Lymphatic system identification, pathophysiology and therapy in the cardiovascular diseases

Journal of Molecular and Cellular Cardiology 133 (2019) 99–111

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Review article

Lymphatic system identification, pathophysiology and therapy in the cardiovascular diseases

T

Dan Hua,1, Long Lia,1, Sufang Lia, Manyan Wua, Nana Geb, Yuxia Cuia, Zheng Liana, ⁎ Junxian Songa, Hong Chena, a Department of Cardiology, Beijing Key Laboratory of Early Prediction and Intervention of Acute Myocardial Infarction, Center for Cardiovascular Translational Research, Peking University People's Hospital, Beijing, China b Department of Geriatrics, Beijing Renhe Hospital, Beijing, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Lymphatic vessels Identification Lymphangiogenesis Atherosclerosis Myocardial infarction

The mammalian circulatory system comprises both the cardiovascular system and the lymphatic system. In contrast to the closed, high-pressure and circular blood vascular circulation, the lymphatic system forms an open, low-pressure and unidirectional transit network from the extracellular space to the venous system. It plays a key role in regulating tissue fluid homeostasis, absorption of gastrointestinal lipids, and immune surveillance throughout the body. Despite the critical physiological functions of the lymphatic system, a complete understanding of the lymphatic vessels lags far behind that of the blood vasculatures due to the challenge of their visualization. During the last 20 years, discoveries of underlying genes responsible for lymphatic vessel biology, combined with state-of-the-art lymphatic function imaging and quantification techniques, have established the importance of the lymphatic vasculature in the pathogenesis of cardiovascular diseases including lymphedema, obesity and metabolic diseases, dyslipidemia, hypertension, inflammation, atherosclerosis and myocardial infraction. In this review, we highlight the most recent advances in the field of lymphatic vessel biology, with an emphasis on the new identification techniques of lymphatic system, pathophysiological mechanisms of atherosclerosis and myocardial infarction, and new therapeutic perspectives of lymphangiogenesis.

1. Introduction Two vascular systems exist in the vertebrate body: the blood and the lymphatic vasculature. The blood vasculature is a closed circulatory system, which is required for exchange of water, salts, oxygen, nutrients, hormones, and waste products between blood and tissues by blood transporting through the body. In contrast, the lymphatic vasculature that runs in parallel with the blood vascular system, is a blindended, unidirectional system responsible for the reabsorption of interstitial fluid leaking out of the blood vasculature into extracellular spaces, complementary to the blood cardiocirculatory system. To date, the lymphatic system has not been well studied compared to the blood vascular system. The lymphatic circulation is still a somewhat forgotten part of the circulatory system, since most research interest is devoted to the blood circulation and related diseases. The recognition of the existence of lymphatic vessels (LVs) has evolved slowly over the course of history, most importantly due to the technical difficulty in visualizing these transparent vessels. In ancient

Greece, Hippocrates (c.460 to c.370 BCE) first described the colorless vessels that are now recognized as may comprising the lymphatic vasculature [1]. In the 17th century, Gaspar Aselli can be credited for being the first officially to depict the lymphatic vasculature [2,3]. The gross anatomy of the LVs was finally settled from the beginning of the 19th century [1,4]. Over the past two decades, the lymphatic research has gained strong momentum, thanks to the discovery of lymphatic endothelium-specific markers, including the lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) [5], vascular endothelial growth factor receptor-3 (VEGFR-3) [6], integral membrane glycoprotein podoplanin (PDPN) [7], prospero homeobox 1 (PROX1) transcription factor [8]. Moreover, since the turn of the present century, new lymphatic-specific conditional knock-outs [9–11] or fluorescent reporter mice [12,13], together with imaging tools [14,15] advances developed for both animals and human, have greatly enhanced our current understanding of LVs. Contrary to the popular belief, LVs do not function simply as a passive transit channel from the extracellular space to the blood circulation, but have been found to actively regulate lots of

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Corresponding author at: Xizhimen South Rd No.11, Xicheng District, Beijing 100044, China. E-mail address: [email protected] (H. Chen). 1 The first two authors contributed equally to this work. https://doi.org/10.1016/j.yjmcc.2019.06.002 Received 24 March 2019; Received in revised form 2 June 2019; Accepted 5 June 2019 Available online 07 June 2019 0022-2828/ © 2019 Published by Elsevier Ltd.

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physiological and pathological processes. Lymph transport throughout the lymphatic network is regulated by a combination of two forces that are passive, extrinsic and active, intrinsic to the LVs. External forces derive from surrounding tissue movements and pressure gradients, including skeletal muscle contractions, thoracic pressure changes included by respiratory cycles, bowel movements, heartbeats and arterial pulsations [16]. Intrinsic forces generate from the contraction of LVs themselves. While external forces can aid in this process, the primary forces for lymph flow are the intrinsic LVs contraction [17,18]. Some anatomic locations of LVs have been identified in organs where they were previously not to thought to exist, such as the eye [19–21] and the central nervous system [22,23]. In this review, we will update the current understanding of LVs and some cardiovascular diseases pathogenesis, with a specific attention to atherosclerosis (AS) and myocardial infraction (MI). Firstly, we will introduce briefly the lymphatic anatomy, function, and composition. Next, we will describe the new identification techniques of LVs in detail. Finally, we will specifically discuss the pathophysiological mechanisms of lymphatic system in AS and MI, as well as therapeutic perspectives of lymphangiogenesis. 2. Description of lymphatic system 2.1. Anatomy of lymphatic system The LVs are a drainage network that begins in the interstitial spaces and ends in the thoracic duct and the right lymphatic trunk. The LVs are found in nearly every vascularized tissue, except avascular tissues such as bone marrow or cartilage. Based on their molecular characteristics, morphology, function, and hierarchy, the LVs can be divided into three different types: lymphatic capillaries (also known as initial LVs), precollecting and collecting LVs (Fig. 1a). Lymphatic capillaries form the blind ends of the lymphatic vasculature, and are responsible for the uptake of interstitial fluid. Lymphatic capillaries are composed of a single thin layer of oak leaf-shaped lymphatic endothelial cells (LECs) forming discontinuous button-like cellcell junction (Fig. 1a) [24], which are characterized by specific expression of LYVE-1 on the surface of LECs [25]. They lack mural cells coverage and have little or no basement membranes. These characteristics likely make lymphatic capillaries highly permeable to interstitial fluid and solutes, allowing the entry of macromolecules such as lipids and even permitting trafficking of immune cells. These button-like junctions display an alternating pattern of the adhesion proteins vascular endothelial (VE) cadherin and platelet endothelial cell adhesion molecule (PECAM-1), forming overlapping flaps between adjacent endothelial cells (EC) [24], and are thought to act as primary valves [26–28]. This primary valve system is required to prevent fluid escape from the initial LVs back into the interstitial space. To prevent the vessels from collapsing or keep the primary lymphatic valves open, LECs in lymphatic capillaries are anchored to the surrounding extracellular matrix by anchoring filaments [29–31]. In addition, lymphatic capillaries are non-contracting vessels with the absence of a muscular layer in the form of specialized, autonomously contracting lymphatic muscle cells (LMCs), a new class of muscle cells [32–34]. The LMCs exhibit tonic and phasic contractions, which are the primary forces to move lymph against a hydrostatic pressure gradient in most regions of the body [35]. The uniqueness of lymphatic contractile function can be directly attributed to the unique ultrastructure of LMCs, which contain both smooth and cardiac muscle components [33]. The LMCs are nonstriated and share biochemical and functional characteristics with both vascular and cardiac muscle cells, but they are unique, a new class of muscle cells that are distinct from smooth and cardiac muscle cells. Like vascular smooth muscle cells, LMCs contraction is regulated primarily by the balance of myosin light chain kinase (MLCK)/myosin light chain phosphatase (MLCP) activity controlling myosin light chain phosphorylation [34]. The contractile activity of LMCs exhibits basal, myogenic

Fig. 1. Anatomy, function and composition of lymphatic system. a Anatomy: the LVs can be classified into lymphatic capillaries, pre-collecting and collecting LVs. Lymphatic capillaries form blind-ended vessels, are covered by a discontinuous basement membrane and composed of a single thin layer of LECs forming discontinuous button-like cell-cell junction. In contrast, collecting LVs exhibit a continuous basement membrane, are surrounded by LMCs, and their LECs are connected by zipper-like junctions. In addition, lymphatic valves of pre-collecting and collecting LVs divide the LVs into lymphangions. b Function: the LVs are critically involved in fluid homeostasis, immune cell trafficking and absorption of dietary fats. c Composition: the lymphatic system contains electrolytes, nutrients, immune cells, antigens, antibodies, macromolecules, lipids, lipoproteins and so on.

tone and myogenic responses to pressure changes [36], as well as being modulated by various neuromodulatory, vasoactive and mechanical factors [37–39]. Like cardiac muscle cells, LMCs express troponin C and I as well as cardiac isoforms of tropomyosin [33]. The uniqueness of LMCs is further highlighted by the presence of several electrophysiological properties with both vascular smooth muscle and cardiac muscle cells. LMCs contraction depends predominantly on Ca2+ influx through L-type or ‘long-lasting’, Ca2+ channels (characteristic of smooth muscle cells) and T-type ‘transient’ Ca2+ channels (implicated as a possible pacemaker component in both cardiac and lymphatic tissues) [40], while the resting membrane potential is influenced substantially by Cl− and voltage-gated K+ channels [41,42]. The mechanism governing LMCs tonic contractions is that Ca2+-dependent activation of myosin light chain (MLC) kinase (MLCK) drives the development of actin-myosin mediated tonic contraction [43]. Rho Kinase (ROCK), which phosphorylates the MLC phosphatase (MLCP) targeting subunit MYPT-1, leading to inactivation of MLCP, also promotes tonic constriction of lymphatics [44,45]. There is also evidence that PKC increases Ca2+-sensitivity by activating CPI-17-mediated inhibition of 100

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2.2. Function of the lymphatic system

MLCP to increase tone [32]. The mechanisms controlling LMCs phasic contractions are less clear. Action potentials and transient increases in intracellular free Ca2+ have been shown to precede phasic contractions of lymphatic vessels [39,46]. The pacemaker may involve Ca2+-activated Cl− currents or hyperpolarization-activated inward current [41,47]. In addition, recent work shows that inhibition of ROCK can prevent phasic contractions while allowing the Ca2+ transients to persist [44]. The shortening velocity of LMCs is much greater than observed in other smooth muscle types and much closer to that of striated muscle [38,48]. This appears to be due to the presence of cardiac muscle contractile proteins in addition to those typically found in smooth muscle. For example, fast isoforms of myosin, such as the smooth muscle B myosin heavy chain (MHC) and the fetal cardiac/ skeletal slow-twitch β-MHC, have been identified within LMCs [33]. Cardiac Troponin-C (cTN-C) and cTN-T may also play a role in the phasic contractile nature of lymphatic vessels [49]. When lymph continues its path from the lymphatic capillaries, it converges into pre-collecting and subsequently collecting LVs. Precolleting LVs contain valves and sparse LMCs coverage, which are a mixture of lymphatic capillaries and collecting LVs. In contrast to lymphatic capillaries, the collecting LVs are characterized by elongated LECs, that are interconnected by tight zipper-like junctions and surrounded by a continuous basement membrane [50] to prevent leakage of lymph during transport (Fig. 1a) [24,51]. Furthermore, collecting LVs are covered by a muscular layer with LMCs, which periodically contract to move lymph forward [17,35,52], and contain luminal valves that are essential for preventing backflow of lymph. Luminal valves divide the collecting LVs into a series of segments, referred to as lymphangions, which act as contractile units that propel lymph forward in a unidirectional manner (Fig. 1a) [16,53,54]. Lymph propulsion requires not only robust contractions of LMCs, but contraction waves that are synchronized over the length of a lymphangion as well as properly functioning intraluminal valves. The anatomical pattern of collecting lymphatics, organized as a chain of lymphangions in series, combined with LMCs-generated contractions, enables collecting lymphatics to work as pumps. Normal lymphatic pump function is determined by the intrinsic properties of LMCs and regulated of pumping by lymphatic preload, afterload, spontaneous contraction rate, contractility and neurohormone influences and so on. Lymphatic pumping has been shown to be very sensitive to changes in transmural pressure. Preload, which is set by end-diastolic pressure of the lymphatic wall during the phase of the contractile cycle, is a significant determinant of lymphatic pump function. Increasing the filling pressure over a certain range enhances pump output, analogous to the Frank–Starling relationship for the heart [55,56]. The lymphatic pump must adapt to elevated outflow pressures resulting from partial outflow obstruction, increased central venous pressure and/or gravitational shifts. LMCs have been shown to undergo an intrinsic increase in contractility when subjected to an elevated afterload [57]. In the heart, cardiac output = stroke volume × heart rate. The analogous expression for the lymphatic pump is pump output = ejection fraction × contraction frequency. The contraction frequency of collecting lymphatics is exquisitely sensitive to pressure, and changes as small as 0.5 cm H2O can double contraction frequency [56]. ‘Contractility’ is often used in a broad sense in the lymphatic literature to describe the enhancement of contraction amplitude or contraction frequency in response to a pressure increase or agonist activation [41,49]. While not required for lymphatic contractions per se, the effects of neurohormone signaling molecules appear to be keenly involved in regulating lymphatic contractions such as sympathetic adrenergic nerve fibres, nitric oxide (NO) and prostanoids [58–60]. With the specific regulation of lymphatic pumping and LMCs contraction, we recommend the reader for reference to several excellent reviews [16,34,61]. In addition, collecting LVs are characterized by expression of podoplanin. Eventually, the collecting LVs converge into the thoracic duct or the right lymphatic trunk, where lymph is reaching the venous circulation via the subclavian vein [3,62].

The principal function of the lymphatic system is to maintain tissue fluid homeostasis by removing the protein-rich lymph from the extracellular space and returning it to the blood circulation [63]. In addition to the regulation of tissue fluid, the lymphatic vasculature is important for transport of immune cell and soluble antigens to lymph nodes (LNs) [64], management of peripheral immune tolerance [65], and absorption of dietary fats in the gastrointestinal organs (Fig. 1b). In most tissues under physiological conditions, whenever blood moves through blood capillaries, there is a constant leakage of the intravascular plasma into extracellular space. The extravasation is estimated by the starling forces and accumulated to a total of 8 L during 24 h [66]. The majority of the extravasated interstitial fluid and macromolecules are absorbed back by the LVs, whereas venous reabsorption plays a small role in most vascular beds [67]. Thus, the lymphatic system is important to regulate the tissue fluid homeostasis [50]. The LVs are essential for trafficking of leukocytes and soluble antigens from peripheral tissues to draining LNs [68]. Under homeostatic conditions, early cannulation studies of LVs conducted in sheep and healthy humans revealed that T lymphocytes are the most common cell types in lymph (80–90%) [69–72]. The majority of these cells are CD4+ effector memory T cells, while CD8+ T cells are only found in small numbers. Dendritic cells (DCs) are also frequently found in cannulated lymph (5–15%) from sheep and humans [69–72]. Meanwhile, other immune cells such as monocytes, neutrophils, eosinophils, basophils and B cells are also routinely detected in steady-state lymph, albeit a very low number [69,70]. Lymphoid structures such as LNs are distributed along the LV network. They are composed of lymphatic and blood vessels spread out inside a parenchyma, subdivided into B-cell follicles and a T-cell area that together form the cortex and the medulla. Upon exposure to inflammatory stimulus and recognition of pathogenassociated molecular patterns, DCs capture antigens in peripheral tissues and migrate through afferent LVs into LNs [73,74]. A fundamental function of the lymph is clearance of tissue invading pathogens [75]. Indeed, by draining through the nodes, the lymphatic system ensures that tissue-invading pathogens do not directing flow into the bloodstream, but are captured by macrophages and DC present in the LNs [74,76]. The lymphatic system serves as the entry point for nearly all dietary lipids, which are taken up by enterocytes (epithelial cells on the lumen of the intestine) and packaged as large lipoproteins chylomicrons for export into the LVs [77,78]. In the mesentery, a network of lacteals (LVs of the gut) associated with the intestinal villi act as the initial destination for chylomicrons, cholesterol, apolipoproteins, and other lipid carrier molecules that are released into the interstitum by mucosal enterocytes [79]. Therefore, the lacteals are essential for the uptake of dietary lipids and fat-soluble vitamins. 2.3. Components of lymphatic fluid Since the early 1970s, it was well known that lymph originates from the interstitial fluid. Lymph contains electrolytes, nutrients, immune cells, antigens, antibodies, macromolecules, lipids, lipoproteins and so on (Fig. 1c). The average lymph chloride and sodium concentrations were higher, where the potassium concentration was lower, when compared with blood. The cardiac lymph showed a significantly higher mean lactate level when compared with the coronary sinus blood samples, and the cardiac lymph PH was situated at around 8.0 or higher [80]. The lymph contains nearly all dietary lipids, such as chylomicrons, cholesterol, apolipoproteins, and other lipid carrier molecules [77,79]. Cholesterol concentration in the lymph is approximately a tenth of the concentration in the plasma [81]. As in the plasma, cholesterol in the lymph is transported by apolipoproteins, such as ApoA1 and ApoB, although Reichl et al. consistently found that concentration of ApoA1 and ApoB were much lower than in plasma [82–84], with all 101

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3.2. In vivo imaging of LVs

ApoB was in LDL [85]. Consistent with Reichl et al. [82–85], lymph was essentially devoid of VLDL. Analysis of human lymph showed that lymph concentration of HDL cholesterol was 30% greater than that of blood [86]. We now know that a broad array of cytokines, proteins, growth factors are contained with lymphatic fluid, which play an important role in metabolism, proliferation, as well as an immunoregulatory role [87]. In the last decade, an increased number of proteomic analyses of the lymphatic fluid have been published. Following on from the original analysis of ovine and bovine lymph, analysis of mouse, rat, and human lymphatic fluid have been performed. Thus far over 2000 proteins have been mapped by proteomic analyses preformed in different species, including humans, mice, ovine, bovine, and swine. Along list of proteins have been reported, as being summarized in reviews [54,88]. For lymph formation, we would like to refer the reader to several excellent reviews [54, 88].

Nowadays, new improvements of sophisticated lymphatic vascular imaging technologies, have greatly enhanced our understanding of LVs. With the cloning of genetically encoded fluorescent proteins in the early 1990s, scientists firstly started to label different cell types in living organisms. The development of fluorescence microscopy, especially the confocal microscope, in combination with specific antibodies designed to recognize LVs, is applied on LVs research. Light sheet-based imaging techniques now offer the ability to live image the lymphatic system in whole organs or even in whole animals during development and in pathological conditions with a satisfactory spatial and temporal resolution. In particular, light sheet fluorescence microscopy-based imaging techniques allow one to achieve almost confocal-like resolution at much higher imaging speeds, while being minimally invasive [107]. However, because the various tissue components possess different refractive indices, it causes light to travel at different speeds and angles within the tissue. As a consequence, light sheet-based imaging techniques have a poor imaging resolution and contrast for the deeper tissues. Recently developed tissue optical clearing techniques, such as dibenzyl ether [108], Clear T [109], three-dimensional imaging of solventcleared organs (3DISCO) [110], CLARITY [111] and passive CLARITY [112], have the ability to substantially reduce light scattering in various tissues and enable one to image cellular details deep within the intact architecture of fixed organ or small animals. The most stunning advances in microscopy in recent years have been made in the area of the super resolution fluorescence microscopy. Super resolution fluorescence microscopy, such as structured illumination microscopy (SIM) [113], photoactivatable localization microscopy (PALM) [114], stochastic optical reconstruction microscopy (STORM) [115], now permits the examination of cellular processes at a near-molecular resolution. It has now become possible to resolve biological structures at the nanometer scale in three dimensions, to measure molecular interactions by multicolor colocalization, and to record dynamic processes in living cells as relatively high speeds. Recently, of special interest to the field of vascular biology, are the recently developed photoacoustic tomography (PAT) and photoacoustic microscopy (PAM) techniques [116]. Both approaches use a pulsed laser to excite the sample and PAT/PAM is especially useful to visualize vessels in vivo because of the fact that vessels have a higher absorption than surrounding tissues and therefore create sufficient endogenous contrast.

3. Research techniques of lymphatic system Morphologically, it is difficult to distinguish LVs from blood vessels. For centuries, dye injection has been a standard technique for identifying LVs and its outflow pathway, including injection with India ink [89] or Evan Blue [90]. Besides, other techniques were also used to visualize LVs, such as hydrogen peroxide techniques [91], and the enzyme histochemical detection of 5′ nucleotidase [92]. In the middle of the 20th century, contrasting techniques, such as phase contrast and differential interference contrast, allowed further advances in LVs research. 3.1. Specific markers of LECs During the late 1990s, the discovery of lymphatic endotheliumspecific markers, including LYVE-1 [5], VEGFR-3 [6], PDPN [7], PROX1 [8], opened up the possibility to study LVs in healthy and diseased tissues and to isolate LECs for transcriptome and proteome analyses. LYVE-1 is an integral membrane glycoprotein and a useful marker to identify lymphatic capillaries. It is an important component of extracellular matrix and a key molecule in cell migration during inflammation, wound healing and in tumorogenesis [5]. Apart from the cytoplasm of LECs, LYVE-1 is also expressed in live and spleen sinusoid endothelium [93] and on certain macrophages [94].VEGFR-3 (previously called FLT4) is the quintessential lymphatic receptor tyrosine kinase binding VEGF-C and VEGF-D [95]. Both in embryos and adults, VEGF-C– VEGFR-3 signaling is crucial for LEC proliferation, migration, and survival [96,97]. In addition to LEC, VEGFR-3 was showed to express in blood vessel endothelium, myoepithelial cells, and some nonendothelial tumor cells [98].PDPN (also known as AGGRUS, gp36, oncofetal antigen M2A and T1A-2 [99]) is frequently used in the immunohistochemical detection of LVs [7]. PDPN was originally identified from cells of osteoblastic lineage [100] and podocytes [101], where it is important for the formation of the glomerular filtration barrier of the kidney [102]. Apart from LECs, it is also expressed by other cells types including tumor cells [99].PROX-1 is a nuclear transcription factor for the early steps of LEC differentiations from the embryonic veins [8] and remains required for lymphatic identify [103]. Immunoreactivity of PROX-1 antibody is nuclear, which is in contrast to the other lymphatic markers. PROX-1 expression has been also observed in lens, heart, liver, pancreas, nervous system [104] and some stem/progenitor cells [105]. Also, other markers such as neuropilin-2, FOXC2, CCL21, D6 and aquaporinm1 appear to be not fully specific for lymphatic endothelium [106]. Despite their expression in other tissues and cell types, the use and combination of these markers nowadays enables the unambiguous molecular distinction between blood and lymphatic vasculature in tissues, in order to avoid false positively/negatively of staining results.

3.3. Transgenic animal models In order to take full advantage of the recent development in imaging techniques to identify lymphatic system, transgenic animal models, that specifically mark LVs' proteins, are indispensable. In the 21st century, molecular genetic studies of developing embryos have revealed more than 50 genes involved in the specification, expansion and maturation of LVs and in lymphovenous separation [117]. And overexpression or knock-out of related genes can bring new insights on the LVs. Currently, the most popular model system for in-vivo imaging of the LVs is zebrafish. A large number of transgenic zebrafish lines that expressing fluorescent reporters or knocking down some genes have been created, such as overexpressed Bmp2b [118] or silencing of miR-126a [119] in zebrafish embryos. The development of transgenic mice has been slightly slower. Several usable transgenic lines already exist, and more being developed. For example, the so-called “Chy mice” for their apparent development of chylous ascites after birth, this mouse model of lymph-edema has an inactivating VEGFR-3 mutation in their germ line, causing a selective incomplete development of LVs dermally and thus swelling of the limb [120]. An additional mouse model has been reported to inhibit the formation of the dermal lymphatic vasculature [121]. Mice expressing soluble VEGFR-3 under the keratin-14 (k14) promoter (k14-vegfr-3-Ig) display a neutralized activity of VEGF-C and VEGF-D in the dermal lymphatic vasculature when expressed in mouse 102

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epidermis. “Soluble VEGFR-3 mice” have impaired LVs in the skin and some more distant organs because they secrete, under the control basal keratinocyte k14- promoter, a chimelic fusion protein which consist of the ligand binging portion of VEGFR-3 extracellular domain and the fragment crystallizable (Fc) domain of immunoglobulin (Ig) γ-chain [120].

in the intima, media, or adventitia), despite the accentuated presence of VEGF-C [135]. In summary, LVs have been readily detected in atherosclerotic and nonatherosclerotic arterial samples in some, such as rats, mice, rabbits, and human, but not in all studies, and seem most present in the adventitial layer. However, what does the present of LVs in the adventitia of aortic wall really mean? Are plaques associated LVs friends or foes in AS? Research groups are actively seeking for the answers, especially through altering lymphatic function in animal models of AS.

3.4. Clinical and preclinical imaging of LVs Nowadays, many lymphatic vascular imaging techniques have been used in clinical or preclinical applications and researches. Two common techniques, lymphangiography and lymphoscintigraphy, have been available for clinical imaging of the LVs [122]. Magnetic resonance lymphangiography provides superior visualization of the LVs, which allows better description of vessel morphology, but in inferior to scintigraphy in the detection of LNs [123]. Moreover, lymphangiography, which involves administration of contrast agents through cannulated LVs, has largely been abandoned because of risk of complications from the contrasting agents and the technical skill requited for vessel cannulation. Lymphoscintigraphy is the gold standard investigation to determine whether limb swelling is the result of lymphatic dysfunction [124]. Lymphoscintigraphy is routinely used for diagnostic imaging of lymphatics and involves injecting radioactive colloid particles intradermally or intraparenchymally, allowing the visualization of their accumulation in the lymphatic plexus and sentinel LNs. However, this technique is not well suited for imaging dynamic lymph flow quantitatively, due to the long integration time of the γ camera, the particle size, and the dermal backflow. Apart from them, a new optical imaging technique with near-infrared fluorescence dyes (mainly indocyanine green) has a clearly higher sensitivity that permits quantitative imaging of lymph flow, especially when highly stable liposomal formulations are used to prevent toxicity [125,126]. However, the near-infrared fluorescence still has some limitations, such as inadequate signal penetration of deep tissues and the possible lymphatic smooth muscle contractions under the indocyanine green dye injection stimulating [127]. Optical frequency-domain imaging, a second-generation optical coherence tomography technology, overcomes the limitations of fluorescent tracers such as tissue penetration and the extravasation through using entirely intrinsic mechanisms of contrast. Optical frequency-domain imaging has been successfully demonstrated to suite for high-resolution, widefield, and deep imaging of tumor microvasculature, as well as functional lymphangiography [128]. Ultrasound array-based real-time photoacoustic microscopy is another highly promising technique, which enables noninvasive high-speed three-dimensional imaging of sentinel LNs and shows promise for imaging lymphatic dynamics in a highly qualitative and quantitative manner [129].

4.1. Lymphatic system involved in reverse cholesterol transport High-density lipoprotein cholesterol (HDL-C) has a variety of important functions, and many human epidemiological studies demonstrate a strong inverse association of plasma levels of HDL-C and coronary artery disease (CAD), which suggest that raising HDL-C levels may decrease risk of CAD [136,137]. However, genetic factors that increase HDL-C are also not associated with reduced CAD [138,139]. In addition, several therapies that raise HDL-C, such as treatment with niacin or CETP inhibitors, have not shown the anticipated clinical benefit based on the epidemiological association [140–142]. Therefore, simply raising plasma HDL-C levels do not capture intended effects and it needs to find new approaches that HDL-C works to reduced CAD, including AS. As we known, the established ability of HDL is to promote the efflux of cholesterol from lipid-laden macrophages for ultimate return to the liver and biliary excretion (reverse cholesterol transport, RCT). RCT is a key player in maintaining peripheral and total-body cholesterol homeostasis, and could lead to regression of AS by reducing the cholesterol content within the plaques. Indeed, the most important site from which HDLs act to promote cholesterol efflux from cells is in the extracellular matrix of tissues, not in plasma. In the interstitial space, HDL becomes loaded with cellular cholesterol, as the first step of RCT, for transport back to the plasma compartment through the lymphatic system. During RCT, the efflux of cholesterol from macrophages requires the action of ATP-binging cassette transporters A1 and G1 (ABCA1, ABCG1). Then, the efflux of cholesterol can occur through uptake by HDL and traveling through the bloodstream to the liver for biliary excretion [143,144]. Recently, it was suggested that in vitro LECs expressed functional HDL transporters, including scavenger receptor class B member (SR-BI) and ABCA1, but not ABCG1 [145]. SR-BI mediates the internalization and transport of HDL, which is important to lymph production. Furthermore, downregulation of SR-BI by small interfering RNAs resulted in 80% inhibition of HDL uptake of LECs in vitro. In vivo, inhibition of SR-BI with blocking antibodies inhibited lymphatic uptake of HDL by as much as 75% [145]. This is a surprising finding, and it would be important to determine why HDL refers LVs instead of the postcapillary venous system to exit the interstitial space. Recent studies suggest the LVs could play an important role in RCT by transporting HDL from ainterstitial tissues to the bloodstream to reduce AS (Fig. 2a) [145,146]. Induction of lymphangiogenesis by the administration of VEGFC into the footpad improved lymphatic function, decreased footpad cholesterol content, and improved RCT in ApoE−/− mice. In contrast, surgical disruption of collecting LVs in the popliteal area reduced RCT from the footpad by as much as 80% [145]. In another study, Martel et al. also found that surgical ablation of LVs in the mouse skin blocked RCT [146]. Simultaneously, they used a genetic model, the so called Chy mice which selectively lack dermal LVs, and showed that RCT from the rear footpad was impaired by 77%. Meanwhile, Martel et al. used a surgical model, where aortic arches with deuterium-labelled cholesterol from ApoE−/− mice were transplanted into ApoE−/− recipient mice that were either treated with anti-VEGFR3 antibody (selective inhibition of the formation of new LVs) or treated with control antibody (regeneration of LVs allowed). It was shown in mice that blocking lymphatic growth with the aortic wall led to greater cholesterol retention in the aortae, because it prevented the cholesterol

4. Lymphatic system in atherosclerosis Despite significant advances in reducing coronary events stemming from AS, AS complications remain a leading cause of morbidity and mortality in the world. Morphological analysis gave the first insight of the association between the lymphatic system and AS. In animal models, LVs have been consistently observed in the adventitial and periadventitial regions of the artery wall [130,131]. In a clinical setting, Drozdz et al. [132] firstly confirmed the present of LVs in the adventitial of human internal carotid arteries and showed that number of adventitial lymphatics increased with severity of AS measured as intimal thickness. The notion that atherosclerotic plaques are accompanied by adventitial lymphatic vessels was further extended by Kholova et al. [133], who demonstrated not only that lymphatics were present in the adventitia of human coronary arteries, but also detected LVs within the atherosclerotic lesions. In contrast to the previous study, other teams observed no [134] or very little [135] LVs in the wall of normal or atherosclerotic human epicardial coronary arteries (whether 103

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Fig. 2. The roles of lymphatic system in AS. a LVs involved in RCT: Lipid free ApoA-I picks up free cholesterol from peripheral cells and further forms HDL. HDL interacts with ABCA1 and ABCG1 and promotes the efflux of cholesterol from lipid-laden macrophages through LVs to return to the liver and biliary excretion. b LVs involved in inflammation: LVs contribute to draining of local inflammatory cells and cytokines in atherosclerotic plaques.

but clear connections between lipid metabolism in AS and lymphatic vessels have not been described. Vuorio et al. crossed two transgenic mice baring lymphatic insufficiency (sVEGFR3 and Chy mice, respectively) with atherosclerotic mice (Ldlr−/−/ApoB100/100 mice), and demonstrated deficiency in adventitial lymphatics of descending aorta [153]. These lymphatic deficient transgenic strains had accelerated AS and increased plasma cholesterol and triglyceride levels compared to lymphatic sufficient controls, directly supporting the role of lymphatic dependent mechanisms in lipids metabolism from atherosclerotic vessels. Moreover, LDLR expression on hepatocytes is crucial for the clearance of circulating LDL and its precursors, intermediate-density lipoproteins (IDL) and very-low density lipoproteins (VLDL) [154]. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a well-established down-regulator of LDLR, which acts by binding the receptor and causes its lysosomal degradation in cells [155,156]. A study employing PCSK9−/− mice suggested that the LDLR could also play a role in lymphatic dysfunction. Lymphatic dysfunction was present before the atherosclerotic lesion formation in a (Ldlr−/−;hApoB100+/+) mouse model. Atherosclerosis-protected PCSK9−/− mice exhibited improved collecting LV function, together with enhanced expression of LDLR on LECs. System treatment with selective VEGFR-3 agonist rescued lymphatic function in pre-atherosclerotic Ldlr−/−;hApoB100+/+ mice [157]. Above researches further emphasize the potential of lymphangiogenic therapy as a tool for regulating lipid metabolism in AS. As hyperlipidemia is a major contributor to atherosclerotic plaque development and coronary heart disease, the role of VEGFs in lipid and lipoprotein metabolism has gained increasing interest. VEGFs and VEGFRs are a family of regulators of lymphangiogenesis [158], and many preclinical studies, as well as clinical trials have shown their potential in the treatment of cardiovascular diseases [159]. Among them, signaling via VEGFC/D and VEGFR3 is perhaps the most central pathway for lymphangiogenesis. Karaman S et al. found that ectopic lipid accumulation was also linked to blockade of VEGF-C, the main lymphangiogenic factor of the VEGF family that functions mainly through third VEGF receptor, VEGFR3. At the same time, VEGF-C overexpression has been shown to increase liver steatosis [160]. The liver is central to lipid and lipoprotein metabolism as CRs (CM remnants) are internalized by hepatocytes via LDLR (low-density lipoprotein receptor), LRP (LDLR related protein), and HSPGs (heparan sulfate proteoglycans) that recognize apoli-poproteins on the surface of the

efflux from the aortic plaques. Simultaneously, there is a study showing that hypercholesterolemic ApoE−/− mice carrying excess cholesterol in VLDL and chylomicron remnant fraction displayed lymphatic structural alterations, with reduced expression of VEGF-C and FOX2. Restoration of lymphatic function with a local injection of recombinant VEGF-C growth factor in ApoE−/− mice, reduced the accumulation of cholesterol in the skin, and improved RCT, further highlighting the importance of LVs function in reducing AS [145]. Another role of LVs on RCT is involved in cholesterol removal from macrophage stores, including hydrolysis, mobilization, and efflux of cholesterol esters to lipoprotein acceptors such as apoA-I [147]. ApoA-I, the main protein constituent of plasma HDL, quickly became a target of interest to reduce AS. Interestingly, subcutaneously injected lipid-free apoA-I has been reported to reduce accumulation of lipid and immune cells within the aortic root of hypercholesterolemic mice without increasing HDL-C concentrations [148,149]. A recent study revealed that a continuous low-dose intradermal injection of diet-fed Ldlr−/−mice with lipid-free apoA-I reversed atherosclerosis-associated collecting LV dysfunction, without significantly affecting plasma or lymph total cholesterol concentrations [150]. The direct effect of apoA-I on LECs combining with its role in platelet activity highlighted the versatility of this apolipoprotein in the modulation of lymphatic function. Altogether, this work suggests that preservation of collecting lymphatic function contributes to the protective effect of apoA-I. Therefore, mice deficient in apoA-I have significantly increased atherosclerotic plague burden and reduced RCT [151,152], further emphasizing the potential of lymphangiogenic therapy as a tool for cholesterol clearance. 4.2. Lymphatic system involved in lipid metabolism Atherosclerosis is a complex process involving many steps and the interplay of systemic and local factors. And multiple lines of evidences, from genetic, experimental, epidemiological, and clinical studies, have converged on plasma cholesterol. LVs are important in lipid abruption, accumulation, clearance and so on. As intestinal lymphatics take up dietary lipids in the form of chylomicrons, large lipoprotein particles, to transport them to the bloodstream. Lymphatic vessels are also found at sites of atherosclerosis, which is associated with lipid accumulation in arterial walls [133]. Previous studies have shown that impairment of lymphatic vessel function causes lymphedema and fat accumulation, 104

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coronary ligation or ischemia– reperfusion injury in rats), tissue edema can additionally be directly quantified using microgravimetry (ratio of wet: dry weight). Myocardial edema is not only a consequence of cardiac injury but also causes microvascular and cardiomyocyte dysfunction and damage. Some studies showed that an increase in tissue fluid content by as little as 2.5%, can lead to 30%–40% reduction in cardiac output [167,170]. Furthermore, myocardial edema can trigger cardiac fibrosis (Fig. 3b) [167,171], as shown by increased collagen synthesis including type I and III by fibroblasts [172,173], contributing to the development of chronic heart failure. Concurrently, there is rapid infiltration of neutrophils, macrophages, and other immune cells, which participate in the inflammatory tissue response to injury (Fig. 3b). These cells perform important beneficial roles such as the removal of dead cells and matrix debris, and the stimulation of reparative or regenerative processes [174,175]. However, inflammatory reactions can also cause deleterious cardiac remodeling, such as cardiac fibrosis [176]. Although resultant fibrotic tissue can compensate for the loss of cardiomyocytes and provide structural support following MI to protect against cardiac rupture, myocardial fibrosis also has negative consequences including hindering interstitial fluid drainage, impairing cardiac function, and contributing to adverse ventricular remodeling.

remnant particles [161]. Furthermore, a new study showed that that VEGF-D knockout mice (VEGF-D−/−LDLR−/−ApoB100/100) had significantly increased plasma cholesterol and triglyceride levels on Western-type high-fat diet [162]. In addition, it revealed that the deletion of VEGF-D leads to the downregulation of a major hepatic HSPG, SDC1 (syndecan 1), and consequently to the accumulation of large lipoprotein particles in the plasma. Taken together, these studies suggest that a beneficial role of peri-adventitial lymphatics through regulating lipid metabolism during atherosclerosis. 4.3. Lymphatic system involved in inflammation AS is a chronic inflammatory disease affecting large- to mediumsized arteries, and characterized by the formation of arterial fatty streaks and plaques loaded with macrophages containing cholesterol (foam cells). Continuous recruitment of monocytes into plaques drives the progression of this chronic inflammatory condition [163]. Xu et al. demonstrated the presence of LVs within the adventitia of the artery wall to be an important factor for the draining of local inflammatory cells and cytokines from peripheral tissues [164]. In the meantime, Susan N. Thomas et al. also found that K14-VEGFR-3-Ig mice, which lack dermal lymphatic capillaries, experience markedly depressed transport of solutes and dendritic cells from the skin to draining LNs [121]. A recent study also showed that the adventitial lymphatic capillary bed was markedly expanded early on in atherogenesis, and surgical interruption of lymphatic plague drainage prior to atherogenesis aggravated AS development, with increased T cell accumulation in plaque and adventitia as most prominent feature [165]. The latter was also observed after systemic inhibition of VEGFR-3 dependent lymphangiogenesis. Interestingly, this study also found that both interventions did not impact lymph capillary bed density in the adventitia or plaque, suggesting that lymphatic expansion is not driven by classical VEGF-C/D independent mechanisms, possibly through the CXCR4/ CXCL12-axis. Indeed, Llodra et al. found that emigration of monocytederived cells through LVs contributed to the regression of plaques that had formed in ApoE−/− and were subsequently transplanted into ApoE+/+ recipient mice [166]. This suggests that lymphatic system indeed contributes to resolution of inflammation in atherosclerotic plaques (Fig. 2b).

5.2. LVs in MI The heart also carries an elaborate lymphatic network, first described by Rudbeck in the 17th century, discussed in Bradham and Parker [177]. Patek revealed that the mammalian cardiac LVs invested all layers of the heart: the subepicardium, the myocardium, the subendocardium [89]. Some studies in dog [89,178] revealed that lymph flow passed from the endocardium after interstitial fluid entered lymphatic capillaries therein, to the epicardium where collecting lymph flow was observed. Similar to other organs, the heart also relies to cardiac LVs to maintain fluid balance and immune surveillance (Fig. 3b). In particular, recent literature also demonstrated that lymphatic drainage plays a major role in MI [179]. MI induced robust, intramyocardial capillary lymphangiogenesis, and adverse remodeling of epicardial pre-collector and collector lymphatics, leading to reduced cardiac lymphatics transport capacity (Fig. 3b) [179]. Ischemic heart exhibits a dysfunctional lymphatic network that participates in the development of chronic myocardial edema and aggravates cardiac dysfunction [179,180]. It is noteworthy that clinically detectably myocardial edema, extending beyond the infarct zone, may persist for up to 6 to 12 months post-MI in humans, which is also suggestive of lymphatic insufficiency [181,182].

4.4. Summary Altogether, the aforementioned studies suggest that enhanced RCT, lipid metabolism and emigration of inflammatory cells through the LVs from arterial walls during atherogenesis contributes to plaque regression (Fig. 3a). In addition, although there is an increase in the density of LVs around AS [132,165], but it is unclear why the increased LVs cannot alleviate the atherosclerotic plaque. Perhaps, newly formed LVs are deficient in function [68], or the number of these newly formed LVs is too low to cause a reversal of stubborn diseases such as AS.

5.3. Lymphangiogenesis in MI As a compensatory response to the myocardiac injury, lymphangiogenesis has been observed after experimental MI in mice [180] and rats [179], and also in postmortem human MI samples [183]. Studies of mouse and rat hearts after MI have shown that cardiac lymphangiogenesis occurs in the infarct zone as well as in the non-infarcted regions of the heart [179,180,183]. The endogenous lymphangiogenic response, driven mainly by increased cardiac expression of VEGFC and VEGFD during the first months after MI, is characterized by lymphatic capillary expansion, especially in the infarct scar. However, lymphatic capillary remodeling is far from the whole story， which could not meet the need of cardiac repair. In fact, MI also rapidly leads to pre-collector and collector lymphatics slimming and rarefaction, which negatively impacts fluid and inflammation drainage from infarcted and adjacent non-infarcted areas [179]. As a consequence, cardiac repair remains severely limited for several months after MI. Thus, it is possible that, although lymphangiogenesis of the small, blind-ended lymphatic capillaries garners attention most readily because this change is most obvious, the most relevant therapeutic target may be sustaining function of pre-collector and collector lymphatics.

5. Lymphatic system in myocardial infarction 5.1. Myocardial edema, fibrosis and inflammation of MI The most important complication of AS is acute coronary syndrome, often culminating to MI. MI, the lack of blood supply to the heart, causes rapid death of cardiomyocytes. At the same time, due to myocardial microvascular permeability and the filtration rate excessing the lymph flow rate, fluid accumulates in the cardiac interstitial space, leading to the myocardial edema (Fig. 3b) [167,168]. Myocardial edema has been demonstrated in the clinic in many different cardiovascular conditions, including acute MI. Clinical detection of myocardial edema is based on cardiac MRI with late gadolinium enhancement T1 mapping, T2-weighted imaging, or more recently, native T2 mapping [169]. In experimental MI (induced by either permanent 105

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Fig. 3. Therapy of lymphatic system in AS and MI. a Therapy of lymphatic system in AS: AS is characterized by the formation of arterial plaques loaded with macrophages containing cholesterol (foam cells). Inducing lymphangiogenesis contributes to plaque regression through enhancing RCT, increasing emigration of inflammatory cells and reducing cholesterol deposition. b Therapy of lymphatic system in MI: MI is associated with adverse ventricular remodeling, including myocardial edema, fibrosis, inflammatory cells infiltration and impairing cardiac function. Meanwhile, MI induces the robust increase of lymphatic capillaries and decrease of pre-collecting and collecting LVs, leading to reduced cardiac lymphatic transport capacity. Inducing cardiac lymphangiogenesis can reduce adverse ventricular remodeling by improving myocardial fluid balance and immune cells draining.

(Fig. 3b) [187].

Inducing lymphangiogenesis therapy appears to be beneficial for post-MI healing, through improving the remodeling of pre-collector and collector lymphatics, leading to accelerated cardiac lymphatic transport capacity and resolution of myocardial edema and inflammation. Stimulation of lymph-angiogenesis has been proposed as a treatment to resolve peripheral edema of different aetiologies, including secondary lymphedema [184]. A study revealed that stimulation of cardiac lymphangiogenesis with VEGF-C improved clearance of the acute inflammatory response after MI by trafficking immune cells to draining mediastinal LNs in a dependent on LYVE-1 [185]. Deletion of LYVE-1 in mice, preventing docking and transit of leukocytes through the lymphatic endothelium, resulted in exacerbation of chronic inflammation and long-term deterioration of cardiac function. Henri et al. observed that intramyocardial-targeted delivery of VEGFR-3-selective designer protein VEGF-CC152S, using albumin-alginate microparticles, accelerated cardiac lymphangiogenesis in a dose-dependent manner and limited pre-collector remodeling post-MI [179]. As a result, myocardial fluid balance was improved, and cardiac inflammation, fibrosis, and dysfunction were attenuated. Moreover, Klotz et al. also showed that treatment with another VEGFR-3 specific recombinant protein VEFFCC156S reduced myocardial edema and pathological remodeling, leading to improving cardiac function [180]. In addition, Tatin et al. found that apelin-deficient mice exhibit abnormal dilated and leaky lymphatic vasculature associated a proinflammatory status after myocardial infarction, while the overexpression of apelin in ischemic heart was sufficient to restore a functional lymphatic vasculature and to reduce matrix remodeling and inflammation [186]. In sum, lymphatic system plays an important role in MI through lymphatic drainage and it alleviates myocardial edema and inflammatory response orchestrated by chemokines and recruited leukocytes elicited following ischemic injury

6. Drug delivery and therapeutic perspectives 6.1. Drug delivery potential of lymphatic system Owing to its specific uptake and transport property, the lymphatic system has become an appealing drug-delivery route. Drugs taken up by the lacteals, especially the lipophilic drugs, can bypass the first-pass metabolism of the liver, which increase the oral bioavailability of rapidly liver-metabolized drugs [188]. The role of lymphatic system in antigen trafficking could serve as sophisticated platform for next-generation vaccines. Specific antigens which are injected in the interstitial fluid, can be transported to the LNs, where the designed immune response can be triggered [189]. Recently, PEGylated polystyrene nanoparticles and PEGylated cationic liposomes are designed to reach the LNs and modulate the immune response [190,191]. This issue deserves attention in future research because of its important implications. On the one hand, drugs engineered to target lipids for transport into the LVs may avoid the liver metabolize and toxicity. On the other hand, the fact that the drugs first gain access to the system circulation, rather than the portal circulation where they could be detoxified by the liver, enhanced the danger they pose to human health. Thus, a better understanding is needed about lymphatic transport mechanism and the drugs properties. 6.2. Molecules in inducing lymphangiogenesis In addition, the lymphatic system plays an important role in the pathogenesis of cardiovascular diseases, such as lymphedema, AS and 106

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experimental models [165,194]. Percutaneous intramyocardial plasmid gene therapy with VEGFC has also been investigated in patients with coronary artery disease [201]. To enable selective stimulation of lymphangiogenesis, without concurrent effects on angiogenesis or on lymphatic vascular permeability, VEGFR3-selective VEGFC designer mutants have been developed [179,185]. Similarly, viral vectors that produce pre-activated recombinant forms of VEGFC or VEGFD (VEGFCΔNΔC and VEGFDΔNΔC, respectively) have been used, which have improved in vivo efficacy [208]. Besides these, other delivery vehicles require further research in the future. For example, modulation of cardiac lymphangiogenesis after MI by apelin [186] or by cell therapy with bone-marrow-derived endothelial progenitor cells [209] has also been reported.

MI. Inducing lymphangiogenesis can bring a serious of therapeutically benefits for these cardiovascular diseases. During the past 2 decades, many lymphangiogenic pathways have been found, including the VEGF family, collagen and calcium-binding EGF domain-containing protein 1 (CCBE), semaphorins (SEMAs) and neuropilins (NPRs), angiopoietins and TIE2, sphingosine 1-phosphate (S1P), BMP9 and ALK1, Notcb1 and ephrin B2 [192]. In addition, many growth factors and new molecules have also been found to stimulate lymphangiogenesis and have highly broadened our knowledge of lymphangiogenesis, such as fibroblast growth factor-2, insulin-like growth factor-1 and -2, hepatocyte growth factor, lymphotoxin-α, platelet-derived growth factor-Β, T box transcription factor Tbx1, claudin- like protein of 24 kDa (Clp24), liprin β1, Aspp1, Emilin1, Spred-1 and -2, the Rho GTPase Rac1, and the scaffold protein synectin [193,194]. In addition to the above-mentioned genes, several new players have been shown to act as regulators of the lymphatic vasculature, for instance, miR-126a [119], Apelin [195], and adrenomedullin [196]. Besides these, other pro-lymphangiogenic factors require further evaluation in the future. An important question is how one can take advantage of these pathways and molecules to manipulate lymphangiogenesis for therapeutic purposes. Most current therapeutic studies have been focused on the VEGFC–VEGFD–VEGFR3 pathway. VEGF-C gene transfer via adenoviruses (Ad), adeno-associated viruses, or naked plasmids, as well as the use of recombinant VEGF-C protein, stimulated the formation of new lymphatic capillaries and reduced edema, AS and MI in several preclinical animal models [165,185,197]. When VEGFC gene therapy was combined with LN transplantation in a mouse model of post-mastectomy lymphedema, the functional outcome was enhanced [198]. However, at present, most of the successful cardiovascular studies have been performed on mice and rat models. Whether can the methods and results from the small animal studies be used to complex and chronic human diseases? To resolve this question, the same treatment strategies can be further employed to treat cardiovascular pathologies in large animal and human models. Nowadays, there have been a few publications that being operated on pig models [199,200], demonstrating the lymphangiogenesis benefits for lymphedema. In pigs, both VEGFC and VEGFD gene therapy induced robust growth of lymphatic vessels in a surgically LN-evacuated groin area, causing a significant improvement in postsurgical lymphatic drainage [200]. Simultaneously, a gene therapy trial in human showed beneficial effects of VEGF-C therapy on angina score [201,202]. A phase I/IIa clinical trial (NCT01002430) is ongoing to evaluate dual angiogenic and lymphangiogenic adenoviral gene therapy with a recombinant VEGFD mutant (VEGFDΔNΔC) in patients with coronary artery disease and refractory angina [203,204]. The safety of the NOGAguided intramyocardial gene delivery has been confirmed, although an increase in anti-adenoviral titres was noted in the treated patients. In the near future, the research progresses of the cardiovascular field on large animals and even to clinical trials are readily available.

6.4. Safety and efficacy of the lymphangiogenic therapy Additional studies are needed to solve important questions concerning therapeutic aspects of lymphatic regulation. One remaining question is whether the pathological changes observed in lymphedema, AS or MI can be reversed by prolymphangiogenic therapy. This question is also intimately associated with the optimal duration of therapy. Indeed, control over growth factor concentrations in tissues and the duration of their expression and distribution are important for safe, therapeutic induction of lymphangiogenesis. Prolymphangiogenic therapy needs to be shut off at the right time to minimize pathological changes and to allow LVs remodeling. The small window of time may favor the application of a recombinant protein rather than viral gene transfer vectors, because the recombinant protein would allow more careful monitoring of dose and treatment duration. Using of the newly described imaging techniques such as near-infrared, optical frequencydomain imaging, or ultrasound array–based real-time photoacoustic microscopy (see above), can monitor of treatment efficacy, which allow quantitative and qualitative dynamic imaging of the LVs. Future therapies will be subject to scrutiny in terms of safety. Because lymphangiogenesis, angiogenesis, and neoplasia share many pathways and mechanisms, agents that target lymphangiogenesis may also affect blood vessels, inflammatory cells, tumor-associated macrophages, and tumor cells. The activation of lymphangiogenesis not only brings the therapeutically benefits, but also results in increasing exposure of LNs to inflammatory mediators, enhancing the immunological rejection and tumor metastasis [210]. Therefore, the safety and efficacy of the lymphangiogenic therapy must be carefully evaluated based on every cardiovascular disease and the designed therapeutic goals. When promoting lymphangiogenesis, the approach should be specific to lymphatic vessels in most cases, employing factors with minimal angiogenic or potentially deleterious activities. 7. Conclusions After years of ignorance, the lymphatic system has become an appealing target for research, and novel findings are being published at an increasing pace. New insights have been provided toward understanding the mechanisms bridging lymphatic system to cardiovascular diseases, such as AS and MI. It has been shown that LVs are present in cardiovascular system and atherosclerotic lesions, and provide an important pathway for cholesterol and immune cells transportation. Therefore, novel lymphangiogenic therapies could accelerate RCT and alleviate inflammatory responses, leading to regression or inhibition of AS, and resolving edema formation, inflammatory cell accumulation, and fibrosis during MI. Notwithstanding of the significant advances, it is clear that much further analysis is needed to dissect the role of the LVs in the pathogenesis and treatment of cardiovascular diseases, including lymphedema, obesity and metabolism diseases, dyslipidemia, hypertension, tissue inflammation, heart transplantation, AS and MI. In the future, the LVs deserve continuously increasing attention and should bring forward

6.3. Delivery of molecules in inducing lymphangiogenesis Over the past 20 years, research has shown that the success of therapeutic angiogenic approaches is largely determined by the mode of growth factor delivery [205]. Arguably, the same might be true for therapeutic lymphangiogenesis. Currently, most used treatment carriers for lymphangiogenesis are recombinant proteins or plasmids, but they have some disadvantages such as short duration and low efficacy. Other treatment vehicles are viral vectors for gene delivery [206] and biodegradable microparticles [207], which both effective carry and transduce the therapeutic factors into target cells, LNs or the myocardial wall. So far, most experimental studies of therapeutic lymphangiogenesis have focused on the delivery of the VEGFC gene or the VEGFC protein, which stimulates both lymphangiogenesis and angiogenesis in its proteolytically processed mature form. Promisingly, VEGFC gene therapy, delivered by adenoviruses (Ad) or adeno-associated viruses, was shown to reduce edema and AS in several preclinical 107

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promising strategies to reduce cardiovascular morbidity and mortality.

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