Altered response of vascular smooth muscle cells to exogenous biochemical stimulation in two- and three-dimensional culture

Removal of vascular smooth muscle cells (SMC) from their native environment alters the biochemical and mechanical signals responsible for maintaining normal cell function, causing a shift from a quiescent, contractile phenotype to a more proliferative, synthetic state. We examined the effect on SMC function of culture on two-dimensional (2D) substrates and in three-dimensional (3D) collagen Type I gels, including the effect of exogenous biochemical stimulation on gel compaction, cell proliferation, and expression of the contractile protein smooth muscle alpha-actin (SMA) in these systems. Embedding of SMC in 3D collagen matrices caused a marked decrease in both cell proliferation and expression of SMA. The presence of the extracellular matrix modulated cellular responses to platelet-derived growth factor BB, heparin, transforming growth factor-beta1, and endothelial cell-conditioned medium. Cell proliferation and SMA expression were shown to be inversely related, while gel compaction and SMA expression were not correlated. Taken together, these results show that SMC phenotype and function can be modulated using biochemical stimulation in vitro, but that the effects produced are dependent on the nature of the extracellular matrix. These findings have implications for the study of vascular biology in vitro, as well as for the development of engineered vascular tissues.     

Smooth muscle cell-driven vascular diseases and molecular mechanisms of VSMC plasticity

Vascular smooth muscle cells (VSMCs) are the major cell type in blood vessels. Unlike many other mature cell types in the adult body, VSMC do not terminally differentiate but retain a remarkable plasticity. Fully differentiated medial VSMCs of mature vessels maintain quiescence and express a range of genes and proteins important for contraction/dilation, which allows them to control systemic and local pressure through the regulation of vascular tone. In response to vascular injury or alterations in local environmental cues, differentiated/contractile VSMCs are capable of switching to a dedifferentiated phenotype characterized by increased proliferation, migration and extracellular matrix synthesis in concert with decreased expression of contractile markers. Imbalanced VSMC plasticity results in maladaptive phenotype alterations that ultimately lead to progression of a variety of VSMC-driven vascular diseases. The nature, extent and consequences of dysregulated VSMC phenotype alterations are diverse, reflecting the numerous environmental cues (e.g. biochemical factors, extracellular matrix components, physical) that prompt VSMC phenotype switching. In spite of decades of efforts to understand cues and processes that normally control VSMC differentiation and their disruption in VSMC-driven disease states, the crucial molecular mechanisms and signalling pathways that shape the VSMC phenotype programme have still not yet been precisely elucidated. In this article we introduce the physiological functions of vascular smooth muscle/VSMCs, outline VSMC-driven cardiovascular diseases and the concept of VSMC phenotype switching, and review molecular mechanisms that play crucial roles in the regulation of VSMC phenotypic plasticity.     

Invited review: cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression.

cGMP is a second messenger that produces its effects by interacting with intracellular receptor proteins. In smooth muscle cells, one of the major receptors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG). PKG has been shown to catalyze the phosphorylation of a number of physiologically relevant proteins whose function it is to regulate the contractile activity of the smooth muscle cell. These include proteins that regulate free intracellular calcium levels, the cytoskeleton, and the phosphorylation state of the regulatory light chain of smooth muscle myosin. Other studies have shown that vascular smooth muscle cells (VSMCs) that are cultured in vitro may cease to express PKG and will, coincidentally, acquire a noncontractile, synthetic phenotype. The restoration of PKG expression to the synthetic phenotype VSMC results in the cells acquiring a more contractile phenotype. These more recent studies suggest that PKG controls VSMC gene expression that, in turn, regulates phenotypic modulation of the cells. Therefore, the regulation of PKG gene expression appears to be linked to phenotypic modulation of VSMC. Because several vascular disorders are related to the accumulation of synthetic, fibroproliferative VSMC in the vessel wall, it is likely that changes in the activity of the nitric oxide/cGMP/PKG pathway is involved the development of these diseases.     

Death in the third dimension: apoptosis regulation and tissue architecture

Tissue development, homeostasis and tumor pathogenesis all depend upon a complex dialogue between multiple cell types operating within a dynamic three-dimensional (3D) tissue extracellular matrix microenvironment. A major issue is whether the spatial organization of a cell within this 3D tissue microenvironment could modulate cell responsiveness to regulate cell fate decisions such as survival, and if so how. Classic developmental model systems and transgenic animals are instructive but pose special challenges for investigators conducting signaling studies and biochemical assays in tissues. As an alternative, 3D culture model systems exist in which cell-adhesion dependent tissue architecture, heterotypic cell-cell interactions and tissue differentiation can be recapitulated with good fidelity. 3D cell culture models are slowly revealing how tissue architecture can dramatically influence how a cell responds to exogenous stimuli to modify its apoptotic behavior and hence should prove instrumental for identifying novel cell death pathways.     

Transduction of cell and matrix geometric cues by the actin cytoskeleton

Engineered culture substrates have proven invaluable for investigating the role of cell and extracellular matrix geometry in governing cell behavior. While the mechanisms relating geometry to phenotype are complex, it is clear that the actin cytoskeleton plays a key role in integrating geometric inputs and transducing these cues into intracellular signals that drive downstream biology. Here, we review recent progress in elucidating the role of the cell and matrix geometry in regulating actin cytoskeletal architecture and mechanics. We address new developments in traditional two-dimensional culture paradigms and discuss efforts to extend these advances to three-dimensional systems, ranging from nanotextured surfaces to microtopographical systems (e.g. channels) to fully three-dimensional matrices.     

Extracellular matrix presentation modulates vascular smooth muscle cell mechanotransduction

The development of atherosclerosis involves phenotypic changes among vascular smooth muscle cells (VSMCs) that correlate with stiffening and remodeling of the extracellular matrix (ECM). VSMCs are highly sensitive to the composition and mechanical state of the surrounding ECM, and ECM remodeling during atherosclerosis likely contributes to pathology. We hypothesized that ECM mechanics and biochemistry are interdependent in their regulation of VSMC behavior and investigated the effect of ligand presentation on certain stiffness-mediated processes. Our findings demonstrate that substrate stiffening is not a unidirectional stimulus—instead, the influence of mechanics on cell behavior is highly conditioned on ligand biochemistry. This “stiffness-by-ligand” effect was evident for VSMC adhesion, spreading, cytoskeletal polymerization, and focal adhesion assembly, where VSMCs cultured on fibronectin (Fn)-modified substrates showed an augmented response to increasing stiffness, whereas cells on laminin (Ln) substrates showed a dampened response. By contrast, cells on Fn substrates showed a decrease in myosin light chain (MLC) phosphorylation and elongation with increasing stiffness, whereas Ln supported an increase in MLC phosphorylation and no change in cell shape with increasing stiffness. Taken together, these findings show that identical cell populations exhibit opposing responses to substrate stiffening depending on ECM presentation. Our results also suggest that the shift in VSMC phenotype in a developing atherosclerotic lesion is jointly regulated by stromal mechanics and biochemistry. This study highlights the complex influence of the blood vessel wall microenvironment on VSMC phenotype and provides insight into how cells may integrate ECM biochemistry and mechanics during normal and pathological tissue function.     

The role of smooth muscle cells in vessel wall pathophysiology and reconstruction using bioactive synthetic polymers

This review summarizes recent trends in the construction of bioartificial vascular replacements, i.e. hybrid grafts containing synthetic polymeric scaffolds and cells. In these advanced replacements, vascular smooth muscle cells (VSMC) should be considered as a physiological component, although it is known that activation of the migration and proliferation of VSMC plays an important role in the onset and development of vascular diseases, and also in restenosis of currently used vascular grafts. Therefore, in novel bioartificial vascular grafts, VSMCs should be kept in quiescent mature contractile phenotype. This can be achieved by (1) appropriate physical and chemical properties of the material, such as its chemical composition, polarity, wettability, surface roughness and topography, electrical charge and conductivity, functionalization with biomolecules and mechanical properties, (2) appropriate cell culture conditions, such as composition of cell culture media and dynamic load, namely cyclic strain, and (3) the presence of a confluent, mature, semipermeable, non-thrombogenic and non-immunogenic endothelial cell (EC) barrier, covering the luminal surface of the graft and separating the VSMCs from the blood. Both VSMCs and ECs can also be differentiated from stem and progenitor cells of various sources. In the case of degradable scaffolds, the material will gradually be removed by the cells and will be replaced by their own new extracellular matrix. Thus, the material component in advanced blood vessel substitutes acts as a temporary scaffold that promotes regeneration of the damaged vascular tissue.     

Responses of vascular smooth muscle cell to extracellular matrix degradation.

Vessels remodel to compensate for increases in blood flow/pressure. The chronic exposure of blood vessels to increased flow and circulatory redox-homocysteine may injure vascular endothelium and disrupt elastic laminae. In order to understand the role of extracellular matrix (ECM) degradation in vascular structure and function, we isolated human vascular smooth muscle cells (VSMC) from normal and injured coronary arteries. The apparently normal vessels were isolated from explanted human hearts. The vessels were injured by inserting a blade into the lumen of the vessel, which damages the inner elastic laminae in the vessel wall and polarizes the VSMC by producing a pseudopodial phenotypic shift in VSMC. This shift is characteristic of migratory, invasive, and contractile nature of VSMC. We measured extracellular matrix metalloproteinases (MMPs), tissue plasminogen activator (tPA), tissue inhibitor of metalloproteinase (TIMP), and collagen I expression in VSMC by specific substrate zymography and Northern blot analyses. The injured and elastin peptide, val-gly-val-ala-pro-gly, treated VSMC synthesized active MMPs and reduced expression of TIMP. The level of tPA and collagen type I was induced in the injured, invasive VSMC and in the val-gly-val-ala-pro-gly treated cells. To demonstrate the angiogenic role of elastin peptide to VSMC we performed in vitro organ culture with rings from normal coronary artery. After 3 days in culture the vascular rings in the collagen gel containing elastin peptide elaborated MMP activity and sprouted and grew. The results suggest that val-gly-val-ala-pro-gly peptide generated at the site of proteolysis during vascular injury may have angiogenic activity.     

Living in three dimensions

Research focused on deciphering the biochemical mechanisms that regulate cell proliferation and function has largely depended on the use of tissue culture methods in which cells are grown on two-dimensional (2D) plastic or glass surfaces. However, the flat surface of the tissue culture plate represents a poor topological approximation of the more complex three-dimensional (3D) architecture of the extracellular matrix (ECM) and the basement membrane (BM), a structurally compact form of the ECM. Recent work has provided strong evidence that the highly porous nanotopography that results from the 3D associations of ECM and BM nanofibrils is essential for the reproduction of physiological patterns of cell adherence, cytoskeletal organization, migration, signal transduction, morphogenesis, and differentiation in cell culture. In vitro approximations of these nanostructured surfaces are therefore desirable for more physiologically mimetic model systems to study both normal and abnormal functions of cells, tissues, and organs. In addition, the development of 3D culture environments is imperative to achieve more accurate cell-based assays of drug sensitivity, high-throughput drug discovery assays, and in vivo and ex vivo growth of tissues for applications in regenerative medicine.     

Microfluidic systems: A new toolbox for pluripotent stem cells

Conventional culture systems are often limited in their ability to regulate the growth and differentiation of pluripotent stem cells. Microfluidic systems can overcome some of these limitations by providing defined growth conditions with user-controlled spatiotemporal cues. Microfluidic systems allow researchers to modulate pluripotent stem cell renewal and differentiation through biochemical and mechanical stimulation, as well as through microscale patterning and organization of cells and extracellular materials. Essentially, microfluidic tools are reducing the gap between in vitro cell culture environments and the complex and dynamic features of the in vivo stem cell niche. These microfluidic culture systems can also be integrated with microanalytical tools to assess the health and molecular status of pluripotent stem cells. The ability to control biochemical and mechanical input to cells, as well as rapidly and efficiently analyze the biological output from cells, will further our understanding of stem cells and help translate them into clinical use. This review provides a comprehensive insignt into the implications of microfluidics on pluripotent stem cell research.     

3-D tissue culture systems for the evaluation and optimization of nanoparticle-based drug carriers

Nanoparticle carriers are attractive vehicles for a variety of drug delivery applications. In order to evaluate nanoparticle formulations for biological efficacy, monolayer cell cultures are typically used as in vitro testing platforms. However, these studies sometimes poorly predict the efficacy of the drug in vivo. The poor in vitro and in vivo correlation may be attributed in part to the inability of two-dimensional cultures to reproduce extracellular barriers, and may also be due to differences in cell phenotype between cells cultured as monolayers and cells in native tissue. In order to more accurately predict in vivo results, it is desirable to test nanoparticle therapeutics in cells cultured in three-dimensional (3-D) models that mimic in vivo conditions. In this review, we discuss some 3-D culture systems that have been used to assess nanoparticle delivery and highlight several implications for nanoparticle design garnered from studies using these systems. While our focus will be on nanoparticle drug formulations, many of the systems discussed here could, or have been, used for the assessment of small molecule or peptide/protein drugs. We also offer some examples of advancements in 3-D culture that could provide even more highly predictive data for designing nanoparticle therapeutics for in vivo applications.     

Modelling tissues in 3D: the next future of pharmaco-toxicology and food research?

The development and validation of reliable in vitro methods alternative to conventional in vivo studies in experimental animals is a well-recognised priority in the fields of pharmaco-toxicology and food research. Conventional studies based on two-dimensional (2-D) cell monolayers have demonstrated their significant limitations: the chemically and spatially defined three-dimensional (3-D) network of extracellular matrix components, cell-to-cell and cell-to-matrix interactions that governs differentiation, proliferation and function of cells in vivo is, in fact, lost under the simplified 2-D condition. Being able to reproduce specific tissue-like structures and to mimic functions and responses of real tissues in a way that is more physiologically relevant than what can be achieved through traditional 2-D cell monolayers, 3-D cell culture represents a potential bridge to cover the gap between animal models and human studies. This article addresses the significance and the potential of 3-D in vitro systems to improve the predictive value of cell-based assays for safety and risk assessment studies and for new drugs development and testing. The crucial role of tissue engineering and of the new microscale technologies for improving and optimising these models, as well as the necessity of developing new protocols and analytical methods for their full exploitation, will be also discussed.     

Use of three-dimensional collagen gels to study mechanotransduction in t47d breast epithelial cells

Several pathological and disease conditions can alter the mechanical properties of the extracellular matrix (ECM). Conversely, some diseases may arise from changes in the density or rigidity of the ECM. This necessitates the use and development of in vitro models to understand how both biophysical and biochemical signals regulate complex cellular behaviors. T47D breast epithelial cells will differentiate into duct-like tubules when cultured in a floating three-dimensional (3D) collagen gel, but not a 3D collagen gel that is left attached to the culture dish. This paper details several protocols we have developed for analyzing breast cell biology in 3D matrices, including culturing cells in 3D collagen gels, immunostaining cellular structures, and performing biochemical procedures directly from cells embedded in collagen gels.     

CCN4 regulates vascular smooth muscle cell migration and proliferation

The migration and proliferation of vascular smooth muscle cells (VSMCs) are essential elements during the development of atherosclerosis and restenosis. An increasing number of studies have reported that extracellular matrix (ECM) proteins, including the CCN protein family, play a significant role in VSMC migration and proliferation. CCN4 is a member of the CCN protein family, which controls cell development and survival in multiple systems of the body. Here, we sought to determine whether CCN4 is involved in VSMC migration and proliferation. We examined the effect of CCN4 using rat cultured VSMCs. In cultured VSMCs, CCN4 stimulated the adhesion and migration of VSMCs in a dose-dependent manner, and this effect was blocked by an antibody for integrin α5β1. CCN4 expression was enhanced by the pro-inflammatory cytokine tumor necrosis factor α (TNF-α). Furthermore, knockdown of CCN4 by siRNA significantly inhibited the VSMC proliferation. CCN4 also could up-regulate the expression level of marker proteins of the VSMCs phenotype. Taken together, these results suggest that CCN4 is involved in the migration and proliferation of VSMCs. Inhibition of CCN4 may provide a promising strategy for the prevention of restenosis after vascular interventions.     

Mimicking dynamic in vivo environments with stimuli-responsive materials for cell culture

Traditional synthetic substrates and matrices for cell culture have proven to be of only limited utility in efforts to understand and control cell behavior, in large part because they fail to capture the multifarious biochemical, mechanical, geometric and dynamic characteristics of in vivo environments. However, recent advances in materials chemistry and engineering have begun to provide researchers with a toolbox to mimic the complex characteristics of natural extracellular matrices (ECMs), providing new pathways to explore cell-matrix interactions and direct cell fate under physiologically realistic conditions. In this review, we describe recent developments in stimuli-responsive materials as dynamic substrates and matrices for cell culture, and highlight their use in furthering our understanding of how cells respond to temporal variations in their environment.     

High-resolution deep imaging of live cellular spheroids with light-sheet-based fluorescence microscopy

Conventional two-dimensional cell monolayers do not provide the geometrical, biochemical and mechanical cues found in real tissues. Cells in real tissues interact through chemical and mechanical stimuli with adjacent cells and via the extracellular matrix. Such a highly interconnected communication network extends along all three dimensions. This architecture is lost in two-dimensional cultures. Therefore, at least in many cases, two-dimensional cell monolayers do not represent a suitable in vitro tool to characterize accurately the biology of real tissues. Many studies performed over the last few years have demonstrated that the differences between three-dimensional and two-dimensional cultured cells are striking at the morphological and molecular levels and that three-dimensional cell cultures can be employed in order to shrink the gap between real tissues and in vitro cell models. End-point and long-term imaging of cellular and sub-cellular processes with fluorescence microscopy provides direct insight into the physiological behavior of three-dimensional cell cultures and their response to chemical or mechanical stimulation. Fluorescence imaging of three-dimensional cell cultures sets new challenges and imposes specific requirements concerning the choice of a suitable microscopy technique. Deep penetration into the specimen, high imaging speed and ultra-low intensity of the excitation light are key requirements. Light-sheet-based fluorescence microscopy (LSFM) offers a favorable combination of these requirements and is therefore currently established as the technique of choice for the study of three-dimensional cell cultures. This review illustrates the benefits of cellular spheroids in the life sciences and suggests that LSFM is essential for investigations of cellular and sub-cellular dynamic processes in three-dimensions over time and space.     

Identification of Serhl, a new member of the serine hydrolase family induced by passive stretch of skeletal muscle in vivo

In response to extended periods of stretch, skeletal muscle typically exhibits cell hypertrophy associated with sustained increases in mRNA and protein synthesis. Several soluble hypertrophic agonists have been identified, yet relatively little is known as to how mechanical load is converted into intracellular signals regulating gene expression or how increased cell size is maintained. In skeletal muscle, hypertrophy is generally regarded as a beneficial adaptive response to increased workload. In some cases, however, hypertrophy can be detrimental as seen in long-term cardiac hypertrophy. Skeletal muscle wasting (atrophy) is a feature of both inherited and acquired muscle disease and normal aging. Elucidating the molecular regulation of cell size is a fundamental step toward comprehending the complex molecular systems underlying muscle hypertrophy and atrophy. Subtractive hybridization between passively stretched and control murine skeletal muscle tissue identified an mRNA that undergoes increased expression in response to passive stretch. Encoded within the mRNA is an open reading frame of 311 amino acids containing a highly conserved type 1 peroxisomal targeting signal and a serine lipase active center. The sequence shows identity to a family of serine hydrolases and thus is named serine hydrolase-like (Serhl). The predicted three-dimensional structure displays a core alpha/beta-hydrolase fold and catalytic triad characteristic of several hydrolytic enzymes. Endogenous Serhl protein immunolocalizes to perinuclear vesicles as does Serhl-FLAG fusion protein transiently expressed in muscle cells in vitro. Overexpression of Serhl-FLAG has no effect on muscle cell phenotype in vitro. Serhl's expression patterns and its response to passive stretch suggest that it may play a role in normal peroxisome function and skeletal muscle growth in response to mechanical stimuli.     

Microarray analysis of shear stressed endothelial cells

The cDNA microarray is an extremely beneficial tool for study of differential gene expression in the cardiovascular system. This technique is used in many different applications including drug discovery, environmental science, and the effects of mechanical forces on vascular cell phenotype. The paper reviews work by others, and describes our study on effects of shear stress on vascular endothelial cells. These microarray studies verified earlier findings using Northern and polymerase chain reaction (PCR) analyses in this area; and also found previously unidentified differentially expressed genes, leading to new hypotheses regarding how cells and tissues respond to biochemical and mechanical stimuli.     

Tetraspanin CD9 Regulates Cell Contraction and Actin Arrangement via RhoA in Human Vascular Smooth Muscle Cells

The most prevalent cardiovascular diseases arise from alterations in vascular smooth muscle cell (VSMC) morphology and function. Tetraspanin CD9 has been previously implicated in regulating vascular pathologies; however, insight into how CD9 may regulate adverse VSMC phenotypes has not been provided. We utilized a human model of aortic smooth muscle cells to understand the consequences of CD9 deficiency on VSMC phenotypes. Upon knocking down CD9, the cells developed an abnormally small and rounded morphology. We determined that this morphological change was due to a lack of typical parallel actin arrangement. We also found similar total RhoA but decreased GTP-bound (active) RhoA levels in CD9 deficient cells. As a result, cells lacking a full complement of CD9 were less contractile than their control treated counterparts. Upon restoration of RhoA activity in the CD9 deficient cells, the phenotype was reversed and cell contraction was restored. Conversely, inhibition of RhoA activity in the control cells mimicked the CD9-deficient cell phenotype. Thus, alteration in CD9 expression was sufficient to profoundly disrupt cellular actin arrangement and endogenous cell contraction by interfering with RhoA signaling. This study provides insight into how CD9 may regulate previously described vascular smooth muscle cell pathophysiology.