Lymphatic Vessel Development Research Articles

Development of the larval lymphatic system in zebrafish.

The lymphatic vascular system is a hierarchically organized complex network essential for tissue fluid homeostasis, immune trafficking and absorption of dietary fats in the human body. Despite its importance, the assembly of the lymphatic network is still not fully understood. The zebrafish is a powerful model organism that enables study of lymphatic vessel development using high-resolution imaging and sophisticated genetic and experimental manipulation. Although several studies have described early lymphatic development in the fish, lymphatic development at later stages has not been completely elucidated. In this study, we generated a new Tg(mrc1a:egfp)y251 transgenic zebrafish that uses a mannose receptor, C type 1 (mrc1a) promoter to drive strong EGFP expression in lymphatic vessels at all stages of development and in adult zebrafish. We used this line to describe the assembly of the major vessels of the trunk lymphatic vascular network, including the later-developing collateral cardinal, spinal, superficial lateral and superficial intersegmental lymphatics. Our results show that major trunk lymphatic vessels are conserved in the zebrafish, and provide a thorough and complete description of trunk lymphatic vessel assembly.

Building the drains: the lymphatic vasculature in health and disease.

The lymphatic vasculature is comprised of a network of endothelial vessels found in close proximity to but separated from the blood vasculature. An essential tissue component of all vertebrates, lymphatics are responsible for the maintenance of fluid homeostasis, dissemination of immune cells, and lipid reabsorption under healthy conditions. When lymphatic vessels are impaired due to invasive surgery, genetic disorders, or parasitic infections, severe fluid build-up accumulates in the affected tissues causing a condition known as lymphedema. Malignant tumors can also directly activate lymphangiogenesis and use these vessels to promote the spread of metastatic cells. Although their first description goes back to the times of Hippocrates, with subsequent anatomical characterization at the beginning of the 20th-century, the lack of identifying molecular markers and tools to visualize these translucent vessels meant that investigation of lymphatic vessels fell well behind research of blood vessels. However, after years under the shadow of the blood vasculature, recent advances in imaging technologies and new genetic and molecular tools have accelerated the pace of research on lymphatic vessel development. These new tools have facilitated both work in classical mammalian models and the emergence of new powerful vertebrate models like zebrafish, quickly driving the field of lymphatic development back into the spotlight. In this review, we summarize the highlights of recent research on the development and function of the lymphatic vascular network in health and disease. WIREs Dev Biol 2016, 5:689-710. doi: 10.1002/wdev.246 For further resources related to this article, please visit the WIREs website.

Structure of the full-length VEGFR-1 extracellular domain in complex with VEGF-A

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases: VEGFR-1, -2, and -3. Partial structures of VEGFR/VEGF complexes based on single-particle electron microscopy, small-angle X-ray scattering, and X-ray crystallography revealed the location of VEGF binding and domain arrangement of individual receptor subdomains. Here, we describe the structure of the full-length VEGFR-1 extracellular domain in complex with VEGF-A at 4 A resolution. We combined X-ray crystallography, single-particle electron microscopy, and molecular modeling for structure determination and validation. The structure reveals the molecular details of ligand-induced receptor dimerization, in particular of homotypic receptor interactions in immunoglobulin homology domains 4, 5, and 7. Functional analyses of ligand binding and receptor activation confirm the relevance of these homotypic contacts and identify them as potential therapeutic sites to allosterically inhibit VEGFR-1 activity.

Regulation of lymphangiogenesis in the diaphragm by macrophages and VEGFR-3 signaling.

Lymphatic vessels play important roles in fluid drainage and in immune responses, as well as in pathological processes including cancer progression and inflammation. While the molecular regulation of the earliest lymphatic vessel differentiation and development has been investigated in much detail, less is known about the control and timing of lymphatic vessel maturation in different organs, which often occurs postnatally. We investigated the time course of lymphatic vessel development on the pleural side of the diaphragmatic muscle in mice, the so-called submesothelial initial diaphragmatic lymphatic plexus. We found that this lymphatic network develops largely after birth and that it can serve as a reliable and easily quantifiable model to study physiological lymphangiogenesis in vivo. Lymphangiogenic growth in this tissue was highly dependent on vascular endothelial growth factor receptor (VEGFR)-3 signaling, whereas VEGFR-1 and -2 signaling was dispensable. During diaphragm development, macrophages appeared first in a linearly arranged pattern, followed by ingrowth of lymphatic vessels along these patterned lines. Surprisingly, ablation of macrophages in colony-stimulating factor-1 receptor (Csf1r)-deficient mice and by treatment with a CSF-1R-blocking antibody did not inhibit the general lymphatic vessel development in the diaphragm but specifically promoted branch formation of lymphatic sprouts. In agreement with these findings, incubation of cultured lymphatic endothelial cells with conditioned medium from P7 diaphragmatic macrophages significantly reduced LEC sprouting. These results indicate that the postnatal diaphragm provides a suitable model for studies of physiological lymphangiogenic growth and maturation, and for the identification of modulators of lymphatic vessel growth.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-016-9523-8) contains supplementary material, which is available to authorized users.

Dendritic cell-derived VEGF-A plays a role in inflammatory angiogenesis of human secondary lymphoid organs and is driven by the coordinated activation of multiple transcription factors.

Lymph node expansion during inflammation is essential to establish immune responses and relies on the development of blood and lymph vessels. Previous work in mice has shown that this process depends on the presence of VEGF-A produced by B cells, macrophages and stromal cells. In humans, however, the cell types and the mechanisms regulating the intranodal production of VEGF-A remain elusive. Here we show that CD11c+ cells represent the main VEGF-A-producing cell population in human reactive secondary lymphoid organs. In addition we find that three transcription factors, namely CREB, HIF-1α and STAT3, regulate the expression of VEGF-A in inflamed DCs. Both HIF-1α and STAT3 are activated by inflammatory agonists. Conversely, CREB phosphorylation represents the critical contribution of endogenous or exogenous PGE2. Taken together, these results propose a crucial role for DCs in lymph node inflammatory angiogenesis and identify novel potential cellular and molecular targets to limit inflammation in chronic diseases and tumors.

Positive Predictive Value of Mammographic Lymphography in the Evaluation of Patients with Breast Cancer: A Preliminary Study

The study aimed to investigate the positive predictive value (PPV) of mammographic lymphography (MLG) for assessing malignant breast disease and lymphatic metastasis in patients in a typical clinical setting. Patients who underwent mammography with Breast Imaging Reporting and Data System (BI-RADS) category 4 or 5 lesions and had abnormal mammographic findings in the upper-outer quadrant of the breast were enrolled. Next, MLG was performed. A water-soluble agent was subcutaneously injected into the upper-outer periareolar region of the bilateral breast, and mammography was then performed. Morphologic characteristics, including lymphatic vessel development, the presence of lymphatic vessel defects, and dilation, were recorded for evaluation. Fifty-one patients with BI-RADS category 4 lesions and 40 patients with BI-RADS category 5 lesions were included in the study. Sixty-one patients were found to have malignant disease, whereas 30 patients were found to have benign disease. Morphologic characteristics were recorded for evaluation. The interobserver agreement was evaluated and was classified as excellent according to kappa analysis. The PPV of MLG characteristics for malignant breast disease and lymphatic metastasis was analyzed by logistic regression, and the presentation of a lymphatic vessel defect was the most predictive characteristic of a malignancy (PPV: 0.89; P value: 0.02) in patients with BI-RADS category 4 lesions. Meanwhile, in patients with malignant breast disease, the PPVs for predicting lymphatic metastasis with lymphatic vessel defect and dilation were 0.50 (P value: 0.02) and 0.67 (P value: <0.01), respectively. The assessment of morphologic characteristics by MLG has the potential to predict malignant breast disease and lymphatic metastasis.

CHD4-regulated plasmin activation impacts lymphovenous hemostasis and hepatic vascular integrity.

The chromatin-remodeling enzyme CHD4 maintains vascular integrity at mid-gestation; however, it is unknown whether this enzyme contributes to later blood vessel or lymphatic vessel development. Here, we addressed this issue in mice harboring a deletion of Chd4 specifically in cells that express lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), which include lymphatic endothelial cells (LECs) and liver sinusoidal endothelial cells. Chd4 mutant embryos died before birth and exhibited severe edema, blood-filled lymphatics, and liver hemorrhage. CHD4-deficient embryos developed normal lymphovenous (LV) valves, which regulate the return of lymph to the blood circulation; however, these valves lacked the fibrin-rich thrombi that prevent blood from entering the lymphatic system. Transcripts of the urokinase plasminogen activator receptor (uPAR), which facilitates activation of the fibrin-degrading protease plasmin, were upregulated in Chd4 mutant LYVE1+ cells, and plasmin activity was elevated near the LV valves. Genetic reduction of the uPAR ligand urokinase prevented degradation of fibrin-rich thrombi at the LV valves and largely resolved the blood-filled lymphatics in Chd4 mutants. Urokinase reduction also ameliorated liver hemorrhage and prolonged embryonic survival by reducing plasmin-mediated extracellular matrix degradation around sinusoidal blood vessels. These results highlight the susceptibility of LV thrombi and liver sinusoidal vessels to plasmin-mediated damage and demonstrate the importance of CHD4 in regulating embryonic plasmin activation after mid-gestation.

Integration-free T cell-derived human induced pluripotent stem cells (iPSCs) from a patient with lymphedema-distichiasis syndrome (LDS) carrying an insertion–deletion complex mutation in the FOXC2 gene

Expanded human T cells from a Japanese male with lymphedema-distichiasis syndrome (LDS) were used to generate integration-free induced pluripotent stem cells (iPSCs) by exogenous expression of four reprogramming factors, OCT3/4, SOX2, cMYC, KLF4, using Sendai virus vector (SeVdp). The authenticity of established iPSC line, LDS-iPSC8, was confirmed by the expression of stem cell markers and the differentiation capability into three germ layers. LDS-iPSC8 may be a useful cell resource for the establishment of in vitro LDS modeling and the study for vascular and lymph vessel development.

Development of the lymphatic system: new questions and paradigms

The lymphatic system is a blind-ended network of vessels that plays important roles in mediating tissue fluid homeostasis, intestinal lipid absorption and the immune response. A profound understanding of the development of lymphatic vessels, as well as of the molecular cues governing their formation and morphogenesis, might prove essential for our ability to treat lymphatic-related diseases. The embryonic origins of lymphatic vessels have been debated for over a century, with a model claiming a venous origin for the lymphatic endothelium being predominant. However, recent studies have provided new insights into the origins of lymphatic vessels. Here, we review the molecular mechanisms controlling lymphatic specification and sprouting, and we discuss exciting findings that shed new light on previously uncharacterized sources of lymphatic endothelial cells.

Structure and decoy-mediated inhibition of the SOX18/Prox1-DNA interaction.

The transcription factor (TF) SOX18 drives lymphatic vessel development in both embryogenesis and tumour-induced neo-lymphangiogenesis. Genetic disruption of Sox18 in a mouse model protects from tumour metastasis and established the SOX18 protein as a molecular target. Here, we report the crystal structure of the SOX18 DNA binding high-mobility group (HMG) box bound to a DNA element regulating Prox1 transcription. The crystals diffracted to 1.75Å presenting the highest resolution structure of a SOX/DNA complex presently available revealing water structure, structural adjustments at the DNA contact interface and non-canonical conformations of the DNA backbone. To explore alternatives to challenging small molecule approaches for targeting the DNA-binding activity of SOX18, we designed a set of five decoys based on modified Prox1-DNA. Four decoys potently inhibited DNA binding of SOX18 in vitro and did not interact with non-SOX TFs. Serum stability, nuclease resistance and thermal denaturation assays demonstrated that a decoy circularized with a hexaethylene glycol linker and terminal phosphorothioate modifications is most stable. This SOX decoy also interfered with the expression of a luciferase reporter under control of a SOX18-dependent VCAM1 promoter in COS7 cells. Collectively, we propose SOX decoys as potential strategy for inhibiting SOX18 activity to disrupt tumour-induced neo-lymphangiogenesis.

Inhibition of endothelial Cdk5 reduces tumor growth by promoting non-productive angiogenesis.

Therapeutic success of VEGF-based anti-angiogenic tumor therapy is limited due to resistance. Thus, new strategies for anti-angiogenic cancer therapy based on novel targets are urgently required. Our previous in vitro work suggested that small molecule Cdk5 inhibitors affect angiogenic processes such as endothelial migration and proliferation. Moreover, we recently uncovered a substantial role of Cdk5 in the development of lymphatic vessels. Here we pin down the in vivo impact of endothelial Cdk5 inhibition in angiogenesis and elucidate the underlying mechanism in order to judge the potential of Cdk5 as a novel anti-angiogenic and anti-cancer target. By the use of endothelial-specific Cdk5 knockout mouse models and various endothelial and tumor cell based assays including human tumor xenograft models, we show that endothelial-specific knockdown of Cdk5 results in excessive but non-productive angiogenesis during development but also in tumors, which subsequently leads to inhibition of tumor growth. As Cdk5 inhibition disrupted Notch function by reducing the generation of the active Notch intracellular domain (NICD) and Cdk5 modulates Notch-dependent endothelial cell proliferation and sprouting, we propose that the Dll4/Notch driven angiogenic signaling hub is an important and promising mechanistic target of Cdk5. In fact, Cdk5 inhibition can sensitize tumors to conventional anti-angiogenic treatment as shown in tumor xenograft models. In summary our data set the stage for Cdk5 as a drugable target to inhibit Notch-driven angiogenesis condensing the view that Cdk5 is a promising target for cancer therapy.

Atypical chemokine receptor ACKR2 controls branching morphogenesis in the developing mammary gland.

Macrophages are important regulators of branching morphogenesis during development and postnatally in the mammary gland. Regulation of macrophage dynamics during these processes can therefore have a profound impact on development. We demonstrate here that the developing mammary gland expresses high levels of inflammatory CC-chemokines, which are essential in vivo regulators of macrophage migration. We further demonstrate that the atypical chemokine receptor ACKR2, which scavenges inflammatory CC-chemokines, is differentially expressed during mammary gland development. We have previously shown that ACKR2 regulates macrophage dynamics during lymphatic vessel development. Here, we extend these observations to reveal a novel role for ACKR2 in regulating the postnatal development of the mammary gland. Specifically, we show that Ackr2−/− mice display precocious mammary gland development. This is associated with increased macrophage recruitment to the developing gland and increased density of the ductal epithelial network. These data demonstrate that ACKR2 is an important regulator of branching morphogenesis in diverse biological contexts and provide the first evidence of a role for chemokines and their receptors in postnatal development processes.

Anatomy and development of the cardiac lymphatic vasculature: Its role in injury and disease.

Lymphatic vessels are present throughout the entire body in all mammals and function to regulate tissue fluid balance, lipid transport and survey the immune system. Despite the presence of an extensive lymphatic plexus within the heart, until recently the importance of the cardiac lymphatic vasculature and its origins were unknown. Several studies have described the basic anatomy of the developing cardiac lymphatic vasculature and more recently the detailed development of the murine cardiac lymphatics has been documented, with important insight into their cellular sources during embryogenesis. In this review we initially describe the development of systemic lymphatic vasculature, to provide the background for a comparative description of the spatiotemporal development of the cardiac lymphatic vessels, including detail of both canonical, typically venous, and noncanonical (hemogenic endothelium) cellular sources. Subsequently, we address the response of the cardiac lymphatic network to myocardial infarction (heart attack) and the therapeutic potential of targeting cardiac lymphangiogenesis.

Hemovascular Progenitors in the Kidney Require Sphingosine-1-Phosphate Receptor 1 for Vascular Development.

The close relationship between endothelial and hematopoietic precursors during early development of the vascular system suggested the possibility of a common yet elusive precursor for both cell types. Whether similar or related progenitors for endothelial and hematopoietic cells are present during organogenesis is unclear. Using inducible transgenic mice that specifically label endothelial and hematopoietic precursors, we performed fate-tracing studies combined with colony-forming assays and crosstransplantation studies. We identified a progenitor, marked by the expression of helix-loop-helix transcription factor stem cell leukemia (SCL/Tal1). During organogenesis of the kidney, SCL/Tal1(+) progenitors gave rise to endothelium and blood precursors with multipotential colony-forming capacity. Furthermore, appropriate morphogenesis of the kidney vasculature, including glomerular capillary development, arterial mural cell coating, and lymphatic vessel development, required sphingosine 1-phosphate (S1P) signaling via the G protein-coupled S1P receptor 1 in these progenitors. Overall, these results show that SCL/Tal1(+) progenitors with hemogenic capacity originate and differentiate within the early embryonic kidney by hemovasculogenesis (the concomitant formation of blood and vessels) and underscore the importance of the S1P pathway in vascular development.

A94: Immunohistochemical expression of Ki-67, CD34 and Podoplanin in hyperkeratosis and squamous cell carcinoma of the lower lip

Background Squamous cell carcinoma is the most common malignancy in the lower lip. Although lower lip squamous cell carcinoma is slow growing, up to 29% of tumors develop metastases to the cervical lymph nodes. Thus, identification of biological markers that could provide prognostic information about the invasive or metastatic potential of these lesions is important. It is known that angiogenesis, lymphangiogenesis and proliferation play an important role in tumor progression. Therefore, the goal of this study was to analyze the immunohistochemical expression of Ki-67, CD34 and Podoplanin in hyperkeratosis and in squamous cell carcinoma of the lower lip. It was intended to assess the possibility of using such markers as indicators of morphological aggressiveness of squamous cell carcinoma of the lower lip (LLSCC). Materials and methods Seventy-one cases of the lower lip lesions, obtained from the files of Novosibirsk Regional Oncology Center were selected for this study. The specimens were divided into three groups: a lower lip hyperkeratosis group consisting of 22 cases; LLSCC with keratinization consisting of 34 cases and LLSCC without keratinization consisting of 15 cases. To analyze angiogenesis, lymphangiogenesis and proliferation, we performed immunostains of the lower lip biopsy material for the CD34, vascular marker, Podoplanin, lymphatic-specific markers and Ki-67, marker of proliferation. Tissue samples were fixed in 10% neutral formalin, processed by standard histological techniques and embedded in paraffin. All steps of the immunohistochemical reaction were performed by using BENCHMARK/XT slide stainer (Ventana). The lymphatic and blood vessels volume density and Ki-67 cells numerical density were morphometrically analyzed in all groups and compared using the non-parametric Mann–Whitney test and the Wilcoxon signed rank test. A level of significance of 5% ( p Results All cases of a lower lip hyperkeratosis and LLSCC were positive for Ki-67, CD34 and Podoplanin. With respect to the pattern of staining, specimens exhibited a predominantly peripheral staining for CD34 and Podoplanin in inflammatory infiltrates and tumor sites. In contrast, staining for Ki-67 was predominantly central in inflammatory infiltrates and tumor sites in hyperkeratosis and LLSCC. When compared to lower lip hyperkeratosis, LLSCC (both with keratinization and without keratinization) showed a higher number of immunopositive Ki-67 cells (by 64% and 77%, respectively, p Conclusion This study has shown the greater development of blood and lymphatic vessels in LLSCC without keratinization in comparison with hyperkeratosis and LLSCC with keratinization, thus contributing to the development of metastasis.

Mafba is a downstream transcriptional effector of Vegfc signaling essential for embryonic lymphangiogenesis in zebrafish

The lymphatic vasculature plays roles in tissue fluid balance, immune cell trafficking, fatty acid absorption, cancer metastasis, and cardiovascular disease. Lymphatic vessels form by lymphangiogenesis, the sprouting of new lymphatics from pre-existing vessels, in both development and disease contexts. The apical signaling pathway in lymphangiogenesis is the VEGFC/VEGFR3 pathway, yet how signaling controls cellular transcriptional output remains unknown. We used a forward genetic screen in zebrafish to identify the transcription factor mafba as essential for lymphatic vessel development. We found that mafba is required for the migration of lymphatic precursors after their initial sprouting from the posterior cardinal vein. mafba expression is enriched in sprouts emerging from veins, and we show that mafba functions cell-autonomously during lymphatic vessel development. Mechanistically, Vegfc signaling increases mafba expression to control downstream transcription, and this regulatory relationship is dependent on the activity of SoxF transcription factors, which are essential for mafba expression in venous endothelium. Here we identify an indispensable Vegfc-SoxF-Mafba pathway in lymphatic development.

Lymph flow regulates collecting lymphatic vessel maturation in vivo.

Fluid shear forces have established roles in blood vascular development and function, but whether such forces similarly influence the low-flow lymphatic system is unknown. It has been difficult to test the contribution of fluid forces in vivo because mechanical or genetic perturbations that alter flow often have direct effects on vessel growth. Here, we investigated the functional role of flow in lymphatic vessel development using mice deficient for the platelet-specific receptor C-type lectin-like receptor 2 (CLEC2) as blood backfills the lymphatic network and blocks lymph flow in these animals. CLEC2-deficient animals exhibited normal growth of the primary mesenteric lymphatic plexus but failed to form valves in these vessels or remodel them into a structured, hierarchical network. Smooth muscle cell coverage (SMC coverage) of CLEC2-deficient lymphatic vessels was both premature and excessive, a phenotype identical to that observed with loss of the lymphatic endothelial transcription factor FOXC2. In vitro evaluation of lymphatic endothelial cells (LECs) revealed that low, reversing shear stress is sufficient to induce expression of genes required for lymphatic valve development and identified GATA2 as an upstream transcriptional regulator of FOXC2 and the lymphatic valve genetic program. These studies reveal that lymph flow initiates and regulates many of the key steps in collecting lymphatic vessel maturation and development.

Lymphatic vessel development: fluid flow and valve-forming cells.

Hemodynamic forces regulate many aspects of blood vessel disease and development, including susceptibility to atherosclerosis and remodeling of primary blood vessels into a mature vascular network. Vessels of the lymphatic circulatory system are also subjected to fluid flow-associated forces, but the molecular and cellular mechanisms by which these forces regulate the formation and maintenance of lymphatic vessels remain largely uncharacterized. This issue of the JCI includes two articles that begin to address how fluid flow influences lymphatic vessel development and function. Sweet et al. demonstrate that lymph flow is essential for the remodeling of primary lymphatic vessels, for ensuring the proper distribution of smooth muscle cells (SMCs), and for the development and maturation of lymphatic valves. Kazenwadel et al. show that flow-induced lymphatic valve development is initiated by the upregulation of GATA2, which has been linked to lymphedema in patients with Emberger syndrome. Together, these observations and future studies inspired by these results have potential to lead to the development of strategies for the treatment of lymphatic disorders.

Lymphangiogenesis and Inflammation-Looking for the "Missing Pieces" of the Puzzle.

Several papers about lymphangiogenesis and inflammation focused on the detailed and complicated descriptions of the molecular pathways accompanying both non-tumor and tumor inflammatory-induced lymphatic vessel development. Many authors are tempted to present inflammatory-induced lymphangiogenesis in pathologic conditions neglecting the role of inflammatory cells during embryonic lymphatic vessel development. Some of the inflammatory cells are largely characterized in inflammatory-induced lymphangiogenesis, while others as mast cells, eosinophils, or plasma cells are less studied. No phenotypic characterization of inflammation-activated lymphatic endothelial cell is available in this moment. Another paradox is related to the existence of few papers regarding lymphangiogenesis inside lymphoid organs and for their related pathology. There are still several "missing pieces of such a big puzzle" of lymphangiogenesis and inflammation, with a direct impact on the ineffectiveness of the anti-inflammatory therapy as lymphangiogenesis inhibitors. The present paper will focus on the controversial issues of lymphangiogenesis and inflammation.

Cdk5 and Foxc2--a new relationship in the lymphatic vasculature.

Lymphatic vessel dysfunction is associated with various pathologic conditions, including immunologic disorders, lymphedema, as well as tumor dissemination. Yet, the knowledge about the regulation of lymphatic vessel development is still limited. Our study elucidates cyclin dependent kinase 5 (Cdk5) as an essential player in the development of lymphatic vessels. Deletion of Cdk5 in the mouse endothelium results in severe lymphedema formation and embryonic lethality. On the mechanistic level, we show that Cdk5 phosphorylates the forkhead transcription factor Foxc2 which regulates Foxc2-dependent transcription. In summary, our study elucidates the Cdk5-Foxc2 interaction as a critical regulator of lymphatic vessel development.