Hemodynamics in the mouse aortic arch as assessed by MRI, ultrasound, and numerical modeling

Mice are widely used to study arterial disease in humans, and the pathogenesis of arterial diseases is known to be strongly influenced by hemodynamic factors. It is, therefore, of interest to characterize the hemodynamic environment in the mouse arterial tree. Previous measurements have suggested that many relevant hemodynamic variables are similar between the mouse and the human. Here we use a combination of Doppler ultrasound and MRI measurements, coupled with numerical modeling techniques, to characterize the hemodynamic environment in the mouse aortic arch at high spatial resolution. We find that the hemodynamically induced stresses on arterial endothelial cells are much larger in magnitude and more spatially uniform in the mouse than in the human, an effect that can be explained by fluid mechanical scaling principles. This surprising finding seems to be at variance with currently accepted models of the role of hemodynamics in atherogenesis and the known distribution of atheromatous lesions in mice.     

Smooth muscle and endothelial cell function in the pathogenesis of atherosclerosis.

Although clinical studies have been very useful in identifying factors that accelerate the development of atherosclerotic vascular disease, the pathogenesis of the vascular lesions remains unknown. Studies carried out in the last 10 years have shown that smooth muscle and endothelial cells of the vascular wall play a very important role in atherogenesis. It has become apparent that these cells are very active metabolically during the initiation and subsequent growth of the plaques, and that the materials that these cells produce and secrete are important in the composition and growth of the plaques. In addition, there are important interactions at the vessel wall-blood interface that involve endothelial cells, hemodynamic forces and many constituents of the blood, including platelets, lipoproteins, coagulation factors, fibrinolytic agents and leukocytes. In this article the numerous functions of both smooth muscle and endothelial cells are discussed and the effects of known atherogenic agents on these cellular functions are reviewed. Emphasis is placed on the important interactions that take place both within the vessel wall and at the vessel wall-blood interface. Understanding of the regulation of smooth muscle and endothelial cell function during the development and subsequent growth of fibrofatty plaques may be useful in designing appropriate therapeutic interventions to control atherosclerotic disease.     

Exogenous and endogenous force regulation of endothelial cell behavior

Endothelial cells live in a dynamic environment where they are constantly exposed to external hemodynamic forces and generate cytoskeletal-based endogenous forces. These exogenous and endogenous forces are critical regulators of endothelial cell health and blood vessel maintenance at all generations of the vascular system, from large arteries to capillary beds. The first part of this review highlights the role of the primary exogenous hemodynamic forces of shear, cyclic strain, and pressure forces in mediating endothelial cell response. We then discuss the emergent role of the mechanical properties of the extracellular matrix and of cellular endogenous force generation on endothelial cell function, implicating substrate stiffness and cellular traction stresses as important mediators of endothelial cell health. The intersection of exogenous and endogenous forces on endothelial cell function is discussed, suggesting some of the many remaining questions in the field of endothelial mechanobiology.     

Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress stimulates an inflammatory response

Atherosclerosis is now viewed as an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions, including oscillatory shear stress (OS), in branched arteries. In contrast, the arterial regions exposed to laminar shear (LS) are relatively lesion-free. The mechanisms underlying the opposite effects of OS and LS on the inflammatory and atherogenic processes are not clearly understood. Here, through DNA microarrays, protein expression, and functional studies, we identify bone morphogenic protein 4 (BMP4) as a mechanosensitive and pro-inflammatory gene product. Exposing endothelial cells to OS increased BMP4 protein expression, whereas LS decreased it. In addition, we found BMP4 expression only in the selective patches of endothelial cells overlying foam cell lesions in human coronary arteries. The same endothelial patches also expressed higher levels of intercellular cell adhesion molecule-1 (ICAM-1) protein compared with those of non-diseased areas. Functionally, we show that OS and BMP4 induced ICAM-1 expression and monocyte adhesion by a NFkappaB-dependent mechanism. We suggest that BMP4 is a mechanosensitive, inflammatory factor playing a critical role in early steps of atherogenesis in the lesion-prone areas.     

Atherogenesis: hemodynamics, vascular geometry, and the endothelium

The evidence for a hemodynamic involvement and possible mechanisms by which hemodynamic-related events could influence the arterial wall, and in particular the vascular endothelium, are reviewed and used to speculate on the role of fluid mechanics in atherogenesis and specifically in lesion localization. The evidence presented suggests that it is vascular geometry, and the way it influences the local detailed flow properties, which is the primary determinant of a hemodynamic effect on the arterial wall and in the initiation of atherosclerosis.     

Platelets: Pleiotropic roles in atherogenesis and atherothrombosis

Platelets are small, anucleate blood elements of critical importance in cardiovascular disease. The ability of platelets to activate and aggregate to form blood clots in response to endothelial injury, such as plaque rupture, is well established. These cells are therefore important contributors to ischaemia in atherothrombosis, and antiplatelet therapy is effective for this reason. However, growing evidence suggests that platelets are also important mediators of inflammation and play a central role in atherogenesis itself. Interactions between activated platelets, leukocytes and endothelial cells trigger autocrine and paracrine activation signals, resulting in leukocyte recruitment at and into the vascular wall. Direct physical interaction may contribute also, through platelet adhesion molecules assisting localization of monocytes to the site of atherogenesis and platelet granule release contributing to the chronic inflammatory milieu which leads to foam cell development and accelerated atherogenesis. Recent studies have shown that antiplatelet therapy in animal models of accelerated atherogenesis can lead to decreased plaque size and improve plaque stability. This review examines the complexity of platelet function and the nature of interactions between activated platelets, leukocytes and endothelial cells. We focus on the growing body of evidence that platelets play a critical role in atherogenesis and contribute to progression of atherosclerosis.     

Oxidation of cholesterol moiety of low density lipoprotein in the presence of human endothelial cells or Cu+2 ions: identification of major products and their effects

Oxidation of lipoproteins is believed to play a key role in atherogenesis. In this study, low density lipoproteins (LDL) was subjected to oxidation in the presence of either human umbilical vein endothelial cells or with Cu+2 ions and the major oxides formed were identified. While cholesterol-alpha-epoxide (C-alpha EP) was the major product of cholesterol peroxidation in the presence of endothelial cells, cholest-3,5-dien-7-one (CD) predominated in the presence of Cu+2 ion. Both steroids were identified by gas chromatography/mass spectrometry. HDL cholesterol was resistant to oxidation. When tested on human skin fibroblasts in culture C-alpha EP (10 micrograms/ml) caused marked stimulation of 14C-oleate incorporation into cholesterol esters, while CD stimulated cholesterol esterification only mildly. These studies show that a) C-alpha EP is the major peroxidation product of LDL cholesterol moiety in the presence of endothelial cells and b) it causes marked stimulation of cholesterol esterification in cells. C-alpha EP may play a key role in increasing cholesterol esterification noted in atherogenesis.     

Matrix-specific p21-activated kinase activation regulates vascular permeability in atherogenesis

Elevated permeability of the endothelium is thought to be crucial in atherogenesis because it allows circulating lipoproteins to access subendothelial monocytes. Both local hemodynamics and cytokines may govern endothelial permeability in atherosclerotic plaque. We recently found that p21-activated kinase (PAK) regulates endothelial permeability. We now report that onset of fluid flow, atherogenic flow profiles, oxidized LDL, and proatherosclerotic cytokines all stimulate PAK phosphorylation and recruitment to cell-cell junctions. Activation of PAK is higher in cells plated on fibronectin (FN) compared to basement membrane proteins in all cases. In vivo, PAK is activated in atherosclerosis-prone regions of arteries and correlates with FN in the subendothelium. Inhibiting PAK in vivo reduces permeability in atherosclerosis-prone regions. Matrix-specific PAK activation therefore mediates elevated vascular permeability in atherogenesis.     

Differentiation of stem/progenitor cells into vascular cells in response to fluid mechanical forces

Vascular functions are regulated not only by chemical mediators, such as hormones, cytokines, and neurotransmitters, but by mechanical hemodynamic forces generated by blood flow and blood pressure. The mechanical force-mediated regulation is based on the ability of vascular cells, including endothelial cells and smooth muscle cells, to recognize fluid mechanical forces, i.e., the shear stress produced by flowing blood and the cyclic strain generated by blood pressure, and to transmit the signals into the cell interior, where they trigger cell responses that involve changes in cell morphology, cell function, and gene expression. Recent studies have revealed that immature cells, such as endothelial progenitor cells (EPCs) and embryonic stem (ES) cells, as well as adult vascular cells, respond to fluid mechanical forces. Shear stress and cyclic strain promote the proliferation and differentiation of EPCs and ES cells into vascular cells and enhance their ability to form new vessels. Even more recently, attempts have been made to apply fluid mechanical forces to EPCs and ES cells cultured on polymer tubes and develop tissue-engineered blood vessel grafts that have a structure and function similar to that of blood vessels in vivo. This review summarizes the current state of knowledge concerning the mechanobiological responses of stem/progenitor cells and its potential applications to tissue engineering.     

Shear stress and cyclic circumferential stretch, but not pressure, alter connexin43 expression in endothelial cells

Hemodynamic forces play a critical role in atherogenesis, as evidenced by the focal pattern of development of atherosclerotic lesions. Whereas disturbed flow in the branches and curved regions of large arteries is proatherogenic, laminar flow in the straight parts of vessels is atheroprotective. In addition, hypertension and age-related changes in arterial stiffness are important risk factors of the disease. Hemodynamic forces induce various changes in the structure and function of vascular endothelium, many of which reflect alterations in gene expression. Endothelial cells are linked by gap junctions, which facilitate the propagation of electrical and chemical signals along the vascular wall. Using an in vitro perfusion system, we investigated the effects of pulsed unidirectional and oscillatory flows in combination with different levels of hydrostatic pressure and circumferential stretch on the expression of Cx43 in endothelial cells. Our results show that shear stress and circumferential stretch, but not pressure, modulate the expression of Cx43. In view of the distribution of this protein along the vascular tree, our findings provide new insights into the role of mechanical forces on gap junctional communication in regions prone to the development of atherosclerosis.     

Shear Stress and Cyclic Circumferential Stretch, But Not Pressure, Alter Connexin43 Expression in Endothelial Cells

Hemodynamic forces play a critical role in atherogenesis, as evidenced by the focal pattern of development of atherosclerotic lesions. Whereas disturbed flow in the branches and curved regions of large arteries is proatherogenic, laminar flow in the straight parts of vessels is atheroprotective. In addition, hypertension and age-related changes in arterial stiffness are important risk factors of the disease. Hemodynamic forces induce various changes in the structure and function of vascular endothelium, many of which reflect alterations in gene expression. Endothelial cells are linked by gap junctions, which facilitate the propagation of electrical and chemical signals along the vascular wall. Using an in vitro perfusion system, we investigated the effects of pulsed unidirectional and oscillatory flows in combination with different levels of hydrostatic pressure and circumferential stretch on the expression of Cx43 in endothelial cells. Our results show that shear stress and circumferential stretch, but not pressure, modulate the expression of Cx43. In view of the distribution of this protein along the vascular tree, our findings provide new insights into the role of mechanical forces on gap junctional communication in regions prone to the development of atherosclerosis.     

Variations in mass transfer to single endothelial cells

Mass transfer between flowing blood and arterial mural cells (including vascular endothelial cells) may play an important role in atherogenesis. Endothelial cells are known to have an apical surface topography that is not flat, and hence mass transfer patterns to individual endothelial cells are likely affected by the local cellular topography. The purpose of this paper is to investigate the relationship between vascular endothelial cell surface topography and cellular level mass transfer. Confluent porcine endothelial monolayers were cultured under both shear and static conditions and atomic force microscopy was used to measure endothelial cell topography. Using finite element methods and the measured cell topography, flow and concentration fields were calculated for a typical, small, blood-borne solute. A relative Sherwood number was defined as the difference between the computed Sherwood number and that predicted by the Leveque solution for mass transfer over a flat surface: this eliminates the effects of axial location on mass transfer efficiency. The average intracellular relative Sherwood number range was found to be dependent on cell height and not dependent on cell elongation due to shear stress in culture. The mass flux to individual cells reached a maximum at the highest point on the endothelial cell surface, typically corresponding to the nucleus of the cell. Therefore, for small receptor-mediated solutes, increased solute uptake efficiency can be achieved by concentrating receptors near the nucleus. The main conclusion of the work is that although the rate of mass transfer varies greatly over an individual cell, the average mass transfer rate to a cell is close to that predicted for a flat cell. In comparison to other hemodynamic factors, the topography of endothelial cells therefore seems to have little effect on mass transfer rates and is likely physiologically insignificant.     

Mechanisms linking angiotensin II and atherogenesis

PURPOSE OF REVIEW: The concept that angiotensin II plays a central role in early atherogenesis, progression to atherosclerotic plaque, and the most serious clinical sequelae of coronary artery disease is the subject of considerable current interest. Results from recent large clinical trials confirm that blunting of the renin-angiotensin system through either angiotensin converting enzyme inhibition or angiotensin II type 1 receptor blockade incurs significant beneficial outcomes in patients with coronary artery disease. The exact mechanisms for these effects are not yet clear, but are suggested by studies demonstrating that suppression of the renin-angiotensin system is associated with muted vascular oxidative stress. RECENT FINDINGS: As most of the biological effects of the renin-angiotensin system occur through stimulation of the angiotensin II type 1 receptor, the focus of this review is on changes in the vascular wall mediated by this receptor and primarily related to endothelial and vascular smooth muscle cells, monocyte/macrophages and platelets. The interactions between angiotensin II and nitric oxide exert particular demands on the vascular capacity to adapt to dyslipidemia, hypertension, estrogen deficiency and diabetes mellitus that appear to exacerbate atherogenesis. Associated with each of these conditions is angiotensin II-mediated stimulation of macrophages, platelet aggregation, plasminogen activator inhibitor 1, endothelial dysfunction, vascular smooth muscle cell proliferation and migration, apoptosis, leukocyte recruitment, fibrogenesis and thrombosis. SUMMARY: Inhibition of the actions of angiotensin II serves a dual purpose: indirectly through reduction of mechanical stress on the vascular wall, and directly by diminished stimulation for vascular restructuring and remodeling. Collectively, data from studies published over the last year confirm and extend the notion that angiotensin II is a true cytokine prevalent at all stages of atherogenesis.     

Human microvessel endothelial cells: Isolation,culture and characterization

Summary Over recent years, interest in endothelial cell biology has increased dramatically with our ability to grow and study endothelial cellsin vitro. While large veins and arteries remain a quick and convenient source of endothelial cells, the great morphological, biochemical and functional heterogeneity that endothelial cells express has necessitated the development of techniques to isolate microvessel endothelial cells from different tissues to create more realisticin vitro models. The majority of isolation procedures employ selective methods to enrich microvessel endothelial cells from tissue homogenates directly, or after a period in culture. These include sieving/filtration, manual weeding, isopycnic centrifugation, selective growth media, and the use of flow cytometry or magnetic beads coupled with specific endothelial cell markers. The establishment of pure endothelial cell populations is important for studying their biochemistry and physiology and there are many morphological, immunological and biochemical criteria which can be used to characterize human endothelial cells. These range from classical markers such as von Willebrand Factor and angiotensin-converting enzyme to novel markers like platelet endothelial cell adhesion molecule-1 (CD31) and the expression of E-selectin on cytokine-activated endothelial cells.     

Mechanical, biochemical, and extracellular matrix effects on vascular smooth muscle cell phenotype.

The vascular smooth muscle cell (VSMC) is surrounded by a complex extracellular matrix that provides and modulates a variety of biochemical and mechanical cues that guide cell function. Conventional two-dimensional monolayer culture systems recreate only a portion of the cellular environment, and therefore there is increasing interest in developing more physiologically relevant three-dimensional culture systems. This review brings together recent studies on how mechanical, biochemical, and extracellular matrix stimulation can be applied to study VSMC function and how the combination of these factors leads to changes in phenotype. Particular emphasis is placed on in vitro experimental studies in which multiple stimuli are combined, especially in three-dimensional culture systems and in vascular tissue engineering applications. These studies have provided new insight into how VSMC phenotype is controlled, and they have underscored the interdependence of biochemical and mechanical signaling. Future improvements in creating more complex in vitro culture environments will lead to a better understanding of VSMC biology, new treatments for vascular disease, as well as improved blood vessel substitutes.     

A stepwise method for the isolation of endothelial cells and smooth muscle cells from individual canine coronary arteries

Methods for the stepwise isolation of endothelial cells and smooth muscle cells from individual canine coronary arteries are described. Both cell types can be isolated in pure culture with high yields. Dogs are a common species used in the study of atherosclerosis and coronary artery disease. Capacity to isolate endothelial cells and smooth muscle cells from individual canine coronary arteries should prove useful in the study of coronary artery disease.     

Separating genetic and hemodynamic defects in neuropilin 1 knockout embryos

Targeted inactivation of genes involved in murine cardiovascular development frequently leads to abnormalities in blood flow. As blood fluid dynamics play a crucial role in shaping vessel morphology, the presence of flow defects generally prohibits the precise assignment of the role of the mutated gene product in the vasculature. In this study, we show how to distinguish between genetic defects caused by targeted inactivation of the neuropilin 1 (Nrp1) receptor and hemodynamic defects occurring in homozygous knockout embryos. Our analysis of a Nrp1 null allele bred onto a C57BL/6 background shows that vessel remodeling defects occur concomitantly with the onset of blood flow and cause death of homozygous mutants at E10.5. Using mouse embryo culture, we establish that hemodynamic defects are already present at E8.5 and continuous circulation is never established in homozygous mutants. The geometry of yolk sac blood vessels is altered and remodeling into yolk sac arteries and veins does not occur. To separate flow-induced deficiencies from those caused by the Nrp1 mutation, we arrested blood flow in cultured wild-type and mutant embryos and followed their vascular development. We find that loss of Nrp1 function rather than flow induces the altered geometry of the capillary plexus. Endothelial cell migration, but not replication, is altered in Nrp1 mutants. Gene expression analysis of endothelial cells isolated from freshly dissected wild-type and mutants and after culture in no-flow conditions showed down-regulation of the arterial marker genes connexin 40 and ephrin B2 related to the loss of Nrp1 function. This method allows genetic defects caused by loss-of-function of a gene important for cardiovascular development to be isolated even in the presence of hemodynamic defects.     

Activation of Rac1 by shear stress in endothelial cells mediates both cytoskeletal reorganization and effects on gene expression

Hemodynamic shear stress is a fundamental determinant of vascular remodeling and atherogenesis. Changes in focal adhesions, cytoskeletal organization and gene expression are major responses of endothelial cells to shear stress. Here, we show that activation of the small GTPase Rac is essential for gene expression and for providing spatial information for shear stress-induced cell alignment. Fluorescence resonance energy transfer (FRET) localizes activated Rac1 in the direction of flow. This directional Rac1 activation is downstream of shear-induced new integrin binding to extracellular matrix. Additionally, Rac1 mediates flow-induced stimulation of nuclear factor kappaB (NF-kappaB) and the subsequent expression of intercellular cell adhesion molecule 1 (ICAM-1), an adhesion receptor involved in the recruitment of leukocytes to atherosclerotic plaque. These studies provide a unifying model linking three of the main responses to shear stress that mediate both normal adaptation to hemodynamic forces and inflammatory dysfunction of endothelial cells in atherosclerosis.     

Optical measurement of age-related calcification in human blood vessels

Vascular calcification is commonly associated with aging. Quantification of calcium accumulation in vessel walls is important in understanding the mechanisms of vascular calcification. To elucidate age-related change of calcification, site dependence of calcification, and the effect of hemodynamic stress on calcification, we measured calcium contents in various blood vessels with atomic emission spectrometry and simulated blood flow in the vessels by computational fluid dynamics. The content of calcium in the arteries increased progressively with aging while there is no change in the veins. The higher accumulation of calcium occurred in the arteries of the lower limb in comparison to the arteries of the upper limb. In the arterial bifurcation, there was the correlation at hemodynamic stress distribution and calcium content. The results of this study quantitatively support clinical findings of nonuniform calcification, and suggest that hemodynamic stress affects vascular calcification.     

Damage and regeneration of the endothelium of the aorta in experimental hypercholesterolemia

In dynamics of the experimental hypercholesterolemia in rabbits, peculiarities of endothelial regeneration have been studied. Comparison of proliferative activity level in endotheliocytes with structural-functional state of the endothelial monolayer at atherogenesis makes it possible to consider, that the lesion of the endothelium cannot be regarded as an initiating factor for formation of atherosclerotic lesions. Formation of the lesions in the internal lining of the arteries is preceded by certain disorders in permeability of the endothelial barrier at increasing concentration of cholesterin in blood plasma, accompanying with a sharp activation of the cell proliferative activity. When lipid plates and atherosclerotic plaques are already formed, the processes of the endothelial damage and regeneration occur in parallel. The regeneration is ensured with an intensive proliferation and growth of endotheliocytes onto deendotheliolized areas of the damaged intima.