Lymphatics in Cardiovascular Physiology

Abstract

The lymphatic vessels play an essential role in maintaining immune and fluid homeostasis and in the transport of dietary lipids. The discovery of lymphatic endothelial cell–specific markers facilitated the visualization and mechanistic analysis of lymphatic vessels over the past two decades. As a result, lymphatic vessels have emerged as a crucial player in the pathogenesis of several cardiovascular diseases, as demonstrated by worsened disease progression caused by perturbations to lymphatic function. In this review, we discuss the major findings on the role of lymphatic vessels in cardiovascular diseases such as hypertension, obesity, atherosclerosis, myocardial infarction, and heart failure.

Cardiovascular diseases (CVDs) are the number one cause of death globally. Central to the development and progression of several CVDs is inflammation and its failure to resolve. Elevated levels of inflammatory cells or markers are predictive of future disease, and experimental models show that targeting immune function alters the course of disease progression (Ruparelia et al. 2017). Increasing evidence over the past decade has implicated lymphatic vessels in the pathophysiology of numerous CVDs including hypertension (Balasubbramanian et al. 2019), atherosclerosis (Lim et al. 2013), and myocardial infarction (MI) (Henri et al. 2016). Lymphatic vessels are a series of blind-ended, unidirectional vessels that play a crucial role in peripheral immune surveillance and fluid homeostasis. The trafficking of infiltrating immune cells out of the tissue interstitium to the draining lymph node is mediated by lymphatic vessels. Immune cells enter initial lymphatic capillaries via button-like junctions, guided by chemokines secreted by lymphatic endothelial cells (LECs), following which they are transported by the pumping action of the collecting lymphatic vessels (Breslin et al. 2018). Inflammation is often accompanied by lymphangiogenesis, the expansion of lymphatic vasculature through the sprouting of existing LECs or the incorporation of lymphatic endothelial progenitor cells usually formed from transdifferentiated macrophages (Kerjaschki et al. 2006; Lee et al. 2010; Hall et al. 2012). This process, now known as inflammation-associated lymphangiogenesis (IAL), has been reported in several chronic inflammatory diseases and CVDs (Kim et al. 2014; Schwager and Detmar 2019). LECs also act as antigen-presenting cells (APCs) themselves, internalizing and presenting antigen to activate T lymphocytes (T cells) (Card et al. 2014). In addition, and relevant to CVDs, lymphatics are involved in maintaining tissue fluid homeostasis, returning roughly 3 liters of extravasated plasma back into circulation each day as lymph. During conditions of volume retention or altered microvascular permeability, a compensatory increase in lymph flow is necessary to restore fluid balance (Wiig and Swartz 2012). Lymphatic dysfunction caused in the course of disease progression could lead to interstitial fluid accumulation or lymphedema (Mortimer and Rockson 2014). In addition to these, lymphatics are also involved in dietary lipid and cholesterol transport (Randolph and Miller 2014). This review aims to provide an overview of the role of lymphatics in commonly manifested CVDs including hypertension, atherosclerosis, obesity, MI, and heart failure.

HYPERTENSION

The maintenance of physiological blood pressure (BP) requires a complex interplay between several systems including the renin-angiotensin-aldosterone system (RAAS), the sympathetic nervous system (SNS), natriuretic peptides, the endothelium, and the immune system. It is owing to this cross talk of numerous factors that it has become challenging to identify a universal and singular cause underlying the pathogenesis of hypertension in most patients. Chronic excess salt intake, overactivation of RAAS and SNS, insufficient vasodilation, and impaired natriuresis by the kidneys, along with genetic factors, could all lead to the onset of hypertension (Harrison et al. 2021). In addition to perturbations affecting hemodynamic and/or volume homeostasis, over the last decade, hypertension has been reported to be mediated in part by inappropriate immune system activation (Lopez Gelston and Mitchell 2017; Rodriguez-Iturbe et al. 2017). Various immune cell subsets, including macrophages (De Ciuceis et al. 2005; Huang et al. 2018), dendritic cells (DCs) (Kirabo et al. 2014; Barbaro et al. 2017; Lu et al. 2020), and T lymphocytes (T cells) (Guzik et al. 2007; Trott et al. 2014; Caillon et al. 2017), participate in the pathogenesis and maintenance of hypertension. Immune cell accumulation occurs in the heart, kidneys, brain, and adventitia of blood vessels, leading to end-organ damage. The prevalence of hypertension is high in patients with autoimmune disease (Shaharir et al. 2015; Taylor and Ryan 2017), and immunosuppression lowers BP in hypertensive subjects (Ferro et al. 2011) and in animal models of hypertension (Rodríguez-Iturbe et al. 2001; Mattson et al. 2006). It is not yet clear what initiates the immune system activation during hypertension; however, oxidation of lipids leads to the formation of isoketals that form adducts to endogenous proteins, thus transforming them into neoantigens. Although the nature of such proteins has not yet been determined, these neoantigens lead to the subsequent activation of adaptive immunity and propagate hypertension in animal models (Kirabo et al. 2014).

Using experimental models of hypertension, our group and others have demonstrated that lymphatic vessel density in the kidney increases during hypertension (Lopez Gelston et al. 2018; Beaini et al. 2019; Balasubbramanian et al. 2020a,b). This lymphangiogenic response closely relates with immune cell accumulation, as spontaneously hypertensive rat (SHR) strains resistant to renal inflammation and injury (SHR-B2) lacked this response. Normotensive Fisher rats that are prone to renal injury with age also demonstrate increased renal lymphangiogenesis compared to young control rats (Kneedler et al. 2017). IAL has also been observed in hearts and skin of rodents during hypertension (Machnik et al. 2009; Yang et al. 2017). Among the myriad of factors regulated during hypertension, it is not clear what signals the lymphatic vessels to grow; however, interstitial salt accumulation has been demonstrated to drive a hypertonicity-driven response in macrophages to secrete vascular endothelial growth factor C (VEGF-C), which then induces lymphangiogenesis (Machnik et al. 2009; Beaini et al. 2019). Indeed, depleting macrophages prevents lymphatic expansion in the skin and raises BP. Additionally, angiotensin II has also been reported to directly induce the proliferation of LECs in vitro, and treatment with losartan, an angiotensin II receptor blocker, reduced lymphangiogenesis in vivo (Lin et al. 2020). Since immune cell accumulation has been observed prior to the onset of hypertension in SHRs (Rodríguez-Iturbe et al. 2004), it is likely that the lymphangiogenesis observed in these models is a consequence of and a compensatory mechanism to reduce immune cell overload. Indeed, genetically augmenting capillary lymphatic density specifically in the kidneys reduced renal proinflammatory immune cell accumulation and prevented the onset of hypertension (Lopez Gelston et al. 2018; Balasubbramanian et al. 2020a,b) representing a safer alternative to immunosuppression. Lymphatics not only reduce immune cell burden, but also alter activation and polarization states of immune cells by the secretion of cytokines and chemokines (Magnusson et al. 2008; Cousin et al. 2021); however, this remains to be demonstrated in the context of hypertension. Similar to our studies, cardiac (and possibly renal) lymphangiogenesis driven by intravenous retrovirus-induced overexpression of VEGF-C_c152s (ΔNΔC/VEGF-C/Cys152Ser, a mutant form of VEGF-C capable of selectively activating VEGF receptor 3 [VEGFR-3]), lowers BP, fibrosis, and inflammation in the heart in rats fed a high-salt diet (HSD) (Yang et al. 2017). In addition to transport of immune cells, lymphatics also participate in sodium homeostasis. Dermal interstitial sodium accumulation that occurs during chronic excess sodium intake requires dermal lymphatics for clearance (Machnik et al. 2009). Expansion of renal lymphatics, while inducing no changes at baseline, increases natriuresis during conditions of salt retention and leads to lowering of BP (Balasubbramanian et al. 2020a). Parallel to capillary lymphatic expansion, collecting vessel function is also altered during hypertension. Following 4 wk of HSD, afferent iliac collecting lymphatic vessels in rats demonstrate an increase in passive diameter, amplitude, and pumping frequency, resulting in an increase in ejection fraction (Mizuno et al. 2015a). It could be presumed that this adaptation helps maintain fluid balance in the face of volume expansion caused by HSD. However, the same group demonstrated that in mice, afferent iliac lymphatic vessels adapt differently to HSD, exhibiting a reduction in amplitude and stroke volume. Moreover, efferent lymphatic vessels from these mice seemed to be resistant to the effects of HSD (Mizuno et al. 2015b). It is possible that this discrepancy is due to strain differences or even due to the lymphatic bed studied since, in the inguinal lymphatic vessels in mice and rats, HSD increases contraction frequency in both afferent and efferent vessels (Kwon et al. 2012). It is also possible that the longer salt-loading period in the study by Mizuno et al. led to an overwhelming collecting of lymphatic function, resulting in reverse adaptations to prolonged increased lymph flow (Gashev et al. 2002; Scallan et al. 2012). Endothelial dysfunction, characterized by increased vessel tone often due to impaired nitric oxide signaling, is a hallmark feature of resistance arterial vessels in hypertension (Brandes 2014). Similar impairment of LEC nitric oxide–mediated relaxation response was also observed in the thoracic ducts of SHRs secondary to an increase in BP (Mukohda et al. 2020). Future studies focusing on mechanisms of collecting lymphatic vessel functional impairment during hypertension and translatable approaches to augment organ-specific lymphatic density might prove beneficial in the treatment of hypertension.

OBESITY

Obesity is a major risk factor for the development of CVD. High body mass index (BMI) contributed to 4 million deaths worldwide in 2015, more than two-thirds of which were caused by CVD (Powell-Wiley et al. 2021). The long-standing definition of obesity is “abnormal or excessive fat accumulation that may impair health.” Although BMI has been classically used to define obesity, it is now recognized that discriminating between fat and lean body mass and taking into account regional fat distribution and different patterns of body composition provide a more comprehensive evaluation of associated risks (Koster et al. 2015; Koliaki et al. 2019). Adipose tissue (AT), once regarded as a long-term energy storage organ, is comprised of not only adipocytes, but also fibroblasts, macrophages, lymphocytes, and vascular cells. Various depots of AT exist within the body including visceral, subcutaneous, and organ-specific, each with a unique metabolomic and secretome profile (Hocking et al. 2010). With the onset of obesity, the cellular compositions of these fat depots change along with the functional phenotypes of the various cell types. An imbalance in secreted adipokines has been demonstrated to have various effects including promoting inflammation, monocyte recruitment, and insulin resistance (Ouchi et al. 2011; Huh et al. 2014; Koliaki et al. 2019). Expansion of AT also leads to cardiovascular adaptations to meet the increased demands of the AT. This includes increased blood volume, heart rate, and cardiac output, eventually leading to left ventricular adaptation and remodeling (Lavie and Messerli 1986). Despite early observations of the role of lymphatic lacteals in lipid transport (Lord 1968), observations surrounding the link between obesity and lymphatic dysfunction are relatively new, nevertheless convincing.

A bidirectional relation has perhaps been best demonstrated between obesity and lymphatic dysfunction. Observations from patients and experimental studies provide evidence that obesity negatively affects lymphatic function and, conversely, that primary lymphatic dysfunction could result in changes in fat accumulation and obesity.

Obesity Leads to Lymphatic Dysfunction

Animal models of obesity, induced by a long-term high-fat diet (HFD), have demonstrated that obesity leads to decreased lymphatic vessel density in the periphery and lymph nodes, leaky dilated capillaries, and reduced frequency of pumping of collecting lymphatic vessels (Savetsky et al. 2014; García Nores et al. 2016; Nitti et al. 2016; Torrisi et al. 2016). Dermal LECs isolated from obese mice display reduced expression of VEGFR-3. The observed lymphatic dysfunction in obese mice often coexists with impaired DC migration and altered local inflammatory responses (Savetsky et al. 2014). Obesity is characterized by chronic low-grade inflammation and secretion of proinflammatory cytokines (Ouchi et al. 2011). This cytokine milieu presumably causes the onset of lymphatic dysfunction during obesity since inhibition of inflammation with topical tacrolimus, an immunosuppressive agent that inhibits T-cell proliferation, restores obesity-induced decreases in lymphatic vessel density and hindlimb lymphatic transport capacity, while treatment with 1400 W, a selective inhibitor of inducible nitric oxide synthase (iNOS), restores lymphatic transport of DCs (Torrisi et al. 2016). In addition, CD4⁺ T-cell-deficient mice fed an HFD are protected from lymphatic dysfunction and perilymphatic accumulation of immune cells despite demonstrating weight gain, suggesting that T cells are necessary for inflammation-induced defects in lymphatic function during obesity (Torrisi et al. 2016). Similarly, Rag1^–/– mice deficient in T and B cells exhibit preserved lymphatic-mediated DC transport during obesity (Weitman et al. 2013). Increasing evidence points to the existence of a subgroup of patients who are obese by definition (high BMI) but lack the metabolic complications of obesity. These “metabolically healthy” obese patients have improved cardiac systolic and diastolic function compared to “metabolically unhealthy” individuals (Dobson et al. 2016). In 2016, García Nores et al. attempted to distinguish the effects of obesity versus chronic HFD exposure on lymphatic function using obesity-prone (C57BL/6J strain) and obesity-resistant mice (BALB/cJ and MSTN^ln strains) (García Nores et al. 2016). As expected, obesity-prone mice demonstrate marked increases in weight gain, AT deposition, increases in fasting serum insulin levels, and glucose intolerance upon HFD, whereas these parameters were not as pronounced in the obesity-resistant mice. Lymphatic vessel density, collecting vessel transport capacity, and DC migration were reduced in the “metabolically unhealthy” obesity-prone mice but not in obesity-resistant mice. Local inflammatory responses were also altered in obesity-prone mice, suggesting that the inflammatory responses are linked to the lymphatic dysfunction. Similarly, studies investigating the effects of metabolic syndrome have found that chronic high fructose diet exposure impairs collecting lymphatic vessel function (Zawieja et al. 2012, 2016). Ex vivo analysis of collecting lymphatics in rats with metabolic syndrome demonstrated impaired pumping, despite short-term increases in pumping frequency and flow upon exposure to low-density lipoprotein (LDL) (Wang et al. 2009). These rats also demonstrated alterations in macrophage polarization in AT and along mesenteric vessels (Zawieja et al. 2016). Given the pathogenic role played by reduced lymphatic density in obesity, inducing adipose lymphangiogenesis would be expected to reduce inflammation and restore metabolic health. Using a mouse model of VEGF-D overexpression-induced de novo AT-specific lymphangiogenesis (Lammoglia et al. 2016), Chakraborty and colleagues demonstrated that when mice are fed an HFD, an expanded adipose lymphatic network improves glucose clearance, reduces systemic insulin resistance, and reduces adipose immune cell accumulation despite no difference in weight gain (Chakraborty et al. 2019). While VEGF-C and VEGF-D have been demonstrated to act as chemoattractants for macrophages (Chakraborty et al. 2020) and blocking VEGF-C/VEGF-D reduces AT inflammation (Karaman et al. 2015, 2016), in the context of an ongoing inflammatory disease such as obesity, increasing adipose-specific lymphatic vessels may help combat inflammation and improve metabolic function.

Lymphatic Dysfunction Leads to Obesity

Ample evidence also exists that lymphatic dysfunction could lead to the onset of obesity. Lymphedema patients in late stages of the disease exhibit progressive AT accumulation (Brorson et al. 2006, 2009; Tashiro et al. 2016). Several studies using animal lymphatic injury models demonstrate that lipid deposition and AT accumulation occurs following lymphatic injury (Rutkowski et al. 2010; Zampell et al. 2012; Tanaka et al. 2016; Tashiro et al. 2016). Lymphatic injury leads to stasis of lymph that contains free fatty acids including oleic acid, α-linoleic acid, and palmitic acid (Escobedo et al. 2016). Exposure to lymph leads to increased expression of adipocyte differentiation genes and promotes adipocyte maturation. That primary congenital lymphatic defects could lead to the development of obesity was demonstrated by observations in mice with haploinsufficiency of Prox1, the master regulator of lymphatic fate specification, which developed adult-onset obesity (Harvey et al. 2005). Prox1^–/– and Prox1^+/– embryos display edema and while most mice die soon after birth, the surviving mice do resolve this edematous state. However, at the adult stage, Prox1^+/– mice display lymphatic patterning defects and have leaky lymphatics. These morphological alterations are evident before the onset of obesity, and even on normal diet, Prox1^+/– mice exhibit an increased weight compared to their wild-type (WT) counterparts. These mice also develop insulin resistance and intra-abdominal and subcutaneous fat accumulation, with no differences in food intake compared to WT mice. Similar observations were made in mice lacking a single copy of VEGFR-3 (Chy mice), which demonstrate an edematous hind paw and AT accumulation (Dellinger et al. 2007; Rutkowski et al. 2010). Importantly, restoring lymphatic function in Prox1^–/– mice by restoring LEC-specific Prox1 levels prevents the onset of obesity in these mice (Escobedo et al. 2016), suggesting that defective lymphatic vasculature increases the predisposition to obesity. Within the intestinal villi, dietary lipids are absorbed as chylomicrons that enter lymphatic lacteals through button-like junctions (Dixon 2010; Cifarelli and Eichmann 2019). These button-like junctions allow for fluid and macromolecule entry in the capillaries as opposed to continuous zipper-like junctions in the collecting vessels (Baluk et al. 2007). However, these junctions display plasticity and are regulated during development and inflammation (Yao et al. 2012; Zhang et al. 2020). Zhang et al. demonstrated that lacteal button-like junctions could be transformed into zipper-like junctions by VEGF-A signaling. By deletion of endothelium-specific VEGFR-1 and Neuropilin-1, a coreceptor for VEGFR-1 and VEGFR-2, the bioavailability of VEGF-A increases, which then signals through VEGFR-2 on both the blood and lymphatic endothelium. While this signaling increases permeability of blood vessels, it induces zippering of lacteals, thus reducing chylomicron uptake and preventing the development of obesity in these mice. Hence, targeting junctional proteins in lacteals may represent a novel approach to reduce dietary lipid uptake and fat accumulation (Zhang et al. 2018).

ATHEROSCLEROSIS

Atherosclerosis manifests as a fatty/fibrous deposition in the intima, the innermost layer of the blood vessel. Formation of atherosclerotic plaques begins with the deposition of cholesterol crystals, usually at sites of endothelial injury and dysfunction caused by turbulent blood flow. With time, the deposition progresses and the plaque grows, encroaching the lumen and obstructing blood flow. Plaques that do not impede blood flow could still rupture and initiate thrombosis that occludes the lumen and blood flow (Libby et al. 2019). LDL particles accumulated in the plaque are susceptible to oxidation by reactive oxygen species (ROS), thus attracting inflammatory cells. Oxidized LDL (oxLDL) also stimulates the expression of adhesion molecules and the secretion of chemokines by endothelial cells. Macrophages that recognize and internalize oxLDL differentiate into foam cells and act as danger-associated molecular patterns (DAMPs), further aggravating inflammation (Moore et al. 2013). Reverse cholesterol transport (RCT) is the process by which cholesterol is removed from peripheral tissues and transported back to the liver for excretion. The major lipoprotein involved in mediating RCT is high-density lipoprotein (HDL). Apolipoprotein A-I (ApoA-I), the major protein component of HDL, is synthesized in the liver, enters into the bloodstream, and interacts with the receptor ATP-binding cassette, subfamily A, member 1 (ABCA1) present on various cells. ABCA1 mediates cholesterol efflux from cells including macrophages. Cholesterol-loaded HDL also interacts with Scavenger receptor class B member 1 (SR-B1) and ATP-binding cassette, subfamily G, member 1 (ABCG1) to incorporate more cholesterol resulting in the formation of a mature HDL particle. Studies have shown that efflux of cholesterol from macrophage foam cells in atherosclerotic plaques, a process commonly termed as macrophage RCT, aids the regression of plaques (Cuchel and Rader 2006), and an inverse relationship exists between cholesterol efflux and atherosclerosis severity (Cuchel and Rader 2006; Rader et al. 2009; Marques et al. 2018).

It was long suspected that lymphatics play a role in RCT, since interstitial fluid and lymph were demonstrated to contain HDL and apo A-I (Sloop et al. 1987). Peripheral lymph lipoproteins differ from plasma lipoproteins in composition and concentration. Total cholesterol concentration in lymph exceeds that of what could be explained by trans-endothelial transfer of HDL from the plasma alone (Nanjee et al. 2001), suggesting that lymph cholesterol is derived from cholesterol efflux from cells. In 2013, a definitive role for lymphatics in the physiological process of RCT was demonstrated (Lim et al. 2013; Martel et al. 2013). Injection of fluorescent cholesterol-loaded macrophages into the footpad of mice enabled tracking of the transport route and temporal dynamics of interstitial cholesterol until excretion. Following injection into the interstitium of the footpad, the first site in which fluorescence is detected is the draining lymph node (at 4 h), followed by the plasma (12 h), liver (48 h), and feces (72 h). Lim et al. then surgically removed the afferent lymphatic vessel draining from the footpad to the popliteal lymph node and noticed that blocking lymphatic drainage significantly impaired RCT upon injection of cholesterol. Similarly, Chy mice that lack lymphatic capillaries in the skin also exhibit impaired RCT (Martel et al. 2013). LECs express ABCA1 and SR-B1 receptors, and blocking LEC SR-B1 receptors by preincubation with SR-B1-specific antibody diminishes HDL transport across an LEC monolayer, demonstrating that lymphatic vessels actively transport HDL through SR-B1 receptors (Lim et al. 2013). Given these observations of the participation of lymphatic vessels in RCT, it would be expected that arterial wall lymphatics play a major role in the progression of atherosclerosis. Lymphangiogenesis has been observed in the adventitia of internal carotid arteries, iliac arteries, and aortic segments of atherosclerotic patients compared to healthy controls (Drozdz et al. 2008; Grzegorek et al. 2014; Rademakers et al. 2017). The thickness of arterial intima, a measure of atherosclerotic severity, correlated with the number of adventitial lymphatics. Protein expression of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1, a marker of LECs), VEGF-C, VEGF-D, and CC-chemokine receptor 7 (CCR7, an immune cell receptor for entry into lymphatics) was also higher in atherosclerotic subjects (Grzegorek et al. 2014), implicating VEGF-C/VEGFR-3-mediated lymphatic proliferation; however, the CXC chemokine CXCL12 interacting with its receptor CXCR4 has also been implicated in atherosclerosis-associated lymphangiogenesis (Rademakers et al. 2017). Lymphatic dysfunction is also observed in mice lacking apolipoprotein E (apoE^–/–) and LDL receptor (Ldlr^–/–), the models of choice for atherosclerosis (Vuorio et al. 2014; Milasan et al. 2019). Initial lymphatics are typically dilated in apoE^–/– mice, accompanied by a decrease in collecting lymphatic function as demonstrated by reduced clearance of injected dye and impaired DC transport. Impaired lymphatic drainage of cholesterol from arterial walls could thus increase susceptibility to atherosclerosis. Martel et al. demonstrated this concept by surgically transplanting atherosclerotic aortae from donor apoE^–/– mice to recipient apoE^–/– mice pretreated with VEGFR-3 antibody to block lymphatic growth. The recipient mice exhibited greater retention of cholesterol within the atherosclerotic plaque, demonstrating that clearance of cholesterol by RCT from the arterial walls is mediated by lymphatic vessels (Martel et al. 2013). Similarly, dissection of plaque-draining lymph nodes and lymphatic vessels increased plaque volume and T-cell numbers within the plaque (Rademakers et al. 2017). The Ldlr^–/–, hApoB100^+/+ mice spontaneously develop atherosclerotic lesions at around 4 mo of age, even on a regular diet. In these mice, lymphatic function and lymphatic vessel–mediated DC transport is impaired at 3 mo of age, before the onset of atherosclerotic lesions, suggesting that an underlying lymphatic defect might predispose to the development of atherosclerosis (Milasan et al. 2016).

Importantly, several studies have demonstrated that atherosclerosis progression can be reversed by improving lymphatic function. Treatment of apoE^–/– mice demonstrating atherosclerotic lesions with VEGF-C_c152s improved lymphatic function and lowered cholesterol accumulation (Milasan et al. 2016). Ezetimibe is a well-known drug that lowers LDL cholesterol by targeting intestinal and biliary cholesterol absorption and induces atherosclerotic plaque regression (Bogiatzi and Spence 2012). Surprisingly, ezetimibe treatment improved functional drainage of aortic adventitial lymphatics during atherosclerosis. Indeed, the efficacy of ezetimibe treatment was dependent on a functional lymphatic network as blocking lymphatic draining led to a larger plaque size and increased content of lipids, CD68⁺ macrophages, collagen, and α-smooth muscle actin within the plaques compared to sham-operated animals (Martel et al. 2013; Yeo et al. 2020). Treatment with ezetimibe also reduced the sprouting of initial lymphatics, demonstrating that compensatory lymphangiogenesis subsides during plaque regression. Similarly, treatment with apoA-I has been reported to have beneficial effects in atherosclerosis by mechanisms that are still being investigated (Wacker et al. 2018; Barrett et al. 2019). It was shown that some of these beneficial effects may be mediated by lymphatic-dependent mechanisms. ApoA-I treatment increased lymphatic vessel density in the aortic sinus and reduced lymphatic vessel leakage when compared to control groups with atherosclerosis. However, no differences were noted in visceral fat accumulation or AT thickness in these mice (Milasan et al. 2017). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that binds to and degrades LDL receptor on cells, thus interfering with the clearance of LDL cholesterol from blood. PCSK9^–/– mice have higher LDL receptor levels on LECs and other tissues, have lower lipid profiles, and are resistant to the development of atherosclerosis. Hence, modulating LDL receptor expression on LECs might be an alternate approach to reduce peripheral cholesterol (Milasan et al. 2016).

MYOCARDIAL INFARCTION

MI is the sudden death of cardiomyocytes due to ischemia caused by a primary event such as atherosclerotic plaque rupture in the coronary artery, or ischemia due to increased oxygen demand or decreased supply. Subsequent reperfusion of the infarcted site is characterized by activation of innate and adaptive immune responses, resulting in inflammation and immune cell infiltration, the hallmark of MI and reperfusion injury. In the initial few hours after reperfusion, the infarcted site is infiltrated by neutrophils (Yellon and Hausenloy 2007). This is followed by monocyte/macrophage infiltration, presumably to aid in the clearing of the necrotic cardiomyocytes and in scar formation and remodeling (Nahrendorf et al. 2007; Gentek and Hoeffel 2017). Cardiac debris is phagocytosed by DCs (Anzai et al. 2012) and processed and presented to naive T cells (Hofmann et al. 2012), which then differentiate into effector or regulatory subtypes, overall contributing to inflammation resolution. In addition to inflammation, MI is also characterized by myocardial edema (Dongaonkar et al. 2012). Myocardial interstitial fluid drains through myocardial lymphatics, and the mammalian heart is invested with an abundance of lymphatic capillaries that keep fluid balance in the myocardium in check (Brakenhielm and Alitalo 2019). MI increases the myocardial microvascular permeability possibly due to endothelial injury. However, lymph flow can increase several-fold in response to increased flow, and edema accumulates in the myocardium only when lymph flow cannot increase at a compensatory rate.

Lymphangiogenesis has been observed in human hearts with infective endocarditis and degenerative calcified stenosis in aortic valves where lymphatics were concentrated around calcified focuses (Kholová et al. 2011). In these conditions, lymphatics were associated with areas rich in extracellular matrix, as is observed in several other fibrotic conditions. Transforming growth factor β (TGF-β)-induced VEGF-C signaling has been implicated in fibrosis-associated lymphangiogenesis in animal models (Suzuki et al. 2012; Kinashi et al. 2013); however, evidence of activity of such pathways remains to be demonstrated in MI. In myocarditis, lymphangiogenesis was observed in areas rich in inflammatory infiltrates. Lymphangiogenesis is also observed along the necrotic edge in acute MI and during remodeling of the heart such as in hypertrophic myocardium and epicardium (Kholová et al. 2011). Following the initiation of MI, VEGFR-3 protein levels increase as early as day 4 and by day 7 following MI, there is an increase in superficial lymphatic density as visualized by LYVE-1/Podoplanin staining. Lymphatic vessels were noticeably dilated by day 14 and remained dilated until day 35 following MI (Klotz et al. 2015; Vieira et al. 2018). However, parallel to an increase in capillary lymphatic density, lymphatic collectors are lost in the scar region along with deleterious remodeling of precollectors, which extends into the noninfarcted region. The overall transport capacity of the lymphatic vessels thus decreases, evidenced by insufficient lymphatic-mediated drainage of fluorescent quantum dots injected into the heart following MI (Henri et al. 2016). VEGF-C-driven lymphangiogenesis has been reported to improve cardiac function following MI, decrease myocardial water content (edema), improve ejection fraction, and lower end systolic volume. The expanded lymphatic network also helps reduce overall immune cell load; however, no differences were noted in macrophage polarization or local cytokine milieu. Interestingly, the immune cell clearance was dependent on LYVE-1 expression as Lyve1^–/– mutants failed to clear immune cells following MI. Overexpression of VEGF-C in Lyve1^–/– mice induced lymphangiogenesis, but did not alter immune cell clearance, leading to deterioration of cardiac function and elevated fibrosis (Vieira et al. 2018). Unexpectedly, Houssari et al. found that delivery of soluble VEGFR-3 (acting as a VEGF-C/VEGF-D trap) 7 d before the initiation of MI limited immediate T-cell infiltration following MI and delayed MI-induced cardiac dysfunction, highlighting the double-edged sword nature of IAL (Houssari et al. 2020). This study also reported that CD4⁺ and CD8⁺ T cells that infiltrate the scar suppress lymphangiogenesis, at least in part, by secreting interferon γ (IFN-γ), and that limiting T-cell infiltration limits remodeling of lymphatics. Depleting CD4⁺ and CD8⁺ T cells or neutralizing IFN-γ prevented the rarefaction of lymphatic capillaries and collectors. Thus, the local cytokine milieu during inflammation may dictate the endogenous lymphatic response and hence the progression of disease.

HEART FAILURE

Venous and lymphatic congestion are the hallmark features of congestive heart failure (CHF), which typically manifests as edema in extremities, pulmonary edema, hepatic congestion, and subsequently ascites and renal failure (Verbrugge et al. 2020). Fluid that leaves the intravascular compartment is normally returned to the heart via the venous and lymphatic circulation. However, when the heart fails as a pump, reabsorption of the fluid is gradually impeded, leading to fluid retention in the extremities, characterized as edema. Edema accumulates at a rate limited only by lymph flow and compliance of the interstitial space. Thus, derangements in the formation and transport of lymph bear direct consequences on the pathogenesis of CHF (Fudim et al. 2021; Itkin et al. 2021). Despite the central role of the lymphatics in maintaining fluid balance, the role of lymphatics and its dysregulation in congestion during CHF has been poorly appreciated and understood.

In the 1960s, studies demonstrated the role of lymphatic drainage in patients with CHF. These patients exhibited classical symptoms of CHF including marked dyspnea, orthopnea, ascites, and peripheral edema. In the absence of benefits from diuretics, low-salt diet, and mechanical removal of intracavitary or peripheral fluid, cannulation of the thoracic duct largely increased lymph flow and diminished symptoms of edema. When lymph flow was returned to normal rate, edema reappeared (Dumont et al. 1963; Witte et al. 1969). A recent study of patients with CHF with preserved ejection fraction (HFpEF) reported that rarefaction of lymphatic vessels is observed in the forearm and calf of these patients, along with a dilation of the existing vessels, reflecting their adaptation to increased fluid load. Despite rarefaction of capillaries leading to reduced surface area for fluid exchange, fluid accumulates in the tissue interstitium at much smaller pressures than healthy controls, reflecting the reduced ability of lymphatic vessels to drain interstitial fluid (Rossitto et al. 2020). Another study has reported that elevated VEGF-D levels were noted in patients with elevated left heart–filling pressures, typically found in states of chronic pulmonary venous congestion. These patients also had a longer duration of CHF diagnosis, and although lymphatic density was not evaluated in this study, it is likely that a lymphatic compensatory mechanism was active in these patients (Houston et al. 2019). Treatment with LCZ696, an angiotensin-neprilysin inhibitor, improves cardiac function and reduces fibrosis in models of CHF. In the transverse-aortic constriction model of CHF, LCZ696 reduced the endogenous lymphangiogenic response and the accumulation of macrophages around lymphatic vessels (Ge et al. 2020). More studies evaluating lymphatic function in animal models of CHF might lead to better mechanical insights and to approaches to improve lymphatic function and aid edema resolution.

CONCLUDING REMARKS

In the face of the overwhelming evidence discussed above, it is obvious that lymphatics are involved in the pathogenesis of CVDs and that modulating lymphatic function could aid in the resolution of CVDs (Fig. 1). In most animal models discussed, VEGF-C therapy seems to induce capillary lymphangiogenesis and ameliorate disease symptoms. The mode of delivery of VEGF-C to achieve organ-specific lymphatic expansion needs to be investigated since any adverse effects of long-term and nontargeted lymphangiogenesis remain to be understood. It must also be noted that efficient lymphatic drainage depends on the coordinated functioning of capillaries and collecting lymphatic vessels, and pharmacological approaches to improve lymphatic pumping might lead to novel approaches for the treatment of hypertension, obesity, atherosclerosis, MI, and CHF.

Figure 1.

Overview of the role of lymphatic vessels in cardiovascular diseases. Increased capillary lymphangiogenesis and/or improved collecting vessel transport enhances lymphatic function. This leads to increased dermal interstitial sodium (Na⁺) clearance, reduced renal immune cell accumulation, and increased natriuresis, all of which has the effect of lowering blood pressure during hypertension. Following myocardial infarction, improved lymphatic function reduces myocardial immune cell accumulation, edema, fibrosis, and improves systolic function. Lymphangiogenesis in the arterial wall increases reverse cholesterol transport and reduces immune cell, collagen, and smooth muscle actin content in atherosclerotic plaques. Improved lymphatic collecting vessel function reduces leakage of fatty acid–containing lymph and adipose accumulation during obesity. (Created with Biorender.com.)