Promoting Lymphangiogenesis and Lymphatic Growth and Remodeling to Treat Cardiovascular and Metabolic Diseases

Abstract

Lymphatic vessels are low-pressure, blind-ended tubular structures that play a crucial role in the maintenance of tissue fluid homeostasis, immune cell trafficking, and dietary lipid uptake and transport. Emerging research has indicated that the promotion of lymphatic vascular growth, remodeling, and function can reduce inflammation and diminish disease severity in several pathophysiologic conditions. In particular, recent groundbreaking studies have shown that lymphangiogenesis, which describes the formation of new lymphatic vessels from the existing lymphatic vasculature, can be beneficial for the alleviation and resolution of metabolic and cardiovascular diseases. Therefore, promoting lymphangiogenesis represents a promising therapeutic approach. This brief review summarizes the most recent findings related to the modulation of lymphatic function to treat metabolic and cardiovascular diseases such as obesity, myocardial infarction, atherosclerosis, and hypertension. We also discuss experimental and therapeutic approaches to enforce lymphatic growth and remodeling as well as efforts to define the molecular and cellular mechanisms underlying these processes.

Graphical Abstract

INTRODUCTION

The lymphatic system is composed of a network of thin-walled initial lymphatics, pre-collecting and collecting vessels, and nodes that run parallel to the blood circulatory system¹. Initial lymphatics are formed by a single layer of lymphatic endothelial cells (LECs) that lack a continuous basement membrane and remain tethered to the interstitium through anchoring filaments. As a result, initial lymphatics are characterized by discontinuous button-like junctions at LEC borders, making them highly permeable and capable of absorbing excess interstitial fluid and macromolecules through passive drainage¹. The fluid and macromolecules that are taken up by the initial lymphatics are collectively referred to as ‘lymph,’ which drains into pre-collecting lymphatic vessels and collecting lymphatic vessels². The pre-collecting and collecting lymphatic vessels are composed of LECs that are tightly connected by zipper-like junctions, unlike the initial lymphatics, and covered with smooth muscle cells and a basement membrane to prevent the leakage of lymph^2-4. Collecting lymphatic vessels also contain intraluminal valves that regulate the unidirectional flow of lymph^{5, 6}. The lymph carried by the collecting lymphatics are transported into the larger thoracic duct and the right lymphatic duct, which eventually drain into the blood vascular system through the subclavian veins^7-10.

Role of Prox1 and VEGF-C/D in Lymphatic Development

Over past few decades, our understanding of lymphatic development and function has greatly improved^{1, 11, 12}. For example, the transcription factor Prospero homeobox 1 (Prox1) has been shown to be one of the key regulators of lymphatic development and function^{13, 14}. It is now well-documented that Prox1 is a master regulator of LEC identity and specification¹⁵. In mice, Prox1 is first expressed around E9.5 in a subgroup of endothelial cells in the cardinal vein¹³. These Prox1 expressing lymphatic progenitor cells commit to a LEC fate, bud off from the cardinal vein, and migrate to form lymph sacs^{13, 16}.

Vascular endothelial growth factor C (VEGF-C) is a major lymphangiogenic factor and harbors multiple lymphangiogenic properties such as promoting LEC proliferation, migration, and tube formation¹⁷. VEGF-D is the second member of the lymphangiogenic growth factor family^{17, 18}. Both VEGF-C and VEGF-D are secreted glycoproteins that can bind and activate VEGF receptors (VEGFR2 and VEGFR3) on the blood and lymphatic endothelium to initiate signaling downstream of VEGFRs^{17, 18}. They are also prominent mitogens for LECs and, as a result, promote the growth and remodeling of blood and lymphatic vessels. Despite the fact that VEGF-D shares many similarities with VEGF-C, VEGF-D possesses a higher binding affinity for VEGFR3 than VEGF-C^{17, 18}. Budding LEC migration responds to a gradient of VEGF-C in the surrounding mesenchyme¹⁹. As previously mentioned, the major receptor for VEGF-C, VEGFR3 is highly expressed in LECs and its activity is required for LEC migration and proliferation¹⁹. Importantly, Prox1 activity is required for lymphatic vasculature maintenance and function throughout life as expression of Prox1 is required to maintain LEC identity, even in adult mice, where the extensive lymphatic vasculature appears to be quiescent in physiological conditions¹⁵. Furthermore, a Prox1-VEGFR3 feedback loop during early lymphatic development is required to intricately regulate the budding of LEC progenitors and to maintain LEC fate²⁰.

The murine lymphatic system further develops from these lymph sacs to form the mature lymphatic vasculature that coordinates specific functions in different organs¹. Many genes and signaling pathways that governing the early steps of lymphatic development have been identified, including Sox18²¹, Ccbe1²², Fat4²³, Adamts3²⁴, Calcrl²⁵, podoplanin²⁶, and Rasip1²⁷. Importantly, a recent study also suggests that mitochondrial function and metabolism regulates Prox1 expression and LEC fate specification^{28, 29}. Although many studies dispute the single venous origin of LECs during mammalian development and alternative sources of LECs from different organ beds have been reported^30-33, additional studies are needed to determine whether non-venous derived LECs play functional roles during development. Nevertheless, it is generally accepted that the majority of mammalian LECs are venous-derived.

Role of Lymphatic System in Physiological and Pathological Conditions

Under physiological conditions, most lymphatic vessels in adults are mature and quiescent, except for in the intestinal lacteals, where constant VEGF-C activity is required to maintain lacteal lymphatic development and function³⁴, and the reproductive organs during ovarian cycles and pregnancy³⁵. In adult lymphatic vessels, the quiescent state can be disrupted by pathological conditions such as inflammation and tumor growth, both of which promote lymphangiogenesis and lymphatic vessel remodeling^{36, 37}. At the same time, several types of cancer cells utilize the lymphatic vasculature to spread from one lymph node to another^37-39. Cancer cells also produce vascular endothelial growth factor-C (VEGF-C) or VEGF-D that induce lymphangiogenesis to facilitate tumor metastasis^37-39. In the context of organ transplantation, increased lymphangiogenesis provokes immune system reactivation in the lymph nodes, which is a major cause of rejection of transplanted organs^{40, 41}. Consequently, anti-lymphangiogenic therapies have been used to prevent and treat cancer metastasis, inflammation, and organ transplantation by suppressing immune reactivation^{36, 41, 42}.

Lymphatic vessels also exert beneficial effects by enhancing the clearance of excessive interstitial fluids and pro-inflammatory mediators along with immune cells⁴³. In view of that, the development of pro-lymphangiogenic therapeutic approaches need to be robustly investigated following detailed characterization of the underlying molecular mechanisms and downstream signaling events. A number of recent studies suggest the therapeutic potential for inducing lymphangiogenesis in various pathological conditions, including cardiovascular and metabolic diseases^{1, 12, 44}. Unfortunately, the causal stimulators of adult lymphangiogenesis remain largely undiscovered and the therapeutic effect of promoting lymphangiogenesis is untested.

Although some studies contend there is no role for injury-induced endogenous lymphangiogenesis in cardiac repair^{45, 46}, other reports indicate that increased lymphangiogenesis can slow or reverse the progression of pathology by facilitating immune cell clearance and fluid transport in a number of cardiac injury models including mice^47-49, rats⁵⁰ and zebrafish^{51, 52}.Intriguingly, a recent study by one of the authors and other investigators has shown the lymphatics actively secrete paracrine factors (i.e., lymphoangiocrine) under pathophysiological conditions in an organ-specific manner⁵³. This study illuminates the processes by which the lymphatics promote cardiac growth, repair, and protection from infarction in mice. For the remainder of this brief review, we provide an overview of this and other studies that investigated the therapeutic potential of enhancing lymphangiogenesis for metabolic disease, hypertension, atherosclerosis, and myocardial infraction (MI).

Enhancing Lymphangiogenesis to Treat Metabolic and Cardiovascular Diseases

Obesity and Diabetes

Obesity is an epidemic-level metabolic syndrome characterized by deficiencies in lymphatic growth and function^54-59. Diet-induced obesity impairs lymphangiogenesis, which is associated with decreased dermal and subcutaneous lymphatic vessel density⁵⁶. Thus, obesity also affects lymphatic transport. At the cellular and molecular levels, high-fat diet-induced obesity resulted in peri-lymphatic inflammation and decreased expression of VEFGR3, which is the critical lymphangiogenesis receptor on LECs. LECs with reduced VEGFR3 expression and signaling are pro-apoptotic⁵⁶. Increased apoptosis of LECs destabilized lymphatic vessels and caused impaired lymphatic function⁵⁶. Impaired lymphangiogenesis is also observed in mouse models of metabolic diseases such as diabetes⁶⁰.

A direct link between obesity and lymphatic defects was initially observed in a mouse model with lymphatic defects⁶¹. Specifically, Prox1 heterozygous mice were shown to develop adult on-set obesity⁶¹. These mice exhibited pronounced fat accumulation with leaky lymphatic vessels. Interestingly, leaky chyle from Prox1 heterozygous mice induced adipocyte hypertrophy and adipogenesis⁶¹. In addition, Chy mice, which lack dermal initial lymphatics and exhibit a lymphedematous phenotype due to inactivating mutations in VEGFR3, was reported to exhibit increased collagen and lipid accumulation in skin^{62, 63}. Notably, the restoration of Prox1 dosage in LECs rescued the obese phenotypes in Prox1 heterozygous mice⁶⁴. Together, these studies provided direct evidence that lymphatic defects lead to obesity. For that reason, the modulation of Prox1 and VEGFR3 activity may have therapeutic potential for obesity as well as metabolic diseases in general.

Consequently, our laboratories have been studying the regulation of VEGFR3 receptor activity in the lymphatic vasculature. We have reported that enhancing VEGFR3 expression and signaling by depleting clathrin-dependent endocytic adaptor proteins called epsins, modulated VEGFR3 levels in isolated LECs and promoted lymphangiogenesis in development and under pathological conditions⁶⁵. We also found genetic deletion of epsins 1 and 2 augmented the flow of lymph in type 2 diabetic mice⁶⁶. Therefore, the depletion of epsins in LECs increased VEGFR3 protein levels by reducing VEGFR3 endocytosis and degradation, resulting in enhanced VEGF-C signaling. When we utilized a skin biopsy wound-healing model and surgically-induced tail lymphedema model, we observed enhanced wound-healing and resolution of tail lymphedema in mice⁶⁶.

Interestingly, an elegant study using transgenic mice with doxycycline-inducible expression of murine VEGF-D in adipose tissues showed that VEGF-D overexpression in adipocytes increased initial lymphatic density, as demonstrated by Lyve1 and Prox1 immunostaining, with a concomitant reduction in local immune cell accumulation and improved systemic metabolic responsiveness; thereby, reducing insulin resistance and increasing insulin sensitivity in HFD-induced obesity in mice⁶⁷. Because there are few lymphatic vessels in the white adipose tissue of mice, de novo lymphangiogenesis enhances insulin sensitivity. Even though the body weights were similar between VEGF-D overexpressing mice and their littermate controls, enhancing lymphatic vessel growth in adipose tissue resulted in increased insulin sensitivity and reduced insulin resistance⁶⁷. This study suggested a functional role for lymphatic vessels in glycerol clearance and removal of infiltrating immune cells to improve metabolism in obese mice.

Atherosclerosis

Atherosclerosis is known to be a vascular inflammatory disease and the major cause of heart attacks and strokes⁶⁸. Atherosclerosis begins with an accumulation of oxidized low-density lipoprotein (oxLDL) in the subendothelial space, which results in endothelial cell activation and macrophage recruitment for oxLDL engulfment. These lipid-laden macrophages become foam cells, produce inflammatory cytokines, and contribute to the formation of atherosclerotic plaques⁶⁹. These plaques consist of fat, cholesterol, and immune cells that reside within the walls of arteries⁷⁰. With increased foam cell accumulation and chronic inflammatory stimulation, these plaques become vulnerable to rupture, which can result in thrombosis and the occlusion of blood flow^{71, 72}. Various strategies have been proposed to treat atherosclerosis, such as minimizing the inflammatory response or reducing plaque burden⁷⁰. The mobilization of cholesterol transport from the blood vessel may also provide a possible solution to alleviate pathological progression of atherosclerosis.

In recent years, a growing body of literature has shown that in mice the lymphatics in the aortic wall are a major route for the transport of cholesterol into the bloodstream—a process referred to as reverse cholesterol transport (RCT). These publications suggest that the lymphatics could represent another means to treat atherosclerosis^{73, 74}. One study showed that damage of lymphatic function by surgical ablation of the lymphatic collecting vessels promoted adventitial inflammation, impaired RCT, and encouraged the development of atherosclerotic plaques in hypercholesterolemic mice⁷³. In addition, the prevention of lymphatic growth using VEGFR3 blocking antibodies or by using Chy mice, which lack functional draining lymphatics due to selective inactivating mutations in Flt4 (VEGFR3), and insufficient coverage of the collecting lymphatic vessels with vascular smooth cells, caused impaired lymphatic function and reduced RCT^{74, 75}. Moreover, inhibition of VEGFR3 signaling or silencing of CXCL12, a lymphangiogenic factor that stimulates lymphatic growth and function, also aggravated atherosclerotic plaque formation by blunting initial lymphatic vessel expansion in the adventitia. The consequence was augmented T cell accumulation that presumably exacerbated inflammation in the atherosclerotic plaque⁷⁶. By measuring aortic lymphatic transport, a recent study showed that lymphatic drainage is largely impaired during atherosclerotic progression and the disruption of aortic lymphatic flow by lymphatic ligation promoted atherosclerotic plaque formation⁷⁷. Taken together, a beneficial role for the peri-adventitial lymphatics to limit cholesterol accumulation and chronic plaque inflammation during pathologic progression of atherosclerosis is evident.

In support of this idea, the restoration of lymphangiogenesis using VEGF-C or VEGF-C152S, a mutant form of VEGF-C that specifically activates VEGFR3 signaling and elevates lymphangiogenesis in mice, can improve cholesterol clearance and RCT transport^{73, 78}. Interestingly, a more recent study reported that arterial delivery of VEGF-C antibodies to pre-existing lesions reversed lipid accumulation, reduced intimal cell death, and stabilized atherosclerotic lesions⁷⁹. These findings indicate that the application of an antibody-based delivery strategy was a promising therapy for the treatment of atherosclerosis. Despite these encouraging findings, critical questions remain to be answered regarding whether induction of local lymphatic growth and function can reduce fat deposition or accelerate lipid removal to lessen tissue inflammation to protect against the progression of atherosclerosis.

Myocardial Infarction

Unlike the collecting lymphatics in distal sites of the body, the cardiac lymphatics are dependent on myocardial contraction to transport lymph from the endocardium to the epicardium. This lymph subsequently drains into the thoracic duct via periaortic and paratracheal mediastinal lymph nodes through connected subepicardial lymphatic trunks^80-82. Cardiovascular pathologies such as pressure overload, myocardial infarction, pulmonary hypertension, and heart failure induce cardiac contractile impairment and microvascular fluid flow disruption, resulting in myocardial edema, inflammation and cardiac fibrosis⁸¹.

Accumulating evidence shows that elevated VEGF-C and VEGF-D during the early stage of myocardial infarction increased endogenous lymphangiogenesis within the infarct zone^{48, 49, 83, 84}. Increased lymphangiogenesis following myocardial infarction is associated with elevated immune cell infiltration, attracted by pro-apoptotic signals released from damaged cells, cell debris, and cytokines from neighboring cells⁴⁸. However, it is debatable whether endogenous lymphangiogenesis is important for the improvement of cardiac function in the early phase of post-infarction. One research group has reported that genetic blockade of lymphangiogenesis through ablation of VEGFR3 signaling using mice deficient in VEGF-C and VEGF-D or VEGFR3 does not impair cardiac function in mice two weeks following MI⁴⁵. While mice with LEC-specific loss of the endothelial adhesion protein VE-cadherin cause lymphatic vessel regression in the injured hearts, the cardiac function in these mutant mice was surprisingly well-preserved⁴⁶. However, the latter mice showed increased cardiac infarct size and fibrosis after MI⁴⁶, suggesting that an impaired lymphatic vasculature may impact long-term heart function following MI and worsen heart failure.

While the above studies show that loss of, or reductions in, the cardiac lymphatics did not impact cardiac functional recovery after initial injury^{45, 46}, another study suggested that the delivery of soluble VEGFR3 to the infarcted heart improves cardiac function in both mice and rats; however, this effect was attributed to the acute suppression of T-cell infiltration⁸⁵. Nevertheless, it is entirely plausible that the maintenance of proper lymphatic function through lymphangiogenesis in the infarcted heart is necessary in the long-term by ensuring fluid drainage in addition to transporting essential lipids, peptides, and reparative myeloid cells.

In support of this contention, one study demonstrated that defects in the cardiac lymphatics were associated with an increased inflammatory response in the injured heart⁸⁶. In this article, the authors showed that mice with Apelin-deficiency exhibit morphological and functional defects in lymphatic vessels and that the defective lymphatics were associated with increased inflammation in the infarcted heart⁸⁶. In addition, several studies in zebrafish indicated the importance of the endogenous lymphatics for cardiac growth and regeneration^{51, 87}. In both papers, it was apparent that zebrafish with blockage or genetic inactivation of VEGF-C/VEGFR3 signaling showed defects in cardiac recovery^{51, 87}. Clearly, more work is needed to confirm if cardiac injury-induced lymphatic vessels are functional and whether lymphatic function is required for cardiac repair.

Because different stages post-MI have been studied, the role of lymphatic function in regulating cardiac repair and regeneration appears quite variable. While the lymphatic vasculature is involved in the pathogenesis of acute cardiac injuries and therapeutic lymphangiogenesis can improve cardiac function following MI, the role of this vascular system in regulating repair and regeneration during the chronic phase (i.e., 6 to 8 weeks post-MI in mice), when inflammation, fibrosis, and impaired cardiac function become established, has only recently been investigated^{85, 88}.

Several recent studies support the contention that enhanced lymphangiogenesis restrains cardiac tissue damage in the chronic phase of recovery in addition to the acute phase^{85, 88}. Moreover, the benefits of cardiac lymphangiogenesis after cardiac injury are evident in numerous studies using a variety of animal models such as mice^47-49, rats⁸⁹, and zebrafish^{51, 87}. In mice, increased lymphangiogenesis through activation of VEGFR3 signaling preserves cardiac function and promotes cardiac recovery post-infarction, indicating the therapeutic potential of enhanced lymphangiogenesis for cardiac repair⁴⁷. Consistent with these results, another report showed that increased cardiac lymphangiogenesis through the stimulation of VEGFR3 signaling by the overexpression of the endogenous epicardial-derived factor adrenomedullin, improved cardiac recovery following MI⁴⁹. Moreover, selectively targeting VEGFR3 in a rat model of ischemia-reperfusion injury promoted lymphangiogenesis in the subepicardial region of the heart in a dose-dependent manner and facilitated cardiac functional recovery after injury⁸⁹. Similarly, activating VEGFR3 signaling in zebrafish improved cardiac regeneration and inflammatory resolution after injury^{51, 87}.

Enhanced lymphangiogenesis and lymphatic remodeling improved cardiac function in these cases, most likely, by facilitating myocardial fluid reabsorption in combination with resolving macrophage infiltration⁴⁸. Aside from the expected immune clearance function, our recent study identified a novel paracrine signal produced by cardiac LECs that regulates cardiac regeneration and repair, further accentuating the essential function of lymphatics in the recovery of cardiac injury⁵³. In this paper, we found that Reelin, an extracellular matrix protein primarily expressed by LECs in the murine heart, regulated embryonic heart development by promoting cardiomyocyte proliferation and survival⁵³. Interestingly, Reelin expression was highly induced in the newly formed lymphatics after MI, and Reelin mutant pups showed impaired cardiac regeneration as a consequence of reduced cardiomyocyte proliferation and increased cardiomyocyte apoptosis⁵³. Furthermore, delivery of Reelin using bioengineered collagen patches into the infarcted adult heart greatly reduced cardiomyocyte apoptosis and promoted cardiac repair post-MI⁵³.

This work provided a novel mechanism describing how cardiac lymphatics could promote cardiac repair. The concept of active paracrine signals from cardiac lymphatics has motivated the field to re-think the functional role of lymphatics in pathologic conditions, not only in heart, but also in other organs. Indeed, more recent studies have reported a similar paracrine function of lymphatics in other vascular beds. In mouse adipose tissue, Neurotensin was identified as an anti-thermogenic peptide produced by LECs and it was found to play an important role in thermogenesis⁹⁰. In the mouse intestine, Wnt signaling and Reelin from intestinal LECs functioned as a central signaling hub to regulate intestinal stem cell activity⁹¹. Taken together, these studies show the importance of the cardiac lymphatics in myocardial injury and suggest the therapeutic potential of promoting lymphangiogenesis to improve cardiac function following acute injury.

Hypertension

Hypertension is associated with renal immune cell infiltration and the production of pro-inflammatory cytokines that alter renal sodium retention. The renal lymphatics, similar to those in other organs, are critical to transport fluid, small molecules, and cells from the renal interstitial space and they play a fundamental role in renal physiology and pathology^92-94. Because the kidney has a unique microenvironment protected by dynamic immune cell subsets and downstream signaling molecules, the lymphatics likely act important conduits for moving antigen-presenting and immune cells from the kidney to the lymph nodes⁹⁴. Hypertension-related renal inflammation and other kidney pathologies exhibit increased renal lymphangiogenesis in a compensatory manner, similar to other tissues^{41, 95-98}.

Selectively promoting initial lymphatic vessel density in the kidney by renal VEGF-D overexpression protected mice from the development of salt-induced hypertension as mice with tetracycline-responsive kidney-specific overexpression of murine VEGF-D (kidney-specific VEGF-D overexpression) enhanced renal lymphatic vessel expansion visualized by Lyve1 immunostaining and prevented hypertension in mice⁹⁹. Furthermore, expanded initial lymphatic vessels in kidney prevented the development of hypertension by inhibiting the elaboration of nitric oxide⁹⁹. These expanded vessels decreased renal immune cell infiltration and lowered blood pressure^99-101. Furthermore, increased lymphangiogenesis also promoted urinary Na excretion, which lowered blood pressure by reducing renal interstitial Na retention¹⁰¹. Interestingly, there are also gender differences in the regulation of lymphangiogenesis in the kidney¹⁰⁰. In female mice, there was increased VEGF-C expression in the kidney during Angiotensin II infusion, which was linked to lower Angiotensin II-induced hypertension in females when compared to males¹⁰⁰. This finding is in line with another study showing that the lymphatic vasculature requires Estrogen Receptor-α to protect against lymphedema¹⁰². Despite this, the exact role of Estrogen Receptor-α in renal lymphangiogenesis has not been fully elucidated.

At the same time, enhanced lymphangiogenesis in the kidney promotes clearance of interstitial immune cells that reduce inflammation regardless of sex. While enhanced renal lymphatics increase Na excretion, high Na promotes lymphangiogenesis and lymphatic contractility in collecting vessels, which enhances the clearance of fluid and infiltrated immune cells to maintain electrolyte homeostasis¹⁰³. Together, promoting renal lymphangiogenesis can prevent salt-sensitive hypertension through augmenting of Na and immune cell clearance in the kidney and represents yet another therapeutic opportunity. Interestingly, a recent report from Helge Wiig’s group using mice with either hypoplastic (Chy), absent (K14-VEGFR3), or hyperplastic (K14-VEGF-C) dermal lymphatic vessels and littermate controls demonstrated lymphatic vessels in skin have minimal impact on blood pressure regulation when the mice were fed with high-salt diet (i.e., 4% NaCl in the chow)¹⁰⁴.

Summary and Future Directions

Here, we have described recent findings that suggest increasing lymphatic function through the promotion of lymphangiogenesis and lymphatic growth and remodeling may represent a treatment for various metabolic disorders as well as myocardial infarction, atherosclerosis, and hypertension (Table 1 and Figure 1). At the same time, our understanding of the molecular mechanisms and signaling pathways involved in controlling the lymphatic vasculature is largely incomplete. Conversely, VEGF-C or VEGF-D therapies have been shown to play an important role in modulating the behavior of immune cells and the inflammatory tumor microenvironment¹⁰⁵. Especially, in an inflammatory skin carcinogenesis model, VEGF-C and VEGF-D blockade reduces infiltrating macrophages and diminishes inflammatory cytokines secreted by these macrophages, suggesting that targeting VEGF-C/VEGF-D using blocking agents holds promise for constraining inflammation in various pathological conditions. As our knowledge of lymphatic biology continues to grow, we expect that the therapeutic potential for targeting this biological system will continue to expand.

Table 1:

Models of metabolic and cardiovascular diseases

Disease model	Approach to promote lymphatic growth	Effects	Ref.
Metabolic Diseases
Obesity induced by HFD in mice	VEGF-D overexpression in adipose tissue	Promoted lymphangiogenesis in adipose tissue; enhanced glucose clearance and reduce insulin and triglyceride levels	67
Stage III type 2 diabetes by streptozotocin injection and HFD in mice	LEC specific Epsins deficient mice to promote VEGF-C/VEGFR3 signaling	Promoted lymphangiogenesis; resolved lymphatic function in diabetic mice.	66
Myocardial Infarction
MI in mice	Recombinant human VEGF-C (C156S), hVEGF-Cc156S) or VEGFR3 adeno-associated viral gene (AAV) delivery	Reduced cardiac inflammation; enhanced post-MI cardiac function; Reduced infarction size and suppressed acute T cell infiltration	85
MI in mice	Recombinant human VEGF-C (C156S)	Improved immune cell clearance; promoted immune trafficking; preserved cardiac function	48
MI in mice	Overexpression of adrenomedullin	Increased connexin 43 localization; improved cardiac function; reduced infarcted size	49
MI in rat	Recombinant human VEGF-C (C152S)	Decreased cardiac inflammation and fibrosis; Increased cardiac function	50
MI in mice	Recombinant human VEGF-C (C156S)	Decreased cardiac fibrosis; Increased cardiac function	47
Hypertension
Hypertension induced by chronic 4% high salt diet in mice	Overexpression VEGF-D in kidney	Reduced blood pressure; increased urinary Na excretion	99
Hypertension induced by angiotensin II infusion in mice	Overexpression VEGF-D in kidney	Reduced blood pressure	98
Hypertension induced by NO inhibitor or high salt diet in mice	Overexpression VEGF-D in kidney	Reduced renal immune cell infiltration; protected nitric oxide synthase inhibition-induced and salt-sensitive hypertension	97

Figure 1. Increasing lymphatic growth and function to treat metabolic and cardiovascular diseases.

A number of recent studies have highlighted the therapeutic potential of stimulating lymphatic function through lymphangiogenesis, lymphatic growth, and lymphatic remodeling for the treatment of obesity, myocardial infarction, atherosclerosis, and hypertension. (A) Metabolic diseases can systemically impair lymphatic function. Promoting lymphatic growth in metabolic tissues (e.g., skeletal muscle, brown adipose tissue [BAT], and white adipose tissue [WAT]) may resolve insulin resistance and enhance insulin sensitivity to protect against disorders such as obesity. (B) Enhancing lymphangiogenesis after myocardial infarction may clear infiltrating immune cells to reduce the elaboration of pro-inflammatory cytokines as well as decreasing edema, inflammation, and fibrosis to increase functional recovery of the injured heart. (C) Halting the progression or reversing growth of atherosclerotic lesions may depend on increasing lymphatic function in the surrounding tissues by blunting inflammation and reducing cholesterol uptake by immune cell clearance. (D) During hypertension, increased immune cell infiltration and inflammation in renal tissue can contribute to elevations in blood pressure. Augmenting renal lymphangiogenesis may promote immune cell clearance to increase kidney function through sodium excretion to reduce blood pressure.

Highlights.

• A brief review of recent research regarding the promotion of lymphatic growth to treat metabolic and cardiovascular diseases

• The diseases discussed in this review include obesity, myocardial infarction, atherosclerosis, and hypertension

• Actual and potential experimental and/or therapeutic approaches that induce lymphatic function and remodeling are presented

• Molecular and cellular mechanisms underlying physiological and pathological lymphangiogenesis and lymphatic growth and remodeling are discussed

Non-standard Abbreviations and Acronyms:

HFD

High-fat diet

LEC

Lymphatic endothelial cell

MI

Myocardial infarction

oxLDL

oxidized low density lipoprotein oxidized

Prox1

Prospero homeobox 1

RCT

Reverse cholesterol transport

VE-cadherin

Vascular endothelial cadherin (Cadherin 5)

VEGF

Vascular endothelial growth factor

VEGFR3

Vascular endothelial growth factor receptor 3