SM Journal of Biomedical Engineering

Review Article

Co-culture Methods Used to Model Atherosclerosis In Vitro Using Endothelial, Smooth Muscle and Monocyte Cells

Kinza Islam1, Sara BH Timraz1, Rasha Nasser1, Deborah L Gater2, Yanthe E Pearson2, N Christoforou1 and Jeremy CM Teo1*

Abstract

Atherosclerosis is a progressive, chronic, inflammatory disease that causes the narrowing and hardening of both the medium and large sized arteries by excessive plaque formation within the artery walls. It is expected that by 2020, atherosclerosis will be the leading cause of death globally. The mechanisms of atherosclerosis are complex and multivariate, not all of which are fully understood. The use of in vitro models is vital to mechanistically predict the main triggers of the disease, disease risk level, determine suitable treatments and to ascertain the efficacy of potential treatment options. This review provides a comprehensive summary of the different studies and co-culture methods used to investigate the mechanisms of atherosclerosis in vitro.

Introduction

Atherosclerosis is the gradual constriction of arteries, brought about by plaque formation within the artery walls [1,2]. Atherosclerosis has a long asymptomatic phase, which tends to be further aggravated by other existing conditions, such as diabetes [3] and hypertension [4-6]. Later stages of atherosclerosis are characterized by the formation of unstable plaques, which are vulnerable to rupture and thus initiate the coagulation cascade, leading to thrombus formation and potentially embolism [7,8]. Thrombosis is the local clotting of blood within the circulatory system, while embolism is the lodging of detached intravascular mass–in this case a blood clot–in a vascular vessel [9]. These two events are precursors to more serious cardiovascular diseases and clinical cardiac events such as Myocardial Infarction (MI) and stroke [2]. Atherosclerosis is a prevalent cause of morbidity and mortality in developed countries [10]. However the incidence of this disease is also increasing in developing countries and it is predicted that, by 2020, atherosclerosis will be the leading cause of death globally [11].

The mechanism by which atherosclerosis is brought about is an extremely complex process involving both biochemical and cellular events [12]. The early events of atherosclerosis occur as a result of injury to the endothelial layer of the vascular wall, which can be triggered by elevated blood pressure or cholesterol [13] (Figure 1A and 1B). This in turn activates monocytes and drives their recruitment to the dysfunctional endothelium (Figure 1C). Research is underway to determine the exact triggers that initiate the onset of atherosclerosis [14,15]. It is hypothesized that toxins from tobacco usage, diabetes, elevated blood pressure or dyslipidemia can lead to irritation and damage of the endothelium. Consequently, permeability of Low-Density Lipoproteins (LDLs) into the intima, or sub-endothelial region, increases. Once inside the intima, LDLs undergo oxidation, which triggers Endothelial Cells (EC) to express cell adhesion molecules such as Vascular Cell Adhesion Molecule-1 (VCAM-1) on their membranes, and simultaneously to secrete chemokines. These secreted chemokines act as signaling cues that attract leukocytes which transmigrate across the blood endothelium [16], while adhesion molecules ensure the arrest of monocytes and T-cells (subsets of leukocytes) to the arterial wall.

Through activation of signaling mechanisms, there is an upregulation of cell adhesion proteins on the activated endothelium as well as on the monocytes as a downstream effect. It is well established that adhesion proteins (L-selectin, P-selectin and PECAM-1) bring blood-suspended monocytes to a tether that rolls along the endothelial surface before endothelial adhesion proteins (ICAM-1 and VCAM-1) completely arrest the immune cells [17]. The final stage here is diapedesis of the monocytes, resulting in their accumulation within the endothelium [18,19]. Localized monocyte activation, transendothelial migration and differentiation within the endothelium are influenced by Macrophage Chemotactic Protein-1 (MCP-1) and macrophage Migratory Inhibitory Factor (MIF) [15], a chemokine produced at the site of injury. MCP-1 deficient mice exhibited attenuated atherosclerosis, indicating the key role of monocytes in disease progression [13]. After the monocytes transmigrate across the endothelial layer, they differentiate into macrophages (Figure 1D). Differentiated macrophages begin to engulf lipids through phagocytosis [14,15] (Figure 1E), and break them down into cholesteryl esters, which form foam-like deposits. Afterwards, cholesteryl esters are broken down into free cholesterol and fatty acids. At this stage, macrophages are known as foam cells due to their lipid-rich foamy-like contents [20] (Figure 1F). These processes are further intensified by an inflammatory responses [14,15].

Endothelial homeostasis is regulated by the endothelium itself, as it releases anti-thrombotic factors that hinder platelet adhesion and aggregation countered by pro-thrombotic release of mediators that encourage platelet activation [21]. Platelet activation not only further encourages a thrombotic environment but it leads to the increase accumulation of Platelet Derived Growth Factor (PDGF), and Lysophosphatic Acid (LPA) which stimulates the migration and proliferation of Smooth Muscle Cells (SMC) from the medial (inner) into the intimal (middle) layer of the vessel wall [22,23]. Endothelial, smooth muscle and macrophage cells at sites of disrupted intima and responding to the various inflammatory cues all amass lipids, through passive and active cell membrane translocation, to form foam cells, resulting in the creation of atherosclerotic plaques [24,25] (Figure 1G). The formation of foam cells is exacerbated by oxidative stress which results in further lipid oxidation [26]. The lipid accumulation alongside the increased proliferation of SMC within the intima creates a growing lesion that radially pushes towards the lumen to cause a decrease in the luminal diameter of the artery and also brings about arterial hardening [27] (Figure 1H).

Figure 1: Accepted sequence for the initiation and progression of atherosclerosis. (A and B) Pathophysiological dysfunction of an otherwise healthy endothelium causes a localized increase in blood vessel permeability and (C) initiation of monocyte recruitment cascade. (C and D) After translocation into the adjacent interstitial space monocyte differentiate into macrophages and (E) proceed with uptake of lipids that also leak into the same compartment. (F) Progressive uptake causes the macrophage to be filled and have foam like content. All this time the localized inflammatory response is intensified and recruiting smooth muscle cells from the intima towards the sites of inflammation. The accumulation of lipids, cells and plaque all contribute towards a growing lesion resulting in a narrowing of the lumen.

Urgent Need for Unified In Vitro Models

The in vivo mechanisms of atherosclerosis are complex, multivariate, and not fully understood [12]. It is vital to increase understanding of the mechanistic progression of atherosclerosis so that improved treatment options can be devised. Animal models have been employed to study disease development. These models are also used prior to clinical trials in order to screen and determine the suitability of various drug candidates as potential treatments [28]. However, the way atherosclerosis manifests within various animal models differs, which prevents representative studies from being carried out [28]. There is evidence to suggest that the response to drugs within animal models can even be influenced by the time of animal sacrifice as well as variations between the sites of lesions tested [29]. Biological differences between species can yield spurious results; that a drug appearing to show positive results in the murine model fails once it reaches clinical trials is a recurring scenario. The efficacy of a candidate drug may be highly dependent on the presence or absence of specific proteins in the corresponding test subjects, and this can only be determined in a clinical setting [30-32]. Unsuitable drug candidates that make their way into clinical trials are unnecessary and ultimately harmful to the patient, expensive, and time consuming.

It is thus necessary to create models that accurately reflect the physiology of atherosclerosis. Appropriate, representative testing with detailed in vitro atherosclerosis models can facilitate prediction of the main triggers of the disease, estimate disease risk level, determine suitable treatments and ascertain the efficacy of potential treatment options [28]. The use of cell co-cultures allows the target drug identification process for atherosclerosis to be carried out under a range of physiological conditions so that changes in the lead molecule activity in response to alterations in these conditions can be ascertained. These models are more representative of the in vivo environment, allowing both normal and diseased conditions to be approximated [33].

Aside from using co-culture based models to study the pathogenesis atherosclerosis, such in vitro models are used to evaluate new technologies for treatment by determining the biocompatibility of new stents / bio-engineered implants such as vessel prototypes [34,35]. Additionally, delivery methods for plasmid DNA (pDNA) [36] and nanoparticles [37,38] for specific targeting of vasculature can be assessed. Better insight into susceptibility to atherosclerosis in patients with pre-existing conditions such as obesity [39], diabetes [3,40-43] and HIV [44] has been gained using in vitro models. Drug testing both for existing medications such as statins [45-49], as well as novel, re-purposed and alternative medicines [50-54] is being carried out using in vitro artery models. Furthermore, the atheroprotective effect of dietary active ingredients [55,56] and diets such as the Mediterranean diet [57,58], are also being gauged using in vitro models.

Despite their extensive use, there is no convergence of arterial in vitro models that facilitates multi-institutional comparison of results to facilitate significant progression in atherosclerosis science. From here on, this review focuses on the biological make up and summarizes current multi-culture approaches to mimic the arterial wall on the bench. Cell types, Extracellular Matrix (ECM) components, biomechanical forces, biochemical cues, and geometry of cell culture receptacle are factors that should be considered for recreating accurate in vitro models.

Role of Vascular Wall Cell Types in Atherosclerosis

The surfaces of blood vessels within the circulatory system are comprised of three main layers; the adventitia, media and intima. The intima is the innermost layer of the blood vessel which is composed of EC; this layer forms the lumen of the vessel, which is in continuous contact with the blood. Between the endothelium and the media is a layer of basement membrane proteins, predominantly laminin and collagen IV. This is followed by a layer of elastin. The primary constituent of the media layer is SMC embedded in a network of connective tissue. This layer regulates blood flow and pressure through vasoconstriction and vasodilation via SMC contraction. The adventitia is the outermost layer of the blood vessel and is made up of loose connective tissue and fibroblasts [33].

The endothelium forms a dynamic layer with the blood, and acts as a barrier between the blood and the surrounding tissues. Additionally, it prevents blood clot formation and leukocyte extravasation when it is in a functional state [59]. Different parts of the circulatory system appear to be either resistant to atherosclerosis (atheroprotective) or prone to atherosclerosis (atheroprone) [60]. Unbranched regions of the cardiovascular network tend to be atheroprotective in nature. Therefore, they are predisposed structurally, biochemically and functionally to resist atherosclerosis. Atheroprotective EC has ellipsoidal cell morphology and aligns them in the primary direction of flow. The steady production of Nitric Oxide (NO) from Nitric Oxide Synthase (eNOS) is important for vascular homeostasis, is anti-atherogenic and is hypothesized to regulate monocyte adhesion [61]. Atherosclerosis often occurs in particular “lesion-prone” areas of the circulatory system. These tend to be where blood vessels branch or curve, creating areas of low time-average shear stress. EC from atheroprone regions tend to have a dysfunctional phenotype. These cells are pro-thrombotic, pro-inflammatory, and have impaired barrier function. They also display a cuboidal morphology, have high cellular turnover, senescence rates and are predisposed to retaining lipoproteins. Cardiovascular risk factors such as hyperlipidemia, oxidative stress and smoking are mediators for endothelial dysfunction.

EC activation is identified by the cytokine induced expression of surface adhesion proteins [62]. This facilitates the recruitment of leukocytes circulating in the blood stream. Mediators of endothelial activation are Advanced Glycation End-Products (AGEs), disturbed blood flow, and pro-inflammatory cytokines, which act through the activation of the transcription factor NF-κB [63]. EC activation can be inhibited through the action of NO. Endothelial dysfunction is worsened by EC activation.

SMC within the medial layer of the blood vessel are involved in maintaining the vessel diameter in response to external stimuli. SMC have three main phenotypes both in vivo and in vitro: synthetic, contractile and quiescent. SMC in a healthy vessel exist in the contractile phenotype where they conduct myogenic autoregulation according to changes in blood pressure or flow. However, in an atherosclerotic environment SMC undergo phenotypic switching from the contractile into the proliferative state. In this state, the myogenic autoregulation function of the SMC becomes impaired and they begin to proliferate and migrate from the medial layer into the intimal layer through both cytoskeletal and ECM remodeling. This process leads to a local thickening of the vessel wall and a decrease in vessel diameter, potentially compromising blood flow. During atherosclerotic plaque formation, Matrix Metalloproteinase (MMP) is produced within the lesions. MMPs contribute to ECM remodeling to enable SMC to proliferate and migrate more within the lesion. The SMC become a major component of the atheroma and form a fibrous cap around it, which provides temporary stability to the plaque.

As described earlier, monocytes circulating in the blood migrate into tissues (particularly at the site of damage or injury) where they differentiate into macrophages [64]. Monocytes have high plasticity that enables them to adapt their phenotype when exposed to different stimuli [65]. There are two main classes of macrophages: classically activated type 1 (M1) and alternatively activated type 2 (M2). M1 macrophages are typically activated post injury or infection and tend to produce inflammatory cytokines, reactive oxygen and nitrogen species [66]. Conversely, M2 macrophages are induced by different stimuli from cytokines and toll like receptors to immune complexes [67]. M2 macrophages also predominately produce antiinflammatory cytokines [68]. M1 macrophages tend to be the most common subtype of macrophages within inflamed atherosclerotic plaques. Macrophage plasticity is also influenced by the presence of Oxidized LDL (oxLDL). OxLDL can accumulate in the vessel walls due to increased influx of LDL in the blood stream through permeable activated endothelium. Once the LDL permeates across endothelium, they undergo oxidation within the vessel wall. Some monocytes uptake oxLDL and undergo foam cell formation, which are commonly found in early atherosclerotic lesions [69].

State of the Art In vitro Co-culture Methods to Study Atherosclerosis

In 1986, co-cultures of endothelial and smooth muscle cells were used by Weinberg et al to create a multilayered, tissue engineered blood vessel model where there were healthy, differentiated EC and SMC, which all exhibited appropriate physical and biosynthetic functions. Investigations carried out by Davies, et al. and Navab, et al. formed the basis of today’s atherosclerosis models [70-72].

Studies that have been carried out to model atherosclerosis can be classified in two main categories: (1) indirect models, which contain two or more cell types without direct cell-cell contacts between cells of different types and (2) direct models, in which the multiple cell types coexist within the same volume. Within these categories there are two main subtypes of model systems: (1) static and (2) dynamic culture systems. Below, we explain the different variations of these models and culture systems in addition to how these models are utilized to study the different aspects of atherosclerosis in vitro.

Indirect Co-culture

Indirect co-culture enables different cell types to be grown in tandem without there being any direct cell-cell interaction. These types of studies are useful to investigate cellular responses between different cell types that make use of secretory pathways and cytokine production. The main variations of the indirect co-culture models are: microcarrier, scaffold, bilayer membrane, Conditioned Media (CM) and Transwell models.

Conditioned Media (CM): Spent media from cell culture contains soluble mediators that have been produced by the cells (Figure 2A). In this method of co-culture, the cell types involved are grown separately and the cell supernatant from one culture is introduced to another cell culture in order to elicit mediator-relayed cell-cell interactions. CM co-cultures enable experimental flexibility since it is possible to freeze the spent media and analyze it well after the experiment has been terminated. The CM model has been used in multiple different types of co-culture studies centered around atherosclerotic related events especially involving the use of SMC. Studies have been carried out using human monocytes and SMC in order to determine the interactions involved in SMC-mediated MMP production [73,74]. Vijayagopal, et al. used both human and bovine SMC and monocyte/ macrophage cultures to study the formation of lipid laden SMC [75]. SMC/macrophage co-cultures were incubated with LDL, Acetylated LDL (acLDL) and Lipoprotein Proteoglycan (PG) complexes obtained from human atherosclerotic lesions, for varying amounts of time. SMC were then isolated and their Cholesterol Ester (CE) levels were quantified. PG complexes that were exposed to SMC macrophage cultures resulted in the formation of aggregated complexes. SMC which were incubated with CM containing aggregated PG-complexes were found to stimulate cholesterol esterification in SMC. This was the same case as with the cells which were grown in SMC macrophage co-cultures but not when the different cell types were grown separately [75]. CM models have also been used to determine the effect of soluble mediators on SMC proliferation [76] and EC TF [77] expression when cells were grown in co-culture with macrophages and SMC respectively. The impact of monocytes on SMC calcification has been investigated. It was found that soluble factors produced by monocytes, namely TNF-α [78,79], enhanced calcification by increasing Alkaline Phosphatase (ALP) activity. Increasing the number monocytes, be it cell line (THP-1) or primary source (Peripheral Blood Mononuclear Cells, PBMC) increased matrix mineralization and ALP activity. ALP activity was not influenced by the addition of oxLDL to the cultures however bacterial Lipopolysaccharide (LPS) [78], Oncostatin M (OSM) and IFN γ [79] when used together resulted in increased ALP activity and in vitro calcification of SMC. Disadvantages of the CM model regard the use of the soluble mediators which may be ineffective in triggering cell response without cell-cell interaction. Additionally, these soluble factors may not be stable in media for a long period of time. Furthermore, when using CM models, the cell response has a uni-directional flow; therefore no feedback signaling is present between the cells types used which makes it an ineffective as a model representative of the cell behavior in vitro.

Microcarriers: The use of microcarriers enables some in vivo complexities to be modeled in vitro while simultaneously maintaining some of the properties of experimental cell culture (Figure 2B). Cells can be grown in close proximity to one another. There is increased surface area between the cell types and easy separation of the cells. Davies, et al used microcarriers to create a co-culture between EC and SMC to determine the influence of the endothelium on LDL metabolism by SMC [70]. A calcium-dependent spheroidal coculture of the two cell types was introduced where SMC organized them into the core of the spheroid with EC encapsulating the SMC core. This model resembles the in vivo angiogenesis of blood vessels. The co-culture of these two cell types resulted in a mature, quiescent EC phenotype. This was demonstrated by an increase in the number of junctional complexes within the EC monolayer, increase in the resistance of EC to apoptose as well as downregulation of PDGF-β expression [80].

Bilayer membranes: Bilayer membrane models, like microcarrier models, enable closer proximity between the cells used in the coculture (Figure 2C). In this model, each cell type is seeded on one side of a porous membrane allowing cell-cell interactions at their basal side. Graham, et al. conducted studies on bilayer co-cultures of EC and SMC to better understand their in vivo interactions. Cells in these membrane systems could be kept in long term cultures, changes in cell-cell interaction up until confluency could be seen, and ease of separation of cells post intervention for the examination of the cells and ECM was observed [81]. Fillinger, et al. compared the co-culture of EC and SMC in a CM model with a bilayer membrane model, finding that, although in both types of models, the SMC demonstrated a similar morphology–spindle like shape with filamentous projections–within the bilayer model these projections transverse the membrane, resulting in contact between the two cell types. At day 14 of the culture, there was a greater effect on protein synthesis and SMC density. Therefore, it was found that bilayers are better models to study EC-SMC interactions than the conditioned media model [82]. Navab, et al. studied monocyte transmigration in EC-SMC co-cultures. It was ascertained that the endothelial layer was the main permeability barrier to the infiltration of monocytes and that monocyte transmigration through the endothelium is possible even without the use of a chemotaxis-inducing agent when EC and SMC were present in co-culture. However, the function of endothelium barrier was not lost upon the transmigration of monocytes [83]. Numerous studies were subsequently conducted in EC-SMC co-cultures to explore the adherence and chemotaxis of monocytes and leukocytes within the culture as well as their impact on the proliferative response of SMC. The presence of TNF-α led to the upregulation of ICAM-1 enabling greater monocyte adhesion and reduction of SMC proliferation, an effect that was mediated by the use of 5 mmol/ L of aspirin. Disadvantages of the bilayer membrane model are that (1) while ECs are found in a planar environment, SMC are usually found in 3D matrices that are not adequately imitated in the bilayer system, (2) the multi-stage seeding technique to coat both sides of the membrane with cells at separate intervals is inconvenient (3) cell interactions influenced by soluble mediators is unidirectional, as is the case with the CM models (4) overgrowth of cells on the pores of the membrane, will cause blockages and reduce or eliminate the cell-cell interactions [84].

Transwell: Transwell co-culture models enable the culture of two or more different cell types. In the Transwell (also known as a Boyden chamber) co-culture model, one cell type is grown on the lower chamber of the culture plate while the other is grown separately on the porous membrane filter insert (Figure 2D). Depending on the design of experiment, a third cell type, usually cells grown in suspension, or a drug candidate, or biochemical cues can be introduced on top of either the upper chamber or lower chamber. At its limit, a further attachment of a fourth cell type is also possible underneath the porous membrane, for a mixture of indirect and direct co-culture. To achieve co-culture, the lower chamber and filter insert can be combined. However, there is no direct cell-cell interaction between the cells within the Transwell insert and the lower chamber. This model type is useful to study cell-cell and cell-substrate interactions with regards to atherosclerosis. A majority of the studies carried out have examined SMC proliferation, calcification and apoptosis. Proudfoot, et al. and Fitzsimmons, et al. studied the influence monocytes and macrophages had on SMC proliferation [85] and procollagen secretion [86]. In this system, monocytes inhibited SMC growth and, while they do not cause procollagen degradation, monocytes do inhibit procollagen secretion by SMC. In co-cultures with EC, SMC and THP-1 cells, SMC proliferation was exacerbated by Advanced Glycation End Products (AGEs) [87]. Boyle, et al. studied SMC apoptosis, finding that macrophages triggered the production of TNF-α through autocrine pathways [88]. However, Cai, et al. showed that when monocytes bound to SMC, they continued to differentiate and apoptosis was stalled [89]. Studies demonstrated that SMC cultured with monocytes underwent calcification [90] and when monocytes developed lipid laden lysosomes, they could transfer these lysosomes into SMC if the two cell types were in direct contact with one another.

Figure 2: Current in vitro models of atherosclerosis. Models of atherosclerosis consist of combinations of: Endothelial Cells (EC), Smooth Muscle Cells (SMC) and monocytes or their respective byproducts, to study their cross talk and behavior during the initiation and progression of atherosclerosis in a mechanistic way. (A) Cells in question can be exposed to secretions of another cell type by means of transfer from a separate receptacle. (B) Bilayer and (C) microcarrier approaches both allow close proximity of two adherent cell types to the extent of allowing direct contact between them. (D) To decouple physical proximity but maintain immediate cell-cell signaling through secreted proteins the Transwell model has been used successfully. Some investigators also directly seed cells on top of each other to ensure cell-cell signaling while others create a biomimetic three-dimensional microenvironment using scaffolds of synthetic and native hydrogels either (E) for anchoring the cells spatially or (F) separating the cells.

EC and monocyte interactions have also been studied in Transwell systems. Westhorpe, et al. exposed PBMCs to confluent layers of Human Umbilical Vein Endothelial Cells (HUVEC) grown to confluency within the upper chamber of the Transwell plate. Different forms of lipoproteins were located in the lower chamber. PBMCs transmigrated past the EC layer into the lower chamber which was activated using TNF-α either prior to or post transmigration. Monocytes were introduced to Transwell upper chambers without an endothelial layer to determine whether the cells would still transmigrate and differentiate. Monocytes transmigrated across the upper chamber regardless of the presence of lipids or endothelium. However, there was a substantial increase in the monocytes which formed foam cells when they were exposed to an activated endothelium [91].

The use of indirect co-culture lends itself to making simple models that are easy to control. Due to the ability to separate the cell types used, specific aspects and mechanisms of cellular behavior and interaction can be studied. The use of indirect co-cultures enables the creation of cellular environments specific to each cell type used within the model. However, because there is no direct cell-cell interaction between the cell types, the only intercellular communication occurs via paracrine signaling through soluble mediators. The disadvantages of using this model type are the necessity for cells to attach to plastic dishes or membrane filters (alone or within Transwell plates), forming non-physiological conditions, and short co-culture times that may result in less-than-adequate development of ECM interactions. Most importantly, the dimension of cellular behavior that is derived from the interaction between cells is not present, which is vital for mimicking and understanding the complexity of cellular mechanisms in vivo [92]. For this reason, it was paramount that direct co-culture systems be used to start modelling atherosclerosis in vitro.

Direct Co-culture

Direct co-culture involves two or more cell types of interest being overlaid upon one another or placed side by side indirect contact with one another. This technique is useful as it involves the three main types of cell interaction, which are signaling via cell-cell adhesion, cell-ECM adhesion and soluble factors.

Scaffold free: In order to study SMC apoptosis, Vasudevan, et al. plated SMC in serum-free media to which freshly isolated PBMCs were added at a 3:1 ratio. Cells were co-cultured for 48h with or without macrophage colony stimulating factor (M-CSF, a growth factor that induces survival, proliferation and differentiation of monocyte cells). Some co-cultures were pre-incubated with antibodies against ICAM- 1. SMC apoptosis was contingent upon binding of monocytes to SMC, mediated by interactions between ICAM-1 and Mac-1 receptors [93]. Monocyte binding to SMC was exacerbated in diabetic conditions [94]. Chaterji, et al. created scaffold-free conditions to mimic both the healthy and diseased states of a blood vessel by altering the seeding densities of EC within EC-SMC co-cultures as well as adding soluble growth factors to induce desired cell phenotypes. The initial seeding densities for SMCs and ECs were 3-5 × 104 cells /cm2 and 8-10 ×104 cells/cm2, respectively. In an SMC monoculture, SMC differentiation markers, smooth muscle α-actin and calponin were upregulated by the addition of transforming growth factor-β1 (TGF-β1) and heparin. However, seeding near-confluent EC concentrations on SMC induced a higher expression of these SMC differentiation markers than the growth factors on their own. These expression levels were further increased when SMC were pre-treated with the soluble factors prior to seeding the EC layer. The soluble factors used were: 2.5 and 5 ng/mL transforming growth factor-β1 (TGF-β1), and 30 μg/ mL heparin in a low-serum environment (1% [v/v] Fetal Bovine Serum [FBS]). Conversely, a hyperplastic state was induced when low concentrations of EC (3 × 104 cells/cm2) were seeded onto the SMC. The study highlights the importance of the growth factors and EC seeding densities used when developing a co-culture model. Chaterji, et al. suggests that in vitro, in order to maintain the proper vascular tissue functionality, it is important to maintain the close proximity between the cell types that is found in vivo. Additionally the study shows that altering this structure is useful in mimicking a diseased state which in turn is invaluable in testing potential therapeutic molecules for treating cardiovascular diseases [95].

Scaffolds: Scaffolds are used in tissue engineering to create a three dimensional (3D) structure on which cells can be seeded and allow tissue formation. Appropriate scaffolds must possess characteristics that best enable cell growth, proliferation and expression of the desired cell phenotype: cell attachment to and migration into the scaffold, delivery of biochemical cues to and from the cells on the scaffold, biodegradability to ultimately enable the absorption and replacement of the scaffold with newly formed neotissue, diffusion of nutrients and cellular products into and out of the scaffold and creating a mechanical environment for the cells to stimulate cell bioconduction and bioinduction.

While different materials can be used to construct scaffolds, the two main categories most often used are biomaterials and synthetic materials. Biomaterials include substances like collagen, chitosan, alginate, gelatin, hyaluronan and fibrin [96]. The most common examples of synthetic materials used in tissue engineering scaffolds are Polylactic Acid (PLA), Polyglycolic Acid (PGA) and Polycaprolactone (PCL). The advantage of using natural polymers is that they are produced in vivo and can be sourced cheaply. Additionally, natural biomaterials have a greater degree of biocompatibility than synthetic materials since they have naturally occurring cell adhesion and binding sites [96]. Disadvantages include batch-to-batch variability, dearth of mechanical properties and the potential for contamination of the raw materials that could illicit immune responses in patients. Synthetic materials on the other hand have high reproducibility; can be produced in bulk and changes in mechanical properties such as degradation and composition can be modified easily. However, biocompatibility is limited due to the absence of cell adhesion and binding sites that are present in naturally occurring biomaterials and immune reactions can still be triggered in patients [96].

Scaffolds can also be two dimensional (2D) layers such as gels which are used to provide a coat on materials that would otherwise not be conducive to cell attachment and proliferation. Scaffolds used in atherosclerosis models have been used for multiple purposes: providing a 3D environment for co-culture of multiple cell types, highly reflective of the native microenvironment; anchorage of cells; creating a selection pressure for cells to express the required phenotype; allowing cells to grow within structures more accurately representing in vivo vessel geometry; and creating a 3D construct for cells to grow on to model the atherosclerotic vessel in vitro.

Gel Separation: VanBuul-Wortelboer, et al. grew SMC within a collagen lattice atop which EC were overlaid in order to understand the role EC play in modulating SMC behavior in co-culture (Figure 2E). In these conditions, SMCs adopted an elongated morphology as opposed to the polygonal shape that occurs when EC are cultured alone or on a Polystyrene (PS) substrate and EC additionally suppressed SMC proliferation [97]. Navab, et al. used an interesting approach to create a bilayer culture used to model monocyte transmigration in EC-SMC co-cultures. SMC were grown on gelatin layers and once sufficient autologously secreted ECM formed, ECs were laid on top [71,72]. LDL was introduced to these co-cultures, which brought about modification of the LDL and resulted in an upregulation of MCP-1. Exposure of monocytes to the co-cultures or CM from the co-cultures resulted in monocyte transmigration into the sub-endothelial space of the co-cultures. However, CM from individual cultures of EC or SMC did not induce monocyte migration. This effect was tempered by 91% with the addition of either antibodies against MCP-1, or HDL alongside LDL [72]. Further studies also showed that LDL, once pre-incubated with leumidins in an in vivo rabbit model resulted in modification of the lipoprotein into a stable complex which, once exposed to the co-cultures did not bring about modification of the LDL thus inducing monocyte transmigration [71].

Co-cultures were carried out by Takuku, et al. to study foam cell formation using rabbit aortic SMC and EC as well as human PBMCs (Figure 2F). MCP-1 was used to induce monocyte transmigration and subsequent differentiation into macrophages. Exposure to modified LDLs brought about foam cell formation [98]. Studies carried out by Wada, et al. corroborated these findings [99]. Phagocytic reverse transmigration across an endothelium, a common occurrence across vascular and/or lymphatic endothelia during atherosclerosis was investigated by Randolph, et al. HUVEC were grown on type I collagen gels upon which PBMCs, either unstimulated or stimulated with LPS, were placed as a control. Monocytes, but not lymphocytes were able to migrate across the endothelial barrier. To test for reverse transmigration through the endothelium, PBMCs were incubated with the endothelial layer for a 1- or 2-h interval in order to facilitate migration of the monocytes into the sub-endothelial collagen. The apical surface of the endothelial layer was washed to remove any monocytes that had failed to transmigrate. Incubation was continued for 24h, and any monocytes that had reversed transmigrated to the apical side of the endothelial layer were washed off. The percentage of reverse transmigrated cells was ascertained by calculating the percentage decrease of monocytes beneath the endothelium at the end of the experiment compared to the number of monocytes present at the initial 2h interval. VIC7 antibodies against TF decreased this reverse transmigration by 77%. It was discovered that TF was not present on resting monocytes, but only on monocytes that had undergone the initial apical to basal transmigration across the endothelium. These findings indicate that monocyte adhesion to endothelium is enabled by the expression of TF on monocytes [100].

Lavender, et al. worked to optimize the conditions for the direct co-culture of EC on SMC and to assess the effect SMC had on EC function. The co-culture consisted of: culture substrate, basal adhesion proteins, a layer of porcine SMC, medial adhesion proteins and a layer of porcine EC. The optimal conditions for this model were: a polystyrene cell substrate, fibronectin basal protein, quiescent SMC with a subconfluent density, and a confluent density for EC. The use of fibronectin, laminin, collagen I and IV as medial adhesion proteins did not have a significant effect on EC adhesion. EC and SMC grew in two distinct layers and EC in co-culture formed tight junctions, however junction formation was not as developed as in EC monocultures. The co-culture was maintained for 10 days. It was demonstrated that EC could adhere to and be grown to confluence on a layer of quiescent SMC cells even when exposed to flow rates of 5 dyne/cm2 for 7.5h. This study demonstrated that EC attached better on quiescent SMC compared to proliferative SMC [101].

Thus far, all the model systems that have been discussed, except the above by Lavender, et al. have been static models. Static cocultures enable the stable growth of several cell types in tandem. While substantial discoveries have been made about atherosclerosis through static models as demonstrated by the large number of studies that has been detailed above, there are a significant number of limitations associated with these types of models. Static models neglect the flow and pulsatile conditions that are inherent in living, in vivo systems. One of the greatest setbacks of using static models for atherosclerosis co-cultures is the inability to account for the impact of shear stress on the different cell types involved. It has been mentioned before the role shear stress has on atherosclerotic regions and how the presence of shear stress influences the phenotypic switching in SMC or the alignment of EC is influenced by blood flow. In order to create more representative models, it was necessary to introduce dynamic parameters into atherosclerosis co-culture models.

Gel anchoring: Uses 3D hydrogels formed most often using either collagen or fibrin which have cells suspended within them. These gels recreate a 3D environment for cells, similar to in vivo conditions where the cells exist in more than one plane, typically within the ECM. Typically, to create a 3D gel, the desired cell type is placed in a gel solution that is left to polymerize. A second cell type can then be placed on top of the gel. A study carried out by Dorweiler, et al. used 3D gels within a static model to create multilayered SMC intima with formation of an ECM on which an endothelial layer was grown. The introduction of monocytes and lipids to this model led to monocyte transmigration and foam cell formation [102]. Subsequent studies have incorporated flow conditions into these models. Chen et al. studied the influence of both disturbed flow on WBC adhesion and transmigration in EC-SMC co-cultures [103], and the soluble mediator production induced by SMC and monocyte interactions [104]. Earlier studies showed the influence of flow and shear stress on the structure and functionality of porcine EC within EC-SMC co-cultures. EC grown in static on top of a porcine SMC layer were elongated and were oriented randomly. However, upon exposure to a shear stress ranging from (10-30 dyne/cm2) within a parallel plate flow chamber, EC aligned them towards the direction of flow after 24 to 48h. This effect on the EC was maintained when they were culture on top of collagen 1 alone without the presence of SMC or flow conditions [105].

Cicha, et al. modelled the non-uniform shear stress profile at arterial bifurcations. HUVECs were seeded on slides and were exposed to 2.5 ng/mL of TNF-α at a flow rate of 9.6 mL/min (equivalent to 10 dyne/cm2) for 2h. Adhesion of THP-1 monocytes was demonstrated using light microscopy. The combination of shear stress with the TNF-α treatment of the HUVEC led to a drastic increase in monocyte recruitment [106]. Li, et al. investigated vulnerable plaque destabilization using calcific nodules to model atherosclerotic plaques. Calcifying Vascular Cells (CVCs) were obtained from bovine ASMC cultures. These nodules were exposed to oxLDL and pulsatile shear stress (23 dyne/cm2) for 2.5h in a pulsatile flow channel. The presence of oxLDL and monocytes increased the amount of CVC destabilization from the substrate as well as an increase in MMP activity, particularly MMP-9 [107].

Robert, et al. created a 3D tissue engineered artery was created under pulsatile flow conditions. The construct contained both HUVEC and human umbilical cord-derived myofibroblasts and was exposed to flow in vitro using a bioreactor. The histological analysis of the tissue demonstrated a basement membrane supporting a tight endothelium and multiple layers of smooth muscle cells. High Density Lipoprotein (HDL) and Low Density Lipoprotein (LDL) were circulated through the construct. These lipoproteins migrated into the endothelium and were recovered both from within the EC as well as in the sub-endothelial intima. The endothelium was activated using TNF-α or LDL. Following that monocytes were introduced into the system and were found to adhere to the activated endothelium and subsequently transmigrate into the intima. This is the first example of a tissue engineered construct which simulates the in vivo physiological conditions [108].

Limitations of Current In Vitro Models

Compatibility between Studies

As discussed herein and summarized in Table 1, there have been many co-culture studies carried out to understand the nature of atherosclerotic mechanisms from monocyte attachment and infiltration of the endothelium to foam cell formation. These studies have been conducted using multiple cell sources: human, rabbit, bovine and porcine aortic cells being the most predominantly used. These studies have been carried out in a multitude of environments from 2D to 3D scaffolds, in gels and with or without the presence of flow. The issues that arise from having such a large variety of studies is that there are often conflicting results or a lack of reproducibility between different types of models. Frequently, there is a variation in the outcomes of direct and indirect model systems, typically with direct cell culture systems demonstrating the more amplified response. Particularly in the case of using cells from non-human species, the data obtained may not be representative of clinically relevant in vivo mechanisms of atherosclerosis. For example, the murine macrophage cell lines such as J774.A1 and RAW264.7 are missing apolipoprotein E expression genes, which are important in the development of atherosclerosis in humans [109]. This proves to be a problem when trying to use such models to gather clinically relevant data that can be used in patient treatment and drug development. Going forward it would be prudent to develop a standardized framework and cell type(s) to ensure compatibility between studies done by different labs worldwide.

Table 1: In vitro co-culture methods to study atherosclerosis using Endothelial Cells (EC), Smooth Muscle Cells (SMC) and monocytes/ macrophages.

References

Source Cell type

Culture system

Focus

Key findings

[70]

Bovine AEC, ASMC

Static – Indirect - Microcarrier

Vascular EC-SMC metabolic interaction

LDL metabolism of SMC was influenced by the presence of endothelium

[115]

Bovine AEC, SMC

Static - Indirect - Scaffold

Construction of in vitro model of blood vessel

Could create multilayered vessel structure, EC and SMC were healthy and well differentiated, EC demonstrated both physical and biosynthetic functions

[97]

EC, SMC

Static - Direct

EC regulation of SMC proliferation in co-culture

EC suppress SMC proliferation

[83]

HAEC, HASMC, HBM

Static - Indirect - Bilayer

Monocyte transmigration into sub endothelial space

Monocyte transmigration occurred in EC-SMC co-culture even in the absence of chemotactic agent. Endothelium is primary permeability barrier to monocyte transmigration

[72]

HAEC, HASMC, HPMB

Static- Direct - Scaffold coated with gelatin

Monocyte transmigration

EC-SMC co-cultures when exposed to LDL and their conditioned media enable monocyte transmigration due to increased MCP-1 production. This effect can be counteracted with HDL and other anti-oxidants

[81]

EC, SMC

Static- Indirect- Bilayer

Bilayer model to study EC-SMC interactions in vivo

Model enables preservation of bilayer morphology, long term culture, and ease of separation of cells within culture post intervention

[71]

HAEC, HASMC, HPBMC

Static- Direct - Scaffold coated with gelatin

Monocyte transmigration

In vivo exposure of LDL to leumidins makes LDL resistant to modification when exposed to EC-SMC co-cultures. Modified LDL did not bring about monocyte transmigration.

[73]

Human saphenous vein SMC, PBMC

Static- Direct- 3D gel or Indirect - Conditioned media

Interactions between human SMC and monocytes causing MMP secretion

Exposure of SMC to monocytes and monocyte conditioned media resulted in secretion of MMP-1 and MMP-3 through the IL-1 dependent pathway

[105]

Porcine AEC, ASMC

Dynamic - Direct- 3D SMC gel, with EC monolayer on top

Regulation of EC structure and function by flow and shear stress within EC-SMC co-culture

EC align with direction of flow when exposed to shear stress. Presence of collagen allowed quiescent endothelium to be maintained in the absence of SMC or flow.

[75]

Bovine ASMC, AEC, P388D1, Human ASMC, AEC, Human monocytes

Static- Direct or Indirect- Conditioned Media

Formation of lipid laden SMC

Exposure of SMC-macrophage co-cultures tolipoprotein PG complexes (but not LDL or acLDL) resulted in CE synthesis and accumulation within SMC

[116]

Bovine ASMC, AEC

Static-Indirect- bilayer

EC influence on SMC phenotype in co-culture

EC influence SMC growth characteristics and phenotype by producing PAI-1

[117]

HAEC, HASMC, HPMBC

Static - Direct

Investigated role of HO in early atherosclerosis

HO-1 is induced by oxidized LDL which in turn reduces monocyte transmigration

[76]

HASMC, PBMC

Static - Indirect - Conditioned media

Effect of soluble macrophage mediators on the proliferation of SMC

SMC exposed to conditioned media from both activated and non-activated macrophages inhibited SMC proliferation

[82]

Bovine ASMC, AEC

Static-Indirect- bilayer and conditioned media

Comparison of conditioned media and bilayer EC-SMC co-culture models

Bilayer model more appropriate to study EC-SMC interactions due to physical proximity and interaction between cells

[100]

HUVEC, PBMC

Static - Direct - 2D gel

Reverse transmigration of phagocytes across endothelium

Adhesion of monocytes to endothelial cells is influenced by TF

[77]

HUVEC, HSMEC, HASMC, HUASMC, HUVSMC

Static- Indirect - Conditioned Media

SMC soluble factors on EC TF expression

Conditioned media from SMC demonstrating synthetic phenotype induced altered TF activity from established inducers of TF activity in EC.

[98]

Rabbit AEC, ASMC, Human PBM

Static - Direct - 2D gel

Foam cell formation

Within EC-SMC co-culture system, addition of monocytes was enhanced using MCP-1. Monocytes differentiated into macrophages. Exposure to modified LDL led to foam cell formation.

[85]

HASMC, PBMC

Static- Indirect - Transwell

Influence of monocytes/ macrophages on SMC growth

SMC growth was inhibited by monocytes

[86]

HASMC, PBMC

Static- Indirect - Transwell

Effect of monocytes on procollagen turnover from SMC

Monocytes inhibited procollagen secretion from SMC without affecting degrading procollagen

[74]

Human VSMC, THP-1 monocytes

Static - Direct or Indirect - Conditioned media/ Transwell

Investigating MMP-1 production from SMC - monocyte co-culture

Both direct co-culture and exposure of SMC to conditioned media resulted in MMP-1 formation. The former resulted in significantly higher MMP-1 production

[118]

Rabbit AEC, ASMC, Human PBMC

Static - Direct - 2D gel

Functional arterial wall model

Arterial wall constructed, HPM demonstrated transmigration and differentiation into macrophages which later formed foam cells within ECM upon exposure to modified LDL.

[119]

HUVEC, HUCSMC, PBL

Dynamic – Indirect- Bilayer - parallel flow plate

Patterns of leukocyte recruitment

Adhesion of lymphocytes was negligible both with EC and EC-SMC co-cultures. TNF-a treated co-cultures demonstrated greater leukocyte recruitment than EC monocultures

[120]

HCAEC, HCMSMC. Monocytes,CD4+ lymphocytes

Static – Indirect- Bilayer

Leukocyte adhesion, chemotaxis and SMC proliferative response

TNF-a up regulation of ICAM-1 on EC/SMC co-cultures studied. Adherence and chemotaxis by monocytes and lymphocytes reduced significantly with 5 mmol/L aspirin. Proliferative response of SMC after lymphocyte attack was also reduced significantly.

[79]

Human VSMC, THP-1, PBMC

Static - Direct or Indirect - Conditioned media/ Transwell

Calcifying phenotype in SMC

Both THP-1 and PBM cells induced ALP activity in SMC which resulted in the calcification of their extracellular matrix

[78]

Bovine CVCs, PBMC

Static - Direct or indirect - Conditioned media or Transwell

Monocyte influence on in vitro vascular calcification

Monocytes enhance vascular calcification both through direct co-culture and production of soluble factors

[121]

VSMC, Monocytes

Static - Direct

SMC apoptosis

Physiological concentrations of M-CSF induced SMC apoptosis

[122]

Human ASMC, PBMC

Static - Direct

SMC proliferation

Monocytes inhibit SMC proliferation

[93]

Human ASMC, Monocytes

Static - Direct

SMC apoptosis

Both M-CSF and monocyte induced SMC apoptosis requires Mac-1 and ICAM-1 mediated binding of monocyte to SMC

[88]

Human PBMCs, carotid, coronary medial, and aortic SMC

Static - Direct or Indirect - Transwell

Influence of TNF-a on SMC apoptosis

Macrophage induced SMC apoptosis is induced by TNF-a using both autocrine and direct pathways

[123]

hASMC, BAEC, BSMC

Dynamic - Scaffold

SMC migration through the vessel wall

PDGF stimulated hASMC migration into the stroma was halted by the induction of TIMP-1 production using TIMP-1 induced SMC

[89]

Human SMC, PBMC, THP-1

Static - Direct or Indirect - Transwell

Monocyte differentiation and apoptosis

Binding of monocytes to SMC increased their survival and differentiation

[124]

HUCEC, HUCSMC

Dynamic/ Static - Indirect- Bilayer

Effect of shear stress on EC-SMC interactions

In co-culture where only EC exposed to flow, SMC oriented perpendicularly to flow at higher shear rates and EC oriented themselves in the direction of flow. Without shear, EC gene expression of MCP-1 and oncogene-a brought about by co-culture with SMC was abated in the presence of shear stress.

[125]

Bovine AEC, SMC

Dynamic/ Static - Indirect

Effect of shear stress on EC-SMC lipoprotein uptake

Regardless of the presence of shear flow, LDL uptake in EC-SMC co-cultures was greater than in monolayers whereas Ac-LDL uptake remained unchanged. In the presence of shear flow, Ac-LDL uptake decreased for both co-cultures and cell monolayers.

[101]

Porcine EC, SMC

Static - Direct - 2D gel

Direct co-culture of EC on SMC and assessment of EC functionality

Confluent, adherent EC can be cultured on a layer of sub-confluent quiescent SMC

[126]

HUCEC, HUCASMC

Dynamic/ Static - Indirect- Bilayer

Inflammation related gene expression in EC due to co-culture with SMC

EC produce pro-inflammatory genes when cultured with SMC. This gene expression is inhibited when EC exposed to shear stress through inhibition of NF-κB activation brought about by SMC. Consequently, THP-1 adhesion was also curtailed.

[103]

PBL, CD14+ monocytes, PBMC, HUCSMC, HUCEC

Dynamic - Direct - 3D gel

Influence of EC-SMC on WBC adhesion and transmigration under disturbed flow

Co-culture significantly increases adhesion and transmigration of all WBC types. WBCs demonstrated different migration patterns.

[127]

HUVEC, HUCSMC

Dynamic - Indirect - Transwell, 2D gel plates/ capillaries

Platelet adhesion to EC within EC-SMC co-culture

Use of TNF-a and TGF-b with EC with a EC-SMC co-culture results in maximal levels of platelet adhesion due to the stimulation of vWF production in EC at low wall shear stress rates of 400 s-1.

[102]

HUASMC, HUVEC, Mono-Mac-6

Static - Direct - 3D gel

In vitro model of muscular artery to study early atherosclerotic events

In vitro model with multilayered SMC intima and endothelium with ECM was made. Addition of monocytes and LDL resulted in lipid insudation, monocyte transmigration and foam cell formation. IL-8 production was also demonstrated.

[128]

PMN, human EC, human SMC

Static - Direct

Sub endothelial infiltration and plaque destabilization

In EC-SMC co-culture, addition of LDL led to PMN adherence, transmigration and infiltration mediated by the IL-8 secretion pathway of SMC. This was followed by release of elastase and MMP-8 inducing EC apoptosis.

[104]

PBMC, PBL, CD14+ monocytes and HUCEC, HUCSMC

Dynamic - Direct - 3D gel

Influence of SMC-monocyte co-culture on soluble mediator production

SMC-monocyte co-cultures contribute to production of pro-inflammatory mediators

[94]

HASMC, THP-1, Mouse ASMC, WEHI78/24 monocyte

Static - Direct

Monocyte binding

Diabetes exacerbates the binding of monocytes to SMC

[129]

HAEC, HASMC

Static/ Dynamic (with pulsatile flow) - Direct/ Indirect (opposite sides of porous membrane)

Quiescent SMC influence on TNF a activated endothelial inflammatory response

EC cultured directly with SMC express fewer surface adhesion proteins after exposure to flow and TNF-a activation than EC cultured alone, or indirectly with SMC.

[95]

HAEC, ASMC, CSMC

Static-Direct

Altering cell concentrations and growth factors to model healthy and diseased blood vessels

Along with using the appropriate soluble factors, seeding near confluent concentrations of EC on SMC and sparse concentrations of EC on SMC can be used to model healthy and diseased states, respectively.

[130]

Human SMC, THP-1, mouse
P388D1 macrophages

Static - Indirect - Transwell

Examined effects of Vitamin D activators on SMC matric calcification

SMC calcification was promoted by monocyte-SMC co-culture. This was inhibited by vitamin D receptor activators

[90]

P388D1 macrophages"

Static - Indirect - Transwell

Examined effects of Vitamin D activators on SMC matric calcification

SMC calcification was promoted by monocyte-SMC co-culture. This was inhibited by vitamin D receptor activators

[131]

Mouse VSMC, mouse bone marrow derived macrophage

Static - Indirect - Conditioned media

Macrophage plasticity

Switching of a macrophage phenotype towards M2 could be atheroprotective

[87]

HUVEC, HAVSMC, THP-1

Static- Indirect- Transwell plate

Influence of AGES on vascular cells

SMC proliferation induced by glycol AGES. Significant increase in cytokine expression levels within co-culture

[106]

HUVEC, THP-1

Dynamic - Direct - Microfluidic slides

Effect of non-uniform shear stress and TNF-a at bifurcations

Exposure of endothelium to shear stress and TNF-a caused increased monocyte recruitment

[91]

HUVEC and PBMCs

Static-Indirect - Transwell plate

Monocyte transmigration and foam cell formation

Monocytes form foam cells in the absence of lipids after transmigrating a TNF alpha activated endothelium

[107]

Bovine aortic
smooth muscle cells, THP-1 monocytes

Dynamic - Direct culture - pulsatile flow channel

Characterizing calcific nodules to better understand vulnerable destabilization

Calcific nodules modelled atherosclerotic plaques. OxLDL and monocytes cause plaque destabilization through action of MMP

[108]

Human UCFMBs, HUVEC and monocytes from patients

Dynamic - Direct co-culture of cells on a 3D scaffold

Tissue engineered artery with 3D structure and pulsatile flow conditions to demonstrate atherosclerotic dysfunctions in vitro

Tight endothelium formation over basement membrane and multiple smooth muscle layers. Endothelium activation with LDL or TNF alpha resulting in monocyte adhesion and transmigration. LDL and HDL injected into the system and recovered within ECand sub-endothelial intima.

[132]

HASMC, PBM, U-937 monocyte like cell line

Static - Direct

Inflammatory mediator production from monocyte-SMC interaction in co-culture

Interaction between activated monocytes and SMC leads to resistin up-regulation in monocytes and ROS production in SMC. Resistin further exacerbates ROS production in SMC

[133]

HAEC, HASMC, PBM, Rat SMC

Static - Direct or Indirect - Conditioned media/ Transwell

Modulation of SMC behaviour by macrophages

Lipid laden lysosomes from macrophages are transferred into SMC in vitro when SMC-macrophages have direct contact

[134]

BAEC, BASMC, PCAEC, PCASMC, HUVEC, HUSMC, HAEC, HASMC

Dynamic - Direct co-culture of cells and indirect culture of cells

Wall shear stress on EC-matric, EC-EC and EC-SMC interactions

Wall shear stress disrupts endothelial monolayer. EC-SMC models more robust. EC-SMC interactions reduce EC-EC interactions and change EC phenotype and response to wall shear stress

Cell Source and Phenotype

Cells that are used to create atherosclerosis models are most often human primary cells. Primary cells are derived from living tissue and are meant to more closely represent the cell behavior in vivo. Primary cells are typically used at earlier passage numbers for a limited number of passages in order to ensure the in vivo phenotype is maintained. Unfortunately, primary cells are not widely available in large supply to carry out controlled studies; rather they are obtained from individual donors. These results in inherent variation in experimental results obtained. It will be challenging to identify a suitable, relevant patient cohort and a standard technique with which to obtain cell samples to be grown to a specific passage number and manipulated in order for them to express the required phenotype. Commercially purchased primary cells are the alternative and standardization of excision procedures provides some uniformity in the cells used for experiments. Cells behave differently in vitro as compared to their in vivo behavior within a native environment. Investigations by Potter et al. show that the glycocalyx-an important structure responsible for ‘sensing’ the hydrodynamic and mechanical environment at the EC surface, is missing from HUVEC and bovine AEC cultured in standard in vitro conditions [110]. The absence of the glycocalyx influences the surface chemistry of the EC which calls into question results obtained from studies that have previously been conducted on topics involving atherosclerosis, and endothelial barrier permeability [110]. Additionally, several different types of macrophages with different polarizations have been used in atherosclerotic models and these studies fail to acknowledge the influence on the model of having macrophages in either the pro-inflammatory M1 or anti-inflammatory M2 state. We too have shown that SMC from commercial source exhibit phenotypic changes through sequential passaging and prolonged culture of cells at a fixed passage number [111]. Thus, an additional challenge would be to optimize cell culture and conditions to suppress phenotype change, and further advocate fixed protocols.

In addition, building patient specific models is a reality considering the possibility of isolating endothelial progenitor and monocytic cells from patient blood. The former have to differentiate into EC and SMC [112,113].

Cell Substrate

When culturing cells in vitro for the purpose of cell expansion prior to actual experiments, cell culture wells, plates and flasks are used. Most often these culture materials are made of polystyrene or polycarbonate. Unlike the native microenvironment, these materials have high stiffness and they are not biomimetic of the ECM. Cell phenotype and behavior is greatly influenced by the stiffness of the cell substrate, particularly for cell proliferation and migration in SMC and cell morphology and tightness of endothelial barrier in EC. Our group has further shown that stiffness of the biomaterial can modulate SMC phenotype [114]. In order to create a more representative environment, cell culture material can be coated with biomaterials such as collagen or fibrin. When using biomaterials such as collagen or fibrin, there is inherent variation in the actual material as well as the composition of the gel solution and the resulting stiffness.

Clinical Applications and Future Trends to Improve Atherosclerosis Models

A majority of the studies that have been undertaken regarding atherosclerosis mechanisms have been carried out using animal studies and in vitro models. So far, the results of these studies have not been completely validated in vivo in humans. Thus, the results from these models cannot as yet be the predominant method used for the development of clinical strategies for disease treatment. Microfluidic devices have been devised for the high throughput testing of drug substances for the study of angiogenesis [33]. It is possible that further development of these techniques could lead to the creation of diagnostic devices for the study of diagnostic drug treatments for atherosclerosis. The major issue in the practical application of such models would be the long culture times required to create a welldeveloped construct that demonstrates the patients’ in vivo blood vessel characteristics.

Concluding Remarks

Both direct and indirect co-culture models have been used to model atherosclerosis in vitro. Earlier studies were done in static culture systems, but in recent years the transition has been made towards modelling flow, pulsatile conditions and shear stress using dynamic modelling systems. The different phenotypes of the cells involved in atherosclerosis have also been described in great depth and have been correlated with cellular processes investigated within the models. Investigations have been carried out to gain better understanding of monocyte transmigration past the endothelium, differentiation and foam cell formation using monocyte and EC cocultures. SMC and monocyte co-cultures were used to understand SMC apoptosis and calcification while EC and SMC cultures were utilized to understand the regulation of cell behavior and proliferation of each cell type by the other. The studies reviewed here provide insight into the relevance of in vitro models for the study of atherosclerosis, detailing the different ways atherosclerosis modelling is used for clinical applications. The various in vitro culture models illustrate the different mechanisms involved in disease progression as well as the importance of understanding cell-cell interactions involved. Overall, this review highlights the importance of in vitro modelling of atherosclerosis and the benefits these models will offer in gaining further understanding of the disease itself in addition to providing invaluable data on clinical screening of disease treatments.

References

  1. Wohlschlaeger J, Bertram S, Theegarten D, Hager T, Baba HA. Coronary atherosclerosis and progression to unstable plaques: Histomorphological and molecular aspects. Herz. 2015; 40: 837-844.
  2. Robinson JG, Fox KM, Bullano MF, Grandy S. Atherosclerosis profile and incidence of cardiovascular events: a population-based survey. BMC Cardiovasc Disord. 2009; 9: 46.
  3. Patibandla PK, Rogers AJ, Giridharan GA, Pallero MA, Murphy-Ullrich JE, Sethu P. Hyperglycemic arterial disturbed flow niche as an in vitro model of atherosclerosis. Anal Chem. 2014; 86: 10948-10954.
  4. Vasdev S, Gill V, Singal P. Role of Advanced Glycation End Products in Hypertension and Atherosclerosis: Therapeutic Implications. Cell Biochem Biophys. 2007; 49: 48-63.
  5. Jankowski P, Bilo G, Kawecka-Jaszcz K. The pulsatile component of blood pressure: its role in the pathogenesis of atherosclerosis. Blood Press. 2007; 16: 238-245.
  6. Su T-C, Chien KL, Jeng JS, Chang CJ, Hsu HC, Chen MF, et al. Pulse pressure, aortic regurgitation and carotid atherosclerosis: a comparison between hypertensives and normotensives. Int J Clin Pract. 2006; 60: 134-140.
  7. Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994; 94: 2493-2503.
  8. Vartak DG, Gemeinhart RA. Matrix metalloproteases: underutilized targets for drug delivery. J Drug Target. 2007; 15: 1-20.
  9. Ridolfi RL, Hutchins GM. The relationship between coronary artery lesions and myocardial infarcts: Ulceration of atherosclerotic plaques precipitating coronary thrombosis. Am Heart J. 1977; 93: 468-486.
  10. Lloyd-Jones D, Adams RJ, Brown TM, Carnethon M, Dai S, De Simone G, et al. Executive summary: heart disease and stroke statistics-2010 update: a report from the American Heart Association. Circulation. 2010; 121: 948-954.
  11. Lam JYT. Atherosclerosis, the Merck Manual for Healthcare Professionals. Whitehouse Station, NJL Merck & Co. Inc. 2008.
  12. Weber B, Hoerstrup SP. Human bioengineered artery models for in vitro atherosclerosis research: fact or fiction? Altern Lab Anim. 2014; 42: 28-32.
  13. Rocha VZ, Libby P. Obesity, inflammation, and atherosclerosis. Nat Rev Cardiol. 2009; 6: 399-409.
  14. Glass CK, Witztum JL. Atherosclerosis. the road ahead. Cell. 2001; 104: 503-516.
  15. Tiwari RL, Singh V, Barthwal MK. Macrophages: an elusive yet emerging therapeutic target of atherosclerosis. Med Res Rev. 2008; 28: 483-544.
  16. Adams WJJ, Zhang Y, Cloutier J, Kuchimanchi P, Newton G, Sehrawat S, et al. Functional Vascular Endothelium Derived from Human Induced Pluripotent Stem Cells. Stem Cell Reports. 2013; 1: 105-113.
  17. Kamei M, Carman CV. New observations on the trafficking and diapedesis of monocytes. Curr Opin Hematol. 2010; 17: 43-52.
  18. Mach F, Schönbeck U, Sukhova GK, Atkinson E, Libby P. Reduction of atherosclerosis in mice by inhibition of CD40 signalling. Nature. 1998; 394: 200-203.
  19. Braunersreuther V, Mach F. Leukocyte recruitment in atherosclerosis: potential targets for therapeutic approaches? Cell Mol Life Sci. 2006; 63: 2079-2088.
  20. Choudhury RP, Lee JM, Greaves DR. Mechanisms of disease: macrophage-derived foam cells emerging as therapeutic targets in atherosclerosis. Nat Clin Pract Cardiovasc Med. 2005; 2: 309-315.
  21. Becker BF, Heindl B, Kupatt C, Zahler S. Endothelial function and hemostasis. Zeitschrift fur Kardiologie. 2000; 89: 160-167.
  22. Schafer A, Bauersachs J. Endothelial Dysfunction, Impaired Endogenous Platelet Inhibition and Platelet Activation in Diabetes and Atherosclerosis. Curr Vasc Pharmacol. 2008; 6: 52-60.
  23. Saini HK, Xu YJ, Arneja AS, Tappia PS, Dhalla NS. Pharmacological basis of different targets for the treatment of atherosclerosis. Journal of Cellular and Molecular Medicine. 2005; 9: 818-839.
  24. Boyle J. Macrophage Activation in Atherosclerosis: Pathogenesis and Pharmacology of Plaque Rupture. Curr Vasc Pharmacol. 2005; 3: 63-68.
  25. Rudijanto A. The role of vascular smooth muscle cells on the pathogenesis of atherosclerosis. Acta medica Indonesiana. 2007; 39: 86-93.
  26. Marcil V, Delvin E, Sané AT, Tremblay A, Levy E. Oxidative stress influences cholesterol efflux in THP-1 macrophages: role of ATP-binding cassette A1 and nuclear factors. Cardiovasc Res. 2006; 72, 473-482.
  27. Crowther MA. Pathogenesis of atherosclerosis. Hematology Am Soc Hematol Educ Program. 2005; 436-441.
  28. Staughton TJ, Weinberg PD. Investigation of the role of endogenous nitric oxide synthesis in determining patterns of arterial wall permeability and diet-include lipid deposition in rabbit. Clark LV, editor. In: Trends in Atherosclerosis Research. Nova Publishers. 2004; 123-144.
  29. Witztum JL. You are right too! J Clin Invest. 2005; 115: 2072-2075.
  30. Tall AR, Yvan-Charvet L, Wang N. The failure of torcetrapib: was it the molecule or the mechanism? Arterioscler Thromb Vasc Biol. 2007; 27: 257-260.
  31. de Haan W, van der Hoogt CC, Westerterp M, Hoekstra M, Dallinga-Thie GM, Princen HM, et al. Atorvastatin increases HDL cholesterol by reducing CETP expression in cholesterol-fed APOE*3-Leiden.CETP mice. Atherosclerosis. 2008; 197: 57-63.
  32. Melchior GW, Castle CK, Murray RW, Blake WL, Dinh DM, Marotti KR. Apolipoprotein A-I metabolism in cholesteryl ester transfer protein transgenic mice. Insights into the mechanisms responsible for low plasma high density lipoprotein levels. J Biol Chem. 1994; 269, 8044-8051.
  33. Truskey GA. Endothelial Cell Vascular Smooth Muscle Cell Co-Culture Assay For High Throughput Screening Assays For Discovery of Anti-Angiogenesis Agents and Other Therapeutic Molecules. Int J High Throughput Screen. 2010; 2010: 171-181.
  34. Li J, Zhang K, Wu J, Liao Y, Yang P, Huang N. Co-culture of endothelial cells and patterned smooth muscle cells on titanium: construction with high density of endothelial cells and low density of smooth muscle cells. Biochem Biophys Res Commun. 2015; 456: 555-561.
  35. Lee C-H, Lin YH, Chang SH, Tai CD, Liu SJ, Chu Y, et al. Local sustained delivery of acetylsalicylic acid via hybrid stent with biodegradable nanofibers reduces adhesion of blood cells and promotes reendothelialization of the denuded artery. Int J Nanomedicine. 2014; 9: 311-326.
  36. Gupta MK, Lee SH, Crowder SW, Wang X, Hofmeister LH, Nelson CE, et al. Oligoproline-derived nanocarrier for dual stimuli-responsive gene delivery. J Mater Chem B. 2015; 3: 7271-7280.
  37. Matuszak J, Zaloga J, Friedrich RP, Lyer S, Nowak J, Odenbach S, et al. Endothelial biocompatibility and accumulation of SPION under flow conditions. J Magn Magn Mater. 2015; 380: 20-26.
  38. Korin N, Kanapathipillai M, Matthews BD, Crescente M, Brill A, Mammoto T, et al. Shear-activated nanotherapeutics for drug targeting to obstructed blood vessels. Science. 2012; 337: 738-742.
  39. Del Turco S, Navarra T, Gastaldelli A, Basta G. Protective role of adiponectin on endothelial dysfunction induced by AGEs: A clinical and experimental approach. Microvasc Res. 2011; 82: 73-76.
  40. Rensing KL, von der Thüsen JH, Weijers EM, Houttuijn Bloemendaal FM, van Lammeren GW, Vink A, et al. Endothelial insulin receptor expression in human atherosclerotic plaques: linking micro- and macrovascular disease in diabetes? Atherosclerosis. 2012; 222: 208-215.
  41. Donnini D, Del Terra E, Ambesi-Impiombato FS, Curcio F. New in vitro model to study high glucose-dependent endothelial dysfunctions. Biochimie. 2003; 85: 701-705.
  42. Carmody BJ, Arora S, Wakefield MC, Weber M, Fox CJ, Sidawy AN. Progesterone inhibits human infragenicular arterial smooth muscle cell proliferation induced by high glucose and insulin concentrations. J Vasc Surg. 2002; 36: 833-838.
  43. Jain SK, Kannan K, Mcvie R. Effect of Hyperketonemia on Blood Monocytes in Type-I Diabetic Patients and Apoptosis in Cultured U937 Monocytes. Antioxidants Redox Signal. 1999; 1: 211-220.
  44. Maisa A, Hearps AC, Angelovich TA, Pereira CF, Zhou J, Shi MD, et al. Monocytes from HIV-infected individuals show impaired cholesterol efflux and increased foam cell formation after transendothelial migration. AIDS. 2015; 29: 1445-1457.
  45. Trion A, Schutte-Bart C, Bax WH, Jukema JW, van der Laarse A. Modulation of calcification of vascular smooth muscle cells in culture by calcium antagonists, statins, and their combination. Mol Cell Biochem. 2008; 308: 25-33.
  46. Yoshida M, Sawada T, Ishii H, Gerszten RE, Rosenzweig A, Gimbrone MA Jr, et al. HMG-CoA reductase inhibitor modulates monocyte-endothelial cell interaction under physiological flow conditions in vitro: Involvement of Rho GTPase-Dependent mechanism. Arterioscler Thromb Vasc Biol. 2001; 21: 1165-1171.
  47. Fujino M, Miura S, Tanigawa H, Matsuo Y, Saku K. Counteracting Effects of High Density Lipoprotein-Cholesterol Subfractions on Statin-Induced Growth Arrest. Cardiovasc Drugs Ther. 2005; 19: 113-118.
  48. Kizu A, Shioi A, Jono S, Koyama H, Okuno Y, Nishizawa Y. Statins inhibit in vitro calcification of human vascular smooth muscle cells induced by inflammatory mediators. J Cell Biochem. 2004; 93: 1011-1019.
  49. Baetta R, Donetti E, Comparato C, Calore M, Rossi A, Teruzzi C, et al. Proapoptotic effect of atorvastatin on stimulated rabbit smooth muscle cells. Pharmacol Res. 1997; 36: 115-121.
  50. Aggarwal BB, Gupta SC, Sung B. Curcumin: an orally bioavailable blocker of TNF and other pro-inflammatory biomarkers. Br J Pharmacol. 2013; 169: 1672-1692.
  51. Wang X, Zhang R, Gu L, Zhang Y, Zhao X, Bi K, et al. Cell-based screening identifies the active ingredients from Traditional Chinese Medicine formula Shixiao San as the inhibitors of atherosclerotic endothelial dysfunction. PLoS One. 2015; 10: e0116601.
  52. Liu L-L, Yan L, Chen YH, Zeng GH, Zhou Y, Chen HP, et al. A role for diallyl trisulfide in mitochondrial antioxidative stress contributes to its protective effects against vascular endothelial impairment. Eur J Pharmacol. 2014; 725: 23-31.
  53. Keller J, Camaré C, Bernis C, Astello-García M, de la Rosa AP, Rossignol M, et al. Antiatherogenic and antitumoral properties of Opuntia cladodes: inhibition of low density lipoprotein oxidation by vascular cells, and protection against the cytotoxicity of lipid oxidation product 4-hydroxynonenal in a colorectal cancer cellular model. J Physiol Biochem. 2015; 71: 577-587.
  54. Wu JM, Hsieh T, Yang C-J, Olson SC. Resveratrol and its metabolites modulate cytokine-mediated induction of eotaxin-1 in human pulmonary artery endothelial cells. Ann N Y Acad Sci. 2013; 1290: 30-36.
  55. Magrone T, Tafaro A, Jirillo F, Panaro MA, Cuzzuol P, Cuzzuol AC, et al. Red Wine Consumption and Prevention of Atherosclerosis: An In Vitro Model Using Human Peripheral Blood Mononuclear Cells. Curr Pharm Des. 2007; 13: 3718-3725.
  56. Huang CY, Sheu WH, Chiang AN. Docosahexaenoic acid and eicosapentaenoic acid suppress adhesion molecule expression in human aortic endothelial cells via differential mechanisms. Mol Nutr Food Res. 2015; 59: 751-762.
  57. Massaro M, De Caterina R. Vasculoprotective effects of oleic acid: Epidemiological background and direct vascular antiatherogenic properties. Nutrition Metabolism and Cardiovascular Diseases. 2002; 12: 42-51.
  58. Massaro M, Carluccio MA, De Caterina R. Direct vascular antiatherogenic effects of oleic acid: a clue to the cardioprotective effects of the Mediterranean diet. Cardiologia. 1999; 44: 507-513.
  59. Tabas I, Garcia-Cardena G, Owens GK. The cell biology of disease: Recent insights into the cellular biology of atherosclerosis. J Cell Biol. 2015; 209: 13-22.
  60. Gimbrone MA, García-Cardeña G. Vascular endothelium, hemodynamics, and the pathobiology of atherosclerosis. Cardiovasc Pathol. 2013; 22: 9-15.
  61. Shaul PW. Regulation of endothelial nitric oxide synthase: location, location, location. Annu Rev Physiol. 2002; 64: 749-774.
  62. Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation. 2002; 105: 1135-1143.
  63. Baeuerle PA. IkappaB-NF-kappaB structures: at the interface of inflammation control. Cell. 1998; 95: 729-731.
  64. Gordon S. The macrophage: Past, present and future. Eur J Immunol. 2007; 37: 9-17.
  65. Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat Rev Immunol. 2005; 5: 953-964.
  66. Gordon S. Alternative activation of macrophages. Nat Rev Immunol. 2003; 3: 23-35.
  67. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol. 2004; 25: 677-686.
  68. Van Ginderachter JA, Movahedi K, Hassanzadeh Ghassabeh G, Meerschaut S, Beschin A, Raes G, et al. Classical and alternative activation of mononuclear phagocytes: picking the best of both worlds for tumor promotion. Immunobiology. 2006; 211: 487-501.
  69. Leitinger N, Schulman IG. Phenotypic Polarization of Macrophages in Atherosclerosis. Arterioscler Thromb Vasc Biol. 2013; 33: 1120-1126.
  70. Davies PF, Truskey Ga, Warren HB, Oconnor SE, Eisenhaure BH. Metabolic Cooperation between Vascular Endothelial-Cells and Smooth-Muscle Cells in Co-Culture: Changes in Low-Density Lipoprotein Metabolism. J Cell Biol. 1985; 101: 871-879.
  71. Navab M, Hama SY, Van Lenten BJ, Drinkwater DC, Laks H, Fogelman AM. A new antiinflammatory compound, leumedin, inhibits modification of low density lipoprotein and the resulting monocyte transmigration into the subendothelial space of cocultures of human aortic wall cells. J Clin Invest. 1993; 91: 1225-1230.
  72. Navab M, Imes SS, Hama SY, Hough GP, Ross LA, Bork RW, et al. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein. J Clin Invest. 1991; 88: 2039-2046.
  73. Lee E, Grodzinsky AJ, Libby P, Clinton SK, Lark MW, Lee RT. Human vascular smooth muscle cell-monocyte interactions and metalloproteinase secretion in culture. Arterioscler Thromb Vasc Biol. 1995; 15: 2284-2289.
  74. Zhu Y, Hojo Y, Ikeda U, Takahashi M, Shimada K. Interaction between monocytes and vascular smooth muscle cells enhances matrix metalloproteinase-1 production. J Cardiovasc Pharmacol. 2000; 36, 152-161.
  75. Vijayagopal P, Luke Glancy D. Macrophages stimulate cholesteryl ester accumulation in cocultured smooth muscle cells incubated with lipoprotein-proteoglycan complex. Arterioscler Thromb Vasc Biol. 1996; 16: 1112-1121.
  76. Halloran BG, Grange JJ, So BJ, Baxter BT. Macrophage Products Inhibit Human Aortic Smooth Muscle Cell Proliferation and Alter 1α (I) Procollagen Expression. Ann Vasc Surg. 1997; 11: 80-84.
  77. Zhang JC, Ruan Q, Paucz L, Fabry A, Binder BR, Wojta J. Stimulation of tissue factor expression in human microvascular and macrovascular endothelial cells by cultured vascular smooth muscle cells in vitro. J Vasc Res. 1999; 36: 126-132.
  78. Tintut Y, Patel J, Territo M, Saini T, Parhami F, Demer LL. Monocyte/Macrophage Regulation of Vascular Calcification in Vitro. Circulation. 2002; 105: 650-655.
  79. Shioi A, Katagi M, Okuno Y, Mori K, Jono S, Koyama H, et al. Induction of Bone-Type Alkaline Phosphatase in Human Vascular Smooth Muscle Cells: Roles of Tumor Necrosis Factor-alpha and Oncostatin M Derived from Macrophages. Circ Res. 2002; 91: 9-16.
  80. Korff T, Kimmina S, Martiny-Baron G, Augustin HG. Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness. FASEB J. 2001; 15: 447-457.
  81. Graham DJ, Alexander JJ, Miguel R. Aortic Endothelial and Smooth Muscle Cell Co-Culture: An In Vitro Model of the Arterial Wall. J Investig Surg. 1991; 4: 487-494.
  82. Fillinger MF, Sampson LN, Cronenwett JL, Powell RJ, Wagner RJ. Coculture of endothelial cells and smooth muscle cells in bilayer and conditioned media models. J Surg Res. 1997; 67: 169-178.
  83. Navab M, Hough GP, Stevenson LW, Drinkwater DC, Laks H, Fogelman AM. Monocyte migration into the subendothelial space of a coculture of adult human aortic endothelial and smooth muscle cells. J Clin Invest. 1988; 82: 1853-1863.
  84. Bogdanowicz DR, Lu HH. Studying cell-cell communication in co-culture. Biotechnol J. 2013; 8: 395-396.
  85. Proudfoot D, Fitzsimmons C, Torzewski J, Bowyer D. Inhibition of human arterial smooth muscle cell growth by human monocyte/macrophages: a co-culture study. Atherosclerosis. 1999; 145: 157-165.
  86. Fitzsimmons C, Proudfoot D, Bowyer DE. Monocyte prostaglandins inhibit procollagen secretion by human vascular smooth muscle cells: implications for plaque stability. Atherosclerosis. 1999; 142: 287-293.
  87. Nam MH, Lee HS, Seomun Y, Lee Y, Lee KW. Monocyte-endothelium-smooth muscle cell interaction in co-culture: proliferation and cytokine productions in response to advanced glycation end products. Biochim Biophys Acta. 2011; 1810: 907-912.
  88. Boyle JJ, Weissberg PL, Bennett MR. Tumor necrosis factor-alpha promotes macrophage-induced vascular smooth muscle cell apoptosis by direct and autocrine mechanisms. Arterioscler Thromb Vasc Biol. 2003; 23: 1553-1558.
  89. Cai Q, Lanting L, Natarajan R. Interaction of monocytes with vascular smooth muscle cells regulates monocyte survival and differentiation through distinct pathways. Arterioscler Thromb Vasc Biol. 2004; 24: 2263-2270.
  90. Khallou-Laschet J, Varthaman A, Fornasa G, Compain C, Gaston AT, Clement M, et al. Macrophage plasticity in experimental atherosclerosis. PLoS One. 2010; 5: e8852.
  91. Westhorpe CLV, Dufour EM, Maisa A, Jaworowski A, Crowe SM, Muller WA. Endothelial cell activation promotes foam cell formation by monocytes following transendothelial migration in an in vitro model. Exp Mol Pathol. 2012; 93: 220-226.
  92. Paschos NK, Brown WE, Eswaramoorthy R, Hu JC, Athanasiou KA. Advances in tissue engineering through stem cell-based co-culture. J Tissue Eng Regen Med. 2015; 9: 488-503.
  93. Vasudevan S, Lopes NH, Seshiah PN, Wang T, Marsh CB, Kereiakes DJ, et al. Mac-1 and Fas activities are concurrently required for execution of smooth muscle cell death by M-CSF-stimulated macrophages. Cardiovasc Res. 2003; 59: 723-733.
  94. Meng L, Park J, Cai Q, Lanting L, Reddy MA, Natarajan R. Diabetic conditions promote binding of monocytes to vascular smooth muscle cells and their subsequent differentiation. Am J Physiol Heart Circ Physiol. 2010; 298: 736-745.
  95. Chaterji S, Park K, Panitch A. Scaffold-free in vitro arterial mimetics: the importance of smooth muscle-endothelium contact. Tissue Eng Part A. 2010; 16: 1901-1912.
  96. Bartis D, Judit P. Three dimensional tissue cultures and tissue engineering. 2011.
  97. van Buul-Wortelboer MF, HJM Brinkman, KP Dingemans, PG De Groot, WG van Aken, JA van Mourik. Reconstitution of the vascular wall in vitro. A novel model to study interactions between endothelial and smooth muscle cells. Exp Cell Res. 1986; 162: 151-158.
  98. Takaku M, Wada Y, Jinnouchi K, Takeya M, Takahashi K, Usuda H, et al. An in vitro coculture model of transmigrant monocytes and foam cell formation. Arterioscler Thromb Vasc Biol. 1999; 19: 2330-2339.
  99. Wada Y, Sugiyama A, Kohro T, Kobayashi M, Takeya M, Naito M, et al. In vitro model of atherosclerosis using coculture of arterial wall cells and macrophage. Yonsei Med J. 2000; 41: 740-755.
  100. Randolph GJ, Luther T, Albrecht S, Magdolen V, Muller WA. Role of tissue factor in adhesion of mononuclear phagocytes to and trafficking through endothelium in vitro. Blood. 1998; 92: 4167-4177.
  101. Lavender MD, Pang Z, Wallace CS, Niklason LE, Truskey GA. A system for the direct co-culture of endothelium on smooth muscle cells. Biomaterials. 2005; 26: 4642-4653.
  102. Dorweiler B, Torzewski M, Dahm M, Ochsenhirt V, Lehr HA, Lackner KJ, et al. A novel in vitro model for the study of plaque development in atherosclerosis. Thromb Haemost. 2006; 95: 182-189.
  103. Chen CN, Chang SF, Lee PL, Chang K, Chen LJ, Usami S, et al. Neutrophils, lymphocytes, and monocytes exhibit diverse behaviors in transendothelial and subendothelial migrations under coculture with smooth muscle cells in disturbed flow. Blood. 2006; 107: 1933-1942.
  104. Chen L, Frister A, Wang S, Ludwig A, Behr H, Pippig S, et al. Interaction of vascular smooth muscle cells and monocytes by soluble factors synergistically enhances IL-6 and MCP-1 production. Am J Physiol Heart Circ Physiol. 2009; 296: 987-996.
  105. Ziegler T, Alexander RW, Nerem RM. An endothelial cell-smooth muscle cell co-culture model for use in the investigation of flow effects on vascular biology. Ann Biomed Eng. 1995; 23: 216-225.
  106. Cicha I, Urschel K, Daniel WG, Garlichs CD. Telmisartan prevents VCAM-1 induction and monocytic cell adhesion to endothelium exposed to non-uniform shear stress and TNF-α. Clin Hemorheol Microcirc. 2011; 48: 65-73.
  107. Li R, Mittelstein D, Lee J, Fang K, Majumdar R, Tintut Y, et al. A dynamic model of calcific nodule destabilization in response to monocyte- and oxidized lipid-induced matrix metalloproteinases. Am J Physiol Cell Physiol. 2012; 302: 658-665.
  108. Robert J, Weber B, Frese L, Emmert MY, Schmidt D, von Eckardstein A, et al. A three-dimensional engineered artery model for in vitro atherosclerosis research. PLoS One. 2013; 8: 79821.
  109. Lindmark H, Rosengren B, Hurt-Camejo E, Bruder CEG. Gene expression profiling shows that macrophages derived from mouse embryonic stem cells is an improved in vitro model for studies of vascular disease. Exp Cell Res. 2004; 300: 335-344.
  110. Potter DR, Damiano ER. The hydrodynamically relevant endothelial cell glycocalyx observed in vivo is absent in vitro. Circ Res. 2008; 102: 770-776.
  111. Timraz SBH, Farhat IAH, Alhussein G, Christoforou N, Teo JCM. In-depth evaluation of commercially available human vascular smooth muscle cells phenotype: Implications for vascular tissue engineering. Exp Cell Res. 2016; 343: 168-176.
  112. Yoder MC. Human endothelial progenitor cells. Cold Spring Harbor Perspectives in Medicine. 2012; 2: 006692.
  113. Ji H, Atchison L, Chen Z, Chakraborty S, Jung Y, Truskey GA, et al. Transdifferentiation of human endothelial progenitors into smooth muscle cells. Biomaterials. 2016; 85: 180-194.
  114. Timraz SBH, Rezgui R, Boularaoui SM, Teo JCM. Stiffness of extracellular matrix components modulates the phenotype of human smooth muscle cells in vitro and allows for the control of properties of engineered tissues. Procedia Eng. 2015; 110: 29-36.
  115. Weinberg CB, Bell E. A blood vessel model constructed from collagen and cultured vascular cells. Science. 1986; 231: 397-400.
  116. Nackman GB, Bech FR, Fillinger MF, Wagner RJ, Cronenwett JL. Endothelial cells modulate smooth muscle cell morphology by inhibition of transforming growth factor-beta 1 activation. Surgery. 1996; 120: 418-425.
  117. Ishikawa K, Navab M, Leitinger N, Fogelman AM, Lusis AJ. Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL. J Clin Invest. 1997; 100: 1209-1216.
  118. Wada Y, Sugiyama A, Kohro T, Kobayashi M, Takeya M, Naito M, et al. In vitro Model of Atherosclerosis Using Coculture of Arterial Wall Cells and Macrophage. Yonsei Med J. 2000; 41: 740-755.
  119. Rainger GE, Stone P, Morland CM, Nash GB. A novel system for investigating the ability of smooth muscle cells and fibroblasts to regulate adhesion of flowing leukocytes to endothelial cells. J Immunol Methods. 2001; 255: 73-82.
  120. Voisard R, Fischer R, Osswald M, Voglic S, Baur R, Susa M, et al. Aspirin (5 mmol/L) inhibits leukocyte attack and triggered reactive cell proliferation in a 3D human coronary in vitro model. Circulation. 2001; 103: 1688-1694.
  121. Seshiah PN, Kereiakes DJ, Vasudevan SS, Lopes N, Su BY, Flavahan NA, et al. Activated Monocytes Induce Smooth Muscle Cell Death: Role of Macrophage Colony-Stimulating Factor and Cell Contact. Circulation. 2002; 105: 174-180.
  122. Kunigal S, Kusch A, Tkachuk N, Tkachuk S, Jerke U, Haller H, et al. Monocyte-expressed urokinase inhibits vascular smooth muscle cell growth by activating Stat1. Blood. 2003; 102: 4377-4383.
  123. Kallenbach K, Fernandez HA, Seghezzi G, Baumann FG, Patel S, Grossi EA, et al. A Quantitative In Vitro Model of Smooth Muscle Cell Migration through the Arterial Wall Using the Human Amniotic Membrane. Arterioscler Thromb Vasc Biol. 2003; 23: 1008-1013.
  124. Chiu JJ, Chen LJ, Chen CN, Lee PL, Lee CI. A model for studying the effect of shear stress on interactions between vascular endothelial cells and smooth muscle cells. J Biomech. 2004; 37: 531-539.
  125. Niwa K, Kado T, Sakai J, Karino T. The effects of a shear flow on the uptake of LDL and acetylated LDL by an EC monoculture and an EC-SMC coculture. Ann Biomed Eng. 2004; 32: 537-543.
  126. Chiu JJ, Chen LJ, Chang SF, Lee PL, Lee CI, Tsai MC, et al. Shear stress inhibits smooth muscle cell-induced inflammatory gene expression in endothelial cells: role of NF-kappaB. Arterioscler Thromb Vasc Biol. 2005; 25: 963-969.
  127. Tull SP, Anderson SI, Hughan SC, Watson SP, Nash GB, Rainger GE. Cellular pathology of atherosclerosis: smooth muscle cells promote adhesion of platelets to cocultured endothelial cells. Circ Res. 2006; 98: 98-104.
  128. Dorweiler B, Torzewski M, Dahm M, Kirkpatrick CJ, Lackner KJ, Vahl CF. Subendothelial infiltration of neutrophil granulocytes and liberation of matrix-destabilizing enzymes in an experimental model of human neo-intima. Thromb Haemost. 2008; 99: 373-381.
  129. Wallace CS, Truskey GA. Direct-contact co-culture between smooth muscle and endothelial cells inhibits TNF-alpha-mediated endothelial cell activation. Am J Physiol Heart Circ Physiol. 2010; 299: 338-346.
  130. Li X, Speer MY, Yang H, Bergen J, Giachelli CM. Vitamin D receptor activators induce an anticalcific paracrine program in macrophages: requirement of osteopontin. Arterioscler Thromb Vasc Biol. 2010; 30: 321-326.
  131. Butoi ED, Gan AM, Manduteanu I, Stan D, Calin M, Pirvulescu M, et al. Cross talk between smooth muscle cells and monocytes/activated monocytes via CX3CL1/CX3CR1 axis augments expression of pro-atherogenic molecules. Biochim Biophys Acta. 2011; 1813: 2026-2035.
  132. Gan AM, Pirvulescu MM, Stan D, Simion V, Calin M, Manduteanu I, et al. Monocytes and smooth muscle cells cross-talk activates STAT3 and induces resistin and reactive oxygen species production [corrected]. J Cell Biochem. 2013; 114: 2273-2283.
  133. Weinert S, Poitz DM, Auffermann-Gretzinger S, Eger L, Herold J, Medunjanin S, et al. The lysosomal transfer of LDL/cholesterol from macrophages into vascular smooth muscle cells induces their phenotypic alteration. Cardiovasc Res. 2013; 97: 544-552.
  134. Shav D, Gotlieb R, Zaretsky U, Elad D, Einav S. Wall shear stress effects on endothelial-endothelial and endothelial-smooth muscle cell interactions in tissue engineered models of the vascular wall. PLoS One. 2014; 9: e88304.

Citation: Islam K, Timraz SBH, Nasser R, Gater DL, Pearson YE, Christoforou N, et al. Co-culture Methods Used to Model Atherosclerosis In Vitro Using Endothelial, Smooth Muscle and Monocyte Cells. SM J Biomed Eng. 2016; 2(1): 1008.

Download PDF