EMILIN1/α9β1 Integrin Interaction Is Crucial in Lymphatic Valve Formation and Maintenance

Lymphatic vasculature plays a crucial role in the maintenance of tissue interstitial fluid balance. The role of functional collecting lymphatic vessels in lymph transport has been recently highlighted in pathologies leading to lymphedema, for which treatments are currently unavailable. Intraluminal valves are of paramount importance in this process. However, valve formation and maturation have not been entirely elucidated yet, in particular, the role played by the extracellular matrix (ECM). We hypothesized that EMILIN1, an ECM multidomain glycoprotein, regulates lymphatic valve formation and maintenance. Using a mouse knockout model, we show that in the absence of EMILIN1, mice exhibit defects in lymphatic valve structure and in lymph flow. By applying morphometric in vitro and in vivo functional assays, we conclude that this impaired phenotype depends on the lack of α9β1 integrin engagement, the specific lymphatic endothelial cell receptor for EMILIN1, and the ensuing derangement of cell proliferation and migration. Our data demonstrate a fundamental role for EMILIN1-integrin α9 interaction in lymphatic vasculature, especially in lymphatic valve formation and maintenance, and underline the importance of this ECM component in displaying a regulatory function in proliferation and acting as a “guiding” molecule in migration of lymphatic endothelial cells.     

Intravital two-photon microscopy of lymphatic vessel development and function using a transgenic Prox1 promoter-directed mOrange2 reporter mouse

Lymphatic vessels, the second vascular system of higher vertebrates, are indispensable for fluid tissue homoeostasis, dietary fat resorption and immune surveillance. Not only are lymphatic vessels formed during fetal development, when the lymphatic endothelium differentiates and separates from blood endothelial cells, but also lymphangiogenesis occurs during adult life under conditions of inflammation, wound healing and tumour formation. Under all of these conditions, haemopoietic cells can exert instructive influences on lymph vessel growth and are essential for the vital separation of blood and lymphatic vessels. LECs (lymphatic endothelial cells) are characterized by expression of a number of unique genes that distinguish them from blood endothelium and can be utilized to drive reporter genes in a lymph endothelial-specific fashion. In the present paper, we describe the Prox1 (prospero homeobox protein 1) promoter-driven expression of the fluorescent protein mOrange2, which allows the specific intravital visualization of lymph vessel growth and behaviour during mouse fetal development and in adult mice.     

Conserved signaling through vascular endothelial growth (VEGF) receptor family members in murine lymphatic endothelial cells

Lymphatic vessels guide interstitial fluid, modulate immune responses by regulating leukocyte and antigen trafficking to lymph nodes, and in a cancer setting enable tumor cells to track to regional lymph nodes. The aim of the study was to determine whether primary murine lymphatic endothelial cells (mLECs) show conserved vascular endothelial growth factor (VEGF) signaling pathways with human LECs (hLECs). LECs were successfully isolated from murine dermis and prostate. Similar to hLECs, vascular endothelial growth factor (VEGF) family ligands activated MAPK and pAkt intracellular signaling pathways in mLECs. We describe a robust protocol for isolation of mLECs which, by harnessing the power of transgenic and knockout mouse models, will be a useful tool to study how LEC phenotype contributes to alterations in lymphatic vessel formation and function.     

Normal lymphatic development and function in mice deficient for the lymphatic hyaluronan receptor LYVE-1

The hyaluronan receptor LYVE-1 is expressed abundantly on the surfaces of lymphatic vessels and lymph node sinus endothelial cells from early development, where it has been suggested to function both in cell adhesion/transmigration and as a scavenger for hyaluronan turnover. To investigate the physiological role(s) of LYVE-1, we generated mice in which the gene for the receptor was inactivated by replacement with a beta-galactosidase reporter. LYVE-1(-/-) mice displayed an apparently normal phenotype, with no obvious alteration in lymphatic vessel ultrastructure or function and no apparent change in secondary lymphoid tissue structure or cellularity. In addition, the levels of hyaluronan in tissue and blood were unchanged. LYVE-1(-/-) mice also displayed normal trafficking of cutaneous CD11c(+) dendritic cells to draining lymph nodes via afferent lymphatics and normal resolution of oxazolone-induced skin inflammation. Finally, LYVE-1(-/-) mice supported normal growth of transplanted B16F10 melanomas and Lewis lung carcinomas. These results indicate that LYVE-1 is not obligatory for normal lymphatic development and function and suggest either the existence of compensatory receptors or a role more specific than that previously envisaged.     

Coxsackie- and adenovirus receptor (CAR) is expressed in lymphatic vessels in human skin and affects lymphatic endothelial cell function in vitro

Lymphatic vessels play an important role in tissue fluid homeostasis, intestinal fat absorption and immunosurveillance. Furthermore, they are involved in pathologic conditions, such as tumor cell metastasis and chronic inflammation. In comparison to blood vessels, the molecular phenotype of lymphatic vessels is less well characterized. Performing comparative gene expression analysis we have recently found that coxsackie- and adenovirus receptor (CAR) is significantly more highly expressed in cultured human, skin-derived lymphatic endothelial cells (LECs), as compared to blood vascular endothelial cells. Here, we have confirmed these results at the protein level, using Western blot and FACS analysis. Immunofluorescence performed on human skin confirmed that CAR is expressed at detectable levels in lymphatic vessels, but not in blood vessels. To address the functional significance of CAR expression, we modulated CAR expression levels in cultured LECs in vitro by siRNA- and vector-based transfection approaches. Functional assays performed with the transfected cells revealed that CAR is involved in distinct cellular processes in LECs, such as cell adhesion, migration, tube formation and the control of vascular permeability. In contrast, no effect of CAR on LEC proliferation was observed. Overall, our data suggest that CAR stabilizes LEC-LEC interactions in the skin and may contribute to lymphatic vessel integrity.     

Mouse chromosome 17 candidate modifier genes for thrombosis

Two overlapping quantitative trait loci (QTLs) for clot stability, Hmtb8 and Hmtb9, were identified on mouse chromosome 17 in an F2 intercross derived from C57BL/6J (B6) and B6-Chr17^A/J (B6-Chr17) mouse strains. The intervals were in synteny with a QTL for thrombotic susceptibility on chromosome 18 in a human study, and there were 23 homologs between mouse and human. The objective of this study was to determine whether any of these genes in the syntenic region are likely candidates as modifiers for clot stability. Seven genes, Twsg1, Zfp161, Dlgap1, Ralbp1, Myom1, Rab31, and Emilin2, of the 23 genes with single nucleotide polymorphisms (SNPs) in the mRNA-UTR had differential expression in B6 and A/J mice. Dlgap1, Ralbp1, Myom1, and Emilin2 also had nonsynonymous SNPs. In addition, two other genes had nonsynonymous SNPs, Lama1 and Ndc80. Of these nine candidate genes, Emilin2 was selected for further analysis since other EMILIN (Elastin Microfibril Interface Located Protein) proteins have known functions in vascular structure and coagulation. Differences were found between B6 and A/J mice in vessel wall architecture and EMILIN2 protein in plasma, carotid vessel wall, and thrombi formed after ferric chloride injury. In B6-Chr17^A/J mice both clot stability and Emilin2 mRNA expression were higher compared to those in B6 and A/J mice, suggesting the exposure of epistatic interactions. Although other homologous genes in the QTL region cannot be ruled out as causative genes, further investigation of Emilin2 as a candidate gene for thrombosis susceptibility is warranted.     

Human lymphatic endothelial cells express multiple functional TLRs

The lymphatic endothelium is the preferred route for the drainage of interstitial fluid from tissues and also serves as a conduit for peripheral dendritic cells (DCs) to reach draining lymph nodes. Lymphatic endothelial cells (LECs) are known to produce chemokines that recruit Ag-loaded DCs to lymphatic vessels and therefore are likely to regulate the migration of DCs to lymph nodes. TLRs are immune receptors that recognize pathogen associated molecular patterns and then signal and stimulate production of inflammatory chemokines and cytokines that contribute to innate and adaptive immune responses. TLRs are known to be expressed by a wide variety of cell types including leukocytes, epithelial cells, and endothelial cells. Because the TLR expression profile of LECs remains largely unexamined, we have undertaken a comprehensive study of the expression of TLR1-10 mRNAs and protein in primary human dermal (HD) and lung LECs as well as in htert-HDLECs, which display a longer life-span than HDLECs. We found that all three cell types expressed TLR1-6 and TLR9. The responsiveness of these LECs to a panel of ligands for TLR1-9 was measured by real-time RT-PCR, ELISA, and flow cytometry, and revealed that the LECs responded to most but not all TLR ligands by increasing expression of inflammatory chemokines, cytokines, and adhesion molecules. These findings provide insight into the ability of cells of the lymphatic vasculature to respond to pathogens and potential vaccine adjuvants and shape peripheral environments in which DCs will acquire Ag and environmental cues.     

Neutrophil elastase-dependent cleavage compromises the tumor suppressor role of EMILIN1

Proteolysis of the extracellular matrix (ECM) is a key event in tumor growth and progression. The breakdown of ECM can lead to the generation of bioactive fragments that promote cell growth and spread. EMILIN1, a multidomain glycoprotein expressed in several tissues, exerts a crucial regulatory function through the engagement of α4/α9 integrins. Unlike the majority of ECM molecules that elicit a proliferative program, the signals emitting from EMILIN1 engaged by α4/α9β1 integrins are antiproliferative. In this study, aimed to demonstrate if the suppressor role of EMILIN1 was related to its structural integrity, we tested the possibility that EMILIN1 could be specifically cleaved. Among the proteolytic enzymes released in the tumor microenvironment we showed that neutrophil elastase cleaved EMILIN1 in three/four major fragments. The consequence of this proteolytic process was the impairment of its anti-proliferative role. Accordingly, EMILIN1 was digested in sarcomas and ovarian cancers. Sarcoma specimens were infiltrated by neutrophils (PMNs) and stained positively for elastase. The present findings highlight the peculiar activity of PMN elastase in disabling EMILIN1 suppressor function.     

Heterogeneous expression of toll-like receptors in lymphatic endothelial cells derived from different tissues

As lymphatic endothelial cells (LECs) express different lymphatic and vascular markers depending on the organ they are derived from, we analysed whether they also show a heterogeneity of response against pathogens. To this end we analysed, for the presence of mRNA encoding for all human toll-like receptor (TLR), LECs isolated from lymph nodes and thymuses. RNA for TLR1-6 and 9 was identified in thymus-derived cells, whereas cells derived from lymph nodes contained mRNA for TLR1-4, 6 and 9, but failed to express mRNA specific for TLR5. The differential expression of TLRs was confirmed by the phosphorylation of nuclear factor-κB p65 only when the two types of LECs were incubated with the appropriate TLR agonists. The stimulation with specific agonists gives rise to a heterogeneous pattern of cytokine and chemokine secretion: thymus-derived LECs produced preferentially interleukin-6, interferon-inducible protein (IP)-10 and tumour necrosis factor-α, whereas cells prepared from lymph nodes mainly released interleukin-8, monocyte chemotactic protein-1, RANTES and (IP)-10. Finally, cells purified from lymph nodes expressed a higher level of intercellular adhesion molecule-1 than did cells prepared from the thymus when stimulated with several TLR agonists. The expression of a large set of TLRs and the responsiveness to specific agonists suggest that LECs are able to respond to pathogens, and the observed differences reflect specialized functions, redundancy and/or roles of LECs of different origin.     

Isolation and characterization of lymphatic microvascular endothelial cells from human tonsils

Human lymphatic endothelial cells (LECs) have isolated prevalently from human derma and tumors. As specialized lymphatic organs within the oropharynx, palatine tonsils are easily obtained and rich in lymphatic venules. Using a two-step purification method based on the sorting of endothelial cells with Ulex Europaeus Agglutinin 1 (UEA-1)-coated beads, followed by purification with monoclonal antibody D2-40, we successfully purified LECs from human palatine tonsils. The LECs were expanded on flasks coated with collagen type 1 and fibronectin for up to 8-10 passages and then analyzed for phenotypic and functional properties. Cultured cells retained the phenotypic pattern of the lymphatic endothelium of palatine tonsils and expressed functional VEGFR-3 molecules. In fact, stimulation with VEGFR-3 ligand, the vascular endothelium grow factor C, induced a marked increase in cell proliferation. Similarly to blood endothelial cells (BECs), LECs were able to form tube-like structure when seeded in Cultrex basement membrane extract. Comparative studies performed on LECs derived from palatine tonsils and iliac lymphatic vessels (ILVs), obtained with the same procedures, showed substantial discrepancies in the expression of various lymphatic markers. This points to the existence of micro- and macrovessel-derived LECs with different phenotypes, possibly involving different biological activities and functions. Palatine tonsil- and ILV-derived LECs may, therefore, represent new models for investigating function and biochemical properties of these lymphatic endothelia.     

Smooth muscle-endothelial cell communication activates Reelin signaling and regulates lymphatic vessel formation

Active lymph transport relies on smooth muscle cell (SMC) contractions around collecting lymphatic vessels, yet regulation of lymphatic vessel wall assembly and lymphatic pumping are poorly understood. Here, we identify Reelin, an extracellular matrix glycoprotein previously implicated in central nervous system development, as an important regulator of lymphatic vascular development. Reelin-deficient mice showed abnormal collecting lymphatic vessels, characterized by a reduced number of SMCs, abnormal expression of lymphatic capillary marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and impaired function. Furthermore, we show that SMC recruitment to lymphatic vessels stimulated release and proteolytic processing of endothelium-derived Reelin. Lymphatic endothelial cells in turn responded to Reelin by up-regulating monocyte chemotactic protein 1 (MCP1) expression, which suggests an autocrine mechanism for Reelin-mediated control of endothelial factor expression upstream of SMC recruitment. These results uncover a mechanism by which Reelin signaling is activated by communication between the two cell types of the collecting lymphatic vessels--smooth muscle and endothelial cells--and highlight a hitherto unrecognized and important function for SMCs in lymphatic vessel morphogenesis and function.     

Mice deficient in galectin-1 exhibit attenuated physiological responses to chronic hypoxia-induced pulmonary hypertension

Pulmonary hypertension (PH) is characterized by sustained vasoconstriction, with subsequent extracellular matrix (ECM) production and smooth muscle cell (SMC) proliferation. Changes in the ECM can modulate vasoreactivity and SMC contraction. Galectin-1 (Gal-1) is a hypoxia-inducible beta-galactoside-binding lectin produced by vascular, interstitial, epithelial, and immune cells. Gal-1 regulates SMC differentiation, proliferation, and apoptosis via interactions with the ECM, as well as immune system function, and, therefore, likely plays a role in the pathogenesis of PH. We investigated the effects of Gal-1 during hypoxic PH by quantifying 1) Gal-1 expression in response to hypoxia in vitro and in vivo and 2) the effect of Gal-1 gene deletion on the magnitude of the PH response to chronic hypoxia in vivo. By constructing and screening a subtractive library, we found that acute hypoxia increases expression of Gal-1 mRNA in isolated pulmonary mesenchymal cells. In wild-type (WT) mice, Gal-1 immunoreactivity increased after 6 wk of hypoxia. Increased expression of Gal-1 protein was confirmed by quantitative Western analysis. Gal-1 knockout (Gal-1(-/-)) mice showed a decreased PH response, as measured by right ventricular pressure and the ratio of right ventricular to left ventricular + septum wet weight compared with their WT counterparts. However, the number and degree of muscularized vessels increased similarly in WT and Gal-1(-/-) mice. In response to chronic hypoxia, the decrease in factor 8-positive microvessel density was similar in both groups. Vasoreactivity of WT and Gal-1(-/-) mice was tested in vivo and with use of isolated perfused lungs exposed to acute hypoxia. Acute hypoxia caused a significant increase in RV pressure in wild-type and Gal-1(-/-) mice; however, the response of the Gal-1(-/-) mice was greater. These results suggest that Gal-1 influences the contractile response to hypoxia and subsequent remodeling during hypoxia-induced PH, which influences disease progression.     

Deficiency of biglycan causes cardiac fibroblasts to differentiate into a myofibroblast phenotype

Myocardial infarction (MI) is followed by extracellular matrix (ECM) remodeling, which is on the one hand required for the healing response and the formation of stable scar tissue. However, on the other hand, ECM remodeling can lead to fibrosis and decreased ventricular compliance. The small leucine-rich proteoglycan (SLRP), biglycan (bgn), has been shown to be critically involved in these processes. During post-infarct remodeling cardiac fibroblasts differentiate into myofibroblasts which are the main cell type mediating ECM remodeling. The aim of the present study was to characterize the role of bgn in modulating the phenotype of cardiac fibroblasts. Cardiac fibroblasts were isolated from hearts of wild-type (WT) versus bgn(-/0) mice. Phenotypic characterization of the bgn(-/0) fibroblasts revealed increased proliferation. Importantly, this phenotype of bgn(-/0) fibroblasts was abolished to the WT level by reconstitution of biglycan in the ECM. TGF-β receptor II expression and phosphorylation of SMAD2 were increased. Furthermore, indicative of a myofibroblast phenotype bgn(-/0) fibroblasts were characterized by increased α-smooth muscle actin (α-SMA) incorporated into stress fibers, increased formation of focal adhesions, and increased contraction of collagen gels. Administration of neutralizing antibodies to TGF-β reversed the pro-proliferative, myofibroblastic phenotype. In vivo post-MI α-SMA, TGF-β receptor II expression, and SMAD2 phosphorylation were markedly increased in bgn(-/0) mice. Collectively, the data suggest that bgn deficiency promotes myofibroblast differentiation and proliferation in vitro and in vivo likely due to increased responses to TGF-β and SMAD2 signaling.     

Regulation of the extrinsic apoptotic pathway by the extracellular matrix glycoprotein EMILIN2

Elastin microfibril interface-located proteins (EMILINs) constitute a family of extracellular matrix (ECM) glycoproteins characterized by the presence of an EMI domain at the N terminus and a gC1q domain at the C terminus. EMILIN1, the archetype molecule of the family, is involved in elastogenesis and hypertension etiology, whereas the function of EMILIN2 has not been resolved. Here, we provide evidence that the expression of EMILIN2 triggers the apoptosis of different cell lines. Cell death depends on the activation of the extrinsic apoptotic pathway following EMILIN2 binding to the TRAIL receptors DR4 and, to a lesser extent, DR5. Binding is followed by receptor clustering, colocalization with lipid rafts, death-inducing signaling complex assembly, and caspase activation. The direct activation of death receptors by an ECM molecule that mimics the activity of the known death receptor ligands is novel. The knockdown of EMILIN2 increases transformed cell survival, and overexpression impairs clonogenicity in soft agar and three-dimensional growth in natural matrices due to massive apoptosis. These data demonstrate an unexpected direct and functional interaction of an ECM constituent with death receptors and discloses an additional mechanism by which ECM cues can negatively affect cell survival.     

Angiopoietin-2 promotes inflammatory lymphangiogenesis and its effect can be blocked by the specific inhibitor L1-10

Angiopoietin (Ang)-2, a ligand of the receptor tyrosine kinase Tie2, is known to be involved in the regulation of embryonic lymphangiogenesis. However, the role of Ang-2 in postnatal pathological lymphangiogenesis, such as inflammation, is largely unknown. We used a combination of imaging, molecular, and cellular approaches to investigate whether Ang-2 is involved in inflammatory lymphangiogenesis. We observed strong and continuous expression of Ang-2 on newly generated lymphatic vessels for 2 wk in sutured corneas of BALB/c mice. This expression was concurrent with an increased number of lymphatic vessels. TNF-α expression also increased, with peak TNF-α expression occurring before peak Ang-2 expression was reached. In vitro experiments showed that TNF-α stimulates Ang-2 and Tie2 and ICAM-1 expression on human lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BECs). Ang-2 alone did not affect the biological behavior of LECs, whereas Ang-2 combined with TNF-α significantly promoted the proliferation of LECs but not BECs. In mouse models, blockade of Ang-2 with L1-10, an Ang-2-specific inhibitor, significantly inhibited lymphangiogenesis but promoted angiogenesis. These results clearly indicate that Ang-2 acts as a crucial regulator of inflammatory lymphangiogenesis by sensitizing the lymphatic vasculature to inflammatory stimuli, thereby directly promoting lymphangiogenesis. The involvement of Ang-2 in inflammatory lymphangiogenesis provides a strong rationale for the exploitation of anti-Ang-2 treatment in the prevention and treatment of tumor metastasis and transplant rejection.     

EMILIN2 Regulates Platelet Activation,Thrombus Formation,and Clot Retraction

Thrombosis, like other cardiovascular diseases, has a strong genetic component, with largely unknown determinants. EMILIN2, Elastin Microfibril Interface Located Protein2, was identified as a candidate gene for thrombosis in mouse and human quantitative trait loci studies. EMILIN2 is expressed during cardiovascular development, on cardiac stem cells, and in heart tissue in animal models of heart disease. In humans, the EMILIN2 gene is located on the short arm of Chromosome 18, and patients with partial and complete deletion of this chromosome region have cardiac malformations. To understand the basis for the thrombotic risk associated with EMILIN2, EMILIN2 deficient mice were generated. The findings of this study indicate that EMILIN2 influences platelet aggregation induced by adenosine diphosphate, collagen, and thrombin with both EMILIN2-deficient platelets and EMILIN2-deficient plasma contributing to the impaired aggregation response. Purified EMILIN2 added to platelets accelerated platelet aggregation and reduced clotting time when added to EMILIN2-deficient mouse and human plasma. Carotid occlusion time was 2-fold longer in mice with platelet-specific EMILIN2 deficiency, but stability of the clot was reduced in mice with both global EMILIN2 deficiency and with platelet-specific EMILIN2 deficiency. In vitro clot retraction was markedly decreased in EMILIN2 deficient mice, indicating that platelet outside-in signaling was dependent on EMILIN2. EMILIN1 deficient mice and EMILIN2:EMILIN1 double deficient mice had suppressed platelet aggregation and delayed clot retraction similar to EMILIN2 mice, but EMILIN2 and EMILIN1 had opposing affects on clot retraction, suggesting that EMILIN1 may attenuate the effects of EMILIN2 on platelet aggregation and thrombosis. In conclusion, these studies identify multiple influences of EMILIN2 in pathophysiology and suggest that its role as a prothrombotic risk factor may arise from its effects on platelet aggregation and platelet mediated clot retraction.     

Spreds are essential for embryonic lymphangiogenesis by regulating vascular endothelial growth factor receptor 3 signaling

Spred/Sprouty family proteins negatively regulate growth factor-induced ERK activation. Although the individual physiological roles of Spred-1 and Spred-2 have been investigated using gene-disrupted mice, the overlapping functions of Spred-1 and Spred-2 have not been clarified. Here, we demonstrate that the deletion of both Spred-1 and Spred-2 resulted in embryonic lethality at embryonic days 12.5 to 15.5 with marked subcutaneous hemorrhage, edema, and dilated lymphatic vessels filled with erythrocytes. This phenotype resembled that of Syk^−/− and SLP-76^−/− mice with defects in the separation of lymphatic vessels from blood vessels. The number of LYVE-1-positive lymphatic vessels and lymphatic endothelial cells increased markedly in Spred-1/2-deficient embryos compared with WT embryos, while the number of blood vessels was not different. Ex vivo colony assay revealed that Spred-1/2 suppressed lymphatic endothelial cell proliferation and/or differentiation. In cultured cells, the overexpression of Spred-1 or Spred-2 strongly suppressed vascular endothelial growth factor-C (VEGF-C)/VEGF receptor (VEGFR)-3-mediated ERK activation, while Spred-1/2-deficient cells were extremely sensitive to VEGFR-3 signaling. These data suggest that Spreds play an important role in lymphatic vessel development by negatively regulating VEGF-C/VEGFR-3 signaling.