Mechanisms of Leptin-Induced Endothelial Dysfunction

Abstract

Purpose of Review

Endothelial dysfunction is a major risk factor for many cardiovascular diseases, notably hypertension. Obesity increases the risk of endothelial dysfunction in association with increasing production of the adipokine leptin. Preclinical studies have begun to unravel the mechanisms whereby leptin leads to the development of endothelial dysfunction, which are sex-specific. This review will summarize recent findings of mechanisms of leptin-induced endothelial impairment in both males and females and in pregnancy.

Recent Findings

Leptin receptors are found in high concentrations in the central nervous system (CNS), via which leptin promotes appetite suppression and upregulates sympathetic nervous system activation. However, leptin receptors are expressed in many other tissues, including the vascular endothelial cells and smooth muscle cells. Recent studies in mice with vascular endothelial or smooth muscle-specific knockdown demonstrate that endothelial leptin receptor activation plays a protective role against endothelial dysfunction in male animals, but not necessarily in females. Clinical studies indicate that women may be more sensitive to obesity-associated vascular endothelial dysfunction. Emerging preclinical data indicates that leptin and progesterone increase aldosterone production and endothelial mineralocorticoid receptor activation, respectively. Furthermore, decades of clinical studies indicate that leptin levels increase in the hypertensive pregnancy disorder preeclampsia, which is characterized by systemic endothelial dysfunction. Leptin infusion in mice induces the clinical characteristics of preeclampsia, including endothelial dysfunction.

Summary

Novel preclinical data indicate that the mechanisms whereby leptin promotes endothelial dysfunction are sex-specific. Leptin-induced endothelial dysfunction may also play a role in hypertensive pregnancy as well.

Introduction

Endothelial dysfunction is a precursor and major contributor to hypertension, atherosclerosis, and coronary artery disease. There are multiple methods to measure clinical endothelial function, the most common of which is flow mediated dilation, a non-invasive technique assessing vascular diameter responses to endothelial-dependent vasodilatory factors. However, even noninvasive measures of endothelial function are not routinely performed in patients considered high risk for cardiovascular diseases, such as obese patients. Therefore, the prevalence and contribution of vascular endothelial dysfunction to cardiovascular diseases in the general population is limited and often extrapolated from diagnoses of hypertension. Obesity significantly increases the risk of both hypertension and endothelial dysfunction via mechanisms not entirely understood. Obesity dramatically increases production of the adipokine leptin, a peptide hormone produced by adipocytes in levels proportionate to their mass. Therefore, cardiovascular risk conferred by obesity and leptin is interchangeable. Over the last 2–3 decades, clinical and preclinical studies demonstrated that leptin is a crucial contributor to cardiovascular risk in obese patients and experimental animal models. However, mechanisms whereby leptin promotes vascular endothelial dysfunction are beginning to be elucidated and demonstrate a significant sex difference between males and females (Figure 1).

Adipose-derived leptin promotes endothelial dysfunction

Leptin receptors are ubiquitously expressed throughout many organ systems, including the central nervous system (CNS) and vasculature (both endothelial and smooth muscle cells). Leptin is an energy homeostasis hormone, whose levels increase in response to growing adipose tissue mass, which is its primary source of production. CNS leptin receptor activation decreases appetite via hypothalamic downstream signaling pathways, and indeed, leptin receptor deficient rodent models (Db/Db mice and Zucker rats) are characteristically hyperphagic and develop pronounced obesity on regular chow diets. Systemically, leptin receptor is a type l cytokine receptor with long and short isoforms but is consistent in structural form as a tyrosine kinase transmembrane receptor. Leptin binding to its receptor induces phosphorylation of kinase Janus kinase 2 (JAK2), activating phosphotyrosines that serve as binding sites for various intracellular signaling pathways (1). Protein tyrosine phosphatase 1B (PTP1B) inhibits the activation of leptin receptor signaling by preventing the JAK2 phosphorylation. In hypothalamic leptin receptor signaling, increased activation of LepR increases intermediary hormones leading to increase MC4R activation. Within the vasculature, leptin receptors are expressed in endothelial cells (EC) and vascular smooth muscle cells (VSMC).

Several studies show that vascular relaxation to acetylcholine, an experimental measure of endothelial function performed via wire myography, decreases in association with increasing blood pressure in Db/Db leptin receptor deficient mice (2, 3) and Zucker leptin receptor deficient rats (4, 5). However, a limitation of both models for the study of leptin-specific mechanisms of vascular function and blood pressure control are development of comorbidities associated with metabolic syndrome, notably extreme hyperglycemia (often >500mg/dL glucose levels) and hyperlipidemia. Hyperphagia induced by deficiency of leptin receptors is mediated in the CNS via stimulation of hypothalamic α-melanocortin stimulating hormone (α-MSH) and subsequent melanocortin 4 receptor (MC4R) activation. Rodents with deficiency of MC4R (MC4R KO) or with overproduction of MC4R antagonist Agouti peptide develop spontaneous obesity, hyperglycemia, and hyperlipidemia, however, maintain leptin receptor signaling (5–8) Despite significant obesity, male MC4R KO mice do not develop endothelial dysfunction (6) or hypertension (6, 7). Female MC4R KO rats (8) and Agouti mice (9), however, demonstrate obesity-associated elevated blood pressure and endothelial dysfunction in contrast to their male counterparts. This is reflected in young clinical populations as well, with autonomic activity strongly correlating with leptin levels in young men, but not in young women (10). Therefore, in males, an intact hypothalamic leptin receptor may mediate endothelial dysfunction by increasing sympathetic activation in response to adiposity. However, in females, there may be a resistance to CNS leptin receptor-induced sympathetic activation in the vasculature to induce endothelial dysfunction and hypertension, however, an increase in susceptibility to such by peripheral leptin receptor activation.

Mice with endothelial-specific leptin receptor deletion (EC LepR KO) demonstrate a worsening of endothelial outcomes in the context of metabolic disorders of high and low leptin levels, notably via studies done in male animals. EC LepR KO male mice resist diet-induced obesity (11) but develop hyperleptinemia, due to decreased leptin uptake in tissues (12). However, in another report EC LepR KO in mice promoted increased adiposity on a high fat diet as well as hyperlipidemia, while also increasing circulating leptin levels independent of adipose mass (13). Therefore, in male mice, EC LepR activation may regulate systemic metabolic function in obesity, albeit current data does not allow for speculation on mechanisms yet. EC LepR KO also increased vasoconstrictor endothelin-1 production and endothelial cell hyperplasia in obese male mice indicating that EC LepR activation may be protective against hyperleptinemia and obesity-associated endothelial dysfunction in male mice. However, it is important to note that endothelial function was not directly assessed in these studies. EC LepR KO in male mice with very low leptin levels (i.e. lipodystrophy) reduces nitric oxide (NO) bioavailability, increases reactive oxygen species (ROS) production, and induces aortic endothelial dysfunction (14, *15). Furthermore, increasing EC LepR activation by selectively deleting EC protein tyrosine phosphatase 1B (PTP1B) ablates endothelial dysfunction in diabetic lean mice (streptozotocin induction) (16, 17). Notably, Atawia et al recently demonstrated that leptin infusion into EC LepR intact and KO mice did not induce endothelial dysfunction (18). Similarly, LepR sensitization by PTP1b, which does not induce hyperphagia/obesity, does not induce endothelial dysfunciton in male mice (9). The lack of endothelial dysfunction in leptin-infused EC LepR KO mice indicates that low activation of EC LepR does not promote endothelial dysfunction per se, but rather that the receptor is protective in the event of a metabolic challenge to endothelial function such as obesity or lipodystrophy. In the VSMC, specific LepR deletion does not affect leptin levels or adiposity in obese male mice (13), however, does promote increased neointima media area indicating that VSMC LepR do not regulate metabolic function but may promote dysfunctional vascular remodeling in obese males. In contradiction, a 2016 study of pooled male and female mice with VSMC LepR deletion demonstrated that VSMC LepR promotes leptin-induced endothelial dysfunction in this cohort, but sex was not separated (19). Therefore, in male mice, vascular LepR may protect from vascular dysfunction, however, vascular LepR may promote endothelial dysfunction in females. More studies, specifically in female mice with EC and VSMC specific LepR deletion, are needed to determine the sex-specific role of vascular LepR on endothelial function.

Leptin-induced endothelial dysfunction is sex dependent

Historically, young women were deemed “protected” from cardiovascular risk, notably hypertension, compared to age-matched men. Many studies report increases in cardiovascular disease prevalence in males ≤55 years of age compared to age-matched females (20–25), however, women “catch up” and surpass men in hypertension and cardiovascular disease prevalence in age groups ≥55 years of age (26). A recent clinical study indicated that small elevations in blood pressure induced a higher hazard ratio for myocardial infarction, heart failure and stroke in women compared to men (27), which was more pronounced even in young women (<52 years of age) than in older women (>52 years of age). In association, it has been shown that microvascular endothelial dysfunction is a more accurate predictor of cardiovascular events in women than in men (28). Therefore, impairments to vascular function that lead to small changes in blood pressure are likely more detrimental to cardiovascular risk in women than in men, particularly at young ages. In both males and females, obesity is a major contributing risk factor to the development of endothelial dysfunction and hypertension (29). However, clinical studies indicate a concerning clinical trend in that obesity ablates the sex difference in hypertension prevalence in young women and men (30), which may be mediated by vascular endothelial dysfunction. Indeed, a recent clinical cohort demonstrates that only in young women, not young men, does an increase in waist to hip ratio, a measure of central adiposity, correlate with worsened vascular function (31). In addition, obesity, rather than age and sex hormone status, may mediate increased endothelial dysfunction risk conferred by post-menopausal status in women. Indeed, menopause has little effect on flow mediated dilation in lean, non-diabetic women (32, 33), in contrast to significant detriment to flow mediated dilation induced by menopause in overweight populations (34). These data imply that either menopause-induced obesity or changes to sex hormone levels/function specific to obesity predispose women to more pronounced endothelial impairment in the menopausal transition.

Whether leptin is a crucial mediator of sex differences in endothelial function in obesity is currently under investigation in preclinical animal models. Our group demonstrated in 2016 that LepR sensitization by PTP1b, which does not induce hyperphagia/obesity, induces endothelial dysfunction only in female mice, which is ablated by leptin receptor antagonist (allo-aca) (9). This study correlates with a 2018 study by Jaffe and colleagues in which they showed that obesity (i.e. high leptin level), but not hyperlipidemia, induces microvascular endothelial dysfunction only in female mice, while males required hyperlipidemia for obesity-associated endothelial dysfunction (35). In addition, studies by our group in both 2015 and 2019 demonstrated that exogenous leptin administration, in the absence of obesity or other comorbidities, induces vascular endothelial dysfunction in female mice (36, 37). These studies imply that leptin is the obesity-associated factor that drives endothelial dysfunction in females, while obesity induces other comorbidities, such as hyperlipidemia, that is a greater contributor to endothelial dysfunction in male mice.

The mechanism(s) whereby leptin induces endothelial damage in females may be an indirect mechanism involving the renin angiotensin aldosterone system (RAAS). Previous studies show that leptin promotes the development of hypertension and endothelial dysfunction in obese males via increased sympathetic activation, however, these sympathetic nervous system mechanisms are not observed in females (18, 37–39). Aldosterone is a critical regulator of blood pressure and vascular tone. Clinical studies indicate that obesity increases circulating leptin levels, however, aldosterone levels are higher per unit of body mass in women than in men, i.e. obesity increases aldosterone more so in women than in men (40). In preclinical female mouse models, exogenous leptin increases plasma aldosterone levels and adrenal aldosterone synthase (CYP11B2) expression in the absence of an increase in angiotensin II (ANGII) or corticosterone (9). In this same study, female obese leptin-deficient (Ob/Ob) mice do not develop obesity-induced increases in plasma aldosterone, which is restored by leptin administration. Mineralocorticoid receptors (MR) are highly expressed in the epithelial cells of the renal distal collecting duct, however, are also highly expressed in vascular endothelial cells (ECMR). ECMR expression is higher in female mice and humans than in males, which we recently showed is driven by endothelial progesterone receptor activation (37). Genetic knockout of ECMR or EC progesterone receptor in mice ablates leptin-induced endothelial dysfunction, indicating the necessity of an intact ECMR and progesterone receptor for leptin-dependent endothelial dysfunction in females (37). Collectively, these data indicate that leptin induces endothelial dysfunction in females via a heightened aldosterone-MR axis.

Leptin in Endothelial Dysfunction of Preeclampsia

Preeclampsia is a cardiovascular pregnancy disease, diagnosed as hypertension presenting at or after the 20^th gestation week in humans alongside a symptom of placental dysfunction. Preeclampsia increases the risk for low birthweight as well as increases cardiometabolic risk for both mother and offspring throughout their lifetimes (41). Endothelial dysfunction is a hallmark of preeclamptic pregnancy (42). Recent clinical data shows that high total vascular peripheral resistance score, but not cardiac output, predicts hypertension and adverse fetal outcomes in hypertensive pregnancy patients more so than blood pressure measure alone (43), indicating that increased vascular resistance due to endothelial dysfunction may play a very significant role in adverse outcomes of preeclampsia patients. Several mechanisms have been proposed to mediate endothelial dysfunction in preeclampsia, including inappropriate RAAS activation, reduced NO bioavailability and increases in the production of the vasoconstrictor endothelin-1 (42). Leptin levels increase in a healthy pregnancy, with the placental trophoblasts increasingly secreting leptin across all trimesters of pregnancy (44). However, both older and recent cohorts repeatedly demonstrate that leptin levels are elevated in preeclamptic patients compared to normotensive healthy pregnant patients (45–48). Leptin levels also positively correlate with PE severity (49) and negatively with fetal weight (50, 51) in preeclamptic patients. Although high BMI/obesity elevates leptin in pregnancy (52), PE patients demonstrate increases in plasma leptin levels that are disassociated with BMI (53–55). These clinical data indicate that leptin plays a role in the pathogenesis of preeclampsia independent of the comorbidities of obesity. Our group recently published a preclinical model of leptin-induced preeclampsia in pregnant mice which begins to shed light on whether leptin is a crucial player and potential therapeutic target for preeclampsia. In this study we showed that exogenous leptin administration to pregnant mice from mid-late gestation induces hypertension as well as fetal growth restriction (i.e. low pup weight) (*56). Notably, these mice developed endothelial dysfunction in response to leptin infusion in both the aorta and the 2^nd order mesenteric resistance arteries. Leptin also increased placental preproendothelin-1 and endothelin converting enzyme-1 mRNA expressions in these pregnant mice, indicating that leptin may stimulate preeclampsia-associated elevations in circulating endothelin-1. In addition, deletion of ECMR ablated the ability of leptin to induce hypertension and fetal growth restriction in pregnant mice, indicating that improving endothelial function with ECMR deletion prevented the development of preeclampsia characteristics. These data indicate that leptin-induced endothelial dysfunction may lead to or propagate preeclampsia pathology in pregnancy. Therefore, changes to endothelial function induced by leptin in preeclampsia may have significant contribution to pregnancy outcomes.

Conclusion and Perspectives

The mechanisms via which leptin induces endothelial dysfunction both in the context of obesity and in its absence are currently being investigated in preclinical animal models. Recent developments in transgenic knockout mouse models of both endothelial and vascular smooth muscle-specific leptin receptors indicates that activation of local leptin receptors plays a protective role against obesity-associated endothelial dysfunction in male animals. However, the role of these receptors in the maintenance of endothelial function in females is likely diverse from that of males, but remains largely unexplored. Recent studies do indicate that females develop a heightened renin-angiotensin aldosterone system activation compared to males. Leptin induced aldosterone production, coupled with increased expression of endothelial MR in females, indicates that the leptin-MR axis leads to obesity associated, leptin-induced vascular endothelial impairment in women. A newer area of study is the role of leptin in the development of endothelial dysfunction and hypertension in preeclampsia. Although studies have long shown an association of leptin with preeclampsia, even in the absence of obesity, a recent study demonstrated for the first time there may be a contributory role for leptin to promote endothelial dysfunction and promote the adverse pregnancy outcomes associated with preeclampsia. Collectively, new insights indicate that leptin is a crucial player in the pathogenesis of endothelial dysfunction in obesity, particularly in women, and the field will need to continue to delve into mechanisms whereby leptin receptor activation induces endothelial cell damage.

Key Points:

• Preclinical and clinical evidence continues to suggest that leptin is a crucial player in obesity-associated endothelial dysfunction and hypertension especially in females

• Endothelial leptin receptor activation may play a protective role from obesity-associated endothelial impairment in males, while its role in females is largely unexplored to-date

• Leptin induces aldosterone production and females have a heightened endothelial MR expression compared to males, therefore, a leptin-aldosterone-endothelial MR axis may predispose females to obesity-associated endothelial dysfunction

• Leptin levels increase in preeclamptic patients and recent mouse models demonstrate that leptin may mediate endothelial dysfunction and increase the risk of adverse pregnancy outcomes in preeclampsia