Fibrinogen: structure, function, and surface interactions.

Fibrinogen plays a central role in the mechanism of coagulation and thrombosis and is partially involved in the development of postintervention restenosis. Because of therapeutic implications, it is convenient for the vascular interventionalist to revisit its structure, function, and relationships within the vascular environment. This review focuses on the molecular structure, mechanisms of polymerization and lysis, and fibrinogen interaction with the platelet alpha(IIb)beta(3) [corrected] integrin. It also addresses the less understood interaction of fibrinogen with artificial surfaces. Glycoprotein IIb-IIIa blockers, targeted to interfere with fibrinogen-platelet interactions, widely used in clinical practice, are discussed, and trials of new drugs are also summarized.

Influence of stent edge angle on endothelialization in an in vitro model.

PURPOSE: To investigate the influence of topographic features in the path of migrating endothelial cells, specifically the effect of edge angle of intravascular metallic material on endothelialization. MATERIALS AND METHODS: Flat 1-cm x 1-cm 316-L pieces of stainless steel were placed on confluent monolayers of human aortic endothelial cells. The thickness of each metal piece was ground to achieve an edge angle of 35 degrees, 70 degrees, 90 degrees, or 140 degrees (n = 6 each) in relation to the endothelial surface. Migration distance and density of endothelial cell coverage on the metal pieces were measured in groups of six each under static conditions at 4, 7, and 11 days and flow conditions (16 dynes/cm(2)) at 4 days. RESULTS: Endothelial cell migration distance along the surface of the pieces with edge angles of 35 degrees was significantly greater than that with those with larger angles (P < .05) under static and flow conditions. The migration distances on the 35 degrees piece were 87.5%, 47.3%, 57.1%, and 66.1% greater than those on the 90 degrees piece at the upstream, downstream, right, and left edges, respectively. There were no significant differences in cell density among different angle groups under flow or static conditions. CONCLUSION: The edge angle of intravascular metallic material has an influence on the rate of endothelialization. A smaller edge angle facilitates endothelialization over metallic material when compared to a larger angle. These results demonstrate the importance of metallic stent profile on endothelialization rate.

Electrostatic forces on the surface of metals as measured by atomic force microscopy.

Electrostatic forces play an important role in modulating the interaction of plasma proteins and blood cellular components with the surface of the vascular endothelium. Based on the concept that electrostatic forces residing on the surface of metal intravascular prostheses, such as the stent, also are critical in influencing blood interactions with those surfaces and the vascular wall, these studies were designed to measure these forces on 4 metals using atomic force microscopy (AFM). AFM measurements performed in a low saline aqueous medium at physiological pH indicate a similar net electronegative surface charge level for gold and 316l stainless steel that is significantly higher than the level measured on an electropolished Nitinol surface. Heat oxidation of the Nitinol surface increased the overall electronegativity and created a more homogeneous surface charge distribution. This study demonstrates that AFM force measurements can be a valuable approach to understanding the electrostatic surface of metallic as well as other biomaterials that may be important in understanding how these surfaces influence vascular healing at intravascular interventional sites.

Protein interactions with endovascular prosthetic surfaces.

Blood protein interaction with prosthetic surfaces seems to be the initial step in the chain of events leading to tissue incorporation of endovascular devices. This paper focuses on the relationship between surface free energy and protein adsorption on metals and polymers commonly used for fabricating vascular prosthetic devices. Our results support a relationship between surface energy and protein adsorption. Albumin was more easily eluted than fibrinogen and fibronectin from most metals and all polymeric surfaces considered. Following elution, metals retained a larger fraction of protein as compared to polymers.

Influence of topography on endothelialization of stents: clues for new designs.

To evaluate the influence of stent design on endothelialization of the stented surface, we placed trapezoidal objects of variable thickness on a confluent culture of endothelial cells and subjected the assembly to flow and shear conditions similar to those found in arteries. After 24 h, we measured and analyzed the area on top of the objects covered by cells and the maximum migration distance from the borders. In addition, we evaluated areas devoid of cells surrounding the objects, which developed after exposure of the assembly to flow. The cell-covered area and migration distance significantly decreased on objects 75 microns thick, and it was nonexistent on objects 250 microns thick. Areas devoid of cells or gaps were largest adjacent to the downflow side of the object, disposed transversely to flow. Cell gaps were smallest along the side aligned with flow. In conclusion, endothelial cell coverage may be impaired by stent wall thickness larger than 75 microns. It is likely that this impairment is related to flow disturbances impairing cell attachment.

Endothelial cell migration onto metal stent surfaces under static and flow conditions.

Restenosis associated with intimal hyperplasia and thrombosis at sites of balloon angioplasty or stent placement remains an important clinical problem. It is likely that loss or damage to the arterial endothelium associated with these interventional procedures as well as the rate of its restoration plays a critical role in the extent of restenosis. Migration of arterial endothelial cells from adjacent intact endothelium is the predominant source of cells involved in re-endothelialization of the injured site. In this paper, we review the influence of hemodynamics on endothelial cell migration, both in vivo and in vitro. In addition, we present recent in vitro studies demonstrating the importance of the nature of metal substrates in modulating endothelial cell migration rate. Finally, we review the cellular and molecular mechanisms likely involved in governing endothelial cell migration, and relate them to a possible scenario of endothelial response to injury at sites of arterial intervention. Understanding the important factors regulating endothelial migration may provide insights that will ultimately lead to methods to accelerate endothelial healing and reduce the occurrence of arterial restenosis.

Mass spectrometric analysis of platelet-activating factor after isolation by solid-phase extraction and direct derivatization with pentafluorobenzoic anhydride.

Platelet-activating factor is the term used to denote a class of extremely potent lipid mediators that consist predominantly of 1-O-alkyl- and 1-O-acyl-2-acetyl-sn-glycero-3-phosphocholines. A method has been devised for rapid isolation of these acetylated phospholipids by solid-phase extraction prior to direct derivatization with pentafluorobenzoic anhydride and analysis by gas chromatography (GC)/electron-capture mass spectrometry. Recovery through the entire method (lipid isolation, derivatization, and purification) typically ranged from 70% to 85%. Using the direct derivatization procedure described here, the practical limit of detection for each of the standard alkyl- and acyl-platelet-activating factor homologs was 1 fmol injected into the GC. Results from the application of the method to the analysis of alkyl and acyl homologs of platelet-activating factor isolated from stimulated human umbilical vein endothelial cells are presented, exhibiting excellent accuracy and precision for a wide range of tissue levels of this class of potent autacoids.

Polytetrafluoroethylene-encapsulated stent-grafts: use in experimental abdominal aortic aneurysm.

PURPOSE: To evaluate expanded polytetrafluoroethylene (ePTFE) encapsulated stents for the treatment of aortic aneurysms with emphasis on the blood and tissue-material interactions. MATERIALS AND METHODS: Experimental aortic aneurysms were created in dogs by enlarging the aortic lumen with an abdominal fascial patch. Twenty animals underwent endoluminal repair after allowing the surgically created aneurysm to heal for 2 months prior to transluminal aneurysmal exclusion. The device used consisted of an 8-cm-long ePTFE encapsulated stent graft. The animals were killed in groups at 1 week and at 1, 2.25, 6, and 12 months. Specimens were processed for histologic and luminal surface studies. RESULTS: Before the animals were killed, aortography demonstrated two thrombosed aortae in the 6-month group and two endoleaks in the 12-month group. Endothelialized neointima extended into the proximal and distal portions of the prosthetic lumen, with minimal cell coverage in the center of the graft. The overall percent surface area covered by endothelialized neointima was 22% +/- 6% at 6 months and 18% +/- 10% by 1 year (P = .75). Histologic examination demonstrated minimal tissue penetration into the ePTFE. CONCLUSION: Transluminal exclusion of abdominal aortic aneurysms by encapsulated stent-graft is easily accomplished. With this device, tissue coverage and penetration of the stent graft is limited and does not tend to increase with time.

Influence of surface topography on endothelialization of intravascular metallic material.

PURPOSE: To determine whether grooves on a metal surface help endothelialization and, furthermore, what groove size is more likely to promote the fastest endothelialization in an in vitro model. Hypothetically, a microscopic pattern of parallel grooves disposed in the direction of flow, on the inner surface of stents, increases endothelial cell migration rates, resulting in decreased time to total coverage of the prosthetic surface. MATERIALS AND METHODS: Square, flat pieces of nitinol were placed level on a monolayer, confluent culture of endothelial cells. The metal pieces were treated to produce parallel grooves on the surface of 1, 3, 15, and 22 microm to be compared to polished, smooth controls. Microscopy images were obtained by digital capture and processed for analysis of migration distance and cell count, density, shape, and alignment. RESULTS: Grooved surfaces promoted increased rate of migration of endothelial cells, up to 64.6% when compared to smooth, control surfaces. Larger grooves resulted in greater migration rates. The cells aligned with the grooves, elongated, and become more numerous on grooved surfaces, particularly with large grooves. CONCLUSION: A pattern of microscopic parallel grooves more than doubles the migration rate of endothelial cells over metallic surfaces ordinarily used for endovascular stents. Future research in this area is aimed at demonstrating the potential effect of grooved endovascular stent surfaces on faster endothelialization times.

Regulation of low shear flow-induced HAEC VCAM-1 expression and monocyte adhesion.

We recently reported that prolonged exposure of human aortic endothelial cells (HAEC) to low shear stress flow patterns is associated with a sustained increase in the activated form of the transcriptional regulator nuclear factor-kappaB (NF-kappaB). Here we investigate the hypothesis that low shear-induced activation of NF-kappaB is responsible for enhanced expression of vascular cell adhesion molecule (VCAM-1) resulting in augmented endothelial cell-monocyte (EC-Mn) adhesion and that this activation is dependent on intracellular oxidant activity. Before exposure to low shear (2 dyn/cm2) for 6 h, HAEC were preincubated with or without the antioxidants pyrrolidine dithiocarbamate (PDTC) or N-acetyl-L-cysteine (NAC). PDTC strongly inhibited low shear-induced activation of NF-kappaB, expression of VCAM-1, and EC-Mn adhesion. Paradoxically, NAC exerted a positive effect on low shear-induced VCAM-1 expression and EC-Mn adhesion and only slightly downregulated NF-kappaB activation. However, cytokine-induced NF-kappaB activation and VCAM-1 expression are blocked by both PDTC and NAC. These data suggest that NF-kappaB plays a key role in low shear-induced VCAM-1 expression and that pathways mediating low shear- and cytokine-induced EC-Mn adhesion may be differentially regulated.

Differential activation of NF-kappa B in human aortic endothelial cells conditioned to specific flow environments.

Endothelial cell-monocyte interaction plays an important role in atherogenesis. The expressions of some endothelial cell adhesion molecules involved in endothelial cell-monocyte interactions are regulated by transcription factor NF-kappa B. Because low shear stress has been known to influence endothelial monocyte adhesion, the differential activation of NF-kappa B under different flow regimens across time (0.5-24 h) was investigated. Nuclear proteins from flow-conditioned human aortic endothelial cells (HAEC) were analyzed by electrophoretic mobility shift assay using [gamma-32P]dATP-labeled NF-kappa B-specific oligonucleotide. Our results demonstrated that NF-kappa B activation was significantly elevated in HAEC exposed to prolonged (> 2 h) steady low shear (2 dyn/cm2) and pulsatile low shear (2 +/- 2 dyn/cm2) compared with HAEC exposed to high shear (16 dyn/cm2). In contrast, at 30 min, high shear-exposed HAEC exhibited an early, transient increase in NF-kappa B activity, relative to low shear-exposed cells, which reversed on continued exposure to high shear. Maximum activity in both low shear- and pulsatile low shear-conditioned HAEC was observed at 16 h compared with HAEC exposed to prolonged high shear. These results indicate that exposure of HAEC to prolonged low shear conditions is associated with significantly increased and prolonged NF-kappa B activity. This observation might provide a mechanism to explain the increased monocyte adhesion in atherosclerosisprone arterial sites exposed to chronic low-shear flow patterns.

Human aortic endothelial cell migration onto stent surfaces under static and flow conditions.

PURPOSE: The objective of the present study is to establish an in vitro model designed to quantitatively define human aortic endothelial cell (HAEC) migration onto stainless steel stent material under both static and flow conditions of high and low wall shear stress. MATERIALS AND METHODS: To simulate implantation of a stent onto the intact arterial wall, HAECs were seeded and grown to confluence on thick, firm collagen gels. Flat 1 x 1-cm square, stainless steel pieces were implanted on this endothelialized surface and migration of HAECs onto the steel surface was monitored, measured, and compared under static and high (15 dynes/cm2) and low (2 dynes/cm2) wall shear stress flow conditions designed to model wall shear stress levels encountered at different sites within the human arterial system. RESULTS: Under no flow, endothelial cell migration occurred uniformly from the periphery, attaining complete confluence over the square surface within 14 days. The initial migratory rate was approximately 10 micrograms/h +/- 0.5 days 1-3 and increased to a rate near 15 micrograms/h +/- 0.5 between days 10 and 14. High shear stress significantly (P < .002) increased HAEC migration rate to 25 micrograms/h +/- 0.8 in the direction of flow, resulting in an increase in total area endothelial coverage from 59% under no flow to 87% under high shear stress flow conditions when compared at 7 days. CONCLUSIONS: These results indicate the rate and extent of endothelial migration onto a prosthetic material surface are influenced by the level of direction of flow-related wall shear stress. Furthermore, these results demonstrate an in vitro model that provides a method to quantitatively evaluate and possibly predict the relative ability of different prosthetic materials to endothelialize under variable in vivo flow conditions.

Inhibition of scavenger receptor-mediated modified low-density lipoprotein endocytosis in cultured bovine aortic endothelial cells by the glycoprotein processing inhibitor castanospermine.

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizidine) is a plant alkaloid that inhibits alpha-glucosidases, including the glycoprotein processing glucosidase I. When endothelial cells were grown for 48 h, or longer, in the presence of this alkaloid, they produced scavenger receptors for modified low-density lipoproteins (LDL) that had mostly Glc3Man7-9(GlcNAc)2 structures rather than the usual complex types of oligosaccharides. Furthermore, growth in the presence of castanospermine resulted in a substantial inhibition in degradation of endocytosed 125I-acetylated LDL, as well as a dose-dependent inhibition of 125I-acetylated LDL binding to these cells. Scatchard analysis of binding curves indicated that the diminished binding was due to a decrease in the number of scavenger receptor molecules at the cell surface rather than to a change in the affinity of the receptors for their ligand. Since castanospermine-treated cells had the same total number of cellular receptor molecules as did controls cells, it seemed likely that castanospermine caused an alteration in receptor targeting, rather than an inhibition in receptor synthesis or a stimulation in receptor degradation. Density gradient fractionation of cell homogenates showed that castanospermine-treated cells did have a much greater percentage of scavenger LDL receptor molecules in the endoplasmic reticulum-Golgi fraction and fewer receptors in the plasma membrane fraction, whereas normal cells showed the opposite distribution.

Flow-related responses of intracellular inositol phosphate levels in cultured aortic endothelial cells.

In vitro and in vivo evidence indicates that hemodynamic wall shear stress evokes a diversity of biological responses in vascular endothelial cells, ranging from cell shape changes to alterations in low density lipoprotein receptor expression. The signal transduction mechanisms by which the level of fluid mechanical shear stress is recognized by the endothelial cell and translated into these diverse biological responses remain to be elucidated. The present study focuses on the association between the onset of elevated shear stress and activation of the phosphoinositide signal transduction pathway, as measured by the intracellular release of inositol phosphates, in cultured bovine aortic endothelial cells (BAECs). BAECs were seeded, grown to confluence on large polyester sheets, and preincubated with 0.3 microCi/ml [3H]inositol for 24 hours before insertion in parallel-plate flow chambers for exposure to high shear stress (HS) at 30 dynes/cm2 or low shear stress (LS) at < 0.5 dyne/cm2 for periods ranging from 15 seconds to 24 hours. The induction of HS was associated with an early, transient but significant increase (142%, HS/LS x 100%) in inositol trisphosphate (IP3) measured at 15 seconds of shear stress exposure followed by a major peak in IP3 (189%) observed at 5 minutes after HS onset. After these initial increases, IP3 levels returned to near resting levels within 30 minutes of continued HS exposure and then continued to decline to significantly lower (75%) levels relative to LS-treated cells within 4 hours and remained lower throughout the remainder of the 24-hour HS exposure. LS-treated cells exhibited no significant changes in inositol phosphate levels throughout the 24-hour exposure periods. Exposure of BAECs to shear stress of 60 dynes/cm2 resulted in an approximately fourfold increase in IP3 levels (396%) measured at 5 minutes, almost double the levels measured in cells exposed to 30 dynes/cm2 for 5 minutes. Pretreatment of BAECs for 30 minutes with 5 mM neomycin, an inhibitor of phosphoinositide metabolism, before HS exposure inhibited both the early increases in inositol phosphates and subsequent cell elongation and alignment observed in untreated BAECs simultaneously exposed to HS without inhibiting protein synthesis. These results indicate that the exposure of cultured BAECs to elevated wall shear stress is associated with an early biphasic IP3 increase followed by a resetting of intracellular inositol phosphate concentrations to levels below that observed in static cultured BAECs. Furthermore, neomycin inhibition of this IP3 response to shear stress is associated with an inhibition of one of the major endothelial biological responses to shear stress, i.e., cell shape change and orientation.(ABSTRACT TRUNCATED AT 400 WORDS)

A modern view of atherogenesis.

Two key events in the atherogenic cascade are the focal influx and accumulation of low-density lipoprotein (LDL) cholesterol at arterial sites having a predilection for atherosclerotic lesion development and the recruitment of blood monocytes to these lesion-prone sites. Both processes are enhanced in the setting of hyperlipidemia and dyslipoproteinemia. The monocytes recruited to the endothelial surface subsequently migrate to the subendothelial space under the directed guidance of chemoattractants, such as monocyte chemotactic protein-1 and oxidatively modified LDL. These cells then undergo activation-differentiation to become macrophages. At the same time, LDL, and probably other lipoproteins such as the small dense LDL particles and lipoprotein (a), traverse the endothelium and undergo oxidative modification by reactive oxygen species. These oxidatively modified lipoproteins are recognizable by the non-down-regulating macrophage scavenger receptor. Their uptake by these receptors results in the formation of the foam cell characteristic of early-stage atherosclerosis. As monocyte recruitment and lipoprotein influx continue, the lesion grows and develops into the fatty streak. Subsequent foam cell necrosis due to the influence of cytotoxic oxidatively modified LDL and increased collagen synthesis by intimal smooth muscle cells lead to the established atherosclerotic lesion referred to as the fibrous plaque. As our understanding of the mechanisms involved in the pathogenesis of atherosclerosis has evolved over the past few years, novel strategies for intervention in the atherogenic process have emerged.

Atherosclerosis. Potential targets for stabilization and regression.

Reviewed are various aspects of atherosclerotic plaque stabilization and regression in humans and experimental animals. Plaque regression is a function of the dynamic balance among initiation, progression, stabilization, and removal of plaque constituents. Pseudoregression, the result of the triad thrombolysis, age- or lesion-dependent arterial dilatation, and relaxation of vasospasm, may readily give rise to angiographic misinterpretation. Although lowering of plasma cholesterol and low density lipoprotein-cholesterol has demonstrated significant clinical benefits in a number of clinical trials, the magnitude of angiographic regressive changes is relatively small despite aggressive lipid-lowering regimens. The emerging need for alternative or complementary therapeutic interventions has been emphasized. In particular, they should be targeted to pivotal cellular or molecular mechanisms in initiation, progression, or stabilization. Potentially important therapeutic targets include the use of antioxidants or free radical scavengers such as Probucol or its analogues, butylated hydroxytoluene, tocopherols, and possibly the tocotrienols. Other therapeutic targets include intimal monocyte-macrophage recruitment, macrophage cholesterol acyltransferase inhibition, stimulation of the high density lipoprotein-mediated reverse cholesterol transport system, smooth muscle cell migration to and proliferation in the arterial intima, and intimal connective tissue synthesis. Whether the isoprenylated proteins associated with the cholesterol biosynthetic pathway will give rise to compounds regulating smooth muscle cell growth has yet to be determined. Because of the importance of thrombosis in the pathogenesis and progression of lesions, the need to develop interventional strategies targeted at endothelial cell thromboresistance and thromboregulation must assume a high priority in future research and development. Other areas of therapeutic promise include the calcium channel blockers and angiotensin converting enzyme inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms in intimal monocyte-macrophage recruitment. A special role for monocyte chemotactic protein-1.

This article briefly addresses the role of the monocyte-macrophage in atherogenesis and the potential mechanisms participating in intimal blood monocyte recruitment. The obligatory components of the recruitment process, i.e., contact with the vascular endothelium, attachment, and migration into the subendothelial space, are addressed with reference to the influence of blood flow, plasma proteins (particularly low density lipoprotein), and inflammatory mediators and cytokines. The potentially important role of monocyte chemotactic protein-1 in regulating monocyte recruitment in plaque pathogenesis is discussed.

Pathogenesis of the atherosclerotic lesion. Implications for diabetes mellitus.

In this review, we have highlighted pivotal cellular and molecular events in the initiation and progression of atherosclerosis. Key components of lesion initiation are an enhanced focal intimal influx and accumulation of lipoproteins, including LDL in hemodynamically determined lesion-prone areas, focal monocyte-macrophage recruitment, intimal generation of ROS, and oxidative modification of lipoproteins (including LDL [Ox-LDL]). Modified lipoproteins are taken up by the non-downregulating macrophage scavenger receptor, with foam cell formation and the development of the so-called fatty streak. One transitional event in lesion progression is foam cell necrosis, likely attributable to the cytotoxicity of both intimal free radicals and Ox-LDL, with development of an extracellular metabolically inert lipid core. Another is the migration to and proliferation within the intima of medial SMCs, leading to the synthesis of plaque collagens, elastin, and proteoglycans. Mural thrombosis plays a significant role in the late-stage progression of lesions. Regression of lesions is considered a function of the dynamic balance among components of initiation, progression, plaque stabilization, and removal of plaque constituents--the so-called regression quartet. Here, we critically examine how components of diabetes mellitus might impact not only lesion development, but also lesion regression. It is concluded that some components of diabetes mellitus augment key mechanisms in lesion initiation and progression and will likely retard the processes of plaque regression. Specifically, we focus on the various influences of diabetes mellitus on lipoprotein influx and accumulation, free radical generation and Ox-LDL, monocyte-macrophage recruitment, thrombosis and impaired fibrinolysis, and the reverse cholesterol transport system. The importance of nonenzymatic protein glycosylation in modifying a number of these processes is emphasized.

Kifunensine inhibits glycoprotein processing and the function of the modified LDL receptor in endothelial cells.

Kifunensine is an alkaloid that is produced by the actinomycete Kitasatosporia kifunense and resembles the cyclic oxamide derivative of 1-aminodeoxymannojirimycin in structure. We previously showed that this compound was a potent inhibitor of the purified glycoprotein processing enzyme, mannosidase I, and caused an almost complete inhibition in the formation of complex types of oligosaccharides with the concurrent accumulation of N-linked oligosaccharides having Man9(GlcNAc)2 structures in influenza virus-infected Madin Darby canine kidney cells. Kifunensine, at concentrations of 1 microgram/ml or higher in the culture medium, caused an almost complete inhibition in the formation of complex types of oligosaccharides by human skin fibroblasts or aortic endothelial cells, with the resulting accumulation of Man9(GlcNAc)2 oligosaccharides on the cell surface N-linked glycoproteins, and more specifically on the scavenger-LDL receptor. When endothelial cells were grown in the presence of 1 microgram/ml of kifunensine, there was a 75% inhibition in the ability of these cells to degrade 125I-labeled acetyl-LDL, but this inhibitor appeared to have little or no effect on the ability of either endothelial cells or fibroblasts to degrade 125I-labeled LDL, even at kifunensine concentrations of 10 micrograms/ml. Kifunensine also decreased the binding of the labeled acetyl-LDL by the scavenger receptor of the endothelial cells, but the amount of this inhibition relative to controls was significantly less than that of the degradation, suggesting that kifunensine affects two different steps of acetyl-LDL metabolism in these cells. Endothelial cells grown in the presence of 10 micrograms/ml of kifunensine had only half the activity of the lysosomal enzymes, beta-hexosaminidase, and proteases, as did control cells, although kifunensine did not affect [3H]leucine incorporation into protein. Thus, kifunensine apparently affects the activity of (some) lysosomal enzymes in an as yet undefined manner.