Sex differences in mechanisms of arterial stiffness

Abstract

Arterial stiffness progressively increases with aging and is an independent predictor of cardiovascular disease (CVD) risk. Evidence supports that there are sex differences in the time course of aging‐related arterial stiffness and the associated CVD risk, which increases disproportionately in postmenopausal women. The association between arterial stiffness and mortality is almost twofold higher in women versus men. The differential clinical characteristics of the development of arterial stiffness between men and women indicate the involvement of sex‐specific mechanisms. This review summarizes the current literature on sex differences in vascular stiffness induced by aging, obesity, hypertension, and sex‐specific risk factors as well as the impact of hormonal status, diet, and exercise on vascular stiffness in males and females. An understanding of the mechanisms driving sex differences in vascular stiffness has the potential to identify novel sex‐specific therapies to lessen CVD risk, the leading cause of death in males and females.

LINKED ARTICLES

This article is part of a themed section on The Importance of Sex Differences in Pharmacology Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.21/issuetoc

Abbreviations

CVD

cardiovascular disease

ECM

extracellular matrix

EC‐MR

endothelial cell mineralocorticoid receptor

ENaC

epithelial sodium channel

ERα

oestrogen receptor‐α

ERβ

oestrogen receptor‐β

HFpEF

heart failure with preserved ejection fraction

HRT

hormone replacement therapy

HT

Hashimoto's thyroiditis

ISH

isolated systolic hypertension

OCP

oral contraceptive pill

PWV

pulse wave velocity

RAAS

renin–angiotensin–aldosterone system

SLE

systemic lupus erythematosus

SMC‐MR

smooth muscle cell mineralocorticoid receptor

VSMC

vascular smooth muscle cell

WD

Western diet

XO

xanthine oxidase

1. INTRODUCTION

Cardiovascular disease (CVD) is the number one cause of death for both men and women. Although premenopausal women are relatively protected from CVD, the incidence increases disproportionately in women after menopause (Coutinho, 2014; Ellekjaer, Holmen, Indredavik, & Terent, 1997; Thrift, Dewey, Macdonell, McNeil, & Donnan, 2000). Arterial stiffening is a risk factor for the development of CVD and for all‐cause mortality independent of the presence of all traditional CVD risk factors (Mitchell, 2009). The association between arterial stiffness and mortality has been shown to be almost twofold higher in women compared to men (Regnault et al., 2012). Women also develop greater increases in pulse pressure and arterial stiffness with aging, at least some of which appears to be independent of body and aortic size (Coutinho, Borlaug, Pellikka, Turner, & Kullo, 2013; Dart et al., 2008; Mitchell et al., 2004). In addition, heart failure with preserved ejection fraction (HFpEF) and coronary microvascular dysfunction are the more commonly diagnosed forms of CVD in women and are associated with elevated arterial stiffness in women (Beale, Meyer, Marwick, Lam, & Kaye, 2018; Coutinho et al., 2013; Coutinho, Yam, Chow, Dwivedi, & Inacio, 2017). Overall, substantial sex differences are observed in the development of arterial stiffness, not only in the timing but also in the setting of CVD risk factors such as diabetes mellitus and obesity. Despite the substantial progress that has been made in narrowing the gender gap in cardiovascular outcomes over the past two decades, our understanding of the mechanistic differences and manifestations of arterial stiffness in men and women remains incomplete. This review summarizes the current information regarding sex differences in arterial stiffness and what is known about potential mechanisms, highlighting the need for further sex‐based research to elucidate the complexity of the impact of sex hormone and sex chromosomal interactions with cardiovascular health.

1.1. Defining and quantifying arterial stiffness

Large conduit arteries are composed of three layers: (a) the outer tunica adventitia, (b) the middle tunica media, and (c) the inner tunica intima, each of which contributes to the overall stiffness of the vessel wall (Figure 1). The tunica adventitia consists primarily of fibroblasts, collagen‐containing matrix tissue, and the external elastic lamina, while the tunica media is primarily made of smooth muscle cells (SMCs) and elastic fibres. The intima is composed of a single layer of endothelial cells with a basement membrane or internal elastic lamina. Large arteries such as the aorta have an elastic nature that allows them to distend and accommodate blood ejected from the heart during systole. With each systolic contraction of the heart, a pulse wave generated by these elastic arteries travels through the circulation. The velocity of this pulse wave is directly related to the stiffness of the arteries, with a higher velocity indicative of stiffer vessels. Thus, vascular stiffness can be measured by the velocity of arterial pulse waves travelling through the arterial tree.

Figure 1.

Basic vascular structure and stiffness‐associated pathology. The three main layers of vascular structure (tunica adventitia, tunica media, and tunica intima) are illustrated, along with pathological changes that occur in each layer with arterial stiffness. AT1R: angiotensin II type 1 receptor; MR: mineralocorticoid receptor; α‐SMA: α smooth muscle actin

Pulse wave velocity (PWV) is a non‐invasive measure of vascular stiffness that can be performed in both humans and animal models. The gold standard technique in humans is carotid‐femoral PWV (Townsend, 2017; Townsend et al., 2015), in which applanation tonometry is used to obtain carotid and femoral artery wave forms and the external distance between the two measurement sites is recorded in order to calculate a velocity (Table 1). Brachial‐ankle PWV is another method of PWV measurement, in which the brachial and tibial arterial waveforms are used for the analysis (Munakata, 2014). Arterial stiffness can also be assessed locally, using the common carotid artery method, in which the carotid artery is imaged via ultrasound and alterations in arterial diameter along with systemic BP are used to calculate the stiffness index beta (Hirai, Sasayama, Kawasaki, & Yagi, 1989; Hougaku et al., 2006; Kawasaki, Sasayama, Yagi, Asakawa, & Hirai, 1987). Pulse wave analysis can also be performed to obtain measures of wave reflection (augmentation index) and central BPs, further indicators of arterial stiffness. This measurement is typically performed on the radial pulse wave but can also be completed on the carotid or femoral waveforms. With each heartbeat, a pulse wave travels through the circulation, and upon reaching the periphery, the wave is reflected back towards the heart (Coutinho, 2014). In young healthy individuals, this reflected wave arrives back to the heart during diastole. However, with aging and enhanced arterial stiffness, this reflected wave travels faster and hence arrives back to the heart closer to systole, thereby augmenting systolic afterload and reducing diastolic coronary perfusion pressure (Luft, 2012). The reader is referred to recent reviews describing in more detail the strengths of each method and clinical correlations of the different methods of quantifying arterial stiffness (Nemcsik, Cseprekal, & Tisler, 2017; Townsend, 2017; Townsend et al., 2015). Aortic PWV is measured by similar methods in rodent models. The most commonly used method is the “time transit” or TT method, in which Doppler ultrasound is used to determine the difference in arrival times of a pulse wave at two locations along the aorta that are a known distance apart (Fry et al., 2016; Kim et al., 2018; Manrique et al., 2016; Weisbrod et al., 2013). The capacity to reproducibly assess arterial stiffness non‐invasively in both humans and animals has contributed substantially to a body of data describing factors that contribute to the development of arterial stiffness, the sex differences, as well as strategies aimed at reducing arterial stiffness, which we summarize here.

Table 1.

Non‐invasive methods of arterial stiffness assessment

	Pulse wave velocity	Pulse wave analysis	Common carotid artery method	Carotid artery compliance
Method of measurement	Applanation tonometry	Applanation tonometry	Ultrasound	Ultrasound + applanation tonometry
Location of measurement	Carotid‐femoral, brachial‐ankle	Radial, femoral, carotid	Carotid	Carotid
Calculated outcomes	Velocity of arterial pulse wave	Wave reflection, central haemodynamics	Stiffness index beta	Arterial compliance

Note. The major methods used to assess arterial stiffness in humans are outlined, including the location of each measurement along with the calculated outcomes of each. Carotid‐femoral pulse wave velocity is considered the gold standard technique for arterial stiffness assessment in humans.

2. THE EFFECT OF SEX HORMONES ON ARTERIAL STIFFNESS

2.1. Oestrogens and arterial stiffness

Due to the past several decades of research, it is now clear that sex steroid hormones and their receptors are, at least in part, determinants of sex differences in cardiovascular outcomes. Specifically, it is well recognized that hormone receptors, including the oestrogen, progesterone, and androgen receptors, are expressed in the vasculature (Karas, Patterson, & Mendelsohn, 1994; Wu et al., 2014) and that the female sex hormone http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1013 is cardio‐protective. Epidemiological studies have revealed that premenopausal women are protected against the development of CVD when compared to age‐matched men. In addition, women present with CVD a decade later than men, a timeframe that coincides with the postmenopausal loss of oestrogen (Mendelsohn & Karas, 1999). The hormone oestrogen acts through two classical oestrogen receptors (ER): http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=622 and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=621. In addition, recent studies suggest that the http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=22 may mediate some of the rapid actions of oestrogen in the vasculature (Zimmerman, Budish, Kashyap, & Lindsey, 2016). These receptors mediate the actions of steroid hormones through both genomic and rapid non‐genomic actions, which have been previously reviewed and are outside the scope of this review (Iorga et al., 2017; Mendelsohn & Karas, 1999, 2005).

The direct effects of oestrogen on arterial stiffness over the lifespan have begun to be explored. Prepubescent females have less compliant arteries, as measured by carotid‐femoral PWV, and higher pulse pressure than their male counterparts (Ahimastos, Formosa, Dart, & Kingwell, 2003). After puberty, arterial stiffness decreases in females and increases in males, providing further evidence supporting sex hormone effects on vascular stiffness in addition to suggesting a potential effect of overall body/aortic growth on arterial stiffness. Some studies reveal an effect of the menstrual cycle phase on arterial stiffness or compliance, supporting the potential of fluctuating female sex hormones to modulate arterial stiffness (Robb et al., 2009; Stamatelopoulos et al., 2012). In contrast, additional studies have not observed alterations in arterial stiffness across the phases of the menstrual cycle, as measured by PWV; however, several of these studies have demonstrated alterations in wave reflection and arterial compliance across the menstrual cycle (Adkisson et al., 2010; Hayashi et al., 2006; Ounis‐Skali, Mitchell, Solomon, Solomon, & Seely, 2006; M. R. Williams et al., 2001). These alterations are likely due to the associated reductions in BP observed during the luteal phase of the menstrual cycle. Studies examining the effects of oestrogen‐containing oral contraceptive pills (OCP) on PWV in young premenopausal women are mixed; one study has supported the concept that OCPs increase arterial stiffness along with systolic BP and pulse pressure when compared to young premenopausal women not taking OCPs (Hickson et al., 2011), while another study has shown an OCP‐associated increase in central and peripheral BPs, but no increase in arterial stiffness (Yu et al., 2014). When taken together, these studies suggest that female sex hormones do have a modulating effect on arterial stiffness, with potential deleterious effects of OCPs on arterial stiffness that are likely due to the associated increase in BP with OCP use rather than a direct effect on the vascular wall to increase stiffness. Additional clinical investigations have identified ERα gene polymorphisms that are associated with CVD phenotypes in middle‐aged and older men and women (Schuit et al., 2004; Shearman et al., 2003). Further studies have determined that the effects of the ERα polymorphisms on arterial stiffness differ between men and women, suggesting an additional genetic component that may contribute to the mechanisms driving sex differences in the development of arterial stiffness (Hayashi et al., 2007). Future mechanistic studies are warranted to determine the precise molecular effects of oestrogens on arterial stiffness and how these mechanisms are altered not only throughout the lifespan but also by long‐term use of oral contraceptives and other hormonal manipulations.

Arterial stiffness increases with age in both men and women, as measured by carotid‐femoral PWV (Mitchell et al., 2004). However, women experience a more rapid increase in stiffening after the onset of menopause, consistent with the idea that the removal of oestrogen contributes to aging‐associated arterial stiffening in females. Indeed, menopausal status has been shown to be an independent factor that augments the age‐related increase in arterial stiffness, as measured by brachial‐ankle PWV (Zaydun et al., 2006), and reductions in arterial compliance are evident throughout the menopausal transition (Hildreth, Kohrt, & Moreau, 2014). Further studies have shown that carotid‐femoral PWV is decreased in postmenopausal women taking hormone replacement therapy (HRT) when compared to age‐matched women not taking HRT (Rajkumar et al., 1997). Similarly, 18 months of oestrogen treatment reduced arterial stiffness in ovariectomized monkeys (Adams et al., 1990). In contrast, several studies have shown that HRT started in women long after menopause or with pre‐existing CVD, does not protect against CVD events, and could even be detrimental. Specifically, the Women's Health Initiative study indicated that HRT increases the risk of coronary heart disease in women starting treatment 10 years or more after the onset of menopause (Anderson et al., 2004; Hulley et al., 1998; Mosca, 2000; Rossouw et al., 2002). These results are likely due to the differences in the timing of initiating HRT and the different characteristics of the populations that were studied, that is, the Women's Health Initiative trials included women with uncontrolled hypertension, obesity, and several other CVD risk factors. Thus, it is clear that the presence of CVD risk factors, in addition to age and timing of HRT, may play a major role in the effect of oestrogens on vascular health.

2.2. Testosterone and arterial stiffness

http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2858 is the main male sex hormone and plays a crucial role in male sexual development and men's overall health. The http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=628, the receptor for testosterone, is also expressed in the blood vessel, where it mediates direct actions of testosterone on blood vessel function (Wu et al., 2014).

Although testosterone was once perceived to play a role only in promoting CVD (Cohen & Hickman, 1987; Sullivan, Martinez, Gennis, & Gallagher, 1998; Thompson et al., 1989), recent studies have highlighted a protective effect of testosterone on cardiovascular health via its influence on vascular reactivity (Webb et al., 2008) and arterial compliance/PWV in men (Dockery, Bulpitt, Donaldson, Fernandez, & Rajkumar, 2003). Serum testosterone levels have been shown to be an independent negative predictor of the development of carotid artery stiffness in men, even after adjusting for risk factors such as age, pulse pressure, body mass index, and total cholesterol (Hougaku et al., 2006). Furthermore, prostate cancer patients undergoing anti‐androgen therapy develop increased arterial wave reflection and augmentation of central pressure within 3 months (Dockery, Bulpitt, Agarwal, Donaldson, & Rajkumar, 2003; Smith et al., 2001), supporting a role for androgens in preventing vascular stiffness. Similarly, there is evidence that testosterone deficiency is associated with increased carotid‐femoral PWV in men without CVD, suggesting that men with testosterone deficiency exhibit signs of early vascular aging via enhanced arterial stiffness (Vlachopoulos et al., 2014). In middle‐aged men who are free of clinical CVD, low testosterone has also been associated with microvascular dysfunction and an increased augmentation index, a measurement of wave reflection indicative of arterial stiffness (Corrigan et al., 2015). In the setting of CVD risk factors, low serum testosterone is also associated with increased arterial stiffness and all‐cause mortality in male haemodialysis patients (Kyriazis et al., 2011). In populations such as hypogonadal men, where the testosterone level is low, the use of testosterone replacement therapy reverses arterial stiffness (Yaron et al., 2009). Thus, ample data suggest a potential protective role of testosterone on arterial stiffness; however, larger studies are needed to fully elucidate the effects of testosterone in the development of arterial stiffness, especially in men with reduced levels of testosterone in the presence and absence of CVD risk factors.

Although testosterone is mainly a male sex hormone, there is evidence indicating that it may play a role in postmenopausal arterial stiffness in women. Elevated levels of serum testosterone and free androgen index have been associated with subclinical atherosclerosis in otherwise healthy but recently menopausal women. Free androgen index was also a significant predictor of arterial stiffness, as measured by carotid‐femoral PWV (Creatsa et al., 2012). Further research is warranted to determine the potential role of testosterone and its effects on arterial stiffness in women with CVD risk factors. Recent studies have also begun to investigate the effects of hormonal therapy on arterial stiffness in transsexual individuals. Female to male transsexuals treated with androgen therapy have increased arterial stiffness, as measured by brachial‐ankle PWV (Emi, Adachi, Sasaki, Nakamura, & Nakatsuka, 2008). In contrast, male to female transsexuals treated with oestrogen, but not oestrogen plus progestin, exhibit decreased wave reflection and brachial‐ankle PWV (Sharula et al., 2012). It is clear that sex hormones substantially impact arterial stiffness, and these effects may differ with age, CVD risk factor presence, and the interaction with the status of other sex hormones in an individual. The determination of the detailed molecular mechanisms that contribute to the effects of altered sex hormone levels on arterial stiffness is warranted and could aid in developing sex‐specific therapeutic strategies to reduce CVD risk and mortality.

3. SEX DIFFERENCES IN THE IMPACT OF CVD RISK FACTORS ON ARTERIAL STIFFNESS

Traditional CVD risk factors, such as aging, hypertension, diabetes, and obesity, have all been associated with elevations in arterial stiffness. However, there is evidence of sex differences in the effects of CVD risk factors on arterial stiffness, especially with regard to clinical pathologies that are more prevalent and/or unique to women, such as pre‐eclampsia and polycystic ovary syndrome.

3.1. Aging

Arterial stiffness, along with systolic BP and pulse pressure, increases with aging in both men and women (Figure 2; Dart & Kingwell, 2001; Mitchell, 2009; Mitchell et al., 2004; 2007). While stiffness and CVD risk increase linearly in men, women experience a curvilinear aging trend, with a flatter curve in young women and a steep increase in arterial stiffness and cardiovascular events after menopause (Lerner & Kannel, 1986; Narkiewicz, Kjeldsen, & Hedner, 2006). Specifically, aging women develop greater increases in pulse pressure and wave reflection compared to age‐matched male counterparts. A component of these differences may be due to women's smaller height and aortic size. Despite this intrinsic size difference, multiple studies have shown that this persists after adjusting for differences in body and aortic size, suggesting that the differences in size do not fully explain the magnitude of differences in pulse pressure and wave reflection (Coutinho et al., 2013; Dart et al., 2008; Mitchell et al., 2004). Thus, the different clinical manifestations of arterial stiffness with regard to aging in men and women suggest that there are distinct sex‐specific mechanisms contributing to this process.

Figure 2.

Cardiovascular disease‐related pathologies and risk factors that lead to arterial stiffness. Aging, hypertension, obesity, and metabolic syndrome contribute to arterial stiffness in both males and females. In males, testosterone deficiency is associated with elevations in arterial stiffness, whereas in females, a number of clinical conditions have been associated with arterial stiffness such as pre‐eclampsia, polycystic ovary syndrome (PCOS), endometriosis, and a variety of autoimmune disorders

There are several mechanisms that are known to be involved in the development of arterial stiffness (Table 2). The most widely studied mechanisms involve structural alterations to the extracellular matrix (ECM), with enhanced collagen deposition and increased elastin breakdown (Aroor et al., 2013; Luft, 2012; Tsamis, Krawiec, & Vorp, 2013; Zieman, Melenovsky, & Kass, 2005). However, these increases in collagen deposition have not been consistent in some preclinical studies of hypertension (Bezie et al., 1998; Koffi et al., 1998; van Gorp et al., 2000), suggesting that there are additional mechanisms contributing to arterial stiffness. Recent studies have emerged implicating a role for the vascular smooth muscle cell (VSMC) as a direct source of enhanced arterial stiffness via alterations in the cytoskeleton and integrin interactions with the ECM (Gao, Saphirstein, Yamin, Suki, & Morgan, 2014; Qiu et al., 2010; Sehgel et al., 2013; Sehgel, Sun, et al., 2015). The detailed role of the VSMC in aging‐associated arterial stiffness has recently been reviewed elsewhere (Lacolley, Regnault, & Avolio, 2018).

Table 2.

Sex differences in mechanisms of arterial stiffness in aging, hypertension, and obesity

Mechanism	Males	Females
	Relevant pathways
ECM alterations	Collagen	Collagen
	Elastin
VSMC stiffening	β1‐integrin Rho kinase	Unknown
Oxidative stress	Superoxide Mitochondrial‐derived ROS NADPH‐oxidase	Superoxide eNOS uncoupling via BH(4) reductions
Inflammation	NF‐κB T‐cell activation	NF‐κB
RAAS signalling	SMC‐MR AT1R activation	EC‐MR ENaC

Note: ECM, extracellular matrix; VSMC, vascular smooth muscle cell; eNOS, endothelial NOS; NADPH, NAD phosphate oxidase; BH(4), tetrahydrobiopterin; SMC‐MR, smooth muscle cell mineralocorticoid receptor; AT1R, angiotensin II type 1 receptor; EC‐MR, endothelial cell mineralocorticoid receptor; ENaC, epithelial sodium channel.

Although the clinical manifestations of arterial stiffness suggest distinct sex‐specific mechanisms, there have been limited preclinical studies to examine mechanistic sex differences. It has been shown that aged male and female monkeys develop similar levels of arterial stiffness and that these changes were associated with alterations in collagen content, but a decrease in elastin was only noted in male monkeys (Qiu et al., 2007). Similarly, aging male mice develop arterial stiffness, as measured by PWV, in addition to an up‐regulation of pro‐fibrotic vascular genes and fibrosis (Kim et al., 2018). In addition to the role for the ECM, recent studies have begun to explore the role for VSMC directly contributing to stiffness (Qiu et al., 2010; Sehgel et al., 2013; Sehgel, Vatner, & Meininger, 2015; Zhu et al., 2012) by utilizing atomic force microscopy probes to nano‐indent the beta surface in order to obtain an estimation of the cell elastic modulus. These preclinical studies in young and aged male monkeys showed that vascular stiffness with aging is also due to intrinsic changes in the stiffness of the SMCs. α‐Smooth muscle actin and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2455&familyId=760&familyType=CATALYTICRECEPTOR#OtherNames expression were increased in VSMCs from the aged male monkeys. α‐Smooth muscle actin is a stress fibre‐specific isoform in the VSMC and plays a major role in mechano‐sensation and transmission through the integrin by transferring force to the ECM, causing ECM remodelling (J. Wang, Zohar, & McCulloch, 2006). Integrins provide a mechanical connection between the VSMCs and ECM environment. The increased expression of β1‐integrin correlated with increased adhesiveness to fibronectin, suggesting a reverse “inside‐out” signalling of VSMCs with the ECM. These data suggest that, in males, β1‐integrin and α‐smooth muscle actin play a crucial role in mediating VSMC stiffness with aging. Additional studies have used a magnetic tweezer apparatus to measure cortical stiffness of the VSMC in aged male rats (Saphirstein et al., 2013). These studies identified the focal adhesion as a cell‐matrix linker and significant component of the development of aortic stiffness with aging. Further studies are warranted in order to fully elucidate the mechanisms contributing to VSMC‐specific stiffness as well as the potential role for VSMC stiffness in female aging, which is relatively understudied.

Additional preclinical studies exploring the mechanisms of large artery stiffening with aging have been performed in males only. These studies have demonstrated a role for vascular oxidative stress derived from mitochondrial sources and increased superoxide production as mechanisms contributing to arterial stiffening with aging in male mice (de Picciotto et al., 2016; Fleenor et al., 2014; Gioscia‐Ryan et al., 2018). Furthermore, renin–angiotensin–aldosterone system (RAAS) signalling contributes to aging‐associated arterial stiffness (Jia, Aroor, Hill, & Sowers, 2018). Activation of the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=34 in the VSMC contributes to the development of arterial stiffness via the accumulation of collagen as well as by contributing to increased vasoconstriction, oxidative stress, and inflammation (Ford, Li, & Pickering, 1999; Yan, Kim, Aizawa, & Berk, 2003; Zieman et al., 2005). Further, there is evidence of sex and aging‐related differences in http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=35 functions in tissues such as the kidney and brain (McCarthy, Widdop, Denton, & Jones, 2013). Specifically, in the kidney, aged female mice have higher gene expression of AT₁ and AT₂ receptors (Mirabito et al., 2014); however, this remains to be studied in the vasculature especially with regard to its impact on aging‐associated arterial stiffness. Another component of the RAAS system, the SMC mineralocorticoid receptor (SMC‐http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=626, the receptor for aldosterone) was recently identified as a contributor to aging‐associated arterial stiffness, vascular pro‐fibrotic gene expression, and vascular fibrosis in male mice (Kim et al., 2018). A role for SMC‐MR in female vascular aging has yet to be explored in vivo. However, in vitro studies have demonstrated that the ERα transcriptionally inhibits some of the actions of the MR (Barrett Mueller et al., 2014), suggesting that females are potentially protected from the deleterious effects of MR until they are postmenopausal. Future studies are warranted to determine the role of vascular MR in female aging as well as the potential interactions of MR and ERα in vascular aging.

Preclinical studies have also identified various therapies that have reversed the aging‐associated arterial stiffening in males, such as curcumin (Fleenor et al., 2013), sodium nitrites (Sindler et al., 2011), mitochondrial antioxidants (Gioscia‐Ryan et al., 2018), nicotinamide mononucleotide (de Picciotto et al., 2016), MR blockade (Kim et al., 2018), and caloric restriction (Donato et al., 2013). Several of these therapies—mitochondrial antioxidants (Rossman et al., 2018), nicotinamide riboside (Martens et al., 2018), and sodium nitrite (DeVan et al., 2016)—have begun to be translated into studies in healthy older human males and females. The clinical results suggest that similar oxidative stress‐derived mechanisms may contribute to aging‐associated arterial stiffness in healthy postmenopausal women. However, these studies may have been underpowered to detect any potential sex differences in the efficacy of such therapies. Additionally, although the women were postmenopausal, the length of the postmenopausal period may have varied between patients, masking potential differences between recently postmenopausal and long‐term postmenopausal women. Several clinical studies have examined mechanisms of arterial stiffness in postmenopausal women; acute administration of http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4781 (Moreau, Gavin, Plum, & Seals, 2005), oral http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5276 (Moreau, Meditz, Deane, & Kohrt, 2012), and TNF‐α inhibitor http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=6789 (Moreau, Deane, Meditz, & Kohrt, 2013) improved carotid artery compliance in oestrogen‐deficient postmenopausal women. These studies suggest that elevated vascular oxidative stress and inflammation contribute to arterial stiffness in postmenopausal women. Furthermore, these results specifically suggest that reductions in NO via elevations in oxidative stress contribute to postmenopausal‐associated arterial stiffness. Future studies are warranted to determine additional mechanisms that may play a role in vascular stiffening in aging women and how these may differ from aging men as such results might suggest novel sex‐specific aging therapies.

3.2. Hypertension

High BP promotes vascular thickening and fibrosis that can contribute to vascular stiffness. At the same time, vascular stiffness contributes to rising BP by increasing pulse pressure and damaging the microvasculature to impair vasodilation, resulting in a positive feedback mechanism that promotes the development and progression of hypertension and stiffness over time (Mitchell, 2014). The order of these events is controversial; some studies in normotensive individuals have demonstrated that arterial stiffness is associated with accelerated BP elevation (Dernellis & Panaretou, 2005; Liao et al., 1999; Najjar et al., 2008). Additionally, the Framingham Heart Study reported that aortic wall stiffness increases from early adulthood and may, in part, contribute to the increase in BP later in life (Kaess et al., 2012). Other studies have noted no association between alterations in BP and arterial stiffness progression (Benetos et al., 2002; Wildman et al., 2005). The controversial relationship between hypertension and arterial stiffness has recently been reviewed further (Franklin, 2005; Mitchell, 2014).

Isolated systolic hypertension (ISH), characterized by increased systolic BP with normal or low diastolic BP, that is, increased pulse pressure, is extremely common with aging and preferentially affects women (D. Martins, Nelson, Pan, Tareen, & Norris, 2001). Arterial stiffening, specifically in the aorta, is thought to be a main underlying cause of ISH (Acelajado & Oparil, 2009; L. C. Martins et al., 2011), as it is mainly influenced by large artery compliance. ISH is more common in older women compared to men at least in part due to greater aortic stiffness. This is compounded by the observation that women are less likely than age‐matched men to achieve optimal control of BP with current therapeutic algorithms; thus, hypertension may compound the stiffness, leading to a feed‐forward cycle of worsening vascular stiffness and rising systolic BP (Keyhani, Scobie, Hebert, & McLaughlin, 2008; Wilkins, Gee, & Campbell, 2012). One potential reason might be that most antihypertensive medications are designed to lower blood volume or peripheral vascular resistance but have little effect on arterial compliance and pulse pressure. Thus, these data suggest that enhanced arterial stiffness in aged women may be a root cause of the higher prevalence of ISH and that further research is needed to determine the mechanistic causes of arterial stiffness in females in order to identify novel therapies to achieve better control of BP in aged women. A recent study also demonstrated that in a large cohort of men and women with elevated BP, increased aortic stiffness and haemodynamic load were associated with concentric left ventricular remodelling in postmenopausal women but not men (Coutinho, Pellikka, Bailey, Turner, & Kullo, 2016). This study suggests that the effects of elevated arterial stiffness and haemodynamic load are exacerbated in aging women compared to men, presenting the need for further study to investigate the potential therapeutic value of controlling for arterial stiffness and haemodynamic load as a sex‐specific therapy to prevent left ventricular remodelling and the associated CVD burden.

Limited preclinical studies have examined potential sex differences in the mechanisms of arterial stiffness due to hypertension. Sex‐specific quantitative trait loci affecting aortic stiffness in the Dahl salt‐sensitive hypertensive rat model have recently been identified (Decano et al., 2016). In male spontaneously hypertensive rats, arterial stiffness was decreased with arotinolol, a nonselective α/β‐adrenoceptor blocker (W. Zhou et al., 2014). This improvement was associated with increased NO production and decreased collagen deposition. In contrast, additional studies in SHRs indicated VSMC stiffness as a mechanism of elevated aortic stiffness in the setting of hypertension, while aortic collagen and elastin were more notably altered with aging (Sehgel et al., 2013; Sehgel, Sun, et al., 2015). Further, http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=289 was recently identified as a regulator of VSMC stiffness in male SHRs via the serum response factor/myocardin pathway (N. Zhou et al., 2017). Vascular oxidative stress has also been shown to promote systemic inflammation leading to T‐cell activation that ultimately contributes to vascular collagen deposition and aortic stiffening in hypertensive male mice (Wu et al., 2016). Despite these recent mechanistic insights, there are limited preclinical studies directly comparing males and females in terms of the effects of hypertension on arterial stiffness. While it is plausible that some of the same mechanisms identified in the aforementioned studies in males may also play a role in female hypertension‐associated arterial stiffness, future studies are warranted in order to determine if the mechanisms are indeed the same or if additional sex‐specific factors may contribute to differential molecular mechanisms.

3.3. Impact of obesity/metabolic syndrome on vascular stiffness in males and females

Obesity is a worldwide public health epidemic that has led to an increase in metabolic syndrome, type 2 diabetes, and CVD. Cardiovascular mortality for overweight individuals is enhanced, particularly in those with systemic hypertension and other components of the metabolic syndrome (Lewis et al., 2009). Arterial stiffness is elevated in patients with obesity and has also been shown to predict future cardiovascular events (Safar et al., 2013; Sutton‐Tyrrell et al., 2001). Furthermore, several large population studies have revealed that women have higher obesity rates than men (Flegal, Kruszon‐Moran, Carroll, Fryar, & Ogden, 2016; Kramer et al., 2016; Ogden, Carroll, Fryar, & Flegal, 2015; Taylor et al., 2008; Yun, Zhu, Black, & Brownson, 2006) and that women with obesity and diabetes have a greater risk for CVD (Mitchell, 2009; Natarajan, Liao, Cao, Lipsitz, & McGee, 2003; Regensteiner et al., 2015). Abdominal adiposity has also been shown to have a greater association with arterial stiffness in young women versus men, suggesting a potentially greater effect of obesity in raising CVD risk in women than in men, thereby eliminating the protection from CVD seen in non‐obese premenopausal women (Scuteri et al., 2012).

Preclinical studies utilizing the high‐fat/high‐fructose or “Western diet” (WD)‐induced obesity mouse model have begun to explore potential mechanisms of obesity‐associated arterial stiffness (Weisbrod et al., 2013). The WD is known to induce arterial stiffness in both male and female mice (Bender et al., 2015; DeMarco et al., 2015). MR blockade prevents WD‐induced arterial stiffness in female mice (DeMarco et al., 2015). Further studies implicate a direct role for endothelial cell MR (EC‐MR) in WD‐induced arterial stiffness in females (Jia et al., 2016). This study utilized in vivo PWV measurements as well as ex vivo atomic force microscopy studies in freshly isolated aortic endothelial cells to demonstrate that EC‐MR promotes aortic vessel stiffness in addition to aortic endothelial cell stiffness in response to a WD. Further mechanistic studies revealed that EC‐MR promotes increased endothelial membrane epithelial Na⁺ channel (http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=122) expression, impaired http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1249 activation, and enhances the inflammatory response and M1/M2 macrophage polarization, all of which likely contribute to aortic remodelling and stiffness observed in response to the WD. A follow‐up study confirmed a role for ENaC by reversing the WD‐induced arterial stiffening in females with chronic http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2421 (ENaC inhibitor) treatment (Martinez‐Lemus et al., 2017). Future research is warranted to determine the role of EC‐MR signalling in WD‐induced arterial stiffness in male mice. Recent studies have examined the role of http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4731 and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2646 in WD‐induced arterial stiffening, as high‐fructose diets are known to increase uric acid levels via activation of XO in the liver and elevations in uric acid levels have been associated with increased vascular stiffness and CVD in women (Fang et al., 2014; Nogi et al., 2015). Uric acid was shown to specifically promote VSMC and endothelial cell stiffness in WD‐fed male mice, and XO inhibition prevented WD‐induced vascular stiffness in male and female mice, suggesting that XO is a source of arterial oxidative stress leading to arterial stiffness in both male and female WD‐fed mice (Aroor et al., 2017; Lastra et al., 2017). Of note, these studies did not examine in vivo PWV measurements and instead used atomic force microscopy to examine cell‐specific stiffness. Thus, the effect of XO inhibition on in vivo aortic stiffness in WD‐fed rodents is unknown. Additional studies utilizing the WD‐fed mouse model reveal a similar role for endothelial cell‐ERα in males and females, with male and female mice lacking the ERα specifically in ECs (EC‐ERα KO) having decreased arterial stiffness in comparison to their EC‐ERα intact WD‐fed littermates (Manrique et al., 2016; Manrique‐Acevedo et al., 2017). Together, these studies suggest that EC‐ERα does not protect against WD‐induced arterial stiffness in females or males and, instead, may potentially contribute to it through different mechanisms. In females, the decrease in vascular stiffness observed with EC‐ERα deletion was not due to a decrease in oxidative stress or inflammation, as 3‐nitrotyrosine (oxidative stress marker) and TGF‐β (inflammation marker) levels in the aorta were similar between groups. In males, it was found that the number of internal elastic lamina fenestrae increased while http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=738 activation decreased with the deletion of ERα, suggesting a paradoxical role of ERα in regard to arterial stiffness in WD‐fed male mice. Further research is warranted to fully understand these mechanisms in addition to examining the effect of ERα in the SMC and its potential role in arterial stiffness. Particularly lacking are studies that compare male and female littermates side by side.

4. SEX‐SPECIFIC EFFECTS OF OTHER CVD‐RELATED PATHOLOGIES ON ARTERIAL STIFFNESS

4.1. Heart failure and coronary artery disease

Heart failure is a major public health concern, affecting nearly 26 million people worldwide (Savarese & Lund, 2017), half of whom have HFpEF. Recently, it has become clear that HFpEF affects twice as many women as men (Beale et al., 2018; Borlaug & Redfield, 2011). As arterial stiffening of the aorta increases with age, cardiac workload and wall stress increase, leading to increased left ventricular systolic stiffness and wall thickness. Increased arterial stiffness and wave reflection have been associated with left ventricular diastolic dysfunction and impaired ventricular‐arterial coupling in women but not men (Coutinho et al., 2013; Goto et al., 2013; Shim et al., 2011), suggesting that arterial stiffness may promote the deleterious alterations in cardiac structure and function known to occur with HFpEF. Further studies are warranted to determine the effect of reducing arterial stiffness on the development and progression of HFpEF in aging women (and men).

In addition, recent studies have demonstrated sex differences in the effects of arterial compliance on the development and impact of coronary artery disease. Specifically, lower arterial compliance (indicative of elevated arterial stiffness) is associated with a higher burden of coronary artery plaque and calcification in women not men (Coutinho et al., 2017). Furthermore, lower arterial compliance was also associated with impaired coronary microvascular function, as measured by myocardial flow reserve in women not men (Coutinho et al., 2018). Coronary microvascular dysfunction is a much more common cause of cardiac ischaemia in females compared to males that is not improved by traditional revascularization therapies (Paul, Sivanesan, & Schulman‐Marcus, 2017; Titterington, Hung, Saraf, & Wenger, 2018). Future studies are warranted to investigate the mechanisms contributing to these sex differences.

4.2. Female‐specific conditions that contribute to vascular stiffness

Pre‐eclampsia is a hypertensive complication of pregnancy, diagnosed by new hypertension and proteinuria that develops during pregnancy and is typically associated with indications of damage to other organ systems such as the liver and kidneys. Pre‐eclampsia contributes to poor outcomes for both the mother and baby (Chen, Jaffe, & Karumanchi, 2014). Women with a history of pre‐eclampsia have a significantly greater risk of developing CVD later in life, but the mechanisms are not known (Bellamy, Casas, Hingorani, & Williams, 2007; McDonald, Malinowski, Zhou, Yusuf, & Devereaux, 2008; Weissgerber et al., 2013). Women exposed to pre‐eclampsia also have increased arterial stiffness and wave reflection (Hausvater et al., 2012) compared to women with normotensive pregnancies. Elevations in arterial stiffness persist in women exposed to pre‐eclampsia even after pregnancy and may contribute to the observed increased risk of developing CVD later in life (Chen et al., 2014). In addition, a small number of studies have reported elevations in arterial stiffness in women with other female‐specific health issues such as endometriosis (Tani et al., 2015) and polycystic ovary syndrome (Armeni et al., 2013; Sasaki et al., 2011). However, the precise mechanisms contributing to arterial stiffness in these conditions are unclear. Future studies are warranted to explore not only the complex mechanisms contributing to arterial stiffness in female‐specific health disorders but also the role of arterial stiffness in the long‐term health outcomes in women with a history of pre‐eclampsia and other hypertensive complications of pregnancy.

4.3. Autoimmune disorders

Autoimmune diseases affect 3–5% of the population (L. Wang, Wang, & Gershwin, 2015), with a heightened gender bias, more females being affected than males (Ngo, Steyn, & McCombe, 2014). Many autoimmune disorders, such as Hashimoto's thyroiditis (HT), are associated with increased CVD risk, even in young premenopausal women, likely at least in part due to chronic inflammation that promotes the development of atherosclerosis (Hollan et al., 2013). In premenopausal women with HT, arterial stiffness, as measured by PWV, is increased to levels similar to that of postmenopausal women with and without HT (Stamatelopoulos et al., 2009). Interestingly, arterial stiffness was independently correlated with age, the presence of HT, and menopausal status in this study. These results suggest that HT may be associated with early vascular aging in premenopausal women and that the effect of HT on vascular stiffness in postmenopausal women may be masked due to the additional presence of CVD risk factors such as age. Systemic lupus erythematosus (SLE) is another autoimmune disorder known to primarily affect women (Yacoub Wasef, 2004). SLE patients, specifically females, have been shown to have increased arterial stiffness (Sabio et al., 2015). Additional investigations have identified a positive correlation between elevations in circulating http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5015 and arterial stiffness in women with SLE, suggesting the potential for leptin to play a role in the development of arterial stiffness in this population (Vadacca et al., 2013). Additional autoimmune disorders that more frequently occur in women and are associated with elevated arterial stiffness include rheumatoid arthritis (Ambrosino et al., 2015; Shin et al., 2015), multiple sclerosis (Irzyk et al., 2015), and Graves' disease (Bodlaj et al., 2007; Inaba et al., 2002). Much of the work examining the presence and severity of arterial stiffness in women with autoimmune disorders has been performed at the clinical level only. Thus, there remains a lack of understanding of the molecular mechanisms contributing to elevations in arterial stiffness observed in these populations.

5. SEX‐SPECIFIC EFFECTS OF LIFESTYLE ON ARTERIAL STIFFNESS

Two major lifestyle determinants of the development of arterial stiffening, especially in regard to aging, are physical activity and dietary habits (Pase, Grima, & Sarris, 2011; Sacre, Jennings, & Kingwell, 2014; Tanaka et al., 2000; Tanaka, DeSouza, & Seals, 1998). Specifically, regular physical activity is associated with lower levels of arterial stiffness and overall reduced CVD risk. A majority of the preclinical studies examining the effects of physical activity and diet on arterial stiffness have primarily been performed in males only; however, as discussed below, there is some clinical evidence of potential sex differences in the influence of physical activity and diet on arterial stiffness.

5.1. Sex‐specific effects of chronic physical activity and exercise training on arterial stiffness

Studies which include both males and females without separate analysis examining sex differences indicate that chronic aerobic exercise reduces arterial stiffness and prevents aging‐associated increases in stiffness, which has been recently reviewed elsewhere (Santos‐Parker, LaRocca, & Seals, 2014). Amongst healthy young men and women, women have lower baseline arterial stiffness, as measured by PWV (Mitchell et al., 2004; Perdomo et al., 2016). Several studies in young healthy populations report a reduction in arterial stiffness in response to acute bouts of aerobic exercise training in men only (Doonan, Mutter, Egiziano, Gomez, & Daskalopoulou, 2013; Perdomo et al., 2016). The lack of effect of exercise on arterial stiffness in young healthy women is likely due to their lower stiffness at baseline compared to age‐matched males, leaving minimal room for exercise‐induced reductions in arterial stiffness. Similarly, in healthy adults, arterial stiffness was inversely associated with cardiorespiratory endurance in men but not in women (Chung, Kim, Jin, Kim, & Hong, 2018). Additionally, 3 months of aerobic exercise did not affect arterial stiffness in postmenopausal women (Seals et al., 2001). Conversely, endurance‐trained women are protected from the aging‐associated increase in pulse pressure observed in their sedentary counterparts (Seals, Stevenson, Jones, DeSouza, & Tanaka, 1999). More recently, 12 weeks of moderate aerobic exercise was shown to decrease carotid artery stiffness in postmenopausal women (Matsubara et al., 2014). The type of exercise chosen combined with the patient characteristics may lead to differential effects of exercise on arterial stiffness in different studies. For example, chronic resistance training is associated with decreased arterial compliance in healthy middle‐aged men (Miyachi et al., 2003) while 12 weeks of low‐intensity resistance training did not reduce arterial stiffness in obese postmenopausal women (Figueroa et al., 2013). In contrast, 16 weeks of resistance training reduced wave reflection in healthy older females but not males (A. D. Williams, Ahuja, Almond, Robertson, & Ball, 2013). The mechanisms by which aerobic exercise improves arterial stiffness, especially with regard to aging, involve reductions in oxidative stress and inflammation, along with alterations in collagen and elastin content (Santos‐Parker et al., 2014). These mechanistic determinations were made via preclinical studies in male rodents; thus, potential sex differences in these mechanisms have yet to be determined.

5.2. Sex‐specific effects of diet on arterial stiffness

Numerous research studies indicate effects of diet on arterial stiffness (Sacre et al., 2014; Seals, Moreau, Gates, & Eskurza, 2006; Zieman et al., 2005). Common dietary interventions such as caloric restriction (weight loss), sodium restriction, and nutrient supplements such as polyphenols have been shown to decrease arterial stiffness in various clinical populations. Evidence for potential sex differences in these effects is currently lacking.

Caloric restriction results in weight loss in both obese men (Balkestein, van Aggel‐Leijssen, van Baak, Struijker‐Boudier, & Van Bortel, 1999) and obese postmenopausal women (Figueroa et al., 2013). These changes in weight were associated with reductions in BP as well as increased carotid artery compliance and reduced arterial stiffness, as measured by brachial‐ankle PWV. Caloric restriction has also been shown to prevent aging‐associated increases in arterial stiffness in male rodents, via the prevention of deleterious changes in collagen and elastin (Ahmet, Wan, Mattson, Lakatta, & Talan, 2005; Donato et al., 2013). Dietary sodium restriction is another lifestyle intervention shown to improve arterial stiffness in older men and women (Cappuccio, Markandu, Carney, Sagnella, & MacGregor, 1997; Gates, Tanaka, Hiatt, & Seals, 2004). More specifically, 3 months of moderate dietary sodium restriction in postmenopausal women resulted in reduced arterial stiffness concomitant with reductions in systolic BP (Seals et al., 2001). Conversely, in a controlled feeding study, a 7‐day high salt diet increased central systolic BP in young and middle‐aged healthy men and women and the middle‐aged adults also exhibited greater forward and reflected wave amplitudes in response to high salt (Muth et al., 2017). These results suggest that with aging, high dietary sodium leads to greater increases in central BP, potentially as a result of the increased wave reflections, with no detectable sex difference. Additionally, habitual lower dietary potassium intake has been associated with greater wave reflection and arterial stiffness in young healthy adults (Lennon‐Edwards et al., 2014), suggesting the potential for increasing dietary potassium to have beneficial effects on arterial stiffness, especially in the setting of a high sodium diet. Future studies are warranted to explore potential sex differences in the balance of dietary sodium and potassium in regard to effects on arterial stiffness. Polyphenol supplementation has also been shown to reduce arterial stiffness in a variety of clinical populations (Dohadwala et al., 2011; Pase et al., 2011; Teede, Giannopoulos, Dalais, Hodgson, & McGrath, 2006), including menopausal and perimenopausal women (Nestel et al., 1997). Potential sex differences in the effects of polyphenols on arterial stiffness have yet to be explored.

Cigarette smoking is associated with an increase in both CVD risk and vascular stiffness/dysfunction (Cacciola, Guarino, & Polosa, 2007; Celermajer et al., 1996; Esen et al., 2004) in addition to being a more potent CVD risk factor in women than in men (Huxley & Woodward, 2011). Sex differences in the effects of smoking on arterial stiffness have recently been observed (Mozos, Maidana, Stoian, & Stehlik, 2017). Duration of smoking was associated with increased arterial stiffness independently of sex; however, the female group had significantly lower duration of chronic smoking. These results suggest that females may be more sensitive to the detrimental vascular effects of smoking, thus, requiring less exposure to smoking in order to observe dysfunctional large artery stiffness. Future studies are warranted to further determine the mechanisms of potential sex differences in the long‐term effects of smoking on arterial stiffness.

6. SUMMARY AND FUTURE DIRECTIONS

Decades of research have made it clear that there are sex differences in the development of CVD and specifically of the timing and degree of arterial stiffening. There are scant mechanistic data determining the factors that contribute to arterial stiffness in female‐specific pathologies or medical conditions more common in females, such as pre‐eclampsia, obesity, and various autoimmune disorders. Given the importance of arterial stiffening in the development of CVD and evidence supporting sex differences in CVD‐related outcomes including HFpEF, coronary microvascular dysfunction, and even mortality, additional preclinical and clinical studies are warranted to further determine sex‐specific molecular mechanisms of arterial stiffening with aging, obesity, and many other clinical pathologies associated with CVD. Specifically, studies investigating sex differences in the role of the RAAS in modulating arterial stiffness, as well as the potential role for VSMC stiffness in females, are warranted to advance the currently known mechanisms contributing to arterial stiffness. Advancement of our understanding of the molecular mechanisms underlying sex differences in the development of arterial stiffness is crucial to the determination of novel sex‐specific pharmacological targets that can then be tested at the clinical level to reduce CVD burden associated with vascular stiffening.

6.1. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018) and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander, Christopoulos, et al., 2017; Alexander, Cidlowski, et al., 2017; Alexander, Fabbro et al., 2017a,b; Alexander, Peters, et al., 2017).

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

DuPont JJ, Kenney RM, Patel AR, Jaffe IZ. Sex differences in mechanisms of arterial stiffness. Br J Pharmacol. 2019;176:4208–4225. 10.1111/bph.14624