Age-related vascular stiffening: causes and consequences

“Different mathematical models are used to fit nanoindentation data and include the Hertz model (Hertz, 1881; Johnson, 1982), the Oliver-Pharr model (Oliver and Pharr, 1992, 2004), and the Johnson–Kendall–Roberts (JKR) adhesion model (Johnson et al., 1971).”

“Unlike AFM which records cantilever displacements, in nanoindentation, an external load is applied to the indenter tip to push the tip into the surface (Mann, 2007).”

“Different mathematical models are used to fit nanoindentation data and include the Hertz model (Hertz, 1881; Johnson, 1982), the Oliver-Pharr model (Oliver and Pharr, 1992, 2004), and the Johnson–Kendall–Roberts (JKR) adhesion model (Johnson et al., 1971).”

“Different mathematical models are used to fit nanoindentation data and include the Hertz model (Hertz, 1881; Johnson, 1982), the Oliver-Pharr model (Oliver and Pharr, 1992, 2004), and the Johnson–Kendall–Roberts (JKR) adhesion model (Johnson et al., 1971).”

“Pulse wave velocity (PWV) measurements are the primary clinical method used to assess arterial stiffening and to help predict CVD events (Laurent et al., 2006; Van Bortel et al., 2012; Protogerou et al., 2014).”

“Unlike AFM which records cantilever displacements, in nanoindentation, an external load is applied to the indenter tip to push the tip into the surface (Mann, 2007).”

“Furthermore, in hypertensive patients, increased arterial stiffness is associated with increased mortality rates (Laurent et al., 2001).”

“During atherosclerosis, cholesterol permeates into the blood vessel wall and must be retained there to elicit the inflammatory reaction resulting in plaque formation (Libby, 2002).”

“Several studies have analyzed the aorta as a two-layer construct consisting of a combined intima-media layer surrounded by the adventitial layer (Holzapfel et al., 2000; Lu et al., 2003).”

“The distinct composition of each artery layer lends itself to layer-specific mechanical properties that can vary from person to person, and also between large and small arteries (Baynes and Thorpe, 1999; Fonck et al., 2009).”

“Many studies treat the artery as an isotropic material andmeasure its properties uniaxially, although it should be noted that anisotropic materials are most accurately tested biaxially (Sacks, 2000; Vande Geest et al., 2005).”

“Decreased arterial compliance is correlated with a higher risk of cardiovascular disease (Zieman et al., 2005).”

“…to collagen crosslinking, AGEs are harmful to vascular health because they reduce nitric oxide availability, an important vasodilator used to maintain vascular tone that also has antiinflammatory effectors on the endothelium (Bucala et al., 1991; De Caterina et al., 1995; Xu et al., 2003, 2005).”

“The healthy human intima has been reported to have an elastic modulus of 34.4 kPa and is considered a compliant material, with an elastic modulus similar to adipose tissue and lower than muscle (Lundkvist et al., 1996; Engler et al., 2004; Patel et al., 2005).”

“Fibroblasts are dispersed within the adventitia, but are generally absent from the intima and media artery layers (Ross and Glomset, 1973).”

“Macroscale arterial mechanics are used clinically to predict the likelihood of cardiovascular disease risk (Blacher et al., 1999), and are also essential to the development of tissue engineered vascular grafts (Ravi and Chaikof, 2010).”

“…and it is well-established that ECs align in the direction of flow, that laminar flow promotes an atheroprotective endothelium, and that atherosclerotic plaques develop predominantly in regions of disturbed flow (Caro et al., 1969; Zarins et al., 1983; Levesque and Nerem, 1985; Tzima et al., 2005).”

“Suction forces as low as 1 nN were used to measure the elastic modulus of ECs, which were reported to be 0.5 kPa (Hochmuth, 2000) and increased with increasing substrate stiffness (Byfield et al., 2009).”

“In the inner adventitia, collagen fibers are oriented, thin, and intermixed with elastin allowing for vessel distension and protection against rupture, whereas the outer adventitia is primarily composed of thick, non-oriented collagen fibers that support the vessel (Chen et al., 2011).”

“Carotid-femoral PWV measurements are correlated with arterial stiffness and can be used to predict cardiovascular disease in patients (Laurent et al., 1994; Safar et al., 2003).”

“…to collagen crosslinking, AGEs are harmful to vascular health because they reduce nitric oxide availability, an important vasodilator used to maintain vascular tone that also has antiinflammatory effectors on the endothelium (Bucala et al., 1991; De Caterina et al., 1995; Xu et al., 2003, 2005).”

“Notably, PWV increases with age for both men and women (Hallock, 1934; van der-Heijden-Spek et al., 2000), and pulse pressure increases by 25% from ages 30 to 60 (Kelly et al., 1989).”

“Recent data suggest numerous cell types respond to matrix stiffness and that stiffness can promote stem cell differentiation, tissue morphogenesis, and tumor malignancy (Paszek and Weaver, 2004; Sieminski et al., 2004; Paszek et al., 2005; Engler et al., 2006).”

“The physical distance between the two catheters is measured on the surface of the body using a tape measure, and the velocity measurement is the ratio of distance traveled to elapsed time (van Popele et al., 2001; Laurent et al., 2006).”

“In the zero-load state, the fibers assume a crimped morphology (Rezakhaniha et al., 2012).”

“…the receptor for advanced glycation end products (RAGE) to have downstream effects that include the production of reactive oxygen species, NF-κB inflammatory signaling, and endothelial hyperpermeability (Mullarkey et al., 1990; Wautier et al., 1996, 2001; Bierhaus et al., 1997; Park et al., 1998).”

“…the receptor for advanced glycation end products (RAGE) to have downstream effects that include the production of reactive oxygen species, NF-κB inflammatory signaling, and endothelial hyperpermeability (Mullarkey et al., 1990; Wautier et al., 1996, 2001; Bierhaus et al., 1997; Park et al., 1998).”

“Calcium in the arterial wall also increases with age facilitating the direct binding of calcium ions to elastin fibers causing calcification (Urry, 1971; Urry and Ohnishi, 1974; Otto et al., 1999).”

“When analyzed as a composite material, large arteries exhibit a non-linear stress-strain pattern, and therefore, are best described in terms of an elastic modulus evaluated at a given physiological stress along the stress-strain curve, termed the incremental elastic modulus (Bergel, 1961).”

“The number of concentric lamellae layers in the media remains constant with age but scales with arterial radius and vessel wall tensional strength (Wolinsky and Glagov, 1967).”

“Collagen fibers are 100–1,000 times stiffer than elastin which causes a sharp increase in the incremental elastic modulus at higher levels of circumferential stretch (Dobrin, 1978; Wagenseil et al., 2005; Lasheras, 2007; Wagenseil and Mecham, 2012).”

“…and it is well-established that ECs align in the direction of flow, that laminar flow promotes an atheroprotective endothelium, and that atherosclerotic plaques develop predominantly in regions of disturbed flow (Caro et al., 1969; Zarins et al., 1983; Levesque and Nerem, 1985; Tzima et al., 2005).”

“In concert with increased collagen concentrations, collagen crosslinking by non-enzymatic glycation increases arterial stiffness with age (Sims et al., 1996; Schleicher et al., 1997).”

“Several macroscale techniques for measuring the mechanical properties of intact arteries have been developed (Figure 2).”

“…the receptor for advanced glycation end products (RAGE) to have downstream effects that include the production of reactive oxygen species, NF-κB inflammatory signaling, and endothelial hyperpermeability (Mullarkey et al., 1990; Wautier et al., 1996, 2001; Bierhaus et al., 1997; Park et al., 1998).”

“At low degrees of stretch, the compliant elastin fibers dominate the mechanics, while at higher levels of deformation, helically oriented collagen fibers are recruited (Roach and Burton, 1957; Schriefl et al., 2012).”

“Higher PWV measurements are associated with a higher risk of stroke, cardiovascular death, coronary heart disease (SuttonTyrrell et al., 2005) and hypertension (Cecelja and Chowienczyk, 2009).”

“Interestingly, VSMCs are durotactic, preferentially migrating in the direction of increased substrate stiffness, but their maximummigration speed depends on both matrix stiffness and ECM chemical cues (Peyton and Putnam, 2005; Isenberg et al., 2009; Sazonova et al., 2014).”

“…and it is well-established that ECs align in the direction of flow, that laminar flow promotes an atheroprotective endothelium, and that atherosclerotic plaques develop predominantly in regions of disturbed flow (Caro et al., 1969; Zarins et al., 1983; Levesque and Nerem, 1985; Tzima et al., 2005).”

“The two primary AFM modes are ‘contact mode,’ where the cantilever tip is dragged along the sample surface, or ‘AC (tapping)’ mode, where the tip oscillates near its resonant frequency close to the sample surface and intermittent indentations are recorded (Zhong et al., 1993).”

“The second intimal layer is musculoelastic and consists of elastin fibers, individual smooth muscle cells, and collagen (Stary et al., 1992).”

“…been shown to alter EC behavior including increased RhoA activity, ICAM-1 expression, proliferation, morphology, and adhesion (Ingber et al., 1990; Tzima et al., 2001, 2002; Young et al., 2007) Recently, it has become apparent that ECs also respond to ECM mechanical cues where increased matrix…”

“The OA measurement was originally developed to understand the stress-free state of tissues (Chuong and Fung, 1986) but can also be used to elucidate the viscoelastic properties of an artery after tissue loading (Rehal et al.”

“During atherogenesis, VSMCs migrate from the media into the intima where their proliferation and ECM secretions increase plaque size (Rudijanto, 2007).”

“Data can be fit to standard mathematical models such as the Hertz model, to provide mechanical properties such as the elastic modulus at the indentation location (Hertz, 1881; Mahaffy et al., 2000; Peloquin et al., 2011).”

“In the inner adventitia, collagen fibers are oriented, thin, and intermixed with elastin allowing for vessel distension and protection against rupture, whereas the outer adventitia is primarily composed of thick, non-oriented collagen fibers that support the vessel (Chen et al., 2011).”

“Given that age is a significant cardiovascular risk factor and that arterial stiffness is known to increase with age (Benetos et al., 2002), both macro- and microscale testing techniques have been developed to evaluate arterial mechanics.”

“Under physiological strain loads, the incremental elastic modulus is a function of strain and the combined contributions of elastin and collagen (Wagenseil and Mecham, 2012).”

“Unlike AFM which records cantilever displacements, in nanoindentation, an external load is applied to the indenter tip to push the tip into the surface (Mann, 2007).”

“Microscale tests are more relevant for studies of cellular mechanotransduction in the vessel wall and investigations of the cellular response to changes in arterial mechanics (Figure 3).”

“Different mathematical models are used to fit nanoindentation data and include the Hertz model (Hertz, 1881; Johnson, 1982), the Oliver-Pharr model (Oliver and Pharr, 1992, 2004), and the Johnson–Kendall–Roberts (JKR) adhesion model (Johnson et al., 1971).”

“The mechanism of AGE crosslinking in vivo is hypothesized to predominately occur between lysine residues on collagen and the AGE N-ε-carboxy-methyllysine (CML) (Ikeda et al., 1996; Schleicher et al., 1997).”

“…activity, ICAM-1 expression, proliferation, morphology, and adhesion (Ingber et al., 1990; Tzima et al., 2001, 2002; Young et al., 2007) Recently, it has become apparent that ECs also respond to ECM mechanical cues where increased matrix stiffness has deleterious effects on endothelial NO…”

“Macroscale arterial mechanics are used clinically to predict the likelihood of cardiovascular disease risk (Blacher et al., 1999), and are also essential to the development of tissue engineered vascular grafts (Ravi and Chaikof, 2010).”

“Interestingly, VSMCs are durotactic, preferentially migrating in the direction of increased substrate stiffness, but their maximummigration speed depends on both matrix stiffness and ECM chemical cues (Peyton and Putnam, 2005; Isenberg et al., 2009; Sazonova et al., 2014).”

“The most common method to determine the elastic modulus of an artery is based on the quotient of stress and strain derived from tensile testing.”

“…the receptor for advanced glycation end products (RAGE) to have downstream effects that include the production of reactive oxygen species, NF-κB inflammatory signaling, and endothelial hyperpermeability (Mullarkey et al., 1990; Wautier et al., 1996, 2001; Bierhaus et al., 1997; Park et al., 1998).”

“…to matrix stiffness and that increased intimal stiffness promotes endothelial dysfunction; therefore the mechanical properties of the intima alone may be important factors in cardiovascular disease progression and warrant further investigation (Reinhart-King et al., 2005; Huynh et al., 2011).”

“Cells sense their local mechanical properties on the order of five microns, suggesting that overall or composite layer artery mechanics are not representative of the microenvironment where ECs reside (Sen et al., 2005; Buxboim et al., 2010).”

“…been shown to alter EC behavior including increased RhoA activity, ICAM-1 expression, proliferation, morphology, and adhesion (Ingber et al., 1990; Tzima et al., 2001, 2002; Young et al., 2007) Recently, it has become apparent that ECs also respond to ECM mechanical cues where increased matrix…”

“At low degrees of stretch, the compliant elastin fibers dominate the mechanics, while at higher levels of deformation, helically oriented collagen fibers are recruited (Roach and Burton, 1957; Schriefl et al., 2012).”

“An important function of the vascular endothelium in atherosclerosis prevention is acting as a protective barrier against cholesterol accumulation and leukocyte transmigration into the intima (Schwenke and Carew, 1989).”

“In the inner adventitia, collagen fibers are oriented, thin, and intermixed with elastin allowing for vessel distension and protection against rupture, whereas the outer adventitia is primarily composed of thick, non-oriented collagen fibers that support the vessel (Chen et al., 2011).”

“Interestingly, recent evidence has shown that ECs are mechanosensitive to matrix stiffness and that increased intimal stiffness promotes endothelial dysfunction; therefore the mechanical properties of the intima alone may be important factors in cardiovascular disease progression and warrant…”

“During atherogenesis, VSMCs migrate from the media into the intima where their proliferation and ECM secretions increase plaque size (Rudijanto, 2007).”

“Cells sense their local mechanical properties on the order of five microns, suggesting that overall or composite layer artery mechanics are not representative of the microenvironment where ECs reside (Sen et al., 2005; Buxboim et al., 2010).”

“Collagen fibers are 100–1,000 times stiffer than elastin which causes a sharp increase in the incremental elastic modulus at higher levels of circumferential stretch (Dobrin, 1978; Wagenseil et al., 2005; Lasheras, 2007; Wagenseil and Mecham, 2012).”

“…proliferation, morphology, and adhesion (Ingber et al., 1990; Tzima et al., 2001, 2002; Young et al., 2007) Recently, it has become apparent that ECs also respond to ECM mechanical cues where increased matrix stiffness has deleterious effects on endothelial NO production and barrier integrity…”

“Amino groups on proteins react with aldehydes or ketones on the reducing sugars to form shift bases that rearrange to Amadori products and are further modified to produce AGEs (Bakris et al., 2004; Sell and Monnier, 2012).”

“Historically, greater emphasis has been placed on macroscale testing because it can be used clinically and is associated with cardiovascular events including aneurism, atherosclerosis, and hypertension (van Popele et al., 2001; Kaess et al., 2012; van Sloten et al., 2014).”

“Unlike AFM which records cantilever displacements, in nanoindentation, an external load is applied to the indenter tip to push the tip into the surface (Mann, 2007).”

“Overall, Type I and Type III collagens account for 60% of the artery wall, and elastin 30% (Rizzo et al., 1989; Powell et al., 1992).”

“Techniques in ultrasound software can also assess other cardiovascular risk factors, such as wall strain (Godia et al., 2007), atherosclerotic plaque location, and intima-media thickness (Selzer et al., 2001; van Popele et al., 2001).”

“Spherical beads of varying diameters up to the microscale can also be placed on the AFM cantilever for force indentation measurements (Radmacher, 1997; Costa and Yin, 1999).”

“In concert with increased collagen concentrations, collagen crosslinking by non-enzymatic glycation increases arterial stiffness with age (Sims et al., 1996; Schleicher et al., 1997).”

“A cantilever with a micron-scale tip is brought in contact with a sample, and as the cantilever deflects, a laser beam focused on the back of the cantilever is also deflected, providing information regarding the depth of indentation (Binnig et al., 1986; Mann, 2007).”

“During atherosclerosis, cholesterol permeates into the blood vessel wall and must be retained there to elicit the inflammatory reaction resulting in plaque formation (Libby, 2002).”

“The emergence of ECM mechanics as an important cue affecting cell function has contributed to the development of new techniques to analyze microscale arterial properties.”

“Carotid-femoral PWV measurements are correlated with arterial stiffness and can be used to predict cardiovascular disease in patients (Laurent et al., 1994; Safar et al., 2003).”

“Fatigue failure from pulsatile wall stress can cause elastin fragmentation throughout the lifetime (O’Rourke, 1976; Avolio et al., 1998; Greenwald, 2007).”

“Overall, Type I and Type III collagens account for 60% of the artery wall, and elastin 30% (Rizzo et al., 1989; Powell et al., 1992).”

“…composition has also been shown to alter EC behavior including increased RhoA activity, ICAM-1 expression, proliferation, morphology, and adhesion (Ingber et al., 1990; Tzima et al., 2001, 2002; Young et al., 2007) Recently, it has become apparent that ECs also respond to ECM mechanical cues…”

“…is the ratio of blood volume to arterial blood pressure, although in practice, the compliance of an artery can be measured as the ratio of change in cross-sectional area to the change in pressure, which is known as the cross-sectional compliance (Reneman et al., 1986; Tozzi et al., 2000).”

“With age, increased activity of the elastases MT1-MMP and MMP-2 has been observed, and MMP-2 has been found near fragmented elastin fibers within the aorta (Wang et al., 2003; Yasmin et al., 2005; Bonnema et al., 2007).”

“Suction forces as low as 1 nN were used to measure the elastic modulus of ECs, which were reported to be 0.5 kPa (Hochmuth, 2000) and increased with increasing substrate stiffness (Byfield et al., 2009).”

“Mechanistically, increased substrate stiffness activates the Rho/Rho kinase pathway to increase cellular contractile forces and compromise cell-cell junction integrity (Huynh et al., 2011; Krishnan et al., 2011).”

“…atherosclerotic plaque development, Frontiers in Genetics | www.frontiersin.org 9 March 2015 | Volume 6 | Article 112 and there is a strong relationship between arteriosclerosis and the calcification and degeneration of elastic fibers in arteries (Blumenthal et al., 1944; Shekhonin et al., 1985).”

“Medial mechanics are dominated by elastin within the lamellar units at physiological pressures (Greenwald et al., 1997).”

“Within the adventitia, collagen I and III deposition by fibroblasts increases with age and is accompanied by vessel stiffening (Fleenor et al., 2010).”

“Amino groups on proteins react with aldehydes or ketones on the reducing sugars to form shift bases that rearrange to Amadori products and are further modified to produce AGEs (Bakris et al., 2004; Sell and Monnier, 2012).”

“Tensile testing has been performed on human arteries to characterize their mechanical changes in a number of conditions, including aging (Vande Geest et al., 2005; Haskett et al., 2010; Shafigh et al., 2013), as well as plaque (Walsh et al., 2014) and aneurism development (Khanafer et al., 2011).”

“As an example, using OA combined with arterial pre-stress measurements, Matsumoto and Hayashi (1996) reported the mean circumferential stress of the thoracic aorta was 339 kPa for hypertensive rats compared to 263 kPa for normotensive controls, and that the circumferential strains were 0.72 and…”

“OA analysis can also be performed after pre-stressing the arteries with a transmural pressure load to cause inflation and deflation and measuring the change in artery diameter and axial force (Matsumoto and Hayashi, 1996; Rachev and Greenwald, 2003).”

“…to collagen crosslinking, AGEs are harmful to vascular health because they reduce nitric oxide availability, an important vasodilator used to maintain vascular tone that also has antiinflammatory effectors on the endothelium (Bucala et al., 1991; De Caterina et al., 1995; Xu et al., 2003, 2005).”

“The dysregulation of MMPs is already known to play a role in the cardiovascular pathologies hypertension and aneurysm (Thompson and Baxter, 1999; Yasmin et al., 2005).”

“As examples, OA measurements have been used to measure the anisotropic response of arteries in hypertensive, diabetic Frontiers in Genetics | www.frontiersin.org 5 March 2015 | Volume 6 | Article 112 and hypoxic conditions (Fung, 1990; Fung and Liu, 1991).”

“Animal studies by Kothapalli et al. (2012) recently demonstrated that arterial stiffening and ECM crosslinking precede cardiovascular disease development.”

“Calcium in the arterial wall also increases with age facilitating the direct binding of calcium ions to elastin fibers causing calcification (Urry, 1971; Urry and Ohnishi, 1974; Otto et al., 1999).”

“Notably, PWV increases with age for both men and women (Hallock, 1934; van der-Heijden-Spek et al., 2000), and pulse pressure increases by 25% from ages 30 to 60 (Kelly et al., 1989).”

“The expansion and contraction of the vessel are normalized to the zeroload outer diameter and measured in conjunction with an ECG measurement that determines heart rate (Fonck et al., 2007).”

“On the subcellular level, AFMhas been used to study the properties of the plasma membrane, ECM, and cytoskeleton (Kasas and Dietler, 2008; Lu et al., 2008).”

“With age, increased activity of the elastases MT1-MMP and MMP-2 has been observed, and MMP-2 has been found near fragmented elastin fibers within the aorta (Wang et al., 2003; Yasmin et al., 2005; Bonnema et al., 2007).”

“OA analysis can also be performed after pre-stressing the arteries with a transmural pressure load to cause inflation and deflation and measuring the change in artery diameter and axial force (Matsumoto and Hayashi, 1996; Rachev and Greenwald, 2003).”

“The emergence of ECM mechanics as an important cue affecting cell function has contributed to the development of new techniques to analyze microscale arterial properties.”

“A proliferative phenotype was observed by VSMCs cultured on stiff collagen fibrils when compared to compliant collagen gels, even though β1 integrin mediated cell-ECM interactions were comparable on both substrates (McDaniel et al., 2007).”

“Stiffer arteries have been shown to exhibit increased permeability in vivo, and therefore are thought to have greater cholesterol uptake and atherosclerotic plaque initiation (Huynh et al., 2011).”

“…including increased RhoA activity, ICAM-1 expression, proliferation, morphology, and adhesion (Ingber et al., 1990; Tzima et al., 2001, 2002; Young et al., 2007) Recently, it has become apparent that ECs also respond to ECM mechanical cues where increased matrix stiffness has deleterious…”

“The stiffness of carotid plaques has also been measured using nanoindentation (Ebenstein et al., 2009).”

“…(Ingber et al., 1990; Tzima et al., 2001, 2002; Young et al., 2007) Recently, it has become apparent that ECs also respond to ECM mechanical cues where increased matrix stiffness has deleterious effects on endothelial NO production and barrier integrity (Huynh et al., 2011; Kohn et al., 2015).”

“In the inner adventitia, collagen fibers are oriented, thin, and intermixed with elastin allowing for vessel distension and protection against rupture, whereas the outer adventitia is primarily composed of thick, non-oriented collagen fibers that support the vessel (Chen et al., 2011).”

“The healthy human intima has been reported to have an elastic modulus of 34.4 kPa and is considered a compliant material, with an elastic modulus similar to adipose tissue and lower than muscle (Lundkvist et al., 1996; Engler et al., 2004; Patel et al., 2005).”

“Several macroscale techniques for measuring the mechanical properties of intact arteries have been developed (Figure 2).”

“The emergence of ECM mechanics as an important cue affecting cell function has contributed to the development of new techniques to analyze microscale arterial properties.”

“Soft biological tissue mechanics are measured using pipette aspiration by applying an aspiration pressure on the inside of the pipette to deform the tissue (Aoki et al., 1997).”

“In the inner adventitia, collagen fibers are oriented, thin, and intermixed with elastin allowing for vessel distension and protection against rupture, whereas the outer adventitia is primarily composed of thick, non-oriented collagen fibers that support the vessel (Chen et al., 2011).”

“For example, measured under axial stretch and using non-axisymmetric deformations, the media is significantly less compliant than the adventitia (Yu et al., 1993; Pandit et al., 2005).”

“…the receptor for advanced glycation end products (RAGE) to have downstream effects that include the production of reactive oxygen species, NF-κB inflammatory signaling, and endothelial hyperpermeability (Mullarkey et al., 1990; Wautier et al., 1996, 2001; Bierhaus et al., 1997; Park et al., 1998).”

“In prior work, our laboratory utilized atomic force microscopy (AFM) to measure the mechanical stiffness of the denuded aortic bovine intima, a composite of surface collagen and underlying elastin (Buxboim et al., 2010; Peloquin et al., 2011).”

“Age-related changes in the arteries caused by ECM rearrangements directly contribute to increased LDL accumulation but they also alter endothelial and VSMC function to contribute to atherogenesis (Tada and Tarbell, 2004; Rudijanto, 2007; Kwon et al., 2008; Huynh et al., 2011).”

“The emergence of ECM mechanics as an important cue affecting cell function has contributed to the development of new techniques to analyze microscale arterial properties.”

“AFM can be used to study cellular mechanics, including EC stiffness (Kusche-Vihrog et al., 2011; Lee et al., 2011).”

“In addition to age-related effects, patients with increased serum cholesterol also have impaired radial artery compliance and distensibility (Giannattasio et al., 1996).”

“On the subcellular level, AFMhas been used to study the properties of the plasma membrane, ECM, and cytoskeleton (Kasas and Dietler, 2008; Lu et al., 2008).”

“Contractile activation of VSMCs has been shown to increase the medial elastic modulus (Dobrin and Rovick, 1969; Tremblay et al., 2010), but separate studies have shown that lamellar mechanical properties are unchanged after VSMC activation with noradrenaline (Berry et al., 1975).”

“However, AFM is advantageous for smaller scale biological applications and has greater versatility in the information it can provide.”

“During testing, a pipette uses suction to pull one end of a cell or matrix into a 0.5–0.8 mm glass tube (Sato et al., 2000; Matsumoto et al., 2002).”

“The extracellular matrix (ECM) proteins collagen and elastin account for approximately half of the vessel dry weight, and play a crucial role in arterymechanics (O’Connell et al., 2008).”

“Interestingly, VSMCs are durotactic, preferentially migrating in the direction of increased substrate stiffness, but their maximummigration speed depends on both matrix stiffness and ECM chemical cues (Peyton and Putnam, 2005; Isenberg et al., 2009; Sazonova et al., 2014).”

“In the inner adventitia, collagen fibers are oriented, thin, and intermixed with elastin allowing for vessel distension and protection against rupture, whereas the outer adventitia is primarily composed of thick, non-oriented collagen fibers that support the vessel (Chen et al., 2011).”

“In the inner adventitia, collagen fibers are oriented, thin, and intermixed with elastin allowing for vessel distension and protection against rupture, whereas the outer adventitia is primarily composed of thick, non-oriented collagen fibers that support the vessel (Chen et al., 2011).”

“Longitudinal tension is primarily assumed by the adventitia which bears ∼75% of the load (Lu et al., 2004).”

“For example, measured under axial stretch and using non-axisymmetric deformations, the media is significantly less compliant than the adventitia (Yu et al., 1993; Pandit et al., 2005).”

“Fatigue failure from pulsatile wall stress can cause elastin fragmentation throughout the lifetime (O’Rourke, 1976; Avolio et al., 1998; Greenwald, 2007).”

“However, AFM is advantageous for smaller scale biological applications and has greater versatility in the information it can provide.”

“In the inner adventitia, collagen fibers are oriented, thin, and intermixed with elastin allowing for vessel distension and protection against rupture, whereas the outer adventitia is primarily composed of thick, non-oriented collagen fibers that support the vessel (Chen et al., 2011).”

“Microscale tests are more relevant for studies of cellular mechanotransduction in the vessel wall and investigations of the cellular response to changes in arterial mechanics (Figure 3).”

“Age-related changes in the arteries caused by ECM rearrangements directly contribute to increased LDL accumulation but they also alter endothelial and VSMC function to contribute to atherogenesis (Tada and Tarbell, 2004; Rudijanto, 2007; Kwon et al., 2008; Huynh et al., 2011).”

“The luminal layer, known as the basal lamina, is comprised of a thin basement membrane with a proteoglycan rich matrix and small amounts of collagen (Palotie et al., 1983; Kramer et al., 1984).”

“The compounds desmosine and isodesmosine are formed from four lysine amino acids and are critical for crosslinking elastin fibers to give them their elastic properties (Davis and Anwar, 1970).”

“AFM has also been used to study the mechanics of atherosclerotic plaques and fibrous caps (Hayenga et al., 2011; Marzec et al., 2014), as well the effects of age and diet on intima stiffness (Huynh et al., 2011; Peloquin et al., 2011; Weisbrod et al., 2013).”

“When the artery is subjected to a transmural pressure, the elastin fibers possessing high entropically driven recoil properties are initially stretched, followed by the stretching of stiffer collagen fibers (Samila and Carter, 1981).”

“Leukocytes exhibit elevated transmigration behavior with increasing stiffness and biphasic migration speeds (Hayenga and Aranda-Espinoza, 2013; Stroka et al., 2013).”

“Healthy human coronary arteries have an elastic modulus of 1.5 MPa, which increases to 3.8 MPa in atherosclerotic vessels (Khanafer et al., 2011).”

“Elastin within the internal elastic lamina is oriented longitudinally in the direction of luminal blood flow, while in the media it is oriented circumferentially (Farand et al., 2007).”

“Simultaneous topography and recognition imaging (TREC) is a recent advancement in AFM technology, where a ligand is functionalized to the AFM cantilever tip to provide simultaneous measurements of material topography and cell-ligand interactions (Preiner et al., 2009).”

“…ECs have morphological changes resembling a VSMC phenotype and express smooth Frontiers in Genetics | www.frontiersin.org 3 March 2015 | Volume 6 | Article 112 muscle alpha actin and collagen I, indicating they may also deposit collagen that contributes to intimal thickening (Fleenor et al., 2012).”

“In the inner adventitia, collagen fibers are oriented, thin, and intermixed with elastin allowing for vessel distension and protection against rupture, whereas the outer adventitia is primarily composed of thick, non-oriented collagen fibers that support the vessel (Chen et al., 2011).”

“AFM has also been used to study the mechanics of atherosclerotic plaques and fibrous caps (Hayenga et al., 2011; Marzec et al., 2014), as well the effects of age and diet on intima stiffness (Huynh et al., 2011; Peloquin et al., 2011; Weisbrod et al., 2013).”

“Similar to migration, VSMC proliferation is also dependent on ECM ligand density, but it has been demonstrated that increased matrix stiffness is the dominant factor affecting proliferation (Vatankhah et al., 2014).”

“Arterial tissue probed with nanoindentation indicates that porcine elastic aortas have elastic moduli between 60 and 70 kPa (Hemmasizadeh et al., 2012).”

“Age-related changes in the arteries caused by ECM rearrangements directly contribute to increased LDL accumulation but they also alter endothelial and VSMC function to contribute to atherogenesis (Tada and Tarbell, 2004; Rudijanto, 2007; Kwon et al., 2008; Huynh et al., 2011).”

“Despite the widespread use of tensile testing, there are more than four definitions of stress and strain in the literature, which leads to differences in the values obtained through mechanical testing results (Khanafer et al., 2013).”

“Notably, even though the absolute elastin content in the aorta remains relatively stable with age, the elastin concentration decreases and is accompanied by a substantial increase in collagen concentration (Myers and Lang, 1946; Kanabrocki et al., 1960; Toda et al., 1980; Fonck et al., 2009).”

“Given the various definitions of stress and strain, both the measurement technique and the associated analysis should be considered to accurately compare the reported mechanical properties.”

“The technique has also been used to measure the mechanical properties of rat arteries, where the effective elastic moduli ranged from 22 to 37 kPa (Hanna et al., 2013).”

“Micropipette aspiration has been used to measure the ECM properties of porcine and rabbit aortas (Sato et al., 2000; Matsumoto et al., 2002; Ohashi et al., 2005).”

“However, AFM is advantageous for smaller scale biological applications and has greater versatility in the information it can provide.”

“Immune cell infiltration into the intima has important implications for driving atherosclerotic plaque formation, and the increased EC-cell junction size in response to substrate stiffness facilitates leukocyte transmigration by the predominant paracellular route (Huynh et al., 2011; Stroka et al.,…”

“Introducing microbubbles into the blood stream can allow for contrast enhanced ultrasound, where specific molecular targets in the vessel are imaged to study disease progression (Shim and Lindner, 2014).”

“underlying mechanisms causative of changes in the physical properties of blood vessels (Kohn et al., 2015).”