Lymphatic Formation Research Articles

Multi-parameter tunable synthetic matrix for engineering lymphatic vessels

Controlling the formation of new lymphatic vessels has been postulated as an innovative therapeutic strategy for various disease phenotypes, including neurodegenerative diseases, metabolic syndrome, cardiovascular disease, and lymphedema. Yet, compared to the blood vascular system, little is known about the molecular regulation that controls lymphatic tube formation in a synthetic matrix. In this study, we utilize hyaluronic acid (HA)-hydrogels to design a novel platform for decoupled investigation into how mechanical and biochemical cues regulate lymphatic vessel formation in a synthetic matrix. Using HA and controlling the degree of modification provides a method to preserve and modulate key lymphatic markers Prox1, LYVE-1, and Pdpn. The chemistry of the system allows for spatial and temporal patterning of specific peptides and substrate stiffnesses, and an MMP-sensitive crosslinker allowed cells to degrade and remodel their matrix. Through systematic optimization of multiple parameters, we have designed a system that allows human lymphatic endothelial cells (LECs) to self-assemble into vessels in vitro within 3 days. These engineered vessels can be cultured for up to 3 weeks and can be used for high-throughput mechanistic studies, or can be implanted into immunodeficient mice where they have demonstrated the ability to integrate and mature. Collectively, these studies report a novel, fully-defined 3D synthetic matrix system capable of generating lymphatic vessels in vitro that provide promise as an in vitro screening platform and as a therapeutic vessel transplant, which to our knowledge, is the first ever 3D lymphatic tissue engineering approach to not require the use of support cells.

Lymphatic-specific methyltransferase-like 3-mediated m6A modification drives vascular patterning through prostaglandin metabolism reprogramming.

Lymphangiogenesis plays a pivotal role in the pathogenesis of various vascular disorders, including ocular vascular diseases and cancers. Deregulation of N 6-methyladenosine (m6A) modification has been identified as a key contributor to human diseases. However, the specific involvement of m6A modification in lymphatic remodeling remains poorly understood. In this study, we demonstrate that inflammatory stimulation and corneal sutures induce elevated levels of methyltransferase-like 3 (METTL3)-mediated m6A modification. METTL3 knockdown inhibits lymphatic endothelial viability, proliferation, migration, and tube formation in vitro. METTL3 knockdown attenuates corneal sutures-induced lymphangiogenesis and intratumoral lymphangiogenesis initiated by subcutaneous grafts, consequently restraining corneal neovascularization, tumor growth, and tumor neovascularization in vivo. Mechanistically, METTL3 knockdown upregulates prostaglandin-endoperoxide synthase 2 expression through an m6A-YTHDF2-dependent pathway, enhancing the synthesis of cyclopentenone prostaglandins (CyPGs). Aberrant CyPG production in lymphatic endothelial cells impairs mitochondrial oxidative phosphorylation, contributing to pathological lymphangiogenesis. Moreover, selective inhibition of METTL3 with STM2457 reduces m6A levels in lymphatic endothelial cells, effectively suppressing pathological lymphangiogenesis. This study provides compelling evidence that lymphatic-specific METTL3 plays a critical role in vascular patterning through prostaglandin metabolism reprogramming. Thus, METTL3 emerges as a promising target for treating lymphangiogenesis-related diseases.

The Study on Lymphatic Cysts Caused by Lymphadenectomy

<i>Background</i>: Traumatic lymphatic cyst, also known as cystic lymphangioma, is a multidisciplinary disease, with congenital lymphatic cysts being more common. Lymphatic cysts lack specific clinical manifestations, and the vast majority of cases are incidentally discovered during auxiliary examinations or surgeries, making diagnosis challenging. Surgery is the most effective treatment for this condition, with a favorable prognosis. Lymphatic cysts caused by lymphatic injury are clinically rare. This study theoretically analyzes the clinical characteristics of lymphatic cysts resulting from lymphatic injury. <i>Objective</i>: To explore the mechanisms by which lymphatic injury leads to lymphatic cyst formation and provide guidance for clinical treatment measures. <i>Methods</i>: The study involves analyzing the principles of lymphatic injury and examining the process of lymphatic cystic transformation and cyst formation. <i>Results</i>: The causes of lymphatic injury include mechanical cutting, thermal energy conduction from electrocautery, needle puncture, and trauma. <i>Conclusion</i>: There are various mechanisms and causes of lymphatic injury, and lymphatic injury is common during surgery. When lymphatic vessels are identified during surgery, they should be managed similarly to blood vessel ligation and disconnection. Lymphatic cysts are prone to recurrence, and complete resection is an effective treatment for this condition. Puncture and subtotal resection have a high recurrence rate.

The Importance of Intestinal Microbiota and Dysbiosis in the Context of the Development of Intestinal Lymphoma in Dogs and Cats.

Recent advancements have significantly enhanced our understanding of the crucial role animal microbiomes play in veterinary medicine. Their importance in the complex intestinal environment spans immune modulation, metabolic homeostasis, and the pathogenesis of chronic diseases. Dysbiosis, a microbial imbalance, can lead to a range of diseases affecting both individual organs and the entire organism. Microbial disruption triggers inflammatory responses in the intestinal mucosa and disturbs immune homeostasis, increasing susceptibility to toxins and their metabolites. These dynamics contribute to the development of intestinal lymphoma, necessitating rigorous investigation into the role of microbiota in tumorigenesis. The principles explored in this study extend beyond veterinary medicine to encompass broader human health concerns. There are remarkable parallels between the subtypes of lymphoproliferative disorders in animals and humans, particularly Hodgkin's lymphoma and non-Hodgkin's lymphoma. Understanding the etiology of a cancer of the lymphatic system formation is critical for developing both preventive strategies and therapeutic interventions, with the potential to significantly improve patient outcomes. The aim of this study is to discuss the optimal composition of the microbiome in dogs and cats and the potential alterations in the microbiota during the development of intestinal lesions, particularly intestinal lymphoma. Molecular and cellular analyses are also incorporated to detect inflammatory changes and carcinogenesis. A review of the literature on the connections between the gut microbiome and the development of lymphomas in dogs and cats is presented, along with potential diagnostic approaches for these cancers.

Multiple cis-regulatory elements control prox1a expression in distinct lymphatic vascular beds.

During embryonic development, lymphatic endothelial cell (LEC) precursors are distinguished from blood endothelial cells by the expression of Prospero-related homeobox1 (Prox1), which is essential for lymphatic vasculature formation in mouse and zebrafish. Prox1 expression initiation precedes LEC sprouting and migration, serving as the marker of specified LECs. Despite its crucial role in lymphatic development, Prox1 upstream regulation in LECs remains to be uncovered. SOX18 and COUP-TFII are thought to regulate Prox1 in mice by binding its promoter region. However, the specific regulation of Prox1 expression in LECs remains to be studied in detail. Here, we used evolutionary conservation and chromatin accessibility to identify enhancers located in the proximity of zebrafish prox1a active in developing LECs. We confirmed the functional role of the identified sequences through CRISPR/Cas9 mutagenesis of a lymphatic valve enhancer. The deletion of this region results in impaired valve morphology and function. Overall, our results reveal an intricate control of prox1a expression through a collection of enhancers. Ray-finned fish-specific distal enhancers drive pan-lymphatic expression, whereas vertebrate-conserved proximal enhancers refine expression in functionally distinct subsets of lymphatic endothelium.

The role of key biomarkers in lymphatic malformation: An updated review.

The lymphatic system, crucial for tissue fluid balance and immune surveillance, can be severely impacted by disorders that hinder its activities. Lymphatic malformations (LMs) are caused by fluid accumulation in tissues owing to defects in lymphatic channel formation, the obstruction of lymphatic vessels or injury to lymphatic tissues. Somatic mutations, varying in symptoms based on lesions' location and size, provide insights into their molecular pathogenesis by identifying LMs' genetic causes. In this review, we collected the most recent findings about the role of genetic and inflammatory biomarkers in LMs that control the formation of these malformations. A thorough evaluation of the literature from 2000 to the present was conducted using the PubMed and Google Scholar databases. Although it is obvious that the vascular endothelial growth factor receptor 3 mutation accounts for a significant proportion of LM patients, several mutations in other genes thought to be linked to LM have also been discovered. Also, inflammatory mediators like interleukin-6, interleukin-8, tumor necrosis factor-alpha and mammalian target of rapamycin are the most commonly associated biomarkers with LM. Understanding the mutations and genes expression responsible for the abnormalities in lymphatic endothelial cells could lead to novel therapeutic strategies based on molecular pathways.

Progenitor Cell Sources for 3D Bioprinting of Lymphatic Vessels and Potential Clinical Application.

The lymphatic system maintains tissue fluid homeostasis and it is involved in the transport of nutrients and immunosurveillance. It also plays a pivotal role in both pathological and regenerative processes. Lymphatic development in the embryo occurs by polarization and proliferation of lymphatic endothelial cells from the lymph sacs, that is, lymphangiogenesis. Alternatively, lymphvasculogenesis further contributes to the formation of lymphatic vessels. In adult tissues, lymphatic formation rarely occurs under physiological conditions, being restricted to pathological processes. In lymphvasculogenesis, progenitor cells seem to be a source of lymphatic vessels. Indeed, mesenchymal stem cells, adipose stem cells, endothelial progenitor cells, and colony-forming endothelial cells are able to promote lymphatic regeneration by different mechanisms, such as direct differentiation and paracrine effects. In this review, we summarize what is known on the diverse stem/progenitor cell niches available for the lymphatic system, emphasizing the potential that these cells hold for lymphatic tissue engineering through 3D bioprinting and their translation to clinical application.

Arsenic Trioxide Decreases Lymphangiogenesis by Inducing Apoptotic Pathways and Inhibition of Important Endothelial Cell Receptors.

Tumor-induced lymphangiogenesis is strongly associated with the formation of tumor metastasis. Therefore, the regulation of lymphangiogenesis offers a promising target in cancer therapy. Arsenic trioxide (ATO) is highly effective in the treatment of patients with acute promyelocytic leukemia (APL). As ATO mediates anti-angiogenic effects on endothelial and tumor cells, we aimed to explore the impact of ATO on lymphangiogenesis in human lymphatic endothelial cells (LEC). The BrdU assay and flow cytometry analysis were used to evaluate the influence of ATO on the proliferation and cell cycle distribution of LECs. The lymphatic suppression effects of ATO were investigated in vitro using the lymphatic tube formation assay. The effects of ATO on apoptosis, mitochondrial membrane potential and endothelial cell receptors were investigated by Western blotting, ELISA, flow cytometry and qRT-PCR. The treatment of LECs with ATO attenuated cell proliferation, blocked tube formation and induced subG0/G1 arrest in LECs, thus suggesting enhanced apoptosis. Although subG0/G1 arrest was accompanied by the upregulation of p21 and p53, ATO treatment did not lead to visible cell cycle arrest in LECs. In addition, ATO caused apoptosis via the release of cytochrome c from mitochondria, activating caspases 3, 8 and 9; downregulating the anti-apoptotic proteins survivin, XIAP and cIAP-2; and upregulating the pro-apoptotic protein Fas. Furthermore, we observed that ATO inhibited the VEGF-induced proliferation of LECs, indicating that pro-survival VEGF/VEGFR signaling was affected by ATO treatment. Finally, we found that ATO inhibited the expression of the important endothelial cell receptors VEGFR-2, VEGFR-3, Tie-2 and Lyve-1. In conclusion, we demonstrate that ATO inhibits lymphangiogenesis by activating apoptotic pathways and inhibiting important endothelial cell receptors, which suggests that this drug should be further evaluated in the treatment of tumor-associated lymphangiogenesis.

Hyaluronic Acid Hydrogels with Phototunable Supramolecular Cross-Linking for Spatially Controlled Lymphatic Tube Formation.

The dynamics of the extracellular matrix (ECM) influences stem cell differentiation and morphogenesis into complex lymphatic networks. While dynamic hydrogels with stress relaxation properties have been developed, many require detailed chemical processing to tune viscoelasticity, offering a limited opportunity for in situ and spatiotemporal control. Here, a hyaluronic acid (HA) hydrogel is reported with viscoelasticity that is controlled and spatially tunable using UV light to direct the extent of supramolecular and covalent cross-linking interactions. This is achieved using UV-mediated photodimerization of a supramolecular ternary complex of pendant trans-Brooker's Merocyanine (BM) guests and a cucurbit[8]uril (CB[8]) macrocycle. The UV-mediated conversion of this supramolecular complex to its covalent photodimerized form is catalyzed by CB[8], offering a user-directed route to spatially control hydrogel dynamics in combination with orthogonal photopatterning by UV irradiation through photomasks. This material thus achieves spatial heterogeneity of substrate dynamics, recreating features of native ECM without the need for additional chemical reagents. Moreover, these dynamic hydrogels afford spatial control of substrate mechanics to direct human lymphatic endothelial cells (LECs) to form lymphatic cord-like structures (CLS). Specifically, cells cultured on viscoelastic supramolecular hydrogels have enhanced formation of CLS, arising from increased expression of key lymphatic markers, such as LYVE-1, Podoplanin, and Prox1, compared to static elastic hydrogels prepared from fully covalent cross-linking. Viscoelastic hydrogels promote lymphatic CLS formation through the expression of Nrp2, VEGFR2, and VEGFR3 to enhance the VEGF-C stimulation. Overall, viscoelastic supramolecular hydrogels offer a facile route to spatially control lymphatic CLS formation, providing a tool for future studies of basic lymphatic biology and tissue engineering applications.

Abstract 13070: Interleukin-19 Regulates Lymphatic Permeability and Induces Prox1-Dependent Lymphangiogenesis

Introduction: Increased lymphangiogenesis is associated with atherosclerosis, but whether it is disease-causative or a compensatory attempt to attenuate disease is currently unresolved. Recent studies suggest lymphatic permeability plays a role in reverse cholesterol transport (RCT) as a possible mechanism for attenuating atherosclerosis. We previously reported that that Interleukin-19 (IL-19), an immunomodulatory cytokine, is unique by inducing angiogenesis while reducing atherosclerosis. The purpose of this study was to test the hypothesis that IL-19 could be atheroprotective by inducing functional lymphangiogenesis and promoting lymphatic flux. Methods & Results: RNA sequencing of IL-19-treated human Lymphatic Endothelial Cells (hLEC) identified induction of chemokines, pro-lymphangiogenic, and permeability-associated genes. Prox1, the master regulator of lymphatic formation, was increased by six-fold, validated by qPCR and western blot. Cellular assays further demonstrated IL-19-associated lymphangiogenesis through hLEC proliferation, migration, and tube formation, each mitigated by Prox1 siRNA knockdown. Angpt2, a regulator of permeability through VE-cadherin, is also upregulated by IL-19 and mitigated by Prox1 siRNA knockdown. Electric Cell-substrate Impedance Sensing (ECIS) showed that IL-19 mitigates oxLDL-associated decreases in permeability, suggestive of preventing lymphatic barrier malfunction during atherosclerosis. Preliminary in vivo lymphatic permeability assays suggest injection of rmIL-19 improves lymphatic function in conditional lymphatic-specific Prox1 heterozygous mice. Conversely, similar experiments show that Il19 / LDLR double knockout mice, which are known to have exacerbated atherosclerosis, also have significantly decreased lymphatic permeability compared to LDLR -/- mice after 16-weeks of high fat diet. Conclusion: These data suggest that IL-19 is a previously unrecognized regulator of lymphatic function as it increases lymphangiogenesis and permeability, with the potential to improve RCT. Together, these effects may regulate atherosclerosis, and influence our understanding of the association between the lymphatic system and atherosclerosis.

Lymphatic Drainage-Promoting Effects by Engraftment of Artificial Lymphatic Vascular Tissue Based on Human Adipose Tissue-Derived Mesenchymal Stromal Cells in Mice

Regenerative medicine using lymphatic vascular engineering is a promising approach for treating lymphedema. However, its development lags behind that of artificial blood vascular tissue for ischemic diseases. In this study, we constructed artificial 3D lymphatic vascular tissue, termed ASCLT, by co-cultivation of ECM-nanofilm-coated human adipose tissue-derived mesenchymal stromal cells (hASCs) and human dermal lymphatic endothelial cells (HDLECs). The effect of hASCs in lymphatic vessel network formation was evaluated by comparison with the tissue based on fibroblasts, termed FbLT. Our results showed that the density of lymphatic vascular network in ASCLT was higher than that in FbLT, demonstrating a promoting effect of hASCs on lymphatic vascular formation. This result was also supported by higher levels of lymphangiogenesis-promoting factors, such as bFGF, HGF, and VEGF-A in ASCLT than in FbLT. To evaluate the therapeutic effects, FbLTs and ASCLTs were subcutaneously transplanted to mouse hindlimb lymphatic drainage interruption models by removal of popliteal and subiliac lymph nodes. Despite the restricted engraftment of lymphatic vessels, ASCLT promoted regeneration of irregular and diverse lymphatic drainage in the skin, as visualized by indocyanine green imaging. Moreover, transplantation of ASCLT to the popliteal lymph node resection area also resulted in lymphatic drainage regeneration. Histological analysis of the generated drainage visualized by FITC-dextran injection revealed that the drainage was localized in the subcutaneous area shallower than the dermal muscle. These findings demonstrate that ASCLT promotes lymphatic drainage in vivo and that hASCs can serve as an autologous source for treatment of secondary lymphedema by tissue engineering.

Abstract 712: IL-19 Is A Lymphokine That Induces Prox1-dependent Lymphangiogenesis And Permeability

Introduction: Atherosclerosis is a vascular inflammatory disease associated with lymphangiogenesis, but whether lymphangiogenesis contributes to disease progression or is simply a compensatory attempt at disease resolution is currently unresolved. Recent studies are revealing a role for lymphatic vessels in reverse cholesterol transport (RCT), which could attenuate atherosclerosis. We have previously reported that Interleukin-19 (IL-19), an anti-inflammatory cytokine, uniquely attenuates atherosclerotic plaque progression while also being pro-angiogenic. This drives our hypothesis that one mechanism whereby IL-19 is atheroprotective is by promoting functional lymphangiogenesis and lymphatic flux. Methods & Results: IL-19 can significantly induce human dermal lymphatic endothelial cell (hdLEC) proliferation, migration, and tube formation, to the same degree as VEGFC, a potent inducer of lymphangiogenesis. RNA sequencing analysis indicates that IL-19 induces an angiogenic transcriptional program through Prox1, a master regulator of lymphangiogenesis. Knockdown of Prox1 led to a significant decrease in IL-19-associated proliferation and migration. IL-19 also increased expression of Angpt2, an endothelial cell protein that induces permeability through regulation of tight junctions, through a Prox1-dependent mechanism. Electric Cell-substrate Impedance Sensing assays showed that IL-19 treatment increased hdLEC permeability and mitigated oxLDL-associated decreased permeability, suggestive of maintaining lymphatic barrier function during atherosclerosis. IL-19 treatment also increased dil-HDL transport across an hdLEC barrier. Preliminary in vivo data suggests injection of rmIL-19 in Il19 -/- mice increases lymphatic permeability and flux through increased lymphatic uptake of Evans blue dye. Conclusion: These data suggest that IL-19 increases lymphangiogenesis, permeability, and may increase RCT to plausibly decrease atherosclerosis. These results can influence our understanding of the association between the lymphatic system and atherosclerosis.

In vivo vascularized scaffold with different shear-exposed models for lymphatic tissue regeneration

Current clinical treatments on lymphedema provide promising results, but also result in donor site morbidities. The establishment of a microenvironment optimized for lymphangiogenesis can be an alternative way to enhance lymphatic tissue formation. Hemodynamic flow stimuli have been confirmed to have an influential effect on angiogenesis in tissue engineering, but not on lymphatic vessel formation. Here, the three in vivo scaffolds generated from different blood stimuli in the subcutaneous layer, in the flow through pedicle, and in an arterio-venous (AV) loop model, were created to investigate potential of lymphangiogenesis of scaffolds containing lymphatic endothelial cells (LECs). Our results indicated that AV loop model displayed better lymphangiogenesis in comparison to the other two models with slower flow or no stimuli. Other than hemodynamic force, the supplement of LECs is required for lymphatic vessel regeneration. The in vivo scaffold generated from AV loop model provides an effective approach for engineering lymphatic tissue in the clinical treatment of lymphedema.

Hsa_circ_0000437 promotes pathogenesis of gastric cancer and lymph node metastasis.

Cellular communication between gastric cancer (GC) cells with different metastatic potentials and microenvironments and resultant cancer progression is not fully understood. Circular RNAs (circRNAs) and exosomal circRNAs are known to play extremely important regulatory roles in GC occurrence and progression. Here, we revealed significant differences in coronin-like actin-binding protein 1C (CORO1C) derived circRNA hsa_circ_0000437 between GC and para-cancer tissues. Hsa_circ_0000437 regulated GC cell proliferation, invasion, migration and apoptosis by targeting Ser/Arg-rich splicing factor 3 (SRSF3) and inhibiting programmed cell death 4 (PDCD4). The ectopic expression of hsa_circ_0000437 dramatically promoted tumor growth in nude mice in vivo. Furthermore, both gain-of-function and loss-of-function experiments demonstrated that hsa_circ_0000437 promoted human lymphatic endothelial cells (HLECs) invasion, migration, and tube formation in vitro and also promoted lymphangiogenesis and lymph node metastasis (LNM) in popliteal LNM model in vivo, when it was enriched in GC-secreted exosomes and transferred into HLECs. Mechanistically, exosomal hsa_circ_0000437 induced LNM via HSPA2-ERK signaling pathway independent of VEGF-C. Clinical data showed that exosomal hsa_circ_0000437 was enriched in the serum of GC patients, which was associated with LNM. In summary, these findings highlight the potential role of hsa_circ_0000437 as an outcome biomarker in GC patients with LNM, which may provide a novel target for GC therapy.

Enhancement of Lymphangiogenesis by Human Mesenchymal Stem Cell Sheet.

Preparation of human mesenchymal stem cell (hMSC) suspension for lymphedema treatment relies on conventional enzymatic digestion methods, which severely disrupts cell-cell and cell-extracellular matrix (ECM) connections, and drastically impairs cell retention and engraftment after transplantation. The objective of the present study is to evaluate the ability of hMSC-secreted ECM to augment lymphangiogenesis by using an in vitro coculturing model of hMSC sheets with lymphatic endothelial cells (LECs) and an in vivo mouse tail lymphedema model. Results demonstrate that the hMSC-secreted ECM augments the formation of lymphatic capillary-like structure by a factor of 1.2-3.6 relative to the hMSC control group, by serving as a prolymphangiogenic growth factor reservoir and facilitating cell regenerative activities. hMSC-derived ECM enhances MMP-2 mediated matrix remodeling, increases the synthesis of collagen IV and laminin, and promotes lymphatic microvessel-like structure formation. The injection of rat MSC sheet fragments into a mouse tail lymphedema model confirms the benefits of the hMSC-derived ECM by stimulating lymphangiogenesis and wound closure.

Paneth Cells Regulate Lymphangiogenesis under Control of Microbial Signals during Experimental Portal Hypertension.

Intestinal microbiota can modulate portal hypertension through the regulation of the intestinal vasculature. We have recently demonstrated that bacterial antigens activate Paneth cells (PCs) to secrete products that regulate angiogenesis and portal hypertension. In the present work we hypothesized that Paneth cells regulate the development of lymphatic vessels under the control of intestinal microbiota during experimental portal hypertension. We used a mouse model of inducible PCs depletion (Math1Lox/LoxVilCreERT2) and performed partial portal vein ligation (PPVL) to induce portal hypertension. After 14 days, we performed mRNA sequencing and evaluated the expression of specific lymphangiogenic genes in small intestinal tissue. Intestinal and mesenteric lymphatic vessels proliferation was assessed by immunohistochemistry. Intestinal organoids with or without PCs were exposed to pathogen-associated molecular patterns, and conditioned media (CM) was used to stimulate human lymphatic endothelial cells (LECs). The lymphangiogenic activity of stimulated LECs was assessed by tube formation and wound healing assays. Secretome analysis of CM was performed using label-free proteomics quantification methods. Intestinal immune cell infiltration was evaluated by immunohistochemistry. We observed that the intestinal gene expression pattern was altered by the absence of PCs only in portal hypertensive mice. We found a decreased expression of specific lymphangiogenic genes in the absence of PCs during portal hypertension, resulting in a reduced proliferation of intestinal and mesenteric lymphatic vessels as compared to controls. In vitro analyses demonstrated that lymphatic tube formation and endothelial wound healing responses were reduced significantly in LECs treated with CM from organoids without PCs. Secretome analyses of CM revealed that PCs secrete proteins that are involved in lipid metabolism, cell growth and proliferation. Additionally, intestinal macrophages infiltrated the ileal mucosa and submucosa of mice with and without Paneth cells in response to portal hypertension. Our results suggest that intestinal microbiota signals stimulate Paneth cells to secrete factors that modulate the intestinal and mesenteric lymphatic vessels network during experimental portal hypertension.

TGF-β1 mediates pathologic changes of secondary lymphedema by promoting fibrosis and inflammation.

BackgroundSecondary lymphedema is a common complication of cancer treatment, and previous studies have shown that the expression of transforming growth factor‐beta 1 (TGF‐β1), a pro‐fibrotic and anti‐lymphangiogenic growth factor, is increased in this disease. Inhibition of TGF‐β1 decreases the severity of the disease in mouse models; however, the mechanisms that regulate this improvement remain unknown.MethodsExpression of TGF‐β1 and extracellular matrix molecules (ECM) was assessed in biopsy specimens from patients with unilateral breast cancer‐related lymphedema (BCRL). The effects of TGF‐β1 inhibition using neutralizing antibodies or a topical formulation of pirfenidone (PFD) were analyzed in mouse models of lymphedema. We also assessed the direct effects of TGF‐β1 on lymphatic endothelial cells (LECs) using transgenic mice that expressed a dominant‐negative TGF‐β receptor selectively on LECs (LECDN‐RII).ResultsThe expression of TGF‐β1 and ECM molecules is significantly increased in BCRL skin biopsies. Inhibition of TGF‐β1 in mouse models of lymphedema using neutralizing antibodies or with topical PFD decreased ECM deposition, increased the formation of collateral lymphatics, and inhibited infiltration of T cells. In vitro studies showed that TGF‐β1 in lymphedematous tissues increases fibroblast, lymphatic endothelial cell (LEC), and lymphatic smooth muscle cell stiffness. Knockdown of TGF‐β1 responsiveness in LECDN‐RII resulted in increased lymphangiogenesis and collateral lymphatic formation; however, ECM deposition and fibrosis persisted, and the severity of lymphedema was indistinguishable from controls.ConclusionsOur results show that TGF‐β1 is an essential regulator of ECM deposition in secondary lymphedema and that inhibition of this response is a promising means of treating lymphedema.

Th2 Cytokines IL-4, IL-13, and IL-10 Promote Differentiation of Pro-Lymphatic Progenitors Derived from Bone Marrow Myeloid Precursors.

Myeloid-lymphatic endothelial cell progenitors (M-LECP) are a subset of bone marrow (BM)-derived cells characterized by expression of M2-type macrophage markers. We previously showed significant contribution of M-LECP to tumor lymphatic formation and metastasis in human clinical breast tumors and corresponding mouse models. Since M2 type is induced in macrophages by immunosuppressive Th2 cytokines IL-4, IL-13, and IL-10, we hypothesized that these factors might promote pro-lymphatic specification of M-LECP during their differentiation from BM myeloid precursors. To test this hypothesis, we analyzed expression of Th2 cytokines and their receptors in mouse BM cells under conditions leading to M-LECP differentiation, namely, CSF-1 treatment followed by activation of TLR4. We found that under these conditions, all three Th2 receptors were strongly upregulated in >95% of the cells that also secrete endogenous IL-10, but not IL-4 or IL-13 ligands. However, addition of any of the Th2 factors to CSF-1 primed cells significantly increased generation of myeloid-lymphatic progenitors as indicated by co-induction of lymphatic-specific (eg, Lyve-1, integrin-a9, collectin-12, and stabilin-1) and M2-type markers (eg, CD163, CD204, CD206, and PD-L1). Antibody-mediated blockade of either IL-10 receptor (IL-10R) or IL-10 ligand significantly reduced both immunosuppressive and lymphatic phenotypes. Moreover, tumor-recruited Lyve-1+ lymphatic progenitors in vivo expressed all Th2 receptors as well as corresponding ligands, including IL-4 and IL-13, which were absent in BM cells. This study presents original evidence for the significant role of Th2 cytokines in co-development of immunosuppressive and lymphatic phenotypes in tumor-recruited M2-type myeloid cells. Progenitor-mediated increase in lymphatic vessels can enhance immunosuppression by physical removal of stimulatory immune cells. Thus, targeting Th2 pathways might simultaneously relieve immunosuppression and inhibit differentiation of pro-lymphatic progenitors that ultimately promote tumor spread.

Endothelial Microparticle-Mediated Transfer of microRNA-19b Inhibits the Function and Distribution of Lymphatic Vessels in Atherosclerotic Mice

In recent years, the function of the lymphatic system in atherosclerosis has attracted attention due to its role in immune cell trafficking, cholesterol removal from the periphery, and regulation of the inflammatory response. However, knowledge of the mechanisms regulating lymphangiogenesis and lymphatic function in the pathogenesis of atherosclerosis is limited. Endothelial microparticles carrying circulating microRNA (miRNA)s are known to mediate cell–cell communication, and our previous research showed that miRNA-19b in EMPs (EMPmiR-19b) was significantly increased in circulation and atherosclerotic vessels, and this increase in EMPmiR-19b promoted atherosclerosis. The present study investigated whether atherogenic EMPmiR-19b influences pathological changes of the lymphatic system in atherosclerosis. We first verified increased miR-19b levels and loss of lymphatic system function in atherosclerotic mice. Atherogenic western diet-fed ApoE-/- mice were injected with phosphate-buffered saline, EMPs carrying control miRNA (EMPcontrol), or EMPmiR-19b intravenously. The function and distribution of the lymphatic system was assessed via confocal microscopy, Evans blue staining, and pathological analysis. The results showed that lymphatic system dysfunction existed in the early stage of atherosclerosis, and the observed pathological changes persisted at the later stage, companied by an increased microRNA-19b level. In ApoE-/- mice systemically treated with EMPmiR-19b, the distribution, transport function, and permeability of the lymphatic system were significantly inhibited. In vitro experiments showed that miRNA-19b may damage the lymphatic system by inhibiting lymphatic endothelial cell migration and tube formation, and a possible mechanism is the inhibition of transforming growth factor beta receptor type II (TGF-βRII) expression in lymphatic endothelial cells by miRNA-19b. Together, our findings demonstrate that atherogenic EMPmiR-19b may destroy lymphatic system function in atherosclerotic mice by downregulating TGF-βRII expression.

Abstract 497: IL-19 Induces Lymphangiogenesis Through A Prox1-dependent Mechanism

Introduction: The role of lymphangiogenesis in atherogenesis is currently under-characterized and controversial. It has been suggested that lymphangiogenesis is either protective by promoting plaque regression through reverse cholesterol transport (RCT) or deleterious by promoting plaque instability through white blood cell trafficking. We have previously reported that Interleukin-19 (IL-19), an anti-inflammatory cytokine, uniquely attenuates atherosclerotic plaque progression while also being pro-angiogenic. This drives our hypothesis that one mechanism whereby IL-19 is atheroprotective is by promoting lymphangiogenesis. Methods & Results: IL-19 can significantly induce primary human lymphatic endothelial cell (LEC) proliferation, migration, and tube formation, all angiogenic assays, to the same degree as VEGF-C, a known mediator of lymphangiogenesis. RNA sequencing (RNAseq) analysis indicates that IL-19 induces expression of chemokines as well as several angiogenic transcription factors in LECs. Notably, it induces expression of Prox1, a master transcription factor of lymphangiogenesis, 6.2-fold. The increases seen in chemokine and transcription factor expression from RNAseq were verified through qPCR analysis. Subsequent Prox1 siRNA knockdown prevented IL-19 associated LEC proliferation and migration. We then utilized an LDL Receptor Knockout mouse model fed a high fat diet for 16-weeks with daily injections of either recombinant mouse IL-19 or saline. Preliminary results suggest an increase in lymphatic vessel presence is associated with the known decrease IL-19 causes in atherosclerotic plaque progression. Transendothelial resistance assays were performed to determine IL-19's effect on lymphatic cell permeability. Preliminary data suggests that IL-19 alters LEC resistance, indicative of permeability and possibly RCT. We will also continue to evaluate RCT and inflammatory cell trafficking via intravital microscopy. Conclusion: These data suggest that IL-19 increases lymphatic vessel formation, likely through a Prox1-dependent mechanism. These results have the potential to shift our understanding toward a positive view of lymphangiogenesis and attenuation of atherosclerosis.