Lymphatic Function and Dysfunction in the Context of Sex Differences

Abstract

Endothelial cells are the building blocks of the blood vascular system and exhibit well-characterized sexually dimorphic phenotypes with regard to chromosomal and hormonal sex, imparting innate genetic and physiological differences between male and female vascular systems and cardiovascular disease. However, even though females are predominantly affected by disorders of lymphatic vascular function, we lack a comprehensive understanding of the effects of sex and sex hormones on lymphatic growth, function, and dysfunction. Here, we attempt to comprehensively evaluate the current understanding of sex as a biological variable influencing lymphatic biology. We first focus on elucidating innate and fundamental differences between the sexes in lymphatic function and development. Next, we delve into lymphatic disease and explore the potential underpinnings toward bias prevalence in the female population. Lastly, we incorporate more broadly the role of the lymphatic system in sex-biased diseases such as cancer, cardiovascular disease, reproductive disorders, and autoimmune diseases to explore whether and how sex differences may influence lymphatic function in the context of these pathologies.

Introduction

The lymphatic vasculature maintains fluid homeostasis, regulates immune cell trafficking, and absorbs fat.^1,2 These vessels form a blind-ended system of capillary and collecting vessels.³ Excess interstitial fluid, immune cells, lipids, protein, and other macromolecules are collectively drained as lymph by small lymphatic capillary vessels in the interstitial space and delivered to larger lymphatic collecting vessels, where lymph is eventually returned to the venous circulatory system through the thoracic or right lymphatic duct. This circulatory system can be exploited by pathogens and infectious agents, thus innate immune cells, concentrated in lymph nodes, are designed to destroy pathogens and mount an immune response.^4,5 Both capillary and collecting vessels are lined by a single layer of lymphatic endothelial cells (LECs). The junctional composition of LECs defines their identity. Lymphatic capillaries have no basement membrane and are characterized by overlapping “button-like” junctions that spread apart in response to pressure fluctuations for optimal fluid drainage;^6,7 the endothelial cells are tethered to fibrillin anchoring filaments that attach the plasma membrane to the extracellular matrix (Figure 1).⁸ Collecting vessels are aligned in tighter “zipper-like” junctions encased in a basement membrane and smooth muscle cell layer for optimal lymphatic flow.⁹ Connections between LECs as well as communication between LECs and the extracellular matrix profoundly affect drainage and flow of lymph by regulating efficient transport of interstitial fluid and lipids, a process of delivering nutrients and removing waste between vasculature and cells.^10,11 Specialized lymphatic capillaries, called lacteals, form in the villi and absorb nearly all dietary lipids to transport the fat as chylomicrons out of the small intestine via larger lymphatic collecting vessels and back to the bloodstream.^11,12 Each organ’s lymphatic system has unique genetic signature, origin, and function, contributing to nearly every biological process in the body and many disease pathologies.¹³⁻¹⁵

Figure 1.

Basic lymphatic capillary structure.

Deficits in lymphatic function can have catastrophic consequences.^16,17 Lymphatic vessels permeate nearly every organ system while also regulating the fluid and cellular components of the human interstitium, which was recently characterized in detail using advanced microscopy technology, even being described as a new organ system.¹⁸ The pathogenesis of cancer, cardiovascular disease, and autoimmune diseases, some of the most prevalent diseases, can often be linked to lymphatic defects and often result in lymphedema, for which there is no cure.^16,19−21

The importance of sex differences in disease is widely appreciated, with new FDA regulations requiring sex dependent testing and dosing; however, clinical studies still carry a large male bias.²²⁻²⁴ Bias also plays a role in the healthcare system at large and often affects how women and men are differentially treated and diagnosed. Genetic studies often neglect sex chromosomes in their studies, with less than 33% of genome-wide association studies (GWAS) including the X chromosome in their analyses.^25,26 Even basic research often fails to distinguish between male and female studies in mice, cells, and other model organisms. Recent requirements to include sex as a biological variable in grants funded by the U.S. National Institutes of Health as well as some scientific journals have led to improvements. Consequently, sex differences in the context of cancer, cardiovascular disease, and autoimmune disorders have been appreciated clinically, and continue to represent a growing area of scientific exploration. Yet, although there is also a well-characterized prevalence of lymphatic disorders in females, our basic understanding of lymphatic development, signaling pathways, and physiology often fails to incorporate an analysis of sexual dimorphisms imparted by sex chromosomes or sex steroids. This review aims to highlight the important sex dependent differences specific to lymphatic biology and lymphatic disease and offer numerous areas for future exploration and focus.

Lymphatic Function, Structure, and Development

Lymphatic Growth Factors

Most LECs differentiate from venous progenitors during development.²⁷ These cells are generally marked by expression of lymphatic regulator, prospero homeobox protein-1 (PROX1), which is turned on by transcription factors Sox18 and Coup-TFII.²⁸⁻³⁰ Gradually, PROX1+ cells migrate away from the cardinal vein to form the initial rudimentary lymphatics, the jugular lymph sac.^31,32 Migration of LECs away from the cardinal vein is controlled by a gradient of vascular endothelial growth factor-C (VEGF-C)/vascular endothelial growth factor receptor-3 (VEGFR-3) signaling, a hallmark of lymphatic signaling and development,³³ that is differentially expressed by the sexes.³⁴ Other important lymphatic factors and signaling pathways include adrenomedullin (Adm = gene; AM = protein)/calcitonin receptor-like receptor (Calcrl = gene; CLR = protein)/RAMP2, hyaluronic acid (HA)/lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), collagen and calcium binding EGF domains 1 (CCBE1)/ADAMTS3, ANG/TIE, Podoplanin, FOXC2, etc.³⁵⁻⁴⁵ These signaling pathways have sexual dimorphisms, discussed in more detail below. Each tissue develops its own lymphatic vasculature at unique developmental stages via lymphangiogenesis, the growth of new vessels from existing vessels.⁴⁶

MicroRNAs and Lymphatics

In recent years, microRNAs (miRNAs) have emerged as important molecules regulating multiple levels of physiology, development, metabolism, disease, etc.⁴⁷ Hormones and X-linked genes can have profound effects on the expression and regulation of miRNAs, thus many miRNAs display functionally important sexual dimorphisms.^48,49 For example, the 3′-UTR of Adm, an endogenous lymphangiogenic factor, contains the binding site for miR-879, the expression of which is regulated via estrogen.⁵⁰ When the native 3′-UTR of Adm is replaced and that binding site is lost, AM expression in females is higher than in males, suggesting a vital role of estrogen in regulating lymphangiogenesis via AM signaling through miRNAs.

Many other miRNAs are also regulated via estrogen and estrogen metabolites, including miR-466 and miR-181a (Figure 2).^51,52 PROX1 directly interacts with estrogen receptor alpha (ERα) and may form a complex with ERα and PGC-1α to inhibit ERα-activated genes.⁵³ PROX1 has a very long 3′-UTR, susceptible to miRNA inhibition.⁵⁴ For example, miR-466 can reduce lymphangiogenesis via PROX1 binding.⁵⁵ Additionally, miR-181a can directly bind and inhibit the expression of PROX1, resulting in reprogramming of LECs toward a blood endothelial cell (BEC) fate.⁵⁶ Both miR-466 and miR-181a have also been shown to inhibit corneal lymphangiogensis.⁵⁵ Another miRNA, miR-31 has been shown to have a direct effect on lymphatic specification and development by repressing LEC-specific genes, including FOXC2.⁵⁷ In another study, miR-31 was upregulated in female mice in certain disease states.⁵⁸ BMP2 signaling may negatively regulate LEC identity via miR-31 and miR-181a.⁵⁹ Post-transcriptional regulation is incredibly complex but significant when considering regulation of genetic identity of LECs as well as development, function, and disease pathogenesis. The importance of miRNAs cannot be overlooked, especially within sex dependent implications of lymphatic transcription activity.

Figure 2.

Effects of estrogen on microRNAs.

Junctional Proteins

The barrier integrity of lymphatic vessels is vital to their proper function, and many key proteins involved in lymphatic barrier function are differentially regulated in males and females. Vascular barrier function in the microvasculature is regulated via cAMP and cGMP, cyclic nucleotides hydrolyzed via phosphodiesterases (PDEs).^60,61 PDEs are a class of enzymes differentially expressed by sex in endothelial cells, specifically PDE1 and PDE3; for example, PDE3 inhibitor, cilostazol, reduces barrier function in XY but not XX cells, indicating sex chromosome specific targets related to barrier function of endothelial cells.⁶⁰ Zona-ocluden 1 (ZO-1) is a core component of tight junctions in LECs, and there is evidence that estrogen may downregulate expression of ZO-1 leading to overall lower levels of ZO-1 in females.⁶² Aquaporin (AQ) channels allow for the passage of water between cells and the environment; Aquaporin-1 (AQ1) has been shown to be involved in water transport between lymph nodes and veins, an essential function of the basic lymphatic system.⁶³ Men have been shown to express higher levels of AQ1, which may be indirectly regulated via testosterone levels.⁶⁴

Gap junctions, comprising connexin (Cx) proteins, are another critical component of lymphatic junctions and general intercellular communication. Cx43, Cx37, and Cx47 are all critical both in the development and maintenance of lymphatic vessels and lymphatic valves, essential in preventing back-flow.⁶⁵⁻⁶⁷ Human mutations in Cx43, Cx47, and Cx37 have been shown to lead to lymphatic disease or susceptibility to lymphedema in some cases.⁶⁸⁻⁷¹ Although sex dependent differences in lymphatic connexins have been largely unexplored, studies in the heart illuminate important sex dependent differences of Cx43, a protein known to interact with ZO-1.⁷² Female cardiomyocytes have higher levels of Cx43 both under basal and pathological states.⁷³ It is possible that LECs may also have sex dimorphisms in Cx protein expression. Notably, differential expression of Cx proteins even within cardiomyocytes could have indirect effects on the lymphatic system, especially within cardiac lymphatics that require cardiac contractions to propel cardiac lymphatic flow.⁷⁴⁻⁷⁷ Sex differences exist in key components of cellular connections between LECs, and because proper maintenance and function of lymphatics depends on junctional integrity and permeability of lymphatic vessels, further studies are warranted to fully understand biological differences of the sexes in lymphatic barrier and permeability.

Estrogen Receptors (ERs) in Lymphatics

LECs, like BECs, express nuclear ERα, but unlike BECs, they do not express estrogen receptor beta (ERβ) as highly.⁷⁸⁻⁸⁰ Another estrogen receptor (ER), plasma membrane receptor G-protein coupled receptor 30 (GPR30), also has innate sex-dependent differences.^81,82 Receptor activity modifying protein 3 (RAMP3), a protein upregulated via estrogen, has a high affinity for GPR30 as a coreceptor of estrogen; the binding of estrogen to RAMP3/GPR30 indirectly increases expression of GPR30 creating an estrogen-dependent cycle of receptor expression.⁸¹

The effects of estrogen mediated signaling on endothelial cells has been widely appreciated and well-characterized. For example, endothelial cells produce NO to maintain vasodilation, a tightly regulated and important biological process with far reaching effects that can be activated via estrogen.⁸³ Endothelial nitric oxide synthase (eNOS) production is further regulated by estradiol (E2) levels via phosphorylation of proto-oncogene tyrosine-protein kinase Src (c-Src), which activates eNOS.⁸⁴ Estrogen also regulates phosphorylation of ERK and AKT in endothelial cells via membrane ER variants to activate mitogenic and survival signaling pathways.⁸⁵ Moreover, estrogen activation of GPR30/RAMP3 induces the phosphoinositide 3-kinase (PI3K) pathway to regulate the cell cycle.⁸⁶ Thus, estrogen can regulate proliferation, dilation, eNOS production, and signaling pathways within vascular endothelial cells. However, additional studies carefully distinguishing lymphatic endothelium from vascular endothelium identity could offer many new insights into lymphatic function.

Although men and women may express equivalent amounts of the ERα receptor, the main ligand and most potent of estrogens, E2, differs between the sexes.⁸⁷ E2 is primarily synthesized and secreted from the ovaries of premenopausal women as a circulating hormone, while extragonadal tissues also secrete lower levels of noncirculating E2, the primary estrogen source for men and postmenopausal women.⁸⁸In vitro studies have demonstrated to be a powerful tool in examining the effects of sex steroid on endothelial cells. For example, testosterone, administered in culture, can reach toxicity in cultured vascular endothelial cells at certain concentrations,⁸⁹ while estrogen treatment of vascular endothelial cells is generally protective.^87,90 Additionally, subtle sex dimorphisms in size and shape have been observed in aortic endothelial cells when plated on gelatin, female cells spread more than male cells, leading to more cells required for full skeletal muscle capillary coverage in males compared to females.⁹¹ It is not unreasonable to assume LECs could have morphological differences between the sexes in basal and/or challenged conditions. However, the current predominant commercial source of primary, dermal LECs is neonatal foreskin collected at the time of circumcision, thereby skewing the currently available data toward male sex.

ERα activation in the cytoplasm via estrogen ligands causes the receptor to dimerize and enter the nucleus thus activating gene expression as a transcription factor via binding to DNA estrogen response elements (EREs).⁹² E2 has been shown to directly target transcription of lymphatic genes via EREs: VEGFR-3, VEGF-D, LYVE-1, and hyaluronic acid synthases (HASs) via EREs.⁹³ Furthermore, endothelial cell deletion of ERα caused physical lymphatic defects and decreased expression of lymphatic genes.⁹³ LYVE-1, a common lymphatic marker, is a receptor for HA, a ligand whose synthesis can be influenced by E2 levels.⁹⁴ Estrogen can also activate expression of Adm, Calcrl (the receptor for AM), RAMP2 (a coreceptor for AM), and RAMP3 (a coreceptor for both GPR30 and CLR).^95,96 Additionally, gonadal lymphangiogenesis has clear sexual dimorphisms; testicular lymphangiogenesis begins in mice in late gestation, while ovarian lymphangiogenesis in mice begins postnatally and is regulated via sex hormones, follicle stimulating hormone (FSH), and E2.^97,98 Therefore, as summarized in Table 1, estrogen has profound effects on the genes and signaling pathways known to regulate lymphatic function and development. How estrogen ligands and receptors affect lymphatics and regulate levels of VEGF-C in the progression of diseases remains mostly unknown, thus careful sex-dependent studies would be incredibly valuable to the field, especially since females have endogenously higher levels of serum VEGF-C.³⁴

Table 1. Summary of Lymphatic-Associated Effects of Estrogen.

estrogen effects	genetic regulation	reference
Lymphatic Factors
VEGF-D	↑	(69)
VEGFR-3	↑	(69)
RAMP2/CLR	↑	(72)
RAMP3/GPR30	↑	(40, 72)
AM	↑	(71, 72)
LYVE-1	↑	(69)
HAS	↑	(69, 70)
Disease
cancer	↑	(134, 135, 153−155)
CXCR4	↑	(153, 154)
CXCR7	↑	(154)
endometriosis	↑	(191)
LAM	↑	(131, 132)
Cell Signaling and Function
NO	↑	(46)
ERK	↑	(48)
PI3K/AKT	↑	(48)
ZO-1	↓	(90)

Blood Pressure and Lymphatics

Starling forces regulate fluid homeostasis within the body via hydrostatic and oncotic pressure gradients maintaining a careful balance, a process that requires both proper blood and lymphatic vasculature function.⁹⁹ Although men and women anatomically contain similar vasculature systems, the pressure within these interstitial spaces, vessels, and fluids maintaining homeostasis can vary by sex. In women, arterial blood pressure is innately lower, while diastolic blood pressure differs by sex with age, resulting in lower capillary pressure and lower venous pressure in women overall.^100−104 The mechanisms regulating gender differences of blood pressure and hypertension are not completely understood, but lymphatic differences are not to be overlooked. These sex-dependent pressure differences result in functional lymphatic differences in how lymph is drained and absorbed to maintain overall fluid homeostasis. High-salt diets can cause hypertension, which can trigger lymphangiogenesis as a means to maintain homeostasis.^105,106 Individuals with hypertension often show increased levels of VEGF-C, a lymphangiogenic growth factor.^106,107 Yet, men and women respond differently to low-salt and high-salt interventions; women respond with more significant changes in blood pressure, which may indicate more stress on their lymphatic systems.¹⁰⁸ Dermal lymphatics may also play a role in regulation of blood pressure in the skin, independent of fluid retention, by increasing lymphatic flow in response to sodium accumulation, an interesting study that was only performed in male rats.¹⁰⁹

In a study measuring intraluminal lymphatic pumping pressure in lymphatic collector vessels in the legs of both men and women of all ages, a marked decrease in pumping pressure correlated with increase in age.¹¹⁰ The decrease, attributed to age-related lymphatic sclerosis, was significantly worse in female patients. Aging, another innately sex-dependent process, in regards to lymphatics is vastly understudied. Differences in age-related sexual dimorphisms may be partially explained by a decrease in estrogen production with age, resulting in nitric oxide (NO) dependent contractile forces in the endothelium being dampened.^111,112 An extensive study to assess functional changes in lymphatics with age was restricted to only males.¹¹³ Further studies to assess directly how blood pressure and hypertension differences in males and females affect lymphatics and vice versa are certainly warranted.

X-Linked Factors

Over 4% of protein-coding genes exist on the X chromosome, yet the X chromosome is often neglected in genetic studies of disease, even though X-linked variants are more likely to result in disparity of traits between males and females.^25,26,114 Using software XWAS, a tool designed to identify associations between disease and X-linked genes, scientists identified associations between core 1 β3GalT specific molecular chaperone (Cosmc) and inflammatory bowel disease (IBD).¹¹⁵ IBD generally affects both men and women with equal frequency, however disease physiology, symptoms, clinical manifestations, and treatments differ significantly between men and women,¹¹⁶ and the Cosmc variant showed significant differences in effect size of risk between males and females.¹¹⁵ Cosmc is a chaperone protein required for T synthase expression and subsequent O-glycan biosynthesis, Cosmc deletion results in loss of T synthase and major vasculature disruption and hemorrhage in developing embryos, however phenotypes do differ by sex.¹¹⁷ O-glycans are highly expressed in endothelial cells and in mice lacking T synthase (encoded by Cosmc) in endothelial and hematopoietic cells, embryos died from disorganized and blood-filled lymphatics; the same study showed that O-glycans were required for lymphangiogensis.¹¹⁸ Although the pathophysiology of IBD is not completely understood, lymphatic dysfunction is well-established as a feature of IBD, and the targeting of the lymphatic system has been explored as a potential treatment option for IBD patients.¹¹⁹ Understanding how X-linked genes contribute to development of lymphatic vessels and the incidence and severity of lymphatic-associated diseases could lead to new therapeutic targets and breakthroughs in understanding sex differences in disease as a whole.

Lymphatic Disease

Perhaps the clearest example of sex dependent differences within lymphatic biology is related to lymphatic disorders themselves. Generalized edema or excess fluid accumulation is a hallmark of many disease states.¹²⁰ Lymphatic diseases overall are much more common in women, but the mechanisms regulating this phenomenon remain unclear.^121−123 A clearer understanding of the pathologies of lymphatic disorders between the sexes could ultimately lead to new and improved treatments or even a cure for these devastating diseases, while also illuminating deficiencies in the understanding of other disease states.

Lymphedema

Lymphedema affects over 200 million people worldwide.¹²⁴ Primary lymphedema, a rare genetic disease characterized by impaired lymph flow resulting in extreme swelling, accumulation of fat, and fibrosis in the extremities is three times more likely to occur in women.⁹³ Only nine causal genes have been identified for lymphedema, leaving much to be discovered regarding the etiology of primary lymphatic disease.¹²⁰ Milroy disease, a form of primary lymphedema, is caused by mutations in the VEGF-C receptor, VEGFR-3, resulting in the failure of initial lymphatic fluid absorption¹²⁵.¹²⁶ Clinical studies have shown sexual dimorphism of VEGF-C/VEGF-D levels, with females having endogenously higher levels of VEGF-C and VEGF-D overall.³⁴ Lymphedema-distichiasis syndrome results from mutations in FOXC2, resulting in faulty lymphatic valves in lymphatic collectors and subsequent lymphatic reflux.¹²⁷ Lymphedema praecox or “Meige’s disease” refers to lymphedema with early onset, before age 35, but it is most common in women entering puberty.¹²⁸ Meige’s disease presents with a small and insufficient lymphatic system resulting in lymphedema and many other debilitating symptoms. Mutations in Cx43 and Cx47 can also cause lymphedema.⁶⁹⁻⁷¹ Turner Syndrome, a disease of chromosomal aneuploidy (45,X), often presents with congenital lymphedema of the hands, neck, and feet caused by an undefined failure of initial lymphatic function and only affects females.¹²⁹

Secondary lymphedema accounts for 99% of lymphedema cases overall and is characterized as lymphedema resulting from trauma, surgery, or other direct damage to the lymphatic system.^130,131 Genetic differences may predispose certain populations to secondary lymphedema and some lymphatic-associated genes have been identified to correlate with higher risk of developing secondary edema,^68,71,132 but conclusive biomarkers identifying high-risk patients remain elusive.⁷¹ The most common cause of secondary lymphedema is lymphatic filariasis, a condition infecting over 120 million people worldwide, with 1.25 billion people at risk across 72 countries.¹³³ Lymphatic filariasis, a parasitic lymphatic infection spread by mosquitoes, is significantly less prevalent in reproductive-aged women, independent of exposure bias.¹³⁴ Cancer treatments, including surgery and radiation, account for most of the secondary lymphedema observed in the industrialized world, particularly female breast cancer patients. Between 25 and 50% of cancer survivors overall are at risk of developing secondary lymphedema and approximately 20% of breast cancer survivors develop lymphedema phenotypes.^135−139 In a mouse model of secondary lymphedema, E2 improved lymphedema.⁹³ Interestingly, in an experiment with profound ramifications in the treatment of breast cancer, treatment of these mice with tamoxifen exacerbated lymphedema.⁹³ Increased knowledge of the pathologies of lymphedema is instrumental in curing this disease and in the prevention of secondary lymphedema. Furthermore, understanding the discrepancies in the formation of secondary lymphedema, particularly in cancer patients, could help improve treatments, identify high-risk patients, and even change the approach to cancer treatments.

Lipedema

Lipedema, a chronic disease condition, affects only women and presents during extreme hormonal shifts in a woman’s life such as puberty, childbirth, or menopause.¹²² Often mistaken for lymphedema or misdiagnosed, lipedema presents with painful swelling and symmetrical accumulation of subcutaneous adipose tissue in the limbs and trunk, sparing the hands and feet.¹⁴⁰ The disease stems from an inability to drain interstitial adipose tissue and fluid; the lymphatics cannot return the fluid to the venous circulation leading to stagnation and “persistent fat.” Lymphatic flow rates are often reduced in lipedema patients.¹⁴¹ The disease is poorly understood, but correlates with low risk of diabetes and hypertension, despite patients having obese body mass index (BMI).¹²² Lifestyle and diet changes are ineffective in reducing fat accumulation that eventually leads to joint problems, immobility, fatigue, secondary lymphedema, psychological problems, venous disease, and lymphatic abnormalities. Currently, the only treatments that exist include liposuction, surgical intervention, and manual lymphatic drainage. The disease is not well-understood, and the mechanisms leading to the susceptibility of only women remain unknown and warrant further study.

Lymphangioleiomyomatosis (LAM)

Lymphangioleiomyomatosis (LAM) is a rare and deadly disease that primarily affects reproductive-aged women, characterized by an invasion of smooth muscle-like and epithelioid-like cells (LAM cells) into the lung tissue.¹²³ It is linked to causative mutations in tumor suppressing genes, tuberous sclerosis proteins 1 and 2 (TS1 and TS2) often leading to constitutive activation of the mTOR pathway, resulting in unregulated cell proliferation and growth.^142,143 LAM presents with uninhibited lung lymphangiogenesis and secretion of VEGF-D, a lymphangiogenic growth factor, by LAM cells.¹⁴⁴ Interestingly, VEGF-D maps to the X chromosome.¹⁴⁵ High serum VEGF-D levels correlate with lymphatic involvement and dysfunction in patients with LAM, and has proven to be an effective new biomarker for diagnosis and prognosis.¹⁴⁶ However, LAM remains difficult to conclusively diagnose and no cures currently exist.¹⁴⁷ Estrogen and progesterone receptors are expressed on LAM cells, and high levels of estrogen enhance proliferation, migration, and metastasis of LAM cells as well as general progression of LAM, yet the mechanisms and sexual dimorphism of the disease are not fully understood.^148,149

Lymphatic-Associated Disease

Lymphatic complications in disease are well-appreciated. Lymphatics can affect acute and chronic inflammation, graft vs host disease, glaucoma, viral infection, liver disease, cancer progression, cardiovascular disease, arthritis, hypertension, obesity, and recently, neurodegenerative diseases.^19,150−155 Most diseases have innate sex differences. However, sex differences within lymphatic function, structure, and development in the context of disease are often overlooked. Here we explore specific sex differences of the lymphatic system and how that may play a role in the pathophysiology of cancer and cardiovascular disease. Other diseases with huge disparities between numbers of males and females affected, like reproductive disorders and autoimmune disease require further investigation into the lymphatic-specific differences between the sexes.

Cancer

The primary method of cancer metastases is via lymphatic vessels.¹⁵⁶ Gap junction coupling between LECs is required for tumor cell migration through the endothelium, a process regulated via AM signaling, an estrogen-regulated hormonal peptide.¹⁵⁷ Breast cancer commonly spreads to the lymph nodes; breast cancers that express ERs or progesterone receptors are over 80% more likely to spread via lymph nodes, and estrogen may play a direct role in the path of metastasis.¹⁵⁸ Tumor growth and internal tumor lymphangiogenesis is estrogen-dependent, yet tumor cells change within the microenvironment of the lymphatics and lymph nodes and may become hormone resistant. Tumor cells migrating from primary tumor to lymphatics are enriched for CD44, a homologue of LYVE-1.^159,160 Innately, breast lymphatic drainage does not significantly differ between men and women, yet breast cancer is much more prevalent in women.¹⁶¹

Cancer cells from primary prostate tumors similarly metastasize via lymph nodes and lymphatic vessels in men.¹⁶² Prostate cancer is commonly treated with androgen deprivation therapy with high success rates, but distant prostate cancer is aggressive, and survival rates decrease from almost 100% to just 30% upon metastasis. Lyphophosphatidic acid (LPA) is associated with malignant cancers by promoting cell migration, invasion, proliferation, and survival; it has recently been shown that LPA may promote cancer progression via activation of VEGF-A and VEGF-C.^163,164 A comprehensive understanding of how cancer cells migrate initially into lymphatics and spread could lead to new treatments and therapies.

Ovarian cancer is the leading cause of death in women with gynecological malignancies, and the risk dramatically increases in postmenopausal women, correlating with drops in estrogen and increases in FSH and luteinizing hormone, hormones known to induce VEGF-C levels via LEDGF/p75.¹⁶⁵ VEGF-C levels correlate with worse overall survival and increased intraperitoneal metastases.¹⁶⁶ Conversely, CCBE1, a lymphangiogenic factor known to promote lymphangiogenesis by activating VEGF-C, is found to be reduced in some ovarian cancers in women.¹⁶⁷

Lymphatic tumors can form in patients with primary or secondary lymphedema (lymphangiosarcoma), but the pathogenesis remains unknown.^168,169 Karposi’s sarcoma (KS) is an angiogenic tumor that has more lymphatic properties than blood vasculature with known sex dimorphisms.^170,171 Classic KS is much more common in men,^172−174 while AIDS-related KS is much more severe in women.¹⁷⁵ Cancerous endothelial cells from Karposi sarcoma tumors express Calcrl, a G-protein coupled receptor (GPCR), that is part of the AM signaling axis, an integral part of lymphatic development and function.¹⁷⁶ LECs secrete chemokines, such as CXCL12 that signals through CXCR4 and CXCR7.¹⁷⁷ This signaling axis is known to promote a lymphatic microenvironment that promotes tumor growth. Additionally, estrogen can upregulate both CXCR4 and CXCR7 expression, further fueling the tumor microenvironment.^178,179 Sex hormones play a pivotal role in cancer biology and metastasis;¹⁸⁰ lymphatic studies in both sexes could offer new insights to cancer leading to new treatments or medicines.

Cardiovascular Disease

Cardiovascular disease remains the number one cause of death worldwide.¹⁸¹ It is well-established that premenopausal women are at a substantially lower risk for cardiovascular disease, but the cardioprotective mechanisms are not completely understood.^182,183 Reproductive-aged women are also innately protected from ischemic cardiac injury, yet women die more often than men as a result of myocardial infarction (MI).¹⁸⁴ The deaths are often associated with complications during or after surgery, drugs that are more suited for men, and tools like vascular assist devices (VAD) that are designed to fit in the larger chest cavity of men over women.^185,186 Most cardioprotective phenotypes in women are attributed to estrogen, yet hormone replacement therapy in postmenopausal women actually increases the risk of cardiovascular disease.¹⁸⁷ Estrogen activation of RAMP3/GPR30 may explain some aspects of cardioprotection observed in women.⁸¹ In mice, RAMP3 levels increase in females in models of hypertension and cardiac hypertrophy. Male mice with RAMP3 deletion develop more severe cardiovascular functional deficiencies compared to females in a model of hypertension.⁸²

Recent work has highlighted the importance of cardiac lymphatics in the healing and remodeling process post-MI.^{13,76,188,189} However, until recently cardiac lymphatics had been largely unexplored compared to lymphatics of other organ systems. Cardiac edema is extremely common and even mild edema can have profound detrimental long-term consequences on cardiovascular health and function.^190,191 General cardiovascular health plays an important role in cardiac lymphatics, because unlike other lymphatic systems, cardiac lymphatics generally lack contractile lymphatic smooth muscles cells thus lymph flow relies heavily on extrinsic cardiac contractile forces and central venous pressures.⁷⁴⁻⁷⁷ There are many innate sex-specific physiological differences in the cardiovascular system that may indirectly affect cardiac lymphatic function: men have a larger left ventricular mass, women have increased contractility compared to age-matched men, cardiac apoptosis associated with aging is three-times higher in men than women, women have lower blood pressure but higher heart rates, and women have smaller coronary arteries.^{101,192−194} New studies in mice show that males and females have innate differences in cardiac lymphatic development; women have more cardiac lymphatic vessels than males, which is even further exacerbated with Adm overexpression.¹⁸⁸ This may be a new explanation for some of the innate cardioprotective phenotypes observed in women. VEGF-C and AM can both improve cardiac function and improve edema clearance after MI via cardiac lymphangiogenesis.^13,76 LYVE-1 is essential for proper immune cell clearance in the heart following MI via cardiac lymphatics,¹⁹⁵ and sex differences have been observed in noncardiac LYVE-1 expressing tissue, female spleen tissue has much higher levels of LYVE-1 compared to male spleen tissue in mice.¹⁹⁶ Acute Kawasaki cardiomyopathy, a disease marked by increased VEGF-D expression, hyperplastic lymphatics, and myocardial edema is more prevalent in males.^197−199 Even slight decreases in cardiac lymphatic drainage may result in myocardial edema or prolonged/exacerbated inflammation and fibrosis leading to decreases in cardiac pumping function.^190,191 Sex-dependent differences in cardiac lymphatics may be the key to understanding previously unexplained cardiovascular risk differences between the sexes.

Men are more susceptible to atherosclerosis at an earlier age compared to women.²⁰⁰ Perivascular lymphatics in the adventitia of arteries may participate in reverse cholesterol transport from the artery wall affecting atherosclerotic plaques.^201,202 Estrogen may promote an antiatherosclerotic phenotype in macrophages.²⁰⁰ Generally, estrogen is thought to promote vasodilation and dampen oxidative stress in plaques, while androgens may promote endothelial cell apoptosis, formation of foam cells, and overall induce overall expression of atherogenic genes.^203−206 Additionally, within the gut, lymphatics play a vital a role in absorbing lipids, and therefore affect overall fat deposition.¹¹ In general, stagnation of lymph near arterial vessel walls may contribute to atherogenic plaque deposition.^207,208 The current knowledge in the field fails to completely explain how sex differences contribute to atherosclerosis pathogenesis and progression, so further studies are certainly warranted.

Understudied Systems

Female Reproduction

The lymphatic system is also an essential component of the female reproductive system, both in normal physiology and disease. Lymphangiogenesis occurs in ovaries and the uterus.^209−212 Endometriosis, an often painful and devastating disease affecting 10–15% of reproductive-aged women, is characterized by endometrium-like tissue growing outside the uterus.^213,214 Endometriosis is fueled by estrogenic signaling via ERα and ERβ, but estrogen inhibition can be ineffective and have negative side-effects.²¹⁵ The lymphatic spread theory states that the lymphatic system may be responsible for the spread and dissemination of endometrial tissue to the rest of the body.²¹³ A better understanding of the lymphatic system in women, particularly in reproductive organs, could lead to new and more effective treatments for endometriosis, a disease with no current cure.²¹⁶ Lymphatic factors affect pregnancy and other gynecological processes. For example, mutations in the AM receptor, Calcrl, can result in miscarriages as a result of nonimmune hydrops fetalis (fluid accumulation in the fetal compartment),²¹⁷ while exogenous AM has shown to improve fertility.²¹⁸ AM levels increase during pregnancy and endometriosis, while VEGF-C levels decrease during pregnancy and increase in patients with endometrial cancer.^219−222 Lymphatic factors are important regulators of reproductive processes and disease and warrant further study.

Autoimmune Disease

Autoimmune diseases, found 78% more commonly in women, can be linked to the lymphatic system.²²³ In fact, after cancer and cardiovascular disease, it is the most prevalent disease in women.²²⁴ Without functional lymphatics, the body cannot mount a proper primary immune response.^120,225 Infections may leave long-lasting “scarring” in lymph nodes that manifest as chronic immune dysfunction.²²⁶ In response to an inflammatory assault or infection, women tend to produce significantly more antibodies than men, while men have a more extreme inflammatory response.²²⁷ The accumulation of antibodies in women may help explain the discrepancy in the prevalence of autoimmune disease between men and women, but we know little about how lymphatics affect these discrepancies. Men and women are known to have different cytokine profiles within their lymph.²²⁸ Furthermore, macrophages, important regulators of lymphangiogenesis and the immune response, have many sex dependent differences.^229−231 Macrophages can change with age, believed to correlate with drops in estrogen production in females. Furthermore, macrophages in females have increased expression of IL-4Rα, ERα, and toll-like receptors as well as enhanced phagocytosis. Rheumatoid arthritis, an inflammatory autoimmune disease, affecting nearly 1% of the population is more prevalent in females and is associated with slow decline of lymphatic drainage from chronic inflammation and eventual collapse of draining lymph nodes.^232,233 There remain many areas of study to explore at the intersection between autoimmune diseases and lymphatics.

Conclusions

Lymphatics have important implications in many diseases, yet no FDA-approved drugs exist to target lymphangiogenesis.²³⁴ In phase 1 clinical trials, a drug targeting VEGFR-3 proved safe but ineffective at preventing tumor progression.²³⁵ Therefore, a more complete comprehension of how sex affects lymphatic endothelial cell growth, function and dysfunction in disease states would be invaluable and could lead to new and improved treatments, including pharmacological interventions and better biomarkers. Future studies in both male and female model organisms, cell lines, and patients are necessary to advance the field in exciting new and therapeutically focused directions.

The authors declare no competing financial interest.