Functions and Regeneration of Mature Cardiac Lymphatic Vessels in Atherosclerosis,Myocardial Infarction,and Heart Failure

Abstract

Cardiac lymphatic vessels play a vital role in maintaining cardiac homeostasis both under physiological and pathological conditions. Clearer illustration of the anatomy of cardiac lymphatics has been achieved by fluorescence exhibition comparing to dye injection. Besides, identification of specific lymphatic markers in recent decades paves the way for researches in development and regeneration of cardiac lymphatics, such as VEGF-C/VEGFR-3, EphB4/ephrin-B2, Prox-1, Podoplanin, and Lyve-1. Knocking out each of these markers in mice model also reveals the signaling pathways instructing the formation of cardiac lymphatics. In the major cardiovascular disease series of atherosclerosis, myocardial infarction (MI), and heart failure, cardiac lymphatics regulate the transportation of extravasated proteins and lipids, inflammatory and immune responses, as well as fluid balance. Elementary intervention methods, such as lymphatic factor protein injection VEGF-C, are applied in murine MI models to restore or enhance functions of lymphatic vessels, achieving improvements in cardiac function. Also, data from our laboratory showed that intramyocardial EphB4 injection also improved lymphatic regeneration in mouse MI model. Therefore, we believe that enhancing functions and regeneration of mature cardiac lymphatic vessels in cardiovascular diseases is of great potential therapeutic meaning in the future.

Researches in cardiac lymphatic vessels tend to prosper in recent years. The confirmation of specific or relevant lymphatic markers accelerates the identification of lymphatic endothelial cells (LEC) and helps track the development and regeneration of lymphatic vessels. Those markers includes vascular endothelial growth factor C (VEGF-C)/vascular endothelial growth factor receptor 3 (VEGFR-3),^1,2 ephrin type-B receptor 4 (EphB4)/ephrin-B2,^3,4 prospero homeobox 1 (Prox-1),^5–7 podoplanin,^8,9 and lymphatic vessel endothelial hyaluronan receptor 1 (Lyve-1).^10,11 At the same time, the pathogenesis of an increasing number of cardiovascular diseases is found to connect with inflammatory or immunological etiologies, such as coronary atherosclerosis and myocardial infarction (MI) involving dendritic cells, monocytes, macrophages, and various cytokines.^12–14 Cardiac lymphatic system is an emerging hotspot in connecting pathogenesis of cardiovascular diseases with inflammatory or immunological etiologies. In this study, we summarize recent studies in cardiac lymphatic vessels, provide a comprehensive overview, and propose some prospective from basic researches to clinical applications.

Introduction of Mature Cardiac Lymphatic Vessels

Mammalian cardiac lymphatic vessels spread in cardiac walls, including the subendocardium, myocardium, and subepicardium, and even in the semilunar and atrioventricular valves.^15,16 The human left ventricular wall possesses about 30 lymphatic vessels/mm² making up about 0.53%–1.57% of all vessels.¹⁷ Cardiac lymphatic capillaries drain into lymphatic vessels, which converge into right and left cardiac lymphatic trunk and then unify as one main lymphatic vessel toward the root of aorta as the major pattern found in mammalian heart.¹⁸ Lymphatic capillaries are net-like in subendocardium, myocardium, and subepicardium, and the drainage direction is from subendocardium through myocardium to subepicardium.^18,19

The macroscopic detection methods of cardiac lymphatic vessels evolve from direct injection of dye to the living mammalian heart to using hydrogen peroxide, which dilates the subepicardial lymphatic vessels to distinguish from other tissues in subepicardium.^15,18,19 A recent study performed invasive cardiac lymphangiography by injecting fluorescent quantum dots intramyocardially in the apex of the heart along with intravenous injection of FITC-dextran to visualize blood vessels, and acquired lymphatic and blood vessels distribution by macroconfocal imaging,²⁰ which appears to be a better method to distinguish distribution of lymphatic vessels from blood vessels.

Major Lymphatic Factors in Lymphatic Development and Lymphangiogenesis

Lymphangiogenesis is an essential part in embryonic development, and participates in adult pathological process, including lymphedema, inflammatory disease, and tumors.²¹ Up to now, the most accepted origin of LEC is embryonic veins,²² while in some organs lymphatic vessels may derive differently, such as lumbar and dorsal skin regions.²³ In heart, Klotz et al.²⁴ indicates that mouse cardiac lymphatic vessels might have a heterogeneous cellular origin that part of the cardiac lymphatic network sprouts from a group of naive hematopoietic cells independently from veins, but their method of Pdgfrb-cre lineage tracing is challenged by Ulvmar et al. due to its unspecific targeting at neither yolk sac hemogenic endothelium nor yolk sac-derived erythromyeloid progenitors.²⁵ Therefore, the identity of cardiac LEC progenitor still remains to be clarified.²⁶

The identification of lymphatic system-specific markers such as VEGF-C/VEGFR-3,^1,2 EphB4/ephrin-B2,^3,4 Prox-1,^5–7 podoplanin,^8,9 and Lyve-1,^10,11 as well as advanced imaging methods such as confocal and multiphoton imaging, facilitates the recognition and research on embryology, anatomy, and physiology of lymphatic vessels in many organs,²⁷ including skin, mesentery, liver, and heart.^20,23,24 Roles of these markers in mammalian cardiac lymphatic vessels are discussed below (Table 1).

Table 1.

Lymphatic Markers and Their Main Roles or Applications in Cardiac Lymphatics Research

Marker	Murine model ^a	Intervention	Remark	References ^b
VEGF-C/VEGFR-3	Vegfr3^+/neo	VEGF-C protein injection post-MI	Intervention induced stronger lymphangiogenesis	24,20
EphB4/Ephrin-B2	None used in cardiac research	EphB4 protein injection post-MI	Intervention enhanced VEGFR-3 expression	Our preliminary data
Prox-1	Cardiac-specific Prox-1 knockout	None	Impaired cardiac growth, dilated cardiomyopathy	41,53
Podoplanin	None used in cardiac research	None	Relative to LEC-generating cells and profibrotic cells in cardiac tissues post-MI	56
Lyve-1	None used in cardiac research	None	Not required for formation or function of mature lymphatic vessels	46,47

Only referring to models used in cardiac researches.

Reference numbers are consistent with the main text.

LEC, lymphatic endothelial cell; MI, myocardial infarction.

Lymphatic development

VEGF-C/VEGFR-3

In embryonic lymphangiogenesis, VEGFR-3 interacts with VEGF-C or vascular endothelial growth factor D (VEGF-D) to induce the sprouting of lymphatic vessels from venous system.²⁸ In adult stage, VEGFR-3 is predominantly expressed in LECs and VEGF-C is essential for subsequent lymphatic vessel development, while VEGF-D seems not to participate in mature lymphatic vessel function or regeneration.^2,29 Hemizygous Vegfc^+/− mouse model has lymphatic vessel defects leading to cutaneous lymphatic vessel hypoplasia and abdominal chylous ascites, while nullizygous Vegfc^−/− mice all die embryonically.²⁸ Vegfr^3+/neo mouse model is a conditional heterozygous knockout model, similar to Vegfc^+/− mouse model, presenting with abdominal chylous ascites in adulthood, while Vegfr3^neo/neo phenotype is embryonically lethal.²⁸

EphB4/ephrin-B2

Ephrin system is another focus in lymphangiogenesis, and its bidirectional signaling pathway renders its mechanism more complicate.³⁰ Ephrin-B2, encoded by the gene Efnb2, is a transmembrane ligand binding its receptor EphB4, a tyrosine kinase receptor, which forms a unique bidirectional signaling pathway.³¹ The EphB4-dependent forward signaling pathway is initiated by ephrin-B2-binding EphB4, leading to autophosphorylation of EphB4 and activation of downstream signaling pathways.^32,33 The ephrin-B2-dependent reverse signaling pathway performs as the ephrin-B2 C-terminal PDZ-binding motif signaling back to the host cell with ephrin-B2.^3,34 Ephrin-B2 is highly expressed in LECs of collecting lymphatic vessel valves, and it functions in regulating formation and morphology of lymphatic valves as well as sprouting of lymphatic capillary.^4,35–37 The complete deletion of efnb2 in mature lymphatic vessels results in decreased number and deformation of the lymphatic luminal valves.³⁸ However, whether the forward or reverse signaling pathway plays a role in lymphangiogenesis remains controversial.^4,39

Prox-1

Prox-1 is a lymphatic-specific marker that works as a transcription factor in LEC derivation from blood endothelial cells.⁵ The Prox-1 heterozygous (Prox-1^+/−) mouse model is lethal in 2–3 days after birth, with chylous ascites, suggesting dysfunctional lymphatic vessels; while the knock out mouse model Prox-1^−/− is lethal at E14.5 without any lymphatic vessels.^5,40 Mouse embryos with cardiac-specific Prox-1 mutation exhibited cardiac growth impairment, fetal cardiomyocyte hypertrophy reduction, and ventricular septal defects.⁴¹

Podoplanin

Podoplanin is widely used as a marker in mature LECs.⁸ Separation of lymphatic and blood vessels is incomplete in podoplanin (gene T1α) knockout embryos with blood-filled lymphatics.^9,42 T1α/podoplanin^+/− mice slightly differ from wild-type mice, and their lymphatic vessels are compact, well-organized, and effective in lymphatic transportation.⁴³ Nullizygous T1α/podoplanin^−/− mice die at birth due to loss of T1α in alveolar type 1 cells causing respiratory failure.⁴⁴ Although podoplanin deficiency does not affect development of lymphatic system, it plays a vital role in later stage of formation of lymphatic networks.³¹ Podoplanin defects result in attenuated capability of lymphatic transport, diminished small lymphatic capillaries, and deficient absorption of dietary lipids due to absent intestinal lymphatic lacteals,³¹ while podoplanin overexpression in lymphatic cells led to long LEC extensions, increased migration, adhesion, and tubule formation.⁴³

Lyve-1

Lyve-1 is a lymphatic-specific marker that intensely expressed on lymphatic vessels, but in a bipolar distribution pattern of both the luminal and contraluminal surface of the lymphatic endothelium.¹⁰ Lyve-1 assists in lymphatic cell migration by binding to and then internalizing hyaluronan before transporting it to lymph nodes or liver for degradation.^10,11,45 Gale et al. reported that LYVE-1-deficient (LYVE-1^−/−) mice have a normal and functional lymphatic system, maybe due to the compensation by its close homologue CD44.⁴⁶ Luong et al. generated LYVE-1/CD44 double knockout mice, only to find that this model exhibited increased edema under carrageenan-induced paw inflammation condition compared with wild-type mice, otherwise the lymphatic systems are indistinguishable.⁴⁷ Therefore, Lyve-1 is a good marker for lymphatic vessel, but not required for formation or function of mature lymphatic vessels.

Lymphangiogenesis in mature heart

VEGF-C/VEGFR-3

Some studies used Vegfc^+/− mouse model to observe the adult cardiac lymphatic vessels and their functions or regeneration after injuries. Klotz et al. treated Vegfr3^lacZ/+ reporter mice with recombinant VEGF-C (C156S) post-MI, in VEGF-C-treated groups finding a stronger lymphangiogenic response in the border zone of the injured myocardial area in comparison to vehicle-treated controlled groups on day 7 post-MI; moreover, the cardiac function was significantly improved in VEGF-C-treated mice.²⁴ Furthermore, Henri et al. found that VEGF-C (C152S) enhanced cardiac lymphangiogenesis in a dose-dependent way and limited precollector remodeling post-MI by intramyocardial-targeted delivery using albumin-alginate microparticles.²⁰ Preliminary data from our laboratory also showed that after intramyocardial injection of protein VEGF-C in mouse MI model, compared with the control groups (sham-operation or MI only), the expression of Lyve-1 and VEGFR-3 increases in the border zone of infarcted myocardium 3 weeks post-MI. In human, it is well established that VEGF-C/VEGFR-3 can induce lymphangiogenesis and a missense mutation in VEGFR-3 causes lymphatic hypoplasia in primary human lymphedema.^48–50 Therapeutic value of VEGF-C/VEGFR-3 injection for cardiac lymphangiogenesis in human remains a promising pathway in treating cardiovascular diseases mentioned later in this review.

EphB4/ephrin-B2

Wang et al. suggested a link between EphB4/ephrin-B2 and VEGF-C/VEGFR-3 signaling systems.³ Stimulation of Eph-ephrin signaling with soluble recombinant ephrin-B2/Fc or EphB4/Fc fusion proteins could lead to the internalization of VEGFR-3 in cultured LECs.³ Also, VEGF-C-induced VEGFR-3 endocytosis was compromised in mouse Efnb2 knockout endothelial cell cultures, and VEGFR-3 downstream pathways, including Rac1, Akt, and Erk, were also diminished.³ The potential linking between ephrin-B2 and VEGFR-3 on LEC was illustrated by Koltowska et al.⁵¹ Our preliminary results showed that after intramyocardial injection of protein EphB4 in mouse MI model, compared with the control groups (sham-operation or MI only), the expression of VEGFR-3 increased evidently in the border zone of infarcted myocardium 3 weeks post-MI. However, whether the cardiac function improvement is mainly due to angiogenesis or lymphangiogenesis or both remains to be discovered. We summarize the potential signaling pathways considering VEGF-C/VEGFR-3 and ephrin-B2/EphB4 systems, and we believe that continuous in vitro study can help explore the combined therapeutic values of the two signaling systems and the mechanisms of their interactions (Fig. 1).

FIG. 1.

Simplified schematic illustration of potential signaling interactions between VEGF-C/VEGFR-3 and EphB4/ephrin-B2 systems. After injuries such as myocardial infarction, mature cardiac lymphatic endothelial cells may restart lymphangiogenesis through VEGFR-3 and ephrin-B2 downstream signaling pathways. The external interventions such as injection of proteins VEGF-C or EphB4 into borderline area of the infarcted myocardium could enhance the lymphangiogenesis process by VEGFR-3 internalization and/or phosphorylation of downstream signaling molecules such as Rac1, Akt, and ERK.

Prox-1

Bianchi et al. created a transgenic Prox-1-Cre-tdTomato reporter mouse model that made Prox-1 in an inducible manner for lymphatic vessel research.⁶ This model was used to trace the dendritic cell migration into lymphatic vessels and describe the lymphatic vasculature in situation of skin inflammation.⁶ Such inducible mouse models may provide a chance for studying mature cardiac lymphatic vessels in cardiovascular diseases. In the meanwhile, Prox-1 is also expressed in hepatocytes, cardiomyocytes, and pancreatic epithelial cells.^7,52 A number of cardiac-specific Prox-1 knockout mice survived, but with significant overexpression of fast-twitch genes in hearts and development of a deadly postnatal dilated cardiomyopathy.⁵³ Thus, costaining for both Prox-1 and CD31 is more convincing to localize lymphatic vessels, thanks to the coexpression of Prox-1 and CD31/PECAM-1, which present on both lymphatic and blood vessels.^54,55

Podoplanin

Podoplanin may not specifically express in mature cardiac lymphatic vessels. Cimini et al. used permanent coronary artery ligation mouse model to examine spatiotemporal expression of lymphatic markers under the circumstances of acutely and chronically MI.⁵⁶ The result suggested that podoplanin-positive cells in MI had a phenotypic and structural heterogeneity.⁵⁶ The podoplanin-positive cardiac cells may contribute to generating both LEC and profibrotic cells in scar formation.⁵⁶

Lyve-1

Although Lyve-1 is not required for formation or function of mature lymphatic vessels, stain with anti-Lyve-1 antibodies assists in revealing lymphatic vessels in the cornea, inflammatory liver disease, skin, and infarcted myocardium.^20,57–59

Roles of Lymphatic Vessels in Cardiovascular Disease

Generally, mature lymphatic vessels function in maintenance of body fluid balance, transportation of lipids, and circulation of immune cells. Recent studies reveal that functional cardiac lymphatic vessels play a vital role in pathogenesis of atherosclerosis, MI, heart failure, and many other cardiovascular diseases.^60,61 Atherosclerosis could be defined as arterial fatty streaks and plaques infused with macrophages-engulfed cholesterol.⁶² Atherosclerotic plaque progresses along with recruitment of monocyte-derived macrophages.⁶³ Plaque rupture could lead to occlusion of blood vessels such as coronary arteries.⁶⁴ Approximately 75% coronary thrombi result from plaque rupture.⁶⁵ One of the severe consequences is MI, with death of cardiac cells and formation of fibrosis scar.⁶⁶ On the one hand, inflammatory cells function in removing dead cells and connective debris away from the infarcted areas, and secrete cytokines and chemokines for tissue regeneration; on the other hand, postinfarction inflammation can also lead to cardiac remodeling resulting in deteriorative cardiac function.⁶⁷ Pathologic left ventricular remodeling could result in heart failure, a chronic inflammation state with high levels of circulating inflammation cells and cytokines.^68,69 To conclude, metabolic waste accumulation in the endothelium of coronary arteries induces atherosclerotic plaques, rupture of which leads to cardiac ischemia; meanwhile, metabolic wastes accumulation due to cardiac lymphatic vessel injuries is prone to induce proliferation of fibroblasts, which results in fibrosis after MI and cardiac dysfunction ending up as heart failure.^18,60,70

In cardiovascular diseases focused on atherosclerosis, MI, and heart failure, we summarize cardiac lymphatic vessel functions as three parts in detail: extravasated proteins and cholesterol transport, inflammation and immune response, and fluid balance.

Extravasated proteins and cholesterol transport

A potential treatment for atherosclerosis is to reduce cholesterol-loaded macrophage burden within artery wall by reverse cholesterol transport (RCT).⁶² RCT means the transport of cholesterol on HDL particles from extravascular tissues to plasma and the return to the liver for bile excretion, and macrophage RCT refers specifically to the removal of cholesterol from macrophage cholesterol ester stores.^63,71 In the process of RCT, pre-β-HDL in apolipoprotein A1 (APOA1), through the action of ABCA1 and ABCG1 transporters, removes cholesterol from the macrophages in the affected site through blood to the liver.⁷²

Recent studies indicated that lymphatic vessels could transport HDL from interstitial tissues to the bloodstream.^63,73 Martel et al. quantitatively tracked RCT by injection of [³H]-cholesterol-loaded macrophages upstream of blocked or absent lymphatic vessels using a surgical model and a genetic model (Chy mice, carrying one mutant allele of the VEGFR-3) and found that macrophage RCT was markedly impaired.⁶³ They also demonstrated that [²H]-Cholesterol was retained in the aorta of anti-VEGFR3 antibody-treated ApoE-deficient mice.⁶³ Lim et al. treated ApoE^−/− mice with a local injection of recombinant VEGF-C, resulting in reduced accumulation of cholesterol in the skin and enhanced RCT.⁷³ Therefore, lymphatic vessels play a vital role in cholesterol clearance. So, improving RCT to reduce macrophage burden and cholesterol accumulation within the artery wall is a promising treatment option for atherosclerosis.⁶²

Less lymphangiogenesis is observed in coronary atherosclerosis, which leads to interstitial fluid accumulation in blood vessel walls, thus affecting oxygen supplement and microcirculation that contributes to atherosclerotic plaque rupture.⁷⁴ Alternatively, factors that promote lymphangiogenesis are released under the situation of atherosclerosis-induced blood vessel injury.⁷⁵ These evidences indicate that lymphatic vessels may contribute to alleviating the process of atherosclerosis by improving RCT or other undiscovered mechanisms. It is worth researching on lymphangiogenesis in atherosclerotic models.

Inflammation and immune response

Besides macrophage-recruited cholesterol core, the atherosclerotic plaques also contain dendritic cells, monocytes, macrophages, and T-cells, secreting or responding to cytokines such as IFN-γ, IL-4, IL-10, IL-13, and so on.^12–14,76 IFN-γ-induced proinflammatory M1 macrophages and IL-4/IL-13-induced anti-inflammatory macrophages are identified in atherosclerotic plaques.⁷⁷ Studies showed that, among the cytokines and chemokines, plaque-destabilizing factors might include IFN-γ, IL-12, IL-18, macrophage migration inhibitory factor, and monocyte chemotactic protein-1, while stabilizing factors were similar to IL-10 and TGF-β.^78–80 Dendritic cells, as antigen-presenting cells, are also involved in the process of atherosclerosis, contributing to early foam cell formation, regulating lipid metabolism and controlling T-cell responses.⁸¹ Initiation of inflammation cascade produces vulnerable plaques, and its rupture may lead to MI, which suggests that inflammation and immune response play a critical role in atherosclerosis.^82,83

In peripheral tissues, lymphatic vessels are crucial part for trafficking of leukocytes and soluble antigens to their draining lymph nodes.⁸⁴ The new lymphatic vessels transport the inflammatory cells and cytokines to lymph node. The inflammatory cells are sensitized in lymph nodes and then circulate in lymphatics and blood vessels. The activated inflammatory cells can be back transported into the injured site to form a circle of immune response, which magnifies the function of inflammatory cells and cytokines, leading to atherosclerosis, persistent endothelium proliferation, and reconstitution of blood vessels.⁸⁴ We assume that this process also exists in mature cardiac environment, as lymphatic vessels participate in the pathogenesis of atherosclerosis and MI through the role in the process of inflammation and immune response.

After MI, cardiac remodeling is in a sequence of myocardium necrosis, granulation tissue formation, and fibrosis. In the early stage, recruited inflammatory cells clear the dead cells and matrix debris and stimulate regenerative processes, but also cause adverse cardiac remodeling.⁶⁷ Some studies reported that the regeneration of lymphatic vessels was later than blood vessels in the early stage of MI.⁸⁵ In the early stage of granulation tissue formation, neolymphatic vessels bud from existing lymphatic vessels, but the origin of the LECs remains controversial. During the stage of fibrosis and scar formation, neolymphatic vessels carry the major responsibility of draining interstitial fluid and proteins due to lack of blood vessels. Some studies indicated that improvement of lymphatic vessels after MI could assist in reducing interstitial edema and eliminating metabolic wastes of hypoxemia, thus helping maintain cardiac function.⁸⁶ Hence, to promote the lymphangiogenesis may be an ideal therapy to improve cardiac function after MI.⁸⁷

Fluid balance

Physiologically, water and electrolytes filter out of the blood capillaries into myocardial interstitium to form interstitial fluid and lymphatic vessels recycle water, lipids, and proteins back to the blood capillaries to maintain dynamic equilibrium of interstitial fluid metabolism.^60,84 Diseases such as tumors, inflammation, trauma, and ischemic cardiac disease can obstruct the lymphatic vessels, resulting in interstitial fluid accumulating in the cardiac interstitium that causes myocyte edema and pericardial effusions.^19,88 Edematous myocardium may compress the cardiac conduction system to induce arrhythmias, and the cardiac contractility reduction aggravates edema in a vicious cycle.^19,89 Now studies in cardiac lymphatic vessel of mammalian animals mainly measure cardiac muscle edema using MRI to describe the function of lymphatic vessels grossly, which is a nonspecific method.²⁴ We suppose that there will be more specific methods to measure functions of cardiac mature lymphatic vessels macroscopically, as blood vessel catheterization does.

What We Expect

Mature lymphatic vessels carry the role of removing interstitial fluid, inflammatory cells, cytokines, chemokines, and metabolic wastes from the injured tissue sites. In cardiovascular diseases such as atherosclerosis and MI, previous studies focused more on the resupply of oxygen or nutrients to promote the regeneration of blood vessels or cardiomyocytes, but insufficient attention was put on the process of wastes or debris transport. Lymphatic vessels could play a critical role in the part of removal. A few studies lead a pace in demonstrating that enhancing lymphangiogenesis through VEGF-C/VEGFR-3 pathway may promote cardiac recovery from MI.^20,24 But the mechanisms behind are to be discovered. Lymphatic system is important in inflammation and immune response.⁹⁰ And in recent days, pathogenesis of cardiovascular diseases involving inflammation and immune system is emerging in a large number. Building a connection between these two phenomena could be an exciting discovery in the big picture of pathogenesis of cardiovascular diseases, especially for atherosclerosis, MI, and heart failure.

For the therapeutic perspective, whether endothelial progenitor cell transplantation or improvement factor supplementation is effective remains to be studied. For cell therapy, in vitro studies showed that endothelial progenitor cells could differentiate into lymphatic LECs.^23,91,92 Transplantation of mesenchymal endothelial progenitor cells to mice could reduce myocardial edema and fibrosis, thus improving cardiac contractility; however, evidences lack in lymphangiogenesis from these cells in border zone of infarcted myocardium.⁹³ There are more convincing research results from lymphatic-inducing protein treatments in MI. Mice treated with abdominal injection of VEGF-C had significant lymphangiogenesis after MI and improvement of the cardiac function.²⁴ And in rats, intramyocardial injection of VEGF-C contained in microparticles also showed significant lymphangiogenesis, reduction in myocardial edema, relief of inflammation, and fibrosis, thus resulting in an improvement of cardiac function.²⁰ Besides VEGF-C, results from our laboratory suggested that intramyocardial injection of protein EphB4 also promoted the lymphatic vessel regeneration post-MI, offering a potential new choice of lymphatic-inducing protein that may exert usefulness in therapeutics. However, cardiac function improvement after EphB4 injection needs further researches to distinguish between the effects of angiogenesis or lymphangiogenesis. A few studies described that local injection of adenoviruses encoding VEGF-C and VEGF-D could treat lymphedema.^94,95 The same method could be applied to intramyocardial injection facilitated by NOGA (Biosense-Webster) electroanatomical mapping and injection catheter for detecting and targeting the border areas of the infarction and delivering the protein treatment simultaneously in the future.⁹⁶

Footnotes

Acknowledgments

This work was supported by the Shanghai Health and Family Planning Commission (20164Y0036); Young Investigator Fund of Zhongshan Hospital, Fudan University (2016ZSQN05).

Author Disclosure Statement

No competing financial interests exit for all the authors.

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