Simvastatin Ameliorates Matrix Stiffness-Mediated Endothelial Monolayer Disruption

Arterial stiffening accompanies both aging and atherosclerosis, and age-related stiffening of the arterial intima increases RhoA activity and cell contractility contributing to increased endothelium permeability. Notably, statins are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors whose pleiotropic effects include disrupting small GTPase activity; therefore, we hypothesized the statin simvastatin could be used to attenuate RhoA activity and inhibit the deleterious effects of increased age-related matrix stiffness on endothelial barrier function. Using polyacrylamide gels with stiffnesses of 2.5, 5, and 10 kPa to mimic the physiological stiffness of young and aged arteries, endothelial cells were grown to confluence and treated with simvastatin. Our data indicate that RhoA and phosphorylated myosin light chain activity increase with matrix stiffness but are attenuated when treated with the statin. Increases in cell contractility, cell-cell junction size, and indirect measurements of intercellular tension that increase with matrix stiffness, and are correlated with matrix stiffness-dependent increases in monolayer permeability, also decrease with statin treatment. Furthermore, we report that simvastatin increases activated Rac1 levels that contribute to endothelial barrier enhancing cytoskeletal reorganization. Simvastatin, which is prescribed clinically due to its ability to lower cholesterol, alters the endothelial cell response to increased matrix stiffness to restore endothelial monolayer barrier function, and therefore, presents a possible therapeutic intervention to prevent atherogenesis initiated by age-related arterial stiffening.     

Indentation measurements of the subendothelial matrix in bovine carotid arteries

Artery biomechanics are an important factor in cardiovascular function and atherosclerosis development; as such, the macro-mechanics of whole arteries are well-characterized. However, much less is known about the mechanical properties of individual layers in the blood vessel wall. Since there is significant evidence to show that cells can sense the mechanical properties of their matrix, it is critical to characterize the mechanical properties of these individual layers at the scale sensed by cells. Here, we measured subendothelium mechanics in bovine carotid arteries using atomic force microscopy (AFM) indentation. To specifically indent the subendothelium, we evaluated three potential de-endothelialization methods: scraping, paper imprinting, and saponin incubation. Using scanning electron microscopy, histology stains, immunohistochemistry, and multiphoton microscopy, we found that scraping was the only effective de-endothelialization method capable of removing endothelial cells and leaving the subendothelial matrix largely intact. To determine the indentation modulus of the subendothelial matrix, both untreated and scraped (de-endothelialized) bovine carotid arteries were indented with a spherical AFM probe and the data were fit using the Hertz model. Both the endothelium on the untreated artery and the en face subendothelium had similar indentation moduli: E=2.5 ± 1.9 and 2.7 ± 1.1 kPa, respectively. These measurements are the first to quantify the micro-scale mechanics of the subendothelial layer, and constitute a critical step in understanding the relationship between altered subendothelial micromechanics and disease progression.     

Proliferation of anchorage-dependent contact-inhibited cells. II: experimental results and validation of the theoretical models

This study establishes that the cellular automata models developed in an earlier article capture the essential features of the proliferation process for anchorage-dependent contact-inhibited cells. Model predictions are in excellent agreement with experimental data obtained with bovine pulmonary artery endothelial cells. The models are particularly suitable for predictive purposes since they have no adjustable parameters. All model parameters can be easily obtained from a priori measurements. Our studies also show that proliferation rates are very sensitive to the spatial distributions of seed cells. The adverse effects of seeding heterogeneities become more pronounced as a cell population approaches confluency and they should be accounted for in experimental studies attempting to assess the response of cells to external stimuli.     

Cell mechanics studied by a reconstituted model tissue

Tissue models reconstituted from cells and extracellular matrix (ECM) simulate natural tissues. Cytoskeletal and matrix proteins govern the force exerted by a tissue and its stiffness. Cells regulate cytoskeletal structure and remodel ECM to produce mechanical changes during tissue development and wound healing. Characterization and control of mechanical properties of reconstituted tissues are essential for tissue engineering applications. We have quantitatively characterized mechanical properties of connective tissue models, fibroblast-populated matrices (FPMs), via uniaxial stretch measurements. FPMs resemble natural tissues in their exponential dependence of stress on strain and linear dependence of stiffness on force at a given strain. Activating cellular contractile forces by calf serum and disrupting F-actin by cytochalasin D yield "active" and "passive" components, which respectively emphasize cellular and matrix mechanical contributions. The strain-dependent stress and elastic modulus of the active component were independent of cell density above a threshold density. The same quantities for the passive component increased with cell number due to compression and reorganization of the matrix by the cells.     

Basal lamina formation by cultured microvascular endothelial cells

The production of a basal lamina by microvascular endothelial cells (MEC) cultured on various substrata was examined. MEC were isolated from human dermis and plated on plastic dishes coated with fibronectin, or cell-free extracellular matrices elaborated by fibroblasts, smooth muscle cells, corneal endothelial cells, or PF HR9 endodermal cells. Examination of cultures by electron microscopy at selected intervals after plating revealed that on most substrates the MEC produced an extracellular matrix at the basal surface that was discontinuous, multilayered, and polymorphous. Immunocytochemical studies demonstrated that the MEC synthesize and deposit both type IV collagen and laminin into the subendothelial matrix. When cultured on matrices produced by the PF HR9 endodermal cells MEC deposit a subendothelial matrix that was present as a uniform sheet which usually exhibited lamina rara- and lamina densa-like regions. The results indicate that under the appropriate conditions, human MEC elaborate a basal lamina-like matrix that is ultrastructurally similar to basal lamina formed in vivo, which suggests that this experimental system may be a useful model for studies of basal lamina formation and metabolism.     

Polar secretion of von Willebrand factor by endothelial cells

Human umbilical vein endothelial cells cultured on a collagen lattice were used to study the polarity of von Willebrand factor (vWF) secretion. Endothelial cells cultured under these conditions allow direct measurements of substances released at both the apical and basolateral surface. The constitutive secretion of vWF was compared to the release of vWF from their storage granules after stimulation (regulated secretion). The basal, constitutive release of vWF occurs into both the apical and subendothelial direction. The rate of accumulation of vWF to the subendothelial direction is about three times higher than the amount of vWF secreted into the lumenal medium per unit of time. However, upon stimulation of confluent endothelial cell monolayers with phorbol myristate acetate, endothelial cells predominantly secrete vWF at the lumenal surface. Under these conditions, vWF does not accumulate in the collagen matrix. Thus, endothelial cells are able to organize themselves into a polarized monolayer, in such a way that vWF secreted by the regulated pathway accumulates at the lumenal site, whereas resting endothelial cells release vWF predominantly at the opposite, basolateral surface.     

Alterations in endothelial cell proteinase and inhibitor polarized secretion following treatment with interleukin-1, phorbol ester,and human melanoma cell conditioned medium

Polarized secretion of matrix metalloproteinases and plasminogen activators by monkey aortic endothelial cells was studied in vitro, using transwell inserts. The endothelial cells constitutively expressed matrix metalloproteinase-2, tissue inhibitors of metalloproteinases 1 and 2, urokinase, and tissue plasminogen activator, all with basal preference. Matrix metalloproteinase-9 activity was induced by phorbol 12-myristate 13-acetate (apical), interleukin-1α (basal), and by conditioned medium from DX3 human melanoma cells (basal). The DX3 melanoma conditioned medium also stimulated basal secretion of matrix metalloproteinase-2, urokinase, tissue plasminogen activator, and tissue inhibitors of metalloproteinases. The rise in proteolytic activity in the basal direction was reflected by increased capacity to degrade subendothelial basement membrane type IV collagen, shown immunohistologically, using monkey kidney tissue sections and basement membrane deposited by endothelial cells into the transwell membrane. Thus, IL-1α and DX3 melanoma conditioned medium can stimulate endothelial cells in vitro to concentrate secretion of proteinases spatially onto the underlying basement membrane. We suggest that the stimulation of endothelial cell proteinase activity by tumor cells may facilitate tumor cell extravasation. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

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.     

Differential adhesion of metastatic rat mammary carcinoma cells to organ-derived microvessel endothelial cells and subendothelial matrix

The in vitro adhesion rates of rat 13762NF mammary adenocarcinoma cell clones of different spontaneous metastatic potentials to cloned microvessel endothelial cell monolayers and their subendothelial extracellular matrix were investigated. In this system, high rates of adhesion of the cloned tumor cell lines to syngeneic target (lung) organ-derived subendothelial matrix correlated with spontaneous metastatic potential, whereas adhesion to the lung microvessel endothelial cell apical surfaces occurred at lower rates and was not highly significantly different among the tumor cell lines. Adhesion rates to bovine aortic large vessel, and human brain and human meningeal microvessel endothelial cell monolayers were, in general, lower than those found with syngeneic lung microvessel endothelial cells, and did not correlate with spontaneous metastatic potential. Growth of endothelial cells in fetal bovine serum or platelet-poor horse serum did not affect the results, suggesting that in this system metastasis-associated organ-adhesive specificity is determined at the level of the subendothelial matrix.     

The synthesis of subendothelial matrix by bovine aortic endothelial cells in culture

Bovine aortic endothelial cells cultured on collagenous or plastic substrata continuously synthesize and deposit a subendothelial matrix, independently of whether the cells are in the logarithmic or the stationary phase of growth. This subendothelial matrix contains fibrillar and amorphous elements comparable with those observed in the subendothelium in vivo. Deposition of subendothelial matrix on a collagen gel substratum both started earlier and progressed at approximately double the rate than that on denatured collagen. The relative composition of the subendothelial matrix was assessed by sequential incubation with trypsin, elastase and collagenase (Jones et al., 1979). The subendothelial matrix deposited on collagen gels by early confluent cultures and late post-confluent cultures differed in their enzyme sensitivity. These age-related changes in the enzyme sensitivity of the subendothelial matrix were characteristic for each cloned cell population examined. Comparable variations in the composition of the subendothelial matrix were not observed when the cells were cultured on plastic or gelatin-coated dishes; the subendothelial matrix deposited on these two substrata contained considerably more trypsin-sensitive material and less elastase and collagenase-sensitive material than the matrix deposited on native collagen gels. Age-related changes in the enzyme sensitivity of the subendothelial matrix deposited on collagen gels was found to be a function of the time elapsed since confluence and it was not related to the time elapsed since plating or to the number of cells present.     

Eigenstrain as a mechanical set-point of cells

Cell contraction regulates how cells sense their mechanical environment. We sought to identify the set-point of cell contraction, also referred to as tensional homeostasis. In this work, bovine aortic endothelial cells (BAECs), cultured on substrates with different stiffness, were characterized using traction force microscopy (TFM). Numerical models were developed to provide insights into the mechanics of cell–substrate interactions. Cell contraction was modeled as eigenstrain which could induce isometric cell contraction without external forces. The predicted traction stresses matched well with TFM measurements. Furthermore, our numerical model provided cell stress and displacement maps for inspecting the fundamental regulating mechanism of cell mechanosensing. We showed that cell spread area, traction force on a substrate, as well as the average stress of a cell were increased in response to a stiffer substrate. However, the cell average strain, which is cell type-specific, was kept at the same level regardless of the substrate stiffness. This indicated that the cell average strain is the tensional homeostasis that each type of cell tries to maintain. Furthermore, cell contraction in terms of eigenstrain was found to be the same for both BAECs and fibroblast cells in different mechanical environments. This implied a potential mechanical set-point across different cell types. Our results suggest that additional measurements of contractility might be useful for monitoring cell mechanosensing as well as dynamic remodeling of the extracellular matrix (ECM). This work could help to advance the understanding of the cell-ECM relationship, leading to better regenerative strategies.     

Is cytoskeletal tension a major determinant of cell deformability in adherent endothelial cells?

We tested the hypothesis that mechanical tension in thecytoskeleton (CSK) is a major determinant of cell deformability. To confirm that tension was present in adherent endothelial cells, weeither cut or detached them from their basal surface by a microneedle. After cutting or detachment, the cells rapidly retracted. This retraction was prevented, however, if the CSK actin lattice was disrupted by cytochalasin D (Cyto D). These results confirmed thatthere was preexisting CSK tension in these cells and that the actinlattice was a primary stress-bearing component of the CSK. Second, todetermine the extent to which that preexisting CSK tension could altercell deformability, we developed a stretchable cell culture membranesystem to impose a rapid mechanical distension (and presumably a rapidincrease in CSK tension) on adherent endothelial cells. Altered celldeformability was quantitated as the shear stiffness measured bymagnetic twisting cytometry. When membrane strain increased 2.5 or 5%,the cell stiffness increased 15 and 30%, respectively. Disruption ofactin lattice with Cyto D abolished this stretch-induced increase instiffness, demonstrating that the increased stiffness depended on theintegrity of the actin CSK. Permeabilizing the cells with saponin andwashing away ATP and Ca²⁺ did notinhibit the stretch-induced stiffening of the cell. These resultssuggest that the stretch-induced stiffening was primarily due to thedirect mechanical changes in the forces distending the CSK but not toATP- or Ca²⁺-dependent processes.Taken together, these results suggest preexisting CSK tension is amajor determinant of cell deformability in adherent endothelial cells.

     

Effect of membrane stiffness and cytoskeletal element density on mechanical stimuli within cells: an analysis of the consequences of ageing in cells

A finite element model of a single cell was created and used to compute the biophysical stimuli generated within a cell under mechanical loading. Major cellular components were incorporated in the model: the membrane, cytoplasm, nucleus, microtubules, actin filaments, intermediate filaments, nuclear lamina and chromatin. The model used multiple sets of tensegrity structures. Viscoelastic properties were assigned to the continuum components. To corroborate the model, a simulation of atomic force microscopy indentation was performed and results showed a force/indentation simulation with the range of experimental results. A parametric analysis of both increasing membrane stiffness (thereby modelling membrane peroxidation with age) and decreasing density of cytoskeletal elements (thereby modelling reduced actin density with age) was performed. Comparing normal and aged cells under indentation predicts that aged cells have a lower membrane area subjected to high strain as compared with young cells, but the difference, surprisingly, is very small and may not be measurable experimentally. Ageing is predicted to have a more significant effect on strain deep in the nucleus. These results show that computation of biophysical stimuli within cells are achievable with single-cell computational models; correspondence between computed and measured force/displacement behaviours provides a high-level validation of the model. Regarding the effect of ageing, the models suggest only small, although possibly physiologically significant, differences in internal biophysical stimuli between normal and aged cells.     

OxLDL and substrate stiffness promote neutrophil transmigration by enhanced endothelial cell contractility and ICAM-1

Elevated levels of oxLDL in the bloodstream and increased vasculature stiffness are both associated with cardiovascular disease in patients. However, it is not known how oxLDL and subendothelial matrix stiffness together regulate an immune response. Here, we used an in vitro model of the vascular endothelium to explore the combined effects of oxLDL and subendothelial matrix stiffening on neutrophil transmigration. We prepared fibronectin-coated polyacrylamide gels of varying stiffness and plated human umbilical vein endothelial cells (ECs) onto the gels. We observed that oxLDL treatment of the endothelium promoted neutrophil transmigration (from <1% to 26% on soft 0.87kPa substrates), with stiffer substrates further promoting transmigration (54% on 5kPa and 41% on 280kPa). OxLDL exposure enhanced intercellular adhesion molecule-1 (ICAM-1) expression on the endothelium, which was likely responsible for the oxLDL-induced transmigration. Importantly, inhibition of MLCK-mediated EC contraction reduced transmigration to ～9% on all substrates and eliminated the effects of subendothelial matrix stiffness. In addition, large holes, thousands of square microns in size, formed in monolayers on stiff substrates following transmigration, indicating that oxLDL treatment and subsequent neutrophil transmigration caused serious damage to the endothelium. Our results reveal that an interplay between ICAM-1 and MLCK-dependent contractile forces mediates neutrophil transmigration through oxLDL-treated endothelium. Thus, microvasculature stiffness, which likely varies depending on tissue location and health, is an important regulator of the transmigration step of the immune response in the presence of oxLDL.     

Transmission of Mechanical Stresses within the Cytoskeleton of Adherent Cells: A Theoretical Analysis Based on a Multi-Component Cell Model

How environmental mechanical forces affect cellular functions is a central problem in cell biology. Theoretical models of cellular biomechanics provide relevant tools for understanding how the contributions of deformable intracellular components and specific adhesion conditions at the cell interface are integrated for determining the overall balance of mechanical forces within the cell. We investigate here the spatial distributions of intracellular stresses when adherent cells are probed by magnetic twisting cytometry. The influence of the cell nucleus stiffness on the simulated nonlinear torque-bead rotation response is analyzed by considering a finite element multi-component cell model in which the cell and its nucleus are considered as different hyperelastic materials. We additionally take into account the mechanical properties of the basal cell cortex, which can be affected by the interaction of the basal cell membrane with the extracellular substrate. In agreement with data obtained on epithelial cells, the simulated behaviour of the cell model relates the hyperelastic response observed at the entire cell scale to the distribution of stresses and strains within the nucleus and the cytoskeleton, up to cell adhesion areas. These results, which indicate how mechanical forces are transmitted at distant points through the cytoskeleton, are compared to recent data imaging the highly localized distribution of intracellular stresses.     

Validation of Reliable Reference Genes for Real-Time PCR in Human Umbilical Vein Endothelial Cells on Substrates with Different Stiffness

Background

The mechanical properties of cellular microenvironments play important roles in regulating cellular functions. Studies of the molecular response of endothelial cells to alterations in substrate stiffness could shed new light on the development of cardiovascular disease. Quantitative real-time PCR is a current technique that is widely used in gene expression assessment, and its accuracy is highly dependent upon the selection of appropriate reference genes for gene expression normalization. This study aimed to evaluate and identify optimal reference genes for use in studies of the response of endothelial cells to alterations in substrate stiffness.

Methodology/Principal Findings

Four algorithms, GeNorm^PLUS, NormFinder, BestKeeper, and the Comparative ΔCt method, were employed to evaluate the expression of nine candidate genes. We observed that the stability of potential reference genes varied significantly in human umbilical vein endothelial cells on substrates with different stiffness. B2M, HPRT-1, and YWHAZ are suitable for normalization in this experimental setting. Meanwhile, we normalized the expression of YAP and CTGF using various reference genes and demonstrated that the relative quantification varied according to the reference genes.

Conclusion/Significance:

Consequently, our data show for the first time that B2M, HPRT-1, and YWHAZ are a set of stably expressed reference genes for accurate gene expression normalization in studies exploring the effect of subendothelial matrix stiffening on endothelial cell function. We furthermore caution against the use of GAPDH and ACTB for gene expression normalization in this experimental setting because of the low expression stability in this study.     

Fibrin-enhanced endothelial cell organization

We examined the synthesis of extracellular matrix macromolecules by human microvascular endothelial cells isolated from the dermis of neonatal (foreskin) and adult (abdominal) skin. Electron microscopy showed that both cell types produced an extracellular matrix that was strictly localized to the subendothelial space. The subendothelial matrices were initially deposited as a single discontinuous layer of filamentous, electron-dense material that progressively became multilayered. Biosynthetic studies indicated that 2-4% of the newly synthesized protein was deposited in the subendothelial matrices by both cell types. Approximately 15-20% of the radiolabeled protein was secreted into the culture medium, and the remainder was confined to the cellular compartment. Biochemical and immunochemical analyses demonstrated the extracellular secretion of type IV collagen, laminin, fibronectin, and thrombospondin by the newborn and adult cells. Whereas type IV collagen was the predominant constituent of the matrix, fibronectin was secreted into the medium, with only small amounts being deposited in the matrix. Thrombospondin was a major constituent of the matrix produced by the newborn foreskin cells but was virtually absent in the matrix elaborated by the adult cells. However, both cell types did release comparable amounts of thrombospondin into their medium. Immunoperoxidase staining for type IV collagen revealed a fibrillar network in the subendothelial matrices produced by both adult and neonatal cells. In contrast, thrombospondin, which was detected only in the matrix of newborn cells, exhibited a spotty and granular staining pattern. The results indicate that the extracellular matrices synthesized by cultured human microvascular endothelial cells isolated from anatomically distinct sites and different stages of development and age are similar in ultrastructure but differ in their macromolecular composition.     

VE-Cadherin-Independent Cancer Cell Incorporation into the Vascular Endothelium Precedes Transmigration

Metastasis is accountable for 90% of cancer deaths. During metastasis, tumor cells break away from the primary tumor, enter the blood and the lymph vessels, and use them as highways to travel to distant sites in the body to form secondary tumors. Cancer cell migration through the endothelium and into the basement membrane represents a critical step in the metastatic cascade, yet it is not well understood. This process is well characterized for immune cells that routinely transmigrate through the endothelium to sites of infection, inflammation, or injury. Previous studies with leukocytes have demonstrated that this step depends heavily on the activation status of the endothelium and subendothelial substrate stiffness. Here, we used a previously established in vitro model of the endothelium and live cell imaging, in order to observe cancer cell transmigration and compare this process to leukocytes. Interestingly, cancer cell transmigration includes an additional step, which we term ‘incorporation’, into the endothelial cell (EC) monolayer. During this phase, cancer cells physically displace ECs, leading to the dislocation of EC VE-cadherin away from EC junctions bordering cancer cells, and spread into the monolayer. In some cases, ECs completely detach from the matrix. Furthermore, cancer cell incorporation occurs independently of the activation status and the subendothelial substrate stiffness for breast cancer and melanoma cells, a notable difference from the process by which leukocytes transmigrate. Meanwhile, pancreatic cancer cell incorporation was dependent on the activation status of the endothelium and changed on very stiff subendothelial substrates. Collectively, our results provide mechanistic insights into tumor cell extravasation and demonstrate that incorporation is one of the earliest steps.     

Characterizing the Mechanical Variations of Human Femoropopliteal Artery During Aging Process

Vascular diseases during aging process are closely correlated to the age-related changes of mechanical stimuli for resident cells. Characterizing the variations of mechanical environments in vessel walls with advancing age is crucial for a better understanding of vascular remodeling and pathological changes. In this study, the mechanical stress, strain, and wall stiffness of the femoropopliteal arteries (FPAs) were compared among four different age groups from adolescent to young, middle-aged, and aged subjects. The material parameters and geometries adopted in the FPA models were obtained from published experimental results. It is found that high mechanical stress appears at different layers in young and old FPA walls respectively. The characteristics of the middle-aged FPA wall suggests that it is the most capable of resisting high blood pressures and maintaining a mechanical homeostasis during the entire life span. It is demonstrated that the variations of stress and strain rather than that of wall stiffness can be used as an indicator to illustrate the profile of FPA aging. Our results could serve as an age-specific mechanical reference for vascular mechanobiological studies, and allow further exploration of cellular dysfunctions in vessel walls during aging process.