Sex Differences In Cardiovascular Disease Research Articles

Sex-specific lifetime risk of cardiovascular events: the European Prospective Investigation into Cancer-Norfolk prospective population cohort study.

Better understanding of sex differences in cardiovascular disease (CVD) is essential in tailoring appropriate preventative strategies. Using a large population-based study with follow-up >25 years, we aimed to determine sex-specific lifetime risks of incident CVD and cardiovascular (CV) mortality amongst populations with and without prevalent CVD. Participants were drawn from the European Prospective Investigation into Cancer-Norfolk and followed up for a median of 26.2 years. Sex-specific lifetime risks were ascertained accounting for the competing risk of death. Models were adjusted for ethnicity and time-updated covariates: material deprivation, CV risk factors, lifestyle factors, comorbidities, and medication. A total of 23 859 participants [54.5% women; mean age (standard deviation) 59.2 (9.3) years at baseline] were included. Adjusted lifetime risks of incident CVD were higher in men than in women (69.1 vs. 57.7% at age 75): cause-specific hazard ratio (cHR) (99% confidence interval)-1.49 (1.41-1.57), while the risks of CV mortality at age 75 were 4.4% (men) and 3.1% (women): cHR-1.42 (1.31-1.54). Myocardial infarction was the predominant first presentation in men until the eighth decade. In women, the first CVD manifestations after their sixth decade were predominantly atrial fibrillation and stroke. The male-associated excess relative risks of incident CVD and CV mortality were halved in people with prevalent CVD. We characterized the sex-specific lifetime CV risks in a large cohort. Men had substantially higher risk of incident CVD and CV mortality than women, which was attenuated amongst people with prevalent CVD. Our findings provide an evidence base for sex-specific CV prevention.

Abstract 15923: Sexually Dimorphic Heart Phenotypes in Mice Lacking Chromatin Factors in Cardiomyocytes

Introduction: Sex differences in cardiovascular disease have been attributed to differences in lifestyle or sex hormones; less is known of genetic components that cause differences in heart disease between men and women. We have characterized a role for the ZMYND8 chromatin reader in maintaining cardiac function through modulation of enhancer activity in cardiomyocytes. ZMYND8 has been reported to interact with H3K4 histone demethylases KDM5C and KDM5D, which are encoded from genes residing on sex chromosomes ( Kdm5c on X and Kdm5d on Y). KDM5C and KDM5D have diverged over millions of years of sex chromosome evolution and their functional congruency is unknown. Hypothesis: We hypothesize that sex differences in cardiovascular disease are due, at least in part, to differences in functionality of genes residing on the sex chromosomes. Methods: We constructed Zmynd8-cKO mice using the cardiomyocyte-specific Myh6-Cre Tg/0 line and an established Zmynd8 flox allele. We also introduced a Kdm5c flox allele to the Myh6-Cre Tg/0 line to generate Kdm5c-cKO females and Kdm5c-cKO hemizygous males. These mice were evaluated by lifespan analyses, echocardiography, and molecular analyses (RNA-seq and ChIP-seq). Results: While both male and female Zmynd8 - cKO mice develop enlarged hearts, reduced contractility, and illegitimate expression of certain genes normally found in skeletal muscle, female mice have a 10% reduction in lifespan compared to males; this sex difference in lifespan is not seen in Cre-only mice. We tested the necessity for Kdm5c in cardiomyocytes and found that its deletion also leads to premature death in a dosage dependent manner compared to Cre-only mice. Interestingly, Myh6-Cre Tg/0 ; X 5c-flox /Y hemizygous males have a 20% shorter lifespan than Myh6-Cre Tg/0 ; X 5c-flox /X + heterozygous females, suggesting differences between KDM5C and KDM5D functionality. Conclusions: Our results provide evidence of sexually dimorphic heart defects linked to chromatin regulators, including a gene-pair found on the sex chromosomes. We are deliniating the molecular underpinnings of these observations including changes to histone marks and gene expression in these knockout models.

Sex Differences in Autonomic Blood Pressure Regulation: Sex Chromosome Complement and Hormonal Involvement

Although several lines of evidence from different studies highlight sex differences in cardiovascular diseases, to date, most studies have been focused on males, with the idea that males and females are similar, differing only in the magnitude of the response. However, the principles learned in male models cannot and should not be extrapolated to women and, therefore, it is important to study in greater detail not only the differences between the sexes but also the physiological intertwining of the underlying genetic and hormonal mechanisms of sexual dimorphism. This review explores the sex disparities in the autonomic nervous system regulation of blood pressure (particularly baroreceptor function), with special emphasis on sex hormones and sex chromosome complement factors involved in sexually dimorphic autonomic blood pressure regulation. A more detailed understanding of the sources of physiological disparities between the sexes may also help in understanding the differences between the sexes in rates of cardiovascular disease and may also aid in designing future improvements for sex-tailored therapeutic treatments.

Association Between Risk Factors in Childhood and Sex Differences in Prevalence of Carotid Artery Plaques and Intima-Media Thickness in Mid-Adulthood in the Childhood Determinants of Adult Health Study.

Background The origins of sex differences in cardiovascular diseases are not well understood. We examined the contribution of childhood risk factors to sex differences in adult carotid artery plaques and intima-media thickness (carotid IMT). Methods and Results Children in the 1985 Australian Schools Health and Fitness Survey were followed up whentheywereaged 36 to 49 years (2014-19, n=1085-1281). Log binomial and linear regression examined sex differences in adult carotid plaques (n=1089) or carotid IMT (n=1283). Childhood sociodemographic, psychosocial, and biomedical risk factors that might contribute to sex differences in carotid IMT/plaques were examined using purposeful model building with additional adjustment for equivalent adult risk factors in sensitivity analyses. Women less often had carotid plaques (10%) than men (17%). The sex difference in the prevalence of plaques (relativerisk[RR] unadjusted 0.59 [95% CI, 0.43 to 0.80]) was reduced by adjustment for childhood school achievement and systolic blood pressure (RRadjusted 0.65 [95% CI, 0.47 to 0.90]). Additional adjustment for adult education and systolic blood pressure further reduced sex difference (RR adjusted 0.72 [95% CI, 0.49 to 1.06]). Women (mean±SD 0.61±0.07) had thinner carotid IMT than men (mean±SD 0.66±0.09). The sex difference in carotid IMT (β unadjusted -0.051 [95% CI, -0.061 to -0.042]) reduced with adjustment for childhood waist circumference and systolic blood pressure (β adjusted -0.047 [95% CI, -0.057 to -0.037]) and further reduced with adjustment for adult waist circumference and systolic blood pressure (β adjusted -0.034 [95% CI, -0.048 to -0.019]). Conclusions Some childhood factors contributed to adult sex differences in plaques and carotid IMT. Prevention strategies across the life course are important to reduce adult sex differences in cardiovascular diseases.

Supplemental Materials for Dei Cas A (JCEM 2023) Sex differences in cardiovascular disease and cardiovascular risk estimation in patients with type 1 diabetes.

Supplemental Materials for Dei Cas A (JCEM 2023) Sex differences in cardiovascular disease and cardiovascular risk estimation in patients with type 1 diabetes.

Supplemental Materials for Dei Cas A (JCEM 2023) Sex differences in cardiovascular disease and cardiovascular risk estimation in patients with type 1 diabetes.

Supplemental Materials for Dei Cas A (JCEM 2023) Sex differences in cardiovascular disease and cardiovascular risk estimation in patients with type 1 diabetes.

Sex and Gender and Allostatic Mechanisms of Cardiovascular Risk and Disease.

Cardiovascular diseases are leading causes of mortality and morbidity in adults worldwide. Multiple studies suggest that there are clinically relevant sex differences in cardiovascular disease. Women and men differ substantially in terms of prevalence, presentation, management, and outcomes of cardiovascular disease. To date, however, little is known about why cardiovascular disease affects women and men differently. Because many studies do not differentiate the concept of sex and gender, it is sometimes difficult to discriminate sociocultural vs biological contributors that drive observed clinical differences. Female sex has some biological advantages in relation to cardiovascular disease, but many of these advantages seem to disappear as soon as women develop cardiovascular risk factors (eg, type 2 diabetes, hypertension, dyslipidemia). Furthermore, stress and allostatic load could play an important role in the relationship between sex/gender and cardiovascular diseases. In this narrative review, we argue that chronic stress and psychosocial factors might better encompass the patterns of allostatic load increases seen in women, while biological risk factors and unhealthy behaviours might be more important mechanisms that drive increased allostatic load in men. Indeed, men show allostatic load patterns that are more associated with impaired anthropometric, metabolic, and cardiovascular functioning and women have greater dysregulation in neuroendocrine and immune functioning. Thus gender-related factors might contribute to the pathogenesis of cardiovascular disease especially through stress mechanisms. It is important to continue to study the mechanisms by which gender influences chronic stress, because chronic stress could influence modifiable gendered factors to promote cardiovascular disease prevention.

Emerging vascular cell-specific roles for mineralocorticoid receptor: implications for understanding sex differences in cardiovascular disease.

As growing evidence implicates extrarenal mineralocorticoid receptor (MR) in cardiovascular disease (CVD), recent studies have defined both cell- and sex-specific roles. MR is expressed in vascular smooth muscle (SMC) and endothelial cells (ECs). This review integrates published data from the past 5 years to identify novel roles for vascular MR in CVD, with a focus on understanding sex differences. Four areas are reviewed in which there is recently expanded understanding of the cell type- or sex-specific role of MR in 1) obesity-induced microvascular endothelial dysfunction, 2) vascular inflammation in atherosclerosis, 3) pulmonary hypertension, and 4) chronic kidney disease (CKD)-related CVD. The review focuses on preclinical data on each topic describing new mechanistic paradigms, cell type-specific mechanisms, sexual dimorphism if addressed, and clinical implications are then considered. New data support that MR drives vascular dysfunction induced by cardiovascular risk factors via sexually dimorphic mechanisms. In females, EC-MR contributes to obesity-induced endothelial dysfunction by regulating epithelial sodium channel expression and by inhibiting estrogen-induced nitric oxide production. In males with hyperlipidemia, EC-MR promotes large vessel inflammation by genomic regulation of leukocyte adhesion molecules, which is inhibited by the estrogen receptor. In pulmonary hypertension models, MRs in EC and SMC contribute to distinct components of disease pathologies including pulmonary vessel remodeling and RV dysfunction. Despite a female predominance in pulmonary hypertension, sex-specific roles for MR have not been explored. Vascular MR has also been directly implicated in CKD-related vascular dysfunction, independent of blood pressure. Despite these advances, sex differences in MR function remain understudied.

LDL-C control according to risk: Is there a gender paradox?

Background and Aims : Background Low density lipoprotein cholesterol (LDL-C) treatment goals used for cardiovascular disease (CVD) prevention. Sex differences in CVD have been reported and women are less likely to receive statin therapy for secondary prevention. Nevertheless, little is known about gender according to risk categories Aims

Sex/Gender- and Age-Related Differences in β-Adrenergic Receptor Signaling in Cardiovascular Diseases.

Sex differences in cardiovascular disease (CVD) are often recognized from experimental and clinical studies examining the prevalence, manifestations, and response to therapies. Compared to age-matched men, women tend to have reduced CV risk and a better prognosis in the premenopausal period. However, with menopause, this risk increases exponentially, surpassing that of men. Although several mechanisms have been provided, including sex hormones, an emerging role in these sex differences has been suggested for β-adrenergic receptor (β-AR) signaling. Importantly, β-ARs are the most important G protein-coupled receptors (GPCRs), expressed in almost all the cell types of the CV system, and involved in physiological and pathophysiological processes. Consistent with their role, for decades, βARs have been considered the first targets for rational drug design to fight CVDs. Of note, β-ARs are seemingly associated with different CV outcomes in females compared with males. In addition, even if there is a critical inverse correlation between β-AR responsiveness and aging, it has been reported that gender is crucially involved in this age-related effect. This review will discuss how β-ARs impact the CV risk and response to anti-CVD therapies, also concerning sex and age. Further, we will explore how estrogens impact β-AR signaling in women.

Illuminating the Mechanisms Underlying Sex Differences in Cardiovascular Disease.

Sex is a key risk factor for many types of cardiovascular disease. It is imperative to understand the mechanisms underlying sex differences to devise optimal preventive and therapeutic approaches for all individuals. Both biological sex (determined by sex chromosomes and gonadal hormones) and gender (social and cultural behaviors associated with femininity or masculinity) influence differences between men and women in disease susceptibility and pathology. Here, we focus on the application of experimental mouse models that elucidate the influence of 2 components of biological sex-sex chromosome complement (XX or XY) and gonad type (ovaries or testes). These models have revealed that in addition to well-known effects of gonadal hormones, sex chromosome complement influences cardiovascular risk factors, such as plasma cholesterol levels and adiposity, as well as the development of atherosclerosis and pulmonary hypertension. One mechanism by which sex chromosome dosage influences cardiometabolic traits is through sex-biased expression of X chromosome genes that escape X inactivation. These include chromatin-modifying enzymes that regulate gene expression throughout the genome. The identification of factors that determine sex-biased gene expression and cardiometabolic traits will expand our mechanistic understanding of cardiovascular disease processes and provide insight into sex differences that remain throughout the lifespan as gonadal hormone levels alter with age.

Sex differences in cardiovascular medication prescription: an interview with Dr Sanne Peters.

Bibliography Dr Sanne Peters is an Associate Professor at The George Institute for Global Health and a Senior Lecturer at Imperial College London. She is also Associate Professor at the University Medical Center Utrecht. After completing her MSc in Epidemiology in 2009, she obtained a PhD in Epidemiology from Utrecht University in 2012. Her PhD focused on subclinical imaging measures of atherosclerosis as a means to identify people at high-risk for future cardiovascular disease. As part of her PhD training, she spent 6 months at The George Institute in Sydney. This is where she started her research on sex differences in cardiovascular disease. In 2013, she was awarded a postdoctoral fellowship, which enabled her to continue her work on sex differences at the University of Cambridge. In 2014, she was offered a position at the George Institute offices at the University of Oxford (now in collaboration with Imperial College London). In 2018, she relocated back to the Netherlands and was also appointed as Associate Professor in Utrecht.

Sex Differences in Cardiovascular Diseases: A Matter of Estrogens, Ceramides, and Sphingosine 1-Phosphate.

The medical community recognizes sex-related differences in pathophysiology and cardiovascular disease outcomes (CVD), culminating with heart failure. In general, pre-menopausal women tend to have a better prognosis than men. Explaining why this occurs is not a simple matter. For decades, sex hormones like estrogens (Es) have been identified as one of the leading factors driving these sex differences. Indeed, Es seem protective in women as their decline, during and after menopause, coincides with an increased CV risk and HF development. However, clinical trials demonstrated that E replacement in post-menopause women results in adverse cardiac events and increased risk of breast cancer. Thus, a deeper understanding of E-related mechanisms is needed to provide a vital gateway toward better CVD prevention and treatment in women. Of note, sphingolipids (SLs) and their metabolism are strictly related to E activities. Among the SLs, ceramide and sphingosine 1-phosphate play essential roles in mammalian physiology, particularly in the CV system, and appear differently modulated in males and females. In keeping with this view, here we explore the most recent experimental and clinical observations about the role of E and SL metabolism, emphasizing how these factors impact the CV system.

Abstract P219: Associations Between Endogenous Sex Hormone Levels And Adipokine Levels In The Multi-ethnic Study Of Atherosclerosis (MESA)

Background: Differences in sex hormone levels contribute to sex differences in cardiovascular disease (CVD). Adipokines, which are central to cardiometabolic disease, differ by sex and have divergent associations with CVD. However the link between sex hormones and adipokines is not well established. We hypothesized that an androgenic sex hormone profile is associated with higher leptin & resistin and lower adiponectin levels among postmenopausal women and men. Methods: We performed an analysis of 1822 adults in MESA who had sex hormones and adipokines measured. Sex hormones [Testosterone (T), estradiol (E2), sex hormone binding globulin (SHBG)] were measured at exam 1; free T was estimated. Serum adipokines [leptin, resistin, adiponectin] were measured at visits 2 or 3, an average of 2.6 yrs later. We used multivariable linear regression to examine the cross-sectional associations between sex hormones and adipokines. Results: The mean age was 63 (10), 48% were women; 41% White, 13% Chinese American, 20% Black, 26% Hispanic. Leptin: After adjusting for demographics (model 1), higher free T & E2 and lower SHBG were associated with greater leptin levels in women and men; higher DHEA was associated with lower leptin in men ( Table ). With full covariate adjustment (model 3), this remained statistically significant for E2 & SHBG in women and for DHEA in men. Adiponectin: With demographic adjustment, higher free T, E2, DHEA & lower SHBG were associated with lower adiponectin in women, and higher free T & lower SHBG with lower adiponectin in men. After full covariate adjustment, these same relationships were slightly attenuated but remained significant for women and men. Resistin: no association in either sex. Conclusion: A more androgenic profile (higher free T & lower SHBG) was independently associated with lower adiponectin in men and women. Higher E2 & lower SHBG in women, and lower DHEA in men, were associated with leptin. These findings may provide mechanistic insight into the interplay between sex hormones, adipokines and CVD risk.

Update on Management of Cardiovascular Diseases in Women.

Cardiovascular diseases (CVD) have a lower prevalence in women than men; although, a higher mortality rate and a poorer prognosis are more common in women. However, there is a misperception of CVD female risk since women have commonly been considered more protected so that the real threat is vastly underestimated. Consequently, female patients are more likely to be treated less aggressively, and a lower rate of diagnostic and interventional procedures is performed in women than in men. In addition, there are substantial sex differences in CVD, so different strategies are needed. This review aims to evaluate the main gender-specific approaches in CVD.

Role of Biological Sex in the Cardiovascular-Gut Microbiome Axis.

There has been a recent, unprecedented interest in the role of gut microbiota in host health and disease. Technological advances have dramatically expanded our knowledge of the gut microbiome. Increasing evidence has indicated a strong link between gut microbiota and the development of cardiovascular diseases (CVD). In the present article, we discuss the contribution of gut microbiota in the development and progression of CVD. We further discuss how the gut microbiome may differ between the sexes and how it may be influenced by sex hormones. We put forward that regulation of microbial composition and function by sex might lead to sex-biased disease susceptibility, thereby offering a mechanistic insight into sex differences in CVD. A better understanding of this could identify novel targets, ultimately contributing to the development of innovative preventive, diagnostic and therapeutic strategies for men and women.

Sex Differences in the Co-Occurrence of PTSD and Cardiovascular Disease

Posttraumatic stress disorder (PTSD) is a highly impairing psychiatric condition, and women are twice as likely as men to receive a diagnosis. It is thought that chronic arousal and physiological stress place individuals with PTSD at greater risk for cardiovascular disease (CVD). Sex differences in CVD are also well established. For example, premenopausal women have lower CVD risk compared to men, and this is partly explained by higher levels of estradiol in premenopausal women. Estradiol is considered to be a protective factor against PTSD as well; therefore, it is important to understand the role of sex hormones in PTSD and CVD, including whether or not there is shared impact of sex hormones on the co-occurrence of these diseases. In this review, we discuss sex differences in PTSD and CVD, as well as the brain-heart connection in the PTSD-CVD link and current treatment approaches, considering sex as a biological variable. [ Psychiatr Ann . 2022;52(1):26–30.]

Sex Differences in Cumulative Exposure to Metabolic Risk Factors Before Hypertension Onset: The Cohort of the Tehran Lipid and Glucose Study.

BackgroundPrevious studies have shown a sex difference in the association between hypertension and cardiovascular disease; however, the precise mechanism remains unclear. Because there are strong associations between metabolic risk factors (MRFs) and hypertension, a sex‐specific analysis of MRFs before hypertension onset could offer new insights and expand our understanding of sex differences in cardiovascular disease. We evaluated cumulative exposure to major MRFs and rate of change of those factors, including body mass index, waist circumference, systolic blood pressure, diastolic blood pressure, fasting plasma glucose, total cholesterol, triglycerides, and high‐density lipoprotein cholesterol among individuals who did and did not develop hypertension at follow‐up.Methods and ResultsWe included 5374 participants (2191 men) initially without hypertension with age range of 20–50 years at baseline who participated in the Tehran Lipid and Glucose Study, and had been examined at least 3 times during the study period (1999–2018). In both sexes, the cumulative exposure to all MRFs (except for fasting plasma glucose and high‐density lipoprotein cholesterol in men) were higher in those who developed hypertension, compared with those who did not develop hypertension. However, women experienced greater cumulative exposure to major MRFs, compared with their male counterparts. Also, they experienced a faster increase in waist circumference, systolic blood pressure, diastolic blood pressure, and high‐density lipoprotein cholesterol than men. Furthermore, rapid increase in systolic blood pressure began earlier in women than men, at the age of 30 years. We also found that those men who developed hypertension experienced unfavorable change in major MRFs during young adulthood (<50 years of age).ConclusionsWomen exhibited more metabolic disturbances than men before onset of hypertension, which may explain the stronger impact of hypertension for major types of cardiovascular disease in women, compared with men.

Associations of endogenous sex hormone levels with the prevalence and progression of valvular and thoracic aortic calcification in the Multi-Ethnic Study of Atherosclerosis (MESA)

Background and aimsSex hormones (SH) may contribute to sex differences in cardiovascular disease (CVD). High free testosterone (T) and low sex hormone binding globulin (SHBG) have been associated with progression of coronary artery calcification in women. We now examined the association of SH with extra-coronary calcification (ECC) prevalence and progression among MESA participants. MethodsWe studied 2,737 postmenopausal women and 3,130 men free of clinical CVD with baseline SH levels. ECC measurements [ascending and descending thoracic aortic calcification (ATAC, DTAC), mitral annular calcification (MAC), aortic valve calcification (AVC)] were obtained by computed tomography at baseline and after 2.4 ± 0.9 years. We used multivariable Poisson regression to evaluate associations with ECC prevalence and incidence (Agatston scores >0) and linear mixed effects models for ECC progression, per 1-SD increment in log(SH) in women and men separately. ResultsThe mean age was 65 ± 9 and 62 ± 10 years for women and men, respectively. In women, greater free T and lower SHBG were associated with MAC incidence in a demographic-adjusted model only. In men, lower free T was associated with MAC prevalence, DTAC incidence and progression, while greater SHBG was associated with MAC prevalence and DTAC progression after further adjusting for CVD risk factors. ConclusionsIn this diverse cohort free of CVD, we found some associations of SH with ECC measures. In particular, free T was inversely associated with prevalent MAC and DTAC progression in men independent of CVD risk factors. SH may influence vascular calcification, but further work is needed to understand clinical implications of these findings.

Sex modifies the association between body mass index and carotid artery intima media thickness in the multi-ethnic Singapore population

Abstract Background and aims Globally cardiovascular disease (CVD) risk is higher in men than in women in the younger age group but the risk is attenuated in later life with an unexpected higher risk in women in some populations. A better understanding of sex differences in CVD is needed. Carotid artery intima-media thickness (CIMT) has been proposed for CVD risk assessment in individuals at intermediate risk. We study the gender interactions in the associations of the traditional cardiovascular risk factors with CIMT. Methods We performed carotid ultrasonography in the large population health study-HELIOS conducted in Singapore. Measurements were performed as per Mannheim consensus, at the far wall of bilateral common carotid artery at 8 sites in each participant. We report the average of all measurements (avgCIMT) and maximum value (maxCIMT). We analysed 2061 healthy participants recruited between 2018–2020. Inclusion criteria for this analysis: non-smokers, not known to have diabetes, hypertension, hyperlipidemia or ischemic heart disease. Individuals with systolic blood pressure &amp;gt;160 mm Hg or fasting glucose &amp;gt;7.0 mmol/L on recruitment were excluded. Multivariable linear regression analysis was performed to examine the association between CIMT and cardiovascular risk factors in the healthy population adjusting for age, sex and ethnicity. All analysis was performed using Stata version 16.0. Results After exclusion, 1407 healthy participants were included in the analysis. Median (IQR) avgCIMT: 0.56 (0.50, 0.65) in men; 0.54 (0.50, 0.58) in women. Median (IQR) maxCIMT: 0.64 (0.57, 0.76) in men; 0.61 (0.54, 0.70) in women. The avgCIMT and the maxCIMT were higher in males when compared to women and all traditional cardiovascular risk factors associated with CIMT after adjustment for age, sex and ethnicity (P&amp;lt;0.05) (Table 1). Interaction tests in multivariable model adjusted for age, ethnicity, pulse pressure, non HDL-C and HbA1c showed a significant interaction between sex and body mass index (BMI) with predicted effects on avgCIMT and maxCIMT being relatively higher in males at the same BMI when compared to females (P-interaction &amp;lt;0.01) (Figure 1). Conclusion Sex modifies the relationship between BMI and CIMT in Singapore's multi-ethnic population. Understanding the intermediary mechanisms involved will help in developing personalized preventive strategies. Funding Acknowledgement Type of funding sources: None. Figure 1