpRS exerts its anti-angiogenic activity has not previously been identified. Here T2-TrpRS was shown to bind at intercellular junctions of endothelial cells (ECs). Using genetic knock-outs, binding was established to depend on VE-cadherin, a calcium-dependent adhesion molecule, which is selectively expressed in ECs, concentrated at adherens junctions, and is essential for normal vascular development. In contrast, T2-TrpRS binding to EC junctions was not dependent on platelet endothelial cell adhesion molecule type-1, another adhesion molecule found at EC junctions. Pull-down assays confirmed direct complex formation between T2-TrpRS and VE-cadherin. Binding of T2-TrpRS inhibited VEGF-induced ERK activation and EC migration. Thus, a VE-cadherin-dependent pathway is proposed to link T2-TrpRS to inhibition of new blood vessel formation.     

An Arg-Gly-Asp sequence within thrombin promotes endothelial cell adhesion

Thrombin, in addition to its central role in hemostasis, possesses diverse cellular bioregulatory functions implicated in wound healing, inflammation, and atherosclerosis. In the present study we demonstrate that thrombin molecules modified either at the procoagulant or catalytic sites induce endothelial cell (EC) adhesion, spreading, and cytoskeletal reorganization. The most potent adhesive thrombin analogue (NO2-alpha-thrombin) was obtained by nitration of tyrosine residues. The cell adhesion promoting activity of NO2-alpha-thrombin was blocked upon the formation of thrombin-antithrombin III (ATIII) complexes and by antiprothrombin antibodies, but was unaffected by hirudin. Arg-Gly-Asp-containing peptides, fully inhibited EC adhesion to NO2-alpha-thrombin, while synthetic peptides corresponding to thrombin "Loop B" mitogenic site and the thrombin-derived chemotactic fragment "CB67-129", were uneffective. Immunofluorescence studies indicated that EC adhesion to NO2-alpha-thrombin was followed by cell spreading, actin microfilament assembly, and formation of focal contacts. By the use of specific antibodies, the vitronectin (vn) receptor (alpha v beta 3) was found to be localized in clusters upon cell adhesion to NO2-alpha-thrombin. An anti alpha v beta 3 antibody blocked EC adhesion and spreading while antifibronectin (fn) receptor (alpha 5 beta 1) antibodies were uneffective. While native thrombin exhibited a very low cell attachment activity, thrombin that was incubated at 37 degrees C before coating of plastic surfaces induced EC attachment and spreading. We propose that under certain conditions the naturally hindered RGD domain within thrombin is exposed for interaction with alpha v beta 3 on EC. This in turn promotes cell adhesion, spreading, and reorganization of cytoskeletal elements, which may altogether contribute to repair mechanisms in the disturbed vessel wall. This study defines a new biological role of thrombin and characterizes a new recognition mechanism on EC for this molecule.     

Mathematical modeling of vascular endothelial layer maintenance: the role of endothelial cell division, progenitor cell homing, and telomere shortening

Maintenance of the endothelial cell (EC) layer of the vessel wall is essential for proper functioning of the vessel and prevention of vascular disorders. Replacement of damaged ECs could occur through division of surrounding ECs. Furthermore, EC progenitor cells (EPCs), derived from the bone marrow and circulating in the bloodstream, can differentiate into ECs. Therefore, these cells might also play a role in maintenance of the endothelial layer in the vascular system. The proliferative potential of both cell types is limited by shortening of telomeric DNA. Accelerated telomere shortening might lead to senescent vascular wall cells and eventually to the inability of the endothelium to maintain a continuous monolayer. The aim of this study was to describe the dynamics of EC damage and repair and telomere shortening by a mathematical model. In the model, ECs were integrated in a two-dimensional structure resembling the endothelium in a large artery. Telomere shortening was described as a stochastic process with oxidative damage as the main cause of attrition. Simulating the model illustrated that increased cellular turnover or elevated levels of oxidative stress could lead to critical telomere shortening and senescence at an age of 65 yr. The model predicted that under those conditions the EC layer could display defects, which could initiate severe vascular wall damage in reality. Furthermore, simulations showed that 5% progenitor cell homing/yr can significantly delay the EC layer defects. This stresses the potential importance of EPC number and function to the maintenance of vascular wall integrity during the human life span.     

Analysis of early embryonic great-vessel microcirculation in zebrafish using high-speed confocal μPIV

In the developing cardiovascular system, hemodynamic vascular loading is critical for angiogenesis and cardiovascular adaptation. Normal zebrafish embryos with transgenically-labeled endothelial and red blood cells provide an excellent in vivo model for studying the fluid-flow induced vascular loading. To characterize the developmental hemodynamics of early embryonic great-vessel microcirculation in the zebrafish embryo, two complementary studies (experimental and numerical) are presented. Quantitative comparison of the wall shear stress (WSS) at the first aortic arch (AA1) of wild-type zebrafish embryos during two consecutive developmental stages is presented, using time-resolved confocal micro-particle image velocimetry (μPIV). Analysis showed that there was significant WSS difference between 32 and 48 h post-fertilization (hpf) wild-type embryos, which correlates with normal arch morphogenesis. The vascular distensibility of the arch wall at systole and the acceleration/deceleration rates of time-lapse phase-averaged streamwise blood flow curves were also analyzed. To estimate the influence of a novel intermittent red-blood cell (RBC) loading on the endothelium, a numerical two-phase, volume of fluid (VOF) flow model was further developed with realistic in vivo conditions. These studies showed that near-wall effects and cell clustering increased WSS augmentation at a minimum of 15% when the distance of RBC from arch vessel wall was less than 3 μm or when RBC cell-to-cell distance was less than 3 μm. When compared to a smooth wall, the WSS augmentation increased by a factor of ~1.4 due to the roughness of the wall created by the endothelial cell profile. These results quantitatively highlight the contribution of individual RBC flow patterns on endothelial WSS in great-vessel microcirculation and will benefit the quantitative understanding of mechanotransduction in embryonic great vessel biology, including arteriovenous malformations (AVM).     

Modulation of the susceptibility of human erythrocytes to snake venom myotoxic phospholipases A(2): role of negatively charged phospholipids as potential membrane binding sites.

Cerrophidion (Bothrops) godmani myotoxins I (CGMT-I) and II (CGMT-II), Asp-49 and Lys-49 phospholipases A(2) (PLA2s), which drastically differ in enzymatic activity, were devoid of direct hemolytic effects on erythrocytes (RBC) from different species despite the fact that enzymatically active CGMT-I was able to hydrolyze RBC membrane phospholipids and disrupt liposomes prepared from RBC lipids. Human RBC did not become susceptible to the toxins after treatment with neuraminidase or after altering membrane fluidity with cholesterol or sublytic concentrations of detergent. Unlike normal RBC, significant hemolysis was induced by CGMT-II and another similar Lys-49 isoform, B. asper MT-II (BAMT-II), in RBC enriched with phosphatidylserine (PS). Hemolysis was greater in RBC preincubated with pyridyldithioethylamine (PDA), a potent inhibitor of aminophospholipid transport. RBC enriched with phosphatidic acid (PA) also became susceptible to the myotoxins but was unaffected by PDA. Cells enriched with phosphatidylcholine (PC) remained resistant to the action of the toxins. BAMT-II also induced damage in black lipid membranes prepared with PS but not PC alone. When RBC binding of BAMT-II was measured by enzyme-linked immunosorbent assay, it was observed that PS- and PA-enriched erythrocytes were always able to capture more toxin than normal and PC-enriched RBC. This effect was significantly improved by PDA (in the case of PS) and it was observed either in the presence or in the absence of calcium in the medium. These data suggest that negatively charged lipids in the outer leaflet of cell membranes constitute myotoxic PLA2 binding sites. The scarcity of anionic phospholipids in the outer leaflet of RBC could explain their resistance to the action of these PLA2s.     

Effects of flowing RBCs on adhesion of a circulating tumor cell in microvessels

Adhesion of circulating tumor cells (CTCs) to the microvessel wall largely depends on the blood hydrodynamic conditions, one of which is the blood viscosity. Since blood is a non-Newtonian fluid, whose viscosity increases with hematocrit, in the microvessels at low shear rate. In this study, the effects of hematocrit, vessel size, flow rate and red blood cell (RBC) aggregation on adhesion of a CTC in the microvessels were numerically investigated using dissipative particle dynamics. The membrane of cells was represented by a spring-based network connected by elastic springs to characterize its deformation. RBC aggregation was modeled by a Morse potential function based on depletion-mediated assumption, and the adhesion of the CTC to the vessel wall was achieved by the interactions between receptors and ligands at the CTC and those at the endothelial cells forming the vessel wall. The results demonstrated that in the microvessel of \(15\,\upmu \hbox {m}\) diameter, the CTC has an increasing probability of adhesion with the hematocrit due to a growing wall-directed force, resulting in a larger number of receptor–ligand bonds formed on the cell surface. However, with the increase in microvessel size, an enhanced lift force at higher hematocrit detaches the initial adherent CTC quickly. If the microvessel is comparable to the CTC in diameter, CTC adhesion is independent of Hct. In addition, the velocity of CTC is larger than the average blood flow velocity in smaller microvessels and the relative velocity of CTC decreases with the increase in microvessel size. An increased blood flow resistance in the presence of CTC was also found. Moreover, it was found that the large deformation induced by high flow rate and the presence of aggregation promote the adhesion of CTC.     

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease², stroke³ and heart failure⁴. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species⁵. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers⁶ however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration^7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry¹⁰. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model⁹ which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter¹¹ as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.