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Effects of Membrane Cholesterol and Substrate Stiffness on Vascular Smooth Muscle Cell Adhesion Mechanics

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ABSTRACT

Atherosclerosis remains a major cause of cardiovascular disease (CVD). As a major contributor to the cause of atherosclerosis cholesterol causes fatty deposition in the atherosclerotic lesion, and may also play a critical role in the vascular smooth muscle cell (VSMC) migration toward the intima of the blood vessel wall. In addition, the arterial wall experiences stiffening with the progression of atherosclerosis altering the micromechanical environment of VSMCs modifying cell stiffness, adhesion, and phenotype. Migration of VSMCs in the atherosclerotic environment is a dynamic and complex process including proliferation and phenotypic switching of VSMCs, thus contributing too many changes in cell membrane adhesion molecules. Therefore, this study examines the important role that membrane cholesterol may play in VSMCs α5β1-integrin mediated adhesion, and alteration in the sensory function of VSMCs to ECM mechanical properties. Cholesterol manipulation was achieved using methyl-β-cyclodextrin, and gel substrates with varying stiffness were used to mimic changing environmental mechanical properties in atherosclerosis. Atomic force microscopy (AFM) was used to determine integrin-fibronectin adhesion force and cell stiffness. Additionally, image processing was performed to visualize changes in cytoskeletal actin orientation and area fraction. Our findings show a significant decrease in α5β1-integrin adhesion of VSMCs upon membrane cholesterol depletion. Furthermore, mechanotransduction of VSMCs upon cholesterol depletion is less efficient. Image processing revealed that cholesterol depletion and softer gel substrates caused actin filaments to disorganize in orientation. Collectively these data indicate that cell membrane cholesterol and extracellular mechanical signals may synergistically regulate cellular mechanical functions of VSMCs and their migration in the progression of atherosclerosis.

This abstract is approved as to form and content. I recommend its publication.

ACKNOWLEDGEMENTS

 

I am very grateful for my advisor, Dr. Zhongkui Hong

TABLE OF CONTENTS

Page

Committee signature page…………………………………………iii

Abstract……………………………………………………….iv

Acknowledgements………………………………………………v

Table of contents………………………………………………..vii

List of Figures…………………………………………………..xi

List of Tables…………………………………………………..xii

1. General Introduction…………………………………………….1

2. Background

  1. Disease Description…………………………………..3
    1.      Defining the environment………………………….3
    2.      Lipoproteins
    3.      Pathophysiology of Atherosclerosis……………………4
    4.      Phenotypic Switch………………………………………..6
  2. Biomechanics……………………………………………………………………………………………….10
    1.      Fibronectin………………..………………………………………………………11
    2.      Integrin…………………………..12
    3.      Actin……………………………………….14
    4.      Adhesion Mechanics………………………………15
    5.      AFM……………………………………….18
  3. Specific Aims…………….20

3. Substrate Engineering

4. Cholesterol Manipulation

5. Biomechanical Analysis

6. Actin Cortex Analysis

7. Discussion

  1.            References………………………………………………..58

LIST OF FIGURES

LIST OF TABLES

Table 1: PA Substrate Stiffness Analysis………………………………28

CHAPTER 1

Introduction

 

Much research has been dedicated to the study and cure of atherosclerosis, the major cause of cardiovascular disease (CVD). However, it still remains the primary issue resulting in more deaths worldwide than any other disease [1]. Atherosclerosis is a complex process and the exact cause of the disease is unclear, however factors that damage the inner arterial wall, such as smoking, high blood pressure, and high amounts of sugar or cholesterol in the blood, have been shown to accelerate the process [23]. Atherosclerotic plaque is built up in the arterial wall restricting and possibly blocking blood flow [4]. This can lead to, depending on the location of the plaque and the diseased artery, heart attack, stroke, chronic kidney disease, and aneurysms [5]. CVD effects 50% of males and 33% of females worldwide aged 40 and above with total direct medical costs for treatment reaching $273 billion in 2010, and a projected $818 billion in 2030 [6]. It is, therefore, essential to gain a better understanding of the mechanisms underlying the progression of atherosclerosis in order to bring forth a cure.

As atherosclerosis progresses high concentrations of cholesterol, or more specifically low-density lipoprotein (LDL), enter the tunica intima of the arterial wall as a result of endothelial dysfunction [7]. Reactive oxygen species (ROS) oxidize LDL producing oxidized LDL (oxLDL) eliciting an immune response. Monocytes infiltrate the vascular wall becoming macrophages, engulfing large amounts of oxLDL, and leading to foam cell formation [8], which functions as a constituent of the atherosclerotic plaque. However, a recent study suggests that cholesterol accumulation in vascular smooth muscle (VSMCs), located in the tunica media of the arterial wall, is larger than previously recognized, and approximately 40% of total foam cells present in the atherosclerotic plaque are VSMC-derived [9]. VSMCs make up a larger portion of the artery and are known to undergo a phenotypic switch from a contractile to synthetic cell. The synthetic cell promotes secretion of fibrous extracellular matrix (ECM) [10] causing the artery to stiffen altering the cellular environment, or may act macrophage-like migrating towards the atherosclerotic plaque. Little is known about the mechanical mechanisms involved with the migration process of the lipid-laden synthetic cells, which contribute to plaque formation, or the cellular effects of a stiffening microenvironment

Biomechanical analysis offers unique insight into understanding the underlying mechanisms involved with atherosclerosis. This project aims to advance understanding of how cholesterol affects the response of VSMCs to the alterations in ECM mechanical properties and offer a unique insight into the pathomechanical role of VSMCs in atherosclerosis. This thesis details the biomechanical analysis of adhesion between α5β1-Integrin and fibronectin (FN). In addition, the changes in cell elasticity as well as actin cytoskeletal remodeling are examined to study the combined effects of ECM stiffness and cellular cholesterol on the biomechanical and physiological functions of VSMCs.

CHAPTER 2

Background

2.1 Disease Description

The development and progression of atherosclerosis is a complex process involving a myriad of different factors. Occurring in the intima of the artery wall, due to the accumulation of excess low density lipoproteins (LDL), the native cells experience a plethora of changes as the microenvironment is progressively altered.

2.1.1 Defining the Environment

 Arteries are blood vessels that carry oxygenated blood away from the heart, with the exception of the pulmonary artery. Arteries are thicker than veins in order to accommodate pulsating blood flow. There are three main layers that make up an artery, moving from the inner layer to the outer layer with respect to the lumen, the tunica intima, tunica media, and tunica adventitia (Figure 1). The intima is made up of a monolayer of endothelial cells and collagen fibrils. SMCs, and elastic and collagen fibrils make up the media which is divided into transversely isotropic units by an elastic laminae [11]. Elastic fibers allow the artery to resist plastic deformation becoming flexible, while the outer collagen fibers organized in helical structures found in the adventitia provide strength, protection, and rigidity [12].  The endothelia cells are in contact with blood and form a monolayer lining the lumen of the blood vessel separating the arterial wall from the circulating blood and associated components [7]. The endothelium directs the action of the SMCs, which make up the majority of the blood vessel and are involved with dilation and contraction of the blood vessel, by releasing various vasoactive factors.

A

 

B

C

 

D

 

E

 

F

Figure 1: Cross section, black square, showing components of an artery. The inner most layer with respect to the lumen is the Tunica Intima consists of the (A) endothelium, (B) basement membrane, and (C) internal elastic lamina. The medial layer consists of (D) vascular smooth muscle cells, and (D) external elastic lamina. The outer most layer, the Tunica Adventitia, is made up of (F) connective tissue. Blood flows through the lumen, which is the hollow portion of the artery.

In a diseased environment however, excess LDL molecules enter the tunica intima of the arterial wall causing inflammation and the formation of an atheroma. The atheroma is generally made up of a fibrous cap, macrophage-derived foam cells, apoptotic bodies, and lipid –rich necrotic core (Figure 2). The fibrous cap consists of SMCs, type I and III collagen, and proteoglycans, which covers the necrotic core preventing plaque rupture and thrombosis [13]. The lipid-rich core is avascular, devoid of supporting collagen, and soft. An early necrotic core usually exhibits proteoglycans and hyaluronan. Whereas in the late necrotic core these ECM constituents are no longer present due to degradation from metalloproteinases. Also, a higher concentration of free cholesterol is observed most likely from cell death of macrophages and SMCs [14]. The size of the core is dependent on the stability of the plaque [5], which  in turn depends on the integrity and thickness of the fibrous cap.

Lumen

Endothelium

Fibrous cap

Necrotic core

Macrophage-foam cell

Intima

Media

Figure 2: Basic anatomy of the atheroma

2.1.2 Lipoproteins

Cholesterol, a sterol lipid, circulated throughout the blood vessels of the cardiovascular system is delivered to cells in fat and protein transport vesicles. Fat and protein transport vesicles consist of four different types, chylomicron, very dense lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL), with the amount of protein increasing and fat decreasing within each listed lipoprotein, respectively. Ingested fats arrive in the small intestine as lipid droplets, which are emulsified and absorbed into simple columnar epithelial cells that line the lumen of the small intestine, and are broken down to monoglycerides and fatty acids for packaging with cholesterol and apoproteins forming chylomicrons. Chylomicrons are absorbed into the blood vessel via the lymphatic system transporting fats to tissues as an energy source [15]. The remnants circulate through the circulatory system until arriving at the liver. The liver produces HDL and VLDL, HDL collects excess cholesterol and is commonly referred to as “good cholesterol”. VLDL receives the chylomicron remnants through the Golgi apparatus, and then transports the fatty acids to tissues [15]. Once liberating triglycerides, via lipases, VLDL becomes LDL commonly referred to as “bad cholesterol”. LDL is primarily responsible for transporting cholesterol to tissues, which is necessary for synthesis of hormones, transcription factors, and maintenance of plasma membrane integrity. In a non-diseased environment LDL returns to the liver undergoing endocytosis, where the lipoprotein is recycled or excreted in bile along with excess cholesterol [16].

In a diseased environment resulting from many different risk factors, such as obesity, smoking, genetic disorders, fatty diet, and high blood pressure, the lipid metabolism pathway becomes disrupted resulting in higher amounts of LDL present in the blood. Some studies have also suggested the atherogenic potential of VLDL [17]. Therefore, as cholesterol is regarded as the sine qua non of atherosclerosis, it is imperative to understand how this lipid affects key contributors to disease progression.

2.1.3 Pathophysiology of Atherosclerosis

Atherosclerosis is a life threatening disease in which deposits of fatty material from excess cholesterol, called plaque, build up inside the walls of arteries reducing or in some cases blocking blood flow.  This process usually takes place in atherosusceptible areas of low shear stress with oscillating flow, typically in branching or areas of curvature.[18]. Studies suggests that the cause of atherosclerosis is due to endothelial dysfunction [1920], which occurs due to vascular injury causing increased endothelial permeability (Figure 3) and decreased nitric oxide (NO) bioavailability. This is known as the Original theory.

Upon vascular injury and endothelial dysfunction circulating LDLs move down the concentration gradient through the permeable endothelial monolayer into the tunica intima, and are retained in the proteoglycans produced by SMCs. Proteoglycans exist in an extended conformation due to being negatively charged from the attached glycosaminoglycans (GAGs) [21]. The extended conformation allows the glycoprotein and GAGs to act, in a sense like glue. It is thought that LDL particles interact with reactive oxygen species (ROS) in the intima of the arterial wall undergoing oxidation, thus exacerbating the inflammatory response [522]. The activated endothelium produce chemoattractants and express adhesion receptors, primarily vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1), for recruitment of T cells and monocytes [23] . As monocytes move through the endothelial monolayer they differentiate to macrophages, which have scavenger receptors that recognize and take up the excess oxidized LDL (oxLDL) [17] (Fig. 3-B).  Oxidized modification of the LDL molecule prevents macrophages from down regulating scavenger receptors to avoid taking in large amounts of cholesterol via the sterol regulatory element binding protein (SREBP) pathway. Therefore, the scavenger receptors  up-regulate resulting in avid phagocytosis of oxLDL resulting in foam cell formation [22], the accumulation of which produces a fatty streak. It should be noted that the mention of LDL oxidation does not refer to the LDL oxidation hypothesis linking oxidized lipoproteins to the originating cause of inflammation, which although theoretically compelling remains unproven [17].

Inflammatory signals emitted from T cells and foam cells, as well as reduced NO levels, stimulate pro-migratory and pro-proliferator [24] effects on SMC leading to increased synthesis of dense collagen [10] forming an atheroprotective cap (Fig. 3-C). Lipid-laden foam cells undergo senescence and die releasing free cholesterol and proinflammatory cytokines. Activated T cells, the second largest leukocyte population in atherosclerosis after monocytes, enter the plaque releasing type II interferon, which recruits additional T cells [25], increases inflammation, inhibits VSMC collagen secretion, and activates macrophages. Activated macrophages in the atheroma are capable of physically disrupting the plaque by producing proteolytic enzymes. These enzymes degrade ECM proteins such as collagen, thus thinning of the fibrous cap resulting in plaque rupture and thrombosis formation (Fig. 3-D). Thrombosis causes local coagulation of platelets and clotting factors, and the development of a thrombus impeding blood flow.

Figure 3: Schematic of the progression of atherosclerosis. (A) Shows the native blood vessel prior to injury. (B) The infiltration of T cells and monocytes, and the formation of macrophage-derived foam cells. (C) The migration and proliferation of SMCs, and the formation of the fibrous cap. (D) Plaque rupture into the lumen forming a thrombus [4].

2.1.4 Phenotypic Switch 

The central dogma that atherosclerotic plaque is predominantly made up of macrophages and macrophage-derived foam cells relative to SMCs, and that SMCs are regarded as having atheroprotective properties stabilized the plaque, whereas macrophages are viewed as atheropromoting causing plaque de-stabilize, is not entirely correct. A growing body of evidence has revealed the ever expanding role of SMCs in the pathogenesis of atherosclerosis [426-28].

As atherosclerosis progresses SMCs undergo a phenotypic switch from a contractile phenotype to a synthetic phenotype [29]. The synthetic SMC migrates from the media of the arterial wall towards the diseased area, in the intima, either secreting atherogenic ECM proteins such as hyaluronan promoting SMC migration [3031], atheroprotective proteins such as collagen forming a fibrous cap [32], or acting macrophage-like phagocytizing oxLDL [27](Figure 4-A). SMC-specific conditional lineage tracing studies have provided evidence of phenotypic switching in vivo [2933-35], and a recent study reported that approximately 40% of total foam cells in the atherosclerotic plaque were SMC-derived [9].

Foam cell formation, illustrated in Figure 4-B, is thought to occur as synthetic SMC-derived ECM proteoglycans facilitate the retention of LDL upon endothelial dysfunction [14]. The accumulated ECM-lipoproteins are oxidized, while SMC receptors specific for cholesterol phagocytosis are up-regulated for macrophage-like cholesterol engulfment. As mentioned previously and similar to macrophages, the modified oxLDL avoids recognition from the SREBP pathway, thus preventing SMCs from up-regulating cholesterol efflux proteins. This results in large amounts of cholesterol intake within the SMC leading to possible apoptosis, or foam cell formation. The apoptotic cell and/or foam cell release cytokines exacerbating inflammation. The presence of the foam cells and the additional inflammation contribute to plaque formation and plaque de-stabilization.

Figure 4: (Fig. 4-A) Schematic of the current understanding of VSMCs and the possible derivatives in the progression of atherosclerosis [29]. The solid lines represent known pathways, whereas dotted lines and “?” represent supposed pathways. (Fig. 4-B) A representation of VSMC-derived foam cell formation [36]. (1) A phenotypic switch occurs within the SMC. (2) Secretion of ECM. (3) The ECM retains lipoproteins. (4) Lipoproteins are oxidized and engulfed by the SMC leading to the formation of a foam cell. (5) Accumulation of cholesterol can also induce apoptosis, necrosis. (6) The resulting processes can contribute to increased inflammation releasing cytokines.

Although, the exact cause of phenotypic switching and the distinction between which SMC derivative, atheroprotective or atheropromotive, will occur is currently unknown. Biomechanical and biochemical alterations in the diseased microenvironment influence the SMC. Notably, loading of cholesterol into the SMC induces expression of macrophage markers, and causes phagocytic activity [37]. Similarly, arterial VSMCs isolated from cholesterol-fed atherosclerotic rabbits showed a progressive elevation in the membrane unesterified cholesterol-phospholipid mole ratio [3839]. Studies have shown that cholesterol may exhibit regulatory capabilities on cell mechanics [40-42], spreading [4344], and migration [45]. Additionally, SMCs experiences reorganization and remodeling of actin cytoskeletal structure due to disease induced alteration in the cell microenvironment [46]. A recent publication linked cytoskeletal remodeling in SMCs to extracellular stimuli [47], suggesting that cholesterol may be able to influence this process. These findings demonstrate the potential biomechanical regulatory role of cholesterol in altering SMC membrane structure and organization in vivo, modifying the actin cytoskeleton, and influencing cell function.

2.2 Biomechanics

Cell biomechanics focuses on the mechanical properties and behavior of living cells. Cells experience and maintain mechanical stimuli as their normal physiology, and are a critical intermediate amid molecular mechanotransduction and tissue function [48]. Cells, via integrins, detect and respond to mechanical signals from the ECM through cytoskeletal re-organization and force generation. This mechanosensing process directly affects cell biomechanics. In a diseased state, disruptions in cytoskeletal architecture or adherence mechanisms may alter the elastic and adhesive mechanical properties of the cell. Significant changes in cell mechanics affects cell function, which can affect higher levels of organization eventually affecting major organs or systems (Figure 5). The study of cell biomechanics can provide an in-depth knowledge of the mechanisms underlying the progression of atherosclerosis.

Figure 5: Illustration of the hierarchical organization of integrins, SMCs, and the aorta [49].

2.2.1 Fibronectin

The ECM of the arterial wall experiences progressive stiffening with hypertension, which may play a regulatory role altering the cell mechanical environment accelerating the progression of atherosclerosis [50]SCMs sense and respond to mechanical changes in ECM proteins, which consist of more than 300 proteins, 200 glycoproteins, and 30 proteoglycans [21]. Therefore, considering the complexity, the ECM serves not just for structural support, but rather for biochemical and biomechanical instruction influencing cell behavior [48].

Prior to collagen expression during atherosclerosis and hypertension, FN is expressed by SMCs as an ECM protein upon vascular injury [2851]. FN is a glycoprotein consisting of two ~250 kDa polypeptide chains with 3 different repeats, FN-1, FN-II, and FN-III [52]. There are two forms of FN, plasma and cellular [53]. Hepatocytes produce plasma FN, while SMCs primarily produce cellular FN. Increased cellular FN provides an avenue for SMC migration and increased proliferation [54], and has been associated with promoting atherosclerosis as well as promoting formation of the fibrous cap [27].  SMCs in the native environment exist on a basement membrane of type IV collagen possibly providing a migratory “brake” [55]; during disease progression the inflammatory response produces ECM degrading enzymes such as metalloproteinase. These enzymes degrade the basement membrane [56] causing SMCs exposure to interstitial ECM such as types I and II collagen, and FN, which bind to avb3 and a5b1integrins, respectively.  Interestingly, a recent study reported that the deletion of plasma FN reduced the amount of SMCs found in the atherosclerotic plaque [57]. These findings imply the importance of FN-binding cell receptors in relation to atherosclerosis.

2.2.2 Integrin

The cells ability to sense and respond to external stimuli is known as mechanotransduction, and integrins are an intrinsic part of this process. Integrins are heterodimers of non-covalently associated subunits, α and β. The integrin family is made up of 24 different heterodimer combinations (18 α and 8 β) [4758], with varying ligand specificity (Figure 6-A).  Although little is known about the cytoplasmic tail of the α subunit, the extracellular domain determines the specificity of the ECM ligand that binds to integrin heterodimer [58]. The cytoplasmic tail of the β subunit connects, through a protein complex, to the actin cytoskeleton transmitting mechanical forces into biomechanical signals.

Integrin activation is a complex process requiring signal transduction for a meaningful interaction to occur [59]. It is thought that in an inactive state integrin assumes a bent conformation with the association of the transmembrane (TM) domain of the two subunits [59-61]. Integrin activation may occur in a bidirectional process, and binding of the talin protein to the cytoplasmic tail of the β subunit is key for activation to take place [62]. Outside-in activation is achieved through ligand binding causing a conformational change increasing the distance between the cytoplasmic tails of the integrin subunit, thus increasing ligand affinity. Inside-out activation begins with talin prior to ligand binging. Although competitive binding takes place between the cytoplasmic α tail and talin, structural studies suggest that talin has two binding sites for the β subunit tail, subsequently increasing the binding affinity  [6364]. In each case, activation results in extracellular-integrin extension, the result of which produces a signaling cascade involving phosphorylation of integrin and many signaling proteins not detailed in this thesis. Figure 6-B shows the individual domains of each integrin subunit.

B

A

Figure 6: (A) The integrin families and corresponding ligands for each receptor group. (B) Arrangement of integrin domains for each subunit. The subunit is propeller is made up of seven blades, which is connected to the thigh domain. Calf-1 and Calf-2 domains tether the extracellular integrin to the transmembrane domain and cytoplasmic tail. Although sown here the αI domain is not present on all α subunits. The β subunit contains a plexin-sempahorin (PSI) domain, a hybrid (H) domain, and βI domain tethered to four repeating epidermal growth factors (EL-4), which connect to the TM domain and cytoplasmic tail [58].

SMCs that have undergone a phenotypic switch migrate in a macrophage-mimicking manor from the media towards the diseased area contributing to plaque formation. The α5β1-integrin is one of the prominent adhesion molecules involved with SMC migration and proliferation [54]. Up-regulation of α5β1-integrin has been reported with diseases causing vascular injury [546566]. This suggests a direct correlation between the α5β1-integrin and atherosclerosis, which experiences increased expression of FN providing an avenue for SMC migration and proliferation [54]. As stated previously the ECM protein FN functions as a ligand for α5β1-integrin. The integrin protein mechanically links the ECM to the cell’s actin cytoskeleton, thus providing a continuous bridge of communication.

2.2.3 Actin

As one of the most ample proteins in SMCs, actin, along with ancillary proteins, forms a filamentous mesh network throughout the cell [67]. The building block of actin is globular-actin (G-actin), which is a chain with a length of 375 amino acids and a molecular mass of 42 kDa. G-actin can assemble into longer semiflexible strands termed filamentous action (F-actin). F-actin has a width of 8 nm with a mass per unit length of 16 kDa/nm [6768], and a persistence length of ~ 20 μm [69]. Polarized actin provides a mechanism by which the SMC can rapidly re-organize in order to migrate, or alter shape.

Mechanically, actin is vital for SMCs. Forces exerted on the cell from the surrounding microenvironment are sustained by the actin cytoskeleton. This is possible due to the ~200 nm thick actin-mesh network that exists directly below the plasma membrane providing the cell with mechanical integrity [70]. Within SMCs exists an inherent pre-stressed environment produced from NMM-II motors that link F-actin, called stress fibers, generating a pulling force [69]. The potential regulating capabilities of cholesterol on cytoskeletal remodeling [47], and the stiffening ECM could disrupt the flow of forces experienced by the cell. Stresses induced by the ECM propagate into the SMC through integrin along stress fibers, eventually to the nucleus where membrane spanning linker receptors [71] connect actin to the nucleus possibly exposing transcription sites affecting gene activation [72] (Figure 7). Therefore, cholesterol-mediated alterations from an atherosclerotic environment could upset force transmission across the cell influencing cell function.

Figure 7: Schematic of how forces experienced by F-actin can be transmitted to the nucleus. Nesprin proteins bind to actin and are linked to SUN via KASH domain proteins. Emerin and SUN proteins may interact with chromatin exposing DNA for transcription as a result of force propagation [73].

2.2.4 Adhesion Mechanics

SMC adhesion mechanics are a dynamic and complex process through which mechanical homeostasis is maintained. This involves ECM proteins, such as collagen and FN, which transfer forces from the tissue to cell transmembrane receptors, integrins [48]. The integrin protein connects to the actomyosin cytoskeleton via talin and vinculin transmitting the force related signal. Actin filaments, NMM-II, and associated proteins relay the signal throughout the cell. SMCs, in a sense, feel the mechanical state of the microenvironment and preserve it to attain homeostasis. Once that desired state is lost pathogenesis, fibrosis, or mechanical failure may occur. Thus, mechanical loads sensed through cell adhesion can greatly influence disease progression.

In order to sense the ECM stiffness adhesion sites start to form at the leading edge of SMCs termed nascent adhesions. As integrin binds the FN, talin and vinculin link integrin to the actin cytoskeleton increasing the pulling force exerted by the cell. NMM-II binds to actin creating an actomyosin complex, creating a pulling force in a ratcheting motion. Within stiffer environments the ECM is able to resist deformation pulling back with an equal and opposite force resulting in the cell moving forward. However, when the ECM is soft deformation occurs and the FN-ECM protein moves backward with little to no forward cellular motion; similar to a car trying to accelerate too quickly in snow. It is through this process that SMCs remodel the ECM in an inside-out mechanism organizing the environment. SMC-ECM rearrangement is critical to their ability to create an environment that can resist the net forces of cyclic stretching from pumping blood. Expression of ECM proteins and/or ECM-remodeling enzymes secreted by the SMC are turned on or off depending on the mechanical forces experienced by the cell [74]. Therefore, stiffness mechanosensing and ECM-remodeling capabilities of the integrin adhesion complex may allow the substratum to regulate FN-integrin timing and interactions, leading to variations in signaling [74].

As the atherogenic SMC migrates towards the plaque it must grab and pull on the ECM in order to propel forward. To accomplish this, the cell extends the plasma membrane forward through actin nucleation and elongation creating lamellipodia, which are actin protein projections at the leading edge of a migrating cell. Projections beyond the lamellipodia are termed filopodia, which are also composed of F-actin. As previously mentioned, the α5β1-integrin also acts as a receptor during migration by binding to the Arg-Gly-Asp (RGD) motif of the tenth FN-III repeat in a fashion thought to be mechanically similar to timber tongs. Early nascent adhesions form an anchor at the leading edge of the cell on which the cell can exert a backward pulling force. Continued force exertion leads to adhesion maturation into focal complexes, which mature further into focal adhesions (FA) [75]. Although this maturating process is not fully understood, it is thought that increased force exposes cryptic sites allowing for additional binding of proteins to integrins. As the adhesion site matures additional integrins are recruited forming the FA, which binds to stress fibers [76]. Migration and adhesion cause rapid actin cytoskeleton remodeling [74] due to the dynamic nature of the leading and lagging edges. The lagging edge experiences actin depolymerization as adhesion sites release ligands allowing for detachment of the trailing end.  Figure 9 shows a summary of possible outcomes from adhesion mechanics.

Figure 8: Summary of the many possible outcomes resulting from the integrin-mediated adhesion biomechanics. Blue represents intracellular events, while orange represents extracellular events [77].

Understanding the importance of adhesion mechanics to cell function, and how the possible atherogenic/regulating effects of cholesterol and ECM stiffness influence that process, is imperative to learning about the SMC mechanisms underlying atherosclerosis. The adhesion site between α5β1-integrin and FN is an ideal location for achieving this goal.  Fortunately, there are a myriad of biomechanical measuring techniques available, such as the atomic force microscope (AFM), to characterize adhesion mechanical properties and cell E-modulus [78]. Interdisciplinary research joining disciplines such as biology, physics, and engineering is the new gold standard in furthering the understanding of the role of cell biomechanics in disease progression.

2.2.5 AFM

The AFM is a dynamic instrument capable of numerous techniques, such as 3D imaging down to the nanometer scale [79], detecting sample elasticity, and measuring pico newton sized tip-sample interactions via force curve analysis. SMCs have an extremely low elastic modulus. Therefore, indentations applied to the cell will range from micro to nano meters, whereas measuring forces exerted by the cell will most likely range from nano to pico newtons. When considering the compliance, size, timescale of mechanical measurements, and environmental conditions of SMCs, the AFM is an ideal experimental tool for biomechanical analysis. Although the many complexities of the AFM are not in the scope of this thesis, an example of basic AFM components (Fig. 9, A-E) and detailed feedback loop (Fig. 9, F) are shown in Figure 9 below.

F

Figure 9: Example of basic AFM components. (A) Overview of entire AFM mounted on an inverted phase contrast microscope. (B) AFM head showing nano-positioning system or z-piezo. (C) AFM head showing laser reflecting off of the cantilever. (D) Cantilever holder that is mounted into the AFM head. (E) Scanning electron microscope (SEM) image of AFM chip with extended rectangle and triangle shaped cantilevers. Although the cantilever tip is not visible, it would be located on the end of the cantilever and oriented perpendicularly. (F) AFM constant force feedback loop. As contact mode takes place the tip moves across the sample surface in a raster motion. The set point (SP) is set, which is the force applied (in voltage) to the sample that the AFM maintains as constant. Bending from the tip is detected by the photodiode (PD) from the reflection of the laser. The PD signal represents the real-time force acting on the cantilever between the tip and the sample. Due to hardware components that do not react instantaneous there is a small delay in force adjustment. This delay is found by subtracting the signal from the SP voltage (the force that we want to hold constant) and the PD. In order to decrease the error a parameter called the gain can be adjusted, however to much gain increase can cause ringing to occur causing the tip to become oversensitive. The error signal and the gain undergo a mathematical operation from which the output (done by operational amplifiers) goes in two directions. One direction is to the computer to produce the image of the z positon, while the other direction passes through a high voltage amplifier and goes up to the scanner causing the cantilever to move up and down by applying the necessary voltage to a piezoelectric diode.  The voltages that drive the AFM are ±10V, but the scanner needs higher voltages, thus the amplifier increases the voltage to ±150V.

Imaging of the leading edge of SMCs via AFM in constant force contact mode produces a high resolution 3D topographic image. The constant force applied by the tip across the SMC indents at a depth of ~200 nm. Thus, the images obtained show the topography and orientation of the actin cortex and stress fibers. Additionally, AFM force curve analysis can allow for biomechanical analysis of adhesion between α5β1-Integrin and FN as well as the SMC E-modulus.  The AFM has been used to study the effect of varying substrate stiffness on actin orientation [80], measure the E-modulus of SMCs [81], and measure the adhesion mechanics of the integrin-FN interaction [8283].  However, the combined effects of substrate stiffness and cellular cholesterol on SMC actin orientation, E-modulus, and adhesion mechanics have yet to be investigated.

In order to effectively understand the mechanisms underlying atherosclerosis, SMCs need to be examined in a state that more closely represents atherosclerotic mechanics. Determining the effect of substrate stiffness on adhesion mechanics or actin organization is helpful, but it does not take into account the possible effects that cholesterol may have on SMCs. Examining the combined effects proposed in this study allows for a more complete view of the possible regulatory roles of cholesterol on SMC biomechanics. The AFM provides the techniques necessary to begin to unravel the mechanical mechanisms underlying an atherosclerotic environment.

2.3 Specific Aims

Four specific aims were established in order to achieve the research goal of understanding how cholesterol affects the response of SMCs to the alterations in ECM mechanical properties. The first specific aim was to fabricate a substrate with controlled stiffness ranges. By fabricating substrates with varying stiffness the varying elasticity of the arterial wall in a diseased state can be modeled. In the atherosclerotic environment type I collagen expression increases. Therefore, the substrates were crosslinked with type I collagen in order to represent the augmented presence of the protein. The AFM was used to measure the stiffness of the substrates. Before and after images were taken using the AFM to visualize any topographic variation after collagen coating.

The second specific aim of this thesis was to quantify SMC cholesterol manipulation. In the diseased environment SMCs experience cholesterol loading, phenotypic switching acting macrophage-link phagocytizing cholesterol, and differentiate into foam cells. Cholesterol manipulation was quantified using a cholesterol quantitation kit in order to verify SMC cholesterol enrichment and cholesterol depletion upon treatment. The vehicle used to manipulate membrane cholesterol was methylated β-cyclodextrin, which has been used in previous experiments [84-86].

The third specific aim studies the combined effects of varying substrate stiffness and cellular cholesterol on the biomechanical analysis of adhesion between α5β1-Integrin and FN. Integrin links the cell to the ECM, where, in atherosclerosis, the presence of FN is atheropromoting. The combination of cholesterol manipulation and varying substrate stiffness create a unique environment in which mechanosensory functions were evaluated. The AFM was used to quantify adhesive force variations between integrin and FN. Additional parameters were also obtained through the AFM force curve analysis, such as adhesion probability, loading rate, relative rupture distance, and total adhesion force.

The fourth and final aim examined cell elasticity and actin cytoskeletal remodeling upon ECM stiffness variation and cellular cholesterol manipulation. Similar to aim three, the AFM was used to complete this analysis. Additionally, image processing was done to quantify actin orientation and area fraction from AFM images as well as confocal microscopy images. This was done in order to verify any correlation between adhesion mechanics and/or SMC E-modulus and actin organization.

Through these specific aims, new insight on the synergistic effects of substrate stiffness and cholesterol on biomechanical and physiological functions of SMCs, adhesion mechanics, and actin organization was gained, potentially offering new therapeutic targets for controlling SMC migration.

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