Cardiac Lymphatics Research Articles

Lymphatic-macrophage crosstalk during neonatal mouse heart regeneration and transition to fibrotic repair

Abstract Background In adult mice, myocardial infarction (MI) activates the cardiac lymphatics, which undergo sprouting angiogenesis (lymphangiogenesis) and function to drain interstitial fluid and traffic macrophages to mediastinal lymph nodes (MLNs). This prevents oedema and reduces inflammatory immune cell content to improve cardiac function. Purpose Given the importance of the adult cardiac lymphatics in macrophage clearance after injury, we investigated their role across the neonatal "regenerative window". At post-natal day 1 (P1) neonatal mice fully regenerate their heart following MI, in a macrophage-dependent manner, whereas equivalent injury at day 7 (P7) leads to scarring driven by pro-fibrotic macrophages. We hypothesised that lymphatics respond and function differently during this "window," to clear macrophage-specific subtypes depending upon their requirement for regeneration versus fibrotic repair. Methods & Results Extensive lymphatic growth and sprouting was evident in intact neonatal hearts until P16, with strain-dependent developmental differences. The response to injury revealed limited lymphangiogenesis and minimal clearance of macrophages from P1 compared to P7 infarcted hearts, as determined by adoptive transfer experiments and monitoring of cleared macrophages to MLNs. This is coincident with maturation of lymphatic endothelial cell junctions across the neonatal period via transition from "zipper" (impermeable) to "button" (permeable) -type junctions. To gain molecular insight into the mechanisms underpinning lymphatic endothelium macrophage interactions at P1 versus P7, we generated unbiased single cell RNA sequencing datasets from neonatal heart samples after MI and observed altered signalling between lymphatic endothelial cells and macrophages across the neonatal period, including altered expression of the lymphangiocrine factor Reelin (RELN). Finally, in mice lacking the lymphatic endothelial receptor-1 (LYVE1), that exhibit impaired transmigration of interstitial macrophages to lymphatic vessels, magnetic resonance imaging (MRI) revealed a surprising impaired functional outcome in P1 mice 28 days post-MI. Given our observations that pro-regenerative macrophages at P1 are not trafficked, this suggested a distinct role for LYVE1 in tissue-resident (TR) macrophages, consistent with its expression in developing and post-natal hearts. Macrophage-specific deletion of Lyve1 during neonatal heart injury revealed impaired heart regeneration, characterised by reduced neovascular response and function, suggesting a hitherto unappreciated role for LYVE1 in regulating the pro-regenerative function of TR macrophages. Conclusions Collectively, we reveal that cardiac lymphatics are developmentally compromised for immune cell clearance in early neonates, which enables retention of pro-regenerative TR macrophages, and that LYVE1 plays an essential role in TR macrophages enabling heart regeneration via the induction of coronary angiogenesis.

Secretomes from human epicardial adipocytes modulate atrial lymphatics and atrial fibrosis

Abstract Background Cardiac lymphatics maintains myocardial fluid and immune cell homeostasis. Downregulation of cardiac lymphatics leads to cardiac dysfunction through impairment of inflammation and edema. On the other hand, it is well known that adipose tissue accumulation causes lymphatic dysfunction and impairs lymphedema. Purpose We evaluated the expression of atrial lymphatics in human left atrial appendage (LAA) histologically and biologically. Subsequently, we examined secretomes from epicardial adipose tissue (EAT)-derived adipocytes and validated if they change atrial lymphatics expression using ex vivo Organo-culture system. Methods Human LAA samples were collected from forty-two patients during cardiovascular surgery. They were categorized into the sinus rhythm (SR) group (n=16, 73.1±10.4 years), the paroxysmal AF (PAF) group (n=13, 69.8±12.6 years) and the persistent AF (PeAF) group (n=13, 71.2±6.5 years). Human EAT were also excised from patients with and without AF (SR; n=5, AF; n=11) during cardiovascular surgery. Preadipocytes were extracted from EAT by homogenization and collagenase treatment, and cultured to confluence. After differentiation and maturation to adipocytes, cell culture supernatant (CCS) was collected, and was dropped onto the epicardial side of left atrial tissues excised from 8 weeks old male rats for 7 days using an ex vivo organo-culture system. Results In human LAA samples, mRNA expression of lymphangiogenesis-related genes (LYVE1, PROX1, VEGFC, and VEGFR3) was significantly lower in the PeAF group compared to the SR group (p=0.0370, <0.0001, 0.0157, and 0.0009). The number of LYVE1-positive atrial lymphatic endothelial cells (LECs) was also significantly lower in the PeAF group compared to the SR group and the PAF group (p=0.0186 and 0.038). There was a negative correlation between the number of LYVE1-positive atrial LECs and the area percentage of atrial myocardial fibrosis (r=-0.5350, p<0.001). In the experiment using organo-culture system, lymphangiogenesis-related genes expression (Lyve1, Prox1, Vegfc, and Vegfr3) of rat atria treated with CCS of adipocytes from AF patients was significantly lower than those from SR patients (p=0.0391, 0.0100, 0.0034, and 0.0338). Furthermore, the CCS of adipocytes from AF patients contained higher protein levels of leptin, which is known to suppress lymphangiogenesis (p=0.0357). Leptin treatment (100ng/ml) on rat atria also decreased lymphangiogenesis-related genes expression such as Lyve1, Prox1, Vegfc, and Vegfr3 (p=0.0413, < 0.0001, <0.0001, and 0.0190). Conclusions Our study with human LAA demonstrated that atrial lymphatics interact with atrial myocardial fibrosis/progression of AF. Furthermore, our findings indicate that secretomes from EAT-derived adipocytes regulate atrial lymphatics expression, and leptin plays an important role in modulating atrial lymphangiogenesis.

Disease-Specific Alteration of Cardiac Lymphatics: A Review from Animal Disease Models to Clinics.

For many years, the significance of cardiac lymphatic vessels was largely overlooked in clinical practice, with little consideration given to their role in the pathophysiology or treatment of cardiac diseases. However, recent research has brought renewed attention to these vessels, progressively illuminating their function and importance within the realm of cardiovascular science. Experimental studies, particularly those utilizing animal models of cardiac disease, have demonstrated a clear relationship between cardiac lymphatic vessels and both the pathogenesis and progression of these conditions. These findings have prompted a growing interest in potential therapeutic applications that specifically target the cardiac lymphatic system. Conversely, while clinical investigations into cardiac lymphatics remain limited, recent studies have begun to explore their identification through specific surface markers, as well as the expression dynamics of lymphangiogenic factors. These studies have increasingly highlighted associations of lymphatic dysfunction with inflammation and fibrosis, both of which negatively impact cardiac function and remodeling across various pathological states. Despite these advances, comprehensive reviews of the current knowledge regarding the cardiac lymphatic vasculature, particularly within specific disease contexts, remain scarce. This review aims to address this gap by providing a detailed synthesis of existing reports, encompassing both animal model research and studies on human clinical specimens, with a special focus on the role of cardiac lymphatic vessels in different disease states.

Cardiac lymphatics undergo distinct remodeling during hypertrophic and non-hypertrophic pregnancy.

Lymphatic vessels of the heart undergo dynamic remodeling in response to physiological and pathological cardiovascular events such as development, adult cardiac maintenance and injury repair. During pregnancy, the cardiovascular system undergoes physiological changes to meet the increased demand of blood supply to the fetus. These extensive physiologic changes make pregnancy and delivery a high-risk period in a woman's life. However, whether and how cardiac lymphatics change during pregnancy is largely undefined. Therefore, we used whole mount immunofluorescent labeling and quantitative morphometric analysis to characterize the changes in cardiac lymphatic vasculature during pregnancy using two genetically distinct inbred mouse strains, C57BL/6J and BALB/cJ. Compared to age-matched, non-pregnant C57BL/6J control mice, the hearts of C57BL/6J dams in late-pregnancy (gestation day 17.5 (G17.5)) undergo physiologic hypertrophy. However, there were no significant changes in the cardiac lymphatic vasculature. In contrast, BALB/cJ mice do not exhibit pregnancy-induced cardiac hypertrophy at G17.5 compared to age-matched, non-pregnant mice. Yet interestingly, the cardiac lymphatic vasculature of pregnant BALB/cJ dams undergoes extensive morphological changes, including decreased lymphatic length, number of endpoints, and vessel branchpoint junctions on the ventral side of the heart. These findings underscore the complexity of genetic and physiologic factors that contribute to the heterotypic remodeling of cardiac lymphatics during late pregnancy.

The lymphatic drainage of the goat heart.

The detailed knowledge of the morphological structure, drainage pathways and patterns, the first tier lymph node of the cardiac lymphatic and its relationship with the circulatory system has not yet been completed. Although, the cardiac lymphatics had been described with renewed interest in past years, which was attributed to the transparent nature of lymphatic vessels that are difficult to be observed. In this study, cardiac lymphatics of the goat heart were perfused by a direct microinjecting technique with a radiopaque mixture. This demonstrated the subepicardial and subendocardial lymph capillary networks communicating with transmyocardial lymph vessels and then entering to subepicardial collecting lymph vessels that were directed toward the atrio-ventricular sulcus where they form a confluence from which the main cardiac lymph channels. We also found that: 1) the quantity and caliber of collecting lymph vessels varied in each goat heart; 2) drainage patterns of lymph vessels in the goat heart were different in individuals; 3) the first tier lymph node that each major lymph vessel drained to was different; and 4) multiple lymphatic-venous anastomosis sites have been confirmed to exist in the subepicardium of the left and right ventricles of each goat heart, which may be the morphological structure to accelerate the return of intercellular fluid to the venous system during excessive exercise of the heart. Therefore, the information may provide reference for further study in physiological and pathological conditions of the human heart.

Cardiac Lymphatics and Therapeutic Prospects in Cardiovascular Disease: New Perspectives and Hopes.

The lymphatic system is the same reticular fluid system as the circulatory system found throughout the body in vascularized tissues. Lymphatic vessels are low-pressure, blind-ended tubular structures that play a crucial role in maintaining tissue fluid homeostasis, immune cell transport, and lipid absorption. The heart also has an extensive lymphatic network, and as research on cardiac lymphatics has progressed in recent years, more and more studies have found that cardiac lymphangiogenesis may ameliorate certain cardiovascular diseases, and therefore stimulation of cardiac lymphangiogenesis may be an important tool in the future treatment of cardiovascular diseases. This article briefly reviews the development and function of cardiac lymphatic vessels, the interaction of cardiac lymphatic vessels with cardiovascular diseases (including atrial fibrillation, coronary atherosclerosis, and heart failure), and finally discusses the therapeutic potential of targeted cardiac lymphatic therapy for cardiovascular diseases.

Abstract We072: Effects of electronic cigarettes on mouse cardiac lymphatic vessels after TAC surgery

Introduction: Heart failure is a public health concern worldwide. It is known that tobacco smoking is a risk factor of heart failure. Tobacco smoking also causes inflammation reaction and lymphatic dysfunction in the lung. However, the effects of electronic cigarettes (ECIG), as a new method of smoking, on the heart and cardiac lymphatic vessels (lymphatics) are not clear yet. The lymphatic vessels play an important role in maintaining cardiac function by modulating fluid homeostasis and transporting macromolecules. It has been reported that cardiac lymphatic vessel formation limits pressure overload-induced heart failure in C57Bl/6 but not in Balb/c mice. How cardiac lymphatics might contribute to the pathogenesis induced by ECIG is not known. Research Questions: The roles of cardiac lymphatic vessels in responses to ECIG exposure are not investigated. I hypothesize that the toxic effects of ECIG on the heart are partially caused by damaging the cardiac lymphatic vessels. Aim: To determine the effects of ECIG exposure on cardiac lymphatics after Transverse aortic constriction (TAC). Methods: I perform TAC surgery on mice as a heart hypertrophy and chronic heart failure model. Mice after TAC or Sham surgery are treated with different vaping components from ECIG: ECIG, propylene glycol and glycerin (PGVG) as vehicle, and air. After 4 weeks of treatment, hearts were harvested for histological analyses. The lymphatic vessel coverage percentage and morphology were examined by immunostaining. Results: Prior research suggested that genetic background in mice might affect the responses of cardiac lymphatic vessels to TAC-induced heart failure. I have established the TAC model using CD-1 mice. My results showed that CD-1 mice treated with PGVG after TAC do not show a significant difference in cardiac lymphatic vessel density compared to Sham group (p value=0.5951). ECIG treatment also did not affect the lymphatic density significantly compared to PGVG treated mice after sham operation (p value=0.8937). ECIG treated mice after TAC surgery showed a trend of increased lymphatic coverage and more mice are still being processed and will be presented at the conference. Conclusion: PGVG treated CD-1 mice do not show changes in lymphatic vessel density 4 weeks post TAC. Longer time points and more mouse numbers are needed to compare the effects of PGVG and ECIG after TAC induced hypertrophy and heart failure. This research is supported by California TRDRP grant.

Abstract 1071: TGFβ Signaling Negatively Regulates Cardiac Lymphangiogenesis Following Myocardial Ischemia

Cardiovascular disease is the leading cause of death worldwide. Despite high prevalence, standard treatments have made little progress, and developing novel therapeutic treatments to improve cardiac diseases is the top priority of the field. Lymphatics are essential in maintaining tissue-fluid homeostasis, clearing cell debris, and trafficking immune cells. Over recent years, cardiac lymphatics have emerged as a potential target for treatment of cardiovascular diseases, as therapeutic lymphangiogenesis is shown to improve cardiac function following myocardial infarction (MI). However, pathologic lymphangiogenesis induced by MI is reported to be dysfunctional, suggesting cellular or molecular changes occurring in MI directly impact the structure and function of cardiac lymphatics. However, the underlying mechanisms leading to these changes have not been fully explored. In this study we propose TGFβ, a profibrotic signaling molecule increased in ischemic heart diseases, negatively regulates cardiac lymphangiogenesis and lymphatic function following MI. We generated mice with conditional deletion of TGFβ receptor in Lymphatic endothelial cells (LECs) and found that these conditional null (LEC-DKO) mice showed improved post-MI cardiac function and increased lymphangiogenesis compared to controls. Mechanistically, we found that LECs with TGFβ treatment showed increased Endothelial to Mesenchymal Transition (EndMT), and impaired junction integrity. Bulk RNAseq results confirmed these findings as genes involved in EndMT were highly upregulated, and LEC marker related genes were significantly downregulated in LECs in the presence of TGFβ. More importantly, GSEA revealed metabolic reprogramming in LECs treated with TGFβ, with significantly increased expression in glycolysis related genes, but significant downregulation of genes related to fatty acid oxidation. These data provide a possible mechanism by which TGFβ signaling induces LEC metabolic switch to impede lymphangiogenesis and promote cardiac dysfunction following MI, and inhibition of TGFβ signaling in LECs could provide a novel therapeutic strategy to improve lymphatic vascular integrity and lymphangiogenesis, thereby improving cardiac function following MI.

Role of Lymphangiogenesis in Cardiac Repair and Regeneration.

This article highlights the importance of the structure and function of cardiac lymphatics in cardiovascular diseases and the therapeutic potential of cardiac lymphangiogenesis. Specifically, we explore the innate lymphangiogenic response to damaged cardiac tissue or cardiac injury, derive key findings from regenerative models demonstrating how robust lymphangiogenic responses can be supported to improve cardiac function, and introduce an approach to imaging the structure and function of cardiac lymphatics.

Tbx1 orchestrates an immune niche that safeguards a broken heart

Cardiac lymphatics cooperate with the reparative immune response in myocardial healing after infarction. In this issue of Immunity, Wang and colleagues discover a mechanism underlying this cooperation, dependent on the transcription factor Tbx1 and responsible for the creation of an immunosuppressive niche that mitigates autoimmunity.

Single-nuclei multiomic analyses identify human cardiac lymphatic endothelial cells associated with coronary arteries in the epicardium

Cardiac lymphatic vessels play important roles in fluid homeostasis, inflammation, disease, and regeneration of the heart. The developing cardiac lymphatics in human fetal hearts are closely associated with coronary arteries, similar to those in zebrafish hearts. We identify a population of cardiac lymphatic endothelial cells (LECs) that reside in the epicardium. Single-nuclei multiomic analysis of the human fetal heart reveals the plasticity and heterogeneity of the cardiac endothelium. Furthermore, we find that VEGFC is highly expressed in arterial endothelial cells and epicardium-derived cells, providing a molecular basis for the arterial association of cardiac lymphatic development. Using a cell-type-specific integrative analysis, we identify a population of cardiac lymphatic endothelial cells marked by the PROX1 and the lymphangiocrine RELN and enriched in binding motifs of erythroblast transformation specific (ETS) variant (ETV) transcription factors. We report the invivo molecular characterization of human cardiac lymphatics and provide a valuable resource to understand fetal heart development.

Three-dimensional spatial quantitative analysis of cardiac lymphatics in the mouse heart.

Three-dimensional (3D) microscopy and image data analysis are necessary for studying the morphology of cardiac lymphatic vessels (LyVs) and their association with other cell types. We aimed to develop a methodology for 3D multiplexed lightsheet microscopy and highly sensitive and quantitative image analysis to identify pathological remodeling in the 3D morphology of LyVs in young adult mouse hearts with familial hypertrophic cardiomyopathy (HCM). We developed a 3D lightsheet microscopy workflow providing a quick turn-around (as few as 5-6 days), multiplex fluorescence detection, and preservation of LyV structure and epitope markers. Hearts from non-transgenic and transgenic (TG) HCM mice were arrested in diastole, retrograde perfused, immunolabeled, optically cleared, and imaged. We built an image-processing pipeline to quantify LyV morphological parameters at the chamber and branch levels. Chamber-specific pathological alterations of LyVs were identified, and significant changes were seen in the right atrium (RA). TG hearts had a higher volume percent of ER-TR7+ fibroblasts and reticular fibers. In the RA, we found associations between ER-TR7+ volume percent and both LyV segment density and median diameter. This workflow and study enabled multi-scale analysis of pathological changes in cardiac LyVs of young adult mice, inviting ideas for research on LyVs in cardiac disease.

The Lymphatic Vasculature in Cardiac Development and Ischemic Heart Disease.

In recent years, the lymphatic system has received increasing attention due to the fast-growing number of findings about its diverse novel functional roles in health and disease. It is well documented that the lymphatic vasculature plays major roles in the maintenance of tissue-fluid balance, the immune response, and in lipid absorption. However, recent studies have identified an additional growing number of novel and sometimes unexpected functional roles of the lymphatic vasculature in normal and pathological conditions in different organs. Among those, cardiac lymphatics have been shown to play important roles in heart development, ischemic cardiac disease, and cardiac disorders. In this review, we will discuss some of those novel functional roles of cardiac lymphatics, as well as the therapeutic potential of targeting lymphatics for the treatment of cardiovascular diseases.

Cardiac Lymphatic Insufficiency Leads to Diastolic Dysfunction Via Myocardial Morphologic Change

Although cardiac lymphatic vessels have received increasing attention in recent years, there is still a knowledge gap between cardiac lymphatics and heart homeostasis in a normal heart. In the present study, we established a mouse model of cardiac lymphatic insufficiency ablating cardiac lymphatic collector vessels to reveal the crucial role of cardiac lymphatic vessels in maintaining cardiac homeostasis and the impact on cardiac function both in physiological and pathologic settings. Furthermore, therapeutic lymphangiogenesis improved the adverse effect on cardiac morphologic changes and functions. These findings suggest that the cardiac lymphatic system would be a novel therapeutic target for heart disease.

Lymphatic insufficiency leads to distinct myocardial infarct content assessed by magnetic resonance TRAFFn, T1ρ and T2 relaxation times

The role of cardiac lymphatics in the pathogenesis of myocardial infarction (MI) is unclear. Lymphatic system regulates cardiac physiological processes such as edema and tissue fluid balance, which affect MI pathogenesis. Recently, MI and fibrosis have been assessed using endogenous contrast in magnetic resonance imaging (MRI) based on the relaxation along a fictitious field with rank n (RAFFn). We extended the RAFFn applications to evaluate the effects of lymphatic insufficiency on MI with comparison to longitudinal rotating frame (T1ρ) and T2 relaxation times. MI was induced in transgenic (TG) mice expressing soluble decoy VEGF receptor 3 that reduces lymphatic vessel formation and their wild-type (WT) control littermates for comparison. The RAFFn relaxation times with rank 2 (TRAFF2), and rank 4 (TRAFF4), T1ρ and T2 were acquired at time points 0, 3, 7, 21 and 42 days after the MI at 9.4 T. Infarct sizes were determined based on TRAFF2, TRAFF4, T1ρ and T2 relaxation time maps. The area of differences (AOD) was calculated based on the MI areas determined on T2 and TRAFF2, TRAFF4 or T1ρ relaxation time maps. Hematoxylin–eosin and Sirius red stained histology sections were prepared to confirm MI locations and sizes. MI was detected as increased TRAFF2, TRAFF4, T1ρ and T2 relaxation times. Infarct sizes were similar on all relaxation time maps during the experimental period. Significantly larger AOD values were found together with increased AOD values in the TG group compared to the WT group. Histology confirmed these findings. The lymphatic deficiency was found to increase cardiac edema in MI. The combination of TRAFF2 (or TRAFF4) and T2 characterizes MI and edema in the myocardium in both lymphatic insufficiency and normal mice without any contrast agents.

The ebb and flow of cardiac lymphatics: a tidal wave of new discoveries.

The heart is imbued with a vast lymphatic network that is responsible for fluid homeostasis and immune cell trafficking. Disturbances in the forces that regulate microvascular fluid movement can result in myocardial edema, which has profibrotic and proinflammatory consequences and contributes to cardiovascular dysfunction. This review explores the complex relationship between cardiac lymphatics, myocardial edema, and cardiac disease. It covers the revised paradigm of microvascular forces and fluid movement around the capillary as well as the arsenal of preclinical tools and animal models used to model myocardial edema and cardiac disease. Clinical studies of myocardial edema and their prognostic significance are examined in parallel to the recent elegant animal studies discerning the pathophysiological role and therapeutic potential of cardiac lymphatics in different cardiovascular disease models. This review highlights the outstanding questions of interest to both basic scientists and clinicians regarding the roles of cardiac lymphatics in health and disease.

Lymphatic endothelial sphingosine 1-phosphate receptor 1 enhances macrophage clearance via lymphatic system following myocardial infarction.

Lymphatic endothelial cell homeostasis plays important roles in normal physiological cardiac functions, and its dysfunction significantly influences pathological cardiac remodeling after myocardial infarction (MI). Our results revealed that sphingosine 1-phosphate receptor 1 (S1pr1) expression in cardiac lymphatic endothelial cells (LECs) was sharply changed after MI. It has been shown that S1pr1 tightly controlled LEC functions and homeostasis. We thus hypothesized that lymphatic endothelial S1pr1 might be involved in post-MI cardiac remodeling. We generated LEC-conditional S1pr1 transgenic mice, in which S1pr1 expression was reduced in cardiac LECs. We performed the left anterior descending coronary artery (LAD) ligation operation to induce MI in these mice. Cardiac functions and remodeling were examined by echocardiography analysis and serial histological analysis. Meanwhile, we performed adoptive cell transfer experiments to monitor macrophage trafficking in post-MI myocardium and their draining lymphatic system. Furthermore, in vitro cell culture experiments and mechanism studies were undertaken to uncover the molecular mechanism by which LEC-S1pr1 regulated cardiac inflammation and remodeling after MI. Our results showed that S1pr1 expression significantly decreased in cardiac LECs after MI. Our in vivo experiments showed that the reduced expression of LEC-S1pr1 deteriorated cardiac function and worsened pathological cardiac remodeling after MI. Our further results demonstrated that the reduced expression of LEC-S1pr1 did not influence macrophage infiltration in an early inflammatory phase of MI, but significantly affected macrophages clearance in the later phase of MI via afferent cardiac lymphatics, and thus influenced inflammatory responses and cardiac outcome after MI. Further study showed that S1P/S1pr1 activated ERK signaling pathway and enhanced CCL2 expression, which promoted macrophage trafficking in a paracrine manner. This study reveals that cardiac lymphatic endothelial cells tightly control macrophage trafficking via lymphatic vessels in injured hearts via S1P/S1pr1/ERK/CCL2 pathway and thus regulate post-MI immune modulation and heart repair. This study highlights the importance of cardiac lymphatic vessel system in orchestrating post-MI immune responses and cardiac remodeling by regulating macrophage transit in injured hearts. Our finding implies that a feasible modulation of S1pr1 signaling in LECs might provide a promising target to resolve excessive inflammation and to ameliorate adverse cardiac remodeling after MI.

Abstract P3069: The Impact Of Lymphatic Vasculature Post-Cardiac Transplantation On Allograft Function, Fibrotic Remodeling, And Lymphatic Drainage

Lymphatic drainage inherently modulates cardiac function by maintaining the immune response and tissue-fluid homeostasis. During cardiac transplantation, the lymphatic collecting vessels are severed at the time of donor heart excision and not surgically reconstructed in the recipient. We hypothesize severed cardiac lymphatics contribute to transplant rejection and reduced cardiac function by influencing myocardial edema and immune cell transit. Methods: A heterotopic abdominal heart transplant (HAHT) rodent model permitted the immunologic evaluation of transplant rejection as supported in past literature. Echocardiography confirmed allograft survival via active contraction, while histologic quantification of lymphatic vessels and fibrosis were obtained for native and transplanted rodent hearts (n=3). Lymphatic drainage was assessed via intramyocardial injection of fluorescein isothiocyanate (FITC)-dextran. Results: Electrocardiographic impulses detected during the echocardiograms revealed additional QRS complexes attributed to contractions of the transplanted hearts. Histologic analysis of fibrosis in rodent allografts at an early timepoint exhibited increases in total tissue accumulation (1.48 – 5.15 fold) and significant increases in various focal regions (left ventricle, ***p < 0.001; interventricular septum, ****p < 0.0001) compared to the native hearts. Furthermore, large amounts of cellular infiltrate and marginal increases in cardiac diameter (1.10 – 1.42 fold) were observed in the transplants. While the scarcity of immunofluorescence signal for lymphatic markers has caused cardiac lymphatic quantification to be inconclusive, evaluation of lymphatic transport revealed fluctuating time-dependent circulating FITC levels (0.002 - 0.010 mg/mL; *p < 0.05) suggesting multi-teared compartmental release properties between the microcirculatory system, lymphatic system, and cardiac tissue. Conclusion: The HAHT rodent model provides a promising modality to assess both lymphatic transport and transplant rejection. Moreover, these studies will progress understanding of transplant rejection’s pathological intricacies and provide the groundwork for lymphangiogenic-based therapeutic intervention.

Regulation and impact of cardiac lymphangiogenesis in pressure-overload-induced heart failure.

Lymphatics are essential for cardiac health, and insufficient lymphatic expansion (lymphangiogenesis) contributes to development of heart failure (HF) after myocardial infarction. However, the regulation and impact of lymphangiogenesis in non-ischaemic cardiomyopathy following pressure-overload remains to be determined. Here, we investigated cardiac lymphangiogenesis following transversal aortic constriction (TAC) in C57Bl/6 and Balb/c mice, and in end-stage HF patients. Cardiac function was evaluated by echocardiography, and cardiac hypertrophy, lymphatics, inflammation, oedema, and fibrosis by immunohistochemistry, flow cytometry, microgravimetry, and gene expression analysis. Treatment with neutralizing anti-VEGFR3 antibodies was applied to inhibit cardiac lymphangiogenesis in mice. We found that VEGFR3-signalling was essential to prevent cardiac lymphatic rarefaction after TAC in C57Bl/6 mice. While anti-VEGFR3-induced lymphatic rarefaction did not significantly aggravate myocardial oedema post-TAC, cardiac immune cell levels were increased, notably myeloid cells at 3 weeks and T lymphocytes at 8 weeks. Moreover, whereas inhibition of lymphangiogenesis did not aggravate interstitial fibrosis, it increased perivascular fibrosis and accelerated development of left ventricular (LV) dilation and dysfunction. In clinical HF samples, cardiac lymphatic density tended to increase, although lymphatic sizes decreased, notably in patients with dilated cardiomyopathy. Similarly, comparing C57Bl/6 and Balb/c mice, lymphatic remodelling post-TAC was linked to LV dilation rather than to hypertrophy. The striking lymphangiogenesis in Balb/c was associated with reduced cardiac levels of macrophages, B cells, and perivascular fibrosis at 8 weeks post-TAC, as compared with C57Bl/6 mice that displayed weak lymphangiogenesis. Surprisingly, however, it did not suffice to resolve myocardial oedema, nor prevent HF development. We demonstrate for the first time that endogenous lymphangiogenesis limits TAC-induced cardiac inflammation and perivascular fibrosis, delaying HF development in C57Bl/6 but not in Balb/c mice. While the functional impact of lymphatic remodelling remains to be determined in HF patients, our findings suggest that under settings of pressure-overload poor cardiac lymphangiogenesis may accelerate HF development.

Regulation and impact of cardiac lymphangiogenesis in pressure-overload-induced heart failure

Lymphatics are essential for cardiac health, and insufficient lymphatic expansion (lymphangiogenesis) contributes to development of heart failure (HF) after myocardial infarction. However, the regulation and impact of lymphatics in non-ischemic cardiomyopathy induced by pressure-overload remains to be determined. Investigate cardiac lymphangiogenesis following transverse aortic constriction (TAC) in adult male or female C57Bl/6J or Balb/c mice, and in patients with end-stage HF. Cardiac function was evaluated by echocardiography, and cardiac hypertrophy, lymphatics, inflammation, edema, and fibrosis by immunohistochemistry, flow cytometry, microgravimetry, and gene expression analysis, respectively. Treatment with neutralizing anti-VEGFR3 antibodies was applied to inhibit cardiac lymphangiogenesis in mice. The gender- and strain-dependent mouse cardiac hypertrophic response to TAC, especially increased ventricular wall stress, led to lymphatic expansion in the heart. Our experimental findings that ventricular dilation triggered cardiac lymphangiogenesis was mirrored by observations in clinical HF samples, with increased lymphatic density found in patients with dilated cardiomyopathy. Surprisingly, the striking lymphangiogenesis observed post-TAC in Balb/c mice, linked to increased cardiac Vegfc, did not suffice to resolve myocardial edema, and animals progressed to dilated cardiomyopathy and HF. Conversely, selective inhibition of the essentially Vegfd-driven capillary lymphangiogenesis observed post-TAC in male C57Bl/6J mice did not significantly aggravate cardiac edema. However, cardiac immune cell levels were increased, notably myeloid cells at 3 weeks and T lymphocytes at 8 weeks. Moreover, while the TAC-triggered development of interstitial cardiac fibrosis was unaffected by anti-VEGFR3, inhibition of lymphangiogenesis increased perivascular fibrosis and accelerated the development of left ventricular dilation and cardiac dysfunction ( Fig. 1 ). We demonstrate for the first time that endogenous cardiac lymphangiogenesis limits pressure-overload-induced cardiac inflammation and perivascular fibrosis, thus delaying HF development. While these findings remain to be confirmed in a larger study of HF patients, we propose that under settings of pressure-overload poor cardiac lymphangiogenesis may accelerate HF development.