Sex- specific and hormone- related differences in vascular remodelling in atherosclerosis

Atherosclerosis, a lipid- driven inflammatory disease, is the main underlying cause of cardiovascular diseases (CVDs) both in men and women. Sex- related dimorphisms regarding CVDs and atherosclerosis were observed since more than a decade ago. Inflammatory mediators such as cytokines, but also endothelial dysfunction, vascular smooth muscle cell migration and proliferation lead to vascular remodelling but are differentially affected by sex. Each year a greater number of men die of CVDs compared with women and are also affected by CVDs at an earlier age (40– 70 years old) while women develop atherosclerosis- related complications mainly after menopause (60 + years). The exact biological reasons behind this discrepancy are still not well- understood. From the numerous animal studies on atherosclerosis, only a few include both sexes and even less investigate and highlight the sex- specific differences that may arise. Endogenous sex hormones such as testosterone and oestrogen modulate the atherosclerotic plaque composition and the frequency of such plaques. In men, testosterone seems to act like a double- edged sword as its decrease with ageing correlates with an increased risk of atherosclerotic CVDs, while testosterone is also reported to promote inflammatory immune cell recruitment into the atherosclerotic plaque. In premenopausal women, oestrogen exerts anti- atherosclerotic effects, which decline together with its level after menopause resulting in increased CVD risk in ageing women. However, the interplay of sex hormones, sex- specific immune responses and other sex- related factors is still incompletely understood. This review

cular remodelling but are differentially affected by sex.Each year a greater number of men die of CVDs compared with women and are also affected by CVDs at an earlier age (40-70 years old) while women develop atherosclerosis-related complications mainly after menopause (60+ years).The exact biological reasons behind this discrepancy are still not well-understood.From the numerous animal studies on atherosclerosis, only a few include both sexes and even less investigate and highlight the sex-specific differences that may arise.Endogenous sex hormones such as testosterone and oestrogen modulate the atherosclerotic plaque composition and the frequency of such plaques.In men, testosterone seems to act like a double-edged sword as its decrease with ageing correlates with an increased risk of atherosclerotic CVDs, while testosterone is also reported to promote inflammatory immune cell recruitment into the atherosclerotic plaque.In premenopausal women, oestrogen exerts anti-atherosclerotic effects, which decline together with its level after menopause resulting in increased CVD risk in ageing women.However, the interplay of sex hormones, sex-specific immune responses and other sex-related factors is still incompletely understood.This review 1

| INTRODUCTION
Cardiovascular diseases (CVDs) are the leading cause of death globally both in men and women.The most important underlying pathology of CVDs is atherosclerosis, an inflammatory disease, driven by lipids and fostering the development of plaques in the intimal layer of the arterial wall.Atherosclerosis is initiated by endothelial dysfunction, which increases the permeability of the endothelial barrier allowing low-density lipoprotein (LDL) and its modified versions like oxidized LDL (oxLDL) to invade the intimal layer. 1 These modified lipids further promote an inflammatory response and upregulation of adhesion molecules such as intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1) and cytokines like monocyte recruitment protein 1 (MCP-1 also known as CCL2) on the surface of endothelial cells (ECs).Thereby, the recruitment of monocytes and other inflammatory cells namely neutrophils and T cells from the circulation into the arterial wall is promoted. 2Once inside the vessel wall, monocytes differentiate into macrophages, which engulf oxLDL and develop into lipid-laden foam cells.Overloaded foam cells will eventually undergo apoptosis or necrosis forming a necrotic core. 2 More advanced lesions form a fibrous cap, which is a barrier built up by vascular smooth muscle cells (VSMCs) migrating from the media into the intimal layer of the vasculature.These VSMCs mostly reside directly underneath the EC lining and play an important role in plaque stability.In atherosclerosis, VSMCs change their phenotype from contractile, which is essential for vascular tone and function, into a synthetic phenotype, which promotes their migration, proliferation and production of extracellular matrix (ECM), which supports the stability of the plaque. 3ascular wall remodelling is one characteristic of the pathophysiology of atherosclerosis and refers to structural and functional alterations of the vascular wall. 4This vascular remodelling is caused by the interplay of EC dysfunction, VSMC migration and proliferation, foam cell formation and increased presence of inflammatory mediators such as cytokines.The integrity and stability of the atherosclerotic plaque are also affected by adverse vascular remodelling causing plaque rupture fostering thrombus formation.Plaques that are prone to rupture are described to be smaller (30%-40% vessels stenosis) compared with stable lesions and generally contain a large lipid core, a thin fibrous cap and numerous inflammatory cells. 5oreover, also plaque erosion can occur, which mainly occurs in plaques that are characterized by a thick fibrous cap, large amounts of extracellular matrix, presence of neutrophil extracellular traps (NETs) and fewer inflammatory cells within the lesion.Plaque erosion is initiated by shear stress, which induces Toll-like receptor (TLR)-2 expression on ECs causing EC desquamation and apoptosis.EC detachment attracts neutrophils and promotes NET release.These NETs can subsequently trap circulating platelets leading to a platelet-rich 'white' thrombus formation. 5Hence, plaque rupture but also plaque erosion may eventually cause major cardiovascular events like myocardial infarction (MI) or stroke. 6Literature suggests that vascular remodelling is not only affected by classical risk factors like dyslipidemia and age but also depends on sex differences.Therefore, this review aims to summarize differences described in vascular remodelling in atherosclerosis between males and females and the implication of sex hormones in this process.

| Risk factors for atherosclerosis and disparity between men and women
CVD is the leading cause of death for both men and women worldwide. 7According to the World Health Organization (WHO), nearly 18 million people have died from CVD in 2019 and 85% of these deaths were caused by a stroke or heart attack. 80][11] Women usually develop atherosclerosis following menopause, which results in more women suffering from atherosclerotic CVD at an older age compared with men. 11Based on the Global Burden of Cardiovascular Disease study, 9.6 million men and 8.9 million women died worldwide from CVD in 2019. 12There are wellknown risk factors for developing CVD such as smoking, hypertension, dyslipidemia, diabetes, physical inactivity and obesity. 13Already in these risk factors, some sex highlights reported sex differences in atherosclerotic vascular remodelling and the role of endogenous sex hormones in this process.

K E Y W O R D S
atherosclerosis, inflammation, inflammatory mediators, remodelling, sex differences disparity can be observed.For example, smoking is one of the most important CVD risk factors that lead to EC dysfunction.While slightly more men smoke than women (15% vs 13%), smoking is more harmful to the cardiovascular system of women. 14Women smokers have 25% higher risk of coronary heart disease (CHD) compared with men who also smoke.The reason for this sex difference has not been sufficiently investigated. 15,16In addition, compared with men who smoke, female smokers have more than 50% increase in the relative risk of MI as revealed in a prospective study including approximately 25,000 persons followed over 13 years. 17Another leading CVD risk factor is diabetes.Men have a higher prevalence of diabetes mellitus type-2 (T2DM) compared with women (14.6% vs 9.1%). 18,19Nevertheless, T2DM only doubles the risk of CVD mortality from ischaemic heart disease or ischaemic stroke in men while it triples it in women. 20Although women have more favourable CVD profiles without diabetes compared with men, if developing T2DM, women's CVD risk factors seem to worsen more rapidly, including greater changes in blood pressure and worse lipid profiles than men, and thereby lead to a greater CVD-mortality rate in diabetic women. 21,22

| Atherosclerosis and sexual immune dimorphism
The adaptive immune system is involved in T2DM development.Sex dimorphism in lymphocytes including T cell subsets such as CD4+ T cells is already described. 23,24or example, women have higher CD4+ T cell counts and higher CD4/CD8 ratios in circulation compared with age-matched men, while men have greater CD8+ T cell counts.Surprisingly, there is also a large disparity in gene expression in CD4+ T cells between men and women with T2DM. 25 Among patients with coronary artery disease (CAD) and T2DM, men have a higher C-C chemokine receptor type-2 (CCR2) + effector memory (Em), matrix metalloproteinase (MMP)-9 (MMP9) + and programmed death-ligand 1 (PDL1) + Em CD4 T cell frequency compared with women as revealed by single-cell RNA sequencing and CITE sequencing. 25Of note, all of the above-cited CD4+ T cell subsets are significantly lower in patients with CAD than without CAD, independently of sex. 25

| Clinical manifestation
Hypertension also greatly affects atherosclerotic CVD risk.Although generally, men have higher blood pressure at a younger age (adolescence) compared with women, both sexes display an increase in blood pressure during ageing.In women, the increase in blood pressure is particularly apparent from 30 to 60 years of age, while under the age of 55 years old, men have a higher incidence of hypertension. 26Beyond 60 years, hypertension is even more prevalent in women. 27,28A prospective UK Biobank cohort study that enrolled around 500,000 individuals revealed that women with hypertension have 80% higher risk of MI than men with the same condition. 29In addition to these common risk factors, women are subject to other sex-specific risks that greatly increase the possibility of developing atherosclerosis such as polycystic ovary syndrome (PCOS) or preeclampsia-eclampsia. 21In line, based on the Heart Disease and Stroke Statistics, women have a higher lifetime stroke risk compared with men. 7lthough there is growing evidence and awareness of sex differences in the prevalence of CVD development and mortality, women are still subjected to delayed diagnosis and treatment. 30Taken together, young men have a worse risk factor profile compared with young women, resulting in increased adverse CVD events already earlier in life.However, the impact of CVD risk factors once they occur on developing CVD is higher in women. 18

| Atherosclerotic plaque morphology
Moreover, plaque morphology in men and women seems to be significantly different as underlined by earlier work.Burke et al. examined the cause of sudden death in 51 women and revealed that in 35% of the cases plaque erosion (defined as plaque lesion without plaque rupture with a VSMC-rich intima) followed by acute thrombus formation was the underlying cause.Notably, only 15% of deaths were caused by plaque rupture. 31Furthermore, Yahagi et al. noted plaque dimorphisms between men and women. 32Plaques in young women had thicker fibrous caps, while plaques in older women tend to have larger necrotic cores.Moreover, the same study noted that thrombi in 80% of women younger than 50 years old were caused by plaque erosion while in women older than 50 years only 47% of the thrombi were caused by plaque erosion.Overall, when comparing women with men at all ages, plaque erosion was more frequent in women than men (58% vs. 24%) and plaque rupture was more frequent in men than women (71% vs. 33%) (Figure 1). 32

| Insight from clinical studies
Moreover, sexual dimorphism is also observed in the atherosclerotic plaque burden and degree of stenosis.The Brazilian Longitudinal Study of Adult Health (ELSA-Brasil) study revealed that, independent of the race and other risk factors associated with the disease, the carotid intimamedia thickness (cIMT) was greater in men than in women. 33Similarly, the Tromsø study observed a greater plaque number, plaque area and plaque size in men than in age-, body mass index-, blood pressure-, smoking-and diabetes mellitus-matched women. 34Additionally, a recent optical coherence tomography (OCT) study on 103 CAD patients (77 men and 26 women) found that men with nonculprit plaques have a higher lipid index and larger lipid core compared with women. 35Here again, men had a greater number of plaques and women suffering from CAD were on average ten years older than men with the same disease. 35hese findings suggest that men and women suffer from a different pathophysiological modulation, which should also be reflected in the used therapeutic approaches. 18,36One reason for the observed differences could be the differential action of sex hormones such as oestrogen and testosterone. 11Therefore, the following section will describe these hormone-related differences in more detail.

F I G U R E 1
Simplified schematic overview of the main relative sex differences in atherosclerotic plaque remodelling.Female (left): Plaque erosion is more dominant in female patients and is initiated by shear stress promoting activation of surface TLR2 on ECs.EC apoptosis fosters their detachment, which subsequently leads to the exposure of the fibrous cap to circulating neutrophils.This promotes the release of NETs from neutrophils, activation of platelets and platelet-rich 'white' thrombus formation. 6However, due to the thick fibrous cap, vascular integrity remains intact, and the plaque remains stable. 114In addition, atherosclerotic plaques in female humans are characterized by a larger necrotic core compared with males while, in female mice, oestrogen decreases adhesion molecule expression, decreases neutrophil infiltration and pro-inflammatory cytokines such as TNFα, IL-1β and IL-6 secretion and therefore halting atherosclerosis. 6,47,488][39][40] Male (right): In male humans, plaque rupture is the main cause of thrombus formation induced by fibrous cap burst leading to physical disruption of the vascular wall integrity and to the exposure of highly thrombotic components from the plaque to the blood and to a so-called 'red' thrombus formation.This phenomenon occurs when the plaques have a thin fibrous cap and a large lipid core therefore the plaque is more vulnerable and more likely to burst. 115Testosterone also affects plaque remodelling by enhancing neutrophil, mast cell and macrophage infiltration into the intima layer of the vasculature as well as promoting IL-1β, IL-6 and TNFα secretion all fostering atherosclerotic lesion development in animal models [68][69][70] (This figure was made with biore nder.com).

EFFECT ON ATHEROSCLEROSIS
In humans, women tend to develop atherosclerosis at an older age, after menopause, compared with men, making the impact of sex steroid hormones on atherosclerosis an interesting research topic.However, the scientific data on sex hormones are very contradictory and difficult to interpret.8][39][40] However, in the most frequently used atherosclerotic mouse models, Apolipoprotein E knockout (Apoe −/− ) and low-density lipoprotein receptor knockout (Ldlr −/− ), female mice up to 6 months old, fed normal chow or atherosclerotic diet, have in general a greater atherosclerotic lesion size and overall burden compared with their male counterparts. 18onversely, with ageing (>6 months old) male mice (Apoe −/− and Ldlr −/− ) have larger or equally sized plaques, suggesting that older animals should be used to study atherosclerosis in mice to better compare obtained results to the human setting.Unfortunately, studies directly comparing male and female lesion sizes at different time points are rather scarce and therefore these reported differences may be (statistically) overemphasized. 18Moreover and by contrast, in older human subjects, oestrogens tend to be proatherogenic due to pro-inflammatory and vasotoxic effects. 41Testosterone levels also decrease in ageing men and this decrease has been linked to increased CVD risk.However, in atherosclerotic mice, testosterone seems to increase inflammation and promotes inflammatory cell migration, further demonstrating the complex relationship between sex hormones and atherosclerosis and vascular remodelling.
The following paragraphs will describe the role of female and male sex hormones in atherosclerosis-associated vascular remodelling.

| Female sex hormones in atherosclerosis remodelling
Oestrogen, androgen and progesterone hormones can all bind to extracellular and intracellular receptors, which act via ligand-dependent, ligand-independent, genomic or non-genomic mechanisms.Intracellular pathways involve the stimulation or inhibition of gene transcription factors by binding to oestrogen response elements (EREs) or androgen response elements (AREs).Extracellular and intracellular oestrogen receptors (ERs) include ERα, ERβ and G protein-coupled receptor 30 (GPR30) and they are present on both innate and adaptive immune cells such as B cells, T cells and monocytes as well as on cardiovascular cells like vascular ECs, VSMCs, cardiac fibroblasts and cardiomyocytes. 11,42ERs are higher expressed in female coronary artery VSMCs compared with male cells but decrease with age and after menopause. 43For example, oestrogen signalling via ERα is protective against vascular injury, remodelling and fibrosis after MI in a cardiomyocyte-specific ERα overexpression model in female mice compared with male mice. 44Furthermore, oestrogen-replacement therapies genuinely decrease atherosclerotic plaque size and prevent vascular remodelling. 45,46 3.1.1| Effect of oestrogen on the inflammatory immune response in atherosclerosis Signalling through ERβ is also implicated in the regulation of arterial tone and blood pressure. 11 Frthermore, oestrogen decreases the expression of pro-inflammatory TNFα (Figure 1 and Table 1). 47TNFα is a cytokine implicated in promoting vascular remodelling by increasing adhesion molecule expression on ECs as well as EC permeability, upregulation of matrix degradation and VSMCs proliferation. 48In line with this, ovariectomized female control rats had a significant increase in serum TNFα compared with ovariectomized animals with oestrogen-replacement treatment (oestrogen pellet:1.5mg/pellet).Moreover, endothelium-dependent vasorelaxation was reduced in the ovariectomized and hence oestrogen-deficient rats compared with control rats undergoing oestrogenreplacement treatment.Combined, these results support the importance of TNFα in causing vascular dysfunction associated with oestrogen deficiency. 47Two studies using ovariectomized rats, which underwent balloon injury in the carotid arteries and were treated with 17β-estradiol (E2) (daily injection of 20 μg•kg −1 •d −1 ), a sex hormone that also represents oestrogen, could show that this treatment reduced neutrophil and monocyte trafficking and infiltration by attenuating the expression of cytokine-induced neutrophil chemoattractant (CINC-2α) and MCP-1, compared with vehicle, medroxyprogesterone acetate (MPA; inhibits the protective effect of oestrogen on neointima formation) treated rats, 24 h postinjury (Table 1). 38,49Two hours following balloon injury, 17β-estradiol-treated ovariectomized rats showed a significant decrease in mRNA expression of the adhesion molecules ICAM1, VCAM1 and P-selectin as well as a decrease in pro-inflammatory IL-1β and IL-6 in injured carotids arteries. 38IL-1β and IL-6 are both pro-inflammatory cytokines that promote vascular remodelling by increasing EC dysfunction, leukocyte recruitment to the intima, VSMC migration and proliferation and lesional collagen deposition. 48 24 h postinjury, the significant decrease remained only for P-selectin and IL-6 suggesting that oestrogen especially helps to reduce early inflammatory processes. 38Therefore, oestrogen mitigates the initial inflammation process seen in atherosclerosis by decreasing adhesion molecules and pro-inflammatory cytokines and chemoattractant molecule expression as well as decreasing the infiltration of neutrophils and monocytes into the plaque.Oestrogen has also an inhibitory effect on the expression of certain growth factors affecting vascular remodelling such as insulin-like growth factor 1, platelet-derived growth factor (PDGF)-A and its receptor PDGF-Rα on VSMCs in a rat model of aortic injury leading to increased VSMC proliferation and intima/media thickening (Table 1). 50Taken together, premenopausal oestrogen seems to protect against atherosclerotic vascular remodelling by decreasing adhesion molecules expression and therefore inflammatory cell recruitment as well as decreasing the expression of pro-inflammatory mediators such as TNFα, IL-1β and IL-6 (Figure 1).Oestrogen also decreases the negative effect of MMP12, an important elastase that contributes to arterial stiffening (Table 1).Indeed, it has been recently shown in vitro that the uptake of oxLDL promotes the release of MMP12 by macrophages, and treatment with E2 decreases MMP12 gene expression and secretion in human macrophages. 51In the same study, both Ldlr −/− MMP12 +/+ and Ldlr −/− MMP12 −/− female mice fed a highfat diet for 16 weeks had a significant decrease in aortic plaque macrophage content compared with males due to greater oestrogen level in females. 51In line, lower oestrogen levels after menopause are related to altered vascular function, enhanced inflammation and upregulation of other hormonal systems such as the renin-angiotensinaldosterone system and reduced nitric oxide-dependent vasodilation. 52,53

| Clinical manifestation and therapies
Menopause goes hand in hand with 10%-15% higher circulating LDL-cholesterol and triglyceride levels and a reduction in high-density lipoprotein (HDL)-cholesterol.6][57] Endothelial dysfunction starts in early menopause even before signs of subclinical atherosclerosis 58 and while healthy endothelium is sensitive to the vasodilator properties of oestrogens, this reverses when vascular stiffness and atherosclerotic disease develop over time. 59All of these detrimental phenomena contribute to an increased rate of MI in women after menopause. 60Therefore, oestrogen supplementation therapies have been investigated since the early 1990s, but their benefits remain debateable. 11Evidence suggests that hormone therapy can be effective in reducing CVD risk when it is started during or shortly after menopause, 61 but there are side effects to be considered like an increased risk of breast cancer. 62Modern oestrogen supplementation therapies contain lower doses of systemic and vaginal oestrogens 63 but oral, not transdermal oestrogen supplementation increases the risk of venous thromboembolism. 64Hence, careful evaluation is needed to weigh the risks and benefits of oestrogen supplementation to decrease CVD in women after menopause.

| Male sex hormones in atherosclerosis remodelling
Testosterone, the most important sex hormone in men, is a steroid from the androgen hormone family and binds to intracellular androgen receptors (ARs).Testosterone levels in men decrease with ageing and this decrease has been linked with an increase in CVD and CVD-associated mortality. 65Testosterone circulates in two forms in the serum, either in its inactive form, which is bound to sex hormone binding globulin (SHBG) and is unable to bind to ARs (68% of total serum testosterone) or in its active form that can bind to albumin or circulate freely in the blood. 66,67

| Effect of testosterone on the inflammatory immune response in atherosclerosis
Testosterone increases neutrophils, mast cell and macrophage numbers and their activation resulting in foam cell formation in atherosclerotic plaques.Furthermore, testosterone affects vascular remodelling by stimulating the release of pro-inflammatory cytokines IL-1β, IL-6 and TNFα, which are, as mentioned above, also involved in the vascular remodelling process, leading to thrombus formation and MI in men [68][69][70] (Figure 1 and Table 1).Testosterone also induces tissue inhibitor of metalloproteinase-1 (TIMP-1) and hypothalamic serpin A 3n expression, which both are implicated in the balance of degradation and synthesis of the ECM (Table 1). 71Elevated serum levels of TIMP-1 and serpin A 3n have been linked to cardiac fibrosis (Table 1). 72,73On the contrary, testosterone deficiency leads to an increase in atherosclerotic lesion areas, foam cell accumulation, IMT and serum lipid levels in mini pigs fed 12 weeks with a high-fat and highcholesterol diet. 74keda et al. examined the role of AR in angiotensin II (Ang II)-induced vascular remodelling. 75Ang II is an important vasoactive peptide that increases vascular wall tension by causing vasoconstriction.Its stimulation leads to an increase in free radicals via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and thereby promotes vascular remodelling. 76AR knockout (ARKO) and WT mice on a C57BL/6J background were infused with Ang II at 2.0 mg/kg per day for 14 days by a subcutaneously implanted osmotic minipump.Treated ARKO mice showed a significant increase in medial thickness and perivascular fibrosis of the coronary artery and aorta compared with untreated animals. 75In addition, collagen I and collagen III gene expression as well as superoxide production was only increased in the Ang II treated ARKO mice compared with all other groups (Ang II treated wild type [WT] and untreated mice). 75urthermore, Ang II promotes vascular transforming growth factor β (TGFβ) expression in ARKO mice compared with male WT mice.Based on these findings, AR seems to have a vascular protective action and counteracts Ang II-induced vascular remodelling. 75Another study investigated AR deletion in monocytes/macrophages, ECs and VSMCs in Ldlr −/− mice.Only monocyte/ macrophage-deficient ARKO Ldlr −/− mice showed a decrease in atherosclerosis compared with control Ldlr −/− mice after 16 weeks of high-cholesterol diet feeding. 77urthermore, these monocyte/macrophage-deficient ARKO Ldlr −/− mice had a significant decrease in macrophage content and collagen deposition as well as increased VSMC content in the aortic root.However, mice with an AR knockout in ECs or VSMCs did not manifest any differences in lesion size or changes in vascular wall composition.In vitro experiments revealed that AR expression on monocytes promotes their migration, adhesion to ECs and differentiation into foam cells. 77t seems that systemic AR deficiency and monocytes/ macrophage-specific AR deficiency have opposing effects on atherosclerosis suggesting that ARs affect each cell type differently, which also reflects on their contribution to the disease progression. 78 3.2.2| Effect of testosterone on cytokines in atherosclerosis Regarding the cytokine profiles that are also implicated in remodelling such as IL-1β, IL-6 and TNFα, testosterone supplementation seems to decrease the expression of pro-inflammatory cytokines while promoting the production of anti-inflammatory cytokines such as IL-10. 79,80 H1][82][83][84][85] A recent study from Bernardi et al. analysed the cytokine profile of 104 healthy adults (20-49 years old), including proatherogenic cytokines such as IL-1β, IL-6, TNFα and anti-atherogenic cytokines such as IL-10 and IL-1rA (Table 1).They showed that men have higher circulating levels of IL-1β, IL-6 and TNFα compared with age-matched women.These pro-inflammatory cytokines were also significantly associated with testosterone and testosterone/estradiol ratio.This phenomenon was only observed in men, while in women, there was no correlation between their levels of testosterone and proinflammatory cytokines. 86,873.2.3| Clinical manifestation and therapies Testosterone replacement therapy (TRT) is widely investigated to lower atherosclerotic CVD risk in men.However, despite the numerous clinical trials, the results are still unclear.3][94][95][96][97] Regarding adverse atherosclerotic remodelling, several clinical studies have noted that a lower level of androgens correlates with an increase in cIMT in men from 40-70 years of age. 98,99However, in a randomized clinical trial, investigating the long-term effect of testosterone administration on subclinical atherosclerosis in men older than 60 years old, no significant differences in the rate of changes in cIMT after 3 years of daily testosterone treatment compared with placebo were found. 100The TRAVERSE (Testosterone Replacement Therapy for Assessment of Long-term Vascular Events and Efficacy ResponSE in Hypogonadal Men) trial is the latest randomized clinical trial on this topic that is currently ongoing.It started in 2018 and tested a topically administered testosterone gel versus placebo gel on more than 5000 symptomatic hypogonadal men with increased risk for atherosclerotic CVD.The completion of the study was estimated to be due in June 2022 but seems to be delayed. 101To conclude, the detrimental or beneficial effect of TRT is still ambiguous and further investigations are needed.

CONCLUSIONS
It is undeniable that there are sex differences in atherosclerosis and subsequent development of CVDs.However, the scientific data on sex hormones and very contradictory and difficult to interpret, more studies are needed to fully understand the underlying mechanisms that lead to the observed sex dimorphism.Men develop atherosclerosis earlier and have larger plaque size and a higher plaque burden compared with women.Women tend to have a thicker fibrous cap and larger necrotic core, and are more susceptible to suffer from thrombus formation via plaque erosion.On the contrary, men have a thinner fibrous cap and a smaller necrotic core, and plaque rupture is the major cause of arterial thrombosis.Looking at the impact of sex hormones on atherosclerotic vascular remodelling, oestrogen in premenopausal women prevents vascular inflammation and ensures a proper vascular tone.Postmenopause women have a higher risk of atherosclerotic CVDs potentially mainly due to a decrease in oestrogen level.Based on the effect of oestrogen observed in women as well as in studies using animal models, there is a growing interest to investigate hormone therapy to prevent CVDs in ageing women.However, careful evaluation of oestrogen supplementation therapies in women after menopause is needed to weigh out the risks of for example breast cancer and thrombosis against the benefits of CVD risk lowering.Studies on testosterone in human and animal atherosclerotic vascular disease are conflicting on the role of testosterone.The decrease in testosterone levels in ageing men is associated with a higher risk of CVDs and testosterone supplementation therapy was described to correlate with a decrease in inflammation.Yet, adverse effects of testosterone on vascular remodelling by promoting pro-inflammatory cytokine production are also reported.
3][104][105][106] Although the exact reason for this sex-related phenomenon remains unclear.It is hypothesized that in women, it may be due to the general increase in low-grade inflammation that comes with ageing or the accumulation and disbalance of O-GlcN-acylation of certain proteins that would lead to a loss of oestrogen-induced vasoprotection. 107However, how these conditions specifically affect ERE signalling is unknown and this question still needs to be addressed. 107t can also be assumed that life-long testosterone exposure in men differentially impacts on the vascular wall architecture compared with oestrogen exposure of the vessel wall in women.In other words, although oestrogen levels significantly and more rapidly decline in menopausal women, while testosterone levels in men more gradually decrease with age, this variance in hormonal vascular 'imprinting' echoes on CVD risk and lesion composition throughout the entire life of both sexes.
In addition to sex-related dimorphisms in CVD, also gender-related differences need to be considered when diagnosing and treating CVD.The term 'sex' refers to pure biological attributes such as physical and physiological features including hormones but also chromosomes, gene expression and reproductive anatomy.'Gender', on the contrary, refers to the socially constructed roles, behaviours, opportunities, expectations, expressions and identities of females in society, which may also affect disease course. 108ndeed, there is a growing number of gender-related variables that are or may be involved in the prevalence of CVD development and outcome.For example, the relationship between family roles and the prevalence of CHD suggest that women living both with their spouse and children had two times higher risk of CHD compared with women living only with a spouse.While married men have a decreased risk of suffering from MI compared with married women, men living alone have an increased risk of fatal MI. 109,110 Of note, even though men develop CVD earlier in life, women catch up after menopause and therefore the lifetime risk of CVD is similar for both men and women if estimated for a total life span. 111,112Yet, women are five times less likely to have a diagnosis considering heart disease as the main health issue or leading cause of death and they are significantly less likely to have ever received a cardiovascular screening test as well. 113Hence, diagnostic and treatment inequalities between men and women need to be urgently improved.
In conclusion, CVD and atherosclerosis pathophysiological development differ between males and females.There are sex differences based on the effect of hormones on atherosclerotic plaque morphology and arterial wall remodelling as well as gender-based differences in the prevalence of CVD risks.A better understanding of both is essential for a better diagnosis, treatment and further study guidelines.